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International audience; The current study aimed to characterize Staphylococcus aureus isolates from foodstuffs collected from western Algeria. A total of 153 S. aureus isolates from various raw and processed foods were obtained and identified using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. Isolates were characterized by antimicrobial susceptibility testing and toxin gene detection. Methicillin-resistant Staphylococcus aureus (MRSA) isolates were identified by detection of the mecA gene and characterized by staphylococcal cassette chromosome mec (SCCmec) typing. We found that 30.9% (153/495) of food samples were contaminated with S. aureus. Thirty-three (21.5%) S. aureus isolates were identified as MRSA, and 16.9% (26/153) carried the mecA gene. Three SCCmec types were identified of which type IV was the most common (69.2%) followed by type V (15.3%) and type II (7.6%). Two MRSA isolates were not typable with SCCmec typing. None of the examined isolates harbored mecC. Furthermore, 14.3% (22/153) of the isolates were toxigenic S. aureus. The cytotoxin gene pvl was detected in 11.1% of the S. aureus isolates. This gene was more commonly detected (76.4%) in MRSA isolates than in methicillin-suceptible Staphylococcus aureus (MSSA) isolates. The tsst-1 gene coding for toxic shock syndrome toxin was isolated rarely (3.2%) and only in MSSA isolates. According to disk diffusion test results, 70 isolates were resistant to only one antimicrobial drug, and 51 (33.3%) isolates were multidrug resistant. Other 32 isolates were susceptible to all antibiotics. Our study highlights, for the first time, a high prevalence of multidrug-resistant S. aureus isolates carrying pvl or tsst-1 found in food products in Algeria. The risk of MRSA transmission through the food chain cannot be disregarded, particularly in uncooked foods.

International audience; Objectives: In this study, the presence of carbapenemase genes in Shewanella xiamenensis strains isolated from river water in Bejaia, Algeria, was investigated. Methods: Four isolates of S. xiamenensis were isolated from water from Soummam River. The isolates were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) and sequencing of the 16S rRNA and gyrB genes. Isolates were subjected to antimicrobial susceptibility testing. Carbapenemase production was screened using phenotypic tests. PCR and sequencing were used to identify carbapenemase genes in the isolates. The genetic context of the bla(OXA-48-like )gene was investigated by sequencing the whole genome of strain AS58. Results: All four S. xiamenensis strains harboured bla(OXA-48-like) genes. They exhibited different resistance patterns and had imipenem minimum inhibitory concentrations (MICs) of >= 0.5 mg/L. Sequencing of bla(OXA-48-like) genes from the S. xiamenensis isolates showed that two strains harboured bla(OXA-181), one strain harboured bla(OXA-199) and one strain exhibited a new variant of the bla(OXA-48-like) gene, named bla(OXA-538). This new variant shared 98% nucleotide identity with bla(OXA-162), with three amino acid changes (G201A, A213G and I219F). Conjugation assays with Escherichia coli J53 recipient were performed but no transconjugants were obtained. Analysis of the genome of AS58 Touati strain confirmed the chromosomal location of the bla(OXA-538) gene. Conclusion: This study showed that environmental water holds a diversity of S. xiamenensis strains harbouring bla(OXA-48-like) genes and may play an important role in the dissemination and spread of these genes from the environment to humans. (C) 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

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.

Open Access; International audience; We isolated from aerobic and anaerobic blood culture bottles from a febrile patient, a Helicobacter-like Gram negative, rod-shaped bacterium that MALDI-TOF MS failed to identify. Blood agar cultures incubated in a microaerobic atmosphere revealed a motile Gram negative rod, which was oxidase, catalase, nitrate reductase, esterase, and alkaline phosphatase positive. It grew at 42 • C with no detectable urease activity. Antimicrobial susceptibility testing showed that the organism was susceptible to beta-lactams, gentamicin, erythromycin, and tetracycline but resistant to ciprofloxacin. Electronic microscopy analysis revealed a 3 × 0.5 µm curved rod bacterium harboring two sheathed amphitrichous flagella. Whole genome sequencing revealed a genome 1,708,265 base-pairs long with a GC content of 37.80% and a total of 1,697 coding sequences. The genomic analyses using the nucleotide sequences of the 16S rRNA gene, hsp60 and gyrB genes, as well as the GyrA protein sequence, and the results of Average Nucleotide Identity and in silico DNA-DNA hybridization suggest evidence for a novel Helicobacter species close to Helicobacter equorum and belonging to the group of enterohepatic Helicobacter species. As soon as the particular peptide mass fingerprint of this pathogen is added to the spectral databases, MALDI-TOF MS technology will improve its identification from clinical specimens, especially in case of " sterile infection ". We propose to associate the present strain with the Latin name of the place of isolation; Caesarodunum (Tours, France) and suggest " Helicobacter caesarodunensis " for further description of this new bacterium.

In this study, the draft genome sequences of two different carbapenem-resistant Acinetobacter baumannii clinical strains isolated from the same blood culture sample of an Iranian patient were determined. The strain A. baumannii 554S harbouring blaoxa72 gene belonged to ST 307 whereas A. baumannii 554L carrying blaoxa23 gene belonged to ST 2. We found that this sample contains two different isolates of A. baumannii, each phenotypically and genetically different.

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; We performed a detailed whole-genome sequence analysis of Providencia rettgeri H1736, a multidrug-resistant clinical pathogen isolated in Israel in 2011. The objective was to describe the genomic flexibility of this bacterium that has greatly contributed to its pathogenicity. The genome has a chromosome size of 4,609,352 bp with 40.22% GC content. Five plasmids were predicted, as well as other mobile genetic elements (MGEs) including phages, genomic islands, and integrative and conjugative elements. The resistome consisted of a total of 27 different antibiotic resistance genes including bla(NDM-1), mostly located on MGEs. Phenotypically, the bacteria displayed resistance to a total of ten different antimicrobial classes. Various features such as metabolic operons (including a novel carbapenem biosynthesis operon) and virulence genes were also borne on the MGEs, making P. rettgeri H1736 significantly different from other P. rettgeri isolates. A large quantity of the genetic diversity that exists in P. rettgeri H1736 was due to extensive horizontal gene transfer events, leading to an enormous presence of MGEs in its genome. Most of these changes contributed toward the pathogenic evolution of this bacterium.

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.

In this study, the presence of carbapenemase genes in Shewanella xiamenensis strains isolated from river water in Béjaïa, Algeria, was investigated.Four isolates of S. xiamenensis were isolated from water from Soummam River. The isolates were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) and sequencing of the 16S rRNA and gyrB genes. Isolates were subjected to antimicrobial susceptibility testing. Carbapenemase production was screened using phenotypic tests. PCR and sequencing were used to identify carbapenemase genes in the isolates. The genetic context of the blaAll four S. xiamenensis strains harboured blaThis study showed that environmental water holds a diversity of S. xiamenensis strains harbouring bla

International audience; no abstract

International audience

Background It has been suggested that prophages in the ST398 S. aureus clone are responsible for expanding ST398's spectrum of action and increasing its ability to cause human infections. We carried out the first characterization of the various prophages carried by 76 ST398 bloodstream infection (BSI) isolates obtained over 9 years of observation. Results Whole-genome sequencing of 22 representative isolates showed (1) the presence of the φ3-prophage and diverse genetic features typical of animal-associated isolates (i.e., SCCmec XI element, Tn916 transposon and non φ3-prophages) in a majority of BSI isolates, (2) one BSI isolate devoid of the φ3-prophage but otherwise similar to an animal-infecting isolate, (3) 35 prophages carrying numerous genes previously associated with virulence or immune evasion in animal models of staphylococcal infections. The analysis of prophage content in all 76 BSI isolates showed an increasing prevalence of polylysogeny over time. Overall, over the course of the last 10 years, the BSI isolates appear to have acquired increasing numbers of genetic features previously shown to contribute to bacterial adaptation and virulence in animal models of staphylococcal infections. Conclusions We hypothesize that lysogeny has played a significant role in increasing the ability of the ST398 clone to cause infections in humans. Our findings highlight the risk that the ST398 lineage will increase its threat to public health by continuing to acquire virulence and/or multiple antibiotic-resistance genes from hospital-associated clones of Staphylococcus aureus. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3516-x) contains supplementary material, which is available to authorized users.

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.

Strain EB01(T) sp. nov. is the type strain of Bacillus massilioalgeriensis, a new species within the genus Bacillus. This strain, whose genome is described here, was isolated from sediment sample of the hypersaline lake Ezzemoul sabkha in northeastern Algeria. B. massilioalgeriensis is a facultative anaerobic Gram-positive bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 5,269,577 bp long genome contains 5,098 protein-coding and 95 RNA genes, including 12 rRNA genes.

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.

Centre National de Re´fe´rence de la Re´sistance aux Antibiotiques, InstitutPasteur, Paris Cedex, FranceKempf M, Rolain J-M, Azza S, Diene S, Joly-Guillou M-L, Dubourg G, Colson P, Papazian L,Richet H, Fournier P-E, Ribeiro A, Raoult D. Investigation of Acinetobacter baumannii resistanceto carbapenems in Marseille hospitals, south of France: a transition from an epidemic to anendemic situation. APMIS 2012.Carbapenem-resistant Acinetobacter baumannii infections are a worldwide endemic nosocomialthreat. Between December 2010 and April 2011, an increase of carbapenem-resistant A. baumanniiinfections occurred in several Marseille University Hospitals. The aim of this study was to investi-gate the increase of carbapenem-resistant A. baumannii infections and to characterize the mecha-nisms of carbapenem resistance. The increase was detected by a homemade computer surveillanceprogram, known as EPIMIC, that monitors antibiotic resistance profiles on a weekly basis. Duringthis period, positive samples of carbapenem-resistant A. baumannii were retrieved from patients hos-pitalized in different units. Genotyping of the isolates was performed using pulsed-field gel electro-phoresis (PFGE) and multi-locus sequence typing (MLST), and carbapenemase gene analyses wereperformed to detect the presence of carbapenemases and to determine the relationships of theisolates. Carbapenem-resistant A. baumannii were isolated in a total of 11 patients who were hospi-talized in different hospitals units. We identified the presence of the bla

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.

Paucisalibacillus algeriensis strain EB02(T) is the type strain of Paucisalibacillus algeriensis sp. nov., a new species within the genus Paucisalibacillus. This strain, whose genome is described here, was isolated from soil sample from the hypersaline lake Ezzemoul Sabkha in northeastern Algeria. Paucisalibacillus algeriensis is a Gram-positive and strictly aerobic bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,006,766 bp long genome (1 chromosome but no plasmid) exhibits a low G+C content of 36% and contains 3,956 protein-coding and 82 RNA genes, including 9 rRNA genes.

Gram-negative carbapenem-resistant bacteria, in particular those producing New Delhi Metallo-beta-lactamase-1 (NDM-1), are a major global health problem. To inform the scientific and medical community in real time about worldwide dissemination of isolates of NDM-1-producing bacteria, we used the PubMed database to review all available publications from the first description in 2009 up to 31 December 2012, and created a regularly updated worldwide dissemination map using a web-based mapping application. We retrieved 33 reviews, and 136 case reports describing 950 isolates of NDM-1-producing bacteria. Klebsiella pneumoniae (n= 359) and Escherichia coli (n=268) were the most commonly reported bacteria producing NDM-1 enzyme. Several case reports of infections due to imported NDM-1 producing bacteria have been reported in a number of countries, including the United Kingdom, Italy, and Oman. In most cases (132/153, 86.3%), patients had connections with the Indian subcontinent or Balkan countries. Those infected were originally from these areas, had either spent time and/or been hospitalised there, or were potentially linked to other patients who had been hospitalised in these regions. By using Google Maps, we were able to trace spread of NDM-1-producing bacteria. We strongly encourage epidemiologists to use these types of interactive tools for surveillance purposes and use the information to prevent the spread and outbreaks of such bacteria.

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.

Strain G2(T) sp. nov. is the type strain of B. massiliogorillae, a proposed new species within the genus Bacillus. This strain, whose genome is described here, was isolated in France from the fecal sample of a wild western lowland gorilla from Cameroon. B. massiliogorillae is a facultative anaerobic, Gram-variable, rod-shaped bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 5,431,633 bp long genome (1 chromosome but no plasmid) contains 5,179 protein-coding and 98 RNA genes, including 91 tRNA genes.

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.

Background Cystic fibrosis (CF) lung microbiota consists of diverse species which are pathogens or opportunists or have unknown pathogenicity. Here we report the full characterization of a recently described multidrug resistant bacterium, Microbacterium yannicii, isolated from a CF patient who previously underwent lung transplantation. Results Our strain PS01 (CSUR-P191) is an aerobic, rod shaped, non-motile, yellow pigmented, gram positive, oxidase negative and catalase positive bacterial isolate. Full length 16S rRNA gene sequence showed 98.8% similarity with Microbacterium yannicii G72T type strain, which was previously isolated from Arabidopsis thaliana. The genome size is 3.95Mb, with an average G+C content of 69.5%. In silico DNA-DNA hybridization analysis between our Microbacterium yannicii PS01isolate in comparison with Microbacterium testaceum StLB037 and Microbacterium laevaniformans OR221 genomes revealed very weak relationship with only 28% and 25% genome coverage, respectively. Our strain, as compared to the type strain, was resistant to erythromycin because of the presence of a new erm 43 gene encoding a 23S rRNA N-6-methyltransferase in its genome which was not detected in the reference strain. Interestingly, our patient received azithromycin 250 mg daily for bronchiolitis obliterans syndrome for more than one year before the isolation of this bacterium. Conclusions Although significance of isolating this bacterium remains uncertain in terms of clinical evolution, this bacterium could be considered as an opportunistic human pathogen as previously reported for other species in this genus, especially in immunocompromised patients.

International audience; Here, we sequenced the 5,419,609 bp circular genome of an Enterobacter aerogenes clinical isolate that killed a patient and was resistant to almost all current antibiotics (except gentamicin) commonly used to treat Enterobacterial infections, including colistin. Genomic and phylogenetic analyses explain the discrepancies of this bacterium and show that its core genome originates from another genus, Klebsiella. Atypical characteristics of this bacterium (i.e., motility, presence of ornithine decarboxylase, and lack of urease activity) are attributed to genomic mosaicism, by acquisition of additional genes, such as the complete 60,582 bp flagellar assembly operon acquired "en bloc" from the genus Serratia. The genealogic tree of the 162,202 bp multidrug-resistant conjugative plasmid shows that it is a chimera of transposons and integrative conjugative elements from various bacterial origins, resembling a rhizome. Moreover, we demonstrate biologically that a G53S mutation in the pmrA gene results in colistin resistance. E. aerogenes has a large RNA population comprising 8 rRNA operons and 87 cognate tRNAs that have the ability to translate transferred genes that use different codons, as exemplified by the significantly different codon usage between genes from the core genome and the "mobilome." On the basis of our findings, the evolution of this bacterium to become a "killer bug" with new genomic repertoires was from three criteria that are "opportunity, power, and usage" to indicate a sympatric lifestyle: "opportunity" to meet other bacteria and exchange foreign sequences since this bacteria was similar to sympatric bacteria; "power" to integrate these foreign sequences such as the acquisition of several mobile genetic elements (plasmids, integrative conjugative element, prophages, transposons, flagellar assembly system, etc.) found in his genome; and "usage" to have the ability to translate these sequences including those from rare codons to serve as a translator of foreign languages.

Microbacterium yannicii is a Gram-positive, aerobic, yellow-pigmented, rod-shaped, nonmotile, oxidase-negative, and catalase-positive bacterium isolated on Columbia colistin-nalidixic acid (CNA) agar with 5% sheep blood from the sputum of a cystic fibrosis patient. The present study reports the draft genome of a Microbacterium yannicii strain.

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.