耐甲氧西林金黄色葡萄球菌的脉冲场凝胶电泳分型研究

目的　探讨耐甲氧西林金黄色葡萄球菌 (MRSA)的分子流行病学特点。方法　采用脉冲场凝胶电泳 (PFGE)技术对我院在 2 0 0 1年 6月～ 2 0 0 2年 4月临床分离的 5 0株MRSA作同源性分析。结果　 5 0株MRSA的PFGE图谱分为 6个组 (A～F型 )。以A型 (2 7株 )、B型 (10株 )、C型 (10株 )流行为主。 5 0株MRSA中共有 17株和重症监护室 (ICU)相关 ,占 34%。流行菌株在各病房之间传播。结论　ICU是MRSA的高发区域和疫源地 ,神经外科、肝胆胰外科和干部病房中MRSA的分离率也较高。在同一病人不同时间不同部位所采集的菌株同源性不尽相同     

Epidemiological typing of methicillin-resistant Staphylococcus aureus outbreak isolates by pulsed-field gel electrophoresis and antibiogram

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common nosocomial pathogens. In April 1997, there were five MRSA-infected patients among 16 patients in the Neonatal Intensive Care Unit (NICU), Seoul National University Hospital, which is a tertiary-care hospital with 1,500 beds. The infections had spread from twin patients with MRSA who had transferred from Hospital C. MRSA was isolated from the axilla of 15 (94%) of the 16 patients, including the two patients with obvious infections. Three (19%) of 16 doctors and nine (30%) of 30 nurses had MRSA colonization of the anterior nares. Six different PFGE patterns (A through F) were identified in the 53 isolates of MRSA tested. Twelve of 13 isolates from infected sites of five patients showed pattern F. Three MRSA strains obtained from hospital C showed closely or possibly related pattern F. MRSA of type F was isolated from three of 16 patients' axilla, and one of 3 doctors' and three of 30 nurses' nasal swabs. The antibiogram code for 12 of 13 MRSA isolates from five infected patients was 66,754. PFGE patterns of these isolates were either F, F1, F2 or Fa. Only one of three strains isolated from clinical specimens of patients in Hospital C showed the antibiogram code 66754, although they were all PFGE types F1 and Fa. In conclusion, the presumptive sources of the outbreak of MRSA infection in NICU were the twin patients transferred from hospital C. Antibiogram correlated reasonably well to the PFGE type. An effective notification system is needed when a MRSA-infected patient is transferred to another hospital to control the spread of the infection.     

Antimicrobial agent of susceptibilities and antiseptic resistance gene distribution among methicillin-resistant Staphylococcus aureus isolates from patients with impetigo and staphylococcal scalded skin syndrome

The susceptibilities to antimicrobial agents of and distributions of antiseptic resistance genes in methicillin-resistant Staphylococcus aureus (MRSA) strains isolated between 1999 and 2004 in Japan were examined. The data of MRSA strains that are causative agents of impetigo and staphylococcal scalded skin syndrome (SSSS) were compared with those of MRSA strains isolated from patients with other diseases. The susceptibilities to antiseptic agents in MRSA isolates from patients with impetigo and SSSS were higher than those in MRSA isolates from patients with other diseases. The distribution of the qacA/B genes in MRSA strains isolated from patients with impetigo and SSSS (1.3%, 1/76) was remarkably lower than that in MRSA strains isolated from patients with other diseases (45.9%, 95/207). Epidemiologic typings of staphylococcal cassette chromosome mec (SCCmec) and pulsed-field gel electrophoresis (PFGE) showed that MRSA strains isolated from patients with impetigo and SSSS had type IV SCCmec (75/76), except for one strain, and 64.5% (49/76) of the strains had different PFGE types. In addition, the patterns of restriction digestion of all tested qacA/B plasmid in MRSA isolates having different PFGE types were identical. The results showed that a specific MRSA clone carrying qacA/B was not prevalent, but qacA/B was spread among health care-associated MRSA strains. Therefore, it was concluded that the lower distribution rate of qacA/B resulted in higher susceptibilities to cationic antiseptic agents in MRSA isolated from patients with impetigo and SSSS.     

甲氧西林耐药的金黄色葡萄球菌耐药性及分子流行病学调查

目的了解甲氧西林耐药的金黄色葡萄球菌(MRSA)的耐药性及分子流行病学特点。方法采用纸片扩散法及琼脂稀释法检测2002年从北京协和医院住院患者分离的165株MRSA的耐药性；采用脉冲场凝胶电泳(PFGE)技术对其中重症监护病房(ICU)和呼吸重症监护病房(RCU)分离的65株MRSA作同源性分析。结果165株MRSA对万古霉素和替考拉宁的敏感率为100％，对庆大霉素的耐药率为100％；对左氧氟沙星、四环素、红霉素耐药率分别为98．2％、96．3％、93．9％。对甲氧苄啶，磺胺甲噁唑和氯霉素敏感率分别为95．8％及98．8％；ICU和RCU病房分离MRSA的PFGE图谱有5种类型(A—E型)，以A型为主(56株)，A型又包括A1亚型(55株)和A2亚型(1株)。结论医院获得性MRSA是多重耐药菌，在ICU和RCU病房发生了基因型为A1亚型的MRSA菌株暴发流行。     

Molecular epidemiologic analysis of methicillin-resistant Staphylococcus aureus isolates from bacteremia and nasal colonization at 10 intensive care units: multicenter prospective study in Korea

We investigated molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) isolated at 10 intensive care units (ICUs) in Korea. MRSA isolates from bacteremia and nasal colonization were collected prospectively from October 2008 through May 2009 at 10 University-affiliated hospital ICUs. A total of 83 and 175 MRSA strains were isolated from bacteremia and nasal colonization, respectively. Acquired group accounted for 69.9% (n = 58) of bacteremia and 73.1% (n = 128) of nasal colonization. Pulsed-field gel electrophoresis (PFGE) type B (SCCmec type II/ST5) was dominant in the acquired group followed by PFGE type D (SCCmec type IVA/ST72; a community genotype). Seven of 58 (12.1%) acquired bacteremia and 15 of 128 (11.8%) acquired nasal colonizations had SCCmec type IVA/ST72 genotype, which indicated that the community genotype had already emerged as a cause of ICU acquired MRSA infection or colonization. Antibiotic resistance rates to ciprofloxacin, tetracycline, clindamycin and trimethoprim/ sulfamethoxazole were 84.4%, 67.1%, 78.1%, and 12.0%, respectively. Susceptibility to ciprofloxacin best predicted a community genotype (sensitivity 96.5%; specificity 96.9%; odds ratio 861; 95% confidence interval 169-4,390, P < 0.001) and the positive predictive value was 90.2%. Among 23 nasal re-colonized strains, 7 MRSA strains (30.4%) were different from the originally colonized strains on the basis of PFGE types.     

Clonal dissemination of epidemic methicillin-resistant Staphylococcus aureus in Belgium and neighboring countries

Objectives   To determine the diversity of pulsed-field gel electrophoresis (PFGE) types among epidemic strains of methicillin-resistant Staphylococcus aureus (MRSA) recovered in Belgium, France, Germany and The Netherlands over the period 1981–94.
Methods   MRSA strains collected in a multicenter survey in Belgium ( n = 171) and from reference laboratories in neighboring countries ( n = 102) were characterized by PFGE analysis using the Sma I enzyme.
Results   In total, 32 PFGE types were found. Epidemic PFGE type 1, first recognized in 1984, accounted for 82% of Belgian strains (87% of hospitals) and 51% of European MRSA strains. Four other internationally epidemic PFGE types (types 8, 10, 11 and 12) were less widely disseminated and more recently detected (1991–94), each recovered from two or three countries. International spread of two PFGE types was linked to transfer of colonized patients to Dutch hospitals from another country where this type was frequently recovered.
Conclusions   Genotypic analysis indicated widespread distribution of several outbreak-associated MRSA strains over large European regions, which was in some instances related to interhospital patient transfer. These findings underscore the need for standardized international surveillance and control of MRSA transmission between healthcare institutions across Europe.     

Characterization of isolates of methicillin-resistant Staphylococcus aureus from Hong Kong by phage typing, pulsed-field gel electrophoresis, and fluorescent amplified-fragment length polymorphism analysis

The genetic relatedness of 127 methicillin-resistant Staphylococcus aureus (MRSA) isolates, belonging to five major types as identified by pulsed-field gel electrophoresis (PFGE) and antibiotic resistance profiles, was examined further using phage typing and fluorescent amplified fragment length polymorphism (FAFLP). The MRSA isolates were recovered from patients at the Prince of Wales Hospital (PWH), Hong Kong, over a 13-year period, 1988 to 2000. These strains were also compared with representatives of the well-described MRSA international clones and with epidemic MRSA strains (eMRSA) 1 to 16 from the United Kingdom. Phage typing distinguished two major "clones" at this hospital: all of the phage type 1 (PT1) isolates belonged to PFGE types A, C, D, and E, while most of the PT2 isolates were associated with PFGE type B, which exhibited a unique antibiotic resistance profile. MRSA isolates belonging to PFGE subtype A2 were indistinguishable from the British eMRSA-1, while isolates of PFGE type B were closely related to eMRSA-9 by PFGE. Based on FAFLP, all five predominant PFGE types at the PWH belonged to one group and fell into the same cluster as eMRSA-1, -4, -7, -9, and -11 isolates. Multilocus sequence typing and staphylococcal cassette chromosome mec typing classified representatives of our MRSA isolates as members of the same clone (ST239-MRSA-III). Thus, the predominant MRSA isolates frin the PWH in the last decade are closely related to early United Kingdom eMRSA clones 1, 4, and 11 and are members of a lineage that includes the Brazilian MRSA clone.     

Analysis of an outbreak of non-phage-typeable methicillin-resistant Staphylococcus aureus by using a randomly amplified polymorphic DNA assay.

A cluster of methicillin-resistant Staphylococcus aureus (MRSA) infections among patients on an intensive care unit (ICU) was detected by routine infection control surveillance. In the period from 5 January to 22 June 1995, 10 patients on the ICU and a further 6 patients (5 on one ward that had received colonized patients transferred from the ICU) were affected by MRSA strains with the same antibiotic susceptibility patterns. Seven (44%) of these 16 colonized patients developed MRSA bacteremia. MRSA isolates with the same characteristics were also found on the hands of one member of the ICU staff. The isolates were untypeable by phage typing, but 15 of 17 outbreak strains analyzed genetically had identical randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) profiles. A single strain of MRSA that was nontypeable by phage typing and that was isolated on the ICU on 1 January and six nontypeable and epidemiologically unrelated MRSA isolates all had RAPD profiles distinct from that of the outbreak strain. Implementation of strict infection control measures stopped the further spread of MRSA on the ICU, the affected general ward, and seven other wards that received MRSA carriers from the ICU. Although nontypeable by phage typing and not previously recognized as an epidemic strain, this strain of MRSA was readily transmissible and highly virulent. RAPD typing was found to be a simple, rapid, and effective method for the epidemiological investigation of this outbreak, and performance of typing by this method was simpler and less time-consuming than that of typing by PFGE. RAPD typing may have more general application for the study of S. aureus infections in hospitals.     

Emergence of rifampicin resistance in methicillin-resistant Staphylococcus aureus isolated at a Turkish university hospital

Twenty-three rifampicin-resistant methicillin-resistant Staphylococcus aureus (MRSA) isolated in three wards at a university hospital in Turkey between June, 2000, and February, 2001, were studied for their genetic relatedness using a combination of antibiogram, coagulase serotyping, coagulase gene polymorphism (coa-RFLP), and pulsed-field gel electrophoresis (PFGE). They all expressed high-level rifampicin resistance (MIC, >256 mg/L) and were resistant to gentamicin, kanamycin, amikacin, ciprofloxacin, tetracycline, and cadmium acetate and were susceptible to fusidic acid, vancomycin, trimethoprim, and mupirocin. They belonged to the same coagulase serotype (serotype IV) and had identical coa-RFLP patterns. In contrast, PFGE generated nine banding patterns designated type A, types A1-A5, B, C, and D. The most common PFGE pattern (type A) and its subtypes (types A1-A5) were seen in 20 (87%) of the 23 isolates in the three wards. The results demonstrated the acquisition of rifampicin resistance by different MRSA clones and the spread of one clone among patients in the three wards.     

Genomic DNA fingerprinting by pulsed-field gel electrophoresis as an epidemiological marker for study of nosocomial infections caused by methicillin-resistant Staphylococcus aureus.

In this study, we have compared genomic DNA fingerprintings among isolates of methicillin-resistant Staphylococcus aureus (MRSA) by using pulsed-field gel electrophoresis (PFGE). Chromosomal fragments digested with SmaI were most suitable for the PFGE separation. SmaI cut genomic DNA into 15 to 20 fragments whose sizes ranged from about 30 to 1,500 kb. Thirty-one distinctive fragment patterns were identified in 111 infecting and colonizing MRSA isolates from six different hospitals in Japan. On the basis of the genomic typing by PFGE, we performed an epidemiological investigation of an outbreak of nosocomial MRSA infections among inpatients in Nagoya University Hospital. Ten types of chromosomal digestion were identified in the 20 strains isolated from 18 infected patients and 1 from colonized hospital personnel. According to the restriction patterns, we found that four types of these strains had caused epidemic infections among 13 patients in the outbreak. Two types (types 1 and 4) of the strains were involved in the death of five patients. The other infections were sporadic. The clarity and polymorphism of the chromosomal digestion patterns enabled us to discriminate between isolates which could not be differentiated by antibiogram or plasmid analysis. Classification of the genomic DNA fingerprinting patterns by PFGE is therefore proposed as a useful method for investigating the source, transmission, and spread of nosocomial MRSA infections.     

Longitudinal study of the molecular epidemiology of methicillin-resistant Staphylococcus aureus at a university hospital

Surveillance for methicillin-resistant Staphylococcus aureus (MRSA) at the University Hospital of Heidelberg revealed an increase in the numbers of newly detected MRSA isolates in recent years. We conducted a study to assess the dynamics of the changes in the MRSA population. Pulsed-field gel electrophoresis (PFGE) typing of MRSA isolates from all patients at the University Hospital of Heidelberg collected between 1993 and 2004 was performed. The microbiology database contained 1,807 entries for newly detected MRSA isolates from 1,301 patients. A total of 1,252 isolates were available for PFGE typing. The isolates could be classified into 109 different PFGE types. Most PFGE types (n=70) were detected less than five times and showed no evidence of transmission (sporadic strains). They accounted for 8.7% of all isolates, with few variations in frequency over the time. Thirty-seven PFGE types were clustered by time of detection, and transmission of the strains was likely (local epidemic strains). A total of 37.3% of the isolates belonged to this group of strains. The remaining 54.0% of the isolates belonged to only two further PFGE types (endemic strains). One endemic strain accounted for 5.0% of all isolates in 1994 and 68.2% in 2004. A second endemic strain was detected in 1.1% of all isolates in 1998 but in 12.4% in 2004. Statistical analysis of the associations between the kind of strain (sporadic, local epidemic, or endemic) and the patients' characteristics revealed a significant association for age and mode of acquisition. The remarkable increase in the rate of MRSA detection at the University Hospital of Heidelberg is mainly due to the dissemination of two different strains. Infection control measures seemed sufficient to prevent further transmission of some but not all of the strains.     

Molecular fingerprinting of fusidic acid- and rifampicin-resistant strains of methicillin-resistant Staphylococcus aureus (MRSA) from Malaysian hospitals

The emergence and spread of multiresistant methicillin-resistant Staphylococcus aureus (MRSA) strains, especially those resistant to fusidic acid and rifampicin, in Malaysian hospitals is of concern. In this study DNA fingerprinting by PFGE was performed on fusidic acid- and rifampicin-resistant isolates from Malaysian hospitals to determine the genetic relatedness of these isolates and their relationship with the endemic MRSA strains. In all, 32 of 640 MRSA isolates from 9 Malaysian hospitals were resistant to fusidic acid and rifampicin. Seven PFGE types (A, ZC, ZI, ZJ, ZK, ZL and ZM) were observed. The commonest type was type ZC, seen in 72% of isolates followed by type A, seen in 13%. Each of the other types (ZI, ZJ, ZK, ZL and ZM) was observed in a single isolate. Each type, even the commonest, was found in only one hospital. This suggests that the resistant strains had arisen from individual MRSA strains in each hospital and not as a result of the transmission of a common clone.     

Use of pulsed-field gel electrophoresis typing to study an outbreak of infection due to Serratia marcescens in a neonatal intensive care unit.

Serratia marcescens is a well-known cause of nosocomial infections and outbreaks, particularly in critically ill neonates and immunocompromised patients. Numerous methods have been proposed for typing. We used pulsed-field gel electrophoresis (PFGE) typing to analyze an outbreak in a neonatal intensive care unit (NICU). We included 23 patient isolates from an outbreak (March to July 1995), and 10 patient isolates from different wards during the same time period. PFGE of whole-cell DNA digested by SpeI was used as a marker of strain identity. The most common presentation of the infection was sepsis in 18 of 23 (78%) neonates. Only four different biotypes were identified; biotype A8d accounted for 84% of the strains. PFGE typing revealed two clones responsible for two different clonal strain dissemination outbreaks from March to July, with 24 patient isolates being pattern A and 4 patient isolates being pattern E. PFGE typing suggests cross transmission between patients in the NICU and other wards. The isolates from 5 other patients showed distinct PFGE patterns. Extensive investigation and cultures failed to identify any environmental or staff reservoir of S. marcescens. This is one of the first reports applying PFGE to the study of S. marcescens, and this method was a useful marker of strain identity. PFGE typing distinguished strains which appeared to be the same by biotyping.     

Staphylococcus aureus as source of catheter‐related bloodstream infection evaluated by PFGE and rep‐PCR typing in a Brazilian hospital

Staphylococci are a common cause of catheter‐related bloodstream infection (CR‐BSI), and epidemiological typing is an important tool for effective infection control. This study evaluated by PFGE and rep‐PCR whether Staphylococcus aureus strains isolated from skin and catheter tips were related to specimens isolated from blood. A prospective observational study, carried out in a clinical surgical ward at a Brazilian hospital between September 2000 and November 2002, investigated non‐tunneled central venous catheters from 179 patients. S. aureus isolates were mainly obtained from blood (41.4%), while coagulase‐negative staphylococci strains were more often isolated from the skin at the catheter insertion site (49.7%) and from the catheter tip (57.5%). Among the 21 strains isolated from 9 patients at 2 or 3 sites simultaneously, 9 were methicillin‐resistant S. aureus (MRSA) and 12 were methicillin‐susceptible S. aureus (MSSA). Seven patients harbored the same S. aureus strain isolated from the skin, blood and/or catheter tip cultures. MRSA isolates belonged to one PFGE pattern (type A‐ subtypes A₁, A₂ and A₃), and to two rep‐PCR patterns (a and b). MSSA isolates were distinguished in five PFGE (B to F) and in three rep‐PCR (c, d and e) patterns. Both PFGE and rep‐PCR methods indicated that the skin at the catheter insertion site was the origin of CR‐BSI caused by S. aureus.     

Adapting spa typing for national laboratory-based surveillance of methicillin-resistant Staphylococcus aureus

Laboratory-based surveillance of methicillin-resistant Staphylococcus aureus (MRSA) monitors the baseline occurrence of different genotypes and identifies strains and transmission chains responsible for outbreaks. The consequences of substituting pulsed-field gel electrophoresis (PFGE) with spa typing as a first-line typing method were analyzed by typing 589 strains isolated between 1997 and 2006, with a focus on both short- and long-term correspondence between the PFGE and spa typing results. The study, covering these ten years, included all Finnish MRSA blood isolates and representatives of the two most prevalent MRSA strains (PFGE types FIN-4 and FIN-16) in Finland. In addition, all sporadic isolates from 2006 were included. spa typing was more expensive but approximately four times faster to perform than PFGE. Nearly 90% of FIN-4 and FIN-16 isolates showed consistent spa types, t172 and t067, respectively. spa typing predicted the PFGE result of the blood isolates by a Wallace coefficient of 0.9009, recognized internationally successful strains (t041, t067) to be common also in Finland, and identified a separate cluster of isolates, also related in time and place among the FIN-4 strains. Additional typing by another method was needed to provide adequate discrimination or to characterize isolates with a newly recognized spa type in Finland.     

Two clones of methicillin-resistant Staphylococcus aureus in Poland

Objective: To evaluate relatedness among methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in Poland.
Method: Ninety-three MRSA hospital isolates were collected from different regions in Poland from 1990 to 1992. Strains were analyzed with respect to heterogeneity of methicillin resistance, phage types, resistance patterns, crystal violet staining, chromosomal DNA Sma I restriction patterns by PFGE, ERIC1 and ERIC2 AP-PCR types and DNA repeat polymorphism within the protein A gene. Resistance to methicillin was confirmed by the detection of the mecA gene by PCR.
Results: The combined results of typing methods demonstrate that all MRSA strains analyzed could be easily divided into two distinct clones (clonally related strains). The first consisted of strains with clear heterogeneous expression of resistance to methicillin (34 isolates) and the second showed more homogeneous resistance (59 isolates). In this study the best method for epidemiologic analysis of MRSA was found to be PFGE. A good correlation between the epidemic behavior of MRSA and a high number of repetitive DNA units within the protein A gene was observed.
Conclusions: Results show that in Poland two distinct clones of epidemic MRSA have circulated in the past, easily discriminated by pheno and genotyping methods, and both could be found together in a single hospital.     

Differences in methicillin-resistant Staphylococcus aureus strains isolated from pediatric and adult patients from hospitals in a large county in California

Studies of U.S. epidemics of community- and health care-associated methicillin-resistant Staphylococcus aureus (MRSA) suggested differences in MRSA strains in adults and those in children. Comprehensive population-based studies exploring these differences are lacking. We conducted a prospective cohort study of inpatients in Orange County, CA, collecting clinical MRSA isolates from 30 of 31 Orange County hospitals, to characterize differences in MRSA strains isolated from children compared to those isolated from adults. All isolates were characterized by spa typing. We collected 1,124 MRSA isolates from adults and 159 from children. Annual Orange County population estimates of MRSA inpatient clinical cultures were 119/100,000 adults and 22/100,000 children. spa types t008, t242, and t002 accounted for 83% of all isolates. The distribution of these three spa types among adults was significantly different from that among children (χ(2) = 52.29; P < 0.001). Forty-one percent of adult isolates were of t008 (USA300), compared to 69% of pediatric isolates. In multivariate analyses, specimens from pediatric patients, wounds, non-intensive care unit (ICU) wards, and hospitals with a high proportion of Medicaid-insured patients were significantly associated with the detection of t008 strains. While community- and health care-associated MRSA reservoirs have begun to merge, significant differences remain in pediatric and adult patient populations. Community-associated MRSA spa type t008 is significantly more common in pediatric patients.     

Molecular evolution of methicillin-resistant Staphylococcus aureus in the metropolitan area of Cologne, Germany, from 1984 to 1998

To investigate the molecular evolution of methicillin-resistant Staphylococcus aureus (MRSA) in a large metropolitan area in Germany, 398 nonrepetitive MRSA isolates recovered from patients from various teaching and nonteaching hospitals in Cologne between 1984 and 1998 were characterized by pulsed-field gel electrophoresis (PFGE). On this basis, 95 representative isolates were selected and further investigated by multilocus sequence typing (MLST), spa typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Overall, there were 9 MLST types and 16 spa types. The most prevalent sequence types (STs) were ST239 (38% of isolates), ST247 (29%), and ST228 (18%); the most prevalent spa types were 37 (32%) and 51 (29%). ST239 comprised five major PFGE types and various unique PFGE patterns, and ST5 comprised two PFGE types. While the same PFGE pattern was not observed among strains with different STs, spa type 37 was observed among strains representing two different STs (ST239 and ST241), and these belonged to the same clonal complex as single-locus variants. ST239 was the earliest predominant ST, with the highest prevalence from 1984 to 1988 (96%), followed by ST247 from 1989 to 1993 (83%) and ST228 from 1994 to 1998 (40%). Spa type 37 was the most prevalent from 1984 to 1988 (96%), spa type 51 was the most prevalent from 1989 to 1993 (83%), and spa types 1 and 458 were the most prevalent from 1994 to 1998 (26% and 14%, respectively). The prevalence of SCCmec type III decreased from 96% from 1984 to 1988 to 8% from 1989 to 1993, the prevalence of SCCmec type I increased from 4% from 1984 to 1988 to 97% from 1989 to 1993 and decreased to 62% from 1994 to 1998. While the genetic diversity of MRSA increased from 1984 to 1998, one prevalent ST usually accounted for most of the isolates in a given time period.     

Molecular alterations of VanA element in vancomycin-resistant enterococci isolated during a survey of colonized patients in an Italian intensive care unit

To screen for vancomycin-resistant enterococci (VRE) colonization in hospitalized patients and to study molecular evolution and alterations of Tn1546-like elements in VRE among potentially at-risk patients, a 3-year surveillance protocol in an Intensive Care Unit was performed. A total of 397 patients were screened in the period June, 1997-June, 2000, and VRE were isolated from rectal swabs taken at admission, weekly, and when clinically indicated. The susceptibility of the enterococci was determined by the disk diffusion and broth dilution methods. The presence of vancomycin-resistance genes (vanA, vanB, and vanC) was assessed by polymerase chain reaction (PCR); genetic clonality of isolates was assessed by pulsed-field gel electrophoresis (PFGE); Tn1546 types were obtained by restriction fragment length polymorphism (RFLP) analysis of Tn1546 PCR fragments. Thirty-four strains, 31 identified as Enterococcus faecium and 3 strains as E. faecalis, were isolated from 12 of the 397 patients (3.0%); all strains were VanA as assessed by PCR and were resistant to the other antibiotics tested and showed high-level resistance to aminoglycosides. Enterococci isolated during the study period showed that different genetic backgrounds of strains, determined by PFGE combined with RFLP of Tn1546, are present in all the strains isolated in the study. PFGE type B was predominant in 1998 and 1999, and insertion sequence movements were found to have a role in the evolution of VanA resistance elements found in all strains. This study demonstrates that single patients may be colonized by closely related VRE with several PFGE types containing a wide variety of VanA elements. Moreover, isolates with identical PFGE types may contain different VanA elements reflecting rearrangements mediated by insertion sequences in VRE strains during their stay in the gastrointestinal tract.     

Comparison and application of ribosome spacer DNA amplicon polymorphisms and pulsed-field gel electrophoresis for differentiation of methicillin-resistant Staphylococcus aureus strains.

Analysis of sequences in the fragments of the 16S-23S rRNA intergenic spacer region by the ribosome spacer PCR (RS-PCR) can differentiate strains of methicillin-resistant Staphylococcus aureus (MRSA). We compared this technique with pulsed-field gel electrophoresis (PFGE) for typing MRSA strains and its application during an investigation of an outbreak. A total of 180 isolates of MRSA collected from various hospital laboratories within the United Kingdom and elsewhere were typed by PFGE and RS-PCR. PFGE identified 17 different types among the 180 strains examined, and RS-PCR generated 13 different types. PFGE could detect minor genetic variations among the isolates and could identify the variants which were not discriminated by RS-PCR. Four unique strain types detected by PFGE were not detected by RS-PCR. When applied to typing the outbreak-related strains from the vascular surgery unit at the General Infirmary at Leeds, the results of RS-PCR were identical to those of PFGE. Our results have shown that RS-PCR is a rapid, inexpensive technique that is highly reproducible and almost as discriminatory as PFGE for typing MRSA isolates and should be useful in the local investigation of MRSA outbreaks.