Next-generation sequencing as a screening tool for foodborne pathogens in fresh produce.

Next generation sequencing (NGS) approaches are increasingly applied to tracing microbial contaminants entering the food chain due to NGS' untargeted nature and ability to investigate non-culturable (and/or difficult to culture) organisms while yielding genomic information about the microbiota. So far, a plethora of microbes has been shown to be associated with fresh produce, but few studies have utilised NGS to identify contamination with human pathogens. This study aims to establish the limit of detection (LoD) for Salmonella and phage MS2 (a Norovirus surrogate) contamination of fresh produce employing NGS approaches on the Illumina MiSeq: 16S amplicon-sequencing, and RNA-seq, using ScriptSeq (Illumina) and NEBNext (New England BioLabs) kits. ScriptSeq proved the most sensitive approach; delivering an LoD of 104 CFU reaction-1 (Colony Forming Units) for Salmonella and 105 PFU reaction-1 (Plaque Forming Units) for phage MS2. Use of the NEBNext kit resulted in detection of Salmonella at 106 CFU reaction-1 and phage MS2 at 107 PFU reaction-1. 16S amplicon-sequencing yielded a similar LoD of 105 CFU reaction-1 for Salmonella but could not detect MS2. The tested NGS methodologies, in combination with bioinformatics approaches applied, proved less sensitive than conventional microbial detection approaches.

Inactivation of Escherichia coli O157:H7 using ultraviolet light-treated bacteriophages.

Escherichia coli O157:H7 causes serious foodborne infections warranting the development of effective control measures. One control option is to use bacteriophages (phages), which are regarded as safe to humans and an environmentally friendly alternative to chemical antimicrobials. One of the few remaining safety concerns is the potential for phages to facilitate genetic exchange between bacteria so resulting in undesirable mobilisation of genes. UV treatment of phages causes a rapid loss in their ability to replicate, while maintaining their antibacterial activity, and so the use of UV-treated phages could be an alternative to the use of viable phages. Data presented here show the inactivation of E. coli O157:H7 by UV-treated phages in milk and on the surface of raw and cooked meat. A minimum concentration of approximately 10(5) PFU cm(-2) (pre-UV treatment titre) of UV-treated phages was required before inactivation of E. coli O157:H7 on the surface of meat was measurable, and 1-2 log10 CFU cm(-2) reductions were typically obtained at concentrations of around 10(7) UV-treated phages cm(-2) (pre-UV treatment titre). Inactivation of E. coli O157:H7 by UV-treated phages was less than that for untreated phages. The production of UV-treated phages was not optimised and it is possible that better reductions in pathogen concentration could be achieved for the same input UV-treated phages concentrations.

Effect of phage and host concentration on the inactivation of Escherichia coli O157:H7 on cooked and raw beef.

A previously described phage infecting Escherichia coli O157:H7 was added to raw and cooked beef pieces at concentrations ranging from 10(1)-10(8) plaque forming units/cm(2) to either low (<100 CFU/cm(2)) or high (10(4) CFU/cm(2)) concentrations of host bacterial cells. Incubation for up to 24 h was performed at 5 ℃ and 24 ℃ to simulate refrigerated and room temperature storage/temperature abuse. Surviving bacteria were enumerated during the incubation period, with phages being counted at the first and last sampling times. Significant reductions of E. coli O157:H7 of the order of >4 log10 CFU/cm(2) at both temperatures could be achieved compared to phage-free controls. There was a trend for greater inactivation to occur with increasing phage concentration. While re-growth of surviving cells occurred in nearly all samples incubated for 24 h at 24 ℃, these conditions are not typical of those experienced by perishable foods. It was concluded that phages can be used to reduce the concentration of a bacterial pathogen on meat, but the concentration of phages needs to be high (>4-5 log10 plaque forming units/cm(2)) for reductions to occur. A concentration of the order 8 log10 plaque forming units/cm(2) was needed to achieve a 4 log10 CFU/cm(2) reduction.

The limitations of pulsed-field gel electrophoresis for analysis of Yersinia enterocolitica isolates.

This study describes the analysis of 432 isolates of Yersinia enterocolitica by pulsed-field gel electrophoresis (PFGE). PFGE had a high level of discrimination with biotype 1A isolates (Simpson's Diversity Index 0.997), but with the clinically important biotypes 2, 3 and 4, the discriminatory ability of PFGE was so low as to severely limit its usefulness (DI <0.6). For biotypes 2, 3 and 4, 79% or more of isolates of each biotype were of just three different PFGE profiles. Because of this, four known outbreaks of yersiniosis would not have been identified by PFGE analysis. However, a previously unrecognized potential outbreak of yersiniosis caused by biotype 4 isolates was identified on the basis of a rare PFGE genotype with spatial and temporal clustering. We conclude that PFGE has a very limited application to the genotyping of Y. enterocolitica biotypes 2, 3 and 4, and inferences based on finding indistinguishable PFGE profiles among cases or between cases and sources need to be substantiated using alternative typing tools, or strong epidemiological evidence.

Use of a bacteriophage to inactivate Escherichia coli O157:H7 on beef.

A number of outbreaks of Escherichia coli O157:H7 infections involving beef have been reported. Options for controlling bacterial pathogens in raw foods are limited, but one is to use bacteriophages (phages). We describe the isolation and characterisation of phage FAHEc1, which infects E. coli O157, and its ability to kill its host in vitro and on beef. The phage belonged to the family Myoviridae and lysed 28 of 30 E. coli O157 (:H7, :HNM and :H not specified) isolates, only one other non-O157 E. coli serotype (O162:H7), and none of the other 13 bacterial species tested. The phage did not contain stx1, stx2, eae or ehxA virulence genes as assessed by PCR. An approximate 4 log10 inactivation of E. coli O157:H7 occurred at 5 °C in the presence of phage FAHEc1 at >107 PFU/ml in broth in vitro. On thinly sliced beef pieces incubated at 37 °C, a > 2.7 log10 reduction occurred with 3.2 × 107 PFU/4 cm² meat piece. At lower phage concentrations (10³-104 PFU/4 cm² piece) phage replication occurred on beef at 37 °C. When the phage was applied to beef pieces under conditions simulating hot boning and conventional carcass cooling, inactivation of E. coli O157:H7 of approximately 2 log10 was measured under optimal conditions with phages applied at 3.2 × 107 PFU/4 cm² meat piece.

Inhibition of Listeria monocytogenes by Enterococcus mundtii isolated from soil.

Two bacterial isolates with inhibitory activity against Listeria monocytogenes and Enterococcus faecalis were obtained from soil. Genotypic and phenotypic characterization identified them as Enterococcus mundtii, a species whose ability to compete with L. monocytogenes is relatively unexplored compared to other members of the genus. The thermal stability of the inhibitory factor and its sensitivity to proteolytic enzymes indicate that it is most likely a bacteriocin. Both isolates grew at comparable rates to L. monocytogenes at 5 °C and 10 °C in vitro. One isolate killed L. monocytogenes when it reached concentrations of 10(6)-10(8) CFU ml(-1). Minimum inocula of 10(6) and 10(5) CFU ml(-1) of E. mundtii were required to reduce and maintain L. monocytogenes concentrations beneath the level of detection at 5 °C and 10 °C, respectively. In situ experiments at 5 °C showed that E. mundtii inhibited the growth of L. monocytogenes on vacuum-packed cold smoked salmon during its four week shelf life. E. mundtii could, therefore, control the growth of L. monocytogenes at low temperatures, indicating a potential application in controlling this pathogen in chilled foods. To control growth of Listeria, the concentration of E. mundtii needs to be high, but it is possible that a purified bacteriocin could be used to achieve the same effect.

Control of Listeria monocytogenes growth in a ready-to-eat poultry product using a bacteriophage.

A bacteriophage (phage) that infected strains of the species Listeria monocytogenes as well as Listeria ivanovii and Listeria welshimeri, but not Listeria grayi or Listeria innocua, was isolated from sheep faeces. The phage had a contractile tail and an icosohedral head indicating that it was a myovirus, and was morphologically similar to phage A511. At 30 °C, phages added at 5.2 × 107 PFU ml⁻¹ prevented the growth in broth of L. monocytogenes present at approximately twice this concentration for 7 h, but re-growth occurred such that the concentration after 24 h incubation was similar in both control and phage-treated cultures. At the same temperature, but on the surface of vacuum-packed ready-to-eat chicken breast roll, there was an immediate 2.5 log10 CFU cm⁻² reduction in pathogen concentration following addition of phages and then re-growth. However, at a temperature reflecting that at which a chilled food might be held (5 °C), this re-growth was prevented over 21 days incubation. The data suggest a dose-dependent rapid reduction in pathogen concentration followed by no continued phage-mediated effect. These results, alongside other published data, indicate that a high concentration of phages per unit area is required to ensure significant inactivation of target pathogens on food surfaces.

Isolation and characterization of phages infecting Bacillus cereus.

AIM: To isolate and characterize bacteriophages (phages) that infect the foodborne pathogen Bacillus cereus. METHODS AND RESULTS: Two phages were isolated from soil based on their ability to form plaques on four indicator hosts including Bacillus thuringiensis subsp. israelensis, and three isolates of B. cereus. The purified phages were characterized by morphology, host range, single-step growth curves and restriction enzyme digestion profiles. The phages appeared to be of the Myoviridae family based on their structure in electron micrographs. The phages lysed bacteria of several species, produced average burst sizes of 322 and 300 phages per infected cell, and both had genomes over 90 kb. The phages were chloroform-resistant and stable at 4°C. They reduced the concentration of B. cereus in mashed potatoes by >6 log(10) CFU ml(-1) within 24 h at room temperature, when applied at a high concentration. CONCLUSIONS: The relatively narrow host range within B. cereus might mean that these phages need to be used as part of a 'cocktail' of phages for biocontrol, but their efficacy for the control of their host in food was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of biocontrol by phages of B. cereus in food.

Campylobacters and bacteriophages in the surface waters of Canterbury (New Zealand).

AIM: To determine the relationship between the presence of thermotolerant campylobacters and their bacteriophages (phages) in surface waters for the potential to use phages as an indicator of Campylobacter spp. METHODS AND RESULTS: Thermotolerant campylobacters were enumerated in 53 water samples using a three tube most probable number (MPN) series in m-Exeter broth. The presence of phages in the same samples was tested using two approaches: qualitative enrichment with five different Campylobacter hosts and a quantitative membrane concentration method. Phages infecting an Escherichia coli O157:H7 isolate were also enumerated by the membrane concentration method. Campylobacter spp. were isolated from 45/53 (85%) of the samples at 0.4-110 MPN 100 ml(-1). No Campylobacter phages were isolated, but coliphages were present in 43/46 (93%) of samples. CONCLUSIONS: The membrane concentration method recovered >80% of Campylobacter phages from spiked samples. The absence of Campylobacter phages in environmental samples, from both enrichment and concentration methods, suggests that, if present, they are at very low titres. SIGNIFICANCE AND IMPACT OF THE STUDY: Testing for Campylobacter phages as an indicator of Campylobacter spp. presence is not effective. The quantitative data for Campylobacter spp. will be useful for risk assessment purposes.

Detection, isolation and enumeration of Yersinia enterocolitica from raw pork.

The methods available for the isolation of Yersinia enterocolitica from foods are generally considered to be less than optimal, and methods for estimation of numbers are lacking. Such methods are needed to understand better the significance of foodborne yersiniosis and to provide data for exposure assessment. We describe a method for the detection and enumeration of Y. enterocolitica containing the pYV virulence plasmid (YeP+) in samples from pork surfaces. The method uses a multiplex PCR targeting the ail and virF genes to detect Y. enterocolitica after incubation of surface swabs in Yersinia enrichment broth according to Ossmer. Enumeration was achieved by adapting the enrichment to a most probable number (MPN) method format. A presumptive result was available within 24 h of sample receipt, and YeP+ isolates were confirmed within four days. The presence/absence and MPN methods were evaluated in a pilot survey of 34 packs of raw pork meat purchased from retail outlets in Christchurch, New Zealand. YeP+ was detected by PCR on meat from 32% of the packs, and YeP+ isolates were obtained from 18% of the samples. YeP+ were present at numbers ranging from 0.30 to 5.42 MPN/cm(2). This improved method for the detection and enumeration of YeP+ from meat samples can be used for microbiological surveys to obtain data for assessments of consumer exposure to virulent Y. enterocolitica, and in outbreak investigations.

Phage inactivation of foodborne pathogens on cooked and raw meat.

Phages infecting Salmonella Typhimurium PT160 and Campylobacter jejuni were added at a low or high (10 or 10(4)) multiplicity of infection (MOI) to either low or high (<100 or 10(4)cm(-2)) densities of host bacteria inoculated onto raw and cooked beef, and incubated at 5 and 24 degrees C to simulate refrigerated and room temperature storage. Counts of host bacteria were made throughout the incubation period, with phages being counted at the first and last sampling times. Host inactivation was variable and depended on the incubation conditions and food type. Significant host inactivations of the order of 2-3 log(10)cm(-2) at 5 degrees C and >5.9 log(10)cm(-2) at 24 degrees C were achieved compared to phage-free controls using the Salmonella phage under optimal conditions (high host cell density and MOI). These results alongside those already published indicate that phages may be useful in the control for foodborne pathogens.

Enumeration and growth of naturally occurring Listeria spp. in unpackaged ham.

A total of 301 unpackaged retail ham samples were tested for the presence and number of Listeria spp. after 7 days at 5 degrees C to simulate domestic storage. Thirteen samples (4.3%) contained Listeria monocytogenes, with the highest count being 1.6 x 10(3)cfu g(-1). Thirteen samples contained other Listeria spp. Genotyping showed that only one L. monocytogenes isolate from the 14 tested was of a type previously identified in New Zealand human cases. Listeria-contaminated batches were incubated at 5 degrees C over approximately 3 weeks to assess the growth rate of natural contaminants. None contained L. monocytogenes, but growth occurred in one sample containing Listeria welshimeri and four containing Listeria innocua. Growth was usually slow at 0.002-0.004 log h(-1). In one sample, L. innocua grew at 0.02 log h(-1) although the maximum number reached was only 4.0-5.0 x 10(3)cfu g(-1). In five other samples little growth, if any, occurred. Growth of naturally occurring Listeria spp. at 5 degrees C was therefore generally slower than predicted by the Pathogen Modelling Programme (PMP) or did not occur.

Statistical comparison of Campylobacter jejuni subtypes from human cases and environmental sources.

AIM: To analyse Campylobacter jejuni typing data to define statistically which potential reservoirs and transmission sources contain isolates that are most similar to one another and to isolates from human infections. METHODS AND RESULTS: Serotyping and SmaI macrorestriction profiling data for C. jejuni isolates from human campylobacteriosis cases, chicken carcass rinses, duck, sheep, dairy and beef cattle faeces, river water, and sheep, beef and pork offal obtained from a defined rural area of New Zealand were compared using the Czekanowski proportional similarity index. Subtypes of isolates from ruminant animals, whether derived from their faeces or offals, were generally similar to one another. The spectrum of isolate subtypes from human cases was more similar to that from ruminant faeces than the other matrices considered. Isolate subtypes from chicken rinses, pork offal, water and duck faeces were not highly similar to those from other matrices. CONCLUSIONS: Results from a combination of phenotypic and genotypic approaches suggest that, for this rural population, exposures associated with a rural lifestyle may be significant sources of human campylobacteriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The Czekanowski index was applied to subtyping data and supported the greater importance of contact with livestock in campylobacteriosis cases associated with a rural setting, in comparison with urban studies that have identified poultry-related factors.

Campylobacter in chicken livers and their destruction by pan frying.

AIM: To enumerate Campylobacter spp. on the external surface and internal portions of chicken livers, and to assess the cooking required to inactivate naturally present cells. METHODS AND RESULTS: Of 30 livers tested all yielded Campylobacter spp. on their surfaces and 90% were found to contain the organism in internal tissue. Four (13%) livers contained >10(4) MPN campylobacters, and an additional seven (23%) contained >10(3) MPN campylobacters per liver. The internal temperature of pan-fried livers under the conditions used reached a maximum of 70-80 degrees C, and maintaining this temperature for 2-3 min was necessary to inactivate naturally occurring Campylobacter spp. All isolates identified were either C. jejuni or C. coli. CONCLUSIONS: Chicken livers represent a potential source of human campylobacteriosis as they contained >10(4) MPN per liver in 13% of the samples tested. Pan-frying can produce an acceptable product that is safe to eat. SIGNIFICANCE AND IMPACT OF THIS STUDY: The data provided can be used in exposure assessments of Campylobacter in poultry products in terms of both quantitative data and assessing pan-frying and its ability to destroy campylobacters.

Synovial Chlamydia trachomatis up regulates expression of a panel of genes similar to that transcribed by Mycobacterium tuberculosis during persistent infection.

BACKGROUND: Synovial tissues in patients with Chlamydia associated arthritis are persistently infected by C trachomatis, an organism for which genetic manipulation is not possible. M tuberculosis also engages in persistent infection, and because this bacterium is genetically tractable many groups have been able to define transcriptional characteristics of mycobacterial growth and persistence. OBJECTIVE: To investigate whether the pattern of gene expression underlying chlamydial persistence is similar to that underlying mycobacterial persistence. METHODS: 194 genes in M tuberculosis that are transcriptionally up regulated to support in vivo growth and persistence of that organism have previously been identified. Each of those genes was compared with the C trachomatis genome to identify orthologues. Expression of selected chlamydial orthologues so identified was assessed by real time RT-PCR in an in vitro model of chlamydial persistence and synovial tissues from patients who were PCR positive for C trachomatis at that site. RESULTS: 67 C trachomatis genes were identified as being orthologous to mycobacterial persistence related genes, representing 35% of the genes tested. The chlamydial orthologues fell into similar metabolic and other categories as those in M tuberculosis. Expression of a majority of selected chlamydial orthologues was strongly up regulated in an in vitro model of chlamydial persistence and in synovial tissues of relevant patients, compared with their expression during active infection. CONCLUSIONS: These observations provide new insight into the molecular genetic basis underlying chlamydial persistence, and indicate that this information can be obtained, in some instances, by extrapolating observations made in other biological systems and/or organisms.

Microbiological survey of prepackaged pâté and ham in New Zealand.

AIMS: To gauge the effectiveness of pâté and ham manufacturers' management of the microbial safety and quality of their products. METHODS AND RESULTS: A survey of 60 batches of prepackaged pâté showed that 41.7% of the batches had aerobic plate counts (APC) exceeding 10(5) CFU g(-1), one of pâté sample contained a Bacillus cereus count of >5000 CFU g(-1) and another contained 1700 CFU g(-1) of Listeria monocytogenes. No other pathogens were isolated from any of the samples. The survey of prepackaged ham showed that only 1% (1/104) of the ham samples were positive for L. monocytogenes (50 CFU g(-1)). CONCLUSIONS: The presence of microbial hazards in these foods has generally declined since the early 1990s in New Zealand. Noncomplying APC levels may be due to an over-estimation of product shelf life or poor food handling practices during manufacture. SIGNIFICANCE AND IMPACT OF THE STUDY: Few of the samples tested contained pathogens at significant levels. The prevalences of L. monocytogenes in pâté and ham were low. The presence of 1700 CFU g(-1) of L. monocytogenes in a pâté sample indicates that occasionally, the population can be exposed to levels of L. monocytogenes above the zero tolerance level set in New Zealand.

The occurrence of Campylobacter subtypes in environmental reservoirs and potential transmission routes.

AIM: To identify potential reservoirs and transmission routes of human pathogenic Campylobacter spp. METHODS AND RESULTS: An enrichment PCR method for the detection and identification of Campylobacter jejuni and/or Campylobacter coli in faecal, food and river water samples was applied to 1450 samples of 12 matrix types obtained from a defined geographical area. PCR-positive samples were cultured to yield isolates for typing, and the data for 616 C. jejuni isolates obtained. Serotyping and SmaI macrorestriction profiling using pulsed field gel electrophoresis revealed a high level of diversity within the isolates from each matrix. Campylobacter jejuni and C. coli subtypes indistinguishable from those obtained from human cases were detected in most of the matrices examined. No Campylobacter isolates were isolated from possum faeces. CONCLUSIONS: Ten of the 12 matrices examined may be involved in the transmission of human campylobacteriosis as they contained Campylobacter subtypes also isolated from clinical cases. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicate that, for this rural population, a range of potential transmission routes that could lead to campylobacteriosis exist. Their relative importance needs to be assessed from an exposure assessment standpoint.

Bacteriophages as biocontrol agents in food.

Bacteriophages possess attributes that appear to be attractive to those searching for novel ways to control foodborne pathogens and spoilage organisms. These phages have a history of safe use, can be highly host specific, and replicate in the presence of a host. Campylobacter, Salmonella, and Listeria monocytogenes and various spoilage organisms have responded to phage control on some foods. However, the use of phages as biocontrol agents is complicated by factors such as an apparent requirement for a threshold level of host before replication can proceed and by suboptimal performance, at best, at temperatures beneath the optimum for the host. This review is a summary of the information on these issues and includes brief descriptions of alternative phage-based strategies for control of foodborne pathogens.

Campylobacter spp. in New Zealand raw sheep liver and human campylobacteriosis cases.

Sheep liver samples were tested for the presence and numbers of Campylobacter jejuni and C. coli during both spring and autumn. Over the same period, isolates were obtained from human clinical cases from the same geographical area as where the food samples were purchased. A subset of the C. jejuni isolates was typed by both Penner serotyping and pulsed field gel electrophoresis using the restriction enzyme SmaI, to estimate the proportion of liver isolate types that were also isolated from human cases of campylobacteriosis. Of the 272 liver samples tested, 180 (66.2%) contained Campylobacter. Most of the positive samples contained <3 MPN/g of the organism, and only 12 (6.7%) were contaminated at a level exceeding 100 MPN/g. A total of 180 C. jejuni isolates were obtained from sheep liver and another 200 from human faeces. Of these, 212 isolates were randomly selected for typing, half from raw liver and half from human faeces. More than half (61.1%) of the 106 C. jejuni isolates from liver were of subtypes that were also isolated from human cases. While the C. jejuni present in sheep liver were mostly of subtypes also isolated from human cases, the significance of this food as a vehicle of human campylobacteriosis needs to be examined further in respect to other factors such as dose-response information, consumption data, frequency of undercooking and cross contamination.

Growth of enterotoxigenic Staphylococcus aureus in povi masima, a traditional Pacific island food.

AIMS: To obtain preliminary data on the microbiology and hurdles to pathogen growth in the traditional Pacific Island food, povi masima, which is essentially beef brisket cured in brine. METHODS AND RESULTS: Six containers of povi masima were prepared and two were inoculated with five enterotoxigenic strains of Staphyloccocus aureus. The povi masima were divided into two lots each containing two uninoculated control and an inoculated container. Lot 1 was incubated at room temperature (20 degrees C) and lot 2 under refrigeration (4-5 degrees C) for up to 98 days. During storage, samples were removed and tested for aerobic plate count, coagulase-producing Staphylococci, Clostridium perfringens, staphylococcal enterotoxin and various chemical parameters of the food. Coagulase-producing Staphylococci and aerobic plate counts grew to high levels in both the inoculated and uninoculated lots stored at room temperature, but enterotoxin was only detected at one time point in these lots and this may represent a false positive result. The concentration of NaCl in the meat increased with time as concentrations equilibrated, and nitrite was rapidly lost in those lots stored at room temperature. Storage at 4-5 degrees C prevented proliferation of coagulase-producing Staphylococci. CONCLUSIONS: For safe curing and storage, this food should be kept under refrigeration as this prevented growth of staphylococci. Optimum storage would also be achieved with improved attempts to ensure equal distribution of NaCl prior to storage. SIGNIFICANCE AND IMPACT OF THE STUDY: Under conditions traditionally used to cure and store this food, enterotoxigenic staphylococci can grow to numbers where toxigenesis might occur, especially during the early stages of curing where the salt has not diffused from the brine into the meat.