Search

The rise of antibiotic resistance in bacterial pathogens is a major health concern and the determinants of this emergence are actively studied. By contrast, although biofilms are an important cause of infections due to their high tolerance to a broad range of antimicrobials, much less is known on the development of antibiotic resistance within the biofilm environment, an issue potentially aggravating the current antibiotic crisis. Here, we compared the occurrence of resistance mutations in pathogenic Escherichia coli planktonic and biofilm populations exposed to clinically relevant cycles of lethal treatments with the aminoglycoside antibiotic amikacin. This experimental evolution approach revealed that mutations in sbmA and fusA are rapidly selected in biofilm but not in planktonic populations. The apparition of these bona fide resistance —and not tolerance— mutations is favored by the biofilm preexisting tolerance and high mutation rate. Moreover, we showed that while fusA mutations displayed a high fitness cost in planktonic conditions, these mutations were maintained in biofilms, a phenomenon further possibly amplified by the selection of fimH mutations favoring biofilm formation itself. Our study therefore provides new insights into the dynamic evolution of antibiotic resistance in biofilms, which could lead to clinically practical antibiotic regimen limiting biofilm-associated infections, while mitigating the emergence of worrisome antibiotic resistance mutations.

The genus Bordetella includes bacteria that are found in the environment and/or associated with humans and other animals. A few closely related species, including Bordetella pertussis, are human pathogens that cause diseases such as whooping cough. Here, we present a large database of Bordetella isolates and genomes and develop genotyping systems for the genus and for the B. pertussis clade. To generate the database, we merge previously existing databases from Oxford University and Institut Pasteur, import genomes from public repositories, and add 83 newly sequenced B. bronchiseptica genomes. The public database currently includes 2582 Bordetella isolates and their provenance data, and 2085 genomes (https://bigsdb.pasteur.fr/bordetella/). We use core-genome multilocus sequence typing (cgMLST) to develop genotyping systems for the whole genus and for B. pertussis, as well as specific schemes to define antigenic, virulence and macrolide resistance profiles. Phylogenetic analyses allow us to redefine evolutionary relationships among known Bordetella species, and to propose potential new species. Our database provides an expandable resource for genotyping of environmental and clinical Bordetella isolates, thus facilitating evolutionary and epidemiological research on whooping cough and other Bordetella infections.

In 1970, the seventh pandemic of cholera (7 P) reached both Africa and Europe. Between 1970 and 2011, several European countries reported cholera outbreaks of a few to more than 2,000 cases. We report here a whole-genome analysis of 1,324 7 P V. cholerae El Tor (7 PET) isolates, including 172 from autochthonous sporadic or outbreak cholera cases occurring between 1970 and 2011 in Europe, providing insight into the spatial and temporal spread of this pathogen across Europe. In this work, we show that the 7 PET lineage was introduced at least eight times into two main regions: Eastern and Southern Europe. Greater recurrence of the disease was observed in Eastern Europe, where it persisted until 2011. It was introduced into this region from Southern Asia, often circulating regionally in the countries bordering the Black Sea, and in the Middle East before reaching Eastern Africa on several occasions. In Southern Europe, the disease was mostly seen in individual countries during the 1970s and was imported from North and West Africa, except in 1994, when cholera was imported into Albania and Italy from the Black Sea region. These results shed light on the geographic course of cholera during the seventh pandemic and highlight the role of humans in its global dissemination., Since 1970, several cholera outbreaks caused by the “seventh pandemic” (7PET) lineage have been reported in Europe. Here, the authors demonstrate that the outbreaks were caused by repeated introductions of 7PET into Europe, rather than local environmental sources.

Improvements in cost and speed of next generation sequencing (NGS) have provided a new pathway for delivering disease diagnosis, molecular typing, and detection of antimicrobial resistance (AMR). Numerous published methods and protocols exist, but a lack of harmonisation has hampered meaningful comparisons between results produced by different methods/protocols vital for global genomic diagnostics and surveillance. As an exemplar, this study evaluated the sensitivity and specificity of five well-established in-silico AMR detection software where the genotype results produced from running a panel of 436 Escherichia coli were compared to their AMR phenotypes, with the latter used as gold-standard. The pipelines exploited previously known genotype–phenotype associations. No significant differences in software performance were observed. As a consequence, efforts to harmonise AMR predictions from sequence data should focus on: (1) establishing universal minimum to assess performance thresholds (e.g. a control isolate panel, minimum sensitivity/specificity thresholds); (2) standardising AMR gene identifiers in reference databases and gene nomenclature; (3) producing consistent genotype/phenotype correlations. The study also revealed limitations of in-silico technology on detecting resistance to certain antimicrobials due to lack of specific fine-tuning options in bioinformatics tool or a lack of representation of resistance mechanisms in reference databases. Lastly, we noted user friendliness of tools was also an important consideration. Therefore, our recommendations are timely for widespread standardisation of bioinformatics for genomic diagnostics and surveillance globally.

11 páginas, 2 figuras, 1 tabla, Inappropriately high breakpoints have resulted in systematic false-susceptible AST results to anti-TB drugs. MIC, PK/PD and clinical outcome data should be combined when setting breakpoints to minimise the emergence and spread of antimicrobial resistance., I. Comas was supported by PID2019-104477RB-I00 from the Spanish Science Ministry and by ERC (CoG 101001038)

International audience; Intestinal carriage of extended spectrum b-lactamase (ESBL)-producing Escherichia coli is a frequent, increasing, and worrying phenomenon, but little is known about the molecular scenario and the evolutionary forces at play. We screened 45 veal calves, known to have high prevalence of carriage, for ESBL-producing E. coli on 514 rectal swabs (one randomly selected colony per sample) collected over 6 months. We characterized the bacterial clones and plasmids carrying bla ESBL genes with a combination of genotyping methods, whole genome sequencing, and conjugation assays. One hundred and seventy-three ESBL-producing E. coli isolates [bla CTX-M-1 (64.7%), bla CTX-M-14 (33.5%), or bla CTX-M-15 (1.8%)] were detected, belonging to 32 bacterial clones, mostly of phylogroup A. Calves were colonized successively by different clones with a trend in decreasing carriage. The persistence of a clone in a farm was significantly associated with the number of calves colonized. Despite a high diversity of E. coli clones and bla CTX-M-carrying plasmids, few bla CTX-M gene/plasmid/chromosomal background combinations dominated, due to (i) efficient colonization of bacterial clones and/or (ii) successful plasmid spread in various bacterial clones. The scenario "clone versus plasmid spread" depended on the farm. Thus, epistatic interactions between resistance genes, plasmids, and bacterial clones contribute to optimize fitness in specific environments. IMPORTANCE The gut microbiota is the epicenter of the emergence of resistance. Considerable amount of knowledge on the molecular mechanisms of resistance has been accumulated, but the ecological and evolutionary forces at play in nature are less studied. In this context, we performed a field work on temporal intestinal carriage of extended spectrum b-lactamase (ESBL)-producing Escherichia coli in veal farms. Veal calves are animals with one of the highest levels of ESBL producing E. coli fecal carriage, due to early high antibiotic exposure. We were able to show that calves were colonized successively by different ESBL-producing E. coli clones, and that two main scenarios were at play in the spread of bla CTX-M genes among calves: efficient colonization of several calves by a few bacterial clones and successful plasmid spread in various bacterial clones. Such knowledge should help develop new strategies to fight the emergence of antibiotic-resistance.

Highly Resistant Cholera Outbreak Strain in Zimbabwe Whole genomes of 11 Vibrio cholerae O1 isolates from the 2018–2019 outbreak in Zimbabwe were sequenced to investigate the determinants of antimi...

Objectives The occurrence and zoonotic potential of antimicrobial resistance (AMR) in pigs and broilers has been studied intensively in past decades. Here, we describe AMR levels of European pig and broiler farms and determine the potential risk factors. Methods We collected faeces from 181 pig farms and 181 broiler farms in nine European countries. Real-time quantitative PCR (qPCR) was used to quantify the relative abundance of four antimicrobial resistance genes (ARGs) [aph(3′)-III, erm(B), sul2 and tet(W)] in these faeces samples. Information on antimicrobial use (AMU) and other farm characteristics was collected through a questionnaire. A mixed model using country and farm as random effects was performed to evaluate the relationship of AMR with AMU and other farm characteristics. The correlation between individual qPCR data and previously published pooled metagenomic data was evaluated. Variance component analysis was conducted to assess the variance contribution of all factors. Results The highest abundance of ARG was for tet(W) in pig faeces and erm(B) in broiler faeces. In addition to the significant positive association between corresponding ARG and AMU levels, we also found on-farm biosecurity measures were associated with relative ARG abundance in both pigs and broilers. Between-country and between-farm variation can partially be explained by AMU. Different ARG targets may have different sample size requirements to represent the overall farm level precisely. Conclusions qPCR is an efficient tool for targeted assessment of AMR in livestock-related samples. The AMR variation between samples was mainly contributed to by between-country, between-farm and within-farm differences, and then by on-farm AMU.

International audience; Methicillin-resistant Staphylococcus aureus (MRSA) can cause chronic lung infections in patients with Cystic Fibrosis (CF). One option for managing them is the use of linezolid. We hereby report the in-host emergence of linezolid resistance (LR) in MRSA in CF siblings via a population analysis. A collection of 171 MRSA strains from 68 samples were characterized by determining their linezolid Minimal Inhibitory Concentrations (MICs), analyzing the locus of staphylococcal protein A (spa) and whole genome sequencing. Courses of linezolid were retraced. Strains belonged to three spa types (t002, t045, t127) and two sequence types (ST1, ST5). Emergence of LR occurred under treatment, one year apart in both siblings, in the CC5-MRSA-I Geraldine clone harboring the toxic shock syndrome toxin-1-encoding gene. Resistance was related to a G2576T substitution present in a variable number of 23S rRNA gene copies. Susceptible and resistant strains were co-isolated within samples. Single Nucleotide Polymorphism-based analysis revealed complex colonizations by highly diversified, clonally related populations. LR remains rare in MRSA and there are very few longitudinal analyses documenting its emergence. Analyzing a large MRSA collection revealed new aspects of LR emergence: it emerges in specific subclonal lineages resulting from adaptive diversification of MRSA in the CF lung and this heterogeneity of intra-sample resistance may contribute to compromising antibiotic management.

International audience; Several phenanthrolinic analogs of quinolones have been synthesized and their antibacterial activity tested against Mycobacterium tuberculosis, other mycobacterial species and bacteria from other genera. Some of them show high activity (of the range observed for rifampicin) against M. tuberculosis replicating in vitro and in vivo (infected macrophages) conditions. These derivatives show the same activity with all or several M. tuberculosis complex bacterial mutants resistant to fluoroquinolones (FQ). This opens the way to the construction of new drugs for the treatment of FQ resistant bacterial infections, including tuberculosis. Several compounds showed also activity against Staphylococcus aureus and probably other species. These compounds do not show major toxicity. We conclude that the novel phenanthrolinic derivatives described here are potent hits for further developments of new antibiotics against bacterial infectious diseases including tuberculosis in particular those resistant to FQ.

bahar tokman, hrisi/0000-0002-2205-5120 WOS:000525325200020 PubMed ID: 31634565 Objectives: Studies on the antimicrobial susceptibility profile of anaerobic bacteria are underrepresented in the literature. Within this study we aim to give an extensive overview of the differences in antimicrobial susceptibility profiles between different European and surrounding countries. Methods: Minimal inhibitory concentration (MIC) data of different antibiotics were collected from 10 participating laboratories, representing an equal number of countries. All MIC's were determined using Etest, according to the protocol used by the participating laboratory. Anaerobic genera represented by at least 10 clinical isolates were included in the study. Results: Each country tested different antibiotics, sometimes depending on the kind of infection and/or the anaerobic species isolated. All countries tested clindamycin and metronidazole. Resistance rates differed remarkably between the different countries. Especially in Kuwait, resistance was high for all tested antibiotics. Unexpected metronidazole resistance was observed for Finegoldia magna isolates, Peptoniphilus isolates and Eggerthella lenta isolates. Conclusions: Due to the extensive differences in antimicrobial susceptibility profile of anaerobic bacteria isolated within different countries, we strongly recommend to perform this kind of study on a regular basis. (C) 2019 The Authors. Published by Elsevier Ltd.

International audience; Background. Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) has emerged globally over the last decade as a major nosocomial pathogen that threatens patient care. These highly resistant bacteria are mostly associated with a single Kp clonal group, CG258, but the reasons for its host and hospital adaptation remain largely unknown. Methods. We analyzed the in vivo evolution of a colistin-resistant KPC-Kp CG258 strain that contaminated a patient following an endoscopy and was responsible for a fatal bacteremia 4.5 years later. Whole-genome sequencing was performed on 17 KPC-Kp isolates from this patient; single-nucleotide polymorphisms were analyzed and their implication in antimicrobial resistance and bacterial host adaptation investigated. Results. The patient KPC-Kp strain diversified over 4.5 years at a rate of 7.5 substitutions per genome per year, resulting in broad phenotypic modifications. After 2 years of carriage, all isolates restored susceptibility to colistin. Higher expression of the fimbriae conferred the ability to produce more biofilm, and the isolate responsible for a bacteremia grew in human serum. The convergent mutations occurring in specific pathways, such as the respiratory chain and the cell envelope, revealed a complex long-term adaptation of KPC-Kp. Conclusions. Broad genomic and phenotypic diversification and the parallel selection of pathoadaptive mutations might contribute to long-term carriage and virulence of KPC-Kp CG258 strains and to the dissemination of this clone.

National audience

International audience; Background: Postoperative pneumonia (POP) is a severe complication of major lung resection. The objective of this study was to describe the current epidemiology and appropriateness of antibiotic prescriptions in severe POP, 4 years after implementation of an antimicrobial stewardship program that was based on weekly multidisciplinary review of all antibiotic therapies.Methods: This study was a retrospective analysis of a prospectively collected database. It included all cases of severe POP occurring within 30 days after major lung resection of in a 1,500-bed hospital between 2013 and 2015. Criteria for severe POP were acute respiratory failure, severe sepsis, or a rapidly extensive pulmonary infiltrate. The study collected data on incidence, clinical outcomes, and microbiological analyses. Appropriateness of antibiotic prescribing was assessed by quality indicators previously validated in the literature.Results: Over the study period, 1,555 patients underwent major lung surgery. Severe POP occurred in 91 patients (5.8%; confidence interval, 4.7%; 7.0%), with a mortality rate of 9.0% (8 of 91; confidence interval, 3.0%; 14.6%). In POP with positive microbiological results, the proportion of gram-negative bacteria other than Haemophilus was 76% (50 of 66 cases). All patients (91 of 91) had respiratory samples taken within 24 hours after the start of antibiotics; empiric therapy was concordant with the guideline in 80% (69 of 86), and it was switched to pathogen-directed therapy in 74% (46 of 62). In 71 of 91 patients (78%), the antibiotic duration was up to 7 days.Conclusions: This study reported a high proportion of gram-negative bacteria in severe POP. Four years after implementation of the program, quality indicators of antibiotic prescribing were all >70%. The rate of de-escalation to pathogen-directed therapy could be improved, however.

Background Limited epidemiological and antimicrobial resistance data are available on Salmonella enterica from sub-Saharan Africa. We determine the prevalence of resistance to antibiotics in isolates in the Central African Republic (CAR) between 2004 and 2013 and the genetic basis for resistance to third-generation cephalosporin (C3G). Methodology/Principal findings A total of 582 non-duplicate human clinical isolates were collected. The most common serotype was Typhimurium (n = 180, 31% of the isolates). A randomly selected subset of S. Typhimurium isolates were subtyped by clustered regularly interspaced short palindromic repeat polymorphism (CRISPOL) typing. All but one invasive isolate tested (66/68, 96%) were associated with sequence type 313. Overall, the rates of resistance were high to traditional first-line drugs (18–40%) but low to many other antimicrobials, including fluoroquinolones (one resistant isolate) and C3G (only one ESBL-producing isolate). The extended-spectrum beta-lactamase (ESBL)-producing isolate and three additional ESBL isolates from West Africa were studied by whole genome sequencing. The blaCTX-M-15 gene and the majority of antimicrobial resistance genes found in the ESBL isolate were present in a large conjugative IncHI2 plasmid highly similar (> 99% nucleotide identity) to ESBL-carrying plasmids found in Kenya (S. Typhimurium ST313) and also in West Africa (serotypes Grumpensis, Havana, Telelkebir and Typhimurium). Conclusions/Significance Although the prevalence of ESBL-producing Salmonella isolates was low in CAR, we found that a single IncHI2 plasmid-carrying blaCTX-M-15 was widespread among Salmonella serotypes from sub-Saharan Africa, which is of concern., Author summary Salmonella enterica infections are common causes of bloodstream infection in sub-Saharan Africa and associated with a high mortality rate. Levels of multidrug resistance have become alarmingly high. Then, third-generation cephalosporin (C3G) and fluoroquinolones have become standard for first-line empirical treatment. Recently, C3G-resistant Salmonella populations have emerged and spread over all continents. This resistance is mainly mediated by acquired extended-spectrum beta-lactamase (ESBL) genes carried by mobile genetic elements such as plasmids. We report here the prevalence of resistance to antibiotics in isolates in the Central African Republic (CAR) between 2004 and 2013 and the genetic basis for resistance to C3G. Overall, resistance rates to antimicrobials were low during the study period, for all classes other than conventional antimicrobials, confirming recommendations for first-line treatment based on C3G and fluoroquinolones. Only one ESBL-producing isolate was recovered. The ESBL gene and the majority of antimicrobial resistance genes found were present in a large plasmid highly similar to ESBL-carrying plasmids found in East and West Africa, highlighting its significant role in the spread of ESBL genes in Salmonella isolates in sub-Saharan Africa. These finding have implications for treatment of salmonellosis and support the growing necessity for increased microbiological surveillance based on networks of clinical laboratories in order to control dissemination of antibiotic resistance among Salmonella isolates.

Voir aussi le même article publié dans le livre : https://onlinelibrary.wiley.com/doi/book/10.1128/9781683670261 ; Dépôt : https://hal-pasteur.archives-ouvertes.fr/pasteur-03116237; International audience; The endothelium is the layer of endothelial cells lining the inner surface of blood vessels, which span the entire body and ensure the distribution of blood throughout the organism. It is estimated that the human body contains a staggering 100,000 km of blood vessels, more than twice the earth’s circumference. Therefore, a bacterium reaching the circulation is engaged in a network of huge proportions. Moreover, a pathogen traveling through the circulatory system does not encounter a homogeneous environment, as an important feature of the vascular network is its diversity. Although endothelial cells are present in all vessels, the organization of the vessel wall, which is formed by three layers—the tunica intima, media, and adventitia (from the vessel lumen outward)—is different among different vessel types and different organs. Vessels can be first differentiated by the complex extracellular matrix layers surrounding them. For instance, elastic arteries such as the aorta are surrounded by 50 elastic layers, providing them with unique mechanical properties. Second, the cellular content is also different according to vessel type; the walls of arteries and veins contain a layer of smooth muscle cells that gives them the capacity to relax or constrict in response to vasoactive molecules. An additional level of complexity in the network stems from the fact that larger vessels, veins or arteries, are themselves vascularized by smaller vessels, the vasa vasorum.

We compared extended-spectrum β-lactamase-producing Escherichia coli isolates from meat and fish, gut-colonized women, and infected patients in Cambodia. Nearly half of isolates from women were phylogenetically related to food-origin isolates; a subset had identical multilocus sequence types, extended-spectrum β-lactamase types, and antimicrobial resistance patterns. Eating sun-dried poultry may be an exposure route. ispartof: EMERGING INFECTIOUS DISEASES vol:25 issue:1 pages:126-131 ispartof: location:United States status: published

Introduction: The 2018 Global Meningococcal Initiative (GMI) meeting focused on evolving invasive meningococcal disease (IMD) epidemiology, surveillance, and protection strategies worldwide, with emphasis on emerging antibiotic resistance and protection of high-risk populations. The GMI is comprised of a multidisciplinary group of scientists and clinicians representing institutions from several continents. Areas covered: that the incidence and prevalence of IMD continually varies both geographically and temporally, and surveillance systems differ worldwide, the true burden of IMD remains unknown. Genomic alterations may increase the epidemic potential of meningococcal strains. Vaccination and (to a lesser extent) antimicrobial prophylaxis are the mainstays of IMD prevention. Experiences from across the globe advocate the use of conjugate vaccines, with promising evidence growing for protein vaccines. Multivalent vaccines can broaden protection against IMD. Application of protection strategies to high-risk groups, including individuals with asplenia, complement deficiencies and human immunodeficiency virus, laboratory workers, persons receiving eculizumab, and men who have sex with men, as well as attendees at mass gatherings, may prevent outbreaks. There was, however, evidence that reduced susceptibility to antibiotics was increasing worldwide. Expert commentary: The current GMI global recommendations were reinforced, with several other global initiatives underway to support IMD protection and prevention. This paper was in part supported by Sanofi Pasteur. Sí

International audience; Whole-genome sequencing (WGS) has been vital for revealing the rapid temporal and spatial evolution of antimicrobial resistance (AMR) in bacterial pathogens. Some antimicrobial-resistant pathogens have outpaced us, with untreatable infections appearing in hospitals and the community. However, WGS has additionally provided us with enough knowledge to initiate countermeasures. Although we cannot stop bacterial adaptation, the predictability of many evolutionary processes in AMR bacteria offers us an opportunity to channel them using new control strategies. Furthermore, by using WGS for coordinating surveillance and to create a more fundamental understanding of the outcome of antimicrobial treatment and AMR mechanisms, we can use current and future antimicrobials more effectively and aim to extend their longevity.

International audience; Antimicrobial resistance (AMR), associated with a lack of new antibiotics, is a major threat. Some countries have been able to contain resistance, but in most countries the numbers of antibiotic-resistant bacteria continue to increase, along with antibiotic consumption by humans and animals. AMR is a global issue, and concerns all decision-makers worldwide. Some initiatives have been undertaken in the last 15 years, in particular by the WHO, the European Centre for Disease Prevention and Control, and the CDC, but those initiatives were partial and poorly implemented, without coordination. Very recently, some important initiatives have been implemented by the WHO. Since 2009, a US and European joint task force, the Trans-Atlantic Task Force on Antibiotic Resistance, has been working on common recommendations. At a national level, some important initiatives have been implemented, in particular in European countries and in the USA. The Chennai declaration, in India, is also a good example of a multidisciplinary and national initiative that was highly political. Finally, several non-governmental non-profit organizations are also very active, and have helped to raise awareness about the problem of AMR. In the future, this global issue will need political involvement and strong cooperation between countries and between international agencies.

International audience; The bacterium Pseudomonas aeruginosa is well known to have a remarkable adaptive capacity allowing it to colonise many environments. A recent study on environmental isolates from dental unit waterlines (DUWLs) hinted at a genetic clustering into two groups. Isolates from one of these groups, named cluster III, were shown to have unusual phenotypes for environmental isolates, such as an increased biofilm production. To have a better ecological view, more specifically on isolates from cluster III, the complete genomes of 39 isolates including 16 from DUWLs were sequenced. In addition to an investigation of antibiotic resistance and secretion system gene content, a molecular phylogeny allowed confirmation of the split of the 16 environmental isolates in two groups and also sheds light on a correlation between the phylogenetic positions and the serotypes of the isolates. Isolates from cluster III harboured insertion sequences ISPa11 inserted into the O-specific antigen biosynthesis clusters and the gene lasR, encoding for a master regulator of the quorum sensing. Investigation of key regulators revealed another truncated gene, gacS. Alteration in lasR and gacS genes was consistent with phenotypic assays confirming their inactivation. These results bring new perspectives regarding the ecological adaptive potential of P. aeruginosa.

Horizontal gene transfer may occur between distantly related bacteria, thus leading to genetic plasticity and in some cases to acquisition of novel resistance traits . Proteus mirabilis is an enterobacterial species responsible for human infections that may express various acquired β-lactam resistance genes, including different classes of carbapenemase genes. Here we report a Proteus mirabilis clinical isolate (strain 1091) displaying resistance to penicillin, including temocillin, together with reduced susceptibility to carbapenems and susceptibility to expanded-spectrum cephalosporins. Using biochemical tests, significant carbapenem hydrolysis was detected in P. mirabilis 1091. Since PCR failed to detect acquired carbapenemase genes commonly found in Enterobacteriaceae , we used a whole-genome sequencing approach that revealed the presence of bla OXA-58 class D carbapenemase gene, so far identified only in Acinetobacter species. This gene was located on a 3.1-kb element coharboring a bla AmpC -like gene. Remarkably, these two genes were bracketed by putative XerC-XerD binding sites and inserted at a XerC-XerD site located between the terminase-like small- and large-subunit genes of a bacteriophage. Increased expression of the two bla genes resulted from a 6-time tandem amplification of the element as revealed by Southern blotting. This is the first isolation of a clinical P. mirabilis strain producing OXA-58, a class D carbapenemase, and the first description of a XerC-XerD-dependent insertion of antibiotic resistance genes within a bacteriophage. This study revealed a new role for the XerC-XerD recombinase in bacteriophage biology.

International audience; Helicobacter pylori colonizes half of the world population and is associated with gastric cancer. We have previously demonstrated that docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid known for its anti-inflammatory and antitumor effects, directly inhibits H. pylori growth in vitro and in mice. Nevertheless, the concentration of DHA shown to reduce H. pylori mice gastric colonization was ineffective in vitro. Related to the auxotrophy of H. pylori for cholesterol, we hypothesize that other mechanisms, in addition to DHA direct antibacterial effect, must be responsible for the reduction of the infection burden. In the present study we investigated if DHA affects also H. pylori growth, by reducing the availability of membrane cholesterol in the epithelial cell for H. pylori uptake. Levels of cholesterol in gastric epithelial cells and of cholesteryl glucosides in H. pylori were determined by thin layer chromatography and gas chromatography. The consequences of epithelial cells’ cholesterol depletion on H. pylori growth were assessed in liquid cultures. We show that H. pylori uptakes cholesterol from epithelial cells. In addition, DHA lowers cholesterol levels in epithelial cells, decreases its de novo synthesis, leading to a lower synthesis of cholesteryl glucosides by H. pylori. A previous exposition of H. pylori to cholesterol influences the bacterium response to the direct inhibitory effect of DHA. Overall, our results suggest that a direct effect of DHA on H. pylori survival is modulated by its access to epithelial cell cholesterol, supporting the notion that cholesterol enhances the resistance of H. pylori. The cholesterol-dependent resistance of H. pylori to antimicrobial compounds raises new important aspects for the development of new anti-bacterial strategies.

Background Pneumococcus kills over one million children annually and over 90 % of these deaths occur in low-income countries especially in Sub-Saharan Africa (SSA) where HIV exacerbates the disease burden. In SSA, serotype 1 pneumococci particularly the endemic ST217 clone, causes majority of the pneumococcal disease burden. To understand the evolution of the virulent ST217 clone, we analysed ST217 whole genomes from isolates sampled from African and Asian countries. Methods We analysed 226 whole genome sequences from the ST217 lineage sampled from 9 African and 4 Asian countries. We constructed a whole genome alignment and used it for phylogenetic and coalescent analyses. We also screened the genomes to determine presence of antibiotic resistance conferring genes. Results Population structure analysis grouped the ST217 isolates into five sequence clusters (SCs), which were highly associated with different geographical regions and showed limited intracontinental and intercontinental spread. The SCs showed lower than expected genomic sequence, which suggested strong purifying selection and small population sizes caused by bottlenecks. Recombination rates varied between the SCs but were lower than in other successful clones such as PMEN1. African isolates showed higher prevalence of antibiotic resistance genes than Asian isolates. Interestingly, certain West African isolates harbored a defective chloramphenicol and tetracycline resistance-conferring element (Tn5253) with a deletion in the loci encoding the chloramphenicol resistance gene (cat pC194), which caused lower chloramphenicol than tetracycline resistance. Furthermore, certain genes that promote colonisation were absent in the isolates, which may contribute to serotype 1’s rarity in carriage and consequently its lower recombination rates. Conclusions The high phylogeographic diversity of the ST217 clone shows that this clone has been in circulation globally for a long time, which allowed its diversification and adaptation in different geographical regions. Such geographic adaptation reflects local variations in selection pressures in different locales. Further studies will be required to fully understand the biological mechanisms which makes the ST217 clone highly invasive but unable to successfully colonise the human nasopharynx for long durations which results in lower recombination rates. Electronic supplementary material The online version of this article (doi:10.1186/s12879-016-1987-z) contains supplementary material, which is available to authorized users.

International audience; Bacteriophages were discovered in the early 20th century and rapidly used to treat bacterial infections in humans. As the first specific antibacterial agents, they were used worldwide until antibiotics ramped up. Thereafter, rapidly forgotten, they be- came the favorite toolbox for researchers that used them to elucidate some of the most fundamental aspects of the cellular life at the molecular level. Today, facing the threat of antibiotic resistant bacteria, bacteriophages are being reconsidered for their use in medicine. During the past century, knowledge on bacteriophages has improved considerably, nevertheless phage therapy is still in its infancy. Taking two examples of recently published experimental phage therapy results, this article summarizes the hopes but also the challenges that surround the future development of human phage therapy.; -Découverts au début du XX e siècle, les bactériophages ont rapidementété utilisés pour traiter des infections bactériennes, devenant ainsi les premiers antibactériens spécifiquesàêtre employés en médecine. Cependant, l'arrivée des antibiotiques a brutalement réduit leur utilisation, les confinant au rayon ((boîteà outils)) du chercheur. Celui-ci en fît bon usage permettant de dévoiler une très grande partie des secrets moléculaires de la cellule. Aujourd'hui, faceà la menace que représentent les pathogènes bactériens résistants aux antibiotiques, les bactériophages tentent de faire leur retour dans le domaine médical. Bien que la connaissance de ces virus bactériens ait considérablement progressé après presque un siècle d'études, leur utilisation thérapeutique est loin d'être maîtrisée. Cet article relate,à l'aide de deux exemples récents de l'utilisation expérimentale des bactériophages, les espoirs mais aussi les challenges qui entourent la mise en place de la phagothérapie appliquéeà l'Homme. Mots clés : Bactériophages thérapeutiques / infections bactériennes / résistance aux antibiotiques / pneumonie / diarrhée Abstract-Phagotherapy: a nightmare for bacteria, a dream for physicians? Bacteriophages were discovered in the early 20th century and rapidly used to treat bacterial infections in humans. As the first specific antibacterial agents, they were used worldwide until antibiotics ramped up. Thereafter, rapidly forgotten, they became the favorite toolbox for researchers that used them to elucidate some of the most fundamental aspects of the cellular life at the molecular level. Today, facing the threat of antibiotic resistant bacteria, bacteriophages are being reconsidered for their use in medicine. During the past century, knowledge on bacteriophages has improved considerably, nevertheless phage therapy is still in its infancy. Taking two examples of recently published experimental phage therapy results, this article summarizes the hopes but also the challenges that surround the future development of human phage therapy.

International audience; OBJECTIVES:Resistance to fluoroquinolones (FQs) in Mycobacterium tuberculosis (Mtb) is mainly due to mutations in DNA gyrase (GyrA2B2), with the most common substitutions located at positions 90 and 94 in GyrA. Two clinical MDR Mtb (MDR-TB) strains harbouring an A90E or D94N substitution in GyrA were found to be surprisingly susceptible to FQs (ofloxacin MIC ≤2 mg/L). We studied the impact of the additional GyrA substitutions found in these strains (T80A and T80A + A90G, respectively) on FQ susceptibility.METHODS:Mutants of interest were generated by site-specific mutagenesis of GyrA alleles. WT and mutant TB DNA gyrase subunits were overexpressed in Escherichia coli and purified, and the in vitro susceptibility to FQs of their DNA supercoiling reaction was studied.RESULTS:IC50s of mutant gyrase complexes bearing GyrA D94N and A90E were 3- to 36-fold higher than WT IC50s, whereas IC50s of gyrase bearing T80A + A90G + D94N and T80A + A90E were close to the WT IC50s.CONCLUSIONS:We demonstrated that substitutions T80A and A90G restore FQ susceptibility when associated with a substitution implicated in high-level FQ resistance. Line probe assay misclassification of MDR-TB strains as pre-XDR or XDR can be corrected by sequence analysis of gyrA.

Copper-based compounds are widely used in integrated pest management (1PM) programs aiming at controlling agriculturally important plant bacterial pathogens and the latter have adapted in response to this selective pressure. Copper resistance of Xanthomonas citri pv. citri (Xcc), a major citrus pathogen worldwide causing Asialie citrus canker, was first observed in Argentina two decades ago and subsequently reported as a copLAB-based, plasmid-encoded system. The emergence of resistant strains has since been reported in Réunion (South West lndian Ocean) and Martinique (Eastern Caribbean Sea). Disease severity was found markedly increased in groves established with susceptible cultivars and infected with copper-resistant Xcc. Using tandem repeat-based genotyping and copLAB PCR, we demonstrated that the genetic structure of the copperresistant strains from these three regions was made up of two distant clusters and varied for the detection of copLAB amplicons. ln order to investigate this pattern more closely, we sequenced six copper-resistant Xcc strains from Argentina, Martinique and Réunion, together with reference copper-resistant Xanthomonas and Stenotrophomonas strains using long-read sequencing technology. Genes involved in copper resistance were found to be strain-dependent with the nove! identification in Xcc of copABCD and a eus heavy metal efflux resistance-nodulation-division system. The genes providing the adaptive trait were part of a mobile genetic element similar to Tn3-like transposons and included in a conjugative plasmid. The mining of ail bacterial genomes available from public databases suggested that the mobile elements containing copper resistance genes and their plasmid environments were primarily detected in the Xanthomonadaceae family.

International audience; The aim of this study was to estimate the prevalence of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) in faeces of healthy children aged 0-59 months in Bangui (Central African Republic). Stool samples of 134 children, recruited for a matched case-control study, were cultured on a commercial ESBL-selective chromogenic medium (CHROMagar ESBL, France). The phenotypic resistance patterns of isolated strains were investigated, as well as the genetic basis for antibiotic resistance. The factors associated with increased risk for ESBL-E carriage were also studied. The prevalence of ESBL-E carriage was 59% (79/134), one of the highest reported worldwide. The only factor found to be associated with carriage was living in a highest-income family (p=0.03). In all, 83 ESBL-E were recovered as simultaneous carriage of two strains was detected in four children. blaCTX-M-15 was found in all strains except two, frequently associated with qnr (54/81, 66%) and aac(6')-Ib-cr (35/81, 43%) genes. Escherichia coli, the most commonly recovered species (51/83, 61%), was assigned mainly to the pandemic B2-O25b-ST131 group (39/51, 76%). Resistance transfer, which was studied in 20 randomly selected ESBL-E strains, was successful in 13 (13/20, 65%) isolates. In eight of these isolates (8/13, 62%), blaCTX-M-15 genes were found in incompatibility group FIb conjugative plasmids. We found one of the highest prevalence rates of faecal carriage of ESBL-E reported worldwide, highlighting the need to improve control of the distribution of antibiotics in limited-resource countries.

Objectives In November 2015, a plasmid-mediated colistin resistance, MCR-1, was described in animals, food and humans in China, and it was considered as a potential emerging threat to public health. Therefore, we screened for the mcr-1 gene a European collection of colistin-resistant Escherichia coli ( n =218) and Salmonella spp. ( n =74) isolated from diseased food-producing animals between 2004 and 2014 and characterized the mcr-1 -positive clones. Methods Screening for mcr-1 gene was performed by PCR on isolates for which inhibition diameter was E. coli isolates were then characterized by phylogrouping, multilocus sequence typing and pulsed-field gel electrophoresis typing. Antibiotic susceptibility was determined by disk diffusion testing or by broth microdilution. Results Among the collection, 42 E. coli and three Salmonella spp. were positive for mcr-1, with continuous detection since 2004 mainly from bovine and swine digestive infections. Most of the mcr-1 -positive strains were resistant to amoxicillin and cotrimoxazole but remained susceptible to cephalosporins, carbapenems and piperacillin/tazobactam. All but one isolate were resistant to colistin, with a minimum inhibitory concentration of >2 mg/L. Most of the mcr-1 -positive E. coli belonged to the phylogroup A with two prevalent clonal complexes, CC10 and CC165, in which sequence type 10 and sequence type 100 were overrepresented and pulsed-field gel electrophoresis typing revealed a high diversity of pulsotypes. Conclusions MCR-1 was detected yearly in European food-producing animal since 2004 with a high diversity of pulsotypes supporting the dissemination of mcr-1 via plasmids.

On 8 June, 2015, London, a workshop on bacteriophage therapy was organized by the European Medicines Agency (EMA). About 60 delegates from various stakeholders (participants belonged to public bodies such as WHO, ECDC, WAAAR, national health agencies from different European countries, politicians, journalists, clinicians, researchers, including six members of P.H.A.G.E., as well as private companies.) were convened to participate, with the workshop broadcast on the EMA website. Why did EMA organize a workshop on bacteriophage therapy? The answer is the emergency to find solutions to overcome the global antibiotic crisis and the unfortunately corresponding lack of significant interest in bacteriophage therapy. The past two decades, public health agencies reported on the dramatic increase of drug-resistant pathogens, a worrisome situation leading the World Health Organization (WHO) to declare a new ‘preantibiotic era’ in its 2014 surveillance report (http://www.who.int). In the USA, based on data collected in 2009, over 20 000 annual deaths were ascribed to the lack of effective antibiotics. Comparably in Europe, we estimate that more than 100 000 people worldwide die annually. The situation gets worse: a recent study mentions that in France about 13 000 people died in 2012 (http://www.invs.sante.fr). Worldwide, the estimation is rising to 700 000 deaths annually, and therefore G7 Health Ministries recently called for a global ‘One Health’ approach (https://www.g7germany.de). Future predictions are alarming, with 10 million people estimated to die from resistant microorganisms in 2050 if nothing changes (Carlet 2015). Bacteriophage therapy is nearly 100 years old and was first introduced in France and Belgium a decade before the discovery of penicillin by A. Fleming. After an initial worldwide interest (including from the …

How bacteria switch between tracks Bacterial riboswitches prevent the formation of full-length messenger RNA, and hence proteins, via transcriptional termination in response to metabolites. However, identifying riboswitches within the genome has previously required comparative analysis, which may miss species- and environmentally specific responses. Dar et al. developed a method called term-seq to document all riboswitches in a bacterial genome, as well as their metabolite counterparts (see the Perspective by Sommer and Suess). The method revealed a role for pathogenic bacterial riboswitches in antibiotic resistance. Thus, transcription may be one way pathogens fend off antibiotic attack. Science , this issue p. 10.1126/science.aad9822 ; see also p. 144

Identification of clinical isolates of Neisseria meningitidis that are resistant to rifampin is important to avoid prophylaxis failure in contacts of patients, but it is hindered by the absence of a breakpoint for resistance, despite many efforts toward standardization. We examined a large number ( n = 392) of clinical meningococcal isolates, spanning 25 years (1984 to 2009), that were collected in 11 European countries, Argentina, and the Central African Republic. The collection comprises all clinical isolates with MICs of ≥0.25 mg/liter ( n = 161) received by the national reference laboratories for meningococci in the participating countries. Representative isolates displaying rifampin MICs of n = 231). Typing of isolates was performed, and a 660-bp DNA fragment of the rpoB gene was sequenced. Sequences differing by at least one nucleotide were defined as unique rpoB alleles. The geometric mean of the MICs was calculated for isolates displaying the same allele. The clinical isolates displaying rifampin MICs of >1 mg/liter possessed rpoB alleles with nonsynonymous mutations at four critical amino acid residues, D542, H552, S548, and S557, that were absent in the alleles found in all isolates with MICs of ≤1 mg/liter. Rifampin-susceptible isolates could be defined as those with MICs of ≤1 mg/liter. The rpoB allele sequence and isolate data have been incorporated into the PubMLST Neisseria database ( http://pubmlst.org/neisseria/ ). The rifampin-resistant isolates belonged to diverse genetic lineages and were associated with lower levels of bacteremia and inflammatory cytokines in mice. This biological cost may explain the lack of clonal expansion of these isolates.

International audience; OBJECTIVES: Integrons are bacterial genetic elements that can capture and express genes contained in mobile cassettes. Integrons have been described worldwide in Gram-negative bacteria and are a marker of antibiotic resistance. We developed a specific and sensitive Taqman probe-based real-time PCR method with three different primer-probe pairs for simultaneous detection of the three main classes of integron. METHODS: Sensitivity was assessed by testing mixtures of the three targets (intI integrase genes of each integron class) ranging from 10 to 10(8) copies. Specificity was determined with a panel of integron-containing and integron-free control strains. The method was then applied to clinical samples. RESULTS: The PCR method was specific and had a sensitivity of 10(2) copies for all three genes, regardless of their respective quantities. The method was quantitative from 10(3) to 10(7) copies, and was able to detect integrons directly in biological samples. CONCLUSIONS: We have developed a rapid, quantitative, specific and sensitive method that could prove useful for initial screening of Gram-negative isolates, or clinical samples, for likely multidrug resistance.

The objective of this study was to implement a semimechanistic pharmacokinetic-pharmacodynamic (PK-PD) model to describe the effects of ciprofloxacin against Pseudomonas aeruginosa in vitro. Time-kill curves were generated with an initial inoculum close to 5 × 10 6 CFU/ml of P. aeruginosa PAO1 and constant ciprofloxacin concentrations between 0.12 and 4.0 μg/ml (corresponding to 0.5× and 16× MIC). To support the model, phenotypic experiments were conducted with the PAO7H mutant strain, which overexpresses the MexEF OprN efflux pump and phenyl arginine β-naphthylamide (PAβN), a known efflux inhibitor of main Mex multidrug efflux systems. A population approach was used for parameter estimation. At subinhibitory ciprofloxacin concentrations (0.12 and 0.25 μg/ml), an initial CFU decay followed by regrowth was observed, attesting to rapid emergence of bacteria with increased but moderate resistance (8-fold increase of MIC). This phenomenon was mainly due to an overexpression of the Mex protein efflux pumps, as shown by a 16-fold diminution of the MIC in the presence of PAβN in these strains with low-level resistance. A PK-PD model with adaptation development was successfully used to describe these data. However, additional experiments are required to validate the robustness of this model after longer exposure periods and multiple dosing regimens, as well as in vivo.

We report the case of a 55-year-old man who exhibited a nodular pneumonia 4 months after an allogeneic hematopoietic stem cell transplantation. Culture of the bronchoalveolar lavage fluid revealed Nocardia pseudobrasiliensis . This recently described carbapenem-resistant species should be included in the differential diagnosis of fungal infection in this setting.

Background: In developing countries, knowledge of antimicrobial resistance patterns is essential to define empirical therapy. Methodology: All the surgery and intensive care wards of two hospitals in Antananarivo were included to study the antimicrobial susceptibility of the pathogenic bacteria causing nosocomial infections. A repeated cross-sectional survey was conducted between September 2006 and March 2008, one day per week. Isolates were identified using classical methods, and resistance to antibiotics was assessed according to the recommendations of the Antibiogram Committee of the French Microbiology Society. Results: Clinical specimens from 706 from 651 patients were collected. Of the 533 bacterial pathogens, 46.7% were Enterobacteriaceae , 19.3% were Staphylococcus aureus , and 19.1% were pathogens from the hospital environment ( Pseudomonas aeruginosa and Acinetobacter baumannii ).Frequencies of resistance were high, particularly in Enterobacteriaceae ; however, the rate of Staphylococcus aureus isolates resistant to oxacillin (13.6 %) was moderate and all these isolates were susceptible to glycopeptids. The percentages of isolates susceptible to ceftazidim were 81.8% for E. coli , 60.9% for Klebsiella , and 52.5% for Enterobacter spp. Resistance to third-generation cephalosporins was due to extended spectrum betalactamases (ESBL). Multivariate analysis showed that diabetes (adjusted OR: 3.9) and use of an invasive procedures (adjusted OR: 3.5) were independent risk factors for resistance to third-generation cephalosporins. Conclusion: A nationwide surveillance programme is needed to monitor the microbial trends and antimicrobial resistance in Madagascar.

Maintaining quinolone concentrations outside the mutant selection window (MSW) between the MIC and mutant prevention concentration (MPC) was suggested by in vitro and in vivo studies to prevent the selection of resistant mutants. However, selection also may depend on the presence of resistant bacterial mutants at the start of treatment, which is highly dependent on the initial inoculum size. In this study, a mouse thigh bacterial infection model was used to test the influence of different exposures to marbofloxacin on the selection of resistant bacteria after infection with a low (10 5 CFU) or high (10 8 CFU) initial inoculum of Escherichia coli . The inoculum size was shown to influence the exposure to marbofloxacin and the values of pharmacokinetic/pharmacodynamic indices. When the abilities of the indices time within the MSW ( T MSW ), area under the concentration-time curve of 0 to 24 h divided by the MIC, and the maximum concentration of drug in plasma divided by the MIC to predict the selection of resistant bacteria were compared, only T MSW appeared to be a good predictor of the prevention of resistance for values less than 30%. When the T MSW was higher than 34%, the selection of resistant bacteria occurred less often in thighs initially infected with the low inoculum (11/24; 46%) than in those infected with the high inoculum (30/36; 80%), suggesting that the selection of resistant mutants depends on both the T MSW and inoculum size. The relevance of these results merits further investigation to test different strategies of antibiotic therapy depending on the expected bacterial burden at the infectious site.

We demonstrate using an in vitro pharmacodynamic model that the likelihood of selection of Escherichia coli mutants resistant to a fluoroquinolone was increased when the initial size of the bacterial population, exposed to fluoroquinolone concentrations within the mutant selection window, was increased.

Shigella flexneri is the most common cause of bacterial dysentery in low-income countries. Despite this, S. flexneri remains largely unexplored from a genomic standpoint and is still described using a vocabulary based on serotyping reactions developed over half-a-century ago. Here we combine whole genome sequencing with geographical and temporal data to examine the natural history of the species. Our analysis subdivides S. flexneri into seven phylogenetic groups (PGs); each containing two-or-more serotypes and characterised by distinct virulence gene complement and geographic range. Within the S. flexneri PGs we identify geographically restricted sub-lineages that appear to have persistently colonised regions for many decades to over 100 years. Although we found abundant evidence of antimicrobial resistance (AMR) determinant acquisition, our dataset shows no evidence of subsequent intercontinental spread of antimicrobial resistant strains. The pattern of colonisation and AMR gene acquisition suggest that S. flexneri has a distinct life-cycle involving local persistence. DOI: http://dx.doi.org/10.7554/eLife.07335.001, eLife digest Dysentery is a disease in which the intestine becomes inflamed due to infection by bacteria, viruses or other microbes. Of the bacteria that can cause dysentery, bacteria called Shigella are most often responsible. Humans can acquire Shigella through contaminated food or water. Over the last century, improvements to sanitation combined with access to clean drinking water and better food hygiene have decreased the number of cases of dysentery in many countries. However, the disease continues to be common in low-income countries, especially in young children. One species of Shigella bacteria, called S. flexneri, causes far more cases of dysentry than other species of Shigella. Across the world, there are many different strains of S. flexneri, but it is not clear how these strains are related to each other, or how variable the genes that they carry are—known as genetic diversity. Here, Connor, Barker, Baker et al. used a technique called whole genome sequencing to map the evolutionary relationships of over 300 S. flexneri samples collected from around the globe over the past 100 years. This revealed that the bacterial strains can be split into seven groups that each have distinct geographic ranges and combinations of genes that enable the bacteria to infect humans. Many of the strains of bacteria within these groups seem to have colonized, and remained in, quite small geographic areas over long periods of time. This is different to other Shigella species, which appear to have spread between continents far more frequently over much shorter timescales. Connor, Barker, Baker et al.'s findings reveal that S. flexneri is more diverse than other Shigella bacteria, and suggest that the ability of strains to persist in local areas may have contributed to the species' long-term success. These results point towards the importance of the provision of clean water in the fight against S. flexneri, and underline the need for a greater understanding of how disease-causing bacteria colonize and interact with the local environment. DOI: http://dx.doi.org/10.7554/eLife.07335.002

International audience; Trans-translation is a process that recycles ribosomes stalled on problematic mRNAs. tmRNA, coded by the DeltassrA gene, is a major component of trans-translation. Bacteria lacking tmRNA are more sensitive to several inhibitors of protein synthesis when compared to a wild type strain. We measured bacterial growth of the DeltassrA and wild type strains in Escherichia coli in the presence of 14 antibiotics including some that do not target protein synthesis. Both the optical density of the bacterial cultures and the number of viable cells were monitored. For the ribosome-targeted antibiotics, sensitization was observed on erythromycin, chloramphenicol, kanamycin, puromycin and streptomycin. Minor or no effects were observed with clindamycin, tetracycline and spectinomycin. Surprisingly, the DeltassrA strain is more sensitive than wild type to inhibitors of cell wall synthesis: fosfomycin and ampicillin. No growth difference was observed on drugs with other target sites: ofloxacin, norfloxacin, rifampicin and trimethoprim. Sensitization to antibiotics having target sites other than the ribosome suggests that trans-translation could influence antibiotic-induced stress responses. In trans-translation-deficient bacteria, cell death is significantly enhanced by the two aminoglycosides that induce translational misreading, streptomycin and kanamycin.

A DNA macroarray containing 465 intragenic amplicons was designed to identify Staphylococcus aureus at the species level and to type S. aureus isolates. The genes selected included those encoding (i) S. aureus -specific proteins, (ii) staphylococcal and enterococcal proteins mediating antibiotic resistance and factors involved in their expression, (iii) putative virulence proteins and factors controlling their expression, and (iv) proteins produced by mobile elements. The macroarray was hybridized with the cellular DNAs of 80 S. aureus clinical isolates that were previously typed by analyses of their antibiograms and SmaI patterns. The set selected contained unrelated, endemic, and outbreak-related isolates belonging to 45 SmaI genotypes. In a gene content dendrogram, the 80 isolates were distributed into 52 clusters. The outbreak-related isolates were linked in the same or a closely related cluster(s). Clustering based on gene content provided a better discrimination than SmaI pattern analysis for the tested mecA + isolates that were endemic to Europe. All of the antibiotic resistance genes detected could be correlated with their corresponding phenotypes, except for one isolate which carried a mecA gene without being resistant. The 16 isolates responsible for bone infections were distinguishable from the 12 isolates from uninfected nasal carriers by a significantly higher prevalence of the sdrD gene coding for a putative SD (serine-aspartate) adhesin (in 15 and 7 isolates, respectively). In conclusion, the macroarray designed for this study offers an attractive and rapid typing method which has the advantage of providing additional information concerning the gene content of the isolate of interest.

The β-proteobacterial strain ULPAs1, isolated from an arsenic-contaminated environment, is able to efficiently oxidize arsenite [As(III)] to arsenate [As(V)]. Mutagenesis with a lacZ -based reporter transposon yielded two knockout derivatives deficient in arsenite oxidation. Sequence analysis of the DNA flanking the transposon insertions in the two mutants identified two adjacent open reading frames, named aoxA and aoxB , as well as a putative promoter upstream of the aoxA gene. Reverse transcription-PCR data indicated that these genes are organized in an operonic structure. The proteins encoded by aoxA and aoxB share 64 and 72% identity with the small Rieske subunit and the large subunit of the purified and crystallized arsenite oxidase of Alcaligenes faecalis , respectively (P. J. Ellis, T. Conrads, R. Hille, and P. Kuhn, Structure [Cambridge] 9:125-132, 2001). Importantly, almost all amino acids involved in cofactor interactions in both subunits of the A. faecalis enzyme were conserved in the corresponding sequences of strain ULPAs1. An additional Tat (twin-arginine translocation) signal peptide sequence was detected at the N terminus of the protein encoded by aoxA , strongly suggesting that the Tat pathway is involved in the translocation of the arsenite oxidase to its known periplasmic location.

Stable resistance to metronidazole in a nontoxigenic Clostridium difficile strain was investigated at both the genomic and proteomic levels. Alterations in the metabolic pathway involving the pyruvate-ferredoxin oxidoreductase were found, suggesting that reduction of metronidazole, required for its activity, may be less efficient in this strain. Proteomic studies also showed a cellular response to oxidative stress.

International audience; Given the current bacterial resistance crisis, antimicrobial stewardship programmes are of the utmost importance. We present a narrative review of the impact of infectious disease specialists (IDSs) on the quality and quantity of antibiotic use in acute-care hospitals, and discuss the main factors that could limit the efficacy of IDS recommendations. A total of 31 studies were included in this review, with a wide range of infections, hospital settings, and types of antibiotic prescription. Seven of 31 studies were randomized controlled trials, before/after controlled studies, or before/after uncontrolled studies with interrupted time-series analysis. In almost all studies, IDS intervention was associated with a significant improvement in the appropriateness of antibiotic prescribing as compared with prescriptions without any IDS input, and with decreased antibiotic consumption. Variability in the antibiotic prescribing practices of IDSs, informal (curbside) consultations and the involvement of junior IDSs are among the factors that could have an impact on the efficacy of IDS recommendations and on compliance rates, and deserve further investigation. We also discuss possible drawbacks of IDSs in acute-care hospitals that are rarely reported in the published literature. Overall, IDSs are valuable to antimicrobial stewardship programmes in hospitals, but their impact depends on many human and organizational factors.

International audience; The emergence of Staphylococcus aureus strains that are resistant to glycopeptides has led to alarming scenarios where serious staphylococcal infections cannot be treated. The bacterium expresses many small regulatory RNAs (sRNAs) that have unknown biological functions for the most part. Here we show that an S. aureus sRNA, SprX (alias RsaOR), shapes bacterial resistance to glycopeptides, the invaluable treatments for Methicillin-resistant staphylococcal infections. Modifying SprX expression levels influences Vancomycin and Teicoplanin glycopeptide resistance. Comparative proteomic studies have identified that SprX specifically downregulates stage V sporulation protein G, SpoVG. SpoVG is produced from the yabJ-spoVG operon and contributes to S. aureus glycopeptide resistance. SprX negatively regulates SpoVG expression by direct antisense pairings at the internal translation initiation signals of the second operon gene, without modifying bicistronic mRNA expression levels or affecting YabJ translation. The SprX and yabJ-spoVG mRNA domains involved in the interaction have been identified, highlighting the importance of a CU-rich loop of SprX in the control of SpoVG expression. We have shown that SpoVG might not be the unique SprX target involved in the glycopeptide resistance and demonstrated that the regulation of glycopeptide sensitivity involves the CU-rich domain of SprX. Here we report the case of a sRNA influencing antibiotic resistance of a major human pathogen.

Listeriosis is a rare but severe food-borne infection due to Listeria monocytogenes. L. monocytogenes is naturally susceptible to a wide range of antibiotics, including penicillins, aminoglycosides, tetracyclines, macrolides, co-trimoxazole, linezolid, moxifloxacin, glycopeptides, and rifampin, but

Cerulenin is a fungal toxin that inhibits both eukaryotic and prokaryotic ketoacyl-acyl carrier protein synthases or condensing enzymes. It has been used experimentally to treat cancer and obesity, and is a potent inhibitor of bacterial growth. Understanding the molecular mechanisms of resistance to cerulenin and similar compounds is thus highly relevant for human health. We have previously described a Bacillus subtilis cerulenin-resistant strain, expressing a point-mutated condensing enzyme FabF (FabF[I108F]) (i.e. FabF with isoleucine 108 substituted by phenylalanine). We now report the crystal structures of wild-type FabF from B. subtilis, both alone and in complex with cerulenin, as well as of the FabF[I108F] mutant protein. The three-dimensional structure of FabF[I108F] constitutes the first atomic model of a condensing enzyme that remains active in the presence of the inhibitor. Soaking the mycotoxin into preformed wild-type FabF crystals allowed for noncovalent binding into its specific pocket within the FabF core. Interestingly, only co-crystallization experiments allowed us to trap the covalent complex. Our structure shows that the covalent bond between Cys163 and cerulenin, in contrast to that previously proposed, implicates carbon C3 of the inhibitor. The similarities between Escherichia coli and B. subtilis FabF structures did not explain the reported inability of ecFabF[I108F] (i.e. FabF from Escherichia coli with isoleucine 108 substituted by phenylalanine) to elongate medium and long-chain acyl-ACPs. We now demonstrate that the E. coli modified enzyme efficiently catalyzes the synthesis of medium and long-chain ketoacyl-ACPs. We also characterized another cerulenin-insensitive form of FabF, conferring a different phenotype in B. subtilis. The structural, biochemical and physiological data presented, shed light on the mechanisms of FabF catalysis and resistance to cerulenin. Database Crystallographic data (including atomic coordinates and structure factors) have been deposited in the Protein Data Bank under accession codes 4LS5, 4LS6, 4LS7 and 4LS8.

International audience; At present, little is known regarding Listeria mono-cytogenes-associated biliary tract infection, a rare form of listeriosis. In this article, we will study 12 culture-proven cases reported to the French National Reference Center for Listeria from 1996 to 2013 and review the 8 previously published cases. Twenty cases were studied: 17 cholecystitis, 2 cholangitis, and 1 biliary cyst infection. Half were men with a median age of 69 years (32-85). Comorbidities were present in 80%, including cirrhosis, rheumatoid arthritis, and diabetes. Five patients received immunosuppressive therapy, including cortico-steroids and anti-tumor necrosis factor biotherapies. Half were afebrile. Blood cultures were positive in 60% (3/5). Gallbladder histological lesions were analyzed in 3 patients and evidenced acute, chronic, or necrotic exacerbation of chronic infection. Genoserogroup of the 12 available strains were IVb (n ¼ 6), IIb (n ¼ 5), and IIa (n ¼ 1). Their survival in the bile was not enhanced when compared with isolates from other listeriosis cases. Adverse outcome was reported in 33% (5/15): 3 deaths, 1 recurrence; 75% of the patients with adverse outcome received inadequate antimicrobial therapy (P ¼ 0.033). Biliary tract listeriosis is a severe infection associated with high mortality in patients not treated with appropriate therapy. This study provides medical relevance to in vitro and animal studies that had shown Listeria monocytogenes ability to survive in bile and induce overt biliary infections. (Medicine 93(18):e105) Abbreviations: BHI = brain heart infusion, CNS = central nervous system, Lm = Listeria monocytogenes, MIC = minimal inhibitory concentration, MLST = multilocus sequence typing, MN = maternal-neonatal, NRCL = National Reference Center for Listeria, S = septicemia.

International audience; An important question regarding the biologic implications of antibiotic-resistant microbes is how resistance impacts the organism's overall fitness and virulence. Currently it is generally thought that antibiotic resistance carries a fitness cost and reduces virulence. For the human pathogen Pseudomonas aeruginosa, treatment with carbapenem antibiotics is a mainstay of therapy that can lead to the emergence of resistance, often through the loss of the carbapenem entry channel OprD. Transposon insertion-site sequencing was used to analyze the fitness of 300,000 mutants of P. aeruginosa strain PA14 in a mouse model for gut colonization and systemic dissemination after induction of neutropenia. Transposon insertions in the oprD gene led not only to carbapenem resistance but also to a dramatic increase in mucosal colonization and dissemination to the spleen. These findings were confirmed in vivo with different oprD mutants of PA14 as well as with related pairs of carbapenem-susceptible and -resistant clinical isolates. Compared with OprD(+) strains, those lacking OprD were more resistant to killing by acidic pH or normal human serum and had increased cytotoxicity against murine macrophages. RNA-sequencing analysis revealed that an oprD mutant showed dramatic changes in the transcription of genes that may contribute to the various phenotypic changes observed. The association between carbapenem resistance and enhanced survival of P. aeruginosa in infected murine hosts suggests that either drug resistance or host colonization can cause the emergence of more pathogenic, drug-resistant P. aeruginosa clones in a single genetic event.

International audience; Until recently, most reported cases of bacteraemia caused by multidrug-resistant strains of Enterobacteriacae producing an extended-spectrum b-lactamase (ESBL) in Europe have been nosocomial in origin. However, increasing numbers of reports of community-acquired bacteraemia and urinary tract infection caused by ESBL-producing microorganisms suggest that the geographical origin of patients should be taken into account as a risk-factor for possible ESBL production. Early identification of patients at highrisk of infection with ESBL-producing microorganisms, based on their geographical origin and travel history, should help to optimise initial antibiotic treatment strategies for severe urinary tract infections in Europe.