Isolation and lytic activity of the Listeria bacteriophage endolysin LysZ5 against Listeria monocytogenes in soya milk.

The endolysin gene (lysZ5) from the genome of the Listeria monocytogenes phage FWLLm3 was cloned in Escherichia coli and characterized. Comparative sequence analysis revealed that lysZ5 resembled the murein hydrolase ply511 encoded by L. monocytogenes phage A511. The encoded protein LysZ5 had a predicted molecular mass of 35.8 kDa and was expressed in E. coli as an N-terminal fusion protein of 41.5 kDa. Addition of purified fusion protein to lawns of indicator bacteria showed that LysZ5 could lyse L. monocytogenes, Listeria innocua and Listeria welshimeri, but not Staphylococcus aureus or Enterococcus faecalis. The purified protein was able to kill L. monocytogenes growing in soya milk, with the pathogen concentration reduced by more than 4 log10 CFU ml⁻¹ after 3 h incubation at 4 °C. As far as we know, this is the first report of a Listeria phage endolysin to control pathogens in soya milk and to demonstrate endolysin activity in foods at refrigeration temperatures. Moreover, LysZ5 may also be useful for biocontrol in other ready-to-eat foods.

Prevalence and numbers of coliphages and Campylobacter jejuni bacteriophages in New Zealand foods.

Vegetable samples were tested for the presence of coliphages. None of the 55 samples contained these phages at concentrations greater than 10 g(-1) (the limit of detection). Spiking and recovery experiments indicated that the method was efficient at detecting coliphage T4 added to the food, and so it was concluded that phage titres were not being falsely underestimated. In addition 51 samples of chicken skin from retail portions were tested for the presence and numbers of coliphages and for presence only of Campylobacter jejuni phages. Coliphages were isolated from 46 samples (90.2% positive), at up to 2570 PFU 10 g sample(-1) but no C. jejuni phages were isolated. Several other methods were used to isolate C. jejuni phages from retail chicken but none was successful. However, when pooled whole chicken rinses from 39 flocks were tested for the presence of C. jejuni phages, 11 (28.2%) of the flocks were positive. It is possible that phages present on birds at the start of processing were either inactivated or simply diluted out during spin chilling. These data add to the body of information indicating that phages can readily be isolated from certain foods and indicate that consumers are exposed to them on a regular basis.

Presence and significance of Bacillus cereus in dehydrated potato products.

Dehydrated potato contains Bacillus cereus at a prevalences of 10 to 40% and at numbers usually less than 10(3) CFU g(-1). B. cereus in dehydrated potato is likely to be present as spores that are able to survive drying of the raw vegetable and may represent a significant inoculum in the reconstituted (rehydrated) product where conditions favor germination of, and outgrowth from, spores. Holding rehydrated mashed potato alone, or as part of another product (e.g., potato-topped pie), at temperatures above 10 degrees C and below 60 degrees C may allow growth of vegetative B. cereus. Levels exceeding 10(4) CFU g(-1) are considered hazardous to human health and may be reached within a few hours if stored inappropriately between these temperatures. Foods incorporating mashed potato prepared from dehydrated potato flakes have been implicated in B. cereus foodborne illness. This review is a summary of the information available concerning the prevalence and numbers of B. cereus in dehydrated potato flakes and the rate at which growth might occur in the rehydrated product.

Isolation and characterization of bacteriophages infecting Salmonella spp.

Bacteriophages infecting Salmonella spp. were isolated from sewage using soft agar overlays containing three Salmonella serovars and assessed with regard to their potential to control food-borne salmonellae. Two distinct phages, as defined by plaque morphology, structure and host range, were obtained from a single sample of screened sewage. Phage FGCSSa1 had the broadest host range infecting six of eight Salmonella isolates and neither of two Escherichia coli isolates. Under optimal growth conditions for S. Enteritidis PT160, phage infection resulted in a burst size of 139 PFU but was apparently inactive at a temperature typical of stored foods (5 degrees C), even at multiplicity of infection values in excess of 10 000. While neither isolate had characteristics that would make them candidates for biocontrol of Salmonella spp. in foods, phage FGCSSa1 behaved unusually when grown on two Salmonella serotypes at 37 degrees C in that the addition of phages appeared to retard growth of the host, presumably by the lysis of a fraction of the host cell population.

Presence and growth of Bacillus cereus in dehydrated potato flakes and hot-held, ready-to-eat potato products purchased in New Zealand.

Potato products prepared from dehydrated potato flakes have been implicated in foodborne illness incidents involving Bacillus cereus intoxications. B. cereus can survive as spores in potato flakes and can germinate and multiply in the rehydrated product. This study assessed the frequency and concentration of B. cereus in dehydrated potato flakes and hot-held, ready-to-eat mashed potato products. Of 50 packets of potato flakes tested, eight contained greater than 100 CFU/g B. cereus (maximum 370 CFU/g). The temperature of the potato portion of 44 hot-held food products was measured immediately after purchase, and 86% were below the safe hot-holding temperature of 60 degrees C. The potato portions were subsequently tested for B. cereus. Only two of the potato portions contained B. cereus at greater than 100 CFU/g, a potato-topped pastry (1000 CFU/g) and a container of potato and gravy (120 CFU/g). To assess multiplication of B. cereus in this food, we held rehydrated potato flakes with naturally occurring B. cereus at 37, 42, and 50 degrees C and tested them over 6 h. By 6 h, the number of B. cereus in potato stored at 37 degrees C had exceeded 10(3) CFU/g, was greater than 10(4) CFU/g at 50 degrees C, and was close to 10(6) CFU/g at 42 degrees C. Growth data were compared to predictions from the U.S. Department of Agriculture Pathogen Modeling Program (PMP 7.0). The PMP predictions were found to simulate the measured growth better at 42 degrees C than at 37 degrees C. Hot-held potato products should be safe for consumption if held at 60 degrees C or above or discarded within 2 h.

Outbreak of campylobacteriosis following pre-cooked sausage consumption.

OBJECTIVES: A small outbreak of campylobacteriosis involving three cases was investigated in terms of Campylobacter types present in the suspect food (pre-cooked sausages) and clinical samples from the cases. METHOD: Foods and faecal samples from people involved in the incident, which occurred in Christchurch, New Zealand, were tested for the presence of Campylobacter and identification of the species made. Isolates were typed by Penner serotyping and macrorestriction analysis using pulsed-field gel electrophoresis (PFGE). Investigations were conducted as to whether the contamination was on the surface or the interior of the sausages. RESULTS: All isolates from food and faecal samples were identified as C. jejuni and were indistinguishable from one another by the typing methods employed. Only the surfaces of the sausages were contaminated. Three other isolates of an indistinguishable subtype were isolated from campylobacteriosis cases in Christchurch occurring over approximately the same period. CONCLUSIONS AND IMPLICATIONS: Given the rarity of the subtype isolated from the three family members and the three other cases, it is possible that the outbreak was larger than the initial investigation revealed. It is likely that the sausages were contaminated after they had been cooked by the retailer and were not reheated prior to consumption. This report illustrates the role of cross-contamination in an outbreak with an unusual food vehicle for campylobacteriosis. Physical separation of cooked and raw product is necessary to prevent recurrences of outbreaks similar to the one described here.

Listeriaphages and coagulin C23 act synergistically to kill Listeria monocytogenes in milk under refrigeration conditions.

Bacteriophages and bacteriocins are promising biocontrol tools in food. In this work, two Listeria bacteriophages, FWLLm1 and FWLLm3, were assessed in combination with the bacteriocin coagulin C23 to inhibit Listeria monocytogenes. Preliminary results under laboratory conditions demonstrated that both antimicrobials act synergistically when they were applied in suboptimal concentrations. The combined approach was further assessed in milk contaminated with 5×10(4) CFU/ml L. monocytogenes 2000/47 and stored at 4 °C for 10 days. When used alone, phage FWLLm1 added at 5×10(6) PFU/ml, FWLLm3 at 5×10(5) PFU/ml and coagulin C23 at 584 AU/ml kept L. monocytogenes 2000/47 counts lower than the untreated control throughout storage. However, when used in combination, inhibition was enhanced and in the presence of FWLLm1 and coagulin C23, L. monocytogenes 2000/47 counts were under the detection limits (less than 10 CFU/ml) from day 4 until the end of the experiment. Resistant mutants towards phages and coagulin C23 could be obtained, but cross-resistance was not detected. Mutants resistant to FWLLm3 and coagulin C23 were also recovered from surviving colonies after cold storage in milk which may explain the failure of this combination to inhibit L. monocytogenes. Remarkably, the fraction of resistant mutants isolated from the combined treatment was lower than that from each antimicrobial alone, suggesting that synergy between bacteriocins and phages could be due to a lower rate of resistance development and the absence of cross-resistance.

Prevention of bacterial foodborne disease using nanobiotechnology.

Foodborne disease is an important source of expense, morbidity, and mortality for society. Detection and control constitute significant components of the overall management of foodborne bacterial pathogens, and this review focuses on the use of nanosized biological entities and molecules to achieve these goals. There is an emphasis on the use of organisms called bacteriophages (phages: viruses that infect bacteria), which are increasingly being used in pathogen detection and biocontrol applications. Detection of pathogens in foods by conventional techniques is time-consuming and expensive, although it can also be sensitive and accurate. Nanobiotechnology is being used to decrease detection times and cost through the development of biosensors, exploiting specific cell-recognition properties of antibodies and phage proteins. Although sensitivity per test can be excellent (eg, the detection of one cell), the very small volumes tested mean that sensitivity per sample is less compelling. An ideal detection method needs to be inexpensive, sensitive, and accurate, but no approach yet achieves all three. For nanobiotechnology to displace existing methods (culture-based, antibody-based rapid methods, or those that detect amplified nucleic acid) it will need to focus on improving sensitivity. Although manufactured nonbiological nanoparticles have been used to kill bacterial cells, nanosized organisms called phages are increasingly finding favor in food safety applications. Phages are amenable to protein and nucleic acid labeling, and can be very specific, and the typical large "burst size" resulting from phage amplification can be harnessed to produce a rapid increase in signal to facilitate detection. There are now several commercially available phages for pathogen control, and many reports in the literature demonstrate efficacy against a number of foodborne pathogens on diverse foods. As a method for control of pathogens, nanobiotechnology is therefore flourishing.

Proteomic Differences between Listeria monocytogenes Isolates from Food and Clinical Environments.

Listeria monocytogenes is an organism associated with a wide range of foods. It causes listeriosis, a severe illness that mainly affects people with weakened immune systems. Proteomic profiles of three different L. monocytogenes isolates were studied using 1D SDS PAGE, 2DE and mass spectrometry. The protein banding patterns generated by 1D SDS PAGE of three strains of L. monocytogenes were found to be similar. Visual observations from 2DE gel maps revealed that certain spots appeared to have intensity differences. Key differences in proteins synthesis of three strains of L. monocytogenes were found using the PDQest TM 2DE Analysis software. Comparison showed that the clinical isolate (strain SB92/844) had 53.4% and 53.9% protein profile similarity with dairy isolate (strain V7) and seafood isolate (SB92/870), respectively. The identity of selected protein spots was achieved using MALDI-TOF and ion trap mass spectrometry. It was found that certain identified proteins (i.e., a major cold shock protein and superoxide dismutase) were expressed differently between two local strains of L. monocytogenes (SB92/844, SB92/870) and one strain from overseas (V7).

Growth of Yersinia enterocolitica and Y. pseudotuberculosis in Yersinia selective enrichment broth according to Ossmer.

The growth of Yersinia enterocolitica and Yersinia pseudotuberculosis in Yersinia enrichment broth according to Ossmer (YSEO) was investigated. Y. enterocolitica reached a higher concentration than Y. pseudotuberculosis but both always exceeded 10(6)CFU/ml. The medium may be useful for the detection of both species in foods.

Construction of and Comparisons Between Response Surface Models for Aeromonas hydrophila ATCC 7966 and a Food Isolate Under Aerobic Conditions.

Response surface models were constructed for two strains of Aeromonas hydrophila , one the type strain (ATCC 7966) and the other isolated from chilled cooked mussels, for various conditions of temperature, pH, and NaCl concentration. The food-derived strain was found to be better adapted to growth at low temperatures than the type strain. Comparisons between the two strains and with the published model for strain K144, a clinical isolate, showed that the three strains differed in their predicted generation and lag times at refrigeration (5°C) and close to optimum (28°C) temperatures. Comparison of the new models with published data for growth of A. hydrophila on fleshfoods, showed that the model for the food-derived strain best predicted the measured lag times, while the model for the type strain best predicted the generation times.

Incidence of Motile Aeromonads in New Zealand Retail Foods.

A total of 396 food samples (ready-to-eat meat and poultry products; shellfish and finfish products; some raw shell and finfish and meat products; salads and coleslaws; baked confectionary) was purchased from local retail outlets and examined for the presence of motile aeromonads. Of the food categories tested, shellfish had the highest incidence, with 66% positive. Vegetable products, luncheon meat, salami, paté, and poultry products had very low incidences, and no motile aeromonads were found in baked confectionary, some of which contained whipped cream. Beef, pork, lamb, sausage, and finfish products had incidences above 20%. Most of the strains isolated were Aeromonas hydrophila . Given the reported distribution, a public health problem may exist if motile aeromonads do cause gastrointestinal disease.

Growth of Listeria monocytogenes , Aeromonas hydrophila , and Yersinia enterocolitica on Vacuum and Saturated Carbon Dioxide Controlled Atmosphere-Packaged Sliced Roast Beef.

The growth of Listeria monocytogenes , Aeromonas hydrophila and Yersinia enterocolitica on sliced roast beef packaged under vacuum or saturated CO2 controlled atmosphere conditions was measured. At -1.5°C, the pathogens declined in numbers in the controlled atmosphere packs but were able to grow under vacuum packaging. At 3°C, growth occurred, with maximum numbers being reached at the end of the product's storage life. The increased shelf life of sliced roast beef incubated under a carbon dioxide controlled atmosphere compared to vacuum-packaged beef did not, therefore, bring with it a concomitant risk associated with the increased growth of these three pathogens over the extended storage period.

Inhibition of Listeria monocytogenes on Normal Ultímate pH Beef (pH 5.3-5.5) at Abusive Storage Temperatures by Saturated Carbon Dioxide Controlled Atmosphere Packaging.

Beef striploin steaks (each weighing between 60 and 100 g) of normal ultimate pH (average 5.45) were inoculated with a mixture of two food isolates of Listeria monocytogenes , packaged under saturated carbon dioxide controlled atmosphere or vacuum, and stored at either 5 or 10°C. Saturated carbon dioxide packaging but not vacuum packaging inhibited growth of L. monocytogenes . During storage under vacuum at 5°C, numbers increased from 6.2-logl0 CFU per sample to 9.0-log10 CFU per sample in 23 days, and at 10°C numbers increased from 6.3-log10 CFU per sample to 9.5-log10 CFU per sample in 11 days. During storage under saturated carbon dioxide at 5°C, numbers decreased from 5.6-log10 CFU per sample to 5.0-log10 CFU per sample in 18 days, and at 10°C numbers decreased from 6.7-log10 CFU per sample to 5.2- logl0 CFU per sample in 44 days. The use of saturated carbon dioxide controlled atmosphere packaging to extend the storage life of fresh beef results in apparent destruction of L monocytogenes even at abuse temperatures.