Alexander Gill

Mechanisms of bactericidal action of cinnamaldehyde against Listeria monocytogenes and of Eugenol against L. monocytogenes and Lactobacillus sakei

ABSTRACT The spice oil components eugenol and cinnamaldehyde possess activity against both gram-positive and gram-negative bacteria, but the mechanisms of action remain obscure. In broth media at 20°C, 5 mM eugenol or 30 mM cinnamaldehyde was bactericidal (>1-log reduction in the number of CFU per milliliter in 1 h) to Listeria monocytogenes. At a concentration of 6 mM eugenol was bactericidal to Lactobacillus sakei, but treatment with 0.5 M cinnamaldehyde had no significant effect. To investigate the role of interference with energy generation in the mechanism of action, the cellular and extracellular ATP levels of cells in HEPES buffer at 20°C were measured. Treatment of nonenergized L. monocytogenes with 5 mM eugenol, 40 mM cinnamaldehyde, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 5 min prevented an increase in the cellular ATP concentration upon addition of glucose. Treatment of energized L. monocytogenes with 40 mM cinnamaldehyde or 10 μM CCCP caused a rapid decline in cellular ATP levels, but 5 mM eugenol had no effect on cellular ATP. Treatment of L. sakei with 10 mM eugenol prevented ATP generation by nonenergized cells and had no effect on the cellular ATP of energized cells. CCCP at a concentration of 100 μM had no significant effect on the cellular ATP of L. sakei. No significant changes in extracellular ATP were observed. Due to their rapidity, effects on energy generation clearly play a major role in the activity of eugenol and cinnamaldehyde at bactericidal concentrations. The possible mechanisms of inhibition of energy generation are inhibition of glucose uptake or utilization of glucose and effects on membrane permeability.

Micro-array for the identification of Shiga toxin-producing Escherichia coli (STEC) seropathotypes associated with Hemorrhagic Colitis and Hemolytic Uremic Syndrome in humans

A micro-array has been developed, based on the GeneDisc(R) array, for the genetic identification of 12 O-types and 7 H-types of Shiga toxin-producing Escherichia coli (STEC) including the most clinically relevant enterohemorrhagic E. coli (EHEC) serotypes. The genes selected for determination of the O antigens (rfbE(O157), wzx(O26), wzx(O103), wbd1(O111), ihp1(O145), wzx(O121), wzy(O113), wzy(O91), wzx(O104), wzy(O118), wzx(O45), and wbgN(O55)) and H-types (fliC(H2), fliC(H7), fliC(H8), fliC(H11), fliC(H19), fliC(H21), and fliC(H28)) showed a high specificity and concordance with serology. The micro-array also had a high specificity for EHEC-associated virulence factors, including Shiga toxins 1 and 2 (stx1 and stx2), intimin (eae), enterohemolysin (ehxA), serine protease (espP), catalase peroxidase (katP), the type II secretion system (etpD), subtilase cytotoxin (subA), autoagglutinating adhesin (Saa) and type III secreted effectors encoded in the genomic islands OI-122 (ent/espL2, nleB, and nleE) and OI-71 (nleF, nleH1-2, and nleA). The eae gene was detected in all typical EHEC strains, and the pattern of nle genes encoded in OI-71 and OI-122 was found to be closely associated with certain serotypes of typical EHEC and emerging EHEC strains. Virulence plasmid associated genes such as katP, espP, and etpD were more common in EHEC than in STEC strains; this supports their association with virulence. This array constitutes a valuable approach for the identification of STEC strains with a high potential for human virulence.

Surface Application of Lysozyme, Nisin, and EDTA to Inhibit Spoilage and Pathogenic Bacteria on Ham and Bologna

A study was conducted to determine if the effectiveness of an antimicrobial treatment for cooked ham and bologna would be increased or maintained when applied in a surface coating. Cooked 10-g disks of ham and bologna sausage received one of three treatments: no coating (control), coating with 0.2 g of 7% (wt/vol) gelatin gel (gel-control), or coating with 0.2 g of 7% gelatin gel containing 25.5 g/liter of lysozyme-nisin (1:3) plus 25.5 g/liter of EDTA (gel-treated). The samples were then inoculated with one of six test organisms: Brochothrix thermosphacta, Escherichia coli O157:H7, Lactobacillus sakei, Leuconostoc mesenteroides, Listeria monocytogenes, or Salmonella Typhimurium. Inoculated samples were vacuum packed and stored at 8 degrees C for 4 weeks. The antimicrobial gel treatment had an immediate bactericidal effect up to 4 log CFU/cm2 on the four gram-positive organisms tested (B. thermosphacta, Lactobacillus sakei, Leuconostoc mesenteroides, and Listeria monocytogenes) and inhibited the growth of these organisms during the 4 weeks of storage. The antimicrobial gel treatment also had a bactericidal effect on the growth of Salmonella Typhimurium during storage. The numbers of E. coli O157:H7 on ham were reduced by 2 log CFU/cm2 following treatment with both antimicrobial-containing and non-antimicrobial-containing gels during the 4-week storage period. No effect was observed on the growth of E. coli O157:H7 on bologna.

Development of a method for the detection of verotoxin-producing Escherichia coli in food.

The growing recognition of the role of non-O157 verotoxigenic Escherichia coli (VTEC) in foodborne illness underscores the importance of developing methods to detect it in the food supply. We describe here the development of a protocol for the detection, isolation, and characterization of VTEC from foods, designed for the serotype-independent enrichment, detection, and isolation of VTEC, in combination with rapid characterization of VTEC O157, O26, O103, O111, and O145. This study examined the inhibitory concentration of six antimicrobial agents used either singly or in combination for the optimal enrichment of a panel of 18 different O serogroups of VTEC in modified tryptic soy broth. Considerable variability in resistance to the different antimicrobials tested was noted among different VTEC strains. The combination enabling growth of strains of all 18 different O serogroups was vancomycin (10 μg/ml) and cefsulodin (3 μg/ml). A similar combination of antimicrobials formulated in agar plates was found beneficial in the recovery of VTEC strains from enrichment broth cultures. The efficacy of these media in the recovery of selected VTEC (O26, O103, O111, O145, and O157) from ground beef and O157 VTEC from lettuce, spinach, and apple cider was demonstrated. The selective enrichment media described herein would appear suitable for incorporation in methods for the recovery and detection of a wide range of VTEC serogroups.

Evaluation of eight agar media for the isolation of shiga toxin—Producing Escherichia coli

The growth characteristics of 96 shiga toxin-producing Escherichia coli (STEC) strains representing 36 different O-types (including priority O types O26, O45, O103, O111, O121, O145 and O157) on commercial and in-house agar media were studied. The ability of the strains to grow on agar media with varying selective supplement formulations was evaluated using MacConkey Agar (MAC); Rainbow® Agar O157 (RBA); Rainbow® Agar O157 with manufacturer-recommended selective supplements (RBA-NT); Rainbow® Agar O157 with USDA-recommended selective supplements (RBA-USDA); CHROMagar STEC™ (CH STEC); Tryptone Bile agar containing cefixime and tellurite (TBA-CT); Tryptone Bile agar containing cefixime, tellurite, eosin and methylene blue (TBA-EM); and VTEC agar. All of the strains were able to grow on MAC, RBA and VTEC agar, whereas a number of strains (including some non-O157 priority O types) were unable to grow on the highly selective media CH STEC, RBA-NT, RBA-USDA, TBA-EM and TBA-CT. Only RBA-NT and CH STEC exhibited significant inhibition of background flora from ground beef enrichment. Significant inhibition of background flora from beef trim enrichment was observed with RBA-NT, RBA-USDA, CH STEC, TBA-EM and VTEC agar. With exception of E. coli O157, several different colony morphologies were observed on the differential plating media among strains of the same O type, indicating that this colony morphology is not a reliable means of identifying target STEC. These results suggest that an approach to maximize the recovery of target STEC from beef enrichment cultures is dual plating on lesser (RBA, MAC, VTEC agar) and more highly (RBA-NT, CH STEC) selective agars.

Use of low dose e-beam irradiation to reduce E. coli O157:H7, non-O157 (VTEC) E. coli and Salmonella viability on meat surfaces

This study determined the extent that irradiation of fresh beef surfaces with an absorbed dose of 1 kGy electron (e-) beam irradiation might reduce the viability of mixtures of O157 and non-O157 verotoxigenic Escherichia coli (VTEC) and Salmonella. These were grouped together based on similar resistances to irradiation and inoculated on beef surfaces (outside flat and inside round, top and bottom muscle cuts), and then e-beam irradiated. Salmonella serovars were most resistant to 1 kGy treatment, showing a reduction of ≤1.9 log CFU/g. This treatment reduced the viability of two groups of non-O157 E. coli mixtures by ≤4.5 and ≤3.9 log CFU/g. Log reductions of ≤4.0 log CFU/g were observed for E. coli O157:H7 cocktails. Since under normal processing conditions the levels of these pathogens on beef carcasses would be lower than the lethality caused by the treatment used, irradiation at 1 kGy would be expected to eliminate the hazard represented by VTEC E. coli.

A Syst-OMICS Approach to Ensuring Food Safety and Reducing the Economic Burden of Salmonellosis

The Salmonella Syst-OMICS consortium is sequencing 4,500 Salmonella genomes and building an analysis pipeline for the study of Salmonella genome evolution, antibiotic resistance and virulence genes. Metadata, including phenotypic as well as genomic data, for isolates of the collection are provided through the Salmonella Foodborne Syst-OMICS database (SalFoS), at https://salfos.ibis.ulaval.ca/. Here, we present our strategy and the analysis of the first 3,377 genomes. Our data will be used to draw potential links between strains found in fresh produce, humans, animals and the environment. The ultimate goals are to understand how Salmonella evolves over time, improve the accuracy of diagnostic methods, develop control methods in the field, and identify prognostic markers for evidence-based decisions in epidemiology and surveillance.

The Importance of Bacterial Culture to Food Microbiology in the Age of Genomics

Culture-based and genomics methods provide different insights into the nature and behavior of bacteria. Maximizing the usefulness of both approaches requires recognizing their limitations and employing them appropriately. Genomic analysis excels at identifying bacteria and establishing the relatedness of isolates. Culture-based methods remain necessary for detection and enumeration, to determine viability, and to validate phenotype predictions made on the bias of genomic analysis. The purpose of this short paper is to discuss the application of culture-based analysis and genomics to the questions food microbiologists routinely need to ask regarding bacteria to ensure the safety of food and its economic production and distribution. To address these issues appropriate tools are required for the detection and enumeration of specific bacterial populations and the characterization of isolates for, identification, phylogenetics, and phenotype prediction.

Method for the detection of priority Shiga toxin-producing Escherichia coli in beef trim.

A method has been developed for the detection in beef trim of priority Shiga toxin-producing E. coli (STEC) strains, defined as E. coli possessing the virulence factors stx1 and/or stx2 and intimin (eae), with O serogroups O26, O45, O103, O111, O121, O145, or O157. The method is based on recovery of the target bacteria by overnight enrichment in a broth optimized for recovery of O157 and non-O157 STEC, followed by screening using multiplex PCR techniques targeting (i) stx1, stx2, and eae (STE PCR) and (ii) gene sequences associated with the seven priority O serogroups (Poly O PCR), and then direct plating of broth samples positive in both STE and Poly O PCR onto Rainbow agar. Colonies on agar media were screened batchwise for STEC by the STE PCR, and presumptive isolates were characterized using a multiplex PCR and cloth-based hybridization array system targeting key virulence and O serogroup-specific markers. Using one representative strain of each priority O serogroup individually inoculated in beef trim samples, the method exhibited a limit of detection approaching 1 to 2 viable STEC cells per 65 g. None of the uninoculated trim samples produced positive results with either of the screening PCR procedures or on analysis of colonies recovered on plating media. STEC-negative samples were readily identified by screening PCR within 24 h, with a turnaround time of fewer than 4 days for confirmation of positives. The inclusivity and exclusivity characteristics of the screening PCR techniques were verified using a total of 65 different priority STEC strains: 24 nonpriority STEC, 15 non-STEC bacteria, and only those strains bearing the targeted characteristics produced screening PCR-positive results.

Enumeration of Escherichia coli O157 in Outbreak-Associated Gouda Cheese Made with Raw Milk.

In this article, we discuss the enumerative analysis for Escherichia coli O157 in two raw milk Gouda cheese products (A and B), implicated in an outbreak of 29 cases of E. coli O157:H7 illness that occurred across Canada in 2013. Samples were enumerated for E. coli O157 by most probable number (MPN) over a period of 30 to 60 days after the end of the outbreak. Samples (55.55 g) of product A (n = 14) were analyzed at 146 to 180 days postproduction. E. coli O157 was isolated from six samples at 19.9 to 44.6 MPN/kg. The E. coli O157 concentration of product A estimated from the results of all 14 samples was 9.5 MPN/kg. Samples (55.55 g) of product B (n = 20) were analyzed at 133 to 149 days postproduction. E. coli O157 was isolated from four samples at 19.9 MPN/kg. The E. coli O157 concentration of product B estimated from the results of all 20 samples was 3.7 MPN/kg. Analysis of a 305-g sample of product A (n = 1) stored at 4°C until 306 days postproduction revealed that the E. coli O157 concentration had declined to 3.6 MPN/kg. E. coli O157 could not be isolated from 555-g samples of product B (n = 5) after 280 days postproduction. The physicochemical parameters (pH, water activity, percent moisture, and percent salt) of both cheese products were found to be in the normal range for this type of product. The results of this study demonstrate that E. coli O157 could not replicate during storage at 4°C in the products tested but was capable of survival following aging and prolonged storage. This indicates that, if contaminated, the minimum 60-day aging period, which is required for raw milk Gouda cheeses, is not sufficient in all cases to ensure that the product does not contain viable cells of E. coli O157. The results also indicate that samples sizes greater than 100 g may be required to reliably detect E. coli O157 in cheese products associated with outbreaks.