Helicobacterpylori-induced genotoxic damage in human b lymphocytes

Helicobacter pylori [H. pylori] is recognized as the causative agent of peptic and duodenal ulcers, gastric adenocarcinoma, and low-grade mucosa-associated lymphoid tissue [MALT] lymphoma. In the present study, we investigate the genotoxic damage of lysates of H, pylori in human B lymphocytes. Human B lymphocytes were treated with 0, 10, 20, and 30 microg/mL of total protein concentration of lysates obtained from H. pylori isolates from dyspeptic patients. Direct H. pylori-induced DNA damage was investigated by the in vitro cytokinesis-block micronucleus assay which detects chromosomal fragments and maldistributed whole chromosomes. The total mean micronuclei number [tMMN] observed per 1000 binucleus B cells significantly correlated with increasing protein concentration of H pylori lysates [r[2]=0.994, p=0.006]. The highest tMMN [3.81-fold] was observed in cells treated with 30 microg/mL of H, pylori lysate [12.43 +/- 1.91] when compared with the control [3.26 +/- 0.48]. This study provides evidence of the direct effect of H, pylori in chromosomal breakage of human B lymphocytes, which might lead to the development of abnormal B cells. Long-term infection by H. pylori has been implicated in epithelial cell damage as a result of continuous induction of the immune system by bacterial antigens. However, the results of this study propose that persistent H. pylori infection could also directly damage B lymphocyte DNA from which gastric MALT lymphoma arises

Guinea Pig as an appropriate model for colonization of helicobacter pylori

Animal models have been proven useful in elucidating the details of interaction between pathogenic bacteria and their human hosts, in addition to assessing the efficacy of therapeutic compounds and vaccines. In this study, we investigate the colonization of Helicobacter pylori [H. pylori] in experimentally-infected guinea pigs over a six-month period. A bacterial suspension was prepared by mixing four H. pylori isolates obtained from patients diagnosed with gastric ulcer. Within a one week period, five female guinea pigs were dosed orally with bacterial suspension for a total of three times. One control animal was gavaged with normal saline. Stool samples were collected at two-week intervals for six months. We used PCR, the stool antigen test, and indirect immunofluorescence assay [IFA] to assess samples for the presence of H, pylori. Stomachs obtained from two chloroform-killed animals, at 8 and 24 weeks, were investigated for histopathologic changes. H. pylori 16S rDNA was amplified from the stool samples of five guinea pigs. The stool antigen test was also positive in all five animals. IFA demonstrated the presence of H, pylori antigens in the stools from all five animals. PCR, stool antigen test and IFA results showed no H. pylori in the stool of the control animal. We observed infiltration of mature lymphocytes and plasma cells in the stomachs of animals killed at 8 and 24 weeks. The occurrence of H, pylori 16S rDNA and antigens in stool samples of guinea pigs demonstrated persistent colonization off H, pylori in the stomachs of guinea pigs. Histopathological findings have confirmed mild-severe gastritis induced by the bacterial infection. The stomach of a guinea pig is similar to the human stomach, in that it is sterile, lined by glandular epithelium, lacks a vitamin C synthesizing system and produces the cytokine interleukin-8. Accordingly, the guinea pig can be considered an appropriate animal model for long-term experiments to follow the process of H, pylori pathogenesis or to assess the efficacy of antibiotics or vaccines

Anti-Helicobacter pylori activity and structure-activity relationship study of 2-alkylthio-5-[nitroaryl]-1,3,4-thiadiazole derivatives

Nitro-containing heteroaromatic derivatives structurally related to nitroimidazole [Metronidazole] are being extensively evaluated against Helicobacter pylori isolates. On the other hand, 1,3,4-thiadiazole derivatives have also demonstrated promising antibacterial potential. In present study, we evaluated anti-H. pylori activity of novel hybrid molecules bearing nitroaryl and 1,3,4-thiadiazole moieties. Anti-H. pylori activity of novel 5-[5-nitroaryl]-1,3,4-thiadiazole derivatives bearing different bulky alkylthio side chains at C-2 position of thiadiazole ring, were assessed against three different metronidazole resistant H. pylori isolates by paper disk diffusion method. Most of the compounds demonstrated moderate to strong inhibitory response especially at 25 micro g/disk. The structure-activity relationship study of the compounds demonstrated that introduction of different alkylthio moieties at C-2 position of thiadiazole ring alter the inhibitory activity which is mainly dependent on the type of C-5 attached nitrohetercyclic ring. The promising compound of this scaffold, bearing 1-methyl-5-nitroimidazole moiety at C-5 and alpha -methylbenzylthio side chain at C-2 position of thiadiazole ring, showed strong inhibitory response against metronidazole resistant H. pyloriisolates at 12.5 micro g/disk [the inhibition zone diameter at all evaluated concentrations [12.5-100 micro g/disk] is > 50 mm]. Novel 5-[5-nitroaryl]-1,3,4-thiadiazole scaffold bearing different C-2 attached thio-pendant moieties with promising anti-H. pylori potential were identified. Among different nitroheterocycles, 5-nitrofuran and 5-nitroimidazole moieties were preferable for the substitution at C-5 position of 1,3,4-thiadiazole ring. Introduction of different alkylthio side chains at C-2 position of central ring alter the inhibitory activity which is mainly dependent on the type of C-5 attached nitrohetercyclic ring

Comparison of the effect of non-antifungal and antifungal agents on Candida isolates from the gastrointestinal tract

Non-antifungal drugs appear promising in treatment of opportunistic infections of Candida spp. that are often resistant to current antifungals. The broth macrodilution method [NCCLS M27-P document] was used to compare the antifungal activity of trifluoperazine, pro-pranolol, and lansoprazole with that of ketoconazole and amphotericin B, using 50 yeast isolates from the Gl tract. The minimum fungicidal concentrations [MFCs], resistance rates and the time required for fungicidal activity of the drugs [2 - 48 hours] were determined. The most effective antifungal activity was exhibited by trifluoperazine. Its MFC was 32 microg/mL for Candida albicans [3.3% resistance] and Candida spp. [0% resistance] yeasts, and 64 ug/mL for Candida tropicalis with 10% resistance. The MFC for C. albicans and Candida spp. was comparable to that of ketoconazole. However, the time required for the inhibitory effect [6 hr] was shorter than that of ketoconazole [48 hr] or amphotericin B [24 hr]. The time required for the inhibitory activity on C. tropicalis was 24 hr, which was shorter than that of ketoconazole and amphotericin B [48 hr]. A considerable number [40%] of Candida spp. showed resistance to ketoconazole, and 20% of C. tropicalis showed resistance to amphotericin B. Trifluoperazine, an antipsychotic drug, exhibited effective antifungal activity with the MFC, comparable to ketoconazole [32 microg/mL]. Among the three yeast groups, C. tropicalis showed resistance to trifluoperazine and amphotericin B, and Candida spp. was considerably resistant to ketoconazole. Trifluoperazine could be considered as an alternative antifungal when encountering Candida spp. resistant to current antifungals

Increase in resistance rates of H. pylori isolates to metronidazole and tetracycline-comparison of three 3-year studies

Antimicrobials have been useful in the treatment of H.pylori-related dyspeptic diseases. However, emergence of resistant strains often decreases the eradication rates of H.pylori infections. Large-scale use of antimicrobials will lead to the diminishment of susceptible strains while allowing resistant survivors to outgrow and spread resistance genes. The aim of this study was to assess the change in antimicrobial resistance rate of H.pylori isolates from 2005 to 2008 and indicate the consequences of indiscriminate and widespread use of antimicrobials against H. pylori-and non-H.pylori-related infections. A total of 110 H. pylori strains were isolated from dyspeptic patients during 2005 to 2008 and tested for their susceptibility to antimicrobials using the disk diffusion method. MICs were determined for metronidazole [8 micro g/mL], tetracycline [0.5 micro g/mL], clarithromycin [2 micro g/mL], amoxicillin [1 micro g/mL] and furazolidone [0.5 micro g/mL]. Since the rates of resistance to metronidazole and tetracycline were remarkably high, another 50 isolates were tested for their susceptibility to metronidazole at the same MIC [8 micro g/mL] and tetracycline at MICs of 0.5, 1 and 2 micro g/mL. Resistance rates were compared to those obtained in our two previous studies between 1997-2000 and 2001-2004. The resistance rates of 110 H.pylori isolates to clarithromycin, amoxicillin and furazolidone were 7.3%, 7.3%, and 4.5%, respectively. Among 160 H.pylori isolates, 55.6% exhibited resistance to metronidazole and 38.1% to tetracycline. Compared to our two previous studies, the resistance rates of H.pylori isolates to current antimicrobials has changed over time. The change in resistance rates of clarithromycin, amoxicillin and furazolidone was not statistically significant. However, resistance to metronidazole and tetracycline showed a considerable increase from 33-36.3% to 55.6% and 0-0.7% to 38.1%, respectively. Emergence of resistance due to the intensive use of antibiotics has become a global public health problem. It appears that plasmid-carried genes are involved in the spread of resistance traits among bacteria. Results obtained in this study indicate that the increase in resistance rates of H.pylori isolates to metronidazole and tetracycline could be the indication of indiscriminate and frequent use of antibiotics in Iran

Extraction of the outer membrane proteins of H. pylori and evaluation of their presence in stool of the infected individuals

Helicobacter pylori infections are accompanied with the release of some antigenic material into the stool. Outer membranes proteins [OMP] are among the major antigens of bacteria, which in comparison with other bacterial constituents have less cross-reactivity. In this study, OMP of H. pylori were isolated from 11 clinical isolates and rabbit antiserum against them was used to detect possible antigens of H. pylori, which are released into the stool. We used immunoblotting and affinity chromatography to detect such antigens in fecal antigenic extracts of infected individuals. By immunoblotting, we were able to detect a 26 kDa band under reducing and non-reducing conditions, but many more antigens [at least 5 antigens with molecular weights of about 14, 26, 52, 57.5 and 66 kDa] were isolated by affinity chromatography. The 26 kDa antigen had a higher concentration and is seen in nearly all positive samples. Since the 26 kDa antigen is detectable by these two techniques, we suggest that this antigen is one of the major antigens of H. pylori which is released into the stool and can be considered as a candidate diagnostic antigen to be used in diagnostic kit development