Community-acquired methicillin-resistant Staphylococcus aureus clones circulating in Belgium from 2005 to 2009: changing epidemiology.

The present study reports the evolution of the demographic characteristics and the molecular epidemiology of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) in Belgium from 2005 to 2009. Four hundred and ten CA-MRSA isolates were prospectively collected and screened for the presence of Panton-Valentin leucocidin (PVL) and toxic shock syndrome toxin 1 (TSST-1) encoding genes, while clinical information were recorded. PVL- and TSST-1-positive isolates were genotyped by pulsed-field gel electrophoresis (PFGE). Staphylococcal cassette chromosome mec (SCCmec) type, spa type and multilocus sequence type (MLST) were determined on representative isolates. One hundred and fifty-nine (39 %) isolates were PVL-positive. PVL-positive isolates were significantly more frequently isolated from skin or soft tissue than PVL-negative isolates, causing mainly subcutaneous abscesses and furuncles. Patients with PVL-positive CA-MRSA were significantly younger than patients with PVL-negative CA-MRSA. Eighty-seven percent of the PVL-positive isolates belonged to a limited number (n = 7) of PFGE types belonging to sequence types (ST) ST80, ST8, ST30, ST5, ST152, ST338 and a new ST, a single-locus variant of ST1. A temporal evolution of the distribution of these PFGE types was observed, characterised by (1) the dissemination of the ST8-SCCmecIV arcA-positive (USA300) genotype and (2) a genetic diversification. Forty-seven (11 %) strains were TSST-1-positive, of which 65 % clustered into four PFGE types, all belonging to ST5. The epidemiology of CA-MRSA in Belgium is changing, as the rapid diffusion of the USA300 clone seems to occur, together with a clonal diversification.

Pseudo-outbreak of extremely drug-resistant pseudomonas aeruginosa urinary tract infections due to contamination of an automated urine analyzer.

By the end of May 2010, an increase in the number of urine specimens that were culture positive for extremely drug-resistant (XDR) Pseudomonas aeruginosa was observed in our 800-bed university hospital. This led to an infection control alert. No epidemiological link between the patients and no increase in the frequency of XDR P. aeruginosa in non-urine samples were observed. Therefore, a pseudo-outbreak due to analytical contamination in the laboratory was rapidly suspected. A prospective and retrospective search of cases was initiated, and the sampling of the automated urine analyzers used in the laboratory was performed. Antibiotypes were determined by disc diffusion, and genotypes were determined by pulsed-field gel electrophoresis (PFGE). From February to July 2010, 17 patients admitted to 12 different departments and 6 outpatients were included. The mixing device of the cytometric analyzer used for the numeration of urinary particles (Sysmex UF1000i) proved to be heavily contaminated. Isolates recovered from 12 patients belonged to the same antibiotype and PFGE type as the isolate recovered from the analyzer. Extensive disinfection with a broad-spectrum disinfectant and the replacement of the entire tubing was necessary to achieve the complete negativity of culture samples taken from the analyzer. A pseudo-outbreak caused by an XDR P. aeruginosa clone was proven to be due to the contamination of the cytometric analyzer for urinary sediment. Users of such analyzers should be aware that contamination can occur and should always perform culture either before the processing of the urine sample on the analyzer or on a distinct sample tube.

Evolutionary relationships between sporadic and epidemic strains of healthcare-associated methicillin-resistant Staphylococcus aureus.

National surveillance of healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) isolates by pulsed-field gel electrophoresis (PFGE) typing allowed identification of rarely occurring 'sporadic' isolates with patterns significantly distinct from those of major epidemic clones of methicillin-resistant S. aureus (MRSA) circulating in Belgian hospitals. The aim of the present study was to compare the genetic background, antibiotic susceptibility profile and in vitro growth rates of 36 MRSA isolates with either 'epidemic' or 'sporadic' PFGE profiles to identify factors that could be involved in the epidemic behaviour of S. aureus. Sequence analysis of seven housekeeping genes (multilocus sequence typing) and seven surface-associated genes, combined with staphylococcal cassette chromosome mec (SCCmec) typing and spa typing results, segregated sporadic isolates into four groups: (1) isolates phylogenetically distant from epidemic HA-MRSA clones that possessed several properties of community-acquired MRSA strains; (2) isolates derived from the same methicillin-susceptible S. aureus ancestor as epidemic isolates but possessing a distinct type of SCCmec; and (3) and (4) isolates that were closely related to epidemic strains, either as recent descendants of these or as intermediate evolutionary steps between epidemic HA-MRSA strains and their putative ancestors. Sporadic isolates did not show slower growth in vitro than epidemic isolates. These findings suggest that the SCCmec type and insertion/deletion of other mobile genetic elements may be involved in modulating the epidemic behaviour of MRSA strains of similar genetic background, independently of fitness cost.

Genetic relatedness between methicillin-susceptible and methicillin-resistant Staphylococcus aureus: results of a national survey.

OBJECTIVES: Surveillance of hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) infections has shown the emergence and spread of several epidemic MRSA clones over the past 10 years in Belgium. Whether these clones have been imported from abroad or else have arisen locally via staphylococcal cassette chromosome mec (SCCmec) acquisition by successful methicillin-susceptible S. aureus (MSSA) clones is unknown. METHODS: We determined by PFGE, spa typing, multi-locus sequence typing (MLST) and agr group analysis the genetic relatedness of 103 MSSA and 511 MRSA strains from a national survey of patients admitted to 112 Belgian hospitals in 2003. RESULTS: The 103 MSSA strains presented very diverse genetic backgrounds, they were distributed into 40 distinct PFGE types and clustered in 15 distinct MLST CCs. Up to 45% harboured the same genotype as five major epidemic HA-MRSA clones. These MRSA clones all harbour a type IV SCCmec element. CONCLUSIONS: These findings are consistent with multiple recent acquisitions of the more mobile type IV SCCmec by MSSA and suggest that certain genetic backgrounds are conferring a selective advantage, regardless of the resistance profile. However, since the predominant MSSA and MRSA lineages identified in Belgium are disseminated worldwide, importation of epidemic MRSA strains remains an alternative hypothesis.

Validation of pulsed-field gel electrophoresis and spa typing for long-term, nationwide epidemiological surveillance studies of Staphylococcus aureus infections.

Pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments has been used by the Belgian Reference Laboratory for Staphylococci for national hospital surveys of methicillin-resistant Staphylococcus aureus since 1992. The sequencing of the polymorphic X region of the protein A gene (spa typing) offers significant advantages over PFGE in terms of speed, ease of interpretation, and exportability. To validate its potential use for national surveillance, we evaluated the robustness of spa typing compared with that of PFGE based on a collection of 217 S. aureus strains representative of the Belgian S. aureus epidemiology during the last 13 years. spa typing and PFGE both showed high discriminatory power (discriminatory indexes of 0.98 and 0.96, respectively) and achieved high concordance (95.9%) in type classification. Both methods also showed good concordance with multilocus sequence typing (MLST) (95.5%). However, we observed occasional "violations" of MLST clonal complex assignment by spa typing. Our results suggest that both PFGE and spa typing are reliable methods for long-term, nationwide epidemiological surveillance studies. We suggest that spa typing, which is a single-locus-based method, should preferably be used in combination with additional markers, such as staphylococcal cassette chromosome mec typing or resistance or virulence gene detection.

External quality assessment of molecular typing of Staphylococcus aureus isolates by a network of laboratories.

A network of laboratories designated Centres for Molecular Diagnosis was funded in 2000 by Belgian National Health Insurance to provide clinically relevant molecular diagnostic tests. These included typing of nosocomial pathogens as a service to local hospital infection control programs. Two external quality assessment (EQA) surveys were performed in 2001 and 2003 to evaluate the proficiencies of the laboratories at Staphylococcus aureus typing. EQA panels included S. aureus isolates with either indistinguishable, clonally related, or unrelated pulsed-field gel electrophoresis (PFGE) patterns. A hypothetical hospital outbreak problem was also submitted for analysis. Typeability, reproducibility, discrimination (D) index, and epidemiological concordance were evaluated. Ten centers participated in each survey. Seven centers performed PFGE analysis, while others used repetitive-element or randomly amplified polymorphic DNA PCR, amplified fragment length polymorphism, or spa typing. Full typeability (100%) was achieved by all centers, and all but one showed 100% reproducibility. Discrimination was appropriate (D index, >or=96%) for centers performing PFGE analysis but not for all those using other methods (D index range, 72% to 97%). Correct answers to the epidemiological questions were provided by 7/10 and 10/10 centers in 2001 and 2003, respectively. Individual feedback of results was provided to each center together with specific technical recommendations for improving performance. Our findings indicate that surveys of lab proficiency are useful for validation and optimization of molecular typing services to local hospital infection control programs.

New phage type among methicillin-resistant Staphylococcus aureus associated with a local outbreak in Belgium during 2002.

In total, 150 methicillin-resistant Staphylococcus aureus (MRSA) isolates collected during 2002 from a general Belgian hospital were phage-typed at routine test dilution x 100. The majority (45%) belonged to phage group (J)*, while 10% were classified as a new phage type 29/(42E)/54/(D11)*. The isolates belonging to this new type carried the aac(6')-aph(2'') and the aph(3') aminoglycoside resistance genes and showed high-level resistance to oxacillin. Molecular typing revealed that they belonged to the multiresistant clonal pulsed-field gel electrophoresis (PFGE) type D8. PFGE group D, characterised as genotype ST228-MRSA-I, has been present in Belgian hospitals since 1999.

Epidemiological validation of pulsed-field gel electrophoresis patterns for methicillin-resistant Staphylococcus aureus.

To determine the stability of pulsed-field gel electrophoresis (PFGE) patterns of methicillin-resistant Staphylococcus aureus in the nosocomial setting, we analyzed isolates from long-term carriers (>1 month) and from patients involved in well-defined nosocomial epidemics. The number of fragment differences between the first isolate and subsequent isolates in long-term carriers showed a bimodal distribution, with one group having 0 to 6 fragment differences and the other group having 14 to 24 fragment differences. The PFGE patterns of isolates involved in epidemics also presented a similar bimodal distribution of the number of fragment differences. Typing these isolates with another molecular method (inter-IS256 PCR) showed that isolates of the first group (i.e., with 1 to 6 fragment differences) were clonally related, whereas the second group (with 14 to 24 fragment differences) could be considered genetically different. Among long-term carriers with clonally related isolates, 74 of 84 (88%) of consecutive isolates showed indistinguishable patterns, whereas 10 of 84 (12%) showed related patterns differing by one to six fragments. Moreover, the frequency of apparition of related patterns is higher when the time between the first and the subsequent isolate is longer. During seven nosocomial epidemics lasting from 1 to 15 months, only 2 of 120 isolates (1.7%) showed a pattern which was different, although related, from the predominant one involved in each of these outbreaks.

Typing of nosocomial strains of Serratia marcescens: comparison of pulsed-field gel electrophoresis of macrorestriction fragments with biotyping, esterase typing and ribotyping.

Fifty nosocomial isolates of Serratia marcescens, collected in six Belgian hospitals between 1986 and 1990, were characterized by using pulsed-field gel electrophoresis (PFGE) with XbaI. The results were compared with those previously obtained by three other methods: biotyping, esterase electrophoresis typing and ribotyping with EcoRI and HindIII. Macrorestriction analysis (42 PFGE groups) and esterase typing (42 zymotypes) proved to be the most discriminating, followed by ribotyping (28 ribotypes) and biotyping (10 biochemical profiles). Biotyping would serve as a screen to identify isolates, due to its accessibility. Esterase typing provided a reliable tool to make subdivisions within biotypes because of congruence between biochemical groups and esterase patterns. Additional discrimination was still achieved by ribotyping and PFGE. It is concluded that the combined results of these four markers were useful for distinguishing all epidemic and sporadic isolates.

Assessment of resolution and intercenter reproducibility of results of genotyping Staphylococcus aureus by pulsed-field gel electrophoresis of SmaI macrorestriction fragments: a multicenter study.

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures.

Rapid and accurate identification of Staphylococcus species by tRNA intergenic spacer length polymorphism analysis.

PCR-amplified tRNA gene (tDNA) intergenic spacer length polymorphism (tDNA-ILP) was analyzed for its ability to identify to the species level type strains (n = 18) and clinical isolates (n = 163) of staphylococci. Amplified products obtained by PCR with outwardly directed consensus tDNA primers were separated by agarose and polyacrylamide gel electrophoreses. The results were compared with those obtained by biochemical identification and ribotyping. Each type strain presented a specific tDNA-ILP pattern. PCR with fluorescent primers allowed for the detection of labelled DNA fragments on polyacrylamide gels by using an automated laser fluorescence sequencer and provided enhanced pattern resolution in comparison with that by analysis on agarose gels. tDNA patterns indistinguishable from those of the type strains were produced by clinical isolates of all tested species except for some isolates of S. aureus (n = 3) and S. haemolyticus (n = 1), which showed variant patterns. Strains of S. saprophyticus and S. xylosus showed very closely related profiles, and S. cohnii subspecies were indistinguishable. The identities obtained by tDNA-ILP analysis agreed with those obtained by the biochemical method to the species level for 99% (162 of 163) of the strains tested and to the subspecies level for 96% (156 of 163) of the strains tested. These results indicate that fluorescence-labelled PCR analysis of tDNA-ILP provides an accurate and rapid molecular method for the identification of human staphylococci.

Comparison of biotyping, ribotyping, and pulsed-field gel electrophoresis for investigation of a common-source outbreak of Burkholderia pickettii bacteremia.

Over a 3-month period, six immunocompromised patients developed one or more episodes of Burkholderia pickettii bacteremia and/or catheter infection. Vials of a commercially available, "sterile" saline for injection which had been used for flushing the patients' indwelling intravenous devices were implicated as the common source of the organisms. No further cases were diagnosed once the use of this saline was discontinued. Twenty-six isolates, including 9 outbreak-related strains from case patients and contaminated saline as well as 17 control strains, were tested comparatively by biotyping, ribotyping with EcoRI and HindIII, and pulsed-field gel electrophoresis (PFGE) with SpeI. Macrorestriction analysis revealed nine PFGE groups and was more discriminating than ribotyping (seven ribotypes) and biotyping (two biovars). Among the outbreak-related isolates, one B. pickettii type was found by the three typing methods. Furthermore, PFGE was useful for subdividing ribotypes and for distinguishing isolates involved in the outbreak from all epidemiologically unrelated strains.

Molecular epidemiology of an outbreak of multidrug-resistant Enterobacter aerogenes infections and in vivo emergence of imipenem resistance.

Molecular typing was used to investigate an outbreak of infection caused by multidrug-resistant Enterobacter aerogenes (MREA) susceptible only to gentamicin and imipenem in an intensive care unit (ICU). Over a 9-month period, ciprofloxacin-resistant E. aerogenes isolates were isolated from 34 patients, or 4.1% of ICU admissions, compared with a baseline rate of 0.1% in the previous period (P < 0.001). Infection developed in 15 (44%) patients. In vivo emergence of imipenem resistance (MIC, 32 micrograms/ml) of organisms causing deep-seated infection was observed in two (13%) of these patients following prolonged therapy with imipenem and gentamicin. Arbitrarily primed PCR (AP-PCR) analysis with ERIC1R and ERIC2 primers and pulsed-field gel electrophoresis (PFGE) analysis of XbaI macrorestriction patterns concordantly showed that outbreak-associated MREA isolates were clonally related and distinct from epidemiologically unrelated strains. AP-PCR and PFGE showed discrimination indices of 0.88 and 0.98, respectively. Space-time clustering of cases within units suggests that the epidemic-related MREA isolates were transmitted on the hands of the health care personnel. A case-control study and repeated environmental culture surveys failed to identify a common source or procedure associated with transmission. In spite of the early implementation of isolation measures, the incidence of MREA colonization remained stable until all colonized patients were discharged. This study confirms the usefulness of AP-PCR and PFGE analyses for the epidemiological study of E. aerogenes and underscores the difficulty of controlling the spread of multiresistant clones of this organism in the ICU setting. The emergence of imipenem resistance represents a threat because virtually no therapeutic option is available for such strains.

Rapid biochemical screening for Salmonella, Shigella, Yersinia, and Aeromonas isolates from stool specimens.

Four screens for the rapid (4 to 6 h) biochemical detection of pathogens from enteric isolation media are described. The Salmonella screen consisted of Kligler iron agar (KIA), motility-indole-urea-tryptophan-deamination semisolid medium (MIU-TDA), and the o-nitrophenyl-beta-D-galactopyranoside (ONPG) test; the Shigella screen consisted of KIA, MIU-TDA, the ONPG test, and the lysine decarboxylation-indole test; the Yersinia screen consisted of a rhamnose broth; the Aeromonas screen consisted of a xylose agar plate. When tested on 2,102 fresh isolates and 71 stock strains, the screens correctly detected 212 enteric pathogens (sensitivity, 100%), with a specificity of 98.1%.