Diversity of flaA genotypes among Campylobacter jejuni isolated from six niche-market poultry species at farm and processing

PCR-restriction fragment length polymorphism of the flagellin (flaA) gene in Campylobacter jejuni was used to determine the relationships of isolates collected at the farm and throughout processing for six niche-market poultry species. This study focused on two specialty chicken products, poussin and free range, and four other specialty products, squab, duck, guinea fowl, and quail. Cloacal and carcass samples were collected from three flocks from each of the six niche species. Three processing plants in California participated in a 2-year investigation. A total of 773 isolates from farm, posttransport, and the processing plants were genotyped, yielding a total of 72 distinct flaA profiles for the six commodities. Genetic diversity of C. jejuni at the farm was greatest for ducks with up to 12 distinct flaA types in two flocks and least for squab 1 flaA type between two farms. For two of the guinea fowl flocks, one free-range flock, two squab flocks, and all three poussin flocks, the flaA types recovered at the prepackage station matched those from the farm. Cross-contamination of poultry carcasses was supported by the observation of flaA types during processing that were not present at the farm level. New C. jejuni strains were detected after transport in ducks, guinea fowl, and free-range chickens. Postpicker, postevisceration, and prewash sampling points in the processing plant yield novel isolates. Duck and free-range chickens were the only species for which strains recovered within the processing plant were also found on the final product. Isolates recovered from squab had 56 to 93% similarity based on the flaA types defined by PCR-restriction fragment length polymorphism profiles. The 26 duck isolates had genetic similarities that ranged from 20 to 90%. Guinea fowl and free-range chickens each had 40 to 65% similarity between isolates. Poussin isolates were 33 to 55% similar to each other, and quail isolates were 46 to 100% similar. Our results continue to emphasize the need to clean processing equipment and posttransport crates in order to decrease cross contamination between flocks. This study also determined that several strains of C. jejuni had unique flaA types that could only be recovered in their host species.     

Fate of Campylobacter jejuni in butter

An outbreak of Campylobacter enteritis was associated with a restaurant in Louisiana during the summer of 1995. Thirty cases were identified, and four required hospitalization. Campylobacter jejuni was isolated from the patients, and epidemiologic studies revealed illness associated with eating garlic butter served at the restaurant. Three batches of garlic butter prepared by the restaurant associated with the outbreak and a C. jejuni isolate obtained from a patient involved in the outbreak were used for studies to determine the fate of C. jejuni in garlic butter. Studies also were done to determine the efficacy of the heat treatment used by the restaurant to prepare garlic bread to kill C. jejuni. Garlic butter was inoculated with approximately 10(4) and 10(6) CFU/g of C. jejuni and held at 5 or 21 degrees C. Results revealed that the survival of C. jejuni differed greatly, depending on the presence or absence of garlic. At 5 degrees C, C. jejuni populations decreased to an undetectable level (<10 CFU/g) within 3 h for two batches and within 24 h for another batch. In contrast, C. jejuni could survive at 5 degrees C for 13 days in butter with no garlic. At 21 degrees C, C. jejuni populations decreased to an undetectable level within 5 h for two batches and to 50 CFU/g in 5 h for another batch. In contrast, C. jejuni was detected at 500 CFU/g at 28 h after inoculation but was undetectable at 3 days in butter with no garlic held at 21 degrees C. The heating procedure (135 degrees C, 4 min) used to make garlic bread by the implicated restaurant was determined not to be sufficient for killing C. jejuni, with the internal temperature of the buttered bread after heating ranging from 19 to 22 degrees C. This study revealed that C. jejuni can survive for many days in refrigerated butter, but large populations (10(3) to 10(5) CFU/g) are killed within a few hours in butter that contains garlic. Furthermore, the heat treatment used by the restaurant to melt garlic butter in making garlic bread was not adequate to kill C. jejuni.     

Occurrence of Campylobacter jejuni in vegetables

In order to understand the importance of vegetables in the transmission of thermophilic Campylobacter, 56 samples of different vegetables were screened. Out of these, 2 samples (1 spinach and 1 fenugreek) revealed the presence of Campylobacter jejuni biotype I. Both the isolates were enteropathogenic in rat ileal loop test.     

Thermal inactivation of Campylobacter jejuni in broth

New Zealand has a high rate of reported campylobacteriosis compared with other developed countries. One possible reason is that local strains have greater heat tolerance and thus are better able to survive undercooking; this hypothesis is supported by the remarkably high D-values reported for Campylobacter jejuni in The Netherlands. The objective of this study was to investigate the thermal inactivation of isolates from New Zealand in broth, using strains that are commonly found in human cases and food samples in New Zealand. Typed Campylobacter strains were heated to a predetermined temperature using a submerged-coil heating apparatus. The first-order kinetic model has been used extensively in the calculation of the thermal inactivation parameters, D and z; however, nonlinear survival curves have been reported, and a number of models have been proposed to describe the patterns observed. Therefore, this study compared the conventional first-order model with eight nonlinear models for survival curves. Kinetic parameters were estimated using both one- and two-step regression techniques. In general, nonlinear models fit the individual inactivation data sets better than the log-linear model. However, the log-linear and the (nonlinear) Weibull models were the only models that could be successfully fitted to all data sets. For seven relevant New Zealand C. jejuni strains, at temperatures from 51.5 to 60°C, D- and z-values were obtained, ranging from 1.5 to 228 s and 4 to 5.2°C, respectively. These values are in broad agreement with published international data and do not indicate that the studied New Zealand C. jejuni strains are more heat resistant than other strains, in contrast with some reports from The Netherlands.     

Campylobacter jejuni non-culturable coccoid cells.

The behaviour of Campylobacter jejuni in the environment is poorly documented. Rapid loss of viability on culture media is reported. This phenomenon is associated with the development of so-called coccoid cells. It has been suggested that these cells can be infective to animals and man. Results obtained with ATP-measurements of coccoid cells and Direct Viable Count (DVC) support this hypothesis. Introduction of coccoid cells into simulated gastric, ileal and colon environments did not result in the presence of culturable cells. Oral administration to laboratory animals and volunteers caused no typical symptoms of campylobacteriosis. Until 30 days after uptake of the cells antibodies against C. jejuni could not be detected in the blood, and the presence of this microorganism in stool samples could not be demonstrated.     

Effects of short-chain nitrocompounds against Campylobacter jejuni and Campylobacter coli in vitro

ABSTRACT:  Effects of 2-nitro-1-propanol, 2-nitroethanol, nitroethane, and 2-nitro-methyl-propionate (0, 10, and 20 mM) on growth of Campylobacter jejuni were tested during culture in Bolton broth adjusted to pH 5.6, 7.0, or 8.2. The nitrocompounds were similarly tested against C. coli but at pH 8.2 only. Viable cell counts measured during incubation revealed main effects ( P < 0.05) of all nitrocompounds on the survivability of C. jejuni . An effect of pH ( P < 0.05) on the survivability of C. Jejuni during incubation with nitrocompounds was observed, with greater inhibition observed at pH 8.2 than at pH 5.6 or 7.0 for nitroethane, 2-nitro-l-propanol, and 2-nitroethanol, but not for 2-nitro-methyl-propionate, which showed greatest inhibition at pH 5.6. Except for 2-nitro-methyl-propionate, which was ineffective, all nitrocompounds elicited similar effects on C. coli . The effect of nitroethane and 2-nitro-l-propanol (10 mM) on naturally occurring Campylobacter was investigated during incubation of porcine fecal suspensions, where Campylobacter concentrations decreased more rapidly ( P < 0.05) in suspensions with added 2-nitro-l-propanol than in unsupplemented or nitroethane-supplemented suspensions, thus reiterating the superior inhibitory effect of 2-nitro-l-propanol.     

Metronidazole resistance in Campylobacter jejuni from poultry meat

The occurrence of metronidazole resistance was investigated among Campylobacter jejuni in raw poultry meat collected from supermarkets. MICs were determined by the agar dilution procedure in the testing range of 3 to 60 microg/ml metronidazole. The MICs showed a bimodal distribution with a significant proportion of metronidazole-resistant isolates among C. jejuni from raw broiler and turkey meat. Metronidazole resistance occurred most frequently among turkey meat isolates (P < 0.005). This is the first report of foodborne bacteria carrying metronidazole resistance.     

Survival of Campylobacter jejuni in Selected Gaseous Environments

Vacuum-packaged and oxygen-permeable, polyvinyl chloride film wrapped broiler chickens were compared with one another to determine influence on the survival of Campylobacter jejuni. No significant (P<0.01) difference in the survival of C. jejuni was observed, irrespective of the packaging system assessed by either surface rinse or drip sampling. Significantly (P<0.1) greater numbers of the organism were recovered by surface rinse sampling in both packaging systems when compared with the numbers obtained from drip sampling. Recovery of indigenous Campylobacter from red meat carcass samples was greater in the vacuum packaged samples (19/63) than in the oxygen permeable wrapped samples (7/63) after the samples were held at refrigeration temperatures for 1 wk. Significantly (P<0.01) greater survival of C. jejuni in ground beef was demonstrated in a 100% nitrogen atmosphere when compared with a vacuum, a 80% carbon dioxide and 20% nitrogen atmosphere or a 5% oxygen, 10% carbon dioxide and 85% nitrogen atmosphere. These results indicate that the organism survives at variable rates in different atmospheres, but these differences were relatively small and unlikely to impact on the public health.     

Plant-derived compounds inactivate antibiotic-resistant Campylobacter jejuni strains

Sixty-three Campylobacter jejuni isolates were screened for their resistance to the antibiotics ampicillin, cefaclor, ciprofloxacin, erythromycin, gentamycin, tetracycline, and trimethoprim-sulfamethoxazole. Based on this screen, the resistant strains D28a and H2a and the nonresistant strain A24a were selected for evaluation of their resistance and susceptibility to inactivation by cinnamaldehyde and carvacrol, the main constituents of plant-derived cinnamon and oregano oils, respectively. Different concentrations (0.05, 0.1, and 0.2% [vol/vol] in sterile phosphate-buffered saline) of cinnamaldehyde and carvacrol were added to C. jejuni cultures with initial populations of 10(4) CFU/ml. The samples were then mixed thoroughly and incubated at 37 degrees C. Viable bacterial populations were enumerated at incubation periods of 0, 30, 60, and 120 min. The results indicate that the extent of inhibition of microbial survival was related to both the nature and concentration of antimicrobials and the incubation time. Both cinnamaldehyde and carvacrol exhibited rapid antimicrobial activity against both antibiotic-resistant and non-resistant C. jejuni strains, at concentrations of approximately 0.1% and higher. The antimicrobial efficacy of cinnamaldehyde was greater than that of carvacrol. The possible significance of the results for microbiological food safety is discussed.     

Effect of Gamma Radiation on Campylobacter jejuni

Radiation resistance of Campylobacter jejuni in broth, ground beef, and ground turkey meat was determined using dose levels from 0 - 200 Krad at −30 ± 10°C, at 0 - 5°C, and at 30 ± 10°C. Irradiation at −30°C increased radiation resistance of cultures in ground meats; broth cultures were not greatly influenced by temperature. The effect of culture age on radiation resistance was also evaluated using cells in various physiological phases. Age did not have a pronounced effect on radiation resistance. The largest D₁₀ value for C. jejuni was 32 Krad, which was less than D₁₀ values commonly reported for salmonellae.     

空肠弯曲菌致病分子机制研究进展

空肠弯曲菌(Campylobacter jejuni)是人类的食源性病原菌,感染后主要引起急性肠炎,与人类格林-巴利综合症也有密切关系.研究表明,空肠弯曲菌致病性是多种毒力因子共同作用的结果.作者从分子生物学研究水平综述空肠弯曲菌黏附、定植、侵入、产细胞毒素、分子模拟等致病机制.     

多重PCR鉴定动物源空肠弯曲菌和结肠弯曲菌方法的建立

建立鉴定空肠弯曲菌和结肠弯曲菌的多重PCR(mPCR)方法。方法 分别以16S rRNA、马尿酸酶和16S-23S rRNA基因为靶序列设计特异性引物,建立多重PCR方法检测37株菌株样品,同时采用ⁱⁿgene CAM nested PCR检测试剂盒检测验证,进行结果比较分析。结果 该多重PCR方法可扩增出空肠弯曲菌和结肠弯曲菌的特异性条带,其他对照菌株均未扩增出条带,具有较好的特异性;检测敏感性可达0.81pg/μl空肠弯曲菌DNA,0.93pg/μl结肠弯曲菌DNA。多重PCR方法和试剂盒检测结果的符合率为100%,二者与国标GB/T 4789.9—2008方法的符合率达97%以上。结论 本试验建立的多重PCR方法操作快速方便、节约试验成本,具有较好的特异性、敏感性和重复性,可用于弯曲菌的鉴定。     

Fla-DGGE法对食品中空肠弯曲菌和结肠弯曲菌的检测和分型

本文应用鞭毛蛋白基因flaA和flaB的变性梯度凝胶电脉(denaturing gradient gel electrophoresis,DGGE)方法对食品中空肠弯曲菌和结肠弯曲菌进行检测和分型。采用RBB(rpeated bead beating)、CTAB和丙酮-氯仿抽提三种方法提取样品基因组后进行fla-DGGE检测,结果完全一致。10份生鲜鸡、鸭肉样品中有8份含有空肠弯曲菌或结肠弯曲菌,其中3份含有两种或两种以上的空肠弯曲菌或结肠弯曲菌或它们的不同型别。克隆测序后结果表明,有7份样品被同一种空肠弯曲菌所污染,其中3份样品还被同一种结肠弯曲菌所污染,1份样品被污染情况严重,检测到含有一种结肠弯曲菌和三种空肠弯曲菌。Fla-DGGE方法快速、准确、灵敏度高,可用于食品中空肠弯曲菌和结肠弯曲菌的快速检测和分型。     

空肠和结肠弯曲菌鞭毛蛋白基因fla A的检测

目的 运用一种快速、敏感、特异的检测空肠和结肠弯曲菌的方法.方法 以空肠和结肠弯曲菌所共有特异的鞭毛蛋白基因 fla A的一段高度保守序列为引物,用PCR法扩增fla A基因上的一段约1 700 bp的片断.用该引物对空肠和结肠弯曲菌的标准株、福建省的食品分离株进行PER扩增检测,并同时检测该PCR方法的敏感性.结果 扩增片断表现出极好的特异性,2株空肠和结肠弯曲菌标准菌株、8株分离自不同食品样品的空肠穹曲菌和结肠弯曲菌菌株均为阳性,且敏感性实验显示该PCR方法的反应体系最低检出菌量为6 CFU.结论 该方法快速、敏感、特异,可用于突发性食物中毒和暴发感染的调查.     

Media for the aerobic resuscitation of Campylobacter jejuni

The microaerophilic nature of Campylobacter jejuni has complicated its recovery from human and animal sources. In this study, enhancement of the growth and aerotolerance of C. jejuni ATCC 35921 in nutrient broth no. 2 (NB2) was investigated. The efficiency of recovery of C. jejuni in NB2 containing FBP (0.025% [each] ferrous sulfate, sodium metabisulfite, and sodium pyruvate), 5% laked horse blood, hemin, Oxyrase, or activated charcoal in an aerobic atmosphere was compared with that obtained under microaerophilic incubation. The shortest lag time (lamda) for cells grown aerobically was observed with NB2 supplemented with FBP, 5% laked horse blood, 0.01 g/liter of hemin, or 0.15 U/ml of Oxyrase. The efficacy of these media to resuscitate C. jejuni cells in late exponential phase, as well as cells subjected to stress induced by cold, heat, starvation, or acid, was determined in aerobic or microaerobic atmospheres. The h of cells grown aerobically in NB2 containing both FBP and blood was similar to that obtained in the same medium incubated in a microaerobic environment (P > 0.05). However, the X was longer during aerobic growth when low numbers of cells (approximately 1 log CFU/ml) in late exponential phase were used as the initial inoculum. The best recovery of stressed C. jejuni was observed in NB2 supplemented with FBP and blood and incubated aerobically. Enrichment in media incorporating FBP and 5% laked horse blood is a simple, convenient, and time-saving method to replace microaerophilic incubation methods for the resuscitation of C. jejuni.     

Survival of Campylobacter jejuni on various fresh produce

The survival of seven human and two chicken Campylobacter jejuni strains, with known Penner heat-stable (HS) serotypes and pulsed-field gel electrophoresis (PFGE) genotypes, was investigated on fresh-cut iceberg lettuce. In addition, the survival of four selected C. jejuni strains was assessed on cantaloupe pieces, cucumber slices, grated carrot and strawberries. Fresh produce was inoculated with 10(5) to 10(7) colony-forming units (CFU) of C. jejuni per gram, and the bacterium was enumerated using standard procedures after sample storage at 7 and 21 degrees C for 24, 48 and 72 h. The absolute values of the slopes (death rates) of the survival curves (log(10) CFU/g versus time) were calculated and compared. At 7 degrees C, the mean death rates (day(-1)) were 0.44 on cantaloupe, 0.41 on cucumber slices, 0.43 on grated carrot, 0.59 on iceberg lettuce and 1.02 on strawberries. The corresponding death rates (day(-1)) at 21 degrees C were 1.52, 1.55, 2.61, 1.39 and 8.74. The death rate of C. jejuni on strawberries (pH 3.4) was significantly (P<0.05) higher than on other produce. Moreover, the death rate at 21 degrees C as compared with 7 degrees C was significantly higher (P<0.05). Minor differences were observed in the survival of different C. jejuni strains. Our results suggest that after contamination of fresh produce, including strawberries, C. jejuni may survive sufficiently long to pose a risk to the consumer.     

Serotypes and typability of Campylobacter jejuni and Campylobacter coli isolated from poultry products

Campylobacter infection is one of the most common bacterial enteric pathogens. Campylobacter jejuni and Campylobacter coli infections are mostly food- and waterborne and especially poultry is often assumed to be an important source. The heat-stable serotyping system (the 'Penner' scheme) was used to study the serotype distribution of C. jejuni and C. coli isolated from different food products of poultry origin sampled from retail outlets in Denmark. A total of 156 isolates were serotyped, 85% of these were C. jejuni and 15% were C. coli. The most common C. jejuni serotypes were O:2 (30%), O:1,44 (12%) and the O:4-complex (8%). O:46 was the most frequent serotype among C. coli isolates. These serotypes are also common among Danish clinical isolates and isolates from broiler chickens and cattle. Differences in serotype distribution were seen for different kinds of poultry products. Isolates from chicken products covered a large selection of serotypes. In contrast, the majority of the isolates from other product groups (turkey, poussin, wild birds) were concentrated on 1-3 serotypes. Using the standard procedure for antigen preparation and serotyping, 25 of the 156 strains (16%) were nontypable. This rate of nontypable isolates is significantly higher than experienced for isolates from other sources than food products, i.e faecal samples from animals and humans. Subculturing and re-typing of the nontypable isolates improved the typability. After two, five and 10 subcultures 16, six and one isolate became typable, respectively. Only three isolates (2%) remained nontypable after 10 subcultures.     

Survival of Campylobacter jejuni in biofilms isolated from chicken houses

Campylobacter jejuni is a thermophilic and microaerophilic enteric pathogen associated with poultry. Biofilms may be a source of C. jejuni in poultry house water systems since they can protect constituent microorganisms from environmental stress. In this study, the viability of C. jejuni in biofilms of gram-positive chicken house isolates (P1, Y1, and W1) and a Pseudomonas sp. was determined using a cultural method (modified brucella agar) and direct viable count (DVC). Two-day biofilms grown on polyvinyl chloride (PVC) coupons in R2A broth at 12 and 23 degrees C were incubated with C. jejuni for a 6-h attachment period. Media were then refreshed every 24 h for 7 days to allow biofilm growth. Two-day biofilms of P1, Y1, and Pseudomonas spp. enhanced attachment (P < 0.01) of C. jejuni (4.74, 4.62, and 4.78 log cells/cm2, respectively) compared to W1 and controls without preexisting biofilm (4.31 and 4.22 log cells/cm2, respectively). On day 7, isolates P1 and Y1 and Pseudomonas biofilms covered 5.4, 7.0, and 21.5% of the surface, respectively, compared to 4.9% by W1. Viable C. jejuni on the surface decreased (P < 0.05) with time, with the greatest reduction occurring on surfaces without a preexisting biofilm. The number of viable C. jejuni determined by DVC was greater than that determined by the cultural method, indicating that C. jejuni may form a viable but nonculturable state within the biofilm. Both DVC and the cultural method indicate that biofilms enhance (P < 0.01) the survival of C. jejuni during incubation at 12 and 23 degrees C over a 7-day period.