Characterization and presumptive identification of Helicobacter pylori isolates from rhesus monkeys.
ABSTRACT: We characterized 38 Helicobacter isolates, including 22 from gastric biopsy samples obtained from 14 rhesus monkeys and single isolates from 16 monkeys in a different colony. Biochemical profiles of these isolates were nearly identical to that of Helicobacter pylori ATCC 43504. Restriction fragment length polymorphism (RFLP) analysis indicated that each infected monkey harbored one to four strains. The 17 RFLP types found among these 22 isolates differed from all seven RFLPs found among the other 16 isolates. Thus, monkeys within a given colony are more likely to be infected by Helicobacter isolates with the same or a similar RFLP than are monkeys from different colonies. A 16S rRNA gene was amplified by PCR and cloned from the Helicobacter isolate from rhesus monkey 85D08. Ribotyping with this probe demonstrated less diversity among isolates from rhesus monkeys than was reported among isolates of H. pylori from humans, as did RFLP analysis of a PCR fragment of the ureA-ureB gene cluster. The DNA sequence of the cloned 16S rRNA gene was determined and compared with sequences reported for H. pylori and other Helicobacter species. Our analysis of 127 nucleotides (corresponding with residues 1240 to 1366 of the Escherichia coli 16S rRNA gene) indicated that the Helicobacter isolate from monkey 85D08 was 99.2 to 100% homologous to isolates of H. pylori from humans but only 83.5 to 96.9% homologous with other Helicobacter species in this region of the 16S rRNA gene. These data provide strong support for the presumptive identification of these isolates as H. pylori.
Project description:Chronic idiopathic colitis is a common clinical entity in young captive rhesus monkeys. Eight isolates, cultured from five monkeys in colony 1 with endemic diarrhea and three from colony 2 without diarrhea, were grown under microaerobic conditions on selective agar and were classified by full 16S rRNA sequence, biochemical, and phenotypic analysis as a novel helicobacter, "Helicobacter macacae" (proposed name). All eight strains of H. macacae had 99.5% identical 16S rRNA sequences.
Project description:PCR detection of H. pylori in biological specimens is rendered difficult by the extensive polymorphism of H. pylori genes and the suppressed expression of some genes in many strains. The goal of the present study was to (1) define a domain of the 16S rRNA sequence that is both highly conserved among H. pylori strains and also specific to the species, and (2) to develop and validate specific and sensitive molecular methods for the detection of H. pylori. We used a combination of in silico and molecular approaches to achieve sensitive and specific detection of H. pylori in biologic media. We sequenced two isolates from patients living in different continents and demonstrated that a 546-bp domain of the H. pylori 16S rRNA sequence was conserved in those strains and in published sequences. Within this conserved sequence, we defined a 229-bp domain that is 100% homologous in most H. pylori strains available in GenBank and also is specific for H. pylori. This sub-domain was then used to design (1) a set of high quality RT-PCR primers and probe that encompassed a 76-bp sequence and included at least two mismatches with other Helicobacter sp. 16S rRNA; and (2) in situ hybridization antisense probes. The sensitivity and specificity of the approaches were then demonstrated by using gastric biopsy specimens from patients and rhesus monkeys. This H. pylori-specific region of the 16S rRNA sequence is highly conserved among most H. pylori strains and allows specific detection, identification, and quantification of this bacterium in biological specimens.
Project description:During a 6-year period, 64 of 227 commercially reared cats had microaerobic bacteria isolated from their feces. All the isolates were initially identified as Campylobacter-like organisms based on biochemical and phenotypic characteristics. DNA extractions from 51 of these isolates were subjected to PCR using primers specific for Helicobacter spp. and Campylobacter spp. Of the isolates, 92% (47 of 51 isolates) were positive for Campylobacter spp., 41% (21 of 51 isolates) were positive for Helicobacter spp., 33% (17 of 51 isolates) were positive for both genera, 59% (30 of 51 isolates) were positive only for Campylobacter spp., and 8% (4 of 51) were positive only for Helicobacter spp. Sixteen of the 47 Campylobacter-positive cultures were positive for more than one Campylobacter spp. Based on a species-specific PCR assay, 83% of the isolates were identified as Campylobacter helveticus, 47% of the isolates were identified as Campylobacter upsaliensis, and 6% of the isolates were classified as Campylobacter jejuni. The 1.2-kb PCR products of the 16S rRNA genes of 19 Helicobacter species isolates were subjected to restriction fragment length polymorphism (RFLP) analysis. Of the five different RFLP patterns obtained, two clustered with Helicobacter ("Flexispira") taxon 8, one clustered with Helicobacter bilis, one clustered with Helicobacter canis, and the remaining pattern was closely related to a novel Helicobacter sp. strain isolated from a woodchuck. The sequence data for the 16S rRNA genes of 10 Helicobacter spp. validated the RFLP-based identification of these isolates. This study demonstrated that biochemical and phenotypic characteristics of microaerobic organisms in cat feces were insufficient to characterize mixed Helicobacter and Campylobacter infections. Molecular structure-based diagnostics using genus- and species-specific PCR, RFLP analysis, and 16S rRNA sequence analysis enabled the identification of multiple microaerobic species in individual animals. The clinical relevance of enteric Helicobacter and Campylobacter coinfection in cats will require further studies.
Project description:Helicobacter pylori colonization is highly prevalent among humans and causes significant gastric disease in a subset of those infected. When present, this bacterium dominates the gastric microbiota of humans and induces antimicrobial responses in the host. Since the microbial context of H. pylori colonization influences the disease outcome in a mouse model, we sought to assess the impact of H. pylori challenge upon the pre-existing gastric microbial community members in the rhesus macaque model. Deep sequencing of the bacterial 16S rRNA gene identified a community profile of 221 phylotypes that was distinct from that of the rhesus macaque distal gut and mouth, although there were taxa in common. High proportions of both H. pylori and H. suis were observed in the post-challenge libraries, but at a given time, only one Helicobacter species was dominant. However, the relative abundance of non-Helicobacter taxa was not significantly different before and after challenge with H. pylori. These results suggest that while different gastric species may show competitive exclusion in the gastric niche, the rhesus gastric microbial community is largely stable despite immune and physiological changes due to H. pylori infection.
Project description:Low-cost and rescue treatments for Helicobacter pylori infections involve combinations of several drugs including tetracycline. Resistance to tetracycline has recently emerged in H. pylori. The 16S rRNA gene sequences of two tetracycline-resistant clinical isolates (MIC = 64 microg/ml) were determined and compared to the consensus H. pylori 16S rRNA sequence. One isolate had four nucleotide substitutions, and the other had four substitutions and two deletions. Natural transformation with the 16S rRNA genes from the resistant organisms conferred tetracycline resistance on susceptible strains. 16S rRNA genes containing the individual mutations were constructed and tested for the ability to confer resistance. Only the 16S rRNA gene containing the triple mutation, AGA965-967TTC, was able to confer tetracycline resistance on H. pylori 26695. The MICs of tetracycline for the transformed strains were equivalent to those for the original clinical isolates. The two original isolates were also metronidazole resistant, but this trait was not linked to the tetracycline resistance phenotype. Serial passage of several H. pylori strains on increasing concentrations of tetracycline yielded mutants with only a very modest increase in tetracycline resistance to a MIC of 4 to 8 microg/ml. These mutants all had a deletion of G942 in the 16S rRNA genes. The mutations in the 16S rRNA are clearly responsible for tetracycline resistance in H. pylori.
Project description:We recently reported the occurrence of natural infection with H. pylori in a group of cynomolgus monkeys with chronic active gastritis and gastric erosions. The goal of the present study was to characterize and to compare strains isolated from animals originating from two different geographical areas. Gross and microscopic pathology determined at the time of necropsy was similar in all animals. H. pylori were isolated from specimens harvested in five monkeys (four from Vietnam and one from the Philippines) with gastritis. Isolates from monkeys bred in Vietnam had a similar DNA fingerprint pattern, which was distinct from that of isolates from a monkey bred in the Philippines. All strains were of the s1a vacA subtype, but all the 'Vietnamese' strains were cagA+ and all but one were iceA1 whereas the 'Philippino' strains were cagA- and iceA2. The sequences of the 16S rRNA of the Vietnamese and Philippino strains shared 98% homology and both clustered with H. pylori sequences present in the NCBI database. In conclusion, cynomolgus monkeys can be naturally colonized by H. pylori, and the strains isolated from these animals appear to vary according to the geographical origin, thus indicating probable infection prior to importation. Since some of the cynomolgus monkeys developed antral erosions during natural infection, we propose that this animal model may be used to investigate the role of H. pylori in ulcerogenesis.
Project description:Among 12 clarithromycin-resistant Helicobacter pylori strains isolated in Guri, Korea, 8 showed an adenine to guanine mutation at position 2143 (formerly A2144G or E. coli 2059) in the 23S rRNA gene by the PCR-restriction fragment length polymorphism (RFLP) method. The remaining 4 strains, digested by neither BsaI nor BbsI, showed a thymine to cytosine mutation at position 2182 (T2182C) by direct sequencing of the PCR products. The T2182C mutants showed a tendency of higher levels of minimum inhibitory concentration to clarithromycin than the A2143G mutants. In conclusion, either the A2143G or the T2182C mutation was present in 100% of clarithromycin-resistant H. pylori isolates examined. The PCR-RFLP technique with restriction enzymes BbsI and BsaI was a rapid and relatively simple method to detect the clarithromycin resistance. But undigested isolates were quite frequent among our isolates (33.3%), the PCR-RFLP method with restriction enzymes BbsI and BsaI should not be used alone, and development of other rapid detection method for clarithromycin resistance is mandatory.
Project description:BACKGROUND & AIMS: Gastric cancer results from a combination of Helicobacter pylori (H pylori) infection, exposure to dietary carcinogens, and predisposing genetic make-up. Because the role of these factors in gastric carcinogenesis cannot be determined readily in human beings, the present study examined the role of an oral carcinogen and H pylori infection in rhesus monkeys. METHODS: Gastroscopies were performed in 23 monkeys assigned to 4 groups: controls; nitrosating carcinogen ethyl-nitro-nitrosoguanidine administration alone; and inoculation of a virulent H pylori strain alone (H), or in combination with ethyl-nitro-nitrosoguanidine (EH). Follow-up gastroscopies and biopsies were performed at 3-month intervals for 5 years for pathologic and molecular studies. RESULTS: Postinoculation, H and EH groups showed persistent infection and antral gastritis. Starting at 2 and 5 years, respectively, gastric intestinal metaplasia and intraepithelial neoplasia developed in 3 EH monkeys but in no other groups. Transcriptional analysis of biopsies at 5 years revealed group-specific expression profiles, with striking changes in EH monkeys, plus a neoplasia-specific expression profile characterized by changes in multiple cancer-associated genes. Importantly, this neoplastic profile was evident in nonneoplastic mucosa, suggesting that the identified genes may represent markers preceding cancer. CONCLUSIONS: Gastric intraglandular neoplasia is induced in primates when H pylori infection is associated with consumption of a carcinogen similar to the nitrosaminesfound in pickled vegetables, suggesting that H pylori and the carcinogen synergistically induce gastric neoplasia in primates. Monkey arrays used for analysis of the affect of Helicobacter pylori and diet on development of gastric cancer. Each RNA was hybridized against a common reference. Biopsy numbers represent the following in terms of descriptions used in publication of the paper... Each set of numbers/descriptions is organized in the following order: Biopsy#; Monkey#; Group Code on Fig 5 Biopsy #4674; AB24; E; E1 Biopsy #4684; 16G; E; E2 Biopsy #4716; 89G; H; H1 Biopsy #4724; 54H; H; H2 Biopsy #4720; 20G; H; H3 Biopsy #4710; 92F; C; C1 Biopsy #4714; 26G; C; C2 Biopsy #4712; 92G; C; C3 Biopsy #4726; 08G; H; H4 Biopsy #4722; 57G; H; H5 Biopsy #4718; 85G; H; H6 Biopsy #4694; 48H; HE NON; HE1 Biopsy #4686; 59H; HE NON; HE2 Biopsy #4690; 81G; HE NEO; HE3 Biopsy #4696; 36G; HE NEO; HE4 Biopsy #4692; 63G; HE NEO; HE5 Biopsy #4680; 76G; E; E3 Biopsy #4682; 52G; E; E4 Biopsy #4678; 15G; E; E5 Biopsy #4676; 93G; E; E6 Biopsy #4688; 47H; HE NON; HE6 A pathogenicity experiment design type is where an infective agent such as a bacterium, virus, protozoan, fungus etc. infects a host organism(s) and the infective agent is assayed. Infection: Rhesus monkeys were infected with H. pylori (+/-) Compound Based Treatment: Rhesus monkeys were treated with ENNG (N-ethyl-N-nitrosoguanidine) (+/-) Overall design: pathogenicity_design
Project description:The primary mode of transmission of Helicobacter pylori, a human pathogen carried by more than half the population worldwide, is still unresolved. Some epidemiological data suggest water as a possible transmission route. H. pylori in the environment transforms into a nonculturable, coccoid form, which frequently results in the failure to detect this bacterium in environmental samples by conventional culture techniques. To overcome limitations associated with culturing, molecular approaches based on DNA amplification by PCR have been developed and used for the detection of H. pylori in clinical and environmental samples. Our results showed the glmM gene as the most promising target for detection of H. pylori by PCR amplification. Under optimal amplification conditions, glmM-specific primers generated PCR-amplified products that were specific for H. pylori and some other Helicobacter species. Genome sequence analysis revealed the existence of a conserved region linked to a hypervariable region upstream of the 16S rRNA gene of H. pylori. Selective PCR primer sets targeting this sequence were evaluated for the specific detection of H. pylori. One primer set, Cluster2 and B1J99, were shown to be highly specific for H. pylori strains and did not produce any PCR products when other Helicobacter species and other bacterial species were analyzed. In tests with 32 strains of H. pylori, 6 strains of other Helicobacter species, 8 strains of Campylobacter jejuni, and 21 strains belonging to different genera, the primers for glmM were selective for the Helicobacter genus and the primers containing the region flanking the 16S rRNA gene were selective for H. pylori species only. The combination of two sensitive PCR-based methods, one targeting the glmM gene and the other targeting a hypervariable flanking region upstream of the 16S rRNA gene, are complementary to each other. Whereas the glmM-specific primers provide a rapid, sensitive presumptive assay for the presence of H. pylori and closely related Helicobacter spp., the primers for sequences flanking the 16S rRNA gene can confirm the presence of H. pylori and locate the potential source of this bacterium.
Project description:Helicobacter pylori commonly infects the epithelial layer of the human stomach and in some individuals causes peptic ulcers, gastric adenocarcinoma or gastric lymphoma. Helicobacter pylori is a genetically diverse species, and the most important bacterial virulence factor that increases the risk of developing disease, versus asymptomatic colonization, is the cytotoxin associated gene pathogenicity island (cagPAI). Socially housed rhesus macaques are often naturally infected with H. pylori similar to that which colonizes humans, but little is known about the cagPAI. Here we show that H. pylori strains isolated from naturally infected rhesus macaques have a cagPAI very similar to that found in human clinical isolates, and like human isolates, it encodes a functional type IV secretion system. These results provide further support for the relevance of rhesus macaques as a valid experimental model for H. pylori infection in humans.