Genotyping of multidrug-resistant strains of Pseudomonas aeruginosa isolated from burn and wound infections by ERIC-PCR

ABSTRACT PURPOSE: To determine the genetic diversity of MDR P. aeruginosa strains isolated from burn and wound infections in Ahvaz, Iran, by ERIC-PCR. METHODS: From total 99 strains of P. aeruginosa defined as MDR by using drug susceptibility testing, 66 were subjected to ERIC-PCR analysis, comprises 53 strains isolated from burn infection, and 13 randomly selected strains from wound infection with higher resistance to combinations of more numbers of drugs. RESULTS: Eight clusters (I to VIII), and 50 single clones were generated for tested MDR isolates analyzed by ERIC-PCR. The high heterogeneity was observed among the isolates from burn infections including 16 isolates which were categorized in eight clusters and 37 single clones. The isolates in clusters II, III, VI, VIII showed 100% similarity. CONCLUSIONS: The high level of genotypic heterogeneity in P. aeruginosa strains demonstrated no genetic correlation between them. Extremely high drug resistance in isolates from burn, suggests that efficient control measures and proper antibiotic policy should be observed.

Detection of genomic mutations in katG, inhA and rpoB genes of Mycobacterium tuberculosis isolates using polymerase chain reaction and multiplex allele-specific polymerase chain reaction

OBJECTIVE: Isoniazid (INH) and rifampin (RIF) are the most effective first line antibiotics against Mycobacterium tuberculosis. Mutations in several genes determine resistance of M. tuberculosis to INH, with the most common gene target of katG, and resistance to RIF is due to mutation in rpoB gene. The aim of present study was to assess the mutations in the regions related to RIF and INH resistance. METHODS: We characterized 80 clinical isolates of confirmed M. tuberculosis to analyze the most commonly observed INH and RIF mutations. PCR analysis and sequencing were used to detect mutations related to RIF and INH resistance. The multiplex allele-specific-PCR (MAS-PCR) was performed as a comparative assay and for evaluation of this method. RESULTS: The sequencing of the 250-bp region of katG codon 315, revealed point mutations at 5 different codons in 13.7 percent of the M. tuberculosis isolates. The sequencing of the 270-bp central region of the rpoB gene revealed point mutations at 7 different codons in 12 (15 percent) of the M. tuberculosis isolates. The results obtained with MAS-PCR are in accordance with PCR-sequencing with high sensitivity and specificity for katG315, inhA15, and rpoB (531, 516, 526). CONCLUSION: The results of this study suggested that molecular techniques can be used as a rapid tool for the identification of drug resistance in clinical isolates of M. tuberculosis. Both DNA sequencing and MAS-PCR yielded high sensitivity for the detection of RIF and INH mutations and detecting multi-drug resistant tuberculosis cases.

Determination of antibiotic susceptibility pattern in Campylobacter jejuni and Campylobacter coli isolated from children with acute diarrhea.

Background: The emergence of antimicrobial resistant Campylobacter populations, which become an important consideration on the use of antimicrobial agents, especially in veterinary and human medicine, has become a serious concern worldwide. For monitoring the drug resistance, there is a need to develop reliable and reproducible laboratory techniques. There are several methods including disk diffusion, broth micro-dilution, agar dilution, and E-test to determine in-vitro susceptibility profiles of Campylobacter to a range of antimicrobial agents. Objectives: Study the Campylobacter jejuni and Campylobacter coli from children with diarrhea and determine the antimicrobial susceptibilities of the isolates to clinically relevant antimicrobials. Materials and methods: Two hundred twenty stool samples of children with diarrhea were cultured on Preston agar and the isolated campylobacter species were identified by further standard identification test. Susceptibility testing was carried out using Kirby-Bauer disk diffusion method and E-test. Results: Fourteen Campylobacter strains were isolated (6.36%), of which nine (64.3%) were identified as C. jejuni and five (35.7%) as C. coli. Using disk diffusion, all the campylobacter isolates were fully resistant to cephalothin, oxacillin, and ampicillin followed by ceftazidime with resistance rate of 71.42%. Gentamicin and ciprofloxacin were the most effective antibiotics against both isolated campylobacter species. According to E-test results, Campylobacter isolates demonstrated the greatest resistance to cephalothin (92.85%), oxacillin (92.85%), and ampicillin (78.57%). Conclusions: Our study reveals a high-level correlation between the E-test and agar disk diffusion method in evaluating the resistance of Campylobacter species to tested antimicrobial agents. This study also suggests disk diffusion is a reliable and cost effective technique for determining the prevalence of resistance among Campylobacter isolates.