Christine E. R. Dodd

Bacterial Community Structure and Location in Stilton Cheese

ABSTRACT The microbial diversity occurring in Stilton cheese was evaluated by 16S ribosomal DNA analysis with PCR-denaturing gradient gel electrophoresis. DNA templates for PCR experiments were directly extracted from the cheese as well as bulk cells harvested from a variety of viable-count media. The variable V3 and V4-V5 regions of the 16S genes were analyzed. Closest relatives of Lactococcus lactis, Enterococcus faecalis, Lactobacillus plantarum, Lactobacillus curvatus, Leuconostoc mesenteroides, Staphylococcus equorum, and Staphylococcus sp. were identified by sequencing of the DGGE fragments. Fluorescently labeled oligonucleotide probes were developed to detect Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides in fluorescence in situ hybridization (FISH) experiments, and their specificity for the species occurring in the community of Stilton cheese was checked in FISH experiments carried out with reference cultures. The combined use of these probes and the bacterial probe Eub338 in FISH experiments on Stilton cheese sections allowed the assessment of the spatial distribution of the different microbial species in the dairy matrix. Microbial colonies of bacteria showed a differential location in the different parts of the cheese examined: the core, the veins, and the crust. Lactococci were found in the internal part of the veins as mixed colonies and as single colonies within the core. Lactobacillus plantarum was detected only underneath the surface, while Leuconostoc microcolonies were homogeneously distributed in all parts observed. The combined molecular approach is shown to be useful to simultaneously describe the structure and location of the bacterial flora in cheese. The differential distribution of species found suggests specific ecological reasons for the establishment of sites of actual microbial growth in the cheese, with implications of significance in understanding the ecology of food systems and with the aim of achieving optimization of the fermentation technologies as well as preservation of traditional products.

Application of Host-Specific Bacteriophages to the Surface of Chicken Skin Leads to a Reduction in Recovery of Campylobacter jejuni

ABSTRACT Retail poultry products are widely purported as the major infection vehicle for human campylobacteriosis. Numerous intervention strategies have sought to reduce Campylobacter contamination on broiler carcasses in the abattoir. This study reports the efficacy of bacteriophage in reducing the number of recoverable Campylobacter jejuni cells on artificially contaminated chicken skin.

Effect of Flagella on Initial Attachment of Listeria monocytogenes to Stainless Steel

ABSTRACT At 22°C a flagellin mutant of Listeria monocytogeneswas found to attach to stainless steel at levels 10-fold lower than wild-type cells, even under conditions preventing active motility. At 37°C, when flagella are not produced, attachment of both strains was identical. Therefore, flagella per se facilitate the early stage of attachment.

Structure, activity and evolution of the group I thiolactone peptide quorum‐sensing system of Staphylococcus aureus

In Staphylococcus aureus, the agr locus is responsible for controlling virulence gene expression via quorum sensing. As the blockade of quorum sensing offers a novel strategy for attenuating infection, we sought to gain novel insights into the structure, activity and turnover of the secreted staphylococcal autoinducing peptide (AIP) signal molecules. A series of analogues (including the l‐alanine and d‐amino acid scanned peptides) was synthesized to determine the functionally critical residues within the S. aureus group I AIP. As a consequence, we established that (i) the group I AIP is inactivated in culture supernatants by the formation of the corresponding methionyl sulphoxide; and (ii) the group I AIP lactam analogue retains the capacity to activate agr, suggesting that covalent modification of the AgrC receptor is not a necessary prerequisite for agr activation. Although each of the d‐amino acid scanned AIP analogues retained activity, replacement of the endocyclic amino acid residue (aspartate) located C‐terminally to the central cysteine with alanine converted the group I AIP from an activator to a potent inhibitor. The screening of clinical S. aureus isolates for novel AIP groups revealed a variant that differed from the group I AIP by a single amino acid residue (aspartate to tyrosine) in the same position defined as critical by alanine scanning. Although this AIP inhibits group I S. aureus strains, the producer strains possess a functional agr locus dependent on the endogenous peptide and, as such, constitute a fourth S. aureus AIP pheromone group (group IV). The addition of exogenous synthetic AIPs to S. aureus inhibited the production of toxic shock syndrome toxin (TSST‐1) and enterotoxin C3, confirming the potential of quorum‐sensing blockade as a therapeutic strategy.

Development of a single-reaction multiplex PCR toxin typing assay for Staphylococcus aureus strains.

ABSTRACT We describe here the development of a single-reaction multiplex PCR assay for the enterotoxin genes from Staphylococcus aureusthat utilizes a universal toxin gene primer in combination with toxin-specific primers to amplify characteristic toxin gene products. In combination with a new DNA purification method, the assay can detect enterotoxin genes A to E from a pure culture within 3 to 4 h. The test was used to characterize a diverse set of environmental S. aureus isolates, and a 99% correlation with toxin typing using standard immunological tests was found. The design of the assay allows it to be extended to include both newly characterized and as-yet-unknown toxin genes.

Isolation and Characterization of Campylobacter Bacteriophages from Retail Poultry

ABSTRACT The ability of phages to survive processing is an important aspect of their potential use in the biocontrol of Campylobacter in poultry production. To this end, we have developed a procedure to recover Campylobacter bacteriophages from chilled and frozen retail poultry and have validated the sensitivity of the method by using a characterized Campylobacter phage (i.e., NCTC 12674). By using this method, we have shown that Campylobacter phages can survive on retail chicken under commercial storage conditions. Retail chicken portions purchased in the United Kingdom were screened for the presence of endogenous Campylobacter phages. Thirty-four Campylobacter bacteriophages were isolated from 300 chilled retail chicken portions, but none could be recovered from 150 frozen chicken portions. The phage isolates were characterized according to their lytic profiles, morphology, and genome size. The free-range products were significantly more likely to harbor phages (P < 0.001 by single-factor analysis of variance) than were standard or economy products. This study demonstrates that Campylobacter bacteriophages, along with their hosts, can survive commercial poultry processing procedures and that the phages exhibited a wide range of recovery rates from chicken skin stored at 4°C.

Inimical processes : Bacterial self-destruction and sub-lethal injury

It is well recognized that exponentially growing cells are more sensitive than stationary-phase cells to inimical processes such as heating, freezing and the presence of biocides and antibiotics. This difference in resistance is currently explained by the differential expression of biosynthetic pathways, gene regulators and associated enzyme systems that provide an adaptive advantage to the stationary-phase cell. Here we describe an additional and significant element for the differential sensitivity that involves the self-destruction of exponentially growing cells. This may have implications for models that predict bacterial survival during the minimal processing of food.

Bacterial suicide through stress.

Abstract. Outside of the laboratory, bacterial cells are constantly exposed to stressful conditions, and an ability to resist those stresses is essential to their survival. However, the degree of stress required to bring about cell death varies with growth phase, amongst other parameters. Exponential phase cells are significantly more sensitive to stress than stationary phase ones, and a novel hypothesis has recently been advanced to explain this difference in sensitivity, the suicide response. Essentially, the suicide response predicts that rapidly growing and respiring bacterial cells will suffer growth arrest when subjected to relatively mild stresses, but their metabolism will continue: a burst of free-radical production results from this uncoupling of growth from metabolism, and it is this free-radical burst that is lethal to the cells, rather than the stress per se. The suicide response hypothesis unifies a variety of previously unrelated empirical observations, for instance induction of superoxide dismutase by heat shock, alkyl-hydroperoxide reductase by osmotic shock and catalase by ethanol shock. The suicide response also has major implications for current [food] processing methods.

Enzymes from isolates of Pseudomonas fluorescens involved in food spoilage

Aims: Psychrotrophic Gram‐negative bacteria, such as Pseudomonas species, pose a significant spoilage problem in refrigerated meat and dairy products due to secretion of hydrolytic enzymes, especially lipases and proteases. This study characterized the enzymes produced by strains of Pseudomonas fluorescens isolated from pasteurized milk.

The importance of RpoS in the survival of bacteria through food processing

The resistance of bacteria to environmental stresses is recognised as an increasingly important area of microbiology. In particular, the alternative sigma factor RpoS has been shown to produce greater stress resistance in stationary phase cells of Salmonella and Escherichia coli compared with those in exponential phase. Our work has shown that RpoS can be induced in exponential phase in response to a number of inimical processes used in the food industry, including changes in water activity produced using a range of humectants and preservatives. The presence of high levels of competitor cells will also lead to early induction of RpoS in Salmonella by an as yet unknown mechanism. High levels of competitor cells also provide Salmonella with an increased resistance to heat and freeze-thaw injury; the mechanism for this, however, is rpoS independent and has lead to the theory of a holistic mechanism for sub-lethal injury in respiring bacteria--the bacterial suicide response. This hypothesis predicts that sub-lethal injury occurs through the production of free radical species and not by the action of the applied inimical process per se. The demonstration of the production of a free radical burst when cells are subjected to differing types of stresses has been shown by a number of methods.