- Volume 49, Issue 7, 2000

The association between Helicobacter pylori infection and gastric motility abnormalities is still controversial, partly because of the lack of an appropriate animal model. H. heilmannii type 1 (Hh1), a spiral bacterium that infects the stomach of both man and pigs, easily colonises and induces an inflammatory response in the gastric mucosa of rodents. For these reasons, the present study investigated the relationship between gastric motility in rats experimentally infected with Hh1 and correlated the results with serum gastrin and gastric somatostatin concentrations, as these hormones seem to be involved in gastric motility. Ten rats were inoculated with gastric mucus from an Hh1-positive pig and 10 animals with gastric mucus from an Hh1-negative pig (control group). After 56 days, gastric emptying was studied in vivo by scintigraphy. The animals were then killed, blood samples were collected for serum gastrin measurement, strips of the gastric wall were obtained for an in-vitro motor study and fragments of the gastric antrum were obtained for somatostatin content evaluation, Hh1 diagnosis and histological study. There was a significant increase in gastric emptying in the test group compared with the controls as demonstrated by the in-vivo and in-vitro studies. Serum gastrin levels were significantly higher and somatostatin levels were lower in the test group than in the controls. In addition, infected animals showed evidence of gastritis on histological examination. Gastric motility is altered in rats infected with Hh1, a fact possibly related to concurrent abnormalities of gastrin and somatostatin secretion.

The interaction between Clostridium butyricum and Helicobacter pylori was examined in vitro and in vivo. The culture supernate of C. butyricum MIYAIRI 588 inhibited the growth of H. pylori even when its pH was adjusted to 7.4. The bactericidal effect of butyric acid on H. pylori was stronger than that of lactic, acetic or hydrochloric acids. Flow cytometric analysis showed that pre-incubation of gastric epithelial (MKN45) cells with H. pylori and C. butyricum inhibited the adhesion of H. pylori to the cells. Persistent infection with H. pylori in the gastric mucosa of germ-free mice was observed for 5 weeks. Cure of persistent infection with H. pylori in the gnotobiotic mice was demonstrated following infection with C. butyricum. The probiotic agent, C. butyricum MIYAIRI 588 may have some beneficial effects on H. pylori infection.

In Mycobacterium tuberculosis there is a strong correlation between in-vitro resistance to rifampicin (RIF) and pyrazinamide (PZA) and mutations in rpoB and pncA, respectively. Approximately 50 mutations associated with resistance have been reported for rpoB and 70 for pncA, and, theoretically, many more are possible. Therefore, the identification of rpoB and pncA mutations in M. tuberculosis might be used for the simultaneous determination of resistance and for typing multi-drug-resistant (MRD) strains during possible outbreaks. The present study examined four sensitive and six MDR isolates of M. tuberculosis from Turkey and eight isolates from a nosocomial MDR tuberculosis (TB) outbreak in the UK. Gene mutations were identified by the Innogenetics LiPA rpoB assay or automated sequencing, or both. All the sensitive isolates had rpoB and pncA wild-type genotypes, whereas all the RIF- and PZA-resistant isolates had rpoB and pncA mutations. All four mutations seen in rpoB, but none of the six in pncA, had been reported previously. The rpoB and pncA mutations seen in the Turkish isolates defined six distinct genotypes amongst the six MDR isolates, while standard IS6110 typing discriminated only four. All isolates from the single strain MDR-TB outbreak had identical genotypes. Rapid genotyping was performed on the sputum from a patient who presented 2 years after the initial MDR-TB outbreak and this showed rpoB and pncA genotypes identical to the other outbreak isolates. This result was available within 36 h. The analysis of rpoB and pncA is a rapid and practical means of simultaneously identifying and typing MDR isolates of M. tuberculosis.

The gene coding for the DNA polymerase I from Treponema pallidum, Nichols strain, was cloned and sequenced. Depending on which of the two alternative initiation codons was used, the protein was either 997 or 1015 amino acids long and the predicted protein had a molecular mass of either 112 or 114 kDa. Sequence comparisons with other polA genes showed that all three domains expected in the DNA polymerase I class of enzymes were present in the protein (5′–3′ exonuclease, 3′–5′ exonuclease and polymerase domains). Additionally, there were four unique insertions of 20–30 amino acids each, not seen in other DNA polymerase I enzymes. Two of the inserts were near the boundary of the two exonuclease domains and the other two interrupted the 3′–5′ exonuclease domain which is involved in proofreading. The predicted amino-acid sequence had an exceptionally high content of cysteine (2.4% compared with <0.05% for most other sequenced DNA polymerase I enzymes). The polA gene was further cloned into pProEXTMHTa for expression and purification. The transformants expressed a protein of 115 kDa. Antibodies raised against synthetic peptide fragments of the putative DNA polymerase I recognised the 115-kda band in Western blot analysis. No DNA synthesis activity could be demonstrated on a primed single-stranded template. Although significant quantities of the protein were produced in the host Escherichia coli carrying the plasmid, it was not capable of complementing a polA− mutant in the replication of a polA-dependent plasmid.

Glyoxalase enzymes I and II are involved in a detoxification process consisting of conversion of reactive dicarbonyl compounds (e.g., methylglyoxal) to less reactive hydroxy acids. The structural gene for meningococcal glyoxalase I (gloA) was identified by screening an expression library with a rabbit antiserum. The meningococcal gloA gene consisted of 138 deduced amino acids, with a calculated mol. wt of 15.7 kDa. The DNA and deduced protein sequence of gloA was compared to known sequences of glyoxalase I enzymes and showed high homology with gloA of several eukaryotic and prokaryotic species. Insertion of a gloA-containing plasmid in Escherichia coli increa-sed the host organism's tolerance to methylglyoxal from <2 mm to >4 mm, thus demonstrating its functional identity. A databank search also revealed the presence of a putative gloB gene, encoding glyoxalase II (GlxII), in the recently released genomic sequences of Neisseria meningitidis and N. gonorrhoeae.

This study examined whether exposure of pigs to both porcine respiratory coronavirus (PRCV) and bacterial lipopolysaccharide (LPS) can potentiate respiratory disease and lung secretion of tumour necrosis factor-α (TNF-α) and interleukin-1 (IL-1). Caesarian-derived colostrum-deprived pigs were inoculated intratracheally with PRCV, with LPS from Escherichia coli O111:B4 (20 μg/kg), or with a combination of the two, and killed at set times after inoculation. Clinical signs, virus replication and (histo)pathological changes in the lungs, percentage of neutrophils and bioactive TNF-α and IL-1 in broncho-alveolar lavage (BAL) fluids were examined. The effects of separate virus or LPS inoculations were subclinical and failed to induce high and sustained cytokine levels. In a preliminary study, pigs were inoculated with PRCV and then with LPS 24 h later and killed sequentially. Severe respiratory disease and significantly enhanced TNF-α titres (208–3601 U/ml versus 40–89 U/ml after LPS only) were seen during the first 12 h after LPS inoculation. IL-1 levels (106–1631 U/ml versus 28–654 U/ml after LPS only) were also increased, but persisted for longer after clinical recovery than TNF-α. In a second study, pigs were inoculated with PRCV and subsequently with LPS at various time intervals ranging from 0 to 24 h, and killed 5 h after inoculation with LPS. A time interval of at least 12 h between inoculations was necessary for prominent respiratory signs to develop. Production of TNF-α, but not IL-1, was also dependent on the time interval between inoculations and was tightly correlated with disease. Lung neutrophil infiltration and pathological changes were comparable after combined PRCV-LPS and single LPS inoculations, and were not associated with disease. These data show that exposure to high endotoxin concentrations in swine buildings can precipitate respiratory disease in PRCV-infected pigs, and that TNF-α is probably an important mediator of these effects. This is the first in-vivo demonstration of synergy between respiratory viruses and LPS.