Thèses : « Plasmid resistance »

1

Udo, Edet Ekpenyong. « Characterisation and molecular studies of plasmids from Nigerian staphylococci ». Thesis, Curtin University, 1991. http://hdl.handle.net/20.500.11937/1845.

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Fifty three Staphylococcus aureus isolates were obtained from three centres, two hospitals and a private pathology laboratory, and studied for susceptibility to bacteriophages, resistance to antimicrobial agents and plasmid contents.Results of bacteriophage typing revealed that they belonged to a variety of phage types. Eighteen were untypable by any of the International Set of Phages, 16 belonged to phage group 111, nine to group I, four to group 11, two to group IV and two to the miscellaneous group.The isolates were resistant to one or more of methicillin (Mc), benzyl penicillin (Pc), gentamicin (Gm) , kanamycin (Km) , neomycin (Nm) , streptomycin (Sm) , trimethoprim (Tp), sulphonamides (Su), tetracycline (Tc), minocycline (Mn), chloramphenicol (Cm), novobiocin (Nb) and fusidic acid (Fa). Resistance to Pc was due to the production of beta-lactamase (Bla). No resistance to vancomycin, spectinomycin and erythromycin was detected. Resistance was also found to heavy metals such as cadmium (Cd), mercury (Hg), phenyl mercuric acetate (Pma), arsenate (Asa) and to the nucleic-acid binding compounds propamidine isethionate (Pi) and ethidium bromide (Eb).All but one of the isolates harboured plasmids. The number of plasmids the isolates carried varied from one to six and their sizes ranged from < 1.0 kb to c.48 kb.Location of the resistance determinants was ascertained by curing and transfer experiments. Loss of resistance was tested after growth at 43.5°C and transfer of resistance determinants was attempted by transduction, mixed-culture transfer and conjugation. The results revealed that resistance to Mc, Gm, Tp, Mn and Fa was chromosomal in all the resistant isolates and in some isolates Bla and resistance to Sm and Cd were chromosomal as well as plasmid encoded. In the majority of cases Bla and resistance to Km, Nm, Sm, Tc, Cin, Cd, Hg, Asa, Pma, Pi and Eb was encoded by plasmids.Conjugation experiments led to the isolation of three unique conjugative plasmids which have not been found to confer resistance to antimicrobials or to produce haemolysins or diffusible pigment (Dip). The three plasmids, pWBG620, pWBG637 and pWBG661, were indistinguishable by restriction endonuclease analysis and DNA-DNA hybridisation. However pWBG620, unlike pWBG637 and pWBG661, was not detected in the cytoplasm of its host and was only detected in transconjugants after it mobilised a non-conjugative Sm-resistance (SmR) plasmid. Further analysis indicated that it is integrated into the chromosome of its host, excises during conjugation and mobilises the SmR plasmid.These plasmids were studied further using pWBG637 as a representative. It was compared with representatives of the two groups of conjugative plasmids which have been reported in the staphylococci. These are the plasmids which encode resistance to Gm, Km and Nm and those which code for the production of diffusible pigment. The three types of conjugative plasmids were compared by restriction endonuclease analysis and DNA- DNA hybridisation and were found to be different. A preliminary restriction map of pWBG637 has also been constructed.However since pWBG637 has no resistance phenotype direct selection for it was not possible in transfer experiments and for incompatibility (Inc.). To study it further it was necessary to construct resistant derivatives which could be selected for in transfer experiments. This was achieved by labelling pWBG637 with resistance transposons to generate two conjugative plasmids, pWBG636 carrying an insert of Tn3851 (Gm- resistance) and pWBG642 carrying an insert of Tn551 (hn- resistance). It was found that transposon labelling had not changed the incompatibility of pWBG637 and therefore pWBG636 and pWBG642 were used in further experiments in place of pWBG637. Inc. tests with the pWBG637 derivatives revealed that the pWBG637 type of plasmid is not only different from the other two types of conjugative plasmids but is different from any of the described staphylococcal Inc. groups and therefore the pWBG637 type of plasmids represent a new Inc. group 15. The pWBG637 type of plasmids were studied further using plasmids pWBG636 and pWBG642. They were able to transfer conjugatively to a capsulated S.aureus strain either by the polyethylene glycol method or on filter membranes. They also transferred by conjugation to S. epidermidis and Streptococcus faecalis and were able to transfer back from these strains to S.aureus indicating that they also replicate in these hosts. Consequently they have been used to mobilise non-conjugative plasmids from S.epidermidis and non phage typable S.aureus. Both plasmids failed to transfer conjugatively to Bacillus subtilis and Escherichia coli.pWBG637 transferred non-conjugative plasmids by mobilising them in a manner similar to mobilisation (donation) in E.coli or by recombining with them to form new resistance plasmids. In one case, pWBG628 which encodes Bla and resistance to Cd, Km, Nm and Sm and has no homology with pWBG637 recombined with it during conjugation to produce three new conjugative plasmids pWBG629, pWBG630 and pWBG631 carrying resistance determinants from pWBG628. One of these plasmids, pWBG629, was found to be pWBG637 which had acquired a 4.5 kb element encoding resistance to Km, Nm and Sm. This element was shown to be transposable in both rec+ and rec- backgrounds and has been designated Tn3854. It expressed Sm resistance in E.coli and differs on this account from the Gram-negative transposon Tn5 which expresses resistance to Km, Nm and Sm in non-enteric bacteria but only resistance to Km and Nm in E. coli.Where possible the non-conjugative plasmids encoding resistance to antimicrobial agents were compared with phenotypically similar plasmids isolated from other parts of the world. It was found that the Tc and Sm resistance plasmids were closely related to other plasmids with the same phenotype whereas the Cm resistance plasmids were different.Although the majority of the Bla plasmids belonged to Inc. group 1 they demonstrated significant restriction enzyme fragment length polymorphism when compared with other Bla plasmids.This study has provided the first data on the genetics of antimicrobial resistance in Nigerian S.aureus. Although many of the plasmids studied were found to be similar to those previously described the isolates also contained some unique and previously undescribed plasmids.

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2

BERTINI, ALESSIA. « Plasmid-mediated antimicrobial resistance in Gram-negative bacteria ». Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2008. http://hdl.handle.net/2108/641.

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L’epidemiologia dei plasmidi di resistenza è il principale mezzo per descrivere la diffusione delle resistenze agli antimicrobici. L’identificazione di plasmidi correlati associati a specifici geni di resistenza può aiutare a tracciare la disseminazione di plasmidi epidemici, aprendo nuovi scenari epidemiologici sui meccanismi di diffusione delle resistenze agli antimicrobici. Questo aspetto è particolarmente evidente quando si considerano plasmidi che sono associati alla diffusione delle Beta Lattamasi a Spettro Esteso (ESBL) come gli enzimi CTX-M, SHV, TEM. Lo scopo di questa tesi è stato quello di caratterizzare plasmidi che conferiscono resistenza a farmaci rilevanti per la terapia umana in differenti collezioni di Enterobacteriaceae di origine umana e animale. Verranno discussi diversi esempi di plasmidi epidemici quali: i plasmidi IncHI2 che trasportano i geni blaCTX-M-9 o blaCTX-M-2 provenienti da isolati di origine umana e animale di Escherichia coli e Salmonella dalla Spagna, Belgio e Inghilterra; i plasmidi IncI1 caratterizzati da specifici fattori di virulenza, che trasportano i geni blaTEM-52 e blaCTX-M-1 derivanti da isolati di Salmonella e E. coli di origine umana e animale; i plasmidi IncN che trasportano i geni codificanti la metallo-β-lattamasi VIM-1 da isolati umani di Klebsiella pneumoniae and E. coli dalla Grecia; plasmidi IncA/C2 associati a specifici geni di resistenza come blaCMY-2, blaCMY-4, blaVIM-4 e blaVEB-1 provenienti da diverse Enterobacteriaceae isolate in differenti parti del mondo, ed infine, i plasmidi associati all’enzima SHV-12 isolati in diverse Enterobacteriaceae di origine umana e animale. L’analisi mediante comparazione della struttura plasmidica ha permesso di rilevare la diffusione di plasmidi emergenti e consente di tracciare i percorsi evolutivi e di diffusione attraverso l’acquisizione di una vastità di elementi genetici mobili associati ai geni di resistenza.
The epidemiology of resistance plasmids is a major issue for the description of antimicrobial resistance diffusion. The identification of related plasmids associated to specific resistance genes may help to follow the spread of epidemic plasmids, opening new epidemiological scenarios on the mechanism of diffusion of antimicrobial resistance. This aspect is particularly interesting when applied to collections of plasmids that are playing a major role in the diffusions of Extended Spectrum Beta Lactamases (ESBLs) such as CTX-M, SHV, TEM. The aim of this thesis is the molecular characterization of plasmids conferring drug resistances in different collections of Enterobacteriaceae of human and animal origin. Several example of epidemic plasmids will be discussed: the IncHI2 plasmids carrying blaCTX-M-9 or blaCTX-M-2 genes identified from human and animal isolates of Escherichia coli and Salmonella from Spain, Belgium and UK; the IncI1 family of plasmids characterized by specific virulence factors, carrying the blaCMY-2, blaTEM-52 and blaCTX-M-1 genes from Salmonella and E. coli of human and animal origin; the IncN plasmids carrying the gene codifying the metallo-β-lacatamase VIM-1 from human isolates of Klebsiella pneumoniae and E. coli in Greece; the IncA/C2 plasmids carrying specific resistance genes such as blaCMY-2, blaCMY-4, blaVIM-4 and blaVEB-1 from different Enterobacteriaceae isolated worldwide; different plasmid replicons (IncFII, IncA/C1, IncI1) carrying the ESBL SHV-12 from human and animal origin. The comparative analysis of plasmid backbones allowed to ascertain the diffusion of common, emerging plasmids and helped to determine the evolution of these plasmids by acquisition of relevant resistance genes by a panoply of mobile genetic elements and illegitimate recombination events.

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3

Chan, Helena. « Characterisation of a plasmid segregational stability determinant, par, of the staphylococcal multiresistance plasmid, pSK1 ». Thesis, The University of Sydney, 2017. http://hdl.handle.net/2123/17714.

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Staphylococcus aureus is a bacterium that is a common cause of hospital-acquired infections that is also becoming a significant cause of serious community-acquired infections. Many clinical strains of S. aureus carry extrachromosomal DNA elements called plasmids, which often encode genes conferring antimicrobial resistance. Plasmid segregation mechanisms such as active partitioning systems ensure that low copy-number plasmids are accurately segregated and inherited by progeny cells upon division, even in the absence of selection. The staphylococcal multiresistance plasmid, pSK1, contains a gene, par, which enhances the efficiency of plasmid inheritance, possibly via an active plasmid partitioning system. Active plasmid partitioning systems characterised thus far encode two individual proteins – a DNA-binding protein and a force-generating NTPase protein. Although the pSK1 par system is widespread among non-conjugative staphylococcal plasmids, it differs from characterised plasmid partitioning systems by encoding only a single protein, Par. Previous studies have shown that pSK1 Par exhibits both DNA-binding and multimerisation activities that are common to characterised partitioning proteins. Much remains to be elucidated about the mechanism by which pSK1 par facilitates efficient plasmid inheritance. This knowledge gap will be addressed by this experiments described in this thesis, which aim to 1) determine the functional significance the predicted Par domains, particularly the disordered C-terminal domain, 2) identify any host factors that interact with Par, and 3) determine the localisation of Par and plasmid DNA during plasmid segregation in S. aureus. These experiments are designed to provide a broader understanding of the mechanism of par-mediated plasmid inheritance, and provide greater insight into possible methods of disrupting the spread and maintenance of antimicrobial resistance plasmids.

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4

Bottery, Michael J. « The sociality and evolution of plasmid-mediated antimicrobial resistance ». Thesis, University of York, 2017. http://etheses.whiterose.ac.uk/19016/.

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Overuse and misuse of antibiotics has led to the global spread of antimicrobial resistance, threatening our ability to treat bacterial infections. The horizontal acquisition of multidrug resistance (MDR) plasmids, from other bacterial lineages, has been instrumental in spreading resistance. Newly acquired plasmids are often poorly adapted to hosts causing intragenomic conflicts, reducing the competitiveness of plasmid-carrying strains. Costs can be overcome by positive selection for plasmid-encoded adaptive traits in the short-term, or ameliorated by compensatory evolution in the long-term. How the selection and adaptation of MDR plasmids varies with antibiotic treatment remains unclear. First, I demonstrate that the selective conditions for the maintenance of an MDR plasmid are dependent upon the sociality of resistance it encodes. Selection for efflux of antibiotics, a selfish trait, occurred at very low concentrations of antibiotic, far below the minimum inhibitory concentration of sensitive plasmid-free strain. In contrast, selection for inactivation of antibiotics, a cooperative trait, increased the amount of antibiotic required to select for the MDR plasmid, allowing sensitive plasmid-free bacteria to survive high levels of antibiotic. These selection dynamics were only accurately predicted when mathematical models included the mechanistic details of antibiotic resistance. Secondly, I show that the trajectory of evolution following MDR plasmid acquisition varies with antibiotic treatment. Tetracycline treatment favoured a distinct coevolutionary trajectory of chromosomal resistance mutations coupled with plasmid mutations impairing plasmid-borne resistance. This led to high-level, low-cost antibiotic resistance, but also produced an integrated genome of co-dependent replicons that may limit the onward spread of co-adapted MGEs to other lineages. This evolutionary trajectory was strikingly repeatable across independently evolving populations despite the emergence of multiple competing lineages within populations. The results presented here demonstrate that the interaction between positive selection and compensatory evolution can help to explain the persistence of MDR plasmids in the clinic and the environment.

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5

Udo, Edet Ekpenyong. « Characterisation and molecular studies of plasmids from Nigerian staphylococci ». Curtin University of Technology, School of Biomedical Sciences, 1991. http://espace.library.curtin.edu.au:80/R/?func=dbin-jump-full&object_id=15648.

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Fifty three Staphylococcus aureus isolates were obtained from three centres, two hospitals and a private pathology laboratory, and studied for susceptibility to bacteriophages, resistance to antimicrobial agents and plasmid contents.Results of bacteriophage typing revealed that they belonged to a variety of phage types. Eighteen were untypable by any of the International Set of Phages, 16 belonged to phage group 111, nine to group I, four to group 11, two to group IV and two to the miscellaneous group.The isolates were resistant to one or more of methicillin (Mc), benzyl penicillin (Pc), gentamicin (Gm) , kanamycin (Km) , neomycin (Nm) , streptomycin (Sm) , trimethoprim (Tp), sulphonamides (Su), tetracycline (Tc), minocycline (Mn), chloramphenicol (Cm), novobiocin (Nb) and fusidic acid (Fa). Resistance to Pc was due to the production of beta-lactamase (Bla). No resistance to vancomycin, spectinomycin and erythromycin was detected. Resistance was also found to heavy metals such as cadmium (Cd), mercury (Hg), phenyl mercuric acetate (Pma), arsenate (Asa) and to the nucleic-acid binding compounds propamidine isethionate (Pi) and ethidium bromide (Eb).All but one of the isolates harboured plasmids. The number of plasmids the isolates carried varied from one to six and their sizes ranged from < 1.0 kb to c.48 kb.Location of the resistance determinants was ascertained by curing and transfer experiments. Loss of resistance was tested after growth at 43.5°C and transfer of resistance determinants was attempted by transduction, mixed-culture transfer and conjugation. The results revealed that resistance to Mc, Gm, Tp, Mn and Fa was chromosomal in all the resistant isolates and in some isolates Bla and resistance to Sm and Cd were chromosomal as well as plasmid encoded. In the majority of cases Bla and resistance to Km, Nm, Sm, Tc, Cin, Cd, Hg, Asa, Pma, Pi and Eb was encoded ++
by plasmids.Conjugation experiments led to the isolation of three unique conjugative plasmids which have not been found to confer resistance to antimicrobials or to produce haemolysins or diffusible pigment (Dip). The three plasmids, pWBG620, pWBG637 and pWBG661, were indistinguishable by restriction endonuclease analysis and DNA-DNA hybridisation. However pWBG620, unlike pWBG637 and pWBG661, was not detected in the cytoplasm of its host and was only detected in transconjugants after it mobilised a non-conjugative Sm-resistance (SmR) plasmid. Further analysis indicated that it is integrated into the chromosome of its host, excises during conjugation and mobilises the SmR plasmid.These plasmids were studied further using pWBG637 as a representative. It was compared with representatives of the two groups of conjugative plasmids which have been reported in the staphylococci. These are the plasmids which encode resistance to Gm, Km and Nm and those which code for the production of diffusible pigment. The three types of conjugative plasmids were compared by restriction endonuclease analysis and DNA- DNA hybridisation and were found to be different. A preliminary restriction map of pWBG637 has also been constructed.However since pWBG637 has no resistance phenotype direct selection for it was not possible in transfer experiments and for incompatibility (Inc.). To study it further it was necessary to construct resistant derivatives which could be selected for in transfer experiments. This was achieved by labelling pWBG637 with resistance transposons to generate two conjugative plasmids, pWBG636 carrying an insert of Tn3851 (Gm- resistance) and pWBG642 carrying an insert of Tn551 (hn- resistance). It was found that transposon labelling had not changed the incompatibility of pWBG637 and therefore pWBG636 and pWBG642 were used in further experiments in place of pWBG637. Inc. ++
tests with the pWBG637 derivatives revealed that the pWBG637 type of plasmid is not only different from the other two types of conjugative plasmids but is different from any of the described staphylococcal Inc. groups and therefore the pWBG637 type of plasmids represent a new Inc. group 15. The pWBG637 type of plasmids were studied further using plasmids pWBG636 and pWBG642. They were able to transfer conjugatively to a capsulated S.aureus strain either by the polyethylene glycol method or on filter membranes. They also transferred by conjugation to S. epidermidis and Streptococcus faecalis and were able to transfer back from these strains to S.aureus indicating that they also replicate in these hosts. Consequently they have been used to mobilise non-conjugative plasmids from S.epidermidis and non phage typable S.aureus. Both plasmids failed to transfer conjugatively to Bacillus subtilis and Escherichia coli.pWBG637 transferred non-conjugative plasmids by mobilising them in a manner similar to mobilisation (donation) in E.coli or by recombining with them to form new resistance plasmids. In one case, pWBG628 which encodes Bla and resistance to Cd, Km, Nm and Sm and has no homology with pWBG637 recombined with it during conjugation to produce three new conjugative plasmids pWBG629, pWBG630 and pWBG631 carrying resistance determinants from pWBG628. One of these plasmids, pWBG629, was found to be pWBG637 which had acquired a 4.5 kb element encoding resistance to Km, Nm and Sm. This element was shown to be transposable in both rec+ and rec- backgrounds and has been designated Tn3854. It expressed Sm resistance in E.coli and differs on this account from the Gram-negative transposon Tn5 which expresses resistance to Km, Nm and Sm in non-enteric bacteria but only resistance to Km and Nm in E. coli.Where possible the non-conjugative plasmids encoding resistance to ++
antimicrobial agents were compared with phenotypically similar plasmids isolated from other parts of the world. It was found that the Tc and Sm resistance plasmids were closely related to other plasmids with the same phenotype whereas the Cm resistance plasmids were different.Although the majority of the Bla plasmids belonged to Inc. group 1 they demonstrated significant restriction enzyme fragment length polymorphism when compared with other Bla plasmids.This study has provided the first data on the genetics of antimicrobial resistance in Nigerian S.aureus. Although many of the plasmids studied were found to be similar to those previously described the isolates also contained some unique and previously undescribed plasmids.

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Coons, Terry M. « Restriction mapping and expression of recombinant plasmids containing the arsenic resistance genes of the plasmid R45 ». PDXScholar, 1986. https://pdxscholar.library.pdx.edu/open_access_etds/3597.

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The trivalent (arsenite) and pentavalent (arsenate) forms of arsenic are introduced into the environment through the use of arsenic in herbicides, pesticides, fertilizers, and the smelting of arsenic-bearing ores. Bacteria resistant to arsenic are readily isolated from surface waters, sewage, and clinical infections. Although some bacterial resistance is provided by inducible phosphate transport systems that discriminate against arsenate, marked resistance is carried on bacterial plasmids. A 6.9 kilobase fragment previously derived from one such plasmid, R45, and containing the genes for inducible resistance to arsenite and arsenate was ligated into the cloning vectors puce and pUC9 in opposite orientations and transformed into Escherichia coli JM 105. Insertion into the multiple cloning site of the pUC vectors places the inserted fragment under the inducible control of the lac operon promoter. An attempt was made to determine the direction of transcription in the fragment by growth in 10-3 M isopropyl-β-D-thiogalactoside prior to challenge with arsenite.

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7

Tavakoli, Norma Parvin. « Characterisation of the plasmid pUB2380 and in particular its transposition system ». Thesis, University of Bristol, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386071.

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8

Evans, Jane E. « The conjugation system of Staphylococcus aureus ». Thesis, University of Oxford, 1986. https://ora.ox.ac.uk/objects/uuid:1c1f5c11-f854-4af5-b9cf-34fdf279fb28.

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A conjugation system in Staphylococcus aureus has been investigated and shown to be determined, at least in part, by genes carried on plasmids. Conjugation required cell-to-cell contact but not calcium ions. The frequency of conjugation depended on the recipient used and on the incubation conditions. Two conjugative plasmids were mapped by restriction enzyme analysis but experiments to clone the conjugation-determining region were unsuccessful although separate regions specifying gentamicin resistance, ethidium bromide resistance and cadmium resistance were cloned. The gentamicin resistance determinant was probably part of Tn4001. Deletion of various sized pieces of DNA from one of the plasmids resulted in reduction of its ability to specify conjugation but no specific part of this plasmid could be implicated in the process. Further experiments led to the conclusion that this particular plasmid (p8325-4) is probably not self-transmissible but transferred by a phage-mediated system. Strains of Staphylococcus aureus produced a pheromone-like substance that elicited a clumping response in Streptococcus faecalis but no evidence was found for the involvement of staphylococcal conjugative plasmids in this. The conjugative plasmid, p8325-2, mobilized a small plasmid (pT181) but not a chromosomal gene. Insertion of transposon Tn551 was used to produce mutants of the conjugative plasmid p8325-2. Some twenty-six mutants were studied and the position of Tn551 in them mapped. There were preferred regions of insertion for Tn551 and twenty out of the twenty-six mutants had altered ability to conjugate. One showed a significantly higher frequency of conjugation and the other nineteen, all with substantially lower frequencies of conjugation, were mapped to two well-separated regions of the plasmid. Similarity between the locations of these putative regions and those reported for some other conjugative plasmids from staphylococci is striking and suggests a common origin.

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9

Eldek, Ahmed. « Antibiotic-Regulated Plasmid Copy Number Variation : A Driver of Antibiotic Resistance ? » Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-388523.

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Plasmids are small circular DNA molecules within bacterial cells that are separated from the bacterial chromosome and replicate independently. Also, they play a crucial role in the dissemination of antibiotic resistance genes among bacteria through horizontal gene transfer. They can be present in many copies within host cell, which is known as plasmid copy number. Plasmids can regulate their own copy number by different mechanisms. Additionally, the selective pressure can also play a pivotal role in determining plasmid copy number. The presence of antibiotics in the surrounding environment can drive variations of plasmid copy number. In this study, we examined plasmid copy number variations of multidrug resistance plasmids in presence of antibiotics by using EvaGreen® - based multiplexed digital droplet PCR. We could observe that cultures of Klebsiella pneumoniae and Escherichia coli harboring multidrug resistance plasmids grown in presence of sub-MIC concentrations of the antibiotics did not show high variations in plasmid copy numbers. On the other hand, mutants of K. pneumoniae selected for increased antibiotic resistance showed high increases in copy number of a multidrug-resistance plasmid.

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Khan, Azra. « An investigation into the association of plasmid-borne qacAB and antimicrobial resistance in meticillin-resistant Staphylococcus aureus ». Thesis, University of Portsmouth, 2013. https://researchportal.port.ac.uk/portal/en/theses/an-investigation-into-the-association-of-plasmidborne-qacab-and-antimicrobial-resistance-in-meticillinresistant-staphylococcus-aureus(49b2b0fc-936a-412a-b418-8d9d24d3b531).html.

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Meticillin-resistant Staphylococcus aureus (MRSA) is globally recognised as a major causative organism of hospital acquired infection (HAI) and continues to present many challenges for infection prevention and control. Once established within hospitals and healthcare centers, the control of spread of MRSA and therapy is difficult due to resistance to otherwise effective antimicrobials. Government initiatives in the United Kingdom (UK) have led to considerable investments in improving infection control practices, with emphasis on improving hand hygiene compliance of healthcare professionals and hospital environmental cleanliness to control the spread and limit the source of MRSA and other HAIs. This has resulted in the subsequent increase in disinfectant and antiseptic usage containing, quaternary ammonium compounds (QACs), cationic biocides such as chlorhexidine and the bisphenol ether, triclosan, for decontamination of surfaces and disinfection of skin. Thus, there is serious concern that as with antibiotic resistance, continual and intensive exposure of MRSA (and other hospital pathogens) to biocides, may result in the emergence of resistance to these agents with further detrimental consequences and substantial burden for prevention, treatment and control of hospital infections. MRSA carry a number of plasmid-borne qac genes, predominantly qacA, qacB and smr that encode resistance to commonly used antiseptics and disinfectants in hospitals, nursing homes and other healthcare establishments. The proteins encoded by qacA and qacB mediate efflux via active transport; QacA multidrug exporter mediates resistance to monovalent, divalent cationic and lipophilic antimicrobial compounds, whilst the closely related export protein QacB mediates lower levels of resistance to divalent cations. In this research a “snapshot” study of hospital strains of MRSA stored at the Hospital Infection Research Laboratory (HIRL), City Hospital, Birmingham, was carried out to determine the prevalence and distribution of qacAB in these isolates and determine a possible association between presence of these genes and biocide resistance. The intercalating dye, ethidium bromide (EtBr) is a substrate for many S. aureus multi-drug resistant (MDR) efflux pumps and was used in the present study as a marker for detection of efflux pump activity. Previous studies have reported that MRSA strains with an MIC of ≥ 64 mg/L to EtBr have qacAB, however, the present study used a lower baseline value of ≥ 32 mg/L resistance to EtBr to capture any isolates with low MICs that may have qacAB and may be missed. Initially 3,400 MRSA strains collected between October 2002 and October 2006 were screened to identify and select isolates with ≥ 32 mg/L resistance to EtBr. A second MRSA collection stored at the Antimicrobial Chemotherapy Laboratory, City Hospital, Birmingham, comprised 63 isolates that showed MICs of ≥ 64mg/L, were also included in the study. At this stage the study set (Set A) comprised 112 isolates with varying MIC to EtBr ranging from ≥ 32 mg/L to 256 mg/L. At a later date an additional 400 strains were screened from the same stored collection to include strains with lower MICs, i.e. < 32 mg/L. Thus a total of 336 isolates with varying levels of resistance to EtBr were studied. PCR was carried out on all 336 isolates for detection o qacAB, smr, qacG, qacH and qacJ to determine the presence and prevalence of the genes. Set A isolates positive for qacAB were further investigated to differentiate between qacA and qacB. Restriction digestion using the restriction enzyme Rsa1 was carried out on PCR products followed by PCR using specific primers for detection of the two genes. Urease activity and neomycin sensitivity were used as a means of basic characterization applied to all the study isolates. A select number of samples negative for qacA and qacB were typed using spa typing. Transfer studies involving, conjugation, plate mating and transformation on selected strains were carried out to attempt transfer of qacAB using the marker EtBr from a strain of MRSA with an MIC of ≥ 256 mg/L to EtBr and qacAB positive to a strain with < 32mg/L MIC to EtBr and lacking qacAB. Unfortunately, conjugation experiments were not successful in this study. Plasmid curing experiments were also carried out to demonstrate loss of plasmid through continual passaging onto selective plates. A variety of antiseptics and disinfectants are used in hospitals for prevention of HAIs. The present study was limited to carrying out minimum bactericidal concentration (MBC) determinations and MIC of four commonly used hospital biocides against randomly selected strains. The strains reflected ranges of MICs to EtBr and presence or absence of qacAB. These experiments, determined the efficacy of the biocides tested, to effectively destroy MRSA on skin and environment when used in healthcare settings. The results suggest that in the majority of strains showing high MICs to EtBr i.e. ≥ 64 mg/L, qacAB is present and thus, the mechanism of resistance to biocides may be attributed to an efflux protein pump encoded by these genes. Following restriction digestion of qacAB positive strains, with the restriction enzyme Rsa1, 81 of the 112 qacAB positive strains tested positive for qacA, i.e. 90% and 9 (11%) for qacB. The predominant prevalence of the qacA gene indicates that most of these strains are likely to be resistant to organic cationic biocides and intercalating dyes such as EtBr and acriflavine. However, the results of the MIC and MBC determinations carried out on a selection of biocides commonly used in the healthcare environment implies that the four biocides tested are likely to be 99.9% effective at killing the majority of isolates in this study set. However, five isolates demonstrated MBCs to chlorhexidine of > 32 mg/L. Chlorhexidine is a compound that is widely used in hand hygiene and surgical antisepsis products, and the results suggest that solutions containing this compound would be ineffective in removing MRSA from the hands of healthcare workers and skin sites if used. Molecular spa typing of selected samples negative for qacAB revealed that Endemic-MRSA (EMRSA) type 15 was the most frequent spa type identified in this study, followed by EMRSA-16 and EMRSA-1. Three strains identified jointly as EMRSA-3 and EMRSA-1. One strain identified as the Berlin clone. With regards to the challenges presented to infection prevention and control, MRSA has the potential to develop increased tolerance to biocides commonly used in the hospital environment, due to expression of efflux pumps, although currently there is little evidence of this. Further research is required to understand and learn of the various mechanisms of resistance, supported by adherence to control of infection strategies for prevention and spread of infections in healthcare facilities.

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Hamilton, Michelle Ann Elizabeth. « The relationship between plasmid presence, antibiotic resistance and surface structures in Bacteroides ». Thesis, University of Edinburgh, 1988. http://hdl.handle.net/1842/28187.

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12

Moran, Robert A. « Characterisation of plasmids in commensal Escherichia coli from healthy Australian adults ». Thesis, The University of Sydney, 2018. http://hdl.handle.net/2123/18199.

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Commensal Escherichia coli are thought to be an important reservoir of antibiotic resistance determinants, which are disseminated by the actions of translocatable genetic elements and plasmids that can transfer horizontally between cells. However, the plasmids of commensal E. coli and their role in the global spread of resistance genes have not been examined in detail. Here, 16 E. coli strains isolated from a single subject over a 700-day period were added to an existing, diverse collection of E. coli from healthy Australians. The complete collection contains 111 strains, with 51 resistant to one or more antibiotics. The plasmid content of the collection was examined using PCR and sequence-based approaches, revealing that plasmid replicons are far more diverse than previously appreciated. The presence of identical FII, FIB and several different I-complex replicons in unrelated strains from different subjects provided evidence for the dissemination of these types. Furthermore, several different plasmids were shown to have transferred between strains in the gastrointestinal tracts of healthy subjects in the absence of antibiotic selection. The complete sequences of plasmids of various types from this collection that contain antibiotic or heavy metal resistance genes were assembled. Their backbones were defined and used to query the GenBank database, revealing that most were also found in strains associated with infections, and that these plasmids are globally disseminated. The evolution of plasmid lineages via recombination and the actions of translocatable elements was traced, revealing how each lineage acquired and accumulated resistance determinants. This study has demonstrated that plasmids found in commensal E. coli are also found in strains that cause infections. The plasmids are acquiring antibiotic resistance genes and disseminating amongst strains, with the healthy human gastrointestinal tract an important site for their persistence and transfer.

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Saial, Dolores Cristina Conceição. « Use of antimicrobials and cephamicin resistance in companion animals ». Master's thesis, Universidade de Lisboa. Faculdade de Medicina Veterinária, 2013. http://hdl.handle.net/10400.5/6251.

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Dissertação de Mestrado Integrado em Medicina Veterinária
Objectives: This work includes two separate studies. In study 1 the aim was to investigate the use of antimicrobials in companion animals in Portugal while in study 2 the objective was to evaluate and characterize the prevalence of blaCMY-2 gene in Enterobacteriaceae and the phylogenetic relatedness among plasmids from companion animals and humans. Materials and Methods: In study 1 in order to understand the patterns of antimicrobial prescription a national survey was submitted to veterinarians. In study 2 plasmids harboring blaCMY-2 were transferred into GeneHog® E. coli by electroporation and typed by S1 endonuclease pulsed-field gel electrophoresis, PCR-based replicon typing, and plasmid multilocus sequence typing (pMLST). Results: In study 1, the use of amoxicillin-clavulanate (28%) and enrofloxacin (18%) were the most common antimicrobials used in dogs and cats, whereas clindamycin (3%) cefovecin (2%) and pradofloxacin (2%) were the less prescribed. In study 2, twenty three blaCMY-2 genes were plasmid encoded. Replicon typing demonstrated that from animal isolates, thirteen isolates were IncFII plasmids, five isolates were IncI1 plasmid, one isolate carried an A/C plasmid and the remaining isolate was non-typeable by PBRT. Regarding human isolates, one isolate was IncFII, one was IncI1 and the third isolate was also non-typeable. IncI1 blaCMY-2 plasmids showed that three were sequence type (ST2), three were non-typeable and fourteen IncFII plasmids were F2;FIA-;FIB- by pMLST. Conclusions and Clinical Importance: This work showed that in order to understand how antimicrobials are prescribed, further studies and implementation of a surveillance system for antimicrobial usage in these species would be recommended. Plasmid encoded resistant genes are an important factor for selection and dissemination of genes such as blaCMY-2. The transmission of resistant genes in humans and animals is due to plasmid encoding which is of great concern, and further research is still necessary to understand about the mechanisms which have led to the rapid spread of resistant bacteria worldwide.
RESUMO - USO DE ANTIMICROBIANOS E RESISTÊNCIA ÀS CEFAMICINAS EM ANIMAIS DE COMPANHIA - Objetivos: Este trabalho inclui 2 estudos. O objetivo do primeiro estudo consistiu em investigar o uso de antimicrobianos em animais de companhia em Portugal enquanto no segundo estudo, o objetivo consistiu na análise e caraterização da prevalência do gene blaCMY-2 em Enterobacteriaceas, ao mesmo tempo que pretendeu determinar a semelhança filogenética dos respetivos plasmídeos, em animais e humanos. Materiais e Métodos: No estudo 1, para compreender os hábitos de prescrição de antimicrobianos em Portugal foi realizado um inquérito nacional aos Veterinários. No estudo 2, os plasmídeos com o gene blaCMY-2 foram transferidos para uma célula electrocompetente GeneHog® E. coli por electroporação, e caraterizados por S1 endonuclease pulsed-field gel electrophoresis, PCR-based replicon typing e plasmid multilocus sequence typing (pMLST). Resultados: No estudo 1, os antimicrobianos mais utilizados em cães e gatos foram a amoxicilina/acido clavulânico (28%) e enrofloxacina (18%). Clindamicina (3%), cefovecina (2%) e pradofloxacina (2%) foram os menos utilizados em ambas as espécies. No estudo 2, vinte e três genes blaCMY-2 estavam codificados em plasmídeos. De acordo com o método replicon typing, os isolados de origem animal, treze pertenciam ao plasmídeo IncFII, cinco estavam codificados no plasmídeo IncI1, um estava presente no plasmídeo A/C e um isolado foi considerado “non-typeable”. Dos 3 isolados humanos, 1 estava incorporado num plasmídeo IncI1, 1 estava inserido no plasmídeo IncFII e o terceiro foi considerado “non-typeable”. Pelo método pMLST, os plasmídeos IncI1 foram caraterizados como ST2, e três foram considerados “non-typeable”. Catorze plasmídeos IncFII foram caraterizados como sendo F2;FIA-;FIB-. Conclusões e Importância Clínica: Para compreender os hábitos de prescrição de antimicrobianos, seriam recomendáveis estudos complementares e a implementação de um sistema de monitorização para o consumo de antimicrobianos nestas espécies. A presença de genes de resistência em plasmídeos é um fator importante para a seleção e disseminação de genes como o gene blaCMY-2. A transmissão destes genes em humanos e animais é mediada por plasmídeos, o que é preocupante. Investigação contínua é pois necessária para entender quais os principais mecanismos que conduziram à disseminação de bactérias com genes de resistência no mundo.

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Risley, Claire. « The population dynamics of plasmid-mediated antibiotic resistance in salmonella typhimurium in chickens ». Thesis, University of Oxford, 2002. http://ora.ox.ac.uk/objects/uuid:a70bda98-533f-41d0-b9a0-630457b3f982.

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A model of growth and plasmid transfer between strains of Escherichia coli and Salmonella typhimurium was developed with reference to the literature. This was the organising principle for the collection of a complete set of in vitro life history parameters of one S. typhimurium and one E. coli strain. In the course of estimating these parameters two results of note were obtained. Fits of the Lotka-Volterra competition model were obtained for data on S. typhimuiurm growing in competition with E. coli. The first noteworthy discovery was the failure of this model to account for several characteristics of growth of these strains under competition. The growth rates of plasmid-bearing and plasmid-free strains were obtained. The second main result came from examination of the results of the growth rate data, which revealed that the cost to S. typhimuiurm 576 of bearing the resistance plasmid was low (4%). The model was also used to simulate the effect of antibiotic dose on the density of the donor, recipient and transconjugant populations over time. These simulations predicted that there would be a convex relationship between antibiotic dose and transconjugant density (i.e. that the density would first rise, then fall, with increasing dose). Following from this result, laboratory experiments and in vivo experiments in chickens were directed towards obtaining information on the relationship between these two variables. This convex relationship was not demonstrated within a single experiment, although some experimental environments produced an increase in transconjugant density with dose, and others, a decrease. Few transconjugants were formed in vivo. In order to investigate the low cost of resistance and low rate of in vivo transconjugant production, cost of resistance and plasmid transfer rate of this plasmid in several strain combinations of E. coli and S. typhimuiurm was evaluated.

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Armbruster, Steven C. (Steven Christopher). « Characterization of the OCT Plasmid-Encoded Mercury Resistance Genetic Locus in Pseudomonas putida ». Thesis, University of North Texas, 1989. https://digital.library.unt.edu/ark:/67531/metadc500381/.

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A 17.1 Kb genetic element encoding for mercury resistance (OCT-Hg^r) was shown to translocate from its original location on the OCT plasmid to the resistance plasmid, RPl, in Pseudomonas putida. Analysis of RPl-Hg^r recombinant plasmids revealed that insertion of mercury resistance genes into RPl could occur at a variety of sites, with all recombinants having common EcoRI restriction fragments of 9.4, 3.8, 2.3, and 1.6 Kb, derived from the insertion. Hybridization analysis suggested the existence of extensive homology between this insertion and the prototypic mercury resistance transposon, Tn501, as well as the location of a similar merA sequence. Although the overall size was shown to be quite different from Tn501, striking physical similarities are shared between these two elements.

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Tatley, Fernanda Maria Palma Ribeiro da Silva. « Characterisation of a replicon of the conjugative, multiple drug resistance, moderately promiscuous, plasmid pGSH500 ». Thesis, University of Cape Town, 1992. http://hdl.handle.net/11427/25668.

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Gullberg, Erik. « Selection of Resistance at very low Antibiotic Concentrations ». Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-235225.

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The extensive medical and agricultural use and misuse of antibiotics during the last 70 years has caused an enrichment of resistant pathogenic bacteria that now severely threatens our capacity to efficiently treat bacterial infections. While is has been known for a long time that high concentrations of antibiotics can select for resistant mutants, less is known about the lower limit at which antibiotics can be selective and enrich for resistant bacteria. In this thesis we investigated the role of low concentrations of antibiotics and heavy metals in the enrichment and evolution of antibiotic resistance. Selection was studied using Escherichia coli and Salmonella enterica serovar Typhimurium LT2 with different resistance mutations, different chromosomal resistance genes as well as large conjugative multidrug resistance plasmids. Using very sensitive competition experiments, we showed that antibiotic and heavy metal levels more than several hundred-fold below the minimal inhibitory concentration of susceptible bacteria can enrich for resistant bacteria. Additionally, we demonstrated that subinhibitory levels of antibiotics can select for de novo resistant mutants, and that these conditions can select for a new spectrum of low-cost resistance mutations. The combinatorial effects of antibiotics and heavy metals can cause an enrichment of a multidrug resistance plasmid, even if the concentration of each compound individually is not high enough to cause selection. These results indicate that environments contaminated with low levels of antibiotics and heavy metals such as, for example, sewage water or soil fertilized with sludge or manure, could provide a setting for selection, enrichment and transfer of antibiotic resistance genes. This selection could be a critical step in the transfer of resistance genes from environmental bacteria to human pathogens.

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Le, Vien Thi Minh. « Plasmid-mediated quinolone resistance determinants in Enterobacteriaceae isolated in Ho Chi Minh City, Vietnam ». Thesis, University of Oxford, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.559782.

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Fluoroquinolones are amongst the most effective antimicrobials used to treat infectious diseases caused by members of the Enterobacteriaceae. The rapid spread of fluoroquinolone resistant Enterobacteriaceae threatens the future effectiveness of this widely used group of antimicrobial compounds. Fluoroquinolone resistance in Enterobacteriaceae in Vietnam highlights an approaching crisis in antimicrobial therapy of Enterobacteriaceae infections as fluoroquinolone resistant infections may require expensive alternative treatments. Furthermore, a lack of alternative drugs for treating fluoroquinolone resistant bacteria also contributes to the difficulty in treating such infections. The aim of this study was to investigate the prevalence and mechanisms of fluoroquinolone resistance in commensal Enterobacteriaceae isolated from two different populations resident in Ho Chi Minh City; hospitalized patients and healthy volunteers. I investigated the prevalence of mutations in the DNA gyrase and the topoisomerase gene, the target of the fluoroquinolones and the main resistance mechanisms in Enterobacteriaceae. Additionally, I investigated the spectrum of plasmid-mediated quinolone resistance (PMQR) determinants, a more contemporary mechanism of fluoroquinolone resistance. By using whole plasmid sequencing, bioinformatics and a miniaturized DNA nanoarray, an association of PMQR determinants with other antimicrobial resistance genes including blaLAP_2, which confers resistance to ~-lactam antimicrobials, was found. Moreover, diverse patterns of antimicrobial resistance between isolates from hospital and community populations were revealed. An increase in the quantity ofPMQR determinants isolated from rectal swabs of children treated with antimicrobials was also demonstrated using a gene specific real-time PCR. I surmise that variety antimicrobial classes might contribute to an increase in copy number and eo- selection of PMQR determinants in Ho Chi Minh City. Fluoroquinolone resistance in Enterobacteriaceae has increased oyer time and this class of antimicrobial is still commonly used, to treat infection caused by such organisms. Understanding fluoroquinolone resistance mechanisms and their relationship to other antimicrobial resistance mechanisms in Enterobacteriaceae should play an important role in the control of the spread of antimicrobial resistance genes and aid treatment strategies for clinicians in Vietnam.

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Wang, Chien-Sao. « Molecular Cloning and Functional Analysis of Transposable Mercury Resistance Genes Encoded by the OCT Plasmid ». Thesis, University of North Texas, 1991. https://digital.library.unt.edu/ark:/67531/metadc501216/.

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Translocation of a 17.1 kilobase region of the OCT plasmid encoding mercury resistance (mer) in Pseudomonas putida was shown to occur in a recombination-deficient host with plasmid PP1 serving as a recipient replicon. The frequency of transposition in Pseudomonas was estimated at 10^3 -10 -^2, but undetectable in Escherichia soli. ' DNA comprising all of mr as well as subregions there of were cloned and subjected to DNA sequence analysis. Like other transposons, mer was found to contain inverted repeat sequences at its termini. These were similar to, but not identical to the inverted repeat structures found in the prototypical mercury resistance transposon Tn501 from E. aeruginosa.

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Bernardo, Nerea. « Prevention of the spread of antibiotic resistance via the structural and molecular study of pLS20 conjugative plasmid proteins ». Doctoral thesis, Universitat Autònoma de Barcelona, 2020. http://hdl.handle.net/10803/671044.

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La resistència a antibiòtics està considerada una de les principals amenaces a la salut pública del segle XXI per la OMS. A causa del seu consum excessiu i incorrecte, els bacteris han desenvolupat mecanismes per fer-les front y així, ser capaços de sobreviure a la seva presència. Quan un bacteri desenvolupa el gen que li dota de resistència, disposa de diversos mecanismes amb el que pot cedir aquesta resistència a bacteris veïns. Aquest procés és conegut com transferència horitzontal de gens, i és la principal causa de la propagació dels gens de resistència a antibiòtics. Podem distingir entre 3 subtipus, sent la conjugació la més comú. La conjugació és la transferència de material genètic des d’un bacteri donant a un altra receptor i requereix contacte directe entre cèl·lules. La seva característica principal és la presència d’un plasmidi conjugatiu. En aquest treball, s’ha estudiat el plasmidi conjugatiu pLS20, del bacteri Gram positiu Bacillus subtilis. És una elecció d’estudi interessant donant que B. subitlis pertany al filo Firmicutes, sent aquest el filo predominant al intestí humà. El intestí humà funciona com un reservori genètic de resistència a antibiòtics. A més, pLS20 té interès biotecnològic degut a la seva existència en B. sutiblis natto, el quin és important en producció alimentària. Entendre com es regula la conjugació i reunir informació sobre les proteïnes que participen al procés conjugatiu és molt important per poder acabar amb la propagació de la resistència a antibiòtics. Per tant, aquesta tesi està centrada en l’estudi estructural i molecular de diverses proteïnes de pLS20. En primer lloc, el circuit regulador que controla la expressió de gens del principal operon conjugatiu ha estat estudiat, en la quina participen Rco, Rap i Phr*. Hem caracteritzat estructuralment el domini de tetramerització de Rco i hem vist que comparteix similitud amb la família de proteïnes p53. A més, hem determinat que Rco té diferents estats de oligomerizació en funció del pH, probablement a causa de la interfície de tetramerització composta per residus carregats. Així mateix, la unió entre Rap i Rco a diferents estequiometries i l’efecte de Phr* en la formació del complex han sigut analitzats per cromatografia de exclusió molecular. Respecte a Rap, s’ha avaluat la possibilitat de regulació creuada amb altres sistemes de Rap per assajos de unió. En segon lloc, hem treballat amb Reg576, una proteïna reguladora que participa en la regulació de gens involucrats en el establiment del plasmidi una vegada transferit a la cèl·lula receptora. Hem identificat al seu lloc d’unió al ADN y hem mutat un residu conservat per concloure que la unió no es veu afectada. D’altra banda, hem obtingut diferents disposicions y grups espacials de la proteïna a més de la que ja està depositada, revelant que Reg576 cristal·litza amb relativa facilitat. Finalment, hem investigat P34, proteïna involucrada en adhesió cel·lular que té un paper igualment important que la regulació del procés conjugatiu. Hem determinat que és una proteïna TIE (tioèster, Isopeptídic, èster) ja que hem caracteritzat estructuralment el seu domini tioèster, denominat TED. Mitjançant la introducció de la mutació de la cisteïna que participa en el enllaç tioèster, no hem observat cap canvi significatiu en l’estructura de la proteïna, però hem mesurat canvis dràstics en la funcionalitat. En definitiva, aquests resultats suposan un important avanç en la comprensió sobre la regulació de la conjugació i el contacte entre cèl·lules per començar la transferència de gens del plasmidi pLS20. Donada la importància de las proteïnes estudiades, no descartem l’opció de utilitzar aquestes proteïnes com futures dianes de fàrmacs per detenir la propagació de la resistència a antibiòtics.
La resistencia a antibióticos está considerada una de las principales amenazas a la salud pública del siglo XXI por la OMS. Debido a su consumo excesivo e incorrecto, las bacterias desarrollan mecanismos para hacerles frente y así, ser capaces de sobrevivir en su presencia. Cuando una bacteria desarrolla el gen que le dota de resistencia, dispone de varios mecanismos para cederlo a las bacterias colindantes. Este proceso es conocido como transferencia horizontal de genes y es la principal causa de la propagación de los genes de resistencia a antibióticos. Podemos distinguir entre 3 subtipos, siendo la conjugación la más común. La conjugación es la transferencia activa de material genético desde una bacteria donante a otra receptora y requiere contacto directo entre células. La característica primordial es la presencia de un plásmido conjugativo. En este trabajo, se ha estudiado el plásmido conjugativo pLS20, de la bacteria Gram positiva Bacillus subtilis. Es una elección de estudio interesante ya que B. subtilis pertenece al filo Firmicutes, siendo este el filo predominante en el intestino humano. El intestino humano funciona como una reserva genética de resistencia a antibióticos. Además, pLS20 tiene interés biotecnológico debido a su existencia en B. subtilis natto, el cual es importante en producción alimentaria. Entender cómo se regula la conjugación y reunir información sobre las proteínas específicas que participan en el proceso conjugativo es muy importante para poder acabar con la propagación de la resistencia a antibióticos. Así, este trabajo está centrado en el estudio estructural y molecular de varias proteínas de pLS20. En primero lugar, se ha estudiado el circuito regulatorio que controla la expresión de genes del principal operon conjugativo, en el cual participan Rco, Rap y Phr*. Hemos caracterizado estructuralmente el dominio de tetramerización de Rco, descubriendo que comparte similitud con la familia de proteínas p53. Además, hemos determinado que Rco posee distintos estados de oligomerización en función al pH, probablemente debido a su interfaz de tetramerización compuesta de residuos cargados. Asimismo, la unión entre Rap y Rco a distintas estequiometrías y el efecto de Phr* en la formación del complejo han sido analizados por cromatografía de exclusión molecular. Respecto a Rap, se ha evaluado la posibilidad de regulación cruzada con otros sistemas de Rap mediante ensayos de unión. En segundo lugar, hemos trabajado con Reg576, una proteína que participa en la regulación de genes involucrados en el establecimiento del plásmido una vez transferido a la célula receptora. Hemos identificado su sitio de unión en el ADN y mutado un residuo conservado para concluir que la unión no se ve afectada. Hemos obtenido diferentes empaqueteamientos cristalinos y grupos espaciales de la proteína además de la que ya está depositada, lo cual revela que Reg576 es una proteína que cristaliza con relativa facilidad. Finalmente, hemos investigado P34, proteína involucrada en adhesión celular que juega un papel igualmente importante que la regulación en el proceso conjugativo. Hemos determinado que es una proteína TIE (tioéster, isopeptídico, éster) ya que hemos caracterizado estructuralmente su dominio tioéster, denominado TED. Mediante la introducción de la mutación de la cisteína que participa en el enlace tioéster, no hemos observado ningún cambio significativo en la estructura de la proteína, sin embargo, hemos medido cambios drásticos en la funcionalidad. En definitiva, estos resultados suponen un importante avance en la comprensión sobre la regulación de la conjugación y el contacto entre células para comenzar la transferencia de genes del plásmido pLS20. Dada la importancia de las proteínas estudiadas, no descartamos la opción de utilizar estas proteínas como futuras dianas de fármacos para detener la propagación de la resistencia a antibióticos.
Antibiotic Resistance is considered one of the most important public health threats of the 21st century by the WHO. Due to human overuse and misuse of antibiotics, bacteria develop new mechanisms to survive in the presence of antibiotics. Once one bacterium has evolved the gene that endows resistance to it, there are several ways by which this trait can pass to neighbouring bacteria. This process is known as horizontal gene transfer and it is the main reason for the spread of antibiotic resistance genes. We can distinguish between three subtypes, conjugation being the most common one. Conjugation is the active transfer of genetic material from a donor bacterium to a recipient bacterium, involving direct cell-to-cell contact. It is characterized by the presence of a conjugative plasmid. In this work, we have studied the pLS20 conjugative plasmid, from Gram positive bacteria Bacillus subtilis. It is an interesting choice of research as Bacillus subtilis belongs to the phylum Firmicutes, which is the predominant pylum in human gut. The human gut has favourable conditions for conjugative gene exchange and therefore is a pool of antibiotic resistance genes. Also, pLS20 has biotechnological interest because of its occurrence in B. subtilis natto, which is important in food production. Understanding how conjugation is regulated and gathering information of the specific proteins that take part in the conjugative process is of extreme importance in order to stop antibiotic resistance spread. Consequently, this work is focused on the structural and molecular study of various pLS20 proteins. Firstly, the regulatory circuit that controls the expression of the genes of the main conjugation operon has been studied, in which Rco, Rap and Phr* take part. We have structurally characterized the tetramerization domain of Rco and realized it shares high structural resemblance with p53 family proteins. Also, we have determined that Rco has different oligomerization states under distinct pHs, probably due to the charged tetramerization interface. Furthermore, binding between Rapp and Rco at different stoichiometries and the effect of Phr* in the complex formation has been analyzed by size exclusion chromatography. With regard to Rap, the possibility of cross-regulation with other Rap systems has been considered and evaluated by binding assays. Secondly, we have studied Reg576, which is also a transcriptional regulator that controls the transctiption of the genes involved in the establishment of the plasmid once it has been transferred to the recipient cell. We have identified its binding region in DNA and mutated a conserved residue of the protein and determined that binding is not affected. Moreover, we have obtained diverse crystal packings and space groups of the proteins, revealing that Reg576 is a protein that tends to crystallize with relative ease. Finally, we have investigated P34, a protein involved in cell adhesion, which plays an equally important part in the overall success of plasmid transfer as does gene regulation. We have determined it is a TIE (thioester, isopeptide, esther) type of protein as we have structurally characterized its TED domain. Also, from the structure of a mutant where the thioester bond-forming cysteine was mutated to a serine, we conclude that there are no significant structural changes. However, we have observed drastic functionality changes: conjugation is inhibited for the mutant. Together, these results bring new important insights into how conjugation in the pLS20 plasmid is regulated and how cells contact each other to start the transfer of the genes. Given the importance of the proteins characterized, we do not discard the option of using these proteins as future drug targets to stop antibiotic resistance spread.
Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina

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Li, Xinhui. « Effective Control of Antibiotic Resistance in Cheese and Characterization of a Dairy Enterococcus faecium Isolate Carrying a Persistent, TA-independent Tetracycline Resistance-encoding Plasmid ». The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1308294661.

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Massie-Schuh, Ella. « The Processing of Replication Initiation Protein PrgW in Enterococcus faecalis is Necessary for Activity and Stable Maintenance of pCF10 ». Master's thesis, Temple University Libraries, 2013. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/216595.

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Microbiology and Immunology
M.S.
Enterococcus faecalis are Gram-positive bacteria that colonize the gastrointestinal tracts of mammals, birds and invertebrates and are also found in sewage, soil, food and water. In addition to being commensal organisms, Enterococci can also cause nosocomial infections in humans including urinary tract infections, septicemia and endocarditis. Hospital-acquired infections often present a challenge in treatment due to the emergence of multi-drug resistant strains. Enterococcal plasmids may act as extremely stable reservoirs for resistance genes and other virulence factors. Pheromone responsive plasmids such as pCF10 mediate efficient transfer of genetic material within the species E. faecalis but may also be capable of transferring resistance genes across species and genus boundaries. Polymicrobial environments often found in nosocomial infections may expose plasmid-harboring enterococci to pathogenic species, poising cells for this type of promiscuous horizontal gene transfer of resistance determinants. Previous studies showed that prgW, which encodes the pCF10 replication initiation protein PrgW, is the minimal origin of replication for this plasmid. The replicon, which is usually limited to Enterococcal spp., can replicate in Lactococcus lactis if it is engineered to produce pre-cCF10. Three conserved cysteines (C78/C275/C307) are important for plasmid stability and allow for replication of the pCF10 replicon in L. lactis in the absence of pre-cCF10. PrgW has a predicted molecular weight of 38,635. Four polyclonal antibodies targeting PrgW at the N-terminus (aa 1-20), C-terminus (aa 314-333) and two internal regions (aa 64-80 and aa 250-271) were used in current experiments and retrospective studies. When PrgW was overexpressed in E. faecalis, four different apparent approximate molecular weights were detected by Western blotting (p40*, p36*, p24* and p18*), suggestive of processing. In Enterococci where the replicon is active, p36* was consistently detected by all four antisera; when PrgW was overexpressed in Streptococcus mutans where the replicon is non-functional, p49* and p40* were detected but p36* was not observed. PrgW p24* was detected by a mixture of the internally targeting antibodies as well as the C-terminal targeting antibody, but not the N-terminal targeting antibody, suggesting that the N-terminal domain of PrgW has been cleaved off in p24*. The p24* form may play a role in pCF10 stability. Mutations to three cysteines in PrgW (C78/C275/C307), which reduce the stability of pCF10, result in the loss of p24*. Enterococcal conjugative plasmids have been previously implicated in the transfer of antibiotic resistance genes. The pCF10 plasmid contains the conjugative transposon Tn925, which possesses the tetM tetracycline resistance gene. Proximity of donor and recipient cells is a key part of pheromone-responsive conjugation. Aggregation substance allows for formation of clumps of E. faecalis in liquid mating experiments. E. faecalis forms biofilms; in contrast to filter mating experiments, polymicrobial biofilms provide an in vitro model of a natural scenario during which horizontal gene transfer may occur. Rates of cross-genus genetic transfer of tetM between E. faecalis OG1RF(pCF10) donor cells and Staphylococcus aureus recipient cells growing on glass coverslips as mixed-species biofilm populations were determined to be 10-8 after pheromone induction of pCF10 conjugation. This biofilm transfer model also holds potential to test the efficacy of synthetic peptides in the reduction or even prevention of pCF10 transfer, and the consequential dissemination of antibiotic resistance determinants throughout the genus Enterococcus and beyond.
Temple University--Theses

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Morgan, Dale. « Molecular analysis of genes encoding resistance to Cationic Biocides in staphylococci ». Thesis, Curtin University, 2007. http://hdl.handle.net/20.500.11937/2467.

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Bacterial resistance to non-antibiotic agents is being increasingly studied. Plasmid-mediated resistance to cationic agents, which are important biocides, has been described in antibiotic-resistant Staphylococcus aureus. Multi-resistant Staphylococcus aureus (MRSA) are often found to express resistance to a range of cationic biocides including quaternary ammonium compounds (QACs), biguanides, diamidino compounds, cationic dyes and nuclear stains. Three resistance determinants, qacA, qacB and smr genes, have been identified that confer resistance to cationic biocides in staphylococci. These genes encode multi-drug efflux pumps that remove the cationic biocides from the cytoplasm using a membrane bound pumping mechanism dependent on the cell's proton-motive force (PMF). This prevents the build up of lethal concentrations of cationic compounds within the cytoplasm avoiding cell death.This research project has focused on the S. aureus strain WBG4364, a transcipient strain carrying the cationic biocide resistant plasmid pWBG1773. The plasmid encodes resistance to several QACs, including benzalkonium chloride and CTAB, and cationic dyes rhodamine 6G, crystal violet and safranin O but not to the dye ethidium bromide and therefore differing from other cationic biocide resistant plasmids previously identified in staphylococci (Emslie et al. 1986). This unique phenotype was further classified in this study alongside those strains carrying the qac gene families, qacA/B and smr.Plasmid pWBG1773 was cloned, sequenced and analysed to reveal a unique plasmid of 2,916 bp in length. Plasmid pWBG1773 was placed with the pC194 family of rolling-circle replicating plasmids. This family appear to be largely composed of interchangeable cassette structures.The plasmid was found to carry three ORFs, designated ORF1, ORF2 and ORF3. ORF1 was homologous to rep genes of small staphylococcal plasmids belonging to the pC194 rolling-circle replication family and has been redefined as repWBG1773. ORF2 was found to have no similarity to any proteins of known function in the GenBank database whereas ORF3 was found to have homology to the marR gene, a regulator of the multiple antibiotic resistance (mar) operon of Gram-negative organisms. MIC analysis of these ORFs found both ORF2 and ORF3 were essential for expression of resistance to cationic biocides. The exact ORF2 sequence required for resistance to be expressed was reduced to only 141 nt in size. This translated to a 47 aa sequence that contained a hydrophobic C-terminus indicating ORF2 to be a membrane-bound protein. The aa sequence of ORF3 contained a helix-turn-helix motif characteristic of the DNA binding domains of MarR-like proteins. Further analysis of pWBG1773 identified a putative 'marbox', a binding site for the homologous transcriptional activators of mar, within the ORF2 sequence. This indicated that ORF3 was binding to the 'marbox' sequence and activating transcription. Induction studies have not been able to ascertain any compounds capable of interacting with the ORF3 regulatory protein resulting in induction of cationic biocide resistance. Each ORF when analysed alone had no effect on the expression of cationic biocide resistance and it is thought that a efflux pump was not involved. This is further corroborated by the CCCP efflux experiments performed in an attempt to determine the mechanism of resistance. The unique ORFs of plasmid pWBG1773 appears to encode a novel cationic biocide resistance phenotype and mechanism.MRSA strains from all around the world were analysed to determine if they possessed sequences homologous to ORF2 and ORF3. Sequences sharing a high degree of homology to ORF2 and/or ORF3 were detected in several MRSA strains including strains sensitive to all cationic biocides tested. These findings suggest that the appearance of ORF2 and ORF3 sequences in MRSAs was not an isolated event and the fact that some MRSAs do not carry both ORF2 and ORF3 sequences simultaneously indicates that these genes have another role that does not involve expression of resistance to cationic biocides.Bacteria are noteworthy for their remarkable ability to adapt to changes in their environments and possess an impressive set of tools with which to adjust the blueprint of the cell to this change. The acquisition of a single system that may decrease a potential pathogenic organisms susceptibility to a wide range of cationic biocides, such as seen in pWBG1773, poses a clinical threat, one that needs to be thoroughly investigated.

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Freitas, Ana Raquel Pinho. « Ecology and evolution of antimicrobial resistance in Enterococcus : a multilayered molecular approach with emphasys in the plasmid diversity ». Doctoral thesis, Faculdade de Farmácia da Universidade do Porto, 2010. http://hdl.handle.net/10216/63801.

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Saeed, Sadia. « Uncovering the molecular mechanism of ParG dimerization and its role in segrosome assembly of multidrug resistance plasmid TP228 ». Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/uncovering-the-molecular-mechanism-of-parg-dimerization-and-its-role-in-segrosome-assembly-of-multidrug-resistance-plasmid-tp228(76dda7e1-a14c-4d85-9b10-6524e1fb9281).html.

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The multidrug resistance plasmid TP228 replicates at low copy number in Escherichia coli. Stable partitioning of this plasmid is mediated by three essential components: a ParA homologue, ParF; a centromere binding protein, ParG; and a centromere site, parH. ParF and ParG jointly assemble on the parH centromere forming the segrosome complex, and thereby direct intracellular plasmid transport. ParG belongs to the ribbon-helix-helix (RHH) class of dimeric DNA binding proteins. ParG specifically binds the parH site and also is a transcriptional repressor of the parFG genes. Previous studies demonstrated that unstructured N-terminal tails in ParG are not important for dimerization. Instead the tails are implicated in assembly of higher order nucleoprotein complexes essential for transcriptional repression and segrosome assembly, and also influence ParF nucleotide hydrolysis and polymerization. In this study we defined the role of residues in the RHH folded domain that are crucial for ParG dimerization and function. To achieve our goal the two α-helices, the intervening loop, and two C-terminal residues were analyzed fully by alanine scanning mutagenesis. Initially, ParG mutants were constructed and assessed for effects on normal plasmid partition activity and on dimerization. In vivo segregation assays and bacterial two-hybrid studies revealed mutation of residues F49 in α-helix 1 and W71 and L72 in α-helix 2 of ParG each resulted in defective plasmid partition activity and impaired dimerization. In vitro chemical cross-linking of purified proteins ParG-F49A, ParG-W71A and ParG-L72A demonstrated predominant monomeric species whereas wild-type ParG formed dimeric species as noted previously. Multiangle light scattering and sedimentation equilibrium analysis of the mutant proteins showed shifts in molar mass towards monomeric species with increased Kd values for dimerization. Protein-DNA interactions studied by gel retardation assays showed impaired interactions of ParG-F49A, ParG-W71A and ParG-L72A with parH. Results of conserved substitutions at position 71 showed that aromatic substitutions of W71 to Y71 or F71 are tolerated and have no apparent effects on ParG mediated plasmid segregation, but the non-aromatic W71L mutation blocked the segregation. However, a ParG double mutant bearing the ‘reversed’ amino acid pair (W71L-A52Y) retained plasmid segregation activity and behaved like wild-type ParG in dimerization assays in vitro and in vivo. Thus, substitution of W71 by tyrosine or phenylalanine does not disturb the monomer-monomer interface interactions that pack α-helix 2 from one monomer against residues of α-helix 1 and α-helix 2 of the partner monomer. Moreover, the permissible amino acid combinations at interacting positions 52 and 71 in ParG show significant flexibility and reveal key roles for these residues in function and dimerization of ParG. Overall, our in vivo and in vitro interaction studies provide novel information about the role of hydrophobic residues F49, W71 and L72 in ParG dimerization and activity. In the longer term, interference with dimerization by ParG and other centromere binding proteins using artificial ligands may provide a novel strategy for destabilization of antibiotic multiresistance plasmids.

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Krishnan, B. Rajendra Carleton University Dissertation Biology. « The incN antibiotic-resistance plasmid pCU1 : bacterial host-range, nucleotide sequence, deletion and subcloning analysis of the replicon ». Ottawa, 1989.

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Freitas, Ana Raquel Pinho. « Ecology and evolution of antimicrobial resistance in Enterococcus : a multilayered molecular approach with emphasys in the plasmid diversity ». Tese, Faculdade de Farmácia da Universidade do Porto, 2010. http://hdl.handle.net/10216/63801.

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Van, Thi Thu Hao, et thuhao2007@gmail com. « Detection of Enteric Bacteria in Raw Food Samples from Vietnam and Evaluation of Antibiotic Resistance ». RMIT University. Applied Sciences, 2007. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20090407.141836.

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This study was conducted to examine the rate of contamination and molecular characteristics of enteric bacteria isolated from a selection of food sources in Vietnam. One hundred and eighty raw food samples were tested and 60.8% of meat and 18.0% of shellfish samples were found to be contaminated with Salmonella spp. which belonged to variety of serogroups and serotypes. More than 90% of all food sources contained Escherichia coli and 32% of 50 shellfish samples were contaminated with Vibrio parahaemolyticus. PFGE was used to determine the degree of relatedness of Salmonella spp. There were 33 distinct PFGE patterns from 51 Salmonella spp. isolates tested, indicating that PFGE could be used as an alternative method for serotyping for use in epidemiology of Salmonella spp. Susceptibility of the isolates to 15 antimicrobial agents was investigated. Moderate to high frequencies of resistance to antibiotics were observed in Salmonella spp. and E. coli isolates and multi-resistance, defined as resistance to at least 4 antibiotics, was observed. All of the V. parahaemolyticus isolates were resistant to ampicillin/amoxicillin but not to other antibiotics. Betalactam TEM gene and tetracycline resistance tetA, tetB genes were widely distributed in both E. coli and Salmonella spp. isolates. Other resistance genes, including sulI, cmlA, aadA, aphA1, dhfrV, and aac(3)-IV were also present at high to moderate levels. Identification and characterisation of the mobile genetic elements, including identification of class 1 integrons and plasmids were carried out for multi-resistance isolates. The integrons harboured varying gene cassettes, including aadA1, aadA2, aadA5, aacA4, dhfrXII, drfA1 and dhfrA17, blaPSE1 and catB3. Thirty-five percent of Salmonella spp. isolates and 76% of E. coli isolates harboured plasmids of more than 95 kb. Transfer of resistance phenotypes between the isolates via conjugation and phage transduction was also demonstrated. Salmonella genomic island 1 (SGI1), a 43-kb genomic region contains a 13-kb antibiotic resistance gene cluster, has been identified in an isolate of S. Albany from chicken. The presence of Salmonella spp. virulence genes was investigated to examine the pathogenicity potential of the isolates. The invA gene was present in all Salmonella spp. isolates and the plasmid virulence gene spvC was detected in one S. Typhimurium isolate only, on a 95 kb virulence plasmid. Invasion assays performed in vitro demonstrated that all Salmonella isolates were capable of invading human intestine INT407 cells. In addition, the investigation for the presence of 58 selected virulence genes showed that all the tested isolates contained at least one virulence gene and there were 16 genes which are associated with different pathotypes detected. The data obtained in this study indicates that raw food in Vietnam is a potential reservoirs for many pathogenic organisms, and confirms the role of food animals as a reservoir of multidrug resistant E. coli and Salmonella spp.

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Morgan, Dale. « Molecular analysis of genes encoding resistance to Cationic Biocides in staphylococci ». Curtin University of Technology, School of Pharmacy, 2007. http://espace.library.curtin.edu.au:80/R/?func=dbin-jump-full&object_id=17463.

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Bacterial resistance to non-antibiotic agents is being increasingly studied. Plasmid-mediated resistance to cationic agents, which are important biocides, has been described in antibiotic-resistant Staphylococcus aureus. Multi-resistant Staphylococcus aureus (MRSA) are often found to express resistance to a range of cationic biocides including quaternary ammonium compounds (QACs), biguanides, diamidino compounds, cationic dyes and nuclear stains. Three resistance determinants, qacA, qacB and smr genes, have been identified that confer resistance to cationic biocides in staphylococci. These genes encode multi-drug efflux pumps that remove the cationic biocides from the cytoplasm using a membrane bound pumping mechanism dependent on the cell's proton-motive force (PMF). This prevents the build up of lethal concentrations of cationic compounds within the cytoplasm avoiding cell death.This research project has focused on the S. aureus strain WBG4364, a transcipient strain carrying the cationic biocide resistant plasmid pWBG1773. The plasmid encodes resistance to several QACs, including benzalkonium chloride and CTAB, and cationic dyes rhodamine 6G, crystal violet and safranin O but not to the dye ethidium bromide and therefore differing from other cationic biocide resistant plasmids previously identified in staphylococci (Emslie et al. 1986). This unique phenotype was further classified in this study alongside those strains carrying the qac gene families, qacA/B and smr.Plasmid pWBG1773 was cloned, sequenced and analysed to reveal a unique plasmid of 2,916 bp in length. Plasmid pWBG1773 was placed with the pC194 family of rolling-circle replicating plasmids. This family appear to be largely composed of interchangeable cassette structures.The plasmid was found to carry three ORFs, designated ORF1, ORF2 and ORF3. ORF1 was homologous to rep genes of small staphylococcal ++
plasmids belonging to the pC194 rolling-circle replication family and has been redefined as repWBG1773. ORF2 was found to have no similarity to any proteins of known function in the GenBank database whereas ORF3 was found to have homology to the marR gene, a regulator of the multiple antibiotic resistance (mar) operon of Gram-negative organisms. MIC analysis of these ORFs found both ORF2 and ORF3 were essential for expression of resistance to cationic biocides. The exact ORF2 sequence required for resistance to be expressed was reduced to only 141 nt in size. This translated to a 47 aa sequence that contained a hydrophobic C-terminus indicating ORF2 to be a membrane-bound protein. The aa sequence of ORF3 contained a helix-turn-helix motif characteristic of the DNA binding domains of MarR-like proteins. Further analysis of pWBG1773 identified a putative 'marbox', a binding site for the homologous transcriptional activators of mar, within the ORF2 sequence. This indicated that ORF3 was binding to the 'marbox' sequence and activating transcription. Induction studies have not been able to ascertain any compounds capable of interacting with the ORF3 regulatory protein resulting in induction of cationic biocide resistance. Each ORF when analysed alone had no effect on the expression of cationic biocide resistance and it is thought that a efflux pump was not involved. This is further corroborated by the CCCP efflux experiments performed in an attempt to determine the mechanism of resistance. The unique ORFs of plasmid pWBG1773 appears to encode a novel cationic biocide resistance phenotype and mechanism.MRSA strains from all around the world were analysed to determine if they possessed sequences homologous to ORF2 and ORF3. Sequences sharing a high degree of homology to ORF2 and/or ORF3 were detected in several MRSA strains including strains sensitive to all cationic ++
biocides tested. These findings suggest that the appearance of ORF2 and ORF3 sequences in MRSAs was not an isolated event and the fact that some MRSAs do not carry both ORF2 and ORF3 sequences simultaneously indicates that these genes have another role that does not involve expression of resistance to cationic biocides.Bacteria are noteworthy for their remarkable ability to adapt to changes in their environments and possess an impressive set of tools with which to adjust the blueprint of the cell to this change. The acquisition of a single system that may decrease a potential pathogenic organisms susceptibility to a wide range of cationic biocides, such as seen in pWBG1773, poses a clinical threat, one that needs to be thoroughly investigated.

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Muhammad, Ghulam. « Staphylococci of bovine mammary gland : conventional and molecular dynamics of infections, plasmid stability, reproducibility, and interspecific conjugal transfer of antibiotic resistance / ». The Ohio State University, 1992. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487780393268973.

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Barge, Madhuri. « Role of the unstructured N-terminus of the centromere binding protein ParG in mediating segregation of the multidrug resistance plasmid TP228 ». Thesis, University of York, 2015. http://etheses.whiterose.ac.uk/8902/.

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TP228 is a large low copy number plasmid harbouring the parFGH partition cassette. The centromere-like site parH is located upstream of the parFG genes. ParF is a Walker-type ATPase of the ParA superfamily. ParG is a centromere binding protein and a transcriptional repressor of the parFG genes. ParF associates with ParG bound to parH forming the segrosome complex. It has been recently observed that ParF oscillates over the nucleoid in the presence of the entire parFGH system and oscillation is responsible for plasmid segregation. ParG is a dimeric protein: each monomer consists of a folded ribbon-helix-helix domain and an unstructured N-terminal tail. ParG enhances ParF ATPase activity and promotes ParF self-assembly through its flexible N-terminus. In the present study, the role of the ParG N-terminus in plasmid partition was dissected. Residues crucial for plasmid partition were identified and found to form three clusters within the tail. One cluster is located at the extreme tip of the N-terminus that is the most flexible region. The second cluster is present in a linker-type region around amino acids 11-12-13 and the third is positioned in the arginine finger loop. When ParG mutant proteins were purified and characterised, they were all found to be efficient in DNA binding, transcriptional repression and in enhancing ParF polymerization. However, all the ParG mutants were impaired in stimulating ParF ATPase activity. Alteration of the residues in the tip and linker region resulted into a weaker interaction with ParF. The mutants were further investigated by using confocal and super resolution microscopy to visualize protein and plasmid positioning in the cell. Time-lapse experiments showed plasmids were static over time and that ParF oscillation over the nucleoid was abolished in the presence of mutant proteins. All the three clusters of the N-terminal tail are responsible for stimulating ParF ATPase activity and failure to do so may lead to lack of ParF oscillation. It is possible that the residues in the ParG N-terminus are strategically placed to carry out interaction and activation functions towards the common goal of coordinated interplay with ParF for efficient plasmid segregation. The data indicate that, a functional ParG N-terminal tail is a prerequisite for ParF oscillation and plasmid segregation. Based on these findings, a novel plasmid partition model is proposed which may apply to ParA-mediated partition in other plasmid systems.

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Prével, Renaud. « Mécanismes de dissémination des Entérobactéries productrices de bêta-lactamase à spectre élargi en médecine intensive réanimation ». Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0315.

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Introduction : En Europe, les entérobactéries productrices de bêta-lactamases à spectre élargi (E-BLSE) sont les principales bactéries pourvoyeuses d’antibiorésistance, à l’origine de difficutés thérapeutiques, notamment en réanimation. L’étude des mécanismes de dissémination de ces E-BLSE comprenant les mécanismes de transmission de colonisation et de lien colonisation-infection est nécessaire afin d’en maitriser la diffusion. Le premier objectif de cette thèse est d’étudier le rôle des disséminations clonale et plasmidique ainsi que celui du microbiote digestif dans la disséminaion de la colonisation digestive à E-BLSE et dans le lien-colonisation infection.Matériels et Méthodes : Les isolats E-BLSE colonisant et infectant les patients admis en service de Médecine Intensive Réanimation de l’hôpital Pellegrin entre janvier et mai 2015 ont été collectés. L’analyse de la dissémination clonale a été réalisée par pulsed-field gel electrophoresis. L’analyse de la dissémination plasmidique a été réalisée par détermination polymerase-chain reaction des groupes d’incompatibilité plasmidique. L’analyse du microbiote a été faite à partir du recueil de selles par écouvillon rectal. Les régions V3-V4 de la région codant pour l’ARN16S ribosomal et la région ITS2 ont été séquencées. L’assignation a été faite suivant le pipe-line DADA-2 et les analyses à l’aide du logiciel R version 3.6.0.Résultats : Sur 608 patients dépistés, 55 (9%) étaient porteurs rectaux d’une E-BLSE, 49 (8%) dès leur admission et 6 (1%) l’ont acquise en réanimation. Sur ces 6 patients, un seul cas de dissémination clonale a été retrouvé. Sur ces 55 patients colonisés, 38 ont été infectés dont seulement 6 (16%) par une E-BLSE, par le même clone que celui colonisant. Le fait d’être porteur rectal d’E-BLSE avait une valeur prédictive positive de 40% et une valeur prédictive négative de 100% pour le fait d’avoir une pneumopathie acquise sous ventilation mécanique. Les plasmides associés aux gènes codant pour les enzymes BLSE étaient les plasmides classiquement décrits sans qu’il ne soit trouvé de plasmide hégémonique. L’influence des plasmides sur le lien colonisation-infection n’a pas été étudiée. Lors de l’étude du microbiote intestinal, il n’y avait pas de différence observée de diversité alpha ni de diversité bêta entre les bactériobiotes et les mycobiotes des patients colonisés à E-BLSE et ceux des patients non colonisés. Il n’y avait pas non plus de différence de diversité alpha ni de diversité bêta entre les bactériobiotes des patients infectés à E-BLSE par rapport à ceux non infectés à E-BLSE parmi les patients colonisés à E-BLSE. En revanche, il existait une différence statistiquement significative de diversité alpha entre les mycobiotes des patients infectés à E-BLSE par rapport à ceux non infectés à E-BLSE parmi les patients colonisés à E-BLSE.Conclusion : La dissémination clonale semble avoir un rôle limité dans la dissémination de la colonisation digestive à E-BLSE mais participer au lien entre colonisation et infection. Notre étude ne permet pas de conclure sur le rôle de la dissémination plasmidique. Enfin, des altérations qualitatives des bactériobiotes et des mycobiotes digestifs sont associées à la colonisation par une E-BLSE. Le passage de la colonisation à l’infection par une E-BLSE pourrait s’expliquer par l’ajout d’altérations quantitatives des bactériobiotes et mycobiotes digestifs. De futures études incluant de plus nombreux patients et analysant les microbiotes associés à la colonisation par les différentes espèces d’E-BLSE permettrait de préciser les modifications du microbiote associées à la colonisation et aux infections à E-BLSE. En cas de résultat significatif, des tentatives de décolonisation digestive d’E-BLSE pourraient être menées par des probiotiques « sur mesure ».Mots clés : antibiorésistance, bêta-lactamase à spectre élargi, plasmides, microbiote intestinal
Introduction: Extended-spectrum beta-lactamase producing Enterobacteriales (ESBL-E) are a leading cause of antimicrobial resistance dissemination in Europe. ESBL-E can lead to inadequate antimicrobial treatment, especially in intensive care units (ICU). A better understanding of ESBL-E mechanisms of dissemination, including colonization diffusion and the link between colonization and infection, is needed to improve ESBL-E containment. The aims of this work are to investigate the roles of clonal and plasmidic disseminations and of gut microbiota in ESBL-E spread.Materiels and Methods: ESBL-E isolates from ICU patients between January and May 2015 were collected. Clonal dissemination was assessed by mean of pulsed-field gel electrophoresis and plasmidic one by mean of incompatibility groups determination by polymerase-chain reaction. Microbiota analysis were performed on rectal swabs by 16SrRNA coding gene and ITS2 coding gene sequencing. Assignation was performed thanks to DADA-2 pipe-line and statistical analysis on Phyloseq R package (version 3.6.0).Results: Among 508 screened patients, 55 (8%) were ESBL-E fecal carriers, 49 (8%) imported- and 6 (1%) acquired-fecal carriage. Among those 6 patients who acquired ESBL-E fecal carriage in ICU, only one case of cross-transmission was found. Among those 55 ESBL-E fecal carriers, 38 were infected during their stay in ICU but only 6 (16%) had a subsequent ESBL-E infection. To be an ESBL-E fecal carrier had a positive predictive value of 40% and a negative predictive value of 100% to have a subsequent ESBL-E ventilator-associated pneumonia. ESBL genes carrying plasmids were those usually described and we did not find any hegemonic plsmid. The plasmidic impact on the link between ESBL-E colonization and subsequent ESBL-E infection was not assessed. We did not find any difference regarding gut bacteriobiota and mycobiota alpha and beta diversities based on ESBL-E carriage status and regarding gut bacteriobiota based on subsequent ESBL-E infection. We found a statistically significant difference regarding gut mycobiota alpha diversity but not beta diversity based on subsequent ESBL-E infection.Conclusion: Clonal dissemination seems to be involved in the link between ESBL-E carriage and subsequent ESBL-E infection but poorly in ESBL-E cross-transmission. Our results do not permit to draw any conclusion regarding plasmidic dissemination. Qualitative alterations of gut microbiota could participate to ESBL-E fecal carriage but further studies are needed to better understand the underlying mechanisms. Further quantitative alterations seem to be associated with the occurrence of subsequent ESBL-E infections but, once again, further studies are nedded to decipher the causative mechanisms. These studies could pave the way to tailored probiotics to eradicate ESBL-E fecal carriage

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Duplay, Quentin. « La galle du collet chez la vigne : de la diversité des pathogènes à l'étude des plasmides et du quorum sensing d'Allorhizobium vitis S4 ». Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1041/document.

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La galle du collet, caractérisée par la formation d'une tumeur, représente la bactériose la plus répandue chez la vigne et entraine d'importantes pertes économiques à travers le monde. L'agent responsable de cette maladie est le plasmide Ti (pTi) qui confère à ces bactéries hôtes, telles que des Allorhizobium vitis, leur pouvoir pathogène. En plus du pTi, les A. vitis sont aussi les hôtes d'autres plasmides dont les fonctions et les modes de dissémination sont peu documentés. Afin d'étudier l'adaptation particulière des souches d'A. vitis à la vigne, nous nous sommes intéressés à la diversité de ces pathogènes ainsi qu'au rôle de ces plasmides et à la régulation de leur dissémination. Tout d'abord, nous avons analysé la diversité des isolats provenant de vignobles du Maroc atteints par la galle du collet. Nous avons pu mettre en évidence que l'ensemble des isolats pathogènes (e.g. porteur d'un pTi) forme 4 groupes génétiques se distinguant par le type d'opine produit. Nous nous sommes par la suite intéressés à la souche A. vitis S4 qui héberge 5 plasmides dont 3 possèdent un mécanisme de transfert pouvant être régulé par un système de communication bactérienne nommé quorum sensing (QS). Nous avons montré que le système QS du plasmide p130 (renommé pApi) contrôle non seulement le transfert conjugatif du pApi mais aussi d'autres fonctions plasmidiques non caractérisées. Ce système QS nécessite la synthèse d'une molécule signal de type N-acyl-homosérine lactone qui d'après nos travaux de caractérisation possède une structure atypique. De plus, des analyses génomiques couplées à des tests de résistance au cuivre nous ont permis d'identifier, sur le plasmide p79 d'A. vitis S4, un îlot de gènes potentiellement impliqués dans la résistance au cuivre de cette souche. Via des analyses de diversité ainsi que l'étude d'une souche modèle, nos travaux fournissent des connaissances sur l'adaptation d'Allorhizobium vitis à son unique plante hôte, la vigne
Crown gall disease, characterized by the formation of a tumor, represents the most widespread bacteriosis in the vineyard and causes significant economic losses throughout the world. The causing agent is the Ti plasmid (pTi) which confers pathogenicity to their bacterial hosts, such as Allorhizobium vitis. In addition to pTi, A. vitis harbours other plasmids whose functions and ways of dissemination are poorly documented. In order to study the peculiar adaptation of A. vitis strains to grapevine, we studied the diversity of these pathogens as well as the role of plasmids and the regulation of their dissemination.First, we analyzed the diversity of isolates from vineyards of Morocco affected by crown gall. We highlighted that all the pathogenic isolates (e.g. carrying a pTi) form 4 genetic groups that are distinguished by the type of opine produced and the ability to induce a hypersensitive response. We then focused on strain A. vitis S4, which contains 5 plasmids, including 3 possessing a transfer mechanism that can be regulated by a bacterial communication system called quorum sensing (QS). We demonstrated that the QS system of plasmid p130 (renamed pApi) controls not only the conjugative transfer of the pApi but also other uncharacterized plasmid-encoded functions. This QS system requires the synthesis of a N-acyl-homoserine lactone signal molecule with an atypical structure. In addition, genomic analyzes combined with copper resistance assays allowed us to identify, on plasmid p79 of A. vitis S4, a genomic island that is likely involved in copper resistance of this strain.Through analyzes of diversity and study of a model strain, our work provides insight into adaptation of Allorhizobium vitis to its unique host plant, grapevine

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Chisholm, Ian Alexander. « The relationship between copper and nickel resistance and the presence of plasmid DNA in isolates of Thiobacillus ferrooxidans recovered from mine tailings effluents ». Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0025/MQ31418.pdf.

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Gatignol, Anne. « Expression de genes plasmidiques de resistance a la phleomycine chez les eucaryotes ». Toulouse 3, 1987. http://www.theses.fr/1987TOU30257.

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Alexandra, Olivia. « Potential Application of Multiplex Automated Genome Engineering (MAGE) and One-Step Curing Plasmid System for Environmental Cambodian Enterobacterial Isolates ». Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-449082.

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Antimicrobial resistance (AMR) is concerning because it limits antimicrobial drug treatment options. AMR occurs by the overuse and misuse of antimicrobial drugs. In environmental settings, AMR can disseminate from places of high use, which leads to increased exposure to humans and animals. A previous study from our laboratory group showed extended-spectrum cephalosporinase-producing Escherichia coli/Klebsiella pneumoniae were isolated from fecal samples obtained in rural Cambodian community settings. Based on these isolates, this study has two aims. The first aim was characterization of selected Cambodian isolates with random amplification polymorphic DNA (RAPD) and antibiotic susceptibility test. From RAPD, the selected six isolates are diverse, except for C61 and C66 bacteria isolates with potential clonality. Additionally, the selected isolates are multidrug resistant (MDR) with reduced susceptibility to beta-lactams and fluoroquinolones. The second aim was to assess two developed methodologies, multiplex automated genome engineering (MAGE) and One-Step Curing Plasmid, by validation in bacteria laboratory strain and development for six Cambodian isolates. To modify AMR genetic elements, MAGE uses pMA7-SacB for homologous recombination with oligos for chromosomal gene disruption. Meanwhile, One-Step Curing Plasmid uses pFREE with the CRISPR/Cas9 system for plasmid and self-curing. Validation showed that MAGE can modify 8% of E. coli MG1655 with lacZ control screening oligos and almost 90% are cured from pFREE. Selected Cambodian isolates have antibiotic-resistance plasmids of IncR or IncFII replicon. For usage in Cambodian isolates, pFREE was modified to be pCAM-FREE by cloning IncR and IncFII plasmid as gRNA1 and gRNA5, respectively. Sequencing results showed pCAM-FREE have gRNA5. In conclusion, our study managed to characterize selected Cambodian isolates as MDR and diverse. In a laboratory strain, MAGE and One-Step Curing Plasmid are functional methods. Furthermore, pCAM-FREE was constructed to target IncFII and in the future, MAGE and pCAM-FREE could be tested in Cambodian isolates.

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Card, Galen Edward. « The Diversity Found Among Carbapenem-Resistant Bacteria ». BYU ScholarsArchive, 2018. https://scholarsarchive.byu.edu/etd/6949.

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This work will look at two factors that add to the diversity of carbapenem resistant bacteria. First, it focuses on the diversity of carbapenemase resistance plasmids. 446 plasmids were characterized by size, gene content and replicon groups. We identified that on average, over 30% of the encoded proteins on each plasmid have an unknown function. Plasmid sizes ranged from 1.6kb to 500kb, with an average of around 100kb and median of 80kb. Additionally, six replicon groups account for 80% of all the carbapenemase resistance plasmids. We also highlight the lack of data available for carbapenemase carrying plasmids from bacterial genera other than Escherichia and Klebsiella, and plasmids that carry the New Delhi metallo-β- lactamase or the Verona-integron encoded metallo-β-lactamase. Second, we characterized the β-lactamase diversity of a single carbapenemase resistant Klebsiella pneumoniae. This isolate encodes six distinct β-lactamases, all of which are functional, and three of which are redundant. Additionally, we determined that the CTX-M-15 cephalosporinase imparts a greater fitness when grown in aztreonam (a monobactam) than ceftazidime (a cephalosporin). Finally, we show that individually, these β-lactamases do not account for the elevated levels of resistance seen in the parent strain, indicating that the passive resistance mechanisms (i.e. efflux pumps, altered membrane porins) may play a larger role than originally thought.

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Pepper, Karen. « Etude de la localisation et de la dispersion des genes de resistance aux antibiotiques chez les streptocoques et les enterocoques ». Paris 7, 1987. http://www.theses.fr/1987PA077144.

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Sousa, Rafaela Rogério Floriano de. « Pesquisa de genes de resistência a quinolonas em bacilos Gram negativos de origem clínica e ambiental ». Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/6/6135/tde-27032014-091736/.

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Introdução. Quinolonas são antimicrobianos sintéticos que inibem as enzimas DNA-girase e topoisomerase IV resultando na morte bacteriana. São altamente eficazes no tratamento de infecções bacterianas, especialmente causadas por bactérias Gram negativas, e portanto amplamente utilizados na medicina humana e veterinária, na qual também são empregados como profiláticos. Porém, o uso indiscriminado e inadequado levou ao aumento de bactérias resistentes a estes compostos. Esta resistência pode ocorrer devido a mutações nas enzimas DNA-girase e topoisomerase IV, e também por genes contidos em plasmídeos. Estes últimos são os principais responsáveis pela disseminação e circulação da resistência entre o meio ambiente e o ambiente hospitalar. Objetivos. Pesquisar genes de resistência a antimicrobianos do grupo das quinolonas em bactérias Gram negativas de origem clínica e ambiental que apresentam resistência fenotípica a este grupo. Material e Métodos. 73 cepas de Enterobacteriaceae e Aeromonas sp. de origem clínica e ambiental foram selecionadas para o estudo, e avaliadas quanto à sensibilidade aos antimicrobianos do grupo das quinolonas e à pesquisa de genes de resistência a este mesmo grupo e mutações no gene que codifica a enzima DNA-girase por meio de PCR e sequenciamento. Resultados. Das 73 cepas previamente selecionadas para compor o estudo, 65 foram utilizadas, devido à exclusão de perfis clonais similares. Nestas, foram observados os genes, qnrS1 (1,5 por cento ), qnrS2 (26,2 por cento ), qnrB1 (3,1 por cento ), qnrB19 (12,3 por cento ), qnrD1 (1,5 por cento ), aac(6)-Ib-cr (10,8 por cento ), oqxA (43,1 por cento ) e oqxB (41,5 por cento ), e duas variantes determinadas qnrB-like (3,1 por cento ) e qnrB69-like (1,5 por cento ). Os genes qnrA, qnrC e qepA não foram identificados. Mutações na enzima DNA-girase foram observadas em 97,9 por cento das cepas positivas para algum dos genes pesquisados. Em 4 cepas foi possível estabelecer a associação do gene aac(6)-Ib-cr com integron de classe 1. Foi realizado sequenciamento e caracterização do plasmídeo completo onde estava inserido o gene qnrD1. Conclusões. Este estudo relata pela primeira vez no Brasil a ocorrência dos genes qnrS2, oqxA e oqxB, a associação entre o elemento genético integron de classe 1 e o gene aac(6)-Ib-cr, e o gene qnrD1 e a caracterização do plasmídeo completo onde este estava inserido. Os genes qnrB1, qnrB19, e aac(6)-Ib-cr, anteriormente apenas relatados em cepas clínicas, foram observados em cepas ambientais. Os resultados deste estudo mostram alta frequência de genes de resistência a quinolonas tanto em isolados clínicos quanto em isolados ambientais, alertando quanto à disseminação da resistência entre fontes diferentes, e possível manutenção destes genes por cepas ambientais.
Introduction. Quinolones are synthetic antimicrobial agents that inhibit DNA gyrase and topoisomerase IV enzymes resulting in bacterial death. They are highly effective in the treatment of bacterial infections, especially the ones caused by Gram negative bacteria, as well as for prophylaxy. Therefore they are widely used in human and veterinary medicine. However, indiscriminate and improper use led to an increase of bacteria resistance to these compounds. This resistance can be due to mutations in DNA gyrase and topoisomerase IV enzymes and also by genes contained in plasmids, which are mainly responsible for the spread and transmission of resistance between the environment and the hospital set. Objectives. To search for genes of resistance to quinolone antimicrobial agents in Gram-negative bacteria from clinical and environmental strains that present phenotypic resistance to this group. Material and Methods. 73 strains of Enterobacteriaceae and Aeromonas spp., from clinical and environmental origin, were selected for this study, and evaluated for antimicrobial susceptibility of quinolone and search of resistance genes in this same group and also for mutations in the gene encoding the enzyme DNA gyrase by PCR and sequencing. Results. Of the 73 strains previously selected to compose this study, 65 were used, due to the exclusion of similar clonal profiles. In these, genes qnrS1 (1.5 per cent ), qnrS2 (26.2 per cent ) qnrB1 (3.1 per cent ), qnrB19 (12.3 per cent ) qnrD1 (1.5 per cent ) aac(6\')-Ib-cr (10.8 per cent ) oqxA (43.1 per cent ) and oqxB (41.5 per cent ) were observed, and two variants were named as qnrB-like (3.1 per cent ) and qnrB69-like (1.5 per cent ). The qnrA, qnrC and qepA genes were not identified. Mutations in DNA gyrase enzyme were observed in 97.9 per cent of the positive strains for at least one of the genes studied. It was possible to establish the association of aac(6\')-Ib-cr with class 1 integron gene in four strains. Complete sequencing and characterization of plasmid qnrD1, where the gene was inserted, was performed. Conclusions. This study reports, for the first time in Brazil, the occurrence of qnrS2, oqxA and oqxB genes, the association between genetic element integron class 1 gene and the aac (6 \')-Ib-cr, and qnrD1 gene and the characterization of the complete plasmid where this was inserted. qnrB1, qnrB19, and aac(6\')-Ib-cr genes, previously only reported in clinical strains, were observed in environmental strains. The results of this study show a high frequency of quinolone-resistance genes for both clinical and environmental isolates, warning about the spread of resistance through different sources, and the possible maintenance of these genes by environmental strains.

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Dotto, Giorgia. « L'antibiotico-resistenza in ceppi di E. coli isolati da specie aviari e cunicole commerciali e selvatiche ». Doctoral thesis, Università degli studi di Padova, 2013. http://hdl.handle.net/11577/3423395.

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Antimicrobial-resistance is a public health problem world-wide and it is largely attributed to horizontal exchange of transferable genetic ele-ments such as plasmids carrying integrons (Sunde and Norstrom, 2006). The aim of this study was to define the antimicrobial-resistance phenotypes and to characterize class 1 and class 2 integrons in E. coli isolated both from commercial and wild birds and lagomorphs between 2006 and 2012 in Italy. The strains isolated form lagomorphs were also genotyped and screened for Plasmid-Mediated Quinolones Resistance (PMQR) genes and plasmids. Strains were examined for antimicrobial susceptibility by agar disk diffusion method. Class 1 and class 2 integrons were detected by real-time PCR and gene cassettes content identified by DNA sequencing. PMQR genes were screened by PCR and DNA sequencing. Clonal relatedness of the isolates from lagomorphs was assessed by Multilocus Sequence Typing (MLST). Plasmids were characterized by PCR-Based Replicon Typing (PBRT). Class 1 integrons were detected in multi-drug resistant E. coli both from commercial and wild birds and lagomorphs while class 2 integrons were found only in domestic avian species. Different gene cassettes were identified but the most common were combinations of aadA, sat and dfrA, codifying for aminoglycosides and trimethoprim resistance. Of the 172 E. coli isolates from lagomorphs, 12.2% (21/172) carried oqxAB, none other PMQR de-terminants. All but 3 oqxAB positive E. coli strains were recovered from farm rabbits and most of them were associated with the predominant CC17 and carried from 1 to 7 different plasmid types, such as IncF, IncHI1, IncI1, IncR, IncN, IncP, IncX1, IncY, and ColE. This study pro-vides new insights on the prevalence and dissemination of integrons and oqxAB in E. coli from farm and wild animals, suggesting that these species may be reservoir of these genetic determinants in Italy and thus a potential source of multi-drug resistant E. coli for humans
Il fenomeno dell’antibiotico-resistenza rappresenta un grave problema per la sanità pubblica e la sua diffusione è attribuita principalmente alla capacità dei batteri di scambiarsi orizzontalmente materiale genetico attraverso plasmidi, spesso associati agli integroni (Sunde and Nor-strom, 2006). Questo studio nasce con l’obiettivo di definire i profili fe-notipici e genotipici di antibiotico-resistenza, in particolare la presenza e la caratterizzazione degli integroni di classe 1 e 2 in E. coli isolati sia da volatili che da lagomorfi domestici e selvatici campionati in Italia tra il 2006 e il 2012. I ceppi isolati da quest’ultima categoria di animali sono stati sottoposti anche a tipizzazione molecolare mediante Multilocus Se-quence Typing (MLST) e del contenuto plasmidico. Mediante PCR-Based Replicon Typing (PBRT). Inoltre sono stati ricercati i geni Plasmid Mediated Quinolone Resistance (PMQR) responsabili di una ridotta sensibilità ai chinoloni e fluorochinoloni. La presenza degli integroni di classe 1 è stata riscontrata in ceppi con profili di multifarmaco-resistenza isolati sia da volatili che da lagomorfi domestici e selvatici. Gli integroni di classe 2 sono stati invece rilevati soltanto negli E. coli di origine aviare. Le cassette geniche riscontrate di frequente sono state varie combinazioni dei geni aadA, sat e dfrA, responsabili della resistenza agli aminoglicosidi e al trimethoprim. Il 12,2% degli isolati da lagomorfi domestici e selvatici è risultato positivo ai geni oqxAB e, ad eccezione di 3, isolati da conigli di allevamento. La maggior parte dei ceppi oqxAB-positivi apparteneva al CC17 e presentava nel proprio corredo da 1 a 7 classi di plasmidi diversi, quali IncF, IncHI1, IncI1, IncR, IncN, IncP, IncX1, IncY, e ColE. Questo studio fornisce un importante contributo sulla diffusione degli integroni e dei geni PMQR in ceppi isolati da animali sia domestici che selvatici e indica l’importante ruolo giocato da queste specie come reservoir di determi-nanti genetici di antibiotico-resistenza e quali possibili fonti di batteri antibiotico-resistenti per l’uomo

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Jackson, David. « A plasmid-based reverse genetics system for influenza B viruses : studies of resistance to neuraminidase inhibitors and the proteins unique to influenza B viruses, NB and BM2 ». Thesis, University of Reading, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.402916.

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Santos, Andressa Liberal. « Perfil fenotípico e genotípico de enterobactérias resistentes aos beta-lactâmicos ». Universidade Federal de Goiás, 2018. http://repositorio.bc.ufg.br/tede/handle/tede/8780.

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Enterobacteria are microorganisms involved with bacteria and the health of women with care. Treatment of bacterial infections is most often done with the use of antibiotics and one of the major classes of antimicrobials is one of the β-lactams. Among the main mechanisms of resistance to the observational β-lactam antibiotics: alteration of the antimicrobial target; alteration of β-lactam permeability; Flow pumps and the entry of enzymatic signals that destroy the β-lactate totally or with the development of an alternative metabolic pathway. These enzymes are known as beta-lactamases and are encoded by specific genes. Thus, the objective of this study was to correlate the resistance profile of enterobacteria, using phenotype methodologies and identifying 14 genes that encode as beta-lactamase enzymes: blaOXA genes; blaIMP; blaNDM; blaSME; blaDHA; blaCMY, blaTEM, blaKPC, blaSPM, blaCTX-M, blaVIM, blaSIM, blaGim and blaSHV. The phenotypic methodologies used were the Antimicrobial Sensitivity Test for Disk-Diffusion (antibiogram), and complementary tests for the detection of resistance mechanisms of beta-lactamases (ESBL, MBL, AmpC and Carbapenemase). The molecular methodology used was Real Time PCR using the Sybr Green system. Among the results found in the tests it was observed that 74.28% were resistant to ampicillin, 34.28% were resistant to aztreonam, 62.85% were resistant to amoxicillin associated with clavalunate, 51.42% were resistant to ceftazidime, 41 , 42% were resistant to cefoxitin, 54.28% were resistant to cefazolin, 44.28% were resistant to cefepime, 41.42% were resistant to cefuroxime, 8.57% were resistant to cefuroxime, 35.71% were resistant an imipenem and 41.42% were resistant to piperacillin associated with tazobactam. Among the total samples, the mechanism of resistance that presented the highest expression was ESBL (17.14%). The genes studied that were detected in a greater number of genera were blaGIM and blaSIM (66.66% of the samples). The gene that was amplified in a smaller number of samples was blaVIM (16.66%). It is concluded that although there is a low correlation between the methodologies analyzed, the levels of antimicrobial resistance in enterobacteria are high and worrying, and a way to minimize the accelerated emergence of resistance includes the development or improvement of techniques that generate diagnoses with high efficiency and speed.
As enterobactérias são microrganismos envolvidos com infecções bacterianas adquiridas na comunidade e nos ambientes dos cuidados com a saúde. O tratamento das infecções bacterianas na maioria das vezes é realizado com a utilização de antibióticos e uma das maiores classes de antimicrobianos é a dos β-lactâmicos. Entre os principais mecanismos de resistência aos antimicrobianos β-lactâmicos observa-se: alteração do alvo antimicrobiano; alteração da permeabilidade ao β-lactâmico; bombas de e-fluxo e a presença de mecanismos enzimáticos que destroem o β-lactâmico totalmente ou parcialmente com desenvolvimento de uma via metabólica alternativa. Essas enzimas são conhecidas como beta-lactamases e são codificadas por genes específicos. Dessa forma, o objetivo deste estudo foi o de correlacionar o perfil de resistência das enterobactérias, utilizando metodologias fenotípicas e identificar 14 genes que codificam as enzimas beta-lactamases: genes blaOXA; blaIMP; blaNDM; blaSME; blaDHA; blaCMY, blaTEM, blaKPC, blaSPM, blaCTX-M, blaVIM, blaSIM, blaGIM e blaSHV. As metodologias fenotípicas utilizadas foram o Teste de Sensibilidade aos Antimicrobianos por disco-difusão (antibiograma), e testes complementares para detecção dos mecanismos de resistência das beta-lactamases (ESBL, MBL, AmpC e Carbapenemase). A metodologia molecular utilizada foi a PCR em Tempo Real, utilizando o sistema Sybr Green. Entre os resultados fenotípicos encontrados nas bactérias observou-se que 74,28% foram resistentes a ampicilina, 34,28% foram resistentes a aztreonam, 62,85% foram resistentes a amoxicilina associado ao clavalunato, 51,42% foram resistentes a ceftazidima, 41,42% foram resistentes cefoxitina, 54,28% foram resistentes a cefazolina, 44,28% foram resistentes a cefepime, 41,42% foram rsistentes a ceftriaxona, 8,57% foram resistentes a cefuroxima, 35,71% foram resistentes a imipenem e 41,42% foram resistentes a piperacilina associada tazobactam. Entre o total de amostras, o mecanismo de resistência que apresentou maior expressão foi o ESBL (17,14%). Os genes estudados que foram detectados em um maior número de gêneros foram o blaGIM e o blaSIM (66,66% das amostras). O gene amplificado em menor número de amostras foi o blaVIM (16,66%). Conclui-se que apesar de haver baixa correlação entre as metodologias analisadas, os níveis de resistência a antimicrobianos em enterobactérias são altos e preocupantes e uma maneira de minimizar a acelerada emergência de resistência é desnvolver e aprimorar técnicas de diagnósticos com alta eficiência e rapidez.

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GUGLIELMETTI, ELENA. « Antibiotico resistenza in batteri lattici : basi molecolari e trasferibilità ». Doctoral thesis, Università Cattolica del Sacro Cuore, 2009. http://hdl.handle.net/10280/404.

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La scoperta e il successivo uso di antibiotici hanno reso resistenti molte specie batteriche sia di origine animale sia umana. I geni di resistenza agli antibiotici possono essere trasferiti tramite la catena alimentare, a partire dagli animali e alimenti, fino al tratto gastrointestinale degli esseri umani. Il presente studio descrive la proprietà coniugativa di alcuni nuovi plasmidi, in particolare di uno identificato in un ceppo di Lactococcus lactis spp. lactis, isolato dall'intestino di pesce, e di altri plasmidi individuati in ceppi di Lactobacillus brevis, Lb. plantarum e Lb. reuteri, isolati da salame. La trasferibilità dei plasmidi che portano i geni di resistenza per l’eritromicina o tetraciclina è stata valutata con metodi di elettroporazione e coniugazione in vitro. Nello specifico è riportato il trasferimento di tali plasmidi a specie batteriche patogene per l’uomo come Listeria monocytogenes e Staphylococcus spp. e a un agente responsabile di Lactococcosi nei pesci come Lc. garvieae. Dopo lo studio sulle proprietà coniugative si è proceduto alla caratterizzazione di questi elementi extracromosomici con esperimenti di comobilizzazione e stabilità. I dati ottenuti suggeriscono come i LAB possano essere un serbatoio di diffusione dei geni per l’antibiotico resistenza, con gravi rischi per l’allevamento di prodotti ittici e salute umana.
The discovery and subsequent widespread use of antibiotics have rendered many bacterial species of human and animal origin resistant to some antibiotics. Antibiotic resistance gene may be transferred via food chain, from animals into fermented and other food or in the human gastrointestinal tract. The transferability of some plasmids that harbor the tetracycline or erythromycin resistance genes to animal and human pathogens was assessed using electrotrasformation and conjugation. The present study describes the proprieties of some new plasmids, originally isolated from fish intestinal Lactococcus lactis ssp. lactis and from fermented sausage Lactobacillus brevis, Lb. plantarum and Lb. reuteri. In particular, here I report the potentially of transferable antibiotic resistance determinants to human pathogenic bacterial like Listeria monocytogenes and Staphylococcus spp. and to an etiologic agent of Lactococcus infection like Lc. garvieae. The possibility of transferring natural Lactococcus and Lactobacillus plasmids into pathogenic bacterial strains involved the characterization of these elements, like comobilization and plasmid stability. These data suggest that lactic acid bacteria (LAB) might be reservoir organism for acquired resistance genes that can be spread both to fish and human pathogens, posing a risk to aquaculture and human health.

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GUGLIELMETTI, ELENA. « Antibiotico resistenza in batteri lattici : basi molecolari e trasferibilità ». Doctoral thesis, Università Cattolica del Sacro Cuore, 2009. http://hdl.handle.net/10280/404.

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La scoperta e il successivo uso di antibiotici hanno reso resistenti molte specie batteriche sia di origine animale sia umana. I geni di resistenza agli antibiotici possono essere trasferiti tramite la catena alimentare, a partire dagli animali e alimenti, fino al tratto gastrointestinale degli esseri umani. Il presente studio descrive la proprietà coniugativa di alcuni nuovi plasmidi, in particolare di uno identificato in un ceppo di Lactococcus lactis spp. lactis, isolato dall'intestino di pesce, e di altri plasmidi individuati in ceppi di Lactobacillus brevis, Lb. plantarum e Lb. reuteri, isolati da salame. La trasferibilità dei plasmidi che portano i geni di resistenza per l’eritromicina o tetraciclina è stata valutata con metodi di elettroporazione e coniugazione in vitro. Nello specifico è riportato il trasferimento di tali plasmidi a specie batteriche patogene per l’uomo come Listeria monocytogenes e Staphylococcus spp. e a un agente responsabile di Lactococcosi nei pesci come Lc. garvieae. Dopo lo studio sulle proprietà coniugative si è proceduto alla caratterizzazione di questi elementi extracromosomici con esperimenti di comobilizzazione e stabilità. I dati ottenuti suggeriscono come i LAB possano essere un serbatoio di diffusione dei geni per l’antibiotico resistenza, con gravi rischi per l’allevamento di prodotti ittici e salute umana.
The discovery and subsequent widespread use of antibiotics have rendered many bacterial species of human and animal origin resistant to some antibiotics. Antibiotic resistance gene may be transferred via food chain, from animals into fermented and other food or in the human gastrointestinal tract. The transferability of some plasmids that harbor the tetracycline or erythromycin resistance genes to animal and human pathogens was assessed using electrotrasformation and conjugation. The present study describes the proprieties of some new plasmids, originally isolated from fish intestinal Lactococcus lactis ssp. lactis and from fermented sausage Lactobacillus brevis, Lb. plantarum and Lb. reuteri. In particular, here I report the potentially of transferable antibiotic resistance determinants to human pathogenic bacterial like Listeria monocytogenes and Staphylococcus spp. and to an etiologic agent of Lactococcus infection like Lc. garvieae. The possibility of transferring natural Lactococcus and Lactobacillus plasmids into pathogenic bacterial strains involved the characterization of these elements, like comobilization and plasmid stability. These data suggest that lactic acid bacteria (LAB) might be reservoir organism for acquired resistance genes that can be spread both to fish and human pathogens, posing a risk to aquaculture and human health.

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Babosan, Anamaria. « Description d'un mécanisme, à l'origine de l'induction de la réponse SOS par les aminosides chez Escherichia coli, favorisant l'émergence de la résistance aux fluoroquinolones ». Thesis, Reims, 2018. http://www.theses.fr/2018REIMS024/document.

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L’émergence des déterminants de résistances plasmidiques aux quinolones (PMQR), auxquels appartient le gène qnrD, participent de manière significative à la sélection des résistances de haut-niveau aux permet les réparations de l’ADN lors des stress soumis aux bactéries, et d’autre part, que les aminosides, une autre classe d’antibiotiques que les fluoroquinolones, induisaient la réponse SOS chez Escherichia coli. En effet, nous avons montré que les petits plasmides-qnrD chez E. coli, induisent la formation de monoxyde de nitrogène et l’inhibition de la voie de détoxification Hmp-dépendante. Ces processus génèrent des lésions à l’ADN qui s’ajoutent à celles occasionnées par les aminosides concourant à activer la réponse SOS chez E. coli. L’ensemble de nos résultats montrent que l’émergence de la résistance aux fluoroquinolones peut être occasionnée par l’exposition d’E. coli à une autre classe d’antibiotiques, ici les aminosides
The emerging plasmid-mediated quinolones resistance (PMQR) determinants significantly participate in the selection of high-level of resistance to the major antibiotics fluoroquinolones, leading to numerous clinical failures. In this study, we reported for the first time that PMQR expression could be triggered by the fluoroquinolones but also by another major class of antibiotics, the aminoglycosides. We were able to show that this unique cross selection of antibiotic resistance was the consequence of the PMQR determinant qnrD being SOS-regulated in a RecA-LexA dependent manner. We demonstrated that sub inhibitory concentration of aminoglycoside induced nitric oxide formation associated with the repression of the Hmp-mediated detoxification pathway, resulting in the induction of the SOS response and thus up-regulation of the PMQR. Overall, our findings revealed an unexpected antibiotic resistance cross-selection with low aminoglycosides concentrations promoting emergence of fluoroquinolones resistance

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Guerreiro, Lara Sofia Fernandes. « Influência do uso de enrofloxacina no aparecimento de resistência às quinolonas mediada por plasmídeos em Escherichia coli de vitelos ». Master's thesis, Universidade Técnica de Lisboa. Faculdade de Medicina Veterinária, 2012. http://hdl.handle.net/10400.5/4589.

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Dissertação de Mestrado em Segurança Alimentar
O conhecimento sobre a presença e frequência de genes de resistência às quinolonas mediada por plasmídeos (RQMP) em estirpes comensais de Escherichia coli de origem bovina é escasso a nível mundial. Foram estudadas um total de 237 amostras de E. coli de vitelos saudáveis previamente isoladas após pressão selectiva in vivo de enrofloxacina (ENR) e caracterizadas quanto à resistência aos antibióticos: 101, 79 e 57 isolados relativos a T0, T1 (6 semanas após administração de ENR) e T2 (10 semanas após administração de ENR), respectivamente. Os isolados foram caracterizados fenotipicamente por determinação da Concentração Inibitória Mínima (CIM) para os antimicrobianos ácido nalidíxico (AN), ciprofloxacina (CIP) e levofloxacina (LEV) e os resultados interpretados segundo os critérios epidemiológicos (ECOFF) estabelecidos pelo EUCAST. A frequência de genes de RQMP (qnr, aac(6’)-Ib-cr e qepA) foi determinada através de amplificação por PCR e sequenciação nucleotídica. A proporção de isolados de E. coli resistentes ao AN em T0, T1 e T2 foi de, respectivamente: 52,5% (n=53; CIM 64->256 μg/ml), 100% (n=79; CIM 128->256 μg/ml) e 82,5% (n=47; CIM 128->256 μg/ml). A resistência à CIP em T0, T1 e T2 foi de, respectivamente: 52,5% (n=53; CIM 0,125->256 μg/ml), 100% (n=79; CIM 0,25-128 μg/ml) e 89,5% (n=51; CIM 0,25-64 μg/ml). A resistência à LEV em T0, T1 e T2 foi de, respectivamente: 46,5% (n=47; CIM 0,5-64 μg/ml), 100% (n=79; CIM 0,5-64 μg/ml) e 87,7% (n=50; CIM 0,5-32 μg/ml). No que respeita aos determinantes de RQMP nos 237 isolados estudados, foram identificados: 11,8% (n=28) positivos para genes qnr (qnrB2, n=4; qnrD, n=11; qnrS1, n=13); e 0,8% (n=2) isolados positivos para o gene aac(6’)-Ib-cr. Da análise da frequência dos genes de RQMP nos isolados de E. coli observou-se: em T0, 3% de genes qnr (todos qnrS1) e 2% do gene aac(6’)-Ib-cr; em T1, 15,2% de genes qnr (10,1% qnrD e 5,1% qnrS1); em T2, 22,8% de genes qnr (7% qnrB2, 5,3% qnrD e 10,5% qnrS1). Os dados obtidos evidenciam um aumento significativo da prevalência de isolados resistentes ao longo do tempo de colheita, sugerindo que a pressão selectiva imposta pela exposição à ENR tem influência no aparecimento de resistência às quinolonas. Observou-se um aumento significativo da frequência de genes de RQMP ao longo do estudo longitudinal e mais de 80% dos isolados positivos para RQMP foram resistentes às quinolonas. Este é, para o nosso conhecimento, o primeiro estudo que descreve a identificação de resistência às quinolonas por qnrD em isolados de E. coli de bovinos.
ABSTRACT - The current knowledge about the presence and frequency of pasmid-mediated quinolone resistance (PMQR) genes in commensal Escherichia coli strains from cattle is scarce. Two hundred and thirty seven E. coli samples isolated from healthy calves were studied after in vivo enrofloxacin (ENR) selective pressure and previously characterized regarding antimicrobial susceptibility, including: 101, 79 and 57 isolates from T0, T1 (six weeks after ENR administration) and T2 (10 weeks after ENR administration), respectively. The phenotypic characterization was performed using Minimum Inhibitory Concentration (MIC) determinantion for nalidixic acid (NAL), ciprofloxacin (CIP) and levofloxacina (LEV) by the microdilution method and the results were interpreted according to EUCAST definitions of epidemiological cut-off values (ECOFF). PMQR genes (qnr, aac(6’)-Ib-cr and qepA) frequency was performed by PCR amplification and nucleotide sequencing. NAL resistant E. coli isolates in T0, T1 and T2 were, respectively: 52,5% (n=53; MICs 64 to >256 μg/ml), 100% (n=79; MICs 128 to >256 μg/ml) and 82,5% (n=47; MICs 128 to >256 μg/ml). CIP resistant isolates in T0, T1 and T2 were, respectively: 52,5% (n=53; MICs 0,125->256 μg/ml), 100% (n=79; MICs 0,25-128 μg/ml) and 89,5% (n=51; MICs 0,25-64 μg/ml). LEV resistant isolates in T0, T1 and T2 were, respectively: 46,5% (n=47; MICs 0,5-64 μg/ml), 100% (n=79; MICs 0,5-64 μg/ml) and 87,7% (n= 50; MICs 0,5-32 μg/ml). From the 237 E. coli isolates tested: 11,8% (n=28) harboured qnr genes (qnrB2, n=4; qnrD, n=11; and qnrS1, n=13) and 0,8% (n=2) were found positive for the aac(6’)-Ib-cr gene. The analysis of PMQR genes in E. coli at the different sampling times showed that: at T0, qnr genes were detected in 3% of the isolates (all found to be qnrS1) and 2% carried the aac(6’)-Ib-cr gene; at T1, 15,2% of the isolates carried qnr genes (10,1% qnrD and 5,1% qnrS1); and at T2, 22,8% of the isolates were found positive for qnr genes (7% qnrB2, 5,3% qnrD and 10,5% qnrS1). The results reveal an increased prevalence of resistant isolates along the time, suggesting that ENR selective pressure influences the emergence of quinolone resistance. A significant increased frequency of PMQR genes along the longitudinal study was observed and more than 80% of PMQR positive isolates were quinolone resistant. This is to our knowledge the first report on PMQR qnrD gene in E. coli isolates from cattle.

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Drieux, Laurence. « Succès plasmidique : transmission inter-espèce d'un plasmide portant un gène de métallo-bêta-lactamase ». Thesis, Paris 11, 2012. http://www.theses.fr/2012PA114816/document.

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Les métallo-b-lactamases (MBL) acquises constituent une menace sanitaire par risqué d’impasse thérapeutique dans les infections causées par les bacilles à Gram négatif, en particulier lorsque ces bactéries produisent une b-lactamase à spectre étendu (BLSE). La MBL VIM-1 est une carbapénémase qui hydrolyse toutes les β-lactamines, à l’exception des monobactames. Cette enzyme a émergé en Grèce où elle est désormais endémique chez les entérobactéries. Six souches de bacilles à Gram négatif produisant une carbapénémase ont été isolées chez un même patient qui avait été rapatrié de Grèce. Trois de ces souches, Providencia stuartii (Ps), Proteus mirabilis (Pm) et Escherichia coli (Ec), produisaient la MBL VIM-1 et la BLSE SHV-5. Dans chacune de ces trois souches, les gènes blaVIM-1 et blaSHV-5 étaient portés par un plasmide transférable par conjugaison in vitro. Les plasmides extraits des transconjugants présentaient le même profil de restriction et portaient un intégron de classe 1 identique dans lequel le gène blaVIM-1 était intégré. Nous avons émis l’hypothèse qu’un plasmide codant pour VIM-1 et SHV-5 avait été transféré de la souche Ps vers les souches Pm et Ec dans le tube digestif du patient et avons reproduit ce transfert in vivo dans un modèle de souris gnotoxéniques. Au cours de cette expérience, le plasmide codant pour VIM-1 et SHV-5 a été transféré avec succès de la souche Ps vers la souche réceptrice E. coli J53, en dehors de toute pression de sélection par les antibiotiques. Nous avons ensuite analysé la séquence complète du plasmide pTC2 extrait d’un transconjugant obtenu par conjugaion in vivo. Ce plasmide de type co-intégrat (IncA/C, IncR) de 180kb possédait un squelette de type IncA/C et une région de multirésistance (MDR1) au sein de laquelle était intégré un fragment de type IncR de 13kb. L’analyse de cette séquence nous a permis d’identifier un système de transfert de type IncA/C complet et intact et différents types de systèmes de maintien, à la fois au sein du squelette IncA/C et au sein du fragment IncR. La région mosaïque MDR1 contenait neuf séquences d’insertion (sept copies de l’IS26, une IS1 et une IS6100), 10 gènes de résistance aux antibiotiques et l’opéron mer de résistance au mercure qui étaient intégrés dans des transposons unitaires, des transposons composites ou des intégrons. Le plasmide pTC2 cumule des propriétés qui font de lui un véhicule performant de la résistance aux antibiotiques : un large spectre d’hôte, de bonnes capacités de transfert, de bonnes capacités de maintien dans une population bactérienne, une grande plasticité de sa région MDR1 et une grande variété de gènes de résistance
Acquired metallo-b-lactamases (MBLs) represent a threat for the treatment of infections caused by Gram-negative bacteria, particularly by enterobacteria that already produce extended-spectrum b-lactamases (ESBL). VIM-1 MBL, which is a carbapenemase that can hydrolyze all classes of β-lactam antibiotics except monobactams, has emerged in Greece and is now commonly found in Enterobacteriaeae. Six carbapenemase-producing strains of Gram-negative bacilli were isolated from a unique patient transferred from Greece to a French hospital. Three of these strains, Providencia stuartii (Ps), Proteus mirabilis (Pm) and Escherichia coli (Ec) strains, were shown to produce the MBL VIM-1 and the ESBL SHV-5. In each of these three strains, the blaVIM-1 gene was carried by a plasmid transferable by in vitro conjugation. The plasmids extracted from the transconjugants displayed a unique restriction profile and harboured identical VIM-1-containing class 1 integrons. Considering the hypothesis that this VIM-1 plasmid had probably been transferred from the Ps strain to the Ec and Pm strains, we performed in vivo conjugation assays in the digestive tract of gnotobiotic mice colonized with E. coli J53, to demonstrate that the VIM-1 plasmid harboured by strain Ps was transferable in vivo, in absence of antibiotic pressure. We determined the complete nucleotide sequence of the VIM-1-encoding plasmid pTC2, which was isolated in a Greek Providencia stuartii multiresistant strain. This 180-kb plasmid was found to be a multireplicon plasmid (IncA/C, IncR), with a large IncA/C backbone and a mosaic multidrug resistance (MDR1) region, in which was inserted a 13-kb IncR fragment. A CD-search-based annotation of the plasmid allowed the identification of a complete IncA/C-type transfer system and of several putative maintenance modules, either on the IncA/C backbone, and on the IncR fragment. The complex MDR1 region contained nine insertion sequences (seven copies of the IS26, one IS1 and one IS6100), 10 resistance genes and a mercury resistance operon integrated either into unit transposons, composite transposons or integrons. The broad-host range, the transfer capacities, the stability, the high plasticity of the MDR1 region combined to the variety of resistance genes make pTC2 a superspreader of resistance determinants

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Bonot, Sébastien. « Persistance et dissémination du plasmide pB10, vecteur de gènes de résistance aux antibiotiques, dans des biomasses issues de stations d'épuration d'eaux usées urbaines ». Thesis, Nancy 1, 2010. http://www.theses.fr/2010NAN10050/document.

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L’utilisation massive des antibiotiques, depuis les années 50, génère une libération importante de ces molécules dans l’environnement (excrétion via les urines et les fèces) que l’on peut retrouver à des concentrations allant de 1 à 100 ng/L dans les eaux usées urbaines. Parce qu’elle réunit microorganismes résistants et antibiotiques, la station d’épuration d’eaux usées urbaines pourrait être une zone propice au transfert des gènes de résistance. Cependant, avec sa position stratégique à l’interface entre les activités humaines et l’environnement, la station d’épuration pourrait constituer un « rempart » contribuant à limiter leur dissémination dans l’environnement.Les paramètres qui influencent ces transferts dans les stations d’épuration sont encore mal connus, en particulier du fait de limitations méthodologiques. Aussi l’objectif de notre travail était de déterminer les facteurs environnementaux influant sur la stabilité et le transfert d’un élément génétique mobile modèle, le plasmide pB10, dans des communautés bactériennes (biomasses de stations d’épuration et sédiments de rivière) maintenues en microcosmes. Jusqu’à présent, les transferts de gènes de résistance ont été principalement étudiés avec des méthodes reposant sur la culture de microorganismes sur milieux sélectifs, dont nous savons aujourd’hui qu’elles sous-estiment les phénomènes observés. Aussi, nous avons élaboré une approche basée sur la PCR quantitative pour détecter la dissémination d’un ADN mobile modèle amené via une bactérie hôte E. coli DH5α. Les couples amorces/sondes très spécifiques ont pu être élaborés en tirant profit de la structure mosaïque du génome bactérien. L’approche proposée repose sur des mesures comparées du nombre de plasmide pB10 et de son hôte bactérien DH5α au cours du temps, où une augmentation du rapport (pB10/DH5α) implique une dissémination du plasmide vers les bactéries indigènes. Outre l’intérêt du développement méthodologique proposé, cette méthode a permis d’évaluer l’incidence de quelques paramètres environnementaux sur la dissémination d’un ADN au sein de communautés microbiennes complexes. Deux groupes de facteurs ont pu être distingués selon qu’ils influencent la persistance du plasmide pB10 dans les communautés dans son hôte initial (oxygénation/brassage, ajout d’antibiotiques en concentrations sub-inhibitrices comme l’amoxicilline et le sulfaméthoxazole fréquemment retrouvés en station d’épuration) ou/et qu’ils favorisent sa dissémination dans les communautés bactériennes (biofilms, sédiments). Sans induire de transferts génétiques, les antibiotiques testés, même en concentrations sub-létales, pourraient participer à la dissémination de gènes de résistance en favorisant leur persistance
The widespread use of antibiotics since the 50s, generates a significant release of these molecules in the environment (excretion via urine and feces) which can be found at concentrations ranging from 1-100 ng/L in wastewater. Due to the high microbial biomass and the abundance of nutrients, wastewater treatment plants (WWTP) represent a suitable habitat for horizontal gene transfer. Because they occupy a key position between human activities and the environment, WWTP may play a major role in limiting the dissemination of antibiotic resistance genes, therefore contributing to the preservation The parameters which influence these transfers in wastewater treatment plants are still poorly known, especially because of methodological limitations. Therefore the aim of our study was to identify environmental factors affecting the stability and transfer of a mobile genetic element model, the plasmid pB10 in bacterial communities (biomass from wastewater treatment plants and river sediments) maintained in microcosms. So far, the transfer of resistance genes have been studied mainly with methods based on the cultivation of microorganisms on selective media that we know now they underestimate the observed phenomena. Also, an approach based on quantitative PCR was developed for detecting the release of a mobile DNA template from the host bacterium E. coli DH5α. Couples of designed primers/probes were very specific and have been developed by taking advantage of the mosaic structure of the bacterial genome. The proposed approach is based on the over time measurements of the number of plasmids pB10 and its bacterial host DH5α, where an increased ratio (pB10/DH5α) implies a release of the plasmid to the indigenous bacteria. This method was used to assess the impact of some environmental parameters on the release of DNA in complex microbial communities. Two groups of factors could be distinguished according to whether they influence the persistence of plasmid pB10 in communities in microcosms (oxygenation / mixing, addition of antibiotics at sub-inhibitory concentrations as amoxicillin and sulfamethoxazole frequently found in treatment plant) and / or they favor his release in bacterial communities (biofilms, sediments). Without inducing genes transfers, the antibiotics tested, even at sub-lethal concentrations, could participate in the dissemination of resistance genes by facilitating their persistence

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Zwanzig, Martin [Verfasser], Uta [Gutachter] Berger, Uta [Akademischer Betreuer] Berger, Thomas U. [Akademischer Betreuer] Berendonk, Jan-Ulrich [Gutachter] Kreft et Susann [Gutachter] Müller. « Modelling the spread of plasmid-encoded antibiotic resistance in aquatic environments considering evolutionary modifications, individual heterogeneity and complex biotic interactions / Martin Zwanzig ; Gutachter : Uta Berger, Jan-Ulrich Kreft, Susann Müller ; Uta Berger, Thomas U. Berendonk ». Dresden : Technische Universität Dresden, 2020. http://d-nb.info/1227196873/34.

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SANCHEZ-LOPEZ, ROSANA. « Partie i : etude des mecanismes impliques dans la resistance au sel chez une bacterie halophile- partie ii : etude des relations structure-fonction de metalloproteinases de la famille collagenase ». Université Louis Pasteur (Strasbourg) (1971-2008), 1989. http://www.theses.fr/1989STR13020.

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These en deux parties: d'une part etude d'une bacterie en milieu salin. L'addition de proline, choline, permet la croissance de la bacterie. Analyse de la composition proteique de la membrane bacterienne. D'autre part, etude de metalloproteinases et de certaines sequences impliquees dans le site actif de l'enzyme et dans l'activation du zymogene

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Thèses sur le sujet « Plasmid resistance »

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