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The role of gyrA and parC mutations in fluoroquinolones-resistant Pseudomonas aeruginosa isolates from Iran.

ABSTRACT: The aim of this study was to examine mutations in the quinolone-resistance-determining region (QRDR) of gyrA and parC genes in Pseudomonas aeruginosa isolates. A total of 100 clinical P. aeruginosa isolates were collected from different university-affiliated hospitals in Tabriz, Iran. Minimum inhibitory concentrations (MICs) of ciprofloxacin and levofloxacin were evaluated by agar dilution assay. DNA sequences of the QRDR of gyrA and parC were determined by the dideoxy chain termination method. Of the total 100 isolates, 64 were resistant to ciprofloxacin. No amino acid alterations were detected in gyrA or parC genes of the ciprofloxacin susceptible or ciprofloxacin intermediate isolates. Thr-83 ? Ile substitution in gyrA was found in all 64 ciprofloxacin resistant isolates. Forty-four (68.75%) of them had additional substitution in parC. A correlation was found between the number of the amino acid alterations in the QRDR of gyrA and parC and the level of ciprofloxacin and levofloxacin resistance of the P. aeruginosa isolates. Ala-88 ? Pro alteration in parC was generally found in high level ciprofloxacin resistant isolates, which were suggested to be responsible for fluoroquinolone resistance. These findings showed that in P. aeruginosa, gyrA was the primary target for fluoroquinolone and additional mutation in parC led to highly resistant isolates.

SUBMITTER: Nouri R

PROVIDER: S-EPMC5052375 | biostudies-literature | 2016 Oct - Dec

REPOSITORIES: biostudies-literature

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The role of gyrA and parC mutations in fluoroquinolones-resistant Pseudomonas aeruginosa isolates from Iran.

Nouri Roghayeh R Ahangarzadeh Rezaee Mohammad M Hasani Alka A Aghazadeh Mohammad M Asgharzadeh Mohammad M

Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] 20160726 4

The aim of this study was to examine mutations in the quinolone-resistance-determining region (QRDR) of gyrA and parC genes in Pseudomonas aeruginosa isolates. A total of 100 clinical P. aeruginosa isolates were collected from different university-affiliated hospitals in Tabriz, Iran. Minimum inhibitory concentrations (MICs) of ciprofloxacin and levofloxacin were evaluated by agar dilution assay. DNA sequences of the QRDR of gyrA and parC were determined by the dideoxy chain termination method. ...[more]

PMID: 27522930

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Project description:BACKGROUND:Phenotypic fluoroquinolone resistance was first reported in Western Kenya in 2009 and later in Coastal Kenya and Nairobi. Until recently gonococcal fluoroquinolone resistance mechanisms in Kenya had not been elucidated. The aim of this paper is to analyze mutations in both gyrA and parC responsible for elevated fluoroquinolone Minimum Inhibitory Concentrations (MICs) in Neisseria gonorrhoeae (GC) isolated from heterosexual individuals from different locations in Kenya between 2013 and 2017. METHODS:Antimicrobial Susceptibility Tests were done on 84 GC in an ongoing Sexually Transmitted Infections (STI) surveillance program. Of the 84 isolates, 22 resistant to two or more classes of antimicrobials were chosen for analysis. Antimicrobial susceptibility tests were done using E-test (BioMerieux) and the results were interpreted with reference to European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards. The isolates were sub-cultured, and whole genomes were sequenced using Illumina platform. Reads were assembled de novo using Velvet, and mutations in the GC Quinolone Resistant Determining Regions identified using Bioedit sequence alignment editor. Single Nucleotide Polymorphism based phylogeny was inferred using RaxML. RESULTS:Double GyrA amino acid substitutions; S91F and D95G/D95A were identified in 20 isolates. Of these 20 isolates, 14 had an additional E91G ParC substitution and significantly higher ciprofloxacin MICs (p?=?0.0044*). On the contrary, norfloxacin MICs of isolates expressing both GyrA and ParC QRDR amino acid changes were not significantly high (p?=?0.82) compared to MICs of isolates expressing GyrA substitutions alone. No single GyrA substitution was found in the analyzed isolates, and no isolate contained a ParC substitution without the simultaneous presence of double GyrA substitutions. Maximum likelihood tree clustered the 22 isolates into 6 distinct clades. CONCLUSION:Simultaneous presence of amino acid substitutions in ParC and GyrA has been reported to increase gonococcal fluoroquinolone resistance from different regions in the world. Our findings indicate that GyrA S91F, D95G/D95A and ParC E91G amino acid substitutions mediate high fluoroquinolone resistance in the analyzed Kenyan GC.

Project description:Resistance to fluoroquinolone (FQ) antibiotics in Streptococcus pneumoniae has been attributed primarily to specific mutations in the genes for DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). Resistance to some FQs can result from a single mutation in one or more of the genes encoding these essential enzymes. A group of 160 clinical isolates of pneumococci was examined in this study, including 36 ofloxacin-resistant isolates (MICs, > or = 8 micrograms/ml) recovered from patients in North America, France, and Belgium. The susceptibilities of all isolates to clinafloxacin, grepafloxacin, levofloxacin, sparfloxacin, and trovafloxacin were examined by the National Committee for Clinical Laboratory Standards reference broth microdilution and disk diffusion susceptibility testing methods. Among the ofloxacin-resistant strains, 32 of 36 were also categorized as resistant to levofloxacin, 35 were resistant to sparfloxacin, 29 were resistant to grepafloxacin, and 19 were resistant to trovafloxacin. In vitro susceptibility to clinafloxacin appeared to be least affected by resistance to the other FQs. Eight isolates with high- and low-level resistance to the newer FQs were selected for DNA sequence analysis of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE. The DNA and the inferred amino acid sequences of the resistant strains were compared with the analogous sequences of reference strain S. pneumoniae ATCC 49619 and FQ-susceptible laboratory strain R6. Reduced susceptibilities to grepafloxacin and sparfloxacin (MICs, 1 to 2 micrograms/ml) and trovafloxacin (MICs, 0.5 to 1 microgram/ml) were associated with either a mutation in parC that led to a single amino acid substitution (Ser-79 to Phe or Tyr) or double mutations that involved the genes for both GyrA (Ser-81 to Phe) and ParE (Asp-435 to Asn). High-level resistance to all of the compounds except clinafloxacin was associated with two or more amino acid substitutions involving both GyrA (Ser-81 to Phe) and ParC (Ser-79 to Phe or Ser-80 to Pro and Asp-83 to Tyr). No mutations were observed in the gyrB sequences of resistant strains. These data indicate that mutations in pneumococcal gyrA, parC, and parE genes all contribute to decreased susceptibility to the newer FQs, and genetic analysis of the QRDR of a single gene, either gyrA or parC, is not predictive of pneumococcal resistance to these agents.

Project description:BACKGROUND:Fluoroquinolone agents, such as enrofloxacin and marbofloxacin, are commonly used for pseudomonal infection in veterinary medicine. However, the rate of resistance to fluoroquinolones is rapidly increasing, according to multiple studies in various countries. Point mutations in the quinolone resistance-determining region (QRDR) are closely related to the increased fluoroquinolone resistance of Pseudomonas aeruginosa. The aim of this study was to investigate current antimicrobial susceptibility and fluoroquinolone resistance in P. aeruginosa strains isolated from dogs. The presence of point mutations in the QRDR was confirmed by gyrA and parC polymerase chain reaction and nucleotide sequencing analysis. RESULTS:A total of 84 nonduplicated P. aeruginosa strains were obtained from 228 healthy dogs (healthy group) and 260 dogs with clinical signs (infected group). Among these isolates, 38 strains from the healthy group were detected in several sample types, whereas 46 strains from the infected group were obtained mostly from dogs' ears with otitis externa (41/260, 15.8%). All strains were resistant to nalidixic acid, while some were also resistant to enrofloxacin (23/84, 27.4%), marbofloxacin (17/84, 20.2%), levofloxacin (12/84, 14.3%), or ciprofloxacin (11/84, 13.1%). Enrofloxacin resistance was significantly higher in strains from the infected group than in those from the healthy group (p?<?0.05). Among the 23 fluoroquinolone-resistant strains, 8 and 4 different mutations were detected in the gyrA and parC genes, respectively. Mutations in gyrA were significantly common in the infected group (p?<?0.05). Hotspots for the gyrA and parC mutations were Thr83 (34.8%, 8/23) and Pro116 (91.3%, 21/23), respectively. Double and triple mutations were also found in 5 of the strains. CONCLUSION:Novel mutations in the gyrA and parC genes were first found in P. aeruginosa isolated from companion dogs in South Korea. These findings suggest that it is important to encourage prudent use of fluoroquinolone antibiotics in canine pseudomonal infection treatment.

Project description:IntroductionPseudomonas aeruginosa has many mechanisms of resistance to fluoroquinolones. The main mechanism is to change the effect of two enzymes that open the DNA helix - the enzyme DNA gyrase (gyrA) and the topoisomerase IV (parC). In addition, mutations that render the MexAB-oprM pump (mexR) dysfunctional, leading to its overexpression, also enhance resistance to fluoroquinolones. In this study, we aim to detect point mutations of gyrA, parC, and mexR genes that are predicted to be associated with fluoroquinolone resistance in 141 fluoroquinolone-resistant clinical isolates of P. aeruginosa isolated in Vietnam during 2013-2016.MethodsWe tested minimum inhibitory concentrations (MICs) of fluoroquinolone antibiotics in 141 clinical isolates of P. aeruginosa using the VITEK 2 Compact System, followed by PCR assay, to detect and clone the fluoroquinolone resistance-determining region (FRDR) of gyrA, parC, and mexR. Point mutations were analyzed through Sanger sequencing, and the correlation between genetic mutations and phenotypic resistance of 141 clinical isolates was undertaken.ResultsFluoroquinolone-resistant substitution mutations such as Ile for Thr83 and Met for Thr133 in gyrA, Leu for Ser87 in parC, and Val for Glu126 in the repressor of mexR were mainly detected. Comparative analytical data indicated that amino acid alterations within the gyrA and parC genes are highly associated with resistance to ciprofloxacin (CIP) and levofloxacin (LEV) in the isolates, whereas alterations in the efflux regulatory mexR gene are not highly consistent with resistance in these isolates. Moreover, fluoroquinolone-resistant clinical isolates of P. aeruginosa were mainly isolated from pus and sputum specimens.ConclusionIn clinical isolates of P. aeruginosa, a high correlation was observed between MICs of CIP and LEV and alterations in gyrA and parC genes. However, mutations occurring in mexR did not highly correlate with the antibiotic resistance of the bacterium.

Dataset Information

The role of gyrA and parC mutations in fluoroquinolones-resistant Pseudomonas aeruginosa isolates from Iran.

Publications

The role of gyrA and parC mutations in fluoroquinolones-resistant Pseudomonas aeruginosa isolates from Iran.

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