Evaluation of an 'all-in-one' seven-day whole-genome sequencing solution in the investigation of a Staphylococcus aureus outbreak in a neonatal intensive care unit.

BACKGROUND: Meticillin-susceptible and -resistant Staphylococcus aureus (MSSA and MRSA) are responsible for outbreaks in intensive care units. MSSA infections have the same morbidity and mortality rate as MRSA infections but are studied less often. Whole-genome sequencing (WGS) is used increasingly for outbreak monitoring, but still requires specific installation and trained personnel to obtain and analyse the data. AIM: To evaluate the workflow and benefits of EpiSeq solution (bioMérieux, Marcy l'Etoile, France) in exploring the increased incidence of S. aureus bloodstream infections in a neonatal intensive care unit (NICU). METHODS: Four S. aureus bacteraemia isolates and 27 colonization isolates obtained between January and July 2016 were submitted to the 'all in one solution' EpiSeq [WGS, quality data assessment, multi-locus sequence typing (MLST), spa typing, virulome and resistome characterization, and phylogenetic tree construction]. More in-depth analyses were performed (whole-genome MLST and whole-genome single nucleotide polymorphism (wgSNP)] with BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium). FINDINGS: Nine different sequence types and 13 different spa types were found among the 31 isolates studied. Among those isolates, 11 (seven patients) were ST146 spa type t002, five (four patients) were ST30 and four (four patients) were ST398. The 11 ST146 isolates had a maximum of seven pairwise SNP differences. CONCLUSION: Use of EpiSeq solution allowed fast demonstration of the polyclonal profile of the MSSA population in neonates, and enabled the suspicion of a global outbreak to be ruled out. However, wgSNP analysis showed the transmission and persistence of one sequence type for over six months in the NICU, and enabled the infection control team to adapt its response.

Inventory of Extended-Spectrum-ß-Lactamase-Producing Enterobacteriaceae in France as Assessed by a Multicenter Study.

The objective of this study was to perform an inventory of the extended-spectrum-ß-lactamase (ESBL)-producing Enterobacteriaceae isolates responsible for infections in French hospitals and to assess the mechanisms associated with ESBL diffusion. A total of 200 nonredundant ESBL-producing Enterobacteriaceae strains isolated from clinical samples were collected during a multicenter study performed in 18 representative French hospitals. Antibiotic resistance genes were identified by PCR and sequencing experiments. The clonal relatedness between isolates was investigated by the use of the DiversiLab system. ESBL-encoding plasmids were compared by PCR-based replicon typing and plasmid multilocus sequence typing. CTX-M-15, CTX-M-1, CTX-M-14, and SHV-12 were the most prevalent ESBLs (8% to 46.5%). The three CTX-M-type EBSLs were significantly observed in Escherichia coli (37.1%, 24.2%, and 21.8%, respectively), and CTX-M-15 was the predominant ESBL in Klebsiella pneumoniae (81.1%). SHV-12 was associated with ESBL-encoding Enterobacter cloacae strains (37.9%). qnrB, aac(6')-Ib-cr, and aac(3)-II genes were the main plasmid-mediated resistance genes, with prevalences ranging between 19.5% and 45% according to the ESBL results. Molecular typing did not identify wide clonal diffusion. Plasmid analysis suggested the diffusion of low numbers of ESBL-encoding plasmids, especially in K. pneumoniae and E. cloacae However, the ESBL-encoding genes were observed in different plasmid replicons according to the bacterial species. The prevalences of ESBL subtypes differ according to the Enterobacteriaceae species. Plasmid spread is a key determinant of this epidemiology, and the link observed between the ESBL-encoding plasmids and the bacterial host explains the differences observed in the Enterobacteriaceae species.

[The new tools of microbiological diagnosis of tuberculosis]. / Les nouveaux outils de diagnostic microbiologique de la tuberculose maladie.

This review focuses on the role of new tools in the "modern" microbiological diagnosis of tuberculosis. Traditional techniques of microscopy and culture remain essential to diagnostic certainty, but some innovations replace daily the older techniques such as the identification of Mycobacterium tuberculosis complex by immunochromatography or mass spectrometry MALDI-TOF type from positive cultures, or susceptibility testing in liquid medium. New tools that use molecular techniques have become important. They all have in common to optimize the fight against tuberculosis by reducing diagnostic delay. They also allow rapid detection of drug resistance. However, the techniques of gene amplification directly from clinical samples are still less sensitive than culture. Bacteriological diagnosis of tuberculosis disease therefore still relies on the complementarities of different phenotypic and molecular techniques.

The emergence of linezolid resistance among Enterococci in intestinal microbiota of treated patients is unrelated to individual pharmacokinetic characteristics.

Linezolid is an antimicrobial agent for the treatment of multiresistant Gram-positive infections. We assessed the impact of linezolid on the microbiota and the emergence of resistance and investigated its relationship with plasma pharmacokinetics of the antibiotic. Twenty-eight patients were treated for the first time with linezolid administered orally (n = 17) or parenterally (n = 11) at 600 mg twice a day. Linezolid plasma pharmacokinetic analysis was performed on day 7. Colonization by fecal enterococci, pharyngeal streptococci, and nasal staphylococci were assessed using selective media with or without supplemental linezolid. The resistance to linezolid was characterized. The treatment led to a decrease of enterococci, staphylococci, and streptococci in the fecal (P = 0.03), nasal, and pharyngeal (P < 0.01) microbiotas. The appearance of resistant strains was observed only in enterococci from the fecal microbiota between the 7th and 21st days of treatment in four patients (14.3%). The resistance was mainly due for the first time to the mutation G2447T in the 23S rRNA gene. No pharmacokinetic parameters were significantly different between the patients, regardless of the appearance of resistance. The emergence of linezolid resistance during treatment was observed only in the intestinal microbiota and unrelated to pharmacokinetic parameters. However, colonization by Gram-positive bacteria was reduced as a result of treatment in all microbiotas.

Prevalence of resistance to antiseptics and mupirocin among invasive coagulase-negative staphylococci from very preterm neonates in NICU: the creeping threat?

In neonatal intensive care units, topical agents represent an increasing part of the infection control armamentarium. Fifty-one coagulase-negative staphylococci (CNS) isolated from catheter-associated bloodstream infections in very preterm neonates were investigated in this study: 41.2% exhibited decreased susceptibility to at least one antiseptic (chlorhexidine 12%, benzalkonium 24%, acriflavine 33%) and 61% were resistant to mupirocin. QacA/B, mupA and both genes were detected by polymerase chain reaction in 59%, 63% and 49% of CNS, respectively. Seventy-six percent of Staphylococcus epidermidis (5/5 pulsed-field-gel electrophoresis subgroups) and 11% of Staphylococcus capitis (1/3 subgroups) were multi-resistant. Skin antisepsis using low-concentration aqueous formulations and off-label mupirocin indications should benefit from a stewardship programme.

(18)F-FDG PET/CT in tuberculosis: an early non-invasive marker of therapeutic response.

OBJECTIVE: To evaluate the potential of (18)F-fluoro-deoxyglucose positron emission tomography/computed tomography ((18)F-FDG PET/CT) for early therapeutic intervention in patients with probable or confirmed tuberculosis (TB). METHODS: Twenty-one consecutive human immunodeficiency virus negative patients were prospectively included. All patients underwent (18)F-FDG PET/CT before and after 1 month of anti-tuberculosis treatment. The maximum standardised uptake value (SUV(max)) of the most (18)F-FDG avid lesions was recorded. RESULTS: The median age of patients was 36 years (range 18-84); 33.3% were male, 80.9% were born in endemic countries, and 23.8% had a past history of TB. TB was confirmed on culture in 8, on histology in 9 and on the basis of clinical symptoms in 4 patients. (18)F-FDG PET/CT detected active pulmonary TB (n = 1), extra-pulmonary (n = 10) or both (n = 10). The second (18)F-FDG PET/CT showed reduced radiotracer uptake intensity in 19 of 21 patients, with a median percentage decrease of SUV(max) of 31% (range 2-84). Two patients showed no improvement. TB was ruled out in one patient during follow-up; the final diagnosis was a non-Hodgkin's lymphoma. The other patient was smear-positive for 3 months. CONCLUSION: (18)F-FDG PET/CT allows an easy evaluation of early therapeutic response in patients with TB, particularly extra-pulmonary TB.

[Post-examination phase: description and management control of the process]. / Description et maîtrise du processus post-analytique en biologie médicale.

This article defines the scope of the post-analytical process. This process transforms the results after quality control review into validated results, interpreted by authorized "medical biologists", reported and communicated to the clinicians and patients. This phase includes the treatment of the samples after analysis, their storage and disposal and records archiving. This phase is a key step of the examination involved often in dysfunctions that could hardly harm the patient. These errors are usually the consequence of failing in the control of organization, lack of communication with the clinicians and defect of adaptation to their needs. Examples of quality indicators are proposed, as well as a model of clinico-biological contract.

[Guidelines for validation of the results in laboratory medicine]. / Recommandations pour la validation des résultats d'examens de biologie médicale.

The validation of the results is defined as the review and verification of the coherency and likelihood of the whole results of the examination for a patient, taking into account needed clinical data, uncertainty of measurement and anteriority's as well. The signature of the authorized person certifies this validation according to the requirements of the French regulation and ISO standard as well. Recommendations are given for the organization of this step specially for duty periods and in case of utilization of an expert system software. Requirements about the content, the release and the signature of the reports are given. A quality indicator applied to the control of the validation process is proposed.

[Guidelines for advisory services and results interpretation of biological examinations]. / Recommandations concernant les prestations de conseil et l'interprétation des résultats des examens de biologie médicale.

The "medical biologist" has to play a role as a consultant for the relevant use of biological examinations and to provide comments for their interpretation, comprehensible and useful to physicians. Advisory activities of the medical laboratory may help physician in diagnosis or therapeutic algorithm, avoiding ordering incomplete or useless examinations. After presentation of regulation and requirements of the EN ISO 15189 standard, this paper gives proposals for recommendations to apply in the context of accreditation. A proven and regular continuing education program is needed and professional practices must be supported by recognized and recent guidelines. This document provides suggestions for advisory services traceability and reports a list of websites and articles to use in defining standardised comments as well.

[Guidelines for records control and documents retention]. / Recommandations concernant la gestion des enregistrements et l'archivage documentaire.

The quality management system is based on records to maintain, according to the requirements of ISO 15189 standard and those of the French regulation as well, to ensure traceability of data. This article provides the nature of information and documents to be stored by the laboratory and the time they have to be maintained according to the French regulation. Moreover, it provides recommendations for the management and the control of records. Auditing the traceability of activities being a part of the elements of verification by COFRAC evaluators, a frame form for audit is also provided for self-assessment and preparation of accreditation.

Antibacterial resistance, genes encoding toxins and genetic background among Staphylococcus aureus isolated from community-acquired skin and soft tissue infections in France: a national prospective survey.

The epidemiology of staphylococcal community-acquired skin and soft tissues infections (CA-SSTIs) has changed dramatically. We described prospectively the characteristics of the Staphylococcus aureus isolated from 71 non-teaching French hospitals and implicated in CA-SSTIs: antimicrobial susceptibility (mecA polymerase chain reaction [PCR], disk diffusion method), virulence factor gene (sea, tst, pvl) prevalence and genetic background (agr allele). During November 2006, 235 strains were collected (wound infection: 51%, abscess: 21%, whitlow: 8%, diabetic foot: 7%, furunculosis: 3%). sea, tst and pvl were identified in 22.1, 13.2 and 8.9% strains, respectively. agr allele 1 was the most frequently encountered genetic background, whatever the methicillin susceptibility. Among the 34 methicillin-resistant S. aureus (MRSA, 14.5% of all S. aureus), only one strain (2.9%) harboured pvl (belonging to the European ST80 clone), four (11.8%) tst (belonging to two endemic French clones) and 18 (52.9%) sea gene (mainly the Lyon clone). According to their in vitro activity, pristinamycin or trimethoprim/sulfamethoxazole could be considered as first-choice antibiotics. To date, the international pvl-positive MRSA clones have not spread in France. MRSA strains isolated from putative CA-SSTIs exhibited a genetic and phenotypic background of hospital-acquired (HA) clones. National survey should be continued, in order to monitor the emergence of virulent clones.

Performance of a New MicroScan WalkAway PC30 panel and disk diffusion method for detection of oxacillin resistance in Staphylococcus spp.

The performance of the MicroScan WalkAway PC30 panel for detection of oxacillin resistance was evaluated by use of a collection of 420 staphylococcus isolates. The addition of a cefoxitin test (4 mg/liter) to the oxacillin MIC determination increased its raw performance for Staphylococcus aureus; additional data were required for coagulase-negative staphylococci.

[Bordetella holmesii bacteremia in a 26-year-old patient with sickle cell disease]. / Bactériémie à Bordetella holmesii chez un patient drépanocytaire de 26 ans.

Bordetella holmesii is a rare cause of bacteremia. It occurs mainly in hyposplenic patients, such as those affected by sickle cell anemia. The most frequent clinical signs are not very specific: fever, cephalalgia, cough, dyspnea, vomiting, etc. B. holmesii is frequently isolated from blood cultures. We describe the case of a 26-year-old sickle cell patient, presenting with dry cough and fever caused by a B. holmesii blood stream infection, identified by 16S rRNA gene sequencing. The outcome was favorable with amoxicillin. It is useful to know about B. holmesii, especially for physicians managing sickle cell or hyposplenic patients, because of its variable susceptibility to beta-lactams.

In vitro activity of daptomycin against Staphylococci isolated from bacteremia and community-onset skin and soft tissue infections in France: data from two nationwide studies.

Staphylococci are a leading cause of skin and soft tissue infections (SSTIs) and bacteremia in France, a country with a high prevalence of oxacillin resistance. We evaluated the in vitro activity of daptomycin compared with reference compounds against 445 Staphylococcus aureus and 53 coagulase-negative Staphylococci (CNS) collected during two large nationwide studies performed in 2006 and 2007. The percentage of oxacillin resistance among S. aureus was 13.6% (SSTIs) and 30.7% (bacteremia). Daptomycin showed lower MIC(90) levels compared to vancomycin, teicoplanin, and linezolid (0.19 mg/L vs. 2, 1.5, and 1 mg/L, respectively), irrespective of oxacillin susceptibility. Amongst the CNS, 64.2% of the isolates originated from clinical bacteremia were resistant to oxacillin and 24.5% to teicoplanin; all but one Staphylococci were susceptible to daptomycin (MIC = 1.5 mg/l). As with linezolid, daptomycin seems to constitute an alternative option to treat some staphylococcal infections in the French context of high oxacillin resistance prevalence and high glycopeptides MIC.

Clostridium symbiosum as a cause of bloodstream infection in an immunocompetent patient.

Bloodstream infection caused by Clostridium symbiosum was previously reported in only one case, from a highly immunocompromised patient with metastic colon cancer. We describe the second case of clinical bacteraemic sepsis caused by C. symbiosum from a previously healthy man, which underlines the pathogenicity of this species.

Tetracycline resistance mediated by tet(W), tet(M), and tet(O) genes of Bifidobacterium isolates from humans.

MICs of tetracyclines were determined for 86 human Bifidobacterium isolates and three environmental strains. The tet(O) gene was found to be absent in these isolates. tet(W) and tet(M) were found in 26 and 7%, respectively, of the Bifidobacterium isolates, and one isolate contained both genes. Chromosomal DNA hybridization showed that there was one chromosomal copy of tet(W) and/or tet(M).

Inhibitory impact of bifidobacteria on the transfer of beta-lactam resistance among Enterobacteriaceae in the gnotobiotic mouse digestive tract.

While looking for new means to limit the dissemination of antibiotic resistance, we evaluated the role of potentially probiotic bifidobacteria on the transfer of resistance genes between enterobacteria. Transfers of bla genes encoding extended-spectrum beta-lactamases (SHV-5 and CTX-M-15) were studied in the absence or presence of bifidobacteria. In vitro, transfer frequencies of these bla genes decreased significantly in the presence of three of five tested strains, i.e., Bifidobacterium longum CUETM-89-215, Bifidobacterium bifidum CIP-56.7T, and Bifidobacterium pseudocatenulatum CIP-104168T. Four transfer experiments were conducted in the digestive tract of gnotobiotic mice, the first three observing the effect of B. longum CUETM-89-215, B. bifidum CIP-56.7T, and B. pseudocatenulatum CIP-104168T on blaSHV-5 transfer and the fourth experiment studying the effect of B. bifidum CIP-56.7T on blaCTX-M-15 transfer. These experiments revealed significant decreases in the transconjugant levels (up to 3 logs) in mice having received B. bifidum CIP-56.7T or B. pseudocatenulatum CIP-104168T compared to control mice. Bifidobacteria appear to have an inhibitory impact on the transfer of antibiotic resistance genes. The inhibitory effect is associated to specific bifidobacterial strains and may be related to the production of thermostable metabolites by these strains.

[Linezolid: antibacterial activity, clinical efficacy and resistance]. / Le linézolide: activité antibactérienne, intérêts cliniques et résistance.

Linezolid is a synthetic antibiotic, the first available agent in a new class of antibiotic called the oxazolidinones, whose particular mechanism of action consists in inhibiting the initiation of protein synthesis. Its spectrum of in vitro and in vivo activity includes staphylococci, streptococci, enterococci, corynebacteria and some anaerobic bacteria (Peptostreptococcus, Clostridium, and Fusobacterium). The first therapeutic results were very encouraging, leading to the marketing of the product in France in 2002. Linezolid is indicated in the treatment of pneumonia and the complicated infections of the skin. Pharmacocinetics studies have shown that linezolid has an excellent bioavailability allowing a fast relay per os. However, failures of treatment under linezolid were reported and resistant strains of staphylococci and enterococci were obtained in vitro and in vivo after therapeutic use of this antibiotic. Changes in the domain V of 23S rRNA were found in the site of fixation, the most frequent was (G out of U) in position 2576 (numbering E. coli). In a context where resistance to traditional treatments in enterococci, pneumococci and S. aureus do not cease to increase, linezolid can be regarded as a therapeutic alternative to treat the infections with Gram-positive cocci.