The TolC Protein of Legionella pneumophila Plays a Major Role in Multi-Drug Resistance and the Early Steps of Host Invasion

Pneumonia associated with Iegionnaires's disease is initiated in humans after inhalation of contaminated aerosols. In the environment, Legionella pneumophila is thought to survive and multiply as an intracellular parasite within free-living amoeba. In the genome of L. pneumophila Lens, we identified a unique gene, tolC, encoding a protein that is highly homologous to the outer membrane protein TolC of Escherichia coli. Deletion of tolC by allelic exchange in L. pneumophila caused increased sensitivity to various drugs. The complementation of the tolC mutation in trans restored drug resistance, indicating that TolC is involved in multi-drug efflux machinery. In addition, deletion of tolC caused a significant attenuation of virulence towards both amoebae and macrophages. Thus, the TolC protein appears to play a crucial role in virulence which could be mediated by its involvement in efflux pump mechanisms. These findings will be helpful in unraveling the pathogenic mechanisms of L. pneumophila as well as in developing new therapeutic agents affecting the efflux of toxic compounds

Differential Gene Expression by RamA in Ciprofloxacin-Resistant Salmonella Typhimurium

Overexpression of ramA has been implicated in resistance to multiple drugs in several enterobacterial pathogens. In the present study, Salmonella Typhimurium strain LTL with constitutive expression of ramA was compared to its ramA-deletion mutant by employing both DNA microarrays and phenotype microarrays (PM). The mutant strain with the disruption of ramA showed differential expression of at least 33 genes involved in 11 functional groups. The study confirmed at the transcriptional level that the constitutive expression of ramA was directly associated with increased expression of multidrug efflux pump AcrAB-TolC and decreased expression of porin protein OmpF, thereby conferring multiple drug resistance phenotype. Compared to the parent strain constitutively expressing ramA, the ramA mutant had increased susceptibility to over 70 antimicrobials and toxic compounds. The PM analysis also uncovered that the ramA mutant was better in utilization of 10 carbon sources and 5 phosphorus sources. This study suggested that the constitutive expression of ramA locus regulate not only multidrug efflux pump and accessory genes but also genes involved in carbon metabolic pathways

Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events.

The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species

Genetic markers associated with resistance to beta-lactam and quinolone antimicrobials in non-typhoidal Salmonella isolates from humans and animals in central Ethiopia

Abstract Background Beta-lactam and quinolone antimicrobials are commonly used for treatment of infections caused by non-typhoidal Salmonella (NTS) and other pathogens. Resistance to these classes of antimicrobials has increased significantly in the recent years. However, little is known on the genetic basis of resistance to these drugs in Salmonella isolates from Ethiopia. Methods Salmonella isolates with reduced susceptibility to beta-lactams ( n \u2009=\u200943) were tested for genes encoding for beta-lactamase enzymes, and those resistant to quinolones ( n \u2009=\u200929) for mutations in the quinolone resistance determining region (QRDR) as well as plasmid mediated quinolone resistance (PMQR) genes using PCR and sequencing. Results Beta-lactamase genes ( bla ) were detected in 34 (79.1%) of the isolates. The dominant bla gene was bla TEM, recovered from 33 (76.7%) of the isolates, majority being TEM-1 (24, 72.7%) followed by TEM-57, (10, 30.3%). The bla OXA-10 and bla CTX-M-15 were detected only in a single S. Concord human isolate. Double substitutions in gyr A (Ser83-Phe\u2009+\u2009Asp87-Gly) as well as par C (Thr57-Ser\u2009+\u2009Ser80-Ile) subunits of the quinolone resistance determining region (QRDR) were detected in all S. Kentucky isolates with high level resistance to both nalidixic acid and ciprofloxacin. Single amino acid substitutions, Ser83-Phe ( n \u2009=\u20094) and Ser83-Tyr ( n \u2009=\u20091) were also detected in\ua0the gyr A gene. An isolate of S . Miami susceptible to nalidixic acid but intermediately resistant to ciprofloxacin had Thr57-Ser and an additional novel mutation (Tyr83-Phe) in the par C gene. Plasmid mediated quinolone resistance (PMQR) genes investigated were not detected in any of the isolates. In some isolates with decreased susceptibility to ciprofloxacin and/or nalidixic acid, no mutations in QRDR or PMQR genes were detected. Over half of the quinolone resistant isolates in the current study 17 (58.6%) were also resistant to at least one of the beta-lactam antimicrobials. Conclusion Acquisition of bla TEM was the principal beta-lactamase resistance mechanism and mutations within QRDR of gyr A and par C were the primary mechanism for resistance to quinolones. Further study on extended ..

The bile salt glycocholate induces global changes in gene and protein expression and activates virulence in enterotoxigenic Escherichia coli

Pathogenic bacteria use specific host factors to modulate virulence and stress responses during infection. We found previously that the host factor bile and the bile component glyco-conjugated cholate (NaGCH, sodium glycocholate) upregulate the colonization factor CS5 in enterotoxigenic Escherichia coli (ETEC). To further understand the global regulatory effects of bile and NaGCH, we performed Illumina RNA-Seq and found that crude bile and NaGCH altered the expression of 61 genes in CS5 + CS6 ETEC isolates. The most striking finding was high induction of the CS5 operon (csfA-F), its putative transcription factor csvR, and the putative ETEC virulence factor cexE. iTRAQ-coupled LC-MS/MS proteomic analyses verified induction of the plasmid-borne virulence proteins CS5 and CexE and also showed that NaGCH affected the expression of bacterial membrane proteins. Furthermore, NaGCH induced bacteria to aggregate, increased their adherence to epithelial cells, and reduced their motility. Our results indicate that CS5 + CS6 ETEC use NaGCH present in the small intestine as a signal to initiate colonization of the epithelium

Etude de l'implication du système d'efflux EmrAB dans la multirésistance chez Salmonella enterica Typhimuriu

Résumé Salmonella est un responsable majeur des cas de toxi-infection d'origine alimentaire. Malgré la diminution de l’incidence des salmonelloses ces dernières années, une augmentation du nombre des souches résistantes aux antibiotiques est observée. La résistance simultanée à plusieurs familles d’antibiotiques est particulièrement importante chez le sérotype Typhimurium de lysotype DT104. La résistance multiple est due à des mécanismes spécifiques associés et au mécanisme d’efflux actif plurispécifique. L’efflux permet à la bactérie de diminuer sa concentration intracellulaire en composés toxiques et ainsi de survivre dans un environnement hostile. Chez les entérobactéries, parmi les différents transporteurs identifiés, AcrB joue un rôle important dans la résistance multiple en fonctionnant en système d’efflux tripartite AcrAB-TolC. Cependant, chez S. Typhimurium, un autre transporteur que AcrB semble jouer un rôle important, en association avec TolC, dans la résistance aux sels biliaires. EmrB, un transporteur de la famille MFS pourrait jouer ce rôle, ainsi, des mutants inactivés au niveau du gène emrB et du gène codant le régulateur transcriptionnel, emrR ont été construits. Les tests de sensibilité aux antibiotiques et aux sels biliaires n’ont pas montré de différence significative entre la souche parentale et les mutants. La complémentation avec le gène emrB n’a pas permis d’amplifier le fragment attendu, pour chacun des clones testé

Abstracts book : 9. Symposium on Antimicrobial Resistance in Animals and the Environment - ARAE

The emergence of antimicrobial resistance is a seminally important public health concern. Significant progress has been made in recent years regarding an understanding of the genetic and biochemical basis for antimicrobial resistance, the emergence of resistance genes, and factors promoting their widespread dissemination including the role of lateral gene transfer. Nevertheless, there is a dearth of information regarding the key ‘hotspots’ and genetic mechanisms responsible for resistance development, and the exposure routes leading to the failure of antimicrobial agents important in human and animal medicine. There is thus an urgent need for research to provide governments, public health stakeholders, and the agricultural sector the knowledge required to develop policies and practices that effectively mitigate resistance development. This, within a growing recognition that humans, animals and the environment must be considered as intimately linked together if any resistance management strategy is to be successful. Livestock are in close contact with soil and water, natural reservoirs of microbiota harbouring resistance genes. In turn, the use of manures as fertilizers for crop production is a potentially important source for environmental contamination of resistance genes selected for and enriched in the animal. A better understanding of the significance of animal and environmental reservoirs of antimicrobial resistance, and factors leading to the emergence and dissemination of antibioticresistant bacteria in agricultural production systems is a priority. The ARAE symposium, created by scientists from INRAE, is organized since 2005. Since thefirst edition in Lyon, ARAE has been a great success with many scientists from all over the world still participating in this symposium which is now in its ninth edition

Effect of ramR mutations on efflux genes expression and on fluoroquinolone susceptibility in <em>Salmonella enterica</em> serotype Kentucky ST-198

PosterNational audienc

Effect of ramR mutations on efflux genes expression and on fluoroquinolone susceptibility in <em>Salmonella enterica</em> serotype Kentucky ST198

National audienceBackground. Efflux is a mechanism that has been previously reported to increase fluoroquinolone (FQ) resistance levels when it is upregulated in clinical isolates of Salmonella enterica mainly of serotype Typhimurium (1). In this study efflux related genes were investigated in the emerging FQ-resistant epidemic S. enterica serotype Kentucky ST198 clone (2) for the presence of mutations in their regulatory genes, and for their expression levels. Methods. Among a representative panel of thirty serotype Kentucky strains from Egypt or east Africa with decreased FQ susceptibility, three strains overproducing the AcrAB-TolC efflux system were detected and studied (3). Two FQ-resistant strains with substitutions in QRDR and with basal expression level of AcrABTolC and the susceptible reference strain 98K were used as control. Genetic relatedness was determined by XbaI-pulsed field gel electrophoresis and multilocus sequence typing. Presence of mutations in the ramRA, soxRS, marOR loci and acrR, acrS genes were assessed by sequencing. The six strains and their wild-type ramR gene complemented derivatives were analysed by (i) qRT-PCR for gene expression of regulatory and efflux genes and by (ii) MIC determinations of quinolones, FQ and florfenicol as other substrate of AcrAB-TolC. Results. All serotype Kentucky strains studied were of sequence type X1-ST198, excepted the 98K strain which was X4-ST198. Among the thirty S. Kentucky strains, three overproduce AcrAB-TolC (3). All three strains presented different mutations in the ramR gene in comparison to the reference strain 98K. The three detected mutations (deletion of 91 bp, insertion of 1 bp or 4 bp) resulted in frame shift of the ramR gene. All other efflux regulatory genes were not mutated. As confirmed by complementation with a wild-type ramR gene, all three mutations were responsible for increased expression of ramA and acrAB. Increased expression of tolC and acrEF genes was observed in 2 out of the 3 strains. All three mutations were shown to increase two-fold the MICs of FQ and florfenicol in comparison to the ramR complemented derivatives and the reference strain. The two strains with a basal expression level of AcrAB-TolC presented any mutation in sequenced genes. The only strain presenting 3 substitutions in QRDR associated with 1 mutation in ramR upregulating AcrAB-TolC leaded to the higher level FQ resistance Conclusion. Various novel ramR mutations, responsible for increased efflux, were detected in the emerging epidemic serotype Kentucky ST198 clone. As previously reported in other FQ-resistant strains of serotypes Typhimurium or Schwarzengrund, ramR mutations seem to be sporadic (10 % in this study) and contribute only to a little extent to the decreased FQ susceptibilit