Effect of pH and water activity on the growth limits of Listeria monocytogenes in a cheese matrix at two contamination levels.

Listeria monocytogenes can proliferate at the beginning of cheesemaking as the conditions favor growth. The objective of this study was to establish the growth limits of L. monocytogenes in a cheese matrix, in case of potential contamination of the milk prior to cheese manufacture. A semisoft laboratory scale model cheese system was made at different initial pH and water activity (a(w)) levels with a mix of two strains of L. monocytogenes. A factorial design of five pH values (5.6 to 6.5), four a(w) values (0.938 to 0.96), and two L. monocytogenes inoculation levels (1 to 20 CFU/ml and 500 to 1,000 CFU/ml) was carried out. Each combination was evaluated in six independent replicates. In order to determine if there was a dominant strain, isolated colonies from the cheeses were analyzed by pulsed-field gel electrophoresis. The data relating to growth initiation were fitted to a logistic regression model. The a(w) of milk influenced the probability of growth initiation of L. monocytogenes at both low and high contamination levels. The pH, at the concentrations tested, had a lower effect on the probability of growth initiation. At pH 6.5 and a(w) of 0.99 for low contamination levels and pH 6.5 and a(w) of 0.97 for high contamination levels, increases in population of up to 4 and 2 log were observed at low and high contamination levels, respectively. This shows that if conditions are favorable for growth initiation at the early stages of the cheesemaking process, contamination of milk, even with low numbers, could lead to L. monocytogenes populations that exceed the European Union's microbiological limit of 100 CFU/g of cheese.

Towards a one-step Enterobacter sakazakii enrichment.

AIMS: The current international standard method for detection of Enterobacter sakazakii from milk products is by the International Organization for Standardization and the International Dairy Federation documented method, a procedure involving two-step enrichment. This study aimed to assess enrichment of E. sakazakii using a one-step enrichment. METHODS AND RESULTS: Enrichment of four strains of E. sakazakii was compared using five different media, with stressed or unstressed cells, and at three levels of competing microflora, which were included to assess their effects on the positive isolation of E. sakazakii. Enrichment of milk powders, prepared by spray-drying milk seeded with E. sakazakii, was assessed using one-step enrichment for detection of E. sakazakii, followed by confirmation of positive isolates by real-time PCR. CONCLUSIONS: Current media are unsuitable for enrichment and detection of all E. sakazakii isolates, in particular, when high levels of background microflora are present in the sample matrix, and new defined media are needed for successful one-step enrichment. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings provide further analysis of one-step enrichment processes for E. sakazakii in the presence of competing microflora, and show that further formulation is needed for a universal E. sakazakii enrichment medium, with careful selection of both nutrients and selective agents.

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.

Identification of a novel stress resistance mechanism in Campylobacter jejuni.

AIM: To study stress resistance mechanisms in Campylobacter spp. METHODS AND RESULTS: Campylobacter strains were grown to the appropriate phase in Brucella broth. The cells were diluted into either cell-free spent medium (obtained by filtration of a grown culture) or a freshly prepared medium and the pH reduced to 4.5, a lethal pH value. At suitable time intervals survivors were enumerated on Campylobacter blood free selective agar base. The cell-free spent medium from mid-exponential and stationary phase had a protective effect on acid and thermal stress in Campylobacter jejuni CI 120, a natural isolate. The protective effect of the extracellular compound was not significantly inactivated by boiling, but was inactivated by proteinase. CONCLUSIONS: The present study suggests that a protein (or proteins) accumulated by C. jejuni CI 120 during growth may play an active role in the induction of stress responses and that this protein is heat stable. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that C. jejuni CI 120, a natural isolate, has the ability to use extracellular signalling mechanisms to induce tolerance to stress factors. This is a major advancement in the understanding of the physiological basis for survival of C. jejuni in the environment.

Sodium chloride enhances recovery and growth of acid-stressed E. coli O157:H7.

AIMS: Combinations of sodium chloride and acid are frequently used to inhibit growth of spoilage and pathogenic bacteria in food. The influence of differing sodium chloride, lactate and pH values on the growth of stressed and unstressed cells of a non-toxigenic strain of Escherichia coli O157:H7 was studied. METHODS AND RESULTS: At pH 5.5 or 6.0, there was little or no effect on the growth rate in the presence of lactate and/or sodium chloride, but the lag times were longer as the lactate concentration increased. At pH 5.0, in the absence of sodium chloride, increasing the lactate concentration increased the growth rate and the lag time; no growth occurred in the presence of 1.5 g 100 g(-1) lactate. In the presence of 4-6 g 100 g(-1) sodium chloride, growth occurred at 1.5 g 100 g(-1) lactate. The growth rate was similar at all lactate concentrations. CONCLUSION: The results demonstrate that the presence of sodium chloride promoted growth of E. coli O157:H7, especially under stressful conditions of low pH. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings could have implications for the use of acid and sodium chloride as a preservation treatment for the inhibition of E. coli O157:H7 in food.

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

AIMS: The behaviour of Escherichia coli O157:H7 was studied during the manufacture and ripening of a smear-ripened cheese produced from raw milk. METHODS AND RESULTS: Cheese was manufactured on a laboratory scale using milk (20 l) inoculated with E. coli O157:H7, and enumeration was carried out using CT-SMAC. From an initial level of 1.52 +/- 0.03 log cfu ml-1 in the milk (34 +/- 2 cfu ml-1), the numbers increased to 3.4 +/- 0.05 log cfu g-1 in the cheese at day 1. During ripening, the numbers decreased to <1 cfu g-1 and <10 cfu g-1 in the rind and core, respectively, after 21 days, although viable cells were detected by enrichment after 90 days. The presence of E. coli O157:H7 in the cheese was confirmed by latex agglutination and by multiplex PCR. CONCLUSION: The results indicate that the manufacturing procedure encouraged substantial growth of E. coli O157:H7 to levels that permitted survival during ripening and extended storage. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of low numbers of E. coli O157:H7 in milk, destined for raw milk cheese manufacture, could constitute a threat to the consumer.

Cloning, sequencing and expression in Escherichia coli of the primary alcohol dehydrogenase gene from Thermoanaerobacter ethanolicus JW200.

The structural gene, adhA, for a thermostable primary alcohol dehydrogenase was cloned from Thermoanaerobacter ethanolicus JW200. Constitutive expression from its own promoter was observed in Escherichia coli. The nucleotide sequence of adhA corresponded to an open reading frame of 1197 bp, encoding a polypeptide of 399 amino acids with a calculated Mr of 43 192. Amino acid sequence analysis showed 67-69% identity with alcohol dehydrogenases from two archaeal species and 29-37% identity with bacterial type III alcohol dehydrogenases. This represents the first reported cloning of an alcohol dehydrogenase from a bacterial species that is both thermostable and active against primary long-chain alcohols.

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 degrees C, Lact. plantarum DPC2102 at temperatures between 48 and 56 degrees C and Lact. paracasei DPC2103 at temperatures between 50 and 67.5 degrees C was determined. The z-values for Lact. plantarum DPC1919, Lact. Plantarum DPC2102 and Lact. paracasei DPC2103 were 6.7 degrees C, 6.2 degrees C and 5.3 degrees 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.

Survival of low-pH stress by Escherichia coli O157:H7: correlation between alterations in the cell envelope and increased acid tolerance.

Survival of a nontoxigenic isolate of Escherichia coli O157:H7 at low pH (pH 3.0) was examined over prolonged time periods for each of three population types: exponential-phase cells, stationary-phase cells, and acid-adapted exponential-phase cells. In each population, approximately 5 x 10(4) CFU ml-1 were detected after a 24-h incubation at pH 3.0. Even after 3 days at pH 3.0, significant numbers of survivors from each of the three populations could be detected. The high level of acid tolerance exhibited by these survivors was found to be quickly lost once they were transferred to conditions which permitted growth to resume, indicating that they were not mutants. Proton flux measurements on the three populations of cells revealed that the initial rates of viability loss at pH 3.0 correlated well with net proton accumulation. Cells showing a high initial rate of viability loss (exponential-phase cells) accumulated protons at the highest rate, whereas resistant populations (adapted or stationary-phase cells) accumulated protons only slowly. Differences in the protein composition of the cell envelope between the three populations were studied by two-dimensional polyacrylamide gel electrophoresis. Complex differences in the pattern of proteins expressed by each population were uncovered. The implications of these findings are discussed in the context of a possible model accounting for acid tolerance in this important food-borne pathogen.

Osmotic stress on dilution of acid injured Escherichia coli O157:H7.

To determine surviving numbers of Escherichia coli from cultures or food systems, dilution with 0.1% peptone is regularly used. Higher numbers of survivors could be obtained from an acid-treated culture if 0.5 mol l-1 sucrose was added to the 0.1% peptone. Sorbitol, glucose or sodium chloride, but not glycerol, could be used in place of sucrose. Using electron microscopy distinct differences could be seen between acid-treated and untreated cells. The osmolarity of the diluents ranged from 5 to 500 mosmol kg-1 H2O for the 0.5 mol l-1 sugar or glycerol solutions, to about 1000 mosmol kg-1 H2O for the salt solution. Maximum recovery diluent has an osmolarity of about 300 mosmol kg-1 H2O and resulted in recovery of similar numbers of injured cells as a 0.5 mol l-1 solution of sugar in 0.1% peptone. Taking into account the observed damage to acid-treated cells and the differences in osmolarity of the diluents, it is likely that dilution in 0.1% peptone imposed additional stress on the acid-injured cells which caused further cell damage. Dilution in a more osmotically stable solution alleviated this osmotic stress.

Identification of a heroin esterase in Rhodococcus sp. strain H1.

A strain of a Rhodococcus sp. (termed H1) capable of utilizing heroin as its sole carbon and energy source was isolated by selective enrichment. An inducible heroin esterase was partially purified and shown to catalyze the hydrolysis of both of the acetylester groups of heroin. The enzyme displays optimum activity at pH 8.5 and appears to be a trimer of identical subunits with an M(r) or 39,000 and a native M(r) of 120,000.

C Nuclear Magnetic Resonance Studies of Citrate and Glucose Cometabolism by Lactococcus lactis.

C nuclear magnetic resonance (C-NMR) was used to investigate the metabolism of citrate plus glucose and pyruvate plus glucose by nongrowing cells of Lactococcus lactis subsp. lactis 19B under anaerobic conditions. The metabolism of citrate plus glucose during growth was also monitored directly by in vivo NMR. Although pyruvate is a common intermediate metabolite in the metabolic pathways of both citrate and glucose, the origin of the carbon atoms in the fermentation products was determined by using selectively labeled substrates, e.g., [2,4-C]citrate, [3-C]pyruvate, and [2-C]glucose. The presence of an additional substrate caused a considerable stimulation in the rates of substrate utilization, and the pattern of end products was changed. Acetate plus acetoin and butanediol represented more than 80% (molar basis) of the end products of the metabolism of citrate (or pyruvate) alone, but when glucose was also added, 80% of the citrate (or pyruvate) was converted to lactate. This result can be explained by the activation of lactate dehydrogenase by fructose 1,6-bisphosphate, an intermediate in glucose metabolism. The effect of different concentrations of glucose on the metabolism of citrate by dilute cell suspensions was also probed by using analytical methods other than NMR. Pyruvate dehydrogenase (but not pyruvate formate-lyase) was active in the conversion of pyruvate to acetyl coenzyme A. alpha-Acetolactate was detected as an intermediate metabolite of citrate or pyruvate metabolism, and the labeling pattern of the end products agrees with the alpha-acetolactate pathway. It was demonstrated that the contribution of the acetyl coenzyme A pathway for the synthesis of diacetyl, should it exist, is lower than 10%. Evidence for the presence of internal carbon reserves in L. lactis is presented.

Microbial degradation of the morphine alkaloids. Purification and characterization of morphine dehydrogenase from Pseudomonas putida M10.

The NADP(+)-dependent morphine dehydrogenase that catalyses the oxidation of morphine to morphinone was detected in glucose-grown cells of Pseudomonas putida M10. A rapid and reliable purification procedure involving two consecutive affinity chromatography steps on immobilized dyes was developed for purifying the enzyme 1216-fold to electrophoretic homogeneity from P. putida M10. Morphine dehydrogenase was found to be a monomer of Mr 32,000 and highly specific with regard to substrates, oxidizing only the C-6 hydroxy group of morphine and codeine. The pH optimum of morphine dehydrogenase was 9.5, and at pH 6.5 in the presence of NADPH the enzyme catalyses the reduction of codeinone to codeine. The Km values for morphine and codeine were 0.46 mM and 0.044 mM respectively. The enzyme was inhibited by thiol-blocking reagents and the metal-complexing reagents 1,10-phenanthroline and 2,2'-dipyridyl, suggesting that a metal centre may be necessary for activity of the enzyme.

Microbial degradation of the morphine alkaloids: identification of morphine as an intermediate in the metabolism of morphine by Pseudomonas putida M10.

A strain of Pseudomonas putida was isolated by selective enrichment with morphine that was capable of utilising morphine as a primary source of carbon and energy for growth. Experiments with whole cells showed that both morphine and codeine, but not thebaine, could be utilised. A novel NADP-dependent dehydrogenase, morphine dehydrogenase, was purified from crude cell extracts and was shown to be capable of oxidising morphine and codeine to morphinone and codeinone, respectively. This NADP-dependent morphine dehydrogenase was not observed in any other species of pseudomonads examined and was quite distinct from the beta-hydroxysteroid dehydrogenase found in Pseudomonas testosteroni, which had previously been shown to have activity against morphine.

A small, discrete acyl carrier protein is involved in de novo fatty acid biosynthesis in Streptomyces erythraeus.

A heat-stable factor, required for de novo synthesis of fatty acids in the erythromycin-producing organism Streptomyces erythraeus, has been purified to homogeneity and identified as an acyl carrier protein (ACP). We conclude that, contrary to previous belief, fatty acid synthase in S. erythraeus more closely resembles the dissociable complex of E. coli than the tightly associated, multifunctional enzyme complex found in the related actinomycete Mycobacterium smegmatis.