Staphylococcus aureus mutants isolated via exposure to nonfluorinated quinolones: detection of known and unique mutations.

The in vitro development of resistance to the new nonfluorinated quinolones (NFQs; PGE 9262932, PGE 4175997, and PGE 9509924) was investigated in Staphylococcus aureus. At concentrations two times the MIC, step 1 mutants were isolated more frequently with ciprofloxacin and trovafloxacin (9.1 x 10(-8) and 5.7 x 10(-9), respectively) than with the NFQs, gatifloxacin, or clinafloxacin (<5.7 x 10(-10)). Step 2 and step 3 mutants were selected via exposure of a step 1 mutant (selected with trovafloxacin) to four times the MICs of trovafloxacin and PGE 9262932. The step 1 mutant contained the known Ser80-Phe mutation in GrlA, and the step 2 and step 3 mutants contained the known Ser80-Phe and Ser84-Leu mutations in GrlA and GyrA, respectively. Compared to ciprofloxacin, the NFQs were 8-fold more potent against the parent and 16- to 128-fold more potent against the step 3 mutants. Mutants with high-level NFQ resistance (MIC, 32 microg/ml) were isolated by the spiral plater-based serial passage technique. DNA sequence analysis of three such mutants revealed the following mutations: (i) Ser84-Leu in GyrA and Glu84-Lys and His103-Tyr in GrlA; (ii) Ser-84Leu in GyrA, Ser52-Arg in GrlA, and Glu472-Val in GrlB; and (iii) Ser84-Leu in GyrA, Glu477-Val in GyrB, and Glu84-Lys and His103-Tyr in GrlA. Addition of the efflux pump inhibitor reserpine (10 microg/ml) resulted in 4- to 16-fold increases in the potencies of the NFQs against these mutants, whereas it resulted in 2-fold increases in the potencies of the NFQs against the parent.

Activity of non-fluorinated quinolones (NFQs) against quinolone-resistant Escherichia coli and Streptococcus pneumoniae.

The newly developed 8-methoxy, non-fluorinated quinolones (NFQs) were studied to elucidate their enzyme inhibitory activity against wild-type and mutant GyrA (Ser-83-->Trp) forms of Escherichia coli DNA gyrase. Using a DNA supercoiling inhibition assay, the NFQs were found to inhibit 50% (IC50) of the E. coli DNA gyrase activity in the 1.6-3.2 mg/L concentration range and were comparable to ciprofloxacin. However, against the GyrA (Ser-83-->Trp) mutant, the NFQs were approximately 16-fold more potent than ciprofloxacin. Antibacterial potency of the NFQs was investigated using clinical isolates of E. coli and penicillin-resistant Streptococcus pneumoniae (PRSP), including strains with reduced susceptibility to quinolones. Against 20 uncharacterized clinical isolates of E. coli, the MIC90s of the NFQs were in the 0.125-0.25 mg/L range while those of ciprofloxacin, trovafloxacin, gatifloxacin and clinafloxacin were in the 0.016-0.125 mg/L range. Against clinical isolates with characterized mutations in gyrA and parC, PGE9262932, an NFQ, was two- to eight-fold more potent than ciprofloxacin. Against 23 clinical isolates of PRSP, the NFQs (MIC90 0.031-0.125 mg/L) were more potent than ciprofloxacin, trovafloxacin, and gatifloxacin (MIC90 0.25-2.0 mg/L), and at least as potent as clinafloxacin (MIC90 0.125 mg/L). Against S. pneumoniae strains with gyrA and parC mutations, the NFQs (MIC 0.125-1.0 mg/L) were more potent than ciprofloxacin, trovafloxacin and gatifloxacin (MIC 4-32 mg/L), and comparable to clinafloxacin (MIC 0.5-1 mg/L).

Quinolone resistance in Staphylococci: activities of new nonfluorinated quinolones against molecular targets in whole cells and clinical isolates.

The activity of three new, 8-methoxy-nonfluorinated quinolones (NFQs) against multiple-drug-resistant staphylococci was investigated. First, using Staphylococcus aureus strains containing point mutations in the serine 84-80 hot spots of the target genes (gyrA and grlA), cell growth inhibition potencies of the NFQs as a result of DNA gyrase and topoisomerase IV inhibition were estimated and compared with those of known fluoroquinolones. The NFQs and clinafloxacin showed higher affinities toward both the targets than ciprofloxacin, trovafloxacin and gatifloxacin. Furthermore, the ratio of the calculated affinity parameter for DNA gyrase to that for topoisomerase IV was lower in the case of the NFQs, clinafloxacin, and gatifloxacin than in the case of ciprofloxacin and trovafloxacin. These results suggest that the former group of quinolones is better able to exploit both the targets. Next, using clinical isolates of methicillin-resistant S. aureus (MRSA; n = 34) and coagulase-negative staphylococci (CoNS; n = 24), the NFQs and clinafloxacin were shown to be more potent (MIC at which 90% of the isolates are inhibited [MIC90] = 2 microg/ml for MRSA and 0.5 microg/ml for CoNS) than ciprofloxacin, trovafloxacin, and gatifloxacin (MIC90 = 16 to >64 microg/ml for MRSA and 4 to >32 microg/ml for CoNS). Bactericidal kinetics experiments, using two MRSA isolates, showed that exposure to the NFQs at four times the MIC reduced the bacterial counts (measured in CFU per milliliter) by > or =3 log units in 2 to 4 h. Overall, the NFQs and clinafloxacin were less susceptible than the other quinolones to existing mechanisms of quinolone resistance in staphylococci.

Virulence of coccoid and bacillary forms of Helicobacter pylori in gnotobiotic piglets.

This study sought to determine if coccoid forms of Helicobacter pylori are virulent for gnotobiotic piglets. Coccoid forms were generated by maintaining broth cultures of H. pylori under microaerobic conditions for 16 days. The resulting cultures contained bacteria with a coccoid morphology that could not be cultured in vitro. Coccoid H. pylori did not colonize any of 6 gnotobiotic piglets that were inoculated, whereas bacillary H. pylori colonized 6 of 6 inoculated piglets. Piglets colonized by bacillary H. pylori developed lymphocytic gastritis, but no gastritis developed in piglets inoculated with coccoid H. pylori, and coccoid-inoculated piglets were sero-negative for H. pylori-specific antibody. Thus, coccoid H. pylori appears to be a degenerate nonviable morphologic phase.

Characterization of the morphologic conversion of Helicobacter pylori from bacillary to coccoid forms.

Growth studies of Helicobacter pylori were performed involving analysis of the bacterium and its microenvironment, to lend insight into the factors responsible for the morphologic conversion phenomenon. H. pylori converted from bacillary to coccoid forms in broth culture after incubation for 5 days under microaerobic conditions with agitation. This morphologic conversion was paralleled by a dramatic decrease in colony-forming units per milliliter (CFU/ml) and a significant endogenous increase in the pH of the broth culture. In addition, removal of broth cultures from microaerobic conditions after 3 days of incubation resulted in a rapid increase in culture pH, a morphologic conversion, and a concomitant decrease of CFU/ml. These observations suggest an inhibitory effect of basic pH, endogenously produced, on the growth of H. pylori in vitro. Experiments designed to identify the reason for the endogenous increase in culture pH demonstrated that the urease enzyme of H. pylori is not primarily responsible for this phenomenon. Rather, H. pylori appears to produce a deaminase enzyme that is likely responsible for the generation of ammonia, which results in the increase in culture pH, the morphologic conversion, and the loss of culturability observed in vitro. Also indicated is the need for a buffering component (for example, bicarbonate) to maintain pH conditions favorable to the growth of H. pylori.