Kieran Jordan

Contribution of the indigenous microflora to the maturation of cheddar cheese

Abstract Cheddar cheeses were made from raw milk, pasteurised milk (72°C, 15 s) or milk produced from skim milk which had been microfiltered using an Alfa-Laval MFS-1 MF unit and mixed with pasteurised cream (72°C, 30 s). Microfiltration (MF) reduced the total bacterial count (TBC) by > 99% and MF cheesemilk had a lower TBC than pasteurised milk; counts of non-starter lactic acid bacteria (NSLAB) were

Environmental survival mechanisms of the foodborne pathogen Campylobacter jejuni

Campylobacter spp. continue to be the greatest cause of bacterial gastrointestinal infections in humans worldwide. They encounter many stresses in the host intestinal tract, on foods and in the environment. However, in common with other enteric bacteria, they have developed survival mechanisms to overcome these stresses. Many of the survival mechanisms used by Campylobacter spp. differ from those used by other bacteria, such as Escherichia coli and Salmonella spp. This review summarizes the mechanisms by which Campylobacter spp. adapt to stress conditions and thereby increase their ability to survive on food and in the environment.

Genome Sequence of Lactobacillus helveticus, an Organism Distinguished by Selective Gene Loss and Insertion Sequence Element Expansion

Mobile genetic elements are major contributing factors to the generation of genetic diversity in prokaryotic organisms. For example, insertion sequence (IS) elements have been shown to specifically contribute to niche adaptation by promoting a variety of genetic rearrangements. The complete genome sequence of the cheese culture Lactobacillus helveticus DPC 4571 was determined and revealed significant conservation compared to three nondairy gut lactobacilli. Despite originating from significantly different environments, 65 to 75% of the genes were conserved between the commensal and dairy lactobacilli, which allowed key niche-specific gene sets to be described. However, the primary distinguishing feature was 213 IS elements in the DPC 4571 genome, 10 times more than for the other lactobacilli. Moreover, genome alignments revealed an unprecedented level of genome stability between these four Lactobacillus species, considering the number of IS elements in the L. helveticus genome. Comparative analysis also indicated that the IS elements were not the primary agents of niche adaptation for the L. helveticus genome. A clear bias toward the loss of genes reported to be important for gut colonization was observed for the cheese culture, but there was no clear evidence of IS-associated gene deletion and decay for the majority of genes lost. Furthermore, an extraordinary level of sequence diversity exists between copies of certain IS elements in the DPC 4571 genome, indicating they may represent an ancient component of the L. helveticus genome. These data suggest a special unobtrusive relationship between the DPC 4571 genome and its mobile DNA complement.

Review of Shiga-toxin-producing Escherichia coli (STEC) and their significance in dairy production

The involvement of the pathogenic Shiga-toxin-producing Escherichia coli (STEC; also called verocytotoxic-producing E. coli or VTEC) in sporadic cases and disease outbreaks is presently increasing. Infrequent cases are due to ingestion of milk and dairy products. As ruminants are healthy carriers of STEC and most dairy products may provide these bacteria with favourable conditions for their growth, milk and dairy products are a potential source of STEC. But not all STEC serotypes are pathogens; only relatively small numbers in the entire family of STEC are pathogenic. This review focuses on the recent advances in understanding of STEC and their significance in milk and dairy products. It is intended to gather the information that is needed to understand how these bacteria are described, detected and characterised, how they contaminate milk and grow in dairy products, and how the dairy industry can prevent them from affecting the consumer.

Listeria monocytogenes in the Irish dairy farm environment.

Listeria monocytogenes is a potentially lethal foodborne pathogen commonly found in the environment. European Union hygiene legislation places responsibility for safety on primary production facilities, including farms, as part of a policy to introduce traceability throughout the food chain. This study aimed to determine the occurrence of L. monocytogenes in the Irish dairy farm environment and in particular the milking facility. Two hundred ninety-eight environmental samples were collected from 16 farms in the southern region of Ireland. A number of farms within the group supply raw milk to the unpasteurized milk cheese industry. The samples taken included cow feces, milk, silage, soil, water, etc. Samples were enriched in Listeria enrichment broth and incubated for 48 h, followed by plating on chromogenic agar Listeria Ottavani & Agosti and further incubation of the plates for 24 to 48 h. Presumptive L. monocytogenes isolates were purified and confirmed by PCR targeting the hly gene. Overall, 19% of the samples (57 of 298) were positive for L. monocytogenes. These were serotyped using conventional and PCR methods; serotypes 1/2a, 1/2b, and 4b made up 78% of the typeable isolates. A correlation was found between the level of hygiene standards on the farm and the occurrence of L. monocytogenes. There was little difference in the occurrence of L. monocytogenes between farms supplying milk to the unpasteurized milk cheese industry and those supplying milk for processing. This study demonstrates the prevalence of L. monocytogenes in the dairy farm environment and the need for good hygiene practices to prevent its entry into the food chain.

Physiological and Transcriptional Characterization of Persistent and Nonpersistent Listeria monocytogenes Isolates

ABSTRACT This study aimed to characterize physiological differences between persistent and presumed nonpersistent Listeria monocytogenes strains isolated at processing facilities and to investigate the molecular basis for this by transcriptomic sequencing. Full metabolic profiles of two strains, one persistent and one nonpersistent, were initially screened using Biologs Phenotype MicroArray (PM) technology. Based on these results, in which major differences from selected antimicrobial agents were detected, another persistent strain and two nonpersistent strains were characterized using two antimicrobial PMs. Resistance to quaternary ammonium compounds (QACs) was shown to be higher among persistent strains. Growth of persistent and nonpersistent strains in various concentrations of the QACs benzethonium chloride (BZT) and cetylpyridinium chloride (CPC) was determined. Transcriptomic sequencing of a persistent and a presumed nonpersistent strain was performed to compare gene expression among these strains in the presence and absence of BZT. Two strains, designated “frequent persisters” because they were the most frequently isolated at the processing facility, showed overall higher resistance to QACs. Transcriptome analysis showed that BZT induced a complex peptidoglycan (PG) biosynthesis response, which may play a key role in BZT resistance. Comparison of persistent and nonpersistent strains indicated that transcription of many genes was upregulated among persistent strains. This included three gene operons: pdu, cob-cbi, and eut. These genes may play a role in the persistence of L. monocytogenes outside the human host.

Listeria monocytogenes in Irish Farmhouse cheese processing environments.

Sixteen cheesemaking facilities were sampled during the production season at monthly intervals over a two-year period. Thirteen facilities were found to have samples positive for Listeria monocytogenes. Samples were divided into 4 categories; cheese, raw milk, processing environment and external to the processing environment (samples from the farm such as silage, bedding, and pooled water). In order to attempt to identify the source, persistence and putative transfer routes of contamination with the L. monocytogenes isolates, they were differentiated using PFGE and serotyping. Of the 250 isolates, there were 52 different pulsotypes. No pulsotype was found at more than one facility. Two facilities had persistent pulsotypes that were isolated on sampling occasions at least 6 months apart. Of the samples tested, 6.3% of milk, 13.1% of processing environment and 12.3% of samples external to the processing environment, respectively, were positive for L. monocytogenes. Pulsotypes found in raw milk were also found in the processing environment, however, one of the pulsotypes from raw milk was found in cheese on only one occasion. One of the pulsotypes isolated from the environment external to the processing facility was found on the surface of cheese, however, a number of them were found in the processing environment. The results suggest that the farm environment external to the processing environment may in some cases be the source of processing environment contamination with L. monocytogenes.

Growth and survival of E. coli O157:H7 during the manufacture and ripening of a smear-ripened cheese produced from raw milk

M.M. MAHER, K.N. JORDAN, M.E. UPTON AND A. COFFEY. 2001.

Induction of an adaptive tolerance response in the foodborne pathogen, Campylobacter jejuni

In this study we aimed to determine if Campylobacter had the ability to induce an adaptive tolerance response (ATR) to acid and/or aerobic conditions. Campylobacter jejuni CI 120 was grown to the appropriate phase in Brucella broth under microaerobic conditions. Cells were initially adapted to a mild stress (pH 5.5) for 5 h prior to challenge at pH 4.5, a lethal pH. Survival was examined by determining the numbers of viable cells on Campylobacter blood free selective agar base. Stationary phase cells adapted at pH 5.5 induced an ATR that enabled a 100-fold greater survival compared to an uninduced culture. Aerobic adaptation also protected the cells against acid challenge. The cross protection provided a 500-fold increase in survival when compared to unadapted cells. The incorporation of chloramphenicol during the induction period eliminated the ATR and resulted in death kinetics similar to an uninduced culture. These data suggest that Campylobacter spp. have the ability to induce an ATR to sublethal treatments, which increased their ability to withstand subsequent stresses.

Heat resistance of Lactobacillus spp. isolated from Cheddar cheese

Mesophilic Lactobacillus spp. are the dominant organisms in mature Cheddar cheese. The heat resistance of broth grown cultures of Lactobacillus plantarum DPC1919 at temperatures between 50 and 57·5 °C, Lact. plantarum DPC2102 at temperatures between 48 and 56 °C and Lact. paracasei DPC2103 at temperatures between 50 and 67·5 °C was determined. The z‐values for Lact. plantarum DPC1919, Lact. plantarum DPC2102 and Lact. paracasei DPC2103 were 6·7 °C, 6·2 °C and 5·3 °C, respectively. Lactobacillus paracasei DPC2103 showed evidence of injury and recovery, especially at higher temperatures. Milk grown cultures of strains DPC2102 and DPC2103 showed greater heat resistance than broth grown cultures, tailing of the death curves and a nonlinear z‐curve. Of the three strains, Lact. paracasei DPC2103 had the potential to survive pasteurization temperatures, whether grown in milk or broth.