Detection of grlA and gyrA Mutations in 344 Staphylococcus aureus Strains

Mutations in the grlA and gyrA genes of 344 clinical strains of Staphylococcus aureus isolated in 1994 in Japan were identified by combinations of single-strand conformation polymorphism analysis, restriction fragment length analysis, and direct sequencing to identify possible relationships to fluoroquinolone resistance. Five types of single-point mutations and four types of double mutations were observed in the grlA genes of 204 strains (59.3%). Four types of single-point mutations and four types of double mutations were found in the gyrA genes of 188 strains (54.7%). Among them, the grlA mutation of TCC→TTC or TAC (Ser-80→Phe or Tyr) and the gyrA mutation of TCA→TTA (Ser-84→Leu) were principal, being detected in 137 (39.8%) and 121 (35.9%) isolates, respectively. The grlA point mutations of CAT→CAC (His-77 [silent]), TCA→CCA (Ser-81→Pro), and ATA→ATT (Ile-100 [silent]) were novel, as was the GAC→GGC (Asp-73→Gly) change in gyrA. A total of 15 types of mutation combinations within both genes were related to ciprofloxacin resistance (MIC ≥ 3.13 μg/ml) and were present in 193 mutants (56.1%). Strains containing mutations in both genes were highly resistant to ciprofloxacin (MIC at which 50% of the isolates are inhibited [MIC₅₀] = 50 μg/ml). Those with the Ser-80→Phe or Tyr alteration in grlA but wild-type gyrA showed a lower level of ciprofloxacin resistance (MIC₅₀ ≤ 12.5 μg/ml). Levofloxacin was active against 68 of 193 isolates (35.2%) with mutations at codon 80 of grlA in the presence or absence of a concomitant mutation at codon 73, 84, or 88 in gyrA (MIC ≤ 6.25 μg/ml). The new fluoroquinolone DU-6859a showed good activity with 186 of 193 isolates (96.4%) for which the MIC was ≤6.25 μg/ml.     

Mechanism of quinolone resistance in Staphylococcus aureus

The resistance mechanisms to fluoroquinolones in Staphylococcus aureus were clarified by analyzing mutations in the genes encoding target enzymes, and examining the expression of the efflux pump, and determining the inhibitory activities of fluoroquinolones against the altered enzymes. Mutations in the grlA and gyrA genes of 344 clinical strains of S. aureus isolated in 1994 in Japan were identified by combinations of methods – single-strand conformation polymorphism analysis, restriction fragment length analysis, and direct sequencing – to identify possible relationships with fluoroquinolone resistance. Five types of single-point mutations and four types of double mutations were observed in the grlA gene in 204 strains (59.3%). Four types of single-point mutations and four types of double mutations were found in the gyrA gene in 188 strains (54.7%). Among these mutations, the grlA mutation of TCC → TTC or TAC (Ser-80 → Phe or Tyr) and the gyrA mutation of TCA → TTA (Ser-84 → Leu) were the principal ones, being detected in 137 (39.8%) and 121 (35.2%) isolates, respectively. A total of 15 types of mutation combinations within both genes were related to ciprofloxacin resistance (MIC ≧3.13 μg/ml) and were present in 193 mutants (56.1%). Strains containing mutations in both genes were highly resistant to ciprofloxacin (MIC₅₀ =50 μg/ml). Those strains with the Ser-80 → Phe or Tyr alteration in grlA, but wild type in gyrA showed a lower level of ciprofloxacin resistance (MIC₅₀≦12.5 μg/ml). Levofloxacin was active against 68 of 193 isolates (35.2%) with mutations at codon 80 of grlA in the presence or absence of concomitant mutations at codons 73, 84, or 88 in gyrA (MIC ≦6.25 μg/ml). Sitafloxacin (DU-6859a) showed good activity in 186 of 193 isolates (96.4%), with an MIC of ≦6.25 μg/ml. The contribution of membrane-associated multidrug efflux protein (NorA) expression to fluoroquinolone resistance was clarified by the checker-board titration method for determining the MIC of norfloxacin alone and in combination with carbonyl cyanide m-chlorophenylhydrazone. Among 344 clinical isolates, 139 strains (40.4%), in which the MIC of norfloxacin varied from 1.56 to >800 μg/ml, overexpressed the NorA protein. GrlA and GrlB proteins of topoisomerase IV, and GyrA and GyrB proteins of DNA gyrase encoded by genes with or without mutations were purified separately. The inhibitory activities of fluoroquinolones against the topoisomerase IV which contained a single amino acid change (Ser → Phe at codon 80, Glu → Lys at codon 84 of grlA, and Asp → Asn at codon 432 of grlB) were from 5 to 95 times weaker than the inhibitory activities against the non-altered enzyme. These results suggest that the mutations in the corresponding genes may confer quinolone resistance; the active efflux pump, NorA, was considered to be the third quinolone-resistance mechanism. The numerous and complicated mutations seen may explain the rapid and widespread development of quinolone resistance described in S. aureus. Sitafloxacin showed good antibacterial activity against ciprofloxacin- or levofloxacin-resistant mutants because of its high inhibitory activity against both topoisomerase IV and DNA gyrase. Received: March 3, 2000 / Accepted: May 2, 2000     

Efflux Pump-Mediated Quinolone Resistance in Staphylococcus aureus Strains Wild Type for gyrA, gyrB, grlA, and norA

Fluoroquinolone efflux was studied in 47 Staphylococcus aureus clinical strains with MICs of ciprofloxacin (CFX) of ≤2 μg/ml. Forty-three strains were wild type for gyrA, gyrB, and grlA quinolone resistance-determining regions and for norA and its promoter region. Forty of these strains (MICs of CFX, 0.1 to 0.2 μg/ml) did not show efflux of fluoroquinolones. Three strains (MICs of CFX, 1 to 2 μg/ml) showed efflux. These results suggest that efflux can appear in S. aureus clinical strains in the absence of mutations in norA and its promoter.     

Type II Topoisomerase Mutations in Ciprofloxacin-Resistant Strains of Pseudomonas aeruginosa

We determined the sequences of the quinolone resistance-determining regions of gyrA, gyrB, and parC genes for 30 clinical strains of Pseudomonas aeruginosa resistant to ciprofloxacin that were previously complemented by wild-type gyrA and gyrB plasmid-borne alleles and studied for their coresistance to imipenem (E. Cambau, E. Perani, C. Dib, C. Petinon, J. Trias, and V. Jarlier, Antimicrob. Agents Chemother. 39:2248–2252, 1995). In the present study, we found mutations in type II topoisomerase genes for all strains. Twenty-eight strains had a missense mutation in gyrA (codon 83 or 87). Ten of them had an additional mutation in parC (codon 80 or 84), including a novel mutation of Ser-80 to Trp, but all were fully complemented by a plasmid-borne wild-type gyrA allele. The remaining two strains harbored the first gyrB mutation described in P. aeruginosa, leading to the substitution of phenylalanine for serine 464. The strains which had two mutations in type II topoisomerase genes (i.e., gyrA and parC) were significantly more resistant to fluoroquinolones than those with a single mutation in gyrA or gyrB (geometric mean MICs of ciprofloxacin, 39.4 versus 10.9 μg/ml, P < 0.01; geometric mean MICs of sparfloxacin, 64.0 versus 22.6, P < 0.01). No mutant with a parC mutation alone was observed, which favors DNA gyrase being the primary target for fluoroquinolones. These results demonstrate that gyrA mutations are the major mechanism of resistance to fluoroquinolones for clinical strains of P. aeruginosa and that additional mutations in parC lead to a higher level of quinolone resistance.     

Topoisomerase Mutations in Fluoroquinolone-Resistant and Methicillin-Susceptible and -Resistant Clinical Isolates of Staphylococcus aureus

The incidence of the various mutations in the genes encoding topoisomerase IV and DNA gyrase in fluoroquinolone-resistant clinical isolates of Staphylococcus aureus is not known. Using restriction fragment length polymorphism analysis and DNA sequencing, we found that in fluoroquinolone- and methicillin-resistant strains, mutations in grlA and gyrA are quite likely to be present together. For fluoroquinolone-resistant but methicillin-susceptible strains, mutations in grlA alone are more common.     

Mutations in Topoisomerase IV and DNA Gyrase of Staphylococcus aureus: Novel Pleiotropic Effects on Quinolone and Coumarin Activity

Previous studies have shown that topoisomerase IV and DNA gyrase interact with quinolones and coumarins in different ways. The MICs of coumarins (novobiocin and coumermycin) for MT5, a Staphylococcus aureus nov mutant, are higher than those for wild-type strains. Sequencing the gyrB gene encoding one subunit of the DNA gyrase revealed the presence of a double mutation likely to be responsible for this resistance: at codon 102 (Ile to Ser) and at codon 144 (Arg to Ile). For single-step flqA mutant MT5224c9, previously selected on ciprofloxacin, the fluoroquinolone MIC was higher and the coumarin MIC was lower than those for its parent, MT5. Sequencing the grlB and grlA genes of topoisomerase IV of MT5224c9 showed a single Asn-470-to-Asp mutation in GrlB. Genetic outcrosses by transformation with chromosomal DNA and introduction of plasmids carrying either the wild-type or the mutated grlB gene indicated that this mutation causes both increased MICs of fluoroquinolones and decreased MICs of coumarins and that the mutant grlB allele is codominant for both phenotypes with multicopy alleles. Integration of these plasmids into the chromosome confirmed the codominance of fluoroquinolone resistance, but grlB⁺ appeared dominant over grlB (Asp-470) for coumarin resistance. Finally, the gyrA (Leu-84) mutation previously described as silent for fluoroquinolone resistance increased the MIC of nalidixic acid, a nonfluorinated quinolone. Combining the grlA (Phe-80) and grlB (Asp-470) mutations with this gyrA mutation also had differing effects. The findings indicate that alterations in topoisomerases may have pleiotropic effects on different classes of inhibitors as well as on inhibitors within the same class. A full understanding of drug action and resistance at the molecular level must take into account both inhibitor structure-activity relationships and the effects of different classes of topoisomerase mutants.Staphylococcus aureus is a major pathogen, many strains of which are now susceptible to only a few antibiotics. Resistance has begun to compromise the utility of fluoroquinolones as antistaphylococcal agents.Four genes related to fluoroquinolone resistance in S. aureus have been reported so far: norA (24), gyrA (15, 32), gyrB (15), and grlA (8, 25, 41). Resistance involving the norA gene is due to increased expression of the NorA efflux pump. The other resistance genes encode the structural proteins of DNA topoisomerases: gyrA and gyrB encode the structural proteins of DNA gyrase, and grlA (referred to as parC in other species) encodes one of the two subunits of DNA topoisomerase IV. The grlB gene encoding the second subunit of topoisomerase IV has not been previously implicated in fluoroquinolone resistance in S. aureus, but the mutation of its homolog in Escherichia coli and Streptococcus pneumoniae, parE, has been shown to cause resistance (4, 31).The fluoroquinolones act on type 2 topoisomerases by trapping or stabilizing an enzyme reaction intermediate in which both DNA strands are cleaved and covalently linked to the breakage-reunion subunits (GyrA in the case of gyrase and GrlA in the case of topoisomerase IV) (14). The stabilization of this cleavage complex initiates a series of events that result in cell death. In contrast to the findings for E. coli, in staphylococci and other gram-positive bacteria that have been studied, it appears that DNA gyrase is only a secondary target of many fluoroquinolones and that topoisomerase IV is the primary target (8, 25, 27). Mutations in gyrA are found only in more highly resistant clinical strains of S. aureus that also have mutations in grlA, and grlA mutations precede gyrA mutations in single-step resistance (9). In addition, gyrA mutations producing fluoroquinolone resistance in S. aureus are silent in the absence of grlA mutations (25). Mutations in three codons were described for grlA: codon 80 (Ser→Phe or Tyr), 84 (Glu→Lys), and 116 (Ala→Glu or Pro) (9, 25, 41).A quinolone resistance locus referred to as flqA was localized to the A fragment of chromosomal DNA of first-step ciprofloxacin- and ofloxacin-selected resistant mutants digested with SmaI between the thr and trp loci (37). This region has been shown to contain the grlB and grlA genes (25). In a previous work (25), some flqA mutants were shown to have grlA mutations but one single-step flqA mutant selected on ciprofloxacin (MT5224c9) (37) showed no nucleotide change in the quinolone resistance-determining region of grlA, suggesting that additional resistance mutations may occur either in grlA outside the region sequenced, or in the adjacent grlB gene, or in another as yet undefined but linked locus. In addition to quinolone resistance, mutant MT5224c9, which was selected from novobiocin-resistant parent strain MT5 (nov-142), showed a substantial decrease in novobiocin resistance (37).Coumarins such as novobiocin and coumermycin act differently from quinolones: they act by competitive inhibition of ATP hydrolysis by the B subunit of DNA gyrase (10, 12). The gyrase B protein consists of two domains: a 43-kDa N-terminal domain containing the ATPase site as well as the coumarin-binding site and a 47-kDa C-terminal domain responsible for interaction with the A protein and DNA (21). The binding of coumarin and ATP is competitive because of overlap in their binding sites (20). All reported mutations that result in coumarin resistance lie at the periphery of the ATP-binding site of GyrB. In contrast, quinolone resistance mutations in GyrB have been localized to a different subunit domain in a position midway between the amino and carboxy termini.The aim of this study was to determine the origin of the modification of the quinolone and coumarin resistance in the mutant MT5224c9 and to determine if the nov-142 locus was a mutant allele of gyrB. We have found that a novel mutation in the grlB gene resulting in the replacement of an asparagine by an aspartate at position 470 is responsible for the phenotype of MT5224c9. In addition, we found that the nov-142 locus is a novel doubly mutant allele of gyrB.     

Detection of gyrA Mutations among 335 Pseudomonas aeruginosa Strains Isolated in Japan and Their Susceptibilities to Fluoroquinolones

gyrA point mutations in 335 clinical Pseudomonas aeruginosa isolates were examined mainly by nonisotopic single-strand conformation polymorphism analysis and direct sequencing. Seven types of missense gyrA mutations were observed in 70 of 335 strains (20.9%), and ciprofloxacin MICs were ≥3.13 μg/ml for 63 of 70 strains (90.0%). These included two double point mutations and three novel mutations (Ala-67→Ser plus Asp-87→Gly, Ala-84→Pro, and Gln-106→Leu). Thr-83→Ile mutants were predominantly observed (63 of 70 mutants) and showed high-level fluoroquinolone resistance (ciprofloxacin MIC at which 50% of isolates are inhibited, 25 μg/ml).     

Topoisomerase Mutations in Trovafloxacin-Resistant Staphylococcus aureus

A total of 201 Staphylococcus aureus isolates were surveyed for susceptibility to ciprofloxacin and trovafloxacin. Of 66 methicillin-resistant isolates, 89% were ciprofloxacin resistant and 6% were also trovafloxacin resistant. Trovafloxacin-resistant strains had unusual patterns of quinoline resistance mutations in DNA topoisomerase genes, including two mutations in the A subunit (encoded by grlA) of topoisomerase IV.     

Antibacterial Activity of Gatifloxacin (AM-1155, CG5501, BMS-206584), a Newly Developed Fluoroquinolone, against Sequentially Acquired Quinolone-Resistant Mutants and the norA Transformant of Staphylococcus aureus

Alternate mutations in the grlA and gyrA genes were observed through the first- to fourth-step mutants which were obtained from four Staphylococcus aureus strains by sequential selection with several fluoroquinolones. The increases in the MICs of gatifloxacin accompanying those mutational steps suggest that primary targets of gatifloxacin in the wild type and the first-, second-, and third-step mutants are wild-type topoisomerase IV (topo IV), wild-type DNA gyrase, singly mutated topo IV, and singly mutated DNA gyrase, respectively. Gatifloxacin had activity equal to that of tosufloxacin and activity more potent than those of norfloxacin, ofloxacin, ciprofloxacin, and sparfloxacin against the second-step mutants (grlA gyrA; gatifloxacin MIC range, 1.56 to 3.13 μg/ml) and had the most potent activity against the third-step mutants (grlA gyrA grlA; gatifloxacin MIC range, 1.56 to 6.25 μg/ml), suggesting that gatifloxacin possesses the most potent inhibitory activity against singly mutated topo IV and singly mutated DNA gyrase among the quinolones tested. Moreover, gatifloxacin selected resistant mutants from wild-type and the second-step mutants at a low frequency. Gatifloxacin possessed potent activity (MIC, 0.39 μg/ml) against the NorA-overproducing strain S. aureus NY12, the norA transformant, which was slightly lower than that against the parent strain SA113. The increases in the MICs of the quinolones tested against NY12 were negatively correlated with the hydrophobicity of the quinolones (correlation coefficient, −0.93; P < 0.01). Therefore, this slight decrease in the activity of gatifloxacin is attributable to its high hydrophobicity. Those properties of gatifloxacin likely explain its good activity against quinolone-resistant clinical isolates of S. aureus harboring the grlA, gyrA, and/or norA mutations.     

Mutations That Enhance the Ciprofloxacin Resistance of Escherichia coli with qnrA1

Plasmid-mediated qnr genes provide only a modest decrease in quinolone susceptibility but facilitate the selection of higher-level resistance. In Escherichia coli strain J53 without qnr, ciprofloxacin resistance often involves mutations in the GyrA subunit of DNA gyrase. Mutations in gyrA were absent, however, when 43 mutants with decreased ciprofloxacin susceptibility were selected from J53(pMG252) with qnrA1. Instead, in 13 mutants, individual and whole-genome sequencing identified mutations in marR and soxR associated with increased expression of marA and soxS and, through them, increased expression of the AcrAB pump, which effluxes quinolones. Nine mutants had increased expression of the MdtE efflux pump, and six demonstrated increased expression of the ydhE pump gene. Many efflux mutants also had increased resistance to novobiocin, another pump substrate, but other mutants were novobiocin hypersusceptible. Mutations in rfaD and rfaE in the pathway for inner core lipopolysaccharide (LPS) biosynthesis were identified in five such strains. Many of the pump and LPS mutants had decreased expression of OmpF, the major porin channel for ciprofloxacin entry. Three mutants had increased expression of qnrA that persisted when pMG252 from these strains was outcrossed. gyrA mutations were also rare when mutants with decreased ciprofloxacin susceptibility were selected from E. coli J53 with aac(6′)-Ib-cr or qepA. We suggest that multiple genes conferring low-level resistance contribute to enhanced ciprofloxacin resistance selected from an E. coli strain carrying qnrA1, aac(6′)-Ib-cr, or qepA because these determinants decrease the effective ciprofloxacin concentration and allow more common but lower-resistance mutations than those in gyrA to predominate.     

Mutations in gyrA and gyrB among Fluoroquinolone- and Multidrug-Resistant Mycobacterium tuberculosis Isolates

In order to correlate the mutations inside the entire gyrA and gyrB genes with the level of resistance to ofloxacin (OFX) and moxifloxacin (MFX) in isolates of multidrug-resistant Mycobacterium tuberculosis (MDR-TB), a total of 111 isolates were categorized into OFX-susceptible (MIC, ≤2 μg/ml) and low-level (MIC, 4 to 8 μg/ml) and high-level (MIC, ≥16 μg/ml) OFX-resistant isolates and MFX-susceptible (MIC, ≤0.5 μg/ml) and low-level (MIC, 1 to 2 μg/ml) and high-level (MIC, ≥4 μg/ml) MFX-resistant isolates. Resistance-associated mutations inside the gyrA gene were found in 30.2% of OFX-susceptible and 72.5% and 72.2% of low-level and high-level OFX-resistant isolates and in 28.6% of MFX-susceptible and 58.1% and 83.9% of low-level and high-level MFX-resistant isolates. Compared with OFX-susceptible isolates, low-level and high-level OFX-resistant isolates had a significantly higher prevalence of mutations at gyrA codons 88 to 94 (17.0%, 65.0%, and 72.2%, respectively; P < 0.001) and a higher prevalence of the gyrB G512R mutation (0.0%, 2.5%, and 16.7%, respectively; P = 0.006). Similarly, compared with MFX-susceptible isolates, low-level and high-level MFX-resistant isolates had a significantly higher prevalence of mutations at gyrA codons 88 to 94 (14.3%, 51.6%, and 80.6%, respectively; P < 0.001) as well as a higher prevalence of the gyrB G512R mutation (0.0%, 0.0%, and 12.9%, respectively; P = 0.011). D94G and D94N mutations in gyrA and the G512R mutation in gyrB were correlated with high-level MFX resistance, while the D94A mutation was associated with low-level MFX resistance. The prevalence of mutations at gyrA codons 88 to 94 and the gyrB G512R mutation were higher among fluoroquinolone (FQ)-susceptible East Asian (Beijing) and Indo-Oceanic strains than they were among Euro-American strains, implying that molecular techniques to detect FQ resistance may be less specific in areas with a high prevalence of East Asian (Beijing) and Indo-Oceanic strains.     

gyrA Mutations Associated with Fluoroquinolone Resistance in Eight Species of Enterobacteriaceae

Fluoroquinolone resistance (FQ-R) in clinical isolates of Enterobacteriaceae species has been reported with increasing frequency in recent years. Two mechanisms of FQ-R have been identified in gram-negative organisms: mutations in DNA gyrase and reduced intracellular drug accumulation. A single point mutation in gyrA has been shown to reduce susceptibility to fluoroquinolones. To determine the extent of gyrA mutations associated with FQ-R in enteric bacteria, one set of oligonucleotide primers was selected from conserved sequences in the flanking regions of the quinolone resistance-determining regions (QRDR) of Escherichia coli and Klebsiella pneumoniae. This set of primers was used to amplify and sequence the QRDRs from 8 Enterobacteriaceae type strains and 60 fluoroquinolone-resistant clinical isolates of Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, E. coli, K. pneumoniae, Klebsiella oxytoca, Providencia stuartii, and Serratia marcescens. Although similarity of the nucleotide sequences of seven species ranged from 80.8 to 93.3%, when compared with that of E. coli, the amino acid sequences of the gyrA QRDR were highly conserved. Conservative amino acid substitutions were detected in the QRDRs of the susceptible type strains of C. freundii, E. aerogenes, K. oxytoca (Ser-83 to Thr), and P. stuartii (Asp-87 to Glu). Strains with ciprofloxacin MICs of >2 μg/ml expressed amino acid substitutions primarily at the Gly-81, Ser-83, or Asp-87 position. Fluoroquinolone MICs varied significantly for strains exhibiting identical gyrA mutations, indicating that alterations outside gyrA contribute to resistance. The type and position of amino acid alterations also differed among these six genera. High-level FQ-R frequently was associated with single gyrA mutations in all species of Enterobacteriaceae in this study except E. coli.     

Moxifloxacin Retains Antimycobacterial Activity in the Presence of gyrA Mutations

Moxifloxacin-resistant Mycobacterium tuberculosis mutants were selected in vitro using different concentrations of moxifloxacin. gyrA mutations at codons 88 and 94 were associated with resistance (defined as an MIC of ≥2 μg/ml) (P < 0.0001 and P = 0.0053, respectively). Despite the presence of gyrA mutations, moxifloxacin significantly impedes bacterial growth, supporting its use for the treatment of ofloxacin-resistant M. tuberculosis.     

Effects of Mutations in GrlA of Topoisomerase IV from Staphylococcus aureus on Quinolone and Coumarin Activity

The grlA genes of Staphylococcus aureus ISP794 (wild type), MT5224c4 (grlA [Phe-80]), MT5224c2 (grlA [Pro-116]), and MT111 (grlA [Glu-116]) were cloned in pSK950, a shuttle vector, and introduced into S. aureus strains derived from strain RN4220. The mutations at position 116 of GrlA (Ala→Pro or Glu) caused an increase in the level of fluoroquinolone resistance and a decrease in the level of coumarin susceptibility, whereas the mutation at position 80 (Ser→Phe) caused only an increase in the level of fluoroquinolone resistance. In multicopy alleles, both types of mutations were codominant for fluoroquinolone resistance, and mutations at position 116 were also codominant for coumarin resistance.     

Fluoroquinolone Resistance Mutations in the parC, parE, and gyrA Genes of Clinical Isolates of Viridans Group Streptococci

The nucleotide sequences of the quinolone resistance-determining regions (QRDRs) of the parC and gyrA genes from seven ciprofloxacin-resistant (Cp^r) isolates of viridans group streptococci (two high-level Cp^r Streptococcus oralis and five low-level Cp^r Streptococcus mitis isolates) were determined and compared with those obtained from susceptible isolates. The nucleotide sequences of the QRDRs of the parE and gyrB genes from the five low-level Cp^r S. mitis isolates and from the NCTC 12261 type strain were also analyzed. Four of these low-level Cp^r isolates had changes affecting the subunits of DNA topoisomerase IV: three in Ser-79 (to Phe or Ile) of ParC and one in ParE at a position not previously described to be involved in quinolone resistance (Pro-424). One isolate did not show any mutation. The two high-level Cp^r S. oralis isolates showed mutations affecting equivalent residue positions of ParC and GyrA, namely, Ser-79 to Phe and Ser-81 to Phe or Tyr, respectively. The parC mutations were able to transform Streptococcus pneumoniae to ciprofloxacin resistance, while the gyrA mutations transformed S. pneumoniae only when mutations in parC were present. These results suggest that DNA topoisomerase IV is a primary target of ciprofloxacin in viridans group streptococci, DNA gyrase being a secondary target.     

In vitro and in vivo profiles of ACH-702, an isothiazoloquinolone, against bacterial pathogens

ACH-702, a novel isothiazoloquinolone (ITQ), was assessed for antibacterial activity against a panel of Gram-positive and Gram-negative clinical isolates and found to possess broad-spectrum activity, especially against antibiotic-resistant Gram-positive strains, including methicillin-resistant Staphylococcus aureus (MRSA). For Gram-negative bacteria, ACH-702 showed exceptional potency against Haemophilus influenzae, Moraxella catarrhalis, and a Neisseria sp. but was less active against members of the Enterobacteriaceae. Good antibacterial activity was also evident against several anaerobes as well as Legionella pneumophila and Mycoplasma pneumoniae. Excellent bactericidal activity was observed for ACH-702 against several bacterial pathogens in time-kill assays, and postantibiotic effects (PAEs) of >1 h were evident with both laboratory and clinical strains of staphylococci at 10× MIC and similar in most cases to those observed for moxifloxacin at the same MIC multiple. In vivo efficacy was demonstrated against S. aureus with murine sepsis and thigh infection models, with decreases in the number of CFU/thigh equal to or greater than those observed after vancomycin treatment. Macromolecular synthesis assays showed specific dose-dependent inhibition of DNA replication in staphylococci, and biochemical analyses indicated potent dual inhibition of two essential DNA replication enzymes: DNA gyrase and topoisomerase IV. Additional biological data in support of an effective dual targeting mechanism of action include the following: low MIC values (≤0.25 μg/ml) against staphylococcal strains with single mutations in both gyrA and grlA (parC), retention of good antibacterial activity (MICs of ≤0.5 μg/ml) against staphylococcal strains with two mutations in both gyrA and grlA, and low frequencies for the selection of higher-level resistance (<10⁻¹⁰). These promising initial data support further study of isothiazoloquinolones as potential clinical candidates.     

Detection of ciprofloxacin resistance mutations in Campylobacter jejuni gyrA by nonradioisotopic single-strand conformation polymorphism and direct DNA sequencing

A total of 27 strains of Campylobacter jejuni (24 clinical strains and three laboratory strains) were examined for the presence of point mutations in the quinolone resistance determining region (QRDR) of gyrA gene by nonradioisotopic single-strand conformation polymorphism (non-RI SSCP) analysis with silver stain. Direct DNA sequencing of the polymerase chain reaction (PCR)-amplified DNA fragments confirmed the results obtained by non-RI SSCP analysis and revealed that in clinical strains high-level quinolone resistance [minimal inhibitory concentration (MIC) to ciprofloxacin ≥ 16 μg/ml] was closely associated with one type of single-point mutation at codon 86 (Thr-lle). Two strains with MICs of 8 and 1 μg/ml showed point mutations at codons 86 and 70, respectively. Furthermore, transitions at codon 119 of the gyrA QRDR were identified in 17 strains. Six types of bands were separated in a single electrophoretic step with silver stain within 2 hours after PCR amplification of the gyrA QRDR as follows: type I associated to mutation at codon 70 (Ala-Thr), type II to mutation at codon 90 (Asp-Asn), type III to variant with transition at 119, type IV to wild-type, type V to mutation at codon 86 (Thr-lle), and type VI to mutation at codon 86 (Thr-lle) and transition at codon 119. Using four DNA extracts from Cambylobacter coli organisms as templates for amplification of the gyrA QRDR, no PCR products were obtained. Non-RI SSCP was proved to be a simple, rapid, and useful screening method for detecting gyrA mutations associated with ciprofloxacin resistance in C. jejuni. © 1996 Wiley-Liss, Inc.     

Identification of ciprofloxacin resistance by SimpleProbe™, High Resolution Melt and Pyrosequencing™ nucleic acid analysis in biothreat agents: Bacillus anthracis,Yersinia pestis and Francisella tularensis

The potential for genetic modification of biological warfare agents makes rapid identification of antibiotic resistant strains critical for the implementation of suitable infection control measures. The fluorinated quinolone, ciprofloxacin, is an antibiotic effective for treating bacterial infections by inhibiting the enzyme activity of the DNA type II topoisomerases DNA gyrase and topoisomerase IV. The genes that encode the subunits of DNA gyrase (gyrA and gyrB) and topo IV (par C and parE) contain hotspots within an area known as the quinolone resistance-determining region (QRDR). Base pair changes within this region give rise to mutations that cause resistance to the antibiotic by altering amino acids within the enzymes. Ciprofloxacin-resistant (cipro^r) strains of Bacillus anthracis, Yersinia pestis, and Francisella tularensis with one or more known mutations within the QRDR of gyrA, gyrB, parC, and parE genes were tested with SimpleProbe? and High Resolution Melt (HRM) dye chemistries and Pyrosequencing? genetic analysis to evaluate the ability to rapidly detect ciprofloxacin-induced mutations. While SimpleProbe? and Pyrosequencing? successfully identified all known mutants, the HRM assay identified all but those resulting from G ? C or A ? T substitutions.     

Greater Ciprofloxacin Tolerance as a Possible Selectable Phenotype Underlying the Pandemic Spread of the H30 Subclone of Escherichia coli Sequence Type 131

Minimum bactericidal concentrations (MBCs) for ciprofloxacin were significantly higher among 41 members of the H30 subclone within Escherichia coli sequence type 131 than among 48 other fluoroquinolone-resistant E. coli isolates. This MBC difference, which was not explained by ciprofloxacin MICs, gyrA, parC, and parE mutations, the presence of aac(6′)-Ib-cr, or organic solvent tolerance (a surrogate for efflux pump activity), conceivably could have promoted the pandemic emergence of the H30 sequence type 131 subclone.     

Activities of Newer Fluoroquinolones against Streptococcus pneumoniae Clinical Isolates Including Those with Mutations in the gyrA, parC, and parE Loci

Resistance to fluoroquinolone (FQ) antibiotics in Streptococcus pneumoniae has been attributed primarily to specific mutations in the genes for DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). Resistance to some FQs can result from a single mutation in one or more of the genes encoding these essential enzymes. A group of 160 clinical isolates of pneumococci was examined in this study, including 36 ofloxacin-resistant isolates (MICs, ≥8 μg/ml) recovered from patients in North America, France, and Belgium. The susceptibilities of all isolates to clinafloxacin, grepafloxacin, levofloxacin, sparfloxacin, and trovafloxacin were examined by the National Committee for Clinical Laboratory Standards reference broth microdilution and disk diffusion susceptibility testing methods. Among the ofloxacin-resistant strains, 32 of 36 were also categorized as resistant to levofloxacin, 35 were resistant to sparfloxacin, 29 were resistant to grepafloxacin, and 19 were resistant to trovafloxacin. In vitro susceptibility to clinafloxacin appeared to be least affected by resistance to the other FQs. Eight isolates with high- and low-level resistance to the newer FQs were selected for DNA sequence analysis of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE. The DNA and the inferred amino acid sequences of the resistant strains were compared with the analogous sequences of reference strain S. pneumoniae ATCC 49619 and FQ-susceptible laboratory strain R6. Reduced susceptibilities to grepafloxacin and sparfloxacin (MICs, 1 to 2 μg/ml) and trovafloxacin (MICs, 0.5 to 1 μg/ml) were associated with either a mutation in parC that led to a single amino acid substitution (Ser-79 to Phe or Tyr) or double mutations that involved the genes for both GyrA (Ser-81 to Phe) and ParE (Asp-435 to Asn). High-level resistance to all of the compounds except clinafloxacin was associated with two or more amino acid substitutions involving both GyrA (Ser-81 to Phe) and ParC (Ser-79 to Phe or Ser-80 to Pro and Asp-83 to Tyr). No mutations were observed in the gyrB sequences of resistant strains. These data indicate that mutations in pneumococcal gyrA, parC, and parE genes all contribute to decreased susceptibility to the newer FQs, and genetic analysis of the QRDR of a single gene, either gyrA or parC, is not predictive of pneumococcal resistance to these agents.