Dissertations / Theses on the topic 'Staphylococcus aureus plasmids'

S. aureus 1054. The recombination occurs by a novel method. The data show that pSl or pΔD contribute the site for recombination and that the gene(s) for the protein(s) involved in recombination are encoded on pOX1054 or the 1054 chromosome. Integration of the plasmids into the chromosome of 1054 was not detected.

Staphylococcus aureus is a Gram positive potentially pathogenic bacterium which has a propensity to gather resistance determinants. Mupirocin is a novel topical antibiotic active against many Gram positive species, including staphylococci and effective in the treatment and prevention of staphylococcal infections. Mupirocin acts by competitively inhibiting the charging of isoleucyl tRNA-synthetase (IRS) with isoleucine. Resistance has been observed in Staphylococcus aureus and coagulase negative staphylococci. Intermediate-level resistance (MIC >8μg ml^-1 >512μg ml^-1) is thought to be due to spontaneous mutations in the native IRS. High-level resistance (>512μg ml^-1) is conferred by a second IRS protein, encoded by mupA which has a much lower affinity for mupirocin than isoleucine. The mupirocin resistance gene (mupA) is usually found on a 4.05kb EcoRI fragment of plasmids of otherwise varied EcoRI restriction pattern which are easily transferred between strains by filter mating. Prior to the onset of these studies, mupirocin resistance had not been found linked to another resistance determinant. Initial investigations intended to identify mechanisms of gene flux resident on mupA plasmids revealed a family of related mupA plasmids, the p3356 family which includes three plasmid types: p3356, p3356D and p3358. p3356 contains a single copy mupA flanked by direct repeats of the staphylococcal insertion sequence IS257. p3356D is identical to p3356 except for the duplication of a "mupA-IS257" cassette in tandem repeat. p3358 is related to p3356D by the insertion of a pT181-like plasmid (tetracycline resistant) accompanied by the duplication of an IS257 in direct repeat to flank the inserted pT181; thus p3358 is the first documented example of linked resistance between mupirocin and another resistance determinant, namely tetracycline. IS257 has been implicated as the recombinogenic site in the gene duplication event involved in the evolution of p3356D from p3356. IS257 co-integrative transposition has been demonstrated to allow the co-integration of pOX7 with p3356 to generate a p3358-type plasmid in which pT181 is replaced by pOX7. Therefore, it is concluded that IS257 transposition and recombination is a mechanism by which staphylococcal replicons can evolve to form multiply resistant replicons.

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

pC221 belongs to a family of staphylococcal plasmids, including pT181, pS194, pC223, pUB112 and pCW7 . All possess open reading frames with 70-80% homology to the pC221 rapD gene. REP D has sequence specific topoisomerase activity at the pC221 origin (or iD) which is thought to be involved in replication initiation. DNase I footprinting has been carried out, showing that REP D binds to a region of oriD downstream of the nick site. The pattern of DNase I cleavage suggests that REP D contacts one face of the DNA helix, which may be bent around the protein. Extracts of S. aureus support incorporation of radioactive dNTPs into pC221 in the presence of REP D. Labelling with a[32P] dATP shows that replication initiates within the region containing oriD and proceeds in the direction expected for elongation of a 3' OH generated by nicking at oriD. With supercoiled DNA, REP D initiates replication of other members of this plasmid family in Vitro. However, with relaxed DNA, REP D is specific for oriD, suggesting that a change in the DNA, stabilised by supercoiling of the DNA or by binding of REP D, may be required for nicking. Of three inverted repeat sequences (ICRI, II & III) at the origin, ICRII has the greatest predicted hairpin stability and is almost totally conserved. Nicking takes place within the loop of this proposed hairpin. Disruption of base pairing within this hairpin has been investigated by mutagenesis of cloned oriD and using oligonucleotides based on the ICRII sequence. These experiments show that the 3' side of ICRII is more important for nicking than the 5' side. This is in agreement with footprinting data which shows that REP D binds the 3' side of ICRII, along with the whole of ICRIII. However, there is no evidence for hairpin formation at ICRII being required for nicking.

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

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

Introducción Las primeras cepas de Staphylococcus aureus resistentes a la meticilina (SARM) se detectaron en el Reino Unido en 1960. Posteriormente, se diseminaron por todo el mundo. Al principio, el SARM era un patógeno nosocomial; pero, progresivamente, se fue extendiendo a pacientes no ingresados, la mayoría de ellos relacionados con el sistema sanitario, como es el caso de los residentes geriátricos. A partir del año 2000, se detectaron cepas de SARM comunitario, con características clínicas y microbiológicas peculiares, como la presencia de genes codificantes de la leucocidina de Panton-Valentine (LPV). Uno de los antimicrobianos alternativos para tratar el SARM es la linezolida, pero se han detectado cepas de estafilococos resistentes a este antimicrobiano. Objetivos Estudiar la relación clonal de los aislados de SARM detectados en el Hospital Universitari Son Dureta-Son Espases, hospital de referencia de las Islas Baleares, durante cuatro períodos: 1999-2000, 2002-2004, 2008 y 2012-2013. Comparar la relación clonal de los aislados de SARM del hospital de referencia con los de otros hospitales de Mallorca. Determinar la frecuencia de LPV. Estudiar la prevalencia de la colonización por SARM en exudados nasales y de úlcera de los residentes en el mayor centro geriátrico de Mallorca. Determinar los factores de riesgo de colonización y evaluar su evolución temporal. Estudiar la epidemiología molecular y el mecanismo de resistencia a la linezolida en cepas de SARM, Staphylococcus epidermidis y Staphylococcus hominis, resistentes a este antimicrobiano detectadas en dos hospitales de Mallorca. Metodología Se documentaron todos los pacientes con SARM en muestras clínicas en los cuatro períodos de estudio. La relación clonal se determinó mediante electroforesis en campo pulsado y multilocus sequence typing. Se realizaron diversos ensayos de PCR para la detección de los genes de LPV, tipificación del casete cromosómico estafilocócico mec (SCCmec) y subtipificación del SCCmec tipo IV. En el estudio de prevalencia de SARM en geriátricos, se recogieron muestras de exudado nasal y de úlcera durante los meses de octubre y noviembre de 2005. Para determinar los factores de riesgo de colonización, se cumplimentó un formulario estandarizado con datos clínicos de cada participante. Se realizó seguimiento clínico y de la colonización a los 8, 12 y 18 meses, tanto en los pacientes colonizados por SARM (casos), como en un grupo de residentes no colonizados (controles). En los aislados de SARM, S. epidermidis y S. hominis resistentes a la linezolida, se realizaron diversos ensayos de PCR y de transferencia del plásmido. Se procedió a la caracterización del plásmido de multirresistencia (pERGB) tras la clonación de los distintos fragmentos en pUCP24. Resultados y conclusiones La situación epidemiológica de SARM en nuestro hospital se caracterizó por la presencia endémica de 3 clones mayoritarios en 1999-2004 (ST125-IVc, ST228-I y ST22-IVh). Los clones ST125-IVc y ST228-I predominaban también en muchos hospitales españoles, mientras que el ST22-IVh (EMRSA-15), prevalente en el Reino Unido, era prácticamente inexistente en el territorio peninsular español. Estos tres clones predominaban en otros hospitales de Mallorca. En 2008, un 7% de las cepas de SARM hospitalarias fueron productoras de LPV. La prevalencia de SARM en exudados nasales de los residentes geriátricos fue del 8%, relativamente baja y generalmente transitoria. Los factores de riesgo asociados con la colonización fueron el ingreso hospitalario previo, la enfermedad vascular, la diabetes, la presencia de úlceras de decúbito y el tratamiento antibiótico previo. La gran mayoría de residentes colonizados no desarrollaron una infección subsiguiente por SARM. Se detectó la presencia de dos clones distintos, también encontrados en el hospital de referencia. Por primera vez, se describe un plásmido de multirresistencia conjugativo portador de los genes cfr, ant(4’)-Ia, tet(L) y dfrK en S. aureus y S. epidermidis que determina la resistencia a la linezolida y a otros antibióticos. Todas las cepas de S. hominis resistentes a la linezolida pertenecían al mismo clon y presentaban la mutación G2576T en el gen ARNr 23S.
Introducció Les primeres soques de Staphylococcus aureus resistents a la meticil·lina (SARM) es detectaren al Regne Unit l’any 1960. Posteriorment, es disseminaren per tot el món. Al començament, el SARM era un patogen nosocomial; però, progressivament, es va estendre a malalts no ingressats, la majoria d’ells relacionats amb el sistema sanitari, com ara els residents geriàtrics. A partir de l’any 2000, es detectaren soques de SARM comunitari, amb característiques clíniques i microbiològiques peculiars, com la presència de gens codificants de la leucocidina de Panton-Valentine (LPV). Un dels antimicrobians alternatius per tractar el SARM és la linezolida, encara que també s’han detectat soques d’estafilococs resistents a aquest antimicrobià. Objectius Estudiar la relació clonal dels aïllats de SARM detectats a l’Hospital Universitari Son Dureta-Son Espases, hospital de referència de les Illes Balears, durant quatre períodes: 1999-2000, 2002-2004, 2008 i 2012-2013. Comparar la relació clonal dels aïllats de SARM de l’hospital de referència amb els d’altres hospitals de Mallorca. Determinar la freqüència de LPV. Estudiar la prevalença de la colonització per SARM en exsudats nasals i d’úlcera dels residents al centre geriàtric més gran de Mallorca. Determinar els factors de risc d’aquesta colonització i avaluar-ne l’evolució temporal. Estudiar l’epidemiologia molecular i el mecanisme de resistència a la linezolida en soques de SARM, Staphylococcus epidermidis i Staphylococcus hominis, resistents a aquest antimicrobià detectades a dos hospitals de Mallorca. Metodologia Es documentaren tots els malalts amb SARM detectat en mostres clíniques durant els quatre períodes d’estudi. La relació clonal es determinà mitjançant electroforesi en camp polsant i multilocus sequence typing. Es dugueren a terme diferents assajos de PCR per a la detecció dels gens de LPV, tipificació del casset cromosòmic estafilocòccic mec (SCCmec) i subtipificació de l’SCCmec tipus IV. A l’estudi de prevalença de SARM en geriàtrics, es recolliren exsudats nasals i d’úlcera durant els mesos d’octubre i novembre de 2005. Per determinar els factors de risc de colonització, s’emplenà un formulari estandarditzat amb les dades clíniques de cada participant. Es va fer el seguiment clínic i de la colonització als 8, 12 i 18 mesos, tant en els malalts colonitzats per SARM (casos), com en un grup de residents no colonitzats (controls). En els aïllats de SARM, S. epidermidis i S. hominis resistents a la linezolida, s’efectuaren diversos assajos de PCR i de transferència del plasmidi. Es procedí a la caracterització del plasmidi de multiresistència (pERGB) després de la clonació dels diferents fragments en pUCP24. Resultats i conclusions La situació epidemiològica de SARM al nostre hospital es caracteritza per la presència endèmica de 3 clons majoritaris en 1999-2004 (ST125-IVc, ST228-I i ST22-IVh). Els clons ST125-IVc i ST228-I predominaven també en molts hospitals espanyols mentre que el ST22-IVh (EMRSA-15), prevalent al Regne Unit, era pràcticament inexistent en el territori peninsular espanyol. Aquests tres clons van ser els més freqüents en els altres hospitals de Mallorca. L’any 2008, un 7% de les soques de SARM de l’hospital foren productores de LPV. La prevalença de SARM en exsudats nasals dels residents geriàtrics fou del 8%, relativament baixa i generalment transitòria. Els factors de risc associats amb la colonització foren l’ingrés hospitalari previ, la malaltia vascular, la diabetis, la presència d’úlceres de decúbit i el tractament antibiòtic previ. La gran majoria dels residents colonitzats no desenvoluparen una infecció subsegüent per SARM. Es detectà la presència dos clons diferents, trobats també a l’hospital de referència. Per primera vegada, es descriu un plasmidi de multiresistència conjugatiu portador dels gens cfr, ant(4’)-Ia, tet(L) i dfrK en S. aureus i S. epidermidis que determina la resistència a la linezolida i a altres antimicrobians. Totes les soques de S. hominis resistents a la linezolida pertanyien al mateix clon i presentaven la mutació G2576T al gen ARNr 23S.
Introduction Methicillin-resistant Staphylococcus aureus (MRSA) strains were first detected in the United Kingdom in the sixties of the past century, and then spread all over the world. At the beginning, MRSA behaved as nosocomial pathogen but progressively was detected outside the hospital, mostly in health-care associated patients. Since year 2000, community-acquired MRSA strains showing a particular clinical and microbiological profile emerged; most of these strains typically contain two genes encoding the Panton-Valentine leucocidin (PVL). Linezolid is a useful alternative for treating patients with staphylococcal infections but resistance to this antimicrobial has arisen. Objectives To study the clonal relatedness of MRSA isolates detected at the Hospital Universitari Son Dureta-Son Espases, along four periods: 1999-2000, 2002-2004, 2008, and 2012-2013; to compare the clonal relatedness of these MRSA isolates with those from other Majorcan hospitals; to determine the frequency of PVL gene detection in the MRSA strains. To study the prevalence of MRSA colonization (nasal and ulcer swabs) in the residents admitted at the major geriatric center of Majorca. To determine the risk factors for colonization, and to evaluate its evolution in over time. To study the molecular epidemiology and the mechanisms of resistance in linezolid-resistant MRSA, Staphylococcus epidermidis and Staphylococcus hominis strains detected at two Majorcan hospitals. Methodology Clonal relatedness was determined by pulsed-field gel electrophoresis and multilocus sequence typing. Several PCR assays were carried out for detection of PVL genes, typing of staphylococcal chromosomal cassette mec (SCCmec), and subtyping of SCCmec type IV isolates. Regarding the prevalence of MRSA carriage in geriatric patients, nasal and ulcer swabs were collected from the study participants in October-November 2005. In order to determine the risk factors for MRSA colonization, a standardized questionnaire with clinical data from each resident was completed. Two cohorts of residents (MRSA carriers and non-carriers) were followed up to 18 months, with nasal cultures performed every six months. PCR detection of several genes and plasmid transfer assays were done in MRSA, S. epidermidis and S. hominis linezolid-resistant isolates to decipher the determinants of this resistance. The multidrug resistance plasmid (pERGB) was characterized after cloning different gene fragments in pUCP24. Results and conclusions The epidemiologic profile of MRSA strains from our hospital was characterized for the endemic presence of 3 major clones during 1999-2004 (ST125-IVc, ST228-I and ST22-IVh). The ST125-IVc and ST228-I clones were also predominant in many Spanish hospitals at that time, whereas the ST22-IVh (EMRSA-15), the most prevalent in British hospitals, was almost nonexistent in centers of the Iberian Peninsula in that period. These three clones were also predominant in the others Majorcan hospitals. In 2008, 7% of MRSA isolates were PVL-producers. MRSA carriage in participants living in the geriatric facility was 8%, lower in comparison with other studies, and generally intermittent. Previous hospital admission, vascular disease, diabetes mellitus, presence of decubitus ulcers and previous antibiotic treatment were the risk factors for MRSA colonization. Most of the residents carrying MRSA did not develop subsequent MRSA infections. MRSA isolates belonged to two different clones, both found also in the reference hospital. A multidrug resistance conjugative plasmid was described for the first time carriyng cfr, ant(4’)-Ia, tet(L) and dfrK genes driving resistance to linezolid and other antimicrobials in S. aureus and S. epidermidis strains. All the linezolid-resistant S. hominis strains belonged to the same clone and presented the G2576T mutation at the 23S rRNA gene.

L'ARN antisens CopA régule le taux de réplication du plasmide bactérien R1 en contrôlant la synthèse de la protéine initiatrice de la réplication, RepA. CopA se fixe à sa séquence complémentaire (CopT) dans la région 5' non traduite de l'ARNm repA. Cette interaction induit principalement une inhibition de la traduction de l'ARNm repA et favorise sa dégradation par la RNase III. L'efficacité du contrôle est directement reliée à la vitesse de formation du complexe CopA-CopT. Nous avons montré que les deux ARN interagissent via une interaction de type boucle-boucle, mais que celle-ci doit être rapidement convertie pour former un complexe irréversible et fonctionnel. Celui-ci n'est pas un duplexe étendu mais contient une jonction à quatre hélices stabilisée par une longue hélice intermoléculaire. Plusieurs intermédiaires réactionnels menant au complexe stable ont été caractérisés, ainsi que les déterminants structuraux de CopA et de CopT nécessaires à cette conversion qui est essentielle au contrôle. Ainsi, nous proposons un mécanisme de formation du complexe stable qui implique plusieurs étapes dans un ordre hiérarchique. Ce mode d'appariement ARN-ARN insoupçonné apparaît être une règle plutôt qu'une exception. En effet, nous avons montré qu'il est conservé dans de nombreux plasmides homologues à R1. L'ARN-III contrôle l'expression des gènes de virulence chez Staphylococcus aureus. Cette deuxième partie de mon travail de thèse a eu pour but de déterminer la structure secondaire de cet ARN en solution et in vivo, et de définir des domaines fonctionnels. En combinant différentes approches in vitro, nous avons établi que l'ARN-III contient 14 structures en tige-boucle et trois interactions à longue distance. Nous avons également identifié un sous domaine fonctionnel impliqué dans le contrôle de la synthèse de la protéine A.

博士
國立臺灣大學
醫學檢驗暨生物技術學研究所
107
Staphylococcus aureus is a worldwide pathogen. Antibiotic resistance in S. aureus is a serious problem. Recently, our group reported the most frequent erythromycin resistance genes in blood-isolated methicillin-susceptible S. aureus (MSSA) is ermB in Taiwan. Although most ermB-positive MSSA carried mobile element structure (MES)-like structures, which has been previously reported in sequence type 59 (ST59) methicillin-resistant S. aureus (MRSA), we also identified a novel structure of Enterococcus faecium-originated ermB-positive Tn6636 in MSSA and MRSA. The transposase gene tnp of Tn6636 is identical to Tn1546. Since vancomycin-resistant S. aureus (VRSA) is due to acquisition of the vanA operon, carried by transposon Tn1546, from Enterococcus spp., the emergence of Tn6636 in S. aureus is alarming. In the present study, we examined ermB-carried 112 MSSA and 224 MRSA in blood-isolated during a 17-year period, 2000 to 2016. The results showed 10 MSSA and 10 MRSA carrying tnp gene of Tn6636. There were four ST types, ST7 (n=6), ST5 (n=3) and ST59 (n=1) in MSSA. Two ST types, ST188 (n=8) and ST965 (n=2) were found in Tn6636-carrying MRSA. PCR mapping showed that the above-described 10 MSSA and 10 MRSA carried similar structure of Tn6636. The results of S1-PFGE with Southern blot showed the Tn6636 of above-described 10 MSSA and 10 MRSA located on plasmids with estimated sizes ranging from 23.1 to 48.5 kb. Isolates of the same ST harbored similar size of plasmids. The conjugation test showed plasmids of ST5 MSSA, ST7 MSSA, ST59 MSSA and ST965 MRSA could transfer to recipient strain RN2677. We used inverse PCR and long and accurate (LA) PCR to determine the sequence of the entire plasmids in each ST. The pBS5-3 of ST5 MSSA is a mosaic plasmid with the 14-kb Tn6636 inserted into the 27-kb pWBG744 of ST5 MSSA. The pBS7-2 of ST7 MSSA and pBR188-6 of ST188 MRSA were mosaic plasmids, the Tn6636 inserted into the 20-kb pSaa6159 of ST93 MRSA. The Tn6636 of pBR965-1 of ST965 MRSA inserted into the 25-kb pN315 of ST5 MRSA. The 47-kb pBS59-1 of ST59 MSSA was a unique plasmid. There was no nucleotide sequence in the NCBI database homologous to pBS59-1 except the region of Tn6636. Only amino acids sequence was available in the NCBI database. In addition, we also screened the presence of Tn6636 in 230 erythromycin-resistant MSSA from non-blood specimens. During two collection periods, April to June in 2016 and Aug 2017 to Feb in 2018 of 33 ermB-positive isolates, there were 11 isolates harboring Tn6636. The 11 Tn6636 carrying isolates belonged to six ST types, ST7 (n=3), ST59 (n=2), ST88 (n=2), ST188 (n=2), ST398 (n=1) and ST2592 (n=1). The results of S1-PFGE with Southern blot showed the Tn6636 of 11 MSSA located on plasmids. There were two sizes of plasmid in three ST7 isolates. One plasmid of ST88 isolate could be transferred to recipient strain RN2677. The entire plasmids of pnBS7-1, pnBS59-2, pnBS88-1, pnBS398-1 and pnBS2592-1 were determined by PCR mapping, inverse PCR and LA-PCR. There were three novel Tn6636-encoding plasmids. This is the first report that E. faecium-originated Tn6636 is present in S. aureus. This Tn6636 harboring erythromycin resistance gene ermB and gentamicin and kanamycin resistance gene aacA-aphD. This Tn6636 located on mobile plasmids. Bacteria could improve the drug resistance by horizontal gene transfer. The emergence of the novel Tn6636 needs to pay close attention.

碩士
國立中興大學
食品科學系
86
Methicillin抗藥性金黃色葡萄球菌(methicillin-resistant S.aureus, MRSA) 為醫院中主要的院內感染菌之一。在台灣地區，MRSA之分離率逐年 增加，因此本研究針對中部地區的醫院於1992-1998年分離出之198株MRSA 菌株，探討其抗藥性、產毒特性並配合質體圖譜及PFGE分析方法了解其感 染分佈情形。另外，亦對食品來源之金黃色葡萄球菌S. aureus產脫皮毒 素分佈加以調查，以了解此類毒素在食品病原菌之存在。利用聚合酉每 鏈鎖反應及免疫分析套組，分別針對MRSA菌株之腸毒素，脫皮毒素及中毒 性休克症候群毒素(TSST-1)基因及毒素之存在進行檢測。以腸毒素之PCR 檢測而言，198株MRSA中，有172株具腸毒素基因，其中有43株具A型腸毒 素基因，8株產B型，5株產D型，53株同時具A B型，26株具A D型，7株具 BD型，30株具A B D型，上述產毒菌在SET-RPLA的檢測中有59株產毒株， 其中有27株具A型腸毒素基因，5株產B型，3株產D型，12株同時具A B型 ，5株具A D型，1株具BD型，5株具A B D型，與PCR結果有明顯不符。在脫 皮毒素檢測方面，PCR結果顯示有22株具ETA基因，沒有菌株具ETB基因； 在RPLA結果方面，有4株產ETA毒素，只有2株與PCR結果相符，49株產ETB 毒素，23株同時產脫皮毒素A、B型，與PCR結果皆不相符。需要再設計實 驗確認之。至於TSST-1毒素，本研究中並未檢測出。而食品來源之150株 金黃色葡萄球菌脫皮毒素之PCR檢測，有38株帶有脫皮毒素基因，其中13 株具ETA型，4株具ETB型，21株同時具ETA、ETB型基因。在RPLA檢測方面 ，共有29株產毒株，產ETA有8株，ETB有8株，同時產ETA及ETB有13株。在 抗生素敏感性試驗方面，90%以上的MRSA菌株對clindamycin、 erythromycin、gentamycin、kanamycin、methicillin、oxacillin、 penicillin、streptomycin、trimethoprim及tetracycline具有抗性；沒 有對vancomycin產生抗藥性的菌株。兩醫院之間MRSA菌株之抗生素抗藥性 圖譜差異不大。本研究分離之MRSA菌株，大多帶有兩個或以上的質體，質 體大小為35.2Kb到1.1kb，只有19株菌不帶質體。可將MRSA分為27種質體 圖譜類型。兩醫院之間沒有完全相同的質體圖譜。因此可與其他分類方法 ，如PFGE，來調查MRSA菌株之間的相關性。在PFGE分析結果方面 ，1992-1993年MRSA菌株以type S 2居多，而type S 5則同為1996-1998年 分離自榮總及中山醫學院MRSA菌株之主要PFGE type。若配合抗生素圖譜 ，質體圖譜及PFGE分析，可將198株MRSA分成112個次分類型(subtype)。 其中有9個次分類型共同存在於1996-1997及1998年分離自榮總的MRSA菌株 中，此九個次分類型中有8株屬於PFGE type S5，1株屬於type S6，因此 這些MRSA菌株之間有高度種源相關性。而兩醫院之間並沒有發現完全相同 的次分類型。本研究結果可為MRSA提供流行病學研究之參考。 Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major nosocomial pathogens in community hospitals. In Taiwan, the incidence of MRSA strains isolation is increasing by years. In this study, the antibiotic susceptibility and distributions of the toxin types for MRSA strains isolated from two hospitals, VGH and CSH, were investigated. In addition, the plasmid profile and PFGE analysis are used to trace the MRSA infection. Not only for MRSA, exfoliative toxins was also investigated for S. aureus strains isolated from food samples. The gene coding for staphylococcal enterotoxin , exfoliative toxin and TSST-1 toxin could be detected by PCR method. Results obtained from PCR were also compared with those from RPLA method. PCR assay showed that 172 of 198 MRSA strains possess enterotoxigenic gene, including 43 type A , 8 type B , 5 type D , 53 type AB , 26 type AD , 7 type BD and 30 type ABD strains. RPLA assay showed that only 59 strains are enterotoxigenic, including 27 type A, 5 type B, 3 type D, 12 type AB, 5 type AD, 1 type BD and 5 type ABD strains, although 172 strains indicate the possession of enterotoxigenic gene. On detection of exfoliative toxin , PCR assay showed that 22 strains pocess exfoliative toxin A (ETA) gene but none for the exfoliative toxin B (ETB) gene. In comparison with the results from RPLA assay which show that 5 strains produce ETA, 49 strains produce ETB and 23 strains produced ETA and ETB toxin, significant discrepancy was found, for the results from and PCR method. Therefore more experiments may be required to check the exfoliative toxin gene of MRSA strains isolated for this study. No TSST-1 toxin strain was found in this study by both the RPLA and the PCR method. ETA and ETB detection for S. aureus strains isolated from food sample by PCR method showed that 38 strains have exfoliative toxin gene, including 13 ETA strains, 4 ETB strains and 21 ETA and ETB strains. In comparison with the result from RPLA assay, 29 strains have positive result, including 8 ETA , 8 ETB, 13 ETA and ETB strains. The antibiotic susceptibility tests showed that no MRSA was resistant to vancomycin. The major antibiotic resistance type for all MRSA strain was resistant to clindamycin, erythromycin, gentamycin, kanamycin, methicillin, oxacillin, ofloxacin, penicillin, streptomycin, trimethoprim, and tetracycline ; There is no significant difference in the antibiogram of MRSA isolated from both hospitals. Plasmid analysis showed the 27 subtypes were found. Most of the MRSA strains bring 2-4 plasmids, the size range is 35.2-1.1Kb. Only 19 MRSA strains was without plasmid. No identical type between the two hospitals were observed.The plasmid profile can be combined for the investigation of the relatedness of MRSA isolates with other typing methods such as PFGE analysis. DNA fingerpriting analysis by PFGE revealed that the predominate PFGE type for MRSA strains isolated from VGH in 1992-1993 is type S2 . Type S5 is the major type for MRSA strains isolated from both hospitals in 1996-1998. When antibiograms , plasmid profiles and PFGE types were combined for subtyping, 112 subtypes were found for the 198 MRSA. Of these subtypes, 9 subtypes (8 in PFGE type S5, 1 in PFGE type S6) coexist between the two groups of MRSA strains isolated from VGH in two different periods (1996-1997 and 1998). These MRSA strains in PFGE type S5 and S6 are high clonally related. No identical subtype was found between VGH and CSH strains, ie, strains from these two hospitals. The results obtained may be used as the reference for epidemiology study of MRSA infection.

碩士
大葉大學
生物產業科技學系
93
ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major pathogens infected in community hospitals, and the incidence of MRSA strains is increasing and becomes a common problem among the hospitals in Taiwan recently. In this study, the polymer chain reaction (PCR) was used to detect the mecA resistant gene, and the Sma I restriction enzyme was used to digest the chromosomal DNA of MRSA. In addition, the plasmid profile and the pulsed field gel electrophoresis (PFGE) analysis were used to investigate the distribution of the toxin types for MRSA strains from Veterinary General Hospital (VGH) in Taichung and Center for Disease Control, Taiwan, ROC. (TCDC), and trace the MRSA infection in the hospitals. The PCR results showed that 45 (59.21 %) clinical strains from VGH and 23 (16.3 %) human S. aureus strains from food-poisoning cases provided by TCDC possessed mecA resistant gene. The ratio of MRSA strains from hospitals was higher than that from food-poisoning cases. As for the distribution for enterotoxin types, it was found that the major enterotoxin types for the strains from hospitals and food-poisoning cases were the enterotoxin A and B, respectively. From the results of the plasmid profiles for MRSA strains, totally 29 distinct plasmid types were found. Of them, no co-shared types were found in the isolates from hospitals and food-poisoning cases. The PFGE results showed that 18 PFGE types were found in the 68 MRSA strains from hospitals and food-poisoning cases in 2003, and the major types for the strains from hospitals were different from those for the strains from food-poisoning cases. In addition, the clinical strains isolated between 1998 and 2003 were found high similarity in PFGE type. The results of this study reveal that the MRSA with same PFGE type still prevail in hospitals.