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BackgroundElizabethkingia anophelis is an emerging Gram-negative nonlactose fermenter in the health care setting, where it causes life-threatening infections in immunocompromised patients. We aimed to characterize the molecular mechanisms of antimicrobial resistance and evaluate the utility of contemporary antibiotics with the intent to offer targeted therapy against an uncommonly encountered pathogen.MethodsWhole-genome sequencing (WGS) was conducted to accurately identify isolate species and elucidate the determinants of β-lactam resistance. Antimicrobial susceptibility testing was performed using broth microdilution and disk diffusion assays. To assess the functional contribution of the major metallo-β-lactamase (MBL) encoding genes to the resistance profile, blaBlaB was cloned into pBCSK(-) phagemid vector and transformed into Escherichia coli DH10B.ResultsWGS identified the organism as E. anophelis. MBL genes blaBlaB-1 and blaGOB-26 were identified, in addition to blaCME-2, which encodes for an extended-spectrum β-lactamase (ESBL). Plasmids were not detected. The isolate was nonsusceptible to all commonly available β-lactams, carbapenems, newer β-lactam β-lactamase inhibitor combinations, and to the combination of aztreonam (ATM) with ceftazidime-avibactam (CAZ-AVI). Susceptibility to the novel siderophore cephalosporin cefiderocol was determined. A BlaB-1 transformant E. coli DH10B isolate was obtained and demonstrated increased minimum inhibitory concentrations to cephalosporins, carbapenems, and CAZ-AVI, but not ATM.ConclusionsUsing WGS, we accurately identified and characterized an extensively drug-resistant E. anophelis in an immunocompromised patient. Rapid evaluation of the genetic background can guide accurate susceptibility testing to better inform antimicrobial therapy selection.

International audience; Background: Whole-genome sequencing is one of the best ways to investigate resistance mechanisms of clinical isolates as well as to detect and identify circulating multi-drug-resistant (MDR) clones or sub-clones in a given hospital setting. Methods: Here, we sequenced 37 isolates of Acinetobacter baumannii, 10 Klebsiella pneumoniae, and 5 Pseudomonas aeruginosa collected from the biobank of the hospital setting of the King Fahad Medical City. Complete phenotypic analyses were performed, including MALDI-TOF identification and antibiotic susceptibility testing. After the genome assembly of raw data, exhaustive genomic analysis was conducted including full resistome determination, genomic SNP (gSNP) analysis, and comparative genomics. Results: Almost all isolates were highly resistant to all tested antibiotics, including carbapenems and colistin. Resistome analysis revealed many antibiotic resistance genes, including those with resistance to β-lactams, aminoglycosides, macrolides, tetracyclines, sulfamids, quinolones, and phenicols. In A. baumannii isolates, the endemic carbapenemase blaOXA-23 gene was detected in 36 of the 37 isolates. Non-synonymous mutations in pmrB were detected in almost all of the isolates and likely mediated colistin resistance. Interestingly, while classical analyses, such as MLST, revealed the predominance of an ST2 clone in A. baumannii isolates, the genomic analysis revealed the presence of five circulating sub-clones and identified several isolate transmissions between patients. In the 10 K. pneumoniae isolates, several resistance genes were identified, and the observed carbapenem resistance was likely mediated by overexpression of the detected extended-spectrum-β-lactamase (ESBL) genes associated with low membrane permeability as few carbapenemase genes were detected with just blaOXA-48 in three isolates. Colistin resistance was mediated either by non-synonymous mutations in the MgrB regulator, PmrA, PmrB, and PhoQ proteins or the presence of the MCR-1 protein. Here, gSNP analysis also revealed the existence of bacterial clones and cases of isolate transmissions between patients. The five analyzed P. aeruginosa isolates were highly resistant to all tested antibiotics, including carbapenems mediated by loss or truncated OprD porin, and colistin resistance was associated with mutations in the genes encoding the PmrA, PmrB, or PhoQ proteins. Conclusion: We demonstrate here the usefulness of whole-genome sequencing to exhaustively investigate the dissemination of MDR isolates at the sub-clone level. Thus, we suggest implementing such an approach to monitor the emergence and spread of new clones or sub-clones, which classical molecular analyses cannot detect. Moreover, we recommend increasing the surveillance of the endemic and problematic colistin resistance mcr-1 gene to avoid extensive dissemination.

International audience

The spread of vancomycin-resistant Enterococci (VRE) in Algerian hospital settings is poorly reported. Since the first report in 2006, few data have been available on the molecular mechanism of this resistance across the country. In this study, we investigate the frequency and antibiotic resistance mechanisms of Enterococci strains isolated from hospitalised patients in the Tlemcen university hospital. 191 Enterococcus spp. strains were collected from various clinical samples and were identified using MALDI-TOF-MS. The presence of van genes was investigated by standard PCR and sequencing. Results revealed that E. faecium and E. faecalis strains are the main pathogens identified in the study. Antibiotic susceptibility testing revealed that the resistance rate was high for the majority of antibiotic classes, including glycopeptides, and only linezolid was effective on all strains. Molecular analysis revealed that 52.2% of strains from intensive care unit (ICU) were positive for the vanA gene, including 44.44% E. faecium, 5.55% E. faecalis and 2.22% E. avium. 25.5% of these isolates co-harboured both the vanA and vanC genes, including E. gallinarum (n = 16) and E. faecium (n = 6). In surgical wards (SW) 29.70% of strains harboured the van genes, including 4.90% of E. faecalis harbouring the vanB gene, and of the rest of strains, (24.80%) harboured the vanC genes. Indeed, 9.90% E. gallinarum and 4.90% E. faecalis were positive for vanC1 and 9.90% of E. casseliflavus were positive for the vanC2/C3 gene. The glycopeptide resistance rate was higher among strains from the ICU and was mainly composed by E. faecium strains compared with surgical wards where resistant E. faecalis strains were predominant.

Staphylococcus aureus causes a wide range of life-threatening infections. In this study, we determined its prevalence in the hospital environment and investigated nasal carriage among healthcare workers and patients admitted to a hospital in western Algeria. A total of 550 specimens were collected. An antibiogram was performed and the genes encoding resistance to methicillin, inducible clindamycin and toxins were sought among the 92 S. aureus isolates. The spread of clones with a methicillin- and/or clindamycin-resistance phenotype between these ecosystems was studied using genomic analysis. A prevalence of 27%, 30% and 13% of S. aureus (including 2.7%, 5% and 1.25% of MRSA) in patients, healthcare workers and the hospital environment were observed, respectively. The presence of the mecA, erm, pvl and tsst-1 genes was detected in 10.9%, 17.4%, 7.6% and 18.5% of samples, respectively. Sequencing allowed us to identify seven sequence types, including three MRSA-IV-ST6, two MRSA-IV-ST80-PVL+, two MRSA-IV-ST22-TSST-1, two MRSA-V-ST5, and one MRSA-IV-ST398, as well as many virulence genes. Here, we reported that both the hospital environment and nasal carriage may be reservoirs contributing to the spread of the same pathogenic clone persisting over time. The circulation of different pathogenic clones of MRSA, MSSA, and iMLSB, as well as the emergence of at-risk ST398 clones should be monitored.

Human microbiomes are complex ecosystems involving bacteria, viruses, archaea or eukaryotes that are co-evolving in an environment subject to various selective pressures, such as antibiotic administration, diet and/or lifestyle. In this sympatric lifestyle, competition is hard and the synthesis of antibiotic molecules and/or antibiotic resistance genes (ARGs) is one solution that was developed by the organisms to survive. This environment becomes a large source of ARGs for pathogenic bacteria, leading to the risk of infection due to multidrug resistant bacteria. Culture and metagenomics are two complementary methods developed to study these microbiomes in order to better understand the type of bacteria and ARGs present in the human body, as well as the factors that modulate the abundance and variety of these ARGs. The objective of this review was to identify factors that influence the colonization and propagation of multidrug resistant bacteria and/or ARGs, and to define resistance genes and multidrug resistant bacteria that have already been isolated from the human microbiota using culturomics and metagenomics techniques. Keywords: Microbiota, Resistance genes, Sympatric life, Gene transfer, Metagenomic, Culture

International audience; Here, we aim to determine the prevalence of Salmonella contamination of poultry meat from butcheries of the province of Skikda and to investigate antibiotic resistance. Salmonella spp. isolates were screened from 70 samples, including chicken breasts (n = 40 samples) and chicken thighs (n = 30 samples) collected from 14 butcheries. All suspected Salmonella colonies from selective media were confirmed by MALDI-TOF MS and serotyped. The susceptibility profile to 16 antibiotics was studied. According to the antibiotic susceptibility results, resistance genes were investigated by standard PCR targeting various genes such as blaSHV, blaTEM, aac3, aac6-Ibcr, aad, qnrA and qnrB. Of the 14 butcheries studied, samples from eight butcheries were contaminated with Salmonella (57.14%). 19 Salmonella strains were isolated, including five serotypes with a predominance of Kentucky serotype (n = 9), Enteridis (n = 3), followed by Heidelberg (n = 3), Virchow (n = 3), and Manhattan (n= 1). All isolates were resistant to Rifampicin (100%; n = 19), and to other antibiotics such as Ciprofloxacin (47.36%), Amoxicillin-clavulanic acid (47.36%; n = 9), Amoxicillin, (47.36%; n = 9), Ticarcillin-clavulanic acid (47.36%; n = 9), and Gentamycin (47.36%; n = 9). All isolates showing multidrug resistance (47.36%; n = 9) were positive by PCR to the blaTEM-1 β-lactamase gene, from which 8 strains carried the aminoglycoside resistance aad7 gene. However, none was positive for the tested blaSHV, Aac3, Aac6-Ibcr, qnrA, qnrB, ArmA and ArmB genes. Our findings show a worrying rate of Salmonella contamination of poultry meats.

International audience; ABSTRACT Fosfomycin is a decades-old antibiotic, currently reused because of its activity against multidrug-resistant bacteria. Here, we used a combined in vitro/in silico approach to search for fosfomycin resistance determinants in 25 new bacterial species isolated from the human microbiota. Putative resistance genes were cloned into a susceptible Escherichia coli strain. MIC values increased from 1 μg/ml to 1,024 μg/ml. Here, we report a new family of potential chromosomal fosfomycin resistance genes, named fosM .

Bacterial infections resulting from wars and natural disasters represent a major public health problem. Over the past 50 years, Asia and the Middle East have suffered several wars. Moreover, East-A...

ObjectivesThe antiviral zidovudine has been recently identified as an active drug against resistant Enterobacteriaceae, but prevalence of resistance to this compound remains unknown. The aim was to estimate the prevalence of clinical Escherichia coli isolates resistant to zidovudine and to decipher the mechanism of zidovudine resistance.MethodsWe screened 537 isolates on zidovudine-containing agar plates and studied their thymidine kinase (tdk) gene sequences, the putative target involved in zidovudine resistance. Moreover, sequence analysis of 633 complete genomes of E. coli was performed to investigate mutation in the tdk gene. A comparative genomic analysis was done on an in vitro zidovudine-resistant mutant.ResultsAfter screening on our medium containing 2.7 mg/L (10 μM) zidovudine, nine strains had a zidovudine MIC >26.7 mg/L. The gene was absent in three isolates, inactivated by an IS (IS1X2 and ISApl1) in two isolates and mutated in four isolates. A genomic analysis of 633 E. coli genomes showed heterogeneity of the tdk gene sequence, with 27 different sequences. Among them, three genomes showed an inactivation of the gene (IS, stop codon and no tdk gene sequence). The in vitro mutant E. coli had 27 SNPs in eight genes of the core genome compared with the initial strain.ConclusionsOur study reports zidovudine-resistant clinical isolates of E. coli, presumably related to tdk inactivation. Diversity of Tdk in bacterial genomes can be large. Other mechanisms need to be considered in zidovudine resistance. The use of zidovudine in antibiotic-resistant infections needs to be in combination and should be tested before clinical administration.

Carbapenemase-producing Alcaligenes species has been described in only few studies, with none so far from the African continent. Here, we report the whole genome sequence of Peanalcaligenes suwonensis bearing bla(VIM-5) metallo-beta-lactamase and first detection of carbapenemase producing Alcaligenes faecalis isolated from patients attending tertiary healthcare facilities in Nigeria. The isolates were identified by MALDI-TOF Mass Spectrometry. Antibiotic susceptibility assay, modified Carba NP test and genomic investigation revealed that two isolates of Alcaligenes faecalis and an isolate of Paenalcaligenes suwonensis harboured bla(VIM-5) gene. The genome sequence analysis of the P. suwonensis 191B isolate, responsible for acute gastroenteritis, reveal the presence of 18 antibiotic resistance genes coding for resistance to five different classes of antibiotics. Three of the genes (bla(OXA-368), bla(CARB-4) and bla(VIM-5)) codes for resistance to beta-lactam antibiotics. To our best knowledge, we describe here the first genome sequence of P. suwonensis species and the first detection of class B carbapenemase bla(VIM-5) in a clinical isolate of P. suwonensis species and Alcaligenes faecalis in Nigeria. The finding of this study is of concern, as lateral dissemination of the genes into clinically important Gram-negative pathogens is highly likely.

The present study aimed to describe the emergence of carbapenem-resistant Pseudomonas aeruginosa isolated from clinical Lebanese patients. The resistance of these isolates is due to the presence of the plasmid-encoded blaVIM-2 gene. We provide its first description in Lebanon, as well as a description of disruption of the oprD gene by mutations. Keywords: Carbapenem resistance, oprDgene, Pseudomonas aeruginosa, VIM-2

β-Lactamase enzymes have attracted substential medical attention from researchers and clinicians because of their clinical, ecological, and evolutionary interest. Here, we present a comprehensive online database of β-lactamase enzymes. The current database is manually curated and incorporates the primary amino acid sequences, closest structural information in an external structure database (the Protein Data Bank [PDB]) and the functional profiles and phylogenetic trees of the four molecular classes (A, B, C, and D) of β-lactamases. The functional profiles are presented according to the MICs and kinetic parameters that make them more useful for the investigators. Here, a total of 1,147 β-lactam resistance genes are analyzed and described in the database. The database is implemented in MySQL and the related website is developed with Zend Framework 2 on an Apache server, supporting all major web browsers. Users can easily retrieve and visualize biologically important information using a set of efficient queries from a graphical interface. This database is freely accessible at http://ifr48.timone.univ-mrs.fr/beta-lactamase/public/.

The emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) represent a major clinical problem and raise serious health concerns. The present study aimed to investigate and ascertain the occurrence of CRE among hospitalized patients of Mohamed VI University Hospital, Marrakech, Morocco. Biological samples were collected over a one-year period (2018). The bacterial isolates were identified by MALDI-TOF-MS. Antibiotic susceptibility testing was performed using disc diffusion and Etest. The modified Hodge test and combined disc diffusion test were used for phenotypic detection. CRE hydrolyzing enzyme encoding genes: blaOXA-48, blaKPC, blaIMP, blaVIM, and blaNDM were characterized by PCR and DNA sequencing. In total, 131 non-duplicate CRE clinical strains resistant to Ertapenem were isolated out of 1603 initial Enterobacteriaceae. Klebsiella pneumoniae was the most common species (59%), followed by Enterobacter cloacae (24%), E. coli (10%), Citrobacter freundii (3%), Klebsiellaoxycota (2%), Serratia marcescens (1%), and Citrobacter braakii (1%). Of these, 56.49%, 21.37%, 15.27%, 3.38%, and 3.05% were collected from blood, urine, pus, catheters and respiratory samples, respectively. Approximately 85.5% (112/131) of the isolates were carbapenemase producers (40 blaOXA-48, 27 blaNDM, 38 blaOXA-48 + blaNDM and 7 blaVIM). All metallo-β-lactamases isolates were NDM-1 and VIM-1 producers. This is the first documentation of blaOXA-48 genes from C. freundii and C. braakii in Morocco.

Emergence and spread of carbapenemase-producing Enterobacteriaceae (CPE) are two of the major problems currently threatening global public health. In Nigeria, interest in CPE is recent. In Sokoto, northwest Nigeria, there are no data on the prevalence and mechanism underlying carbapenem resistance. In this study, we aimed to investigate the presence of clinical carbapenems-resistant Enterobacteriaceae isolates in two leading hospitals in Sokoto, northwest Nigeria. A total of 292 non-duplicate Enterobacteriaceae isolated from clinical specimens processed in the diagnostic laboratories of two hospitals between January and June 2019 were collected. Of these, 129 (44.2 %) and 19 (6.5%) were resistant to third-generation cephalosporin and carbapenems, respectively. RT-PCR revealed that 10 (7.8%), 19 (14.7%) and 46 (35.7%) of the third-generation cephalosporin-resistant isolates harboured bla(SHV), bla(TEM) and bla(CTX-M) genes, respectively. The modified Carba NP test result showed that only 7 (36.8 %) of the 19 carbapenem-resistant isolates were carbapenemase producing; among them, bla(NDM-5) and bla(OXA-181) genes were identified in five and two isolates, respectively. However, none of the carbapenemase genes investigated, including bla(VIM), bla(KPC) and bla(IMP), was detected in the remaining carbapenem-resistant isolates, suggesting a non-enzymatic mechanism. This study reports for the first time, the emergence of CPE in Sokoto state and the detection of NDM-producing Citrobacter freundii in Nigeria. The observed CPE in this study is a concern in a country where alternative antibiotics are rarely available. (C) 2020 The Authors. Published by Elsevier Ltd.

International audience; Carriage and invasion balance in the pathogenesis of Neisseria meningitidis was analyzed during a recent clonal outbreak of meningococcal B in Normandy, France, that offered the opportunity to compare six isolates undistinguable by conventional typing (B:14:P1.7,16:F3-3/ST-32) isolated from invasive disease or pharyngeal asymptomatic carriage. Data from animal model (transgenic mice rendered susceptible to N. meningitidis infection) showed an absence of virulence for two non-capsulated carriage isolates, an intermediate virulence for two capsulated carriage isolates and a marked virulence for two capsulated invasive isolates. This differential pathogenesis well correlated with whole genome sequencing analysis that clustered both isolates of each group together, forming their own arm within the Norman cluster. Gene-by-gene analysis specified that genes involved in iron acquisition were among the elements differentially represented in cluster of invasive isolates compared to cluster of capsulated carriage isolates. The hemoglobin receptor encoding gene hmbR was in an ON-phase in the capsulated invasive isolates while carriage capsulated isolates were in an OFF-phase. An ON-phase variant of a capsulated carriage isolate showed enhanced virulence. These data underline the role of phase variation (ON/OFF) of HmbR in the balance between disease isolates/carriage isolates.

International audience; Objectives: Group B Streptococcus (GBS) emerged in the 1970s as a major cause of neonatal infections, and has been increasingly associated with infections in adults since the 1990s. Prophages have been suspected to have driven these epidemiological trends. We have characterized the prophages harboured by 275 human GBS isolates belonging to the major lineages.& para;& para;Methods: We applied whole genome sequencing (WGS) to 14 isolates representative of the diversity within GBS species, located and identified their prophages. Using prediction tools, we searched for prophage elements potentially involved with the ability of GBS to infect humans. Using the data obtained by WGS, we designed a PCR-based tool and studied the prophage content of 275 isolates.& para;& para;Results: WGS of the 14 isolates revealed 22 prophages (i) distributed into six groups (A-F), (ii) similar to phages and prophages from GBS and non-GBS streptococci recovered from livestock, and (iii) carrying genes encoding factors previously associated with host adaptation and virulence. PCR-based detection of prophages revealed the presence of at least one prophage in 72.4% of the 275 isolates and a significant association between neonatal infecting isolates and prophages C, and between adult infecting isolates and prophages A.& para;& para;Conclusions: Our results suggest that prophages (possibly animal-associated) have conditioned bacterial adaptation and ability to cause infections in neonates and adults, and support a role of lysogeny with the emergence of GBS as a pathogen in human. (C) 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

International audience; Staphylococcus saprophyticus is one of the leading causes of urinary tract infections (UTI). In December 2014, our surveillance system identified an abnormal increase in S. saprophyticus causing UTIs in four university hospitals in Marseille, indicating a suspected community S. saprophyticus UTI outbreak. This was detected by our surveillance system BALYSES (Bacterial real-time Laboratory-based Surveillance System). S. saprophyticus/ Escherichia coli UTI ratio increased three-fold from 0.0084 in 2002 to 0.025 in December 2015 in Marseille with an abnormal peak in December 2014, and with an annual estimated ratio trend of 5.10(-6) (p-value < 10(-3)). Matrix-Assisted Laser Desorption Ionisation-Time of Flight Mass Spectrometry (MALDI-TOF MS) spectral analysis of strains was used to analyse strains cluster expansion, comparing strains from Marseille to those from Nice during the same period. MALDI-TOF MS spectral analysis revealed a geographical restricted clonal expansion of the strains clusters in Marseille as compared to Nice. Our finding suggests (i) a geographically restricted expansion of a specific S. saprophyticus strain clusters circulating in Marseille, and (ii) MALDI-TOF MS can be used as a cost-effective tool to investigate an outbreak.

International audience; The emergence of colistin-resistant Klebsiella pneumoniae (CRKP) is a major public health concern worldwide. In this study, the prevalence and molecular basis of colistin resistance in CRKP isolated from healthy individuals and patients in Lao PDR, Thailand, Nigeria and France were investigated. Stool samples were screened by culture for the presence of colistin-resistant Klebsiella spp. Whole-genome sequence analysis was used to decipher the molecular mechanism of colistin resistance in a blaNDM-1-positive in vitro-selected CRKP mutant. PCR amplification and sequencing of the mgrB genetic environment was performed for all CRKP isolates as well as control colistin-susceptible K. pneumoniae (CSKP) isolates recovered from the same stools. A total of 869 stool samples were screened for colistin-resistant Klebsiella spp., yielding 32 CRKP and 2 colistin-resistant Klebsiella oxytoca. Comparative whole-genome sequence analysis revealed that an in vitro-selected CRKP mutant had an insertion sequence in its mgrB gene, as well as missense mutations in other selected clones. Of the 34 colistin-resistant Klebsiella spp. isolates, 14 (41.2%; 13 CRKP and 1 K. oxytoca) from the four countries also had various defects in their mgrB genes, but no such defects were found in the CSKP controls (P

Methicillin-resistant Staphylococcus aureus (MRSA) resistant to decolonization agents such as mupirocin and chlorhexidine increases the need for development of alternative decolonization molecules. The absence of reported severe adverse reactions and bacterial resistance to polyhexanide makes it an excellent choice as a topical antiseptic. In the present study, we evaluated the in vitro and in vivo capacity to generate strains with reduced polyhexanide susceptibility and cross-resistance with chlorhexidine and/or antibiotics currently used in clinic. Here we report the in vitro emergence of reduced susceptibility to polyhexanide by prolonged stepwise exposure to low concentrations in broth culture. Reduced susceptibility to polyhexanide was associated with genomic changes in the mprF and purR genes and with concomitant decreased susceptibility to daptomycin and other cell wall-active antibiotics. However, the in vitro emergence of reduced susceptibility to polyhexanide did not result in cross-resistance to chlorhexidine. During in vivo polyhexanide clinical decolonization treatment, neither reduced polyhexanide susceptibility nor chlorhexidine cross-resistance was observed. Together, these observations suggest that polyhexanide could be used safely for decolonization of carriers of chlorhexidine-resistant S. aureus strains; they also highlight the need for careful use of polyhexanide at low antiseptic concentrations.

International audience; no abstract

International audience; Reports of carbapenemase-producing Enterobacteriaceae in Africa remain rare and assess mostly blaOXA-48-producing isolates from Mediterranean countries and South Africa. We identified blaNDM-7-producing Enterobacteriaceae in Gabon in 2016. The isolates contained blaNDM-7 IncX3 plasmids that were unusual and similar to the one described in a colistin-resistant Klebsiella pneumoniae SZ04 isolate from China.

We report here the emergence of VIM-2 and IMP-15 carbapenemases in a series of clinical isolates of carbapenem-resistant Pseudomonas aeruginosa in Lebanon. We also describe the disruption of the oprD gene by either mutations or insertion sequence (IS) elements ISPa 1328 and ISPre 2 isoform. Our study reemphasizes a rapid dissemination of the VIM-2 carbapenemase-encoding gene in clinical isolates of P. aeruginosa in the Mediterranean basin.

Objective: To analyze the resistome and virulence genes of Morganella morganii F675, a multidrug-resistant clinical isolate using whole genome sequencing (WGS). Methods: M. morganii F675 was isolated from a patient from Jerusalem, Israel. WGS was performed using both 454 and SOLiD sequencing technologies. Analyses of the bacterial resistome and other virulence genes were performed in addition to comparison with other available M. morganii genomes. Results: The assembled sequence had a genome size of 4,127,528 bp with G+C content of 51%. The resistome consisted of 13 antibiotic resistance genes including blaNDM-1 located in a plasmid likely acquired from Acinetobacter spp. Moreover, we characterized for the first time the whole lipid A biosynthesis pathway in this species along with the O-antigen gene cluster, the urease gene cluster and several other virulence genes. Conclusion: The WGS analysis of this pathogen further provides insight into its pathogenicity and resistance to antibiotics.

Clinical isolates of Pseudomonas aeruginosa exhibiting high-level resistance to carbapenems were recovered from a French patient with cystic fibrosis (CF) who had not received carbapenem therapy. This study was conducted to investigate the molecular mechanism conferring the carbapenem-resistant phenotype in clinical isolates of P. aeruginosa recovered from the same CF patient chronically colonised since 2005. Investigation of imipenem resistance of P. aeruginosa strain_02 isolated in May 2011 showed no carbapenemase activity. However, amplification and sequencing of the oprD porin gene revealed disruption of this gene by an insertion sequence (IS) element of 1337 bp that contained a novel transposase of 1227 bp (ISPa46) bordered by two terminal imperfect inverted repeats of 28 bp, which was associated with carbapenem resistance. Retrospective analysis of five additional strains of P. aeruginosa isolated before May 2011 from the same patient revealed that all isolates were likely to be the same clone by multilocus sequence typing analysis (ST540/551), but one of the five isolates was imipenem-susceptible. Although it was possible to demonstrate the presence of ISPa46 in all strains by PCR, this IS was transposed in the oprD gene only for imipenem-resistant isolates. Therefore, this study reports a novel IS element (ISPa46) in P. aeruginosa clinical isolates of a CF patient in Marseille, France, that was associated with carbapenem resistance and was selected in the absence of carbapenem treatment.

Elizabethkingia miricola is a Gram-negative non-fermenting rod emerging as a life-threatening human pathogen. The multidrug-resistant (MDR) carbapenemase-producing clinical isolate E. miricola EM_CHUV was recovered in the setting of severe nosocomial pneumonia. In this study, the genome of E. miricola EM_CHUV was sequenced and a functional analysis was performed, including a comparative genomic study with Elizabethkingia meningoseptica and Elizabethkingia anophelis. The resistome of EM_CHUV revealed the presence of a high number of resistance genes, including the presence of the blaGOB-13 and blaB-9 carbapenemase-encoding genes. Twelve mobility genes, with only two of them located in the proximity of resistance genes, and four potential genomic islands were identified in the genome of EM_CHUV, but no prophages or CRISPR sequences. Ten restriction-modification system (RMS) genes were also identified. In addition, we report the presence of a putative conjugative plasmid (pEM_CHUV) that does not encode any antibiotic resistance genes. Altogether, these findings point towards a limited number of DNA exchanges with other bacteria and suggest that multidrug resistance is an intrinsic trait of E. miricola owing to the presence of a high number of resistance genes within the bacterial core genome.

Streptococcus tigurinus is responsible for severe invasive infections such as infective endocarditis, spondylodiscitis and meningitis. As described, S. tigurinus isolates AZ_3aT and AZ_14 were highly virulent (HV phenotype) in an experimental model of infective endocarditis and showed enhanced adherence and invasion of human endothelial cells when compared to low virulent S. tigurinus isolate AZ_8 (LV phenotype). Here, we sought whether genetic determinants could explain the higher virulence of AZ_3aT and AZ_14 isolates. Several genetic determinants specific to the HV strains were identified through extensive comparative genomics amongst which some were thought to be highly relevant for the observed HV phenotype. These included i) an iron uptake and metabolism operon, ii) an ascorbate assimilation operon, iii) a newly acquired PI-2-like pilus islets described for the first time in S. tigurinus, iv) a hyaluronate metabolism operon, v) an Entner-Doudoroff pathway of carbohydrates metabolism, and vi) an alternate pathways for indole biosynthesis. We believe that the identified genomic features could largely explain the phenotype of high infectivity of the two HV S. tigurinus strains. Indeed, these features include determinants that could be involved at different stages of the disease such as survival of S. tigurinus in blood (iron uptake and ascorbate metabolism operons), initial attachment of bacterial pathogen to the damaged cardiac tissue and/or vegetation that formed on site (PI-2-like pilus islets), tissue invasion (hyaluronate operon and Entner-Doudoroff pathway) and regulation of pathogenicity (indole biosynthesis pathway).

Previous investigations of community-associated methicillin-resistant Staphylococcus aureus(CA-MRSA) isolates have revealed a wide diversity of genetic backgrounds, with only sporadic occurrence of ST8-USA300, in Geneva, Switzerland. We conducted a molecular epidemiologic analysis to identify the origin of a sudden increase of ST8 PVL-positive isolates in Geneva during 2013.

International audience; A 79-year-old French woman living in a nursing home was brought to the hospital emergency room for indwelling urinary catheter re-insertion following accidental removal. Despite no clinical signs suggesting infection, urine was sent to the laboratory for microbiological and cytological analysis. An Escherichia coli bacteriuria (104 CFU/mL) was found, but no leukocyturia. Antibiotic susceptibility testing revealed an isolate with resistance to penicillins, cephalosporins, aztreonam, ertapenem, nalidixic acid, fluoroquinolones, tetracycline, aminoglycosides, trimethoprim/sulfonamides and temocillin.

For the first time, we report the whole-genome sequence analysis ofChryseobacterium oranimenseG311, a multidrug-resistant bacterium, from a cystic fibrosis patient in France, including resistance to colistin. Whole-genome sequencing ofC. oranimenseG311 was performed using Ion Torrent PGM, and RAST, the EMBL-EBI server, and the Antibiotic Resistance Gene-ANNOTation (ARG-ANNOT) database were used for annotation of all genes, including antibiotic resistance (AR) genes. General features of theC. oranimenseG311 draft genome were compared to the other available genomes ofChryseobacterium gleumandChryseobacteriumsp. strain CF314.C. oranimenseG311 was found to be resistant to all β-lactams, including imipenem, and to colistin. The genome size ofC. oranimenseG311 is 4,457,049 bp in length, with 37.70% GC content. We found 27 AR genes in the genome, including β-lactamase genes which showed little similarity to the known β-lactamase genes and could likely be novel. We found the type I polyketide synthase operon followed by a zeaxanthin glycosyltransferase gene in the genome, which could impart the yellow pigmentation of the isolate. We located the O-antigen biosynthesis cluster, and we also discovered a novel capsular polysaccharide biosynthesis cluster. We also found known mutations in the orthologs of thepmrA(E8D),pmrB(L208F and P360Q), andlpxA(G68D) genes. We speculate that the presence of the capsular cluster and mutations in these genes could explain the resistance of this bacterium to colistin. We demonstrate that whole-genome sequencing was successfully applied to decipher the resistome of a multidrug resistance bacterium associated with cystic fibrosis patients.

The purpose of this study was to analyze the molecular mechanisms of ampicillin-resistant Haemophilus influenzae isolated in Geneva, Switzerland. We investigated the association between specific patterns of amino acid substitutions in penicillin-binding protein 3 (with or without β-lactamase production) and β-lactam susceptibility. Another main focus for this study was to compare the accuracy of disk diffusion and Etest methods to detect resistance to ampicillin and amoxicillin/clavulanic acid. The antibiotic susceptibility to β-lactam antibiotics of 124 H. influenzae isolates was determined by disk diffusion and Etest methods, and interpreted by European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) breakpoints. Alterations in PBP3 were investigated by sequencing the ftsI gene. Of the 124 clinical isolates analyzed, ampicillin resistance was found in 36% (45 out of 124). The rate of resistance to amoxicillin/clavulanic acid was 9% and 0.8%, using EUCAST and CLSI breakpoints respectively. For the 78 β-lactamase negative ampicillin-susceptible (BLNAS) isolates for which the Etest method indicated a high degree of susceptibility (MIC ≤ 1 mg/L), the disk diffusion method revealed resistance to ampicillin and amoxicillin/clavulanic acid in 33 cases (42%). Most common amino acid substitutions were Asn526Lys and Val547Ile, followed by Asp569Ser, Ala502Val, Asp350Asn, Met377Ile, Ile449Val, and Arg517His. The patterns observed were classified into six groups (IIa, IIb, IIc, IId, III-like, and miscellaneous). Continued characterization of both invasive and respiratory H. influenzae isolates is necessary in order to observe changes in the microbiology and epidemiology of this pathogen that could lead to clinical failure when treated by empirical antibiotic therapy.

Objective To investigate the potential roles of PBPs, efflux pumps and slow drug influx for imipenem heteroresistance in nontypeable Haemophilus influenzae (NTHi). Methods Fifty-nine NTHi clinical isolates examined in this study were collected at Geneva University Hospitals between 2009 and 2014. Alterations in PBPs were investigated by gene sequencing. To evaluate the affinities of the PBPs to imipenem, steady-state concentration–response experiments were carried out using imipenem in a competition assay with Bocillin-FL. The effect of the carbonyl cyanide m -chlorophenylhydrazone (CCCP) on imipenem susceptibility was assessed using broth dilution and viable cell counting. Using whole-genome sequencing, we explored the potential roles of outer membrane protein P2 (OmpP2), LytM proteins and the dcw gene cluster in imipenem heteroresistance. Results All 46 imipenem-heteroresistant isolates (IMI hR ) harboured amino acid substitutions in the ftsI gene, which encodes PBP3, corresponding to 25 different mutation patterns that varied from the ftsI gene mutation patterns found in imipenem-susceptible isolates. Among all PBPs, the highest affinity to imipenem was documented for PBP3 (IC 50 , 0.004 μg/mL). Different amino acid substitutions and insertions were noted in OmpP2, suggesting a relationship with imipenem heteroresistance. The IMI hR isolates were affected by CCCP differently and displayed a higher percentage of killing by imipenem in CCCP-treated cells at concentrations ranging between 0.5 and 8 μg/mL. Conclusions The present study provides robust evidence indicating that in combination with the altered PBP3, the slowed drug influx and its enhanced efflux due to the loss of regulation led to the development of imipenem heteroresistance in NTHi.

In this study, we report the development of a rapid real-time polymerase chain reaction (PCR) assay with TaqMan probe to detect the New Delhi metallo-β-lactamase (NDM-1)-encoding gene directly from bacterial isolates. The specificity of the assay was verified in silico as well as with a large panel of 84 clinically relevant bacteria, including the Klebsiella pneumoniae NCTC 13443 NDM-1-positive reference strain. Using this assay retrospectively on a local series of 44 K. pneumoniae isolates from Marseille Hospitals (France), it was possible to detect and identify an NDM-1-producing K. pneumoniae strain isolated from bronchoalveolar lavage in April 2010 from a French patient repatriated from India after a motorbike accident. Standard PCR amplification and sequencing of the entire NDM-1 gene from this isolate was also performed and the amino acid sequence showed 100% homology with the NDM-1 protein from the K. pneumoniae reference strain. We believe that this real-time PCR assay would be a powerful tool that could be added to other molecular detection assays such as detection of KPC- or OXA-encoding genes for rapid screening and/or identification of carbapenem-resistant bacterial isolates from patients returning from the Asian continent that could be implemented in a point-of-care strategy.

ARG-ANNOT (Antibiotic Resistance Gene-ANNOTation) is a new bioinformatic tool that was created to detect existing and putative new antibiotic resistance (AR) genes in bacterial genomes. ARG-ANNOT uses a local BLAST program in Bio-Edit software that allows the user to analyze sequences without a Web interface. All AR genetic determinants were collected from published works and online resources; nucleotide and protein sequences were retrieved from the NCBI GenBank database. After building a database that includes 1,689 antibiotic resistance genes, the software was tested in a blind manner using 100 random sequences selected from the database to verify that the sensitivity and specificity were at 100% even when partial sequences were queried. Notably, BLAST analysis results obtained using the rmtF gene sequence (a new aminoglycoside-modifying enzyme gene sequence that is not included in the database) as a query revealed that the tool was able to link this sequence to short sequences (17 to 40 bp) found in other genes of the rmt family with significant E values. Finally, the analysis of 178 Acinetobacter baumannii and 20 Staphylococcus aureus genomes allowed the detection of a significantly higher number of AR genes than the Resfinder gene analyzer and 11 point mutations in target genes known to be associated with AR. The average time for the analysis of a genome was 3.35 ± 0.13 min. We have created a concise database for BLAST using a Bio-Edit interface that can detect AR genetic determinants in bacterial genomes and can rapidly and easily discover putative new AR genetic determinants.

Mimiviruses are giant viruses that infect phagocytic protists, including Acanthamoebae spp., which were discovered during the past decade. They are the current record holder among viruses for their large particle and genome sizes. One group is composed of three lineages, referred to as A, B and C, which include the vast majority of the Mimiviridae members. Cafeteria roenbergensis virus represents a second group, though the Mimiviridae family is still expanding. We analyzed the codon and amino acid usages in mimiviruses, as well as both the transfer RNA (tRNA) and amino acyl-tRNA synthetases. We confirmed that the codon and amino acid usages of these giant viruses are highly dissimilar to those in their amoebal host Acanthamoeba castellanii and are instead correlated with the high adenine and thymine (AT) content of Mimivirus genomes. We further describe that the set of tRNAs and amino acyl-tRNA synthetases in mimiviruses is globally not adapted to the codon and amino acid usages of these viruses. Notwithstanding, Leu(TAA)tRNA, present in several Mimivirus genomes and in multiple copies in some viral genomes, may complement the amoebal tRNA pool and may contribute to accommodate the viral AT-rich codons. In addition, we found that the genes most highly expressed at the beginning of the Mimivirus replicative cycle have a nucleotide content more adapted to the codon usage in A.castellanii.

The increase and spread of multidrug-resistant (MDR) Gram-negative bacteria, including Enterobacteriaceae, Pseudomonas and Acinetobacter species, have become major concerns worldwide. Although the frequent misuse of antibiotic drugs has greatly contributed to worldwide antibiotic resistance by causing a large dispersal of resistance determinants, recent studies demonstrate that these resistance determinants could have emerged from ancient or environmental sources. Moreover, during the last 10 years, we have been witnessing the emergence and development of technologies for high-throughput sequencing, coinciding with an exponential increase in the number of bacterial genomes sequenced. These sequencing technologies allow a complete study of MDR bacterial genomes and are the best way to investigate the genetic determinants of antimicrobial resistance. Accordingly, studies using genome sequencing to decipher resistance determinants in Enterobacteriaceae, Pseudomonas and Acinetobacter species have demonstrated several advantages including, among others: an exhaustive identification of resistance determinants from any clinical, epidemiological or environmental MDR bacterium; identification of the acquisition mechanisms for resistance determinants exchanged between bacterial species through mobile genetic elements and elucidation and understanding, in record time (less than 1 week), of some critical or epidemic events caused by particular pathogenic bacteria. Therefore, it is clear today that the bacterial genome sequencing approach has revolutionized all fields of scientific research and represents a powerful tool to explore the world of microorganisms.

International audience

We compare the whole-genome sequences of two multidrug-resistant clinical Acinetobacter baumannii isolates recovered in the same patient before (ABIsac_ColiS susceptible to colistin and rifampin only) and after (ABIsac_ColiR resistant to colistin and rifampin) treatment with colistin and rifampin. We decipher all the molecular mechanisms of antibiotic resistance, and we found mutations in the rpoB gene and in the PmrAB two-component system explaining resistance to rifampin and colistin in ABIsac_ColiR, respectively.

The aim of this study was to determine the susceptibility to imipenem (IPM) of Acinetobacter baumannii isolates from different countries and to characterise the carbapenemase-encoding genes in IPM-resistant isolates. A total of 12 A. baumannii strains collected in Belgium (n=2), Iraq (n=8) and Georgia (n=2) were included in the study. Identification of the isolates was confirmed using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility testing was performed by the disk diffusion method, and Etest was used to determine the IPM minimum inhibitory concentrations (MICs) of resistant isolates. The presence of carbapenemase-encoding genes was investigated by polymerase chain reaction (PCR). All A. baumannii isolates were eventually identified by MALDI-TOF MS with high score values. Amongst the 12 strains, 6 were found to be resistant to IPM (MICs ≥16 μg/mL), comprising clinical isolates from wound infections of soldiers who were injured either during the Iraq war in 2007 (5 isolates) or during the Georgian-Russian war in 2008 (1 isolate from Georgia). All isolates contained ISAba1 and bla(OXA-51-like), but isolates from Iraq contained the bla(OXA-23) gene located on a plasmid whereas the isolate from Georgia contained the bla(OXA-24) gene located on the chromosome. None of the IPM-resistant isolates contained the bla(OXA-58)- or bla(NDM-1)-encoding genes. In conclusion, these results re-emphasise the worldwide dissemination of OXA carbapenemase genes in multidrug-resistant clinical isolates of A. baumannii and, to the best of our knowledge, report the first IPM-resistant A. baumannii strain isolated from a patient during the Georgian-Russian war with the bla(OXA-24) gene located on the chromosome.

Molecular epidemiological surveys of CA-MRSA revealed a wide diversity of genetic backgrounds with only sporadic identification of USA300 isolates during the period 1994-2012.

International audience; BACKGROUND: Klebsiella pneumoniae is one of the most important pathogens responsible for nosocomial outbreaks worldwide. Epidemiological analyses are useful in determining the extent of an outbreak and in elucidating the sources and the spread of infections. The aim of this study was to investigate the epidemiological spread of K. pneumoniae strains using a MALDI-TOF MS approach.METHODS: Five hundred and thirty-five strains of K. pneumoniae were collected between January 2008 and March 2011 from hospitals in France and Algeria and were identified using MALDI-TOF. Antibiotic resistance patterns were investigated. Clinical and epidemiological data were recorded in an Excel file, including clustering obtained from the MSP dendrogram, and were analyzed using PASW Statistics software.RESULTS: Antibiotic susceptibility and phenotypic tests of the 535 isolates showed the presence of six resistance profiles distributed unequally between the two countries. The MSP dendrogram revealed five distinct clusters according to an arbitrary cut-off at the distance level of 500. Data mining analysis of the five clusters showed that K. pneumoniae strains isolated in Algerian hospitals were significantly associated with respiratory infections and the ESBL phenotype, whereas those from French hospitals were significantly associated with urinary tract infections and the wild-type phenotype.CONCLUSIONS: MALDI-TOF was found to be a promising tool to identify and differentiate between K. pneumoniae strains according to their phenotypic properties and their epidemiological distribution. This is the first time that MALDI-TOF has been used as a rapid tool for typing K. pneumoniae clinical isolates.

The emergence and rapid spread of carbapenemases in Enterobacteriaceae, Pseudomonas and Acinetobacter (EPA) species is becoming a major public health crisis worldwide, and is responsible for large number of hospital-acquired and nosocomial infections. In this article, we review the current knowledge on the classification, phylogeny and genetic platforms of the main carbapenemases already described in Gram-negative bacteria.