Emergence and spread of a mupirocin-resistant variant of the European epidemic fusidic acid-resistant impetigo clone of Staphylococcus aureus, Belgium, 2013 to 2023.

BackgroundAntimicrobial resistance to mupirocin and fusidic acid, which are used for treatment of skin infections caused by Staphylococcus aureus, is of concern.AimTo investigate resistance to fusidic acid and mupirocin in meticillin-susceptible S. aureus (MSSA) from community-acquired skin and soft tissue infections (SSTIs) in Belgium.MethodsWe collected 2013-2023 data on fusidic acid and mupirocin resistance in SSTI-associated MSSA from two large Belgian laboratories. Resistant MSSA isolates sent to the Belgian Staphylococci Reference Centre were spa-typed and analysed for the presence of the eta and etb virulence genes and the mupA resistance gene. In addition, we whole genome sequenced MSSA isolates collected between October 2021 and September 2023.ResultsMupirocin resistance increased between 2013 and 2023 from 0.5-1.5% to 1.7-5.6%. Between 2018 and 2023, 91.4% (64/70) of mupirocin-resistant isolates were co-resistant to fusidic acid. By September 2023, between 8.9% (15/168) and 10.1% (11/109) of children isolates from the two laboratories were co-resistant. Of the 33 sequenced isolates, 29 were sequence type 121, clonal and more distantly related to the European epidemic fusidic acid-resistant impetigo clone (EEFIC) observed in Belgium in 2020. These isolates carried the mupA and fusB genes conferring resistance to mupirocin and fusidic acid, respectively, and the eta and etb virulence genes.ConclusionWe highlight the spread of a mupirocin-resistant EEFIC in children, with a seasonal trend for the third quarter of the year. This is of concern because this variant is resistant to the two main topical antibiotics used to treat impetigo in Belgium.

Persistence of the Staphylococcus aureus epidemic European fusidic acid-resistant impetigo clone (EEFIC) in Belgium.

OBJECTIVES: In August 2018, a public health alert was issued in Belgium regarding clusters of impetigo cases caused by the epidemic European fusidic acid-resistant impetigo clone (EEFIC) of Staphylococcus aureus. As a result, the Belgian national reference centre (NRC) was commissioned to update the epidemiology of S. aureus causing community-onset skin and soft tissues infection (CO-SSTI) to assess the proportion of EEFIC among them. METHODS: For 1 year, Belgian clinical laboratories were asked to send their first three S. aureus isolated from CO-SSTI each month. Isolates were tested for antimicrobial susceptibility to oxacillin, mupirocin and fusidic acid. Resistant isolates were also spa typed and tested for the presence of the genes encoding the Panton-Valentine leucocidin, the toxic shock syndrome toxin and the exfoliatins A and B. MLST clonal complexes were deduced from the spa types. RESULTS: Among the 518 S. aureus strains analysed, 487 (94.0%) were susceptible to oxacillin. Of these, 79 (16.2%) were resistant to fusidic acid, of which 38 (48.1%) belonged to the EEFIC. EEFIC isolates were mostly isolated from young patients with impetigo and showed a seasonal late summer peak. CONCLUSIONS: These results suggest the persistence of EEFIC in Belgium. Furthermore, its prevalence may lead to reconsideration of the treatment guidelines for impetigo.

Incidence and clinical and microbiological features of invasive and probable invasive streptococcal group A infections in children and adults in the Brussels-Capital Region, 2005-2020.

Assess the incidence, risk factors, clinical and microbiological features, and outcome of both probable invasive and invasive group A Streptococcus (GAS) infections in children and adults in the BrusselsCapital Region between 2005 and 2020. A retrospective, multicentric study was performed in three university hospitals in Brussels. Patients were identified through the centralized laboratory information system. Epidemiological and clinical data were collected from patients' hospital records. A total of 467 cases were identified. Incidence has increased from 2.1 to 10.9/100,000 inhabitants between 2009 and 2019 in non-homeless adults while it was above 100/100,000 on homeless in years with available denominators. Most of GAS were isolated from blood (43.6%), and the most common clinical presentation was skin and soft tissue infections (42.8%). A third of all the patients needed surgery, a quarter was admitted to the intensive care unit, and 10% of the adult patients died. Wounds and chickenpox disease were the main risk factors for children. Tobacco, alcohol abuse, wounds or chronic skin lesion, being homeless, and diabetes were identified as major predisposing factors for adults. The most common emm clusters were D4, E4, and AC3; 64% of the isolates were theoretically covered by the 30-valent M-protein vaccine. The burden of invasive and probable invasive GAS infections is on the rise in the studied adult population. We identified potential interventions that could contribute to decrease this burden: appropriate care of wounds, specifically among homeless and patients with risk factors such as diabetes and systematic chickenpox vaccination for children.

External Quality Assessment of SARS-CoV-2 Sequencing: an ESGMD-SSM Pilot Trial across 15 European Laboratories.

This first pilot trial on external quality assessment (EQA) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequencing, initiated by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Genomic and Molecular Diagnostics (ESGMD) and the Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing. Ten samples with various viral loads were sent out to 15 clinical laboratories that had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centers were compared were the identification of (i) single nucleotide polymorphisms (SNPs) and indels, (ii) Pango lineages, and (iii) clusters between samples. The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to various depths (up to a 100-fold difference across centers). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignments. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data. The pilot EQA was overall a success. It was able to show the high quality of participating laboratories and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.

Characterization of hypervirulent Klebsiella pneumoniae isolates in Belgium.

Hypervirulent Klebsiella pneumoniae (hvKp) raised concern worldwide. We studied 22 hvKp clinical invasive isolates referred to the Belgian national reference laboratory between 2014 and 2020. Sixty-four percent of the isolates expressed K2 capsular serotype and belonged to 7 different MLST lineages, while 32% expressed K1 (all belonging to ST23) and were associated with liver abscesses. Primary extra-hepatic infections were reported in 36% and sepsis for 95% of the patients with 30% of deaths. Improved clinical and microbiological diagnostics are required as hvKp may represent an underestimated cause of community-acquired invasive infections in Belgium.

Consolidation of Clinical Microbiology Laboratories and Introduction of Transformative Technologies.

Clinical microbiology is experiencing revolutionary advances in the deployment of molecular, genome sequencing-based, and mass spectrometry-driven detection, identification, and characterization assays. Laboratory automation and the linkage of information systems for big(ger) data management, including artificial intelligence (AI) approaches, also are being introduced. The initial optimism associated with these developments has now entered a more reality-driven phase of reflection on the significant challenges, complexities, and health care benefits posed by these innovations. With this in mind, the ongoing process of clinical laboratory consolidation, covering large geographical regions, represents an opportunity for the efficient and cost-effective introduction of new laboratory technologies and improvements in translational research and development. This will further define and generate the mandatory infrastructure used in validation and implementation of newer high-throughput diagnostic approaches. Effective, structured access to large numbers of well-documented biobanked biological materials from networked laboratories will release countless opportunities for clinical and scientific infectious disease research and will generate positive health care impacts. We describe why consolidation of clinical microbiology laboratories will generate quality benefits for many, if not most, aspects of the services separate institutions already provided individually. We also define the important role of innovative and large-scale diagnostic platforms. Such platforms lend themselves particularly well to computational (AI)-driven genomics and bioinformatics applications. These and other diagnostic innovations will allow for better infectious disease detection, surveillance, and prevention with novel translational research and optimized (diagnostic) product and service development opportunities as key results.

Large diversity of linezolid-resistant isolates discovered in food-producing animals through linezolid selective monitoring in Belgium in 2019.

BACKGROUND: Linezolid is a critically important antibiotic used to treat human infections caused by MRSA and VRE. While linezolid is not licensed for food-producing animals, linezolid-resistant (LR) isolates have been reported in European countries, including Belgium. OBJECTIVES: To: (i) assess LR occurrence in staphylococci and enterococci isolated from different Belgian food-producing animals in 2019 through selective monitoring; and (ii) investigate the genomes and relatedness of these isolates. METHODS: Faecal samples (n = 1325) and nasal swab samples (n = 148) were analysed with a protocol designed to select LR bacteria, including a 44-48 h incubation period. The presence of LR chromosomal mutations, transferable LR genes and their genetic organizations and other resistance genes, as well as LR isolate relatedness (from this study and the NCBI database) were assessed through WGS. RESULTS: The LR rate differed widely between animal host species, with the highest rates occurring in nasal samples from pigs and sows (25.7% and 20.5%, respectively) and faecal samples from veal calves (16.4%). WGS results showed that LR determinants are present in a large diversity of isolates circulating in the agricultural sector, with some isolates closely related to human isolates, posing a human health risk. CONCLUSIONS: LR dedicated monitoring with WGS analysis could help to better understand the spread of LR. Cross-selection of LR transferable genes through other antibiotic use should be considered in future action plans aimed at combatting antimicrobial resistance and in future objectives for the rational use of antibiotics in a One Health perspective.

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.

Multi-step optimization of the filtration method for the isolation of Campylobacter species from stool samples.

The filtration method (FM) is the most effective isolation technique for Epsilobacteriaceae from stool samples. FM's different adaptations make it difficult to compare data between studies. This study was performed in three phases to optimize FM from a routine laboratory perspective. In July-September 2014 (part I), FM was performed on Mueller-Hinton agar containing 5% sheep blood and Columbia agar containing 5% sheep blood. In July 2016 (part II), FM was performed using 0.60-µm pore size polycarbonate filters (0.6-PC filter) and 0.45-µm pore size cellulose acetate filters (0.45-AC filter); in January 2018 (part III), the addition of hydrogen to incubators was studied. On 1146 stools analyzed in part I, the positive samples that showed no growth on the Butzler medium (n = 22/72, 30.6%) had improved growth of Epsilobacteriaceae when using the Columbia instead of the Mueller-Hinton medium (21/22 strains vs. 11/22, p < 0.05). In part II, on 718 stools, 91 strains grew with FM (12.7%), more with 0.6-PC filter (90/91) than with 0.45-AC filter (44/91) (p < 0.05). In part III, 578 stools were cultured, 98 Epsilobacteriaceae strains grew with FM, and 7% hydrogen finding significantly more Epsilobacteriaceae than without hydrogen (90/98, 91.8%, vs. 72/98, 73.5%; p < 0.05). The use of a Columbia medium containing 5% sheep blood with 0.6-PC filters incubated at 37 °C in a 7% hydrogen-enriched atmosphere led to an almost fourfold increase in the isolation rate of Epsilobacteriaceae among the studied combinations. Reference centers for Campylobacter should use standardized protocols to enable the comparison of prevalence in space and time.

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.

Comparison of the antibiotic activities of Daptomycin, Vancomycin, and the investigational Fluoroquinolone Delafloxacin against biofilms from Staphylococcus aureus clinical isolates.

Biofilm-related infections remain a scourge. In an in vitro model of biofilms using Staphylococcus aureus reference strains, delafloxacin and daptomycin were found to be the most active among the antibiotics from 8 different pharmacological classes (J. Bauer, W. Siala, P. M. Tulkens, and F. Van Bambeke, Antimicrob. Agents Chemother. 57:2726-2737, 2013, doi:10.1128/AAC.00181-13). In this study, we compared delafloxacin to daptomycin and vancomycin using biofilms produced by 7 clinical strains (S. aureus epidemic clones CC5 and CC8) in order to rationalize the differences observed between the antibiotics and strains. The effects of the antibiotics on bacterial viability (resazurin reduction assay) and biomass (crystal violet staining) were measured and correlated with the proportion of polysaccharides in the matrix, the local microenvironmental pH (micro-pH), and the antibiotic penetration in the biofilm. At clinically meaningful concentrations, delafloxacin, daptomycin, and vancomycin caused a ≥25% reduction in viability against the biofilms formed by 5, 4, and 3 strains, respectively. The antibiotic penetration within the biofilms ranged from 0.6 to 52% for delafloxacin, 0.2 to 10% for daptomycin, and 0.2 to 1% for vancomycin; for delafloxacin, this was inversely related to the polysaccharide proportion in the matrix. Six biofilms were acidic, explaining the high potency of delafloxacin (lower MICs at acidic pH). Norspermidine and norspermine (disassembling the biofilm matrix) drastically increased delafloxacin potency and efficacy (50% reduction in viability for 6 biofilms at clinically meaningful concentrations) in direct correlation with its increased penetration within the biofilm, while they only modestly improved daptomycin efficacy (50% reduction in viability for 2 biofilms) and penetration, and they showed marginal effects with vancomycin. Delafloxacin potency and efficacy against biofilms are benefited by its penetration into the matrix and the local acidic micro-pH.

Detection of Herpesviridae in whole blood by multiplex PCR DNA-based microarray analysis after hematopoietic stem cell transplantation.

Viral infections are important causes of morbidity and mortality in patients after hematopoietic stem cell transplantation. The monitoring by PCR of Herpesviridae loads in blood samples has become a critical part of posttransplant follow-up, representing mounting costs for the laboratory. In this study, we assessed the clinical performance of the multiplex PCR DNA microarray Clart Entherpex kit for detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6) as a screening test for virological follow-up. Two hundred fifty-five blood samples from 16 transplanted patients, prospectively tested by routine PCR assays, were analyzed by microarray. Routine PCR detected single or multiple viruses in 42% and 10% of the samples, respectively. Microarray detected single or multiple viruses in 34% and 18% of the samples, respectively. Microarray results correlated well with CMV and EBV detections by routine PCR (kappa tests = 0.79 and 0.78, respectively), whereas a weak correlation was observed with HHV-6 (0.43). HHV-7 was also detected in 48 samples by microarray. In conclusion, the microarray is a reliable screening assay for a posttransplant virological follow-up to detect CMV and EBV infections in blood. However, positive samples must be subsequently confirmed and viral loads must be quantified by PCR assays. Limitations were identified regarding HHV-6 detection. Although it is promising, is easy to use as a first-line test, and allows a reduction in the cost of analysis without undue delay in the reporting of the final quantitative result to the clinician, some characteristics of this microarray should be improved, particularly regarding quality control and the targeted virus panel, such that it could then be used as a routine test.

High genetic diversity of methicillin-susceptible Staphylococcus aureus (MSSA) from humans and animals on livestock farms and presence of SCCmec remnant DNA in MSSA CC398.

OBJECTIVES: To investigate the genetic diversity of methicillin-susceptible Staphylococcus aureus (MSSA) carriage isolates from animals and humans on pig, veal, dairy, beef and broiler farms. METHODS: S. aureus isolates were genotyped using spa typing and multilocus sequence typing (MLST). Antimicrobial susceptibility phenotypes and genotypes were determined. The presence of staphylococcal cassette chromosome mec (SCCmec)-associated DNA was characterized by PCR and sequencing among isolates of clonal complex (CC) 398. RESULTS: Overall, 41 MSSA isolates in humans and 141 in animals were found, originating from all farm types. These MSSA were mainly assigned to CC398, CC1, CC5, CC9, CC30, CC97, CC133 and CC705/151. MSSA CC398 showed resistance to tetracycline, trimethoprim, macrolides and/or lincosamides, aminoglycosides, ciprofloxacin, spectinomycin and quinupristin/dalfopristin, whereas non-CC398 MSSA showed considerably less resistance. Three porcine MSSA CC398-t011 isolates harboured remnant DNA of a composite SCCmec V(5C2&5)c element that lacked the mec gene complex. This resulted from an MRSA-to-MSSA conversion due to recombination between the ccrC genes flanking the mec gene complex. The SCC remnant still contained an intact J1 region harbouring czrC and tet(K), encoding zinc and tetracycline resistance, respectively, thereby illustrating the capacity of S. aureus CC398 to adapt to different antibiotic selection pressures in the farming environment. Processes such as mec gene complex deletion probably contribute to the enormous diversity of SCC(mec) elements observed in staphylococci. CONCLUSIONS: MSSA CC398 precursors from which MRSA CC398 might (re)emerge were present on pig, veal and broiler farms, all of which are livestock sectors commonly known to be affected by MRSA CC398. The multiresistance phenotype of S. aureus CC398 appears to be independent of methicillin resistance.

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.

Surveillance of the respiratory syncytial virus outside infancy: impact of testing methods, a retrospective observational study.

Background: The European Medicines Agency has approved several vaccines to protect the elderly against respiratory syncytial virus (RSV) infections. However, differences in performance between antigen and PCR tests, especially in adults, can make monitoring RSV difficult. This study aims to assess the impact of the chosen diagnostic methods on the surveillance of RSV. Methods: RSV and influenza test results obtained from July 2022 to June 2023 in a consolidated clinical laboratory in Brussels, Belgium, were collected. These results included antigen tests, quadruplex PCR tests and viral cultures on respiratory samples. Epidemiological trends related to the age of patients and the diagnostic methods were analysed. Results: Among 14 761 RSV tests, the overall number of positive tests for infants until 1 year of age peaked on 5 November 2022 (67 per 7 days) whereas it peaked on 22 December 2022 for adults (33 per 7 days). Positive antigen tests peaked on 7 November 2022 (56 per 7 days) whereas positive PCRs peaked on 19 December 2022 (36 per 7 days). Nevertheless, the positivity rate of RSV PCRs had peaked 1 month previously. Infants were mainly diagnosed through antigen testing, contrary to older patients. The influenza epidemic was probably the cause of the increased use of a quadruplex PCR, leading to a delayed increase in the absolute number of PCRs positive for RSV. Conclusion: This study shows that the use of different diagnostic methods could lead to an erroneous representation of RSV epidemiology in adults due to the lack of sensitivity of antigen detection. RSV surveillance in the elderly should rely rather on molecular methods.

Multiple interspecies recombination events documented by whole-genome sequencing in multidrug-resistant Haemophilus influenzae clinical isolates.

Introduction: Haemophilus influenzae (Hi) was long known as an easy-to-treat bacterium, but increasing resistance against beta-lactams and other critically important antibiotics is now a growing concern. We describe here the whole-genome sequencing (WGS) analysis of three non-typeable Hi isolates received in 2018-2019 by the Belgian National Reference Centre (NRC) for Haemophilus influenzae, as they presented an unusual multi-resistant profile. Methods: All three isolates were sequenced by WGS and mapped to the reference isolate Hi Rd KW20. Shorten uptake signal sequences (USSs) known to be associated with homologous recombination were sought in ftsI, murE and murF genes, and inner partial sequences were compared against the blast nucleotide database to look for similarity with other Haemophilus species. Their antimicrobial resistance (AMR) genotype was studied. Core-genome multilocus sequence typing (MLST) was performed on the NTHi database pubMLST to place our isolates in the actual worldwide epidemiology. Results: The isolates also harboured interspecies recombination patterns in the murF-murE-ftsI region involved in cell wall synthesis. The three isolates were multidrug resistant and two of them were also resistant to amoxicillin-clavulanic acid and showed a reduced susceptibility to meropenem. All three isolates belonged to the MLST clonal complex (CC) 422, and WGS revealed that the three were very similar. They harboured mobile genetic elements (carrying blaTEM-1B, mefA and msrD genes associated with resistance), mutations in gyrA and parC linked to fluoroquinolone resistance as well as remodelling events in ompP2 that might be related to lower carbapenem susceptibility. Conclusion: The Hi evolution towards antimicrobial multiresistance (AMR) is a complex and poorly understood phenomenon, although probably linked to a large degree to the presence of USSs and exchange within the family Pasteurellaceae. To better understand the respective roles of clonal expansion, horizontal gene transfers, spontaneous mutations and interspecies genetic rearrangements in shaping Hi AMR, both analysis of Hi communities over time within individuals and worldwide monitoring of non-typeable Hi causing infections should be conducted.

From Investigating a Case of Cellulitis to Exploring Nosocomial Infection Control of ST1 Legionella pneumophila Using Genomic Approaches.

Legionella pneumophila can cause a large panel of symptoms besides the classic pneumonia presentation. Here we present a case of fatal nosocomial cellulitis in an immunocompromised patient followed, a year later, by a second case of Legionnaires' disease in the same ward. While the first case was easily assumed as nosocomial based on the date of symptom onset, the second case required clear typing results to be assigned either as nosocomial and related to the same environmental source as the first case, or community acquired. To untangle this specific question, we applied core-genome multilocus typing (MLST), whole-genome single nucleotide polymorphism and whole-genome MLST methods to a collection of 36 Belgian and 41 international sequence-type 1 (ST1) isolates using both thresholds recommended in the literature and tailored threshold based on local epidemiological data. Based on the thresholds applied to cluster isolates together, the three methods gave different results and no firm conclusion about the nosocomial setting of the second case could been drawn. Our data highlight that despite promising results in the study of outbreaks and for large-scale epidemiological investigations, next-generation sequencing typing methods applied to ST1 outbreak investigation still need standardization regarding both wet-lab protocols and bioinformatics. A deeper evaluation of the L. pneumophila evolutionary clock is also required to increase our understanding of genomic differences between isolates sampled during a clinical infection and in the environment.

Staphylococcus aureus causing tropical pyomyositis, Amazon Basin, Peru.

We studied 12 Staphylococcus aureus isolates causing tropical pyomyositis in the Amazon Basin of Peru. All isolates were methicillin-susceptible; 11 carried Panton-Valentine leukocidin-encoding genes, and 5 belonged to multilocus sequence type 25 and possessed an extensive set of enterotoxins. Our findings suggest sequence type 25 is circulating in tropical areas of South America.

Prevalence, risk factors and genetic diversity of methicillin-resistant Staphylococcus aureus carried by humans and animals across livestock production sectors.

OBJECTIVES: This study aimed to assess the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in animals and humans on veal, dairy, beef and broiler farms and to compare the risk for human MRSA carriage with that of strictly horticulture farmers. The genetic background, resistance phenotypes and genotypes and toxin gene content of the isolated MRSA strains were compared with MRSA collected on MRSA clonal complex (CC)398-positive pig farms. METHODS: MRSA carriage isolates were genotyped (spa, SCCmec and multilocus sequence typing), resistance to 16 antimicrobials was determined and resistance and toxin genes were detected. RESULTS: MRSA carriage rates were higher (P<0.01) on veal farms (calves, 64%; farmers, 72%) compared with on dairy (cows, 1%), beef (cows, 5%; farmers, 11%), broiler (pooled broths, 5%; farmers, 3%) and horticulture (farmers, 3%) farms. The intensity of animal contact was identified as a risk factor for human MRSA carriage. The vast majority of MRSA (n=344), including those from pigs, were CC398 (98%). SCCmec V(5C2), V(5C2&5)c, IV(2B) and IV(2B&5) predominated. MRSA CC130 and CC599 carrying mecC were detected in beef and dairy cattle. MRSA from veal calves were significantly more resistant than MRSA from pigs (P<0.01). A few isolates, including mecC-carrying MRSA, harboured pyrogenic superantigen toxins. Human- and animal-derived MRSA from individual farms showed similar characteristics. CONCLUSIONS: This systematic cross-sector survey revealed a high prevalence of multiresistant livestock-associated MRSA on Belgian veal calf farms as compared with other farm types. MRSA harbouring mecC was detected at a low frequency in beef and dairy cows, but not in humans.

Next-generation sequencing: what are the needs in routine clinical microbiology? A survey among clinicians involved in infectious diseases practice.

Background: The translation of Next-Generation Sequencing (NGS) from research to clinical microbiology is increasing rapidly, but its integration into routine clinical care struggles to catch-up. A challenge for clinical laboratories is that the substantial investments made in the required technologies and resources must meet both current and forthcoming needs. Methods: To get a clinical perspective of these needs, we have sent a survey to infectious diseases clinicians of five hospitals, covering the following topics: NGS knowledge, expected syndromes and patients foreseen to benefit from NGS, and expected impact on antimicrobial prescription. Results: According to clinicians, benefits of NGS are mostly expected in neurological and respiratory infections diagnostics. Conclusion: A better dialog between microbiologists and clinicians about hopes and limits of NGS in microbiology may help identifying key investments needed for clinical laboratories, today and tomorrow.