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.

The genetic variability and population structure of Plasmodium falciparum are key factors in malaria control strategies. Studies have suggested no P. falciparum population structure although linkage disequilibrium was observed in some African areas. We have assessed length polymorphism at 6-22 microsatellites in four urban and rural sites (Djibouti, Dakar, Niamey, and Zouan-Hounien, n = 240 blood samples). Results have shown a P. falciparum population structure in Africa (Fst = 0.17-0.24), lower genetic diversity in Djibouti (He = 0.53) than in the other sites (He = 0.73-0.76), and 3) significant linkage disequilibrium in Djibouti. These results could be related to geographic isolation and low flow of parasites between sites. They also suggest a potential effect of rural suburbs to generate genetic diversity in towns. This could affect the dispersal of selected drug resistance and should be considered when adapting urban malaria control strategies.

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.

Careful surveys for these clones need to be conducted, but at present they play only a minor role in the overall epidemiology of meningococcal meningitis.

Over a 3-year follow-up, 30 out of the 318 unique Mycobacterium tuberculosis complex isolates recovered in the Republic of Djibouti had a smooth-type morphology and were Niacine-negative, the characteristics of 'Mycobacterium canettii' strains. Unlike M. tuberculosis, 'M. canettii' grew on nutrient-poor media at 30°C, and possessed characteristic lipids. They were isolated from respiratory and extra-respiratory sites from patients with typical forms of tuberculosis. Most cases resolved with antibiotic therapy but in two human immunodeficiency virus-positive patients 'M. canettii' infection led to septicaemia and death. No cases of human-to-human transmission were observed. The proportion of tuberculosis cases caused by 'M. canettii' was higher among French patients than among Djiboutian patients. Patients with 'M. canettii' were significantly younger than those with tuberculosis caused by other M. tuberculosis complex strains. Smooth tubercle bacilli could be misidentified as non-tuberculous mycobacteria and appear to be limited to the Horn of Africa. Their characteristics are consistent with the existence of non-human sources of infection.

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.

BackgroundThe 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/SignificanceFor 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.

Multidrug resistant bacteria have been a worldwide concern for decades. Though new molecules that effectively target Gram-positive bacteria are currently appearing on the market, a gap remains in the treatment of infections caused by Gram-negative bacteria. Therefore, new strategies must be developed against these pathogens. The aim of this study was to select an antibiotic for which a bacterium is naturally resistant and to use an escort molecule to restore susceptibility, similarly to the model of β-lactam/ β-lactamase inhibitors. High-content screening was performed on the reference strain PA01, allowing the selection of four polyamino-isoprenic compounds that acted synergistically with doxycycline. They were assayed against clinical isolates and Multi-Drug-Resistant strains. One of these compounds was able to decrease the MIC of doxycycline on the reference strain, efflux pump overproducers and clinical isolates of P. aeruginosa, to the susceptibility level. Similar results were obtained using chloramphenicol as the antibiotic. Membrane permeation assays and real-time efflux experiments were used to characterize the mechanism of doxycycline potentiation.The results showed that the selected compound strongly decreases the efficiency of glucose-triggered efflux associated with a slight destabilization of the outer membrane. According to these data, targeting natural resistance may become an interesting way to combat MDR pathogens and could represent an alternative to already devised strategies.