Concurrent parC and gyrA fluoroquinolone resistance mutations and associated strains in Mycoplasma genitalium in Queensland, Australia.
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ABSTRACT:
SUBMITTER: Ertl NG
PROVIDER: S-EPMC10832590 | biostudies-literature | 2024 Feb
REPOSITORIES: biostudies-literature
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Concurrent parC and gyrA fluoroquinolone resistance mutations and associated strains in Mycoplasma genitalium in Queensland, Australia.
Ertl Nicole G NG Anderson Taylah K TK Pardo Carolyn J CJ Maidment Toby I TI Murray Gerald L GL Bradshaw Catriona S CS Whiley David M DM Sweeney Emma L EL
The Journal of antimicrobial chemotherapy 20240201 2
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Project description:BackgroundPhenotypic 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.MethodsAntimicrobial 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.ResultsDouble 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.ConclusionSimultaneous 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.
| S-EPMC6454682 | biostudies-literature
Project description:BackgroundTraditional drug susceptibility testing cannot be performed in clinical laboratories due to the slow-growing characteristics of Mycoplasma genitalium when cultured in vitro. Sanger sequencing is the standard method for detecting drug resistance-associated mutations. It has been used in some laboratories to guide the choice of macrolide antibiotics for Mycoplasma genitalium infected patients. Furthermore, resistance to fluoroquinolone has become another emerging clinical challenge.ObjectiveSequencing analysis can detect unknown mutations, but it is time-consuming, requires professional analytical skills and the appropriate testing equipment. The main objective of this study was to establish a nested real-time PCR method for the simultaneous detection of 23S rRNA and parC genotypes in relation to the macrolide and fluoroquinolone resistance.Results105 MG-positive samples and 27 samples containing other pathogens were used for validation. The limit of the nested real-time PCR detection was 500 copies/reaction and there was no cross-reaction with Ureaplasma urealyticum, Mycoplasma hominis, Chlamydia trachomatis, Neisseria gonorrhoeae, Human papillomavirus, Herpes simplex virus, Candida albicans and Ureaplasma parvum, but the 23S rRNA assay cross-reacted with Mycoplasma pneumoniae. Compared with sequencing results, the sensitivity of 23S rRNA was 100% (95% CI; 93.3 -100), the specificity was 94.3% (95% CI; 79.4 - 99.0), the overall consistency was 98% (95% CI; 92.5 - 99.7) and kappa value was 0.96 (P < 0.001); the sensitivity of parC was 100% (95% CI; 93.4 - 100), the specificity was 89.7% (95% CI; 71.5 - 97.3) and the overall consistency was 96.9% (95% CI; 90.7 - 99.2) with a kappa value of 0.92 (P < 0.001).ConclusionsThe results of this sensitive and rapid alternative for identifying resistant genotypes of Mycoplasma genitalium are intuitive and easy to interpret, especially for mixed MG populations. Although the relevant 23S rRNA primers need further adjustment, this reliable method would provide an effective diagnostic tool for the selection of antibiotics in clinical practice.
| S-EPMC10623348 | biostudies-literature
Project description:AimTo investigate the resistance of Helicobacter pylori (H. pylori) to ciprofloxacin (CIP), levofloxacin (LVX) and moxifloxacin (MOX) in the Beijing area and to elucidate the resistance mechanisms.MethodsSeventy-nine H. pylori clinical strains, isolated from patients who had undergone upper gastrointestinal endoscopy in Peking University First Hospital from 2007 to 2009, were tested for their susceptibility to CIP, LVX and MOX using the E-test method. H. pylori strain 26695 was included in the susceptibility testing as a control strain. According to the minimal inhibitory concentration (MIC) values, a strain was classified as resistant to CIP, LVX or MOX when the MIC was > 1 microg/mL. We amplified by polymerase chain reaction (PCR) and sequenced the quinolone resistance-determining regions of the gyrA and gyrB genes from 29 quinolone-resistant and 16 quinolone-susceptible H. pylori strains selected at random.ResultsIn this study, the resistance rates of H. pylori to CIP, LVX or MOX were 55.7% (44/79), and the primary resistance rates were 26.6% (21/79). Patients with secondary resistance had received LVX in previous eradication treatments, but not MOX or CIP. Forty-five strains, including 29 CIP, LVX or MOX-resistant strains (MIC: 1.5-32 microg/mL) and 16 susceptible strains, were selected randomly from the 79 strains and used in PCR analysis. Among these 45 strains, 27 resistant strains had mutations in the gyrA gene, including 11 strains with mutations corresponding to Asp-91 (MIC: 2-32 microg/mL), one of which also had a mutation corresponding to Val-150, and 16 strains had mutations at Asn-87 (MIC: 4-32 microg/mL), three of which also had mutations corresponding to Arg-140 or Val-150. In addition, Arg-140, Val-150 or Ala-97 mutations were separately detected in three susceptible strains. Analysis of the gyrB gene showed that one strain of low resistance had a mutation corresponding to Ser-457 that coexisted with an Asp-91 mutation. There was a significant difference in the occurrence of mutations in the gyrA gene between CIP, LVX and MOX-resistant and -susceptible strains (P < 0.05), but 2 resistant strains were found to possess no quinolone resistance-determining region mutations.ConclusionResistance is primarily mediated through point mutations in gyrA. Whether other mechanisms are responsible for resistance in strains without mutations in the QRDR should be detected.
| S-EPMC2868221 | biostudies-literature
Project description:The nucleotide sequences of the quinolone resistance-determining regions (QRDRs) of the parC and gyrA genes from seven ciprofloxacin-resistant (Cpr) isolates of viridans group streptococci (two high-level Cpr Streptococcus oralis and five low-level Cpr Streptococcus mitis isolates) were determined and compared with those obtained from susceptible isolates. The nucleotide sequences of the QRDRs of the parE and gyrB genes from the five low-level Cpr S. mitis isolates and from the NCTC 12261 type strain were also analyzed. Four of these low-level Cpr isolates had changes affecting the subunits of DNA topoisomerase IV: three in Ser-79 (to Phe or Ile) of ParC and one in ParE at a position not previously described to be involved in quinolone resistance (Pro-424). One isolate did not show any mutation. The two high-level Cpr S. oralis isolates showed mutations affecting equivalent residue positions of ParC and GyrA, namely, Ser-79 to Phe and Ser-81 to Phe or Tyr, respectively. The parC mutations were able to transform Streptococcus pneumoniae to ciprofloxacin resistance, while the gyrA mutations transformed S. pneumoniae only when mutations in parC were present. These results suggest that DNA topoisomerase IV is a primary target of ciprofloxacin in viridans group streptococci, DNA gyrase being a secondary target.
| S-EPMC105945 | biostudies-literature
Project description:High-level quinolone resistance in Enterococcus faecium was associated with mutations in both gyrA and parC genes in 10 of 11 resistant strains. On low-level resistant strain without such mutations may instead possess an efflux mechanism or alterations in the other subunits of the gyrase or topoisomerase IV genes. These findings are similar to those for other gram-positive bacteria, such as Enterococcus faecalis.
| S-EPMC89232 | biostudies-literature
Project description:Escalating resistance to azithromycin and moxifloxacin is being reported for Mycoplasma genitalium in the Asia-Pacific region. Analyzing 140 infections, we found pretreatment fluoroquinolone-resistance mutations in parC (13.6%) and gyrA (5%). ParC S83 changes were associated with moxifloxacin failure. Combined macrolide/fluoroquinolone-resistance mutations were in 8.6% of specimens, for which recommended therapies would be ineffective.
| S-EPMC5403035 | biostudies-literature
Project description:Alterations in the target proteins of fluoroquinolones, especially in GyrA and ParC, are known to cause resistance. Here, we investigated environmental Escherichia communities to explore the possible link between the abundance of mutations, and the exposure to fluoroquinolones. Sediment samples were collected from a relatively pristine lake, up and downstream from a sewage treatment plant, and from several industrially polluted sites. The quinolone resistance-determining regions of gyrA and parC were analyzed using amplicon sequencing of metagenomic DNA. Five non-synonymous substitutions were present in all samples, and all of these mutations have been previously linked to fluoroquinolone resistance in Escherichia coli. In GyrA, substitutions S83L and D87N were on average detected at frequencies of 86 and 32%, respectively, and 31% of all amplicons encoded both substitutions. In ParC, substitutions S80I, E84G, and E84V were detected in 42, 0.9, and 6.0% of the amplicons, respectively, and 6.5% encoded double substitutions. There was no significant correlation between the level of fluoroquinolone pollution and the relative abundance of resistance mutations, with the exception of the most polluted site, which showed the highest abundance of said substitutions in both genes. Our results demonstrate that resistance mutations can be common in environmental Escherichia, even in the absence of a fluoroquinolone selective pressure.
| S-EPMC4673309 | biostudies-literature
Project description:We have analyzed genetically three clinical isolates (3180, 3870, and 1244) of Streptococcus pneumoniae with high-level ciprofloxacin resistance. Isolates 3180 and 3870 were atypical because of their insolubility in deoxycholate. However, they hybridized specifically with pneumococcal autolysin and pneumolysin gene probes and have typical pneumococcal atpC and atpA gene sequences. Analysis of the complete sequences of the parC and gyrA genes revealed total variations of 8 and 8.7% (isolate 3180) and 7.4 and 3.6% (isolate 3870), respectively, compared to the wild-type strain R6 sequence. The variations observed between the sequences of R6 and isolate 1244 were less than 0.9%. The structure of the gyrA and parC genes from isolates 3180 and 3870 was organized in sequence blocks that show different levels of divergence, suggesting a pattern of recombination. These results are evidence for recombination at the fluoroquinolone target genes in clinical isolates of S. pneumoniae. The genetically related viridans group streptococci could act as a reservoir for fluoroquinolone resistance genes.
| S-EPMC89780 | biostudies-literature
Project description:BackgroundRiemerella anatipestifer is one of the most serious infectious disease-causing pathogens in the duck industry. Drug administration is an important method for prevention and treatment of infection in duck production, leading to widespread drug resistance in R. anatipestifer.MethodsFor a total of 162 isolates of R. anatipestifer, the MICs were determined for a quinolone antimicrobial agent, namely, nalidixic acid, and three fluoroquinolones, namely, ciprofloxacin, enrofloxacin and ofloxacin. The gyrA, parC, and parE gene fragments were amplified by PCR to identify the mutation sites in these strains. Site-directed mutants with mutations that were detected at a high frequency in vivo were constructed (hereafter referred to as site-directed in vivo mutants), and the MICs of these four drugs for these strains were determined.ResultsIn total, 100, 97.8, 99.3 and 97.8% of the 137 R. anatipestifer strains isolated between 2013 and 2018 showed resistance to nalidixic acid, ciprofloxacin, enrofloxacin, and ofloxacin, respectively. The high-frequency mutation sites were detected in a total of 162 R. anatipestifer strains, such as Ser83Ile and Ser83Arg, which are two types of substitution mutations of amino acid 83 in GyrA; Val799Ala and Ile811Val in ParC; and Val357Ile, His358Tyr, and Arg541Lys in ParE. MIC analysis results for the site-directed in vivo mutants showed that the strains with only the Ser83Ile mutation in GyrA exhibited an 8-16-fold increase in MIC values, and all mutants showed resistance to ampicillin and ceftiofur.ConclusionsThe resistance of R. anatipestifer to quinolone agents is a serious problem. Amino acid 83 in GyrA is the major target mutation site for the fluoroquinolone resistance mechanism of R. anatipestifer.
| S-EPMC6892153 | biostudies-literature
Project description:The DNA genome of Mycoplasma genitalium currently represents the smallest of all known human bacterial pathogens. Despite their clinical importance in sexually transmitted infection and relevance as model bacterial pathogens, genomic diversity among M. genitalium strains worldwide is unknown. Herein we present the complete draft genome sequences of four geographically diverse strains of M. genitalium.
| S-EPMC3486117 | biostudies-literature