Quinolone Resistance-determining Regions Of parC Research Articles

Molecular characterization of quinolone resistance and antimicrobial resistance profiles of Klebsiella pneumoniae and Escherichia coli isolated from human and broiler chickens

ABSTRACT This study characterized quinolone (Q) resistance determinants in a series of Klebsiella pneumoniae (n = 26) and Escherichia coli (n = 19) isolates of human and animal origin. The presence of plasmid-mediated quinolone resistance (PMQR) and carabpenemase genes was examined by PCR. The quinolone resistance-determining regions (QRDRs) of gyrA and parC genes were sequenced. Thirty-three isolates had ciprofloxacin MIC≥8 mg/l. About 34.6% and 10.5% of K. pneumoniae and E. coli isolates were ESBL producers respectively. The PMQR genes were detected in 77% (n = 35) of isolates. The oqxAB was the most prevalent PMQR gene being identified in all K. pneumoniae isolates, followed by aac(6ʹ)-Ib-cr (34.6%), qnrS (23%) and qnrB (7.7%). The most frequently detected gene among E. coli isolates was qnrS (36.8%) followed by aac(6′)-Ib-cr (10.5%) and qepA (5.2%). All Q resistant isolates harbored amino acid substitutions in both GyrA and ParC QRDRs. High prevalence of PMQR genes among food-producing animal isolates is an issue of great concern.

A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of gyrA and parC Mutations Associated With Fluoroquinolone Resistance in Acinetobacter baumannii.

BackgroundMutations in the quinolone resistance-determining regions (QRDRs) of Acinetobacter baumannii DNA gyrase (gyrA) and topoisomerase IV (parC) are linked to fluoroquinolone (FQ) resistance. We developed a mismatched PCR-restriction fragment length polymorphism (RFLP) assay to detect mutations in the gyrA and parC QRDRs associated with FQ resistance in A. baumannii.MethodsBased on the conserved sequences of A. baumannii gyrA and parC, two primer sets were designed for mismatched PCR-RFLP to detect mutations in gyrA (codons 83 and 87) and parC (codons 80 and 84) by introducing an artificial restriction enzyme cleavage site into the PCR products. This assay was evaluated using 58 A. baumannii strains and 37 other Acinetobacter strains that have been identified by RNA polymerase β-subunit gene sequence analysis.ResultsPCR amplification of gyrA and parC was successful for all A. baumannii strains. In 11 FQ -susceptible strains, the gyrA and parC PCR products were digested by the selected restriction enzymes at the site containing gyrA (codons 83 and 87) and parC (codons 80 and 84). PCR products from 47 FQ-resistant strains containing mutations in gyrA and parC were not digested by the restriction enzymes at the site containing the mutation. As for the non-baumannii Acinetobacter strains, although amplification products for gyrA were obtained for 28 strains, no parC amplification product was obtained for any strain.ConclusionsThis assay specifically amplified gyrA and parC from A. baumannii and detected A. baumannii gyrA and parC mutations with FQ resistance.

Occurrence of plasmid-mediated quinolone resistance determinants among Escherichia coli strains isolated from animals in Tunisia: Specific pathovars acquired qnr genes.

The aim of this study was to characterise Escherichia coli strains harbouring plasmid-mediated quinolone resistance (PMQR) genes recovered from various samples (n = 116) from healthy and diarrhoeic animals in Tunisia. All nalidixic acid-resistant E. coli isolates were screened for the presence of PMQR genes. Isolates positive for PMQR genes were investigated by PCR for chromosomal mutations in the quinolone resistance-determining regions (QRDRs) of GyrA and ParC, the presence of class 1 and class 2 integrons, genes encoding tetracycline and sulfonamide resistance, genes encoding virulence factors, and phylogenetic group. Genetic relationships was determined by pulsed-field gel electrophoresis (PFGE). Amongst 51 nalidixic acid-resistant isolates, 9 harboured PMQR genes (5 co-harbouredqnrS1 and qnrB1, 3 harboured qnrS1 and 1 harboured qnrB1). Two types of mutation in the QRDR of GyrA were observed: S83L and D87N (eight isolates) and S83L (one isolate). For the QRDR of ParC, the substitution S80I was observed in four isolates. A class 1 integron was found in six isolates. The tetA or tetB gene was observed in six isolates and both tetA and tetB were co-harboured by two isolates. The sul1, sul2 and sul3 genes were detected in six, four and one isolates, respectively. According to the presence of specific virulence genes, the nine strains were classified as UPEC (5), EAEC (3) and EPEC (1). Three isolates from turkey faeces were clonally related by PFGE. These findings highlight the plausible role of the avian industry as a reservoir of human pathogenic E. coli strains.

Genetic mutations in the quinolone resistance-determining region are related to changes in the epidemiological profile of methicillin-resistant Staphylococcus aureus isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important causative agent of nosocomial infections. Mutations in the quinolone resistance-determining regions (QRDRs) of the gyr and par genes have been described. This study aimed to characterise phenotypic and genotypic fluoroquinolone resistance in 69 MRSA isolates of different clonal lineages from hospitals in Rio de Janeiro, Brazil. QRDR mutations in the gyrA, gyrB, parC and parE genes were detected by DNA sequencing. Minimum inhibitory concentrations (MICs) for ciprofloxacin and moxifloxacin were determined by broth microdilution. The occurrence of associations between mutations and MICs among the different clonal lineages of MRSA isolates was then verified. Most isolates from the USA400/ST1/SCCmec IV lineage, but mainly USA100/ST5/SCCmec II isolates, which have been more recently found in Rio de Janeiro hospitals, showed different patterns of mutations, including double mutation in the QRDR of parC (Ser-80⿿⿿⿿Tyr and Glu-84⿿⿿⿿Lys/Gly) and/or gyrA (Ser-84⿿⿿⿿Leu and/or Glu-88⿿⿿⿿Lys) associated with higher moxifloxacin and ciprofloxacin MICs (MIC90, ⿥8⿿mg/L and 256⿿mg/L, respectively). On the other hand, all USA800/ST5/SCCmec IV and the BEC/ST239/SCCmec III isolates, which have disappeared from hospitals, showed single mutations in parC (Ser-80⿿⿿⿿Phe) and gyrA (Ser-84⿿⿿⿿Leu or Glu-88⿿⿿⿿Gly) and lower fluoroquinolones MICs (MIC90, ⿥2⿿mg/L and ⿥16⿿mg/L). This study highlights an increase in the number and types of mutations in the QRDRs ofgyrA and parC associated with high fluoroquinolones MICs that may be related to changes in the epidemiological profile of MRSA isolates from hospitals in Rio de Janeiro.

Microbial virulence, molecular epidemiology and pathogenic factors of fluoroquinolone-resistant Haemophilus influenzae infections in Guangzhou, China

BackgroundFluoroquinolone-resistant Haemophilus influenzae (FRHI) has been reported worldwide but remain unclear in China.MethodsA total of 402 H. influenzae isolates collected from 2016 to 2017 were included. Antimicrobial susceptibility on 10 antibiotics was performed, and minimum inhibitory concentration of ciprofloxacin- and nalidixic acid-resistant strains were further determined by E-test strips, with risk factors also evaluated. Strains with resistance or reduced susceptibility to ciprofloxacin were subjected to sequencing of the quinolone resistance-determining regions (QRDR) and plasmid-mediated quinolone resistance genes by sequencing, with multi-locus sequence typing.Results2.2% of H. influenzae strains were non-susceptible (7/402, 1.7%) or susceptible (2/402, 0.5%) to ciprofloxacin but NAL-resistant by E-test, and multidrug resistance was more common in fluoroquinolones non-susceptible H. influenzae group (p = 0.000). Infection risk factors included invasive procedure (p = 0.011), catching cold/previous contact with someone who had a cold (p = 0.019), fluoroquinolones use during previous 3 months (p = 0.003). With none of mutations obtained in gyrB, parE and other plasmid-mediated quinolone resistance genes, 7 and 4 strains were found for Ser-84-Leu substitutions in gyrA and one amino acid substitution in the QRDR of gyrA linked with one amino acid substitution in the QRDR of parC, respectively. In addition, five sequence types (ST) were identified, with ST1719 firstly found.ConclusionsFor the first time, this study has reported the incidence, risk factors, molecular determinants on fluoroquinolones resistance and ST of FRHI strains in mainland China, representing the first evidence of mutation of gyrA and parC in China and the new ST1719 worldwide.

Mutations in ParC and GyrA of moxifloxacin-resistant and susceptible Mycoplasma genitalium strains.

Macrolide or fluoroquinolone-resistant Mycoplasma genitalium is spreading worldwide. We aimed to determine the influence of single nucleotide polymorphisms (SNPs) in the quinolone resistance determining regions (QRDR) of parC and gyrA in cultured M. genitalium strains. In addition, we examined the prevalence of macrolide- and fluoroquinolone resistance mediating mutations in specimens collected from Japanese male patients with urethritis in two time-periods between 2005–2009 and 2010–2017, respectively, by sequencing the QRDR of parC and gyrA and domain V of the 23S rRNA gene. The minimum inhibitory concentrations (MIC) of moxifloxacin, sitafloxacin, ciprofloxacin, levofloxacin, doxycycline, minocycline, azithromycin and clarithromycin were determined in 23 M. genitalium strains. Three cultured strains had elevated MICs for moxifloxacin at 16, 4 and 2 mg/L and had SNPs with the amino-acid change Ser83→Ile in ParC (p<0.001) and 3 kinds of SNPs with amino-acid changes Asp99→Asn, Gly93→Cys and Met95→Ile in GyrA, respectively. Among a total of 148 M. genitalium positive urine specimens, the prevalence of A2058G and A2059G SNPs in the 23S rRNA gene and any SNPs in ParC increased from 4.8% and 22.6% in 2005–2009 to 42.2% and 53.1% in 2010–2017, respectively. If M. genitalium is considered multi-drug resistant in clinical specimens carrying SNPs in the 23S rRNA gene and Ser83→Ile in ParC, the prevalence of multi-drug resistance is 12.5% in 2010–2017 in Japan. In conclusion, the SNP resulting in Ser83→Ile in ParC is closely related to moxifloxacin resistance even though other factors may also affect treatment outcomes by moxifloxacin. The prevalence of circulating multi-drug resistant M. genitalium strains with macrolide- and fluoroquinolone-resistance is dramatically increasing in Japan.

Identification and Characterization of Fluoroquinolone Non-susceptible Streptococcus pyogenes Clones Harboring Tetracycline and Macrolide Resistance in Shanghai, China.

Streptococcus pyogenes, also known as group A Streptococcus (GAS), is one of the top 10 infectious causes of death worldwide. Macrolide and tetracycline resistant GAS has emerged as a major health concern in China coinciding with an ongoing scarlet fever epidemic. Furthermore, increasing rates of fluoroquinolone (FQ) non-susceptibility within GAS from geographical regions outside of China has also been reported. Fluoroquinolones are the third most commonly prescribed antibiotic in China and is an therapeutic alternative for multi-drug resistant GAS. The purpose of this study was to investigate the epidemiological and molecular features of GAS fluoroquinolone (FQ) non-susceptibility in Shanghai, China. GAS (n = 2,258) recovered between 2011 and 2016 from children and adults were tested for FQ-non-susceptibility. Efflux phenotype and mutations in parC, parE, gyrA, and gyrB were investigated and genetic relationships were determined by emm typing, pulsed-field gel electrophoresis and phylogenetic analysis. The frequency of GAS FQ-non-susceptibility was 1.3% (30/2,258), with the phenotype more prevalent in GAS isolated from adults (14.3%) than from children (1.2%). Eighty percent (24/30) of FQ-non-susceptible isolates were also resistant to both macrolides (ermB) and tetracycline (tetM) including the GAS sequence types emm12, emm6, emm11, and emm1. Genomic fingerprinting analysis of the 30 isolates revealed that non-susceptibility may arise in various genetic backgrounds even within a single emm type. No efflux phenotype was observed in FQ non-susceptible isolates, and molecular analysis of the quinolone resistance-determining regions (QRDRs) identified several sequence polymorphisms in ParC and ParE, and none in GyrA and GyrB. Expansion of this analysis to 152 publically available GAS whole genome sequences from Hong Kong predicted 7.9% (12/152) of Hong Kong isolates harbored a S79F ParC mutation, of which 66.7% (8/12) were macrolide and tetracycline resistant. Phylogenetic analysis of the parC QRDR sequences suggested the possibility that FQ resistance may be acquired through inter-species lateral gene transfer. This study reports the emergence of macrolide, tetracycline, and fluoroquinolone multidrug-resistant clones across several GAS emm types including emm1 and emm12, warranting continual surveillance given the extensive use of fluoroquinolones in clinical use.

The Mutations of Topoisomerase Genes and Their Effect on Resistance to Fluoroquinolones in Extended-Spectrum β-Lactamase-Producing Escherichia coli

Background: Fluoroquinolones have been used for empirical treatment of urinary tract infections in recent years. This study aimed at identifying mutations in gyrA and parC genes and their correlation with fluoroquinolone minimal inhibitory concentration (MIC) among extended-spectrum β-lactamase-producing (ESBL) Escherichia coli. Methods: A total of 240 E. coli were isolated from urine of patients during 2014 and 2015 in the west of Iran. The isolates were screened for ESBL-producing phenotype using combined disc diffusion test. The susceptibility of ESBL isolates to ciprofloxacin and levofloxacin was determined by disk diffusion and microdilution assay. PCR, sequencing and bioinformatics analysis were performed to identify mutations in gyrA and parC genes. Results: Of 240 isolates, 66 (27.5%) were ESBL positive. Of them, 45 (68.1%) isolates were resistant to both ciprofloxacin and levofloxacin. Sequence analysis showed mutations in the quinolone resistance determining region (QRDR) in the 2 codons (Ser-83, Asp-87) of gyrA and 2 codons (Ser-80, Glu-84) of parC. One mutation at codon of Ala-192 was found outside the QRDR in parC. Conclusions: Isolates with mutations in QRDR of parC and gyrA had the higher MIC level compared to isolates with mutations only in gyrA. The highest level of resistance was detected in isolates with accumulation of mutations in gyrA and parC. A high frequency of fluoroquinolone resistance among ESBL producing E. coli isolates indicated the clustered transmission of resistance genes in this bacterium. A new mutation outside the QRDR of parC was detected, which may play a role in fluoroquinolone resistance.

Prevalence of gyrA and parE mutations in clinical isolates of Streptococcus pneumoniae with decreased susceptibilities to different Fluoroquinolones

Introduction: Streptococcus pneumoniae is a major Gram-positive pathogen responsible for pneumonia, bactermia, otitis media, and meningitis leading to considerable morbidity and mortality among children and elderly individuals. The primary goals of antibiotic treatment of respiratory tract infections are clinical efficacy of treatment, pathogen eradication, and prevention of resistance development. Resistance to fluoroquinolones in S. pneumoniae arises in a stepwise fashion and results from alterations in the target binding site due to the acquisition of spontaneous mutations in the quinolone resistance-determining regions (QRDRs) of the topoisomerase IV and DNA gyrase genes. Although mutations usually occur in the QRDRs of parC and gyrA, a role for mutations in the parE subunit in low-level resistance has been reported. Aim of the work: The aim of this study was to determine the prevalence of fluoroquinolone resistance Streptococcus pneumoniae (FQRSP) and to examine the genetic relatedness of pneumococcal isolates with parE and gyrA genes mutations in different specimens in Sohag University Hospital. Patients and Methods: This study was prospectively conducted over a period of 24 months between October 2015 and September 2017, at Sohag university hospital. During the study period, 78 patients hospitalized for a syndrome consistent with a diagnosis of community acquired pneumonia (CAP ) included in this study with a mean age of 34.5 years (range, 2 to 67), 60% of whom were males. A CAP syndrome was defined as a newly recognized pulmonary infiltrate together with 2 of the following findings: subjective fever or documented temperature 37.4 °C, increased cough, sputum production, or shortness of breath, pleuritic chest pain, confusion, rales, leukocytosis, (according to age) (1). Patients who had taken antibiotic treatment within 3 days prior to initial visit were excluded from this study. Results: Our study illustrate the role of mutation in the gyrA&parE genes and the effect of mutations in the both genes in fluoroquinolone resistance among S. pneumoniae isolates. Conclusion: The present study provide an opportunity to view the predominant mutations conferring reduced susceptibility to FQs in clinical pneumococcal isolates. There is a strong relationship between these mutations and decrese susceptibility to the most fameous FQs to some extent, although this varies between strains and for each drug.

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.

In Vitro Activity of Sitafloxacin and Additional Newer Generation Fluoroquinolones Against Ciprofloxacin-Resistant Neisseria gonorrhoeae Isolates.

Emergence of antimicrobial resistance in Neisseria gonorrhoeae is a major public health concern globally, and new antimicrobials for treatment of gonorrhea are imperative. In this study, the in vitro activity of sitafloxacin, a fluoroquinolone mainly used for respiratory tract or urogenital infections in Japan, and additional newer generation fluoroquinolones were determined against ciprofloxacin-resistant N. gonorrhoeae isolates. Minimum inhibitory concentrations (MICs) of ciprofloxacin, levofloxacin, moxifloxacin, sitafloxacin, pazufloxacin, and tosufloxacin against 47 N. gonorrhoeae isolates cultured in 2009 in Japan were determined by agar dilution method. The quinolone resistance-determining region (QRDR) of gyrA and parC was sequenced. The in vitro potency of sitafloxacin was substantially higher compared with all other tested fluoroquinolones. The MICs of sitafloxacin ranged from 0.03 to 0.5 mg/L for 35 ciprofloxacin-resistant N. gonorrhoeae isolates (ciprofloxacin MICs from 2 to 32 mg/L). No identified mutations in GyrA and ParC QRDR resulted in higher sitafloxacin MIC than 0.5 mg/L. Sitafloxacin had a high activity against N. gonorrhoeae isolates, including strains with mutations in DNA gyrase and topoisomerase IV, resulting in high-level resistance to ciprofloxacin and all other newer generation fluoroquinolones examined. However, it was still to a lower extent affected by GyrA and ParC QRDR mutations resulting in sitafloxacin MICs of up to 0.5 mg/L. This indicates that sitafloxacin should not be considered for empirical first-line monotherapy of gonorrhea. However, sitafloxacin could be valuable in a dual antimicrobial therapy and for cases with ceftriaxone resistance or allergy.

Molecular Basis of Resistance to Selected Antimicrobial Agents in the Emerging Zoonotic Pathogen Streptococcus suis.

Characterization of 227 Streptococcus suis strains isolated from pigs during 2010 to 2013 showed high levels of resistance to clindamycin (95.6%), tilmicosin (94.7%), tylosin (93.8%), oxytetracycline (89.4%), chlortetracycline (86.8%), tiamulin (72.7%), neomycin (70.0%), enrofloxacin (56.4%), penicillin (56.4%), ceftiofur (55.9%), and gentamicin (55.1%). Resistance to tetracyclines, macrolides, aminoglycosides, and fluoroquinolone was attributed to the tet gene, erm(B), erm(C), mph(C), and mef(A) and/or mef(E) genes, aph(3')-IIIa and aac(6')-Ie-aph(2″)-Ia genes, and single point mutations in the quinolone resistance-determining region of ParC and GyrA, respectively.

Co-existence of plasmid-mediated quinolone resistance determinants and mutations in gyrA and parC among fluoroquinolone-resistant clinical Enterobacteriaceae isolated in a tertiary hospital in Warsaw, Poland

Plasmid-mediated quinolone resistance (PMQR) determinants and the distribution of mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC were investigated in 215 ciprofloxacin-resistant (MIC>1mg/L) clinical Enterobacteriaceae collected during a 6-month prospective study in a tertiary hospital in Warsaw, Poland. PMQR determinants were present in 49 isolates (22.8%), among which aac(6′)-Ib-cr and qnrB1 predominated (85.7% and 26.5%, respectively). Mutations in gyrA and parC QRDRs were detected among 89.8% of isolates (MIC≥4mg/L). Changes in Ser-83, Ala-84 and Asp-87 in GyrA and Ser-80 and Glu-84 in ParC were detected. Five isolates with ciprofloxacin MICs in the range 1.5–16mg/L were found to have unaltered QRDRs, with PMQR as the only fluoroquinolone (FQ) resistance trait detected. The remaining 44 PMQR-positive isolates were found to carry altered QRDRs. Three substitutions (two in GyrA and one in ParC) were detected in 23 isolates, whilst 8 isolates carried four mutations (two in GyrA and two in ParC). One isolate of Klebsiella pneumoniae with two amino acid substitutions in the ParC QRDR in the presence of aac(6′)-Ib-cr and qnrB1 had a ciprofloxacin MIC of 16mg/L. The results presented here show that FQ resistance in these clinical Enterobacteriaceae is a complex interplay between PMQR determinants and mutations in gyrA and parC rather than a single stepwise accumulation of mutations in the gyrase and topoisomerase subunits. In addition, these results show the role of PMQR determinants in promoting QRDR mutations and the acquisition of high-level FQ resistance in clinical settings.

Streptococcus parauberis의 퀴놀론 내성 증가와 Topoisomerase 유전자에서의 돌연변이 신속 분석

To investigate the acquisition of quinolone resistance, we examined mutations in the quinolone resistance-determining region (QRDR) of type II topoisomerase genes in ciprofloxacin (CIP)-resistant clinical isolates and in vitro mutants of Streptococcus parauberis. The CIP-resistant clinical isolates had one base change responsible for a Ser-79<TEX>${\rightarrow}$</TEX>Thr in the QRDR of parC. However, the CIP-resistant in vitro mutants had an altered QRDR of parC (Ser-79<TEX>${\rightarrow}$</TEX>Ile) that differed from that of the isolates. None of the CIP-resistant S. parauberis clinical isolates or in vitro mutants exhibited amino acid changes in gyrA or gyrB. However, even though involvement in the increased resistance was not clear, an Arg-449<TEX>${\rightarrow}$</TEX>Ser mutation outside of the QRDR of parE was detected in CIP-resistant mutant 2P1. These results suggest that the topoisomerase IV gene, parC (and possibly parE, as well), is the primary ciprofloxacin target in S. parauberis. Additionally we established a high-resolution melting (HRM) assay capable of detecting the dominant mutation in four type II topoisomerase genes conferring ciprofloxacin resistance. These rapid and reliable assays may provide a convenient method of surveillance for genetic mutations conferring antibiotic resistance.

Fluoroquinolone resistance and molecular characterization of gyrA and parC quinolone resistance-determining regions in Escherichia coli isolated from poultry

Escherichia coli are a common inhabitant of the gastrointestinal tract of mammals and birds; nevertheless, they may be associated with a variety of severe and invasive infections. Whereas fluoroquinolones (FQ) have been banned in the United States for use in poultry production, the use of these antimicrobials in poultry husbandry is still possible in the European Union, although with some restrictions. The aim of this study was to investigate the FQ resistance of 235 E. coli isolates recovered from chickens and turkeys. Minimum inhibitory concentrations were determined by a microdilution method, whereas mutations in the quinolone resistance-determining regions of the target genes, gyrA and parC, were detected by a PCR-based method. High resistance rates (>60%) were observed for nalidixic acid, flumequine, and difloxacin, whereas resistance to ciprofloxacin, danofloxacin, enrofloxacin, marbofloxacin, and sarafloxacin was less frequently reported (<40%). Sixty-four isolates (27.2%) showed full susceptibility toward the tested FQ, but 57 isolates (24.2%) were resistant to all tested FQ. The remaining 114 E. coli isolates (48.5%) were grouped in 5 different resistance patterns. Isolates resistant only to flumequine or nalidixic acid or both possessed 1 gyrA mutation, whereas isolates with further resistance to enrofloxacin, difloxacin, danofloxacin, and sarafloxacin had in addition 1 or 2 parC substitutions. Two gyrA mutations coupled with 1 substitution in parC were detected in isolates resistant to all tested FQ. The number of mutations and their correlation with the in vitro activity of FQ reflected the currently accepted model, according to which a single gyrA substitution is associated with resistance or decreased susceptibility to older quinolones, whereas further gyrA or parC substitutions are needed for a higher level of resistance.

Molecular Characterization of Acquired Enrofloxacin Resistance in Mycoplasma synoviae Field Isolates

The in vitro activity of enrofloxacin against 73 Mycoplasma synoviae field strains isolated in Israel and Europe was determined by broth microdilution. Decreased susceptibility to enrofloxacin was identified in 59% of strains, with the MICs ranging from 1 to >16 μg/ml. The estimated MIC50 and MIC90 values for enrofloxacin were 2 and 8 μg/ml, respectively. Moreover, this study showed that 92% of recent Israeli field isolates (2009 to 2011) of M. synoviae have MICs of ≥ 2 μg/ml to enrofloxacin. Comparison of the quinolone resistance-determining regions (QRDRs) in M. synoviae isolates revealed a clear correlation between the presence of one of the amino acid substitutions Asp79-Asn, Thr80-Ala/Ile, Ser81-Pro, and Asp84-Asn/Tyr/His of the ParC QRDR and decreased susceptibility to enrofloxacin (MIC, ≥ 1 μg/ml). Amino acid substitutions at positions GyrA 87, GyrB 401/402, and ParE 420/454 were also identified, but there was no clear-cut correlation with susceptibility to enrofloxacin. Comparison of vlhA molecular profiles revealed the presence of 9 different genotypes in the Israeli M. synoviae field isolates and 10 genotypes in the European isolates; only one vlhA genotype (type 4) was identified in both cohorts. Based on results of vlhA molecular typing, several mechanisms for emergence and dissemination of Israeli enrofloxacin-resistant M. synoviae isolates are suggested.

Contribution of Topoisomerase IV Mutation to Quinolone Resistance in Mycoplasma genitalium

The mechanism of quinolone resistance in Mycoplasma genitalium remains poorly understood due to difficulties with in vitro culture, especially of clinical isolates. In this study, to confirm the association between mutations in topoisomerases and antimicrobial susceptibilities to quinolones, ciprofloxacin-resistant mutant strains were selected using the cultivable type strain ATCC 33530. Sequence analysis revealed that the mutant strains harbored mutations in topoisomerase IV: Gly81Cys in ParC, Pro261Thr in ParC, or Asn466Lys in ParE. The MICs of all quinolones tested against the mutant strains were 2- to 16-fold higher than those against the wild-type strain. No cross-resistance was observed with macrolides or tetracyclines. We determined the inhibitory activities of quinolones against DNA gyrase and topoisomerase IV in order to investigate the correlation between antimicrobial susceptibility and inhibitory activity against the target enzymes, considered the primary targets of quinolones. Furthermore, using enzymatic analysis, we confirmed that Gly81Cys in the ParC quinolone resistance-determining region (QRDR) contributed to quinolone resistance. This is the first study to isolate quinolone-resistant mutant strains of M. genitalium harboring substitutions in the parC or parE gene in vitro and to measure the inhibitory activities against the purified topoisomerases of M. genitalium.

Molecular epidemiology of fluoroquinolone resistance in invasive clinical isolates of Streptococcus pneumoniae in Seville

Due to the emergence of drug-resistant pneumococcal isolates, new fluoroquinolones have been recommended for the treatment of pneumococcal infections. The purpose of this study was to establish surveillance, and to conduct molecular characterization, of fluoroquinolone-resistant Streptococcus pneumoniae in Seville. Norfloxacin-resistant S. pneumoniae isolates were characterized by quinolone resistance-determining region (QRDR) substitutions, reserpine-sensitive efflux, serotype and by pulsed-field gel electrophoresis (PFGE) patterns. Fourteen isolates (5.1%) showed an MIC>16 μg/ml to norfloxacin. Eight of 10 adult isolates were susceptible to levofloxacin. The 4 infant isolates with norfloxacin MIC>16 μg/ml were susceptible to levofloxacin. Seven of these 12 low-level-resistant isolates had mutations in ParC, while mutations both in ParC and GyrA genes were only detected in one of the two high-level-resistant isolates. All the isolates without QRDR substitutions that remained norfloxacin-resistant were positive for reserpine-inhibited efflux. The serotyping and PFGE revealed significant heterogeneity. We obtained 9 different profiles, 3 of which had two isolates each. Two of the isolates with the same pulsotype were from the same patient. The first isolate showed a mutation in the QRDR of ParC, and the second one had an additional GyrA mutation. In our study a levofloxacin resistance rate of 0.7% was found among invasive isolates. Although resistance level is low, surveillance is necessary, especially to prevent cases of in vivo resistance development as reported.

First description of the qnrS-like ( qnrS5) gene and analysis of quinolone resistance-determining regions in motile Aeromonas spp. from diseased fish and water

Antimicrobial resistance patterns in a collection of 33 motile Aeromonas species were described in this study. Quinolone has been frequently employed for treatment of Aeromonas-related diseases, and prolonged use of antimicrobial compounds has led to development of resistant strains. In a sample of diseased fish and environmental water, we evaluated nalidixic acid ( n = 19) and ciprofloxacin ( n = 4) resistance via minimum inhibitory concentration (MIC) assays and the genetic basis was also investigated. Among the isolated Aeromonas spp., 17 strains encoded for chromosomal mutations of quinolone resistance-determining regions (QRDRs) in gyrA, 11 strains encoded for mutations of QRDRs in parC, 1 strain harbored plasmid-mediated quinolone resistance (PMQR) qnrS1-like gene and 4 strains harbored the PMQR qnrS2 gene. In particular, the new variant ( qnrS1-like) differed from qnrS1 by 6 amino acid substitutions at positions 5 (Asn 5→Arg 5), 120 (Ser 120→Thr 120), 148 (Asn 148→His 148), 206 (Leu 206→Glu 206), 207 (Ile 207→ Leu 207), and 216 (Tyr 216→Phe 216), and the gene was designated qnrS5. These resistant strains may function as reservoirs of quinolone resistance.

Antimicrobial Resistance and Multilocus Sequence Typing of Vancomycin-ResistantEnterococcus faeciumIsolated from the Chungcheong Area

Background: Enterococcus faecium has emerged as an important nosocomial pathogen worldwide, and this trend has been associated with the dissemination of a genetic lineage designated clonal complex 17 (CC17). In the present study, characterization of the glycopeptide resistance mechanism, genetic relatedness, and pathogenicity in isolates of vancomycin-resistant E. faecium in the Chungcheong area were investigated. Methods: A total of 37 consecutive, non-duplicate, vancomycin-resistant E. faecium were isolated at three university hospitals in the Chungcheong area. The mechanism of glycopeptide resistance and pathogenicity factors were studied using PCR, and the genetic relatedness was determined via multilocus sequence type and esp repeat profile analysis. Additionally, the quinolone resistance-determining regions of parC and gyrA were sequenced to identify mutations involved in ciprofloxacin resistance. Results: Two genotypes of VRE were confirmed: VanAphenotype vanA genotype VRE (25 isolates) and VanB-phenotype vanA genotype VRE (12 isolates). MLST analysis revealed five sequence types. A significant result was that ST414 and CNS4 (4-11-1-1-1-1) were considered as belonging to CC17. The esp and hyl genes were found in 100% and 86.4% of the isolates, respectively. A total of 37 isolates showed genetic mutations in parC and gyrA. Conclusion: All isolated strains in the present study belonged to one of the CC17 genotypes including ST414 and CNS4 (4-1-1-1-1-1-1), which were not previously detected in Korea. The combination of MLST and the esp gene repeat profiles can be useful for genetic characterization of VREF isolates with regard to the evolutionary process and epidemiology of the clones. (Korean J Clin Microbiol 2011;14:6066)