Lynn M. McMullen

Occurrence of Pathogens in Raw and Ready-to-Eat Meat and Poultry Products Collected from the Retail Marketplace in Edmonton, Alberta, Canada

A total of 800 meat and poultry products were purchased from the retail marketplace in Edmonton, Alberta, Canada. The products consisted of raw ground beef, chicken legs, pork chops, and ready-to-eat fermented sausage, roast beef, processed turkey breast, chicken wieners, and beef wieners. The samples were analyzed to determine the prevalence of Shiga toxin-producing Escherichia coli, Salmonella, Campylobacter spp., and Listeria monocytogenes. Shiga toxin-producing E. coli 022: H8 was found in one raw ground beef sample. Salmonella and Campylobacter were found in 30 and 62% of raw chicken legs, respectively. L. monocytogenes was found in 52% of raw ground beef, 34% of raw chicken legs, 24% of raw pork chops, 4% of fermented sausages, 3% of processed turkey breast, 5% of beef wieners, and 3% of chicken wieners. The occurrence of pathogens in this study is similar to that in retail products in many other international locales.

Isolation and characterization of carnocyclin a, a novel circular bacteriocin produced by Carnobacterium maltaromaticum UAL307.

ABSTRACT 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.

Origin of contamination and genetic diversity of Escherichia coli in beef cattle.

ABSTRACT The possible origin of beef contamination and genetic diversity of Escherichia coli populations in beef cattle, on carcasses and ground beef, was examined by using random amplification of polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the fliC gene. E. coli was recovered from the feces of 10 beef cattle during pasture grazing and feedlot finishing and from hides, carcasses, and ground beef after slaughter. The 1,403 E. coli isolates (855 fecal, 320 hide, 153 carcass, and 75 ground beef) were grouped into 121 genetic subtypes by using the RAPD method. Some of the genetic subtypes in cattle feces were also recovered from hides, prechilled carcasses, chilled carcasses, and ground beef. E. coli genetic subtypes were shared among cattle at all sample times, but a number of transient types were unique to individual animals. The genetic diversity of the E. coli population changed over time within individual animals grazing on pasture and in the feedlot. Isolates from one animal (59 fecal, 30 hide, 19 carcass, and 12 ground beef) were characterized by the PCR-RFLP analysis of the fliC gene and were grouped into eight genotypes. There was good agreement between the results obtained with the RAPD and PCR-RFLP techniques. In conclusion, the E. coli contaminating meat can originate from cattle feces, and the E. coli population in beef cattle was highly diverse. Also, genetic subtypes can be shared among animals or can be unique to an animal, and they are constantly changing.

Microbial ecology of fresh pork stored under modified atmosphere at −1, 4.4 and 10°C

Abstract The prevalent bacteria on fresh pork packaged in modified atmosphere with elevated CO2 were determined by selection of representative colonies from the greatest dilution of meat samples. The pork samples were stored in two packaging films of different oxygen permeability at three storage temperatures. Strains were classified and those identified as lactic acid bacteria were screened for production of inhibitory substances. The types of bacteria isolated from samples stored in the two packaging films were similar. Storage temperature influenced the type of bacteria that dominated the microbial population. At 10°C the prevalent microflora consisted of aeromonads, Enterobacteriaceae and lactic acid bacteria but at 4.4 and −1°C, aeromonads, Brochothrix thermosphacta and lactic acid bacteria dominated. Listeriae were detected as part of the prevalent microflora on samples stored at −1°C, but not on samples stored at 4.4 or 10°C. Species of lactic acid bacteria dominating the microflora were influenced by growth medium. The majority of isolates taken from Plate Count agar were carnobacteria whereas those from Lactobacilli MRS agar were homofermentative lactic acid bacteria. Of the 538 lactic acid bacteria isolates screened for production of inhibitory substances, 162 strains showed deferred inhibition toward a range of lactic acid bacteria and nonlactic acid bacteria indicator strains.

Evaluation of Detection Methods for Screening Meat and Poultry Products for the Presence of Foodborne Pathogens

Rapid and molecular technologies such as enzyme-linked immunosorbent assay (ELISA), PCR, and lateral flow immunoprecipitation can reduce the time and labor involved in screening food products for the presence of pathogens. These technologies were compared with conventional culture methodology for the detection of Salmonella, Campylobacter, Listeria, and Escherichia coli O157:H7 inoculated in raw and processed meat and poultry products. Recommended protocols were modified so that the same enrichment broths used in the culture methods were also used in the ELISA, PCR, and lateral flow immunoprecipitation assays. The percent agreement between the rapid technologies and culture methods ranged from 80 to 100% depending on the pathogen detected and the method used. ELISA, PCR, and lateral flow immunoprecipitation all performed well, with no statistical difference, compared with the culture method for the detection of E. coli O157:H7. ELISA performed better for the detection of Salmonella, with sensitivity and specificity rates of 100%. PCR performed better for the detection of Campylobacter jejuni, with 100% agreement to the culture method. PCR was highly sensitive for the detection of all the foodborne pathogens tested except Listeria monocytogenes. Although the lateral flow immunoprecipitation tests were statistically different from the culture methods for Salmonella and Listeria because of false-positive results, the tests did not produce any false negatives, indicating that this method would be suitable for screening meat and poultry products for these pathogens.

Genotypic analysis of Escherichia coli recovered from product and equipment at a beef-packing plant

Aims:  To identify sources of Escherichia coli on beef by characterizing strains of the organism on animals, equipment and product at beef‐packing plant.

Interactions between meat proteins and barley (Hordeum spp.) β-glucan within a reduced-fat breakfast sausage system.

Barley β-glucan, a soluble fibre component with health benefits, has the potential to be used as a fat replacer in meat systems. Interactions between meat proteins and β-glucan gum were examined in reduced-fat (12%, w/w) sausages formulated with β-glucan at 0.3% (w/w) (0.3β-gl) and 0.8% (w/w) (0.8β-gl) levels, as well as high- and reduced-fat controls. Cooking loss results indicated that β-glucan gum held more water in cooked sausages than control gum (carboxymethyl cellulose), due to its ability to form a tighter network within the protein matrix, as shown by scanning electron microscopy. In a raw system, β-glucan gum was not as effective at retaining moisture as a stable protein network, formed by heating. Differential scanning calorimetry results showed that sausages with a higher gum to protein ratio required additional energy for protein denaturation to occur. Findings indicate that β-glucan gum increases the amount of moisture held in a cooked meat protein system, due to the physical entrapment of water, when compared to the high-fat control, but is similar to the reduced-fat formulation with added water.

Characterization of an extremely heat‐resistant Escherichia coli obtained from a beef processing facility

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.

Genetic determinants of heat resistance in Escherichia coli

Escherichia coli AW1.7 is a heat resistant food isolate and the occurrence of pathogenic strains with comparable heat resistance may pose a risk to food safety. To identify the genetic determinants of heat resistance, 29 strains of E. coli that differed in their of heat resistance were analyzed by comparative genomics. Strains were classified as highly heat resistant strains, exhibiting a D60-value of more than 6 min; moderately heat resistant strains, exhibiting a D60-value of more than 1 min; or as heat sensitive. A ~14 kb genomic island containing 16 predicted open reading frames encoding putative heat shock proteins and proteases was identified only in highly heat resistant strains. The genomic island was termed the locus of heat resistance (LHR). This putative operon is flanked by mobile elements and possesses >99% sequence identity to genomic islands contributing to heat resistance in Cronobacter sakazakii and Klebsiella pneumoniae. An additional 41 LHR sequences with >87% sequence identity were identified in 11 different species of β- and γ-proteobacteria. Cloning of the full length LHR conferred high heat resistance to the heat sensitive E. coli AW1.7ΔpHR1 and DH5α. The presence of the LHR correlates perfectly to heat resistance in several species of Enterobacteriaceae and occurs at a frequency of 2% of all E. coli genomes, including pathogenic strains. This study suggests the LHR has been laterally exchanged among the β- and γ-proteobacteria and is a reliable indicator of high heat resistance in E. coli.

Biochemical, structural, and genetic characterization of tridecaptin A₁, an antagonist of Campylobacter jejuni.

Bacillus circulans NRRL B‐30644 (now Paenibacillus terrae) was previously reported to produce SRCAM 1580, a bacteriocin active against the food pathogen Campylobacter jejuni. We have been unable to isolate SRCAM 1580, and did not find any genetic determinants in the genome of this strain. We now report the reassignment of this activity to the lipopeptide tridecaptin A1. Structural characterization of tridecaptin A1 was achieved through NMR, MS/MS and GC‐MS studies. The structure was confirmed through the first chemical synthesis of tridecaptin A1, which also revealed the stereochemistry of the lipid chain. The impact of this stereochemistry on antimicrobial activity was examined. The biosynthetic machinery responsible for tridecaptin production was identified through bioinformatic analyses. P. terrae NRRL B‐30644 also produces paenicidin B, a novel lantibiotic active against Gram‐positive bacteria. MS/MS analyses indicate that this lantibiotic is structurally similar to paenicidin A.