Heat stress adaptation induces cross-protection against lethal acid stress conditions in Arcobacter butzleri but not in Campylobacter jejuni

The ability of many bacteria to adapt to stressful conditions may later protect them against the same type of stress (specific adaptive response) or different types of stresses (multiple adaptive response, also termed cross-protection). Arcobacter butzleri and Campylobacter jejuni are close phylogenetic relatives that occur in many foods of animal origin and have been linked with human illness (mainly diarrhoea). In the present study, sublethal stress adaptation temperatures (48 °C and 10 °C) and mild and lethal acid conditions (pH 5.0 and pH 4.0) were determined for A. butzleri and C. jejuni. In addition, it was evaluated whether these sublethal stress adaptations cause specific adaptive responses or cross-protection against subsequent mild or lethal acid stresses in these bacteria. The studies were conducted in broth adjusted to the different conditions and the results were determined by the dilution series plating method. It was shown that heat stress adapted A. butzleri (incubated for 2 h at 48 °C) were significantly more resistant to subsequent lethal acid stress (pH 4.0) than non-adapted cells at the 1 h time-point (p < 0.01 in Wilcoxon rank sum test). No specific adaptive responses against the stresses in A. butzleri or C. jejuni and no cross-protection in C. jejuni were found. The ability of heat stressed A. butzleri to tolerate later lethal acid conditions should be taken into account when designing new food decontamination and processing strategies.     

Biofilm formation by Campylobacter jejuni in controlled mixed-microbial populations

This study was to screen the ability of biofilm formation by Campylobacter jejuni strains found in New Zealand, and investigate the biofilm growth of C. jejuni in a controlled mixed-microbial population that includes five different bacteria. The ability of C. jejuni to form a biofilm in monoculture and mixed-microbial populations was measured in a laboratory assay using a microtiter plate screening assay. The optical density of the biofilm and cell growth from mixed-microbial populations was converted to a Biofilm Formation Index (BFI). This index was used to standardize the biofilm formation in the mixed-microbial populations. High BFI was observed for Enterococcus faecalis (2.30) and Staphylococcus simulans (3.75) when they were grown with C. jejuni multilocus sequence type ST-474: a dominant poultry and human-associated type in New Zealand. C. jejuni cells were recovered from most of the biofilms containing E. faecalis and/or S. simulans. These results suggest that E. faecalis and S. simulans may play a role in biofilm formation in the poultry environment as both of these microorganisms are found in poultry processing environments and were able to form a biofilm in association with C. jejuni under microaerobic conditions. Understanding the relationships among C. jejuni, E. faecalis and S. simulans in poultry processing plants and farms may help in the design of strategies to reduce the reservoir of contamination of these bacteria and reduce the incidence of campylobacteriosis.     

Quantitative analysis of viable,stressed and dead cells of Campylobacter jejuni strain 81-176

Campylobacter jejuni is an important foodborne gastrointestinal pathogen and highly sensitive to environmental stresses. Research has shown that changes in culturability, cell morphology, and viability occur when C. jejuni cells are subjected to stresses. In this study, real-time PCR, ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR), BacLight bacterial viability staining, and agar plate counting methods were used to quantitatively analyze viable, stressed, and dead C. jejuni strain 81-176. The real-time PCR assay provides highly sensitive and specific quantification of total genome copies of C. jejuni culture in different growth phases. Our results also reveal that real-time PCR can be used for direct quantification of Campylobacter genome release into Phosphate Buffered Saline (PBS) as an indicator of cell lysis. Using EMA-PCR, we obtained a dynamic range of greater than 3 logs for differentiating viable vs. dead cells. The viability and morphological characteristics of the stressed cells after one-week incubation at 25 °C, in air, and under nutrient-poor conditions were investigated. Our results indicated that, over 99% of the stressed cells were converted from the spiral to the coccoid form and became non-culturable. However, more than 96% of the coccoid cells retained their membrane integrity as suggested by both the BacLight staining and EMA-PCR analyses. Thus, to detect C. jejuni under stress conditions, conventional culturing method in conjunction with EMA-PCR or BacLight staining might be a more appropriate approach.     

Development and evaluation of aptamer magnetic capture assay in conjunction with real-time PCR for detection of Campylobacter jejuni

A prototype method for the concentration and detection of Campylobacter jejuni was developed using a previously reported biotinylated DNA aptamer in conjunction with qPCR. The so-called aptamer-based magnetic capture-qPCR (AMC-qPCR) assay was compared to a similar immunomagnetic separation (IMS)-qPCR assay. In small volume experiments (300 μl) applied to serially diluted C. jejuni suspended in buffer containing a mixed culture of other common food borne pathogens, the lower detection limit of the AMC-qPCR method was 1.1 log₁₀/300 μl C. jejuni cells, one log₁₀ better (lower) than that of IMS-qPCR (2.1 log₁₀ CFU/300 μl). AMC-qPCR capture efficiency was 10–13% at assay detection limit. In 10 ml scale-up experiments, the lower detection limit of AMC-qPCR was 2.0 log₁₀ CFU/10 ml with corresponding capture efficiency of 4–7%. Nucleic acid aptamers are promising alternatives to antibodies for magnetic bead-based capture followed by qPCR detection.     

The survival of Campylobacter jejuni in red meats stored at different temperatures

The survival of 4 strains of Campylobacter jejuni was studied in raw minced beef and raw pork sausage mixture stored in plastic stomacher bags at freezer temperatures (−19°C) for up to 10 weeks, refrigirator temperatures (< 10°C) for 6 days and 22°C for 24 h. At each of the 3 storage temperatures survival was better in minced beef. Similarly, there was less variation in percentage survival between the 4 strains in minced beef than in sausage mixture after storage at each temperature. Detailed studies were carried out with one strain of C. jejuni. Viable counts were relatively unchanged in minced beef at refrigerator temperatures and 22°C, but showed a decrease in corresponding samples of sausage mixture. At freezer temperatures decreases in count of approximately 1 log unit were observed during the first week for both meats followed by a more gradual decrease. The effect of desiccation by exposure was studied in minced beef and lamb outer carcass meat (breast) at refrigerator temperatures (≤ 10°C). Decreases in viable count were observed in lamb carcass meat after 32 h although large variations were sometimes observed between duplicate samples for the same strain of C. jejuni. Counts were unchanged in exposed minced beef after storage for 48 h.     

Model of inactivation of Campylobacter jejuni in poultry scalding

The purpose of this study was to develop a model of inactivation of Campylobacter jejuni in industrial scalding of chickens. Models can be used as a guide for broiler slaughterhouse operations for reducing levels of C. jejuni contamination on broiler carcasses. Mean concentrations of C. jejuni in terms of colony forming units (CFU) in scald tank water and in carcass rinse solution after scalding were 2.90 ± 0.07 and 3.86 ± 0.11 LogCFU/mL, respectively. Scald tank water temperature, flow rate, pH, and total solids in scalding process water were 54.15 ± 0.2 °C, 172.0 ± 8.4 L/min, 8.0 ± 0.01, and 2565 ± 114.3 mg/L, respectively. Inactivation models were developed by using mass balances and literature data for inactivation kinetics, of Campylobacter and the Arrhenius equation. Results of the inactivation models of scalding process indicate that high temperature and short time (less than 2 min) of scalding process were effective in reducing the number of viable cells. For this experimental data more than 50% of the Campylobacter are inactivated on surfaces of the chickens. The model fits the experimental data well and the values of the estimated parameters provide insight for this process. The model can be used for process design and potential process modifications.     

Survival of Campylobacter jejuni on beef and pork under vacuum packaged and retail storage conditions: examination of the role of natural meat microflora on C. jejuni survival

The ability of Campylobacter jejuni ATCC 11168 to survive on beef and pork stored under chilled, vacuum packaged and retail display conditions were examined. In addition, the effect of natural microflora on commercial beef and pork on the survival of C. jejuni under these storage conditions was examined. When sterile cores of beef and pork were inoculated with ∼10⁵ to 10⁶ cfu cm⁻²C. jejuni, and were stored under aerobic or vacuum packaged conditions at −1.5 or 4 °C, its numbers dropped significantly and C. jejuni could not be enumerated by direct plating after 21 d of the 6 wks study. In contrast, survival of C. jejuni on commercial vacuum packaged beef and pork was significantly enhanced, resulting in only 1 log cfu cm⁻² reduction at the end of 6 wks. During 7 d of display in a retail case, numbers of C. jejuni dropped quickly, but could be enumerated by direct plating even after the 7 d. The presence of high numbers of inoculated C. jejuni on beef and pork had no significant effect on the natural microflora numbers compared to uninoculated controls when the meat was stored either in vacuum or in a retail display case. These results show that natural microflora on vacuum packaged meat afford enhanced survival of C. jejuni present on the surfaces of both beef and pork when stored at refrigeration temperatures. Hence, strict hygienic practices or the implementation of decontamination technologies are recommended to ensure safety of meat with respect to this pathogen.     

A “successful allele” at Campylobacter jejuni contingency locus Cj0170 regulates motility; “successful alleles” at locus Cj0045 are strongly associated with mouse colonization

Campylobacter jejuni is an important foodborne pathogen of humans and its primary reservoir is the gastrointestinal (GI) tract of chickens. Our previous studies demonstrated that phase variation to specific “successful alleles” at C. jejuni contingency loci Cj0045 (successful alleles carry 9G or 10G homopolymeric tracts) and Cj0170 (successful allele carries a 10G homopolymeric tract) in C. jejuni populations is strongly associated with colonization and enteritis in C57BL/6 IL-10 deficient mice. In the current study, we strengthened the association between locus Cj0170, Cj0045, and mouse colonization. We generated 8 independent strains derived from C. jejuni 11168 strain KanR4 that carried a Cj0170 gene disruption and these were all non motile. Two randomly chosen strains with the Cj0170 gene disruption (DM0170-2 and DM0170-6) were gavaged into mice. DM0170-2 and DM0170-6 failed to colonize mice while the control strain that carried a “successful” Cj0170 10G allele was motile and did colonize mice. In parallel studies, when we inoculated C. jejuni strain 33292 into mice, the “unsuccessful” Cj0045 11G allele experienced phase variation to “successful” 9G and 10G alleles in 2 independent experiments prior to d4 post inoculation in mice while the “successful” 9G allele in the control strain remained stable through d21 post inoculation or shifted to other successful alleles. These data confirm that locus Cj0170 regulates motility in C. jejuni strain KanR4 and is a virulence factor in the mouse model. The data also support a possible role of locus Cj0045 as a virulence factor in strain 33292 in infection of mice.     

Survival of Vibrio parahaemolyticus under environmental stresses as influenced by growth phase and pre-adaptation treatment

In this study, the susceptibility of Vibrio parahaemolyticus in different growth phases after exposure to lethal stresses including 47 °C and 8% ethanol was first investigated. The effect of a culture's growth phase on both the heat and ethanol shock response of V. parahaemolyticus was then examined. It was found that cells of V. parahaemolyticus in the mid-exponential phase, regardless of adaptation, were most susceptible to environmental stresses, while cells in the stationary phase were least susceptible to the lethal stresses examined. Adaptation with heat shock at 42 °C for 45 min or ethanol shock with 5% ethanol for 60 min induced an increased resistance of V. parahaemolyticus to subsequent lethal stresses at 47 °C and 8% ethanol. While the adaptation treatments resulted in a reduced resistance of the test organism to pH 4.4 and 20% NaCl. Generally, the extent of changes in the resistance of V. parahaemolyticus to lethal stresses between the adapted and control cells was found to be growth phase dependent. Compared with the respective control cells, the adapted late-exponential phase cells exhibited the greatest extent of change, while the adapted stationary phase cells showed the least change in their resistance to the lethal stresses examined.     

Campylobacter jejuni and Campylobacter coli as surface contaminants of fresh and frozen poultry carcasses

Campylobacter jejuni and Campylobacter coli were isolated as surface contaminants from 216 (31.3%) of 691 freshly eviscerated poultry carcasses which were examined just prior to packaging in a highly automatized poultry processing plant. The highest overall contamination rate was recorded among turkey carcasses (56.7%), followed by hens (48.2%), and broiler chickens (13.8%). A total of 245 carcasses were re-examined after they had been processed in the plant and kept frozen (−25°C) for 1 to 15 weeks. The results indicate that campylobacters may survive frozen storage for at least four weeks. Selective enrichment proved essential for isolation from frozen carcasses; the procedure employed enabled a 108.3% increase in isolation rate, compared with direct plating. Colistin-amphotericin-keflin agar was superior to Skirrow's agar for isolation from poultry cascasses. The most common biotype was C. jejuni biotype 1 (82.0%), followed by C. coli (16.9%), and C jejuni biotype 2 (1.1%). It was possible to serotype 129 (72.9%) of 177 campylobacters by means of heat-stable antigens. The most common serotype was LAU 2 which comprised 21.5% of all isolates, followed by LAU 1 with 13.6%. Six additional serotypes occurred regularly (3.4 to 6.2%), while nine serotypes were rare. The serotypes found to be most prevalent in poultry (LAU 1 and LAU 2) were the same ones as recovered most frequently from Norwegian patients.     

一起空肠弯曲菌引起食源性疾病事件的病原学研究及溯源分析

目的 对一起食源性疾病事件进行病原菌检测,了解病原菌毒力基因携带情况并进行溯源分析。方法 对事件采集的样本经FilmArray多重PCR系统进行快速初筛,同时进行细菌分离培养鉴定。使用PCR检测技术对分离菌株进行毒力基因检测,采用16S rRNA基因序列分析与PFGE分型方法对分离菌株进行同源性分析。结果 2份患者肛拭子样本和4份食堂厨工肛拭子样本检出空肠弯曲菌,检出菌株均携带flaA、cadF、imaA、cdtA、cdtB、cdtC等毒力基因。16S rRNA基因序列分析表明6株分离菌株均为空肠弯曲菌,1株菌株与其他5株菌株分子发育距离稍远。6株菌株经PFGE分型可分为3种带型,3株菌和2株菌分别呈现同一带型,2种带型相似性为52.2%;另1株菌为另一带型,与其他菌株带型相似性仅为26.7%。结论 实验室结果表明这是一起由不同克隆株的空肠弯曲菌感染引起的食源性疾病事件。     

Effect of crust freezing applied alone and in combination with ultraviolet light on the survival of Campylobacter on raw chicken

The application of crust freezing (CF) applied as a stand-alone treatment or in combination with ultraviolet (UV) light for reducing the level of artificially inoculated Campylobacter jejuni on raw chicken was investigated. CF air temperatures of −5, −15 and −27 °C (±3 °C) with freezing times of 70, 15 and 6 min, respectively, were used. The level of C. jejuni on chicken was also examined following subsequent refrigerated (0–4 °C) storage at 3 and 7 days. All CF treatments resulted in significant reductions compared to untreated controls (P < 0.05). Although combining CF with UV also resulted in significant reductions for C. jejuni, the combined treatments were generally no more effective than treatment by CF alone. Overall, the color of chicken drumsticks was not affected by CF treatments (P ≥ 0.05). In general, CF resulted in increased drip loss (P < 0.05), which increased over storage time and was greater at higher CF temperatures. The current study indicates that CF has potential for reducing the levels of C. jejuni by between 0.5 and 1.5 log₁₀ CFU/g and impacts minimally on the color of treated skin.     

Survival of Pseudomonas fluorescens on beef carcass surfaces in a commercial abattoir

The influence of a commercial chilling process (18 h at 10 °C followed by up to 78 h at 2 °C) on Pseudomonas fluorescens inoculated on beef carcass surfaces at four sites, neck (NE), outside round (OR), brisket (BR) and foreshank/brisket (FB) before chilling (“hot inoculated”) or after chilling for 24 h (“cold inoculated”) was investigated. Pseudomonas counts increased significantly at all sites on “hot inoculated” carcasses during storage, but on “cold inoculated” carcasses, counts declined or remained unchanged. On hot and cold inoculated carcasses, differences in Pseudomonas growth or survival were demonstrated between sites. No clear relationships were observed between Pseudomonas growth or survival and chiller relative humidity (RH) or surface water activity (a_w) at the different sites. These results were unexpected, and are discussed in relation to environmental factors that affect the growth/survival of P. fluorescens on carcass surfaces during chilling i.e. temperature, RH, and the relationship of these parameters to surface water activity (a_w).     

Survival of human derived Lactobacillus plantarum in fermented cereal extracts during refrigerated storage

The aim of this study was to investigate the survival of a potentially probiotic Lactobacillus plantarum strain in barley, wheat and barley malt extracts. The extracts were produced from three flour/water suspensions, i.e., 5%, 20%, 30% w/w. After inoculation, the cultures were incubated for 24 h at 37 °C, and were subsequently stored at 4 °C for up to seventy days. The lactic acid and reducing sugar concentrations at the beginning of storage were significantly different between the fermented media, ranging from 0.5 g/L to 17 g/L and from 0.8 g/L to 6.5 g/L respectively, while the pH ranged between 2.9 and 3.4. It was observed that the cells survived much better in the malt extracts compared to barley and wheat extracts during refrigerated storage. Based on the results from a study using model media and supplemented cereal extracts it was derived that this was most likely due to their higher sugar concentration and the presence of protective unidentified compounds, albeit the fact that the malt extracts contained higher amounts of lactic acid.     

Synthesis of cyclopropane fatty acids in Lactobacillus helveticus and Lactobacillus sanfranciscensis and their cellular fatty acids changes following short term acid and cold stresses

An implemented GC method to separate and quantify the cell cyclopropane fatty acids lactobacillic (C19cyc11) and dehydrosterculic (C19cyc9) was used to study the adaptive response to sublethal acid and cold stresses in Lactobacillus helveticus and Lactobacillus sanfranciscensis. The comparison of the composition of cellular fatty acids of the two strains and their changes after 2 h of stress exposure under micro-aerobic and anaerobic conditions indicated that the aerobic biosynthetic pathway for unsaturated fatty acids is prevalent in L. sanfranciscensis, while the anaerobic pathway is prevalent in L. helveticus. Indeed in the latter strain, in the presence of a source of oleic acid and under micro-aerobic conditions, C18:1n11 and its post-synthetic derivative C19cyc11 accounted for overall proportion ranging from 52 to 28% of the total FAs. On the other hand L. sanfranciscensis synthesizes by aerobic pathway C18:1n9 and transforms it to C19cyc9. However in this species the cumulative level of these two FAs did not exceed 30%. The relevant proportion of dodecanoic acid in the latter species suggests that carbon chain shortening is the principal strategy of L. sanfranciscensis to modulate fluidity or chemico-physical properties of the membranes.     

High prevalence,low counts and uncommon serotypes of Listeria monocytogenes in linguiça, a Brazilian fresh pork sausage

Linguiça is a highly popular and appreciated pork product in Brazil, frequently consumed undercooked. Aiming at collection of data for a future risk assessment, this study evaluated the prevalence and counts of Listeria monocytogenes in linguiça samples collected at retail level in Sao Paulo, SP, Brazil. ISO methods were used for detection and enumeration of the pathogen (11290-1 and 11290-2, respectively). Isolates were submitted to Simplex-PCR for hlyA gene and those with biochemical features of L. monocytogenes and hlyA positive were serotyped using a Multiplex PCR. Ninety percent of the samples were positive for Listeria spp., and L. monocytogenes was detected in 42% of the samples, with counts below 10² CFU/g in all samples. A prevalence of uncommon serotypes 4a and 4c was observed.     

Survival and reproduction of Tribolium castaneum (Herbst), Rhyzopertha dominica (F.) and Sitophilus oryzae (L.) following periods of starvation

This study determined the starvation tolerance of Tribolium castaneum (Herbst), Rhyzopertha dominica (F.) and Sitophilus oryzae (L.) in terms of both adult survival and reproduction, the impact of starvation on reproduction not having been studied before. Experiments were conducted at 30 °C and 55% or 70% r.h. using a laboratory strain and a field strain of each species. The number of progeny was a better indicator of the impact of starvation on a species than adult survival. Tribolium castaneum was the most tolerant species, requiring up to 35 d starvation before no progeny were produced. Rhyzopertha dominica and S. oryzae required up to 8 d starvation before no progeny were produced. The results suggest that hygiene will have a greater impact on populations of S. oryzae and R. dominica than T. castaneum.