Quinolone-susceptible Strains Research Articles

Apigenin as an anti-quinolone-resistance antibiotic

We previously reported the first ‘reverse antibiotic’ (RA), nybomycin (NYB), which showed a unique antimicrobial activity against Staphylococcus aureus strains. NYB specifically suppressed the growth of quinolone-resistant S. aureus strains but was not effective against quinolone-susceptible strains. Although NYB was first reported in 1955, little was known about its unique antimicrobial activity because it was before the synthesis of the first quinolone (‘old quinolone’), nalidixic acid, in 1962. Following our re-discovery of NYB, we looked for other RAs among natural substances that act on quinolone-resistant bacteria. Commercially available flavones were screened against S. aureus, including quinolone-resistant strains, and their minimum inhibitory concentrations (MICs) were compared using the microbroth dilution method. Some of the flavones screened showed stronger antimicrobial activity against quinolone-resistant strains than against quinolone-susceptible ones. Amongst them, apigenin (API) was the most potent in its RA activity. DNA cleavage assay showed that API inhibited DNA gyrase harbouring the quinolone resistance mutation gyrA(Ser84Leu) but did not inhibit ‘wild-type’ DNA gyrase that is sensitive to levofloxacin. An API-susceptible S. aureus strain Mu50 was also selected using agar plates containing 20mg/L API. Whole-genome sequencing of selected mutant strains was performed and frequent back-mutations (reverse mutations) were found among API-resistant strains derived from the API-susceptible S. aureus strains. Here we report that API represents another molecular class of natural antibiotic having RA activity against quinolone-resistant bacteria.

Effects of Natural Mutations in the ramRA Locus on Invasiveness of Epidemic Fluoroquinolone-Resistant Salmonella enterica Serovar Typhimurium Isolates

Fluoroquinolone (FQ) resistance is increasing worldwide among Salmonella species. Among the mechanisms involved, increased efflux via the tripartite AcrAB-TolC efflux system is mainly modulated through control of expression via the ramRA regulatory locus gene products. Interestingly, in some reference strains these have also been experimentally shown to regulate cell invasion-related genes of the type III secretion system 1 (T3SS-1). In this study, we investigated whether natural mutations occurring in this locus in FQ-resistant S. enterica serovar Typhimurium epidemic clones resulted in the same effects. Quantitative reverse transcription polymerase chain reaction and cell invasion assays were used to study 3 clinical FQ-resistant S. Typhimurium isolates representative of the DT104 and DT204 epidemic clones. For comparison, 3 control reference quinolone-susceptible strains were included. As previously shown, the investigated mutations altering RamR or its DNA-binding site increased expression of efflux genes dependently on ramA. However, the decreased expression of T3SS-1 genes previously reported was not always observed and seemed to be dependent on the genetic background of the FQ-resistant isolate. Indeed, a ramA-dependent decreased invasion of intestinal epithelial cells was only observed for a particular clinical ramR mutant. ramRA mutations occurring in clinical FQ-resistant S. Typhimurium isolates may negatively modulate their invasiveness but this is strain-dependent.

Plasmid-mediated quinolone resistance in expanded spectrum beta lactamase producing enterobacteriaceae in Morocco

Although independently acquired, plasmid-mediated quinolone resistance appears to be linked with extended-spectrum or AmpC-type beta-lactamases. Since no data are available in African countries, the prevalence of qnr genes at the University Hospital Ibn Rochd, Casablanca, Morocco, was investigated. Between October 2006 and March 2007, the following 39 randomly selected non-duplicate Enterobacteriaceae producing an extended-spectrum beta-lactamase (ESBL), representing 20% of ESBL strains with respect to species and ward origin, were collected: Escherichia coli (n = 16); Klebsiella spp (n = 14); Enterobacter cloacae (n = 8); Proteus mirabilis (n = 1). Antibiotic susceptibility testing was performed according to CLSI guidelines. ESBL detection was performed by the double disc diffusion test. A multiplex PCR was conducted to detect qnrA, qnrB and qnrS genes that were confirmed by sequencing of the PCR product. The estimated overall prevalence of qnr reached 36% (n = 14; qnrA, 10.25%; qnrB, 23.07%; qnrS, 2.56%). Genes were identified in E. coli, Klebsiella and Enterobacter with a respective prevalence of 18.7%, 50% and 62.5%. The qnr genes were detected in nine wards and qnrA1, qnrB1-B2-B4 and qnrS1 variants were identified. Three genes were identified among nalidixic acid susceptible strains (n = 6); three of those were also susceptible to ciprofloxacin. Among nalidixic acid and ciprofloxacin resistant strains, all strains had qnrB. This study highlights the high prevalence of qnr genes among ESBL strains in the Ibn Rochd CHU, Casablanca. Moreover, qnr were present in quinolone-susceptible strains which could lead to in vivo selection of ciprofloxacin-resistant strains.

Antimicrobial activity of DW286 against Streptococcus pneumoniae

DW286 is a novel broad-spectrum fluoroquinolone with excellent antipneumococcal activity. The in vitro activity of DW286 was evaluated against quinolone-susceptible and -resistant Streptococcus pneumoniae and was compared with the activities of reference compounds. Among the tested agents, DW286 showed the most potent antibacterial activity against 94 quinolone-susceptible strains [minimum inhibitory concentration (MIC) 0.008–0.03 mg/L]. Against 23 quinolone-resistant S. pneumoniae with known resistance mechanisms, DW286 also had the lowest MICs of all the tested quinolones [MIC at which 90% of isolates were inhibited (MIC 90) = 0.5 mg/L], followed by ciprofloxacin, sparfloxacin, moxifloxacin and gemifloxacin. The in vivo activity of DW286 against penicillin-susceptible and -resistant S. pneumoniae was more effective than that of gemifloxacin.

Prevalence of enterotoxins amongEscherichia coliisolates causing bacteraemia

The most frequent cause of bacteraemia among Gram-negative bacteria is Escherichia coli. Analysis of the genes encoding the Shigella enterotoxin 1 (ShET-1), ShET-2, enteroaggregative heat stable toxin 1 (EAST-1) toxins and AggR factor in E. coli strains causing bacteraemia revealed that set1 genes were presented significantly more frequently among quinolone-susceptible strains (P<0.0001), in phylogenetic group B2 (P=0.0004) and in biofilm strains (P=0.02). In contrast, sen genes were significantly more frequent among nalidixic acid-resistant isolates (15% vs. 6%, P=0.046) and in phylogenetic group B1 (P=0.0001). This is the first study in which ShET1, ShET2 and EAST-1 have been found in E. coli collected from blood.

Quinolone-resistance in Salmonella is associated with decreased mRNA expression of virulence genes invA and avrA, growth and intracellular invasion and survival

A variety of environmental factors, such as oxygen, pH, osmolarity and antimicrobial agents, modulate the expression of Salmonella pathogenicity islands (SPI) genes. This study investigated SPI-1 gene expression and the pathogenicity of quinolone-resistant Salmonella. mRNA expression levels of the invA and avrA genes, located in SPI-1, in quinolone-susceptible and quinolone-resistant Salmonella strains were determined using real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR). Twenty-five quinolone-resistant Salmonella mutants were derived from quinolone-susceptible strains by multiple-passage selection through increasing concentrations of ciprofloxacin in vitro, while an additional 15 strains were quinolone-resistant Salmonella clinical isolates. Sequence analysis showed no gene deletion or point mutations of nine SPI-1 genes (including invA and avrA) occurred in either the selected or clinical quinolone-resistant strains, while a single gyrA point mutation (S83F) was observed in all 40 quinolone-resistant strains. The mRNA expression levels of invA and avrA were significantly decreased ( P < 0.005) in quinolone-resistant strains (clinically acquired or experimentally selected in vitro), compared to the quinolone-susceptible strains. The resistant strains also had a slower growth rate combined with decreased epithelial cell invasion and intracellular replication in epithelial cells and macrophages. The results suggest that quinolone-resistance may be associated with lower virulence and pathogenicity than in quinolone-susceptible strains.

Dual-targeting properties of the 3-aminopyrrolidyl quinolones, DC-159a and sitafloxacin, against DNA gyrase and topoisomerase IV: contribution to reducing in vitro emergence of quinolone-resistant Streptococcus pneumoniae

DC-159a (a novel quinolone) and sitafloxacin (DU-6859a) are structurally related quinolones, bearing a 3-aminopyrrolidyl substitution. We investigated the relationship between the target preferences of these 3-aminopyrrolidyl quinolones, in vitro potencies and emergence of quinolone-resistant mutants in Streptococcus pneumoniae, compared with other quinolones. MICs, resistance frequencies and mutant prevention concentrations (MPCs) were determined using quinolone-susceptible strains and first-step parC mutant strains of S. pneumoniae. Target preferences were tested by the following two methods: antibacterial activities against gyrA or parC mutants and in vitro enzyme assays for the determination of 50% inhibition (IC(50)) values. DC-159a and sitafloxacin exhibited potent antibacterial activities, low frequencies of mutant selection, low MPCs and narrow mutant selection windows against both quinolone-susceptible strains and first-step parC mutants of S. pneumoniae, compared with gatifloxacin, moxifloxacin and other quinolones tested. DC-159a and sitafloxacin showed relatively low MIC ratios against single gyrA or parC mutants relative to the wild-type strain and low IC(50) ratios against DNA gyrase and topoisomerase IV. DC-159a and sitafloxacin demonstrated a more balanced dual-targeting activity than gatifloxacin, moxifloxacin and other quinolones tested. In addition, DC-159a and sitafloxacin have a lower propensity for selecting first- and second-step resistant mutants.

Quinolones Induce Partial or Total Loss of Pathogenicity Islands in Uropathogenic Escherichia coli by SOS-Dependent or -Independent Pathways, Respectively

Escherichia coli is the most common microorganism causing urinary tract infections. Quinolone-resistant E. coli strains have fewer virulence factors than quinolone-susceptible strains. Several urovirulence genes are located in pathogenicity islands (PAIs). We investigated the capacity of quinolones to induce loss of virulence factors such as hemolysin, cytotoxic necrotizing factor 1, P fimbriae, and autotransporter Sat included in PAIs in three uropathogenic E. coli strains. In a multistep selection, all strains lost hemolytic capacity at between 1 and 4 passages when they were incubated with subinhibitory concentrations of ciprofloxacin, showing a partial or total loss of the PAI containing the hly (hemolysin) and cnf-1 (cytotoxic necrotizing factor 1) genes. RecA(-) mutants were obtained from the two E. coli strains with partial or total loss of the PAI. The inactivation of the RecA protein affected only the partial loss of the PAI induced by quinolones. No spontaneous loss of PAIs was observed on incubation in the absence of quinolones in either the wild-type or mutant E. coli strains. Quinolones induce partial or total loss of PAIs in vitro in uropathogenic E. coli by SOS-dependent or -independent pathways, respectively.

Pharmacodynamic evaluation of tosufloxacin against Streptococcus pneumoniae in an in vitro model simulating serum concentration

We compared the antibacterial effects and the emergence of resistance to tosufloxacin or levofloxacin for Streptococcus pneumoniae by simulating the serum concentration according to the Japanese clinical regimens using an in vitro pharmacokinetic-pharmacodynamic model. For quinolone-susceptible strain ATCC49619, tosufloxacin showed bactericidal activity, given that both the AUC(0-24h)/MIC ratios at the dosage of 150 mg t.i.d. and 300 mg b.i.d. of tosufloxacin tosilate were 138 and 193, and the C(max)/MIC ranges were 7.93-10.2 and 15.9-17.6, respectively, which were greater than those of levofloxacin (100 mg t.i.d. and 200 mg b.i.d.). The greater area above the killing curves (AAKCs) or shorter time to achieve 99.9% killing (99.9% KT) in both models of tosufloxacin than those of levofloxacin was related to their larger AUC(0-24h)/MIC and C(max)/MIC. Exposure of only 100 mg t.i.d. of levofloxacin led to outgrowth of the parC mutants, which were twofold less susceptible to levofloxacin than the parent strain. Neither of the tosufloxacin tosilate regimens resulted in isolation of resistant mutants of this strain. For the parC mutant strain D-3197, both the AUC(0-24h)/MIC and C(max)/MIC ratios of tosufloxacin were greater than those of levofloxacin, which resulted in comparable or better bactericidal activity as compared to those of levofloxacin. However, both fluoroquinolones and both regimens led to outgrowth of resistant mutants, which possessed a mutation in gyrA in addition to parC. In conclusion, tosufloxacin is superior to levofloxacin in bactericidal activity against S. pneumoniae in the Japanese clinical regimens, especially in the quinolone-susceptible strain, without emergence of resistant subpopulations.

Antistaphylococcal Activity of DX-619, a New Des-F(6)-Quinolone, Compared to Those of Other Agents

The in vitro activity of DX-619, a new des-F(6)-quinolone, was tested against staphylococci and compared to those of other antimicrobials. DX-619 had the lowest MIC ranges/MIC(50)s/MIC(90)s (microg/ml) against 131 Staphylococcus aureus strains (</=0.002 to 2.0/0.06/0.5) and 128 coagulase-negative staphylococci (0.004 to 0.25/0.016/0.125). Among strains tested, 76 S. aureus strains and 51 coagulase-negative staphylococci were resistant to ciprofloxacin. DX-619 had the lowest MIC(50)/MIC(90) values against 127 quinolone-resistant staphylococci (0.125/0.5), followed by sitafloxacin (0.5/4), moxifloxacin (2/8), gatifloxacin (4/16), levofloxacin (16/>32), and ciprofloxacin (>32/>32). Raised quinolone MICs were associated with mutations in GyrA (S84L) and single or double mutations in GrlA (S80F or Y; E84K, G, or V) in all S. aureus strains tested. A recent vancomycin-resistant S. aureus (VRSA) strain (Hershey) was resistant to available quinolones and was inhibited by DX-619 at 0.25 microg/ml and sitafloxacin at 1.0 microg/ml. Vancomycin (except VRSA), linezolid, ranbezolid, tigecycline, and quinupristin-dalfopristin were active against all strains, and teicoplanin was active against S. aureus but less active against coagulase-negative staphylococci. DX-619 produced resistant mutants with MICs of 1 to >32 microg/ml after <50 days of selection compared to 16 to >32 microg/ml for ciprofloxacin, sitafloxacin, moxifloxacin, and gatifloxacin. DX-619 and sitafloxacin were also more active than other tested drugs against selected mutants and had the lowest mutation frequencies in single-step resistance selection. DX-619 and sitafloxacin were bactericidal against six quinolone-resistant (including the VRSA) and seven quinolone-susceptible strains tested, whereas gatifloxacin, moxifloxacin, levofloxacin, and ciprofloxacin were bactericidal against 11, 10, 7, and 5 strains at 4x MIC after 24 h, respectively. DX-619 was also bactericidal against one other VRSA strain, five vancomycin-intermediate S. aureus strains, and four vancomycin-intermediate coagulase-negative staphylococci. Linezolid, ranbezolid, and tigecycline were bacteriostatic and quinupristin-dalfopristin, teicoplanin, and vancomycin were bactericidal against two, eight, and nine strains, and daptomycin and oritavancin were rapidly bactericidal against all strains, including the VRSA. DX-619 has potent in vitro activity against staphylococci, including methicillin-, ciprofloxacin-, and vancomycin-resistant strains.

Activities of ciprofloxacin and moxifloxacin against Stenotrophomonas maltophilia and emergence of resistant mutants in an in vitro pharmacokinetic-pharmacodynamic model.

A two-compartment in vitro pharmacokinetic-pharmacodynamic model, with full computer-controlled devices, was used to accurately simulate human plasma pharmacokinetic profiles after multidose oral regimens of ciprofloxacin (750 mg every 12 h) and moxifloxacin (400 mg every 24 h) during 48 h. Pharmacodynamics of these drugs was investigated against three quinolone-susceptible strains of Stenotrophomonas maltophilia (MICs of ciprofloxacin and moxifloxacin of 0.5 to 2 and 0.0625 to 0.5 microg/ml, respectively). The first dose of ciprofloxacin and moxifloxacin reduced the bacterial count by 1 and 2 log CFU/ml, respectively, prior to a bacterial regrowth that reached the plateau value of the growth control curve at 13 to 24 h versus 24 to 36 h and persisted despite repeated administration of both drugs. The surviving bacterial cells were quinolone-resistant mutants (2 to 128 times the MIC) that exhibited cross-resistance to unrelated antibiotics. Their antibiotic resistance probably resulted from the overproduction of different multidrug resistance efflux system(s). C(max)/MIC and area under the concentration-time curve from 0 to 24 h (AUC(0-24))/MIC values were at least threefold higher for moxifloxacin than for ciprofloxacin. Moreover, integral parameters of ciprofloxacin and moxifloxacin, in particular the area under the killing and regrowth curve from 0 to 48 h (AUBC(0-48), 342.3 to 401.3 versus 295.2 to 378.7 h x log CFU/ml, respectively) and the area between the control growth curve and the killing and regrowth curve from 0 to 48 h (ABBC(0-48), 40.4 to 101.1 versus 72.9 to 144.7 h x log CFU/ml, respectively), demonstrated a better antibacterial effect of moxifloxacin than ciprofloxacin on S. maltophilia. However, selection of resistant mutants by both fluoroquinolones, although delayed with moxifloxacin, emphasizes the need to use maximal dosages and combined therapy in the treatment of systemic S. maltophilia infections.

Analysis of the mechanisms of quinolone resistance in clinical isolates of Citrobacter freundii.

The presence of gyrA, gyrB and/or parC mutations, quinolone uptake, outer membrane protein profiles and epidemiological relationship were studied in 12 clinical isolates of Citrobacter freundii. No alterations were observed in the gyrB gene of any of the strains, or gyrA or parC of the four quinolone-susceptible strains (nalidixic acid MIC of 2-4 mg/L, and a ciprofloxacin MIC of 0.006-0.06 mg/L). The quinolone-resistant strains were classified into two groups: one group (group A) composed of strains resistant to nalidixic acid but not to ciprofloxacin and another (group B) including those resistant to both antibiotics with a mutation at codon 83 of the gyrA gene (Thr-->Ile), but no alteration in either parC or gyrB genes. In group B, three of the four resistant isolates, with a nalidixic acid MIC > 1024 mg/L and ciprofloxacin MIC of 8-32 mg/L, showed concomitant mutations at codons 83 and 87 of the gyrA gene (Thr-->Ile and Asp-->Tyr, respectively) as well as a single mutation in codon 80 of the parC gene (Ser-->Ile). The fourth isolate did not possess the mutation at codon 87 of gyrA. Two strains belong to the same clone and, although they had the same type of mutations in the gyrA and parC genes, showed different MICs of ciprofloxacin. This difference was related to an efflux pump mechanism. Mutations in the gyrA and parC genes play the main role in quinolone resistance development in Citrobacter freundii, although other factors such as overexpression of efflux pumps can play a complementary role and thus modulate the final quinolone MIC.

Antimicrobial activity and accumulation of moxifloxacin in quinolone-susceptible bacteria.

The antibacterial activity of moxifloxacin, compared with that of ciprofloxacin, was determined for five strains of Staphylococcus aureus, including one NorA-overproducing strain, two quinolone-susceptible strains of Streptococcus pneumoniae, four quinolone-susceptible strains of Haemophilus influenzae, and one strain each of quinolone-susceptible Escherichia coli, Pseudomonas aeruginosa and Moraxella catarrhalis. In addition, the accumulation of moxifloxacin and ciprofloxacin by the NCTC type strain of S. pneumoniae, H. influenzae, S. aureus, E. coli and P. aeruginosa was determined by a fluorescence method. For all strains, moxifloxacin accumulated to a lower concentration than ciprofloxacin. The concentrations of moxifloxacin accumulated ranged from 12 to 44 ng/mg dry cells. The lowest concentration was accumulated by S. pneumoniae NCTC 7465 and the highest concentration by S. aureus NCTC 8532. Increased expression of norA in S. aureus had no effect on the accumulation of moxifloxacin. Despite differences in the concentration of moxifloxacin accumulated by the different species, there was little difference between the MICs of this agent for each strain (0.06-0.5 mg/L), suggesting that the concentration accumulated by wild-type bacteria has little effect on the MIC.

Inhibitory activities of quinolones against DNA gyrase and topoisomerase IV purified from Staphylococcus aureus.

In order to clarify the mechanism of action of quinolones against Staphylococcus aureus, GrlA and GrlB proteins of topoisomerase IV encoded by genes with or without mutations were purified separately as fusion proteins with maltose-binding protein in Escherichia coli. The reconstituted enzymes showed ATP-dependent decatenation and relaxing activities but had no supercoiling activity. The inhibitory effects of quinolones on the decatenation activity of topoisomerase IV were determined by quantitative electrophoresis with kinetoplast DNA as a substrate. The 50% inhibitory concentrations (IC50s) of levofloxacin, DR-3354, DU-6859a, DV-7751a, ciprofloxacin, sparfloxacin, and tosufloxacin against topoisomerase IV of S. aureus FDA 209-P were 2.3, 97, 0.45, 1.5, 2.5, 7.4, and 1.8 microg/ml, respectively, and were correlated well with their MICs. The IC50s of these drugs were from 2 to 20 times lower than those for the DNA gyrase. These results support genetic evidence that the primary target of new quinolones is topoisomerase IV in quinolone-susceptible strains of S. aureus. Three altered proteins of topoisomerase IV containing Ser-->Phe changes at codon 80 or Glu-->Lys changes at codon 84 of grlA, or both, were also purified. The inhibitory activities of quinolones against the topoisomerase IV which contained a single amino acid change were from 8 to 95 times weaker than those against the nonaltered enzyme. These results suggest that the mutations in the corresponding genes confer quinolone resistance.

Detection of mutations in the gyrA and parC genes in quinolone-resistant clinical isolates of Enterobacter cloacae.

We have determined partial sequences of the gyrA and parC genes of Enterobacter cloacae type strain including the regions analogous to the quinolone resistance-determining region of the Escherichia coli gyrA gene. The deduced 65- and 49-amino acid sequences of the determined regions of the E. cloacae gyrA and parC genes were identical to the corresponding regions of the E. coli GyrA and ParC proteins, respectively. We examined 40 clinical strains of E. cloacae isolated from patients with urinary tract infection for susceptibilities to nalidixic acid and ciprofloxacin. Based on the nalidixic acid and ciprofloxacin MICs, these isolates were divided into 19 quinolone-susceptible strains (MICs of nalidixic acid, 3.13-25 mg/L; MICs of ciprofloxacin, < or = 0.025 mg/L) and 21 quinolone-resistant strains (MICs of nalidixic acid, 400 to > 800 mg/L; MICs of ciprofloxacin, 0.39-100 mg/L). We analysed five quinolone-susceptible and 21 quinolone-resistant strains for alterations in GyrA and ParC. The five quinolone-susceptible strains had amino acid sequences in GyrA and ParC identical to those of type strain. Of the 21 quinolone-resistant isolates, three (MICs of nalidixic acid, 400 to > 800 mg/L; MICs of ciprofloxacin, 0.39-3.13 mg/L) had a single amino acid change at the position equivalent to Ser-83 in the E. coli GyrA protein and no alterations in ParC; one (MIC of nalidixic acid, > 800 mg/L; MIC of ciprofloxacin, 3.13 mg/L) had a single amino acid change at Ser-83 in GyrA and a single amino acid change at the position equivalent to Glu-84 in the E. coli ParC protein; two (MIC of nalidixic acid, > 800 mg/L; MIC of ciprofloxacin, 25 mg/L) had double amino acid changes at Ser-83 and Asp-87 in GyrA and no alterations in ParC; and 15 (MICs of nalidixic acid, > 800 mg/L; MICs of ciprofloxacin, 25-100 mg/L) had double amino acid changes at Ser-83 and Asp-87 in GyrA and a single amino acid change at Ser-80 or Glu-84 in ParC. This study suggests, that in clinical isolates of E. cloacae, DNA gyrase is a primary target of quinolones, that only a single amino acid change at Ser-83 in GyrA is sufficient to generate high-level resistance to nalidixic acid and to decrease susceptibility to ciprofloxacin, and that the accumulation of amino acid changes in GyrA and the simultaneous presence of the ParC alterations play a central role in developing high-level resistance to ciprofloxacin.

Rapid decline in penicillinase-producing Neisseria gonorrhoeae in Hong Kong associated with emerging 4-fluoroquinolone resistance.

OBJECTIVE--To study the changes in penicillinase-producing (PPNG) and high-level tetracycline resistant (TRNG) Neisseria gonorrhoeae isolated in Hong Kong associated with emerging quinolone resistance (QRNG) over a two year period from November 1992 to October 1994. MATERIALS AND METHODS--Four thousand and eighty-six strains of Neisseria gonorrhoeae isolated, of which 432 were PPNG, were examined for susceptibilities to penicillin and tetracycline by an agar dilution method using the breakpoint minimum inhibitory concentrations (MICs) of 1 and 10 mg/1 respectively. Ofloxacin susceptibility was done using 0.1 and 1 mg/l. Penicillinase production was detected by performing the chromogenic cephalosporin nitrocefin test on all penicillin resistant (MIC > 1 mg/l) strains. RESULTS--Three thousand and eighty (75.4%) and 79 (1.9%) strains were found to be penicillin resistant and TRNG (MIC > 10 mg/l) respectively. Sixty-nine strains (1.7%) were resistant to both, of which 54 (1.3%) were PPNG. Three strains were multiply-resistant to penicillin, tetracycline and ofloxacin; however, none was PPNG. While the percentage of penicillin resistant strains remained stable (mean 75.5%, SD 7.0), TRNG decreased from 4.5% to 2.1%. The most dramatic change was the sharp decline of PPNG from 25.5% in January 1993 to 4.3% in October 1994, concurrent with a linear increase in strains with ofloxacin MIC > 0.1 mg/l. Significant clinical failure was seen in strains having ofloxacin MIC > 1 mg/l (QRNG), which increased drastically from 0.5% to 10.4% during the study period. Selection against PPNG and TRNG strains appeared to occur only when fully quinolone-susceptible strains first become less susceptible (MIC > 0.1 mg/l), but not when these less susceptible strains become fully resistant (MIC > 1 mg/l). CONCLUSION--PPNG is now no longer hyperendemic in Hong Kong. Emergence of QRNG is associated with rapid decline of both PPNG and TRNG. This is the first report of plasmid-curing effect of the 4-fluoroquinolones occurring on an ecological scale.

Mechanisms of 4-quinolone resistance in quinolone-resistant and methicillin-resistant Staphylococcus aureus isolates from Japan and China

Ninety-two and 33 methicillin-resistant Staphylococcus aureus (MRSA) strains were isolated in Japan and China respectively. They were categorised as ofloxacin-susceptible (MIC < 12.5 mg/L), moderately (MIC 12.5-25 mg/L) or highly (MIC > or = 50 mg/L) ofloxacin-resistant. 4-Quinolone concentrations required to inhibit purified DNA gyrase from the moderately and highly quinolone-resistant MRSA were at least 20 times higher than those required to inhibit the equivalent enzyme from quinolone-susceptible strains. Reconstitution assays demonstrated that the 4-quinolone-resistant MRSA had a mutation in subunit A of DNA gyrase. A portion of the gyrA gene from amino acids codons 40-115 was sequenced. Four moderately resistant and seven highly resistant MRSA contained a Ser-->Leu substitution at amino acid 84; one moderately and one highly resistant MRSA and one moderately resistant methicillin-susceptible S. aureus (MSSA) strain contained a Glu-->Lys substitution at amino acid 88. Eight MRSA, including one quinolone-susceptible strain and one MSSA contained a silent mutation at amino acid 86. Uptake of ofloxacin in moderately resistant strains was almost the same in the presence or absence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), whereas in highly resistant strains, uptake increased when CCCP was added. Restriction fragment length analysis of the norA gene with the restriction endonuclease SfcI showed a mutation of nucleotide position 1085 in all MRSA strains tested except for one highly quinolone-resistant strain. Thus the mechanisms of 4-quinolone-resistance in these MRSA isolates involved alterations in both DNA gyrase and antimicrobial uptake and efflux.

Identification of DNA gyrase A mutations in ciprofloxacin-resistant isolates of Salmonella typhimurium from men and cattle in Germany.

Six multiply resistant isolates of Salmonella typhimurium var. copenhagen with high-level resistance to fluoroquinolones (e.g., MIC of ciprofloxacin: 32 micrograms/ml) were isolated from human patients (n = 3) and from cattle (n = 3). The isolates were examined by complementation tests using a set of broad-host-range plasmids, which carry either the gyrA+ or the gyrB+ genes or a combination of both from Escherichia coli K-12. The results indicated a combination of gyrA and gyrB mutations in all isolates. Subsequent direct sequencing of PCR-generated internal DNA fragments of gyrA revealed an identical double mutation in all six isolates (Ser-83-->Ala and Asp-87-->Asn). In addition, the results of phenotypic (i.e., phagetype, biotype, serotype) and genotypic characterization [i.e., ribotyping and polymerase chain reaction fingerprinting (PCR-fingerprinting)] were identical for all six isolates and were distinguishable from a quinolone-susceptible strain of the same serovar and an unrelated isolate of S. typhimurium. These data indicate the clonal identity of the fluoroquinolone-resistant strains of S. typhimurium isolated from men and cattle in Germany.

In vitro activity of novobiocin against multiresistant strains of Enterococcus faecium

Sixty strains of vancomycin- and ampicillin-resistant Enterococcus faecium were evaluated for their susceptibilities to novobiocin in vitro. In Mueller-Hinton broth, novobiocin inhibited all strains when it was used at a concentration of < or = 2 microgram/ml and 40 of 60 strains when it was used at a concentration of < or = 0.5 micrograms/ml. MICs were 8- to 16-fold higher in 50% serum. Novobiocin alone resulted in 2-log-unit killing at 24 h. Combinations of novobiocin and a fluoroquinolone (either ciprofloxacin or ofloxacin) were additive and bactericidal for quinolone-susceptible strains in either broth or 50% serum. Gentamicin did not affect novobiocin activity, and rifampin and doxycycline were antagonistic.

Lack of effect of carbonyl cyanide m-chlorophenylhydrazone on KB-5246 accumulation by Staphylococcus aureus.

The accumulation of KB-5246 in a quinolone-susceptible strain of Staphylococcus aureus was about 70 times that of norfloxacin. Carbonyl cyanide m-chlorophenylhydrazone increased the accumulation of norfloxacin about eightfold, but it did not influence that of KB-5246. The low efflux of KB-5246 from S. aureus may contribute to its potent antibacterial activity.