Change In GyrA Research Articles

Prevalence of extended-spectrum beta-lactamase-producing Escherichia coli isolates in faecal samples of broilers

Seventy-six faecal samples were obtained from broilers at slaughterhouse level in Portugal. Samples were inoculated on cefotaxime-supplemented Levine agar plates. Cefotaxime-resistant Escherichia coli isolates were recovered from 32 samples (42.1%), obtaining a total of 34 E. coli isolates (one or two isolates per sample). Susceptibility to 16 antibiotics was studied by disk diffusion method, and 85% of the isolates presented a phenotype of multi-resistance that included antimicrobial agents of at least four different families. Extended-spectrum-beta-lactamases (ESBL) of the TEM and CTX-M groups were detected in 31 ESBL-positive E. coli isolates. Twenty-six isolates harboured the bla TEM-52 gene and two of them also harboured bla TEM-1b. The bla CTX-M-14 gene was identified in three isolates (in association with bla TEM-1b in one of them), and bla CTX-M-32 was demonstrated in two additional isolates. Three of the 34 cefotaxime-resistant isolates (9%) did not produce ESBLs, and two of them presented mutations at positions −42 (C → T), −18 (G → A), −1 (C → T), and +58(C → T) of the promoter/attenuator region of ampC gene. tet(A) and/or tet(B) genes were detected in all 34 tetracycline-resistant isolates, aadA in all 26 streptomycin-resistant isolates; cmlA in 3 of 6 chloramphenicol-resistant isolates, and aac(3)-II or aac(3)-I + aac(3)-IV genes in all 4 gentamicin-resistant isolates. Different combinations of sul1, sul2 and sul3 genes were demonstrated among the 22 trimethoprim–sulfamethoxazole-resistant isolates. Amino acid changes in GyrA and ParC proteins were identified in all 18 ciprofloxacin-resistant isolates. The results of this study indicate that the intestinal tract of healthy poultry is a reservoir of ESBL-positive E. coli isolates.

In vitro-selected resistance to fluoroquinolones in two Brucella strains associated with mutational changes in gyrA

In the present study, Brucella melitensis biovar Abortus 2308 and Brucella abortus 3196 biotype 5 reference strains, which are susceptible to fluoroquinolones, became in vitro-resistant to fluoroquinolones by culture in trypticase soy agar. The quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes of the two reference strains were analysed by polymerase chain reaction sequencing analysis to obtain the wild-type sequence. These sequences were then compared with the corresponding sequences of four in vitro-selected fluoroquinolone-resistant mutants to characterise mutations associated with resistance. Sequencing of the ofloxacin-selected resistant mutant 2308 revealed a transition of GAT to AAT (corresponding to position 87 of Escherichia coli gyrA), leading to substitution of Asp91 → Asn, whilst at the same position the ciprofloxacin-selected resistant mutant 2308 revealed a transition of GAT to TAT (corresponding to the same position of E. coli as above), leading to substitution of Asp91 → Tyr. The ofloxacin-selected resistant mutant 3196 had a transition of GCT to GTT, generating an amino acid change of Ala87 → Val. Amino acid changes were detected in the portion of the Brucella gyrA gene (Ala71 to Gln110) corresponding to the E. coli gyrA QRDR region (Ala67 to Gln110). Amino acid changes were also detected in Ser83, corresponding to the region where fluoroquinolone-associated amino acid changes are most commonly found in other bacterial species.

Prevalence of antimicrobial resistance and resistance genes in faecal Escherichia coli isolates recovered from healthy pets

Faecal samples of healthy dogs ( n = 39) and cats ( n = 36) obtained in Northern Portugal were seeded on Levine agar plates, and two Escherichia coli isolates per sample were recovered (78 of dogs and 66 of cats). The susceptibility to 16 antimicrobial agents was tested in this series of 144 E. coli isolates. Almost 20% of them showed tetracycline resistance and 12 and 15% presented ampicillin or streptomycin resistance, respectively. The percentage of resistance to the other antimicrobial agents was in all cases below 4%, and no resistant isolates were detected for ceftazidime, imipenem, cefoxitin or amikacin. Two isolates (from one dog) showed cefotaxime-resistance and harboured both the CTX-M-1 and OXA-30 β-lactamases. A bla TEM gene was detected in 12 of 17 ampicillin-resistant isolates, the aac(3)-II gene in the three gentamicin-resistant isolates, aadA in 7 of 22 streptomycin-resistant isolates, and tet(A) and/or tet(B) gene in all 28 tetracycline-resistant isolates. The gene encoding class 1 integrase was detected in six E. coli isolates, including the four trimethoprim-sulfamethoxazole-resistant isolates and those two harbouring CTX-M-1 and OXA-30 β-lactamases; different gene cassette arrangements were identified: dfrA1 + aadA1 (two isolates), dfrA12 + orfF + aadA2 (two isolates) and bla OXA30 + aadA1 (two isolates). One amino acid change in GyrA protein (Ser83Leu or Asp87Tyr) was detected in four nalidixic acid-resistant and ciprofloxacin-susceptible isolates and two amino acid changes in GyrA (Ser83Leu + Asp87Asn) and one in ParC (Ser80Ile) were identified in one nalidixic acid- and ciprofloxacin-resistant isolate. Faecal E. coli isolates of healthy pets could be a reservoir of antimicrobial resistance genes.

Mutant-prevention concentrations of enrofloxacin for Escherichia coli isolates from chickens

To investigate the development of enrofloxacin resistance among Escherichia coli isolates obtained from chickens by determining mutant-prevention concentrations (MPCs) and sequence the quinolone resistance-determining regions (QRDRs) of gyrA and parC genes in selected isolates. 15 chicken-derived E coli isolates. For all isolates, MPC and minimal inhibition concentration (MIC) of enrofloxacin were determined. The MPCs and maximum serum drug concentrations attained with enrofloxacin doses recommended for treatment of E coli infections in chickens were compared. Mutation frequencies and QRDR sequence changes in gyrA and parC were also determined. In 2 of 15 E coli strains, MPCs were low (0.016 and 0.062 microg/mL), MPC:MIC ratios were 2 and 4, and the GyrA and ParC proteins had no mutations. In 9 susceptible isolates with a GyrA point mutation, MPCs ranged from 2 to 16 microg/mL. For isolates with double mutations in GyrA and a single mutation in ParC, MPCs were > 32 microg/mL (several fold greater than the maximal plasma concentration of enrofloxacin in chickens); mutation frequencies were also much lower, compared with frequencies for single-mutation isolates. For E coli infections of chickens, MPC appears to be useful for determining enrofloxacin-dosing strategies. The high MPC:MIC ratio may result in enrofloxacin-treatment failure in chickens infected with some wild-type gyrA E coli isolates despite the isolates' enrofloxacin susceptibility (MICs 0.125 to 1 microg/mL). For infections involving isolates with high MPCs, especially those containing mutations in gyrA and parC genes, treatment with combinations of antimicrobials should be adopted.

Mutations in the gyrA and parC genes associated with fluoroquinolone resistance in clinical isolates of Citrobacter freundii.

We determined partial sequences of the gyrA and parC genes of Citrobacter freundii type strain, and then examined 38 C. freundii clinical strains isolated from patients with urinary tract infections for the association of alterations in GyrA and ParC with susceptibility to fluoroquinolones. Our results suggest that in C. freundii DNA gyrase may be a primary target of quinolones, that an amino acid change at Thr-83 or Asp-87 in GyrA is sufficient to decrease susceptibility to fluoroquinolones, and that accumulation of changes in GyrA with the simultaneous presence of an alteration at Ser-80 or Glu-84 in ParC may be associated with the development of high-level fluoroquinolone resistance in C. freundii clinical isolates.

In Vitro Assessment of the Further Potential for Development of Fluoroquinolone Resistance in Neisseria meningitidis

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.

Mutant prevention concentrations for single-step fluoroquinolone-resistant mutants of wild-type, efflux-positive, or ParC or GyrA mutation-containing Streptococcus pneumoniae isolates.

Three fluoroquinolone-susceptible and five fluoroquinolone-resistant (two with ParC Ser79Phe mutations, one with a GyrA Ser81Phe mutation, and two that were efflux positive) Streptococcus pneumoniae isolates were exposed to one, two, four, eight, and sixteen times the MICs of ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin. Mutational frequencies were calculated at each multiple of the MIC for which growth was observed. Mutant prevention concentrations (MPCs) and the multiple of the MIC at the MPC (MP(MIC)) were evaluated. All resulting mutants were sequenced for quinolone resistance-determining region changes in GyrA and ParC and were evaluated for reserpine-sensitive efflux. The MPC order was generally ciprofloxacin > levofloxacin > gatifloxacin > moxifloxacin > gemifloxacin. The MP(MIC) order varied depending on the genetic constitution of the original isolates from which the mutants were generated. For those mutants created from fluoroquinolone-susceptible isolates (those that had wild-type ParC and GyrA and were efflux negative), the MP(MIC) order was ciprofloxacin = moxifloxacin > gemifloxacin > levofloxacin > gatifloxacin. The MP(MIC)s of each fluoroquinolone for mutants created from isolates with a ParC mutation (with wild-type GyrA and efflux negative) were similar. A similar occurrence was observed with the mutants created from the efflux-positive isolates (with wild-type ParC and GyrA). The MP(MIC) order for the mutants created from the isolate with a GyrA mutation (with wild-type ParC and efflux negative) was ciprofloxacin = gemifloxacin > levofloxacin = moxifloxacin > gatifloxacin. Gatifloxacin, levofloxacin, and moxifloxacin may be intrinsically more able to prevent the development of resistance by fluoroquinolone-susceptible isolates, isolates that are efflux positive, or isolates that carry a GyrA mutation. However, once a ParC mutation is present, the MPC increases dramatically for all fluoroquinolones.

Patterns of quinolone susceptibility in Campylobacter jejuni associated with different gyrA mutations

To investigate the diversity of genetic mutation in the quinolone resistance-determining region (QRDR) of gyrA in clinical isolates and laboratory-derived mutants of Campylobacter jejuni resistant to ciprofloxacin (CipR) and to determine the influence of this mutation on the susceptibility of the organisms to different quinolone antibiotics. Laboratory-derived CipR mutants were obtained from C. jejuni NCTC 11 168 and six quinolone-sensitive faecal isolates (parent prototypes) grown in sub-inhibitory concentrations of ciprofloxacin. Initial mutants found to be CipR were designated 'primary mutants' and subjected to a repeat of this process to select 'secondary mutants' with increased resistance. The susceptibility of the mutants and an additional six clinical isolates of CipR C. jejuni to seven quinolone antibiotics was determined by measuring their MICs. The QRDR of gyrA in all strains was amplified by PCR, sequenced and compared with that of the L04566 C. jejuni gyrA gene. All six CipR clinical isolates contained a Thr-86-Ile mutation. This was also the commonest mutation found amongst the laboratory derived CipR strains. Other derived mutations in the in vitro derived CipR group included Asp-90-Asn, Thr-86-Ala, and a previously unreported double mutation, Asp-85-Tyr and Thr-86-Ile. Strains with the Thr-86-Ile mutation had the highest MICs to seven different quinolones. CipR strains with other single mutations had a lower range of MICs. There were no additional QRDR mutational changes detected in secondary mutants even where MICs to the fluoroquinolones were higher than in primary mutants. Thr-86-Ile mutations were common in both clinical and laboratory derived CipR strains. Other mutations found amongst the latter strains were more sensitive to the fluoroquinolones. Different QRDR changes in gyrA differentially affected the susceptibility of CipR C. jejuni to the various fluoroquinolones.

Laboratory detection of Haemophilus influenzae with decreased susceptibility to nalidixic acid, ciprofloxacin, levofloxacin, and moxifloxacin due to GyrA and ParC mutations.

The detection of clinical isolates with decreased fluoroquinolone susceptibilities and a resistance mechanism is of epidemiological and clinical interest. We studied the susceptibilities of 62 clinical isolates and 2 American Type Culture Collection reference strains of Haemophilus influenzae to ciprofloxacin, levofloxacin, moxifloxacin, and nalidixic acid by the microdilution and disk diffusion methods. The ciprofloxacin MICs for 34 of the isolates were >/=0.12 micro g/ml (range, 0.12 to 32 micro g/ml), and the ciprofloxacin MICs for 28 matched control isolates were </=0.06 micro g/ml. In addition, we sequenced the quinolone resistance-determining regions (QRDRs) of gyrA and parC of all strains. The log(2) MICs of all quinolones were plotted against the inhibition zone diameters. The MICs and inhibition zone diameters selected to screen for the resistance mechanism were based on the susceptibility distribution data and the presence or absence of amino acid changes in the QRDRs of GyrA and ParC. Strains for which ciprofloxacin MICs were </=0.06 micro g/ml, levofloxacin and moxifloxacin MICs were </=0.03 micro g/ml, and nalidixic acid MICs were </=2.0 micro g/ml lacked modifications in the QRDR of GyrA. In contrast, all strains for which ciprofloxacin, levofloxacin, and moxifloxacin MICs were >/=0.5 micro g/ml and the vast majority of those for which nalidixic acid MICs were >/=32 micro g/ml exhibited amino acid changes in GyrA and ParC. Nalidixic acid and the other three fluoroquinolones studied could be used to screen H. influenzae isolates for the detection of decreased susceptibilities to quinolones due to the acquisition of two amino acid changes in the QRDRs of GyrA and ParC (sensitivity, >95%; specificity, >80%).

Fluoroquinolone-resistant Neisseria gonorrhoeae isolates from Russia: molecular mechanisms implicated

During a longitudinal study of the prevalence of antimicrobial resistance in Neisseria gonorrhoeae, a number of high-level fluoroquinolone-resistant isolates were obtained from the sexually transmitted diseases clinic in the Moscow region in 2002. The aim of the present study was to determine the molecular mechanisms of resistance and to assess the clonal relationship of these strains For the 32 clinical strains of N. gonorrhoeae studied, the MIC values were determined for four fluoroquinolones. The gyrA, parC, por and mtrR genes were studied for the presence of mutations associated with fluoroquinolone resistance. We detected strains of N. gonorrhoeae showing high-level resistance to fluoroquinolones (21 strains, with MICs 1-32 mg/L). Mutations in gyrA and parC known to cause fluoroquinolone resistance were detected in a majority of strains. There were four strains (among 21) without known changes in gyrA and parC. However, amino acid changes in the Por protein and mutations in the promoter or encoding region of the mtrR gene were detected in three of them. One strain had no alteration in gyrA, parC, por or mtrR. The present study documents the first case of fluoroquinolone-resistant N. gonorrhoeae in Russia.

Effect of the efflux pump inhibitor Phe-Arg- -naphthylamide on the MIC values of the quinolones, tetracycline and chloramphenicol, in Escherichia coli isolates of different origin

Sir, The main mechanisms of quinolone resistance in Escherichia coli include mutations in the gyrA and parC target genes and decreased intracellular accumulation.1 Active efflux pumps are important for intrinsic and acquired antibiotic resistance, and overexpression of efflux pumps affecting quinolones is becoming increasingly common in E. coli. Recently, efflux pump inhibitors have been investigated with a view to improving and potentiating the activity of exported antibiotics.2 Phe-Arg-β-naphthylamide has been described as a broad-spectrum efflux pump inhibitor in Pseudomonas aeruginosa.2 The effect of this inhibitor on fluoroquinolone susceptibilities in Enterobacter aerogenes,3 E. coli,4 P. aeruginosa,2 Acinetobacter baumannii5 and Stenotrophomonas maltophilia5 has also been studied, but not for nalidixic acid and other antimicrobial agents in E. coli. Therefore in this study, we analysed the influence of the Phe-Arg-β-naphthylamide (Sigma) inhibitor on the activity of quinolones, chloramphenicol and tetracycline against a collection of 60 E. coli isolates previously characterized with respect to amino acid changes in both GyrA and ParC proteins.6,7 The collection includes the strain Nor5, an in vitro selected quinolone-resistant mutant, in which the overexpression of MarA, SoxS and AcrA has been shown (J. Vila, B. Peter, J. Sanchez-Cespedes & M. Tario, unpublished data). The remaining 59 isolates were recovered from foods (n = 11), humans (n = 25) and animals (n = 23). Susceptibilities to nalidixic acid, norfloxacin, chloramphenicol, tetracycline and ciprofloxacin were studied by agar dilution in the presence or absence of 20 mg/L of Phe-Arg-β-naphthylamide, in accordance with NCCLS guidelines. All of the 60 E. coli isolates showed an MIC value of >256 mg/L to Phe-Arg-β-naphthylamide. Table 1 shows the effect of 20 mg/L of the inhibitor Phe-Arg-β-naphthylamide on the MIC values of the five tested antibiotics in 60 E. coli isolates with different numbers of amino acid changes in GyrA and T ab le 1 . M IC v al ue s (i n m g/ L ) of th e fi ve te st ed a nt ib io tic s in th e pr es en ce o r ab se nc e of 2 0 m g/ L o f P he -A rg β -n ap ht hy la m id e in a s er ie s of 6 0 E . c ol i i so la te s or ga ni ze d

Mutations in gyrA and parC genes in nalidixic acid-resistant Escherichia coli strains from food products, humans and animals.

Mutations in quinolone targets were analysed in 80 unrelated nalidixic acid-resistant (NALR) Escherichia coli strains whose nalidixic acid and ciprofloxacin MICs ranged from 32 to >256 mg/L and 0.03-64 mg/L, respectively. These strains were isolated from food products (23) and faecal samples from humans (15) and healthy animals (42). Thirteen nalidixic acid-susceptible (NALS) E. coli strains were also analysed. Mutations in gyrA and parC genes were studied by PCR and sequencing. No amino acid changes were detected in GyrA or ParC proteins of the 13 NALS strains. A single change in the GyrA protein was detected in all 61 NALR strains with ciprofloxacin MICs </= 2 mg/L with the following substitutions (number of strains): Ser-83-->Leu (54), Ser-83-->Ala (one), Ser-83-->Val (one), Asp-87-->Asn (two), Asp-87-->Tyr (two) and Asp-87-->Gly (one). A double change in GyrA was found in 18 of 19 NALR strains with ciprofloxacin MICs >/= 4 mg/L. Amino acid substitutions were Ser-83-->Leu, with an additional change [Asp-87-->Asn (15), Asp-87-->Tyr (two) or Asp-87-->His (one)]. The remaining strain (ciprofloxacin MIC 4 mg/L) showed a single Ser-83-->Leu substitution. In respect of the ParC protein, a single change at Ser-80 or Glu-84 was found in 25 of 42 strains, with ciprofloxacin MICs ranging from 0.5 to 32 mg/L. A double substitution (Ser-80-->Ile and Glu-84-->Gly) was found in one strain (ciprofloxacin MIC 64 mg/L). No amino acid changes were detected in the GyrB protein of 18 NALR strains.

Antimicrobial susceptibility and molecular characterization of community-acquired methicillin-resistant Staphylococcus aureus

Community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infections are increasing. Since most published data are on nosocomial MRSA, our goal was to identify the antimicrobial susceptibility profile and resistance mechanisms of pretreatment MRSA isolates obtained from adult subjects participating in recent clinical treatment trials of community respiratory infections. Out of 465 S. aureus isolates, 43 were identified as MRSA. Antimicrobial susceptibility testing indicated susceptibility rates to: vancomycin (100%), gentamicin (86%), clindamycin (39%), quinolones (49%), and erythromycin (12%). Among our MRSA isolates, the MLS constitutive phenotype and ermA were more prevalent than the MLS inducible phenotype and ermC. No isolates had ermB or msrA. All ciprofloxacin resistant isolates had an amino acid change in GyrA and GrlA. The relatedness of our MRSA strains was assessed by ribotyping. Our results indicate that MRSA from adult subjects with community respiratory infections have similar antimicrobial susceptibility profiles and resistance mechanisms as nosocomial MRSA, and represent a genetically diverse group.

Selection and genetic characterization of Streptococcus pneumoniae mutants resistant to the des-F(6) quinolone BMS-284756.

Existing quinolones are known to target the type II topoisomerases in bacteria. In order to determine which of these targets are of key importance in Streptococcus pneumoniae treated with BMS-284756 (T-3811ME), a novel des-F(6) quinolone, resistant mutants were selected in several steps of increasing resistance by plating pneumococci on a series of blood agar plates containing serial twofold-increasing concentrations of drug. After incubation, colonies that arose were selected and passaged twice on antibiotic-containing media at the selection level. Mutants generally showed increases in resistance of four- to eightfold over the prior level of susceptibility. Mutants in the next-higher level of resistance were selected from the previous round of resistant mutants. Subsequently, chromosomal DNA was prepared from parental (R6) pneumococci and from at least three clones from each of four levels of increasing antibiotic resistance. Using PCR primers, 500- to 700-bp amplicons surrounding the quinolone resistance determining regions (QRDR) of gyrA, gyrB, parC, and parE genes were prepared from each strain. Internal primers were used to sequence both DNA strands in the regions of approximately 400 bp centered on the QRDR. Mutations identified with increasing levels of resistance included changes in GyrA at Ser-81 and Glu-85 and changes in ParC at Ser-79 and Asp-83. Changes in GyrB and ParE were not observed at the levels of resistance obtained in this selection. The resistance to comparator quinolones (levofloxacin, ciprofloxacin, and moxifloxacin) also increased in four- to eightfold steps with these mutations. The intrinsically greater level of antibacterial activity and thus lower MICs of BMS-284756 observed at all resistance levels in this study may translate to coverage of these resistant pneumococcal strains in the clinic.

Correlation of in vitro susceptibilities to newer quinolones of naturally occurring quinolone-resistant Neisseria gonorrhoeae strains with changes in GyrA and ParC.

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 microg/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 microg/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 microg/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.

Cloning and Nucleotide Sequence of the DNA Gyrase ( gyrA ) Gene from Mycoplasma hominis and Characterization of Quinolone-Resistant Mutants Selected In Vitro with Trovafloxacin

We report the cloning and characterization of the gyrA gene of the Mycoplasma hominis DNA gyrase, which was previously shown to be associated with quinolone resistance in this organism. The 2,733-bp gyrA gene encodes a protein of 911 amino acids with a calculated molecular mass of 102.5 kDa. As expected, M. hominis GyrA exhibits higher homology with the GyrA subunits of the gram-positive bacteria Clostridium acetobutylicum, Bacillus subtilis, Streptococcus pneumoniae, and Staphylococcus aureus than with its Escherichia coli counterpart. Knowing the entire sequence of the gyrA gene of M. hominis could be very useful for confirming the role of the GyrA subunit in fluoroquinolone resistance. Twenty-nine mutants of M. hominis were selected stepwise for resistance to trovafloxacin, a new potent fluoroquinolone, and their gyrA, gyrB, parC, and parE quinolone resistance-determining regions were characterized. Three rounds of selection yielded 3 first-step, 12 second-step, and 14 third-step mutants. The first-step mutants harbored a single substitution, Glu460-->Lys (E. coli coordinates), in ParE. GyrA changes, Ser83-->Leu, Glu87-->Lys, and Ala119-->Glu or Val, were found only in the second round of selection. At the third step, additional substitutions, at ParC Ser80, Ser81, and Glu84 and ParE Leu440, associated with high-level resistance to fluoroquinolones, appeared. Thus, high-level resistance to trovafloxacin required three steps and was associated with alterations in both fluoroquinolone targets. According to these genetic data, in M. hominis, as in Staphylococcus aureus and Streptococcus pneumoniae, topoisomerase IV seems to be the primary target of trovafloxacin.

In vitro selection of fluoroquinolone-resistant Neisseria gonorrhoeae harboring alterations in DNA gyrase and topoisomerase IV.

We attempted to select increasingly fluoroquinolone-resistant strains of Neisseria gonorrhoeae in vitro and to assess whether selected mutants harbored alterations in the GyrA subunit of DNA gyrase and the ParC subunit of DNA topoisomerase IV, which were analogous to those in fluoroquinolone-resistant clinical isolates. A fluoroquinolone-susceptible strain was exposed to norfloxacin in vitro. Selected mutants were sequentially exposed to norfloxacin, and this procedure was repeated. For 11 mutants, minimum inhibitory concentrations (MICs) of antimicrobial agents were determined, and mutations in the region corresponding to the quinolone resistance-determining region (QRDR) of the Escherichia coli gyrA gene and the analogous region of the parC gene were analyzed. Mutants obtained in one step exhibited significantly increased MICs of norfloxacin, ofloxacin and ciprofloxacin and had a single amino acid change in GyrA. Two-step mutants exhibited significantly higher norfloxacin MICs. Three of four two-step selected strains had single amino acid changes in both GyrA and ParC. Three-step mutants exhibited further increases in fluoroquinolone MICs and were assigned to the ciprofloxacin-resistant category. Two had a double amino acid change in GyrA, and one had a double GyrA change and a single amino acid change in ParC. We selected fluoroquinolone-resistant strains that carried GyrA and ParC alterations analogous to those in clinical isolates. The serial accumulation of changes in the QRDR of GyrA and the analogous region of ParC was associated with a stepwise increase in fluoroquinolone resistance, although the development of additional alterations in other regions of GyrA and ParC or other mechanisms of fluoroquinolone resistance also might contribute to the enhancement in fluoroquinolone resistance. The clinical emergence of fluoroquinolone-resistant strains may be due to in-vivo stepwise selection of strains with genetic alterations in GyrA and ParC, as observed here in the in-vitro selection of fluoroquinolone-resistant mutants.

IN VITRO SELECTION OF FLUOROQUINOLONE-RESISTANT NEISSERIA GONORRHOEAE HARBORING ALTERATIONS IN DNA GYRASE AND TOPOISOMERASE IV

IN VITRO SELECTION OF FLUOROQUINOLONE-RESISTANT NEISSERIA GONORRHOEAE HARBORING ALTERATIONS IN DNA GYRASE AND TOPOISOMERASE IV

Roles of gyrA mutations in resistance of clinical isolates and in vitro mutants of Bacteroides fragilis to the new fluoroquinolone trovafloxacin.

We determined whether gyrA mutations were present in fluoroquinolone-resistant laboratory mutants derived from the Bacteroides fragilis reference strain ATCC 25285 and in clinical isolates of B. fragilis. The two first-step mutants selected on ciprofloxacin (CIP) were devoid of gyrA mutations, whereas two of the three CIP-selected second-step mutants studied presented the same gyrA mutation leading to a Ser82Phe change. Unusual GyrA alterations, Asp81Asn or Ala118Val, were detected in two of the three first-step mutants selected on trovafloxacin (TRO), Mt3 and Mt1, respectively. The Ala118Val change had no effect on the susceptibility of Mt1 to CIP. No second-step mutant could be obtained with TRO as a selector. For the 12 clinical isolates studied, a Ser82Phe change in GyrA was found only in the 3 strains which showed the highest levels of TRO resistance (MIC, 4 microgram/ml). Thus, the resistance phenotypes and genotypes observed in fluoroquinolone-resistant clinical isolates of B. fragilis were similar to those found in CIP-selected laboratory mutants, whereas peculiar mutational events could be selected in vitro with TRO.

Alterations in Topoisomerase IV and DNA Gyrase in Quinolone-Resistant Mutants of Mycoplasma hominis Obtained In Vitro

Mycoplasma hominis mutants were selected stepwise for resistance to ofloxacin and sparfloxacin, and their gyrA, gyrB, parC, and parE quinolone resistance-determining regions were characterized. For ofloxacin, four rounds of selection yielded six first-, six second-, five third-, and two fourth-step mutants. The first-step mutants harbored a single Asp426-->Asn substitution in ParE. GyrA changes (Ser83-->Leu or Trp) were found only from the third round of selection. With sparfloxacin, three rounds of selection generated 4 first-, 7 second-, and 10 third-step mutants. In contrast to ofloxacin resistance, GyrA mutations (Ser83-->Leu or Ser84-->Trp) were detected in the first-step mutants prior to ParC changes (Glu84-->Lys), which appeared only after the second round of selection. Further analysis of eight multistep-selected mutants of M. hominis that were previously described (2) revealed that they carried mutations in ParE (Asp426-->Asn), GyrA (Ser83-->Leu) and ParE (Asp426-->Asn), GyrA (Ser83-->Leu) and ParC (Ser80-->Ile), or ParC (Ser80-->Ile) alone, depending on the fluoroquinolone used for selection, i.e., ciprofloxacin, norfloxacin, ofloxacin, or pefloxacin, respectively. These data indicate that in M. hominis DNA gyrase is the primary target of sparfloxacin whereas topoisomerase IV is the primary target of pefloxacin, ofloxacin, and ciprofloxacin.