Expression and characterization of cell-signalling molecules in Campylobacter jejuni.

AIMS: This study investigated the production and effects of cell-signalling compounds on selected survival and virulence mechanisms of Campylobacter jejuni. METHODS AND RESULTS: The production of Autoinducer 1 (AI-1) compounds by Camp. jejuni was investigated in-vitro using a variety of available AI-1 bioassays. We further examined the role of a range of commercially available homoserine lactones (HSL) and a novel compound (cjA) isolated from Camp. jejuni. The selected attributes included the transformation to a viable but nonculturable (VBNC) state, biofilm formation, interleukin 8 (IL-8) stimulation in INT-407 cells and virulence gene expression as determined by qRT-PCR. This study is the first to report an HSL or HSL mimic produced by Camp. jejuni. Short chained HSLs and the novel compound cjA prolonged the delay to a VBNC state as well as inhibiting biofilm formation and the majority of HSLs examined and the HSL mimic cjA significantly affected virulence gene expression as well as increasing the production of IL-8 in challenged INT-407 cells. CONCLUSIONS: Despite the lack of a homologous HSL kinase or sensor, Camp. jejuni appears to produce, as well as detect, exogenous signalling molecules and respond accordingly to aid in the survival and virulence capabilities of this micro-organism. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that Camp. jejuni is able to detect and utilize as well as possibly produce cell-signalling molecules that enhance both survival and virulence attributes. This possibility opens a new field in the search for Camp. jejuni reduction and elimination strategies.

Combined antimicrobial effect of nisin and a listeriophage against Listeria monocytogenes in broth but not in buffer or on raw beef.

The effect of nisin and listeriophage LH7, alone and in combination, on the growth and survival of two strains of Listeria monocytogenes in broth and two model food systems, with appropriate controls, was determined. Growth curves for both bacterial strains in tryptic soy broth incubated at 7 or 30 degrees C, and with the addition of nisin and/or listeriophage at lag, mid-exponential or early stationary phase, were obtained by measuring absorbance at 550 nm. Numbers of mixed populations of both L. monocytogenes strains in phosphate buffered saline (pH 5.5) and on vacuum-packaged fresh beef, both stored for 4 weeks at 4 degrees C, and with the addition of nisin and/or listeriophage, were determined. This was achieved by plating appropriately diluted samples on both Tryptic Soy Agar and Modified Oxford Agar to determine both L. monocytogenes numbers and the presence of sub-lethal injury. In broth nisin alone, reduced levels or prevented growth of the two strains under the conditions studied, but regrowth to levels equivalent to those of untreated cells, occurred. Listeriophage LH7 alone, on the other hand, had no effect in broth under the conditions studied. Notably, however, a mixture of nisin and listeriophage displayed a combined effect in broth and reduced levels of cells substantially without regrowth under the conditions studied. In both model food systems only nisin appeared to be active, in a manner consistent with existing literature, and no combined action was apparent. The use of nisin and listeriophage has potential to control L. monocytogenes in foods but a further understanding of the interactions in this complex system needs to be achieved before it could be applied practically.

Survival of Campylobacter jejuni on beef trimmings during freezing and frozen storage.

AIMS: To investigate the survival of two animal isolates of Campylobacter jejuni on beef trimmings during freezing and frozen storage. METHODS AND RESULTS: Meat packs inoculated with 10(3) or 10(6) cfu g(-1) of either strain of C. jejuni were frozen to -18 degrees C, and sampled at regular intervals over 112 d storage to determine Campylobacter numbers and sublethal injury. For both strains and inoculation levels the numbers of Campylobacter decreased in the first 7 d of storage by ca. 0.6-2.2 log cfu g(-1) and then remaining constant over the remainder of the storage trial, with neither isolate exhibiting sublethal injury. CONCLUSIONS: Despite an initially significant decrease in number, these pathogens were able to survive standard freezing conditions in meat, but did not exhibit sublethal injury. SIGNIFICANCE AND IMPACT OF THE STUDY: Strict hygiene and/or the implementation of decontamination technologies are recommended as a means to assure the safety of meat with respect to this pathogen.

Survival of Escherichia coli O157:H7 and Salmonella on chill-stored vacuum or carbon dioxide packaged primal beef cuts.

The ability of two Escherichia coli O157:H7 strains (E27, a cattle isolate, and B6-914 gfp-91, a fluorescent marker strain) and two Salmonella serotypes (S. typhimurium and S. brandenberg) to survive on chilled preservatively packaged primal beef cuts was examined. Each of the strains was inoculated separately at two dilution levels (10(3) and 10(5) cfu g(-1)) onto 500 g beef steaks, packaged under vacuum or 100% carbon dioxide, and stored, with uninoculated controls, for 6 weeks at - 1.5 degrees C, then for 2 weeks at 4 degrees C. Bacterial numbers were determined by dilution and incubation at 37 degrees C for 24 h on either Sorbitol McConkey Agar or Xylose Lysine Desoxycholate Agar for E. coli O157:H7 and Salmonella samples, respectively. Counts were corrected for background growth and their accuracy checked using immunological tests. Fluorescent E. coli O157:H7 B6-914 gfp-91 was also counted under ultra-violet light. No significant changes in numbers of the E. coli O157:H7 or Salmonella strains occurred during storage at either - 1.5 or 4 degrees C packaged under either vacuum or carbon dioxide. The ability of these pathogens to survive standard preservative packaging conditions is different from that reported from their generic counterparts and therefore a cause for public health concern.

The role of L-carnitine and glycine betaine in the survival and sub-lethal injury of non-growing Listeria monocytogenes cells during chilled storage.

AIMS: To determine the role played by previous growth in the presence of osmolytes on the subsequent survival and sub-lethal injury of L. monocytogenes during long-term chilled storage in a model buffer system. METHODS AND RESULTS: Four Listeria monocytogenes strains were grown separately to stationary phase in Listeria minimal medium (DM) alone or in DM with 4% NaCl alone, or both these media supplemented with 1 mM L-carnitine and/or 1 mM glycine betaine. Cells were resuspended in phosphate buffered saline (pH 5.5) and stored for four weeks at 4 degrees C. Initially, and at weekly intervals, samples were plated on both Tryptic Soy Agar and Tryptic Soy Agar with 4% NaCl to determine total numbers and degree of sub-lethal injury in the populations. The numbers of cells within all strains after growth to stationary phase, except one which increased ( approximately 2 log cfu ml-1, P < 0.05) in the presence of NaCl, were not influenced significantly by previous growth conditions (P > 0.05). During subsequent chilled storage, however, numbers of all strains grown in the presence of NaCl remained constant while those grown in its absence decreased. The rate and magnitude of the decrease in cell numbers was strain dependent. The initial percentage of sub-lethal injury increased significantly in all strains when grown previously in the presence of L-carnitine (P < 0.05). During subsequent chilled storage sub-lethal injury increased for all strains in a manner that was strain dependent, but not related to the previous growth conditions. CONCLUSION: Previous growth in the presence of osmolytes of NaCl, but not osmolytes alone, increases the subsequent survival, but not percentage sub-lethal injury, of L. monocytogenes during subsequent chilled storage in buffer. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that risks associated with L. monocytogenes in chilled food may be influenced by the individual life histories of the cells.

Survival of osmotic and acid stress by Listeria monocytogenes strains of clinical or meat origin.

The ability of 30 Listeria monocytogenes strains, 15 of meat origin and 15 of clinical origin, to use carnitine as an osmoprotectant and to resist acid stress was determined. All strains examined were able to use carnitine as an osmoprotectant, indicating the importance of this characteristic to the survival of L. monocytogenes in natural environments. Clinical and meat strains, however, differed with respect to this characteristic. Specifically, 73% of meat strains reached a lower maximum cell density in the presence of carnitine with osmotic stress than in its absence with no stress. Only 33% of clinical strains displayed the same feature whereas the remaining clinical strains reached a higher maximum cell density in the presence of carnitine with osmotic stress than in its absence with no stress. The physiological reasons and advantage of this difference are unclear. When exposed to conditions of severe acid stress (pH 2.5) for 2 h, only two L. monocytogenes strains (L66 and L78), both of meat origin, displayed significant reductions (P < 0.05) in number (3.51 and 2.79 log cfu, respectively). Acid-sensitive strains were not found among the clinical isolates examined, highlighting the importance of acid stress resistance in the infection process.

Psychrotrophic clostridia mediated gas and botulinal toxin production in vacuum-packed chilled meat.

A cocktail of washed spores from six psychrotrophic Clostridium strains isolated from blown vacuum-packed meats was inoculated onto lamb chumps. A second washed spore cocktail of four toxigenic reference Cl. botulinum strains, types A, B (two strains) and E, and a Cl. butyricum type E strain, was similarly inoculated onto lamb chumps. All inoculated lamb chumps were individually vacuum-packed and placed into storage at various temperatures typical of good to grossly abusive chilled storage (-1 degree C to 15 degrees C). All packs were observed for gas production (pack-'blowing') over a 12 week storage period. On gas production, or after 12 weeks of storage, packs were examined by mouse bioassay for botulinum toxin production. The packs inoculated with the meat isolate cocktail showed evidence of gas production earlier than packs inoculated with reference strains. No botulinum toxin was recovered from the meat isolate inoculated packs, while botulinal toxin was detected in reference strain inoculated packs down to a nominal storage temperature of 2 degrees C.

Comparison of aerobic and anaerobic methods for the microbiological monitoring of chilled packaged meat during storage.

Aerobic and anaerobic plate counts were compared for routine monitoring of the microflora, dominated by lactic acid bacteria, developing on vacuum- and carbon dioxide-packaged raw meat during chilled storage. No statistical differences were observed between aerobic and anaerobic enumerations, made on plate count and blood agar plates, of the microflora developing on beef striploins packaged under vacuum or carbon dioxide during 14 weeks' storage at 0 degree C. With both techniques the spoilage microflora development differed between the two packaging regimes. The results indicate that there is no necessity for aerobic plate counts to be replaced by anaerobic plate counts in the routine microbiological examination of the spoilage microflora developing on chilled meats packaged under anoxic modified atmospheres.

Insufficient antilisterial capacity of low inoculum Lactobacillus cultures on long-term stored meats at 4 degrees C.

Two of the 210 lactobacilli strains isolated from chilled meats produced antilisterial bacteriocins: Lactobacillus sake 265 (Lb 265) and Lactobacillus casei 52 (Lb 52). Factors affecting antilisterial effectiveness of these and two other bacteriocin-producing (Bac+) strains (Lactobacillus sake 706, Lb 706; and Lactobacillus sake 148, Lb 148) at refrigeration temperature (4 degrees C) were studied in laboratory media and meat systems. At both 4 degrees C and 25 degrees C, these Bac+ strains grown in buffered MRS broths (pH 5.4 or 6.5) showed longer lag phases and shorter generation times than Listeria monocytogenes (mixture of strains NCTC 7973 and two food derived strains, L70 and L72) when grown in buffered BHI broths at the same pH values. These differences were more significant at 4 degrees C than at 25 degrees C. The highest concentrations of bacteriocin in MRS broth were produced at 25 degrees C and 4 degrees C by strain Lb 265 and Lb 706, respectively. Generally, production of bacteriocins was more efficient at lower pH (in buffered MRS broths of pH 5.4 and unbuffered MRS broths), than at higher pH (in buffered broths of pH 6.5). On vacuum packaged, raw beef (pH 5.3-5.4) initial numbers of L. monocytogenes (10(3)/g) did not change significantly during 23-days storage at 4 degrees C, when inoculated either alone or in the presence of Bac+ strains inoculated at initial levels of 10(3)/g. On vacuum packaged emulsion-type of sausages (pH 6.4) inoculated with L. monocytogenes and stored at 4 degrees C for 23 days growth was not significantly affected by addition of Bac+ strains at initial levels of 10(3)/g. These results indicated that amounts of bacteriocins produced in situ by low initial numbers (10(3)/g) of the protective strains tested were not sufficient to inhibit and/or reduce L. monocytogenes on these chilled meats, where high initial numbers of lactic acid bacteria are not desirable for product quality resons. To achieve these effects, higher concentrations of active (free) bacteriocins in meats must be provided.

The retail display life of steaks prepared from chill stored vacuum and carbon dioxide-packed sub-primal beef cuts.

Chilled striploins and cube rolls from ten Australian steers (grain-fed for 150 days) were trimmed of external fat and cut transversely into portions approximately 10 cm thick, each weighing between 750 and 1000 g. These 'retailer-ready' cuts were each wrapped in drip saver pads and slid inside a plastic sleeve before being individually placed into a clear plastic high oxygen barrier film, metallized film or conventional vacuum bag. Cuts in clear plastic and metallized film packs were packaged in an oxygen-free saturated carbon dioxide atmosphere (CO(2)-CAP), those in vacuum bags were conventionally vacuum-packed. All packs were returned to the chiller for further cooling. After 24 hr, half the clear plastic and metallized CO(2)-CAP packs were carbon dioxide master-packed in groups of eight. Retailer-ready cuts in both clear plastic and metallized film single unit and master-packed CO(2)-CAP packs were air freighted to New Zealand and sea freighted to Japan for assessment. The control vacuum packs were all consigned to New Zealand. Assessments in both countries after 39-89 days storage at between 0 °C and -1.0 °C indicated that fat colour stability limited the retail display life of steaks cut from meat in these retailer-ready packs to approximately 48 hr. In this regard, meat from single unit CO(2)-CAP, master pack CO(2)-CAP and vacuum packs performed similarly. Lean meat colour and sensory attributes remained acceptable for up to 48 hr after displayed product was rejected because of grey-green fat discoloration. The microbiological status of retailer-ready cuts removed from CO(2)-CAP packs after 89 days chilled storage was superior to that of cuts from vacuum packs. Clear plastic and metallized film CO(2)-CAP packs performed comparably.

The chilled storage life and retail display performance of vacuum and carbon dioxide packed hot deboned beef striploins.

Two cooling regimes that complied with the New Zealand meat hygiene requirement that hot deboned meat be chilled to +7 °C or less within 24 hr of leaving the slaughter floor were evaluated for the production of chilled table meats. Electrically stimulated hot deboned bull beef half striploins were either vacuum or carbon dioxide packed before being cooled in accordance with either Regime 1 (cool at +5 °C for 24 hr, transfer to chiller operating at -1.0 ± 0.5 °C) or Regime 2 (cool at +5 °C for 24 hr, hold at 5 °C for 6 days, transfer to chiller operating at -1.0 ± 0.5 °C). Striploins were removed from -1.0 °C storage 8, 28, 42, 56, 70, 84 and 98 days after slaughter and subjected to microbiological, tenderness, sensory and retail display performance evaluations. Both Regimes 1 and 2 produced meat of acceptable mean tenderness, 8 kgF (MIRINZ Tenderometer) in either vacuum or carbon dioxide packs within 28 and 8 days of slaughter, respectively. However, 70 days after slaughter the first signs of over-ageing became apparent. Steaks from Regimes 1 and 2 maintained acceptable visual appearance during retail display at 5 °C for 48 hr and 24 hr, respectively. After these times, the product was judged by the panel to be unacceptable because of its dull dark lean tissue and grey to green discoloration of the fat. Poor colour stability during retail display was mirrored by deterioration of sensory attributes, particularly aroma which is indicative of incipient spoilage. While carbon dioxide packaging in combination with Regime 1 offered an initial microbiological advantage over vacuum packaging, this advantage was not, however, carried over into retail display. Poor colour and sensory stability during retail display suggest that chilled table cuts derived from hot deboned bull beef are more suited to the Hotel-Restaurant-Institutional (HRI) trade than supermarket retailing. To serve the HRI, vacuum packed hot deboned bull beef primal cuts processed by Regime 1 appear to be the combination of choice. This combination would enable commercial processors to produce quality table beef with a chilled storage life of up to 70 days.