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 Denmark surveillance of the in vitro susceptibility to ciprofloxacin of Neisseria gonorrhoeae was established in 1990. The proportion of N. gonorrhoeae strains with decreased susceptibility or resistance to ciprofloxacin (MIC ≥ 0.06 μg/ml) was low (0.3 to 2.3%) up to 1995. Between 1995 and 1998 the rate of less-susceptible and resistant strains rose from 6.9 to 13.2%. Among ciprofloxacin-resistant strains (MIC ≥ 1 μg/ml), 81% were highly resistant (MIC ≥ 4 μg/ml). Thirty-five N. gonorrhoeae strains (40 isolates) for which ciprofloxacin MICs were 4 to 32 μg/ml were investigated for the frequency and patterns of mutations within the gyrA and parC genes. The quinolone resistance-determining regions of the gyrA and parC genes were amplified by PCR, and the amplicons were directly sequenced. Alterations at Ser-91 and Asp-95 in GyrA and a single or double alteration in ParC were identified in 32 strains (91%). Ser-91-to-Phe and Asp-95-to-Gly alterations in GyrA were detected in 28 strains (80%). The most common ParC alteration, Asp-86 to Asn, was found in 19 strains (54%). The strains were analyzed for genetic relationship by pulsed-field gel electrophoresis (PFGE). The analysis showed that nine strains with the same mutation pattern in the gyrA and parC genes, originating from different geographical areas over 3 years, had the same PFGE patterns after SpeI as well as NheI digestion (only one strain with one band difference in the NheI pattern), suggesting that a resistant clone had spread worldwide. The results from this study strongly suggest that double gyrA mutations plus a parC mutation(s) play an important role in the development of high-level fluoroquinolone resistance in N. gonorrhoeae.
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.
The in vitro activities of ciprofloxacin, trovafloxacin, moxifloxacin, and grepafloxacin against 174 strains of Neisseria gonorrhoeae isolated in Sydney, Australia, were determined. The strains included 84 quinolone-less-sensitive and -resistant N. gonorrhoeae (QRNG) strains for which ciprofloxacin MICs were in the range of 0.12 to 16 μg/ml. The QRNG included strains isolated from patients whose infections were acquired in a number of countries, mostly in Southeast Asia. The gyrA and parC quinolone resistance-determining regions (QRDR) of 18 selected QRNG strains were sequenced, and the amino acid mutations observed were related to the MICs obtained. The activities of moxifloxacin and grepafloxacin against QRNG were comparable to that of ciprofloxacin. Trovafloxacin was more active than the other quinolones against some but not all of the QRNG strains. Increments in ciprofloxacin resistance occurred in a step-wise manner with point mutations initiated in gyrA resulting in amino acid alterations Ser91-to-Phe, Ser91-to-Tyr, Asp95-to-Gly, and Asp95-to-Asn. Single gyrA changes correlated with ciprofloxacin MICs in the range 0.12 to 1 μg/ml. The Ser91 changes in GyrA were associated with higher MICs and further QRDR changes. QRNG strains for which ciprofloxacin MICs were greater than 1 μg/ml had both gyrA and parC QRDR point mutations. ParC alterations were seen in these isolates only in the presence of GyrA changes and comprised amino acid changes Asp86-to-Asn, Ser87-to-Asn, Ser87-to-Arg, Ser88-to-Pro, Glu91-to-Lys, and Glu91-to-Gln. QRNG strains for which MICs were in the higher ranges had double GyrA mutations, but again only with accompanying ParC alterations. Not only did the nature and combination of GyrA and ParC changes influence the incremental increases in ciprofloxacin MICs, but they seemingly also altered the differential activity of trovafloxacin. Our findings suggest that the newer quinolones of the type examined are unlikely to be useful replacements for ciprofloxacin in the treatment of gonorrhea, particularly where ciprofloxacin MICs are high or where resistance is widespread.
BACKGROUND AND OBJECTIVES: Recently a rapid decrease in the susceptibility of Neisseria gonorrhoeae isolates to fluoroquinolones has occurred and gonococcal fluoroquinolone resistance is now a significant problem in the treatment of gonorrhoea in Japan. Thus, in order to investigate the quinolone resistance mechanisms in clinical isolates of N gonorrhoeae we studied an alteration in the DNA gyrase subunit A (GyrA) which is well-known as a common mechanism of bacterial quinolone resistance. MATERIALS AND METHODS: Four clinical isolates of N gonorrhoeae resistant to norfloxacin and 5 strains susceptible to norfloxacin, including 2 clinical isolates and 3 WHO reference strains, were tested in this study. To identify mutations in the GyrA genes of gonococcal strains, polymerase chain reaction and direct DNA sequencing were performed. RESULTS: A single base change (serine codon TCC changed to phenylalanine codon TTC), which resulted in an amino acid change in GyrA at position 91, was identified in all 4 norfloxacin-resistant strains for which the MICs of norfloxacin ranged from 1.0 to 8.0 micrograms/ml, while no mutation within GyrA was detected in 5 norfloxacin-susceptible strains for which the MICs of norfloxacin ranged from 0.004 to 0.063 microgram/ml. CONCLUSIONS: The results from this study suggest that the serine-91 to phenylalanine substitution in GyrA is probably an essential mutation in fluoroquinolone resistance in clinical isolates of N gonorrhoeae.
Despite the rapid spread of antibiotic resistance among gonococci worldwide, limited reports are available from Brazilian locations. In the present study, 25 quinolone-resistant Neisseria gonorrhoeae (QRNG) strains isolated in Rio de Janeiro, Brazil, were characterized by phenotypic and molecular methods, including analysis of mutations in the gyrA and parC genes. They represented 16.5% of the N. gonorrhoeae isolates obtained during a survey performed from 2006 to 2010. A trend for increasing resistance to ciprofloxacin was observed in the period investigated. The most prevalent pattern of mutation observed among QRNG isolates, Ser-91 to Phe and Asp-95 to Gly in gyrA and Ser-87 to Arg in parC, was detected in 40% of the isolates exhibiting MICs ranging from 4 to >32 μg/ml. Rare types of mutations were found in the gyrA gene (Gln-102 to His [12%] and Asp-95 to Tyr [4%]) and in the parC gene (Ser-88 to Thr [4%]). The genetic relationship of the QRNG isolates, evaluated by pulsed-field gel electrophoresis, suggested that the increase in the frequencies of the QRNG isolates in Rio de Janeiro, Brazil, may have arisen as a result of simultaneous spread of two clonal groups. The results also indicate that fluoroquinolones may no longer be used as first line antibiotics for the treatment of gonorrhea in Rio de Janeiro, and that programs for antimicrobial susceptibility surveillance of N. gonorrhoeae should also be implemented in other regions of Brazil.
A mismatch amplification mutation assay (MAMA) was developed for identification of point mutations in quinolone resistance-determining region (QRDR) of gyrA at codons 91 and 95. MAMA PCR was used to detect mutations at codons 91 and 95 of gyrA in 117 Neisseria gonorrhoeae isolates (with ciprofloxacin MICs of 0.004 to >32 μg/ml) from Bangladesh during 1997 to 2001. The QRDR regions of the gyrA genes from 31 randomly selected isolates were sequenced, and the results were compared with those of MAMA PCR. Using mismatch PCR, a mutation at Ser91 could be detected in all 27 (resistant and intermediate) isolates, and an Asp95-to-Gly95 mutation could be detected in all 15 isolates, as detected by sequencing. MAMA PCR offers a simple, inexpensive, rapid, and easier alternative for detection of point mutations in fluoroquinolone resistance in N. gonorrhoeae.
Decreased susceptibility to fluoroquinolones has become a major problem for the successful therapy of human infections caused by Salmonella enterica, especially the life-threatening typhoid and paratyphoid fevers.
By using Luminex xTAG beads, we developed a rapid, reliable and cost-effective multiplexed genotyping assay for simultaneously detecting 11 mutations in gyrA, gyrB and parE of S. enterica serovars Typhi and Paratyphi A that result in nalidixic acid resistance (NalR) and/or decreased susceptibility to fluoroquinolones.
This assay yielded unambiguous single nucleotide polymorphism calls on extracted DNA from 292 isolates of Salmonella Typhi (NalR = 223 and NalS = 69) and 106 isolates of Salmonella Paratyphi A (NalR = 24 and NalS = 82). All of the 247 NalR Salmonella Typhi and Salmonella Paratyphi A isolates were found to harbour at least one of the target mutations, with GyrA Phe-83 as the most common one (143/223 for Salmonella Typhi and 18/24 for Salmonella Paratyphi A). We also identified three GyrB mutations in eight NalS Salmonella Typhi isolates (six for GyrB Phe-464, one for GyrB Leu-465 and one for GyrB Asp-466), and mutations GyrB Phe-464 and GyrB Asp-466 seem to be related to the decreased ciprofloxacin susceptibility phenotype in Salmonella Typhi. This assay can also be used directly on boiled single colonies.
The assay presented here would be useful for clinical and reference laboratories to rapidly screen quinolone-resistant isolates of Salmonella Typhi and Salmonella Paratyphi A, and decipher the underlying genetic changes for epidemiological purposes.
genotyping; DNA gyrase; mechanisms of resistance; Salmonella
Mutations in the gyrA gene resulting in amino acid changes at Ser-91 and Asp-95 are significantly associated with decreased susceptibilities to quinolones in Neisseria gonorrhoeae. To detect these mutations, we developed a rapid and simple assay based on amplification of the region of the gyrA gene containing the mutation sites by PCR and digestion of the PCR product with a restriction enzyme. A naturally occurring HinfI restriction site was present in the region containing the Ser-91 codon, and an artificial HinfI restriction site was created in the region containing the Asp-95 codon by the method of primer-specified restriction site modification. The mutations generating alterations at Ser-91 and Asp-95 were detected as restriction fragment length polymorphisms of the PCR products digested with HinfI. Fifty-five clinical strains of N. gonorrhoeae were examined for mutations in the gyrA gene by this method. Mutations at Ser-91 and/or Asp-95 were detected in all the 31 strains in which the mutations had been confirmed by DNA sequencing. Our method allows simultaneous testing of a large number of strains and provides results within 8 h. This rapid and simple assay could be a useful screening device for genetic alterations associated with decreased susceptibilities to quinolones in N. gonorrhoeae and could facilitate epidemiological studies on clinical isolates of N. gonorrhoeae with decreased susceptibilities to quinolones.
Ciprofloxacin, 500 mg, was introduced as the first-line therapy for gonorrhea at St. Mary’s Hospital, London, in 1989, when a surveillance program was initiated to detect the emergence of resistance. Isolates of Neisseria gonorrhoeae from consecutive patients attending the Jefferiss Wing, Genitourinary Medicine Clinic at St. Mary’s Hospital, between 1989 and 1997 have been tested for susceptibility to ciprofloxacin by using an agar dilution breakpoint technique. Isolates considered potentially resistant (MIC, >0.12 μg/ml) were further characterized by determination of the MICs of ciprofloxacin, nalidixic acid, and penicillin, auxotyped and serotyped, and screened for mutations in the DNA gyrase gene, gyrA, and the topoisomerase IV gene, parC. A total of 4,875 isolates were tested. While the majority of isolates were highly susceptible (MIC, ≤0.008 μg of ciprofloxacin/ml), there was a drift toward reduced susceptibility in N. gonorrhoeae isolated between 1993 and 1996 (P < 0.001). In 1997 this drift was reduced but remained above pre-1993 levels. Isolates from 18 patients were classed as potentially resistant (MIC, >0.12 μg/ml); all of these belonged to serogroup B, and NR/IB-1 was the most common auxotype/serovar class. The infections in 14 of the 18 patients were known to be acquired abroad, and 5 were known to result in therapeutic failure. The surveillance program has established that ciprofloxacin is still a highly effective antibiotic against N. gonorrhoeae in this population. However, it has identified a drift in susceptibility which may have resulted from increased usage of ciprofloxacin. High-level resistance has now emerged, although treatment failure is still uncommon.
BACKGROUND AND OBJECTIVES: Gonococcal fluoroquinolone resistance is now a significant problem in Japan. We generated gonococcal mutants resistant to norfloxacin in vitro from norfloxacin sensitive isolates and analysed the contribution of three known mechanisms of quinolone resistance in Neisseria gonorrhoeae. MATERIALS AND METHODS: Three clinical isolates of N gonorrhoeae susceptible to norfloxacin were exposed to increasing concentrations of norfloxacin. To identify mutations in the gyrA and parC genes of the gonococcal mutants, the quinolone resistance determining regions of the gyrA and parC genes were polymerase chain reaction (PCR) amplified and the PCR products were directly sequenced. Norfloxacin accumulation in the gonococcal cells was also measured. RESULTS: The MICs of norfloxacin for three variants containing a single GyrA mutation were 16-fold higher than that for their parent isolates. A variant showing reduced norfloxacin accumulation in the cells, without mutations in the GyrA or ParC proteins, was also less sensitive to norfloxacin, with a 16-fold increase in the MIC, compared with the parent strain. The MIC of norfloxacin for a variant which contained a single GyrA mutation with reduced norfloxacin accumulation in the cells was 128-fold higher than for the parent strain. A variant containing mutations in both GyrA and ParC proteins with reduced accumulation of norfloxacin in the cells showed a 256-fold increase in the norfloxacin MIC compared with the parent strain. There was no variant containing a ParC mutation without the simultaneous presence of a GyrA mutation. CONCLUSIONS: The results from this study suggest that not only a mutation in the gyrA gene but also reduced drug accumulation in cells contributes to the development of fluoroquinolone a mutation in the gyrA gene contributes to a high level of fluoroquinolone resistance in gonococci with decreases in accumulation in cells having an additional but lesser effect.
Five commercial broiler flocks were treated with a fluoroquinolone for a clinically relevant infection. Fresh feces from individual chickens and environmental samples were cultured for campylobacters before, during, and weekly posttreatment until slaughter. Both Campylobacter jejuni and C. coli were isolated during all treatment phases. An increased proportion of quinolone-resistant strains was seen during treatment, and these strains persisted posttreatment. One quinolone-resistant isolate of each species, each serotype, and each phage type from each sample at all treatment phases was examined for its phenotype and mechanism of resistance. Two resistant phenotypes were isolated: Nalr Cipr and Nalr Cips. The majority (269 of 290) of fluoroquinolone-resistant isolates, whether they were C. jejuni or C. coli, had a mutation in gyrA that resulted in the substitution Thr-86→Ile. The other gyrA mutations detected were Thr-86→Ala (n = 17) and Asp-90→Asn (n = 10). The genotypic variation, based on the silent mutations in gyrA identified by the denaturing high-performance liquid chromatography pattern and DNA sequencing, was used to supplement typing data and provided evidence for both the spread of preexisting resistant strains and the selection of spontaneous resistant mutants in treated flocks. Multidrug resistance was significantly (P < 0.01) associated with resistance to ciprofloxacin. Twenty-five percent (73 of 290) of ciprofloxacin-resistant isolates but only 13% (24 of 179) of susceptible isolates were resistant to three or more unrelated antimicrobial agents. In conclusion, quinolone-resistant campylobacters were isolated from commercial chicken flocks in high numbers following therapy with a veterinary fluoroquinolone. Most ciprofloxacin-resistant isolates had the GyrA substitution Thr-86→Ile. Resistant isolates were isolated from the feces of some flocks up to the point of slaughter, which may have consequences for public health.
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.
OBJECTIVES: To establish the in vitro susceptibility of gonococci isolated in the London area to antibiotics in current therapeutic use and to establish a sentinel surveillance system for monitoring trends in antibiotic resistant gonorrhoea in London. METHODS: Isolates of Neisseria gonorrhoeae from consecutive patients attending genitourinary medicine clinics at 10 hospitals in the London area were collected over a 3 month period, May to July 1997. The susceptibility to penicillin, ciprofloxacin, tetracycline, and spectinomycin was determined for each isolate. Isolates exhibiting either plasmid or chromosomally mediated resistance were additionally tested for susceptibility to agents used as alternative treatments including azithromycin, ceftriazone, and ofloxacin. The resistant isolates were also tested for plasmid profiles (penicillinase producing N gonorrhoeae, PPNG), type of tetM determinant (tetracycline resistant N gonorrhoeae, TRNG), and presence of gyrA and parC mutations (quinolone resistant N gonorrhoeae, QRNG). RESULTS: A total of 1133 isolates were collected which represents > 95% of the total gonococci isolated in the 3 months. Plasmid mediated resistance was exhibited by 48 (4.2%) isolates; six (0.5%) were PPNG, 15 (1.3%) were PP/TRNG, and 27 (2.4%) were TRNG. The majority of PPNG (18 of 20 tested) carried the 3.2 MDa penicillinase plasmid whereas the two types of tetM determinant were more evenly distributed. High level resistance to ciprofloxacin was detected in four (0.4%) isolates and double mutations were found in the quinolone resistance determining region (QRDR) of the gyrA gene in three QRNG with MICs of 16 mg/l and a single mutation in one isolate with a MIC of 1 mg/l to ciprofloxacin. No parC mutations were found. Of the remaining 1081 isolates, 86 (8.0%) were chromosomally mediated resistant N gonorrhoeae (CMRNG). CONCLUSIONS: A unique collection of gonococcal isolates has been established which can be used as a baseline for surveillance of susceptibility to antibiotics and for epidemiological purposes.
Forty-two men with gonococcal urethritis were treated with an oral dosage of 200 mg of pazufloxacin, a new fluoroquinolone, three times daily for 3 days. Only 28 of the 42 men (66.7%) had negative culture results for Neisseria gonorrhoeae during follow-up. Of the 42 isolates, 41 could be recultured for antibiotic susceptibility testing and DNA sequencing. In 26 of the 41 isolates (63.4%), GyrA mutations with or without ParC mutations were identified. Among the 26 isolates, 23 contained a single GyrA mutation, 1 contained two GyrA mutations, and 2 contained three mutations including double GyrA and single ParC mutations. A single Ser-91-to-Phe mutation, which was detected in 14 of the 26 isolates, was the most common GyrA mutation, followed by an Ala-75 to Ser mutation and an Asp-95 to Asn or Gly mutation in GyrA. All three isolates with two or three mutations contained the Ser-91-to-Phe GyrA mutation. Eleven of the 14 isolates with the single Ser-91-to-Phe mutation within GyrA and all 3 isolates with two or three mutations persisted after pazufloxacin treatment. On the other hand, all 15 wild-type and 9 mutant isolates with a substitution at codon Ala-75 or Asp-95 were eradicated. The mean MIC of pazufloxacin for mutants with the single Ser-91-to-Phe mutation in GyrA was 66-fold higher than that for the wild type. The results obtained in this study suggest that a high prevalence of fluoroquinolone-resistant gonococcal isolates with the Ser-91-to-Phe mutation in GyrA reduced the efficacy of pazufloxacin as treatment for gonococcal urethritis.
A highly discriminative and objective genetic characterization of N. gonorrhoeae, which increases our knowledge of strain populations in different geographic areas, is crucial for the development of improved control measures. In the present study, conventional phenotypic characterization and genetic characterization by means of pulsed-field gel electrophoresis (PFGE), sequencing of the entire porB gene, N. gonorrhoeae multiantigen sequence typing (NG-MAST), and pyrosequencing of a quinolone resistance determining region (QRDR) of the gyrA gene of Swedish ciprofloxacin-resistant N. gonorrhoeae serovar IB-10 isolates (n=45) were performed. The genetic characterization identified one widely spread ciprofloxacin-resistant N. gonorrhoeae ST147 strain. In addition, isolates with slightly different genetic characteristics, which presumably reflect the ongoing evolution only, were also identified. All the isolates contained single nucleotide polymorphisms in QRDR of the gyrA gene that are highly correlated with ciprofloxacin resistance. Consequently, comprehensive characterization identified the first confirmed large domestic transmission, mainly among young heterosexuals, of one ciprofloxacin-resistant N. gonorrhoeae strain in Swedish society during 2002–2003. In conclusion, a precise, i.e. genetic, characterization for identification of individual strains is a very valuable support to the crucial active surveillance of the epidemiological characteristics and the antibiotic susceptibility of N. gonorrhoeae in the effective treatment of gonorrhoea.
Neisseria gonorrhoeae; ciprofloxacin resistance; molecular epidemiology; porB gene; NG-MAST
Fluoroquinolones acting equally through DNA gyrase and topoisomerase IV in vivo are considered desirable in requiring two target mutations for emergence of resistant bacteria. To investigate this idea, we have studied the response of Staphylococcus aureus RN4220 to stepwise challenge with sparfloxacin, a known dual-target agent, and with NSFQ-105, a more potent sulfanilyl fluoroquinolone that behaves similarly. First-step mutants were obtained with both drugs but only at the MIC. These mutants exhibited distinctive small-colony phenotypes and two- to fourfold increases in MICs of NSFQ-105, sparfloxacin, and ciprofloxacin. No changes were detected in the quinolone resistance-determining regions of the gyrA, gyrB, grlA, or grlB gene. Quinolone-induced small-colony mutants shared the delayed coagulase response but not the requirement for menadione, hemin, or thymidine characteristic of small-colony variants, a subpopulation of S. aureus that is often defective in electron transport. Second-step mutants selected with NSFQ-105 had gyrA(S84L) alterations; those obtained with sparfloxacin carried a gyrA(D83A) mutation or a novel gyrB deletion (ΔRKSAL, residues 405 to 409) affecting a trypsin-sensitive region linking functional domains of S. aureus GyrB. Each mutation was associated with four- to eightfold increases in MICs of NSFQ-105 and sparfloxacin, but not of ciprofloxacin, which we confirm targets topoisomerase IV. The presence of wild-type grlB-grlA gene sequences in second-step mutants excluded involvement of topoisomerase IV in the small-colony phenotype. Growth revertants retaining mutant gyrA or gyrB alleles were quinolone susceptible, indicating that resistance to NSFQ-105 and sparfloxacin was contingent on the small-colony mutation. We propose that small-colony mutations unbalance target sensitivities, perhaps through altered ATP or topoisomerase levels, such that gyrase becomes the primary drug target. Breaking of target parity by genetic or physiological means eliminates the need for two target mutations and provides a novel mechanism for stepwise selection of quinolone resistance.
The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has resulted in increased interest in the fluoroquinolones (FQs) as antituberculosis agents. To investigate the frequency and mechanisms of FQ resistance in M. tuberculosis, we cloned and sequenced the wild-type gyrA and gyrB genes, which encode the A and B subunits of the DNA gyrase, respectively; DNA gyrase is the main target of the FQs. On the basis of the sequence information, we performed DNA amplification for sequencing and single-strand conformation polymorphism analysis to examine the presumed quinolone resistance regions of gyrA and gyrB from reference strains (n = 4) and clinical isolates (n = 55). Mutations in codons of gyrA analogous to those described in other FQ-resistant bacteria were identified in all isolates (n = 14) for which the ciprofloxacin MIC was > 2 micrograms/ml. In addition, we selected ciprofloxacin-resistant mutants of Mycobacterium bovis BCG and M. tuberculosis Erdman and H37ra. Spontaneously resistant mutants developed at a frequency of 1 in 10(7) to 10(8) at ciprofloxacin concentrations of 2 micrograms/ml, but no primary resistant colonies were selected at higher ciprofloxacin concentrations. Replating of those first-step mutants selected for mutants with high levels of resistance which harbored gyrA mutations similar to those found among clinical FQ-resistant isolates. The gyrA and gyrB sequence information will facilitate analysis of the mechanisms of resistance to drugs which target the gyrase and the implementation of rapid strategies for the estimation of FQ susceptibility in clinical M. tuberculosis isolates.
The MICs of ofloxacin for 743 strains of Escherichia coli isolated from 1988 to 1994 were determined by testing. The strains were from patients with urinary tract infections complicated by functional or anatomical disorders of the urinary tract. Those determined to be ofloxacin resistant (MIC, > or =12.5 microg/ml) comprised 3 of 395 strains (1.3%) from the 1988 to 1990 group, 2 of 166 strains (1.2%) from the 1991 to 1992 group, and 7 of 182 strains (3.8%) from the 1993 to 1994 group. The incidence of resistant strains increased significantly during this period. The percentage of isolates with moderately decreased susceptibilities to ofloxacin (MIC, 0.39 to 3.13 microg/ml) also rose during the same period. To determine the incidence of gyrA mutations in urinary-tract-derived strains of E. coli, we developed a simple and rapid assay based on PCR amplification of the region of the gyrA gene containing the mutation sites followed by digestion of the PCR product with a restriction enzyme. Using this assay, we examined all 182 strains isolated in 1993 and 1994 for the presence of mutations at Ser-83 and Asp-87 in the gyrA gene. Of these strains, 33 (18.1%) had mutations in the gyrA gene. The incidences of mutations at Ser-83, at Asp-87, and at both codons were 10.4 (19 strains), 4.4 (8 strains), and 3.3% (6 strains), respectively. To determine the correlation of the mutations in the gyrA gene with susceptibilities to quinolones (nalidixic acid, ofloxacin, norfloxacin, and ciprofloxacin), we further examined 116 strains for which the MICs of ofloxacin were > or =0.2 microg/ml that were chosen from the isolates in the 1988 to 1992 group. The MICs of nalidixic acid for the strains without mutations at either Ser-83 or Asp-87 were < or =25 microg/ml, whereas those for the strains with single mutations or double mutations were from 50 to >800 microg/ml. For the fluoroquinolones, significant differences in the distributions of the MICs were observed among the strains without mutations, with single mutations, and with double mutations. The accumulation of mutations in the gyrA gene was associated with an increase in fluoroquinolone resistance. Ofloxacin MICs for the majority of the strains with single and double mutations were 0.39 to 3.13 and 6.25 to 100 microg/ml, respectively. This study demonstrates a chronological increase in the percentage of not only highly fluoroquinolone-resistant strains, corresponding to those with double mutations in the gyrA gene, but also strains with moderately decreased susceptibilities to fluoroquinolones, corresponding to those with single mutations. This increase in the incidence of strains with a single mutation in the gyrA gene portends a further increase in the incidence of strains with clinically significant resistance to fluoroquinolones.
The Pseudomonas aeruginosa DNA gyrase gyrA gene was cloned and sequenced from strain PAO1. An open reading frame of 2,769 bp was found; it coded for a protein of 923 amino acids with an estimated molecular mass of 103 kDa. The derived amino acid sequence shared 67% identity with Escherichia coli GyrA and 54% identity with Bacillus subtilis GyrA, although conserved regions were present throughout the sequences, particularly toward the N terminus. Complementation of an E. coli mutant with a temperature-sensitive gyrA gene with the PAO1 gyrA gene showed that the gene is expressed in E. coli and is able to functionally complement the E. coli DNA gyrase B subunit. Expression of PAO1 gyrA in E. coli or P. aeruginosa with mutationally altered gyrA genes caused a reversion to wild-type quinolone susceptibility, indicating that the intrinsic susceptibility of the PAO1 GyrA to quinolones is comparable to that of the E. coli enzyme. PCR was used to amplify 360 bp of P. aeruginosa gyrA encompassing the so-called quinolone resistance-determining region from ciprofloxacin-resistant clinical isolates from patients with cystic fibrosis. Mutations were found in three of nine isolates tested; these mutations caused the following alterations in the sequence of GyrA: Asp at position 87 (Asp-87) to Asn, Asp-87 to Tyr, and Thr-83 to Ile. The resistance mechanisms in the other six isolates are unknown. The results of the study suggested that mechanisms other than a mutational alteration in gyrA are the most common mechanism of ciprofloxacin resistance in P. aeruginosa from the lungs of patients with cystic fibrosis.
We examined the potential for the development of fluoroquinolone resistance in Neisseria meningitidis by cultivating two clinical isolates of meningococci in the presence of concentrations of ciprofloxacin at and about the predetermined MIC. The quinolone resistance determining regions (QRDRs) of gyrA and parC of 50 stable quinolone-resistant mutants derived in vitro were sequenced and compared with QRDR alterations reported in clinical isolates of quinolone-resistant meningococci and gonococci. MICs to ciprofloxacin and trovafloxacin were determined and sequence changes were correlated with quinolone MICs. Ciprofloxacin and trovafloxacin MICs of the in vitro-derived quinolone-resistant mutants ranged up to 16 mg/liter. Single GyrA alterations were the first change detected and were accompanied by raised MICs, followed by double GyrA changes and still higher MICs. MICs increased further as single ParC substitutions appeared and these were always in the presence of a single or double GyrA change. GyrA changes occurred at positions 91 and 95 with substitutions of Asp-95→Asn and Thr-91→Ala and Ile. Changes in the parC QRDR occurred at positions 85, 86, and 91 with four substitutions, Gly-85→Asp, Asp-86→Asn, Glu-91→Gly, and Glu-91→Lys, detected. The nature of the individual QRDR substitution appeared to influence the level of quinolone resistance expressed, and this varied with the quinolone agent examined. Close similarities occurred between the sequence and nature of QRDR changes in clinical and in vitro-generated quinolone-resistant mutants and with those previously reported for clinical and in vitro-generated quinolone-resistant gonococci. This suggests that quinolone resistance in meningococci may arise in the same manner and reach similar levels in vivo to those seen in quinolone-resistant Neisseria gonorrhoeae.
We have previously described a clinical isolate of Escherichia coli (Q2) that is highly resistant to fluoroquinolones (MIC of ciprofloxacin, 16 micrograms/ml) but susceptible to nalidixic acid (MIC of nalidixic acid, 4 micrograms/ml) (N. Moniot-Ville, J. Guibert, N. Moreau, J.F. Acar, E. Collatz, and L. Gutmann, Antimicrob. Agents Chemother. 35:519-523, 1991). Transformation of strain Q2 with a plasmid carrying the wild-type gyrA gene from E. coli K-12(pAFF801) resulted in a 32-fold decrease in the MIC of ciprofloxacin, suggesting that at least one mutation in gyrA was involved in the resistance of Q2. Intragenic gyrA fragments of 668 and 2,500 bp from strain Q2 were amplified by the polymerase chain reaction. We sequenced the 668-bp fragment and identified a single novel point mutation (transition from G to A at position 242), leading to an amino acid substitution (Gly-81 to Asp) in the gyrase A subunit. We constructed hybrid plasmids by substituting either the 668-bp fragment or the 2,500-bp fragment from Q2 DNA, both of which contained the gyrA point mutation, for the corresponding fragments in wild-type gyrA (2,625 bp) of E. coli K-12. When introduced into E. coli KNK453 (gyrA temperature sensitive), both plasmids conferred an eightfold increase in the MIC of ciprofloxacin, but only a twofold increase in the MIC of nalidixic acid. When introduced into E. coli Q2, neither plasmid conferred any change in the MICs of ciprofloxacin or nalidixic acid, suggesting that only the point mutation found in gyrA was involved in the resistance that we observed.
A LightCycler-based PCR-hybridization gyrA mutation assay (GAMA) was developed to rapidly detect gyrA point mutations in multiresistant (MR) Salmonella enterica serotype Typhimurium DT104 with decreased susceptibility to ciprofloxacin (MIC, 0.25 to 1.0 mg/liter). Ninety-two isolates (49 human, 43 animal) were tested with three individual oligonucleotide probes directed against an Asp-87-to-Asn (GAC→AAC) mutation, an Asp-87-to-Gly (GAC→GGC) mutation, and a Ser-83-to-Phe (TCC→TTC) mutation. Strains homologous to the probes could be distinguished from strains that had different mutations by their probe-target melting temperatures. Thirty-seven human and 30 animal isolates had an Asp-87-to-Asn substitution, 6 human and 6 animal isolates had a Ser-83-to-Phe substitution, and 5 human and 2 animal isolates had an Asp-87-to-Gly substitution. The remaining six strains all had mismatches with the three probes and therefore different gyrA mutations. The sequencing of gyrA from these six isolates showed that one human strain and two animal strains had an Asp-87-to-Tyr (GAC→TAC) substitution and two animal strains had a Ser-83-to-Tyr (TCC→TAC) substitution. One animal strain had no gyrA mutation, suggesting that this isolate had a different mechanism of resistance. Fifty-eight of the strains tested were indistinguishable by several different typing methods including antibiograms, pulsed-field gel gel electrophoresis, and plasmid profiling, although they could be further subdivided according to gyrA mutation. This study confirmed that MR DT104 with decreased susceptibility to ciprofloxacin from humans and food animals in England and Wales may have arisen independently against a background of clonal spread of MR DT104.
In the study, the ciprofloxacin resistance rate was 100%. High-level ciprofloxacin resistance rate was 63.55%. Sixteen different mutation patterns involved in the formation of ciprofloxacin resistance were identified. The most prevalent were patterns P7 (25.2%), P8 (15.0%), P9 (11.2%), P1 (10.3%), and P5 (10.3%). All of the 107 NG isolates analyzed for mutations in the study have demonstrated a change of Ser-91 → Phe in the gyrA gene, and all except one have demonstrated a change in position 95 of the amino acid sequence. All of the 68 high-level QRNG isolates had double mutations in gyrA gene combined with a single or two mutations in parC gene. It is most important that a new mutation site of Ile-97 → Met in gyrA and a new mutation of Leu-106 → Ile in parC were found in the study, both leading to high-level ciprofloxacin resistance (MIC values, 8 μg/mL, 32 μg/mL, respectively). Therefore, we confim that gyrA mutations are necessary for the fluoroquinolone resistance phenotype and parC mutations are correlated intimately with high-level fluoroquinolone resistance. In China fluoroquinolone resistance in Neisseria gonorrhoeae strains is very serious and the new mutation sites in the fluoroquinolone resistance-determining regions emerge more and more quickly. Hence, in China fluoroquinolones, which are used to treat gonorrhoea presently, should be substituted by a new antibiotics.
Neisseria gonorrhoeae; ciprofloxacin resistance; high-level; molecular analysis
A PCR-sequencing assay was evaluated for direct detection of mutations in the quinolone resistance-determining region (QRDR) of gyrase A (gyrA) gene in fluoroquinolone-resistant Mycobacterium tuberculosis in respiratory specimens. As determined by gyrA QRDR analysis, complete concordance of genotypic and phenotypic fluoroquinolone resistance was demonstrated. Our results indicate that the assay is a rapid and reliable method for the diagnosis of fluoroquinolone-resistant tuberculosis, facilitating timely clinical management and public health control. Using the assay, we detected a novel gyrA Ala74Ser mutation in M. tuberculosis directly from sputum specimens. The functional effect of the Ala74Ser mutant was verified through the study of the DNA supercoiling inhibitory activity of fluoroquinolones against the recombinant gyrase. The drug-mediated gyrase-DNA cleavage complex model suggests perturbation of the gyrA-gyrA dimer interface caused by the Ala74Ser mutation probably disturbs the putative quinolone binding pocket and leads to the reduction of the drug binding affinity. A number of gyrA mutations (Glu21Gln, Ser95Thr, and Gly668Asp) were also characterized to be natural polymorphisms not associated with fluoroquinolone resistance.