Search

Meticillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare-associated infections in Europe. Many examples have demonstrated that the spread of MRSA within healthcare settings can be reduced by targeted infection control measures. The aim of this systematic literature analysis and review was to summarise the evidence for the use of bacterial cultures for active surveillance the benefit of rapid screening tests, as well as the use of decolonisation therapies and different types of isolation measures. We included 83 studies published between 2000 and 2012. Although the studies reported good evidence supporting the role of active surveillance followed by decolonisation therapy, the effectiveness of single-room isolation was mostly shown in non-controlled studies, which should inspire further research regarding this issue. Overall, this review highlighted that when planning the implementation of preventive interventions, there is a need to consider the prevalence of MRSA, the incidence of infections, the competing effect of standard control measures (e.g. hand hygiene) and the likelihood of transmission in the respective settings of implementation.

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.

This study aimed to estimate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) carriage upon hospital admission and to study the molecular epidemiology of MRSA in order to assess the proportion of Panton-Valentine leukocidin (PVL)-positive community-associated (CA) and livestock-associated (LA) MRSA strains. Epidemiological data on MRSA carriage upon hospital admission (2006-2009) were collected in a compulsory, continuous, national MRSA surveillance in Belgian acute-care hospitals. Additionally, 328 MRSA strains in 2005 and 314 strains in 2008 were collected in a separate, multicenter microbiological survey. Spa-typing, SCCmec-typing and MLST were performed; toxin genes were detected by PCR. The overall prevalence of MRSA carriage upon hospital admission was 8.9 cases/1,000 admissions between 2006 and 2009. Of MRSA carriers, 37.5% had a known MRSA history, 39.4% had stayed in a care facility, 12.2% reported no contact with healthcare. Over 90% of MRSA belonged to five healthcare-associated clones. Of these, MRSA spa-CC038-ST45-IV was in decline, mainly in favor of spa-CC008-ST8-IV. MRSA spa-CC002-ST5-IV, spa-CC002-ST5-II and spa-CC032-ST22-IV remained relatively stable. The proportion of PVL-positive CA-MRSA and LA-MRSA ST398 was below 2% of all MRSA. The extra-hospital MRSA reservoir in Belgium mainly consists of persons with previous healthcare exposure. PVL-positive CA-MRSA and LA-MRSA strains remained infrequent among hospitalized patients.

The purpose of this study was to assess the accuracy of the Xpert MRSA assay (XP) for the detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage upon hospital admission. Nasal swabs were prospectively collected for MRSA screening from 1,891 patients admitted to a teaching hospital. XP results were compared to chromogenic agar culture results. MRSA was cultured in 61 specimens (3%). Compared with culture, XP had a sensitivity, specificity, positive, and negative predictive value of 60.7, 97.3, 37.8, and 98.9%, respectively. The median turnaround time (TAT) for the results was 3 h. Of 24 MRSA isolated from XP-negative samples, three harbored composite SCCmec. Among 61 samples with culture-negative but XP-positive results, 15 methicillin-susceptible S. aureus (MSSA) isolates tested positive by XP on pure colony lysates. These MSSA included: (i) strains with SCCmec deletion encompassing mecA and (ii) multilocus sequence typing (MLST) clonal complex (CC) 1 strains harboring a chromosomal sequence homologous to one of the orfX-SCCmec junction sequences targeted by XP. On account of the low sensitivity and positive predictive value in a hospital patient population with moderate prevalence of MRSA, culture still appears to be necessary in order to confirm polymerase chain reaction (PCR) results. The emergence of new SCCmec variants and the presence of MSSA harboring cross-reactive SCCmec-like elements may challenge the successful implementation of such detection systems.

The prevalence, distribution, and antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) in Belgian pig farms has been investigated. To that end, nasal samples were collected from 1,500 pigs on 50 farms randomly selected over Belgium. Both closed (breeding or farrow-to-finish) and open (fattening) farms were included. Within closed farms different age groups were investigated. A total number of 663 (44%) pigs belonging to 34 (68%) farms carried MRSA. According to their management practice, MRSA was detected on 94% of the open farms and 56% of the closed farms. Focusing on the in-herd prevalence among fattening pigs for both management systems, a significantly higher rate was found in open farms (72%) compared to closed farms (26%). Within the closed farms, piglets (41%) showed a higher MRSA prevalence than sows (26%) and fattening pigs (26%). All strains tested were ST398 and showed mainly spa-type t011, as commonly found on pig herds in Europe. Less dominating spa-types were t034, t567, and t2970. The MRSA strains carried two SCCmec-types, type IVa or V. All 643 MRSA strains were resistant to tetracycline and additional resistances to trimethoprim (97%), lincosamides (73%), macrolides (56%), aminoglycosides (48%), and fluoroquinolones (32%) were found. Multiresistance (defined as resistance to four or more non-β-lactam antimicrobial classes) was found in 63% of the tested strains. In conclusion, a high prevalence of MRSA was found in Belgian pig farms, with the highest prevalence in open farms. In accordance with other European countries, age-related and management-related differences in MRSA prevalence were observed that should be considered when control strategies are outlined.

Background: Molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) has documented the diversity of the genetic background of strains associated with healthcare (HA-MRSA), community (CA-MRSA) and livestock (LA-MRSA). The accessory and core-variable genomes of those strains however remain largely unknown., Objective: To compare the genetic background and accessory and core variable gene content of ST398 LA-MRSA strains with those of HA-and CA-MRSA strains from the same region., Methods: Representative strains of HA- (n=21), CA- (n=13) and ST398 LA-MRSA (n=18) were selected from Belgian National Reference Laboratory collections. The accessory and core-variable genomes of these strains were characterized by a DNA-microarray composed of oligonucleotide probes targeting ~400 resistance, adhesion and virulence associated genes., Results: ST398 strains displayed very homogenous hybridization profiles irrespective of their host origin. This ST398 genomic profile was moderately related to that of certain human HA- or CA-lineages but distinctively lacked several virulence- and colonization-associated genes implicated in carriage in humans, such as proteases and adhesins. No enterotoxin gene was found among ST398 strains. Differences were observed in the mobile resistance gene content of ST398 strains, including antibiotic resistance determinants., Conclusion: LA-MRSA strains represent a homogenous lineage distinct from co-local HA- and CA-MRSA strains, especially in its accessory genome content characterized by a lack of human-associated virulence and adhesion determinants. The absence of detectable enterotoxin gene among ST398 LA-MRSA strains from a wide host range is reassuring regarding their foodborne pathogenic potential., (Copyright © 2010 Elsevier B.V. All rights reserved.)

To estimate the proportion of methicillin-resistant Staphylococcus aureus (MRSA) isolates from humans that were sequence type (ST) 398, we surveyed 24 laboratories in 17 countries in Europe in 2007. Livestock-associated MRSA ST398 accounted for only a small proportion of MRSA isolates from humans; most were from the Netherlands, Belgium, Denmark, and Austria.

Methicillin-resistant Staphylococcus aureus (MRSA) isa major cause of healthcare- and community-associated infections worldwide. Within the healthcare setting alone, MRSA infections are estimated to affect more than 150,000 patients annually in the European Union (EU), resulting in attributable extra in-hospital costs of EUR 380 million for EU healthcare systems. Pan-European surveillance data on bloodstream infections show marked variability among EU Member States in the proportion of S. aureus that are methicillin-resistant, ranging from less than 1% to more than 50%. In the past five years, the MRSA bacteraemia rates have decreased significantly in 10 EU countries with higher endemic rates of MRSA infections. In addition to healthcare-associated infections, new MRSA strains have recently emerged as community and livestock-associated human pathogens in most EU Member States. The prevention and control of MRSA have therefore been identified as public health priorities in the EU. In this review, we describe the current burden of MRSA infections in healthcare and community settings across Europe and outline the main threats caused by recent changes in the epidemiology of MRSA. Thereby, we aim at identifying unmet needs of surveillance, prevention and control of MRSA in Europe.

Rapid and reliable detection of methicillin-resistant Staphylococcus aureus (MRSA) carriers is crucial for control of MRSA nosocomial transmission. We aimed to evaluate the performance of the GeneXpert real-time PCR system using the Xpert MRSA assay on a collection of 40 representative Belgian MRSA strains and for MRSA screening of geriatric inpatients. Double nasal swabs were used: the first swab for the Xpert MRSA assay and the second for culture onto chromogenic selective medium and enrichment broth. All but 1 of the 40 collection strains were recognized as MRSA by the Xpert MRSA assay. Nares swabs were prospectively collected from 246 inpatients including 25 nasal MRSA carriers. Compared with enriched cultures, the sensitivity, the specificity, and the positive and negative predictive values of the Xpert MRSA assay were 69.2%, 97.7%, 78.3%, and 96.3% respectively. The 7 evaluable false-negative results according to the assay were due to its possible lack of sensitivity (n = 3) and to the occurrence of a Belgian MRSA clone carrying a particular staphylococcal chromosomal cassette mec (SCCmec) type IV variant (n = 4) not targeted by the current Xpert MRSA assay. Because of the evolution of SCCmec in MRSA, new primers should be designed and further studies are warranted to ensure continuous monitoring of this assay.

Objectives: Approximately 75% of the genome of Staphylococcus aureus (the 'core' genome) is highly conserved between strains, whereas the remaining 25% (the 'accessory' genome) is composed of mobile genetic elements (MGEs), containing virulence and resistance genes. We developed a composite microarray focused on resistance and virulence genes located on the accessory or core-variable genome to characterize a collection of Belgian community-acquired methicillin-resistant S. aureus (CA-MRSA) strains., Methods: Oligonucleotide probes targeting 403 genes encoding antimicrobial resistance (35%), virulence (28%) and adhesion (31%) factors were designed among eight S. aureus sequenced genomes. The StaphVar Array was validated by testing five of the strains used for the design and utilized to characterize 13 CA-MRSA strains representative of the multilocus sequence typing (MLST) sequence types circulating in Belgium., Results: Analysis of the gene content of the five reference strains by the StaphVar Array matched 90% to 97% of the theoretical results. Analysis of CA-MRSA strains showed that 54.4% of the genes tested were strain-dependent. Strains presented specific exotoxin, enterotoxin, cytolysin and adhesin gene profiles by MLST lineage. One exception to these 'lineage-specific' profiles was the variable presence of the arginine catabolic mobile element (characteristic of the USA300 clone) within ST8 strains., Conclusions: The StaphVar Array enables the characterization of approximately 400 variable resistance and virulence determinants in S. aureus. CA-MRSA strains displayed extensive diversity in virulence and resistance profiles. The presence of the USA300 clone in Belgium was confirmed. Although mainly located on MGEs, associations of virulence genes were highly conserved within strains of the same MLST lineage.

This study compares the performance of three chromogenic culture agar plates, chromID MRSA, MRSA-Screen and MRSA-Select, by challenging with a collection of Staphylococcus aureus strains and screening samples obtained from hospitalised patients. All chromogenic media showed excellent sensitivity (>95%) and specificity after 18 h on the methicillin-resistant Staphylococcus aureus (MRSA) collection strains, but the specificity of MRSA-Screen decreased markedly after 42 h. Sixty-eight of 1,002 screening specimens yielded MRSA on at least one medium. The sensitivity of all media to detecting MRSA after 18 h was <50%, but this increased to 75% (chromID MRSA), 81% (MRSA-Screen) and 72% (MRSA-Select) after 42 h and 85% after enrichment and plating on the same media. The specificity at 18 h was excellent, but was significantly lower for MRSA-Screen after 42 h and enrichment. In conclusion, all media showed equivalent sensitivities after 18 h of incubation and performed better when enriched before inoculation. MRSA-Screen was more sensitive but less specific than the two other media after 42 h of incubation.

A multicenter preclinical evaluation was conducted to evaluate the performance of two Cepheid Xpert assays for detection of methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus. Sensitivity was 97.1% and 98.3% for MRSA in wound and blood culture specimens, respectively. Sensitivity was 100% for S. aureus from both specimen types.

The public health burden caused by methicillin-resistant Staphylococcus aureus (MRSA) infections is now widely recognized, and is a cause of public alarm. Effective MRSA risk management in the healthcare system as well as in the community should rely on accurate detection of reservoirs and sources of transmission, as well as on close monitoring of the impact of interventions on disease incidence and bacterial dissemination. MRSA carrier screening and disease surveillance, coupled with molecular typing, are key information tools for integrated MRSA control and individual risk assessment. These tools should be tailored to the distinct needs of local interventions and national prevention programmes. Surveillance schemes should primarily inform local staff and serve as quality assurance about MRSA risk management. New technologies, including the use of selective culture media and real-time PCR assays, allow faster detection of MRSA carriers upon admission or during stay in healthcare institutions. More research is needed to ascertain their cost-effectiveness for MRSA control. Likewise, tremendous progress has been made concerning molecular typing methods, with optimization and standardization of sequence-based technologies offering broad applicability and high throughput. However, no single S. aureus typing method is yet providing fully reliable information within the range of discrimination needed for public health action. Further refinement of genotyping methods and international harmonization of surveillance and typing schemes must be achieved to facilitate global MRSA control.

Methicillin-resistant Staphylococcus aureus (MRSA) infections do occur in equine patients. Little is known, however, about their origin and the general equine MRSA colonization status. In West European horses in particular, neither the colonization rate nor the present strains or their antimicrobial susceptibility patterns are known. In the present study, a sample of 110 (Belgian, French, Dutch and Luxemburg) horses presented at a Belgian equine clinic was screened for nasal MRSA carriage. An indirect culturing protocol using a 0.001% colistin and nalidixic acid containing broth was compared to a direct agar method. Phenotypic identification following growth on a chromogenic MRSA screening agar (ChromID MRSA) was combined with genotypic analysis (PCR, PFGE, SCCmec, spa, and MLST typing). Antimicrobial susceptibility was tested through disk diffusion. Twelve (10.9%) horses carried MRSA, with the enrichment protocol resulting in a significantly higher isolation rate. None of the isolated strains were typeable through SmaI PFGE. They all harboured SCCmec type IVa or V and belonged to spa type t011 or t1451 of the ST398 lineage. All isolates were tetracycline resistant and sulfonamide and enrofloxacin susceptible. Macrolide, lincosamide, trimethoprim and aminoglycoside susceptibility varied and in total five different antimicrobial resistance patterns were distinguished. These results show that ST398 is certainly present in West European horses. Due to its known interspecies transmission and the structure of the equine industry, the presence of this clone in horses poses a substantial health hazard for both animals and humans.

A 63-year-old female pig farmer was referred to our department with a protracted course of otomastoiditis with destruction of the tympanic roof and cerebrospinal fluid leakage. The patient underwent a cortical mastoidectomy with closure of a large dural defect. Cultures of the middle ear effusion yielded a methicillin-resistant Staphylococcus aureus (MRSA), which upon further analysis was found to be from porcine origin. To our knowledge, this is the first report of a complicated case of otomastoiditis caused by a pig-type MRSA.

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.

Objectives: Epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is poorly defined in cystic fibrosis (CF) patients, and S. aureus detection may be hampered by the presence of small colony variants (SCVs). We conducted a multicentre survey to determine the prevalence of S. aureus and MRSA colonization in Belgian CF patients and characterize the phenotype and clonal distribution of their staphylococcal strains., Methods: S. aureus isolated from CF patients attending nine CF centres were collected. Oxacillin resistance was detected by oxacillin agar screen and mecA PCR. Antibiotic susceptibility was tested by microdilution. MRSA strains were genotyped by PFGE and SCCmec typing and compared with hospital-associated MRSA strains., Results: Laboratories used a diversity of sputum culture procedures, many of which appeared substandard. S. aureus was isolated from 275/627 (44%) CF patients (20% to 72% by centre). The prevalence of SCV colonization was 4%, but SCVs were almost exclusively recovered from patients in two centres performing an SCV search. Phenotypically, 14% of S. aureus isolates were oxacillin-resistant: 79% carried mecA and 19% were SCVs lacking mecA. The mean prevalence of 'true' MRSA colonization was 5% (0% to 17% by centre). By PFGE typing, 67% of CF-associated MRSA were related to five epidemic clones widespread in Belgian hospitals., Conclusions: This first survey of S. aureus colonization in the Belgian CF population indicated a diversity in local prevalence rates and in proportion of oxacillin-resistant and SCV phenotypes, probably related to variation in bacteriological methods. These findings underscore the need for standard S. aureus detection methods and MRSA control policies in Belgian CF centres.

Major regional variations in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) are observed across Europe. This study investigated hospital MRSA prevalence in relation to patterns of antimicrobial use and infection control policies in an observational, cross-sectional study that used retrospective data from 2001 and linear regression to model relationships. MRSA prevalence (median 20.8%, n = 173 hospitals) and antimicrobial consumption (median 55.2 defined daily doses/100 bed-days, n = 140 hospitals) both varied significantly according to geographical region (p <0.001). MRSA prevalence and antimicrobial consumption data were provided by 128 hospitals, and showed a strong statistical relationship between macrolide use and MRSA prevalence. Use of (i) third-generation cephalosporins, (ii) all antimicrobial agents, and (iii) all antimicrobial agents except glycopeptides was also associated with MRSA prevalence. Up to 146 hospitals provided data on MRSA prevalence and key infection control parameters. Adjusted linear regression modelling provided strong evidence that infection control policy recommendations associated with lower MRSA prevalence rates were (i) use of alcohol-based solutions for hand hygiene (mean difference 10.3%, 99% CI 1.2-10.3), and (ii) placement of MRSA patients in single rooms (mean difference 11.2%, 99% CI 1.4-20.9). Hospitals with problems in implementing isolation policies had higher resistance levels (mean difference 12%, 99% CI 3.8-20.1). Additional recommendations showed less evidence of association with a low MRSA prevalence. Overall, this study highlighted significant associations between MRSA prevalence, antimicrobial use and various key infection control parameters, all of which showed significant individual variations according to geographical region.

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.

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.

Objectives: Worldwide spread of a limited number of Panton-Valentine leucocidin (PVL) -producing methicillin-resistant Staphylococcus aureus (MRSA) clones has been reported in various communities. The objective of this study was to describe the molecular characteristics of the first PVL-positive MRSA strains isolated in Belgium., Methods: Clinical MRSA isolates (n = 41) collected from 2002 to 2004 from Belgian patients were investigated for the PVL gene by PCR. PVL-positive isolates were genotyped by PFGE, staphylococcal cassette chromosome mec (SCCmec) typing, spa sequence typing, accessory gene regulator (agr) polymorphism and multi-locus sequence typing (MLST). Susceptibility to 14 antimicrobials was determined by the disc diffusion method. Genes encoding resistance to tetracyclines, aminoglycosides and macrolide-lincosamide-streptogramin were determined by PCR., Results: Sixteen isolates carried lukS-lukF genes that encode the PVL toxin. All but one isolate were community-acquired. Three patients reported recent travel to North Africa and South America. They were associated with skin or soft tissue infections, bacteraemia and peritonitis. By molecular typing, they belonged to five genotypes: ST80-SCCmec IV, ST8-SCCmec IV, ST30-SCCmec IV, ST153-SCCmec IV and ST88-SCCmec IV. They belonged to the agr type 3 except for ST8 strains, which showed agr type 1. All isolates were susceptible to fluoroquinolones. Approximately, half of them were resistant to tetracycline, fusidic acid and kanamycin. Tetracycline-resistant strains harboured the tet(K) gene and resistance to kanamycin was associated with the aph3'-IIIa gene. The single erythromycin-resistant isolate harboured msr(A/B) genes conferring the M resistance phenotype., Conclusions: These results indicate the recent emergence and sporadic importation into Belgium of PVL-positive community-associated MRSA strains belonging to five distinct clones.

Staphylococcus aureus strains with decreased susceptibility to glycopeptides (GISA) have been associated with increased risk of glycopeptide treatment failure. To assess the prevalence of these strains in hospitalised patients in Belgium, 455 methicillin-resistant S. aureus (MRSA) isolates collected in 2001 were screened by two assays: (i) growth on vancomycin agar screen (VAS; brain heart infusion agar (BHI) + vancomycin 6 mg/L); and (ii) a synergy/antagonism test with aztreonam/cefazolin on Mu3 agar (BHI + vancomycin 3 mg/mL). Isolates growing on VAS or Mu3 agar were characterised further by analysis of population susceptibility profiles. MICs of glycopeptides were determined by agar dilution, broth microdilution and Etest (low and high inocula) methods. The isolates were genotyped by pulsed-field gel electrophoresis (PFGE) and determination of staphylococcal cassette chromosome mec (SCCmec) type. No GISA isolates were found. Three (0.7%) hetero-vancomycin intermediate S. aureus (hVISA) and ten (2.2%) hetero-teicoplanin intermediate S. aureus (hTISA) isolates were identified by population analysis. All but one hetero-GISA isolate belonged to either epidemic PFGE group A/SCCmec type I (69%) or PFGE group D/SCCmec type I (23%), both of which were resistant to gentamicin. The sensitivity and specificity for the detection of hetero-GISA by the two assays were 15.4% and 99.8%, respectively, for VAS, and 84.6% and 95.9%, respectively, for Mu3. The data indicated that hetero-GISA strains were uncommon among Belgian MRSA isolates from hospitalised patients. Use of Mu3 agar was more sensitive, but less specific, than VAS as a screening method.

We evaluated the clinical usefulness of a PCR assay that discriminates Staphylococcus aureus from coagulase-negative staphylococci and detects methicillin resistance on blood cultures by measuring the adaptation of antimicrobial therapy based on the PCR results. Only 7 of 28 patients (25%) benefited from a modification of antibiotic therapy based on the PCR results, since empirical therapy was appropriate in a majority of cases.

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.

Rapid and efficient epidemiologic typing systems would be useful to monitor transmission of methicillin-resistant Staphylococcus aureus (MRSA) at both local and interregional levels. To evaluate the intralaboratory performance and interlaboratory reproducibility of three recently developed repeat-element PCR (rep-PCR) methods for the typing of MRSA, 50 MRSA strains characterized by pulsed-field gel electrophoresis (PFGE) (SmaI) analysis and epidemiological data were blindly typed by inter-IS256, 16S-23S ribosomal DNA (rDNA), and MP3 PCR in 12 laboratories in eight countries using standard reagents and protocols. Performance of typing was defined by reproducibility (R), discriminatory power (D), and agreement with PFGE analysis. Interlaboratory reproducibility of pattern and type classification was assessed visually and using gel analysis software. Each typing method showed a different performance level in each center. In the center performing best with each method, inter-IS256 PCR typing achieved R = 100% and D = 100%; 16S-23S rDNA PCR, R = 100% and D = 82%; and MP3 PCR, R = 80% and D = 83%. Concordance between rep-PCR type and PFGE type ranged by center: 70 to 90% for inter-IS256 PCR, 44 to 57% for 16S-23S rDNA PCR, and 53 to 54% for MP3 PCR analysis. In conclusion, the performance of inter-IS256 PCR typing was similar to that of PFGE analysis in some but not all centers, whereas other rep-PCR protocols showed lower discrimination and intralaboratory reproducibility. None of these assays, however, was sufficiently reproducible for interlaboratory exchange of data.

Cultures of blood obtained from a patient with Staphylococcus epidermidis prosthetic valve endocarditis yielded 15 strains of S. epidermidis. Genome macrorestriction and amplified fragment-length polymorphism analyses of these strains showed that they belonged to 4 different, very closely related clones, suggesting that they were the result of genetic variability of an infecting strain during the infectious episode. In vivo experiments in a rat model for foreign body infections using 1 of the S. epidermidis strains from the patient showed genetic variability similar to that of the infecting strain. In the rat model, we also detected the simultaneous presence of different clones that were identical to those isolated from our patient, thus confirming the possibility of genetic variability. It is important to note that the 4 clones isolated from our patient presented with 2 different antibiograms. Therefore, in cases of foreign device-related infections due to coagulase-negative staphylococci, the possibility of polyclonal infection has to be taken into account, particularly as regards differences in antibiotic susceptibility.

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 SmaI 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.

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.

Forty-three methicillin-resistant Staphylococcus aureus (MRSA) isolates with known genetic and epidemiological relatedness and different degrees of transmission were analyzed by antibiotyping, protein A gene polymorphism analysis, and coagulase gene polymorphism analysis. The three typing systems were evaluated for their performance and convenience to define clones and to discriminate between epidemic MRSA (EMRSA) and sporadic MRSA (SMRSA). Antibiotyping and AluI restriction fragment length polymorphism analysis of the coagulase gene were able to define clones in the same way as DNA macrorestriction analysis (SmaI). However, both techniques presented disadvantages, making neither of them useful as a single typing method. Protein A gene polymorphism analysis appeared to be of no value for clonal analysis. None of the three typing methods was able to differentiate between EMRSA and SMRSA.

A commercial system for the rapid detection of methicillin-resistant Staphylococcus aureus, the BBL Crystal MRSA test (C-MRSA ID; Becton Dickinson, USA), was evaluated prospectively and compared with a polymerase chain reaction test for the presence of the mecA gene. Ten European centres tested a total of 676 isolates of Staphylococcus aureus from blood cultures. The system correctly identified 661 (97.8%) isolates within 4 h. All but three mecA gene-negative isolates (99.4% specificity) yielded a negative C-MRSA ID reaction, and 158 of 170 mecA gene-positive isolates were accurately detected (92.9% sensitivity). After repeated testing of discrepant results, sensitivity and specificity increased to 99% and 100%, respectively.

Objectives: To describe the Belgian methicillin-resistant Staphylococcus aureus (MRSA) surveillance network, the evolution of methods used in Belgian hospitals for MRSA detection and control, and MRSA incidence from 1994 to 1995., Design, Setting, and Participants: Questionnaire surveys; infection control physicians from acute-care hospitals in Belgium., Intervention: Publication of national guidelines for MRSA control in 1993., Results: The participation rate in surveys ranged from 42% to 57% of hospitals. In 1995, 88% of participants detected MRSA strains by disk diffusion tests, with little improvement in standardization since 1991. More centers employed the oxacillin agar screen method (27%), automated systems (29%), or a combination of methods (29%) than in 1991 (P < .005). Between 1991 and 1995, the proportion of hospitals reporting MRSA control measures increased from 68% to 95% (P < .01). Practices that were used increasingly included patient placement in private room (from 50% to 93%, P < .01) and hand decontamination with antiseptic (from 43% to 87%, P < .01). The proportion of centers that reported screening MRSA carriers and treating them topically increased two- and threefold, respectively (P < .05). Surveillance data from 1994 to 1995 showed that MRSA represented a mean of 21.3% of S aureus clinical isolates (range, 1.6% to 62.4%). The median incidence of nosocomial MRSA acquisition was 2.8 per 1,000 admissions, with a wide range (0 to 13.7 per 1,000 admissions) across hospitals of all sizes. The median incidence decreased over the first three semesters of surveillance in hospitals with continuous participation., Conclusion: MRSA detection and control measures have improved in Belgian hospitals after publication of national guidelines. However, MRSA incidence rates show the persistence of nosocomial transmission, with large variations between centers. The national MRSA surveillance network should indicate whether control efforts eventually will curb the problem.

A questionnaire survey of Belgian acute care hospitals was conducted to determine the methods used for detection of methicillin-resistant Staphylococcus 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 heterogenous resistance. Among 36 hospitals reporting complete susceptibility data of Staphylococcus 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.