accounts for a significant number of foodborne illnesses around the world.
is microaerophilic and typically does not survive efficiently in oxygen-rich conditions. We recently reported that ...hyper-aerotolerant (HAT)
are highly prevalent in retail poultry meat. To assess the capabilities of HAT
in foodborne transmission and infection, in this study, we investigated the prevalence of virulence genes in HAT
and the survival in poultry meat in atmosphere at a refrigeration temperature. When we examined the prevalence of eight virulence genes in 70
strains from raw poultry meat, interestingly, the frequencies of detecting virulence genes were significantly higher in HAT
strains than aerosenstive
strains. This suggests that HAT
would potentially be more pathogenic than aerosensitive
. Under aerobic conditions, aerosensitive
survived at 4°C in raw poultry meat for 3 days, whereas HAT
survived in poultry meat for a substantially extended time; there was a five-log CFU reduction over 2 weeks. In addition, we measured the effect of other gas conditions, including N
and CO
, on the viability of HAT
in comparison with aerosensitive and aerotolerant strains. N
marginally affected the viability of
. However, CO
significantly reduced the viability of
both in culture media and poultry meat. Based on the results, modified atmosphere packaging using CO
may help us to control poultry contamination with HAT
.
In this manuscript, we report the design and development of a fast, reliable instrument to run gel-based cassette polymerase chain reactions (PCR). Here termed the GelCycler Mark II, our instrument ...is a miniaturized molecular testing system that is fast, low cost and sensitive. Cassette PCR utilizes capillary reaction units that carry all reagents needed for PCR, including primers and Taq polymerase, except the sample, which is loaded at the time of testing. Cassette PCR carries out real time quantitative PCR followed by melt curve analysis (MCA) to verify amplicon identity at the expected melt temperature (Tm). The cassette PCR technology is well developed, particularly for detecting pathogens, and has been rigorously validated for detecting pathogenic Escherichia coli in meat samples. However, the work has been hindered by the lack of a robust and stable instrument to carry out the PCR, which requires fast and accurate temperature regulation, improved light delivery and fluorescent recording, and faster PCR reactions that maintain a high sensitivity of detection. Here, we report design and testing of a new instrument to address these shortcomings and to enable standardized testing by cassette PCR and commercial manufacture of a robust and accurate instrument that can be mass produced to deliver consistent performance. As a corollary to our new instrument development, we also report the use of an improved design approach using a machined aluminum cassette to meet the new instrument standards, prevent any light bleed across different trenches in each cassette, and allow testing of a larger number of samples for more targets in a single run. The GelCycler Mark II can detect and report E. coli contamination in 41 minutes. Sample positives are defined in as having a melt curve comparable to the internal positive control, with peak height exceeding that of the internal negative control. In a fractional analysis, as little as 1 bacterium per capillary reaction unit is directly detectable, with no enrichment step, in 35 cycles of PCR/MCA, in a total time of 53 minutes, making this instrument and technology among the very best for speed and sensitivity in screening food for pathogenic contamination.
Aims: This study aimed to determine the survival of Escherichia coli strains during steam and lactic acid decontamination interventions currently used by the beef-processing industry, and to ...determine their heat resistance. Methods and Results: Strains were grouped into cocktails of five strains each differing in their RAPD patterns for subsequent identification. Steam and lactic acid treatments on meat reduced cell counts of E. coli strain cocktails by 90-99%. The 20 slaughter plant isolates exhibited only minor variation in their resistance to steam and lactic acid treatments but were more resistant than reference strains (three strains) or isolates from live cattle (seven strains). D₆₀ values of strains from live cattle, and reference strains ranged from 0·1 to 0·5 min, in keeping with literature data. However, D₆₀ values of current slaughter plant isolates ranged between 15 for E. coli DM18.3 and 71 min AW 1.7. Cell counts of E. coli AW 1.7 were reduced by <5 log₁₀ CFU g⁻¹ in ground beef patties cooked to an internal temperature of 71°C. Conclusions: Strains of E. coli that survive cooking of ground beef to the recommended internal temperature of 71°C can be isolated from beef-processing facilities. Significance and Impact of the Study: Pathogen interventions in current commercial beef slaughter may select for extremely heat-resistant strains of E. coli.
Pressure treatment of ready-to-eat (RTE) meats extends the shelf life and reduces risks associated with
. However, pressure reduces numbers of
on ham by less than 5 log (CFU/g) and pressure effects ...on other meat microbiota are poorly documented. This study investigated the impact of pressure and RTE meat microbiota, with or without nisin and rosemary oil, on survival of
after refrigerated storage. Ham was inoculated with a 5-strain cocktail of
alone or with a cocktail of RTE meat microbiota consisting of
,
,
, and
. Products were treated at 500 MPa at 5°C for 1 or 3 min, with or without rosemary extract or nisin. Surviving cells were differentially enumerated after pressure treatment and after 4 weeks of refrigerated storage. After 4 weeks of storage, products were also analyzed by high throughput sequencing of 16S rRNA amplicons. Pressure treatment reduced counts of
by 1 to 2 log (CFU/g); inactivation of RTE meat microbiota was comparable. Counts of
increased by 1-3 log (CFU/g) during refrigerated storage. RTE meat microbiota did not influence pressure inactivation of
but prevented growth of
during refrigerated storage. Rosemary extract did not influence bacterial inactivation or growth. The combination of nisin with pressure treatment for 3 min reduced counts of
and meat microbiota by >5 log (CFU/g); after 4 weeks of storage, counts were below the detection limit. In conclusion, pressure alone does not eliminate
or other microbiota on RTE ham; however, the presence of non-pathogenic microbiota prevents growth of
on pressure treated ham and has a decisive influence on post-pressure survival and growth.
Over a one year period, swabs of 820 beef carcasses were tested for the presence of Shiga toxin-producing Escherichia coli by performing Polymerase Chain Reaction (PCR) in a novel technology termed ..."cassette PCR", in comparison to conventional liquid PCR. Cassette PCR is inexpensive and ready-to-use. The operator need only add the sample and press "go". Cassette PCR can simultaneously test multiple samples for multiple targets. Carcass swab samples were first tested for the presence of STEC genes (O157, eae, stx1 and stx2). Samples were considered to be pathogenic if positive for eae plus stx1 and/or stx2. For samples scored as pathogenic, further testing screened for 6 additional high frequency O-antigens (O26, O45, O103, O111, O121, and O145).
Of the 820 samples, 41% were pathogenic and 30% were O157 positive. Of these, 19% of samples were positive for O157 and carried potentially pathogenic E. coli (eae plus stx1 and/or stx2). Of all samples identified as carrying pathogenic E. coli, 18.9, 38.8, 41.4, 0, 36.1, and 4.1% respectively were positive for O26, O45, O103, O111, O121, and O145. To validate cassette PCR testing, conventional PCR using STEC primers was performed on each of the 820 samples. Only 148 of 3280 cassette PCR tests were discordant with conventional PCR results. However, further fractional testing showed that 110 of these 148 PCRs reflected low numbers of E. coli in the enrichment broth and could be explained as due to Poisson limiting dilution of the template, affecting both cassette PCR and conventional PCR. Of the remaining 38 discordant tests, 27 initial capillary PCRs and 10 initial conventional tests were nominally discordant between cassette and conventional PCR, perhaps reflecting human/technical error on both sides of the comparison.
Contaminated beef carcass swabs were often complex, likely harboring more than one strain of pathogenic E. coli. Cassette PCR had 98.8% concordance with parallel conventional PCR for detection of STEC genes. This indicates that cassette PCR is highly reliable for detecting multiple pathogens in beef carcass swabs from processing plants.
Carnobacterium maltaromaticum UAL307, isolated from fresh pork, exhibits potent activity against a number of gram-positive organisms, including numerous Listeria species. Three bacteriocins were ...isolated from culture supernatant, and using matrix-assisted laser desorption ionization-time of flight mass spectrometry and Edman sequencing, two of these bacteriocins were identified as piscicolin 126 and carnobacteriocin BM1, both of which have previously been described. The remaining bacteriocin, with a molecular mass of 5,862 Da, could not be sequenced by traditional methods, suggesting that the peptide was either cyclic or N-terminally blocked. This bacteriocin showed remarkable stability over a wide temperature and pH range and was unaffected by a variety of proteases. After digestion with trypsin and α-chymotrypsin, the peptide was de novo sequenced by tandem mass spectrometry and a linear sequence deduced, consisting of 60 amino acids. Based on this sequence, the molecular mass was predicted to be 5,880 Da, 18 units higher than the observed molecular mass, which suggested that the peptide has a cyclic structure. Identification of the genetic sequence revealed that this peptide is circular, formed by a covalent linkage between the N and C termini following cleavage of a 4-residue peptide leader sequence. The results of structural studies suggest that the peptide is highly structured in aqueous conditions. This bacteriocin, named carnocyclin A, is the first reported example of a circular bacteriocin produced by Carnobacterium spp.
To examine the results of a randomized clinical trial (RCT) of Teen Online Problem Solving (TOPS), an online problem solving therapy model, in increasing problem-solving skills and decreasing ...depressive symptoms and global distress for caregivers of adolescents with traumatic brain injury (TBI).
Families of adolescents aged 11-18 who sustained a moderate to severe TBI between 3 and 19 months earlier were recruited from hospital trauma registries. Participants were assigned to receive a web-based, problem-solving intervention (TOPS, n = 20), or access to online resources pertaining to TBI (Internet Resource Comparison; IRC; n = 21). Parent report of problem solving skills, depressive symptoms, global distress, utilization, and satisfaction were assessed pre- and posttreatment. Groups were compared on follow-up scores after controlling for pretreatment levels. Family income was examined as a potential moderator of treatment efficacy. Improvement in problem solving was examined as a mediator of reductions in depression and distress.
Forty-one participants provided consent and completed baseline assessments, with follow-up assessments completed on 35 participants (16 TOPS and 19 IRC). Parents in both groups reported a high level of satisfaction with both interventions. Improvements in problem solving skills and depression were moderated by family income, with caregivers of lower income in TOPS reporting greater improvements. Increases in problem solving partially mediated reductions in global distress.
Findings suggest that TOPS may be effective in improving problem solving skills and reducing depressive symptoms for certain subsets of caregivers in families of adolescents with TBI.
The locus of heat resistance (LHR) confers extreme heat resistance in
. This study explored the role of the LHR in heat and pressure resistance of
, as well as its relationship with protein folding ...and aggregation
. The role of LHR was investigated in
MG1655 and the pressure resistant
LMM1010 expressing an
fusion protein to visualize inclusion bodies by fluorescence microscopy. The expression of proteins by the LHR was determined by proteomic analysis; inclusion bodies of untreated and treated cells were also analyzed by proteomics, and by fluorescent microscopy. In total, 11 proteins of LHR were expressed: sHSP20, ClpK
, sHSP, YdfX1 and YdfX2, HdeD, KefB, Trx, PsiE, DegP, and a hypothetical protein. The proteomic analysis of inclusion bodies revealed a differential abundance of proteins related to oxidative stress in strains carrying the LHR. The LHR reduced the presence of inclusion bodies after heat or pressure treatment, indicating that proteins expressed by the LHR prevent protein aggregation, or disaggregate proteins. This phenotype of the LHR was also conferred by expression of a fragment containing only sHSP20, ClpK
, and sHSP. The LHR and the fragment encoding only sHSP20, ClpK
, and sHSP also enhanced pressure resistance in
MG1655 but had no effect on pressure resistance of
LMM1010. In conclusion, the LHR confers pressure resistance to some strains of
, and reduces protein aggregation. Pressure and heat resistance are also dependent on additional LHR-encoded functions.
Fast molecular detection methods benefit from ready-to-run lab-on-a-chip molecular assays with minimum preparation time. Detection efficiency of such methods can improve if multiple targets are ...detected simultaneously per given reaction. Detection of food pathogens, i.e. Escherichia coli (E. coli), is generally performed in two stages with the detection of multiple targets in each stage.With simultaneous testing, screening for pathogens is fast and efficient.
In this study, we show the application of multiplex PCR performed on a ready-made cassette to detect 10 targets each for eight samples known to harbor E. coli. In cassette PCR, the aluminum cassette (38.6 mm × 31.4 mm) contains 10 trenches having a total of 50 capillaries with microliter volumes of desiccated acrylamide gels holding all reagents required for the PCR including internal positive and negative controls. The gel contains LCGreen dye to detect double stranded DNA. Fluorescence monitoring allows the detection of the amplified products by melt curve analysis. In this application, each of the five capillaries in a given trench contains two of the primer sets for the detection of 10 targets in pathogenic E. coli, namely, O157, Eae, Stx1, Stx2 and six O-antigen genes. Primer specificity was confirmed. Each trench tests one sample. Eight minimally processed enriched beef carcass swab samples were analyzed for parallel detection of 10 targets within 1 h and 15 min. Samples were delivered to the capillaries by capillary forces thereby hydrating the gels. Multiplex cassette PCR results were confirmed with conventional multiplex PCRs performed in a commercial real-time PCR system.
Cassette PCR technology is ideally suited to multi-target detection of pathogens in food products. The cassette performs multiple PCR reactions in parallel, with multiplex detection of targets within each reaction unit. Cassette PCR/ melt curve analysis results for the simultaneous detection of 10 targets of pathogenic E.coli in beef carcass swab samples were confirmed with a conventional real-time PCR/ melt curve analysis as well as with agarose gel electrophoresis. Although designed for the detection of E. coli, this multiplex cassette PCR technique can be applied to any other assay where the fast detection of multiple targets is required.
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