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The objective of this study was to determine the effect of antibiotics on Chlamydia trachomatis viability by using a quantitative real-time PCR assay that measured DNA replication and mRNA transcription of the structural omp1 and omp2 genes, 16S rRNA and the groEL1 gene with and without antibiotics. Ofloxacin, moxifloxacin, azithromycin and doxycycline were tested against the serovar D and L2 reference strains and a derivative mutant resistant to fluoroquinolones, L2-OFXR, obtained by in vitro selection. Using DNA quantification, the antibiotic MIC was calculated when the number of DNA copies was equal to that of the chlamydial inoculum at time zero. This method allowed the easy determination of MICs by DNA quantification of the four selected genes and gave similar results to those obtained by immunofluorescence staining without biased interpretation. By using cDNA quantification, the lowest antibiotic concentration for which no RNA was transcribed corresponded to the minimum bactericidal concentration. C. trachomatis still transcribed the16S rRNA and groEL1 genes, even at concentrations well above the MIC, showing a bacteriostatic effect for all antibiotics tested. This method allows the study of antibiotic activity on growth and viability of C. trachomatis by DNA and RNA quantification at the same time without additional cell-culture passaging.

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.

Objectif: Faire le point sur les concepts de synergie et d'antagonisme lors de l'utilisation d'une association d'antibiotiques. Méthode: Revue de la littérature sur les aspects in vitro et in vivo de la synergie et de l'antagonisme. Discussion: La synergie et l'antagonisme peuvent se voir de différentes façons : du point de vue du bactériologiste avec des définitions bien claires ou du point de vue du clinicien qui va rechercher une efficacité accrue du traitement. Cela entraîne des divergences sur la ou les définitions à retenir. Parallèlement, alors qu'il existe des certitudes sur l'aspect in vitro, peu d'études cliniques ont validé ces notions de l'association in vivo. Les cliniciens ont transposé des résultats bruts, sans toujours avoir démontré l'intérêt pour le patient. Alors que, sur la paillasse, l'association est antagoniste, il ne semble pas y avoir de conséquence clinique. Pour certaines associations, même les résultats in vitro sont discordants entre différentes équipes. Conclusion: En essayant de faire le point sur cette question de tous les jours, on s'aperçoit qu'il n'existe que peu de certitudes. Cela doit renforcer le lien entre les bactériologistes et les cliniciens, en particulier lors de l'utilisation d'association inhabituelle mais aussi pour la l'indication et l'interprétation des tests in vitro, qui pourraient avoir une conséquence sur la prise en charge du patient.

International audience; Background: Meningococcal meningitis requires rapid diagnosis and immediate management which is enhanced by the use of PCR for the ascertainment of these infections. However, its use is still restricted to reference laboratories. Methods: We conducted an inter-laboratory study to assess the implementation and the performance of PCR in ten French hospital settings in 2010. Results: Our data are in favour of this implementation. Although good performance was obtained in identifying Neisseria meningitidis positive samples, the main issue was reported in identifying other species (Streptococcus pneumoniae and Haemophilus influenzae) which are also involved in bacterial meningitis cases. Conclusions: Several recommendations are required and, mainly, PCR should target the major etiological agents (N. meningitidis, S. pneumonia, and H. influenzae) of acute bacterial meningitis. Moreover, PCR should predict the most frequent serogroups of Neisseria meningitidis according to local epidemiology. (Clin. Lab. 2012;58:343-346)

International audience; Gene cassettes of class 1 integrons may be differently expressed depending on the Pc promoter variant as well as occasionally from a second promoter located downstream of Pc, named P2. So far, the distribution of the variants has only been described in an in silico study. In this study, the prevalence of these variants in vivo was analysed in a population of 85 Escherichia coli strains from a variety of phylogenetic groups isolated from healthy subjects and clinical samples in Spain and France from 2004 to 2007. The weakest variants (PcW and PcH1) prevailed (variants associated with the integrase having the most efficient excision activity), whilst the two strongest variants, PcW(TGN-10) and PcS, were less frequent. Furthermore, a new variant of P2 associated with PcW was characterised in one integron (harbouring the gene cassette bla(OXA-1)-aadA1) from a French strain of a healthy subject. This variant was hereafter named P2m3 and shows a G→A substitution in its -10 element (TACAGT to TACAAT), a mutation that doubled the strength of P2 and approached the level of expression of the strong PcW(TGN-10) variant. When the correlation between the Pc variants and the origin of the strains was analysed, no significant difference (P

International audience; Class 1 integrons are widespread genetic elements responsible for dissemination of antibiotic resistance among Gram-negative bacteria. Integrons allow bacteria to capture and express gene cassettes (GCs) via an integrase (IntI1) and a promoter (Pc) contained in the integron functional platform. GCs are transcribed from Pc, of which 13 variants of different strengths have been described, or, occasionally, from both Pc and a second promoter (P2). The intI1 promoter (PintI1) is repressed by LexA, which is the transcriptional repressor of the global regulatory SOS response network. Moreover, PintI1 lies face to face with Pc and overlaps P2, both configurations being propitious to transcriptional interference (TI). In this study, we analyzed possible transcriptional interference by quantifying transcripts produced from Pc, P2, and PintI1. We found that the Pc promoter interferes with the level of intI1 transcription but that this effect depends on the Pc variant: the strong Pc variant prevents intI1 expression, in contrast to the other variants. Although P2 formation results in LexA binding site disruption and thus prevents SOS regulation of intI1 expression, P2 does not interfere with PintI1. These findings reveal a tight relationship between GC and integrase expression.

International audience; Objectives Cytomegalovirus (CMV) drug resistance is a therapeutic challenge in the transplant setting. No longitudinal cohort studies of CMV resistance in a real-life setting have been published in the valganciclovir era. We report findings for a French multicentre prospective cohort of 346 patients enrolled at initial diagnosis of CMV infection (clinical trial registered at clinicaltrials.gov: NCT01008540). Patients and methods Patients were monitored for detection of CMV infection for ≥2 years. Real-time detection of resistance by UL97 and UL54 gene sequencing and antiviral phenotyping was performed if viral replication persisted for >21 days of appropriate antiviral treatment. Plasma ganciclovir assays were performed when resistance was suspected. Results Resistance was suspected in 37 (10.7%) patients; 18/37 (5.2% of the cohort) had virological resistance, associated with poorer outcome. Most cases involved single UL97 mutations, but four cases of multidrug resistance were due to UL54 mutations. In solid organ transplant recipients, resistance occurred mainly during primary CMV infection (odds ratio 8.78), but also in two CMV-seropositive kidney recipients. Neither CMV prophylaxis nor antilymphocyte antibody administration was associated with virological resistance. Conclusions These data show the feasibility of surveying resistance. Virological resistance was frequent in patients failing antiviral therapy. More than 1/5 resistant isolates harboured UL54 mutations alone or combined with UL97 mutations, which conferred a high level of resistance and sometimes were responsible for cross-resistance, leading to therapeutic failure.

Background: Integrons have a well-established role in the dissemination of resistance among Gram-negative pathogens and are thus a useful marker of antibiotic resistance. Shigellae are noteworthy for their multiple drug resistance, having gradually acquired resistance to most widely use and inexpensive antimicrobial drugs. Methodology: A total of 32 Shigella strains belonging to serotypes flexneri , dysenteriae , and boydii 20, a new Shigella serovar, resistant to at least four antibiotics were analyzed by molecular techniques. Results: Class 1 integrons were the most prevalent (92.8%); class 2 integrons were found in 16 strains (57.1%). Fifty percent of the strains harboured both class 1 and 2 integrons ( intI1 and intI2 genes); this combination of integrase genes was most prevalent in S. boydii 20 and S . dysenteriae strains. The class 1 integrons detected contained dfr and aadA cassettes, alone or in combination ( dfrA5 / dfrA15 , or dfrA15 - aadA1 , dfrA1 - aadA2 ), and an atypical cassette array with an insertion sequence ( oxa30 - aadA1- IS 1 ). For class 2 integrons, we detected either the same cassettes as those found in Tn 7 ( dfrA1 - sat1 - aadA1 - orfX ) or truncated class 2 integrons without aadA1 or orfX . The tns genes were absent from all class 2 integrons. The distribution of integrons among RAPD profiles and serotypes revealed a clonal spread of integrons into serotypes and a transfer of integrons between different serotypes. Conclusions: The detection of integrons in a new Shigella serovar, in addition with a high integron prevalence among Shigella strains, confirms the propensity of shigellae to acquire and disseminate resistance determinants.

Class 1 integrons are widespread genetic elements that allow bacteria to capture and express gene cassettes that are usually promoterless. These integrons play a major role in the dissemination of antibiotic resistance among Gram-negative bacteria. They typically consist of a gene (intI) encoding an integrase (that catalyzes the gene cassette movement by site-specific recombination), a recombination site (attI1), and a promoter (Pc) responsible for the expression of inserted gene cassettes. The Pc promoter can occasionally be combined with a second promoter designated P2, and several Pc variants with different strengths have been described, although their relative distribution is not known. The Pc promoter in class 1 integrons is located within the intI1 coding sequence. The Pc polymorphism affects the amino acid sequence of IntI1 and the effect of this feature on the integrase recombination activity has not previously been investigated. We therefore conducted an extensive in silico study of class 1 integron sequences in order to assess the distribution of Pc variants. We also measured these promoters' strength by means of transcriptional reporter gene fusion experiments and estimated the excision and integration activities of the different IntI1 variants. We found that there are currently 13 Pc variants, leading to 10 IntI1 variants, that have a highly uneven distribution. There are five main Pc-P2 combinations, corresponding to five promoter strengths, and three main integrases displaying similar integration activity but very different excision efficiency. Promoter strength correlates with integrase excision activity: the weaker the promoter, the stronger the integrase. The tight relationship between the aptitude of class 1 integrons to recombine cassettes and express gene cassettes may be a key to understanding the short-term evolution of integrons. Dissemination of integron-driven drug resistance is therefore more complex than previously thought., Author Summary Integrons are widespread bacterial genetic elements able to capture and express gene cassettes that often encode antibiotic resistance determinants. Gene cassettes are usually promoterless and are transcribed from a common promoter, Pc. Pc is located within the coding sequence of the integron integrase, IntI, which is the key element catalyzing the integration and excision of gene cassettes. Several Pc variants, associated with different integrase amino acid sequences, have been described, but the influence of these differences on integrase activity has never been investigated. Here, we show that Pc is highly polymorphic, conferring a wide range of antibiotic resistance. Furthermore, we found that different Pc variants are associated with different integrase excision activities: the weaker the Pc variant, the more active the integrase. These results point to evolutionary compromises between the expression and mobility of drug resistance determinants located on integrons.

www.invs. sante.fr/behweb/2010/01/r-1.htm; National audience; La surveillance nationale de l'hépatite B chronique a été mise en place en 2008, en collaboration avec les pôles de ré- férence (services hospitalo-universitaires d'hépatologie), pour décrire les caractéristiques épidémiologiques et clinico- biologiques des patients nouvellement pris en charge pour une hépatite B chronique. L'hépatite B chronique est définie par le portage de l'antigène HBs (AgHBs) depuis plus de six mois. Les caractéristiques des patients qui sont recueillies incluent les modalités de découverte de l'AgHBs et leurs expositions à risque vis-à-vis du virus de l'hépatite B (VHB). Nous présentons certaines caractéristiques des patients naïfs de traitement antiviral, selon le niveau de prévalence de l'AgHBs du pays de naissance (endémicité faible vs. moyenne/forte). Entre janvier 2008 et août 2009, 1 016 patients ont été pris en charge, dont 78% sont nés dans une zone de moyenne ou forte endémicité VHB. La découverte de l'AgHBs est fortuite pour 69% des patients. Le délai entre le dépistage et la prise en charge, déterminé pour 837 patients, est supérieur à trois ans pour 41% des patients nés en zone de faible endémicité et pour 22% de ceux nés ailleurs. Ces résultats préliminaires suggèrent des pratiques de dépistage de l'AgHBs imparfaites et un retard à la prise en charge des personnes dépistées, en particulier celles nées dans une zone de faible endémicité VHB.

International audience; In a previous study, we identified 12 conserved domains within pUL89, the small terminase subunit of the human cytomegalovirus. A latter study showed that the fragment pUL89(580-600) plays an important role in the formation of the terminase complex by interacting with the large terminase subunit pUL56. In this study, analysis was performed to solve the structure of pUL89(568-635) in 50% H2O/50% acetonitrile (v/v). We showed that pUL89(568-635) consists of four alpha helices, but we did not identify any tertiary structure. The fragment 580-600 formed an amphipathic alpha helix, which had a hydrophobic side highly conserved among herpesviral homologs of pUL89; this was not observed for its hydrophilic side. The modeling of pUL89(457-612) using the recognition fold method allowed us to position pUL89(580-600) within this domain. The theoretical structure highlighted three important features. First, we identified a metal-binding pocket containing residues Asp(463), Glu(534), and Glu(588), which are highly conserved among pUL89 homologs. Second, the model predicted a positively charged surface able to interact with the DNA duplex during the nicking event. Third, a hydrophobic patch on the top of the catalytic site suggested that this may constitute part of the pUL89 site recognized by pUL56 potentially involved in DNA binding.

International audience; We have studied 457 Streptococcus pneumoniae isolated in 2007 from adults and children. For all isolates, both latex agglutination and molecular capsular typing were performed. Antibiotic resistance patterns were determined. S. pneumoniae 19A was the most frequently isolated serotype (34.7%) both in children and adults. It represented 12.8% of the strains isolated from invasive infections in adults and 27.0% in children and 63.6% (110/173) of strains isolated from acute otitis media. Between children and adults, no difference concerning antibiotic susceptibility was observed for penicillin, amoxicillin, cefotaxime, and erythromycin in strains isolated from invasive diseases. Comparing antibiotic susceptibilities according to the serotype, the 19A isolates appeared to be the least susceptible to penicillin (3.2%) and erythromycin (4.5%), followed by serotypes 19F and 14. We confirm the predominance of serotype 19A among S. pneumoniae responsible for invasive and noninvasive diseases either in children or adults in France.

Analysis of 15 European clinical Enterobacteriaceae isolates showed that differences in the genetic context of bla CMY-2 -like genes reflected the replicon type, usually IncA/C or IncI1. These bla CMY-2 loci may originate from the same IS Ecp1 -mediated mobilization from the Citrobacter freundii chromosome as structures described in earlier studies.

International audience; The qnr genes are plasmid-borne fluoroquinolone-resistance determinants widespread in Enterobacteriaceae. Three families of qnr determinants (qnrA, B and S) have been described, but little is known about their expression and regulation. Two new determinants, qnrC and qnrD, have been found recently. Here, we describe the characterization of the qnrB2 promoter and the identification of a LexA-binding site in the promoter region of all qnrB alleles. LexA is the central regulator of the SOS response to DNA damage. We show that qnrB2 expression is regulated through the SOS response in a LexA/RecA-dependent manner, and that it can be induced by the quinolone ciprofloxacin, a known inducer of the SOS system. This is the first description of direct SOS-dependent regulation of an antibiotic-resistance mechanism in response to the antibiotic itself.

International audience; Integrons are found in the genome of hundreds of environmental bacteria but are mainly known for their role in the capture and spread of antibiotic resistance determinants among Gram-negative pathogens. We report a direct link between this system and the ubiquitous SOS response. We found that LexA controlled expression of most integron integrases and consequently regulated cassette recombination. This regulatory coupling enhanced the potential for cassette swapping and capture in cells under stress, while minimizing cassette rearrangements or loss in constant environments. This finding exposes integrons as integrated adaptive systems and has implications for antibiotic treatment policies.

Centre Hospitalier Universitaire (CHU) Limoges, Centre National de Reference du CMV, Laboratoire de Bactériologie-Virologie-Hygiène, Limoges, France; International audience; BACKGROUND: The polymorphism of genes encoding CMV envelope protein is used for strain classification and may influence pathogenesis and/or infectivity. CMV genotyping is usually based on sequencing or acrylamide gel-RFLP, but these methods are not suited to rapid screening of large populations. OBJECTIVES: We developed a high-throughput method to analyze CMV strains diversity and to detect multiple-strain infection in a large population of toddlers (six daycare centers (DCC) and an emergency unit (EU)). METHODS: We developed a new PCR-RFLP method coupled with capillary electrophoresis fragment detection for UL55-gB, UL75-gH and UL73-gN genotyping. To detect gB recombinants, gpUL55 typing was applied to two variable zones (NTerminal and central). We applied this method to 212 CMV-positive saliva samples and controlled the results by direct sequencing of PCR products. RESULTS: We identified 112 strains, that fell into eight groups in UL55-gB, two groups in UL75-gH, and seven groups in UL73-gN. The 79 samples from the emergency unit contained 30 strains, 28 children harboring 2 strains. The samples (n=133) from the six daycare centers contained respectively 4, 1, 6, 1 and 11 strains. Fifteen percent of strains were UL55-gB recombinants. CONCLUSION: Our new method can simultaneously determine gB, gH and gN genotypes and offers more precise classification of CMV strains than previous RFLP-based methods. This could constitute the basis for a new classification, particularly in UL55-gB. Easy direct identification of multiple strains and recombinants in pathological samples could facilitate large epidemiologic studies.

Objectives: Biocides are used intensively in a large variety of applications, usually with inanimate objects or on the skin. The general consumer markets led to increase their use by the general public and the risk assessment should be affected by the wide frequency of contact with these products for humans or environment. Risks: Increasingly accurate legislation takes into account physical and chemical risks and the best prevention is the respect of security rules. The evaluation of biological risks is not so easy because antibacterial products activity is linked either to major agents (disinfectant) or to additional agents used as preservatives. Mode of action and biological risk: Some similarities between these antibacterial compounds and antibiotic action have been described maintaining some fears on development or induction of a coping mechanism that should result in a new or increased antibiotic resistance. In fact, these resistances are described as increase in minimal inhibitory concentration (MIC) of antibiotics and not as loss of efficacy at usual concentrations. Resistance and survival: Mainly, there is an over way for microorganisms to survive like very well organized communities manned "biofilms" which preserve organisms of adverse effects including biocide action and present real health and environmental risks.

International audience; La résistance du cytomégalovirus aux antiviraux est un véritable problème en transplantation, mais aussi chez d'autres patients immunodéprimés, conduisant parfois à l'impasse thérapeutique. Elle concerne près de 5 % des patients receveurs d'organe ou de cellules souches hématopoïétiques, et représente un facteur d'évolution défavorable après greffe. Elle est la conséquence de l'usage intensif, en prévention ou en traitement prolongé, sur un terrain favorable à la réplication virale, des molécules disponibles. Ces molécules sont le ganciclovir et sa prodrogue le valganciclovir, le cidofovir et le foscarnet, qui toutes ciblent l'ADN polymérase virale. La détection des souches résistantes, indiquée lorsque la réplication virale persiste au-delà de trois semaines, repose sur le phénotype qui nécessite l'isolement de la souche, et le génotype, qui correspond à la détection des mutations responsables de résistance, le plus souvent par séquençage des gènes concernés : UL97 et UL54. Les mutations de UL97 sont les premières à apparaître et confèrent une résistance au ganciclovir. Les mutations de UL54, plus tardives, peuvent conduire à une résistance croisée au ganciclovir et au cidofovir, à une résistance au foscarnet ou à une résistance croisée aux trois antiviraux. L'ajustement des doses d'antiviral, disponible pour le ganciclovir, la réduction de l'immunosuppression, le changement d'antiviral voire l'utilisation de nouveaux antiviraux guidée par le génotype et le phénotype sont associés dans la prise en charge des patients en cas de résistance.

International audience

International audience