Comparison of three enrichment media for the isolation of Campylobacter spp. from foods

AIM: This study compared the performance of three Campylobacter enrichment broths: Bolton broth (BB), Campylobacter Enrichment broth (CEB) and Preston broth (PB). METHODS AND RESULTS: Pure cultures of target and competitor organisms, and naturally-contaminated food samples, were used to establish the performance of these media. In pure culture the PB supported the growth of the greatest number of strains of Campylobacter spp. but failed to inhibit some competitor organisms. The CEB showed the opposite result, inhibiting all 15 competitor organisms used but failing to support the growth of five Campylobacter strains. By comparison, BB showed the best compromise between inhibition of competitors and growth of Campylobacter. CONCLUSIONS: Plates inoculated with BB and CEB food enrichments resulted in more Campylobacter growth than those inoculated with PB, which supported significantly less typical growth (P < or = 0.001). The most common competitor organism isolated from PB was Escherichia coli, and Pseudomonas spp. were frequently isolated from BB and CEB. Both BB and CEB were better than PB for the isolation of Campylobacter from naturally-contaminated foods, although BB yielded more confirmed Campylobacter growth than CEB. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted differences in performance of media used to isolate Campylobacter spp. from foods.     

Evaluation of a commercial diagnostic PCR for the identification of Campylobacter jejuni and Campylobacter coli

AIMS: DuPont Qualicon recently developed a new PCR assay for the identification of Campylobacter jejuni and Campylobacter coli. We evaluated the selectivity and utility of this assay compared with a PCR method already in use in our laboratory. METHODS AND RESULTS: A group of 133 Campylobacter isolates from poultry carcass rinse samples were screened using the commercial PCR and standard PCR. Identical results were found for 89.5% (119/133) of the isolates. However, 10.5% (14/133) gave conflicting results suggesting mixed cultures. These 14 strains were retested by both PCR methods. Of these, 78.6% (11/14) showed identical results for both PCR methods after retesting; the results for the remaining 21.4% (3/14) again indicated mixed cultures. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: The new multiplex PCR is a rapid and accurate alternative to more conventional PCR methods. The persistence of mixed Campylobacter cultures noted in this study suggests certain strains may be very difficult to isolate clonally by standard culture methods.     

Evaluation of selective media for Campylobacter isolation when cycloheximide is replaced with amphotericin B

AIMS: Laboratory media for the isolation of Campylobacter usually contain various selective agents, designed to allow these bacteria to grow whilst suppressing that of other organisms. For example, cycloheximide has often been incorporated into Campylobacter media to inhibit the growth of fungi. The production and availability of cycloheximide, however, has recently decreased due to concerns relating to its potential toxicity for mammalian cells and the compound has also become more expensive as a result. An alternative antifungal agent is necessary, and to address this, the effect of using amphotericin B in place of cycloheximide in Campylobacter selective broths and agars was examined. METHODS AND RESULTS: The growth of Campylobacter strains and the suppression of potential competitor organisms in selective broths or on selective agars containing either amphotericin B or cycloheximide was quantified, using pure microbial cultures. The results were then confirmed by testing food and water samples that contained high levels of micro-organisms, including Campylobacter. CONCLUSIONS: The results of this study indicate that amphotericin B is a suitable replacement for cycloheximide in Campylobacter selective media.     

The use of latex agglutination tests for determining Campylobacter species

A comparison was made between three commercially available latex agglutination tests for the detection of Campylobacter. All tests showed clear agglutination with pure cultures of several Campylobacter strains in both the spiral and coccoid form. The Microscreen test was able to detect 10 times less cells than the Campyslide and Meritec tests. The latex tests were also applied to enrichment broth cultures of chicken products. Sixty-nine per cent of the Campylobacter positive enrichment broth cultures were positive with the Microscreen test. The Meritec test detected 63% of the positive samples. The Campyslide test detected only 15% of the positive samples and often showed non-specific agglutination.     

139株空肠弯曲菌耐药性分析

空肠弯曲菌(Campylobacter jejuni)是最常见的食源性病原菌之一。本研究采用微量肉汤稀释法对分离得到的139株空肠弯曲菌(117株为禽源样本分离株,22株为人源样本分离株)进行耐药性检测。通过对最小抑菌浓度(MIC)的判定结果得出:120株(86. 33%)空肠弯曲菌分离株对6类9组临床常用的抗生素表现出不同程度的耐药,其中禽源空肠弯曲菌耐药率为83. 76%,22株人源空肠弯曲菌均表现出耐药性。对喹诺酮类抗生素表现出高度耐药(环丙沙星80. 58%,萘啶酸77. 70%);对四环素类表现为中等耐药(四环素53. 24%);对部分大环内酯类、氨基糖苷类、林可酰胺类表现为低耐药(庆大霉素7. 19%,阿奇霉素5. 76%,克林霉素6. 47%);对酰胺醇类、部分大环内酯类表现为敏感(氟苯尼考0%,红霉素0%、泰利霉素0%)。139株空肠弯曲菌共产生14种耐药谱型,以TET-CIP-NAL谱型最多,占比38. 13%,耐三重及以上抗生素的多重耐药菌株占比53. 24%。禽源菌株中多重耐药占比46. 15%,人源菌株中多重耐药占比90. 91%。研究结果显示空肠弯曲菌耐药现状不容乐观,尤其对喹诺酮类与四环素类抗生素耐药性较为突出,且过半数菌株为多重耐药。本研究为食源性空肠弯曲菌的防控及临床用药提供参考。     

Specific detection and confirmation of Campylobacter jejuni by DNA hybridization and PCR.

Conventional detection and confirmation methods for Campylobacter jejuni are lengthy and tedious. A rapid hybridization protocol in which a 1,475-bp chromogen-labelled DNA probe (pDT1720) and Campylobacter strains filtered and grown on 0.22-micron-pore-size hydrophobic grid membrane filters (HGMFs) are used was developed. Among the environmental and clinical isolates of C. jejuni, Campylobacter coli, Campylobacter jejuni subsp. doylei, Campylobacter lari, and Arcobacter nitrofigilis and a panel of 310 unrelated bacterial strains tested, only C. jejuni and C. jejuni subsp. doylei isolates hybridized with the probe under stringent conditions. The specificity of the probe was confirmed when the protocol was applied to spiked skim milk and chicken rinse samples. Based on the nucleotide sequence of pDT1720, a pair of oligonucleotide primers was designed for PCR amplification of DNA from Campylobacter spp. and other food pathogens grown overnight in selective Mueller-Hinton broth with cefoperazone and growth supplements. All C. jejuni strains tested, including DNase-producing strains and C. jejuni subsp. doylei, produced a specific 402-bp amplicon, as confirmed by restriction and Southern blot analysis. The detection range of the assay was as low as 3 CFU per PCR to as high as 10(5) CFU per PCR for pure cultures. Overnight enrichment of chicken rinse samples spiked initially with as little as approximately 10 CFU/ml produced amplicons after the PCR. No amplicon was detected with any of the other bacterial strains tested or from the chicken background microflora. Since C. jejuni is responsible for 99% of Campylobacter contamination in poultry, PCR and HGMF hybridization were performed on naturally contaminated chicken rinse samples, and the results were compared with the results of conventional cultural isolation on Preston agar. All samples confirmed to be culture positive for C. jejuni were also identified by DNA hybridization and PCR amplification, thus confirming that these DNA-based technologies are suitable alternatives to time-consuming conventional detection methods. DNA hybridization, besides being sensitive, also has the potential to be used in direct enumeration of C. jejuni organisms in chicken samples.     

SIMPLIFIED CAPACITANCE MONITORING FOR THE DETERMINATION OF CAMPYLOBACTER SPP. GROWTH RATES

Capacitance monitoring is commonly used as an efficient means to generate growth curves of bacterial pathogens. However, the use of capacitance monitoring with Campylobacter spp. was previously determined to be difficult due to the complexity of the required media. We investigated capacitance monitoring using a simplified medium for the efficient and reproducible construction of growth curves for Campylobacter spp . It was determined that Campylobacter spp. should initially be propagated on Mueller Hinton plates in a microaerobic atmosphere (5% O₂, 10% CO₂, 85% N₂) at 37C for 24 h, followed by transfer to Mueller Hinton biphasic cultures for 6 h at 37C in a microaerobic atmosphere. Serial dilutions of the Campylobacter spp. cultures should be used for inoculation of Bactometer wells that contain 1 mL Mueller Hinton broth supplemented with 0.1 M sodium pyruvate for the completion of Campylobacter spp. growth curves with the Bactometer.

PRACTICAL APPLICATIONS

Growth rates can vary greatly among Campylobacter isolates; therefore, the rapid and reproducible methods described will prove useful in studies where the generation of growth curves is critical for subsequent experimental analyses.     

Survival of Campylobacter jejuni strains of different origin in drinking water

AIMS: The aim of the study was to measure the survival of 19 Campylobacter jejuni strains of different origins, including two reference strains, four poultry-derived isolates, nine human isolates and four water isolates, in sterilized drinking water. METHODS AND RESULTS: Pure cultures of 19 C. jejuni strains were inoculated in sterile drinking water and incubated at 4 degrees C for 64 days. Survival was determined by culturability on both selective (Karmali agar) and non-selective [Columbia blood agar (CBA)] media. Culturability was shown to be strain and origin-dependent. Campylobacter jejuni showed prolonged survival on a non-selective than on a selective medium. CONCLUSIONS: The origin of the strain is a determining factor for the survival of C. jejuni in drinking water at 4 degrees C. Poultry isolates showed a prolonged survival, which could be an indication that these strains could play an important role in the transmission of campylobacteriosis through water. In addition, culture conditions are an important factor for evaluating the survival of C. jejuni in drinking water at 4 degrees C. The non-selective agar (CBA) allowed growth of C. jejuni over a longer period of time than the selective agar (Karmali). Furthermore, an enrichment broth (Bolton) allowed the recovery of all 19 C. jejuni strains during the 64 days of incubation at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted differences in culturability depending on culture conditions and on strain origin.     

Factors affecting the pressure resistance of some Campylobacter species

AIMS: To compare pressure resistance between strains of Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter fetus, and to investigate the effect of suspending medium on pressure resistance of sensitive and more resistant strains. METHODS AND RESULTS: Six strains of C. jejuni and four each of C. coli, C. lari and C. fetus were pressure treated for 10 min at 200 and 300 MPa. Individual strains varied widely in pressure resistance but there were no significant differences between the species C. jejuni, C. coli and C. lari. Campylobacter fetus was significantly more pressure sensitive than the other three species. The pressure resistance of C. jejuni cultures reached a maximum at 16-18 h on entry into stationary phase then declined to a minimum at 75 h before increasing once more. Milk was more baroprotective than water, broth or chicken slurry but did not prevent inactivation even of a resistant strain at 400 MPa. CONCLUSIONS: Pressure resistance varies considerably between species of Campylobacter and among strains within a species, and survival after a pressure challenge will be markedly influenced by culture age and food matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: Despite the strain variation in pressure resistance and protective effects of food, Campylobacter sp. do not present a particular problem for pressure processing.     

Advantages of peptide nucleic acid oligonucleotides for sensitive site directed 16S rRNA fluorescence in situ hybridization (FISH) detection of Campylobacter jejuni, Campylobacter coli and Campylobacter lari

Traditionally fluorescence in situ hybridization (FISH) has been performed with labeled DNA oligonucleotide probes. Here we present for the first time a high affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting thermotolerant Campylobacter spp. using FISH. Thermotolerant Campylobacter spp, including the species Campylobacter coli, Campylobacter jejuni and Campylobacter lari, are important food and water borne pathogens. The designed PNA probe (CJE195) bound with higher affinity to a previously reported low affinity site on the 16S rRNA than the corresponding DNA probe. PNA also overcame the problem of the lack of affinity due to the location of the binding site and the variation of the target sequence within species. The PNA probe specificity was tested with several bacterial species, including other Campylobacter spp. and their close relatives. All tested C. coli, C. jejuni and C. lari strains were hybridized successfully. Aging of the Campylobacter cultures caused the formation of coccoid forms, which did not hybridize as well as bacteria in the active growth phase, indicating that the probe could be used to assess the physiological status of targeted cells. The PNA FISH methodology detected C. coli by membrane filtration method from C. coli spiked drinking water samples.     

The epidemiological aspects of campylobacteriosis in children in arid area]

Specific epidemiological features of Campylobacter infection in children in Samarkand Province during the period of 1987-1990 are discussed. The specific proportion of this infection in the total structure of acute enteric infections was, on the average, 8.5%, and Campylobacter carriership among healthy children, 7.0%. Among children with Campylobacter infection, children aged up to 1 year constituted 71.5%. The results of the study of the biological properties of Campylobacter strains isolated from sick and healthy children and their difference from strains isolated in other regions are discussed.     

Growth of Bacillus coagulans in Chemically Defined Media

A nutritional study was made of five strains of Bacillus coagulans obtained from various culture collections. These five strains were descendants of two original isolates; three had been derived from one parent culture in years past and the other two were transfers from another parent culture. Therefore, the five cultures should have represented two distinct groups of genetically identical cultures. Three of the strains obtained from one culture collection had become methyl red-negative and sorbitol-negative and had gained abilities to hydrolyze gelatin and ferment arabinose. Nutritional requirements of the five cultures, determined at 37, 45, and 55 C, differed considerably among strains; however, thiamine and biotin were required by all cultures at all temperatures. Aspartic acid was stimulatory at 37 C and was required at 45 C; folic acid, basic amino acids, and certain other nutrilites were required at 55 C. Adenine supplementation was necessary for two strains at 55 C to prevent autolysis; this phenomenon is discussed. The response of these organisms to both serine and the basic amino acids at the three growth temperatures seems especially significant. The media devised for the growth of the five strains of B. coagulans used in this study permit excellent growth at three incubation temperatures.     

Sensitivity of the pathogen of Campylobacter infection to various antibiotics]

Antibiotic sensitivity of 21 Campylobacter strains isolated from humans, monkeys and birds with diarrhea was assayed and the findings are presented. It was shown that all the isolates were highly sensitive to ofloxacin, pefloxacin, ciprofloxacin, josamycin, pipemidinic acid and amoxyclav (a combination of amoxycillin and clavulanic acid). The predominating majority of the strains were resistant to rifampicin, amoxycillin and phosphomycin. The majority of the cultures were sensitive to erythromycin and roxithromycin while some of the isolates were resistant to them which could be used as an additional biological characteristic of Campylobacter cultures and considered in choosing agents for antibiotic therapy.     

The adhesive properties of bacteria of intestinal origin]

Differences between strains of nonpathogenic Escherichia and lactobacilli, as well as some pathogenic bacteria of enteric origin (Escherichia, Shigella, Campylobacter), in their capacity to adhesion to rat enteric and colonic cells have been shown in vitro. The strains under study have been found to possess more pronounced adhesiveness with respect to colonic cells, which is indicative of their higher receptive capacity in comparison with enteric cells. In the absence of normal microflora lactobacilli and Escherichia exhibit increased adhesiveness with respect to enteric cells. Escherichia enterotoxigenic strains, Yersinia enterocolitica and Salmonella typhimurium virulent strains, Campylobacter jejuni clinical isolates possess more pronounced capacity for adhesion to enteric cells of Peyer's plaques than to other types of epithelial cells, which may be of importance in the pathogenesis of these infections.     

Effect of various gas atmospheres on the growth and survival of Campylobacter jejuni on beef

Pieces of fresh beef were inoculated with three strains of Campylobacter jejuni. The meat was then allocated to three treatments: (a) vacuum packaged, (b) packaged in an atmosphere of 20% CO2 + 80% N2, and (c) packaged into sterile Petri dishes in anaerobic cultivation boxes, which were filled with a gas mixture of 5% O2 + 10% CO2 + 85% N2. The packaging material in the first two treatments was PA 80/PE 100-PE 100/PA 80/PE 100. The survival of Campylobacter cells was followed at 37 degrees C, 20 degrees C and 4 degrees C for 48 h, 4 days and 25 days, respectively. At 37 degrees C the counts of two Campylobacter strains increased in each package treatment for 48 h. At 20 degrees C and at 4 degrees C the counts of the same two strains decreased by 1 to 2 log units and 0.5 to 1 log unit, respectively, during storage. The survival of the two strains was about the same in all package treatments. The third strain was the most sensitive of the strains studied. At 37 degrees C its numbers increased only in the optimal gas atmosphere; at 20 degrees C the strain was not detectable after 24 to 48 h storage and at 4 degrees C after 4 days storage. The aerobic plate counts were determined for all samples at the same time as Campylobacter counts. The high indigenous bacterial numbers of the meat samples did not appear to have a great effect on the survival or growth of campylobacters.     

Discrimination of enterobacterial repetitive intergenic consensus PCR types of Campylobacter coli and Campylobacter jejuni by Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FT-IR) has been used together with pattern recognition methodology to study isolates belonging to the species Campylobacter coli and Campylobacter jejuni and to compare FT-IR typing schemes with established genomic profiles based on enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Seventeen isolates were cultivated under standardized conditions for 2, 3, and 4 days to study variability and improve reproducibility. ERIC-PCR profiles and FT-IR spectra were obtained from strains belonging to the species Campylobacter coli and C. jejuni, normalized, and explored by hierarchical clustering and stepwise discriminant analysis. Strains could be differentiated by using mainly the first-derivative FT-IR spectral range, 1,200 to 900 cm(-1) (described as the carbohydrate region). The reproducibility index varied depending on the ages of the cultures and on the spectral ranges investigated. Classification obtained by FT-IR spectroscopy provided valuable taxonomic information and was mostly in agreement with data from the genotypic method, ERIC-PCR. The classification functions obtained from the discriminant analysis allowed the identification of 98.72% of isolates from the validation set. FT-IR can serve as a valuable tool in the classification, identification, and typing of thermophilic Campylobacter isolates, and a number of types can be differentiated by means of FT-IR spectroscopy.     

Acanthamoeba-Campylobacter coculture as a novel method for enrichment of Campylobacter species

In this study, we present a novel method to isolate and enrich low concentrations of Campylobacter pathogens. This method, Acanthamoeba-Campylobacter coculture (ACC), is based on the intracellular survival and multiplication of Campylobacter species in the free-living protozoan Acanthamoeba polyphaga. Four of the Campylobacter species relevant to humans and livestock, Campylobacter jejuni, C. coli, C. lari, and C. hyointestinalis, were effectively enriched by the coculture method, with growth rates comparable to those observed in other Campylobacter enrichment media. Studying six strains of C. jejuni isolated from different sources, we found that all of the strains could be enriched from an inoculum of fewer than 10 bacteria. The sensitivity of the ACC method was not negatively affected by the use of Campylobacter-selective antibiotics in the culture medium, but these were effective in suppressing the growth of seven different bacterial species added at a concentration of 10(4) CFU/ml of each species as deliberate contamination. The ACC method has advantages over other enrichment methods as it is not dependent on a microaerobic milieu and does not require the use of blood or other oxygen-quenching agents. Our study found the ACC method to be a promising tool for the enrichment of Campylobacter species, particularly from water samples with low bacterial concentrations.     

Fumarate metabolism and the microaerophily of Campylobacter species.

(1) The role of fumarate metabolism in the microaerophily of the Campylobacter genus and the effects of therapeutic agents against it were investigated. (2) NMR spectroscopy was employed to determine the properties of Campylobacter fumarase (Fum) and fumarate reductase (Frd). Radiotracer analysis was used to determine the production of carbon dioxide by Campylobacter cells. Standard microbiological techniques were used to measure the effects of environmental conditions and inhibitors on bacterial growth. (3) All Campylobacter species tested showed both Fum and Frd activities. Frd activity was observed with or without the addition of an exogenous electron donor in the particulate fractions obtained from lysates. Fumarate was oxidized to carbon dioxide via the acetyl-CoA cleavage pathway. The genes encoding proteins involved in fumarate metabolism were identified in the Campylobacter jejuni genome. Cells grew better in atmospheres with 5 and 10% oxygen levels. Fum activity was the same in cultures grown under different oxygen tensions and did not vary with the age of cultures. Frd activity was higher in cultures which grew at faster rates and decreased with the age of cultures. Four Frd inhibitors showed bactericidal effects against Campylobacter spp. with different potencies. The relative strengths of inhibition of the compounds followed the same order as the bactericidal effects. (4) The results suggested that Frd and Fum are constitutive and play a fundamental role in these microaerophiles which show characteristics of anaerobic metabolism, and that the Frd inhibitors tested would not be of therapeutic use.     

Genotypic and phenotypic properties of cattle-associated Campylobacter and their implications to public health in the USA

Since cattle are a major source of food and the cattle industry engages people from farms to processing plants and meat markets, it is conceivable that beef-products contaminated with Campylobacter spp. would pose a significant public health concern. To better understand the epidemiology of cattle-associated Campylobacter spp. in the USA, we characterized the prevalence, genotypic and phenotypic properties of these pathogens. Campylobacter were detected in 181 (19.2%) out of 944 fecal samples. Specifically, 71 C. jejuni, 132 C. coli, and 10 other Campylobacter spp. were identified. The prevalence of Campylobacter varied regionally and was significantly (P<0.05) higher in fecal samples collected from the South (32.8%) as compared to those from the North (14.8%), Midwest (15.83%), and East (12%). Pulsed Field Gel Electrophoresis (PFGE) analysis showed that C. jejuni and C. coli isolates were genotypically diverse and certain genotypes were shared across two or more of the geographic locations. In addition, 13 new C. jejuni and two C. coli sequence types (STs) were detected by Multi Locus Sequence Typing (MLST). C. jejuni associated with clinically human health important sequence type, ST-61 which was not previously reported in the USA, was identified in the present study. Most frequently observed clonal complexes (CC) were CC ST-21, CC ST-42, and CC ST-61, which are also common in humans. Further, the cattle associated C. jejuni strains showed varying invasion and intracellular survival capacity; however, C. coli strains showed a lower invasion and intracellular survival potential compared to C. jejuni strains. Furthermore, many cattle associated Campylobacter isolates showed resistance to several antimicrobials including ciprofloxacin, erythromycin, and gentamicin. Taken together, our results highlight the importance of cattle as a potential reservoir for clinically important Campylobacter.     

Prevalence and antimicrobial resistance of Campylobacter in US dairy cattle

AIMS: To obtain an overview of the prevalence and antimicrobial resistance of Campylobacter in faeces of US dairy cows in 2002. METHODS AND RESULTS: Faeces from 1435 cows, representing 96 dairy operations in 21 US states, were collected for the culture of Campylobacter. A total of 735 Campylobacter strains were isolated (51.2% positive samples) with 94 operations positive (97.9%) for Campylobacter. From this collection, 532 isolates (473 Campylobacter jejuni and 59 Campylobacter coli) were randomly selected for susceptibility testing to eight antimicrobials: azithromycin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, nalidixic acid and tetracycline. The C. jejuni isolates exhibited resistance to tetracycline (47.4%), nalidixic acid (4.0%) and ciprofloxacin (2.5%), while the C. coli strains exhibited some resistance to all antimicrobials except chloramphenicol and ciprofloxacin. Only 3.6% of the C. jejuni isolates were resistant to two or more antimicrobials but 20.3% of the C. coli strains were multiresistant. CONCLUSIONS: On most operations, at least one cow was positive for Campylobacter and more than half of the cows sampled were shedding Campylobacter. The C. coli isolates had significantly higher levels of resistance to macrolides and to tetracycline compared with the C. jejuni strains, but were susceptible to ciprofloxacin. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated a high prevalence of Campylobacter on US dairy operations; however, US dairy cattle have not been recognized as a major source of human infection compared with poultry. Campylobacter coli appears to develop antimicrobial resistance more readily than C. jejuni from the same environment.