Detection of Campylobacter species by using polymerase chain reaction and nonradioactive labeled DNA probe]

We have detected Campylobacter species which are now recognized as major pathogens of acute diarrheal disease in humans using polymerase chain reaction (PCR) and a nonradioactive labeled DNA probe. Diagnosis of Campylobacter enteritis without doing culture from stool samples is of great benefit in the laboratory. Two oligonucleotide primers (20 mer) complementary to a unique sequence of the DNA encoding ribosomal RNA (rRNA) of Campylobacter jejuni for PCR were synthesized by solid-phase phosphoamidite method. Amplified target DNA of 275 base pairs could be resolved on ethidium bromide-stained gels, and hybridized with an oligodeoxynucleotide probe (28 mer) conjugated to alkaline phosphatase. In identification experiments, it was shown that the nonradioactive probe was hybridized to clinical strains of C. jejuni (104), C. coli (5), C. laridis (5), C. hyointestinalis (1) and C. fetus subsp. fetus (1) with an accuracy of 99-100%, while it was not for Helicobacter pylori. Further, there was no evidence of amplification in strains of K. pneumoniae, S. marcescens and E. coli. Using direct detection to stool specimens, this method could be performed in C. jejuni in 39 of 43 culture-positive specimens (91%), and in 19 of 141 culture-negative specimens (13.5%), respectively. The results of this comparative study suggested that the DNA probe assay became a rapid and reliable technique to confirm culture of Campylobacter species.     

Specific detection of Campylobacter jejuni and Campylobacter coli by using polymerase chain reaction.

Development of a routine detection assay for Campylobacter jejuni and Campylobacter coli in clinical specimens was undertaken by using the polymerase chain reaction (PCR). An oligonucleotide primer pair from a conserved 5' region of the flaA gene of C. coli VC167 was used to amplify a 450-bp region by PCR. The primer pair specifically detected 4 strains of C. coli and 47 strains of C. jejuni; but it did not detect strains of Campylobacter fetus, Campylobacter lari, Campylobacter upsaliensis, Campylobacter cryaerophila, Campylobacter butzleri, Campylobacter hyointestinalis, Wolinella recta, Helicobacter pylori, Escherichia coli, Shigella spp., Salmonella spp., Vibrio cholerae, Citrobacter freundii, or Aeromonas spp. By using a nonradioactively labeled probe internal to the PCR product, the assay could detect as little as 0.0062 pg of purified C. coli DNA, or the equivalent of four bacteria. In stools seeded with C. coli cells, the probe could detect between 30 and 60 bacteria per PCR assay. The assay was also successfully used to detect C. coli in rectal swab specimens from experimentally infected rabbits and C. jejuni in human stool samples.     

Development and evaluation of immunochromatographic assay for simple and rapid detection of Campylobacter jejuni and Campylobacter coli in human stool specimens

An immunochromatographic assay (Campy-ICA) using a newly generated single monoclonal antibody against a 15-kDa cell surface protein of Campylobacter jejuni was developed. When cell suspensions of 86 C. jejuni strains and 27 Campylobacter coli strains were treated with a commercially available bacterial protein extraction reagent and the resulting extracts were tested with the Campy-ICA, they all yielded positive results. The minimum detectable limits for the C. jejuni strains ranged from 1.8 x 10(4) to 8.2 x 10(5) CFU/ml of cell suspension, and those for the C. coli strains ranged from 1.4 x 10(5) to 4.6 x 10(6) CFU/ml of cell suspension. All 26 non-Campylobacter species tested yielded negative results with the Campy-ICA. To evaluate the ability of the Campy-ICA to detect C. jejuni and C. coli in human stool specimens, suspensions of 222 stool specimens from patients with acute gastroenteritis were treated with the bacterial protein extraction reagent, and the resulting extracts were tested with the Campy-ICA. The Campy-ICA results showed a sensitivity of 84.8% (28 of 33 specimens) and a specificity of 100% (189 of 189 specimens) compared to the results of isolation of C. jejuni and C. coli from the stool specimens by a bacterial culture test. The Campy-ICA was simple to perform and was able to detect Campylobacter antigen in a fecal extract within 15 min. These results suggest that Campy-ICA testing of fecal extracts may be useful as a simple and rapid adjunct to stool culture for detecting C. jejuni and C. coli in human stool specimens.     

Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni,C. coli,C. lari,C. upsaliensis,and C. fetus subsp. fetus

A multiplex PCR assay was used to simultaneously detect genes from the five major clinically relevant Campylobacter spp. Those genes selected were hipO and 23S rRNA from Campylobacter jejuni; glyA from each of C. coli, C. lari, and C. upsaliensis; and sapB2 from C. fetus subsp. fetus. The assay was evaluated with 137 clinical and environmental isolates and was found to be rapid and easy to perform and had a high sensitivity and specificity for characterizing isolates, even in mixed cultures.     

Dark-field microscopy of human feces for presumptive diagnosis of Campylobacter fetus subsp. jejuni enteritis.

To determine the value of direct dark-field microscopy for diagnosing enteritis due to Campylobacter fetus subsp. jejuni, we examined 1,377 human fecal specimens for bacteria with typical Campylobacter darting motility, leukocytes, and erythrocytes. Eighty-four specimens (6.1%) grew C.fetus subsp. jejuni. Of the 48 specimens showing Campylobacter motility, 30 (62%) grew C. fetus subsp. jejuni. The sensitivity, specificity, and predictive value of observing Campylobacter motility were 36%, 99%, and 62%, respectively. The predictive value of detecting Campylobacter motility was improved if the specimens were diarrheal (23 of 31, 74%), leukocytes were present (25 of 33, 76%), erythrocytes were present (22 of 27, 81%), or if all of the above findings were present (18 of 20, 90%). The sensitivity of detecting Campylobacter darting motility was highest if specimens were examined within 2 h of arrival in the laboratory (15 of 30, 50%) as opposed to after 2 h (15 of 53, 28%; P less than 0.01). Prompt dark-field microscopic examination of diarrheal stool specimens is valuable for the presumptive diagnosis of Campylobacter enteritis.     

Evaluation of a PCR/DNA Probe Colorimetric Membrane Assay for Identification of Campylobacter spp. in Human Stool Specimens

DNA was extracted from 50 human stool specimens using the QIAamp DNA stool minikit. PCR amplification was followed by post-PCR hybridization to DNA probes specific for the Campylobacter genus, Campylobacter jejuni, and Campylobacter coli in a colorimetric membrane assay. Thirty-two of 38 culture-positive specimens were PCR/DNA probe positive for C. jejuni. The assay is rapid and simple and can be applied to stool specimens for the detection of Campylobacter.     

Detection of Campylobacter species by using polymerase chain reaction and nonradioactive DNA probes. II. PCR direct sequencing of the Campylobacter DNA]

We have developed a sensitive DNA hybridization assay for the detection and identification of Campylobacter species which are recognized as important pathogens of acute diarrheal disease in humans. This technique utilizes DNA probes complementary to nucleotide sequences present in 16S ribosomal RNA (rRNA) of C. jejuni, C. coli, C. laridis, C. fetus, C. fetus subsp. fetus, C. fetus subsp. venerealis and C. hyointestinalis, and polymerase chain reaction. The partial sequence of DNAs encoding the Campylobacter rRNA was first analyzed by direct solid phase sequencing in order to select suitable DNA probes. Amplified target DNA of 240 base pairs could be resolved on ethidium bromide-stained gels, and hybridized with DNA probes conjugated to alkaline phosphatase. In identification experiments, one of the 10 probes tested here gave a positive hybridization reaction with C. jejuni, C. coli and C. hyointestinalis but not with other Campylobacter species. The other was specifically reactive with C. fetus, C. fetus subsp. fetus and C. fetus subsp. venerealis. When applied to stool specimens, a good correlation was found between the results obtained by the present assay and by biochemical tests. These findings suggest that the nonradioactive probe assay can be used as the practical criterion for differentiating Campylobacter species.     

实时荧光PCR方法快速检测空肠弯曲菌方法的建立

目的建立实时荧光PCR快速检测空肠弯曲菌的方法。方法以空肠弯曲杆菌HipO基因的保守序列为模板设计特异性引物探针,建立一种能快速检测样本中空肠弯曲杆菌的实时荧光PCR方法;对方法的特异性和敏感性进行评价,并以正常人粪便为空白样本,添加一定量空肠弯曲菌标准株菌液进行检测,以对方法的检测效果进行初步评价。结果该实时荧光PCR方法只对空肠弯曲杆菌进行特异扩增,同种属的结肠弯曲菌及其他常见食源性病原菌均不能扩增;整个检测过程只需要80min,对空肠弯曲菌菌悬液可检测至5个细菌,对加标粪便样本可检测至10-100个细菌。结论本研究建立的实时荧光PCR检测空肠弯曲菌方法不仅能实现对空弯菌的快速检测,而且还为空弯菌的快速诊断及其引起的食源性疾病的监控溯源提供有意义的参考。     

Uncommon Campylobacter species in infant Macaca nemestrina monkeys housed in a nursery.

Studies were conducted to characterize 18 isolates of Campylobacter spp. that could not be identified as either Campylobacter jejuni or C. coli. The isolates were cultured from specimens from 13 of 18 infant nonhuman primates during a prospective epidemiologic study reported previously. Phenotypic tests, DNA hybridization, and analysis of DNA coding for rRNA identified the isolates as C. butzleri (seven isolates), C. hyointestinalis (seven isolates), and C. fetus subsp. fetus or C. fetus subsp. fetus-like organisms (four isolates). Ribotype and polyacrylamide gel electrophoresis patterns indicated that there was heterogeneity among the isolates of C. butzleri and C. fetus subsp. fetus-like organisms.     

Comparison of rectal swabs and stool cultures in detecting Campylobacter fetus subsp. jejuni.

Rectal swabs and stool specimens were compared for the detection of Campylobacter fetus subsp. jejuni in marmosets. Rectal swabs were superior to stool specimens for detection of Campylobacter fetus subsp. jejuni (P = 0.016). Preliminary human data are also presented.     

Cellular fatty acid composition of Campylobacter fetus.

The cellular fatty acid composition of 29 human and animal isolates of Campylobacter fetus was determined with gas-liquid chromatography. All strains contained small amounts of 3-hydroxytetradecanoic acid, suggesting the presence of lipid A. Each of 13 different blood or stool isolates of C. fetus subsp. jejuni obtained from humans or fowl contained a 19-carbon cyclopropane acid which was not present in C. fetus subsp. fetus (6 strains from cattle) or C. fetus subsp. intestinalis (10 strains from humans and cattle). These findings indicate that C. fetus subsp. jejuni can be rapidly differentiated from other C. fetus by gas-liquid chromatography analysis of cellular fatty acids.     

Rapid detection and differentiation of pathogenic Campylobacter jejuni and Campylobacter coli by real-time PCR

A two-tube real-time assay, developed in a LightCycler, was used to detect, identify and differentiate Campylobacter jejuni and Campylobacter coli from all other pathogenic members of the family Campylobacteriaceae. In the first assay, continuous monitoring of the fluorescence resonance energy transfer (FRET) signal acquired from the hybridisation of two adjacent fluoroprobes, a specific FITC probe 5'-GTGCTAGCTTGCTAGAACTTAGAGA-FITC-3') and a universal downstream probe Cy5 (5'-Cy5-AGGTGITGCATGGITGTCGTTGTCG-PO(4)-3'), to the 681-base pair 16S rRNA gene amplicon target (Escherichia coli position 1024-1048 and 1050-1075, respectively) produced by the primer pair, F2 (ATCTAATGGCTTAACCATTAAAC, E. coli position 783) and Cam-Rev (AATACTAAACTAGTTACCGTC, E. coli position 1464), detected C. coli, C. lari and C. jejuni. As expected, a Tm of 65 degrees C was derived from the temperature-dependent probe DNA strand disassociation. In the second assay, an increase in fluorescence due to binding of the intercalating dye SYBR Green I to the DNA amplicons of the hippuricase gene (hipO) (produced by the primer pair hip2214F and hip2474R) was observed for C. jejuni but not for C. coli which lacks the hipO gene. A Tm of 85+/-0.5 and 56 degrees C determined from temperature-dependent dye-DNA disassociation identified C. jejuni and the non-specific PCR products, respectively, in line with our expectation. The two-tube assay was subsequently used to identify and differentiate the 169 Campylobacteriaceae isolates of animal, human, plant and bird origin held in our culture collection into C. coli (74 isolates), C. jejuni (86 isolates) and non-C. coli-C. jejuni (9 isolates). In addition, the method successfully detected C. jejuni, C. coli and C. lari from 24-h enrichment cultures initiated from 30 commercial chicken samples.     

Oligonucleotide probe for detection and identification of Campylobacter pylori.

We have developed a novel and practical DNA-RNA hybridization assay for the detection and identification of Campylobacter pylori in the gastric mucosa. This technique utilizes a [32P]ddATP-labeled synthetic oligonucleotide probe complementary to a nucleotide sequence present in C. pylori 16S rRNA. This probe is very sensitive and reacted with all 23 strains of C. pylori tested. It is also highly specific, since there was no cross-reactivity with the heterologous organisms Campylobacter coli, C. fetus subsp. fetus, C. jejuni, and C. laridis or with Escherichia coli. Hybridization of the oligonucleotide probe with C. pylori RNA was completely inhibited by treatment of the membrane filters with RNase but not DNase. Although a gastric mucosa tissue homogenate slightly inhibited the hybridization, as few as 10(4) C. pylori cells could be detected even in the presence of 5 mg of gastric mucosa. Gastric biopsy specimens obtained from patients referred for upper gastrointestinal tract endoscopy were tested for C. pylori infection by direct oligonucleotide hybridization, and the results were compared with those of bacteriological cultures, the urease test, and histological observations. A comparison of the urease test and the oligonucleotide hybridization results showed an excellent correlation between the two methods. The clinical usefulness of this oligonucleotide-RNA hybridization method is discussed.     

Comparison of a blood-free medium and a filtration technique for the isolation of Campylobacter spp. from diarrhoeal stools of hospitalised patients in central Australia.

Single specimens of diarrhoeal stool from 676 patients, mostly aboriginals aged less than 5 years, admitted to Alice Springs Hospital, central Australia, for diarrhoea between Sept. 1988 and Feb. 1989, were examined for Campylobacter spp. by culture on a blood-free medium with selective supplement (BFM; Oxoid) and blood agar overlaid with a membrane filter (FM). Campylobacter spp. were isolated on either BFM or FM or both from 225 patients. Campylobacter spp. were isolated on BFM alone from 75 patients and on FM alone from 213 patients (p less than 0.001; chi 2 test). Most campylobacters isolated on BFM were C. jejuni. All C. jejuni subsp. doylei, all "C. upsaliensis" except one, all C. laridis, C. fetus subsp. fetus and several uncharacterised Campylobacter isolates were isolated on FM only. C. jejuni was isolated on BFM but not FM from several patients, and vice versa. Serotyping of C. jejuni and C. coli isolated from both media showed the serotypes recovered from the two media to be different in some patients. In some patients concurrent infection with several species or serotypes (up to five) of Campylobacter, or both, was shown for the first time by the use of FM. We conclude that the use in combination of a selective medium and a non-selective medium with a filtration technique are better than either medium alone for the isolation of Campylobacter spp.     

Evaluation of culture methods and a DNA probe-based PCR assay for detection of Campylobacter species in clinical specimens of feces

Campylobacter species are the leading agents of bacterial gastroenteritis in developed countries. In this study 320 specimens of feces from patients with symptoms of acute gastroenteritis were cultured for Campylobacter species by direct plating on modified charcoal cefoperazone deoxycholate agar and by enrichment in modified Preston broth, with or without blood added, for 48 h at 37 degrees C prior to plating. A 16S/23S PCR/DNA probe membrane-based colorimetric detection assay was evaluated on a subset of the feces (n = 127), including 18 culture-positive and 109 culture-negative specimens. DNA was extracted directly from the fecal specimens by using the QIAamp DNA stool Minikit for the DNA probe-based PCR assay (PCR/DNA probe assay). A second PCR/DNA probe assay based on the 16S rRNA gene in Campylobacter spp. was applied to all specimens that were culture negative, PCR/DNA positive on initial analysis. Campylobacter species were cultured in 20 of the 320 specimens. The 16S/23S PCR/DNA probe assay detected campylobacter DNA in 17 of 18 (94% sensitivity) culture-positive specimens and in 41 (38%) culture-negative specimens. The presence of campylobacter DNA in 35 of 41 culture-negative specimens was confirmed by the 16S PCR/DNA probe assay. DNA sequence analysis of seven 16S/23S PCR products and five 16S PCR products amplified from a selection of these specimens confirmed the presence of campylobacter DNA and more specifically Campylobacter jejuni, C. concisus, C. curvus, and C. gracilis DNA in these specimens. The molecular assays described in this study are rapid methods for the detection and identification of Campylobacter species in fecal specimens. The finding of Campylobacter spp. DNA in a large number of specimens of feces from patients with no other identified cause of diarrhea may suggest that Campylobacter spp. other than C. jejuni and C. coli may account for a proportion of cases of acute gastroenteritis in which no etiological agent is currently identified.     

Evaluation of the ProSpecT Microplate Assay for detection of Campylobacter: a routine laboratory perspective

Objectives  To evaluate the use of the new enzyme-linked immunosorbent assay, the ProSpecT Campylobacter Microplate Assay (Alexon-Trend, Minneapolis, MN, USA), which allows 2-h detection of both Campylobacter jejuni and Campylobacter coli antigen directly in stool specimens.
Methods  Over 4 months, all stool samples preserved in Cary–Blair medium, or fresh specimens, from non-hospitalized children and HIV-infected patients (adults and children), submitted to our laboratory were evaluated with the ProSpecT Campylobacter Microplate Assay. Results were compared with those obtained by routine culture methods using both a specific medium and a filtration method for the recovery of Campylobacter spp.
Results  Of the 1205 stool specimens cultured, 101 were found to be positive for either C. jejuni or C. coli , giving an overall recovery rate of 8.38%. Ninety samples were positive by both culture and ProSpecT Campylobacter Microplate Assay, and 11 were positive by culture only, giving a sensitivity of 89.1%. In addition, of 1104 samples negative by culture, 25 were initially positive by ProSpecT Campylobacter Microplate Assay. We found no cross-reaction with other bacterial enteropathogens isolated from stool specimens. These results thus confirm a high specificity (97.7%) for both C. jejuni and C. coli. The positive and negative predictive values found were 78.3% and 99%, respectively. There was no statistically significant difference in sensitivity and specificity if the stool was fresh or preserved with Cary–Blair medium.
Conclusion  These data suggest that the ProSpecT Campylobacter Microplate Assay is a rapid and easy-to-use test for the detection of both C. jejuni and C. coli in stool specimens. It could be used for patients for whom early antibiotic therapy is needed or for epidemiologic studies.     

Hydrogenase activity in catalase-positive strains of Campylobacter spp.

A rapid hydrogenase assay has been developed which may be useful in separating the species Campylobacter jejuni and C. coli from the subspecies C. fetus subsp. fetus and C. fetus subsp. venerealis. This assay employs the impermeant redox dye benzyl viologen, and positive determinations can be made within 20 min. All strains of C. jejuni and C. coli were found to be strongly hydrogenase positive. All strains of C. fetus subsp. fetus and C. fetus subsp. venerealis were negative for hydrogenase when the assay was performed at a benzyl viologen concentration of 2 mM and an incubation temperature of 30 degrees C. Some strains of C. fetus had low levels of hydrogenase as determined with cell extracts but were hydrogenase negative by the benzyl viologen assay. Since there are few rapid diagnostic tests available for screening Campylobacter isolates, we hope that the rapid hydrogenase assay will prove useful.     

Campylobacter fetus subspecies fetus infection

Summary During a six-year period five patients withCampylobacter fetus subspecies fetus infections were seen at the Mayo Clinic in Rochester, Minnesota. Bacteremia was observed in two patients, one presenting with aortic valve endocarditis and the other with abdominal atherosclerotic aortic aneurysm.C. fetus subsp. fetus was isolated from tibial tissue of a patient with osteomyelitis. Diarrhea was the main complaint of two further patients, and was also mentioned by the patient with the aortic aneurysm. Despite the use of incubation conditions and selective media geared to detect onlyCampylobacter jejuni, C. fetus subsp. fetus was isolated from stool specimens of the two patients with gastrointestinal symptoms. The fact that three of fiveC. fetus subsp. fetus infections observed in this study were associated with intestinal symptoms further supports the importance of the gastrointestinal tract in the pathogenesis ofC. fetus subsp. fetus infections.     

Uneven distribution of the luxS gene within the genus Campylobacter

Polymerase chain reaction (PCR) amplification was performed on 20 isolates of five Campylobacter species using a degenerate primer pair designed in silico to generate a product of the luxS gene or its homologue from Campylobacter organisms. Although the primer pair successfully amplified products of approximately 500 base pairs (bp) with the eight isolates of C. jejuni and C. coli and some of C. upsaliensis and C. fetus, it failed to amplify fragments with all four isolates of C. lari (two urease-negative C. lari; two urease-positive thermophilic campylobacters). When Southern blot hybridisation analysis was carried using the mixed luxS gene fragments prepared from the C. jejuni, C. coli, C. upsaliensis and C. fetus strains as a probe, all C. jejuni, C. coli, C. upsaliensis and C. fetus isolates gave positive signals, but no positive signal was detected with any C. lari isolate. These results clearly indicate that C. jejuni, C. coli, C. upsaliensis and C. fetus carry the luxS gene or its homologue. However, no luxS gene or its homologue was identified to occur in the C. lari genome. Although autoinducer-2 assays were positive in C. jejuni, C. coli, C. upsaliensis and C. fetus isolates, it was negative with all the C. lari isolates examined. In addition, a biofilm formation assay demonstrated that biofilm formation in the C. lari species does not appear to correlate with the occurrence of the luxS gene because biofilm formation occurred among some isolates of C. lari.     

Evaluation of media for isolation of Campylobacter fetus subsp. jejuni from fecal specimens.

Isolation rates of Campylobacter fetus subsp. jejuni from human fecal specimens were equivalent after broth enrichment (thioglycolate medium containing antibiotics) and direct inoculation on two brucella blood agar media containing ferrous sulfate, sodium metabisulfite, and sodium pyruvate, identical concentrations of vancomycin and trimethoprim, and different concentrations of polymyxin B and cephalothin. Studies with clinical isolates of C. fetus subsp. jejuni demonstrated temperature-dependent activity of polymyxin E (colistin) and substantial inhibition of growth on Thayer-Martin and Martin-Lewis media.