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.     

Prevalence and characterization of Campylobacter jejuni isolated from pasture flock poultry

The growing interest in organic and natural foods warrants a greater need for information on the food safety of these products. In this study, samples were taken from 2 pasture flock farms (N = 178; feed, water, drag swabs, and insect traps), pasture flock retail carcasses (N = 48) and 1 pasture flock processing facility (N = 16) over a period of 8 mo. A total of 105 Campylobacter isolates were obtained from 53 (30%), 36 (75%), and 16 (100%) samples from the farms, retail carcasses, and processing facility, respectively. Of the 105 isolates collected, 65 were C. jejuni, 31 were C. coli, and 9 were other Campylobacter spp. Using PCR, the C. jejuni isolates were further analyzed for virulence genes involved in colonization and survival (flaA, flaC, cadF, dnaJ, racR, cbrR), invasion (virB11, ciaB, pldA), protection against harsh conditions (sodB, htrA, clpA), toxin production (cdtA, cdtB, cdtC), siderophore transport (ceuE), and ganglioside mimicry (wlaN). In addition, the short variable region of the flaA locus (flaA SVR) was sequenced to determine the genetic diversity of the C. jejuni isolates. The flaA SVR diversity indices increased along the farm to carcass continuum. PCR-based analysis indicated a low prevalence of 5 genes involved in colonization (dnaJ, ciaB, pldA, racR, virB11). The results of this survey indicate that the prevalence of Campylobacter on organic retail carcasses is similar to prevalence reports of Campylobacter on conventional retail carcasses. However, the genetic diversity of the flaA SVR genotypes increased along the farm to carcass continuum that contrasted with conventional poultry studies. PRACTICAL APPLICATION: Campylobacter jejuni is a leading cause of foodborne illness with poultry and poultry products being leading sources of infection. Free-range and pasture flock chickens are becoming more popular; however, there is an inherent biosecurity risk that can increase the prevalence of foodborne pathogens in these flocks. This study aimed to determine sources and characterize C. jejuni isolated from pasture flocks.     

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.     

海洋多黏类芽孢杆菌L1-9 菌株抗菌蛋白的分离纯化及其抗菌作用

通过硫酸铵分级沉淀、DEAE Sepharose Fast Flow 柱层析、Sephadex G-200 柱层析,以小麦蠕孢病菌和金黄色葡萄球菌为指示菌采用抑菌活性追踪和SDS-PAGE 跟踪检测,从分离自连云港海域、对多种病原真菌具有抑菌作用的多黏类芽孢杆菌L1-9 菌株发酵液中分离纯化得到一种对金黄色葡萄球菌和小麦蠕孢病菌的菌丝生长及孢子萌发具有抑制作用的抗菌蛋白,分子质量约31kD。     

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.     

Antimicrobial edible apple films inactivate antibiotic resistant and susceptible Campylobacter jejuni strains on chicken breast

Abstract: Campylobacter jejuni is the leading cause of bacterial diarrheal illness worldwide. Many strains are now becoming multidrug resistant. Apple‐based edible films containing carvacrol and cinnamaldehyde were evaluated for bactericidal activity against antibiotic resistant and susceptible C. jejuni strains on chicken. Retail chicken breast samples inoculated with D28a and H2a (resistant strains) and A24a (a sensitive strain) were wrapped in apple films containing cinnamaldehyde or carvacrol at 0.5%, 1.5%, and 3% concentrations, and then incubated at 4 or 23 °C for 72 h. Immediately after wrapping and at 72 h, samples were plated for enumeration of viable C. jejuni. The antimicrobial films exhibited dose‐ and temperature‐dependent bactericidal activity against all strains. Films with ≥1.5% cinnamaldehyde reduced populations of all strains to below detection at 23 °C at 72 h. At 4 °C with cinnamaldehyde, reductions were variable for all strains, ranging from 0.2 to 2.5 logs and 1.8 to 6.0 logs at 1.5% and 3.0%, respectively. Films with 3% carvacrol reduced populations of A24a and H2a to below detection, and D28a by 2.4 logs at 23 °C and 72 h. A 0.5‐log reduction was observed for both A24a and D28a, and 0.9 logs for H2a at 4 °C at 3% carvacrol. Reductions ranged from 1.1 to 1.9 logs and 0.4 to 1.2 logs with 1.5% and 0.5% carvacrol at 23 °C, respectively. The films with cinnamaldehyde were more effective than carvacrol films. Reductions at 23 °C were greater than those at 4 °C. Our results showed that antimicrobial apple films have the potential to reduce C. jejuni on chicken and therefore, the risk of campylobacteriosis. Possible mechanisms of antimicrobial effects are discussed. Practical Application: Apple antimicrobial films could potentially be used in retail food packaging to reduce C. jejuni commonly present on food.     

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.     

Cu~(2+)对多粘类芽孢杆菌增殖及其转化人参皂苷的影响

目的:研究Cu~(2+)对多粘类芽孢杆菌增殖及转化人参皂苷的影响。方法:采用酶标仪和高效液相色谱仪分别测定加入Cu~(2+)后多粘类芽孢杆菌的增殖及转化人参皂苷情况。结果:Cu~(2+)促进多粘类芽孢杆菌增殖适宜质量浓度为10~50 mg/L,Cu~(2+)质量浓度为30 mg/L时促进多粘类芽孢杆菌增殖效果最好,增殖时间最短,与对照组相比提前1 d,生物量显著增加;转化体系中Cu~(2+)质量浓度为10~50 mg/L时,促进多粘类芽孢杆菌转化人参皂苷,Cu~(2+)质量浓度为30 mg/L时,多粘类芽孢杆菌转化人参皂苷能力最强;但Cu~(2+)质量浓度超过100 mg/L时反而抑制多粘类芽孢杆菌转化人参皂苷,并且随Cu~(2+)质量浓度增大而抑制作用增强。结论:Cu~(2+)质量浓度为30 mg/L时,可促进多粘类芽孢杆菌增殖及其转化人参皂苷。     

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.     

Bacillus and Paenibacillus species associated with extended shelf life milk during processing and storage

Characterisation of spore formers associated with extended shelf life milk was performed by analysing the bacteriological quality of milk samples collected at various processing stages and during storage. Isolates were identified with MALDI‐TOF‐MS. Milk had spore counts <2 log₁₀ cfu/mL and 4 log₁₀ cfu/mL during processing and storage, respectively. Bacillus pumilus dominated the bacterial population. Bacterial species were inoculated into sterile milk for a shelf life study, and the population change was observed over 42 days at 7 °C. Although the extended shelf life milk process was effective in reducing bacterial counts and species diversity, the presence of Bacillus cereus shows a potential safety problem in extended shelf life milk.     

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.     

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.     

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。