Emergence of livestock-associated MRSA isolated from cystic fibrosis patients: Result of a Belgian national survey.

BACKGROUND: This study aims to determine the prevalence and characteristics of Staphylococcus aureus in Belgian cystic fibrosis (CF) patients. METHODS: Non-duplicate respiratory samples from 510 CF-patients (2012-2013) were examined. One isolate per patient was analysed unless different phenotypes were recovered. Isolates were investigated for mecA/mecC, toxins presence, spa-typing, MLST and SCCmec-typing. Potential livestock-associated (LA) isolates were examined for their immune-evasion-cluster (IEC) genes. RESULTS: S. aureus (n = 380), including 41 small-colony variants (SCVs), were isolated from 66.7% patients. The prevalence of methicillin-resistant S. aureus (MRSA) colonization was 4.9%. Two MRSA isolates carried toxic shock syndrome toxin 1 (TSST-1). Most MRSA (65%) belonged to two nosocomial epidemic clones (CC5, CC8) widespread in Belgium. Methicillin susceptible S. aureus (MSSA) showed great genetic diversity. Five of 33 isolates belonging to potential LA-lineages were IEC negative, including three methicillin-resistant isolates, suggesting an animal origin. CONCLUSIONS: The MRSA-prevalence in Belgian CF-patients remained constant (2001-2013), but SCV-prevalence increased. Most MRSA belonged to health-care-associated clones. Three patients carrying LA-MRSA were found, requiring further investigation to determine the risk factors for LA-MRSA acquisition.

Genetic Diversity among Staphylococcus aureus Isolates Showing Oxacillin and/or Cefoxitin Resistance Not Linked to the Presence of mec Genes.

Methicillin-resistant Staphylococcus aureus isolates lacking mec genes (n = 32), collected from Belgian hospitals, were characterized for their ß-lactamase production and the presence of mutations in pbp genes, the pbp4 promoter, and genes involved in penicillin-binding protein 4 overproduction (gdpP and yjbH). Twelve isolates were ß-lactamase hyperproducers (BHPs), while 12 non-BHP isolates might produce an incomplete GdpP protein. Most isolates showed nucleotide missense mutations in pbp genes. A few isolates also showed mutations in the pbp4 promoter.

CC398 Staphylococcus aureus subpopulations in Belgian patients.

Studies based on genome-wide single nucleotide polymorphisms (SNPs) supported the existence of two subpopulations in clonal complex (CC) 398 Staphylococcus aureus: an ancestral human-adapted clade (HC) and an animal-associated clade (AC). In this study, we have investigated the occurrence of genetic markers that allow discrimination of these subpopulations among CC398 isolates collected during 2014 to 2016 from human patients in Belgium. A collection of isolates was investigated by means of spa-typing and 16S-mecA-nuc PCR. CC398 isolates were classified as belonging to the human or the animal clade by using a canonical SNPs PCR and further studied by antimicrobial susceptibility and the presence of toxins, immune evasion cluster (IEC), and resistance genes. A total of 124 (7.8%) human isolates belonged to CC398. They were grouped into HC (n = 58) or AC (n = 66). The genes erm(T), pvl, chp, and scn were predominantly found in HC-CC398, while AC-CC398 isolates carried more frequently than the mecA, erm(C), tet(K), tet(M), and tet(L) genes. Different combinations of gene profiles were observed according to the clade. CC398 isolates from Belgian patients belonged to different subpopulations including typical HC and AC-isolates. Few HC-strains with mecA and AC-isolates harboring IEC were found. CC398 isolates from Belgian patients belonged to different subpopulations including typical HC and AC-isolates, as well as new emerging subpopulations that underline the ability of this lineage to acquire resistance and virulence genes. Further research is needed to evaluate the emergence of these subpopulations in the clinical setting.

Prospective evaluation of a high multiplexing real-time polymerase chain reaction array for the rapid identification and characterization of bacteria causative of nosocomial pneumonia from clinical specimens: a proof-of-concept study.

The purpose of this study was evaluation of the VAPChip assay based on the "Rapid-Array-PCR-technology" which targets 13 respiratory pathogens and 24 ß-lactam resistance genes directly on respiratory clinical specimens. The first step included analysis of 45 respiratory specimens in order to calibrate and determine the threshold for target genes. The second prospective step involved 85 respiratory samples from patients suspected of nosocomial pneumonia collected in two academic hospitals over an 8-month period. Results of the VAPChip assay were compared to routine methods. The first step showed a large proportion of positive signals for H. influenzae and/or S. pneumoniae. For identification, discrepancies were observed in seven samples. Thresholds were adapted and two probes were re-designed to create a new version of the cartridge. In the second phase, sensitivity and specificity of the VAPchip for bacterial identification were 72.9% and 99.1%, respectively. Seventy (82%) pathogens were correctly identified by both methods. Nine pathogens detected by the VAPChip were culture negative and 26 pathogens identified by culture were VAPChip negative. For resistance mechanisms, 11 probes were positive without identification of pathogens with an antimicrobial-susceptibility testing compatible by culture. However, the patient's recent microbiological history was able to explain most of these positive signals. The VAPChip assay simultaneously detects different pathogens and resistance mechanisms directly from clinical samples. This system seems very promising but the extraction process needs to be automated for routine implementation. This kind of rapid point-of-care automated platform permitting a syndromic approach will be the future challenge in the management of infectious diseases.

Mutations at the Ribosomal S10 Gene in Clinical Strains of Staphylococcus aureus with Reduced Susceptibility to Tigecycline.

Mutations on the tip of the extended loop of the ribosomal S10 protein have been associated to tigecycline (TGC) resistance in passaged mutants of different bacteria species. This study described the first two clinical TGC-resistant Staphylococcus aureus isolates with these mutations. One strain (TGC MIC = 2 mg/liter) had a 12-nucleotide deletion affecting residues 56 to 59 (HKYK) of the S10 protein. The second strain (TGC MIC = 1 mg/liter) had amino acid substitutions (K57M and Y58F) previously described in S. aureus passaged mutants.

In vitro activity of ceftaroline against clinical Staphylococcus aureus isolates collected during a national survey conducted in Belgian hospitals.

OBJECTIVES: The aim of this study was to estimate the in vitro activity of ceftaroline against clinical Staphylococcus aureus isolates collected during national surveillance in Belgian acute-care hospitals. Ceftaroline-resistant isolates were further investigated for their resistance mechanisms. METHODS: From October 2013 to March 2014, 155 laboratories of Belgian acute-care hospitals were invited to send to the National Reference Centre-Staphylococcus aureus (Belgium) up to five non-duplicate S. aureus including three MRSA and two MSSA from hospitalized patients. Isolates were analysed by spa typing, SCCmec typing (for MRSA) and PCR for detection of 16S-mecA-nuc and 16S-mecC. MICs of oxacillin, cefoxitin and ceftaroline were determined by the broth microdilution method. The nucleotide sequences of mecA, native pbp and gdpP genes of isolates with reduced susceptibility to ceftaroline were analysed for the presence of mutations responsible for amino acid substitutions. RESULTS: Ninety-nine percent of isolates, including MRSA (n = 284) and MSSA (n = 131), were susceptible to ceftaroline. Only four MRSA isolates showed resistance to ceftaroline (MIC = 2 mg/L). These four isolates belonged to lineages CC5 (n = 1), CC22 (n = 2) and CC8 (n = 1). Two isolates (CC22 and CC8) carried mutations in mecA, as well as in other pbp genes. The remaining isolates carried mutations in native pbp genes or in gdpP. CONCLUSIONS: This is the first Belgian in vitro survey on ceftaroline activity against S. aureus. This antibiotic showed excellent activity against MRSA and MSSA, and only a few MRSA isolates with resistance were found. Reduced susceptibility to ceftaroline seems a complex phenomenon due to the accumulation of mutations in genes involved in ß-lactam tolerance.

Low occurrence of the new species Staphylococcus argenteus in a Staphylococcus aureus collection of human isolates from Belgium.

Staphylococcus argenteus is a novel Staphylococcus species closely related to Staphylococcus aureus that has been recently described. In this study, we investigated the proportion and the characteristics of S. argenteus recovered from humans in Belgium. S. aureus. human isolates collected in Belgium from 2006 to 2015 (n = 1,903) were retrospectively characterised via the presence of non-pigmented colonies on chocolate agar, spa typing and rpoB sequencing to determine if some of them were in fact S. argenteus. Out of 73 strains non-pigmented on chocolate plates, 3 isolates (0.16 %) showed rpoB sequences, in addition to spa and sequence types (ST2250/t5787, ST2250/t6675, ST3240/t6675), related to S. argenteus. Two of them were methicillin-resistant, harbouring a SCCmec type IV. The three S. argenteus isolates carried genes (sak, scn) of the immune evasion cluster. This first Belgian nationwide analysis showed a low occurrence of S. argenteus. Further studies should be conducted to identify the distribution range and the clinical impact of this new species.

Evaluation of Verigene Gram-Positive Blood Culture Assay performance for bacteremic patients.

The Verigene Gram-Positive Blood Culture (BC-GP) Assay (Nanosphere Inc., Northbrook, IL) is a microarray-based test designed to rapidly identify directly from positive blood cultures multiple bacterial species and their antimicrobial resistance markers. Nonduplicate blood cultures from 118 patients admitted to Erasme Hospital were prospectively enrolled. All but six organisms were members of the panel (95.6 %). For the identification of pathogens and detection of the mecA gene, the agreement with routine methods was 87.6 % and 97.7 %, respectively. The performance of the BC-GP assay was lower with polymicrobial than with monomicrobial blood cultures. Another concern of the BC-GP assay was the misidentification of Streptococcus mitis as S. pneumoniae (3/8). The BC-GP assay is a rapid and accurate tool for the simultaneous detection of multiple sepsis-causing bacteria and resistant genes from blood cultures, which could have an impact on patient management and healthcare cost.

Comparative epidemiology of Staphylococcus epidermidis isolates from patients with catheter-related bacteremia and from healthy volunteers.

Staphylococcus epidermidis is a major cause of catheter-related bloodstream infections (CRBSIs). Recent studies suggested the existence of well-adapted, highly resistant, hospital-associated S. epidermidis clones. The molecular epidemiology of S. epidermidis in Belgian hospitals and the Belgian community has not been explored yet. We compared a set of 33 S. epidermidis isolates causing CRBSI in hospitalized patients with a set of 33 commensal S. epidermidis isolates. The factors analyzed included resistance to antibiotics and genetic diversity as determined by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and SCCmec typing. Additionally, the presence of virulence-associated mobile genetic elements, the ica operon and the arginine catabolic mobile element (ACME), was assessed and compared against clinical data. CRBSI S. epidermidis isolates were significantly resistant to more antibiotics than commensal S. epidermidis isolates. The two populations studied were very diverse and genetically distinct as only 23% of the 37 PFGE types observed were harbored by both CRBSI and commensal isolates. ACME was found in 76% of S. epidermidis strains, regardless of their origin, while the ica operon was significantly more prevalent in CRBSI isolates than in commensal isolates (P < 0.05). Nine patients presented a clinically severe CRBSI, eight cases of which were due to an ica-positive multiresistant isolate belonging to sequence type 2 (ST2) or ST54. S. epidermidis isolates causing CRBSI were more resistant and more often ica positive than commensal S. epidermidis isolates, which were genetically heterogeneous and susceptible to the majority of antibiotics tested. Clinically severe CRBSIs were due to isolates belonging to two closely related MLST types, ST2 and ST54.

Dynamic pattern and genotypic diversity of Staphylococcus aureus nasopharyngeal carriage in healthy pre-school children.

OBJECTIVES: It is common wisdom that persistent carriage of Staphylococcus aureus is more frequent in young children than in adults. The objectives of this study were to assess the S. aureus temporal carriage pattern among a healthy community of pre-school children, with concomitant description of genotype diversity, toxin-encoding genes and antibiotic resistance. METHODS: Among 333 children 3-6 years of age, S. aureus nasopharyngeal carriage was assessed over one school year by culture of three sequential nasopharyngeal aspirates. Identification, methicillin resistance and toxin production profile were determined by PCR. Genotyping was performed by spa sequencing and multilocus sequence typing (MLST). RESULTS: Out of 830 samples collected, 286 (34%) yielded S. aureus from 185 carriers (55%). Based on consecutive genotype analysis, only 40/268 (15%) children could be classified as persistent carriers, and the remaining 118 (44%) showed intermittent carriage. spa typing revealed 82 types clustered into 13 spa clonal complexes (CCs). Fourteen strains isolated from 11 (3%) children were methicillin-resistant S. aureus (MRSA), half of these strains belonged to the commonly hospital-associated spa t008-ST8-SCCmec IV. Methicillin-susceptible S. aureus (MSSA) were genotypically more diverse. Toxic shock syndrome toxin and egc1/2 complexes were highly prevalent (24%). Contrastingly, Panton-Valentine leucocidin (PVL) was carried only by three MSSA strains (0.6% of children). Exfoliative toxins were detected in 10 (3.5%) MSSA strains, of which 5 were related to the impetigo clone CC121. CONCLUSIONS: Although S. aureus nasopharyngeal carriage was high among healthy pre-school children, persistent carriage seems to be less frequent than previously reported. The prevalence of MRSA carriage was 3%, but was not associated with PVL.

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.

Previous healthcare exposure is the main antecedent for methicillin-resistant Staphylococcus aureus carriage on hospital admission in Belgium.

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.

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.

Heterogeneity of disease and clones of community-onset methicillin-resistant Staphylococcus aureus in children attending a paediatric hospital in Belgium.

The increase in the number of methicillin-resistant Staphylococcus aureus (MRSA) infections in children has prompted paediatricians to broaden th empirical treatment of common community-onset (CO) infections in children in several countries. Most European countries have reported low rates of CO-MRSA infection, but limited data on paediatric CO-MRSA infections are available. A prospective study was conducted from January 2002 to December 2004 in Brussels. CO-MRSA was defined as MRSA first detected by culture within 48 h of admission or in outpatients. Clinical and epidemiological data were recorded. CO-MRSA strains were genotyped by pulsed-field gel electrophoresis and multilocus sequence typing. Staphylococcal chromosomal cassette mec, toxin (Panton-Valentin leukocidin (PVL), toxic shock syndrome toxin 1, and Eta/b), enterotoxin and antibiotic resistance genes were detected by PCR. The antibiotic resistance phenotype was determined by disk diffusion. S. aureus was isolated in 1681 children. Among these, 107 harboured MRSA. Fifty-one children were colonized or infected by CO-MRSA, 20% of whom had no healthcare exposure. Twelve infants <3 months old and five cystic fibrosis patients were colonized. None of the 22 infected patients (59% with acute otitis media and 36% with skin and soft tissue infections (SSTIs)) required hospitalization. Two-thirds of them failed to respond to empirical antibiotic therapy. The 37 characterized CO-MRSA strains were genetically diverse. Most of them had healthcare-associated genotypes. Only six strains were PVL-positive, all of which were ciprofloxacin-susceptible and more common in children with SSTIs (p 0.001). CO-MRSA remains uncommon in our paediatric population. So far, there is no need to modify the empirical treatment of common S. aureus infections. Monitoring of MRSA rates in S. aureus CO infections remains mandatory, and further investigation is warranted to establish the source of colonization in young infants.

Positive predictive value of the Xpert MRSA assay diagnostic for universal patient screening at hospital admission: influence of the local ecology.

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.

Comparison of three chromogenic media and enrichment broth media for the detection of methicillin-resistant Staphylococcus aureus from mucocutaneous screening specimens : Comparison of MRSA chromogenic media.

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.

Clostridium difficile colitis in cystic fibrosis patients with and without lung transplantation.

BACKGROUND: Despite a large carriage rate of Clostridium difficile among cystic fibrosis (CF) patients, C. difficile-associated disease (CDAD) is rather rare. In case of lung transplantation, the incidence and clinical aspects of CDAD in this patient population are not well known. METHODS: We reviewed the medical files of all CF patients who presented with symptomatic C. difficile infection from January 1998 to December 2004 and compared the incidence, clinical aspects, severity of disease, and clinical outcome between non-transplanted and transplanted CF patients. RESULTS: Between 1998 and 2004, 106 adult CF patients were followed at our clinic. Forty-nine patients underwent lung transplantation; 15 before 1998 and 34 after 1998. The incidence density of CDAD was higher in transplanted CF patients as compared with non-transplanted CF patients (24.2 vs. 9.5 episodes/100,000 patient-days; risk ratio: 2.93 [1.41-6.08]; P=0.0044). Diarrhea was a very frequent feature, but was notably absent in 20% of the cases. Rates of moderate and severe colitis were similar in both groups. However, only transplanted patients developed complicated colitis. CT scan and endoscopy were performed more frequently in the transplant group. Two transplant recipients died because of CDAD. CONCLUSION: CF patients who undergo lung transplantation are at a higher risk of developing CDAD and seem to present more often atypical and/or complicated disease. CDAD should be part of the differential diagnosis in case of digestive symptoms, even in the absence of diarrhea, and requires early treatment.

Outbreak of hospital-adapted clonal complex-17 vancomycin-resistant Enterococcus faecium strain in a haematology unit: role of rapid typing for early control.

OBJECTIVES: To describe the investigation and molecular characterization of a vancomycin-resistant Enterococcus faecium (VREF) strain responsible for a nosocomial outbreak in the haematology unit of a tertiary-care university hospital. PATIENTS AND METHODS: Two patients admitted to the haematology unit developed infection/colonization with VREF over a 3 month period when compared with none in the 2 previous years. On the basis of the identification of a clonal link between these two strains, weekly rectal screening was implemented for all patients in the haematology unit and contact precautions were extended to VREF carriers. In the following 6 month period, 11 patients colonized with VREF were detected. No further case was detected in the following 1 year period. RESULTS: VREF isolates from the haematology unit carried the vanA gene and were multiresistant to antimicrobial agents, including high-level resistance to vancomycin, teicoplanin and ampicillin. This resistance profile restricted the choice of antimicrobial therapy to linezolid or investigational drugs such as tigecycline. Molecular analysis showed that 11 of 13 (85%) VREF isolates belonged to pandemic clonal complex-17 carrying the esp and hyl virulence genes. CONCLUSIONS: Rapid typing and infection control measures, including early reinforcement of barrier precautions combined with weekly rectal surveillance cultures, were followed by control of nosocomial spread of this VREF clone.