Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus.

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.

Action of quinolones against Staphylococcus aureus topoisomerase IV: basis for DNA cleavage enhancement.

Topoisomerase IV is the primary cellular target for most quinolones in Gram-positive bacteria; however, its interaction with these agents is poorly understood. Therefore, the effects of four clinically relevant antibacterial quinolones (ciprofloxacin, and three new generation quinolones: trovafloxacin, levofloxacin, and sparfloxacin) on the DNA cleavage/religation reaction of Staphylococcus aureus topoisomerase IV were characterized. These quinolones stimulated enzyme-mediated DNA scission to a similar extent, but their potencies varied significantly. Drug order in the absence of ATP was trovafloxacin > ciprofloxacin > levofloxacin > sparfloxacin. Potency was enhanced by ATP, but to a different extent for each drug. Under all conditions examined, trovafloxacin was the most potent quinolone and sparfloxacin was the least. The enhanced potency of trovafloxacin correlated with several properties. Trovafloxacin induced topoisomerase IV-mediated DNA scission more rapidly than other quinolones and generated more cleavage at some sites. The most striking correlation, however, was between quinolone potency and inhibition of enzyme-mediated DNA religation: the greater the potency, the stronger the inhibition. Dose-response experiments with two topoisomerase IV mutants that confer clinical resistance to quinolones (GrlA(Ser80Phe) and GrlA(Glu84Lys)) indicate that resistance is caused by a decrease in both drug affinity and efficacy. Trovafloxacin is more active against these enzymes than ciprofloxacin because it partially overcomes the effect on affinity. Finally, comparative studies on DNA cleavage and decatenation suggest that the antibacterial properties of trovafloxacin result from increased S. aureus topoisomerase IV-mediated DNA cleavage rather than inhibition of enzyme catalysis.

Quinolones inhibit DNA religation mediated by Staphylococcus aureus topoisomerase IV. Changes in drug mechanism across evolutionary boundaries.

Quinolones are the most active oral antibacterials in clinical use and act by increasing DNA cleavage mediated by prokaryotic type II topoisomerases. Although topoisomerase IV appears to be the primary cytotoxic target for most quinolones in Gram-positive bacteria, interactions between the enzyme and these drugs are poorly understood. Therefore, the effects of ciprofloxacin on the DNA cleavage and religation reactions of Staphylococcus aureus topoisomerase IV were characterized. Ciprofloxacin doubled DNA scission at 150 nM drug and increased cleavage approximately 9-fold at 5 microM. Furthermore, it dramatically inhibited rates of DNA religation mediated by S. aureus topoisomerase IV. This inhibition of religation is in marked contrast to the effects of antineoplastic quinolones on eukaryotic topoisomerase II, and suggests that the mechanistic basis for quinolone action against type II topoisomerases has not been maintained across evolutionary boundaries. The apparent change in quinolone mechanism was not caused by an overt difference in the drug interaction domain on topoisomerase IV. Therefore, we propose that the mechanistic basis for quinolone action is regulated by subtle changes in drug orientation within the enzyme.drug.DNA ternary complex rather than gross differences in the site of drug binding.

Activities of trovafloxacin compared with those of other fluoroquinolones against purified topoisomerases and gyrA and grlA mutants of Staphylococcus aureus.

Frequencies of mutation to resistance with trovafloxacin and four other quinolones were determined with quinolone-susceptible Staphylococcus aureus RN4220 by a direct plating method. First-step mutants were selected less frequently with trovafloxacin (1.1 x 10(-10) at 2 to 4x the MIC) than with levofloxacin or ciprofloxacin (3.0 x 10(-7) to 3.0 x 10(-8) at 2 to 4x the MIC). Mutants with a change in GrlA (Ser80-->Phe or Tyr) were most commonly selected with trovafloxacin, ciprofloxacin, levofloxacin, or pefloxacin. First-step mutants were difficult to select with sparfloxacin; however, second-step mutants with mutations in gyrA were easily selected when a preexisting mutation in grlA was present. Against 29 S. aureus clinical isolates with known mutations in gyrA and/or grlA, trovafloxacin was the most active quinolone tested (MIC at which 50% of isolates are inhibited [MIC(50)] and MIC(90), 1 and 4 microg/ml, respectively); in comparison, MIC(50)s and MIC(90)s were 32 and 128, 16 and 32, 8 and 32, and 128 and 256 microg/ml for ciprofloxacin, sparfloxacin, levofloxacin, and pefloxacin, respectively. Strains with a mutation in grlA only were generally susceptible to all of the quinolones tested. For mutants with changes in both grlA and gyrA MICs were higher and were generally above the susceptibility breakpoint for ciprofloxacin, sparfloxacin, levofloxacin, and pefloxacin. Addition of reserpine (20 microg/ml) lowered the MICs only of ciprofloxacin fourfold or more for 18 of 29 clinical strains. Topoisomerase IV and DNA gyrase genes were cloned from S. aureus RN4220 and from two mutants with changes in GrlA (Ser80-->Phe and Glu84-->Lys). The enzymes were overexpressed in Escherichia coli GI724, purified, and used in DNA catalytic and cleavage assays that measured the relative potency of each quinolone. Trovafloxacin was at least five times more potent than ciprofloxacin, sparfloxacin, levofloxacin, or pefloxacin in stimulating topoisomerase IV-mediated DNA cleavage. While all of the quinolones were less potent in cleavage assays with the altered topoisomerase IV, trovafloxacin retained its greater potency relative to those of the other quinolones tested. The greater intrinsic potency of trovafloxacin against the lethal topoisomerase IV target in S. aureus contributes to its improved potency against clinical strains of S. aureus that are resistant to other quinolones.

Anti-Toxoplasma gondii activities and structure-activity relationships of novel fluoroquinolones related to trovafloxacin.

Eleven novel fluoroquinolones closely related to trovafloxacin were evaluated for their in vitro activity against Toxoplasma gondii, and their structure-activity relationships were examined. The 50% inhibitory concentration (IC50) of trovafloxacin against T. gondii was 2.93 microM; the IC50 of the 11 analogs ranged from 0.53 to 14. 09 microM. Six analogs had IC50s lower than that of trovafloxacin. Examination of the structure-activity relationships of the compounds revealed that addition of a -CH3 at C-5 of the 1,8-naphthyridone ring, at C-2 of the azabicyclohexane ring, or on the -NH2 at the 6 position of the azabicyclohexane ring resulted in a four- to sixfold increase in activity. Moreover, replacement of 2,4-difluorophenyl by cyclopropyl at N-1 of the 1,8-naphthyridone ring increased activity twofold, and moving the -NH2 one atom further away from the azabicyclohexane ring decreased activity. There was no difference between the naphthyridone and quinolone analogs. These results indicate that structure-activity studies of compounds related to drugs active against T. gondii may be useful in producing compounds with more potent activities against the parasite.

Bacteriological activity of trovafloxacin, a new quinolone, against respiratory tract pathogens.

The use of established fluoroquinolones, such as ciprofloxacin and ofloxacin, as empirical therapy for the treatment of moderate-to-severe respiratory tract infections is limited by their poor activity against gram-positive and atypical pathogens. Data from in vitro susceptibility studies and in vivo animal protection models suggest that the new fluoroquinolone, trovafloxacin, compared with ciprofloxacin and ofloxacin offers equivalent activity against gram-negative pathogens and improved activity against gram-positive pathogens. In particular, susceptibility data indicate that trovafloxacin is at least 16-fold more potent than either ciprofloxacin or ofloxacin against penicillin-susceptible and penicillin-resistant strains of Streptococcus pneumoniae. Other susceptible pathogens include Streptococcus pyogenes, vancomycin-susceptible Enterococcus faecalis and the atypical respiratory pathogens Legionella pneumophila, Mycoplasma pneumoniae and Chlamydia pneumoniae. In vivo studies involving models of protection against acute systemic infection and pneumococcal pneumonia in mice, and Legionnaires' disease in guinea pigs, indicate that the antibacterial spectrum observed for trovafloxacin in vitro extends to the in vivo setting. Together, these findings suggest that trovafloxacin may offer clinical efficacy against respiratory pathogens superior to that of ciprofloxacin and of ofloxacin, and may find a useful role as empiric therapy in both the community and hospital setting.

Topoisomerase IV catalysis and the mechanism of quinolone action.

Topoisomerase IV is a bacterial type II topoisomerase that is essential for proper chromosome segregation and is a target for quinolone-based antimicrobial agents. Despite the importance of this enzyme to the survival of prokaryotic cells and to the treatment of bacterial infections, relatively little is known about the details of its catalytic mechanism or the basis by which quinolones alter its enzymatic functions. Therefore, a series of experiments that analyzed individual steps of the topoisomerase IV catalytic cycle were undertaken to address these critical mechanistic issues. The following conclusions were drawn. First, equilibrium levels of DNA cleavage mediated by the bacterial enzyme were considerably (>10-fold) higher than those observed with its eukaryotic counterparts. To a large extent, this reflected decreased rates of DNA religation. Second, the preference of topoisomerase IV for catalyzing DNA decatenation over relaxation reflects increased rates of strand passage and enzyme recycling rather than a heightened recognition of intermolecular DNA helices. Third, quinolones stimulate topoisomerase IV-mediated DNA cleavage both by increasing rates of DNA scission and by inhibiting religation of cleaved DNA. Finally, quinolones inhibit the overall catalytic activity of topoisomerase IV primarily by interfering with enzyme-ATP interactions.

New quinolone compounds from Pseudonocardia sp. with selective and potent anti-Helicobacter pylori activity: taxonomy of producing strain, fermentation, isolation, structural elucidation and biological activities.

Eight novel quinolones with anti-Helicobacter pylori activity were isolated from the actinomycete Pseudonocardia sp. CL38489. The quinolones were very potent against H. pylori with MICs up to 0.1 ng/ml. The quinolones appear to be specific for H. pylori, since they did not show antimicrobial activity when tested against a panel of other microorganisms.

CJ-12,954 and its congeners, new anti-Helicobacter pylori compounds produced by Phanerochaete velutina: fermentation, isolation, structural elucidation and biological activities.

Seven new phthalide compounds with anti-Helicobacter pylori activities were isolated from the basidiomycete Phanerochaete velutina CL6387. The two most potent phthalide compounds, CJ-12,954 and CJ-13,014, have MICs of 5 ng/ml. The structure-activity relationship shows that the presence of a spiroketal part in addition to the phthalide part, greatly enhances the activity. The phthalide compounds appear to be specific for H. pylori, since they did not show antibacterial activities when tested against a panel of other microorganisms.

The chemistry and biological profile of trovafloxacin.

The fluoroquinolone antibacterials are noted for their activity after oral administration and potent activity against Gram-negative pathogens. Trovafloxacin (CP-99,219) is a new quinolone antibacterial characterized by a novel 3-azabicyclo[3.1.0]hexyl substituent at the C-7 position, which was discovered in the course of a programme targeting improved activity compared with ciprofloxacin against Gram-positive aerobic organisms and anaerobes, as well as an extended elimination half-life. An overview of the chemical properties of trovafloxacin is given. Trovafloxacin exhibits excellent potency against Gram-positive organisms and anaerobes, while retaining the potent Gram-negative activity of ciprofloxacin. Its pharmacokinetic properties in humans have been shown to be compatible with a once-daily dosing regimen. The combined spectrum and pharmacokinetics of trovafloxacin have been demonstrated to result in excellent efficacy in both animal models of infections and human clinical trials. Phase II and Phase III programmes have been completed.

In-vitro and in-vivo activity of trovafloxacin against Streptococcus pneumoniae.

Trovafloxacin had greater in-vitro activity than comparative fluoroquinolone agents against penicillin-sensitive pneumococci in studies from the USA, UK, Slovakia, Czech Republic, Sweden and South Africa. This activity was maintained against penicillin-resistant strains, with MIC90 values of < or = 0.25 mg/L observed for both groups. Bactericidal activity appeared to occur within one or two dilutions of the MIC and, in the limited number of strains studied, the MIC was independent of the medium tested and pH over the range pH 5-8. Mutation to decreased susceptibility to trovafloxacin occurred in vitro at a low frequency in the pneumococcus (< or = 8.9 x 10(-9)). Mutants with changes in the topoisomerase IV A subunit (GrlA) were still inhibited by 0.5 mg/L of trovafloxacin. Trovafloxacin was more efficacious than ciprofloxacin, temafloxacin or ofloxacin in mouse pneumonia models for both penicillin-susceptible and penicillin-resistant pneumococci. Trovafloxacin was also highly efficacious in a rabbit pneumococcal meningitis model. These data suggest that the clinical efficacy of trovafloxacin against pneumococci should be evaluated further.

Activity of the new fluoroquinolone trovafloxacin (CP-99,219) against DNA gyrase and topoisomerase IV mutants of Streptococcus pneumoniae selected in vitro.

The MICs of trovafloxacin, ciprofloxacin, ofloxacin, and sparfloxacin at which 90% of isolates are inhibited for 55 isolates of pneumococci were 0.125, 1, 4, and 0.5 microgram/ml, respectively. Resistant mutants of two susceptible isolates were selected in a stepwise fashion on agar containing ciprofloxacin at 2 to 10 times the MIC. While no mutants were obtained at the highest concentration tested, mutants were obtained at four times the MIC of ciprofloxacin (4 micrograms/ml) at a frequency of 1.0 x 10(-9). Ciprofloxacin MICs for these first-step mutants ranged from 4 to 8 micrograms/ml, whereas trovafloxacin MICs were 0.25 to 0.5 microgram/ml. Amplification of the quinolone resistance-determining region of the grlA (parC; topoisomerase IV) and gyrA (DNA gyrase) genes of the parents and mutants revealed that changes of the serine at position 80 (Ser80) to Phe or Tyr (Staphylococcus aureus coordinates) in GrlA were associated with resistance to ciprofloxacin. Second-step mutants of these isolates were selected by plating the isolates on medium containing ciprofloxacin at 32 micrograms/ml. Mutants for which ciprofloxacin MICs were 32 to 256 micrograms/ml and trovafloxacin MICs were 4 to 16 micrograms/ml were obtained at a frequency of 1.0 x 10(-9). Second-step mutants also had a change in GyrA corresponding to a substitution in Ser84 to Tyr or Phe or in Glu88 to Lys. Trovafloxacin protected from infection mice whose lungs were inoculated with lethal doses of either the parent strain or the first-step mutant. These results indicate that resistance to fluoroquinolones in S. pneumoniae occurs in vitro at a low frequency, involving sequential mutations in topoisomerase IV and DNA gyrase. Trovafloxacin MICs for wild-type and first-step mutants are within clinically achievable levels in the blood and lungs of humans.

Pharmacokinetics of trovafloxacin (CP-99,219), a new quinolone, in rats, dogs, and monkeys.

The pharmacokinetics of trovafloxacin [CP-99,219; 7-(3-azabicyclo[3.1.0]hexyl)-naphthyridone] were studied in rats, dogs, and monkeys following oral and intravenous administration. After intravenous dosing, the systemic clearances of trovafloxacin in rats, dogs, and monkeys were 12.5, 11.1, and 7.2 ml/min/kg of body weight, respectively, and the respective volumes of distribution were 0.9, 1.7, and 4.3 liters/kg, with corresponding elimination half-lives of 0.7, 1.8, and 7.0 h. After the administration of oral doses of 50, 20, and 20 mg/kg to rats, dogs, and monkeys serum trovafloxacin concentrations reached a maximum at 0.6, 2.3, and 2.3 h, respectively, with respective maximum concentrations of trovafloxacin in serum of 11.5, 3.5, and 5.2 micrograms/ml; the corresponding elimination half-lives were 2.2, 2.5, and 7.5 h. The oral bioavailability of trovafloxacin was 68, 58, and 85% in rats, dogs, and monkeys, respectively. The binding of trovafloxacin to serum proteins was concentration independent, averaging 92, 75, and 66% for rats, dogs, and monkeys, respectively. Trovafloxacin penetrated well into tissues in dogs. The urinary recoveries of unchanged drug were less than 5% in dogs and monkeys, with or without incubation with alkali or Glusulase (beta-glucuronidase and sulfatase). In rats, 99.8% of the orally administered radioactivity was recovered in feces, while 20.6, 3.4, and 67.1% of the radioactive dose in bile duct-cannulated rats were recovered in feces, urine, and bile, respectively. These results suggest that the elimination of trovafloxacin from rats, and possibly from dogs and monkeys, is primarily through biliary excretion.

In vivo efficacy of trovafloxacin (CP-99,219), a new quinolone with extended activities against gram-positive pathogens, Streptococcus pneumoniae, and Bacteroides fragilis.

The interesting in vitro antimicrobial activity and pharmacokinetics of the new quinolone trovafloxacin (CP-99,219) warranted further studies to determine its in vivo efficacy in models of infectious disease. The significance of the pharmacokinetic and in vitro antimicrobial profiles of trovafloxacin was shown through efficacy in a series of animal infection models by employing primarily oral therapy. Against acute infections, trovafloxacin was consistently more effective than temafloxacin, ciprofloxacin, and ofloxacin against Streptococcus pneumoniae and other gram-positive pathogens while maintaining activity comparable to that of ciprofloxacin against gram-negative organisms. In a model of murine pneumonia, trovafloxacin was more efficacious than temafloxacin, while ciprofloxacin failed against S. pneumoniae (50% protective doses, 2.1, 29.5, and >100 mg/kg, respectively). In addition to its inherent in vitro potency advantage against S. pneumoniae, these data were supported by a pharmacokinetic study that showed levels of trovafloxacin in pulmonary tissue of S. pneumoniae-infected CF1 mice to be considerably greater than those of temafloxacin and ciprofloxacin (twice the maximum drug concentration in serum; two to three times the half-life, and three to six times the area under the concentration-time curve). Against localized mixed anaerobic infections, trovafloxacin was the only agent to effectively reduce the numbers of recoverable CFU of Bacteroides fragilis ( >1,000-fold), Staphylococcus aureus (1,000-fold), and Escherichia coli ( >100-fold) compared with ciprofloxacin, vancomycin, metronidazole, clindamycin, cefoxitin, and ceftriaxone. The in vitro and in vivo antimicrobial activities of trovafloxacin and its pharmacokinetics in laboratory animals provide support for the ongoing and planned human phase II and III clinical trials.

Analysis of eukaryotic topoisomerase II cleavage sites in the presence of the quinolone CP-115,953 reveals drug-dependent and -independent recognition elements.

The quinolone derivative CP-115,953 [6,8-difluoro-7-(4-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid] has been shown to induce eukaryotic topoisomerase II-mediated breaks in DNA, producing cleavage patterns that are distinct from those induced by the anticancer drugs amsacrine, etoposide, and teniposide. High levels of the quinolone have been found to inhibit topoisomerase II activity via an interaction with the enzyme and not by DNA unwinding. Topoisomerase II cleavage sites were analyzed on nine DNA fragments, and 85 quinolone-induced sites were sequenced, as well as 86 amsacrine and 134 teniposide sites. A consensus sequence was derived for the quinolone sites that is different from those reported for other drugs; however, because topoisomerase II cleavage sites are double-stranded but not palindromic, different consensus sequences are not easily compared. For this reason, a new, double-stranded, consensus sequence method, the "unique-base analysis," was developed; this was applied to the quinolone sites as well as six other large sets of topoisomerase II sites determined in the absence or presence of drugs. For each of the seven sets of sites, conserved bases were found in the 16-base region spanning positions -6 to +10, relative to the enzyme cleavage site (DNA breakage between -1 and +1). The conserved bases were virtually identical in the regions flanking the cleavage site for all seven data sets. In contrast, the base preferences identified proximal to the cleavage sites were unique to the drug tested. These observations suggest that the selection of cleavage sites by topoisomerase II involves both enzyme-dependent and drug-dependent recognition elements. The single most preferred base in the quinolone sites was a cytosine at -1; the same preference was found with teniposide, and 60 of the 85 quinolone sites co-localized with teniposide sites.

In vitro activity of CP-99,219, a novel 7-(3-azabicyclo[3.1.0]hexyl) naphthyridone antimicrobial.

The in vitro activity of CP-99,219 was compared with that of ciprofloxacin and sparfloxacin against 814 clinical bacterial isolates using a microdilution method with brain-heart infusion broth. CP-99,219 was the most potent agent tested against methicillin-resistant, ciprofloxacin-susceptible staphylocci (minimum inhibitory concentration [MIC]90 < or = 0.25 microgram/ml). CP-99,219 was 32-fold and fourfold more potent than ciprofloxacin and sparfloxacin, respectively, against Streptococcus pneumoniae, including strains resistant to penicillin G and erythromycin (MIC90 < or = 0.25 microgram/ml). CP-99,219 was also the most potent agent tested against S. pyogenes and Enterococcus faecalis (MIC90 < or = 0.5 microgram/ml). The activity of CP-99,219 against Enterobacteriaceae was comparable to that of sparfloxacin, with 90% of Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Citrobacter freundii, C. diversus, Helicobacter pylori, and K. oxytoca being inhibited by < or = 0.5 microgram/ml. Serratia marcescens, Morganella morganii, and Pseudomonas aeruginosa were less susceptible, with MIC90 values to CP-99,219 of 4, 2, and 2 micrograms/ml, respectively. The MIC90 for Bacteroides fragilis was 0.39 microgram/ml for CP-99,219 compared with 12.5 micrograms/ml for ciprofloxacin. CP-99,219 was highly bactericidal at 1 x to 4 x MIC against both Gram-positive and Gram-negative organisms; its activity was similar in nutrient, trypticase soy, and cation-supplemented Mueller-Hinton broths. The spectrum and potency observed with CP-99,219 warrant further testing with this novel quinolone.

Immunological and molecular characterization of Helicobacter felis urease.

Urease activity has recently been shown to be an important virulence determinant for Helicobacter pylori, allowing it to survive the low pH of the stomach during colonization. Experimental murine infection with Helicobacter felis is now being used as a model for H. pylori infection to study the effects of vaccines, antibiotics, and urease inhibitors on colonization. However, little information comparing the ureases of H. felis and H. pylori is available. Urease was partially purified from the cell surface of H. felis ATCC 49179 by A-5M agarose chromatography, resulting in an eightfold increase in specific activity over that of crude urease. The apparent Km for urea for the partially purified urease was 0.4 mM, and the enzyme was inhibited in a competitive manner by flurofamide (50% inhibitory concentration = 0.12 microM). Antiserum to whole cells of H. pylori recognized both H. pylori and H. felis urease B subunits. Antiserum raised against H. felis whole cells recognized the large and small autologous urease subunits and the cpn60 heat shock molecule in both H. felis and H. pylori. However, this antiserum showed only a weak reaction with the B subunit of H. pylori urease. Two oligomeric DNA sequences were used as probes to evaluate the relatedness of H. felis and H. pylori urease gene sequences. One 30-mer from the ureA sequence, which had been shown previously to be specific for H. pylori, failed to hybridize to H. felis genomic DNA. A probe to the putative coding sequence for the active site of the H. pylori ureB subunit hybridized at low intensity to a 2.8-kb fragment of BamHI-HindIII-digested H. felis DNA, suggesting that the sequences were homologous but not identical, a result confirmed from the recently published sequences of ureA and ureB from H. felis.

Bisulfite or sulfite inhibits growth of Helicobacter pylori.

Bisulfite or sulfite was found to be inhibitory to Helicobacter pylori growth. A modified version of Brucella broth (BB), bisulfite-less BB (BLBB), supported rapid, robust, and consistent growth of H. pylori. We suggest that BLBB simply be called "Pylori broth" for distinction from Brucella broth.