Amino Acid Substitutions In ParC Research Articles

Macrolide and fluoroquinolone associated mutations in Mycoplasma genitalium in a retrospective study of male and female patients seeking care at a STI Clinic in Guangzhou, China, 2016-2018

BackgroundAntimicrobial resistance in M. genitalium is a growing clinical problem. We investigated the mutations associated with macrolide and fluoroquinolone resistance, two commonly used medical regimens for treatment in China. Our aim is to analyze the prevalence and diversity of mutations among M. genitalium-positive clinical specimens in Guangzhou, south China.MethodsA total of 154 stored M. genitalium positive specimens from men and women attending a STI clinic were tested for macrolide and fluoroquinolone mutations. M. genitalium was detected via TaqMan MGB real-time PCR. Mutations associated with macrolide resistance were detected using primers targeting region V of the 23S rRNA gene. Fluoroquinolone resistant mutations were screened via primers targeting topoisomerase IV (parC) and DNA gyrase (gyrA).Results98.7% (152/154), 95.5% (147/154) and 90.3% (139/154) of M. genitalium positive samples produced sufficient amplicon for detecting resistance mutations in 23S rRNA, gyrA and parC genes, respectively. 66.4% (101/152), 0.7% (1/147) and 77.7% (108/139) samples manifested mutations in 23S rRNA, gyrA and parC genes, respectively. A2072G (59/101, 58.4%) and S83I (79/108, 73.1%) were highly predominating in 23S rRNA and parC genes, respectively. Two samples had amino acid substitutions in gyrA (M95I and A96T, respectively). Two samples had two amino acid substitutions in parC (S83I + D87Y). 48.6% (67/138) of samples harbored both macrolide and fluoroquinolone resistance-associated mutations. The most common combination of mutations was A2072G (23S rRNA) and S83I (parC) (40/67, 59.7%). One sample had three amino acid changes in 23S rRNA, gyrA and parC genes (A2072G + A96T + S83I).ConclusionsThe high antimicrobial resistance rate of M. genitalium in Guangzhou is a very worrying problem and suggests that antimicrobial resistance testing and the development of new antibiotic regimens are crucially needed.

GyrA and parC mutations in fluoroquinolone-resistant Neisseria gonorrhoeae isolates from Kenya

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.

Ciprofloxacin and nalidixic acid resistance of Salmonella spp. isolated from retail food in Poland

Distribution of amino acid substitutions in the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, parE and determinants of plasmid-mediated quinolone resistance (PMQR) were investigated among quinolone-resistant Salmonella spp. strains isolated from retail food in Poland in the years 2008–2013.Ten different amino acid substitutions were identified in QRDRs. Five different amino acid substitutions were identified in gyrA: Ser83Tyr, Ser83Phe, Asp87Tyr, Asp87Asn, Asp87Gly, two amino acid substitutions in parC: Thr57Ser, Ser80Ile and in parE: Leu445Phe, Arg511Ser. One substitution — Ser464Phe — was detected within gyrB. In gyrA a single substitution (Ser83Tyr) was identified the most frequently – 34.8% (63/181). Second most frequently identified variant (21.0%–38/181) was a co-existence of two single substitutions in gyrA: Ser83Tyr and parC: Thr57Ser. In four isolates co-existed three substitutions in three different genes: gyrA: Ser83Tyr + parC: Thr57Ser + parE: Leu445Phe (two isolates), gyrA: Ser83Phe + parC: Thr57Ser + parE: Leu445Phe, and gyrA: Ser83Tyr + parC: Thr57Ser + parE: Arg511Ser. In the two isolates four substitutions were identified – in gyrA: Ser83Phe + Asp87Tyr and in parC: Thr57Ser + Ser80Ile.Among resistant isolates, MIC values varied between 32 and 2048 mg/L (nalidixic acid) and between 0.125 and 16 mg/L (ciprofloxacin).MIC values of two isolates harboring qnrS1without any substitutions were 32 mg/L (NA) and 0.5–1.0 mg/L (CIP). The highest MIC values for NA and CIP were observed in two isolates of Salmonella spp. carrying double substitutions in gyrA: Ser83Phe + Asp87Tyr and parC: Thr57Ser + Ser80Ile. MIC value for NA was 2048 mg/L while for CIP – 16 mg/L.

Molecular Characterization of Italian Isolates of Fluoroquinolone-Resistant Streptococcus agalactiae and Relationships with Chloramphenicol Resistance.

A total number of 368 clinical isolates of Streptococcus agalactiae (group B Streptococcus, GBS) were collected in 2010-2016 from three hospitals in a region of central Italy. Fluoroquinolone (FQ)-resistant isolates were selected using levofloxacin. Levofloxacin-resistant (LR) strains (11/368, 2.99%) were characterized for several features, and their FQ resistance was analyzed phenotypically and genotypically using seven additional FQs. Their gyrA and parC quinolone resistance-determining regions were sequenced. Of the 11 LR isolates, 10 showed high-level and 1 low-level resistance. The former isolates exhibited higher minimal inhibitory concentrations also of the other FQs and all shared one amino acid substitution in ParC (Ser79Phe) and one in GyrA (Ser81Leu); only Ser79Phe in ParC was detected in the low-level LR isolate. The 11 LR strains exhibited distinctive relationships between their susceptibilities to non-FQ antibiotics and typing data. Remarkably, despite the very rare occurrence of chloramphenicol resistance in S. agalactiae, no <4 of the 11 LR isolates were chloramphenicol-resistant. Studies of GBS resistance to FQs in Europe remain scarce, notwithstanding the emergence of multidrug-resistant isolates. The incidence of LR GBS isolates is still limited in Italy, consistent with the moderate (though growing) rates reported in Europe, and much lower than the very high rates reported in East Asia. The intriguing relationships between FQ and chloramphenicol resistance deserve further investigation.

Antibiotic Susceptibility and Sequence Type Distribution of Ureaplasma Species Isolated from Genital Samples in Switzerland.

Antibiotic resistance in Ureaplasma urealyticum/Ureaplasma parvum and Mycoplasma hominis is an issue of increasing importance. However, data regarding the susceptibility and, more importantly, the clonality of these organisms are limited. We analyzed 140 genital samples obtained in Bern, Switzerland, in 2014. Identification and antimicrobial susceptibility tests were performed by using the Mycoplasma IST 2 kit and sequencing of 16S rRNA genes. MICs for ciprofloxacin and azithromycin were obtained in broth microdilution assays. Clonality was analyzed with PCR-based subtyping and multilocus sequence typing (MLST), whereas quinolone resistance and macrolide resistance were studied by sequencing gyrA, gyrB, parC, and parE genes, as well as 23S rRNA genes and genes encoding L4/L22 ribosomal proteins. A total of 103 samples were confirmed as positive for U. urealyticum/U. parvum, whereas 21 were positive for both U. urealyticum/U. parvum and M. hominis. According to the IST 2 kit, the rates of nonsusceptibility were highest for ciprofloxacin (19.4%) and ofloxacin (9.7%), whereas low rates were observed for clarithromycin (4.9%), erythromycin (1.9%), and azithromycin (1%). However, inconsistent results between microdilution and IST 2 kit assays were recorded. Various sequence types (STs) observed previously in China (ST1, ST2, ST4, ST9, ST22, and ST47), as well as eight novel lineages, were detected. Only some quinolone-resistant isolates had amino acid substitutions in ParC (Ser83Leu in U. parvum of serovar 6) and ParE (Val417Thr in U. parvum of serovar 1 and the novel Thr417Val substitution in U. urealyticum). Isolates with mutations in 23S rRNA or substitutions in L4/L22 were not detected. This is the first study analyzing the susceptibility of U. urealyticum/U. parvum isolates in Switzerland and the clonality outside China. Resistance rates were low compared to those in other countries. We hypothesize that some hyperepidemic STs spread worldwide via sexual intercourse. Large combined microbiological and clinical studies should address this important issue.

Amino Acid Substitutions in GyrA and ParC are Associated with Fluoroquinolone Resistance in &lt;i&gt;Mycoplasma bovis&lt;/i&gt; Isolates from Japanese Dairy Calves

We investigated the contribution of quinolone resistance-determining region (QRDR) mutations to fluoroquinolone (enrofloxacin, orbifloxacin and danofloxacin) susceptibility in 58 Mycoplasma bovis isolates from dairy cattle in Japan. Fluoroquinolone non-resistant isolates (fluoroquinolone-MICs≤2 μg/ml) possessed no QRDR mutations (19 isolates) or Ser83Leu in GyrA (32 isolates). Fluoroquinolone-resistant isolates (fluoroquinolone-MICs≥4 μg/ml) possessed Ser83Leu in GyrA and Ser81Pro in ParC (3 isolates) or Ser83Phe in GyrA and Ser80Ile in ParC (4 isolates). Laboratory-derived fluoroquinolone-resistant mutants selected from 2 isolates (possessing Ser83Leu in GyrA) had an amino acid substitution in ParC at the same position (Ser80Ile or Ser81Tyr) as fluoroquinolone-resistant isolates, suggesting a concurrent amino acid substitution in ParC (Ser80 or Ser81) is important in fluoroquinolone resistance in M. bovis isolates.

Prevalence and Mechanism of Antimicrobial Resistance in Staphylococcus aureus Isolates from Diseased Cattle, Swine and Chickens in Japan

Antimicrobial administration is essential for the control and treatment of diseases in animals, but the emergence and prevalence of antimicrobial-resistant Staphylococcus aureus is a significant concern during animal production. Here we investigated the antimicrobial susceptibility of S. aureus from diseased food-producing animals and molecularly characterized the methicillin-resistant and fluoroquinolone-resistant isolates. A total of 290 S. aureus isolates obtained from cattle (n=246), swine (n=16), and chickens (n=28) between 2003 and 2009 were examined for antimicrobial susceptibility against 9 antimicrobials using an agar dilution method. Resistance to penicillin (PC) was most frequently found (24.8%), followed by oxytetracycline (OTC, 10.0%), dihydrostreptomycin (4.1%), erythromycin (EM, 3.1%), enrofloxacin (ERFX, 2.1%), and kanamycin (1.7%). The PC resistance rate was significantly higher in swine than in cattle (P<0.01) and chickens (P<0.01). The resistance rates to OTC, EM and ERFX were significantly higher in swine and chickens than in cattle (P<0.05). Methicillin-resistant S. aureus (MRSA) was recovered from milk derived from a cow with mastitis in 2003; sequence type 8, SCCmec type IV and spa type t024. In the six ERFX-resistant strains isolated after 2003, amino acid substitutions in ParC with/without GyrA were detected. As the prevalence of MRSA and FQ-resistant S. aureus in the animals should be noticed, continuous monitoring is necessary to control resistance to clinically important antimicrobials in S. aureus from food-producing animals.

Rapid Screening of Fluoroquinolone Resistance Determinants in Streptococcus pneumoniae by PCR-Restriction Fragment Length Polymorphism and Single-Strand Conformational Polymorphism

A rapid method, using PCR-restriction fragment length and single-strand conformation polymorphism (SSCP), was applied to screen for mutations of the fluoroquinolone resistance determinants in Streptococcus pneumoniae. One hundred nonduplicate Streptococcus pneumoniae isolates with ciprofloxacin MICs of > or = 4.0 microg/ml from the Prince of Wales Hospital, Hong Kong, years 2000 to 2003, were examined. For each isolate, PCR amplicons of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE genes were digested with AluI, HinfI, Sau3AI, and MspI, respectively, and analyzed by SSCP. Each SSCP pattern was given a number, and each isolate obtained a four-digit code, e.g., 1111, that represented the SSCP profile. The SSCP patterns were correlated to mutations characterized from sequence analyses of PCR amplicons. The most common SSCP profile obtained was no. 5232 (40%), which included strains with two amino acid substitutions in the ParC (Lys-137-Asn) and ParE (Ile-460-Val) genes, followed by the SSCP profile 5223 (17%), which included strains with amino acid substitutions in the ParE (Ile-460-Val) gene only. Ten isolates (10%) with amino acid substitutions at GyrA and ParE (+/-ParC) genes were resistant to levofloxacin with a MIC of > or = 16 microg/ml. Other SSCP profiles were unique in distinguishing the common amino acid substitutions in GyrA (Ser-81-Phe) and ParC (Lys-137-Asn, Ser-79-Phe plus Lys-137-Asn, Asp-83-Asn plus Lys-137-Asn, Ser-79-Phe, and Glu-96-Asp). SSCP analysis of restricted fragments generated patterns that were highly discriminative for mutations present in the QRDRs of gyrA, gyrB, parC, and parE. This method provides a database of high resolution profiles on these mutations and allows rapid screening for new mutations of the fluoroquinolone resistance genes.

Are fluoroquinolone-susceptible isolates of Streptococcus pneumoniae really susceptible? A comparison of resistance mechanisms in Canadian isolates from 1997 and 2003

To assess the prevalence of efflux and amino acid substitutions in ParC and GyrA in Canadian clinical isolates of fluoroquinolone-susceptible Streptococcus pneumoniae with levofloxacin MICs of 1 mg/L collected before the introduction of the respiratory fluoroquinolones (1995-1997) and after 7 years of use (2003). Quinolone resistance determining regions of parC and gyrA were sequenced for 111 clinical isolates collected from 1995 to 1997 and 665 isolates collected in 2003. Efflux was assessed using a reserpine agar dilution method. No isolates exhibited efflux. No significant increase in isolates harbouring amino acid substitutions was observed over time (0.9% in 1995-1997 to 2.1% in 2003, P = 0.32). However, the proportion of isolates with a ciprofloxacin MIC = 2 mg/L and a levofloxacin MIC = 1 mg/L versus ciprofloxacin MIC = 1 mg/L and a levofloxacin MIC = 1 mg/L increased over time (3.6% to 6.5%, P = 0.0021). No increase in prevalence of first-step parC mutations was observed among all fluoroquinolone-susceptible clinical isolates of S. pneumoniae with levofloxacin MICs of 1 mg/L after the introduction of the respiratory fluoroquinolones; however, fluoroquinolones appear to be selecting for isolates with elevated ciprofloxacin MICs.

Molecular characterization of fluoroquinolone resistant Streptococcus pneumoniae clinical isolates obtained from across Canada

There is little published data detailing fluoroquinolone resistance in clinical isolates of S. pneumoniae. The purpose of this study was to characterize the resistance mechanisms of 34 fluoroquinolone-resistant S. pneumoniae clinical isolates obtained from medical centers in 8 of 10 Canadian provinces between 1997 and 2000. The quinolone resistance determining regions of gyrA, parC, and parE from the isolates were sequenced. The isolates were evaluated for reserpine-sensitive efflux of ciprofloxacin and the new fluoroquinolones: gatifloxacin, gemifloxacin, levofloxacin and moxifloxacin. The isolates were typed using pulsed field gel electrophoresis. The majority of the isolates were genetically unrelated. Lower level fluoroquinolone resistance (ciprofloxacin MIC 4-8μg/ml) was associated with amino acid substitutions in ParC, while higher level resistance (ciprofloxacin MIC ≥16μg/ml) was associated with amino acid substitutions in both ParC and GyrA. ParE substitutions were not associated with clinical resistance. Twelve of 34 (35%) isolates demonstrated reserpine-sensitive efflux of ciprofloxacin. Efflux alone conferred low level ciprofloxacin resistance in 3 isolates. Significant reserpine-sensitive efflux of the new fluoroquinolones was not observed.

Fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae: contributions of type II topoisomerase mutations and efflux to levels of resistance.

We report on amino acid substitutions in the quinolone resistance-determining region of type II topisomerases and the prevalence of reserpine-inhibited efflux for 70 clinical isolates of S. pneumoniae for which the ciprofloxacin MIC is >/=4 microgram/ml and 28 isolates for which the ciprofloxacin MIC is </=2 microgram/ml. The amino acid substitutions in ParC conferring low-level resistance (MICs, 4 to 8 microgram/ml) included Phe, Tyr, and Ala for Ser-79; Asn, Ala, Gly, Tyr, and Val for Asp-83; Asn for Asp-78; and Pro for Ala-115. Isolates with intermediate-level (MICs, 16 to 32 microgram/ml) and high-level (MICs, 64 microgram/ml) resistance harbored substitutions of Phe and Tyr for Ser-79 or Asn and Ala for Asp-83 in ParC and an additional substitution in GyrA which included either Glu-85-Lys (Gly) or Ser-81-Phe (Tyr). Glu-85-Lys was found exclusively in isolates with high-level resistance. Efflux contributed primarily to low-level resistance in isolates with or without an amino acid substitution in ParC. The impact of amino acid substitutions in ParE was minimal, and no substitutions in GyrB were identified.

Role of mutation in the gyrA and parC genes of nalidixic-acid-resistant salmonella serotypes isolated from animals in the United Kingdom.

To answer the question as to whether isolates of salmonella from animals with decreased susceptibility to quinolones possess the same mechanism of resistance as isolates from humans, the polymerase chain reaction (PCR) was used to amplify a 347 base pair fragment equivalent to codons 39-159 covering the quinolone resistance determining region (QRDR) of the gyrA gene of Salmonella typhimurium NCTC 74, and 196 veterinary isolates of 26 serotypes of salmonella (109 from turkeys, 29 from chickens, the remainder from cattle, parrots, pigs, dogs, a pigeon, an exotic bird, a duck, a partridge, a sheep, a horse and environmental samples including litter, a nest and water). All PCR products were electrophoresed for single-stranded conformational polymorphism (SSCP). The DNA sequence of all eight novel SSCP patterns was determined. Nucleotide substitutions were identified in the gyrA gene of all except two isolates; all substitutions conferred a substitution at serine 83 or aspartate 87, as has previously been shown for isolates from humans. No mutations conferring an amino acid substitution in parC (the gene encoding the secondary target for quinolones) were revealed in the 48 isolates requiring 0.5-1 mg/L ciprofloxacin for inhibition, or the isolates for which no mutation in gyrA was revealed. The accumulation of enrofloxacin and ciprofloxacin was determined for 30 representative isolates (of which five were multiply drug resistant). Five isolates accumulated one-quarter to one-half the concentration achieved by the corresponding wild-type serotype. For two of these five isolates no mutation in gyrA was revealed, and one lacked OmpF.