M J Struelens

Publications of M J Struelens

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

  Authors: S Vandendriessche, M Hallin, B Catry, B Jans, A Deplano, C Nonhoff, S Roisin, R De Mendonça, M J Struelens, O Denis

  European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology. 03/2012;

  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 assessThis 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.

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

  Authors: S Roisin, C Laurent, C Nonhoff, A Deplano, M Hallin, B Byl, M J Struelens, O Denis

  European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology. 08/2011;

  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 swabsThe 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.

• Diversity of accessory genome of human and livestock-associated ST398 methicillin resistant Staphylococcus aureus strains.

  Authors: M Hallin, R De Mendonça, O Denis, A Lefort, F El Garch, P Butaye, K Hermans, M J Struelens

  Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases. 12/2010; 11(2):290-9.

  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) andMolecular 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. 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. 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. 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. 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.

• Evaluation of the Xpert MRSA assay for rapid detection of methicillin-resistant Staphylococcus aureus from nares swabs of geriatric hospitalized patients and failure to detect a specific SCCmec type IV variant.

  Authors: C Laurent, P Bogaerts, D Schoevaerdts, O Denis, A Deplano, C Swine, M J Struelens, Y Glupczynski

  European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology. 08/2010; 29(8):995-1002.

  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 theRapid 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.

• Molecular characterization of AmpC-producing Escherichia coli clinical isolates recovered at two Belgian hospitals.

  Authors: P Bogaerts, H Rodriguez-Villalobos, C Bauraing, A Deplano, C Laurent, C Berhin, M J Struelens, Y Glupczynski

  Pathologie-biologie. 11/2009;

  OBJECTIVE: To characterize the genetic determinants associated with an AmpC phenotype in clinical Escherichia coli isolates. METHODS: E. coli strains recovered at two Belgian hospitals between 2004OBJECTIVE: To characterize the genetic determinants associated with an AmpC phenotype in clinical Escherichia coli isolates. METHODS: E. coli strains recovered at two Belgian hospitals between 2004 and 2006 were selected on the basis of an AmpC-producing phenotype. Plasmid-mediated cephalosporinases coding genes and the sequence of chromosomal ampC genes were identified by PCR/sequencing. The isolates were submitted to phylotyping and genotyping analysis using rep-PCR (Diversilab) and PFGE. A novel chromosomal ampC gene was cloned. RESULTS: Eighty-three out of 6850 E. coli isolates were selected. Seventy-two isolates were found to overexpress their chromosomal cephalosporinases while 11 contained plasmid-mediated cephalosporinases. Among chromosomal AmpC overproducers, 12 were extended-spectrum AmpC (ESAC) expressing isolates which all displayed reduced susceptibility to cefepime. Cloning of a new ESAC allele suggested that L293P mutation was responsible of the extension of the hydrolysis spectrum to cefepime. AmpC overproducers, including ESAC producers, predominantly belonged to phylogenetic group A and B1, while plasmid-mediated AmpC-producing isolates preferentially belong to phylogroup B2 and D. According to rep-PCR, the majority of the E. coli isolates belonging to phylogroup A were clonally related which was further confirmed by PFGE for the 11 ESAC expressing isolates. CONCLUSIONS: Chromosomal AmpC overproduction was the most common resistance mechanism, and the occurrence of ESAC was found to be as frequent as plasmid-mediated cephalosporinases. The detection of a new ESAC allele, of an ESAC producing strain belonging to phylogroup D and the existence of a clonal relationship between ESAC producing strains underline the need for study of the clinical relevance of this mechanism of resistance.

• StaphVar-DNA microarray analysis of accessory genome elements of community-acquired methicillin-resistant Staphylococcus aureus.

  Authors: F El Garch, M Hallin, R De Mendonça, O Denis, A Lefort, M J Struelens

  The Journal of antimicrobial chemotherapy. 06/2009; 63(5):877-85.

  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 mobileOBJECTIVES: 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.

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

  Authors: M Hallin, O Denis, A Deplano, R De Ryck, S Crèvecoeur, S Rottiers, R De Mendonça, M J Struelens

  Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases. 08/2008; 14(7):659-69.

  National surveillance of healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) isolates by pulsed-field gel electrophoresis (PFGE) typing allowed identification of rarelyNational 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.

• Intensive care unit outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae controlled by cohorting patients and reinforcing infection control measures.

  Authors: C Laurent, H Rodriguez-Villalobos, F Rost, H Strale, J L Vincent, A Deplano, M J Struelens, B Byl

  Infection control and hospital epidemiology : the official journal of the Society of Hospital Epidemiologists of America. 07/2008; 29(6):517-24.

  OBJECTIVE: To describe an outbreak of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in the intensive care units (ICUs) of a hospital and the impact of routine and reinforcedOBJECTIVE: To describe an outbreak of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in the intensive care units (ICUs) of a hospital and the impact of routine and reinforced infection control measures on interrupting nosocomial transmission. DESIGN: Outbreak report. SETTING: A 31-bed intensive care department (composed of 4 ICUs) in a university hospital in Belgium. INTERVENTION: After routine infection control measures (based on biweekly surveillance cultures and contact precautions) failed to interrupt a 2-month outbreak of ESBL-producing K. pneumoniae, reinforced infection control measures were implemented. The frequency of surveillance cultures was increased to daily sampling. Colonized patients were moved to a dedicated 6-bed ICU, where they received cohorted care with the support of additional nurses. Two beds were closed to new admissions in the intensive care department. Meetings between the ICU and infection control teams were held every day. Postdischarge disinfection of rooms was enforced. Broad-spectrum antibiotic use was discouraged. RESULTS: Compared with a baseline rate of 0.44 cases per 1,000 patient-days for nosocomial transmission, the incidence peaked at 11.57 cases per 1,000 patient-days (October and November 2005; rate ratio for peak vs baseline, 25.46). The outbreak involved 30 patients, of whom 9 developed an infection. Bacterial genotyping disclosed that the outbreak was polyclonal, with 1 predominant genotype. Reinforced infection control measures lasted for 50 days. After the implementation of these measures, the incidence fell to 0.08 cases per 1,000 patient-days (rate ratio for after the outbreak vs during the outbreak, 0.11). CONCLUSION: These data indicate that, in an intensive care department in which routine screening and contact precautions failed to prevent and interrupt an outbreak of ESBL-producing K. pneumoniae, reinforced infection control measures controlled the outbreak without major disruption of medical care.

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

  Authors: A Deplano, O Denis, C Nonhoff, F Rost, B Byl, F Jacobs, V Vankerckhoven, H Goossens, M J Struelens

  The Journal of antimicrobial chemotherapy. 11/2007; 60(4):849-54.

  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 ofOBJECTIVES: 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.

• [The threat of antibiotic resistance: origin, impact and public health perspectives]

  Authors: M J Struelens, O Denis, H Rodriguez-Villalobos, M Hallin

  Revue médicale de Bruxelles. 10/2007; 28(4):381-4.

  Bacterial resistance to antibiotics is as old as the living planet, i.e. over 3 billion years ago when bacterial life emerged together with its biological warfare weapons, the antibiotics. BacteriaBacterial resistance to antibiotics is as old as the living planet, i.e. over 3 billion years ago when bacterial life emerged together with its biological warfare weapons, the antibiotics. Bacteria can readily develop a diverse array of antibiotic resistance mechanisms, including selection of mutants which produce modified low-affinity drug targets, exhibit reduced drug permeability or increased drug efflux. Another pathway is through acquisition of mobile genetic determinants of drug inactivating enzymes, target protection or bypass mechanisms. Antibiotic resistance has accelerated and spread across the community of human and animal bacteria following massive use of antibiotics for over half-a century in human and veterinary medicine and agriculture. Today, the increasing prevalence of resistance among many bacterial pathogens is no longer counter-balanced by the launching of innovative and active antibiotics. This imbalanced "arms race" undermines the escalating use of broad-spectrum antibiotics and increases the risk of therapeutic failure, drug side effets and excess costs of care. The accumulation of antibiotic resistance genes and their epidemic dissemination is fuelled by the selective pressure associated with excess and inappropriate use of antibiotics combined with insufficient infection prevention and control measures. Confronting this threat effectively requires new strategies to be implemented globally, nationally, and locally across all healthcare and agricultural sectors.

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

  Authors: C Theunissen, C Knoop, C Nonhoff, B Byl, M Claus, C Liesnard, M J Estenne, M J Struelens, F Jacobs

  Transplant infectious disease : an official journal of the Transplantation Society. 08/2007;

  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, theBackground. 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.

• National survey of molecular epidemiology of Staphylococcus aureus colonization in Belgian cystic fibrosis patients.

  Authors: A Vergison, O Denis, A Deplano, G Casimir, G Claeys, F DeBaets, K DeBoeck, N Douat, H Franckx, J Gigi, M Ieven, C Knoop, P Lebeque, F Lebrun, A Malfroot, F Paucquay, D Pierard, J Van Eldere, M J Struelens

  The Journal of antimicrobial chemotherapy. 06/2007; 59(5):893-9.

  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 smallOBJECTIVES: 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.

• Prospective evaluation of imipenem/EDTA combined disc and Etest for detection of metallo-beta-lactamase-producing Pseudomonas aeruginosa.

  Authors: L Bergès, H Rodriguez-Villalobos, A Deplano, M J Struelens

  The Journal of antimicrobial chemotherapy. 05/2007; 59(4):812-3.

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

  Authors: M Hallin, O Denis, A Deplano, R De Mendonça, R De Ryck, S Rottiers, M J Struelens

  The Journal of antimicrobial chemotherapy. 04/2007; 59(3):465-72.

  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 10OBJECTIVES: 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.

• Emergence and dissemination of multidrug resistant clones of Pseudomonas aeruginosa producing VIM-2 metallo-beta-lactamase in Belgium.

  Authors: A Deplano, H Rodriguez-Villalobos, Y Glupczynski, P Bogaerts, D Allemeersch, A Grimmelprez, G Mascart, L Bergès, B Byl, C Laurent, M J Struelens

  Euro surveillance : bulletin européen sur les maladies transmissibles = European communicable disease bulletin. 02/2007; 12(1):E070118.2.

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

  Authors: M Hallin, A Deplano, O Denis, R De Mendonça, R De Ryck, M J Struelens

  Journal of clinical microbiology. 02/2007; 45(1):127-33.

  Pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments has been used by the Belgian Reference Laboratory for Staphylococci for national hospital surveys ofPulsed-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.

  Authors: A Deplano, R De Mendonça, R De Ryck, M J Struelens

  Journal of clinical microbiology. 09/2006; 44(9):3236-44.

  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 includedA 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.

• High interlaboratory reproducibility of DNA sequence-based typing of bacteria in a multicenter study.

  Authors: M Aires de Sousa, K Boye, H de Lencastre, A Deplano, M C Enright, J Etienne, A Friedrich, D Harmsen, A Holmes, X.W. Huijsdens [......] J K Rasheed, E. Spalburg, B Strommenger, M J Struelens, F C Tenover, J Thomas, U Vogel, H Westh, J Xu, W Witte

  Journal of clinical microbiology. 03/2006; 44(2):619-21.

  Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureusCurrent DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without extensive harmonization of protocols for 30 blind-coded S. aureus DNA samples sent to 10 laboratories. Specialized software for automated sequence analysis ensured a common typing nomenclature.

• High interlaboratory reproducibility of DNA sequence-based typing of bacteria in a multicenter study

  Authors: M. ires-de-Sousa, K. Boye, Lencastre H. de, A. Deplano, M.C. Enright, J. Etienne, A. Friedrich, D. Harmsen, A. Holmes, X.W. Huijsdens [......] J.K. Rasheed, E. Spalburg, B. Strommenger, M.J. Struelens, F.C. Tenover, J. Thomas, U. Vogel, H. Westh, J. Xu, W. Witte

  J.Clin.Microbiol. 02/2006; 44.

  Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureusCurrent DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without extensive harmonization of protocols for 30 blind-coded S. aureus DNA samples sent to 10 laboratories. Specialized software for automated sequence analysis ensured a common typing nomenclature

• Polyclonal emergence and importation of community-acquired methicillin-resistant Staphylococcus aureus strains harbouring Panton-Valentine leucocidin genes in Belgium.

  Authors: O Denis, A Deplano, H De Beenhouwer, M Hallin, G Huysmans, M G Garrino, Y Glupczynski, X Malaviolle, A Vergison, M J Struelens

  The Journal of antimicrobial chemotherapy. 01/2006; 56(6):1103-6.

  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.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.