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Analysis of integrons and associated gene cassettes in clinical isolates of multidrug resistant Pseudomonas aeruginosa from Southwest Nigeria.

ABSTRACT: BACKGROUND: Multidrug 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. METHODOLOGY: Plasmid 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 DH5? was performed to demonstrate transferability of integrons and gene cassettes. RESULT: Agarose 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. CONCLUSION: This 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.

SUBMITTER: Odumosu BT

PROVIDER: S-EPMC3842740 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Analysis of integrons and associated gene cassettes in clinical isolates of multidrug resistant Pseudomonas aeruginosa from Southwest Nigeria.

Odumosu Bamidele T BT Adeniyi Bolanle A BA Chandra Ram R

Annals of clinical microbiology and antimicrobials 20131021

<h4>Background</h4>Multidrug 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.<h4>Methodology</h4>Plasmid 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 is ...[more]

PMID: 24143920

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Project description:Purpose:The integrons, as the mobile exogenous elements, play a prominent role in the spreading of antimicrobial resistance genes from Pseudomonas aeruginosa clinical isolates to other bacteria. This study aimed to investigate the frequency of class 1 integrons andresistance gene cassettes carrying by them in clinical isolates as well as multidrug resistant P. aeruginosa. Materials and Methods:A total of 100 clinical isolates of P. aeruginosa were collected from 5 hospitals in Mazandaran province, north Iran. The antibiotic susceptibility pattern of the isolates was evaluated using the disk agar diffusion method. Genomic DNAs were extracted and then the presence of class 1 integrons was detected by the PCR test. All PCR products of the positive isolates were sequenced for the detection of resistance gene cassettes by the Sanger method. Results:Forty-one percent of the clinical isolates were multi-drug resistant. Also, 42% of the isolates were contained class 1 integron, and 61.9% of the integron positive isolates were detected as MDR. We detected 10 different gene cassettes sizing from 0.6 to 3.5 kb in the present study. The sequencing analysis of the internal variable regions of the class 1 integrons showed that the 0.75 kb gene cassette (aadB) was the most frequent resistance gene (54.76%) among all clinical isolates, as well as the MDR isolates. Other resistance genes detected in this study were included: aadA6-orfD (35.71%), aacA4-bla OXA-10 (21.42%), aadB-aacA4-bla OXA-10 (19.04%), bla OXA-10-aacA4-VIM1 (11.9%), aacA4-catB10 (7.14%), aacA5-aadA1-cmlA5 (7.14%), bla OXA31-aadA2 (4.76%), and aac(3)-Ic-aacA5-cmlA5 (4.76%). To the best of our knowledge, bla OXA-10 -aacA4-VIM1 cassette array is detected for the first time in this study. Conclusion:The treatment of infections caused by P. aeruginosa in this region of Iran is a major problem due to the high prevalence of class 1 integrons. It seems that the high prescription of beta-lactams and aminoglycosides for the treatment of these infections may be replaced by other combination therapy stewardships.

Project description:The multifactorial nature of Pseudomonas aeruginosa biofilm development and genomic variabilities implicates its resistance to conventional antimicrobials and virulence. Therefore, genetic determinants need to be extensively studied to block the early steps of biofilm or already formed biofilms. In this study, a total of 20 multidrug resistant (MDR) clinical P. aeruginosa isolates were evaluated for their biofilm forming abilities and related genes. Of the isolates tested, all of them showed surface attachment tendencies in nutrient limiting conditions, and classified as strong (SBF = 45%), moderate (MBF = 30%) and weak (WBF = 25%) biofilm formers. Complete genome sequencing of representative strong (DMC-27b), moderate (DMC-20c) and weak biofilm former (DMC-30b) isolates was performed. Analysis of biofilm related genes in the sequenced genomes revealed that, 80 of the 88 biofilm related genes possess 98-100% sequence identity to the reference PAO1 strain. Complete and partial sequence data of LecB proteins from tested isolates indicate that isolates containing PA14-like LecB sequences produced strong biofilms. All of the 7 pel operon protein coding genes in weak biofilm former isolate 30b showed significant nucleotide sequence variation with other tested isolates, and their corresponding proteins are 99% identical with the pel operon proteins of PA7. Bioinformatics analyses identified divergent sequence and structural features that separate PA7 like pel operon proteins from reference PAO1-like pel operon. Congo red and pellicle forming assays revealed that the sequence and structure variations may have interfered with the Pel production pathway and resulted in impaired Pel production in isolate 30b that has a PA7 like pel operon. Expression analysis also showed that both pelB and lecB genes were about 5 to 6 folds upregulated after 24 h in SBF 27b in comparison with WBF 30b. Our findings indicate significant genomic divergence in biofilm related genes of P. aeruginosa strains that affect their biofilm phenotypes.

Dataset Information

Analysis of integrons and associated gene cassettes in clinical isolates of multidrug resistant Pseudomonas aeruginosa from Southwest Nigeria.

Publications

Analysis of integrons and associated gene cassettes in clinical isolates of multidrug resistant Pseudomonas aeruginosa from Southwest Nigeria.

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