Paenibacillus polymyxa purified bacteriocin to control Campylobacter jejuni in chickens

Campylobacter spp. cause numerous foodborne diseases. Poultry is thought to be a significant source of this zoonosis. Although many interventions designed to control this agent have been researched, none have succeeded. We evaluated a bacteriocin-based treatment to reduce Campylobacter jejuni colonization in poultry. A previously described purified bacteriocin (class IIa; molecular mass, 3,864 Da), secreted by Paenibacillus polymyxa NRRL-B-30509, was microencapsulated in polyvinylpyrrolidone, and 0.25 g of the purified bacteriocin was incorporated into 1 kg of chicken feed. One-day-old chickens were orally challenged and colonized with one of four isolates of C. jejuni, then reared in isolation facilities. Birds were provided ad libitum access to standard broiler starter feed and water for 7 days until 3 days before sampling, when only the treated groups of birds were provided the bacteriocin-emended feed described. In each of the eight (four by two replicates) trials, significant reductions in colonization by C. jejuni were observed (P < or = 0.05). As an example of this highly consistent data, in the first trial, 10 untreated 10-day-old chickens were colonized at a mean log 7.2 + 0.3 CFU/g of feces, whereas none of the 10 bacteriocin-treated 10-day-old chickens were colonized with detectable numbers of C. jejuni. Bacteriocin treatment dramatically reduced both intestinal levels and frequency of chicken colonization by C. jejuni. Feeding bacteriocins before poultry slaughter appears to provide control of C. jejuni to effectively reduce human exposure. This advance is directed toward on-farm control of pathogens, as opposed to the currently used chemical disinfection of contaminated carcasses.     

Potential competitive exclusion bacteria from poultry inhibitory to Campylobacter jejuni and Salmonella

The objective of this study was to isolate from chickens potential competitive exclusion bacteria (CE) that are inhibitory to Campylobacter jejuni or Salmonella, or to both, for subsequent development of a defined CE product for use in poultry. Adult chickens from family farms, commercial farms, and broiler chicken research centers were sampled to identify and select C. jejuni-free donor chickens. A challenge treatment, which included administering perorally 106 CFU C. jejuni per chicken and determining undetectable cecal shedding of campylobacters at 4 weeks, was important for identifying the best CE donor chickens. Screening of bacterial colonies obtained from nine donor chickens by using selective and nonselective media yielded 636 isolates inhibitory to six C. jejuni strains in vitro, with 194 isolates being strongly inhibitory. Of the 194 isolates, 145 were from ceca, and 117 were facultative anaerobic bacteria. One hundred forty-three isolates were inhibitory to six strains of Salmonella (including five different serotypes) in vitro. Of these, 41 were strongly inhibitory to all C. jejuni and Salmonella strains evaluated, and most were Lactobacillus salivarius. A direct overlay method, which involved directly applying soft agar on plates with discrete colonies from mucus scrapings of gastrointestinal tracts, was more effective in isolating CE than was the frequently practiced isolation method of picking and transferring discrete colonies and then overlaying them with soft agar. The best approach for obtaining bacteria highly inhibitory to Salmonella and C. jejuni from chickens was to isolate bacteria from ceca under anaerobic conditions. Free-range chickens from family farms were better donors of potential CE strongly inhibitory to both Salmonella and Campylobacter than were chickens from commercial farms and broiler chicken research centers.     

耐胃肠道环境芽孢杆菌菌株的筛选

以来源于健康动物肠道的芽孢杆菌为出发菌株,通过模拟体内高胆盐环境(胆盐含量0.3%),来监测菌株的耐受能力;测定在人工胃液、肠液环境中作用不同时间后的存活菌数的方法,来初步筛选能够作为益生菌的芽孢杆菌。实验结果表明,有3株芽孢杆菌分别为B5329、B22、B544,在胆酸盐、人工胃液、人工肠液中表现出了很强的耐受能力。     

Prevalence of potentially neuropathic Campylobacter jejuni strains on commercial broiler chicken products

Campylobacteriosis is the most common antecedent infection leading to the development of inflammatory neuropathies including Guillain Barré syndrome (GBS) and Miller Fisher syndrome (MFS), with alterations in surface proteins and genetic polymorphisms conferring increased risk. Poultry is the most common source of C. jejuni infection in industrialized countries, including the US. There are no data on the prevalence on consumer poultry products of various strains of C. jejuni, including those hypothesized to be associated with neuropathy. To study this, C. jejuni was isolated from fresh broiler chicken products purchased from grocery stores in the Baltimore area. LOS subtypes and specific genetic polymorphisms were determined by PCR and DNA sequencing. The observed relative proportions of LOS subtypes and genetic polymorphisms in the cstII gene (encoding bacterial sialyltransferases involved in LOS synthesis in C. jejuni) were characterized and compared to those reported in published studies of patients with GBS, MFS and uncomplicated enteritis. Commercial poultry products carry a relatively high prevalence of C. jejuni strains that have been associated with neuropathic sequelae. The relative proportions of LOS classes in poultry isolates were similar to those reported in isolates from human enteritis cases, and in some instances also similar to isolates from patients diagnosed with neuropathic disease. In terms of cstII polymorphisms, there were also similarities between isolates from poultry and those from patients with GBS and MFS.     

Isolation and polymerase chain reaction-based detection of Campylobacter jejuni and Campylobacter coli from poultry in the Philippines.

The polymerase chain reaction (PCR) and the conventional culture method of detecting thermophilic Campylobacter species in duck and chicken samples from two locations in the province of Laguna, Philippines, were compared. Three Campylobacter jejuni and five C. coli strains were isolated from a total of 135 duck and chicken samples from both methods. The PCR technique, however, was found to be more sensitive, accurate and rapid than the conventional culture method. The specificity of two sets of published primers, C442-C490 (specific for C. jejuni, C. coli and C. lari) and CL2-CR3 (specific for C. jejuni) were confirmed with reference and field strains. To improve detection, a lysate was prepared by boiling cells in Triton X-100, and then used as template for PCR to detect Campylobacter from spiked and naturally contaminated chicken rinse. For spiked chicken samples, a 17-h Meuller-Hinton Broth enrichment for the chicken rinse resulted in an improved sensitivity at 31.7 CFU/g using C442-C490. This enrichment-PCR tandem also detected thermophilic Campylobacter from 1 out of 21 native chicken samples from a wet market. To our knowledge, this is the first report of thermophilic Campylobacter isolation from poultry in the Philippines. The approaches described here could serve as a basis for future surveillance and/or epidemiological studies on this emerging foodborne pathogen.     

Morphological and physiological responses of Campylobacter jejuni to stress

Under conditions of stress, cells of Campylobacter assume a coccoid shape that may be an evolutionary strategy evolved by the organism to enable survival between hosts. However, the physiology of Campylobacter as it devolves from spiral to coccoid-shaped morphology is poorly understood. In this study, conditions influencing the survival of Campylobacter jejuni ATCC 35921 in broth were determined. Cells in late log phase were resuspended in broth at 4 or 60 degrees C. The culturability of these cold- or heat-stressed cell suspensions was determined by spread plate counts and the activity of cells by the direct viable count technique and 5-cyano-2,3-ditolyltetrazolium chloride staining. C. jejuni changed form completely from culturable to viable but nonculturable cells (VBNC) within 25 days at 4 degrees C, and 15 min at 60 degrees C. Light microscopy of C. jejuni VBNC cells showed that the spiral-shaped cells became coccoid, and transmission electron microscopy of C. jejuni VBNC cells showed that the outer membrane was lost in aging cell suspensions. Furthermore, a limited proteomic study was carried out to compare C. jejuni proteins that exhibited increased or decreased synthesis on exposure to 60 degrees C.     

Campylobacter fetus ssp. jejuni: Recovery Methodology and Isolation from Lamb Carcasses

Campylobacter fetus ssp. jejuni has increasingly been implicated as the causative agent in human cases of gastroenteritis. The first account of C. jejuni isolation from red meats—lamb carcasses—is reported. Recovery of Campylobacter from meat surfaces was tested as follows: selective agar plates consisted of tryptic soy agar, 5% lysed defibrinated horse blood, 10 mg/L vancomycin, 2500 units/L polymyxin B, 5 mg/L trimethoprim lactate, and 15 mg/L cephalothin (Keflin; VPTK: plates). These VPTK plates were incubated at 35°C for 72 hr in a gas atmosphere of 5% O₂, 10% CO₂, and 85% N₂. With these selective elements, recovery from meat surfaces inoculated with a calculated 32 Campylobacter cells per cm² was accomplished in 5 of 5 replicate tests. At levels of 3.2 and 0.3 cells of Campylobacter per cm², recoveries were accomplished in 2 of 5 replicate tests at each inoculum level.     

Resistance of Campylobacter jejuni to heat and to pulsed electric fields

The resistance of Campylobacter jejuni NCTC 11351 to heat and to pulsed electric fields (PEF) was studied at different treatment intensities (temperatures between 52 and 60 °C, and electric field strengths between 15 and 35 kV/cm, respectively). The influence of the growth phase, the pH of the treatment medium and the presence of sodium pyruvate in the recovery medium was also examined. A model based on the Weibull distribution was used to describe the inactivation curves, and times for the first decimal reduction were calculated (δ values). C. jejuni cells did not increase their resistance to heat nor to PEF upon entrance into stationary phase. The acidification of the treatment medium from 7.0 to 4.5 caused a sensitization of C. jejuni to heat (δ value at 55 °C × 1/4); on the contrary, resistance to PEF was increased (δ value at 25 kV/cm × 2.5). The absence of pyruvate in the recovery medium prevented recovery of a high percentage of heat-treated cells, but did not affect PEF survival. Whereas C. jejuni can be considered a heat sensitive organism (δ value at 55 °C and buffer of pH 7.0 of 2 min, z value 4.40 °C), it showed a relatively high resistance to PEF as compared to other vegetative cells (δ value at 25 kV/cm and buffer of pH 7.0 of 7 pulses, z_PEF value 8.20 kV/cm). Results obtained in this investigation indicate that Campylobacter spp. should be taken into account for the design of PEF treatments for food hygienization.Industrial relevanceBefore PEF can be commercially implemented it is necessary to determine its efficacy on pathogenic microorganisms of interest in order to ensure safety of food. There is no data available about the resistance of C. jejuni to pulsed electric fields, although it is now recognised as the leading cause of bacterial food-borne gastroenteritis throughout the world. In this research we characterize the resistance to heat and to PEF of C. jejuni NCTC 11351. Physiological factors affecting its survival to both agents are also explored.     

Isolation of Salmonella spp. from lettuce and evaluation of its susceptibility to novel bacteriocins of Bacillus thuringiensis and antibiotics

In this study, 13% of fresh lettuce (Lactuca sativa) samples collected from markets and supermarkets in two cities of Mexico were contaminated with Salmonella spp. From those samples, amplicons of ～300 base pairs (bp) were amplified, corresponding to the expected size of the invasion (invA) and internal transcribed spacer regions of the 16S and 23S rRNA genes of Salmonella spp. Additionally, Salmonella strains were isolated and harbored plasmids ranging from ～9 to 16 kbp. From these strains, 91% were resistant to ampicillin and nitrofurantoin, whereas 55% were resistant to cephalothin and chloramphenicol. No resistance was detected to amikacin, carbenicillin, cefotaxime, gentamicin, netilmicin, norfloxacin, and sulfamethoxazole-trimethoprim. When Salmonella isolates were tested against novel bacteriocins (morricin 269, kurstacin 287, kenyacin 404, entomocin 420, and tolworthcin 524) produced by five Mexican strains of Bacillus thuringiensis, 50% were susceptible to these antimicrobial peptides. This is the first report showing that Salmonella strains isolated from lettuce are susceptible to bacteriocins produced by the most important bioinsecticide worldwide, suggesting the potential use of these antibacterial peptides as therapeutic agents or food preservatives to reduce or destroy populations of Salmonella spp.     

Isolation and characterization of mutants of Propionibacterium strains

Procedures were developed to isolate and characterize mutants of strains of dairy propionibacteria. These procedures included the construction of minimal defined media to support growth of the strains, optimization of conditions of exposure of the strains to nitrosoguanidine, and identification of the phenotypes of the mutants that were generated. The minimal defined medium contained inorganic salts, adenosine, three vitamins, and sodium lactate as the carbon source, with cysteine, methionine, or cysteine plus methionine added as required by some strains. For mutagenesis, cells were exposed to either 100, 200, or 1000 micrograms/ml nitrosoguanidine, depending on the sensitivity of the strain, for 60 min at 35 degrees C. At least nine stable mutants were isolated and characterized for each of the five strains under study. The most frequent mutations generated were requirements for arginine, histidine, methionine, and uracil and alteration in pigment production.     

Isolation and characterization of benzene-tolerant Rhodococcus opacus strains

Twenty-two benzene-utilizing bacteria were isolated from soil samples. Among them, three isolates were highly tolerant to benzene. They grew on benzene when liquid benzene was added to the basal salt medium at 10--90% (v/v). Taxonomical analysis identified the benzene-tolerant isolates as Rhodococcus opacus. One of the benzene-tolerant isolates, designated B-4, could utilize many aromatic and aliphatic hydrocarbons including benzene, toluene, styrene, xylene, ethylbenzene, propylbenzene, n-octane and n-decane as sole sources of carbon and energy. Strain B-4 grew well in the presence of 10% (v/v) organic solvents that it was capable of using as growth substrates. Genetic analysis revealed the benzene dioxygenase pathway is involved in benzene catabolism in strain B-4. A deletion-insertion mutant defective in the benzene dioxygenase large and small subunits genes (bnz A 1 and bnz A 2) was as tolerant to organic solvents as the wild-type strain B-4, suggesting that utilization or degradation of organic solvents is not essential for the organic solvent tolerance of R. opacus B-4.     

Sensitivity of cold-stressed Campylobacter jejuni to solid and liquid selective environments

Either freezing (?20°C) or refrigeration (4°C) for 2 days inflicted sublethal injury among survivors of Campylobacter jejuni strains. The injured cells developed sensitivity to a mixture of antibiotics and probably to high incubation temperature (42°C). However, the degree of sensitivity when the injured survivors were exposed to these selective environments was greater in a liquid medium (≥99%) than in a solid medium (≤45%). Blood in the solid medium probably afforded some protection to injured survivors against the selective environments.     

多粘芽孢杆菌发酵培养基优化及发酵特性研究

采用正交实验方法,对多粘芽孢杆菌的发酵培养基进行优化,通过统计学分析,确定发酵培养基,并利用30L发酵罐进行实验,测定发酵过程中相关参数,确定生长代谢曲线。结果表明,发酵效价由优化前的8.6×104U/mL提高到12.9×104U/mL,提高近1.5倍。     

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.     

Occurrence and resistance to antibiotics of Campylobacter jejuni and Campylobacter coli in animals and meat in northeastern Italy

A study was carried out in northeastern Italy during 2000 and 2001 to investigate the occurrence of Campylobacter jejuni and Campylobacter coli in animals, cattle, pigs, and broilers, and raw meat, beef, pork, and chicken. Campylobacter spp. were detected in 53.9% of the cattle, 63.5% of the pigs, and 82.9% of the broilers examined. Chicken meat was frequently contaminated (81.3%), while lower rates were found in pork meat (10.3%) and beef (1.3%). The resistance to antibiotics of the strains was also investigated, and compared to that of human clinical isolates. C. coli was generally more resistant than C. jejuni. Resistance to quinolones was frequently observed in C. coli isolated in chicken meat (78.6%); slightly lower rates were found in C. jejuni isolated in broilers (42.2%), chicken meat (52.8%), and humans (38.2%). C. coli was also frequently resistant to tetracycline in all sources, while resistance to streptomycin was most frequently observed in pig isolates (89.4%).     

Amplified fragment length polymorphism, serotyping, and quinolone resistance of Campylobacter jejuni and Campylobacter coli strains from chicken-related samples and humans in Taiwan

The high-resolution genotyping method of amplified fragment length polymorphism (AFLP) analysis was used to study the genetic relationships between Campylobacter jejuni isolates from chicken-related samples (n = 32) and humans (n = 27) as well as between Campylobacter coli isolates from chicken-related samples (n = 27) and humans (n = 5). These isolates were collected between 1994 and 2003 in Taiwan. All C. jejuni and C. coli isolates showed highly heterogeneous fingerprints. C. jejuni isolates were separated in two distinct genetic clusters (A and B) at 40% genetic similarity and 42 different AFLP types at 90% similarity. However, three clusters at 40% genetic similarity and 33 different AFLP types at 90% similarity were observed in C. coli isolates. These results showed that AFLP analysis could be used to identify individual isolates of two Campylobacter species. Among C. jejuni isolates, the predominant AFLP type 1 was observed in five (7.9%) isolates, and types 5 and 12 in four (6.3%) isolates each. Cluster B consisted of 10 isolates, while the majority of isolates (n = 53) belonged to cluster A. In some AFLP types (1, 5, 12, 14 and 31), AFLP fingerprints of chicken-related isolates were closely related genetically to those of isolates from humans with gastroenteritis. The predominant serotypes in C. jejuni isolates were B:2 and Y:37. All isolates belonging to serotype O:19 grouped into one single AFLP type. Some chicken samples yielded multiple isolates of Campylobacter harboring simultaneously quinolone-resistant and quinolone-sensitive isolates attributed to the same species, or harboring C. jejuni and C. coli that have the characteristics of quinolone resistance.     

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。     

Isolation of Campylobacter jejuni and coliform bacilli from bile and liver obtained from slaughter cattle in Western Japan

A total of 290 bile samples from 143 Japanese Brown, 97 Japanese Black, and 50 Holstein cattle, and a total of 148 liver samples from 81 Japanese Brown, 49 Japanese Black, and 18 Holstein cattle were examined for the presence of Campylobacter jejuni by direct plating. The bile samples were also subjected to enumeration of coliform bacilli. Sixty-eight (23%) bile samples and 2 (1.4%) liver samples were positive for C. jejuni. A significantly higher isolation rate was observed from bile samples from Holstein (50%) than from Japanese Black (22%) and Japanese Brown (15%) cattle. C. jejuni was isolated from 52 of 232 bile samples that contained < 30 CFU/ml (under the detection threshold) of coliform bacilli. The presence of C. jejuni from bile was observed throughout the year. Fifty-four of the 68 bile isolates were serologically typed into eight groups. Serotypes O:4 complex (28 isolates) and O:2 (11 isolates), which were commonly isolated from human patients in Japan, accounted for 57% of the isolates. These observations suggest that bile can be a cause of contamination with C. jejuni even though it contains only a low number of coliforms.     

Antagonistic activities of several bacteria on in vitro growth of 10 strains of Campylobacter jejuni/coli

Chicken meat contaminated with Campylobacter jejuni can be the source of human enteritis. To decrease the risk of human infection, Campylobacter should be controlled at farm levels. Orally given probiotic bacteria could prevent colonisation of chicken with pathogenic bacteria like Campylobacter. The aim of this study was to investigate the effect of different bacteria on Campylobacter growth. Our results demonstrated that bacteria isolated from conventional chicken had potential inhibitory activities against Campylobacter. Other bacteria not isolated from chickens but with known antagonistic capacities, e.g. Enterococcus (56 strains) and Escherichia coli (20 strains), did not show any negative effect on Campylobacter. Interestingly, one Lactobacillus (P93) strain isolated from the chicken gut showed bactericidal activity against all tested Campylobacter. The bactericidal effect was characterised as the production of organic acids in combination with probably production of an anti-Campylobacter protein. In a co-culture study of Campylobacter and Lactobacillus (P93), the culturability of Campylobacter was under the detection limit after 48 h of incubation. A chicken experiment is needed to further evaluate the effect of the promising probiotic bacteria against Campylobacter colonisation in chicken.