A total of 88 salmonella isolates (72 clinical isolates for which the ciprofloxacin MIC was >0.06 μg/ml, 15 isolates for which the ciprofloxacin MIC was ≤0.06 μg/ml, and Salmonella enterica serotype Typhimurium ATCC 13311) were studied for the presence of genetic alterations in four quinolone resistance genes, gyrA, gyrB, parC, and parE, by multiplex PCR amplimer conformation analysis. The genetic alterations were confirmed by direct nucleotide sequencing. A considerable number of strains had a mutation in parC, the first to be reported in salmonellae. Seven of the isolates sensitive to 0.06 μg of ciprofloxacin per ml had a novel mutation at codon 57 of parC (Tyr57→Ser) which was also found in 29 isolates for which ciprofloxacin MICs were >0.06 μg/ml. Thirty-two isolates had a single gyrA mutation (Ser83→Phe, Ser83→Tyr, Asp87→Asn, Asp87→Tyr, or Asp87→Gly), 34 had both a gyrA mutation and a parC mutation (29 isolates with a parC mutation of Tyr57→Ser and 5 isolates with a parC mutation of Ser80→Arg). Six isolates which were isolated recently (from 1998 to 2001) were resistant to 4 μg of ciprofloxacin per ml. Two of these isolates had double gyrA mutations (Ser83→Phe and Asp87→Asn) and a parC mutation (Ser80→Arg) (MICs, 8 to 32 μg/ml), and four of these isolates had double gyrA mutations (Ser83→Phe and Asp87→Gly), one parC mutation (Ser80→Arg), and one parE mutation (Ser458→Pro) (MICs, 16 to 64 μg/ml). All six of these isolates and those with a Ser80→Arg parC mutation were S. enterica serotype Typhimurium. One S. enterica serotype Typhi isolate harbored a single gyrA mutation (Ser83→Phe), and an S. enterica serotype Paratyphi A isolate harbored a gyrA mutation (Ser83→Tyr) and a parC mutation (Tyr57→Ser); both of these isolates had decreased susceptibilities to the fluoroquinolones. The MICs of ciprofloxacin, levofloxacin, and sparfloxacin were in general the lowest of those of the six fluoroquinolones tested. Isolates with a single gyrA mutation were less resistant to fluoroquinolones than those with an additional parC mutation (Tyr57→Ser or Ser80→Arg), while those with double gyrA mutations were more resistant.
Paratyphoid fever is considered an emerging systemic intracellular infection caused by Salmonella enterica serotypes Paratyphi A, B, and C. We performed in vitro time-kill studies on three clinical isolates of nalidixic acid-resistant Salmonella serotype Paratyphi (NARSP) with different concentrations of ciprofloxacin and cefotaxime to identify combinations of antibiotics with synergistic activity against paratyphoid fever. Furthermore, we identify the frequency of mutations to ciprofloxacin, cefotaxime, and rifampin resistance and also sequenced the gyrA, gyrB, parC, and parE genes to identify the cause of resistance in NARSP. When the activity of ciprofloxacin at 0.75× MIC (0.012 to 0.38 μg/ml) with cefotaxime at the MIC (0.125 to 0.25 μg/ml) against all three NARSP isolates was investigated, synergy was observed at 24 h, and the bacterial counts were reduced by >3 log10 CFU/ml. This synergy was elongated for up to 72 h in two out of three isolates. When ciprofloxacin at 0.75× MIC (0.012 to 0.38 μg/ml) was combined with cefotaxime at 2× MIC (0.25 to 0.50 μg/ml), synergy was prolonged for up to 72 h in all three isolates. Both Salmonella serotype Paratyphi A isolates carried single mutations in codon 83 of the gyrA gene and codon 84 of the parC gene that were responsible for their reduced susceptibility to ciprofloxacin, while no mutations were found in the gyrB or parE gene. The ciprofloxacin-plus-cefotaxime regimen was very effective in reducing the bacterial counts at 24 h for all three isolates, and this combination therapy may be helpful in reducing the chance of the emergence of fluoroquinolone-resistant mutants in patients with severe paratyphoid fever.
Strains of the multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium isolated in Japan were examined for high-level fluoroquinolone resistance. Since the first isolation in 2000 (described in reference 13), we have identified 12 human and 5 nonhuman isolates with high-level fluoroquinolone-resistance (ciprofloxacin MIC of 24 μg/ml or more). Most of these isolates shared some features including definitive phage type (DT12/193), resistance type (ACSSuTNCp; resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, nalidixic acid, and ciprofloxacin), and genotype on pulsed-field gel electrophoresis that were different from those of the MDR S. enterica Typhimurium DT104. Mutations in quinolone resistance-determining regions of gyrA and parC were also conserved in almost all of the isolates despite the absence of any apparent epidemiological relationships among cases. This suggests that a specific clonal group of the serovar Typhimurium with high levels of fluoroquinolone resistance is disseminating among animals and humans in Japan.
Fluoroquinolones commonly are used to treat adult Salmonella infections. Fluoroquinolone treatment has failed for persons infected with nalidixic acid-resistant Salmonella. From 1996 to 2003, state public health laboratories forwarded 12,252 non-Typhi Salmonella enterica isolates to the Centers for Disease Control and Prevention for antimicrobial susceptibility testing; 203 (1.6%) of the isolates were nalidixic acid resistant, and 14 (7%) of those were ciprofloxacin resistant. Resistance to nalidixic acid significantly increased from 0.4% in 1996 to 2.3% in 2003. All ciprofloxacin-resistant isolates had at least one point mutation in the quinolone resistance determining region (QRDR) of gyrA and did not harbor qnr or have point mutations in the QRDR of gyrB, parC, or parE. Continued surveillance of antimicrobial resistance among non-Typhi S. enterica isolates is needed to mitigate the increasing prevalence of nalidixic acid resistance.
We report 9 ciprofloxacin-resistant Salmonella enterica serotype Typhi isolates submitted to the US National Antimicrobial Resistance Monitoring System during 1999–2008. The first 2 had indistinguishable pulsed-field gel electrophoresis patterns and identical gyrA and parC mutations. Eight of the 9 patients had traveled to India within 30 days before illness onset.
Salmonella Typhi; ciprofloxacin; antimicrobial drug resistance; fluoroquinolones; typhoid fever; bacteria; dispatch
We report two cases of infection with clonally unrelated, high-level ciprofloxacin-resistant, β-lactamase–producing strains of Salmonella enterica Typhimurium. Resistance was caused by four topoisomerase mutations, in GyrA, GyrB, and ParC and increased drug efflux. Ciprofloxacin treatment failed in one case. In the second case, reduced susceptibility to third-generation cephalosporins occurred after initial treatment with these drugs and may explain the treatment failure with ceftriaxone.
The occurrence of active efflux and cell wall modifications were studied in Salmonella enterica serovar Typhimurium mutants that were selected with enrofloxacin and whose phenotypes of resistance to fluoroquinolones could not be explained only by mutations in the genes coding for gyrase or topoisomerase IV. Mutant BN18/21 exhibited a decreased susceptibility to ciprofloxacin (MIC = 0.125 μg/ml) but did not have a mutation in the gyrA gene. Mutants BN18/41 and BN18/71 had the same substitution, Gly81Cys in GyrA, but exhibited different levels of resistance to ciprofloxacin (MICs = 2 and 8 μg/ml, respectively). None of the mutants had mutations in the parC gene. Evidence for active efflux was provided by a classical fluorimetric method, which revealed a three- to fourfold decrease in ciprofloxacin accumulation in the three mutants compared to that in the parent strain, which was annuled by addition of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone. In mutant BN18/71, a second fluorimetric method also showed a 50% reduction in the level of accumulation of ethidium bromide, a known efflux pump substrate. Immunoblotting and enzyme-linked immunosorbent assay experiments with an anti-AcrA antibody revealed that the resistance phenotype was strongly correlated with the expression level of the AcrAB efflux pump and suggested that decreased susceptibility to ciprofloxacin due to active efflux probably related to overproduction of this pump could occur before that due to gyrA mutations. Alterations were also found in the outer membrane protein and lipopolysaccharide profiles of the mutants, and these alterations were possibly responsible for the decrease in the permeability of the outer membrane that was observed in the mutants and that could act synergistically with active efflux to decrease the level of ciprofloxacin accumulation.
Mutations in the quinolone resistance-determining regions (QRDR) in chromosomal gyrA and parC genes and fluoroquinolone susceptibility profiles were investigated in quinolone-resistant Enterobacteriaceae isolated from community and hospitalized patients in the Brazilian Southeast region. A total of 112 nalidixic acid-resistant enterobacterial isolates collected from 2000 to 2005 were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the QRDR regions. Susceptibility to fluoroquinolones was tested by the agar dilution method. Amongst the 112 enterobacterial isolates, 81 (72.3%) were resistant to ciprofloxacin and 5 (4.5%) showed reduced susceptibility. Twenty-six (23.2%) were susceptible to ciprofloxacin. Several alterations were detected in gyrA and parC genes. Escherichia coli isolates (47.7%) showed double mutations in the gyrA gene and a single one in the parC gene. Two unusual aminoacid substitutions are reported, an Asp87-Asn in a Citrobacter freundii isolate with reduced susceptibility to fluoroquinolones and a Glu84-Ala in one E. coli isolate. Only a parC gene mutation was found in fluoroquinolone-susceptible Enterobacter aerogenes. None of the isolates susceptible to ciprofloxacin presented mutations in topoisomerase genes. This comprehensive analysis of QRDRs in gyrA and parC genes, covering commonly isolated Enterobacteriaceae in Brazil is the largest reported up to now.
quinolone resistance-determining region; mutations; gyrA; parC; Enterobacteriaceae
We characterized 208 human Salmonella isolates from 2006 to 2007 and 27 human Salmonella enterica serovar Typhimurium isolates from 1987 to 1993 from Henan Province, China, by serotyping, by antimicrobial susceptibility testing, and, for the most common serovars, by pulsed-field gel electrophoresis (PFGE). The most common serovars among the 2006-2007 isolates were S. enterica serovar Typhimurium (27%), S. enterica serovar Enteritidis (17%), S. enterica serovar Derby (10%), S. enterica serovar Indiana (6%), and S. enterica serovar Litchfield (6%). A high percentage of the isolates were multiple-drug resistant, and 54% were resistant to both nalidixic acid and ciprofloxacin. Of these, 42% were resistant to a high level of ciprofloxacin (MIC > 4 μg/ml), whereas for the remaining isolates, the MICs ranged from 0.125 to 2 μg/ml. Five isolates (2%) were ceftiofur resistant and harbored blaCTX-M14 or blaCTX-M15. With the possible exception of the quinolones and cephalosporins, the 1987-1993 S. enterica serovar Typhimurium isolates were almost as resistant as the recent isolates. PFGE typing of S. enterica serovar Typhimurium showed that the most common cluster predominated over time. Two other clusters have emerged, and another cluster has disappeared.
This study is focused on real-time detection of gyrA mutations and of the presence of class I integrons in a panel of 100 veterinary isolates of Salmonella enterica from farm animals. The isolates were selected on the basis of resistance to nalidixic acid, representing a variety of the most prevalent serotypes in England and Wales. In addition, organic solvent (cyclohexane) resistance in these isolates was investigated in an attempt to elucidate the presence of efflux pump mechanisms. The most prevalent mutation among the isolates studied was Asp87-Asn (n = 42), followed by Ser83-Phe (n = 38), Ser83-Tyr (n = 12), Asp87-Tyr (n = 4), and Asp87-Gly (n = 3). Two distinct subpopulations were identified, separated at the 1-mg/liter breakpoint for ciprofloxacin: 86% of isolates with mutations in codon 83 showed MICs of ≥1 mg/liter, while 89.8% of isolates with mutations in codon 87 presented MICs of ≤0.5 mg/liter. Cyclohexane resistance was more prevalent among Ser83 mutants than among Asp87 mutants (34.7 and 4%, respectively), and in 79% of isolates that presented both gyrA mutations and cyclohexane resistance, the level of ciprofloxacin resistance was ≥2.0 mg/liter. Thirty-four isolates contained class I integrons, with 71% of the S. enterica serovar Typhimurium isolates and 6.9% of isolates belonging to other serotypes containing such elements. The methods used represent sensitive ways of investigating the presence of gyrA mutations and of detecting class-I integrons in Salmonella isolates. The results can be obtained in less than 1 h from single colonies without the need for purifying DNA.
Aim: To analyse mutations in the gyrA and parC genes leading to possible increase in ciprofloxacin resistance (high MIC values for ciprofloxacin) in clinical isolates of Neisseria gonorrhoeae in Delhi, India.
Method: MIC of ciprofloxacin for 63 clinical isolates of N gonorrhoeae were examined by the Etest method. Subsequently, gyrA and parC genes of these isolates were amplified and sequenced for possible mutations.
Results: Out of the 63 clinical isolates tested, only five (8%) isolates were found to be susceptible to ciprofloxacin (MIC <0.06 µg/ml). DNA sequence analysis of the gyrA and the parC genes of all these isolates (n = 63) revealed that all isolates which were not susceptible to ciprofloxacin (n=58) had mutation(s) in gyrA and parC genes. 12 isolates (19%) exhibited high resistance with an MIC for ciprofloxacin of 32 µg/ml. Two out of these 12 isolates (UD62 and UD63), harboured triple mutations (Ser-91 to Phe, Asp-95 to Asn and Val-120 to Leu) in the gyrA gene. The third mutation of Val-120 to Leu, lies downstream of the quinolone resistance determining region (QRDR) of the gyrA and has not been described before in gonococcus. In addition, both these isolates had a Phe-100 to Tyr substitution in the parC, a hitherto unknown mutation.
Conclusions: Emergence of ciprofloxacin resistance with high levels of MIC values (up to 32 µg/ml) in India is alarming. Double and triple mutations in gyrA alone or together in gyrA and parC could be responsible for such a high resistance.
Salmonella enterica isolates (n = 182) were examined for mutations in the quinolone resistance-determining region of gyrA, gyrB, parC, and parE. The frequency, location, and type of GyrA substitution varied with the serovar. Mutations were found in parC that encoded Thr57-Ser, Thr66-Ile, and Ser80-Arg substitutions. Mutations in the gyrB quinolone resistance-determining region were located at codon Tyr420-Cys or Arg437-Leu. Novel mutations were also found in parE encoding Glu453-Gly, His461-Tyr, Ala498-Thr, Val512-Gly, and Ser518-Cys. Although it is counterintuitive, isolates with a mutation in both gyrA and parC were more susceptible to ciprofloxacin than were isolates with a mutation in gyrA alone.
The aim of this study was to determine the distribution of the antimicrobial resistance phenotypes (R types), the phage types and XbaI-pulsed-field gel electrophoresis (PFGE) types, the genes coding for resistance to β-lactams and to quinolones, and the class 1 integrons among a representative sample of Salmonella enterica serotype Typhimurium isolates collected from humans in 2002 through the French National Reference Center for Salmonella (NRC-Salm) network. The trends in the evolution of antimicrobial resistance of serotype Typhimurium were reviewed by using NRC-Salm data from 1993, 1997, 2000, and 2003. In 2002, 3,998 isolates of serotype Typhimurium were registered at the NRC-Salm among 11,775 serotyped S. enterica isolates (34%). The most common multiple antibiotic resistance pattern was resistance to amoxicillin, chloramphenicol, streptomycin and spectinomycin, sulfonamides, and tetracycline (ACSSpSuTe R type), with 156 isolates (48.8%). One isolate resistant to extended-spectrum cephalosporins due to the production of TEM-52 extended-spectrum β-lactamase was detected (0.3%), and one multidrug-resistant isolate was highly resistant to ciprofloxacin (MIC > 32 mg/liter). We found that 57.2% of the isolates tested belonged to the DT104 clone. The main resistance pattern of DT104 isolates was R type ACSSpSuTe (83.2%). However, evolutionary changes have occurred within DT104, involving both loss (variants of Salmonella genomic island 1) and acquisition of genes for drug resistance to trimethoprim or to quinolones. PFGE profile X1 was the most prevalent (74.5%) among DT104 isolates, indicating the need to use a more discriminatory subtyping method for such isolates. Global data from the NRC-Salm suggested that DT104 was the main cause of multidrug resistance in serotype Typhimurium from humans from at least 1997 to 2003, with a roughly stable prevalence during this period.
Molecular characterization of fluoroquinolone-resistant Streptococcus pneumoniae in Canada was conducted from 1997 to 2005. Over the course of the study, 205 ciprofloxacin-resistant isolates were evaluated for ParC and GyrA quinolone resistance-determining region (QRDR) substitutions, substitutions in the full genes of ParC, ParE, and GyrA, reserpine sensitivity, and serotype and by pulsed-field gel electrophoresis. Rates of ciprofloxacin resistance of S. pneumoniae increased significantly, from less than 1% in 1997 to 4.2% in 2005. Ciprofloxacin resistance was greatest in people >64 years of age and least in those <16 years of age. Significant increases were also noted in rates of resistance to gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin, to the current rates of 1.6%, 1.0%, 1.1%, and 1.0%, respectively. The most common genotype observed consisted of QRDR substitutions in GyrA (Ser81Phe) and ParC (Ser79Phe). Substitutions outside the QRDR of GyrA, ParC, and ParE were not associated with fluoroquinolone resistance in this study. Overall, 21% of isolates were reserpine-sensitive and were thus assumed to be efflux positive. The ciprofloxacin-resistant isolates belonged to 35 different serotypes, but 10 (19F, 11A, 23F, 6B, 22F, 12F, 6A, 14, 9V, and 19A) accounted for 72% of all isolates. The majority of the isolates were found to be genetically unrelated by pulsed-field gel electrophoresis. Within the observed clusters, there was considerable genetic heterogeneity with regard to fluoroquinolone resistance mechanisms and serotypes. Continued surveillance and molecular analysis of fluoroquinolone-resistant S. pneumoniae in Canada are essential for appropriate empirical treatment of infections and early detection of novel resistance mechanisms.
In recent years, an increase in the occurrence of antimicrobial resistance among Salmonella enterica has been observed in several countries, which is worrisome because S. enterica is one of the most common causes of human gastroenteritis worldwide. The aim of this study was to characterize class 1 integrons and antibiotic resistance genotypes in Salmonella enterica isolates recovered from foodstuff and related sources. Nineteen multidrug-resistant (MDR) Salmonella enterica isolates were recovered. Higher resistance rates to tetracycline (90%), streptomycin (80%), sulfamethoxazole-trimethoprim (80%), ampicillin (60%) and nalidixic acid (70%) were related to the presence of the tetA, aadA, sul1/sul2, blaTEM-1 genes, and a codon mutation at position 83 of the gyrA gene, respectively. Class 1 integrons harboring aadA, blaTEM-1, sul1 or dhfr1 genes were detected in nine (45%) Salmonella enterica strains belonging to serotypes Brandenburg, Panama, Agona, Mbandaka and Alachua. Finally, clonal dissemination of S. Panama, S. Derby and S. Mbandaka was confirmed by PFGE. Detection of clonally related MDR Salmonella enterica suggests that endemic serotypes can be supported by class 1 integron-borne gene cassettes and/or mutations in drug targets. Emergence and dissemination of multidrug-resistant Salmonella enterica can have a major public health impact in an environment where large-scale suppliers ship their products.
Salmonella; multidrug-resistant; class 1 integrons; foodstuff; Brazil
Nontyphoidal Salmonella enterica strains with a nonclassical quinolone resistance phenotype were isolated from patients returning from Thailand or Malaysia to Finland. A total of 10 isolates of seven serovars were studied in detail, all of which had reduced susceptibility (MIC ≥ 0.125 μg/ml) to ciprofloxacin but were either susceptible or showed only low-level resistance (MIC ≤ 32 μg/ml) to nalidixic acid. Phenotypic characterization included susceptibility testing by the agar dilution method and investigation of efflux activity. Genotypic characterization included the screening of mutations in the quinolone resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE by PCR and denaturing high-pressure liquid chromatography and the amplification of plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB, qnrS, qnrD, aac(6′)-Ib-cr, and qepA by PCR. PMQR was confirmed by plasmid analysis, Southern hybridization, and plasmid transfer. No mutations in the QRDRs of gyrA, gyrB, parC, or parE were detected with the exception of a Thr57-Ser substitution within ParC seen in all but the S. enterica serovar Typhimurium strains. The qnrA and qnrS genes were the only PMQR determinants detected. Plasmids carrying qnr alleles were transferable in vitro, and the resistance phenotype was reproducible in Escherichia coli DH5α transformants. These data demonstrate the emergence of a highly mobile qnr genotype that, in the absence of mutation within topoisomerase genes, confers the nontypical quinolone resistance phenotype in S. enterica isolates. The qnr resistance mechanism enables bacteria to survive elevated quinolone concentrations, and therefore, strains carrying qnr alleles may be able to expand during fluoroquinolone treatment. This is of concern since nonclassical quinolone resistance is plasmid mediated and therefore mobilizable.
The occurrence of mutations in the genes coding for gyrase (gyrA and gyrB) and topoisomerase IV (parE and parC) of Salmonella typhimurium experimental mutants selected in vitro and in vivo and of 138 nalidixic acid-resistant Salmonella field isolates was investigated. The sequencing of the quinolone resistance-determining region of these genes in highly fluoroquinolone-resistant mutants (MICs of 4 to 16 μg/ml) revealed the presence of gyrA mutations at codons corresponding to Gly-81 or Ser-83, some of which were associated with a mutation at Asp-87. No mutations were found in the gyrB, parC, and parE genes. An assay combining allele-specific PCR and restriction fragment length polymorphism was developed to rapidly screen mutations at codons 81, 83, and 87 of gyrA. The MICs of ciprofloxacin for the field isolates reached only 2 μg/ml, versus 16 μg/ml for some in vitro-selected mutants. The field isolates, like the mutants selected in vivo, had only a single gyrA mutation at codon 83 or 87. Single gyrA mutations were also found in highly resistant in vitro-selected mutants (MIC of ciprofloxacin, 8 μg/ml), which indicates that mechanisms other than the unique modification of the intracellular targets could participate in fluoroquinolone resistance in Salmonella spp. A comparison of experimental mutants selected in vitro, field strains, and mutants selected in vivo suggests that highly fluoroquinolone-resistant strains are counterselected in field conditions in the absence of selective pressure.
Mechanisms of antibiotic resistance were examined in nalidixic acid-resistant Salmonella enterica serovar Enteritidis field isolates displaying decreased susceptibility to ciprofloxacin and in in vitro-derived ciprofloxacin-resistant mutants (104-cip and 5408-cip). All field isolates harbored a single gyrA mutation (D87Y). Deletion of acrB and complementation with wild-type gyrA increased quinolone susceptibility. Selection for ciprofloxacin resistance was associated with the development of an additional gyrA (S83F) mutation in 104-cip, novel gyrB (E466D) and parE (V461G) mutations in 5408-cip, overexpression of acrB and decreased susceptibility to nonquinolone antibiotics in both mutants, and decreased OmpF production and altered lipopolysaccharide in 104-cip. Complementation of mutated gyrA and gyrB with wild-type alleles restored susceptibility to quinolones in 104-cip and significantly decreased the ciprofloxacin MIC in 5408-cip. Complementation of parE had no effect on quinolone MICs. Deletion of acrB restored susceptibility to ciprofloxacin and other antibiotics tested. Both soxS and marA were overexpressed in 104-cip, and ramA was overexpressed in 5408-cip. Inactivation of each of these global regulators lowered ciprofloxacin MICs, decreased expression of acrB, and restored susceptibility to other antibiotics. Mutations were found in soxR (R20H) and in soxS (E52K) in 104-cip and in ramR (G25A) in 5408-cip. In conclusion, both efflux activity and a single gyrA mutation contribute to nalidixic acid resistance and reduced ciprofloxacin sensitivity. Ciprofloxacin resistance and decreased susceptibility to multiple antibiotics can result from different genetic events leading to development of target gene mutations, increased efflux activity resulting from differential expression of global regulators associated with mutations in their regulatory genes, and possible altered membrane permeability.
Blood isolates of Salmonella enterica serovar Typhi from two recently returned Bangladeshi patients in Kuwait were ciprofloxacin resistant, with ciprofloxacin MICs of 12 mg/liter for both isolates. Both isolates had three novel gyrA mutations (55-Leu→Trp, 87-Asp→Ala, and 106-Gln→Arg) and three novel parC mutations (84-Glu→Lys, 106-Trp→Gly, and 128-Tyr→Asp).
An oligonucleotide biochip that specifically detects point mutations in the gyrA and parC genes of Neisseria gonorrhoeae was designed and subsequently evaluated with 87 untreated clinical specimens. The susceptibilities of the N. gonorrhoeae strains were tested to determine the prevalence of ciprofloxacin-resistant strains in Anhui Province, People's Republic of China. Conventional DNA sequencing was also performed to identify mutations in gyrA and parC and to confirm the biochip data. The study demonstrates that all of the point mutations in the gyrA and parC genes of N. gonorrhoeae were easily discriminated by use of the oligonucleotide biochip. Fifteen different alteration patterns involved in the formation of ciprofloxacin resistance were identified by the biochip assay. Double mutations in both Ser91 and Asp95 of the GyrA protein were seen in all nonsensitive isolates. Double mutations in Ser91 and Asp95 of GyrA plus mutation of Glu91 or Ser87 of the ParC protein lead to significant high-level resistance to ciprofloxacin in N. gonorrhoeae isolates. The results obtained by use of the oligonucleotide biochip were identical to those obtained by use of DNA sequencing. In conclusion, the oligonucleotide biochip technology has potential utility for the rapid and reliable identification of point mutations in the drug resistance genes of N. gonorrhoeae.
In a previous study, four Salmonella isolates from humans in the Henan province of China showed reduced susceptibility to ciprofloxacin (MIC, 0.125 to 0.25 μg/ml) but were susceptible to nalidixic acid (MIC, 4 to 8 μg/ml). All isolates were negative for known qnr genes (A, B, and S), aac(6′)Ib-cr, and mutations in gyrA and parC. Plasmid DNA was extracted from all four isolates and transformed into Escherichia coli TG1 and DH10B cells by electroporation, and transformants were selected on 0.06 μg/ml ciprofloxacin containing brain heart infusion agar plates. Resistance to ciprofloxacin could be transferred by electroporation, and a similar 4,270-bp plasmid was found in all transformants. By sequence analysis, the plasmid was found to carry an open reading frame that had similarities to other qnr genes and that encoded a 214-amino-acid pentapeptide repeat protein. This gene, designated qnrD, showed 48% similarity to qnrA1, 61% similarity to qnrB1, and 41% similarity to qnrS1. Further subcloning of the qnrD coding region into the constitutively expressed tetA gene of vector pBR322 showed that the gene conferred an increase in the MIC of ciprofloxacin by a factor of 32 (from an MIC of 0.002 to an MIC of 0.06 μg/ml). For comparison, qnrA1 and qnrS1 were also subcloned into pBR322 and transformed into DH10B cells, conferring MICs of 0.125 and 0.5 μg/ml, respectively. A phylogenetic analysis of all known qnr sequences was performed and showed that qnrD was more closely related to the qnrB variants but formed an independent cluster. To our knowledge, this is the first description of this qnrD gene.
Twenty-one clinical isolates of Streptococcus pneumoniae showing reduced susceptibility or resistance to fluoroquinolones were characterized by serotype, antimicrobial susceptibility, and genetic analyses of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE. Five strains were resistant to three or more classes of antimicrobial agents. In susceptibility profiles for gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, ofloxacin, sparfloxacin, and trovafloxacin, 14 isolates had intermediate- or high-level resistance to all fluoroquinolones tested except gemifloxacin (no breakpoints assigned). Fluoroquinolone resistance was not associated with serotype or with resistance to other antimicrobial agents. Mutations in the QRDRs of these isolates were more heterogeneous than those previously reported for mutants selected in vitro. Eight isolates had amino acid changes at sites other than ParC/S79 and GyrA/S81; several strains contained mutations in gyrB, parE, or both loci. Contributions to fluoroquinolone resistance by individual amino acid changes, including GyrB/E474K, ParE/E474K, and ParC/A63T, were confirmed by genetic transformation of S. pneumoniae R6. Mutations in gyrB were important for resistance to gatifloxacin but not moxifloxacin, and mutation of gyrA was associated with resistance to moxifloxacin but not gatifloxacin, suggesting differences in the drug-target interactions of the two 8-methoxyquinolones. The positions of amino acid changes within the four genes affected resistance more than did the total number of QRDR mutations. However, the effect of a specific mutation varied significantly depending on the agent tested. These data suggest that the heterogeneity of mutations will likely increase as pneumococci are exposed to novel fluoroquinolone structures, complicating the prediction of cross-resistance within this class of antimicrobial agents.
The genes encoding the ParC and ParE subunits of topoisomerase IV of Streptococcus pneumoniae, together with the region encoding amino acids 46 to 172 (residue numbers are as in Escherichia coli) of the pneumococcal GyrA subunit, were partially characterized. The gyrA gene maps to a physical location distant from the gyrB and parC loci on the chromosome, whereas parC is closely linked to parE. Ciprofloxacin-resistant (Cpr) clinical isolates of S. pneumoniae had mutations affecting amino acid residues of the quinolone resistance-determining region of ParC (low-level Cpr) or in both quinolone resistance-determining regions of ParC and GyrA (high-level Cpr). Mutations were found in residue positions equivalent to the serine at position 83 and the aspartic acid at position 87 of the E. coli GyrA subunit. Transformation experiments suggest that ParC is the primary target of ciprofloxacin. Mutation in parC appears to be a prerequisite before mutations in gyrA can influence resistance levels.
The mutations that are responsible for fluoroquinolone resistance in the gyrA, gyrB, parC, and parE genes of Salmonella enterica serovar Typhi and serovar Paratyphi A were investigated. The sequences of the quinolone resistance-determining region of the gyrA gene in clinical isolates which showed decreased susceptibilities to fluoroquinolones had a single mutation at either the Ser-83 or the Asp-87 codon, and no mutations were found in the gyrB, parC, and parE genes.
Fluoroquinolone resistance in Streptococcus pyogenes has been described only anecdotally. In this study we describe two invasive ciprofloxacin-resistant S. pyogenes isolates (ciprofloxacin MICs, 8 mg/liter), one of which shows evidence of interspecies recombination. The quinolone resistance-determining regions of gyrA and parC were sequenced. In both isolates, there was no evidence for an efflux pump and no mutation in gyrA. Both isolates had an S79F mutation in parC that is known to confer fluoroquinolone resistance. In addition, a D91N mutation in parC, which is not related to fluoroquinolone resistance but is a feature of the parC sequence of Streptococcus dysgalactiae, was found in one isolate. The parC nucleotide sequence of that isolate showed greater diversity than that of S. pyogenes. A GenBank search and phylogenetic analysis suggest that this isolate acquired resistance by horizontal gene transfer from S. dysgalactiae. Statistical testing for recombination confirmed interspecies recombination of a 90-bp sequence containing the S79F mutation from S. dysgalactiae. For the other isolate, we could confirm that it acquired resistance by spontaneous mutation by identifying the susceptible ancestor in an outbreak setting.