Combined effect of sodium nitrite with high-pressure treatments on the inactivation of Escherichia coli BW25113 and Listeria monocytogenes NCTC 11994.

The bactericidal effect of acidified sodium nitrite alone or when combined with high hydrostatic pressure (HHP) treatment was examined with Escherichia coli BW25113 and Listeria monocytogenes NCTC 11994. A powerful synergistic effect of HHP plus nitrite was observed at pH 4·0, but not at higher pH values. Escherichia coli hmpA and norV mutants lacking defences against nitrosative stress were more sensitive to pressure combined with acidified sodium nitrite than the wild-type strain, suggesting an involvement of nitric oxide in the bactericidal effect.

Emergence of variants with altered survival properties in stationary phase cultures of Campylobacter jejuni.

During the stationary phase of Campylobacter jejuni NCTC 11351 viable numbers fluctuate in a characteristic fashion. After reaching the maximum cell count (ca. 2 x 10(9) CFU/ml) in early stationary phase (denoted phase 1), viable numbers subsequently decrease to about 10(6) CFU/ml after 48 h and then increase again to about 10(8) CFU/ml (denoted phase 2) before decreasing once more to a value intermediate between the previous maximum and minimum values. To investigate whether the increase in viable numbers following the initial decline was due to the emergence of a new strain with a growth advantage in stationary phase analogous to the 'GASP' phenotype described in Escherichia coli [Science 259 (1993) 1757], we conducted mixed culture experiments with cells from the original culture and antibiotic-resistant marked organisms isolated from the re-growth phase. In many experiments of this type, strains isolated from phase 2 failed to out-compete the original strain and we have thus been unable to demonstrate a convincing GASP phenotype. However, strains isolated from phase 2 showed a much lower rate of viability loss in early stationary phase and a small increase in resistance to aeration, peroxide challenge and heat, indicating that the emergent strain was different from the parent. These results support the view that dynamic population changes occur during the stationary phase of C. jejuni that may play a role in the survival of this organism.

Enhanced inactivation of Listeria monocytogenes by nisin in the presence of ethanol.

AIMS: The effect of combinations of nisin and ethanol on the survival of Listeria monocytogenes was investigated. METHODS AND RESULTS: Killing by nisin was enhanced during simultaneous exposure to ethanol (2-7% v/v). For example, while 10 IU ml(-1) nisin reduced viability by 1 log unit in 20 min, a combination of this antimicrobial peptide and 5% ethanol, reduced numbers of surviving cells by 3 log units. Increasing the concentrations of either ethanol (2-7%) or nisin (10-50 IU ml(-1)) led to increased cell death with synergy being demonstrated for all combinations tested and at a range of temperatures from 5 to 37 degrees C. CONCLUSIONS: Ethanol can act synergistically with nisin to reduce the survival of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Combinations of ethanol and nisin may be feasible as an effective way of controlling this pathogen in the food processing environment.

Variation in resistance to high hydrostatic pressure and rpoS heterogeneity in natural isolates of Escherichia coli O157:H7.

Several natural isolates of Escherichia coli O157:H7 have previously been shown to exhibit stationary-phase-dependent variation in their resistance to inactivation by high hydrostatic pressure. In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure in E. coli O157:H7 and in a commensal strain. Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains. Heterogeneity was noted in the rpoS alleles of the natural isolates that may explain the differences in RpoS activity. These results are consistent with a role for rpoS in mediating resistance to high hydrostatic pressure in E. coli O157:H7.

Sensitization of Listeria monocytogenes to low pH, organic acids, and osmotic stress by ethanol.

The killing of Listeria monocytogenes following exposure to low pH, organic acids, and osmotic stress was enhanced by the addition of 5% (vol/vol) ethanol. At pH 3, for example, the presence of this agent stimulated killing by more than 3 log units in 40 min of exposure. The rate of cell death at pH 3.0 was dependent on the concentration of ethanol. Thus, while the presence 10% (vol/vol) ethanol at pH 3.0 stimulated killing by more than 3 log units in just 5 min, addition of 1.25% (vol/vol) ethanol resulted in less than 1 log unit of killing in 10 min. The ability of 5% (vol/vol) ethanol to stimulate killing at low pH and at elevated osmolarity was also dependent on the amplitude of the imposed stress, and an increase in the pH from 3.0 to 4.0 or a decrease in the sodium chloride concentration from 25 to 2.5% led to a marked reduction in the effectiveness of 5% (vol/vol) ethanol as an augmentative agent. Combinations of organic acids, low pH, and ethanol proved to be particularly effective bactericidal treatments; the most potent combination was pH 3.0, 50 mM formate, and 5 % (vol/vol) ethanol, which resulted in 5 log units of killing in just 4 min. Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane.

Survival of Campylobacter jejuni during stationary phase: evidence for the absence of a phenotypic stationary-phase response.

When Campylobacter jejuni NCTC 11351 was grown microaerobically in rich medium at 39 degrees C, entry into stationary phase was followed by a rapid decline in viable numbers to leave a residual population of 1% of the maximum number or less. Loss of viability was preceded by sublethal injury, which was seen as a loss of the ability to grow on media containing 0.1% sodium deoxycholate or 1% sodium chloride. Resistance of cells to mild heat stress (50 degrees C) or aeration was greatest in exponential phase and declined during early stationary phase. These results show that C. jejuni does not mount the normal phenotypic stationary-phase response which results in enhanced stress resistance. This conclusion is consistent with the absence of rpoS homologues in the recently reported genome sequence of this species and their probable absence from strain NCTC 11351. During prolonged incubation of C. jejuni NCTC 11351 in stationary phase, an unusual pattern of decreasing and increasing heat resistance was observed that coincided with fluctuations in the viable count. During stationary phase of Campylobacter coli UA585, nonmotile variants and those with impaired ability to form coccoid cells were isolated at high frequency. Taken together, these observations suggest that stationary-phase cultures of campylobacters are dynamic populations and that this may be a strategy to promote survival in at least some strains. Investigation of two spontaneously arising variants (NM3 and SC4) of C. coli UA585 showed that a reduced ability to form coccoid cells did not affect survival under nongrowth conditions.

Roles of Fe superoxide dismutase and catalase in resistance of Campylobacter coli to freeze-thaw stress.

We demonstrated that oxidative stress plays a role in freeze-thaw-induced killing of Campylobacter coli following analysis of mutants deficient in key antioxidant functions. Superoxide anions, but not H(2)O(2), were formed during the freeze-thaw process. However, a failure to detoxify superoxide anions may lead to spontaneous disproportionation of the radicals to H(2)O(2).

Localized reversible frameshift mutation in the flhA gene confers phase variability to flagellin gene expression in Campylobacter coli.

Phase variation of flagellin gene expression in Campylobacter coli UA585 was correlated with high-frequency, reversible insertion and deletion frameshift mutations in a short homopolymeric tract of thymine residues located in the N-terminal coding region of the flhA gene. Mutation-based phase variation in flhA may generate functional diversity in the host and environment.

Direct detection of Prevotella intermedia and P. nigrescens in suppurative oral infection by amplification of 16S rRNA gene.

A specific 16S rDNA PCR and subsequent hybridisation reaction was designed to discriminate between strains of Prevotella intermedia (n = 15) and P. nigrescens (n = 15). This technique was then used to detect the presence of these two bacterial species in acute suppurative oral infection. A total of 36 pus samples aspirated from 26 peri-apical abscesses, three root canals, three periodontal abscesses, two cases of refractory periodontitis, one cyst and one haematoma was examined. A portion of the pus sample was processed by PCR and the remainder of the specimen was subjected to routine culture. The PCR-based technique gave an identical pattern of detection of P. intermedia or P. nigrescens to that obtained by culture for 30 of the 36 specimens. Either P. intermedia or P. nigrescens was present in 14 samples and neither species was detected in 16 samples. In the remaining six samples the PCR method indicated the presence of one (n = 3) or both (n = 3) of the Prevotella species but neither or only one species was isolated by culture. It is concluded that the presence of P. intermedia and P. nigrescens in pus can be detected rapidly and specifically by direct PCR amplification of 16S rDNA. P. nigrescens was detected more frequently than P. intermedia in suppurative peri-apical infection both by culture and PCR.

Generation of a superoxide dismutase (SOD)-deficient mutant of Campylobacter coli: evidence for the significance of SOD in Campylobacter survival and colonization.

The microaerophilic nature of Campylobacter species implies an inherent sensitivity towards oxygen and its reduction products, particularly the superoxide anion. The deleterious effects of exposure to superoxide radicals are counteracted by the activity of superoxide dismutase (SOD). We have shown previously that Campylobacter coli possesses an iron cofactored SOD. The sodB gene of C. coli UA585 was insertionally inactivated by the site-specific insertion of a tetO cassette. Organisms harboring the inactivated gene failed to produce a biologically functional form of the enzyme. While the ability of this mutant to grow in aerobic conditions was unchanged relative to the parental strain, its survival was severely compromised when nongrowing cells were exposed to air. Accordingly, the SOD-deficient mutant was unable to survive for prolonged periods in model foods. Furthermore, inactivation of the sodB gene decreased the colonization potential in an experimental infection of 1-day-old chicks. In contrast, strain CK100, which is deficient in catalase activity, showed the same survival and colonization characteristics as the parental strain. These results indicate that SOD, but not catalase, is an important determinant in the ability of C. coli to survive aerobically and for optimal colonization within the chicken gut.

The use of hipO, encoding benzoylglycine amidohydrolase (hippuricase), as a reporter of gene expression in Campylobacter coli.

A novel integrative promoter probe vector which utilizes the Campylobacter jejuni hipO gene as a reporter of gene expression was developed as a genetic tool in Campylobacter coli. The utility of the system was demonstrated by coupling expression of the hipO reporter to the promoters of flaA, flaB and katA. Subsequently, expression of these genes could be monitored accurately from chromosomally-borne transcriptional fusions using a simple non-destructive colorimetric assay. The system should serve as a useful tool for studying the response of Camp. coli to environmental stresses.

Augmentation of killing of Escherichia coli O157 by combinations of lactate, ethanol, and low-pH conditions.

The acid tolerance of Escherichia coli O157:H7 strains can be overcome by addition of lactate, ethanol, or a combination of the two agents. Killing can be increased by as much as 4 log units in the first 5 min of incubation at pH 3 even for the most acid-tolerant isolates. Exponential-phase, habituated, and stationary-phase cells are all sensitive to incubation with lactate and ethanol. Killing correlates with disruption of the capacity for pH homeostasis. Habituated and stationary-phase cells can partially offset the effects of the lowering of cytoplasmic pH.

Identification of a gene encoding a methyl-accepting chemotaxis-like protein form Campylobacter coli and its use in a molecular typing scheme for campylobacters.

Using PCR amplification with degenerate primers, a gene (tlpA) form Campylobacter coli encoding a putative 63x0 kDa polypeptide which exhibited significant identity with bacterial methyl-accepting chemotaxis proteins (MCPs) was identified. A mutant containing an inactivated copy of the tlpA A gene showed a wild-type chemotactic response to all of the chemo-attractants tested. A DNA probe based on the Highly Conserved Domain (HCD) of TlpA revealed the presence of multiple copies of genes encoding MCP-like proteins in both Camp. coli and Camp. jejuni. The arrangement of restriction sites within, and proximal to, genes with homology to the HCD probe varied among strains, resulting in a high degree of polymorphism. It is demonstrated here that a DNA probe compromising the HCD region of MCP-like proteins can be used, in Southern hybridization-based assays, to provide novel information which allows the discrimination of individual strains of Camp. coli and Camp. jejuni.

Molecular characterization of a strain-specific sequence in Campylobacter jejuni.

Campylobacter jejuni strains 81116 and NCTC 11392 were shown to contain a region of DNA that was not present in other strains. This region was cloned from the chromosome of strain 81116 and the nucleotide sequence determined. It was found to contain an insert of 742 bp which was flanked by direct repeats of 45 bp. Although the nature of this sequence is unknown at present, it is clear that it only presents in certain strains of Camp. jejuni and it may therefore be useful as an epidemiological tool.

Molecular characterization of pldA, the structural gene for a phospholipase A from Campylobacter coli, and its contribution to cell-associated hemolysis.

A gene (pldA) encoding a 35.0-kDa protein with significant homology to the Escherichia coli outer membrane phospholipase was identified upstream of an operon encoding an enterochelin transport system in Campylobacter coli. The results of this study suggest that this gene encodes an outer membrane phospholipase A in C. coli. First, expression of the pldA gene product in a PldA-deficient mutant of E. coli led to the restoration of phospholipase A activity. The recombinant product also partitioned to the outer membrane, suggesting that it may be similarly located in C. coli. Second, heterologous overexpression in E. coli, followed by in vitro folding and purification of C. coli PldA, resulted in pure protein which displayed calcium-dependent lysophospholipase and phospholipase A activities in vitro. Finally, mutants of C. coli in which the pldA gene had been inactivated by allelic exchange were deficient in phospholipase A activity. Phospholipases are associated with lysis of erythrocytes by a number of bacterial pathogens. The pldA mutant was shown to have a reduced hemolytic activity compared to the wild-type strain, suggesting a role for the phospholipase A in the lysis of erythrocytes by C. coli. Since hemolysins are intimately associated with the disease-causing potential of a number of bacterial pathogens, it is likely that the phospholipase A plays some role in Campylobacter virulence.

Specific identification of the enteropathogens Campylobacter jejuni and Campylobacter coli by using a PCR test based on the ceuE gene encoding a putative virulence determinant.

A PCR method for the rapid identification and discrimination of thermophilic Campylobacter jejuni and Campylobacter coli was developed by using a gene encoding a protein involved in siderophore transport (ceuE). A nucleotide sequence divergence of approximately 13% in the ceuE genes of C. jejuni and C. coli facilitated the design of two species-specific PCR primer sets. The specificity of the PCR amplification reactions was confirmed by using two nonradioactively labelled species-specific internal oligonucleotide hybridization probes for each of these species.

Integration of heterologous plasmid DNA into multiple sites on the genome of Campylobacter coli following natural transformation.

The efficiency of homologous recombination in Campylobacter coli following the introduction of DNA by natural transformation was determined by using a series of nonreplicating integrative vectors containing DNA fragments derived from the C. coli catalase gene. Homologous recombination occurred with as little as 286 homologous bp present and was not detected when 270 bases of homology was provided. Instead, when plasmids with little or no homology to the chromosome were introduced by natural transformation, the vector DNA became chromosomally integrated at random sites scattered throughout the C. coli genome. Southern analysis and nucleotide sequencing revealed that recombination had occurred between nonhomologous sequences and can therefore be described as illegitimate. There were at least five different recombination sites on plasmid pSP105. The ability of C. coli to acquire heterologous plasmids by natural transformation, and maintain them by chromosomal integration following illegitimate recombination, has fascinating implications for the genomic diversity and evolution of this species.

Enterochelin acquisition in Campylobacter coli: characterization of components of a binding-protein-dependent transport system.

Siderophore-mediated iron uptake systems play a central role in the pathogenesis of infection for many bacterial pathogens. Campylobacter species are not thought to produce siderophores, yet they are able to utilize both ferrichrome and enterochelin as sources of iron. Part of an operon named ceuBCDE, encoding components of a periplasmic binding-protein-dependent transport (PBT) system for the uptake of a ferric siderophore from Campylobacter coli, was cloned directly into Escherichia coli using a plasmid rescue technique. Phenotypic and genetic analyses of this system showed it to comprise two hydrophobic integral membrane proteins, CeuB (35.5 kDa) and CeuC (34.8 kDa), which may form the cytoplasmic membrane permease, an ATP-binding protein, CeuD (28.8 kDa), and a periplasmic substrate-binding protein, CeuE (34.5 kDa). In vivo labelling studies using [3H]palmitate demonstrated that CeuE, the periplasmic binding protein, is expressed as a lipoprotein in C. coli, which is unusual for a Gram-negative PBT system. Mutants of C. coli, defective in components of the transport mechanism, were severely impaired in the ability to utilize enterochelin as an iron source suggesting that this siderophore is a substrate for the transport system. This is the first molecular characterization of a PBT system in Campylobacter species.

Targeted and random mutagenesis of the Campylobacter coli chromosome with integrational plasmid vectors.

A number of integrational vectors were developed for use as genetic tools in the food-borne pathogen Campylobacter coli. Integration of the plasmids occurred following genetic recombination via a Campbell-like mechanism. For an integrative plasmid containing a DNA fragment internal to the C. coli catalase gene, the insertion was mutagenic and led to a catalase-deficient phenotype. A procedure for generating random mutations in the C. coli chromosome, with these suicide-plasmids, was developed. In addition, the construction and utility of an integrable plasmid for generating transcriptional fusions to a cat reporter gene is described.

Molecular characterization of a Campylobacter jejuni lipoprotein with homology to periplasmic siderophore-binding proteins.

A genomic library of Campylobacter jejuni (NCTC 11351) was used to identify genes which could confer a hemolytic phenotype to Escherichia coli. Accordingly, when transformants were screened on blood plates, hemolytic colonies appeared at a frequency of 3 x 10(-4). The gene conferring the hemolytic activity was identified by subcloning and was found to be responsible for the phenotype of all hemolytic transformants isolated. The open reading frame conferring this activity encodes a protein of 36,244 Da with a typical endopeptidase type II leader sequence. The protein is modified with palmitic acid when it is processed in E. coli, confirming that it is a typical lipoprotein. The deduced gene product of 329 amino acids has significant homology to the group of solute binding proteins from periplasmic-binding-protein-dependent transport systems for ferric siderophores, including the FatB protein from Vibrio anguillarium and the FhuD protein from Bacillus subtilis. In particular, the protein contained the signature sequence for siderophore-binding proteins, suggesting that the protein may be the siderophore-binding protein component of an iron acquisition system of C. jejuni.