Epidemiology of the Staphylococcus aureus CA-MRSA USA300 in Belgium.

The methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 8 Panton-Valentine toxin (PVL)-positive USA300 clone has a worldwide distribution. The USA300 North American (NA) variant, harbouring the arginine catabolic mobile element (ACME), is predominant in the USA while the Latin American (LV) variant is predominant in Northern South America. Both variants have failed to become endemic in Europe. We examined here the epidemiology of the USA300 clone in Belgium from 2006 to 2019. A total of 399 clonal complex 8 PVL-positive MRSA isolates received between 2006 and 2019 by the Belgian National Reference Laboratory for S. aureus were investigated for the presence of ACME. Selected ACME-positive (n=102) and ACME-negative (n=16) isolates were sequenced, characterized for the presence of several resistance and virulence molecular markers and subjected to phylogenetic analysis. A total of 300 isolates were USA300-NA (ACME-positive), while only 99 were ACME-negative. Most USA300-NA interspersed in the phylogeny analysis with isolates from other countries, suggesting multiple introductions. However, two big clades were maintained and spread over a decade, peaking between 2010 and 2017 to finally decrease. Few ACME-negative isolates, mainly related to trips to South America, were identified as USA300-LV. The remaining ACME-negative isolates were ST8 SCCmec IVb or ST923 SCCmec IVa (COL923). Two clades of the USA300-NA clone have successfully spread in Belgium, but seem to currently decrease. Related South American variants have been detected for the first time in Belgium, including the emerging COL923 clone.

Multidrug resistance of the extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolated in Tizi-Ouzou (Algeria).

The emergence and spread of multidrug-resistant bacteria is a major public health concern. This study sought to investigate the phenotypic and genotypic characteristics of clinical isolates of ESBL-producing Klebsiella pneumoniae, at University Hospital of Tizi-Ouzou.  Antibiotic susceptibility testing of the strains was carried out by the disc diffusion method, the ESBL production was screening by the Double Disc Synergy Test and  confirmed by the Phenotypic Confirmatory Disc Diffusion Test. Genomic DNA was extracted using the  Qiagen DNeasy Blood & Tissue Kit  mini kit (Qiagen) according to the manufacturer's instructions.PCR targeting the genes  blaCTX-M, blaTEM, blaSHV, blaVEB, blaGES, blaPER, blaBEL, blaVIM, blaIMP, blaKPC, blaNDM and blaOXA48 was performed. A CTX-M PCR-based grouping method was carried out using primers specific to the groups 1, 2 and 9. Conjugative transfer of plasmids was carried out using sodium azide-resistant recipient strain Escherichia coli K12. The phylogenetic relationship was determined by ERIC-PCR. All strains of K. pneumoniae tested shared ESBL producer's genes belonging to the CTX-M group 1. These strains showed a high level of resistance to ß-lactams, aminoglycosides, fluoroquinolones and trimethoprim/ sulfamethoxazole. Resistance to fosfomycin was also detected in one strain of K. pneumoniae. Only one carbapenem-resistant strain was isolated. Phylogenetic analysis showed 49 different genetic profiles of K. pneumoniae strains, showing the absence of clonality. This study revealed a high prevalence of ESBL belonging to the CTX-M group 1 in K. pneumoniae tested. The emergence of resistance to carbapenem and fosfomycin, could seriously limits the therapeutic choices options.

Atopobium vaginae intrapartum bacteremia: A case report with a literature review.

Atopobium vaginae is an anaerobic Gram-positive bacterium recognized as a causative agent of bacterial vaginosis and associated with preterm delivery. Invasive infection and bacteremia have been rarely reported. We describe the case of a woman expecting her firstborn child who presented with a A. vaginae bacteremia during labor. Identification was performed using 16S rRNA gene sequencing. Both maternal and fetal outcomes were favorable due to the maternal treatment with amoxicillin-clavulanic acid. We identified three other cases in the literature with different fetal outcome. The genetic diversity of A. vaginae should be further explored in order to reveal potential strains with differential pathogenic potential.

National surveillance of Staphylococcus epidermidis recovered from bloodstream infections in Belgian hospitals.

OBJECTIVES: The objectives of this study were: (i) to determine the species diversity of CoNS isolated from bloodstream infections collected during a national surveillance study; and (ii) to examine the antimicrobial resistance and genomic diversity among Staphylococcus epidermidis isolates. METHODS: Eighty CoNS were identified by MALDI-TOF. Antimicrobial resistance determination, molecular characterization of resistance and virulence genes, and molecular typing were performed for S. epidermidis isolates. RESULTS: The majority (76%) of CoNS were identified as S. epidermidis. Among these S. epidermidis, 77% were resistant to methicillin [methicillin-resistant S. epidermidis (MRSE)] and showed multiresistance to other antimicrobials. Genes implicated in resistance were erm(C), erm(A) and msr(A) for erythromycin, aacA-aphD and aadC for aminoglycosides, tet(K) for tetracycline and mupA for high-level resistance to mupirocin. Molecular typing showed that 34/40 MRSE isolates (85%) belonged to clonal complex (CC) 2 that could be subdivided into CC2-I (ST2) and CC2-II (ST5, ST59 and ST88). In contrast, methicillin-susceptible S. epidermidis displayed high genomic diversity. The majority (70%) of S. epidermidis isolates contained an icaA or arcA gene. The icaA gene was found in the CC2-I subgroup, whereas arcA was more common in methicillin-susceptible S. epidermidis. CONCLUSIONS: S. epidermidis was frequently recovered among CoNS isolated from bloodstream infections with a high proportion of MRSE being multiresistant. A large number of S. epidermidis belonged to CC2, a clone that is disseminated worldwide. More studies are needed to understand its clonal evolutionary success.

Prospective Two-Center Comparison of Three Chromogenic Agars for Methicillin-Resistant Staphylococcus aureus Screening in Hospitalized Patients.

Three chromogenic media, chromID MRSA SMART (SMART), chromID MRSA first generation (chromID), and Brilliance MRSA (OX2), were evaluated for methicillin-resistant Staphylococcus aureus (MRSA) screening using 1,220 samples. The sensitivity at 24 h was significantly better with the SMART agar (66.4%) than that with chromID agar (50.5%). Enrichment and incubation until 48 h are still needed for an optimal yield.

Evaluation of the automated Vitek 2 system for detection of various mechanisms of macrolide and lincosamide resistance in Staphylococcus aureus.

We evaluated the performance of the automated Vitek 2 system against disk diffusion for susceptibility testing of Staphylococcus aureus strains showing various resistance mechanisms to macrolides and lincosamides (ML). The Vitek 2 system showed 100% concordance with the D-zone test in detection of the most common resistance mechanisms to ML, including methylase and efflux systems.

Performance of the Verigene Gram-negative blood culture assay for rapid detection of bacteria and resistance determinants.

Nonduplicate blood cultures that were positive for Gram-negative bacilli (n = 125) were tested by the Verigene Gram-negative blood culture (BC-GN) assay; 117 (90.7%) isolates were members of the panel. For identification and resistance markers, the agreements with routine methods were 97.4% (114/117) and 92.3% (12/13). The BC-GN assay is a rapid and accurate tool for the detection of pathogens from blood cultures and could be integrated alongside conventional systems to enable faster patient management, but the clinical benefits should be further evaluated.

Genetic diversity among methicillin-resistant Staphylococcus aureus isolates carrying the mecC gene in Belgium.

OBJECTIVES: A mecA homologue gene, named mecC, has been reported in methicillin-resistant Staphylococcus aureus (MRSA) isolates from humans and from diverse animal species. We investigated the proportion, and the phenotypic and genotypic characteristics, of mecC-carrying MRSA recovered from humans in Belgium. METHODS: A total of 4869 S. aureus isolates, collected by the National Reference Centre from 2003 to 2012, were retrospectively analysed for the presence of mecC. The mecC-carrying MRSA isolates were tested for phenotypic resistance and the presence of toxin genes. Genotyping was performed using spa typing and multilocus sequence typing. RESULTS: Nine S. aureus isolates, mecA negative but cefoxitin resistant (MIC 16-64 mg/L), were found to carry the mecC gene. Among these, eight showed resistance to oxacillin (MIC 4-64 mg/L). These isolates remained fully susceptible to all non-ß-lactam antimicrobials. Although the proportion of mecC-carrying MRSA in Belgium was low (<1% per year), mecC-MRSA were assigned to three distinct genetic lineages corresponding to clonal complex (CC) 130, CC49 and CC1943. CONCLUSIONS: This first Belgian nationwide analysis showed a low occurrence of mecC-MRSA. Further studies should be conducted to better understand the reservoirs and risk factors for mecC-MRSA acquisition.

Fourth Belgian multicentre survey of antibiotic susceptibility of anaerobic bacteria.

OBJECTIVES: To collect recent data on the susceptibility of anaerobes to antimicrobial agents with known activity against anaerobes, and to compare them with results from previous Belgian multicentre studies. METHODS: Four hundred and three strict anaerobic clinical isolates were prospectively collected from February 2011 to April 2012 in eight Belgian university hospitals. MICs were determined by one central laboratory for 11 antimicrobial agents using Etest methodology. RESULTS: According to EUCAST breakpoints, >90% of isolates were susceptible to amoxicillin/clavulanate (94%), piperacillin/tazobactam (91%), meropenem (96%), metronidazole (92%) and chloramphenicol (98%), but only 70% and 40% to clindamycin and penicillin, respectively. At CLSI recommended breakpoints, only 71% were susceptible to moxifloxacin and 79% to cefoxitin. MIC50/MIC90 values for linezolid and for tigecycline were 1/4 and 0.5/4 mg/L, respectively. When compared with survey data from 2004, no major differences in susceptibility profiles were noticed. However, the susceptibility of Prevotella spp. and other Gram-negative bacilli to clindamycin decreased from 91% in 1993-94 and 82% in 2004 to 69% in this survey. Furthermore, the susceptibility of clostridia to moxifloxacin decreased from 88% in 2004 to 66% in 2011-12 and that of fusobacteria from 90% to 71%. CONCLUSIONS: Compared with previous surveys, little evolution was seen in susceptibility, except a decline in activity of clindamycin against Prevotella spp. and other Gram-negative bacteria, and of moxifloxacin against clostridia. Since resistance was detected to all antibiotics, susceptibility testing of anaerobic isolates is indicated in severe infections to confirm appropriateness of antimicrobial therapy.

Genetic characteristics and antimicrobial resistance of Staphylococcus epidermidis isolates from patients with catheter-related bloodstream infections and from colonized healthcare workers in a Belgian hospital.

BACKGROUND: Staphylococcus epidermidis is a pathogen that is frequently encountered in the hospital environment. Healthcare workers (HCWs) can serve as a reservoir for the transmission of S. epidermidis to patients. METHODS: The aim of this study was to compare and identify differences between S. epidermidis isolated from 20 patients with catheter-related bloodstream infections (CRBSIs) and from the hands of 42 HCWs in the same hospital in terms of antimicrobial resistance, biofilm production, presence of the intercellular adhesion (ica) operon and genetic diversity (pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and staphylococcal cassette chromosome (SCC) mec typing). RESULTS: S. epidermidis isolates that caused CRBSI were resistant to significantly more non-betalactam drugs than were isolates collected from HCWs. Among the 43 mecA positive isolates (26 from HCWs), the most frequent SCCmec type was type IV (44%). The ica operon was significantly more prevalent in CRBSI isolates than in HCWs (P < 0.05). Weak in vitro biofilm production seemed to correlate with the absence of the ica operon regardless of the commensal or pathogenic origin of the isolate. The 62 isolates showed high diversity in their PFGE patterns divided into 37 different types: 19 harbored only by the CRBSI isolates and 6 shared by the clinical and HCW isolates. MLST revealed a total of ten different sequence types (ST). ST2 was limited to CRBSI-specific PFGE types while the "mixed" PFGE types were ST5, ST16, ST88 and ST153. CONCLUSION: One third of CRBSI episodes were due to isolates belonging to PFGE types that were also found on the hands of HCWs, suggesting that HCW serve as a reservoir for oxacillin resistance and transmission to patients. However, S. epidermidis ST2, mecA-positive and icaA-positive isolates, which caused the majority of clinically severe CRBSI, were not recovered from the HCW's hands.

Evaluation of Clearview Exact PBP2a, a new immunochromatographic assay, for detection of low-level methicillin-resistant Staphylococcus aureus (LL-MRSA).

We evaluated the performance of a new immunochromatographic assay (ICA), the Clearview Exact PBP2a, for rapid detection of penicillin-binding protein 2a (PBP2a) in a challenge set of Staphylococcus aureus strains showing MICs to oxacillin of ≤16 mg/liter. The sensitivity and specificity of the ICA were 96.6% and 100%, respectively.

Multicentre interlaboratory analysis of routine susceptibility testing with a challenge panel of resistant strains.

OBJECTIVES: In order to elaborate a new national challenge panel of resistant Gram-negative bacilli and Gram-positive cocci strains for the validation of routine antimicrobial susceptibility testing (AST) methods, an interlaboratory evaluation was organised. METHODS: The results of 12 well-characterised multidrug-resistant strains tested by nine laboratories using local disk diffusion (DD) and automated AST (AUST) methods were compared with the reference broth microdilution method. RESULTS: Overall categorical agreement ranged from 70% to 100% both for DD and AUST and was >90% for all but one strain for all antibiotics. CONCLUSION: Our multicentre AST study showed good reproducibility and the panel can be used as national resistant reference strains for routine AST validation.

Epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) among residents of nursing homes in Belgium.

OBJECTIVES: A national survey was conducted to determine the prevalence, risk factors and molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) carriage among nursing home (NH) residents in Belgium. METHODS: A random stratified, cross-sectional prevalence survey was conducted in NH residents who were screened for MRSA carriage by multisite enriched culture. Characteristics of NHs and residents were collected by a questionnaire survey and analysed by two-stage logistic regression modelling. MRSA isolates were genotyped by PFGE, staphylococcal cassette chromosome mec (SCCmec) typing, multilocus sequence typing (MLST) and resistance genes. RESULTS: Of 2953 residents screened in 60 NHs, 587 (19.9%) were MRSA carriers. Risk factors included hospital contact, antibiotic exposure, impaired mobility and skin lesions at the resident level, and lack of MRSA surveillance, lack of antibiotic therapeutic formulary and the combination of less-developed infection control activities and a high ratio of physicians to residents at the institution level. MRSA isolates showed eight major types, three of which were predominant: B2-ST45-SCCmec IV (49%; where ST stands for sequence type); A21-ST8-SCCmec IV (13%); and A20-ST8-SCCmec IV (10%). Each was recovered in 55, 21 and 25 NHs, respectively. The geographical distribution of NH genotypes paralleled that of acute-care hospitals. CONCLUSIONS: A high prevalence of MRSA carriage in NH residents was associated with hospital care, co-morbidities and less-developed coordination of institutional care. The predominant MRSA strains from NH residents and hospitalized patients of the same area were identical. Strengthening and coordination of MRSA surveillance and control activities are warranted within and between NHs and hospitals.

Prevalence of multidrug-resistant organisms in nursing homes in Belgium in 2015.

OBJECTIVES: Following two studies conducted in 2005 and 2011, a third prevalence survey of multidrug-resistant microorganisms (MDRO) was organised in Belgian nursing homes (NHs) using a similar methodology. The aim was to measure the prevalence of carriage of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), extended-spectrum ß-lactamase producing Enterobacteriaceae (ESBLE) and carbapenemase-producing Enterobacteriaceae (CPE) in NH residents. Risk factors for MDRO carriage were also explored. METHODS: Up to 51 randomly selected residents per NH were screened for MDRO carriage by trained local nurses between June and October 2015. Rectal swabs were cultured for ESBLE, CPE and VRE, while pooled samples of nose, throat and perineum and chronic wound swabs were obtained for culture of MRSA. Antimicrobial susceptibility testing, molecular detection of resistance genes and strain genotyping were performed. Significant risk factors for MDRO colonization MDRO was determined by univariate and multivariable analysis. RESULTS: Overall, 1447 residents from 29 NHs were enrolled. The mean weighted prevalence of ESBLE and MRSA colonization was 11.3% and 9.0%, respectively. Co-colonization occurred in 1.8% of the residents. VRE and CPE carriage were identified in only one resident each. Impaired mobility and recent treatment with fluoroquinolones or with combinations of sulphonamides and trimethoprim were identified as risk factors for ESBLE carriage, while for MRSA these were previous MRSA carriage/infection, a stay in several different hospital wards during the past year, and a recent treatment with nitrofuran derivatives. Current antacid use was a predictor for both ESBL and MRSA carriage. CONCLUSIONS: In line with the evolution of MRSA and ESBL colonization/infection in hospitals, a decline in MRSA carriage and an increase in ESBLE prevalence was seen in Belgian NHs between 2005 and 2015. These results show that a systemic approach, including surveillance and enhancement of infection control and antimicrobial stewardship programs is needed in both acute and chronic care facilities.

Evaluation of new Vitek 2 card and disk diffusion method for determining susceptibility of Staphylococcus aureus to oxacillin.

Detection of methicillin resistance in Staphylococcus aureus is a challenge, especially low-level resistance, which is often misdiagnosed. The aim of this study was to compare the diagnostic accuracies of the automated Vitek 2 system and disk diffusion tests, using cefoxitin and moxalactam, for the detection of methicillin resistance in S. aureus strains. Four sets of genotypically diverse isolates were selected from a national reference collection, including mecA-negative S. aureus isolates (n = 56), hospital-acquired (n = 88) and community-acquired (n = 40) S. aureus isolates, and heterogeneous methicillin-resistant S. aureus isolates (n = 29). Oxacillin susceptibility was tested by the Vitek 2 system with the AST P549 card and by disk diffusion methods using 10, 30, and 60 microg cefoxitin and 30 microg moxalactam. Oxacillin resistance was confirmed by PCR for the mecA gene. The overall sensitivities for oxacillin resistance detection were 97.5% for the Vitek 2 automated system, 98.7% for 60-microg cefoxitin and moxalactam disk diffusion, and 99.6% for 10- and 30-microg cefoxitin disks, respectively. Methicillin-susceptible S. aureus isolates were correctly reported as susceptible by all methods. The median times for methicillin testing were 7 h for the Vitek 2 system versus 24 h for disk diffusion methods. In conclusion, the cefoxitin and moxalactam disk diffusion methods and the Vitek 2 automated system are highly accurate methods for methicillin resistance detection, including a range of representative Belgian methicillin-resistant S. aureus strains and unusual strains exhibiting cryptic or low-level oxacillin resistance.

Evaluation of disc diffusion methods and Vitek 2 automated system for testing susceptibility to mupirocin in Staphylococcus aureus.

OBJECTIVES: To compare the performance of the automated Vitek 2 system, the disc diffusion method and a home-made mupirocin screen agar (MSA) to detect mupirocin resistance in methicillin-resistant Staphylococcus aureus (MRSA) isolates. METHODS: A total of 125 MRSA isolates were tested. The level of mupirocin resistance was determined by agar dilution and Etest techniques (gold standard), by the Vitek 2 system, on MSA (Mueller-Hinton + mupirocin 4 mg/L) and with the disc diffusion method using 10 microg mupirocin Neo-Sensitabs (MUP-10) and mupirocin paper discs of 5, 20 and 200 microg (MUP-5, MUP-20 and MUP-200). High-level mupirocin resistance (HLMR) was confirmed by PCR for the mupA gene. RESULTS: Thirty-two MRSA isolates showed HLMR (MIC > or = 512 mg/L) and harboured the mupA gene, 39 strains showed low-level mupirocin resistance (LLMR) (8-32 mg/L) without the mupA gene and 54 were susceptible without the mupA gene. The sensitivity and the specificity of the Vitek 2 system and the screening medium (MSA) for the detection of mupirocin resistance was 100%. The diffusion method using 5 and 10 microg discs demonstrated a sensitivity of 100% and a specificity of 98.1% and 100%, respectively. Using interpretative criteria of 6 and 17 mm, the MUP-20 disc showed the best classification concordance with reference methods. CONCLUSIONS: The diffusion method using low-content discs or the Vitek 2 microdilution system showed excellent agreement with MICs and PCR results to separate mupirocin-susceptible from -resistant MRSA strains. Disc diffusion with MUP-20 or combined use of low and high mupirocin content discs enabled the classification of susceptibility categories (susceptible, LLMR and HLMR) but required overnight incubation compared with 12 h for the Vitek 2 system.

Bacteria from Animals as a Pool of Antimicrobial Resistance Genes.

Antimicrobial agents are used in both veterinary and human medicine. The intensive use of antimicrobials in animals may promote the fixation of antimicrobial resistance genes in bacteria, which may be zoonotic or capable to transfer these genes to human-adapted pathogens or to human gut microbiota via direct contact, food or the environment. This review summarizes the current knowledge of the use of antimicrobial agents in animal health and explores the role of bacteria from animals as a pool of antimicrobial resistance genes for human bacteria. This review focused in relevant examples within the ESC(K)APE (Enterococcus faecium, Staphylococcus aureus, Clostridium difficile (Klebsiella pneumoniae), Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae) group of bacterial pathogens that are the leading cause of nosocomial infections throughout the world.

Multicentre evaluation of the BYG Carba v2.0 test, a simplified electrochemical assay for the rapid laboratory detection of carbapenemase-producing Enterobacteriaceae.

Rapid detection of carbapenemase-producing Enterobacteriaceae (CPE) represents a major challenge for microbiology laboratories. We evaluated the BYG Carba v2.0 using a simplified protocol, which detects CPE in less than 30 minutes. This new procedure reduces the hands-on-time from 5 to one minute and only requires a limited amount of material (one to three colonies) thereby preventing the need for subculturing bacterial isolates to reach a larger amount of pure biomass. This multicentre study involved four European reference laboratories. For the 1181 isolates tested across four centres, BYG Carba v2.0 yielded overall sensitivity and specificity of 96.3% (CI95: 94.5-97.5) and 99.7% (CI95: 98.6-100) respectively. Considering only the 670 consecutive isolates tested prospectively, the BYG Carba v2.0 displayed overall positive and negative predictive values of 99.7% (CI95: 95.4-98.9) and 97.5% (CI95: 94.9-98.8). Regarding time to positivity, 85% of CPE detected were positive within ten minutes. The BYG Carba v2.0 is a new highly simplified, rapid and accurate electrochemical assay discriminating between CPE and non-CPE in less than 30 min. The real-time quantified signal allows objective and traceable interpretation of the results.