Inhibition of Listeria monocytogenes by Enterococcus mundtii isolated from soil.

Two bacterial isolates with inhibitory activity against Listeria monocytogenes and Enterococcus faecalis were obtained from soil. Genotypic and phenotypic characterization identified them as Enterococcus mundtii, a species whose ability to compete with L. monocytogenes is relatively unexplored compared to other members of the genus. The thermal stability of the inhibitory factor and its sensitivity to proteolytic enzymes indicate that it is most likely a bacteriocin. Both isolates grew at comparable rates to L. monocytogenes at 5 °C and 10 °C in vitro. One isolate killed L. monocytogenes when it reached concentrations of 10(6)-10(8) CFU ml(-1). Minimum inocula of 10(6) and 10(5) CFU ml(-1) of E. mundtii were required to reduce and maintain L. monocytogenes concentrations beneath the level of detection at 5 °C and 10 °C, respectively. In situ experiments at 5 °C showed that E. mundtii inhibited the growth of L. monocytogenes on vacuum-packed cold smoked salmon during its four week shelf life. E. mundtii could, therefore, control the growth of L. monocytogenes at low temperatures, indicating a potential application in controlling this pathogen in chilled foods. To control growth of Listeria, the concentration of E. mundtii needs to be high, but it is possible that a purified bacteriocin could be used to achieve the same effect.

Control of Listeria monocytogenes growth in a ready-to-eat poultry product using a bacteriophage.

A bacteriophage (phage) that infected strains of the species Listeria monocytogenes as well as Listeria ivanovii and Listeria welshimeri, but not Listeria grayi or Listeria innocua, was isolated from sheep faeces. The phage had a contractile tail and an icosohedral head indicating that it was a myovirus, and was morphologically similar to phage A511. At 30 °C, phages added at 5.2 × 107 PFU ml⁻¹ prevented the growth in broth of L. monocytogenes present at approximately twice this concentration for 7 h, but re-growth occurred such that the concentration after 24 h incubation was similar in both control and phage-treated cultures. At the same temperature, but on the surface of vacuum-packed ready-to-eat chicken breast roll, there was an immediate 2.5 log10 CFU cm⁻² reduction in pathogen concentration following addition of phages and then re-growth. However, at a temperature reflecting that at which a chilled food might be held (5 °C), this re-growth was prevented over 21 days incubation. The data suggest a dose-dependent rapid reduction in pathogen concentration followed by no continued phage-mediated effect. These results, alongside other published data, indicate that a high concentration of phages per unit area is required to ensure significant inactivation of target pathogens on food surfaces.

Isolation and characterization of phages infecting Bacillus cereus.

AIM: To isolate and characterize bacteriophages (phages) that infect the foodborne pathogen Bacillus cereus. METHODS AND RESULTS: Two phages were isolated from soil based on their ability to form plaques on four indicator hosts including Bacillus thuringiensis subsp. israelensis, and three isolates of B. cereus. The purified phages were characterized by morphology, host range, single-step growth curves and restriction enzyme digestion profiles. The phages appeared to be of the Myoviridae family based on their structure in electron micrographs. The phages lysed bacteria of several species, produced average burst sizes of 322 and 300 phages per infected cell, and both had genomes over 90 kb. The phages were chloroform-resistant and stable at 4°C. They reduced the concentration of B. cereus in mashed potatoes by >6 log(10) CFU ml(-1) within 24 h at room temperature, when applied at a high concentration. CONCLUSIONS: The relatively narrow host range within B. cereus might mean that these phages need to be used as part of a 'cocktail' of phages for biocontrol, but their efficacy for the control of their host in food was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of biocontrol by phages of B. cereus in food.

Phage inactivation of foodborne pathogens on cooked and raw meat.

Phages infecting Salmonella Typhimurium PT160 and Campylobacter jejuni were added at a low or high (10 or 10(4)) multiplicity of infection (MOI) to either low or high (<100 or 10(4)cm(-2)) densities of host bacteria inoculated onto raw and cooked beef, and incubated at 5 and 24 degrees C to simulate refrigerated and room temperature storage. Counts of host bacteria were made throughout the incubation period, with phages being counted at the first and last sampling times. Host inactivation was variable and depended on the incubation conditions and food type. Significant host inactivations of the order of 2-3 log(10)cm(-2) at 5 degrees C and >5.9 log(10)cm(-2) at 24 degrees C were achieved compared to phage-free controls using the Salmonella phage under optimal conditions (high host cell density and MOI). These results alongside those already published indicate that phages may be useful in the control for foodborne pathogens.

Selection for plasmid post-segregational killing depends on multiple infection: evidence for the selection of more virulent parasites through parasite-level competition.

Is the virulence of parasites an outcome of optimized infection? Virulence has often been considered an inevitable consequence of parasite reproduction when the cost incurred by the parasite in reducing the fitness of its current host is offset by increased infection of new hosts. More recent models have focused on how competition occurring between parasites during co-infection might effect selection of virulence. For example, if co-infection was common, parasites with higher intrinsic growth rates might be selected, even at the expense of being optimally adapted to infect new hosts. If growth rate is positively correlated with virulence, then competition would select increased virulence. We tested these models using a plasmid-encoded virulence determinant. The virulence determinant did not contribute to the plasmid's reproduction within or between hosts. Despite this, virulent plasmids were more successful than avirulent derivatives during selection in an environment allowing within-host competition. To explain these findings we propose and test a model in which virulent parasites are selected by reducing the reproduction of competitors.

Postsegregational killing does not increase plasmid stability but acts to mediate the exclusion of competing plasmids.

Postsegregational killing (PSK) systems consist of a tightly linked toxin-antitoxin pair. Antitoxin must be continually produced to prevent the longer lived toxin from killing the cell. PSK systems on plasmids are widely believed to benefit the plasmid by ensuring its stable vertical inheritance. However, experimental tests of this "stability" hypothesis were not consistent with its predictions. We suggest an alternative hypothesis to explain the evolution of PSK: that PSK systems have been selected through benefiting host plasmids in environments where plasmids must compete during horizontal reproduction. In this "competition" hypothesis, success of PSK systems is a consequence of plasmid-plasmid competition, rather than from an adaptive plasmid-host relationship. In support of this hypothesis, a plasmid-encoded parDE PSK system mediated the exclusion of an isogenic DeltaparDE plasmid. An understanding of how PSK systems influence plasmid success may provide insight into the evolution of other determinants (e.g., antibiotic resistance and virulence) also rendering a cell potentially dependent on an otherwise dispensable plasmid.

New hypotheses on the material nature of horizontally mobile genes.

The evolutionary history of organisms is often assumed to be recorded in the structure of important molecules, such as DNA sequences. Whereas the structure of these molecules does sometimes affirm other evidence of ancestry, like fossil records, it sometimes does not. Horizontal gene transfer can distort perceptions of ancestry. Determining the impact of horizontal gene transfer on evolution has been limited by the crude tools available to detect it. Physical and genetic vectors are now known to conduct genes between organisms, even between biological kingdoms of organisms. The effects are being noticed in important molecules preserved in the genomes of organisms. This article will review the systematic bias in using molecular morphology, like DNA sequences, to infer ancestry and how this bias is the unavoidable result of the way that experimental genetics itself evolved. We present the novel hypothesis that genes usually called epigenes, like methylation patterns and prions, are infectiously transferred, sometimes using DNA as a vector, but not as a gene.

Transfer of conjugative plasmids and bacteriophage lambda occurs in the presence of antibiotics that prevent de novo gene expression.

Plasmids transferred between bacteria prevented from expressing genes by the presence of bacteriostatic antibiotics. Whereas it has long been known that de novo gene expression is not required in donor cells for conjugation, the observations reported here extend the autonomy of plasmid transfer to the early events of establishment in recipients. In addition, this phenomenon was extended to bacteriophage lambda.

Genetic evidence of protein transfer during bacterial conjugation.

DNA can be transferred from eubacteria to at least plants, fungi, and all other eubacteria by related, plasmid-mediated conjugation. Little is known about the biochemistry of intraspecies or interspecies DNA transfer. Even less is known about what other molecules may accompany the DNA, or the direct or inheritable effects on recipients of these escort molecules. This report describes a genetic assay for detecting protein transfer during conjugation. The assay monitored phage lambda released from lysogenic recipients as a result of the concomitant delivery of the Escherichia coli RecA protein and plasmid DNA. The heretofore unexpected transfer of a donor chromosome-encoded protein initiates a heritable change in the recipient without altering its genetic make-up. The mechanism of transfer could be independent of transferred DNA.

Doing the conjugative two-step: evidence of recipient autonomy in retrotransfer.

Bidirectional exchange of genetic information, called retrotransfer, during bouts of bacterial conjugation has drawn the interest of those concerned with the risk of releasing genetically engineered microbes, the fluidity of genes among species, and the mechanism of DNA transport between cells. The phenomenon has generated two models in explanation, both of which yield highly testable predictions. The first model, called the one-step, predicts that the flow of genes from recipient bacteria to donor bacteria is mechanistically distinct from, but dependent on, conjugation between donors and recipients. The second model, called the two-step, predicts that the same genetic requirements and mechanistic constraints apply to the process of gene flow from recipients to donors as for gene flow from donors to recipients. The requirement for expression of at least 10 plasmid-encoded genes in recipients, sensitivity of the reverse flow (recipient to donor) to restriction of DNA transferring from the donor, and the requirement of an additional 30-90 min for DNA to flow from recipients back to donors are predictions of the two-step model and directly refute the one-step model. Retrotransfer of genes to donors during conjugation remains genetically and physically indistinguishable from two successive rounds of conjugation between neighbors.

Isolation of a conditional suppressor of leucine auxotrophy in Saccharomyces cerevisiae.

Phenotypically and genotypically (leu2-3, 112) Leu- cells of Saccharomyces cerevisiae gave rise to small colonies on medium devoid of leucine. This only occurred on plates with a high density of Leu- cells or on medium supplemented with limiting quantities of leucine. Cells from these small colonies retained a growth advantage over their parent on limiting leucine supplements even after growth in a non-selecting (rich) medium. Therefore, the growth variants had acquired a heritable change. The phenotype was recessive and due to a change in a nuclear gene unlinked to the LEU2 locus. The phenotype provided a growth advantage only during leucine starvation; growth of the variants was indistinguishable from their parent on medium lacking the other essential supplements (histidine and uracil) required for the growth of the strain. [14C]Leucine uptake assays demonstrated that the variants were better able than their parents to accumulate leucine from their environments, and this ability extended to other hydrophobic amino acids. These results suggest that in the variants an amino acid uptake system has been derepressed rather than there having been reversion or extragenic suppression of the mutation in leucine biosynthesis. We designate the mutant gene responsible for the phenotype lup1 (for leucine uptake). The transport characteristics of the lup1 mutants suggested that LUP1 is a regulatory component of an ammonium-regulated hydrophobic amino acid uptake system.

Retrotransfer of IncP plasmid R751 from Escherichia coli maxicells: evidence for the genetic sufficiency of self-transferable plasmids for bacterial conjugation.

Gene transfer between organisms is a prime contributor to evolution. Bacterial conjugation is probably the most important mechanism by which genes are spread among prokaryotes and perhaps also contributes to eukaryotic evolution. Conjugation is mediated by plasmids. The mechanism of conjugation remains ill-understood despite progress in the identification, mapping and sequencing of genes required for plasmid transmission. All conjugation-specific genes (those required only for DNA transfer and establishment) identified to date map to plasmids. We found that IncP plasmids could enter and subsequently convert maxicells, which are trapped in a metabolic state that prevents de novo expression of chromosomal genes, into conjugative donors. This suggests that IncP plasmids encode not only necessary functions but indeed all functions specific to DNA transmission. Thus, like viruses, plasmids can convert non-viable cells into gene vectors.

Retrotransfer in Escherichia coli conjugation: bidirectional exchange or de novo mating?

DNA can be transferred among eubacteria and to plants and fungi by related, plasmid-mediated processes collectively referred to as bacterial conjugation. Conjugation occurs between cells in contact with one another and results in the unidirectional delivery of DNA from a bacterial donor to a recipient. Recent experiments that have reexamined the directionality of DNA flow during conjugation have come to different conclusions, some suggesting that genetic material also flows from recipient cells into the donor and that this process, termed retrotransfer, is likewise directed by donor-encoded functions. Given that bacteria are perhaps united with all living creatures by conjugation, the possibility of gene flow into donor bacteria during conjugation raises interesting evolutionary and biocontainment issues. Here we report that plasmid transmission from bacterial recipients to donors is not a donor-mediated event. Movement of genetic material from recipients to donors was inhibited by streptomycin, which does not inhibit the conjugative donor, indicating that retrotransfer requires gene expression in recipients. Furthermore, retrotransfer was reduced in matings mediated by plasmids that encode strong entry exclusion, to a similar degree as matings between two donors. Therefore we suggest that retrotransfer is in fact newly initiated conjugation between transconjugants and donors.