Catrenich CE, Makin KM. Characterization of the morphologic conversion of Helicobacter pylori from bacillary to coccoid forms. Scand J Gastroenterol1991,26(suppl 181), 58-64Growth 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.Scand J Gastroenterol Downloaded from informahealthcare.com by Thomas Jefferson University on 03/11/15For personal use only.Scand J Gastroenterol Downloaded from informahealthcare.com by Thomas Jefferson University on 03/11/15For personal use only.
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.
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 ؋ 10 ؊8 and 5.7 ؋ 10 ؊9 , respectively) than with the NFQs, gatifloxacin, or clinafloxacin (<5.7 ؋ 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 g/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 g/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.Bacterial infections caused by multidrug-resistant pathogens are a major global problem, especially for nosocomial infections (6). Methicillin-resistant Staphylococcus aureus (MRSA) is one such pathogen against which options for effective antibacterial therapies are already limited (2). While certain newly developed drugs have promising activity against MRSA (1, 9), their relatively narrow spectra of activity could limit their clinical use in empirical therapy.Recently, a series of 8-methoxy, nonfluorinated quinolones (NFQs) (Fig. 1) F-1509, p. 210, 2000). On the basis of in vitro potency data, these compounds are more potent against MRSA and coagulase-negative staphylococci than several fluoroquinolones and have activities comparable to that of clinafloxacin (10). An apparent advantage of the NFQs against S. aureus lies in their ability to (i) better utilize both DNA gyrase and topisomerase IV as dual targets than certain quinolones, such as ciprofloxacin and trovafloxacin, and (ii) largely circumvent existing mutations in serine and glutamate "hot spots" of the target genes, gyrA and grlA, commonly associated with quinolone resistance (10, 11). However, it is imperative to ascertain the potential for development of de novo resistance to the NFQs in these pathogens. This report describes the in vitro isolation of S. aureus mutants with reduced susceptibilities to the NFQs and other quinolones by two approaches: stepwise isolation of mutants and spiral plater-based serial passage. MATERIALS AND METHODSMaterial...
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 coagulasenegative staphylococci (CoNS; n ؍ 24), the NFQs and clinafloxacin were shown to be more potent (MIC at which 90% of the isolates are inhibited [MIC 90 ] ؍ 2 g/ml for MRSA and 0.5 g/ml for CoNS) than ciprofloxacin, trovafloxacin, and gatifloxacin (MIC 90 ؍ 16 to >64 g/ml for MRSA and 4 to >32 g/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 >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.
A high-throughput screen (HTS)was developed and used to identify inhibitors of bacterial DNA gyrase. Among the validated hits were 53 compounds that also inhibited mammalian topoisomerase II with IC50 values of <12.5 µg/mL for 51 of them. Using computational methods, these compounds were subjected to cluster analysis to categorize them according to their chemical and structural properties. Nine compounds from different clusters were tested for their whole-cell inhibitory activity against 3 cancer cell lines—NCI-H460 (lung), MCF7 (breast), and SF-268 (CNS)—at a concentration of 100 µM. Five compounds inhibited cell growth by >50% for all 3 cell lines tested. These compounds were tested further against a panel of 53 to 57 cell lines representing leukemia, melanoma, colon, CNS, ovarian, renal, prostate, breast, and non–small cell lung cancers. In this assay, PGE-7143417 was found to be the most potent compound, which inhibited the growth of all the cell lines by 50% at a concentration range of 0.31 to 2.58 µM, with an average of 1.21 µM. An additional 17 compounds were also tested separately against a panel of 10 cell lines representing melanoma, colon, lung, mammary, ovarian, prostate, and renal cancers. In this assay, 4 compounds—PGE-3782569, PGE-7411516, PGE-2908955, and PGE-3521917—were found to have activity with concentrations for 50% cell growth inhibition in the 0.59 to 3.33, 22.5 to 59.1, 7.1 to >100, and 24.7 to >100 µM range. ( Journal of Biomolecular Screening 2003:157-163)
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