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This study aimed to evaluate the routine identification tools available in Lebanon for differentiation of Escherichia coli and Shigella spp. The identification of 43 isolates defined as Shigella spp. by Api 20E was accessed using MALDI-TOF, serological testing, duplex PCR targeting ipaH (present in Shigella spp. and enteroinvasive E. coli “EIEC”) and lacY (found in E. coli including EIEC but not Shigella spp.) as well as gyrB gene sequencing. Antibiotic susceptibility was investigated as well as Shiga-toxin production. All isolates were identified as E. coli by MALDI-TOF while the PCR showed a disparate group of 26 EIEC, 11 Shigella spp., 5 E. coli and 1 inactive E. coli. However, the sequencing of gyrB gene, which was recently described as a suitable marker for distinguishing E. coli and Shigella spp., identified all isolates as E. coli. Antibiotic resistance was noticeable against ß-lactams, rifampicin, trimethoprim-sulfamethoxazole, gentamicin, and ciprofloxacin. The most common variants of beta-lactamase genes were blaTEM-1, blaCTX-M-15, and blaCTX-M-3. A great discordance between the used methods in identification was revealed herein. An accurate identification technique able to distinguish E. coli from Shigella spp. in routine laboratories is a pressing need in order to select the appropriate treatment and assess the epidemiology of these bacteria.

Since 2015, new worrying colistin resistance mechanism, mediated by mcr-1 gene has been reported worldwide along with eight newly described variants but their source(s) and reservoir(s) remain largely unexplored. Here, we conducted a massive bioinformatic analysis of bacterial genomes to investigate the reservoir and origin of mcr variants. We identified 13’658 MCR-1 homologous sequences in 494 bacterial genera. Moreover, analysis of 64’628 bacterial genomes (60 bacterial genera and 1’047 species) allows identifying a total of 6’651 significant positive hits (coverage >90% and similarity >50%) with the nine MCR variants from 39 bacterial genera and more than 1’050 species. A high number of MCR-1 was identified in Escherichia coli (n = 862). Interestingly, while almost all variants were identified in bacteria from different sources (i.e. human, animal, and environment), the last variant, MCR-9, was exclusively detected in bacteria from human. Although these variants could be identified in bacteria from human and animal sources, we found plenty MCR variants in unsuspected bacteria from environmental origin, especially from water sources. The ubiquitous presence of mcr variants in bacteria from water likely suggests another role in the biosphere of these enzymes as an unknown defense system against natural antimicrobial peptides and/or bacteriophage predation.

This study aimed to determine the incidence and the molecular mechanisms of carbapenem-resistant Enterobacteriaceae in patients from the Setif University Hospital, Algeria. Nonduplicate clinical bacterial isolates recovered from patients attending the University Hospital of Setif were collected between April and October 2018. Species identification was performed by MALDI-TOF/MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) method. The susceptibility of the isolates to carbapenems was determined using the disc diffusion method. The carbapenem resistant isolates were screened for the presence of common carbapenemase genes (blaKPC, blaOXA-48, blaVIM, blaIMP, and blaNDM) and extended-spectrum β-lactamase (blaCTX, blaTEM, and blaSHV) using PCR and sequencing technique. A total of 123 nonrepetitive Enterobacteriaceae isolates were obtained. Klebsiella pneumoniae (n = 52/42.28%), Escherichia coli (n = 24/19.51%), and Enterobacter cloacae (n = 19/15.45%) were the most prevalent species. The Carba-NP test showed that 6 out of 123 isolates carried carbapenemase enzymes. OXA-48 was found in five isolates (four K. pneumoniae and one E. coli) and NDM-5 in one E. cloacae isolate. We reported for the first time in Algeria the presence of NDM-5 carbapenemase enzyme in a clinical E. cloacae isolate.

The current study is about genomic characterisation of an atypical multidrug-resistant Escherichia coli harbouring two mobilised colistin resistance (mcr) genes isolated from pigs in France. Stool samples taken from a pig farm in Avignon in the department of the Vaucluse were subjected to a molecular screening for the detection of mcr gene variants. These samples were cultured on selective LBJMR medium. Growing bacteria were identified using MALDI-TOF, followed by antibiotic susceptibility testing (AST). Whole genome sequencing (WGS) and bioinformatic genome analysis was performed. The selective culture of stools revealed the presence of an E. coli strain named Q4552 which was simultaneously positive for the mcr-1.1 and mcr-3.5 genes. This strain exhibited resistance phenotype to fourteen antibiotics, including colistin. Genome sequencing revealed a circular chromosome and eight plasmids. Genomic analysis revealed a chromosomic integration of a mobile genetic element (MGE) harbouring the mcr-1.1 gene, while the mcr-3.5 gene was plasmidic (i.e., an IncFII plasmid). Its resistome exhibited twenty-two resistance genes, explaining its multidrug resistance phenotype. The Q4552 strain is an ST-843 clone belonging to the clonal complex Cplx-568 and is the only ST type of this cplx-568 which has been isolated from animals, humans, and the environment. Here, we report the first co-occurrence of the mcr-1 and mcr-3 genes in France from a pathogenic E. coli strain isolated from a pig farm. Since this clone (ST-843) has been reported in zoonotic transmissions, programs to monitor such colistin resistant bacterium are urgently required to avoid its spread and zoonotic transmission to humans.

Strain Marseille-P3519T isolated from the fecal flora of a 25-year-old healthy French woman was a Gram-positive anaerobic bacterium, non-motile and non-spore forming. The 16S rRNA gene sequence of Marseille-P3519 showed 97.73% of sequence similarity with Limosilactobacillus reuteri DSM 20016, the closest species, phylogenetically. Furthermore, the average nucleotide identity of strain Marseille-3519 with its closest related species was 75.8% that was very below the recommended threshold (>95–96%). Its genome had 2 237 367 bp with 45.42 mol% of G + C content. Major fatty acids were C16:0 (50.8%), C18:1n9 (18.0%), C18:2n6 (9.8%) and C19:1n9 (8.9%). It was catalase negative and fermented glycerol, glucose, fructose, D-maltose, lactose and mannose. These findings support that strain Marseille-P3519 ( = CSURP3519 = CECT 30110) is a new member of the genus Limosilactobacillus for which the name Limosilactobacillus caccae sp. nov., is proposed.

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.

Background Acinetobacter baumannii is an opportunistic pathogen causing various nosocomial infections. The spread of multidrug-resistant A. baumannii is a major public health problem. The aim of this study was to investigate the molecular epidemiology and the genetic support of multidrug-resistant A. baumannii isolates collected from Saint-Georges Hospital in Lebanon. Methods Between January and August 2016, 31 A. baumannii isolates were collected from sputum samples of patients infected with ventilator-associated pneumonia (VAP) and treated with colistin-carbapenem combination therapy. Antibiotic susceptibility testing was performed using the disk diffusion method. Carbapenemases, extended spectrum β-lactamases encoding genes and mcr-1/2 genes were investigated by RT-PCR and standard PCR. The epidemiological relatedness of the strains was studied using MLST analysis. Results Most of the isolates exhibited multidrug-resistant phenotypes. All the isolates were carbapenem-resistant and among them, 30 carried the class D carbapenemase bla oxa-23 gene while one isolate carried bla oxa-72 gene. MLST results revealed three sequence types, namely ST2, ST699, and ST627. Isolates having ST2 were the most prevalent clone (29/31, 93.5%). Conclusions This study shows a nosocomial spread of multidrug-resistant A. baumannii ST2 having bla OXA-23 gene in Saint-George in Lebanon. Monitoring and control measures need to be adopted to avoid the spread of A. baumannii to patients.

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; CC17 Streptococcus agalactiae carrying group-A prophages is increasingly responsible for neonatal infections. To investigate the impact of the genetic features of a group-A prophage, we first conducted an in silico analysis of the genome of 12/111phiA, a group-A prophage carried by a strain responsible for a bloodstream infection in a parturient. This revealed a Restriction Modification system, suggesting a prophage maintenance strategy and five ORFs of interest for the host and encoding a type II toxin antitoxin system RelB/YafQ, an endonuclease, an S-adenosylmethionine synthetase MetK, and an StrP-like adhesin. Using the WT strain cured from 12/111phiA and constructing deleted mutants for the ORFs of interest, and their complemented mutants, we demonstrated an impact of prophage features on growth characteristics, cell morphology and biofilm formation. Our findings argue in favor of 12/111phiA domestication by the host and a role of prophage features in cell autoaggregation, glycocalyx and biofilm formation. We suggest that lysogeny may promote GBS adaptation to the acid environment of the vagina, consequently colonizing and infecting neonates.

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 .

The Shigella genus includes serious foodborne disease etiologic agents, with 4 species and 54 serotypes. Identification at species and serotype levels is a crucial task in microbiological laboratories. Nevertheless, the genetic similarity between Shigella spp. and Escherichia coli challenges the correct identification and serotyping of Shigella spp., with subsequent negative repercussions on surveillance, epidemiological investigations, and selection of appropriate treatments. For this purpose, multiple techniques have been developed historically ranging from phenotype-based methods and single or multilocus molecular techniques to whole-genome sequencing (WGS). To facilitate the selection of the most relevant method, we herein provide a global overview of historical and emerging identification and serotyping techniques with a particular focus on the WGS-based approaches. This review highlights the excellent discriminatory power of WGS to more accurately elucidate the epidemiology of Shigella spp., disclose novel promising genomic targets for surveillance methods, and validate previous well-established methods.

β-lactam antibiotics have a well-known activity which disturbs the bacterial cell wall biosynthesis and may be cleaved by β-lactamases. However, these drugs are not active on archaea microorganisms, which are naturally resistant because of the lack of β-lactam target in their cell wall. Here, we describe that annotation of genes as β-lactamases in Archaea on the basis of homologous genes is a remnant of identification of the original activities of this group of enzymes, which in fact have multiple functions, including nuclease, ribonuclease, β-lactamase, or glyoxalase, which may specialized over time. We expressed class B β-lactamase enzyme from Methanosarcina barkeri that digest penicillin G. Moreover, while weak glyoxalase activity was detected, a significant ribonuclease activity on bacterial and synthetic RNAs was demonstrated. The β-lactamase activity was inhibited by β-lactamase inhibitor (sulbactam), but its RNAse activity was not. This gene appears to have been transferred to the Flavobacteriaceae group especially the Elizabethkingia genus, in which the expressed gene shows a more specialized activity on thienamycin, but no glyoxalase activity. The expressed class C-like β-lactamase gene, from Methanosarcina sp., also shows hydrolysis activity on nitrocefin and is more closely related to DD-peptidase enzymes. Our findings highlight the need to redefine the nomenclature of β-lactamase enzymes and the specification of multipotent enzymes in different ways in Archaea and bacteria over time.

International audience; Long considered to be a consequence of human antibiotics use by deduction, antibiotic resistance mechanisms appear to be in fact a much older phenomenon as antibiotic resistance genes have previously been detected from millions of year-old permafrost samples. As these specimens guarantee the viability of archaic bacteria, we herein propose to apply the culturomics approach to recover the bacterial content of a Siberian permafrost sample dated, using the in situ-produced cosmogenic nuclide chlorine36 (36 Cl), at 2.7 million years to study the dynamics of bacterial evolution in an evolutionary perspective. As a result, we cultured and sequenced the genomes of 28 ancient bacterial species including one new species. To perform genome comparison between permafrost strains and modern isolates we selected 7 of these species (i.e., Achromobacter insolitus, Bacillus idriensis, Brevundimonas aurantiaca, Janibacter melonis, Kocuria rhizophila, Microbacterium hydrocarbonoxydans and Paracoccus yeei). We observed a high level of variability in genomic content with a percentage of shared genes in the core genomes ranging from 21.23% to 55.59%. In addition, the Single Nucleotide Polymorphism (SNP) comparison between permafrost and modern strains for the same species did not allow a dating of ancient strains based on genomic content. There were no significant differences in antibiotic resistance profiles between modern and ancient isolates of each species. Acquired resistance to antibiotics was phenotypically detected in all gram-negative bacterial species recovered from permafrost, with a significant number of genes coding for antibiotic resistance detected. Taken together, these findings confirm previously obtained data that antibiotic resistance predates humanity as most of antimicrobial agents are natural weapons used in inter-microbial conflicts within the biosphere.

The probability of the evolution of a character depends on two factors: the probability of moving from one character state to another character state and the probability of the new character state fixation. More the evolution of a character is probable more convergent evolution will be witnessed, consequently, convergent evolution could mean that the convergent character evolution result as a combination of these two factors. We investigate this phenomenon by studying the convergent evolution of biochemical functions. We use for the investigation the case of β-lactamases. β-lactamases hydrolyzes β-lactams which are antimicrobials able to block the DD-peptidases involved in bacterial cell wall synthesis. β-lactamase activity is present in two different superfamilies: the metallo-β-lactamase and the serine β-lactamase superfamily. The mechanism used to hydrolyze the β-lactam is different for the two superfamilies. We named this kind of evolution an allo-convergent evolution. We further show that the β-lactamase activity evolved several times within each superfamily, a convergent evolution type that we named iso-convergent evolution. Both types of convergent evolution can be explained by the two evolutionary mechanisms discussed above. The probability of moving from one state to another is explaining the promiscuous β-lactamase activity present in the ancestral sequences of each superfamily, while the probability of fixation is explained in part, by positive selection as the organisms having β-lactamase activity allows them to resist to organism secreting β-lactams. Indeed a mutation increasing the β-lactamases activity will be selected as the organisms having this activity will have an advantage over the others.

The increasingly wide knowledge of the microbiota, as well as its continuous relation with several pathologies, favours the interest, discovery and classification of all the bacteria that are part of it. The isolation of strain P3519T is possible thanks to the application of new strategies such as culturomics and taxono-genomics. Strain Marseille-P3519T isolated from the fecal flora of a 25-year-old healthy French woman was a Gram-positive anaerobic bacterium, non-motile and non-spore-forming. The 16S rRNA gene sequence of Marseille-P3519 showed 98.57% of sequence similarity with Lactobacillus caviae MOZM2, the closest species, phylogenetically. Its genome had 2,237,367 bp with 45.42 mol% of G+C content. These findings support that strain Marseille-P3519 is a new member of the genus Lactobacillus for which the name Lactobacillus caccae sp. nov., is proposed.

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.

The objective of this study was to investigate the occurrence of multidrug-resistant Escherichia coli in cows with clinical mastitis in 42 different dairy farms located in the Bordj Bou Arreridj region of Algeria. Milk samples were cultured on Columbia blood agar, and isolates were then identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. In total, 200 samples were screened and 52 E. coli strains confirmed as causative agents were obtained. The antimicrobial susceptibility testing was performed by disk diffusion method. Antibiotic resistance genes, including those conferring resistance to extended-spectrum beta-lactamases (i.e., bla(TEM), bla(SHV), and bla(CTX-M)), tetracyclines (tetA, tetB, tetC, and tetJ), aminoglycosides [aph(30), aac(30), aac(60), ant, aad, and armA], and quinolones (qnrA and qnrB) were amplified by standard PCR and sequenced when positive. Transferability of resistance genes has been investigated by conjugation experiments and multilocus sequence typing. The most frequently observed resistance was to amoxicillin (86.5%), followed by tetracycline (75%), amoxicillin-clavulanic acid (59.6%), trimethoprim-sulfamethoxazole (36.5%), doxycycline (13.5%), and ciprofloxacin (13.5%). Multidrug resistance was observed in 38.4% of isolates. Genotypic characterization showed that tetA (44.2%) and bla(TEM-1) (30.7%) genes were the most prevalent. Screening for plasmid-mediated quinolone resistance genes demonstrated that seven isolates (13.5%) expressed qnrB and one isolate (1.9%) harbored qnrA. In addition, aminoglycoside resistance determinants including aadA1 and aac(3)-Id were detected in seven and two isolates, respectively. Moreover, blaTEM, tetA, tetB, qnrB, and aadA1 were successfully transferred horizontally to transconjugant strains. The multilocus sequence typing revealed the presence of three different sequence types (ST162, ST371, and ST 949).

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.

Background We investigate here the presence of multidrug-resistant bacteria isolated from stool samples of yellow-legged gulls and chickens (n = 136) in urban parks and beaches of Marseille, France. Bacterial isolation was performed on selective media, including MacConkey agar with ceftriaxone and LBJMR medium. Antibiotic resistance genes, including extended-spectrum β-lactamases (ESBL) (i.e. blaCTX-M, blaTEM and blaSHV), carbapenemases (blaKPC, blaVIM, blaNDM, blaOXA-23, blaOXA-24, blaOXA-48 and blaOXA-58) and colistin resistance genes (mcr-1 to mcr-5) were screened by real-time PCR and standard PCR and sequenced when found. Results Of the 136 stools samples collected, seven ESBL-producing Gram-negative bacteria (BGN) and 12 colistin-resistant Enterobacteriaceae were isolated. Among them, five ESBL-producing Escherichia coli and eight colistin-resistant Hafnia alvei strains were identified. Four blaTEM-1 genes were detected in yellow-legged gulls and chickens. Three CTX-M-15 genes were detected in yellow-legged gulls and pigeons, and one CTX-M-1 in a yellow-legged gull. No mcr-1 to mcr-5 gene were detected in colistin-resistant isolates. Genotyping of E. coli strains revealed four different sequence types already described in humans and animals and one new sequence type. Conclusions Urban birds, which are believed to have no contact with antibiotics appear as potential source of ESBL genes. Our findings highlight the important role of urban birds in the proliferation of multidrug-resistant bacteria and also the possible zoonotic transmission of such bacteria from wild birds to humans.

International audience

International audience; Staphylococcus lugdunensis is a commensal bacterium of human skin that has emerged as a virulent Coagulase-Negative Staphylococcus in both community-acquired and healthcare associated infections. Genotyping methods have shown a clonal population structure of this pathogen but failed to identify hypervirulent lineages. Here, complete genomes of three pathogenic and three carriage S. lugdunensis strains were obtained by Single-Molecule sequencing (PacBio) and compared to 15 complete genomes available in GenBank database. The aim was to identify (i) genetic determinants specific to pathogenic or carriage strains or specific to clonal complexes (CCs) defined by MultiLocus Sequence Typing, and (ii) antibiotic resistance genes and new putative virulence factors encoded or not by mobile genetic elements (MGE). Comparative genomic analysis did not show a strict correlation between gene content and the ability of the six strains to cause infections in humans and in a Galleria mellonella infection model. However, this study identified new MGEs (five prophages, two genomic islands and one plasmid) and genetic variations of some putative virulence-associated loci, especially in CC3 strains. For a clonal population, high variability and eight CC-dependent genetic organizations were observed for the ess locus, which encodes a putative type VII secretion system (T7SS) homologous to that of S. aureus. Further phenotypic and functional studies are needed to characterize this particular CC3 and to evaluate the role of T7SS in the virulence of S. lugdunensis.

Objectives We aimed to develop here a specific real-time PCR assay with TaqMan® probe to detect efficiently bacterial strains harboring the new plasmid mediated-colistin resistance mcr-8 gene. Methods Specific primers and probe for mcr-8 gene were designed from sequences alignment of all mcr genes variants. Specificity of the designed primers and probe were first checked par BlastN analysis and by in silico PCR. The analytical sensitivity and specificity tests were performed in vitro on a panel of 290 genomic DNA of Gram-negative bacteria and 250 metagenomic DNA from human stool samples. Whole genome sequencing (WGS) was performed here using MiSeq technology. Results Designed primers and probe were 100% specific tomcr-8 gene by BlastN and in silico PCR analysis. Real-time PCR screening of a collection of clinical isolates resulted to one positive Klebsiella pneumoniae isolate (KP95). WGS confirmed that this isolate harbored the mcr-8 gene and other resistance genes such as blaOXA-48, blaCTX-M-15 β-lactamases. Our real-time PCR was highly sensitive on a 10-fold dilution serie from a calibrated inoculum at 108 CFU/mL with a limit of detection at 55 CFU/mL. Conclusion To the best of our knowledge, we propose here, the first real-time PCR assay targeting mcr-8 gene with high specificity and sensitivity, able to detect mcr-8 gene in less than 2 h from any DNA sample. This real-time PCR assay allowed the first description of a clinical K. pneumoniae strain harboring the mcr-8 gene in Algeria.

β-lactams targeting the bacterial cell wall are not active on archaea. Here, we figure out that annotation of genes as β-lactamase in Archeae on the basis of homologous genes, initially annotated β-lactamases, is a remnant of the identification of the original activities of this group of enzymes, which in fact, have multiple functions including nuclease, ribonuclease, β-lactamase, or glyoxalase; which may specialized over time. We expressed a class B β-lactamase enzyme from Methanosarcina barkeri that digest penicillin G. Moreover, while a weak glyoxalase activity was detected, a significant ribonuclease activity on bacterial and synthetic RNAs was demonstrated. The β-lactamase activity was inhibited by a β-lactamase inhibitor (sulbactam), but its RNAse activity was not. This gene appears to has been transferred to the Flavobacteriaceae group including Elizabethkingia genus in which the expressed gene shows a more specialized activity toward resistance to tienanmicin but no glyoxalase activity. The expressed class C-like β-lactamase gene, also from Methanosarcina sp., shows also hydrolysis activity and was more closely related to DD-peptidase enzymes than known bacterial class C β-lactamases. Our findings highlight the requalification needness of annotated enzymes as β-lactamases and the specification overtime of multipotent enzymes in different ways in Archaea and bacteria.

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

Antibiotic resistance (AR) is a worldwide concern and the description of AR have been discovered mainly because of their implications in human medicine. Since the recent burden of whole-genome sequencing of microorganisms, the number of new AR genes (ARGs) have dramatically increased over the last decade. Areas covered: In this review, we will describe the different methods that could be used to characterize new ARGs using classic or innovative methods. First, we will focus on the biochemical methods, then we will develop on molecular methods, next-generation sequencing and bioinformatics approaches. The use of various methods, including cloning, mutagenesis, transposon mutagenesis, functional genomics, whole genome sequencing, metagenomic and functional metagenomics will be reviewed here, outlining the advantages and drawbacks of each method. Bioinformatics softwares used for resistome analysis and protein modeling will be also described. Expert opinion: Biological experiments and bioinformatics analysis are complementary. Nowadays, the ARGs described only account for the tip of the iceberg of all existing resistance mechanisms. The multiplication of the ecosystems studied allows us to find a large reservoir of AR mechanisms. Furthermore, the adaptation ability of bacteria facing new antibiotics promises a constant discovery of new AR mechanisms.

Purpose. The aim was to evaluate several microbiological tools for the identification of non-gonococcal Neisseria spp. isolated from semen samples from Lebanese men and to determine the putative link between the presence of Neisseria commensal species and infertility. Methodology. Within a cross-sectional retrospective study design, the whole population included in this investigation was divided in 2 categories: 173 patients with symptoms of infertility and 139 patients with normal seminograms. Epidemiological and microbiological investigations were performed for 59 strains of Neisseria through several phenotypic and genotypic tools, including seminograms, an analytical profile index of Neisseria and Haemophilus (API-NH), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), porA PCR, 16S rRNA and rplF gene sequencing, and antimicrobial susceptibility testing. Results. The risk of Neisseria infection was twice as high in infertile patients compared to the control group [odds ratio (OR): 1.95, confidence interval (CI): 1.05–3.65, P =0.03]. Unreliable diagnosis of Neisseria urogenital infection has serious health and social consequences. Our findings showed that API-NH and 16S rRNA sequencing are poor tools to identify Neisseria at the species level. Therefore, reliable diagnosis of cases using MALDI-TOF MS and/or rplF sequencing is needed to provide critical treatment decisions and prevent antimicrobial resistance spreading in the community. Conclusion. This work predicted a strong and significant association between the presence of Neisseria spp. in semen and male infertility among the Lebanese population. For a better understanding of this association, it is recommended that more genomic and large-scale epidemiological investigations are undertaken to reach definitive conclusions.

Infections with Staphylococcus aureus are being spread through contact with the community environment, but the role of wastewater treatment plants in the transmission routes is not defined. This study investigated the prevalence, types, genetic elements, and potential for transmission of S. aureus by these engineered systems. Synchronized sampling events at two wastewater treatment plants were conducted with isolates of S. aureus obtained by a selective enrichment method using acriflavine that suppressed Staphylococcus epidermidis growth. DNA was extracted from a subset of the S. aureus isolates, checked by PCR to assure the absence of S. epidermidis, and sequenced to determine the multilocus sequence type, spa type, and carriage of the methicillin resistance and Panton-Valentine leukocidin genetic elements. Sequences were analyzed for single nucleotide polymorphism differences in pairwise comparison of isolates. There were two dominant S. aureus clonal complexes identified in the isolates, one commonly identified as hospital-related (CC5) and one community-related (CC8). Both types of isolates were found at both treatment facilities, even though only one facility had significant hospital sewage inputs. The presence of S. aureus persisted through treatment, with some isolates recovered from the final processes showing genetic diversity. The presence of the Panton-Valentine leukocidin genetic element was greater than the 1-5% expected from global reports. Our results suggest that treatment provides an opportunity for genetic shift, while the persistence and release of evolved strains of S. aureus may provide an environmentally relevant pathway to new hosts in the environment.

Genomic and phenotypic characterization of resistance mechanisms to carbapenems and colistin in Klebsiella pneumoniae strains isolated from the Shaare Zedek Medical Center (Jerusalem, Israel).The 15 K. pneumoniae isolates studied present a high level of resistance to the antibiotics tested. Microbiological tests revealed production of carbapenemase enzymes by all isolates. ABI SOLiD sequencing of K. pneumoniae genomes generated between 5,033,665 and 8,876,861 million reads per genome. The genomic study revealed that carbapenem resistance was mediated by production of the KPC-3 enzyme in 13 isolates and NDM-1 enzyme in the remaining 2 isolates. In addition, colistin resistance was induced either by a missense mutation in the mgrB gene or inactivation of mgrB by an IS5-like insertion sequence. The mobile genetic element, transposon Tn4401, was identified in all genomes harboring the blaIn this study, we report the usefulness of whole-genome sequencing in detection of antibiotic resistance mechanisms and in highlighting the emergence of the carbapenem and colistin-resistant K. pneumoniae clone ST512 in Israeli hospitals.

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.

The production of extended-spectrum beta-lactamase (ESBL) by Enterobacteriaceae is a global public health problem. The present study was carried out on 156 strains of enteric bacteria isolated from urinary and cervicovaginal fluid samples. Identification of the strains was performed using MALDI-TOF MS and antibiotic susceptibility tests by disk diffusion method on Mueller Hinton agar in accordance with the recommendations of the Antibiogram Committee of the French Society for Microbiology. ESBL genes were sought by real time-polymerase chain reaction (RT-PCR) and by gel-based PCR. Gel-based PCR products were used for sequencing of the resistance genes, which were analyzed in the NCBI and Arg Annot databases. Results showed a predominance of Escherichia coli both in the urinary and cervicovaginal fluid samples. Klebsiella pneumoniae was the second most isolated bacterium in the specimens. Sensitivity to antibiotics revealed high levels of cephalosporin resistance but low resistance to carbapenem. No resistance was noted to colistine. The bla-TEM gene was present in Escherichia coli, while bla-SHV was found in Klebsiella pneumoniae and bla-CTX-M was recovered in both strains. Analysis of the sequences revealed that bla-Tem1 was predominant in bla-TEM and bla-CTX-M-15 was most represented by bla-CTX-M. This study confirms the presence of ESBL-producing Enterobacteria in Benin. This was an epidemiological study aimed at detecting cephalosporin resistance in gram-negative Bacillus isolated from urinary tract and genital infections developed by women. Since the advent of molecular biology techniques for the identification of resistance in bacteria including determination of ESBL resistance genes (i.e., TEM, SHV, CTX-M), no study has been conducted to identify the different variants that circulate in Benin by sequencing these resistance genes. This sequencing is essential in order to differentiate the non-ESBL parental enzymes, which is not possible with the commonly used PCR techniques that do not permit differentiation of the point generating different variants of the ESBL genes. The present study then helped to identify those variants, in particular Tem1, SHV1, and CTX-M15, which are most encountered in Benin and around the world.

International audience; This study investigates the molecular mechanisms of colistin and carbapenem resistance in Klebsiella pneumoniae ST101 strains. The three K. pneumoniae carried bla(CTX-M-15), bla(TEM-183), and bla(SHV-106) genes and two coharbored bla(OXA-48). As for colistin resistance, the isolates had amino acid substitutions in PmrA/B and a truncated mgrB gene in one isolate.

International audience

Multidrug-resistant Acinetobacter baumannii infection has recently emerged as a worldwide clinical problem and colistin is increasingly being used as last resort therapy. Despite its favorable bacterial killing, resistance and heteroresistance to colistin have been described. Mutations in the PmrAB regulatory pathway have been already associated with colistin resistance whereas the mechanisms for heteroresistance remain largely unknown. The purpose of the present study is to investigate the role of PmrAB in laboratory-selected mutants representative of global epidemic strains. During brief colistin exposure, colistin resistant and colistin heteroresistant mutants were selected in a one-step strategy. Population Analysis Profiling (PAP) was performed to confirm the suspected phenotype. Upon withdrawal of selective pressure, compensatory mutations were evaluated in another one-step strategy. A trans-complementation assay was designed to delineate the involvement of the PmrAB regulatory system using qPCR and PAP. Mutations in the PmrAB regulatory pathway were associated with colistin resistance and colistin heteroresistance as well. The transcomplementation assay provides a proof for the role played by changes in the PmrAB regulatory pathway. The level of colistin resistance is correlated to the level of expression of pmrC. The resistance phenotype was partially restored since the complemented strain became heteroresistant. This report shows the role of different mutations in the PmrAB regulatory pathway and warns on the development of colistin heteroresistance that could be present but not easily detected with routine testing.

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; 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.

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

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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.

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.