Eric Garnotel

Membrane Permeability and Regulation of Drug “Influx and Efflux” in Enterobacterial Pathogens

In Enterobacteriaceae, membrane permeability is a key in the level of susceptibility to antibiotics. Modification of the bacterial envelope by decreasing the porin production or increasing the expression of efflux pump systems has been reported. These phenomena are frequently associated with other resistance mechanisms such as alteration of antibiotics or modification of the drug targets, in various clinical isolates showing a Multi Drug Resistant phenotype (MDR). In Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae and Salmonella enterica several genes and external factors are involved in the emergence of MDR isolates. These bacterial isolates exhibit a noticeable reduction of functional porins per cell due to a decrease, a complete shutdown of synthesis, or the expression of an altered porin and a high expression of efflux systems (e.g. overexpression of the pump). The combined action of these mechanisms during an infection confers a significant decrease in bacterial sensitivity to antibiotherapy ensuring dissemination and colonization of the patient and favours the acquisition of additional mechanisms of resistance. MarA and ramA are involved in a complex regulation cascade controlling membrane permeability and actively participate in the triggering of the MDR phenotype. Mutations in regulator genes have been shown to induce the overproduction of efflux and the down-regulation of porin synthesis. In addition, various compounds such as salicylate, imipenem or chloramphenicol are able to activate the MDR response. This phenomenon has been observed both in vitro during culture of bacteria in the presence of drugs and in vivo during antibiotic treatment of infected patients. These effectors activate the expression of specific global regulators, marA, ramA, or target other genes located downstream in the regulation cascade.

Significance of the Identification in the Horn of Africa of an Exceptionally Deep Branching Mycobacterium tuberculosis Clade

Molecular and phylogeographic studies have led to the definition within the Mycobacterium tuberculosis complex (MTBC) of a number of geotypes and ecotypes showing a preferential geographic location or host preference. The MTBC is thought to have emerged in Africa, most likely the Horn of Africa, and to have spread worldwide with human migrations. Under this assumption, there is a possibility that unknown deep branching lineages are present in this region. We genotyped by spoligotyping and multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) 435 MTBC isolates recovered from patients. Four hundred and eleven isolates were collected in the Republic of Djibouti over a 12 year period, with the other 24 isolates originating from neighbouring countries. All major M. tuberculosis lineages were identified, with only two M. africanum and one M. bovis isolates. Upon comparison with typing data of worldwide origin we observed that several isolates showed clustering characteristics compatible with new deep branching. Whole genome sequencing (WGS) of seven isolates and comparison with available WGS data from 38 genomes distributed in the different lineages confirms the identification of ancestral nodes for several clades and most importantly of one new lineage, here referred to as lineage 7. Investigation of specific deletions confirms the novelty of this lineage, and analysis of its precise phylogenetic position indicates that the other three superlineages constituting the MTBC emerged independently but within a relatively short timeframe from the Horn of Africa. The availability of such strains compared to the predominant lineages and sharing very ancient ancestry will open new avenues for identifying some of the genetic factors responsible for the success of the modern lineages. Additional deep branching lineages may be readily and efficiently identified by large-scale MLVA screening of isolates from sub-Saharan African countries followed by WGS analysis of a few selected isolates.

Identification and Evolution of Drug Efflux Pump in Clinical Enterobacter aerogenes Strains Isolated in 1995 and 2003

Background The high mortality impact of infectious diseases will increase due to accelerated evolution of antibiotic resistance in important human pathogens. Development of antibiotic resistance is a evolutionary process inducing the erosion of the effectiveness of our arsenal of antibiotics. Resistance is not necessarily limited to a single class of antibacterial agents but may affect many unrelated compounds; this is termed ‘multidrug resistance’ (MDR). The major mechanism of MDR is the active expulsion of drugs by bacterial pumps; the treatment of Gram negative bacterial infections is compromised due to resistance mechanisms including the expression of efflux pumps that actively expel various usual antibiotics (ß-lactams, quinolones, …). Methodology/Principal Findings Enterobacter aerogenes has emerged among Enterobacteriaceae associated hospital infections during the last twenty years due to its faculty of adaptation to antibiotic stresses. Clinical isolates of E. aerogenes belonging to two strain collections isolated in 1995 and 2003 respectively, were screened to assess the involvement of efflux pumps in antibiotic resistance. Drug susceptibility assays were performed on all bacterial isolates and an efflux pump inhibitor (PAßN) previously characterized allowed to decipher the role of efflux in the resistance. Accumulation of labelled chloramphenicol was monitored in the presence of an energy poison to determine the involvement of active efflux on the antibiotic intracellular concentrations. The presence of the PAßN-susceptible efflux system was also identified in resistant E. aerogenes strains. Conclusions/Significance For the first time a noticeable increase in clinical isolates containing an efflux mechanism susceptible to pump inhibitor is report within an 8 year period. After the emergence of extended spectrum ß-lactamases in E. aerogenes and the recent characterisation of porin mutations in clinical isolates, this study describing an increase in inhibitor-susceptible efflux throws light on a new step in the evolution of mechanism in E. aerogenes.

Efflux pumps of gram-negative bacteria, a new target for new molecules.

Antibiotic resistance mechanisms reported in Gram-negative bacteria are a worldwide health problem. The continuous dissemination of multi-drug resistant (MDR) bacteria drastically reduces the efficacy of our antibiotic “arsenal” and consequently increases the frequency of therapeutic failure. In MDR bacteria the over-expression of efflux pumps expel structurally-unrelated antibiotics decreasing their intracellular concentration. It is necessary to clearly define the molecular and genetic bases of the efflux pump in order to combat this mechanism. The characterization of efflux pumps, from genetic to structural studies, allows the definition of a new, original antiresistance bullet, the efflux pump inhibitor (EPI). This new class of antibacterial molecules may act conjointly to the usual antibiotic in order to restore its activity. Several families of EPIs have been now reported and described. The use of these EPIs promotes a significant increase of susceptibility to one or more antibiotics in strains or clinical isolates which were initially resistant. These EPIs may target different efflux targets, (i) the expression of genes that induces MDR, the transporters that pump the antibiotic out of bacterium, (ii) the assembly of membrane transporter complex involved in drug efflux, (iii) the energy involved in this active transport, (iv) the inhibition of the flux of molecules inside the efflux channel by competition or blocking (via steric hindrances). With the recent thorough characterization of the efflux pump AcrB at its structural and physiological level including the identification of drug affinity sites and kinetic parameters for some antibiotics, it is now possible to rationally develop an improved new generation of EPIs.

Involvement of the efflux pumps in chloramphenicol selected strains of Burkholderia thailandensis: proteomic and mechanistic evidence.

Burkholderia is a bacterial genus comprising several pathogenic species, including two species highly pathogenic for humans, B. pseudomallei and B. mallei. B. thailandensis is a weakly pathogenic species closely related to both B. pseudomallei and B. mallei. It is used as a study model. These bacteria are able to exhibit multiple resistance mechanisms towards various families of antibiotics. By sequentially plating B. thailandensis wild type strains on chloramphenicol we obtained several resistant variants. This chloramphenicol-induced resistance was associated with resistance against structurally unrelated antibiotics including quinolones and tetracyclines. We functionally and proteomically demonstrate that this multidrug resistance phenotype, identified in chloramphenicol-resistant variants, is associated with the overexpression of two different efflux pumps. These efflux pumps are able to expel antibiotics from several families, including chloramphenicol, quinolones, tetracyclines, trimethoprim and some β-lactams, and present a partial susceptibility to efflux pump inhibitors. It is thus possible that Burkholderia species can develop such adaptive resistance mechanisms in response to antibiotic pressure resulting in emergence of multidrug resistant strains. Antibiotics known to easily induce overexpression of these efflux pumps should be used with discernment in the treatment of Burkholderia infections.

First Case of Emergence of Atovaquone-Proguanil Resistance in Plasmodium falciparum during Treatment in a Traveler in Comoros

Atovaquone-proguanil (Malarone; GlaxoSmithKline) is now commonly used for the treatment and prophylaxis of falciparum malaria in France. We report here a treatment failure of atovaquone-proguanil in a patient who was infected during a 33-day visit without antimalarial prophylaxis in Comoros. The patient presented with fever 10 days after the end of his trip, and a diagnosis of falciparum malaria was made. Treatment with atovaquone-proguanil was well tolerated. Isolated fever in association with falciparum parasites appeared 23 days after therapy. The patient was successfully treated with quinine. In vitro susceptibility tests performed on blood samples from day 0 and day 23 showed a 50% inhibitory concentration (IC50) value for atovaquone that was more than 100-fold greater on day 23 than on day 0 (Table ​(Table1).1). In addition, the IC50 for cycloguanil was increased by 18-fold. TABLE 1. In vitro drug susceptibility profiles and changes in genotyping profiles of the day 0 and day 23 P. falciparum parasitesa Sequencing of the cyt b gene, encoding the atovaquone target (12), showed a wild-type P. falciparum strain on day 0 and a Y268S mutation on day 23. Genotyping of the dhfr gene, encoding the proguanil target (5), showed a double mutation, C59R and S108N, on day 0, while a triple mutation, N51I, C59R, and S108N, was observed on day 23. However, proguanil likely does not act by itself in atovaquone-proguanil treatment but only facilitates the atovaquone activity (11). Genotyping of the Pfcrt gene (wild type, K76), encoding a transport protein involved in chloroquine resistance, and of the dhps gene (wild type, S436, A437, K540, A581, and A613), encoding the sulfadoxine target (5), showed wild-type, identical alleles. The genotyping of the two isolates, using three of six microsatellite loci (7A11, Pf2802, C4M79, Pf2689, TRAP, and C4M69) (1), msp1, and msp2 (5), showed differences between days 0 and 23 (Table ​(Table11). The day 23 parasites presented a high IC50 for atovaquone associated with a Y268S mutation in Cyt b. Since 2002, fewer than 20 cases of genetically confirmed clinical resistance to atovaquone-proguanil had been reported (2-4, 6, 7, 9, 13, 14). Clinical failures were associated with in vitro-increased IC50s for atovaquone between day 0 and the failure day only in five isolates (4, 7, 9). In some cases, the increased IC50 was moderate (7, 8). An in vitro atovaquone threshold of 1,900 nM was recommended to discriminate resistant isolates (10). Considering our results, this cutoff must be adjusted to >350 nM. We were unable to detect Cyt b mutations on codon 268 and high IC50 to atovaquone in the pretreatment isolate. Reinfection was excluded because the patient was treated after returning to France. The atovaquone-resistant strain was probably present in the initial isolate but in the minority, making it undetectable by classical genotyping methods and in vitro testing. The isolate was polyclonal on day 0 and monoclonal on day 23. This is the first observation of the clinical failure of atovaquone-proguanil treatment of P. falciparum infection in a traveler in Comoros, an area where the in vitro prevalences of isolates with reduced susceptibilities to classical antimalarial drugs were <7% (12). Although clinical failures of atovaquone-proguanil therapy remain rare in travelers, an increased vigilance is required during their treatment followup, and surveillance of the parasite population should be reinforced as well.

Highly structured genetic diversity of the Mycobacterium tuberculosis population in Djibouti.

Djibouti is an East African country with a high tuberculosis incidence. This study was conducted over a 2-month period in Djibouti, during which 62 consecutive patients with pulmonary tuberculosis (TB) were included. Genetic characterization of Mycobacterium tuberculosis, using mycobacterial interspersed repetitive-unit variable-number tandem-repeat typing and spoligotyping, was performed. The genetic and phylogenetic analysis revealed only three major families (Central Asian, East African Indian and T). The high diversity and linkage disequilibrium within each family suggest a long period of clonal evolution. A Bayesian approach shows that the phylogenetic structure observed in our sample of 62 isolates is very likely to be representative of the phylogenetic structure of the M. tuberculosis population in the total number of TB cases.

Delayed Onset of Plasmodium falciparum Malaria after Doxycycline Prophylaxis in a Soldier Returning from the Central African Republic

Emilie Javelle, Marylin Madamet, Tiphaine Gaillard, Guillaume Velut, Corinne Surcouf, Rémy Michel, Eric Garnotel, Fabrice Simon, Bruno Pradines Service de Maladies Infectieuses et Tropicales, Hôpital d’Instruction des Armées Laveran, Marseille, France; Aix Marseille Université, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UM 63, CNRS 7278, IRD 198, Inserm 1095, Marseille, France; Equipe Résidente de Recherche en Infectiologie Tropicale, Institut de Recherche Biomédicale des Armées, Hôpital d’Instruction des Armées Laveran, Marseille, France; Centre National de Référence du Paludisme, Marseille, France; Fédération des Laboratoires, Hôpital d’Instruction des Armées Saint Anne, Toulon, France; Service de Santé Publique, Centre d’Epidémiologie et de Santé Publique des Armées, Marseille, France; Fédération des Laboratoires, Hôpital d’Instruction des Armées Laveran, Marseille, France; Service de Surveillance Epidémiologique, Centre d’Epidémiologie et de Santé Publique des Armées, Marseille, France; Ecole du Val-de-Grâce, Paris, France; Unité de Parasitologie et d’Entomologie, Département des Maladies Infectieuses, Institut de Recherche Biomédicale des Armées, Brétigny sur Orge, France

Pulmonary Isolation of Multidrug resistant “ Mycobacterium simulans” and Mycobacterium tuberculosis from a patient in the Horn of Africa

In low-income countries of the Horn of Africa, pulmonary infections are usually considered as tuberculosis, which diagnosis relies on clinical data and positive microscopic observation. This strategy allows non-tuberculous mycobacteria to escape detection, facilitating their emergence in populations. A non-tuberculous mycobacterium strain FB-527 was unexpectedly cultured from the sputum of a Djiboutian patient otherwise diagnosed with multi-drug resistant (MDR) tuberculosis. The sequencing of the rpoB and 16S rRNA genes showed that the isolate was identical to strain FI-09026 previously named “Mycobacterium simulans” and reported only once from a Somali patient. Strain FB-527 mimicked Mycobacterium tuberculosis colonies and enzymatic profile using API ZYM strip and was in vitro resistant to rifampicin and isoniazid. Isolation of two MDR mycobacteria complicated the diagnosis and therapeutic management of the patient. We here report on the complete description of strain FB-527 and strain FI-09026 including genome sequencing, finalizing the description of the proposed new species “Mycobacterium simulans”.

Sensibilité aux antibiotiques d’Escherichia coli isolé des infections urinaires communautaires : étude AFORCOPI-BIO, 2015

Resume Objectifs Etudier la sensibilite des Escherichia coli isoles des urines de medecine de ville dans l’ensemble de la population (femmes, hommes, enfants). Patients et methodes Il s’agit d’une etude multicentrique retrospective sur 121 sites de prelevements draines sur 7 grands plateaux techniques repartis dans la moitie sud de la France. Il est effectue un recueil exhaustif des donnees d’antibiogramme des Escherichia coli isoles des urines d’origine communautaire durant l’annee 2015. Resultats Les aminopenicillines (sensibilite (Se): 51 %) et l’amoxicilline + acide clavulanique (Se: 69,3 %) ont une activite insuffisante pour une utilisation en antibiotherapie probabiliste. Les taux de sensibilite aux fluoroquinolones continuent de diminuer (Se de la ciprofloxacine : 87,8 %). Les cepholosporines de 3e generation gardent une excellente activite. Les antibiotiques specifiques du traitement des infections urinaires, fosfomycine (Se: 98,7 %), nitrofuranes (Se : 98,7 %) et mecillinam (Se : 92,4 %), ont les meilleurs taux de sensibilite meme en presence de souches produisant une β-lactamase a spectre etendu. On observe une diminution de l’activite des antibiotiques avec l’âge des patients. Conclusion La surveillance de la resistance aux antibiotiques est essentielle pour guider les recommandations de l’antibiotherapie probabiliste. Les donnees collectees en 2015 sont en adequation avec les recommandations actuelles.