Detection of Mycobacterium tuberculosis group organisms in human and mouse joint tissue by reverse transcriptase PCR: prevalence in diseased synovial tissue suggests lack of specific association with rheumatoid arthritis.

Infection with mycobacterial species, including Mycobacterium tuberculosis, has long been implicated in the etiopathology of rheumatoid arthritis (RA) on the basis of clinical and pathological similarities between tuberculosis and RA. Despite evidence of immune responses to mycobacterial antigens in RA patient synovial fluid, cross-reactivity between these and host joint antigens, and the presence of M. tuberculosis protein antigen in RA synovial fluid, a definite causal association with RA has not been shown. Previous studies from our laboratory using reverse transcriptase PCR (RT-PCR) of bacterial rRNAs have shown RA synovium to be colonized by a diverse range of bacteria, most of commensal origin. However, M. tuberculosis group organism (MTG) RNA sequences were found in one RA patient tissue. Since this was considered of sufficient interest to warrant further investigation, we devised a M. tuberculosis-specific nested RT-PCR test which could be used for detection of MTG in a mixed pool of bacterial crDNAs. This test was used to investigate the distribution of MTG in RA synovial tissue and also non-RA arthritis and healthy control tissues and was also used to examine the tissue distribution of MTG in an acute and chronic model of M. tuberculosis infection in the BALB/c mouse. MTG sequences were found in a high proportion of RA patient synovial tissues but also in non-RA arthritis control tissues at lower frequency. This likely reflects trafficking of persistent M. bovis BCG to inflamed joint tissue, irrespective of cause. MTG were not found in healthy synovial tissue or the tissue of patients with undifferentiated arthritis. In both the acute and chronic models of infection in BALB/c mice, M. tuberculosis was also found to have trafficked to joint tissues, however, no signs of inflammation, arthritis, or pathology associated with M. tuberculosis infection was seen. These combined results would argue against a specific causal role of MTG in RA-like arthritis; however, their role as adjuvant in immune dysfunction in an innately susceptible host cannot be excluded.

Iron acquisition and virulence in Helicobacter pylori: a major role for FeoB, a high-affinity ferrous iron transporter.

The genome sequence of Helicobacter pylori suggests that this bacterium possesses several Fe acquisition systems, including both Fe2+- and Fe3+-citrate transporters. The role of these transporters was investigated by generating insertion mutants in feoB, tonB, fecA1 and fecDE. Fe transport in the feoB mutant was approximately 10-fold lower than in the wild type (with 0.5 microM Fe), irrespective of whether Fe was supplied in the Fe2+ or Fe3+ form. In contrast, transport rates were unaffected by the other mutations. Complementation of the feoB mutation fully restored both Fe2+ and Fe3+ transport. The growth inhibition exhibited by the feoB mutant in Fe-deficient media was relieved by human holo-transferrin, holo-lactoferrin and Fe3+-dicitrate, but not by FeSO4. The feoB mutant had less cellular Fe and was more sensitive to growth inhibition by transition metals in comparison with the wild type. Biphasic kinetics of Fe2+ transport in the wild type suggested the presence of high- and low-affinity uptake systems. The high-affinity system (apparent Ks = 0.54 microM) is absent in the feoB mutant. Transport via FeoB is highly specific for Fe2+ and was inhibited by FCCP, DCCD and vanadate, indicating an active process energized by ATP. Ferrozine inhibition of Fe2+ and Fe3+ uptake implied the concerted involvement of both an Fe3+ reductase and FeoB in the uptake of Fe supplied as Fe3+. Taken together, the results are consistent with FeoB-mediated Fe2+ uptake being a major pathway for H. pylori Fe acquisition. feoB mutants were unable to colonize the gastric mucosa of mice, indicating that FeoB makes an important contribution to Fe acquisition by H. pylori in the low-pH, low-O2 environment of the stomach.

Helicobacter pylori possesses two CheY response regulators and a histidine kinase sensor, CheA, which are essential for chemotaxis and colonization of the gastric mucosa.

Infection of the mucous layer of the human stomach by Helicobacter pylori requires the bacterium to be motile and presumably chemotactic. Previous studies have shown that fully functional flagella are essential for motility and colonization, but the role of chemotaxis remains unclear. The two-component regulatory system CheA/CheY has been shown to play a major role in chemotaxis in other enteric bacteria. Scrutiny of the 26695 genome sequence suggests that H. pylori has two CheY response regulators: one a separate protein (CheY1) and the other (CheY2) fused to the histidine kinase sensor CheA. Defined deletion mutations were introduced into cheY1, cheY2, and cheA in H. pylori strains N6 and SS1. Video tracking revealed that the wild-type H. pylori strain moves in short runs with frequent direction changes, in contrast to movement of cheY2, cheAY2, and cheAY2 cheY1 mutants, whose motion was more linear. The cheY1 mutant demonstrated a different motility phenotype of rapid tumbling. All mutants had impaired swarming and greatly reduced chemotactic responses to hog gastric mucin. Neither cheY1 nor cheAY2 mutants were able to colonize mice, but they generated a significant antibody response, suggesting that despite impaired chemotaxis, these mutants were able to survive in the stomach long enough to induce an immune response before being removed by gastric flow. Additionally, we demonstrated that cheY1 failed to colonize gnotobiotic piglets. This study demonstrates the importance of the roles of cheY1, cheY2, and cheA in motility and virulence of H. pylori.

Characterization of Helicobacter pylori PldA, a phospholipase with a role in colonization of the gastric mucosa.

BACKGROUND & AIMS: Phospholipase activity may play a role in the pathogenicity of Helicobacter pylori. Furthermore, some drugs that are effective against H. pylori infection are phospholipase inhibitors. Scrutiny of the H. pylori 26695 genome sequence revealed the presence of a putative protein with homology to Esherichia coli outer membrane phospholipase A (PldA). The aim of this study was to investigate the role of this putative PldA in the pathogenicity of H. pylori. METHODS: An isogenic pldA mutant was constructed and analyzed for in vitro phospholipase A(2) and hemolytic activity. Adherence of the mutant to human gastric adenocarcinoma cells and the ability to colonize mice were also investigated. RESULTS: The pldA mutant showed a marked reduction in phospholipase A(2) and hemolytic activity compared with the wild-type strain. The mutant was unable to colonize mice at 2 and 8 weeks, but it did induce a significant immune response. In contrast, the ability of the mutant to adhere to human gastric adenocarcinoma cells was unaffected. CONCLUSIONS: The results suggest a role for PldA in colonization of the gastric mucosa and possibly tissue damage after colonization.

Phase variation in Helicobacter pylori lipopolysaccharide due to changes in the lengths of poly(C) tracts in alpha3-fucosyltransferase genes.

The lipopolysaccharide (LPS) of Helicobacter pylori expresses the Lewis x (Lex) and/or Ley antigen. We have shown previously that H. pylori LPS displays phase variation whereby an Lex-positive strain yields variants with different LPS serotypes, for example, Lex plus Ley or nonfucosylated polylactosamine. H. pylori has two alpha3-fucosyltransferase genes that both contain poly(C) tracts. We now demonstrate that these tracts can shorten or lengthen randomly, which results in reversible frameshifting and inactivation of the gene products. We provide genetic and serological evidence that this mechanism causes H. pylori LPS phase variation and demonstrate that the on or off status of alpha3-fucosyltransferase genes determines the LPS serotypes of phase variants and clinical isolates. The role of the alpha3-fucosyltransferase gene products in determining the LPS serotype was confirmed by structural-chemical analysis of alpha3-fucosyltransferase knockout mutants. The data also show that the two alpha3-fucosyltransferase genes code for enzymes with different fine specificities, and we propose the names futA and futB to designate the orthologs of the H. pylori 26695 alpha3-fucosyltransferase genes HP0379 and HP0651, respectively. The data also show that the alpha3-fucosylation precedes alpha2-fucosylation [corrected], an order of events opposite to that which prevails in mammals. Finally, the data provide an understanding at the molecular level of the mechanisms underlying LPS diversity in H. pylori, which may play an important role in adaptation to the host.

Functional analysis of the roles of FliQ and FlhB in flagellar expression in Helicobacter pylori.

Expression of the two Helicobacter pylori flagellin proteins FlaA and FlaB is required for full motility and persistent infection of the gastric mucosa. The mechanisms and regulation of the biosynthesis and export of flagella in H. pylori are still poorly understood. Scrutiny of the H. pylori 26695 genome sequence revealed homologues of FliQ and FlhB. The roles of the fliQ and flhB genes in H. pylori were investigated by the construction and characterisation of defined isogenic mutants. The results indicate that these genes are involved in the flagellar expression, adhesion to and colonisation of the gastric mucosa.

Ranitidine bismuth citrate and clarithromycin, alone or in combination, for eradication of Helicobacter mustelae infection in ferrets.

OBJECTIVE: To determine whether ranitidine bismuth citrate, clarithromycin, or a combination of ranitidine bismuth citrate and clarithromycin would be efficacious in eradication of Helicobacter mustelae infection in ferrets. ANIMALS: 60 seven-month-old ferrets. PROCEDURE: To determine dosages of clarithromycin and ranitidine bismuth citrate that would suppress growth of, but not eradicate infection with, H mustelae, ferrets (n = 6/group) were treated p.o. with clarithromycin or ranitidine bismuth citrate at various dosages. Efficacy of treatment was then determined by treating ferrets with clarithromycin alone, ranitidine bismuth citrate alone, or clarithromycin and ranitidine bismuth citrate. Gastric biopsy specimens were obtained before, during, and at various times after treatment and submitted for quantitative bacterial culture and histologic evaluation. Minimum concentrations of clarithromycin that inhibited 90% of the growth of isolates obtained before and after treatment were determined. RESULTS: Dosages of clarithromycin and ranitidine bismuth citrate that suppressed growth of H mustelae were 12.5 and 24 mg/kg of body weight, p.o., every 8 hours, respectively. Infection was not eradicated in ferrets treated with ranitidine bismuth citrate alone but was eradicated in all 6 ferrets treated with clarithromycin and ranitidine bismuth citrate and in 4 of 6 treated with clarithromycin alone. A decrease in susceptibility to clarithromycin was detected for H mustelae isolates obtained after treatment. Mild or moderate antral gastritis was observed even in ferrets from which infection was eradicated. CONCLUSIONS AND CLINICAL RELEVANCE: A combination of ranitidine bismuth citrate and clarithromycin was efficacious in eradicating H mustelae infection from ferrets.

Nonprimate Animal Models of H. pylori Infection.

Ever since the realization that Helicobacter pylori was intimately associated with the development of gastritis and peptic ulcer disease in humans, there has been a need for a simple animal model in which modes of pathogenicity, transmission, immunization, and chemotherapeutic intervention can be evaluated. Whereas small animals such as mice and rats are particularly well suited as experimental hosts for many infections, early studies suggested that H. pylori had a very narrow host range that did not extend to these species. Although many attempts to infect small laboratory animals with H. pylori were apparently made, these proved generally unsuccessful (1,2) and the view became established rapidly that "H. pylori will not colonize many of the usual laboratory animal species, including conventionally reared rats, mice, rabbits, guinea pigs, specific-pathogen-free pigs, colostrum-deprived piglets, and gnotobiotic rats and mice" (3). An apparent exception was the claim that H. pylori would colonize. Mongolian gerbils particularly after gastric lesions were produced by indomethacin (4); however, this work has never been substantiated nor followed up. Instead, most attention was paid subsequently to the use of naturally occurring Helicobacter mustelae infections of ferrets (5,6), experimental challenge with H. pylori in gnotobiotic piglets (7) and the important development of the Helicobacter felis model in mice and rats (8,9).

Ranitidine bismuth citrate: a novel anti-ulcer agent with different physico-chemical characteristics and improved biological activity to a bismuth citrate-ranitidine admixture.

BACKGROUND: Ranitidine bismuth citrate (RBC) is a new chemical entity for the treatment of peptic ulcer disease. RESULTS: RBC is freely soluble in water (more than 600 mg/mL at pH 4.6), whereas an equimolar admixture of its component molecules, bismuth citrate and ranitidine, formed an almost totally insoluble suspension. Even at very low pH values (around 2.0), the solubility of bismuth in ranitidine bismuth citrate was at least two-fold better than in the admixture. Comparison of several physico-chemical characteristics indicated that RBC possessed significantly different melting point properties, X-ray powder diffraction patterns, infra-red spectra and 13C-NMR solid-state spectra to the admixture. Ranitidine bismuth citrate inhibited human pepsin isoenzymes 1, 2, 3 and 5 but the admixture was inactive. RBC showed approximately two-fold greater anti-Helicobacter pylori activity in vitro than the admixture (geometric mean minimum inhibitory concentrations of 12.5 and 25.7 mg/L, respectively) and was more rapidly bactericidal. In a mouse model of gastric H. pylori colonization, 200 mg/kg of bismuth, given as RBC, eradicated the organism from all mice while only 10% of infections were eradicated by equivalent levels of bismuth in admixture form. CONCLUSION: It is believed that the significantly greater solubility of RBC, especially at lower pH values, is highly relevant to its better antipepsin and anti-H. pylori action compared to the insoluble admixture of bismuth citrate and ranitidine.

Gastric anti-secretory, mucosal protective, anti-pepsin and anti-Helicobacter properties of ranitidine bismuth citrate.

Ranitidine bismuth citrate is a novel compound formed from ranitidine and a bismuth citrate complex. In conscious dogs, ranitidine bismuth citrate had similar activity to ranitidine hydrochloride as an inhibitor of histamine-induced gastric acid secretion when oral doses containing equivalent amounts of ranitidine base (0.1 or 0.3 mg/kg) were compared. In the rat, ranitidine bismuth citrate (3-30 mg/kg p.o.) prevented gastric mucosal damage induced by ethanol (fundic damage) and indomethacin (antral damage). Ranitidine hydrochloride and tripotassium dicitrato bismuthate were also effective against indomethacin-induced damage, but were both significantly less potent than ranitidine bismuth citrate in this model. Ranitidine hydrochloride was inactive against ethanol-induced damage. In vitro, ranitidine bismuth citrate (1 mmol/L) inhibited human pepsin isoenzymes 1, 2, 3 and 5. Pepsin 1 was inhibited to a similar extent by ranitidine bismuth citrate, bismuth citrate and tripotassium dicitrato bismuthate at concentrations equivalent to 1 mmol/L bismuth, but ranitidine (1 mmol/L) was inactive. Ranitidine bismuth citrate was more potent than tripotassium dicitrato bismuthate as an inhibitor of pepsins 2, 3 and 5. Ranitidine bismuth citrate inhibited both Helicobacter pylori (effective concentration 4-32 micrograms bismuth/ml) and H. mustelae (1-4 micrograms bismuth/ml); similar results were obtained with tripotassium dicitrato bismuthate. Bismuth citrate was slightly less effective, and ranitidine hydrochloride was inactive (> 125 micrograms/ml). In ferrets naturally colonized with H. mustelae, oral treatment with ranitidine bismuth citrate, 12 or 24 mg/kg twice daily for 4 weeks, caused a dose related clearance of H. mustelae. Qualitatively similar results were obtained in a small study with tripotassium dicitrato bismuthate and bismuth citrate.

Development of a 14C-urea breath test in ferrets colonised with Helicobacter mustelae: effects of treatment with bismuth, antibiotics, and urease inhibitors.

A 14C-urea breath test analogous to its clinical counterpart is described for use in ferrets naturally or experimentally infected with Helicobacter mustelae. The test is performed within a sealed glass metabolism chamber through which air is drawn at a constant rate and expired breath collected into sodium hydroxide. Peak 14CO2 production occurred approximately 1 hour after substrate administration. Both inter- and intra-animal responses were highly reproducible, with mean coefficients of variation less than 10%. Other than enhancing peak 14CO2 levels very slightly, fasting had little influence on the response. In infant animals challenged with H mustelae, breath test activity increased linearly with the total count of culturable bacteria isolated from the antrum. Treatment of established infections with colloidal bismuth subcitrate (DeNol) for 4 weeks resulted in clearance of all detectable bacteria but retention of some breath test activity. Subsequent regrowth of bacteria was parallelled by an increase in the breath test response. Inclusion of amoxycillin and metronidazole in the treatment regimen, however, eradicated all the bacteria and almost totally eliminated 14CO2 production. This response parallels the clinically observed suppressive effect on H pylori achieved with bismuth alone relative to the total eradication seen with triple therapy. A single oral dose of the urease inhibitor, flurofamide, inhibited over 90% of the response for at least 24 hours. Acetohydroxamic acid was less effective. These findings suggest that in the ferret H mustelae model, breath test analysis can be a useful, non-invasive alternative to endoscopy for evaluation of agents affecting either growth of the organism or urease activity.

A morphological comparison of treatment with different beta-lactam antibiotics on experimental staphylococcal endocarditis and aortitis.

The effect of treatment of staphylococcal endocarditis and aortitis with five different beta-lactam antibiotics (ceftazidime, cephaloridine, cefotaxime, methicillin and flucloxacillin) was evaluated by light and electron microscopy. It was found that therapy with all five antibiotics produced similar morphological changes. At 3 and 8 h, the bacterial colonies showed zonal changes with the bacteria furthest from the lumen exhibiting less severe damage while the outer region consisted largely of lysed cells. However, in the outer zone a few apparently viable, thick-walled persistent bacteria were observed. At 24 and 48 h, many colonies consisted of large masses of lysed bacteria with only a few thick-walled persistent bacteria. In all cases, therapy was associated with an increased host inflammatory cell response resulting in invasion of leucocytes through the aortic wall or vegetation towards and engulfing the colonies. However, even at 48 h the inflammatory cells had not reached all the deep-seated colonies. It would appear that all the antibiotics reached bactericidal concentrations within the lesions. However, the eradication of the few 'persistent' bacteria was delayed by the inability of the inflammatory cells to reach all the colonies.

Differential response to benzylpenicillin in vivo of tolerant and non-tolerant variants of Streptococcus sanguis II.

A variant that was highly tolerant to benzylpenicillin was obtained from a non-tolerant clinical isolate of Streptococcus sanguis II by repeated exposure to penicillin. The rabbit model of endocarditis was used to investigate the efficacy of a high dose regimen of benzylpenicillin (250 mg/kg; peak serum concentration c. 25 mg/l) in the prophylaxis and treatment of endocarditis during challenge or infection with the non-tolerant parent strain or its tolerant variant. The two strains exhibited a similar capacity to initiate infection. A single dose of penicillin administered 0.5 h before bacterial challenge protected six of nine rabbits infected with the non-tolerant parent strain, but none of nine infected with the tolerant variant. Treatment of established infection with penicillin administered twice daily for four days cured eight of 13 (61%) rabbits infected with the non-tolerant parent strain, but only one of 14 (7%) rabbits infected with the tolerant variant. These results support the view that tolerance to penicillin has therapeutic implications.

A morphological study of the effect of treatment with the antibiotic ceftazidime on experimental staphylococcal endocarditis and aortitis.

The morphological effects of antibiotic therapy with either single or repeated (8 hourly) injections of ceftazidime were examined in rabbits with experimentally induced staphylococcal endocarditis and aortitis. At 3 h after initiating treatment, many of the bacteria, irrespective of the location of the colony, showed evidence of abnormal ultrastructural changes of the cytoplasm and/or cell wall. By 8 h many degenerate lysed bacteria were present. By 24 h, in rabbits which received a single injection, bacterial colonies contained many normal and dividing bacteria. In comparison, bacterial colonies at 24 h in rabbits receiving repeated injections consisted of large masses of lysed bacteria with only a few viable appearing thick-walled 'persistent' cells. At 48 and 72 h, no viable appearing bacteria were observed although they could be isolated by culture methods. Treatment was associated with an increased inflammatory cell response at the surface of the vegetation and within the vasculature. In the later stages there was evidence of healing with endothelialization of the lesions. It would appear, therefore, that ceftazidime penetrates efficiently into the vegetations with only a short transitory phase at sub-bactericidal concentrations. The few 'persistent' bacteria appear to be protected from the host defences by the surrounding thrombus which prevents their eradication.

Penetration of ceftazidime into the rabbit respiratory tract.

A procedure is described for the direct collection of bronchial or tracheal fluid samples on to paper discs. Using the procedure, the pharmacokinetics of ceftazidime in rabbit bronchial and tracheal fluids were compared with those in the respective wall tissue samples and in lung tissue. Concentrations appearing in lung tissue were approximately half those seen in bronchial or tracheal fluid or bronchial and tracheal wall tissue. Concentrations in these latter compartments were, in turn, four- to six-fold lower at all times than simultaneously-measured serum levels. The shape of the concentration/time curves were similar for all compartments sampled. The half life values were 63 to 64 min for respiratory tract concentrations and 59 min for serum levels. Percentage penetration from serum into the various compartments was 20.8% for bronchial fluid, 19.9% for tracheal fluid, 22.4% for bronchial wall, 20.0% for tracheal wall and 11.3% for lung tissue.

Evaluation of ceftazidime in experimental Klebsiella pneumoniae pneumonia: comparison with other antibiotics and measurement of its penetration into respiratory tissues and secretions.

The activity of ceftazidime was examined in a murine model of Klebsiella pneumoniae pneumonia in which the antibiotic was administered subcutaneously 6 h after intranasal infection and then twice daily for the next three days (i.e. seven doses). In a series of experiments using this test, the dose of ceftazidime giving 50% survival relative to controls (SD50) ranged from 1.0-9.0 mg/kg/dose while the dose required to reduce the log10 cfu/lung by 50% (CD50) ranged from 24-64 mg/kg/dose. Ceftazidime was considerably more effective than cefotiam, amoxycillin-clavulanic acid or kanamycin in the test. Pharmacokinetic studies with ceftazidime showed that no differences in respiratory tract penetration existed between uninfected mice and mice infected for 48 h with K. pneumoniae. The percentage penetration of ceftazidime from serum was 73% for pleural fluid, 44% for tracheal fluid, 27% for tracheal wall tissue and 17% for whole lung tissue after a subcutaneous injection of 100 mg/kg. At this dose, ceftazidime remained at supra-MIC concentrations for 2-3 h in all compartments examined.

A morphological study of experimental rabbit staphylococcal endocarditis and aortitis. I. Formation and effect of infected and uninfected vegetations on the aorta.

In this study the development of sterile thrombic vegetations on the aorta resulting from catheterization and the effect of subsequent infection with Staphylococcus aureus were examined by light and electron microscopy. Thrombi of various sizes, comprising fibrin, platelets and a few leucocytes and erythrocytes, develop on the damaged surface of the aorta with minimal changes in the underlying aortic wall. After intravenous inoculation of Staph. aureus most vegetations become infected, as shown by the presence of bacterial colonies, and the underlying aortic wall is markedly inflamed. The inflammatory cells invade the wall from the base of the aorta and cause swelling plus disruption of the elastic laminae with ulceration of the luminal surface in some cases. This structural damage appears to be a direct result of the bacterial infection of the lesions on the luminal surface.

A morphological study of experimental staphylococcal endocarditis and aortitis. II. Inter-relationship of bacteria, vegetation and cardiovasculature in established infections.

The inter-relationship of the bacteria, vegetations and cardiovasculature was studied by light and electron microscopy in experimental staphylococcal endocarditis and aortitis in acute and fatal infections. A specific spatial relationship was observed with the majority of bacterial colonies located along the junction between the cardiovasculature and the overlying thrombic vegetation. The bacterial colonies within the vegetations on the ventricular and aortic walls were smaller and embedded in a thick layer of thrombus with certain colonies showing evidence of bacterial cell death. By comparison, the lesions on the aortic valve consisted of large masses of bacteria with little or no thrombic coating. The structural damage to the aortic valve appeared to be the direct result of bacterial invasion into the connective tissue of the cusp. The acute nature of the disease may be related to bacterial destruction of the aortic valve whereas the colonies within the aortic wall vegetations are probably the source of 'resistance' to antibiotic treatment.

Comparative penetration of ceftazidime into cardiac and skeletal muscle in rabbits.

The concentrations of ceftazidime attained in cardiac and leg muscles of rabbits were compared after a single intramuscular injection of 100 mg/kg. Simultaneous measurements of muscle and muscle fluid over an 8-hour period showed that ceftazidime penetrated heart muscle more rapidly and to a greater extent than leg muscle. Although the half-lives did not vary, the areas under the time/concentration curves for heart muscle and heart muscle fluid were significantly greater than those for leg muscle and leg muscle fluid. It is suggested that an increased fluid content and vascularity of cardiac muscle may explain these results. This study also confirms previous reports that whole muscle tissue levels of beta-lactam antibiotics substantially underestimate the actual concentrations present in the extracellular fluid.