Controlled performance evaluation of the DiversiLab repetitive-sequence-based genotyping system for typing multidrug-resistant health care-associated bacterial pathogens.

Fast, reliable, and versatile typing tools are essential to differentiate among related bacterial strains for epidemiological investigation and surveillance of health care-associated infection with multidrug-resistant (MDR) pathogens. The DiversiLab (DL) system is a semiautomated repetitive-sequence-based PCR system designed for rapid genotyping. The DL system performance was assessed by comparing its reproducibility, typeability, discriminatory power, and concordance with those of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and by assessing its epidemiological concordance on well-characterized MDR bacterial strains (n = 165). These included vanA Enterococcus faecium, extended-spectrum ß-lactamase (ESBL)-producing strains of Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii, and ESBL- or metallo-ß-lactamase (MBL)-producing Pseudomonas aeruginosa strains. The DL system showed very good performance for E. faecium and K. pneumoniae and good performance for other species, except for a discrimination index of <95% for A. baumannii and E. coli (93.9% and 93.5%, respectively) and incomplete concordance with MLST for P. aeruginosa (78.6%) and E. coli (97.0%). Occasional violations of MLST assignment by DL types were noted for E. coli. Complete epidemiological concordance was observed for all pathogens, as all outbreak-associated strains clustered in identical DL types that were distinct from those of unrelated strains. In conclusion, the DL system showed good to excellent performance, making it a reliable typing tool for investigation of outbreaks caused by study pathogens, even though it was generally less discriminating than PFGE analysis. For E. coli and P. aeruginosa, MLST cannot be reliably inferred from DL type due to phylogenetic group violation or discordance.

Epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) among residents of nursing homes in Belgium.

OBJECTIVES: A national survey was conducted to determine the prevalence, risk factors and molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) carriage among nursing home (NH) residents in Belgium. METHODS: A random stratified, cross-sectional prevalence survey was conducted in NH residents who were screened for MRSA carriage by multisite enriched culture. Characteristics of NHs and residents were collected by a questionnaire survey and analysed by two-stage logistic regression modelling. MRSA isolates were genotyped by PFGE, staphylococcal cassette chromosome mec (SCCmec) typing, multilocus sequence typing (MLST) and resistance genes. RESULTS: Of 2953 residents screened in 60 NHs, 587 (19.9%) were MRSA carriers. Risk factors included hospital contact, antibiotic exposure, impaired mobility and skin lesions at the resident level, and lack of MRSA surveillance, lack of antibiotic therapeutic formulary and the combination of less-developed infection control activities and a high ratio of physicians to residents at the institution level. MRSA isolates showed eight major types, three of which were predominant: B2-ST45-SCCmec IV (49%; where ST stands for sequence type); A21-ST8-SCCmec IV (13%); and A20-ST8-SCCmec IV (10%). Each was recovered in 55, 21 and 25 NHs, respectively. The geographical distribution of NH genotypes paralleled that of acute-care hospitals. CONCLUSIONS: A high prevalence of MRSA carriage in NH residents was associated with hospital care, co-morbidities and less-developed coordination of institutional care. The predominant MRSA strains from NH residents and hospitalized patients of the same area were identical. Strengthening and coordination of MRSA surveillance and control activities are warranted within and between NHs and hospitals.

In vitro activities of ceftobiprole, tigecycline, daptomycin, and 19 other antimicrobials against methicillin-resistant Staphylococcus aureus strains from a national survey of Belgian hospitals.

The in vitro activities of 22 antimicrobial agents, including ceftobiprole, daptomycin, and tigecycline, against 511 methicillin-resistant Staphylococcus aureus (MRSA) isolates from 112 Belgian hospitals were studied by using the CLSI agar dilution method. Isolates were characterized by pulsed-field gel electrophoresis (PFGE) analysis and by PCR detection of determinants of resistance to aminoglycosides, macrolides-lincosamides-streptogramins, and tetracyclines. A representative set of isolates with different PFGE genotypes was further characterized by multilocus sequence typing, determination of staphylococcal cassette chromosome mec (SCCmec) type, and multiplex PCR for toxic shock syndrome type 1 (TSST-1) and Panton-Valentine leukocidin genes. MRSA isolates belonged to nine epidemic MRSA clones, of which sequence type 45 (ST45)-SCCmec IV and ST8-SCCmec IV were predominant, accounting for 49 and 20% of isolates, respectively. The distribution of antimicrobial resistance and TSST-1 genes was strongly linked to clonal types. Ceftobiprole, daptomycin, and tigecycline showed high activity against all isolates of these sporadic and epidemic MRSA clones, as indicated by MIC(90)s of 2 mg/liter, 0.5 mg/liter, and 0.25 mg/liter, respectively. The MIC distribution of daptomycin and tigecycline was not different in isolates with decreased susceptibility to glycopeptides or tetracyclines, respectively. Ceftobiprole MICs were not correlated with oxacillin and cefoxitin MICs. These data indicate excellent activity of the newly developed agents ceftobiprole, daptomycin, and tigecycline against MRSA isolates recently recovered from hospitalized patients in Belgium, supporting their therapeutic potential for nosocomial MRSA infections.

National surveillance of methicillin-resistant Staphylococcus aureus in Belgian hospitals indicates rapid diversification of epidemic clones.

Methicillin-resistant Staphylococcus aureus (MRSA) strains (n = 455) collected in 2001 from 100 Belgian hospitals were characterized by molecular typing and by resistance gene distribution to macrolides-lincosamides-streptogramins and to aminoglycoside antibiotics. Rapid diversification of MRSA clones, compared with results of previous surveys, was evidenced by the broad geographic distribution of seven major clones belonging to the pandemic MRSA clonal complexes 5, 8, 22, 30, and 45 by multilocus sequence typing.

Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains.

Pulsed-fieldgel electrophoresis (PFGE) is the most common genotypic method used in reference and clinical laboratories for typing methicillin-resistant Staphylococcus aureus (MRSA). Many different protocols have been developed in laboratories that have extensive experience with the technique and have established national databases. However, the comparabilities of the different European PFGE protocols for MRSA and of the various national MRSA clones themselves had not been addressed until now. This multinational European Union (EU) project has established for the first time a European database of representative epidemic MRSA (EMRSA) strains and has compared them by using a new "harmonized" PFGE protocol developed by a consensus approach that has demonstrated sufficient reproducibility to allow the successful comparison of pulsed-field gels between laboratories and the tracking of strains around the EU. In-house protocols from 10 laboratories in eight European countries were compared by each center with a "gold standard" or initial harmonized protocol in which many of the parameters had been standardized. The group found that it was not important to standardize some elements of the protocol, such as the type of agarose, DNA block preparation, and plug digestion. Other elements were shown to be critical, namely, a standard gel volume and concentration of agarose, the DNA concentration in the plug, the ionic strength and volume of running buffer used, the running temperature, the voltage, and the switching times of electrophoresis. A new harmonized protocol was agreed on, further modified in a pilot study in two laboratories, and finally tested by all others. Seven laboratories' gels were found to be of sufficiently good quality to allow comparison of the strains by using a computer software program, while two gels could not be analyzed because of inadequate destaining and DNA overloading. Good-quality gels and inclusion of an internal quality control strain are essential before attempting intercenter PFGE comparisons. A number of clonally related strains have been shown to be present in multiple countries throughout Europe. The well-known Iberian clone has been demonstrated in Belgium, Finland, France, Germany, and Spain (and from the wider HARMONY collection in Portugal, Slovenia, and Sweden). Strains from the United Kingdom (EMRSA-15 and -16) have been identified in several othercountries, and other clonally related strains have also been identified. This highlights the need for closer international collaboration to monitor the spread of current epidemic strains as well as the emergence of new ones.

Emergence and spread of gentamicin-susceptible strains of methicillin-resistant Staphylococcus aureus in Belgian hospitals.

The aim of this study was to follow the evolution of the clonal distribution and antimicrobial susceptibility of clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) recovered from Belgian hospitals between 1995 and 1997-1998. MRSA strains were genotyped by inter-IS256 spacer length polymorphism PCR and SmaI macrorestriction analysis. MICs of 18 antimicrobials were determined by the agar dilution method. MRSA strains from the 1997-1998 survey were further tested by vancomycin screen agar, E-test, broth microdilution methods, and population analysis. Between 1995 and 1997-1998, epidemic group A strains decreased in proportion from 73% to 44%, whereas MRSA Group B and C strains increased from 17% to 38% and from 5% to 8%. The proportion of strains susceptible to gentamicin increased between the surveys from 22% to 48%. This was associated with a higher proportion of group B and C strains in the last survey. Heterogeneous vancomycin-intermediate S. aureus (hetero-VISA) strains were found in 2% isolates from 1997 to 1998. These hetero-VISA isolates were genotypically related to the MRSA group A strains and were resistant to gentamicin. In conclusion, two emerging epidemic MRSA genotypes, susceptible to gentamicin, have spread among Belgian hospitals during the 1990s. Hetero-VISA were present at low frequency among MRSA strains belonging to a widespread endemic genotype.