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1
Alfredson, David, and n/a. "Characterisation of the B-Lactamase Gene From Campylobacter Jejuni." Griffith University. Institute for Glycomics, 2005. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20060111.172351.
Full textAbstract:
Thermophilic Campylobacter species such as Campylobacter jejuni and Campylobacter coli are recognised worldwide as major causes of acute gastroenteritis in humans. Campylobacteriosis is frequently a mild to moderate self-limited illness and most cases do not require antimicrobial therapy; antimicrobial therapy is necessary for patients with systemic Campylobacter infections, for patients with severe disease, or for immunosuppressed patients. Antimicrobial susceptibility testing of Campylobacter species using disk diffusion currently is not standardised by the National Committee for Clinical Laboratory Standards (NCCLS), however, in order to monitor the prevalence of antimicrobial resistance in Campylobacter species, there is a need for standardised or calibrated methods of susceptibility testing. Initially, 90 human clinical isolates of thermophilic Campylobacter species from Southeast Queensland, Australia, were screened for resistance to ampicillin, erythromycin and tetracycline using the disk diffusion susceptibility testing method. Levels of resistance were then determined using E test MIC and agar dilution methods to determine the reliability of disk diffusion results. Results of the disk diffusion testing showed 87 (97%) isolates resistant to ampicillin, 14 (16%) isolates were resistant to tetracycline and three (3.4%) isolates were resistant to erythromycin. Results of disk diffusion testing showed 100% correlation (+1 log2 dilution) with agar dilution for erythromycin and tetracycline, and 77% for ampicillin. E test showed 68% correlation with agar dilution for ampicillin, 100% for erythromycin and 64% for tetracycline. These data suggest that disk diffusion susceptibility testing may be used to screen thermophilic Campylobacter spp. for putative resistance to erythromycin and tetracycline and that the incidence of resistance of Campylobacter spp. to erythromycin and tetracycline is low in Southeast Queensland, Australia. Agar dilution remains the most accurate method for determination of ampicillin susceptibility. Numerical analyses of restriction endonuclease (RE) fragment profiles were performed to elucidate relatedness of the antibiotic resistant isolates and the results suggested a high level of isolate variation. The role of the B-lactamase in the resistance of C. jejuni to various B-lactams has been well documented and B-lactamase production in C. jejuni has been reported in 83-93% of strains. The expression and characterisation of the Campylobacter B-lactamase, however, has not been described. In this work, standard cloning techniques utilising a high-copy number E. coli cloning vector and a previously described E. coli-Campylobacter shuttle cloning vector were unsuccessful in isolation and expression of the C. jejuni B-lactamase gene in E. coli, possibly due to a lack of expression of the campylobacter gene in its host or low efficiency of transformation. Therefore, in order to facilitate the isolation, expression and characterisation of the C. jejuni B-lactamase gene, it was necessary to construct a new E.coli-Campylobacter shuttle cloning vector for the purposes of expressing the C. jejuni B-lactamase in Campylobacter. To aid in the construction of the vector, the sequence and genetic organisation of a 4.0-kb cryptic plasmid, termed pCJ419, identified in a human clinical isolate of C. jejuni was determined. Plasmid pCJ419 is a circular molecule of 4013 bp and contains four open reading frames (ORFs), the products of which share significant sequence similarity with putative proteins from known C. jejuni and C. coli plasmids. ORF-1 encodes a putative mobilisation protein (Mob); ORF-2 and ORF-3 encode proteins which have high identity to putative RepA and RepB proteins, respectively, of known C. jejuni and C. coli plasmids. ORF-4 encodes a protein which has high identity to a hypothetical protein of unknown function, Cjp32, previously described in a pVir plasmid of C. jejuni. Tandem repeating sequences typical of a plasmid replication origin (ori) were identified upstream of the DNA sequences encoding putative replication initiation proteins RepA and RepB. An E. coli-Campylobacter shuttle cloning vector, pGU0202, was constructed using plasmid pMW2 which harbours a Campylobacter-derived kanamycin-resistance gene, aphA(3)-III. The sequences encoding pCJ419 mob, repA and repB were inserted upstream of aphA(3)-III resulting in a stable construct of 6174 bp that was used successfully to transform both E. coli and Campylobacter. Subsequently, a novel molecular class D ?-lactamase gene, blaOXA-61, from a B-lactamase-positive, ampicillin-resistant (MIC 64 mg l-1), clinical strain of Campylobacter jejuni, strain GC015 was isolated, cloned and characterized using the newly constructed shuttle vector pGU0202. An open reading frame of 774 bp was identified on a ClaI genomic fragment of 2.2 kb and encodes a protein of 257 amino acids. Conserved motifs composed of identical amino acids typical of penicillin-recognising proteins and specific class D motifs were identified. blaOXA-61 was cloned into the shuttle cloning vector pGU0202 and expressed in B-lactamase-negative, ampicillin-susceptible C. jejuni and E. coli. A conserved 122-bp sequence directly upstream of blaOXA-61 was identified and shown to be required in cis for high-level resistance of Campylobacter to the penicillins although blaOXA-61 expressed only at low levels in E.coli. Southern hybridisation analysis demonstrated that the bla gene was chromosomally encoded and present on the same BglII and ClaI-digested genomic DNA fragments from various strains of Campylobacter with ampicillin MICs of between 4 and 64 mg l-1. In addition, DNA fragments encoding two putative zinc-dependent hydrolases from the metallo-B-lactamase superfamily, designated GLX2-1 and GLX2-2, were identified in a clinical isolate of Campylobacter jejuni, strain 012, cloned and sequenced. A strictly conserved motif, -H-X-H-X-D-, characteristic of the metallo- B-lactamase superfamily of proteins, including the class B metallo- B-lactamases, was identified in both proteins although functional B-lactamase could not be expressed in either E. coli or C. coli transformed using the C. jejuni hydrolase-containing shuttle vector pGU0202. Further work is warranted to determine the exact function of these proteins.
2
Alfredson, David. "Characterisation of the B-Lactamase Gene From Campylobacter Jejuni." Thesis, Griffith University, 2005. http://hdl.handle.net/10072/367271.
Full textAbstract:
Thermophilic Campylobacter species such as Campylobacter jejuni and Campylobacter coli are recognised worldwide as major causes of acute gastroenteritis in humans. Campylobacteriosis is frequently a mild to moderate self-limited illness and most cases do not require antimicrobial therapy; antimicrobial therapy is necessary for patients with systemic Campylobacter infections, for patients with severe disease, or for immunosuppressed patients. Antimicrobial susceptibility testing of Campylobacter species using disk diffusion currently is not standardised by the National Committee for Clinical Laboratory Standards (NCCLS), however, in order to monitor the prevalence of antimicrobial resistance in Campylobacter species, there is a need for standardised or calibrated methods of susceptibility testing. Initially, 90 human clinical isolates of thermophilic Campylobacter species from Southeast Queensland, Australia, were screened for resistance to ampicillin, erythromycin and tetracycline using the disk diffusion susceptibility testing method. Levels of resistance were then determined using E test MIC and agar dilution methods to determine the reliability of disk diffusion results. Results of the disk diffusion testing showed 87 (97%) isolates resistant to ampicillin, 14 (16%) isolates were resistant to tetracycline and three (3.4%) isolates were resistant to erythromycin. Results of disk diffusion testing showed 100% correlation (+1 log2 dilution) with agar dilution for erythromycin and tetracycline, and 77% for ampicillin. E test showed 68% correlation with agar dilution for ampicillin, 100% for erythromycin and 64% for tetracycline. These data suggest that disk diffusion susceptibility testing may be used to screen thermophilic Campylobacter spp. for putative resistance to erythromycin and tetracycline and that the incidence of resistance of Campylobacter spp. to erythromycin and tetracycline is low in Southeast Queensland, Australia. Agar dilution remains the most accurate method for determination of ampicillin susceptibility. Numerical analyses of restriction endonuclease (RE) fragment profiles were performed to elucidate relatedness of the antibiotic resistant isolates and the results suggested a high level of isolate variation. The role of the B-lactamase in the resistance of C. jejuni to various B-lactams has been well documented and B-lactamase production in C. jejuni has been reported in 83-93% of strains. The expression and characterisation of the Campylobacter B-lactamase, however, has not been described. In this work, standard cloning techniques utilising a high-copy number E. coli cloning vector and a previously described E. coli-Campylobacter shuttle cloning vector were unsuccessful in isolation and expression of the C. jejuni B-lactamase gene in E. coli, possibly due to a lack of expression of the campylobacter gene in its host or low efficiency of transformation. Therefore, in order to facilitate the isolation, expression and characterisation of the C. jejuni B-lactamase gene, it was necessary to construct a new E.coli-Campylobacter shuttle cloning vector for the purposes of expressing the C. jejuni B-lactamase in Campylobacter. To aid in the construction of the vector, the sequence and genetic organisation of a 4.0-kb cryptic plasmid, termed pCJ419, identified in a human clinical isolate of C. jejuni was determined. Plasmid pCJ419 is a circular molecule of 4013 bp and contains four open reading frames (ORFs), the products of which share significant sequence similarity with putative proteins from known C. jejuni and C. coli plasmids. ORF-1 encodes a putative mobilisation protein (Mob); ORF-2 and ORF-3 encode proteins which have high identity to putative RepA and RepB proteins, respectively, of known C. jejuni and C. coli plasmids. ORF-4 encodes a protein which has high identity to a hypothetical protein of unknown function, Cjp32, previously described in a pVir plasmid of C. jejuni. Tandem repeating sequences typical of a plasmid replication origin (ori) were identified upstream of the DNA sequences encoding putative replication initiation proteins RepA and RepB. An E. coli-Campylobacter shuttle cloning vector, pGU0202, was constructed using plasmid pMW2 which harbours a Campylobacter-derived kanamycin-resistance gene, aphA(3’)-III. The sequences encoding pCJ419 mob, repA and repB were inserted upstream of aphA(3’)-III resulting in a stable construct of 6174 bp that was used successfully to transform both E. coli and Campylobacter. Subsequently, a novel molecular class D ?-lactamase gene, blaOXA-61, from a B-lactamase-positive, ampicillin-resistant (MIC 64 mg l-1), clinical strain of Campylobacter jejuni, strain GC015 was isolated, cloned and characterized using the newly constructed shuttle vector pGU0202. An open reading frame of 774 bp was identified on a ClaI genomic fragment of 2.2 kb and encodes a protein of 257 amino acids. Conserved motifs composed of identical amino acids typical of penicillin-recognising proteins and specific class D motifs were identified. blaOXA-61 was cloned into the shuttle cloning vector pGU0202 and expressed in B-lactamase-negative, ampicillin-susceptible C. jejuni and E. coli. A conserved 122-bp sequence directly upstream of blaOXA-61 was identified and shown to be required in cis for high-level resistance of Campylobacter to the penicillins although blaOXA-61 expressed only at low levels in E.coli. Southern hybridisation analysis demonstrated that the bla gene was chromosomally encoded and present on the same BglII and ClaI-digested genomic DNA fragments from various strains of Campylobacter with ampicillin MICs of between 4 and 64 mg l-1. In addition, DNA fragments encoding two putative zinc-dependent hydrolases from the metallo-B-lactamase superfamily, designated GLX2-1 and GLX2-2, were identified in a clinical isolate of Campylobacter jejuni, strain 012, cloned and sequenced. A strictly conserved motif, -H-X-H-X-D-, characteristic of the metallo- B-lactamase superfamily of proteins, including the class B metallo- B-lactamases, was identified in both proteins although functional B-lactamase could not be expressed in either E. coli or C. coli transformed using the C. jejuni hydrolase-containing shuttle vector pGU0202. Further work is warranted to determine the exact function of these proteins.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
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3
Mossakowska, Danuta Ewa Irena. "Molecular characterisation of the OXA-2 beta-lactamase." Thesis, University of Surrey, 1988. http://epubs.surrey.ac.uk/847832/.
Full textAbstract:
The DNA sequence of the gene coding for the OXA-2 beta-lactamase has been completed. The primary amino acid sequence deduced from the DNA sequence was used to study homologies with other beta-lactamases; no good homologies were observed with any class of beta-lactamase. A more detailed analysis has revealed from comparison of both primary and predicted secondary structure, that the OXA-2 beta-lactamase may be more closely related to class A enzymes. Confirmation that the OXA-2 beta-lactamase is a serine enzyme has come from the DNA sequence and the interaction with mechanism based inactivators. Clavulanic acid and a novel beta-lactamase inhibitor, BRL36148, both interact specifically with the OXA-2 beta-lactamase by branched pathway mechanisms. Analysis of inactivated enzymes by peptide mapping and isoelcetric focusing have revealed that more than one inactive enzyme species is formed with each inactivator. A specific DNA probe was designed to come entirely from within the coding sequence of the OXA-2 beta-lactamase gene. This probe was found to interact only with plasmids specifying the OXA-2 or OXA-3 type enzymes. These results confirm earlier observations that OXA-2 and OXA-3 beta-lactamases are related. Another probe which comprised the whole of the OXA-2 beta-lactamase gene as well as segments of DNA on either side of the gene, was found to hybridise with a number of resistance plasmids. This interaction suggests that multi resistance plasmids carry common segments of DNA. Characterisation of the physical properties of the OXA-2 enzyme by analytical ultracentrifugation have not only confirmed the dimeric nature of this beta-lactamase but have also shown that this enzyme forms aggregates at high protein concentrations. Probing of the enzyme structure with trypsin, strongly points to the OXA-2 beta-lactamase having a domain structure.
4
Ali, Norryai A. "Expression and secretion of OXA-2 beta-lactamase by Streptomyces lividans." Thesis, University of Surrey, 1986. http://epubs.surrey.ac.uk/847196/.
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The OXA-2 beta-lactamase gene was first found on a conjugative plasmid R46 from a clinical isolate of Salmonella typhimurium. To test the expression and secretion of OXA-2 beta-lactamase in Streptomyces lividans a shuttle plasmid (pSU101) was created by fusing an Escherichia coli plasmid (pSU8) carrying the OXA-2 beta-lactamase gene with the S. lividans vector pLJ61. The OXA-2 beta-lactamase gene specified by the hybrid plasmid PSU101 was expressed in S. lividans, although at a lower level than in coli. Almost all the beta-lactamase activity was found in the culture supernatant of S. lividans, whereas in E. coli the enzyme was almost wholly cell associated. The identity of the enzyme was established by substrate specificity and isoelectric focusing. The stability and integrity of the plasmid pSU101 in both E. coli and lividans was determined, in comparison with that of pSU8 and pLJ61 plasmids. The promoter regions of the OXA-2 beta-lactamase gene were identified by using promoter-probe plasmid vectors; and by S1 mapping of the transcriptional start-sites coupled to the DNA sequencing of the OXA-2 beta-lactamase gene. Multiple transcriptional start sites were found in both hosts, with the origin of transcription apparently different in the two organisms. Part of this work has been published as a scientific paper which is appended.
5
Doyle, Jamielynn. "Identification of an L2 ß-lactamase gene from Stenotrophomonas maltophilia OR02." Youngstown State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1528369969169163.
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Cooper, Kerri W. "Expression, degradation, and applications of Escherichia coli TolA-beta-lactamase fusion proteins /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/9815.
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Albaaj, Mohammed. "Diversity of β-Lactamase Genes in Gram-Negative Soil Bacteria from Northwest Ohio." Bowling Green State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1566553116919146.
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Henderson, Ian. "Solving the inclusion body problem - a case study : high level expression of TEM-1 #beta#-lactamase in Escherichia coli." Thesis, University of Warwick, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.282432.
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Li, Wai-shan. "Cloning and characterization of chromosomal class C [beta]-Lactamase and its regulatory gene in Laribacter hongkongensis." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B31046393.
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Li, Wai-shan, and 李慧珊. "Cloning and characterization of chromosomal class C {221}-Lactamase and its regulatory gene in Laribacter hongkongensis." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B31046393.
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11
Abuhalfaya, Ali Mohamed. "Recruitment of the gene for teh TEM-1 #beta#-lactamase into the chromosome of Acinetobacter spp." Thesis, University of Bristol, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337485.
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Demolli, Shemsi, Miriam M. Geist, Julia E. Weigand, Nicole Matschiavelli, Beatrix Süß, and Michael Rother. "Development of β-Lactamase as a Tool for Monitoring Conditional Gene Expression by a Tetracycline-Riboswitch in Methanosarcina acetivorans." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-132179.
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The use of reporter gene fusions to assess cellular processes such as protein targeting and regulation of transcription or translation is established technology in archaeal, bacterial, and eukaryal genetics. Fluorescent proteins or enzymes resulting in chromogenic substrate turnover, like β-galactosidase, have been particularly useful for microscopic and screening purposes. However, application of such methodology is of limited use for strictly anaerobic organisms due to the requirement of molecular oxygen for chromophore formation or color development. We have developed β-lactamase from Escherichia coli (encoded by bla) in conjunction with the chromogenic substrate nitrocefin into a reporter system usable under anaerobic conditions for the methanogenic archaeon Methanosarcina acetivorans. By using a signal peptide of a putative flagellin from M. acetivorans and different catabolic promoters, we could demonstrate growth substrate-dependent secretion of β-lactamase, facilitating its use in colony screening on agar plates. Furthermore, a series of fusions comprised of a constitutive promoter and sequences encoding variants of the synthetic tetracycline-responsive riboswitch (tc-RS) was created to characterize its influence on translation initiation in M. acetivorans. One tc-RS variant resulted in more than 11-fold tetracycline-dependent regulation of bla expression, which is in the range of regulation by naturally occurring riboswitches. Thus, tc-RS fusions represent the first solely cis-active, that is, factor-independent system for controlled gene expression in Archaea.
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Ricchiuti, Michelle. "Identification of a putative ampG ampicillin resistance gene in Stenotrophomonas maltophilia OR02." Youngstown State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1484670421517116.
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Weldhagen, Gerhard Frederick. "Laboratory Detection and Gene Cassette Stability of the Novel Extended-Spectrum Beta-Lactamase, GES-2 from Pseudomonas aeruginosa." Thesis, University of Pretoria, 2004. http://hdl.handle.net/2263/29221.
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Extended-spectrum beta-lactamases (ESBLs) in Pseudomonas aeruginosa tend to be geographically scattered, such as GES-2, which partially compromises the efficacy of imipenem. The G170N mutation, ascribed to a CC to AA base pair substitution on positions 493-494 of the blaGES-2 coding region, distinguishes this ESBL from blaGES-1 and the blaIBC-type genes, making it an ideal target for developing a novel sequence-specific, peptide nucleic acid (PNA)-based, multiplex-PCR detection method. Utilizing two primer pairs in conjunction with a PNA probe, this novel method delivered accurate identification of blaGES-2 compared to standard PCR and gene sequencing techniques, when tested against one hundred (n = 100) P. aeruginosa clinical isolates as well as previously published, well-described control strains. This method has the potential to be used in large-scale, cost-effective screening programmes for specific or geographically restricted ESBLs. To date, in addition to being only described in South Africa, GES-2 is notoriously difficult to identify in P. aeruginosa, using standard methodology. A real-time PCR method using the LightCycler™ was compared to a two-step nested-PCR assay for the detection of blaGES and blaIBC genes from one hundred P. aeruginosa clinical isolates collected over a four-year period from two teaching hospitals in Pretoria, South Africa. Real-time PCR amplification was monitored through hybridisation of fluorescently labelled probes followed by melting curve analysis to detect the relevant G170N mutation occurring in the omega loop region of blaGES-2. Nested-PCR products were subjected to automated DNA sequencing and compared to melting point (Tm) analyses results obtained from the LightCycler assay. Real time and nested-PCR assays detected a blaIBC gene product from 83 and 88 clinical isolates respectively, with the LightCycler thus exhibiting a sensitivity of 94.3% compared to the nested-PCR assay. Comparison of Tm and gene sequencing data however revealed 100% specificity for sequence specific detection of blaGES-2 with the LightCycler. One clinical isolate was found to harbour a blaGES-1 gene, making this the first report of this specific ESBL from South Africa. Selective antibiotic pressure has recently been implicated as a possible driving force behind point mutations observed in blaGES–type genes. This part of the study subjected two well-characterized clinical isolates with class 1 integron-borne blaGES-type genes to five days incubation in the presence of sub-inhibitory concentrations of 15 different antibiotics, including beta-lactams, aminoglycosides and quinolones. Restriction enzyme analysis and DNA sequencing of blaGES-1, blaGES-2 and their immediate upstream genetic environments failed to demonstrate any changes compared to non-exposed controls. Short-term exposure to a sub-inhibitory level of a single antimicrobial agent is thus unlikely to select significant mutations in these beta-lactamase genes or their regulatory mechanisms.Thesis (PhD (Medical Microbiology))--University of Pretoria, 2006.
Medical Microbiology
unrestricted
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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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Abda, Ebrahim Mama [Verfasser], and Wolfgang R. [Akademischer Betreuer] Streit. "Identifying molecular keys regulating phenotypic heterogeneity of Stenotrophomonas maltophilia K279a β-lactamase blaL1 and blaL2 gene expression / Ebrahim Mama Abda ; Betreuer: Wolfgang R. Streit." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2018. http://d-nb.info/1161847685/34.
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Marin, Canica Maria Manuela. "Evolution des beta-lactamases tem et resistance aux inhibiteurs des beta-lactamases chez escherichia coli." Paris 11, 1997. http://www.theses.fr/1997PA114859.
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Campbell, Joan Iyabo Amiemenoghena. "β-lactamase genes of gram-negative bacteria." Thesis, University of Edinburgh, 1986. http://hdl.handle.net/1842/15646.
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Two β-lactamase gene sequences encoded by Ps. aeruginosa RMS 149 plasmid and Rps. capsulata sp 108 were investigated. The genes were located using DNA recombinant techniques and their nucleic acid sequences were determined using the Sanger dideoxy-sequencing technique. The amino acid sequences were identified and compared with other characterised β-lactamases. They are both class A enzymes (Ambler classification). The pseudomonad plasmid encoded enzyme is expressed constitutively, but its gene sequence has an attenuator sequence - reminiscent of inducible bacterial synthetic operons. It also has three putative loop-forming sequences in the middle of the gene. RNA mapping studies indicate that the attenuator is read through from an upstream promoter. There is low level initiation from its own promoter and the transcripts sometimes terminate around the second internal stem-loop. The full message is also made. Thus, it is likely that the pseudomonad gene is normally highly regulated. Its constitutive expression may be as a result of some control mutation. The rhodopseudomonad enzyme is unlike other characterised Gram-negative class β-lactamases because it is inducible. Gene hybridization experiments suggest that it may be chromosomally encoded in strain sp 108 as well as in the Pen^S strain sp 109. β-lactamase active bands were also observed in PenS Rps. capsulata St. Louis and Rps. sphaeroides. If this is the usual state of affairs in photosynthetic bacteria which are not normally subject to the selective pressures of the presence of β-lactam antibiotics by virtue of their aquatic habitat, the sp 108 strain may also be producing the enzyme in large quantities due to some control mutation. It is postulated then, that β-lactamase genes in Gram-negative bacteria may be of two kinds - one that is chromosomal and is highly regulated, and another which has lost the regulation and over-expresses the enzyme. The latter may be representative of the common plasmid-borne β-lactamase genes.
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Clímaco, Eduardo Carneiro. "Caracterização molecular de genes blaCTX-M presentes em Klebsiella spp. isoladas em hospital universitário do Brasil." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-28052007-093704/.
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Entre as ß-lactamases, as enzimas CTX-M têm despertado atenção especial pela alta incidência e grande capacidade de propagação. Eventos como recombinação gênica, transferência plasmideal e multirresistência podem ser a razão da manutenção e da ampla disseminação dos genes blaCTX-M. Este é um trabalho retrospectivo que teve como objetivo caracterizar genes blaCTX-M presentes em Klebsiella spp. Foram estudadas 27 linhagens de Klebsiella pneumoniae e 8 linhagens de Klebsiella oxytoca, produtoras de ?-lactamase de espectro estendido, isoladas de pacientes hospitalizados no período de janeiro a junho de 2000. A detecção e identificação dos genes blaCTX-M, assim como dos elementos relacionados com a mobilização destes genes, foi realizada por PCR e seqüenciamento. A localização genética e a mobilidade dos genes blaCTX-M foram pesquisadas por análise plasmideal e hibridação e por conjugação. Os perfis de sensibilidade das linhagens estudadas e das linhagens transconjugantes foram comparados pela determinação da concentração inibitória mínima de antibióticos das classes das cefalosporinas, cefamicinas, aminoglicosídeos e quinolonas. Foram encontrados genes blaCTX-M em plasmídeos conjugativos em 13 (37%) linhagens estudadas: blaCTX-M-9 em 4 K. oxytoca, e blaCTX-M-2 em 9 K. pneumoniae. Os genes blaCTX-M-9 estavam associados ao elemento de inserção ISEcp1, enquanto os genes blaCTX-M-2 estavam associados a integrons de classe I contendo ISCR1. O genes blaCTX-M-2, carreado por plasmídeo, pode estar relacionado com disseminação horizontal entre vários clones de K. pneumoniae, enquanto o gene blaCTX-M-9 foi encontrado sendo carreado por um único clone de K. oxytoca. Este estudo determinou a incidência e a diversidade de enzimas CTX-M no período estudado, além de fornecer dados epidemiológicos que podem explicar a sua prevalência no mundo e contribuir para o entendimento e controle da disseminação deste tipo de resistência.CTX-M enzymes, the world\'s most prevalent ß-lactamases disseminate very easily. Genetic recombination, plasmid transference and multiresistance could be responsible for the wide spread of blaCTM-X genes. This retrospective study aims to characterize blaCTX-M genes found in Klebsiella spp. The strains were isolated in hospital patients from January to June 2000 and consisted of 27 ESBL-producing Klebsiella pneumoniae and 8 ESBL-producing Klebsiella oxytoca. PCR and sequencing were used in the detection and identification of blaCTX-M genes and genetic elements associated with their mobilization. Determination of genetic localization and mobility of blaCTX-M genes was by plasmid analyses, hybridization and transfer assays. The minimal inhibitory concentrations (MICs) of cephalosporins, cefamicins, aminoglycosides and quinolone antimicrobials evaluated the antibiotic susceptibility profile of transconjugants and strains in the study. The blaCTX-M genes were found in 13 strains (37%): blaCTX-M-9 in 4 K. oxytoca and blaCTX-M-2 in 9 K. pneumoniae. The insertion sequence ISEcp1 was associated with blaCTX-M-9 and blaCTX-M-2 was found in a class I integron bearing ISCR1. Plasmid blaCTX-M-2 genes dissemination was due to horizontal transfer among many K. pneumoniae clones, while blaCTX-M-9 dissemination was associated with a particular clone of K. oxytoca. The study characterized incidence and diversity of CTX-M enzymes during the period studied. Moreover it showed epidemiological data, which may explain CTX-M prevalence worldwide and contribute for the understanding and control of the resistance spread.
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Siebor, Eliane. "Caractérisation physicochimique et immunologique de ß-lactamases chez les entérobactéries : un modèle d'étude chez le genre Serratia." Lyon 1, 1989. http://www.theses.fr/1989LYO10004.
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Cette etude a porte sur les cephalosporinases chez le genre serratia. Elles sont inductibles et presentent une grande heterogeneite dans leurs constantes cinetiques et leurs points isoelectriques. Dans un but prospectif, des variants resistants synthetisant une cephalosporinase constitutive ont ete obtenus chez differentes enterobacteries, apres exposition a un agent mutagene. La frequence de ces mutants est la plus elevee pour enterobacter cloacae et citrobacter freundii, aussi bien in vitro qu'in vivo. Ils entrainent une resistance plus ou moins importante a l'ensemble des beta-lactamines, a l'exception du mecillinam, de la nf-thienamycine et du cm 40874. La selection d'un mutant constitutif a haut niveau de serratia liquefaciens a permis de determiner les constantes cinetiques de toutes les beta-lactamines. Les antibiotiques non hydrolyses ont ete testes comme inhibiteurs. Le processus d'inactivation de la cephalosporinase par l'aztreonam et le latamoxef est caracteristique des inhibiteurs suicides. L'etude des inhibiteurs de penicillinases a montre que le sulbactam etait egalement un inhibiteur irreversible des cephalosporinases. Contrairement a l'acide clavulanique, il n'est pas inducteur de cephalosporinase et presente une fixation differente sur les proteines de liaison a la penicilline. Dans le cas des etudes immunologiques, seules les methodes de neutralisation de l'activite enzymatique presentent une haute specificite. L'obtention d'anticorps monoclonaux apporte un nouvel interet dans la comparaison phylogenique de beta-lactamases
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Galetti, Renata. "Estudo de Pseudomonas aeruginosa produtoras de metalo-beta-lactamase e de genes envolvidos na resistência aos carbapenêmicos." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-06102010-154221/.
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As metalo-beta-lactamases (MBL) são carbapenemases pertencentes à classe B de Ambler e ao grupo 3 de Bush-Jacoby, as duas classificações mais utilizadas atualmente. Essas enzimas conferem, às bactérias, resistência às cefalosporinas, penicilinas e carbapenêmicos, mas não conferem resistência ao monobactam aztreonam. Além disso, não são inibidas por inibidores de -lactamases comercialmente disponíveis, porém possuem sensibilidade ao ácido etileno diamino tetracético (EDTA) e ácido mercaptopropiônico (MPA). Atualmente são conhecidas nove subclasses de MBL: IMP,VIM, SPM, GIM, SIM, AIM, KHM, NDM e DIM. Essas MBL têm se tornado clinicamente importantes, principalmente, em Pseudomonas spp., Acinetobacter spp., e alguns gêneros da família Enterobacteriaceae. O principal objetivo desse trabalho foi a caracterização genética e epidemiológica de P. aeruginosa resistentes aos carbapêmicos, produtoras de MBL, isoladas no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo no período de abril a agosto de 2007. Das 54 P. aeruginosa estudadas, 24 foram positivas na triagem fenotípica para MBL e 5 apresentaram o gene blaSPM-1 detectado por PCR e confirmado pelo seqüenciamento. O inibidor mais eficiente na triagem fenotípica foi o EDTA. A baixa correlação entre o teste fenotípico e molecular pode ser explicada pela capacidade que o EDTA possui de aumentar a permeabilidade da membrana celular e assim tornar a bactéria sensível a baixas concentrações do antibiótico. Além disso, a diferença entre o número de isolados resistentes aos carbapenêmicos e/ou ceftazidima e os que apresentaram a MBL podem ser explicados pela associação de outros mecanismos de resistência, como hiperprodução de AmpC, redução da expressão de porinas e/ou aumento do efluxo de antibióticos. De acordo com o perfil de macrorrestrição obtido por eletroforese em campo pulsado as 5 linhagens produtoras de SPM-1 estão agrupadas em 3 perfis clonais distribuídos em diferentes clínicas no hospital não sendo identificado nenhum clone predominante. Finalizando, entre os isolados resistentes aos carbapenêmicos e/ou à ceftazidima a freqüência de P. aeruginosa produtoras de SPM-1 foi de 9,3%, indicando que as medidas aplicadas pela Comissão de Controle de Infecção Hospitalar (CCIH) desta instituição estão sendo eficientes, porém mesmo assim é necessário que a CCIH esteja sempre atenta a relatos de resistência aos carbapenêmicos pois, linhagens produtoras de MBL restringem muito as opções terapêuticas para infecções causas por elas.Metallo-beta-lactamase (MBL) are carbapenemases which belong to the Bush-Jacoby-Medeiros group 3 and to the molecular class B, according Ambler. These two classifications are the most used nowadays. These enzymes confer microorganisms resistant to cephalosporins, penicillins and carbapenems, but not to aztreonam, a monobactam. Moreover, they are not inhibited by commercially available -lactamases inhibitors, but they are susceptible to EDTA and MPA. Currently this is known nine subclasses of MBL: IMP, VIM, SPM, GIM, SIM, AIM, KHM, NDM and DIM. These MBL became clinically important, especially in microorganisms such as Pseudomonas spp., Acinetobacter spp. and genera of the family Enterobacteriaceae. The main objective of this study was genetic and epidemiologic characterization of MBL-producing-P. aeruginosa, isolated in the Hospital of the Faculty of Medicine of Ribeirão Preto - University of São Paulo in the period from April to August 2007. Among the 54 P. aeruginosa studied, 24 were positive for phenotypic MBL screening and 5 presented blaSPM-1 gene, detected by PCR and confirmed by sequencing. The most efficient inhibitor in phenotypic screening was EDTA. The low correlation between phenotypic and molecular testing can be explained by the ability of EDTA to increase the permeability of cell membranes and rendering the bacteria as sensitive to low concentrations of antibiotic. Furthermore, the difference between number of isolates carbapenem resistant and / or ceftazidime resistant and the number of MBL-producing-P. aeruginosa can be explained by the association with other resistance mechanisms, such as AmpC overproduction, reduced expression of porins and / or increased antibiotics efflux. According to profile of macrorestriction after pulsed-field gel electrophoresis, five SPM-1-producing-P. aeruginosa are grouped in three different clonal profiles. These clonal strains were proceeded from different clinics at the hospital and no predominant clone was identified. Finally, among the carbapenems and/or ceftazidime resistant isolates the frequency of SPM-1-producing-P. aeruginosa was 9.3%, suggesting that the hospital care of infection control has being effective.
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Faure, Stéphanie. "Transfert d'un gène de résistance aux beta-lactamines blaCTX-M-9 entre Salmonella et les entérobactéries de la flore intestinale humaine : influence d'un traitement antibiotique." Phd thesis, Université Rennes 1, 2009. http://tel.archives-ouvertes.fr/tel-00449376.
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La dissémination mondiale de la résistance aux β-lactamines est un problème de santé publique majeur. Les voies de transmission de la résistance sont mal connues, néanmoins de nombreux arguments attestent du potentiel transfert de bactéries résistantes et de gènes de résistance entre l'animal et l'homme. L'objectif du présent travail est d'évaluer le transfert du gène de résistance blaCTX-M-9 entre une souche d'origine animale, Salmonella enterica Virchow et les entérobactéries de la flore humaine, et l'impact d'un traitement à une β- lactamine sur ce transfert. Dans un modèle de rat associé à une flore humaine, le transfert du gène de résistance n'est pas détectable entre S. enterica Virchow et les entérobactéries de la flore. Pourtant l'inoculation concomitante d'une souche d'Escherichia coli suffit à observer la diffusion du gène de résistance. La pression de sélection ne permet pas d'augmenter le transfert de gène mais contribue largement à la sélection et à la colonisation de salmonelles résistantes dans le tractus digestif. L'analyse des paramètres pharmacocinétiques et pharmacodynamiques confirme également la pertinence des critères de substitution dans la prédiction de l'efficacité d'un traitement au céfixime en présence de telles souches. L'une des stratégies thérapeutiques proposée consiste à utiliser une combinaison de β-lactamines et d'inhibiteurs de β-lactamases.
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Yamamoto, Masaki. "Regional Dissemination of Acinetobacter Species Harbouring Metallo-β-Lactamase Genes in Japan." Kyoto University, 2013. http://hdl.handle.net/2433/174762.
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East, A. K. "A study of the leader peptides of staphylococcal #BETA#-lactamases." Thesis, University of Oxford, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233462.
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CAVALCANTI, Felipe Lira de Sá. "Detecção de genes de metalo-beta-lactamases em isolados clínicos de Pseudomonas aeruginosa." Universidade Federal de Pernambuco, 2010. https://repositorio.ufpe.br/handle/123456789/2138.
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Previous issue date: 2010Faculdade de Amparo à Ciência e Tecnologia do Estado de Pernambuco
A resistência aos carbapenêmicos pode dar-se através de vários mecanismos, incluindo a expressão de beta-lactamases do tipo carbapenemases. Metalo-beta-lactamases (MBLs) constituem o grupo mais clinicamente importante das carbapenemases, na medida em que elas hidrolisam praticamente todos os beta-lactâmicos e, em alguns casos, também os monobactams (devido a outros mecanismos associados), o que implica em uma vasta redução das opções terapêuticas atualmente disponíveis. O objetivo deste trabalho foi detectar a produção de MBLs em cepas de Pseudomonas aeruginosa resistentes tanto a imipenem quanto a ceftazidima, verificar o perfil de susceptibilidade dos isolados aos mais utilizados grupos de antibióticos comercialmente disponíveis, investigar a ocorrência dos genes blaSPM-1 e blaIMP e realizar a tipagem molecular dos isolados MBL positivos. Foram analisadas 61 amostras do biênio 2002/2003 e 12 amostras do biênio 2008/2009, identificadas em um hospital de ensino. A susceptibilidade aos antimicrobianos foi feita de acordos com os critérios estabelecidos pelo CLSI. A identificação dos isolados MBL positivos seguiu o método de disco-difusão proposto por Arakawa. A detecção dos genes blaSPM-1 e blaIMP foi feita por análise de PCR usando iniciadores específicos. A análise dos fragmentos das macrorestrições do DNA genômico foi feita por PFGE. Os resultados mostraram que 86,3% (63/73) dos isolados resistentes a imipenem e ceftazidima foram confirmados como MBL positivos pelo teste fenotípico. O gene blaSPM-1 foi encontrado em 61 destes isolados. Nenhuma das amostras testadas possuía o gene blaIMP. Quanto aos ensaios de susceptibilidade, foi observado que dos anos 2002 e 2003: 100% dos isolados eram resistentes a ciprofloxacina, gentamicina e amicacina; 44% eram resistentes a piperacilina/tazobactam e 56% mostraram sensibilidade a esta droga. Dos anos 2008 e 2009: 100% dos isolados eram resistentes a ciprofloxacina e gentamicina; 91,6% eram resistentes a amicacina; 8,4% mostraram resistência intermediária a este aminoglicosídeo; 83,3% eram resistentes a piperacilina/tazobactam e 16,7% mostraram sensibilidade a este antimicrobiano. Quando as duas principais drogas para tratamento de infecções causadas por cepas MBL positivas foram analisadas, dos isolados de 2002/2003, apenas 1,63% foram resistentes ao aztreonam; 62,2% tiveram resistência intermediária e 36% mostraram sensibilidade. Dos isolados de 2008/2009, 83,4% foram resistentes e 16,6% apresentaram resistência intermediária, com nenhum isolado mostrando sensibilidade. Quanto à polimixina B, todos os isolados deste trabalho eram sensíveis. A análise dos fragmentos das macrorestrições dos isolados mostrou um único tipo de PFGE (A), com sete subtipos (A1 a A7). Estes achados sugerem que a produção de MBLs mediada por um clone único epidêmico continua sendo um importante mecanismo de resistência aos carbapenems no hospital estudado, como confirmado pela detecção do gene SPM. Além disso, o perfil de pan-resistência encontrado também alerta para a possível presença de múltiplos mecanismos de resistência nos isolados bacterianos, o que sinaliza uma necessidade urgente por estratégias de vigilância e melhoria das práticas de controle de infecções
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Yousef, Langaee Taimour. "Characterization of the regulatory genes of inducible AmpC ß-lactamase of Pseudomonas aeruginosa PAO1." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0019/NQ48992.pdf.
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Dimude, Juachi Uzochukwu. "CTX-M β-lactamases and associated integrons : their dissemination in Gram-negative bacteria." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/11729.
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Gram-negative bacteria are able to cause many infections including blood stream infections (BSI).These bacteria may become resistant to antibiotics, often by acquiring genes in the presence of antibiotic selection pressure. Multi drug resistant Gram-negative bacteria have become an increasing problem worldwide. A study of antibiotic resistance in Gram-negative bacteria isolated from blood cultures from patients in the New Royal Infirmary of Edinburgh (NRIE) was performed. In addition, a study was performed on isolates from patients in an intensive care unit in Egypt. All isolates were investigated for susceptibility to an extensive range of antibiotics. Gram-negative bacteria from Edinburgh found to be resistant to either cefotaxime or ceftazidime were investigated further. Among the cefotaxime/ceftazidime resistant isolates, Polymerase Chain Reaction (PCR) analysis revealed the presence of CTX-M- β-lactamases. Seven E.coli isolates were found to have CTX-M-15 β-lactamases while the CTX-M-14 β-lactamase was detected in six Enterobacter cloacae. The insertion sequence ISEcp1 was detected upstream of the blaCTX-M-15 gene in some isolates while IS26 was found truncating the ISEcp1 in other isolates. Conjugation experiments found the blaCTX-M-15 gene was transferable to E. coli J62-2. All the isolates had detectable plasmids, a plasmid ~260kb carried the blaCTX-M-15 gene. Analysis of the -containing isolates by PFGE shows that those carrying the CTX-M-14 β-lactamase were identical indicating cross infection within the hospital. The CTX-M- 15 β-lactamase-containing isolates showed four isolates had ≥85% similarity but the others were diverse. Class 1 integrons were found in eight of the CTX-M β-lactamase containing isolates with the associated gene cassette and sul1 gene. The isolates from Egypt were found to be resistant to carbapenem, which is the final mainstream antibiotic option in the treatment of multidrug resistant Gram-negative bacteria. Further analysis revealed all carried the CTX-M-14 β-lactamase and two additionally carried the VIM-4 metallo β-lactamase, which accounted for the resistance to the carbapenems. Furthermore, the insertion sequence ISEcp1 was found upstream of the blaCTX-M-14 gene in two of the isolates. The blaVIM-4 gene was found to be part of the gene cassette in the class 1 integron associate with complex ISCR1. Two of the Egyptian isolates had a detectable plasmid, ~300kb in size, which carried both blaCTX-M-14 and blaVIM-4 genes. All the blood culture isolates were examined to ascertain the persistence of sulphonamide resistance despite the long-term prescribing reduction on this antibacterial. PCR was performed to detect sul1, sul2 and sul3 genes in all the isolates. Of the sulphonamide resistant isolates 25 carried the sul1, 27 carried the sul2 and none carried the sul3 genes. Eight isolates had both the sul1 and sul2 genes. Most of the isolates carried sul1 had Int1 as part of the same class 1 integron. Interestingly three isolates were PCR negative for sul1 but positive for sul2 and int1. Int2 and 3 were found in 3 and 2 isolates respectively. The class 1 integron contained different insert gene cassettes; dfrA (dfrA17, dfrA16, dfrA15), aadA (aadA5, aadA2, aadA1) and blaOXA-1 families in addition to the resident sul gene. In conclusion this thesis shows the diversity of the genetic environment and carriers of the CTX-M β-lactamases within the same hospital. Sulphonamide resistance in Gram-negatives persists despite the prescribing reduction of this antibacterial in a Scottish hospital and the recommended constraint on the use of sulphonamide.
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Ensor, V. M. "the molecular epidemiology and genetic mobility of the blaCTX-M extended spectrum beta-lactamase genes." Thesis, University of Birmingham, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.743413.
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Takahashi, Juliana Tiemi. "Detecção e identificação de beta-lactamases de espectro estendido e de genes de resistência às quinolonas em Enterobacteriaceae isoladas de amostras de carnes de frango, suína e bovina destinadas ao consumo humano." Faculdade de Medicina de São José do Rio Preto, 2015. http://hdl.handle.net/tede/260.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Introduction: Bacterial resistance to antimicrobials is an important public health problem, which results in increased treatment period, increase in mortality and in health care costs. This phenomenon is considered a result of the indiscriminate use of antimicrobials in human and veterinary medicine, and along the food chain. In recent years, it has been observed the frequent isolation of resistant bacteria from animal production and food of animal origin. In this context, have been highlighted the resistance to beta-lactâmicos and to quinolones, the first, due to extended-spectrum beta-lactamase (ESBL) production and the second, resultant from the acquisition of qnr genes encoding proteins that interfere in quinolone action on DNA gyrase and topoisomerase IV. Several bacterial species with antimicrobial resistance have been isolated from farm animals. Objective: This study evaluated the antimicrobial susceptibility profile and the presence of resistance genes blaCTX-M, blaSHV, blaTEM, qnrA, qnrB, qnrS and oqxAB in Enterobacteriaceae isolated from meat market in the municipality of São José do Rio Preto- SP, acquired between November 2010 and August 2012. Material and Methods: The antimicrobial susceptibility was determined by disk diffusion according to CLSI. Strains resistant to beta-lactams and quinolones were submitted to PCR using specific primers for detection and sequencing of these genes for identification. Results: A total of 75 isolates were resistant to beta-lactam and 125 to quinolones. Among these, 6.6% blaCTX-M, 6.6% blaTEM, 16% blaSHV, 21.6% had qnr variants and 13.6% oqxAB. In a strain of Aeromonas sobria was detected the coexistence of qnrA and qnrS genes; the qnrB gene was detected in five strains of E. coli, five K.pneumoniae, two Salmonella spp. and 12 Citrobacter spp. The qnrB19 gene was detected in two K. pneumoniae, duas E. coli and one Citrobacter braakii. qnrB2 was detected in three K. pneumoniae. The qnrS1 gene was detected in a strain of K. pneumoniae. The sequencing blaCTX-M revealed blaCTX-M-2, all of which were derived from chicken meat samples. Conclusion: These data demonstrate there nay be association between excessive use of antimicrobials in animal production and isolation of bacteria resistant to these antimicrobials. The presence of bacteria resistant to antimicrobials in meat intended for human consumption consists in a public health problem and can affect commercials relationships of the country, since it the Brazil is one of the world's largest meat exporters.
Introdução: A resistência bacteriana aos antimicrobianos é um importante problema de saúde pública, que prolonga o período de tratamento, aumenta as taxas de mortalidade e custos da assistência à saúde. Este fenômeno pode ter considerado consequência do uso indiscriminado de antimicrobianos na medicina humana e veterinária, e ao longo da cadeia produtiva de alimentos. Nos últimos anos tem sido observado o frequente isolamento de bactérias resistentes a partir de animais de produção e alimentos de origem animal. Neste contexto, têm-se destacado a resistência aos beta-lactâmicos, devido à produção de beta-lactamase de espectro estendido (ESBL) e às quinolonas pela aquisição de genes qnr que codificam proteínas que interferem na ação das quinolonas sobre a DNA-girase e a topoisomerase IV. Diversas espécies bacterianas apresentando resistência aos antimicrobianos têm sido isoladas a partir de animais de produção. Objetivo: Este estudo teve como objetivo isolar enterobactérias resistentes aos beta-lactâmicos e quinolonas e detectar os genes blaCTX-M, blaSHV, blaTEM, qnrA, qnrB, qnrS e oqxAB em Enterobacteriaceae resistentes isoladas de carnes resfriadas de frango, suína e bovina comercializadas no município de São José do Rio Preto-SP, adquiridas entre novembro de 2010 e agosto de 2012. Material e Métodos: A suscetibilidade aos antimicrobianos foi determinada por disco-difusão de acordo com as normas do CLSI. As cepas resistentes aos beta-lactâmicos e às quinolonas foram submetidas à PCR com primers específicos para a detecção destes genes e ao sequenciamento para a identificação dos mesmos. Resultados: Um total de 75 isolados apresentou resistência aos beta-lactâmicos e 125 às quinolonas. Entre estes, 6,6% blaCTX-M, 6,6% blaTEM, 16% blaSHV, 21,6% apresentaram variantes de qnr e 13,6% oqxAB. Em uma cepa de Aeromonas sobria foi detectada a coexistência dos genes qnrA e qnrS; o gene qnrB foi detectado em cinco cepas de E. coli, cinco K.pneumoniae, duas Salmonella spp. e 12 Citrobacter spp. O gene qnrB19 foi detectado em duas K. pneumoniae, duas E. coli e uma Citrobacter braakii. qnrB2 foi detectado em três K. pneumoniae. O gene qnrS1 foi detectado em uma cepa de K. pneumoniae. O sequenciamento de blaCTX-M revelou blaCTX-M-2, sendo que todas foram provenientes de amostras de carne de frango. Conclusão: Esses dados revelam que pode haver associação entre o excessivo uso de antimicrobianos em animais de produção e o isolamento de bactérias resistentes aos mesmos. A presença de bactérias resistentes aos antimicrobianos em carnes destinadas ao consumo humano consiste em um problema de saúde pública e pode afetar as relações comerciais do país, já que o Brasil é um dos maiores exportadores de carne do mundo.
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Belas, Adriana. "Extended–spectrum–beta-lactamases, cephalosporinases and carbapenemase-producing Escherichia coli in the human-dog interface." Doctoral thesis, Universidade de Lisboa, Faculdade de Medicina Veterinária, 2021. http://hdl.handle.net/10400.5/21237.
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Tese de Doutoramento em Ciências Veterinárias na Especialidade de Ciências Biológicas e BiomédicasExtended–spectrum–beta-lactamases (ESBLs), cephalosporinases (encoded by the ESBLs and Ampc genes, respectively) and carbapenemase–producing Escherichia coli have become a major public health concern to both human and animal health. Urinary tract infections (UTI) are one of the most frequent bacterial infections in both human and companion animals. Uropathogenic E. coli (UPEC), belonging to extraintestinal pathogenic E. coli (ExPEC), is the most common bacterium isolated from companion animals. Moreover, the close contact of companion animals with humans creates opportunities for interspecies transmission of resistant bacteria and genes. E. coli from companion animals with UTI were found to harbour important antimicrobial resistance mechanisms and to belong to high-risk human clonal lineages, namely third-generation cephalosporin (3GC)-resistant E. coli O25b:H4-B2-ST131-H30/H30Rx, CC23 and ST648. In this work E. coli O25b:H4-ST131-H30/H30Rx was described for the first time in Europe in companion animals. Furthermore, the blaCMY-2 producing E. coli ST648 is the most common high-risk clonal lineage causing UTI in companion animals from the Lisbon area. Companion animals also seem to be reservoirs of bacteria and clinically important resistance genes, such β-lactams genes (classe A and C) which supports their role as reservoirs. The detection of faecal high-risk clone OXA-181-producing- E. coli ST410 strains that were closely related to uropathogenic clinical human strains was also an important finding and to our best knowledge was the first description in Portugal and Europe. These studies highligth the importance of companion animals as reservoirs of pathogenic E. coli harbouring important antimicrobial resistant genes. The emergence and spread of multidrug-resistant (MDR) E. coli in the natural environment by companion animal faecal contamination is also a concern towards animal and human health. These results point to need for control measures to prevent the dissemination of MDR ESBLs/AmpC and carbapenemases – producing bacteria from companion animals.
RESUMO - Escherichia coli produtora de Beta-lactamases de Espectro Alargado e Carbapenemases na interface Homem-cão - A Escherichia coli produtora de beta-lactamases de espectro alargado (ESBLs / Ampc) e de carbapenemase tornou-se uma grande preocupação de saúde pública em termos de saúde humana e animal. As infeções do trato urinário (ITU) são uma das infeções bacterianas mais frequentes nos humanos e nos animais de companhia. A E. coli uropatogénica pertencente à família da E. coli patogénica extra-intestinal é a bactéria mais comum isolada em animais de companhia. Além disso, o contato próximo dos animais de companhia com os seres humanos permite oportunidades para a transmissão de bactérias resistentes e genes entre as espécies. Descobriu-se que E. coli de animais de companhia com ITU possuiem importantes mecanismos de resistência antimicrobiana e pertencem a linhagens clonais humanas de alto risco, nomeadamente, E. coli resistente as cefalosporinas de terceira geração (3GC) O25b: H4-B2-ST131-H30 / H30Rx, CC23 e ST648. Neste trabalho, a E. coli O25b: H4-ST131-H30 / H30Rx foi descrita pela primeira vez na Europa em animais de companhia. Além disso, a E. coli ST648 produtora de blaCMY-2 é a linhagem clonal de alto risco mais comum que causa ITU em animais de companhia na área de Lisboa. Os animais de companhia podem ser também potenciais reservatórios de bactérias e de genes de resistência clinicamente importantes, como os genes das β-lactamases (classes A e C). A detecção de estirpes fecais de linhagens clonais de alto risco E. coli produtora de OXA-181 ST410, relacionadas com estirpes clínicas uropatógenicas humanas foi também um achado importante e para nosso conhecimento foi a primeira descrição em Portugal e na Europa. Estes estudos destacam a importância dos animais de companhia como reservatórios de E. coli patogénica que contém importantes genes de resistência a antimicrobianos. O aparecimento e disseminação de E. coli multirresistente (MDR) no ambiente natural por contaminação fecal de animais de companhia é também uma preocupação para a saúde humana e animal. Estes resultados apontam para a necessidade de medidas de controlo para prevenir a disseminação de bactérias produtoras de ESBLs / AmpC e carbapenemases por animais de companhia.
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SILVA, Natália Regina Souza da. "Análise da ocorrência do gene da betalactamase em isolados clínicos de staphylococcus spp. resistentes à meticilina procedentes de hospital universitário da cidade do Recife-PE." Universidade Federal de Pernambuco, 2015. https://repositorio.ufpe.br/handle/123456789/17069.
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Dissertação Natália Final.pdf: 1682940 bytes, checksum: 561b828b32fcd807e7f34e4daee835f6 (MD5)Made available in DSpace on 2016-06-10T18:51:32Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Natália Final.pdf: 1682940 bytes, checksum: 561b828b32fcd807e7f34e4daee835f6 (MD5) Previous issue date: 2015-02-24
Staphlylococcus desenvolveu a resistência aos betalactâmicos através de dois principais mecanismos, a expressão das betalactamases e a produção de PBP2a, codificadas pelos genes blaZ e mecA, respectivamente. Apesar da detecção do gene mecA por técnicas moleculares ser considerado o padrão ouro para a identificação das cepas de Staphlylococcus resistentes à meticilina (MRS), alguns estudos têm verificado discrepância entre a apresentação fenotípica da resistência à meticilina nessas cepas e a presença do gene mecA.O presente estudo teve como objetivo verificar se existe diferença de frequência do gene blaZ entre isolados clínicos de Staphylococcus spp. resistentes à meticilina portadores do gene mecA e os não portadores do gene. Foram estudados 47 isolados provenientes de amostras clínicas de hospital universitário da cidade do Recife. Essas amostras foram submetidas aos testes de susceptibilidade antimicrobiana, detecção do gene mecA para separação dos grupos de comparação e posterior detecção da presença de betalactamase através dos testes de cefinase em disco, Clover-Leaf e detecção do gene blaZ por PCR. Desta forma, foram observadas diferenças na frequência quando utilizados os testes fenotípicos para detecção. No teste de cefinase em disco, foram positivos 33,3% dos isolados portadores do gene mecA e 56,25% dos não portadores. No teste de Clover-Leaf, foi detectada a produção de betalactamase em 33,3% dos isolados portadores do gene mecA e 65,62% dos não portadores. Na pesquisa do gene blaZ, no entanto, não foi verificada diferença significativa nas frequências de detecção entre os grupos. Desta forma, indica-se o uso dos testes fenotípicos de detecção de produção de betalactamase, por apresentarem baixo custo e fácil operação, além de demonstrarem boa sensibilidade.
Staphlylococcus developed resistance to beta-lactam via two major mechanisms of beta-lactamase expression and production of PBP2a encoded by gene mecA and blaZ, respectively. Although the detection of the mecA gene by molecular techniques be considered the gold standard for identifying Staphlylococcus of methicillin-resistant strains (MRS), some studies have found discrepancies between the phenotypic presentation of methicillin resistance in these strains and the presence of the mecA gene. This study aimed to verify if there is frequency difference of blaZ gene among clinical isolates of Staphylococcus spp. methicillin-resistant carriers of mecA gene and non-carriers of the gene.47 isolates from clinical samples of University Hospital in Recife were studied.These samples were subjected to antimicrobial susceptibility tests and subsequent detection of mecA gene by PCR for separation of the comparison groups. After this, was performed to detect the production of beta-lactamase using the disk Cefinase tests, Clover Leaf and detection of blaZ gene by PCR. Thus, diferences in frequency were observed when phenotypics tests were used for detection. In Cefinase test disk,were positive 33.3% of the isolates carrying the mecA gene and 56.25% of non-carriers. In Clover-Leaf assay, the production of beta-lactamase was detected in 33.3% of isolates carrying the mecA gene and 65.62% of non-carriers.In search of blaZ gene, however, there was no significant difference in the frequency detection between groups. In this way, it indicates the use of phenotypic tests for detection of beta-lactamase production for having low cost and easy operation as well as demonstrating good sensitivity.
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Whitelaw, Andrew Christopher. "Molecular methods for the detection of TEM- and SHV-related beta lactamase genes in members of the Enterobacteriaceae." Master's thesis, University of Cape Town, 1999. http://hdl.handle.net/11427/25665.
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Bacterial resistance to antibiotics is a common and important clinical problem. Beta lactam resistance in Gram negative bacilli is mediated predominantly by beta lactamases, enzymes able to hydrolyse the beta lactam ring. The commonest plasmid mediated beta lactamases in the Enterobacteriaceae are those related to either TEM-1 or SHY-1. Although TEM-1, TEM-2 and SHY-1 do not have activity against extended spectrum beta lactams, their derivatives (TEM-3 and SHY-2 onwards) are able to confer resistance to one or more of these antibiotics. A problem encountered in clinical microbiology laboratories is the lack of a reliable method for the detection of ESBLs, along with the lack of a quick, reliable method of differentiating TEM-related genes from SHY -related genes. The primary aim of this study was to evaluate two molecular techniques for the detection of SHY and TEM-related genes in clinical isolates. The study sample consisted of 209 clinical isolates of enteric Gram negative bacilli, isolated at Groote Schuur Hospital microbiology laboratory. The isolates had all been selected on the basis of resistance to one or more of the extended spectrum beta lactams. These isolates were all identified, and the susceptibility of each to a variety of beta lactam antibiotics determined. Using this information, 45 isolates, belonging to different genera and with differing antimicrobial sensitivity patterns, were selected for this pilot study. These 45 isolates consisted of 24 Klebsiella spp., 14 Enterobacter spp., 3 Citrobacter spp., 2 Salmonella spp., 1 Pantoea agglomerans and 1 Serratia marcescens.
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Gonçalves, Teresa Maria Pinto. "Caracterização de genes que codificam para beta-lactamases de espectro alargado em Enterobacteriaceae de origem hospitalar." Bachelor's thesis, [s.n.], 2010. http://hdl.handle.net/10284/1889.
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Monografia apresentada à Universidade Fernando Pessoa para obtenção do grau de Licenciada em Ciências FarmacêuticasOs antibióticos do grupo dos β-lactâmicos encontram-se entre os mais utilizados no tratamento de infecções causadas por Enterobacteriaceae. Contudo, a produção de beta-lactamases de espectro alargado (ESBLs) nestas bactérias diminui a sua eficácia clínica, restando poucas alternativas terapêuticas, visto que estas bactérias acumulam frequentemente resistência a outras famílias de antibióticos. A produção de ESBLs constitui por isso um dos mecanismos de resistência com maior importância e impacto clínico em Enterobacteriaceae. É essencial detectar no laboratório de microbiologia clínica todos os isolados produtores de ESBLs, assim como proceder à caracterização dos genes que codificam para estas enzimas, de forma a detectar precocemente a emergência de novos genes de resistência e determinar os genes que se encontram mais disseminados entre diferentes espécies bacterianas. Em Portugal a ocorrência, diversidade e relevância clínica das ESBLs são ainda pouco estudadas, existindo apenas relatos esporádicos em hospitais específicos ou estudos efectuados em períodos de tempo relativamente curtos. Mudanças na epidemiologia das ESBLs impõem novas abordagens terapêuticas e de controlo da infecção, justificando a sua vigilância periódica e o presente estudo. Assim, com esta monografia pretendeu-se investigar a ocorrência e a diversidade de genes que codificam para ESBLs (blaESBL) em Enterobacteriaceae isoladas de um hospital Português entre Junho de 2006 e Junho de 2007, comparando com dados de anos anteriores (2002-2004), assim como avaliar a coresistência a antibióticos não β-lactâmicos nos isolados produtores de ESBLs. Para isso, foram utilizados métodos universalmente aceites para a identificação das espécies e avaliação da susceptibilidade a antibióticos. A caracterização de ESBLs incluiu a realização do teste do duplo sinergismo e a identificação de genes blaESBL (blaTEM/blaSHV/blaCTX-M) por PCR e sequenciação. A presença do gene blaOXA-1 e qepA entre os isolados produtores de ESBLs foi também pesquisada por PCR. A expressão de ESBLs foi observada em 5% (81/1486) dos isolados clínicos analisados, os quais foram identificados como Escherichia coli (n=48), Enterobacter cloacae (n=14), Klebsiella pneumoniae (n=10), Klebsiella oxytoca (n=4), Morganella morganii (n=2), Enterobacter asburiae (n=1), Proteus mirabilis (n=1) e Serratia marcescens (n=1). As ESBLs detectadas incluíram representantes de enzimas do tipo TEM (18/81, 22%), SHV (18/81, 22%) e CTX-M (38/81, 47%), correspondendo a oito tipos de ESBLs distintos: TEM (-10, -52, -116), SHV (-12, -64) e CTX-M (-14, -15, -32). As ESBLs mais comuns foram CTX-M-15 (35/81, 43%) identificada em E. coli, e SHV-12 (17/81, 21%) detectada em diferentes espécies. blaOXA-1 e blaTEM-1 foram detectadas na maioria dos isolados produtores de CTX-M-15 (20/32, 63%). Não foi detectada a presença do gene qepA entre as Enterobacteriaceae produtoras de ESBLs. A co-resistência a antibióticos não β-lactâmicos foi frequentemente observada, maioritariamente a tetraciclinas (86%), canamicina (85%), gentamicina (78%), tobramicina (77%), estreptomicina (61%), ciprofloxacina (65%) e sulfonamidas (59%). Conclui-se que ocorreu um aumento na ocorrência e na diversidade de ESBLs e de espécies produtoras de ESBLs no hospital analisado. CTX-M-15 e SHV-12 foram os tipos de ESBLs mais frequentes, tendo sido a espécie E. coli a mais comummente identificada como produtora de ESBLs. A disseminação de clones e plasmídeos que albergam estas ESBLs e que conferem resistência a antibióticos de outras famílias parecem estar a contribuir para este cenário, embora estudos moleculares mais aprofundados devam ser realizados para confirmar o seu envolvimento na situação epidemiológica actual. β-lactams are among the most widely used antibiotics to treat infections caused by Enterobacteriaceae. However, production of extended-spectrum beta-lactamases (ESBLs) reduces their clinical efficacy, leaving few therapeutic alternatives, since these bacteria often accumulate resistance genes to antibiotics of other groups. Therefore, production of ESBLs constitutes one of the mechanisms of resistance with greater importance and clinical impact in Enterobacteriaceae. It is essential to detect in the clinical microbiology laboratory all isolates producing ESBLs, as well as to characterize the genes coding for these enzymes, in order to early detect the emergence of new resistance genes and determine the genes that are most widespread among different bacterial species. In Portugal the occurrence, diversity and clinical significance of ESBLs are still poorly studied, with only sporadic reports on individual hospitals or studies in relatively short time periods. Shifts in ESBL-epidemiology might impose new therapeutic approaches and infection control measures, justifying their periodic surveillance and the present study. The main objectives of this monograph were to investigate the occurrence and diversity of genes coding for ESBLs (blaESBL) among Enterobacteriaceae recovered at a Portuguese hospital between June 2006 and June 2007, comparing data with previous years (2002-2004), as well as to evaluate the co-resistance to non-β-lactam antibiotics in ESBL-producing isolates. To accomplish these goals, we used standard methods for species identification and evaluation of susceptibility to antibiotics. The characterization of ESBLs included detection of expression by the double disc synergy test and identification of blaESBL genes (blaTEM/blaSHV/blaCTX-M) by PCR and sequencing. The presence of blaOXA-1 and qepA among ESBL-producing isolates was also investigated by PCR. Expression of ESBLs was observed in 5% (81/1486) of the clinical isolates analyzed, which were identified as Escherichia coli (n=48), Enterobacter cloacae (n=14), Klebsiella pneumoniae (n=10), Klebsiella oxytoca (n=4), Morganella morganii (n=2), Enterobacter asburiae (n=1), Proteus mirabilis (n=1) and Serratia marcescens (n=1). The ESBLs detected included enzymes representatives of TEM- (18/81, 22%), SHV- (18/81 22%) and CTX-M-types (38/81, 47%), corresponding to eight different types of ESBLs: TEM (-10, -52, -116), SHV (-12, -64) and CTX-M (-14, -15, -32). The most common ESBLs were CTX-M-15 (35/81, 43%) identified in E. coli, and SHV-12 (17/81, 21%) detected in different species. blaOXA-1 and blaTEM-1 were detected in almost all isolates producing CTX-M-15 (20/32, 63%). The presence of the gene qepA was not detected among Enterobacteriaceae producing ESBLs. Co-resistance to non-β-lactam antibiotics was frequently observed, mostly to tetracycline (86%), kanamycin (85%), gentamicin (78%), tobramycin (77%), streptomycin (61%), ciprofloxacin (65%) and sulfonamides (59%). It is concluded that happened an increase in the occurrence and diversity of ESBLs and ESBLs-producing species in the hospital analyzed. CTX-M-15 and SHV-12 were the most frequent ESBL types and E. coli was the species most commonly identified as ESBL producer. The spread of clones and plasmids harboring ESBLs and conferring resistance to antibiotics of other groups seems to contribute for this scenario, although more detailed molecular studies should be conducted to confirm their involvement in the current epidemiological situation.
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Rodrigues, Ana Catarina Mesquita Montes. "Phenotypical tests to detect antibiotic resistance genes." Master's thesis, Universidade de Aveiro, 2012. http://hdl.handle.net/10773/11300.
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Mestrado em Biotecnologia MolecularA disseminação da resistência a antibióticos é um grave problema de saúde pública que está a tomar proporções com efeitos difíceis de controlar. Nos últimos anos tem-se verificado que esta disseminação está associada com o fluxo de elementos genéticos móveis, como os integrões, os plasmídeos ou os transposões. Associados a estas estruturas, surgem frequentemente genes que codificam para beta-lactamases, cuja expressão confere resistência a antibióticos frequentemente usados na prática clínica. A causa desta disseminação ainda não é totalmente conhecida, contudo especula-se que possa estar associada ao uso de antibióticos, muitas vezes excessivo ou indevido. É um fenómeno atual, o facto de grande parte da população mundial estar envelhecida. Uma consequência óbvia deste facto, é o aumento de população envelhecida infetada com estirpes resistentes, o que dificulta a caracterização dos backgrounds genéticos existentes no hospital, visto este tipo de pacientes ter uma afluência ao hospital muito elevada. O recurso a testes fenotípicos, de resposta rápida e barata, em laboratórios hospitalares, torna-se cada vez mais necessário. Tendo por base a crescente necessidade de obtenção de resultados que levem a um tratamento mais direcionado, esta tese tem como objetivo principal a aplicação de testes fenotípicos para a deteção de beta-lactamases. Adicionalmente, foram comparadas metodologias existentes no mercado para a deteção de beta-lactamases de espectro alargado (ESBL) e beta-lactamases do tipo AmpC. Para este efeito, foram recolhidas estirpes de bactérias de Gram-negativo produtoras de beta-lactamases, de diferentes produtos biológicos, e compararam-se diferentes testes fenotípicos. Os resultados obtidos foram posteriormente confirmados por metodologias moleculares, pesquisando genes que codificam para beta-lactamases. A prevalência de Enterobacteriaceae produtoras de ESBL encontrada neste estudo é de 10% (n=498), que é preocupantemente elevada. Estas bactérias predominam nas mulheres (54%) e em crianças e idosos (num total de 78%). Os resultados obtidos mostraram que os testes fenotípicos que fazem a deteção de beta-lactamases, como o teste CicaBeta (MAST, UK), têm elevada sensibilidade (90%). Com testes fenotípicos que permitem uma identificação do tipo de beta-lactamase, como o MASTDISC Test (MAST, UK) e Etest (AB BioMérieux, Sweden), verificou-se uma sensibilidade de 94% e 96%, respetivamente. Os métodos moleculares confirmaram a presença de uma ESBL em 98% dos isolados, que na maior parte dos casos se verificou, usando metodologias moleculares, ser uma CTX-M-15. Existe uma elevada diversidade genética, tendo sido identificados 16 perfis genéticos diferentes e em 82% das estirpes foi identificado um integrão. A presença de isolados produtores de ESBL tem implicações devastadoras, pondo em causa o tratamento dos pacientes. Assim, a rápida deteção deste tipo de isolados torna-se imperativa, não só para a implementação de uma terapia adequada, mas também para monitorizar a disseminação de resistência.
The dissemination of antibiotic resistance is a serious problem, difficult to control and poses at risk public health. Several studies report that this dissemination is associated to mobile genetic elements flux, such as integrons, plasmids or transposons. Associated to these structures, are frequently genes coding for beta-lactamases, which expression confers resistance to antibiotics usually used in the clinic. The cause of this dissemination still is not fully known, however it is speculated that might be associated to the use and misuse of antibiotics. World population is aging. An obvious consequence of this fact is the increasing number of elderly infected with antibiotic resistant strains that attend to the emergency room (ER). Therefore the use of phenotypical tests, which give a fast and economic answer, in hospital laboratories, becomes increasingly necessary to achieve an effective treatment. The main goal of this thesis was the evaluation of phenotypical tests to detect extended-spectrum beta-lactamases (ESBL) and AmpC-like beta-lactamases. For this purpose, strains with an ESBL-producer phenotype were collected from different biological products, and the performance of different phenotypical tests was compared. The results obtained were subsequently confirmed by molecular methods. The prevalence of ESBL-producers Enterobacteriaceae found in this study of 10% (n=498) is concerningly high. These bacteria are prevalent in women (54%) and in the early and in elderly ages (in a total of 78%). The results obtained revealed that phenotypical tests that screen for beta-lactamases, as CicaBeta Test (MAST, UK), have a high sensitivity of 90%. Phenotypical tests that allow the identification of the type of beta-lactamase produced, such as MASTDISC Test (MAST, UK) and Etest (AB BioMérieux, Sweden) exhibited higher sensitivity, 94% and 96%, respectively. The molecular methods confirmed the presence of an ESBL in 98% of the isolates, which in most cases was found to be a CTX-M-15, using molecular methodologies. There is a high genetic diversity, being identified 16 different genetic profiles and in 82% of the strains was identified an integron. The clinical implications of strains carrying beta-lactamases, especially ESBLs and AmpC, are of enormous relevance and can compromise patient's treatment. Thus, the rapid detection of this type of isolates will allow the implementation of appropriate therapy, as well as monitor resistance dissemination.
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VERAS, Dyana Leal. "Determinação da ação in vitro de antibióticos β-lactâmicos frente isolados multidroga-resistentes de Klebsiella pneumoniae portadores dos genes blakpc, blashv, blatem e blactx-m." Universidade Federal de Pernambuco, 2014. https://repositorio.ufpe.br/handle/123456789/12070.
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O objetivo deste trabalho foi determinar a ação in vitro de antibióticos β-lactâmicos em isolados de Klebsiella pneumoniae portadores dos genes blaKPC, blaSHV, blaTEM ou blaCTX-M, desenvolvendo embasamento teórico para futura proposição de novas combinações terapêuticas. Sete isolados de K. pneumoniae foram utilizados, sendo 3 clínicos, 2 de comunidade e 2 de microbiota. Os isolados clínicos e de comunidade são portadores de genes codificantes de ESBL ou KPC e os de microbiota de -lactamases clássicas. Os genes de resistência foram identificados por PCR e seqüenciados utilizando primers específicos. A primeira fase deste estudo identificou os efeitos ultra-estruturais causados por -lactâmicos in vitro nos isolados clínicos e de microbiota de K. pneumoniae. Os isolados clínicos foram submetidos a concentrações clinicamente relevantes de ceftazidima, cefotaxima, aztreonam e/ou imipenem e os isolados de microbiota a ampicilina e amoxicilina, para análise pela Microscopia Eletrônica de Transmissão (MET) e Varredura (MEV). Diferentes alterações morfológicas e ultra-estruturais foram identificadas, como filamentação celular, perda de material citoplasmático e deformação dos septos de divisão, após submissão aos antibióticos, tanto nos isolados clínicos de K. pneumoniae como nos obtidos de microbiota. A segunda fase deste estudo determinou a influência destes antibióticos na produção de cápsula polissacarídica (CPS) nos isolados clínicos e de comunidade de K. pneumoniae. Os isolados foram submetidos a concentrações clinicamente relevantes de cefotaxima, ceftazidima, aztreonam e/ou imipenem para analise pela MET, utilizando citoquímica de carboidratos. Todos os isolados tiveram confirmação da presença da região promotora do operon envolvido na produção de CPS, identificado através de PCR e sequenciamento. A ceftazidima e o aztreonam foram capazes de inibir a produção de CPS nos isolados de K. pneumoniae produtores de ESBLs mas não no isolado produtor de KPC, o qual teve sua síntese fortemente inibida apenas pelo imipenem. A cefotaxima não interferiu na produção de CPS em nenhum dos isolados analisados. Nossos resultados demonstraram que antibióticos -lactâmicos possuem ação residual in vitro nos isolados analisados, sugerindo que a produção de ESBL e KPC por isolados de K. pneumoniae não são capazes de evitar completamente a interação de antibióticos β-lactâmicos com as PBPs bacterianas. Podemos concluir ainda que a ceftazidima, o aztreonam e o imipenem, podem ser capazes de inibir a produção de CPS em isolados clínicos e de comunidade de K. pneumoniae, contribuindo para diminuir a expressão do fator de virulência mais importante desta espécie bacteriana.
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Mendes, Rodrigo Elisandro [UNIFESP]. "Caracterização dos elementos genéticos moveis responsáveis pela disseminação de genes associados a resistência bacteriana em Pseudomonas spp. isoladas na América Latina." Universidade Federal de São Paulo (UNIFESP), 2005. http://repositorio.unifesp.br/handle/11600/20578.
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Previous issue date: 2005Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
CAPES: BEX0612/02-2
BV UNIFESP: Teses e dissertações
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Salvador, Luján Gina Nilda, and Luján Gina Nilda Salvador. "Identificación de genes de Metalo β-lactamasas en Pseudomonas aeruginosa de aislados clínicos hospitalarios 2016." Master's thesis, Universidad Nacional Mayor de San Marcos, 2017. http://cybertesis.unmsm.edu.pe/handle/cybertesis/6206.
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Identifica la prevalencia de genes que codifican carbapenemasas de tipo metalo β-lactamasas (MBL) en aislados clínicos de P. aeruginosa. Analiza 76 aislados clínicos de P. aeruginosa resistentes a Ceftazidima y “no sensibles” (intermedio o resistentes) a Imipinem y/o Meropem colectados en el Hospital Militar Central de enero a setiembre del 2016. Muestras de secreciones respiratorias, heridas, orinas y hemocultivos de pacientes hospitalizados son procesadas en el Laboratorio de Microbiología. Determina la sensibilidad antimicrobiana por el método de disco de difusión según los criterios del Clinical and Laboratory Standards Institute. Realiza la detección fenotípica de MBL por el test de sinergia de doble disco con imipenem, meropenem y EDTA. La detección genotípica se realiza amplificando por PCR multiplex, los genes blaIMP y blaVIM, mientras que para el gen blaNDM se hizo PCR convencional. Obtiene fenotípicamente 25 de 76 pruebas positivas para MBL, 24(31.58%) de las cuales se confirmaron genéticamente, encontrando el gen blaIMP (23/24, 95.83%) y el gen blaVIM (1/24, 4.17%). La sensibilidad de la prueba fenotípica es del 100% y la especificidad del 98%. Concluye que el 31.58% de los aislamientos clínicos de P. aeruginosa recuperados de pacientes hospitalizados en el HMC, presentan MBL, siendo el gen blaIMP el más prevalente.Tesis
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Mendonça, Nuno Ricardo Furtado Dias. "Molecular diversity of bla genes in Klebsiella pneumoniae and Escherichia coli isolates." Doctoral thesis, FCT - UNL, 2009. http://hdl.handle.net/10362/2046.
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Dissertation presented to obtain a Ph.D. degree in Biology, speciality Microbiology, by Universidade Nova de Lisboa, Faculdade de Ciências e TecnologiaAntimicrobial resistance is a growing problem worldwide, with extended-spectrum β- lactamase (ESBL)-producing organisms remaining an important cause of cephalosporin therapy failure. The main purpose of the work presented in this dissertation was to search for the molecular diversity of Ambler class A β-lactamase encoding genes in clinical Klebsiella pneumoniae and Escherichia coli isolates and its consequences. In a first step, the evaluation of ESBL detection and confirmation methods for K. pneumoniae, using different guidelines, was performed, with nucleotide sequencing allowing the identification of a new ESBL (SHV-55). The enzymatic properties of this new β-lactamase confirmed a higher affinity towards extended-spectrum cephalosporins, which is characteristic of ESBLs, contrasting to the parental enzyme SHV-1. For the new β-lactamase SHV-72, the higher value of 50% inhibitory concentration of clavulanic acid than for SHV-1 correlated with the values of higher afinity towards penicillins,which are characteristic of an inhibitor resistance enzyme. Simulation of molecular dynamics suggested that the Lys234Arg substitution in SHV-72 was responsible for stabilizing an atipical conformation of the Ser130 side chain, which may decrease susceptibility to clavulanic acid by preventing cross-linking between Ser130 and Ser70. Among the collection of K. pneumoniae strains studied, we identified several genes coding for different enzymes belonging to the CTX-M, GES, LEN, OKP, OXA, TEM, and SHV families and, among them, 35 were new β-lactamases. Among ESBL-producing isolates from 1999 and 2006, we detected the presence of CTX-M enzymes only in the latest period. Overall, the complex molecular diversity of the blaSHV genes detected impelled us to propose a classification for this gene family, based on nucleotide synonymous mutations and the presence or absence of the nonsynonymous mutation T92A. Finally, among E. coli isolates colected in Portugal, a predominat multidrug resistant clone, producing TEM, OXA and CTX-M enzymes, was present in different hospital wards and community environments. The high dissemination of the CTX-M enzymes detected could also be associated with horizontal transfer of plasmids or mobile elements, like ISEcp1. In Algeria, we identified the same insertion sequence in clinical E. coli strains producing TEM and either CTX-M-3 or CTX-M-15 β-lactamases. In conclusion, the results from this dissertation extended our knowledge about the most important mechanism of resistance – the β-lactamases production – focusing on the phenotypic characteristics, biochemical properties, structure-function relationships, molecular diversity and dissemination, which are of most importance to the comprehension of the general resistance scenario in Portugal and worldwide.
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Oliveira, Danielle Escudeiro de. "Caracterização, pesquisa dos genes de virulência e beta-lactamases em Aeromonas hydrophila provenientes de esgoto e lodo tratados." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/6/6135/tde-28092011-143221/.
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Introdução: Bactérias do gênero Aeromonas estão presentes em ambientes de água doce, salgada e salobra. O isolamento destes microrganismos já foi relatado em água de abastecimento público e alimentos. Algumas espécies podem ser patogênicas ao homem, causando gastrenterites e outras infecções. Isolados de Aeromonas de fontes diversas expressam resistência a antimicrobianos, especialmente a -lactâmicos, devido à presença de enzimas -lactamases. A patogenicidade das espécies se deve à virulência multifatorial, que compreende a produção de enterotoxinas (Act, Alt e Ast), de elastase, presença de flagelo, entre outros. Objetivo: Isolar, identificar e quantificar Aeromonas hydrophila isoladas de esgoto e lodo tratado; pesquisar a ocorrência dos genes de virulência e resistência a -lactâmicos. Material e Métodos: A detecção e quantificação de Aeromonas hydrophila foram realizadas por meio da técnica de membrana filtrante e meio de cultura específico; a identificação foi realizada por meio da PCR utilizando um par de primers específicos para a espécie. Após a confirmação da espécie foi realizado o antibiograma para conhecer o perfil de resistência aos antibióticos; a pesquisa dos genes de virulência act, alt, ast, ela, lip e fla e genes de resistência a -lactâmicos foi realizada por meio da PCR e seqüenciamento. Resultados: Foram analisadas 15 amostras (seis de esgoto tratado e nove de lodo tratado). Destas, somente nove foram positivas para A. hydrophila, obtendo-se 441 colônias típicas, das quais 348 foram positivas, por PCR para identificação do gênero e 209 para identificação da espécie. Os 209 isolados, sendo 92 do esgoto tratado e 117 do lodo tratado, apresentaram os seguintes valores na pesquisa dos genes de virulência: 36 por cento (act), 40 por cento (ast), 78 por cento (alt), 82 por cento (fla), 86 por cento (lip) e 87 por cento (ela) e 100 por cento dos isolados apresentaram pelo menos um dos genes. Para os testes de sensibilidade aos antibióticos todos os isolados foram resistentes a pelo menos um dos antibióticos. A produção de enzimas MBL, ESBL e AmpC foi detectada em isolados. Também foram encontrados genes de resistência cphA, bla TEM e bla MOX, enquanto que os genes bla VIM, bla IMP, bla e bla não foram detectados. Conclusão: Os resultados sugerem que A. hydrophila pode resistir ao processo de tratamento de esgoto e lodo, além disso, pode apresentar diversos genes de virulência e resistência a antibióticos, motivos pelos quais A. hydrophila pode ser uma ameaça a Saúde Pública, uma vez que estas amostras são reutilizadas para fins urbanos ou agrícolasIntroduction: Bacteria of the genus Aeromonas are present in fresh, brackish and salty waters. The isolation of these microorganisms has been reported in public water supplies and foods. Some species can be pathogenic to humans, causing gastroenteritis and other infections. Aeromonas isolates from different sources express resistance to antimicrobials, especially -lactams, due to the presence of lactamase enzymes. The pathogenicity of the species is due to the multifactorial virulence, wich includes the production of enterotoxins (Act, Alt and Ast) of Elastase and presense of flagello, among others. Objectives: Identify and quantify Aeromonas hydrophila isolated from treated wastewater and sludge, to investigate the occurrence of virulence genes and resistance to -lactams. Material and methods: The detection and quantification of A. hydrophila were made through the membrane filter technique and specific culture medium, the identification was performed by PCR using a pair of primers specific for the species. After confirming the species sensitivity was performed to know the profile of antibiotic resistance, the survey of virulence genes act, alt, ast, ela, lip, fla and resistance to -lactams gene was performed by PCR and sequencing. Results: We analyzed 15 samples (six of nine treated wastewater and sludge). Of these only nine were positive for A. hydrophila, resulting in 441 typical colonies, of wich 348 were positive by PCR to identify the genus and 209 for species identification. The 209 isolates, being 92 and 117 of treated wastewater and treated sludge showed the following values in the study of the virulence genes: 36 per cent (act), 78 per cent (alt), 82 per cent (fla), 86 per cent (lip), 87 per cent (ela) and 100 per cent of the isolates had at least one of the genes. For antibiotic susceptibility testing all isolates were resistant to at least one antibiotic. The production of MBL, ESBL and AmpC enzyme was detected in isolates. It was also found resistance genes cphA, bla TEM and bla MOX, while genes bla VIM , bla IMP , bla and bla FOX CTX-M were not detected. Conclusion: The results suggest that A. hydrophila can resist the process of treating of wastewater and sludge, moreover, may have different virulence genes and antibiotic resistance, which is why A. hydrophila can be a threat to public health, since these samples are reused for agricultural or urban purposes. , bla SHV
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Gaspareto, Patrick Barcelos. "Pesquisa de genes de metalo-beta-lactamases em pseudomonas aeruginosa isoladas em três hospitais universitários de Porto Alegre." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2005. http://hdl.handle.net/10183/6206.
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Objetivos: Padronizar uma técnica molecular, Reação em Cadeia da Polimerase (PCR) para detectar os genes blaSPM-1 , blaVIM-2 e blaIMP-1 que conferem a Pseudomonas aeruginosa resistência a carbapenêmicos (imipenem e meropenem) através da produção das metalo-β-lactamases (MBL) SPM-1, VIM-2 e IMP-1. Comparar os resultados obtidos com a técnica de PCR com os obtidos através de uma técnica fenotípica para detecção desse mecanismo enzimático de resistência. Descrever a prevalência dos genes de MBL. Metodologia: Utilizando 48 amostras clínicas de P. aeruginosa resistentes a ceftazidima e/ou imipenem sendo 37 caracterizadas como produtoras de MBL através de uma técnica fenotípica (aproximação de disco) e 11 como não produtoras pela mesma técnica. Esses isolados foram testados com três primers específicos para os seguintes genes: blaSPM-1 , blaVIM-2 e blaIMP-1 Resultados e Discussão: Dos 37 isolados produtores de metalo-β-lactamase, 29 tiveram resultado positivo na PCR sendo 27 positivos para o gene blaSPM-1 e 2 positivos para o gene blaIMP-1 . Em nenhum isolado foi detectado o gene blaVIM-2 .Oito isolados caracterizados como produtores de MBL não tiveram nenhum produto amplificado com os três pares de primers. Os 11 isolados caracterizados como não produtores de MBL não tiveram produto amplificado. Assim, a sensibilidade da técnica molecular foi de 78,40% e a especificidade 100,0% considerando a técnica fenotípica de aproximação de disco como padrão ouro Conclusões: Foi possível a padronização da técnica de PCR para a detecção dos genes blaSPM-1 , blaVIM-2 e blaIMP-1 bem como foi possível a comparação entre a técnica de PCR e a técnica fenotípica, sendo que a metodologia molecular apresentou 100% de especificidade e 78,40% de sensibilidade. O gene de MBL mais prevalente foi blaSPM-1 encontrado em 27/29 amostras de P. aeruginosa resistentes ao imipenem. O gene blaIMP-1 foi detectado em apenas 2/29 amostras de P. aeruginosa resistentes ao imipenem. O gene blaVIM-2 e não foi detectado nas amostras de P.aeruginosa analisadas neste estudo.
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Castanheira, Mariana [UNIFESP]. "Caracterização de genes que codificam beta-lactamases mediadas por integrons de classe 1 em amostras de Pseudomonas aeruginosa." Universidade Federal de São Paulo (UNIFESP), 2005. http://repositorio.unifesp.br/handle/11600/20570.
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Previous issue date: 2005Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
BV UNIFESP: Teses e dissertações
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Salvador, Luján Gina Nilda. "Identificación de genes de Metalo β-lactamasas en Pseudomonas aeruginosa de aislados clínicos hospitalarios 2016." Master's thesis, Universidad Nacional Mayor de San Marcos, 2017. https://hdl.handle.net/20.500.12672/6206.
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Identifica la prevalencia de genes que codifican carbapenemasas de tipo metalo β-lactamasas (MBL) en aislados clínicos de P. aeruginosa. Analiza 76 aislados clínicos de P. aeruginosa resistentes a Ceftazidima y “no sensibles” (intermedio o resistentes) a Imipinem y/o Meropem colectados en el Hospital Militar Central de enero a setiembre del 2016. Muestras de secreciones respiratorias, heridas, orinas y hemocultivos de pacientes hospitalizados son procesadas en el Laboratorio de Microbiología. Determina la sensibilidad antimicrobiana por el método de disco de difusión según los criterios del Clinical and Laboratory Standards Institute. Realiza la detección fenotípica de MBL por el test de sinergia de doble disco con imipenem, meropenem y EDTA. La detección genotípica se realiza amplificando por PCR multiplex, los genes blaIMP y blaVIM, mientras que para el gen blaNDM se hizo PCR convencional. Obtiene fenotípicamente 25 de 76 pruebas positivas para MBL, 24(31.58%) de las cuales se confirmaron genéticamente, encontrando el gen blaIMP (23/24, 95.83%) y el gen blaVIM (1/24, 4.17%). La sensibilidad de la prueba fenotípica es del 100% y la especificidad del 98%. Concluye que el 31.58% de los aislamientos clínicos de P. aeruginosa recuperados de pacientes hospitalizados en el HMC, presentan MBL, siendo el gen blaIMP el más prevalente.Tesis
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Rottman, Martin. "Phylogenie et variabilite des cephalosporinases chromosomiques au sein du genre enterobacter." Paris 5, 2001. http://www.theses.fr/2001PA05N004.
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Dropa, Milena. "Disseminação da resistência a antimicrobianos em cepas clínicas e ambientais de Enterobacteriaceae: identificação e mapeamento do ambiente genético de genes codificadores de ESBL." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/6/6135/tde-17062014-111143/.
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Introdução. A resistência bacteriana é facilitada pela pressão seletiva do uso de antimicrobianos na clínica e em outras atividades, como a agricultura e pecuária, além de poder ser disseminada para a natureza por meio do lançamento inadequado do esgoto ou pela aplicação do lodo de esgoto na agricultura. As -lactamases de espectro estendido (ESBL) são uma das formas mais prevalentes de resistência em Gram negativos no mundo, e seus genes codificadores são disseminados por meio de diversos elementos genéticos, principalmente transposons e integrons mobilizados para plasmídios. Objetivo. Identificar e caracterizar genes codificadores de ESBL, bem como suas prováveis formas de mobilização, em enterobactérias isoladas de fontes ambientais e clínicas. Material e Métodos. Quarenta e cinco cepas isoladas de um hospital público em 2004 e 2005, responsáveis por infecções hospitalares (14), infecções comunitárias (7) e colonizações (24), e 7 isoladas de estações de tratamento de esgoto (ETE) em 2009, em São Paulo, geneticamente distintas e produtoras de ESBL da família Enterobacteriaceae, foram estudadas. A técnica de PCR seguida de sequenciamento foi utilizada para a identificação dos genes blaESBL, triagem de elementos móveis e mapeamento do ambiente genético de blaESBL. A identificação dos grupos de incompatibilidade plasmidial (Inc) foi realizada pela técnica de PBRT, e a determinação dos tamanhos dos plasmídios pela técnica de S1-PFGE. Resultados. Os genes blaESBL identificados foram: amostras clínicas - blaTEM-15, blaTEM-197, blaSHV-5, blaSHV-12, blaSHV-27, blaSHV-28, blaSHV-45, blaSHV-55, blaSHV-110, blaCTX-M-2, blaCTX-M-59 e blaCTX-M-131; amostras ambientais blaSHV-28, blaCTX-M-15 e blaCTX-M-8. Os genes blaTEM- 15 e blaTEM-197 estavam associados aos elementos Tn2* e Tn3, respectivamente. Os genes blaSHV-5 e blaSHV-12 estavam associados à IS26, e não foi possível determinar o ambiente genético dos demais genes blaSHV. Os genes blaCTX-M-2, blaCTX-M-59 e blaCTXM- 131 estavam inseridos em integrons de classe 1 complexos, blaCTX-M-15 estava associado à ISEcp1 interrompida pela IS26, e blaCTX-M-8 estava associado à IS10, também interrompida pela IS26. Os principais grupos Inc detectados foram IncA/C (37 por cento ) e IncF (30,4 por cento ). Exceto por 7 cepas clínicas, todas apresentavam plasmídios de alto peso molecular, entre 48,5kb e 388kb. Conclusões. Este estudo detectou 15 genes blaESBL diferentes, dos quais dois são genes novos (blaTEM-197 e blaCTX-M-131) e três são inéditos no Brasil (blaTEM-15, blaSHV-55 e blaSHV-110). A maioria das cepas deste estudo possuía genes blaESBL associados a elementos mobilizáveis, bem como continham plasmídios de grupos Inc envolvidos na disseminação da resistência antimicrobiana. Além disso, carreavam plasmídios provavelmente conjugativos. Os resultados deste estudo mostram genes de resistência associados a elementos mobilizáveis em cepas contendo elementos transferíveis. As cepas foram isoladas tanto em uma instituição de saúde como nas ETEs da Grande São Paulo, mostrando o potencial de disseminação da resistência da clínica para o ambiente em nossa região.Introduction. Bacterial resistance is facilitated by selective pressure of antimicrobial use in clinical and other activities, as agriculture and livestock, and can be spread to nature through the inadequate discharge of sewage or by the use of sludge in agriculture. Extended-spectrum -lactamases (ESBL) are the most prevalente forms of resistance in Gram-negative bacteria in the world, and their encoding genes are disseminated through several genetic elements, especially transposons and integrons mobilized to plasmids. Objective. To identify and characterize ESBL-encoding genes, as well as their probable mobilization pathways, in enterobacteria isolated from clinical and environmental sources. Material and Methods. Forty-five strains isolated from a public hospital in 2004 and 2005, responsible for hospital infections (14), community-acquired infections (7) and colonizations (24), and 7 isolated from sewage treatment plants (ETE) in 2009, in São Paulo, genetically distinct and ESBL producers from Enterobacteriaceae family, were studied. PCR technique followed by sequencing was used for blaESBL genes identification, mobile elements screening and blaESBL genetic environment mapping. Plasmid incompatibility groups (Inc) were identified by PBRT technique, and plasmid sizes were determined by S1-PFGE technique. Results. The blaESBL genes identified were: clinical samples - blaTEM-15, blaTEM-197, blaSHV-5, blaSHV-12, blaSHV-27, blaSHV-28, blaSHV-45, blaSHV-55, blaSHV-110, blaCTX-M-2, blaCTX-M-59 and blaCTX-M-131; environmental samples blaSHV-28, blaCTX-M-15 and blaCTX-M-8. Genes blaTEM-15 and blaTEM-197 were associated to the elements Tn2* and Tn3, respectivelly. Genes blaSHV-5 and blaSHV-12 were associated to IS26, and it was not possible to detect the genetic environment of the other blaSHV genes. Genes blaCTX-M-2, blaCTX-M-59 and blaCTX-M-131 were inserted in complex class 1 integrons, blaCTX-M-15 was associated to ISEcp1 interrupted by IS26, and blaCTX-M-8 was associated to IS10, also interrupted by IS26. The most common Inc grups detected were IncA/C (37 per cent ) and IncF (30,4 per cent ). Except for 7 clinical strains, all isolates showed high molecular weight plasmids, rangng from 48,5kb to 388kb. Conclusions. This study detected 15 different blaESBL genes, from which 2 are new genes (blaTEM-197 e blaCTX-M-131) and 3 are still unpublished in Brazil (blaTEM-15, blaSHV-55 and blaSHV-110). Most of the strains from this study had blaESBL genes associated to mobile elements, as well as they had plasmids from Inc groups involved in the spread of antimicrobial resistance. Moreover, the strains probably carried conjugative plasmids. Results from the present work show resistance genes associated to mobile elements in strains carrying transferable elements. The strains were isolated either from a healthcare institution or from ETEs in São Paulo, which shows the spread potential of resistance from the clinic to the environment in our region.
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Silva, Ketrin Cristina da. "Caracterização molecular de plasmídeos carreadores de genes codificadores de beta-lactamases de espectro estendido em Enterobactereaceas isoladas de suínos." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-03062016-151301/.
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A produção de beta-lactamases de espectro estendido (ESBL) tornou-se um desafio em saúde pública por restringir as opções terapêuticas para o tratamento de infecções causadas por bactérias gram-negativas. O objetivo desse estudo foi avaliar a ocorrência de estirpes produtoras de ESBL nas granjas de suínos brasileiras, bem como caracterizar os plasmídeos carreadores dos genes blaESBL quanto ao grupo de incompatibilidade, tamanho e presença de genes de resistência adicionais. As estirpes foram isoladas em meio MacConkey e identificadas por MALDI-TOF. Posteriormente, os valores de concentração inibitória mínima foram determinados por microdiluição e/ou ágar diluição para aminoglicosídeos, carbapenens, cefalosporinas, fluoroquinolonas, tetraciclinas, sulfas e cefalosporinas associadas a inibidores competitivos. Os genes codificadores de beta-lactamases foram identificados por PCR assim como o grupo de incompatibilidade dos respectivos plasmídeos carreadores e o grupo filogenético das estirpes de E. coli. A análise de clonalidade foi realizada por ERIC-PCR e MLST. Finalmente, o ambiente genético do gene blaCTX-M-15 foi determinado por PCR e/ou sequenciamento, sendo que os plasmídeos carreando genes blaESBL foram transferidos às estirpes receptoras E. coli TOP10 e C600 por transformação e conjugação, respectivamente, e parcialmente sequenciados. As estirpes de Escherichia coli produtoras de CTX-M-2 foram as mais prevalentes, sendo endêmicas no estado de Minas Gerais. Além disso, é relatada a presença da enzima CTX-M-15 em estirpes de E. coli (ST224, ST410, ST1284), Klebsiella pneumoniae, Enterobacter cloacae e Pseudomonas aeruginosa (ST3201). O gene blaCTX-M-15 esteve associado a plasmídeos IncF e foi transferido com sucesso para a estirpe receptora E.coli TOP10, plasmídeos IncF também foram associados a presença do gene blaCTX-M-2. O gene blaCTX-M-8 foi detectado em quatro novos STs de E. coli (ST5845, ST5847, ST5848 e ST5350) e não foi adquirido pelas estirpes receptoras. Estes dados indicam que a vigilância de fenótipos resistentes na produção suína deve de ser considerada uma prioridade, assim como a preferência ao uso de antimicrobianos de espectro estrito a fim de evitar a disseminação desses fenótipos nas granjas e sua possível transmissão para população humana.Extended-spectrum beta-lactamase production (ESBL) became a great challenge regarding public health because limit the therapeutic options to treat infections by gram-negative bacteria. The aim of this study were evaluate the occurrence of ESBL producers in Brazilian swine farms and characterize blaESBL-carrying plasmids by sizing and incompatibility group and presence of additional resistance genes. Strains were isolated in MacConkey agar and identified by Maldi-Tof. Next, the minimal inhibitory concentration values were determined by microdilution and/or agar dilution to aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, tetracyclines, sulfas and cephalosporin/inhibitors association. Betalactamase encoding genes, plasmid incompatibility group and Escherichia coli phylogenetic group were determined by PCR. Clonal relatedness was evaluated by ERIC-PCR and MLST. Finally, the blaCTX-M-15 genetic environment was determined by PCR and/or sequencing and blaESBL-carrying plasmids transferred to E. coli TOP10 and C600 receptor strains by transformation and conjugation, respectively, and partially sequenced. CTX-M-2-producing E. coli were the most prevalent phenotype, which were endemic in Minas Gerais State. Moreover, the CTX-M-15 enzyme emerged among E. coli (ST224, ST410, ST1284), Klebsiella pneumoniae, Enterobacter cloacae e Pseudomonas aeruginosa (ST3201) strains. The blaCTX-M-15 was associated with IncF plasmids, which were successfully transferred to E.coli TOP10, similarly, IncF plasmids were found harboring the blaCTX-M-2. The blaCTX-M-8, detected in four novel E. coli sequence types (ST5845, ST5847, ST5848 e ST5350), was not acquired by receptor strains. Thus, the surveillance of resistant phenotypes in swine production must be established as a priority as well as narrow spectrum antimicrobials prescription antimicrobial instead broad spectrum to prevent the dissemination of these phenotypes in farms and their transmission to human population.
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Balsalobre, Livia Carminato. "Pesquisa de genes codificadores de enterotoxinas e B-lactamases em Aeromonas jandaei e Aeromas hydrophila provenientes de ambientes aquáticos." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/6/6135/tde-28092009-162820/.
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O gênero Aeromonas está amplamente distribuído em ambientes aquáticos, e estudos recentes incluem o gênero no grupo de patógenos emergentes, devido à sua freqüente associação com infecções locais e sistêmicas em humanos. Este trabalho foi realizado com o objetivo de pesquisar por meio da PCR e confirmar por meio de seqüenciamento, a ocorrência dos genes de virulência act, alt e ast, e resistência cphA, blaIMP, blaVIM, blaSPM-1, blaCTXM, blaTEM e blaSHV, verificando também o perfil de resistência a partir de antibiogramas, e a ocorrência de plasmídios nas cepas estudadas. A partir dos resultados observou-se que das 100 cepas selecionadas inicialmente, 87 pertenciam às espécies A. jandaei (46) e A. hydrophila (41). Dentre as quais pôde-se observar a ocorrência de act, alt e ast, respectivamente em 70,7 por cento (29), 97,6 por cento (40) e 26,8 por cento (11) das cepas de A. hydrophila, e em 4,4 por cento (2), 0 por cento (0) e 32,6 por cento (15) nas cepas de A. jandaei. Os genes blaIMP, blaVIM, blaSPM-1, blaCTX-M, e blaSHV não foram encontrados em nenhuma cepa. O gene cphA foi encontrado em 97,6 por cento (40) e 100 por cento (46) das cepas de A. hydrophila e A. jandaei, respectivamente e o gene blaTEM foi encontrado em 97,6 por cento (40) das cepas de A. hydrophila e em 85 por cento (39) das cepas de A. jandaei. Foi verificada a presença de plasmídio em 10/41 (24,4 por cento) das cepas de A. hydrophila e em 16/46 (34,9 por cento) das cepas de A. jandaeiThe genus Aeromonas is widely distributed in aquatic environments, and recent studies include the genus in the emergent pathogens group, due to its frequent association with local and systemic human infections. This work was carried out aiming the investigation and sequencing virulence (act, alt and ast) and resistance (cphA, blaIMP, blaVIM, blaSPM-1, blaCTX-M, blaTEM e blaSHV) genes, also verifying the resistance profile of the strains using antibiograms and the occurrence of plasmids. From the 100 strains analyzed in this study, 87 belonged to A. jandaei (46) and A. hydrophila (41) species. Out of which it was observed the occurrence of act, alt and ast, respectively in 70.7 per cent (29), 97.6 per cent (40) and 26.8 per cent (11) of A. hydrophila strains, and in 4.4 per cent (2), 0 per cent (0) e 32.6 per cent (15) of A. jandaei strains. The genes blaIMP, blaVIM, blaSPM-1, blaCTX-M e blaSHV were not found. CphA gene was found in 97.6 per cent (40) and 100 per cent (46) of A. hydrophila and A. jandaei strains, respectively and blaTEM gene was found in 97.6 per cent (40) of A. hydrophila strains and in 85 per cent (39) of A. jandaei strains. Presence of plasmid was found in 10/41 (24.4 per cent) of A. hydrophila strains and in 16/46 (34,9 per cent) of A. jandaei strains
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Costa, Sara Inês Almeida Dias. "Disseminação horizontal de genes que codificam para β-lactamases de espectro alargado em isolados de enterobacteriaceae de origem hospitalar." Master's thesis, [s.n.], 2013. http://hdl.handle.net/10284/4157.
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Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências FarmacêuticasA família Enterobacteriaceae é composta por uma variedade de géneros e espécies de bacilos de Gram negativo que podem ser responsáveis por várias infecções, sendo Escherichia coli e Klebsiella pneumoniae as espécies mais frequentemente envolvidas. Os antibióticos β-lactâmicos e as fluoroquinolonas constituem as principais escolhas terapêuticas para o tratamento das infecções provocadas por estes microrganismos. A resistência aos antibióticos β-lactâmicos em Enterobacteriaceae está fundamentalmente associada a genes que codificam para beta-lactamases de espectro alargado (ESBLs), principalmente genes blaTEM, blaSHV e blaCTX-M. Estes podem ser disseminados por expansão clonal ou por mecanismos de transferência horizontal (THG). A THG consiste na transferência de material genético para outro organismo que não seja seu descendente. Essa transferência ocorre por intervenção de elementos genéticos móveis, assumindo os plasmídeos conjugativos um papel preponderante na sua disseminação. Desta forma, com o presente estudo pretendeu-se avaliar a frequência de dispersão dos genes blaESBLs por conjugação em isolados de Enterobacteriaceae de origem hospitalar de Portugal, bem como comparar essa taxa de transferência entre diferentes espécies e tipos de genes blaESBL. Para isso, foram estudados 210 isolados de Enterobacteriaceae previamente identificados como produtores de ESBLs obtidos de hospitais da região Norte (n=1) e Centro (n=2) de Portugal. A avaliação da transferência de genes blaESBL foi efectuada através de ensaios de conjugação em meio sólido, utilizando como estirpes receptoras Escherichia coli BM21NR ou E. coli HB101. A confirmação da transferência por conjugação dos genes blaESBL foi efectuada através do teste de duplo sinergismo. Foi detectada a transferência de genes blaESBL em 98 dos 210 isolados testados (correspondendo a uma taxa de conjugação de 47%). A transferência de genes blaESBL por conjugação foi observada em E. coli (n=84), K.pneumoniae (n=8), P. mirabilis (n=2), K. oxytoca (n=1), E. cloacae (n=1), E. aerogenes (n=1) e M. morganii (n=1). A espécie onde se observou mais frequentemente a transferência de genes blaESBL por conjugação foi E. coli, apresentando uma taxa de conjugação de 57% (84/147), seguindo-se K.pneumoniae (20%, 8/40) e E. cloacae (8%, 1/12). Entre as ESBLs do tipo TEM foi observada uma taxa de conjugação de 58% [TEM-10 (60%); TEM-52 (67%); TEM-116 (50%); outras TEM (50%)]. A frequência de conjugação foi inferior entre os isolados produtores de CTX-M [52%; CTX-M-1 (33%); CTX-M-14 (83%); CTX-M-15 (51%)] ou SHV [27%; SHV-12 (26%); outras SHV (33%)]. Neste estudo verificou-se que é frequente a disseminação de genes blaESBL por conjugação em isolados de Enterobacteriaceae de origem hospitalar em Portugal. Constatou-se ainda que E. coli foi a espécie em que se observou uma maior taxa de transferência por conjugação de genes blaESBL. As ESBLs do tipo TEM-52 e CTX-M-14 foram as que apresentaram taxas de transferência por conjugação mais elevadas, estando possivelmente associadas a plasmídeos narrow-host range. SHV-12 foi a ESBL cuja transferibilidade esteve associada a maior diversidade de espécies, provavelmente demonstrando o sucesso da disseminação de plasmídeos contendo esta ESBL em Portugal.
Enterobacteriaceae is composed by a variety of genus and species of negative Gram bacteria that may be responsible for many infections, being Escherichia coli and Klebsiella pneumonia the most frequently involved. The β-lactam antibiotics and fluoroquinolones are the main therapeutic choices for the treatment of infections caused by these microorganisms. The resistance to β-lactam antibiotics in Enterobacteriaceae is fundamentally linked to genes encoding Extended-Spectrum Beta-Lactamases (ESBLs), particularly blaTEM, blaSHV and blaCTX-M. These can be spread by clonal expansion or by Horizontal Gene Transfer (HGT) mechanisms. The HGT is the mobilization of genetic material to another organism that is not its offspring and occurs through the intervention of mobile genetic elements, assuming the conjugative plasmids a leading role. The present study aimed to evaluate the dispersion frequency of blaESBLs genes by conjugation in Enterobacteriaceae isolates of hospital origin and to compare the transfer rate between different species and diverse blaESBL genes. For this work, 210 Enterobacteriaceae isolates previously identified as ESBL producers were analysed. They were obtained from hospitals in the Northern (n=1) and Central (n=2) regions of Portugal. The evaluation of blaESBL gene transfer was carried out by conjugation tests on solid medium, using Escherichia coli BM21NR or E. coli HB101 as receptor strains. The transfer by conjugation of blaESBL genes was confirmed using the double disk synergy test (DDST). The transfer of blaESBL genes was detected in 98 of the 210 isolates tested (corresponding to a conjugation rate of 47%). Transfer by conjugation of blaESBL genes was observed in E. coli (n=84), K.pneumoniae (n=8), P. mirabilis (n=2), K. oxytoca (n=1), E. cloacae (n=1), E. aerogenes (n=1) and M. morganii (n=1). The species where the transfer by conjugation of blaESBL genes was more frequently observed was E. coli 57% (84/147), followed by K.pneumoniae (20%, 8/40) and E. cloacae (8%, 1/12). Among the TEM-type ESBLs, a conjugation rate of 58% was observed [TEM-10 (60%); TEM-52 (67%); TEM-116 (50%); other TEM (50%)]. The conjugation frequency was lower among the CTX-M - 52%; CTX-M-1 (33%); CTX-M-14 (83%); CTX-M-15 (51%) or SHV - 27%; SHV-12 (26%); other SHV (33%) – producing isolates. This study confirmed that the dissemination of blaESBL genes by conjugation in Enterobacteriaceae isolates of hospital origin is relatively frequent. E. coli was the species where a higher rate of transfer by conjugation of blaESBL genes was observed. The TEM-52 and CTX-M-14 ESBLs were those with higher transfer rates by conjugation, being possibly associated to narrow-host range plasmids. SHV-12 was the ESBL whose transferability was associated to a larger diversity of species, demonstrating the success of dissemination of plasmids containing this ESBL.
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Ahmed, Aden. "Utveckling av en PCR-baserad metod för detektion av plasmidburna kolistinresistens, mcr-1 och mcr-3 gener i extended-spectrum beta-lactamase (ESBL)-producerande enterobacteriaceae." Thesis, Högskolan Kristianstad, Sektionen för lärande och miljö, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:hkr:diva-17928.
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Kolistin är ett gammalt polypeptidantibiotikum och används som sista utväg för behandling av allvarliga infektioner orsakad av multiresistenta gramnegativa bakterier. Nya studier har påvisat kolistinresistensgener, mcr (mobil colistin resistance), hos extended spectrum beta-lactamase (ESBL)-producerande Enterobacteriaceae. Mcr-genen ligger i plasmider som kan överföras mellan bakterier, vilket innebär att det är mycket svårare att behandla människor och djur vid infektion orsakad av patogen som erhållit denna resistens. Syfte med detta projekt var att utveckla en PCR-baserad metod för detektion av mcr-1 och mcr-3 gener. I denna studie optimerades en PCR-metod och sedan screenades 60 ESBL-isolat från Kristianstads vattenrike. Därefter undersöktes PCR-produkt med hjälp av agarosgelselektrofores. Resultatet visade att 51oC är den optimala annealingtemperaturen vid multiplex-PCR för detektion av mcr-1 och mcr-3. Ingen av mcr-generna kunde detekteras i de 60 ESBL-isolaten. Positiva kontrollstammar med specifika primers kunde detekteras i PCR-analyser som genomfördes i denna studie, vilket tyder på att den optimerade PCR metoden kan vara pålitlig för detektion av mcr-1 och mcr-3 generna.
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Tolentino, Fernanda Modesto [UNESP]. "Detecção e identificação dos genes de beta-lactamases blaSHV, blaTEM e blaCTX-M em Klebsiella penumoniae isoladas em um Hospital Terciário do Estado de São Paulo." Universidade Estadual Paulista (UNESP), 2009. http://hdl.handle.net/11449/94845.
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tolentino_fm_me_sjrp.pdf: 712675 bytes, checksum: a173e0a0b0b13ecd5a1bb07001fdec0b (MD5)A produção de beta-lactamases de espectro estendido (ESBL) por bactérias gram-negativas causadoras de infecções hospitalares é a maior causa de falha terapêutica com cefalosporinas. Klebsiella pneumoniae, um importante causador de infecções hospitalares em todo o mundo apresenta a maior diversidade de fenótipos de resistência associados à produção de ESBL, e são os organismos onde estas enzimas são mais comumente encontradas. As ESBL dos tipos TEM, SHV e CTX-M são as mais prevalentes em K. pneumoniae, e nos últimos anos, CTX-M emergiu como o principal mecanismo de resistência a cefalosporinas de amplo espectro. Surtos de infecção hospitalar causados por esta bactéria estão associados à disseminação de clones resistentes, e a disseminação de genes de resistência entre cepas ecologicamente relacionadas é comum. O Brasil apresenta uma das mais altas frequências de K. pneumoniae produtoras de ESBL do mundo, mas estudos para a caracterização genética das ESBL no país são raros. Este estudo teve como objetivos detectar e identificar os principais genes responsáveis pela produção de ESBL por cepas de K. pneumoniae isoladas de pacientes internados no Hospital de Base de São José do Rio Preto (HB), no período entre dezembro de 2005 e outubro de 2007, e realizar a tipagem molecular destes isolados buscando identificar seu perfil de ocorrência e disseminação dentro do HB. Noventa e quatro isolados clínicos de K. pneumoniae foram estudados. A detecção e identificação de blaCTX-M, blaSHV e blaTEM foi realizada por PCR e sequenciamento. A tipagem molecular dos isolados foi realizada por PFGE e ERIC-PCR. Os genes responsáveis pela produção de ESBL encontrados foram blaSHV-2, blaSHV-2a, blaSHV-5, blaSHV-12, blaSHV-31 blaCTX-M-2, blaCTX-M-59 e blaCTX-M-15. Além destes, foram identificados blaSHV-1, blaSHV-11, blaSHV-38, blaSHV-62, blaSHV-25,...
Antibiotic resistance due to the production of extended-spectrum beta-lactamases (ESBL) is a worldwide increasing problem, leading to therapeutic failure with third-generation cephalosporins. K. pneumoniae, an important nosocomial pathogen, presents the higher diversity of ESBL and resistance phenotypes, and is the organism where ESBL are most frequently identified. The TEM, SHV and CTX-M ESBL types are the most prevalent in K. pneumoniae, and in recent years, CTX-M has emerged as the main mechanism of resistance to third generation cephalosporins. Hospital infections caused by K. pneumoniae are associated to the dissemination of resistant clones, and also to dissemination of resistance genes among ecologically related strains. In Brazil, ESBL producing K. pneumoniae are detected with the highest frequencies of the world, but studies for the genetic characterization of this resistance mechanism in our country are rare. This study aimed to detect and identify the main ESBL genes harbored by K. pneumoniae isolated from patients admitted to the Hospital de Base de São José do Rio Preto (HB), within the period of December/2005 and October/2007. The pattern of dissemination of the ESBL producing isolates inside the institution was also investigated. Ninety four clinical isolates of K. pneumoniae were studied. The detection and identification of blaCTX-M, blaSHV and blaTEM was performed by PCR and sequencing. The molecular typing was performed by PFGE and ERIC-PCR. The ESBL genes detected were blaSHV-2, blaSHV-2a, blaSHV-5, blaSHV-12, blaSHV-31 blaCTX-M-2, blaCTX-M-59 and blaCTX-M-15. Also, other beta-lactamase genes as blaSHV-1, blaSHV-11, blaSHV-38, blaSHV-62, blaSHV-25, blaSHV-108 and blaTEM-1 were identified. The presence of blaCTX-M in 58, 5% of the studied isolates indicate that this is probably the main genetic mechanism of ESBL production... (Complete abstract click electronic access below)
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Schink, Anne-Kathrin [Verfasser]. "Studies on the prevalence, distribution and organization of extended-spectrum β-lactamase genes and transferable (fluoro)quinolone resistance genes among Enterobacteriaceae from defined disease conditions of companion and farm animals / Anne-Kathrin Schink." Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2012. http://d-nb.info/1024516393/34.
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