ABSTRACT
The inhibitory effect of human and porcine bile samples to detect Helicobacter DNA was studied by adding different concentrations of bile samples to PCR mixtures of six thermostable DNA polymerases containing cagA specific primers and Helicobacter pylori DNA. PCR products were amplified by using the Rotorgene system and SYBR Green I. Among the six DNA polymerases tested, rTth had the lowest sensitivity to bile inhibitors, whereas Taq and Tfl had the highest sensitivity. Bile proteins did not inhibit AmpliTaq DNA polymerase, whereas the fraction containing mainly bile acids and their salts inhibited the amplification capacity of AmpliTaq. Heating human bile at 98 degrees C and adding casein and formamide to the reaction mixture reduced the PCR inhibitory effect of bile. Therefore, a pre-PCR treatment based on dilution and heating of bile, adding casein and formamide to the reaction mixture of rTth DNA polymerase was found efficient to amplify DNA directly in bile.
Subject(s)
Bile Acids and Salts/pharmacology , Bile/chemistry , Helicobacter/isolation & purification , Nucleic Acid Synthesis Inhibitors , Polymerase Chain Reaction/methods , Animals , Enzyme Stability , Helicobacter/classification , Helicobacter/genetics , Hot Temperature , Humans , Male , Mice , Mucins/pharmacology , SwineABSTRACT
Rodent models have been developed to study the pathogenesis of diseases caused by Helicobacter pylori, as well as by other gastric and intestinal Helicobacter spp., but some murine enteric Helicobacter spp. cause hepatobiliary and intestinal tract diseases in specific inbred strains of laboratory mice. To identify these murine Helicobacter spp., we developed an assay based on PCR-denaturing gradient gel electrophoresis and pyrosequencing. Nine strains of mice, maintained in four conventional laboratory animal houses, were assessed for Helicobacter sp. carriage. Tissue samples from the liver, stomach, and small intestine, as well as feces and blood, were collected; and all specimens (n = 210) were screened by a Helicobacter genus-specific PCR. Positive samples were identified to the species level by multiplex denaturing gradient gel electrophoresis, pyrosequencing, and a H. ganmani-specific PCR assay. Histologic examination of 30 tissue samples from 18 animals was performed. All mice of eight of the nine strains tested were Helicobacter genus positive; H. bilis, H. hepaticus, H. typhlonius, H. ganmani, H. rodentium, and a Helicobacter sp. flexispira-like organism were identified. Helicobacter DNA was common in fecal (86%) and gastric tissue (55%) specimens, whereas samples of liver tissue (21%), small intestine tissue (17%), and blood (14%) were less commonly positive. Several mouse strains were colonized with more than one Helicobacter spp. Most tissue specimens analyzed showed no signs of inflammation; however, in one strain of mice, hepatitis was diagnosed in livers positive for H. hepaticus, and in another strain, gastric colonization by H. typhlonius was associated with gastritis. The diagnostic setup developed was efficient at identifying most murine Helicobacter spp.
Subject(s)
Helicobacter/isolation & purification , Mice, Inbred Strains/microbiology , Polymerase Chain Reaction/methods , Animals , Base Sequence , Blood/microbiology , DNA Primers , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , DNA, Ribosomal/genetics , Electrophoresis, Polyacrylamide Gel/methods , Feces/microbiology , Helicobacter/genetics , Intestine, Small/microbiology , Liver/microbiology , Mice , Molecular Sequence Data , RNA, Ribosomal, 16S/genetics , Stomach/microbiologyABSTRACT
Over a 1-y period, 26 inpatients at the Jordan University Hospital in Amman were detected with bacteraemia (23 cases) or respiratory tract colonized with B. cepacia (3 cases). A combination of genetic identification and molecular typing has proved that all cases were caused by a single epidemic strain of B. cepacia genomovar IIIa. Nosocomial infections could be documented in 21/26 (81%) patients, mostly with severe underlying or malignant diseases other than cystic fibrosis, but the source of infection was undetected. The overall mortality related to infection with B. cepacia was 42%. All B. cepacia isolates were resistant to ampicillin, amikacin, carbenicillin and gentamicin; and mostly susceptible to piperacillin, chloramphenicol, cotri-moxazole, tetracycline, ceftazidime, and tazocin (62-88%). This study demonstrates the nosocomial and high fatality of B. cepacia genomovar IIIa in Jordanian patients suffering from diseases other than cystic fibrosis.
Subject(s)
Bacteremia/epidemiology , Burkholderia Infections/epidemiology , Burkholderia cepacia/isolation & purification , Cross Infection/epidemiology , Disease Outbreaks , Adolescent , Adult , Age Distribution , Aged , Anti-Bacterial Agents/therapeutic use , Bacteremia/microbiology , Burkholderia Infections/diagnosis , Burkholderia Infections/drug therapy , Child , Child, Preschool , Cohort Studies , Cross Infection/drug therapy , Cross Infection/microbiology , Cystic Fibrosis/diagnosis , Cystic Fibrosis/epidemiology , DNA, Bacterial/analysis , Disease Susceptibility , Female , Humans , Jordan/epidemiology , Male , Microbial Sensitivity Tests , Middle Aged , Polymerase Chain Reaction/methods , Risk Assessment , Severity of Illness Index , Sex Distribution , Survival AnalysisABSTRACT
Helicobacter species are fastidious bacterial pathogens that are difficult to culture by standard methods. A PCR-denaturing gradient gel electrophoresis (PCR-DGGE) technique for detection and identification of different Helicobacter species was developed and evaluated. The method involves PCR detection of Helicobacter DNA by genus-specific primers that target 16S rDNA and subsequent differentiation of Helicobacter PCR products by use of DGGE. Strains are identified by comparing mobilities of unknown samples to those determined for reference strains; sequence analysis can also be performed on purified amplicons. Sixteen DGGE profiles were derived from 44 type and reference strains of 20 Helicobacter species, indicating the potential of this approach for resolving infection of a single host by multiple Helicobacter species. Some more highly related species were not differentiated whereas in highly heterogeneous species, sequence divergence was observed and more than one PCR-DGGE profile was obtained. Application of the PCR-DGGE method to DNA extracted from faeces of zoo animals revealed the presence of Helicobacter DNA in 13 of 16 samples; a correlation was seen between the mobility of PCR products in DGGE analysis and DNA sequencing. In combination, this indicated that zoo animals are colonized by a wide range of different Helicobacter species; seven animals appeared to be colonized by multiple Helicobacter species. By this approach, presumptive identifications were made of Helicobacter bilis and Helicobacter hepaticus in a Nile crocodile, Helicobacter cinaedi in a baboon and a red panda, and Helicobacter felis in a wolf and a Taiwan beauty snake. All of these PCR products ( approximately 400 bp) showed 100 % sequence similarity to 16S rDNA sequences of the mentioned species. These results demonstrate the potential of PCR-DGGE-based analysis for identification of Helicobacter species in complex ecosystems, such as the gastrointestinal tract, and could contribute to a better understanding of the ecology of helicobacters and other pathogens with a complex aetiology.
