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Molecular characterization of selected multidrug resistant Pseudomonas from water distribution systems in southwestern Nigeria.

ABSTRACT: Persistence of antibiotic resistant bacteria, including multidrug resistant (MDR) pseudomonads, is an important environmental health problem associated with drinking water distribution systems (DWDS) worldwide. There is paucity of data on the molecular characteristics of antibiotic resistance genes and their mode of transfer among pseudomonads from DWDS located in resource-challenged areas such as southwestern Nigeria.MDR pseudomonads (n = 22) were selected from a panel of 296 different strains that were isolated from treated and untreated water in six DWDS located across southwest Nigeria. Primarily, the isolated pseudomonads strains were identified by 16S rDNA sequencing and antibiotic-resistance testing was completed using agar breakpoints assays. The final panel of strains of resistant to more than three classes of antibiotics (i.e. MDR), were further characterized by PCR genotyping, Sanger sequencing, and plasmid profiling.Pseudomonad resistance to gentamicin and streptomycin ranged from 22.7 to 54.6 % while resistance to tetracycline, ceftiofur and sulphamethoxazole ranged from 40.9 to 77.3 %. The most commonly detected antibiotic resistance genes were tet(A) (31.8 % of isolates), sul1 (31.8 %), bla TEM (40.9 %) and aph(3?) (c) (36.4 %). Class 1 integron sequences were evident in 27.3 % of isolates and they harbored genes encoding resistance to aminoglycosides (aadA2, aadA1), trimethoprim (dfrA15, dfr7) and sulphonamide (sul1) while the plasmid ranged between 22 and 130 kb.Pseudomonas spp, isolated from these DWDS possess resistance genes and factors that are of public and environmental health significance. Therefore, has the potential of contributing to the global scourge of resistance genes transfer in human, animals and environments, thereby, useful in the epidemiology of antimicrobial resistance.

SUBMITTER: Adesoji AT

PROVIDER: S-EPMC4557310 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Molecular characterization of selected multidrug resistant Pseudomonas from water distribution systems in southwestern Nigeria.

Adesoji Ayodele T AT Ogunjobi Adeniyi A AA Olatoye Isaac O IO

Annals of clinical microbiology and antimicrobials 20150902

<h4>Background</h4>Persistence of antibiotic resistant bacteria, including multidrug resistant (MDR) pseudomonads, is an important environmental health problem associated with drinking water distribution systems (DWDS) worldwide. There is paucity of data on the molecular characteristics of antibiotic resistance genes and their mode of transfer among pseudomonads from DWDS located in resource-challenged areas such as southwestern Nigeria.<h4>Methods</h4>MDR pseudomonads (n = 22) were selected fro ...[more]

PMID: 26328550

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Project description:Antibiotic resistance genes [ARGs] in aquatic systems have drawn increasing attention they could be transferred horizontally to pathogenic bacteria. Water treatment plants (WTPs) are intended to provide quality and widely available water to the local populace they serve. However, WTPs in developing countries may not be dependable for clean water and they could serve as points of dissemination for antibiotic resistant bacteria. Only a few studies have investigated the occurrence of ARGs among these bacteria including tetracycline resistance genes in water distribution systems in Nigeria.Multi-drug resistant (MDR) bacteria, including resistance to tetracycline, were isolated from treated and untreated water distribution systems in southwest Nigeria. MDR bacteria were resistant to >3 classes of antibiotics based on break-point assays. Isolates were characterized using partial 16S rDNA sequencing and PCR assays for six tetracycline-resistance genes. Plasmid conjugation was evaluated using E. coli strain DH5? as the recipient strain.Out of the 105 bacteria, 85 (81 %) and 20 (19 %) were Gram- negative or Gram- positive, respectively. Twenty-nine isolates carried at least one of the targeted tetracycline resistance genes including strains of Aeromonas, Alcaligenes, Bacillus, Klebsiella, Leucobacter, Morganella, Proteus and a sequence matching a previously uncultured bacteria. Tet(A) was the most prevalent (16/29) followed by tet(E) (4/29) and tet30 (2/29). Tet(O) was not detected in any of the isolates. Tet(A) was mostly found with Alcaligenes strains (9/10) and a combination of more than one resistance gene was observed only amongst Alcaligenes strains [tet(A)?+?tet30 (2/10), tet(A)?+?tet(E) (3/10), tet(E)?+?tet(M) (1/10), tet(E)?+?tet30 (1/10)]. Tet(A) was transferred by conjugation for five Alcaligenes and two E. coli isolates.This study found a high prevalence of plasmid-encoded tet(A) among Alcaligenes isolates, raising the possibility that this strain could shuttle resistance plasmids to pathogenic bacteria.

Project description:UnlabelledPseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates.ImportanceP. aeruginosa is both an antibiotic-refractory pathogen and an important model system for type I CRISPR-Cas bacterial immune systems. By combining the genome sequences of 672 newly and previously sequenced genomes, we were able to provide a global view of the phylogenetic distribution, conservation, and potential targets of these systems. This analysis identified a new and putatively mobile P. aeruginosa CRISPR-Cas subtype, characterized the diverse distribution of known CRISPR-inhibiting genes, and provided a potential new use for CRISPR spacer libraries in accessory genome analysis. Our data demonstrated the importance of CRISPR-Cas systems in modulating the accessory genomes of globally distributed strains while also providing substantial data for subsequent genomic and experimental studies in multiple fields. Understanding why certain genotypes of P. aeruginosa are clinically prevalent and adept at horizontally acquiring virulence and antibiotic resistance elements is of major clinical and economic importance.

Project description:BackgroundMultidrug resistant Pseudomonas aeruginosa harbours integrons and other mobile genetic elements such as plasmids and transposons, which easily disseminate antibiotic resistance genes among clinical strains of P. aeruginosa.MethodologyPlasmid extraction of 54 clinical isolates of P. aeruginosa was carried out by alkaline lysis method; and plasmid size estimation was done by using E. coli V517 standard plasmid marker. Fifty-four clinical strains of P. aeruginosa were isolated from 5 hospitals in 3 Southwestern states of Nigeria between March and September 2010. Plasmid extraction of isolates was carried out by alkaline lysis method; and plasmid size estimation was done by using E. coli V517 standard plasmid marker. PCR amplification for the 3 classes of resistance integrons, and gene cassette characterization were carried out using specific primers and by sequencing of PCR products. Conjugal mating of the integron positive P. aeruginosa strains with E. coli DH5α was performed to demonstrate transferability of integrons and gene cassettes.ResultAgarose gel electrophoresis of plasmid DNA revealed that all the 54 P. aeruginosa harboured 1-4 plasmids with sizes ranging from 2.2 - >58 kb. Class 1 integron was identified in 31 (57%) strains; but none of them carried class 2 and class 3 integrons. High prevalence of aadA gene conferring resistance to streptomycin/spectinomycin was detected in the strains positive for class 1 integron. Sequencing of the 1.6 kb and 1.2 kb amplified band of gene cassettes revealed the presence of aadA6-orfD and aadA13 respectively.ConclusionThis study demonstrates the presence of plasmids and integrons harbouring resistance gene cassettes, which may collectively constitute an efficient system for dissemination of resistance genes in P. aeruginosa. Disturbingly, the rapid and unabated spread of class 1 integron-associated multidrug resistant P. aeruginosa in Southwest Nigeria may greatly hamper successful treatment of infections caused by such strains. This necessitates the establishment of functional antimicrobial resistance surveillance programmes in Nigeria.

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Molecular characterization of selected multidrug resistant Pseudomonas from water distribution systems in southwestern Nigeria.

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Molecular characterization of selected multidrug resistant Pseudomonas from water distribution systems in southwestern Nigeria.

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