Subulura eliseae sp. n. (Ascaridida: Subuluroidea), a parasite of Marmosa spp. from Amazon rainforest, Brazil.

The parasite biodiversity of mouse opossums in Brazil remains incompletely explored. We describe a new species of Subulura (Ascaridida: Subuluroidea) from the large intestine of the white-bellied woolly mouse opossum, Marmosa constantiae, based on the results of light and scanning electron microscopy (SEM). We also partially sequenced the mitochondrial cytochrome c oxidase I (MT-CO1) gene of the new species, using molecular phylogenetic analyses to determine its relationships within the Subuluroidea superfamily. As molecular data on subuluroid species are extremely limited, few inferences could be drawn from our phylogenies. Our SEM observations showed the detailed morphology of the cephalic extremity, precloacal pseudo-sucker, caudal papillae, phasmids and vulva. Subulura eliseae sp. n. differs from the other four Subulura parasites species of marsupials by the number of caudal papillae and the structure dimensions, and size of the spicule. Moreover, S. eliseae sp. n. has ten pairs of caudal papillae, which is unique compared to other species. We present morphometric and molecular data on this new species, contributing to future studies on subuluroids.

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.

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.

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.

Pan-genome multilocus sequence typing and outbreak-specific reference-based single nucleotide polymorphism analysis to resolve two concurrent Staphylococcus aureus outbreaks in neonatal services.

We used a two-step whole genome sequencing analysis for resolving two concurrent outbreaks in two neonatal services in Belgium, caused by exfoliative toxin A-encoding-gene-positive (eta+) methicillin-susceptible Staphylococcus aureus with an otherwise sporadic spa-type t209 (ST-109). Outbreak A involved 19 neonates and one healthcare worker in a Brussels hospital from May 2011 to October 2013. After a first episode interrupted by decolonization procedures applied over 7 months, the outbreak resumed concomitantly with the onset of outbreak B in a hospital in Asse, comprising 11 neonates and one healthcare worker from mid-2012 to January 2013. Pan-genome multilocus sequence typing, defined on the basis of 42 core and accessory reference genomes, and single-nucleotide polymorphisms mapped on an outbreak-specific de novo assembly were used to compare 28 available outbreak isolates and 19 eta+/spa-type t209 isolates identified by routine or nationwide surveillance. Pan-genome multilocus sequence typing showed that the outbreaks were caused by independent clones not closely related to any of the surveillance isolates. Isolates from only ten cases with overlapping stays in outbreak A, including four pairs of twins, showed no or only a single nucleotide polymorphism variation, indicating limited sequential transmission. Detection of larger genomic variation, even from the start of the outbreak, pointed to sporadic seeding from a pre-existing exogenous source, which persisted throughout the whole course of outbreak A. Whole genome sequencing analysis can provide unique fine-tuned insights into transmission pathways of complex outbreaks even at their inception, which, with timely use, could valuably guide efforts for early source identification.

When you can't see the wood for the trees. Mucor circinelloides: A rare case of primary cutaneous zygomycosis.

A patient with refractory diffuse lymphoma treated for pulmonary invasive aspergillosis developed a concomitant primary cutaneous mucormycosis. The mucormycete was identified by sequencing as Mucor circinelloides. This case confirms the importance of a rapid pathogen diagnosis in immunocompromised patients and the usefulness of molecular methods for identification of rare fungal species.

Design and validation of a qPCR assay for accurate detection and initial serogrouping of Legionella pneumophila in clinical specimens by the ESCMID Study Group for Legionella Infections (ESGLI).

Prompt detection of Legionella pneumophila is essential for rapid investigation of legionellosis. Furthermore, as the majority of L. pneumophila infections are caused by serogroup 1 (sg1) strains, rapid identification of such strains can be critical in both routine and outbreak scenarios. The ESCMID Study Group for Legionella Infections (ESGLI) was established in 2012 and immediately identified as a priority the validation of a reliable, easy to perform and interpret, cost-effective qPCR assay to standardise the detection of L. pneumophila DNA amongst members. A novel L. pneumophila assay targeting the mip gene was designed and combined with previously published methodologies amplifying the sg1 marker (wzm) and the green fluorescent protein gene (gfp) internal process control. The resulting triplex assay was validated internationally on the three qPCR platforms used by the majority of European Legionella reference laboratories: ABI 7500 (Life Technologies), LightCycler 480 Instrument II (Roche) and Rotor-Gene Q (Qiagen). Clinical and EQA specimens were tested together with a large panel of strains (251 in total) to validate the assay. The assay proved to be 100% specific for L. pneumophila and sg1 DNA both in silico and in vitro. Efficiency values for mip and wzm assays ranged between 91.97 and 97.69%. Limit of detection values estimated with 95% confidence were adopted for mip and wzm assays on all three qPCR platforms. Inhibition was not observed. This study describes a robust assay that could be widely implemented to standardise the molecular detection of L. pneumophila among ESGLI laboratories and beyond.

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.

Validation of carbapenemase and extended-spectrum ß-lactamase multiplex endpoint PCR assays according to ISO 15189.

OBJECTIVES: To validate and accredit a set of three multiplex endpoint PCR assays, targeting the most important carbapenemase and minor extended-spectrum ß-lactamase (ESBL) resistance genes, according to the international ISO 15189 particular requirements for the quality and competence of medical laboratories. METHODS: Specific primers targeting ESBLs and carbapenemases were collected from the literature or designed internally. The multiplex PCRs were validated for sensitivity, specificity, intra- and inter-run reproducibility and accuracy by means of external quality control (EQC) using a collection of 137 characterized and referenced isolates. For each multiplex PCR assay, the presence of an extraction control ruled out false-negative results due to PCR inhibition or extraction faults. Amplicons were separated by capillary electrophoresis (QIAxcel system, Qiagen). The protocols and validation files were reviewed in the setting of an external audit conducted by the Belgian organization for accreditation (BELAC). RESULTS: Sensitivity, specificity and reproducibility for each targeted gene were 100%. All isolates from the three EQC panels were correctly identified by each PCR assay (accuracy 100%). The validation files were controlled by BELAC, and the PCR protocols were accepted as accredited according to ISO 15189. CONCLUSIONS: Three home-made multiplex PCRs targeting the major carbapenemases and four minor class A ESBL genes encountered in Gram-negative bacteria were accredited according to the ISO 15189 standards. This validation scheme could provide a useful model for laboratories aiming to accredit their own protocols.

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.

Analytical validation of a novel high multiplexing real-time PCR array for the identification of key pathogens causative of bacterial ventilator-associated pneumonia and their associated resistance genes.

OBJECTIVES: Rapid diagnosis and appropriate empirical antimicrobial therapy before the availability of conventional microbiological results is of pivotal importance for the clinical outcome of ventilator-associated pneumonia (VAP). We evaluated the VAPChip, a novel, closed cartridge molecular tool aiming to identify directly from clinical samples and within a working day the principal bacteria causative of VAP as well as clinically relevant ß-lactam resistance genes. METHODS: The Real-time Array PCR for Infectious Diseases (RAP-ID) is a novel technology that combines multiplex PCR with real-time microarray detection. The VAPChip is a closed cartridge kit adapted to the RAP-ID instrument that targets 13 key respiratory pathogens causative of VAP and 24 relevant antimicrobial resistance genes that mediate resistance to ß-lactam agents, including extended-spectrum cephalosporins and carbapenems. Analytical validation of the VAPChip was carried out blindly on a collection of 292 genotypically characterized bacterial reference and clinical isolates, including 225 isolates selected on the basis of their species identification and antimicrobial resistance profiles and 67 bacterial isolates belonging to the oropharyngeal flora not targeted by the array. RESULTS: The limit of detection of the assay lies between 10 and 100 genome copies/PCR and the dynamic range is five orders of magnitude permitting at least semi-quantitative reporting of the results. Sensitivity, specificity and negative and positive predictive values ranged from 95.8% to 100% for species identification and detection of resistance genes. CONCLUSIONS: VAPChip is a novel diagnostic tool able to identify resistant bacterial isolates by RAP-ID technology. The results of this analytical validation have to be confirmed on clinical specimens.

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.

Sequence-based typing of Legionella pneumophila serogroup 1 clinical isolates from Belgium between 2000 and 2010.

Sequence-based typing (SBT) is a discriminatory method widely used to genotype Legionella pneumophila strains. A total of 86 clinical L. pneumophila serogroup 1 (sg1) isolates, collected between January 2000 and December 2010 in the two Belgian National Reference Centres for Legionella pneumophila, were genotyped using the internationally standardised SBT protocol of the European Working Group for Legionella Infections (EWGLI). The isolates could be classified into 31 different sequence types (ST, index of diversity: 0.879). The obtained STs were submitted to the EWGLI SBT-database for L. pneumophila. In our study, ST47 (27.9%) and ST1 (19.8%) were the most frequently detected STs. The detected profiles were a combination of both frequently isolated and unique STs, and of both worldwide distributed and more local strains. Two STs, ST880 and ST881, were new to the EWGLI database. In conclusion, we characterised L. pneumophila sg1 isolates with the SBT method, and created a Belgian profile database that will be useful for future epidemiological studies.

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.

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.

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

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.

Trends in production of extended-spectrum beta-lactamases among Enterobacteriaceae of clinical interest: results of a nationwide survey in Belgian hospitals.

OBJECTIVES: to assess the frequency and diversity of extended-spectrum ß-lactamases (ESBLs) in Enterobacteriaceae isolates in Belgium. METHODS: during 2006 and 2008, non-duplicate clinical isolates of Enterobacteriaceae resistant to ceftazidime and/or cefotaxime were collected in 100 Belgian hospitals. ESBL production was confirmed by phenotypic and genotypic tests. MICs of 13 antimicrobial agents were determined by Etest. ESBL-encoding genes were identified by PCR sequencing and the bla(CTX-M) environment was characterized by PCR mapping. Selected isolates were genotyped by PFGE, multilocus sequence typing analysis and phylogenetic grouping by PCR. RESULTS: overall, 733 isolates were confirmed as ESBL producers. Carbapenems and temocillin were active against ≥ 95% of all tested isolates. Co-resistance to co-trimoxazole and to ciprofloxacin was found in almost 70% and 80% of the strains, respectively. Overall, Escherichia coli (49%), Enterobacter aerogenes (32%) and Klebsiella pneumoniae (9%) represented the most prevalent species. Isolates harboured predominantly TEM-24 (30.7%), CTX-M-15 (24.2%) and TEM-52 (12.1%). Compared with 2006, the proportion of CTX-M-type enzymes increased significantly in 2008 (54% versus 23%; P < 10(-6)), mostly linked to a rising proportion of CTX-M-15-producing E. coli. TEM-24 decreased (19% in 2008 versus 43% in 2006; P < 10(-6)) during the same period, while the prevalence of TEM-52 remained unchanged (10% in 2008 versus 14% in 2006; not significant). Over 80% of the CTX-M-15-producing E. coli isolates clustered into a single PFGE type and phylogroup B2, corresponding to the sequence type (ST) 131 clone. Intra- and inter-species gene dissemination (CTX-M-15, CTX-M-2 and CTX-M-9) and wide epidemic spread of the CTX-M-15-producing E. coli ST131 clone in several Belgian hospitals were observed. CONCLUSIONS: the rapid emergence of multiresistant CTX-M-15-producing E. coli isolates is of major concern and highlights the need for further surveillance in Belgium.

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

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.