Molecular analysis of methicillin-resistant Staphylococcus aureus reveals an absence of plasmid DNA in multidrug-resistant isolates

The number, diversity and restriction enzyme fragmentation patterns of plasmids harboured by 44 multidrug-resistant hospital-acquired methicillin-resistant Staphylococcus aureus (MR-HA-MRSA) isolates, two multidrug-resistant community-acquired MRSA (MR-CA-MRSA), 50 hospital-acquired MRSA (HA-MRSA) isolates (from the University Hospital Birmingham, NHS Trust, UK) and 34 community-acquired MRSA (CA-MRSA) isolates (from general practitioners in Birmingham, UK) were compared. In addition, pulsed-field gel electrophoresis (PFGE) type following SmaI chromosomal digest and SCCmec element type assignment were ascertained for each isolate. All MR-HA-MRSA and MR-CA-MRSA isolates possessed the type II SCCmec, harboured no plasmid DNA and belonged to one of five PFGE types. Forty-three out of 50 HA-MRSA isolates and all 34 CA-MRSA isolates possessed the type IV SCCmec and all but 10 of the type IV HA-MRSA isolates and nine CA-MRSA isolates carried one or two plasmids. The 19 non-multidrug-resistant isolates (NMR) that did not harbour plasmids were only resistant to methicillin whereas all the NMR isolates harbouring at least one plasmid were resistant to at least one additional antibiotic. We conclude that although plasmid carriage plays an important role in antibiotic resistance, especially in NMR-HA-MRSA and CA-MRSA, the multidrug resistance phenotype from HA-MRSA is not associated with increased plasmid carriage and indeed is characterised by an absence of plasmid DNA.     

Antimicrobial susceptibility and clonal relatedness between community- and hospital-acquired methicillin-resistant Staphylococcus aureus from blood cultures

We compared the antimicrobial resistance and clonal relationships among the community-acquired (CA) and hospital-acquired (HA) methicillin-resistant Staphylococcus aureus (MRSA) strains that were isolated from blood cultures in a university hospital over a 4-year period. A total of 131 MRSA isolates, including 28 CA-MRSA and 103 HA-MRSA strains, were identified; antimicrobial susceptibility testing indicated that the CA-MRSA isolates were more susceptible to erythromycin (21% vs 6%; P=0.02), clindamycin (46% vs 12%; P=0.01), ciprofloxacin (43% vs 11%; P=0.01), and gentamicin (43% vs 6%; P=0.01) than were the HA-MRSA isolates. Pulsed-field gel electrophoresis (PFGE) typing and antimicrobial resistance profiles separated the 20 CA-MRSA isolates into 14 and 10 different patterns, respectively, and the 53 HA-MRSA isolates were separated into 24 and 7 different patterns, respectively. Twenty-one (40%) of the 53 HA-MRSA isolates belonged to two predominant PFGE types, and most of them showed multi-drug resistant patterns. Four (20%) of the 20 CA-MRSA and 10 (19%) of the 53 HA-MRSA isolates fell into two common PFGE patterns, and each of them showed the same multi-drug resistant pattern. This study suggests that, although the CA-MRSA blood isolates showed diverse PFGE and antimicrobial resistance patterns, some of these isolates may have originated from the HA-MRSA strains.     

Comparison of arbitrarily primed-polymerase chain reaction and pulse-field gel electrophoresis for characterizing methicillin-resistant Staphylococcus aureus

AIMS: The aim of this study was to analyse genotypes for clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), including hetero-vancomycin-resistant Staph. aureus (VRSA), at a Japanese university hospital. METHODS AND RESULTS: Seventy-eight clinical isolates of MRSA were analysed by arbitrarily primed-polymerase chain reaction (AP-PCR) using ERIC2 primer and by pulse-field gel electrophoresis (PFGE) following SmaI digestion. Analyses of the nine genotypes and 28 subtypes defined by PFGE, and of the three genotypes and 22 subtypes defined by AP-PCR, both facilitated epidemiological tracing. Used in combination, AP-PCR and PFGE provided more precise classification than the use of a single genotyping method. The six hetero-VRSA isolates were classified into four genotypes defined by the combination of both methods, but these genotypes contained non-VRSA isolates. CONCLUSIONS: The results suggest that both PFGE and AP-PCR are useful in discriminating MRSA, but not hetero-VRSA, isolates for epidemiological analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Combining the results of PFGE with the results of AP-PCR can provide more detail differentiation of MRSA and hetero-MRSA isolates than either method alone.     

Molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) isolates from major hospitals in Riyadh, Saudi Arabia

The few studies that have reported the incidence of methicillin-resistant Staphylococcus aureus (MRSA) in Saudi Arabia have indicated that a diverse number of circulating MRSA strains have been detected in several major hospitals. Thus, this study was designed to track the presence of MRSA strains in major hospitals in Riyadh, Saudi Arabia, and perform comparative chromosomal DNA analysis of MRSA strains for epidemiological investigation using pulsed-field gel electrophoresis (PFGE). Correlation of the PFGE types generated with microbiological and clinical data of the isolates was attempted. Screening for decreased susceptibility to vancomycin among the isolates was also done. A dendogram was generated using PFGE macrorestriction fragments and 6 types were identified (M1-M6) with M1 being predominant and widespread. A clear link between PFGE types and some clinical and microbiological data available for the strains was found. For example, M1 was statistically associated with male patients, whereas the unique types were associated with female patients, M2 was associated with isolates from wounds and age group <5 years, and M4 was associated with isolates from patients admitted to intensive care units. M5 was highly correlated with low sensitivity to linezolid. No vancomycin-resistant isolates were detected.     

Staphylococcal cassette chromosome mec (Sccmec) classification and typing methods: an overview

Meticillin-resistant Staphylococcus aureus (MRSA) is one of the main causes of hospital-acquired infections, but since late 1990s also the community-acquired. For better understanding of the S.aureus epidemiology there is an urgent need for creation of new typing method for SCCmec element. The molecular typing of MRSA for epidemiological purposes is investigated by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), spa typing and the SCCmec type assignment. In last few years not only new type of SCCmec (VI to XI) have been identified, but also additional subtypes (i.e. IVg-j) and different variants of already existed one (i.e. 5C2&5 and 2B2&5) were discovered. The aim of this review is to briefly summarize current knowledge about SCCmec classification and to discuss advantages and disadvantages of selected SCCmec typing methods.     

Epidemiologic Typing of Methicillin-Resistant Staphylococcus aureus in Neonate Intensive Care Units Using Pulsed-Field Gel Electrophoresis

To elucidate the mode of dissemination of methicillin-resistant Staphylococcus aureus (MRSA) in neonate intensive care units (NICUs), a total of 223 isolates from 3 separate hospitals were investigated between 1994 and 1996 by a DNA fingerprinting technique with pulsed-field gel electrophoresis (PFGE). Exoprotein profiles of some strains were also examined using SDS-polyacrylamide gel-electrophoresis (SDS-PAGE) and the assessment of enzyme/toxin production such as coagulase, enterotoxin and TSST-1. Judging from the strain typing data from PFGE results and the epidemiological data, 2 different types of PFGE patterns (A and B) and their subtypes (A′, A″ and B′) were identified. The A type including A′ and A″ (comprising approximately 95% of the isolates) was markedly dominant. Only 5% of the isolates belonged to type B and subtype B′. On the other hand, MRSA isolated from adult patients admitted to the same hospital showed many different PFGE patterns. The results strongly suggested that some strain(s) with specific PFGE pattern(s) is prevalent in NICUs. Furthermore, isolates which expressed the same PFGE pattern did not always express the same SDS-PAGE pattern. There were some isolates with different abilities to produce coagulase, enterotoxin C and toxic-shock syndrome toxin (TSST)-1, and the abilities had no relation with a particular type of PFGE pattern. Therefore, a combination of PFGE analysis and biochemical analyses of coagulase, enterotoxin C and TSST-1 may provide us with more detailed information for the epidemiological study of MRSA in NICUs.     

Virtual digest identification of secondary enzymes for use in pulsed-field gel electrophoresis of Staphylococcus aureus

Pulsed-field gel electrophoresis (PFGE) is currently the gold standard for methicillin-resistant Staphylococcus aureus (MRSA) typing but only one enzyme, SmaI, is currently used for restriction digest. We report the use of virtual digestion to identify enzymes for S. aureus PFGE. Two enzymes (EagI and SacII) were identified and successfully used to characterize two sets of S. aureus isolates, 12 USA300, and 14 additional MRSA isolates comprised of seven SmaI patterns. Phylogenetic analysis of patterns generated by all enzymes determined that the USA300 MRSAs are identical. In contrast, digestion with EagI or SacII resolved one to two band differences among three MRSA pattern sets that were not detected using SmaI. These results demonstrate that a second enzyme may detect differences in S. aureus isolates not detected by single enzyme digestion. However, because isolates differing by one to two bands are considered identical, such discrimination may not be clinically or epidemiologically relevant.     

Transcriptional analysis of antibiotic resistance and virulence genes in multiresistant hospital-acquired MRSA

The staphylococcal chromosome cassette mec cannot solely explain the multiresistance phenotype or the relatively mild virulence profile of hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA). This study reports that several multiresistant HA-MRSA strains differently expressed genes that may support antibiotic resistance, modify the bacterial surface and influence the pathogenic process. Genes encoding efflux pumps (norA, arsB, emrB) and the macrolide resistance gene ermA were found to be commonly expressed by HA-MRSA strains, but not in the archetypal MRSA strain COL. At equivalent cell density, the agr system was considerably less activated in all MRSA strains (including COL) in comparison with a prototypic antibiotic-susceptible strain. These results are in contrast to those observed in recent community-acquired MRSA isolates and may partly explain how multiresistant HA-MRSA persist in the hospital setting.     

Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolates from clinical specimens of patients with nosocomial infection: are there unnoticed silent outbreaks?

Bacteriological and epidemiological studies were carried out on 90 isolates of methicillin-resistant Staphylococcus aureus (MRSA) at Turgut Ozal Medical Center of In?nü University, (Malatya/Turkey). MRSA isolates were obtained from patients with nosocomial infections. Staphylococcus aureus clinical isolates were collected between May 2004-May 2005. Isolates were tested for resistance to methicillin. Antimicrobial susceptibility testing and slime production evaluation was performed. Genotype studies were carried out by arbitrarily primed polymerase chain reaction (AP-PCR) and consequent cluster analysis. All of the isolates were mecA-positive in a PCR-based assay; all exhibited resistance to oxacillin, by agar dilution (MICs > or = 4 mg/L) and disc diffusion methods, and multiple antibiotics. Most MRSA isolates were collected in intensive care units. Of 90 samples, 53 were found to be unrelated to the others while the remaining 37 strains were either identical or closely related. Dendrogram analysis identified nine major clusters. These data support the opinion that MRSA are significant nosocomial pathogens in intensive care units and that resistant clones may be transmitted between patients. Molecular epidemiological tools are helpful for understanding transmission patterns and sources of infection, and are useful for measuring outcomes of intervention strategies implemented to reduce nosocomial MRSA.     

Rapid identification of coagulase-negative staphylococci by Fourier transform infrared spectroscopy

Coagulase-negative staphylococci (CNS), frequently associated with both community-acquired and nosocomial bloodstream infections, must be distinguished from Staphylococcus aureus for clinical purposes. Conventional methods are too laborious and time-consuming and often lack sensitivity to CNS. Fourier transform infrared (FTIR) spectroscopy combined with the use of a universal growth medium (Que-Bact Universal Medium No. 2) and chemometrics was evaluated for its potential as a rapid and simple clinical tool for making this distinction. FTIR spectra of 11 methicillin-sensitive and 11 methicillin-resistant CNS isolates as well as 25 methicillin-sensitive, 47 methicillin-resistant, 34 borderline oxacillin-resistant and 35 glycopeptide intermediate S. aureus isolates were obtained from dried films of stationary-phase cells grown on the universal medium. Principal component analysis (PCA), self-organizing maps, and the K-nearest neighbor algorithm were employed to cluster the different phenotypes based on similarity of their FTIR spectra. PCA of the first-derivative normalized spectral data from a single narrow region (2888-2868 cm(-1)) yielded complete differentiation of CNS from both methicillin-sensitive and methicillin-resistant S. aureus. The rate of correct classification was somewhat reduced, from 100% to 90%, after inclusion of borderline oxacillin-resistant and glycopeptide intermediate S. aureus strains in the data set. Differentiation based on the data in broader spectral regions was much less reliable. The results of this study indicate that with proper spectral region selection, FTIR spectroscopy and cluster analysis may provide a simple and accurate means of CNS species identification.     

Epidemiological typing of methicillin-resistant Staphylococcus aureus strains by Fourier transform infrared spectroscopy

A rapid and simple typing system is needed for controlling the spread of epidemic methicillin-resistant Staphylococcus aureus (MRSA), currently one of the most widespread multi-resistant nosocomial pathogens in Canadian hospitals. Fourier transform infrared (FTIR) spectroscopy was used to subtype 85 isolates representing five strains of epidemic Canadian MRSA (CMRSA). Spectral fingerprints of whole cells grown on Que-Bact(R) Universal Medium No. 2 were transformed to first derivative peak-height normalized files and examined visually and by singular-value decomposition (SVD). Distinguishing spectral regions were processed by principal component analysis (PCA), self-organizing map and K-nearest neighbor supervised cluster analysis. Among the visually identified regions, 1070-1050 and 1155-1137 cm(-1) were found suitable for discrimination of CMRSA-4 and CMRSA-2 respectively, while CMRSA-1, CMRSA-3, and CMRSA-5 each exhibited distinctive spectral profiles in the 1123-1094 cm(-1) region. The combination, 1123-1094, 1174-1154 and 2904-2864 cm(-1) separated the five CMRSA with 84.6% correct classification by PCA. Five clusters were also obtained using the SVD-selected regions 1096-1066, 1118-1090 and 2914-2880 cm(-1), with 87.8% correct classification based on visual examination of the PCA scores plot and 97% based on supervised cluster analysis. These results demonstrate that FTIR spectroscopy has considerable potential as a rapid (1-hour) and simple method for MRSA strain typing and monitoring in clinical settings.     

Commercially distributed meat as a potential vehicle for community-acquired methicillin-resistant Staphylococcus aureus

The incidence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infection has been increasing; however, the sources of infection remain unclear. Therefore, we investigated the involvement of meat as a possible mediator of CA-MRSA infection. We examined the distribution of MRSA strains in commercially distributed raw meat samples (n = 197) and diarrheal stool samples of outpatients (n = 1,287) that were collected in Oita Prefecture, Japan, between 2003 and 2009 for routine legal inspections. Fourteen MRSA strains were isolated from three meat and 11 stool samples. Among these, seven isolates from three meat and four stool samples exhibited the same epidemiological marker profiles [coagulase type III, staphylococcal enterotoxin C, staphylococcal chromosomal cassette mec (SCCmec) type IV, ST8, spa type 606 (t1767), and toxic shock syndrome toxin-1 (TSST-1) producing type]. Furthermore, of the seven strains, three isolates from two meat samples and one stool sample collected in 2007 exhibited completely identical characteristics with respect to phage open reading frame (ORF) typing, pulsed-field gel electrophoresis, and drug susceptibility profiles. The results suggest that commercially distributed meat could play a role in the prevalence of CA-MRSA in the community.     

Characterisation of Staphylococcus aureus nasal and skin carriage among patients undergoing haemodialysis treatment

The aim of the study was to investigate the rate of Staphylococcus aureus nasal and skin carriage in patients undergoing haemodialysis. The cultured staphylococcal isolates were subsequently characterized by molecular methods. The study group comprised 43 haemodialysed patients from whom nasal and skin swabs from the vascular access sites were collected. The identification of staphylococcal isolates and antibiotic susceptibility testing were performed on the basis of conventional diagnostic procedures. The staphylococci were further characterized using Pulsed-Field Gel Electrophoresis (PFGE). S. aureus was cultured from 12 (27.9%) patients. Only one (8.3%) patient was colonized with the microorganism both in the anterior nares and the vascular access site representing a single strain, as evidenced by PFGE analysis. Antibiotic susceptibility testing identified one (7.6%) methicillin-resistant S. aureus (MRSA) strain. PFGE typing identified several S. aureus genotypes with the lack of one specific strain responsible for colonization. However, it should be noted that among two (A and D) PFGE patterns genetically indistinguishable and closely related isolates (two isolates for each pattern) were identified. The obtained results revealed a relatively low rate of S. aureus carriage accompanied by low methicillin resistance rate and a significant genetic diversity of cultured isolates with the lack of one predominant strain responsible for colonization.     

Enterocolitis caused by methicillin-resistant Staphylococcus aureus: molecular characterization of respiratory and digestive tract isolates.

We investigated the mechanism of outbreak of enterocolitis caused by methicillin-resistant Staphylococcus aureus (MRSA). Five epidemiological markers [coagulase type, enterotoxin type, toxic shock syndrome toxin-1 (TSST-1) production, beta-lactamase production and pulsed-field gel electrophoresis (PFGE)] of 45 strains of MRSA isolated simultaneously from the respiratory tract (nasal cavity and/or pharynx and/or sputum) and stool (plus one sample of gastric juice) in 13 patients (8 males and 5 females, mean age, 77.1 years) were compared retrospectively. Forty-four of the 45 isolates of MRSA were positive for enterotoxin C and TSST-1 production, and the remaining isolate was positive for enterotoxin A and negative for TSST-1 production. All isolates were coagulase type II, and 27 showed beta-lactamase production. The patterns of coagulase type, enterotoxin type, TSST-1 and beta-lactamase production of MRSA isolated from the respiratory tract were similar to those of MRSA isolated from the intestine in 12 of 13 patients. Molecular typing by PFGE demonstrated that the pattern of respiratory tract isolates was identical to those of stool isolates in 9 (69.2%), similar in 3 (23.1 %), and different in 1 (7.7%). The data suggested that enterocolitis might be caused by the MRSA colonized in the respiratory tract and incorporated into the digestive tracts. Therefore, we propose that early eradication of MRSA in the respiratory tract is important for protection of patients against the development of enterocolitis, particularly in susceptible patients, e.g., immunocompromised or pre-operated patients with digestive diseases, especially malignant disease.     

耐甲氧西林金黄色葡萄球菌的分子流行病学研究

了解我院患者耐甲氧西林金黄色葡萄球菌（MRSA）的分子流行病学特点,为临床抗感染治疗提供依据。收集2007年1月~2008年9月我院分离的耐甲氧西林金黄色葡萄球菌共54株,采用PCR进行SCCmec基因分型、葡萄球菌A蛋白（SPA）分型,并检测杀白细胞毒素（PVL）基因,同时应用脉冲场凝胶电泳（PFGE）进行同源性分析。54株MRSA菌株SCCmec基因分型为SCCmecⅡ型17株,SCCmecⅢ型33株,SCCmecⅣ型2株,SCCmecⅤ型2株;SPA基因分型将28株归属为t030,9株为t002,8株为t037,5株为t570,2株为t437,t163和t796各1株;PVL毒素检测只有2株SCCmecⅣ型菌株阳性;PFGE证实院内MRSA感染主要为2种克隆株传播,同时还有其他型别出现。本院MRSA流行传播的SCCmec基因型主要以Ⅲ型占优势,同时发现有携带PVL毒素的CA-MRSA分离株流行,应引起密切关注。     

Molecular typing of Staphylococcus aureus isolates from an intensive care unit

Seventeen S. aureus clinical isolates, collected from an Intensive Care Unit (ICU) during a seven-month period were analyzed to investigate their antimicrobial susceptibility and clonal diversity. Eleven isolates (65%) were found to be resistant to methicillin (MRSA). Pulsed-field gel electrophoresis (PFGE) profiles of genomic DNAs, and analysis of the polymorphisms of the variable regions of the protein A (spa) and coagulase (coa) genes revealed a lower clonal heterogeneity among MRSA than among methicillin-susceptible isolates (MSSA). Two of the MRSA clones were repeatedly isolated in different patients, within a variable period of time, suggesting the presence in the ward of a resident, endemic and multi-drug resistant MRSA population. Our results also emphasize the lower discriminatory power of spa and coa typing compared with PFGE typing.     

Community-acquired methicillin-resistant staphylococcus aureus: diagnosis and treatment update for plastic surgeons

LEARNING OBJECTIVES:: After studying this article, the participant should be able to: 1. Identify risk factors associated with community-acquired methicillin-resistant Staphylococcus aureus. 2. Recognize the clinical presentation of patients with community-acquired methicillin-resistant S. aureus. 3. Understand the treatment and indications for decolonization of patients who have community-acquired methicillin-resistant S. aureus infections. SUMMARY:: Community-acquired methicillin-resistant Staphylococcus aureus has evolved over the past 10 years as a new health threat seen by plastic surgeons and is an increasing cause of soft-tissue infections. This pathogen has several distinct virulence factors and unique antimicrobial susceptibilities that distinguish methicillin-resistant S. aureus from traditional hospital-acquired methicillin-resistant S. aureus. This article reviews the epidemiology, risk factors, clinical presentation, and treatment of community-acquired methicillin-resistant S. aureus.     

Gene sequences and specific detection for Panton-Valentine leukocidin

A new category of methicillin-resistant Staphylococcus aureus (MRSA), called community-acquired MRSA (CA-MRSA), has emerged worldwide. In contrast to previous MRSA, most CA-MRSA carries the Panton-Valentine leukocidin (PVL) genes (lukPVSF) as a virulence genetic trait. Sequence analysis of the lukPVSF gene of a Japanese isolate demonstrated that the gene has more similarity to methicillin-susceptible S. aureus from France than MRSA from the United States. Based on the sequences, we developed a real-time PCR assay for the three key genes of CA-MRSA; that is, lukPVSF, mecA (for methicillin resistance), and spa (for S. aureus). Dual or triple assay for lukPVSF, mecA, and spa in one test tube became possible. The detection limit of the assay with probe and SYBR Green methods was between 2.7 and 2.7 x 10(1) CFU/ml. The assay detected PVL-positive MRSA in clinical (blood) isolates.     

Molecular analysis and antimicrobial susceptibility of methicillin resistant Staphylococcus aureus in one of the hospitals of Tehran University of Medical Sciences: high prevalence of sequence type 239 (ST239) clone

Methicillin-resistant Staphylococcus aureus (MRSA), particularly the multidrug-resistant clones, is an increasing worldwide problem. The average incidence rate of MRSA in Tehran was found to be over 40%. A total of 140 MRSA isolates obtained from patients attending a teaching hospital in Tehran, from May 2009 to December 2009, were included in this study. The antimicrobial susceptibility profile of MRSA isolates was determined by the agar disk diffusion method. Molecular analysis of MRSA strains was accomplished by Pulsed-Field Gel Electrophoresis (PFGE) and Multi-locus sequence typing (MLST). Detection of mecA gene was used to confirm resistance to methicillin among the MRSA isolates. All the MRSA isolates were susceptible to chloramphenicol, teicoplanin, tigecycline and vancomycin. All MRSAisolates were resistant to oxacillin, whilst 139 strains showed resistance against ciprofloxacin, erythromycin, gentamicin, tetracycline and trimethoprim-sulfamethoxazole. PFGE analysis of all the 140 MRSA isolates produced five distinct pulsotypes designated as pulsotypes A-E. Most of the isolates (n=132) were clustered into pulsotype A. The most prevalent sequence type (ST) was ST 239 (pulsotype A) found in 82% (37/45) of the tested isolates. The second most prevalent type was ST 1238 (pulsotypes B, C and D) found in 15% (7/45) of the isolates. The remaining type, ST 8 (pulsotype E) was found in a single isolate. The results of this study indicated that the MRSA clone ST 239 was a major clone in the selected university hospital of Tehran and that it was widely spread among the different wards as well as all the age groups of patients.