Evaluation of the relevance of use of the BD-BACTEC®MycosisIC/F, BD-BACTEC®PlusAerobic/F, BD-BACTEC®Lytic/10 anaerobic/F and BD-BACTEC®PedsPlus/F culture bottle system for fungemia detection: A 4-year retrospective study at the Dijon university hospital, France.

INTRODUCTION: Fungemia is a severe invasive fungal infection that combines rapid progression and a high mortality rate. This type of infection is a vital emergency, and early diagnosis is crucial. Currently, only the BD-BACTEC® Automated Blood Culture System (Becton Dickinson, New Jersey, USA) has a medium specifically dedicated to the detection of fungal agents: the BD-BACTEC®MycosisIC/F bottle. GAP STATEMENT: Thus, it is important to assess the performance of the different bottles offered by the BD-BACTEC® Automated Blood Culture System for the diagnosis of fungemia. AIM: The aim of this study was to evaluate the performance of the BD-BACTEC® MycosisIC/F culture medium in comparison to bacteriologic culture bottle media for the detection of fungemia in different clinical situations. METHODOLOGY: This retrospective study was conducted over a period of 4 years at the Dijon University Hospital. Three hundred and thirty-one pairs of blood cultures (i.e. a BD-BACTEC® MycosisIC/F culture bottle associated with at least one bacteriologic culture media bottle) were included in this study. RESULTS: We showed that the BD-BACTEC® MycosisIC/F culture bottles performed significantly better (i.e. diagnostic advantage either because it was the only positive bottle of the pair or time to positive result was shorter) than the bacteriological culture bottles in 57.7% (191/331) of cases (p <0.01). Multivariate analysis revealed that BD-BACTEC®MycosisIC/F bottles had better diagnostic performance than BD-BACTEC®Bacteriologic bottles in the context of: (i) the initial versus follow-up diagnostic subgroup, (ii) venipuncture or arterial sampling versus other sampling methods, and (iii) detection of filamentous versus yeast fungi. CONCLUSION: We concluded that the use of BD-BACTEC® MycosisIC/F culture bottles is a relevant addition to media optimized for routine bacterial detection.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Rapid Detection of Isolates Belonging to the Epidemic Clones Achromobacter xylosoxidans ST137 and Achromobacter ruhlandii DES from Cystic Fibrosis Patients.

Achromobacter spp. are increasingly reported among cystic fibrosis patients. Genotyping requires time-consuming methods such as multilocus sequence typing or pulsed-field gel electrophoresis. Therefore, data on the prevalence of multiresistant epidemic clones, especially A. xylosoxidans ST137 (AxST137) and the Danish epidemic strain A. ruhlandii (DES), are lacking. We recently developed and published a database for Achromobacter species identification by matrix-assisted laser desorption-ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics). The aim of this study was to evaluate the ability of the MALDI-TOF MS to distinguish these multiresistant epidemic clones within Achromobacter species. All the spectra of A. xylosoxidans (n = 1,571) and A. ruhlandii (n = 174) used to build the local database were analyzed by ClinProTools, MALDI Biotyper PCA, MALDI Biotyper dendrogram, and flexAnalysis software for biomarker peak detection. Two hundred two isolates (including 48 isolates of AxST137 and 7 of DES) were tested. Specific biomarker peaks were identified: absent peak at m/z 6,651 for AxST137 isolates and present peak at m/z 9,438 for DES isolates. All tested isolates were well typed by our local database and clustered within distinct groups (ST137 or non-ST137 and DES or non-DES) no matter the MALDI-TOF software or only by simple visual inspection of the spectra by any user. The use of MALDI-TOF MS allowed us to identify isolates of A. xylosoxidans belonging to the AxST137 clone that spread in France and Belgium (the Belgian epidemic clone) and of A. ruhlandii belonging to the DES clone. This tool will help the implementation of segregation measures to avoid interpatient transmission of these resistant clones.

In vitro antimicrobial activity of daptomycin alone and in adjunction with either amoxicillin, cefotaxime or rifampicin against the main pathogens responsible for bacterial meningitis in adults.

OBJECTIVES: As daptomycin adjunction is currently under clinical evaluation in the multicentre phase II AddaMAP study to improve the prognosis of pneumococcal meningitis, the present work aimed at evaluating the in vitro antimicrobial activity of daptomycin-based combinations against some of the most frequent species responsible for bacterial meningitis. METHODS: Clinically relevant strains of Streptococcus pneumoniae, Listeria monocytogenes, Haemophilus influenzae and Neisseria meningitidis were obtained from National Reference Centers. The antimicrobial activity of amoxicillin, cefotaxime and rifampicin, either alone or in association with daptomycin, was explored through the determination of minimum inhibitory concentration (MIC) and fractional inhibitory concentration index (FICI) as well as time-kill assay (TKA) using the broth microdilution method. RESULTS: All species taken together, the adjunction of daptomycin had no deleterious impact on the antimicrobial activity of amoxicillin, cefotaxime or rifampicin in vitro. Regarding Gram-positive bacteria, FICI and TKA analysis confirmed a global improvement of growth inhibition and bactericidal activity due to the adjunction of daptomycin. The synergistic effect prevailed for L. monocytogenes as demonstrated by FICI mainly <0.5 and a dynamic TKA-based synergy rate >50%. In addition, daptomycin-based associations did not modify the activity of ß-lactam antibiotics or rifampicin against Gram-negative bacteria, notably N. meningitidis. CONCLUSION: These results bring comforting evidence towards the clinical potential of daptomycin adjunction in the treatment of bacterial meningitis, which supports the ongoing AddaMAP clinical trial.

Two new Salmonella genomic islands 1 from Proteus mirabilis and description of blaCTX-M-15 on a variant (SGI1-K7).

Objectives: To characterize the structure of Salmonella genomic islands 1 (SGI1s) from two clinical Proteus mirabilis isolates: one producing an ESBL and the other a penicillinase. Methods: WGS completed by PCR and Sanger sequencing was performed to determine sequences of SGI1s from Pm2CHAMA and Pm37THOMI strains. Results: Two new variants of SGI1 named SGI1-Pm2CHAMA (53.6 kb) and SGI1-K7 (55.1 kb) were identified. The backbone of SGI1-Pm2CHAMA shared 99.9% identity with that of SGI1. Its MDR region (26.3 kb) harboured two class 1 integrons (an In2-type integron and an In4-type integron) containing in particular a qacH cassette (encoding a quaternary ammonium compound efflux pump). These two integrons framed a complex region (harbouring among others blaCARB-4) resulting from transposon insertions mediated by IS26 and successive transposition events of ISs (ISAba14 isoform and the new ISPmi2). The second variant (SGI1-K7) had the same backbone as SGI1-K. Its MDR region (29.7 kb) was derived from that of SGI1-K and was generated by three events. The two main events were mediated by IS26: inversion of a large portion of the MDR region of SGI1-K and insertion of a structure previously reported on plasmids carried by prevalent and successful MDR clones of Enterobacteriaceae. This last event led to the insertion of the blaCTX-M-15 gene into SGI1-K7. Conclusions: This study confirmed the great plasticity of the MDR region of SGI1 and its potential key role for the dissemination of clinically significant antibiotic resistance among Enterobacteriaceae.

"Does the Salmonella Genomic Island 1 (SGI1) confer invasiveness properties to human isolates?"

BACKGROUND: In the eighties, a multidrug resistant clone of Salmonella Typhimurium DT104 emerged in UK and disseminated worldwide. This clone harbored a Salmonella genomic island 1 (SGI1) that consists of a backbone and a multidrug resistant region encoding for penta-resistance (ampicillin, chloramphenicol/florfenicol, streptomycin/spectinomycin, sulphonamides and tetracycline (ACSSuT)). Several authors suggested that SGI1 might have a potential role in enhancement of virulence properties of Salmonella enterica. The aim of this study was to investigate whether nontyphoidal S. enterica isolates carrying SGI1 cause more severe illness than SGI1 free ones in humans. METHODS: From 2011 to 2016, all patients infected with nontyphoidal S. enterica in our hospital were retrospectively included. All nontyphoidal S. enterica isolates preserved in our University Hospital (Dijon, France) were screened for the presence of SGI1. Clinical and biological data of patients were retrospectively collected to evaluate illness severity. Statistical analysis of data was performed by Kruskal-Wallis test or Fisher's exact test for univariate analysis, and by logistic regression for multivariate analysis. RESULTS: A total of 100 isolates of S. enterica (22 serovars) were collected. Twelve isolates (12%) belonging to 4 serovars harbored SGI1: S. Typhimurium, S. Infantis, S. Kentucky, S. St Paul. The severity of the disease was age-related (for invasive infection, sepsis and inflammatory response) and was associated with immunosuppression (for invasive infection, sepsis and bacteremia) but not with the presence of SGI1 or with antimicrobial resistance. CONCLUSION: A rather high proportion (12%) of human clinical isolates belonging to various serovars (for the first time serovar St Paul) and harboring various antimicrobial resistance profile carried SGI1. Diseases due to SGI1-positive S. enterica or to antimicrobial resistant isolates were not more severe than the others. This first clinical observation should be confirmed by a multicenter and prospective study.

Nosocomial Infections with IMP-19-Producing Pseudomonas aeruginosa Linked to Contaminated Sinks, France.

We isolated IMP-19-producing Pseudomonas aeruginosa from 7 patients with nosocomial infections linked to contaminated sinks in France. We showed that blaIMP-19 was located on various class 1 integrons among 8 species of gram-negative bacilli detected in sinks: P. aeruginosa, Achromobacter xylosoxidans, A. aegrifaciens, P. putida, Stenotrophomonas maltophilia, P. mendocina, Comamonas testosteroni, and Sphingomonas sp.

Outbreak of Extended-Spectrum Beta-Lactamase Producing Enterobacter cloacae with High MICs of Quaternary Ammonium Compounds in a Hematology Ward Associated with Contaminated Sinks.

OBJECTIVE: To investigate an outbreak of extended-spectrum beta-lactamase (ESBL) producing Enterobacter cloacae that occurred in the Hematology ward (24-bed unit) of the François Mitterrand University Hospital (Dijon, France) between January 2011 and December 2013. The outbreak involved 43 patients (10 infected and 33 colonized). DESIGN: We performed environmental analysis to detect multiresistant E. cloacae for comparison with clinical isolates (genotyping by pulsed-field gel electrophoresis and MLST as well as ESBL-typing) and determined the MICs of the quaternary ammonium compounds (QACs) alkyldimethylbenzylammonium chloride (ADBAC) and didecyldimethylammonium chloride (DDAC). A bleach-based cleaning-disinfection program was implemented in December 2012 after mechanical removal of the biofilm in all sinks. RESULTS: We have detected 17 ESBL-producing E. cloacae in patients sink drains, shower drains and medical sink drains. Sequencing of the bla genes performed on 60 strains recovered from patients and environment (n = 43 clinical and n = 17 environmental) revealed that bla CTX-M15 was predominant (37 isolates) followed by bla CTX-M9 plus bla SHV-12 (20 isolates). We observed a great diversity among the isolates: 14 pulsotypes (11 STs) in clinical isolates and 9 pulsotypes (7 STs) in environmental isolates. Six pulsotypes were identical between clinical and environmental isolates. MICs of the quaternary ammonium compounds widely used for disinfection were very high in clinical and environmental isolates. Immediately after the implementation of the disinfection program we noticed a substantial fall in cases number. Our findings demonstrate the role of drains as important reservoir of ESBL-producing E. cloacae and highlight the necessity to settle drains accessible to achieve correct cleaning as well as to use disinfectant with proved activity against nosocomial pathogens.

Distribution of the species of Achromobacter in a French Cystic Fibrosis Centre and multilocus sequence typing analysis reveal the predominance of A. xylosoxidans and clonal relationships between some clinical and environmental isolates.

BACKGROUND: Achromobacter spp. are emerging pathogens in Cystic Fibrosis (CF) patients. Recent studies proposed Multilocus Sequence Typing (MLST) scheme and a species-level identification method by nrdA sequencing for this genus. Epidemiological data are needed to assess the species and/or the sequence types (STs) involved and their potential role in CF patients lung function degradation. The aims of this study were i) to describe the distribution of the different species of Achromobacter in our CF centre ii) to detect potential STs more involved in chronic colonisations iii) to detect a potential local or worldwide predominance of some STs among clinical and environmental isolates. METHODS: All the isolates (477) collected in our CF centre from 2007 to 2014 among the 177 patients attending the centre were identified using nrdA sequencing. MLST analysis was performed for 37 clinical and 14 environmental isolates. RESULTS: A total of 47 out of 177 patients presented positive culture(s) with Achromobacter spp., representing 12.7% of the patients of the centre each year. Eleven species were detected, A. xylosoxidans being the most prevalent species (27 patients). Only A. xylosoxidans (>80%) and A. insuavis were involved in chronic colonisation (6.7%). MLST analysis revealed a wide diversity among the isolates (36 STs for 51 isolates). Nevertheless, one third of the isolates belonged to STs previously detected in clinical isolates from other countries. CONCLUSIONS: This study is a first approach in understanding the global epidemiology of Achromobacter species in CF. These results confirm the high prevalence of the species A. xylosoxidans among CF patients, reveal the worldwide distribution of some STs and point out the potential role of environmental sources of contamination. More studies are needed to search for relationships between species and/or ST and pathogenicity.