A probe for the detection of methicillin-resistantStaphylococcus aureus

An approximately 300 base pair DNA fragment for use as a probe was isolated from methicillin-resistantStaphylococcus aureus DNA partially digested withSau3AI. This probe hybridized with 25 methicillin-resistant clinical isolates ofStaphylococcus aureus belonging to 18 different phage types, but not with 41 clinical isolates susceptible to methicillin.     

Strategies for typing and properties of epidemic methicillin-resistantStaphylococcus aureus

Isolates of 17 strains of epidemic methicillin-resistant Staphylococcus aureus from outbreaks in ten hospitals in the UK were investigated with a variety of techniques both to explore their properties and to type them in order to confirm or refute known or suspected epidemiology. The techniques consisted of a biotyping system, peptidogylcan analysis, testing of antibiotic sensitivity to 21 agents, various phage-typing methods including heat shock, plasmid pattern analysis, and heat cure derivation of plasmid-less isogenic strains. All strains resembled those originally isolated in Australia, being in the possession of a large number of chromosomal resistance factors, pigmentation, ability to produce lipase and large molecular weight plasmids (c.15 Md to c.23 Md) which conferred resistance to gentamicin, propamidine, ethidium bromide, cetrimide and chlorhexidine. Some strains also had a c.3 Md plasmid conferring chloramphenicol resistance and others a c.1 Md cryptic plasmid. A large percentage of the population was resistant to 25 mg/l methicillin at 37 °C, an unusual feature. All the strategies, with the exception of peptidoglycan analysis, contributed to typing of the strains.     

Latex agglutination for rapid identification of methicillin-resistantStaphylococcus aureus recovered from selective media

The accuracy of combining latex agglutination with selective media for the identification of methicillin-resistantStaphylococcus aureus (MRSA) was determined. Test strains were identified by latex agglutination on blood agar, the heat-stable thermonuclease test and broth microdilution MICs of oxacillin and included 97 MRSA, 56 methicillin-susceptibleStaphylococcus aureus, 52 methicillin resistant, and 49 methicillin-susceptibleStaphylococcus species. Isolates were grown on trypticase-soy agar with 5 % sheep red blood cells (TSAB), Mueller-Hinton agar (MHA), mannitol-salt agar (MSA), and four media designed for the selective growth of MRSA: TSAB with clindamycin and gentamicin, MHA with oxacillin, MSA with oxacillin, and lipovitellin-salt-mannitol agar (LVSM) with 1 µg oxacillin disks applied. The mean sensitivity, specificity, and positive predictive value for the combination of latex agglutination with selective media for the identification of MRSA was 96 %, 99 % and 98 % respectively.     

Comparison of screening methods to identify methicillin-resistantStaphylococcus aureus

Screening methods to identify methicillin-resistantStaphylococcus aureus (MRSA) were compared using 96 isolates representing 17 distinct clones. The sensitivity of four commercial agglutination tests was determined in comparison to the tube coagulation test, and the results related to the presence of the coagulase gene. The broth screening test, agar dilution test and disc diffusion test were carried out, and the results related to the presence of themecA gene. Mannitol salt agar and Iso-Sensitest agar with varying salt supplements were used. All agglutination tests had high rates of detection ofStaphylococcus aureus (95.8–99.0%). Resistance in mecA gene-positiveStaphylococcus aureus isolates was correctly detected by the oxacillin broth test, the agar dilution test and the disc diffusion test on mannitol salt agar, whereas on Iso-Sensitest agar detection rates were lower (between 68.5% and 94.4%, depending on the salt supplement). Incubation of the Iso-Sensitest plates for 48 hours significantly improved the rate of detection of resistance, but increased the major error rate up to 71.4%.MecA genepositiveStaphylococcus aureus isolates not detected by the disc diffusion test on Iso-Sensitest agar had significantly lower oxacillin minimal inhibitory concentration values and were significantly less resistant to a variety of antibiotics. Thus, mannitol salt agar might be a suitable medium for use in the disc diffusion and agar dilution test to detect resistance to oxacillin inStaphylococcus aureus.     

Control of methicillin-resistantStaphylococcus aureus bacteraemia in high-risk areas

In a 3,000-bed tertiary care hospital, 88 cases of methicillin-resistantStaphylococcus aureus (MRSA) bacteraemia were identified from 22,383 blood cultures (0.39 %) submitted to the microbiology laboratory over a one-year period. Two high-risk areas were identified: the paediatric oncology unit, in which 12 cases of MRSA bacteraemia were identified from 924 blood cultures (1.3 %), and the intensive care unit (ICU), in which 14 cases of MRSA bacteraemia were identified from 1,391 blood cultures (1.0 %). In a one-year targeted intervention programme in which staff and patients were screened for MRSA carriage, patient carriers isolated, and mupirocin and chlorhexidine treatment administered, the number of MRSA bacteraemia cases decreased in these areas to 0 and 4, respectively (p=0.000123 and 0.016), while the incidence of MRSA bacteraemia in non-targeted areas increased from 62 of 20,068 blood cultures (0.3 %) to 82 of 18,784 blood cultures (0.44 %) (p=0.047). In the year post intervention the incidence of MRSA bacteraemia increased to 3 of 815 cultures (0.37 %) in the paediatric oncology unit, 10 of 1,934 cultures (0.5 %) in the ICU, and 112 of 18,977 cultures (0.59 %) in the rest of the hospital (p=0.00004 versus preintervention period). This study demonstrates the efficacy of targeted MRSA control measures in a hospital in which MRSA is endemic.     

Diversity of methicillin-resistantStaphylococcus aureus isolated in a Canadian hospital

Three neonates and three other patients located elsewhere in the hospital became infected withStaphylococcus aureus. Initial automated microdilution susceptibility testing with oxacillin and disk diffusion testing with amoxicillin-clavulanic acid indicated the isolates had borderline oxacillin resistance (MICs 4 µg/ml), presumably due to hyperproduction of -lactamase. Chromosomal DNA restriction fingerprinting and phage typing revealed the neonatal isolates to be identical; whereas, the other patients were infected with three different strains. Further analysis of the four strains by Southern hybridization with amecA specific oligoprobe and a quantitative -lactamase assay demonstrated that two strains carried themecA gene (coding for low affinity penicillin-binding protein 2a), and two strains were hyperproducers of -lactamase, including one which wasmecA gene positive. One strain neither carried themecA gene nor hyperproduced -lactamase. The twomecA gene positive strains displayed oxacillin MICs of 16 µg/ml on dilution susceptibility testing in 4% NaCl supplemented Mueller-Hinton agar. Hence, they were considered intrinsically methicillin-resistantStaphylococcus aureus. Both oxacillin and amoxicillin-clavulanic acid MICs were increased on NaCI supplementation. Results of amoxicillin-clavulanic acid disk diffusion susceptibility testing did not correlate with quantitative -lactamase production. It is recommended that clinical laboratories do not use amoxicillin-clavulanic acid disk diffusion assays to differentiate suspected borderline resistance due to -lactamase hyperproduction frommecA gene expression of PBP-2a since additional mechanisms may account for resistance.     

National survey of methicillin-resistantStaphylococcus aureus in Belgian hospitals: Detection methods,prevalence trends and infection control measures

A questionnaire survey of Belgian acute care hospitals was conducted to determine the methods used for detection of methicillin-resistantStaphylococcus aureus (MRSA), to estimate the prevalence of this organism during the period 1989–1991 and to describe the infection control measures used locally for limiting its spread. Questionnaires were returned by 144 acute care hospitals, with a coverage of 41 to 72 % of hospitals by province. Methods used for detection of MRSA included disk diffusion (91 %), microdilution panels (8 %) and oxacillin agar screen (9 %). Only 34 % of laboratories performed disk diffusion testing under optimal conditions for detection of heterogeneous resistance. Among 36 hospitals reporting complete susceptibility data ofStaphylococcus aureus isolates tested during the study period (n=24,153), a mean MRSA prevalence of 14 % was found (range: 0–70 %). The median prevalence increased from 9.5 % in 1989 to 13.7 % in 1991 and showed a significant linear increase during this period in 30 % of these hospitals (p<0.01). Precautions used for controlling spread of MRSA included hand decontamination using either soap and water or antimicrobial preparations (68 % of hospitals), room decontamination (62 %), patient isolation (55 %) and various barrier precautions (24–49 %). Carrier screening was performed in 37 % of hospitals, but antibiotic decolonization was attempted in only 24 %. This survey identified areas for improvement in MRSA detection methods and underscored the need for multicentric surveillance of MRSA prevalence and a reappraisal of MRSA control strategies in Belgian hospitals.     

Risk factors for nosocomial bacteremia due to methicillin-resistantStaphylococcus aureus

In a prospective surveillance study (February 1990–December 1991) performed at a 1000-bed teaching hospital to identify risk factors for nosocomial methicillin-resistantStaphylococcus aureus (MRSA) bacteremia, 309 patients were found to be colonized (n=103; 33 %) or infected (n=206; 67 %) by MRSA. Sixty-three of them developed bacteremia. Compared with 114 patients who had nosocomial bacteremia caused by methicillin-sensitiveStaphylococcus aureus during the same period of time, MRSA bacteremic patients had more severe underlying diseases (p<0.01), were more often in intensive care units (p<0.01) and had received prior antibiotic therapy more frequently (p<0.01). To further identify risk factors for MRSA bacteremia, univariate and multivariate analyses of this series of 309 patients were performed using the occurrence of MRSA bacteremia as the dependent variable. Among 14 variables analyzed, intravascular catheterization, defined as one or more intravascular catheters in place for more than 48 h, was the only variable selected by a logistic regression model as an independent risk factor (OR=2.7, CI=1.1–6.6). The results of this study reinforce the concept that recent antibiotic therapy may predispose patients to MRSA infection and suggest that among patients colonized or infected by MRSA, those with intravascular catheters are at high risk of developing MRSA bacteremia.     

Distinct genotypic clusters of heterogeneously and homogeneously methicillin-resistantStaphylococcus aureus from a Greek hospital

Methicillin-resistantStaphylococcus aureus (MRSA) strains isolated over a one-year period from a Greek hospital were tested for their levels of resistance to methicillin by population analysis. Heterogeneously resistant strains belonged to classes I, II, and II/III, whereas homogeneously resistant ones belonged to class IV. Strains of all classes possessed the mecA gene. Pulsed-field gel electrophoresis (PFGE) of Smal-digested genomic DNA revealed that all heterogeneously resistant strains were related. Homogeneously resistant strains were also closely related, but in a cluster distinct from the heterogeneous one. The methicillin-sensitive strains displayed a greater variety of PFGE types compared to MRSA isolates.     

Long-term efficacy of a program to control methicillin-resistantStaphylococcus aureus

The long-term efficacy of a program to control methicillin-resistantStaphylococcus aureus (MRSA) was evaluated in a 350-bed university hospital. Three periods were monitored: pre-epidemic (January 1989–November 1989), outbreak (December 1989–June 1990) and control program (July 1990–December 1992) periods. Control measures included cohort isolation, patient care measures and therapy (oral cotrimoxazole plus fusidic acid ointment) of MRSA carriage in patients, roommates and personnel. A total of 117 MRSA-infected patients were detected. For each period respectively, MRSA incidence (number of cases per 1,000 patient-days) was 3.2, 8.2 and 2.0 in the intensive care unit (ICU) and 0.08, 0.23 and 0.26 in the general wards. During the outbreak there was a 2.7-fold overall increase of baseline MRSA incidence (p<0.02). The crude mortality was 68 % and the attributable mortality was estimated to be 50 %. The program was estimated to have prevented 76 % (CI95 28–91, p<0.0001) of expected MRSA cases and 85 % (CI95 62–94, p<0.0001) of expected fatalities due to MRSA in the ICU, but it had no significant effect in the general wards. The program did not control vancomycin consumption.     

Genetic lineages of community-associated methicillin-resistant Staphylococcus aureus in Kuwait hospitals

Twenty-six community-associated methicillin-resistant Staphylococcus aureus (CAMSRA) isolates were characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and screened for accessory gene regulator (agr), capsular polysaccharide (cap), and Panton-Valentine leucocidin (PVL) genes. They exhibited five PFGE patterns (types A to E). The majority were PFGE type A (12 isolates) or type B (8 isolates). MLST showed that PFGE type A isolates belonged to sequence type 80 (ST80), while the PFGE type B isolates were ST30. The ST80 and ST30 clones contained agr allotype 3, cap type 8, and PVL. The results showed that two internationally recognized CAMRSA clones are dominant in Kuwait hospitals.     

Control of epidemic methicillin-resistantStaphylococcus aureus in a Dutch University Hospital

Between 1986 and 1989 a single strain of a methicillin- and multiply-resistantStaphylococcus aureus caused three distinct outbreaks at Utrecht University Hospital, involving 11, 19 and 32 patients, respectively. In all three episodes, members of staff were screened for MRSA carriage, and 58 persons were found to have positive nose cultures. In each outbreak it became necessary to isolate colonized and infected patients on a separate isolation ward. Staff carriers were also treated. Over the 18 months since the last outbreak, no new acquisitions of this epidemic MRSA strain have occurred. Between 1986 and 1989, the strain which caused the three outbreaks was not the only MRSA strain which was introduced into the hospital. Six other strains, which differed from the epidemic strain as shown by phage typing and antimicrobial susceptibility pattern, were found in single patients. The experience at Utrecht University Hospital illustrates the need for strict measures to eradicate epidemic strains of MRSA as well as the differences in epidemicity among various strains of MRSA.     

Laboratory and epidemiologic experience with methicillin-resistantStaphylococcus aureus in the USA

Methicillin-resistant Staphylococcus aureus (MRSA) was a rare occurrence in US hospitals until the mid-1970s. Since that time outbreaks of MRSA infection have been reported in both large and small hospitals, in rehabilitation facilities, and in nursing homes. Transmission has been documented not only between hospitals, but between long-term care facilities and hospitals, and between the community and hospitals. Patient-to-patient spread within hospitals appears to result from transient colonization of the hands of health care workers, with colonized or infected patients being the intrahospital reservoir for the organisms. The best opportunity for control of outbreaks of MRSA infection within hospitals may depend on the rapid recognition of newly admitted patients who are colonized or infected. The laboratory plays a crucial role in this by providing prompt and accurate information indicating the presence of MRSA. Susceptibility test methods found to be most reliable for detecting MRSA in the USA include the broth microdilution MIC determination (performed in salt-supplemented broth), the Bauer-Kirby test with slight modification, or oxacillin-salt agar screening plates.     

Clonal heterogeneity, distribution, and pathogenicity of methicillin-resistantStaphylococcus aureus

Four thousand eighty-eightStaphylococcus aureus isolates obtained from patients hospitalised in a university clinic and four community hospitals over a period of one year were screened for methicillin resistance. A resistance rate of 5% was detected among initial isolates. Distribution of methicillin-resistantStaphylococcus aureus (MRSA) and methicillin-sensitiveStaphylococcus aureus showed an increased prevalence of MRSA in clinically significant specimens such as blood, central venous catheter tips, bronchial secretions, and wound secretions. Typing of 110 MRSA strains (initial isolates) by macrorestriction analysis of chromosomal DNA revealed 26 different genotypes that could be divided into five epidemic and 21 sporadic strains. More than 50% of all isolates belonged to one type that was confirmed to be closely related to the southern-German epidemic strain. Production of virulence factors such as enterotoxin A-D and toxic shock syndrome-toxin 1 among MRSA strains (initial isolates) occurred in ten of 26 different MRSA types. A strong correlation between genotype and toxin production was demonstrated.     

Clinical and epidemiological findings in mechanically-ventilated patients with methicillin-resistantStaphylococcus aureus pneumonia

Over the 5-year period from 1990 to 1994, a prospective cohort study was conducted to define the clinical and epidemiological characteristics of ventilator-associated methicillin-resistantStaphylococcus aureus (MRSA) pneumonia acquired during a large-scale outbreak of MRSA infection. Of 2411 mechanically ventilated patients, 347 (14.4%) acquired MRSA, 220 (63.4%) had MRSA positive respiratory tract samples and 41 (18.6%) developed ventilator-associated MRSA pneumonia. The overall attack rate for ventilator-associated MRSA pneumonia was 1.56 episodes/1000 ventilator days, but annual attack rates varied according to the trend of the outbreak (range 4.9–0.2). In comparison with methicillin-sensitiveStaphylococcus aureus (MSSA), which was implicated in 98 episodes of ventilator-associated pneumonia, MRSA caused exclusively late-onset ventilator-associated pneumonia, while MSSA caused both early-onset [55 of 98 (56.1%) episodes] and late-onset [43 of 98 (43.8%) episodes] ventilator-associated pneumonia. Logistic regression analysis of all patients withStaphylococcus aureus pneumonia revealed intubation for more than 3 days (odds ratio (OR),1.11; confidence interval (CI):1.03–1.18) and prior bronchoscopy (OR,5.8; CI,1.85–18.19) to be independent variables associated with MRSA pneumonia. The results indicate that MRSA ventilator-associated pneumonia is a frequent complication in intensive care patients, manifesting itself as late-onset pneumonia in patients who have been intubated for prolonged periods and/or have often undergoing previous bronchoscopy.