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。     

Culture-based detection of methicillin-resistant Staphylococcus aureus by a network of European laboratories: an external quality assessment study

Twenty-three hospital laboratories from Europe and Israel participated in an external quality assessment (EQA) of the culture-based detection of methicillin-resistant Staphylococcus aureus (MRSA). Participants also reported the MRSA prevalence in clinical cultures and patient screening specimens, as well as the MRSA screening practices employed at their hospitals. An EQA panel of 18 samples consisting of two MRSA harbouring SCCmec IV and I, and one strain each of methicillin-resistant coagulase-negative S. epidermidis, methicillin-sensitive S. aureus and Escherichia coli as pure strains or in mixtures at 10(7)-1 cfu absolute loads was analysed by the 23 participants. Seventeen (74%) participants identified 17 or more samples correctly. Of these, 15 (88%) utilised a chromogenic medium alone (ChromID, bioMérieux; BBL CHROMagar, BD Diagnostics; MRSA Select, Bio-Rad Laboratories) or combined with a conventional medium and up to three confirmatory tests. Proportions of MRSA among S. aureus isolated from clinical cultures varied widely, even among hospitals within countries, ranging from 11-20% to 61-70%. MRSA carriage rates were less variable (0-20%) between countries. Almost all participants (n=22, 96%) screened patients for MRSA carriage during 2009-2010, of which 15 (68%) screened intensive care unit (ICU) patients alone or combined with other targeted high-risk groups, and 10 (45%) combined nasal screening with another body site.     

Methicillin-resistant Staphylococcus caprae in a neonatal intensive care unit

Staphylococcus caprae, a hemolytic coagulase-negative staphylococcus that is infrequently associated with humans, was initially detected in specimens from six infants in our neonatal intensive care unit due to phenotypic characteristics common to methicillin-resistant Staphylococcus aureus. These isolates were subsequently identified as S. caprae by the Automated RiboPrinter microbial characterization system. This prompted an 8-month retrospective investigation in our neonatal intensive care unit. S. caprae was the cause of 6 of 18 episodes of coagulase-negative staphylococcal bacteremia, was the most common coagulase-negative staphylococcus recovered from the nares of 6 of 32 infants surveyed in a methicillin-resistant S. aureus surveillance program, and was isolated from 1 of 37 health care providers' hands. Of 13 neonatal intensive care unit isolates tested, all were methicillin resistant and positive for the mecA gene. All 21 isolates were found to be a single strain by Automated RiboPrinter and pulsed-field gel electrophoresis with ApaI or SmaI digestion; ApaI was more discriminating in analyzing epidemiologically unrelated strains than Automated RiboPrinter or electrophoresis with SmaI. These findings extend the importance of S. caprae, emphasize its similarities to methicillin-resistant S. aureus, and demonstrate its ability to persist in an intensive care unit setting.     

Staphylococcal resistance to aminoglycosides before and after introduction of amikacin in two teaching hospitals

A prospective study was conducted to determine the prevalence of aminoglycoside-resistant Staphylococcus aureus and coagulase-negative staphylococci before and after the introduction of amikacin as the sole aminoglycoside used in our burn unit, adult intensive care unit, and neonatal intensive care unit. Pharyngeal or endotracheal cultures, as well as superficial surveillance cultures, were collected weekly during the following four study periods: all units for 4 months before amikacin introduction, all units 4 to 8 months after, all units 12 to 13 months after, and the neonatal intensive care unit 30 months after. A total of 2,613 strains of coagulase-negative staphylococci and 316 strains of S. aureus were obtained from 916 patients. During the course of the study, amikacin-resistant coagulase-negative staphylococci increased from 0 to 22%, colonizing 43% of patients, whereas no amikacin-resistant S. aureus was detected. During the preamikacin survey, 68% of the coagulase-negative staphylococci and 12% of the S. aureus strains were resistant to tobramycin and gentamicin. This resistance did not decrease after amikacin was introduced. Initially, 83% of the aminoglycoside-resistant coagulase-negative staphylococci were resistant to both tobramycin and gentamicin. During the last surveillance this value dropped to 40%, and 48% of the strains had become resistant to all three aminoglycosides. Resistance to aminoglycosides, including amikacin, develops quickly in coagulase-negative staphylococci from clinical areas where these antimicrobial agents are widely used. However, aminoglycoside resistance in S. aureus is much less frequent.     

The point prevalence and associated factors of nasal methicillin-resistant Staphylococcus aureus colonisation in eight geriatric hospitals in Korea

The prevalence and associated factors of nasal methicillin-resistant Staphylococcus aureus (MRSA) colonisation were investigated among patients in geriatric hospitals in Korea. S. aureus was isolated from 317 (50.2%) of 632 patients. The nasal MRSA colonisation prevalence was 36.1%. In bivariate analysis, stay in an intensive care unit, decreased functional status, recent use of antibiotics, use of urinary catheters and the existence of skin breaks were associated with nasal MRSA colonisation (p < 0.05). Of these factors, only decreased functional status and recent use of systemic antibiotics were associated independently with nasal MRSA colonisation following logistic regression analysis.     

Colonization with vancomycin-intermediate Staphylococcus aureus strains containing the vanA resistance gene in a tertiary-care center in north India

A nasal carriage survey for methicillin-resistant Staphylococcus aureus (MRSA) in an intensive care unit detected four strains of MRSA with reduced susceptibility to vancomycin. The vanA gene was found in two of these vancomycin-intermediate Staphylococcus aureus (VISA) strains. The absence of selective vancomycin pressure might have resulted in reduced expression of the resistant gene.     

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.     

Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein-labeled PNA probe that targets a species-specific sequence of the 16S rRNA of S. aureus. Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible S. aureus species, coagulase-negative Staphylococcus species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the S. aureus-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of S. aureus directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy.     

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     

Quantitative detection of Staphylococcus aureus and Enterococcus faecalis DNA in blood to diagnose bacteremia in patients in the intensive care unit

Direct detection of bacterial DNA in blood offers a fast alternative to blood culture and is presumably unaffected by the prior use of antibiotics. We evaluated the performance of two real-time PCR assays for the quantitative detection of Staphylococcus aureus bacteremia and for Enterococcus faecalis bacteremia directly in blood samples, without prior cultivation. Whole-blood samples for PCR were obtained simultaneously with blood cultures from patients admitted to the intensive care unit of our hospital. After the extraction of DNA from 200 mul of blood, real-time PCR was performed for the specific detection and quantification of S. aureus and E. faecalis DNA. The sensitivity for bacteremia of the S. aureus PCR was 75% and that of the E. faecalis PCR was 73%, and both tests had high specificity values (93 and 96%, respectively). PCR amplification reactions were positive for S. aureus for 10 (7%) blood samples with negative blood cultures, and 7 (4%) PCR reactions were positive for E. faecalis. The majority of these PCR results were likely (50%) or possibly (42%) related to infection with the specific microorganism, based on clinical data and radiological and microbiological investigations. PCR results were concordant for 95% of paired whole-blood samples, and blood culture results were concordant for 97% of the paired samples. We conclude that the detection of S. aureus and E. faecalis DNA in blood by real-time PCR enables a rapid diagnosis of bacteremia and that a positive DNAemia is related to proven or possible infection with the specific microorganism in the majority of patients with negative blood cultures.     

Outbreak of Mupirocin-Resistant Staphylococci in a Hospital in Warsaw, Poland, Due to Plasmid Transmission and Clonal Spread of Several Strains

An outbreak of mupirocin-resistant (MuR) staphylococci was investigated in two wards of a large hospital in Warsaw, Poland. Fifty-three MuR isolates of Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. xylosus, and S. capitis were identified over a 17-month survey which was carried out after introduction of the drug for the treatment of skin infections. The isolates were collected from patients with infections, environmental samples, and carriers; they constituted 19.5% of all staphylococcal isolates identified in the two wards during that time. Almost all the MuR isolates were also resistant to methicillin (methicillin-resistant S. aureus and methicillin-resistant coagulase-negative staphylococci). Seven of the outbreak isolates expressed a low-level-resistance phenotype (MuL), whereas the remaining majority of isolates were found to be highly resistant to mupirocin (MuH). The mupA gene, responsible for the MuH phenotype, has been assigned to three different polymorphic loci among the strains in the collection analyzed. The predominant polymorph, polymorph I (characterized by a mupA-containing EcoRI DNA fragment of about 16 kb), was located on a specific plasmid which was widely distributed among the entire staphylococcal population. All MuR S. aureus isolates were found to represent a single epidemic strain, which was clonally disseminated in both wards. The S. epidermidis population was much more diverse; however, at least four clusters of closely related isolates were identified, which suggested that some strains of this species were also clonally spread in the hospital environment. Six isolates of S. epidermidis were demonstrated to express the MuL and MuH resistance mechanisms simultaneously, and this is the first identification of such dual MuR phenotype-bearing strains. The outbreak was attributed to a high level and inappropriate use of mupirocin, and as a result the dermatological formulation of the drug has been removed from the hospital formulary.     

MRSA-surveillance in Germany: data from the Antibiotic Resistance Surveillance System (ARS) and the mandatory surveillance of MRSA in blood

Data from the German Antibiotic Resistance Surveillance system (ARS) and statutory notification of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures are presented. ARS is a voluntary laboratory-based surveillance system providing resistance data of all clinical pathogens and sample types from hospitals and ambulatory care. Statutory notification includes MRSA detected in blood and cerebrospinal fluid by microbiological laboratories. Resistance data from 2008 to 2010 and MRSA-bacteraemia incidences from 2010 are presented. From 2008 to 2010, resistance data from 70,935 Staphylococcus aureus isolates were transferred to the national health institution. MRSA proportions in hospitals and outpatient care account for 19.2% and 10.6%, respectively. In hospital care high proportions of MRSA were found in nephrological, geriatric, neurological general wards and surgical ICUs (49.4%, 45.8%, 34.2%, and 27.0%, respectively), while in community outpatient care urological practices (29.2%) account for the highest values. In both healthcare settings urinary tract samples stand out with high proportions of MRSA (hospitals, 32.9%; outpatients, 20.5%). In 2010, 3900 cases of MRSA bacteraemia were reported, accounting for an incidence of MRSA bacteraemia of 4.8/100,000 inhabitants/year. Stratification by federal states shows considerable regional differences (range, 1.0-8.3/100,000 inhabitants/year). Vulnerable areas in hospitals and outpatient care have been pointed out as subjects for further inquiries.     

Resistance to desiccation and skin fatty acids in outbreak strains of methicillin-resistant Staphylococcus aureus.

Resistance to desiccation and to skin fatty acids was measured in three groups of methicillin-resistant Staphylococcus aureus (MRSA) strains and a group of control strains. Organisms from a large outbreak on a special care baby unit (SCBU), where MRSA had been isolated from staff hands but not from the environment, were significantly more sensitive to drying than strains from a burns unit where extensive environmental contamination had been demonstrated. MRSA from other wards, in the same hospital but not associated with large outbreaks, gave heterogeneous results. Fatty-acid resistance, determined by an agar dilution method, was not associated with strain origin. Some epidemic strains of MRSA were relatively sensitive to desiccation, and the abilities of such strains to spread widely on a SCBU by the hand-borne route could not be explained by enhanced resistance to skin fatty acids.     

Time-trends for Gram-negative and multidrug-resistant Gram-positive bacteria associated with nosocomial infections in German intensive care units between 2000 and 2005.

This study analysed the time-trends for bacteria associated with nosocomial lower respiratory tract infections (LRTIs), bloodstream infections (BSIs) and urinary tract infections (UTIs) that were reported to the German Nosocomial Infection Surveillance System for intensive care units (ICUs). Data concerning 19 822 nosocomial infections were submitted by 139 ICUs between 2000 and 2005. There was a significant increase in the proportion of Gram-negative bacteria causing LRTIs (from 63.9% to 68.4%) and UTIs (from 65.3% to 68.6%). The proportion of BSIs caused by Gram-negative bacteria declined significantly, from 36.4% to 22.7%. The frequency of methicillin-resistant Staphylococcus aureus among all S. aureus isolates increased from 19.8% to 37.2%.     

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.     

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.     

Long-term Staphylococcus aureus carrier state in hospital patients.

Staphylococcus aureus colonization of 326 predominantly chronic-care (long-term) patients was studied for 24 years. There were 5,827 upper respiratory cultures positive for S. aureus, ranging from 10 to 88 per patient, determined by the number of years (1 to 21; average, 4.6) that the patient was studied. Patients on the average carried 2.8 S. aureus strains. One patient carried eight strains. Each patient had a predominant strain. Predominant strains tended to be permanent, with 87% persistence over the studied years. Lytic group III strains were more than twice as frequent as group I strains and eight times as frequent as group II strains. Patient carriage of multiple S. aureus strains was usual. A collection of large numbers of cultures over an extended period was necessary for this statistical study, since cultures positive for predominant strains were interspersed with negative cultures and cultures positive for minor strains. Thus, persistence of carriage of a predominant S. aureus strain in a patient continues despite frequent negative cultures and cultures positive for minor strains.     

Molecular epidemiology of methicillin-resistant Staphylococcus aureus strains causing neonatal toxic shock syndrome-like exanthematous disease in neonatal and perinatal wards

Neonatal toxic shock syndrome-like exanthematous disease (NTED) is a new neonatal disease caused by toxic shock syndrome toxin 1 (TSST-1). We conducted a prospective surveillance study and characterized the methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from patients with NTED and compared them with the strains from patients with other MRSA infections and asymptomatic carriers. The study was performed in the neonatal intensive care unit and a general neonatal and maternal ward in the Tokyo Women's Medical University Hospital (TWMUH) from September to December 1998. Among 103 patients eligible for the study, MRSA was detected in 62 (60.2%) newborns; of these 62 newborns, 8 (12.9%) developed NTED, 1 (1.6%) had another MRSA infection, and 53 (85.5%) were asymptomatic MRSA carriers. Sixty-nine MRSA strains were obtained from the 62 newborns. DNA fingerprinting by pulsed-field gel electrophoresis showed two clusters: clone A with 8 subtypes and clone B. Sixty-seven of the 69 MRSA strains (97.1%) belonged to clone A, and type A1 was the most predominant (42 of 69 strains; 60.9%) in every neonatal and perinatal ward. All but one of the clone A strains had the TSST-1 and staphylococcal enterotoxin C genes. We also analyzed eight MRSA strains from eight NTED patients in five hospitals in Japan other than TWMUH. All the MRSA strains from NTED patients also belonged to clone A. These results suggest that a single clone that predominated in the neonatal wards of six hospitals might have caused NTED. However, the occurrence of NTED might not be dependent on the presence of an NTED-specific strain.     

Epidemiology of methicillin-resistance among Staphylococci strains isolated in risk units and effects of the vancomycin on the expression of methicillin-resistance

Four hundred Staphylococci strains, isolated from different in intensive care unit hospitalized patients, were analyzed. 53% of all strains were resistant to methicillin. Against methicillin-resistant S. aureus (MRSA), teicoplanin and vancomycin (100% of susceptibility), rifampin (76.3%) and co-trimoxazole (73%) emerged as the most potent drugs tested; the 15% of the strains were susceptible to ciprofloxacin, erythromycin, clindamycin and gentamicin. Only one MRSA strain (0.8%) resulted hetero-resistant to vancomycin. Among 100 strains exposed to serial concentration of vancomycin (0.25-32 mg/L for 30 days), 57 were selected with intermediate-level of resistance to the glycopeptides; the MRSA strains have shown to acquire resistance in vitro more easily than methicillin-susceptible. These results indicate that in the clones of Staphylococci circulating in our region, the evolution of glycopeptides-resistance is not a rapid process and the loss of effectiveness of these antibiotics cannot be predicted to short term. In particular, the restriction profile analysis of chromosomal DNA from MRSA strains, selected in vitro with intermediate -level of vancomycin resistance, demonstrated that at the moment in the hospital departments studied, the diffusion of a clone able to acquire resistance more easily than others is not present.     

Early Screening of oxacillin-resistant Staphylococcus aureus and Staphylococcus epidermidis from blood culture

The timely detection of blood-borne pathogens is one of the most important functions of the microbiology laboratory. Recently, methicillin-resistant staphylococci have become the most important pathogens seen by the laboratory. The purpose of this study was to evaluate Staphy agar, a novel screening medium, for the detection methicillin-resistant Staphylococcus aureus, S. epidermidis, or other coagulase-negative staphylococci (CNS) from positive blood cultures showing Gram-positive cocci in clusters. Eighty-six blood cultures that yielded Gram-positive cocci in clusters were included in this study. The organisms were finally identified by the Vitek system, and oxacillin resistance was confirmed by polymerase chain reaction (PCR)-based mecA gene detection. The identification and oxacillin resistance of all S. aureus strains showed complete agreement with the Vitek and PCR results. The presumptive detection of S. epidermidis and other CNS were consistent with the Vitek system in 94.7%, and the screening of oxacillin resistance was consistent with the result of PCR in 92.1% of 38 strains. The Staphy agar method is reliable and rapid for differentiating Gram-positive cocci in clusters in blood and for determining their methicillin resistance.