Search

Background Extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE) represent a major problem in the management of nosocomial infections. However, ESBL-PE are not systematically monitored in African countries. The aim of this study was to determine ESBL-PE prevalence in patients from three hospitals in N’Djamena, the capital city of Chad, and to characterize the genetic origin of the observed resistance. Methods From January to March 2017, 313 non-duplicate isolates were recovered from various clinical specimens obtained from 1713 patients in the three main hospitals of N’Djamena. Bacterial species were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Susceptibility to 28 antibiotics was tested using the disk diffusion method on Müller-Hinton agar, and ESBL production was confirmed with the double-disc synergy test. The most prevalent ESBL genes associated with the observed resistance were detected using multiplex PCR followed by double-stranded DNA sequencing. Results Among the 313 isolates, 197 belonged to the Enterobacteriaceae family. The overall ESBL-PE prevalence was 47.72% (n = 94/197), with a higher rate among inpatients compared with outpatients (54.13% vs. 34.37%). ESBL-PE prevalence was highest in older patients (≥60 years of age). E. coli was the most common ESBL-producer organism (63.8%), followed by K. pneumoniae (21.2%). ESBL-PE were mainly found in urine samples (75%). The CTX-M-1 group was dominant (96.7% of the 94 ESBL-PE isolates, CTX-M-15 enzyme), followed by the CTX-M-9 group (4.1%). 86% of resistant isolates harbored more than one ESBL-encoding gene. ESBL production was also associated with the highest levels of resistance to non-β-lactam drugs. Conclusions The prevalence of ESBL-PE harboring resistant genes encoding ESBLs of the CTX-M-1 group was high (48%) among clinical isolates of three main hospitals in Chad, suggesting an alarming spread of ESBL-PE among patients. Electronic supplementary material The online version of this article (10.1186/s12879-019-3838-1) contains supplementary material, which is available to authorized users.

Erratum in : Publisher Correction: Definitions and guidelines for research on antibiotic persistence. [Nat Rev Microbiol. 2019] https://doi.org/10.1038/s41579-019-0207-4; International audience; Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-cell analyses have inspired a surge of research into antibiotic persistence. Bacterial persister cells represent a subpopulation of cells that can survive intensive antibiotic treatment without being resistant. Several approaches have emerged to define and measure persistence, and it is now time to agree on the basic definition of persistence and its relation to the other mechanisms by which bacteria survive exposure to bactericidal antibiotic treatments, such as antibiotic resistance, heteroresistance or tolerance. In this Consensus Statement, we provide definitions of persistence phenomena, distinguish between triggered and spontaneous persistence and provide a guide to measuring persistence. Antibiotic persistence is not only an interesting example of non-genetic single-cell heterogeneity, it may also have a role in the failure of antibiotic treatments. Therefore, it is our hope that the guidelines outlined in this article will pave the way for better characterization of antibiotic persistence and for understanding its relevance to clinical outcomes.

The intestinal microbiota is considered to be a major reservoir of antibiotic resistance determinants (ARDs) that could potentially be transferred to bacterial pathogens via mobile genetic elements. Yet, this assumption is poorly supported by empirical evidence due to the distant homologies between known ARDs (mostly from culturable bacteria) and ARDs from the intestinal microbiota. Consequently, an accurate census of intestinal ARDs (that is, the intestinal resistome) has not yet been fully determined. For this purpose, we developed and validated an annotation method (called pairwise comparative modelling) on the basis of a three-dimensional structure (homology comparative modelling), leading to the prediction of 6,095 ARDs in a catalogue of 3.9 million proteins from the human intestinal microbiota. We found that the majority of predicted ARDs (pdARDs) were distantly related to known ARDs (mean amino acid identity 29.8%) and found little evidence supporting their transfer between species. According to the composition of their resistome, we were able to cluster subjects from the MetaHIT cohort (n = 663) into six resistotypes that were connected to the previously described enterotypes. Finally, we found that the relative abundance of pdARDs was positively associated with gene richness, but not when subjects were exposed to antibiotics. Altogether, our results indicate that the majority of intestinal microbiota ARDs can be considered intrinsic to the dominant commensal microbiota and that these genes are rarely shared with bacterial pathogens., The project was funded in part by the European Union Seventh Framework Programme (FP7-HEALTH-2011-single-stage) under grant agreement number 282004, EvoTAR. IRYCIS authors acknowledge the European Development Regional Fund ‘A way to achieve Europe’ for co-founding the Spanish R&D National Plan 2012–2019 Work (PI15-0512), CIBER (CIBERESP; CB06/02/0053) and the Government of Madrid (InGeMICS- B2017/BMD-3691). V.F.L. was further funded by a Research Award Grant 2016 of the European Society for Clinical Microbiology and Infectious Diseases.

Targeted killing of pathogenic bacteria without harming beneficial members of host microbiota holds promise as a strategy to cure disease and limit both antimicrobial-related dysbiosis and development of antimicrobial resistance. We engineer toxins that are split by inteins and deliver them by conjugation into a mixed population of bacteria. Our toxin–intein antimicrobial is only activated in bacteria that harbor specific transcription factors. We apply our antimicrobial to specifically target and kill antibiotic-resistant Vibrio cholerae present in mixed populations. We find that 100% of antibiotic-resistant V. cholerae receiving the plasmid are killed. Escape mutants were extremely rare (10−6–10−8). We show that conjugation and specific killing of targeted bacteria occurs in the microbiota of zebrafish and crustacean larvae, which are natural hosts for Vibrio spp. Toxins split with inteins could form the basis of precision antimicrobials to target pathogens that are antibiotic resistant. Centre National de la Recherche Scientifique CNRS-UMR 3525 French Government Investissement d'Avenir ANR-10-LABX-62-IBEID Agencia Estatal de Investigación TIN2016-81079-R Comunidad de Madrid B2017/BMD-3691 Fondation pour la Recherche Médicale DBF20160635736, DEQ20140329508

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

The aim of the current study is to assess the prevalence ofCampylobacterinfection in broiler chickens, raised in intensive production conditions, and to evaluate the antimicrobial susceptibility of recoveredCampylobacterisolates. A total of 590 cloacal swab samples were taken from 13 broiler chicken flocks in the North East of Tunisia. All samples were tested for the presence of thermophilicCampylobacterby culture and PCR, targeting themapAandceuEgenes, respectively. Susceptibility to antimicrobial drugs was tested against 8 antibiotics. Prevalence ofCampylobacterinfection, relationship with geographic origins and seasons, antimicrobial resistance rates and patterns were analyzed. Total prevalence ofCampylobacterinfection in broiler flocks was in the range of 22.4%, with a predominance ofC. jejuni(68.9%), followed byC. coli(31.1%). Positive association was highlighted between the infection level and the season (P< 0.001), but no link was emphasized considering the geographic origin. Antimicrobial susceptibility testing revealed very high resistance rates detected against macrolide, tetracycline, quinolones, and chloramphenicol, ranging from 88.6% to 100%. Lower resistance prevalence was noticed forβ-lactams (47% and 61.4%) and gentamicin (12.9%). 17 R-type patterns were observed, and a common pattern was found in 30.3% of isolates. This study provides updates and novel data on the prevalence and the AMR of broiler campylobacters in Tunisia, revealing the occurrence of high resistance to several antibiotics and emphasizing the requirement of better surveillance and careful regulation of antimicrobials use.

International audience; Bacterial contamination and subsequent infections are a major threat to human health. An early detection in the food chain, clinics or the environment, is key to limit this threat. We present a new concept to develop low-cost hand-held devices for the ultra-sensitive and specific detection of bacteria in a one-step process of 2–8 h, directly from complex raw samples. This approach is based on a novel microfluidic magnetic fluidized bed. It reaches a 4 CFU (colony forming unit) sensitivity with high quantification accuracy in a large dynamic range of 100–107CFU/mL. The versatility of the approach was demonstrated with the detection of different bacteria strains, among which Salmonella Typhimurium and E. coli O157:H15. Additionally, the method is sensitive to infectious bacteria only, a criterion requested by main applications and currently requiring additional culture steps of one to several days.

The spread of antimicrobial resistance has become a serious public health concern, making once-treatable diseases deadly again and undermining the achievements of modern medicine. Drug combinations can help to fight multi-drug-resistant bacterial infections, yet they are largely unexplored and rarely used in clinics. Here we profile almost 3,000 dose-resolved combinations of antibiotics, human-targeted drugs and food additives in six strains from three Gram-negative pathogens-Escherichia coli, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa-to identify general principles for antibacterial drug combinations and understand their potential. Despite the phylogenetic relatedness of the three species, more than 70% of the drug-drug interactions that we detected are species-specific and 20% display strain specificity, revealing a large potential for narrow-spectrum therapies. Overall, antagonisms are more common than synergies and occur almost exclusively between drugs that target different cellular processes, whereas synergies are more conserved and are enriched in drugs that target the same process. We provide mechanistic insights into this dichotomy and further dissect the interactions of the food additive vanillin. Finally, we demonstrate that several synergies are effective against multi-drug-resistant clinical isolates in vitro and during infections of the larvae of the greater wax moth Galleria mellonella, with one reverting resistance to the last-resort antibiotic colistin.

International audience; Antimicrobial resistance, genotype, and virulence gene content of Escherichia coli isolated from bovine clinical mastitis in Tehran Province, Iran, were studied. Two hundred seven (207) milk samples from individual cows presenting mastitis symptoms collected from different dairy farms were used to determine the presence of specific genes of E. coli responsible for this pathology. Multiplex PCR was used to differentiate E. coli isolates into different phylogenetic groups/subgroups and to detect their virulence and involved resistance genes. All the isolated strains were tested for the susceptibility to 21 antimicrobial agents. The results showed that E. coli was detected in 42 (20.3%) samples and 69% of them belonged to the phylogenetic groups A and B1. The phylogenetic subgroup A1 (31%) and subgroup B1 (28.6%) demonstrated the highest prevalence of virulence genes (f17c-A, and eae (n = 6), f17b-A, and f17d-A (n = 5), afaD-8, afaE-8, aucD, and bfpA (n = 4), clpG and VT (n = 2), and LT and ST genes (n = 1)). The highest antimicrobial resistance was observed for tetracycline (45.2%) followed by streptomycin (26.2%). The antimicrobial resistance genes tetB (31%), tetA (28.6%), and aadA (26.2%) were the most prevalent. Moreover, integron class 1 and 2 were found in 24 (57.1%) and 8 (19%) of the E. coli isolates.

International audience; Among 831 cases of neonatal bacterial meningitis occurring from 2001 to 2013, Neisseria meningitidis was the third most frequent bacterial species found. All cases occurred only in term neonates and were mainly late onset. Serogroup B accounted for 78% of cases. At diagnosis, 27% of cases had at least one sign of disease severity. All strains were susceptible to cefotaxime, but 12% showed intermediate susceptibility to penicillin G and to aminopenicillin.

International audience; Active food packaging films based on crab chitosan and Spirulina extract (SE) were developed. The effects of the SE incorporation at different levels on physical (color, opacity water vapor and oxygen permeability) and mechanical (tensile strength and elongation at break) properties of chitosan films were investigated. FTIR was carried out to observe the potential modifications of the chitosan films when incorporated with SE. The obtained results suggested that incorporation of SE into chitosan films improved mechanical and barrier properties. The antioxidant activity of the chitosan/SE films was characterized by means of three different analytical assays (DPPH, FRAP and FIC). Crab chitosan edible films containing SE showed higher antioxidant activity, regardless concentrations and methods assayed. Furthermore, the antioxidant activity occurred in a concentration-dependent manner. The agar disc diffusion method was used to determine the antibacterial activities of chitosan edible films against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, Bacillus subtilis and Bacillus cereus. The chitosan/SE films were more effective (p\textless0.05) against five of the seven tested bacteria. The obtained crab chitosan edible films incorporated with SE showed great potential to be used for active food packaging due to its excellent antioxidant and antibacterial activities.

In the last few years, different surveillances have been published in Africa, especially in northern countries, regarding antimicrobial resistance among husbandry animals. Information is still scarce, but the available data show a worrying picture. Although the highest resistance rates have been described against tetracycline, penicillins and sulphonamides, prevalence of plasmid-mediated quinolone resistance genes and extended spectrum β-lactamase (ESBL) are being increasingly reported. Among ESBLs, the CTX-M-1 group was dominant in most African surveys. Within this group, CTX-M-15 was the main variant both in animals and humans, except in Tunisia where CTX-M-1 was more frequently detected among Escherichia coli from poultry. Certain blaCTX-M-15-harbouring clones (ST131/B2 or ST405/D) are mainly identified in humans, but they have also been reported in livestock species from Tanzania, Nigeria or Tunisia. Moreover, several reports suggest an inter-host circulation of specific plasmids (e.g. blaCTX-M-1-carrying IncI1/ST3 in Tunisia, IncY- and Inc-untypeable replicons co-harbouring qnrS1 and blaCTX-M-15 in Tanzania and the worldwide distributed blaCTX-M-15-carrying IncF-type plasmids). International trade of poultry meat seems to have contributed to the spread of other ESBL variants, such as CTX-M-14, and clones. Furthermore, first descriptions of OXA-48- and OXA-181-producing E. coli have been recently documented in cattle from Egypt, and the emergent plasmid-mediated colistin resistance mcr-1 gene has been also identified in chickens from Algeria, Tunisia and South Africa. These data reflect the urgent need of a larger regulation in the use of veterinary drugs and the implementation of surveillance programmes in order to decelerate the advance of antimicrobial resistance in this continent.

International audience; OBJECTIVES:Reduced susceptibility to penicillin G in Neisseria meningitidis is mainly due to alterations in PBP2 encoded by the penA gene. However, this phenotype was not associated with reduced susceptibility to third-generation cephalosporins (C3Gs). We aimed to study the emergence of meningococci with reduced susceptibility to C3Gs (MIC >0.06 mg/L) in France since 2012.METHODS:Invasive meningococcal isolates were typed by MLST and penA sequencing and their antibiotic susceptibility was tested.RESULTS:Isolates with reduced susceptibility to C3Gs represented 2% of all invasive isolates from 2012-15, but were absent before. They harboured a new penA allele, penA327, that was also detected in isolates from urethritis cases and in gonococci.CONCLUSIONS:Surveillance of these isolates should be enhanced as they may jeopardize the use of C3Gs in the management of invasive meningococcal disease.

Plasmids play a key role in the horizontal spread of antibiotic resistance determinants among bacterial pathogens. When an antibiotic resistance plasmid arrives in a new bacterial host, it produces a fitness cost, causing a competitive disadvantage for the plasmid-bearing bacterium in the absence of antibiotics. On the other hand, in the presence of antibiotics, the plasmid promotes the survival of the clone. The adaptations experienced by plasmid and bacterium in the presence of antibiotics during the first generations of coexistence will be crucial for the progress of the infection and the maintenance of plasmid-mediated resistance once the treatment is over. Here we developed a model system using the human pathogen Haemophilus influenzae carrying the small plasmid pB1000 conferring resistance to β-lactam antibiotics to investigate host and plasmid adaptations in the course of a simulated ampicillin therapy. Our results proved that plasmid-bearing clones compensated for the fitness disadvantage during the first 100 generations of plasmid-host adaptation. In addition, ampicillin treatment was associated with an increase in pB1000 copy number. The augmentation in both bacterial fitness and plasmid copy number gave rise to H. influenzae populations with higher ampicillin resistance levels. In conclusion, we show here that the modulations in bacterial fitness and plasmid copy number help a plasmid-bearing bacterium to adapt during antibiotic therapy, promoting both the survival of the host and the spread of the plasmid.

International audience; One-hundred-nine samples of 18 different farms (49 of food-vegetables, 41 of soil and 19 of irrigation water) and 45 vegetable food samples of 13 markets were collected in Tunisia. These samples were inoculated in MacConkey agar plates supplemented with cefotaxime (2 μg/ml). ESBL-producing Enterobacteriaceae (ESBL-Eb) were detected in 10 of the 109 farm samples (vegetables, 8.2%; soil, 7.3%; water, 15.8%), and in 4 of 45 vegetables of markets (8.9%), recovering 15 ESBL-Eb. Isolates and ESBL genes detected were: Escherichia coli (n = 8: 5 blaCTX-M-1, 2 blaCTX-M-15 and one blaCTX-M-14), Citrobacter freundii (n = 4: 3 blaCTX-M-15 and one blaSHV-12), Enterobacter hormaechei (n = 2: 2 blaCTX-M-15) and Klebsiella pneumoniae (n = 1, blaCTX-M-15). The ISEcp1 sequence was found upstream of blaCTX-M genes in 13 of 14 strains (in three cases truncated by IS5), and orf477 or IS903 downstream. Class 1 integrons were detected in five strains and contained two gene cassette arrangements (dfrA17-aadA5 and aadA1). Most isolates tested showed a multiresistant phenotype. All blaCTX-M-15-positive strains carried the aac(6′)-1b-cr gene, that affects to amikacin–tobramycin–kanamycin–ciprofloxacin. Five ESBL-Eb strains carried genes of the qnr family. The 8 ESBL-positive E. coli isolates were typed as: ST58/B1 (n = 3) and ST117/D, ST131/B2, ST10/A, ST23/A, and the new ST3496/D (one strain, each). From 1–2 plasmids were detected in all ESBL-positive E. coli isolates (63–179 kb). The ESBL genes were transferred by conjugation in 4 blaCTX-M-1-positive E. coli strains, and transconjugants acquired a 97 kb IncI1 plasmid. ESBL-Eb isolates are frequently disseminated in vegetable farms and potentially could be transmitted to humans through the food chain.

International audience; BACKGROUND: Limitations in treatment of biofilm-associated bacterial infections are often due to subpopulation of persistent bacteria (persisters) tolerant to high concentrations of antibiotics. Based on the increased aminoglycoside efficiency under alkaline conditions, we studied the combination of gentamicin and the clinically compatible basic amino acid L-arginine against planktonic and biofilm bacteria both in vitro and in vivo. METHODS: Using Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli bioluminescent strains, we studied the combination of L-arginine and gentamicin against planktonic persisters through time-kill curves of late stationary-phase cultures. In vitro biofilm tolerance towards gentamicin was assessed using PVC 96 well-plates assays. Efficacy of gentamicin as antibiotic lock treatment (ALT) at 5 mg/mL at different pH was evaluated in vivo using a model of totally implantable venous access port (TIVAP) surgically implanted in rats. RESULTS: We demonstrated that a combination of gentamicin and the clinically compatible basic amino acid L-arginine increases in vitro planktonic and biofilm susceptibility to gentamicin, with 99% mortality amongst clinically relevant pathogens, i.e. S. aureus, E. coli and P. aeruginosa persistent bacteria. Moreover, although gentamicin local treatment alone showed poor efficacy in a clinically relevant in vivo model of catheter-related infection, gentamicin supplemented with L-arginine led to complete, long-lasting eradication of S. aureus and E. coli biofilms, when used locally. CONCLUSION: Given that intravenous administration of L-arginine to human patients is well tolerated, combined use of aminoglycoside and the non-toxic adjuvant L-arginine as catheter lock solution could constitute a new option for the eradication of pathogenic biofilms.

International audience; The rising number of infections caused by biofilm formation and the difficulties associated with their treatment by conventional antimicrobial therapies have led to an intensive search for novel antibiofilm agents. Dermaseptins are antimicrobial peptides with a number of attractive properties that might offer alternative therapies against resistant microorganisms. In this study, we synthesized a set of dermaseptin-derived peptides and evaluated their activities against Gram-positive and Gram-negative bacterial biofilm formation. All dermaseptin-derived peptides demonstrated concentration-dependent antibiofilm activities at microgram concentrations, and their activities were dependent on the nature of the peptides, with the highest levels of activity being exhibited by highly charged molecules. Fluorescent binding and confocal microscopy demonstrated that dermaseptin K4S4, a substituted derivative of the native molecule S4, significantly decreased the viability of planktonic and surface-attached bacteria and stopped biofilm formation under dynamic flow conditions. Cytotoxicity assays with HeLa cells showed that some of the tested peptides were less cytotoxic than current antibiotics. Overall, these findings indicate that dermaseptin derivatives might constitute new lead structures for the development of potent antibiofilm agents.

Sub-inhibitory concentrations (sub-MIC) of antibiotics play a very important role in selection and development of resistances. Unlike Escherichia coli, Vibrio cholerae induces its SOS response in presence of sub-MIC aminoglycosides. A role for oxidized guanine residues was observed, but the mechanisms of this induction remained unclear. To select for V. cholerae mutants that do not induce low aminoglycoside-mediated SOS induction, we developed a genetic screen that renders induction of SOS lethal. We identified genes involved in this pathway using two strategies, inactivation by transposition and gene overexpression. Interestingly, we obtained mutants inactivated for the expression of proteins known to destabilize the RNA polymerase complex. Reconstruction of the corresponding mutants confirmed their specific involvement in induction of SOS by low aminoglycoside concentrations. We propose that DNA lesions formed on aminoglycoside treatment are repaired through the formation of single-stranded DNA intermediates, inducing SOS. Inactivation of functions that dislodge RNA polymerase leads to prolonged stalling on these lesions, which hampers SOS induction and repair and reduces viability under antibiotic stress. The importance of these mechanisms is illustrated by a reduction of aminoglycoside sub-MIC. Our results point to a central role for transcription blocking at DNA lesions in SOS induction, so far underestimated.

International audience; Formation of bacterial biofilm communities leads to profound physiological modifications and increased physical and metabolic exchanges between bacteria. It was previously shown that bioactive molecules produced within the biofilm environment contribute to bacterial interactions. Here we describe new pore-forming colicin R, specifically produced in biofilms formed by the natural isolate Escherichia coli ROAR029 but that cannot be detected under planktonic culture conditions. We demonstrate that an increased SOS stress response within mature biofilms induces SOS-dependent colicin R expression. We provide evidence that colicin R displays increased activity against E. coli strains that have a reduced lipopolysaccharide length, such as the pathogenic enteroaggregative E. coli LF82 clinical isolate, therefore pointing to lipopolysaccharide size as an important determinant for resistance to colicins. We show that colicin R toxicity toward E. coli LF82 is increased under biofilm conditions compared with planktonic susceptibility and that release of colicin R confers a strong competitive advantage in mixed biofilms by rapidly outcompeting sensitive neighboring bacteria. This work identifies the first biofilm-associated colicin that preferentially targets biofilm bacteria. Furthermore, it indicates that the study of antagonistic molecules produced in biofilm and multispecies contexts could reveal unsuspected, ecologically relevant bacterial interactions influencing population dynamics in natural environments.

International audience; In this study we investigated the anti-Helicobacter pylori activity of four isopentenyloxycinnamyl derivatives from the Australian shrub Boronia pinnata Sm. (Rutaceae), structurally related to boropinic acid: (E)-3-(4-(3-methylbut-2-enyloxy)-3,5-dimethoxyphenyl)acrylaldehyde (1), boropinol C (2), boropinal (3) and boropinol A (4). In vitro growth of H. pylori strains 26695 and B128 was analyzed in liquid culture with increasing doses of these compounds. Bacterial morphology was visualized by scanning electron microscopy. The in vivo effects of the two most efficient molecules that reduced bacterial growth in vitro, compounds 3 and 4, were investigated on H. pylori gastric colonization in the mouse model. The presence of these compounds in the bacterial cultures led to alterations of bacterial surface and flagella. In vivo, both compounds 3 and 4 at 250 microM reduced significantly the ability of H pylori to colonize the gastric mucosa of mice, compared with untreated ones. These data indicate that these natural isopentenyloxycinnamyl derivatives related to boropinic acid can be considered as novel antibacterial agents with anti-H. pylori activity.

Seventy-four unrelated clinical isolates of Streptococcus pneumoniae harboring the tet (M) gene were studied. Seven strains with low tetracycline (Tc) MICs (0.25 to 0.5 μg/ml) were found to harbor truncated tet (M) alleles that were inactivated by different frameshift mutations. In contrast, five strains bore deletions in the tet (M) promoter region, among which four displayed increased Tc MICs (16 to 64 μg/ml). The same promoter mutations were detected in Tc-resistant mutants selected in vitro from various susceptible strains. Sequence analysis revealed that these deletions might impede the formation of the transcriptional attenuator located immediately upstream of tet (M). Expression in Enterococcus faecalis of a tet (M) reporter gene transcribed from these promoter mutants conferred a level of Tc resistance similar to that observed in the parental S. pneumoniae strains. These results show that different levels of Tc susceptibility found in clinical isolates of S. pneumoniae can be explained by frameshift mutations within tet (M) and by alterations of the upstream transcriptional attenuator.

High levels of antibiotic tolerance are a hallmark of bacterial biofilms. In contrast to well-characterized inherited antibiotic resistance, molecular mechanisms leading to reversible and transient antibiotic tolerance displayed by biofilm bacteria are still poorly understood. The physiological heterogeneity of biofilms influences the formation of transient specialized subpopulations that may be more tolerant to antibiotics. In this study, we used random transposon mutagenesis to identify biofilm-specific tolerant mutants normally exhibited by subpopulations located in specialized niches of heterogeneous biofilms. Using Escherichia coli as a model organism, we demonstrated, through identification of amino acid auxotroph mutants, that starved biofilms exhibited significantly greater tolerance towards fluoroquinolone ofloxacin than their planktonic counterparts. We demonstrated that the biofilm-associated tolerance to ofloxacin was fully dependent on a functional SOS response upon starvation to both amino acids and carbon source and partially dependent on the stringent response upon leucine starvation. However, the biofilm-specific ofloxacin increased tolerance did not involve any of the SOS-induced toxin–antitoxin systems previously associated with formation of highly tolerant persisters. We further demonstrated that ofloxacin tolerance was induced as a function of biofilm age, which was dependent on the SOS response. Our results therefore show that the SOS stress response induced in heterogeneous and nutrient-deprived biofilm microenvironments is a molecular mechanism leading to biofilm-specific high tolerance to the fluoroquinolone ofloxacin., Author Summary Biofilm surface-attached communities have the capacity to tolerate high concentrations of antibiotics, and bacterial biofilms formed on indwelling medical devices are difficult to eradicate and often lead to the onset of chronic or systemic infections. The physiological heterogeneity of multicellular biofilms has been associated with development of subpopulations highly tolerant to multiple antibiotics. Here we demonstrate that, upon starvation for specific essential growth nutrients, biofilm bacteria become highly tolerant to fluoroquinolone ofloxacin. The SOS response plays a critical role in this phenomenon, while the stringent response plays only a minor role. Taken together, these results support the hypothesis that bacteria localized within nutrient-limited niches of the biofilm structure may temporarily enter a physiological state enabling them to tolerate bactericidal concentrations of antibiotics.

International audience; H. pylori drug-resistant strains and non-compliance to therapy are the major causes of H. pylori eradication failure. For some bacterial species it has been demonstrated that fatty acids have a growth inhibitory effect. Our main aim was to assess the ability of docosahexaenoic acid (DHA) to inhibit H. pylori growth both in vitro and in a mouse model. The effectiveness of standard therapy (ST) in combination with DHA on H. pylori eradication and recurrence prevention success was also investigated. The effects of DHA on H. pylori growth were analyzed in an in vitro dose-response study and n in vivo model. We analized the ability of H. pylori to colonize mice gastric mucosa following DHA, ST or a combination of both treatments. Our data demonstrate that DHA decreases H. pylori growth in vitro in a dose-dependent manner. Furthermore, DHA inhibits H. pylori gastric colonization in vivo as well as decreases mouse gastric mucosa inflammation. Addition of DHA to ST was also associated with lower H. pylori infection recurrence in the mouse model. In conclusion, DHA is an inhibitor of H. pylori growth and its ability to colonize mouse stomach. DHA treatment is also associated with a lower recurrence of H. pylori infection in combination with ST. These observations pave the way to consider DHA as an adjunct agent in H. pylori eradication treatment.

International audience; PURPOSE OF REVIEW:Modeling of antibiotic resistance in pathogenic bacteria responsible for human disease has developed considerably over the last decade. Herein, we summarize the main published studies to illustrate the contribution of models for understanding both within-host and population-based phenomena. We then suggest possible topics for future studies.RECENT FINDINGS:Model building of bacterial resistance has involved epidemiologists, biologists and modelers with two different objectives. First, modeling has helped largely in identifying and understanding the factors and biological phenomena responsible for the emergence and spread of resistant strains. Second, these models have become important decision support tools for medicine and public health.SUMMARY:Major improvements of models in the coming years should take into account specific pathogen characteristics (resistance mechanisms, multiple colonization phenomena, cooperation and competition among species) and better description of the contacts associated with transmission risk within populations.

International audience; We analyzed the prevalence of resistance to extended-spectrum cephalosporins (ESCs) among clinical strains of Salmonella enterica collected by the Laboratory of Clinical Microbiology in the University Clinical Hospital Lozano Blesa in the region of Aragó n (Spain), for which very few epidemiological information exists. A total of 2,092 strains of S. enterica were identified in stool samples from patients with gastroenteritis. Five isolates showed an extended-spectrum beta-lactamase (ESBL) phenotype: four isolates of S. enterica serotype Virchow harbored the ESBL-encoding bla CTX-M-9 gene and an isolate of serotype Enteritidis carried a bla CTX-M-1 gene, which, to the best of our knowledge, is described here for the first time in this serotype of S. enterica. The five ESC-resistant isolates were also resistant to spectinomycin, streptomycin, kanamycin, sulfonamides, tetracycline, and trimethoprim as well as to nalidixic acid. The ESBL isolate of serotype Enteritidis, however, remained susceptible to kanamycin and nalidixic acid. A class 1 integron of 1.5 kb was detected for the four serotype Virchow isolates with the gene cassette dfrA16–aadA2. The bla CTX-M-9 gene was carried by an *300-kb IncHI2 conjugative plasmid in the case of the S. enterica serotype Virchow isolates. The bla CTX-M-1 gene was carried by an *100-kb IncI1-N conjugative plasmid for the serotype Enteritidis ESC-resistant isolate. All the four ESC-resistant strains of S. enterica serotype Virchow clustered together in a XbaI pulsed-field gel electrophoresis, which also revealed a strong similarity between them and some pulsotypes of S. enterica serotype Virchow from France.

To the Editor: Class D OXA β-lactamases are characterized as penicillinases that can hydrolyze oxacillin and cloxacillin and are poorly inhibited by clavulanic acid and EDTA. OXA-48 is one of the few members of this family to possess notable carbapenem-hydrolyzing activity (1). First described in 2004 in Turkey, OXA-48 has recently started to spread in Europe and the Middle East (2). We report the recent emergence of the plasmid-encoded blaOXA-48 gene in multidrug-resistant Enterobacteriaceae recovered from patients in Dakar, Senegal, in hospitals and in the community. From November 2008 through October 2009, 11 Enterobacteriaceae isolates (8 Klebsiella pneumoniae, 1 Escherichia coli, 1 Enterobacter cloacae, and 1 Enterobacter sakazakii) with reduced susceptibility to imipenem were identified at the Institut Pasteur (Dakar, Senegal). Antibacterial drug susceptibility was determined by the disk diffusion method and interpreted according to the European Committee on Antimicrobial Susceptibility Testing guidelines (www.eucast.org). Nine isolates were resistant to expanded-spectrum cephalosporins and also to other antibacterial drug classes. The isolates were recovered from 6 patients with urinary tract infections, 4 patients with surgical infections, and 1 patient with omphalitis. Nine infections were hospital acquired (Le Dantec and Principal Hospitals). Because the patients died before antibacterial drug susceptibility testing could be completed, all 5 patients with surgical infections or omphalitis received only empirical therapy with amoxicillin/clavulanate. One patient with a nosocomial urinary tract infection caused by a co-trimoxazole–susceptible strain was successfully treated with this antibacterial agent. The antibacterial drug regimens of the remaining 4 patients were not known, and they were lost to follow-up. We determined the MICs of imipenem, meropenem, and ertapenem by using the Etest method (AB Biodisk, Solna, Sweden), which showed that 9 isolates were susceptible to imipenem and meropenem but either intermediately susceptible or resistant to ertapenem (Table). The 2 imipenem-nonsusceptible isolates were susceptible or intermediately susceptible to meropenem, and both were resistant to ertapenem. We used previously described PCRs (1,3–7) to screen for carbapenem-hydrolyzing β-lactamase genes (blaVIM, blaIMP, blaKPC, and blaOXA-48), as well as plasmid-encoded blaCTX-M, blaAmpC, blaOXA-1, and blaTEM β-lactamase genes; the aac(6′)-Ib aminoglycoside resistance gene; the quinolone resistance genes qnrA,B,S; the tetracycline resistance genes tetA,B,D; and class 1 integron. The blaOXA-48, blaCTX-M, blaAmpC, and aac(6′)-Ib genes and the variable region of class 1 integron were then characterized by direct DNA sequencing of the PCR products. blaOXA-48 was present in all 11 isolates. blaVIM, blaIMP, and blaKPC were not detected. The qnr genes were present in 7 isolates resistant to ciprofloxacin. The aac(6′)-Ib-cr variant was present in 7 isolates resistant to gentamicin, tobramycin, and ciprofloxacin. The 9 isolates resistant to expanded-spectrum cephalosporins all harbored the blaCTX-M-15 gene. The E. coli isolate also harbored the plasmid-encoded blaAmpC gene ACT-1; blaCTX-M-15, blaOXA-1, blaTEM, and aac(6′)lb-cr were associated in 6 isolates. Long-range PCRs showed that these latter 4 genes were located in the same “multidrug resistance region,” as described in Senegal (6). Positive conjugation experiments with sodium azide–resistant E. coli J53 showed through PCR results, plasmid DNA extraction, and antibiogram patterns of the obtained transconjugants that blaOXA-48 was located on a 70-kb self-conjugative plasmid. The genetic environment of blaOXA-48 was then investigated by PCR with primers specific for insertion sequence IS1999 and for the 5′ part of blaOXA-48 (1). blaOXA-48 was found to be part of a Tn1999 composite transposon composed of 2 copies of the insertion sequence IS1999, as reported (2). Further sequencing of the IS1999 located upstream of blaOXA-48 showed that it was consistently truncated by the insertion sequence IS1R, as initially described in Turkey and more recently in Lebanon and Egypt (2,8). XbaI pulsed-field gel electrophoresis was then used to study the genetic relatedness of the 8 K. pneumoniae isolates. Three isolates had similar restriction profiles and had been recovered from 3 patients concurrently hospitalized at Le Dantec Hospital, suggesting nosocomial transmission. A class 1 integron harboring the dfrA1 trimethoprim-resistance gene was detected in the 3 clonal isolates. Together, these findings show the recent emergence of blaOXA-48 in Senegal in community and hospital settings. They may also suggest the spread of the same major carrying plasmid between the Middle East and Africa. Although 9 of the 11 isolates were found to be susceptible to imipenem on the basis of their MICs, their MICs were nonetheless higher than those of blaOXA-48–negative isolates. This raises 2 issues. First, these strains might go undetected during routine antibacterial drug susceptibility testing, a problem that could be overcome by using ertapenem, a compound more susceptible to carbapenemases. Second, the clinical efficacy of imipenem on such strains is uncertain. The frequency of acquired carbapenemases, which emerged early after imipenem introduction in Senegal (2008), is probably strongly underestimated, partly owing to the limited availability of reliable clinical laboratories (9). Because multidrug resistance is prevalent among Enterobacteriaceae isolated in Dakar hospitals (B. Garin, unpub. data) and in rural communities (6), the emergence of blaOXA-48 is a clear cause for concern. Table Resistance genes and carbapenem MICs of 11 Enterbacteriaceae isolates, Senegal, 2008–2009*

Although polymicrobial infections, caused by combinations of viruses, bacteria, fungi and parasites, are being recognised with increasing frequency, little is known about the occurrence of within-species diversity in bacterial infections and the molecular and evolutionary bases of this diversity. We used multiple approaches to study the genomic and phenotypic diversity among 226 Escherichia coli isolates from deep and closed visceral infections occurring in 19 patients. We observed genomic variability among isolates from the same site within 11 patients. This diversity was of two types, as patients were infected either by several distinct E. coli clones (4 patients) or by members of a single clone that exhibit micro-heterogeneity (11 patients); both types of diversity were present in 4 patients. A surprisingly wide continuum of antibiotic resistance, outer membrane permeability, growth rate, stress resistance, red dry and rough morphotype characteristics and virulence properties were present within the isolates of single clones in 8 of the 11 patients showing genomic micro-heterogeneity. Many of the observed phenotypic differences within clones affected the trade-off between self-preservation and nutritional competence (SPANC). We showed in 3 patients that this phenotypic variability was associated with distinct levels of RpoS in co-existing isolates. Genome mutational analysis and global proteomic comparisons in isolates from a patient revealed a star-like relationship of changes amongst clonally diverging isolates. A mathematical model demonstrated that multiple genotypes with distinct RpoS levels can co-exist as a result of the SPANC trade-off. In the cases involving infection by a single clone, we present several lines of evidence to suggest diversification during the infectious process rather than an infection by multiple isolates exhibiting a micro-heterogeneity. Our results suggest that bacteria are subject to trade-offs during an infectious process and that the observed diversity resembled results obtained in experimental evolution studies. Whatever the mechanisms leading to diversity, our results have strong medical implications in terms of the need for more extensive isolate testing before deciding on antibiotic therapies., Author Summary We investigated whether an infection is a site of pathogen within-species diversity. Our results indicate that there is indeed extensive diversity during human extraintestinal infections by Escherichia coli. This diversity was of two types, not mutually exclusive, as we found that patients were infected either by several distinct E. coli clones or by members of a single clone that exhibit micro-heterogeneity. The high degree of phenotypic diversity, including antibiotic resistance, suggests that there is no uniform selection pressure leading to a single fitter clone during an infection. We discuss a possible mechanism and a mathematical model that explains these unexpected results. Our data suggest that the evolution of diversity in the course of an infection and in in vitro experimental evolution in the absence of host immune selective pressure may have many parallels. Whatever the mechanisms leading to diversity, our results have strong medical implications in terms of the need for more extensive isolate testing before deciding on antibiotic therapies.

International audience; Molecular epidemiology at local scale in Sicily (Italy) of ampicillin resistance in Salmonella spp. isolates from municipal wastewater (n ¼ 64) and clinical specimens (n ¼ 274) is described in comparison with previously examined Escherichia coli isolates (n ¼ 273) from wastewater. High prevalence of antibiotic resistance (28.9%) with highest resistance rates against ampicillin (22.7%) was observed in E. coli isolates. Different resistance rates were observed in Salmonella according to the serovars, with prevalences of the same order in both wastewater and clinical isolates belonging to the same serovar (e.g., 91.7% ampicillin resistance in wastewater isolates vs. 70.8% in clinical isolates of the Salmonella serovar Typhimurium and 0% ampicillin resistance in both wastewater and clinical isolates of the Salmonella serovar Enteritidis). The b-lactam resistance gene bla TEM was present in both wastewater and clinical Salmonella spp. isolates, with the exception of Salmonella enterica serovar Typhimurium isolates with a typical six-drug resistance pattern AmpChlSulTeStrSp that had the bla PSE-1 gene. The bla TEM gene was present in all the E. coli isolates but one had the bla SHV gene. Several E. coli and some Salmonella isolates were positive for class 1 integrons with variable regions of 1.0 or 1.5 kb containing aadA1, dfrA17-aadA5, or dfrA1-aadA1 gene cassettes, whereas Salmonella serovar Typhimurium isolates with the six-drug resistance pattern were positive for both 1.0 and 1.2 kb integrons. Polymerase chain reaction replicon typing demonstrated the presence of multireplicon resistance plasmids in several isolates of E. coli, containing two to four of the replicons IncF, IncI1, IncFIA, and IncFIB, whereas other isolates showed resistance plasmids with only IncF, IncP, or IncK replicons. Replicon IncI1 was detected in one Salmonella isolate, whereas other isolates belonging to different serovars had IncN replicons. Analysis of isolates from wastewater can be a useful epidemiologic tool to monitor the prevalence of antibiotic resistance and genetic elements related to antibiotic resistance in Salmonella clones circulating in the human population.

This work reports the characterization of the first known peptidoglycan hydrolase (Acp) produced mainly during vegetative growth of Clostridium perfringens . Acp has a modular structure with three domains: a signal peptide domain, an N-terminal domain with repeated sequences, and a C-terminal catalytic domain. The purified recombinant catalytic domain of Acp displayed lytic activity on the cell walls of several Gram-positive bacterial species. Its hydrolytic specificity was established by analyzing the Bacillus subtilis peptidoglycan digestion products by coupling reverse phase-high-pressure liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis, which displayed an N -acetylglucosaminidase activity. The study of acp expression showed a constant expression during growth, which suggested an important role of Acp in growth of C. perfringens . Furthermore, cell fractionation and indirect immunofluorescence staining using anti-Acp antibodies revealed that Acp is located at the septal peptidoglycan of vegetative cells during exponential growth phase, indicating a role in cell separation or division of C. perfringens . A knockout acp mutant strain was obtained by using the insertion of mobile group II intron strategy (ClosTron). The microscopic examination indicated a lack of vegetative cell separation in the acp mutant strain, as well as the wild-type strain incubated with anti-Acp antibodies, demonstrating the critical role of Acp in cell separation. The comparative responses of wild-type and acp mutant strains to stresses induced by Triton X-100, bile salts, and vancomycin revealed an implication of Acp in autolysis induced by these stresses. Overall, Acp appears as a major cell wall N -acetylglucosaminidase implicated in both vegetative growth and stress-induced autolysis.

Recent outbreaks of Clostridium difficile infection have been related to the emergence of the NAP1/027 epidemic strain. This strain demonstrates increased virulence and resistance to the C-8-methoxyfluoroquinolones gatifloxacin and moxifloxacin. These antibiotics have been implicated as major C. difficile infection-inducing agents. We investigated by real-time reverse transcription-PCR the impact of subinhibitory concentrations of ampicillin, clindamycin, ofloxacin, and moxifloxacin on the expression of genes encoding three colonization factors, the protease Cwp84, the high-molecular-weight S-layer protein, and the fibronectin-binding protein Fbp68. We have previously shown in six non-NAP1/027 moxifloxacin-susceptible strains that the presence of ampicillin or clindamycin induced an upregulation of these genes, whereas the presence of fluoroquinolones did not. The objective of this study was to analyze the expression of these genes under the same conditions in four NAP1/027 strains, one moxifloxacin susceptible and three moxifloxacin resistant. Two in vitro-selected moxifloxacin-resistant mutants were also analyzed. Moxifloxacin resistance was associated with the Thr82→Ile substitution in GyrA in all but one of the moxifloxacin-resistant strains. The expression of cwp84 and slpA was strongly increased after culture with ampicillin or clindamycin in NAP1/027 strains. Interestingly, after culture with fluoroquinolones, the expression of cwp84 and slpA was only increased in four moxifloxacin-resistant strains, including the NAP1/027 strains and one of the in vitro-selected mutants. The overexpression of cwp84 was correlated with increased production of the protease Cwp84. The historical NAP1/027 moxifloxacin-susceptible strain and its mutant appear to be differently regulated by fluoroquinolones. Overall, fluoroquinolones appear to favor the expression of some colonization factor-encoding genes in resistant C. difficile strains. The fluoroquinolone resistance of the NAP1/027 epidemic strains could be considered an ecological advantage. This could also increase their colonization fitness and promote the infection.

International audience; AbstractIn case-series or cohort studies, we propose a test of independence between the occurrences of two types of recurrent events (such as two repeated infections) related to an intermittent exposure (such as an antibiotic treatment). The test relies upon an extension of a recent method for analysing case-series data, in the presence of one type of recurrent event. The test statistic is derived from a bivariate Poisson generated-multinomial distribution. Simulations for checking the validity of the test concerning the type I error and the power properties are presented. The test is illustrated using data from a cohort on antibiotics bacterial resistance in schoolchildren.