Evaluation of a Preharvest Bacteriophage Therapy for Control of Salmonella within Bovine Peripheral Lymph Nodes.

ABSTRACT: A series of proof-of-concept studies were developed to determine whether a commercial bacteriophage cocktail could be utilized for the mitigation of Salmonella in bovine peripheral lymph nodes (LNs). The first objective sought to determine whether exogenous phage could be isolated from the LNs following administration. If isolation were successful, the second objective was to determine whether the phage in the LNs could effectively reduce Salmonella. Salmonella Montevideo was inoculated intradermally at multiple sites and multiple times, followed by delivery of the phage cocktail subcutaneously in two injections around each of the right and left prescapular and subiliac LNs. At the conclusion of each study, animals were euthanized, and the popliteal, prescapular, and subiliac LNs were examined. The inoculated phage was successfully isolated from the LNs; transmission electron microscopy revealed phages in the LNs of the treated cattle, and these phages were identical to those in the cocktail. Levels of phage were higher (P < 0.01) in the prescapular and subiliac LNs in the phage-treated than in the control cattle. In subsequent studies, the protocols were modified to increase Salmonella and phage levels within the LNs. Compared with the first study, overall Salmonella levels were increased in the LNs, and phage treatment decreased (P < 0.01) Salmonella in the some of the LNs. Phage levels were numerically but not significantly increased (P = 0.12) in the treated cattle. The final study was modified, hypothesizing that a 48-h postmortem period before LN removal would facilitate phage-Salmonella interaction; however, no differences (P > 0.10) in Salmonella levels were found among treatments. Salmonella-specific phages administered to live cattle can translocate to the LNs; however, these phages had limited to no effect on Salmonella in these LNs under these experimental conditions.

Bacteriophage therapy of venous leg ulcers in humans: results of a phase I safety trial.

OBJECTIVE: This phase 1 trial set out to examine the safety of a bacteriophage-based preparation for difficult-to-treat wounds. METHOD: The intention-to-treat sample comprised 42 patients with chronic venous leg ulcers (VLUs); 39 patients completed the trial. The ulcers were treated for 12 weeks with either a saline control or bacteriophages targeted against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli. Follow-up continued until week 24. RESULTS: No adverse events were attributed to the study product. No significant difference (p>0.05) was determined between the test and control groups for frequency of adverse events, rate of healing, or frequency of healing. CONCLUSION: This study found no safety concerns with the bacteriophage treatment. Efficacy of the preparation will need to be evaluated in a phase II efficacy study. DECLARATION OF INTEREST: One of the authors (AS) holds an equity interest in Intralytix. The other authors do not have any interest in commercial activities.

Characterisation of Yersinia pestis isolates from natural foci of plague in the Republic of Georgia, and their relationship to Y. pestis isolates from other countries.

Forty Yersinia pestis isolates from endemic foci of plague in the Republic of Georgia, and six Y. pestis isolates from neighbouring former Soviet Union countries, were analysed for their biochemical and phenotypic properties, and their genetic relatedness was compared with Y. pestis strains KIM and CO92 by pulsed-field gel electrophoresis (PFGE). In addition, 11 Y. pestis isolates from the USA, together with published nucleotide sequences from Y. pestis strains KIM, CO92 and 91001, were compared with the 46 isolates in the present collection using multilocus sequence typing (MLST), based on sequence data for the 16S rRNA, hsp60, glnA, gyrB, recA, manB, thrA and tmk loci. Four virulence gene loci (caf1, lcrV, psaA and pla) were also sequenced and analysed. Two sequence types (ST1 and ST2), which differed by a single nucleotide, were identified by MLST. With the exception of a single isolate (771G), all of the Georgian Y. pestis isolates belonged to ST2. PFGE also grouped the Georgian Y. pestis isolates separately from the non-Georgian isolates. Overall, PFGE discriminated the Y. pestis isolates more effectively than MLST. The sequences of three of the four virulence genes (lcrV, psaA and pla) were identical in all Georgian and non-Georgian isolates, but the caf1 locus was represented by two allele types, with caf1 NT1 being associated with the non-Georgian isolates and caf1 NT2 being associated with the Georgian isolates. These results suggest that Georgian Y. pestis isolates are of clonal origin.

Bacteriophages as therapeutic agents.

The emergence of antibiotic resistance among pathogenic bacteria is one of the most critical problems of modern medicine, and novel, effective approaches for treating infections caused by multidrug-resistant bacteria are urgently required. In this context, one intriguing approach is to use bacteriophages (viruses that kill bacteria) to eliminate specific bacterial pathogens. Bacteriophage therapy was widely used around the world in the 1930s and 1940s, and it is still used in Eastern Europe and the former Soviet Union. However, phage therapy was all but abandoned in the West after antibiotics became widely available. Promising results from recent animal studies using phages to treat bacterial infections, together with the urgent need for novel and effective antimicrobials, should prompt additional rigorous studies to determine the value of this therapeutic approach.

Examination of bacteriophage as a biocontrol method for salmonella on fresh-cut fruit: a model study.

The preparation and distribution of fresh-cut produce is a rapidly developing industry that provides the consumer with convenient and nutritious food. However, fresh-cut fruits and vegetables may represent an increased food safety concern because of the absence or damage of peel and rind, which normally help reduce colonization of uncut produce with pathogenic bacteria. In this study, we found that Salmonella Enteritidis populations can (i) survive on fresh-cut melons and apples stored at 5 degrees C, (ii) increase up to 2 log units on fresh-cut fruits stored at 10 degrees C, and (iii) increase up to 5 log units at 20 degrees C during a storage period of 168 h. In addition, we examined the effect of lytic, Salmonella-specific phages on reducing Salmonella numbers in experimentally contaminated fresh-cut melons and apples stored at various temperatures. We found that the phage mixture reduced Salmonella populations by approximately 3.5 logs on honeydew melon slices stored at 5 and 10 degrees C and by approximately 2.5 logs on slices stored at 20 degrees C, which is greater than the maximal amount achieved using chemical sanitizers. However, the phages did not significantly reduce Salmonella populations on the apple slices at any of the three temperatures. The titer of the phage preparation remained relatively stable on melon slices, whereas on apple slices the titer decreased to nondetectable levels in 48 h at all temperatures tested. Inactivation of phages, possibly by the acidic pH of apple slices (pH 4.2 versus pH 5.8 for melon slices), may have contributed to their inability to reduce Salmonella contamination in the apple slices. Higher phage concentrations and/or the use of low-pH-tolerant phage mutants may be required to increase the efficacy of the phage treatment in reducing Salmonella contamination of fresh-cut produce with a low pH.

Improved pulsed-field gel electrophoresis for typing vancomycin-resistant enterococci.

A rapid protocol for subtyping vancomycin-resistant enterococci by pulsed-field gel electrophoresis is reported. The procedure is simple and potentially cost-effective and allows reproducible subtyping of the strains in approximately 1 day.

Yersiniae other than Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis: the ignored species.

The genus Yersinia is composed of 11 species, of which three (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica) have been exhaustively characterized. The remaining eight species (Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. bercovieri, Y. mollaretii, Y. rohdei, Y. ruckeri, and Y. aldovae) have not been studied extensively and, because of the absence of classical Yersinia virulence markers, are generally considered to be nonpathogenic. However, recent data suggest that some of these eight species may cause disease by virtue of their having virulence factors distinct from those of Y. enterocolitica. These data raise intriguing questions about the mechanisms by which these species interact with their host cells and elicit human disease.

Salmonellosis in the Republic of Georgia: using molecular typing to identify the outbreak-causing strain.

In May 1998, three large outbreaks of salmonellosis, affecting 91 persons, were identified in the Republic of Georgia. Eighteen Salmonella Typhimurium strains were characterized by arbitrary primed polymerase chain reaction and pulsed-field gel electrophoresis; the results suggested that all cases were part of a single outbreak caused by a distinct clonal strain.

Cloning and sequencing of the genes downstream of the wbf gene cluster of Vibrio cholerae serogroup O139 and analysis of the junction genes in other serogroups.

The DNA sequence of the O-antigen biosynthesis cluster (wbf) of a recently emergent pathogen, Vibrio cholerae serogroup O139, has been determined. Here we report the sequence of the genes downstream of the O139 wbfX gene and analysis of the genes flanking the wbf gene cluster in other serogroups. The gene downstream of wbfX, designated rjg (right junction gene), is predicted to be not required for O-antigen biosynthesis but appears to be a hot spot for DNA rearrangements. Several variants of the rjg gene (three different insertions and a deletion) have been found in other serogroups. DNA dot blot analysis of 106 V. cholerae strains showed the presence of the left and right junction genes, gmhD and rjg, respectively, in all strains. Further, these genes mapped to a single I-CeuI fragment in all 21 strains analyzed by pulsed-field gel electrophoresis, indicating a close linkage. The insertion sequence element IS1358, found in both O1 and O139 wb* regions, is present in 61% of the strains tested; interestingly, where present, it is predominantly linked to the wb* region. These results indicated a cassette-like organization of the wb* region, with the conserved genes (gmhD and rjg) flanking the divergent, serogroup-specific wb* genes and IS1358. A similar organization of the wb* region in other serogroups raises the possibility of the emergence of new pathogens by homologous recombination via the junction genes.

Diphtheria in the Republic of Georgia: use of molecular typing techniques for characterization of Corynebacterium diphtheriae strains.

Sixty-six Corynebacterium diphtheriae strains (62 of the gravis biotype and 4 of the mitis biotype) isolated during the Georgian diphtheria epidemic of 1993 to 1998 and 13 non-Georgian C. diphtheriae strains (10 Russian and 3 reference isolates) were characterized by (i) biotyping, (ii) toxigenicity testing with the Elek assay and PCR, (iii) the randomly amplified polymorphic DNA (RAPD) technique, and (iv) pulsed-field gel electrophoresis (PFGE). Fifteen selected strains were ribotyped. Six RAPD types and 15 PFGE patterns were identified among all strains examined, and 12 ribotypes were found among the 15 strains that were ribotyped. The Georgian epidemic apparently was caused by one major clonal group of C. diphtheriae (PFGE type A, ribotype R1), which was identical to the predominant epidemic strain(s) isolated during the concurrent diphtheria epidemic in Russia. A dendrogram based on the PFGE patterns revealed profound differences between the minor (nonpredominant) epidemic strains found in Georgia and Russia. The methodologies for RAPD typing, ribotyping, and PFGE typing of C. diphtheriae strains were improved to enable rapid and convenient molecular typing of the strains. The RAPD technique was adequate for biotype differentiation; however, PFGE and ribotyping were better (and equal to each other) at discriminating between epidemiologically related and unrelated isolates.

Production of enterotoxin by Yersinia bercovieri, a recently identified Yersinia enterocolitica-like species.

Yersinia bercovieri, a recently identified Y. enterocolitica-like species, produces a heat-stable enterotoxin (designated YbST) which has biologic activity in infant mice and increases short circuit current in Ussing chambers. Although YbST has some properties in common with the heat-stable enterotoxins of Y. enterocolitica (YST I and YST II), it appears to be a novel toxin because (i) it was not neutralized by anti-YST I antiserum, (ii) YbST-neutralizing antiserum did not neutralize YST I, and (iii) Y. bercovieri strains did not hybridize with genetic probes for yst I, yst II, and other known enterotoxins.

Analysis of clinical and environment Yersinia isolates in the Republic of Georgia.

The Center for Infectious Diseases Control, Georgian Ministry of Health, isolated 2,493 Yersinia enterocolitica and Y. enterocolitica-like strains, 22 Y. pestis strains, and 21 Y. pseudotuberculosis strains from 130,574 clinical and environmental samples. Analysis of 100 Y. enterocolitica and Y. enterocolitica-like strains showed none to be within traditional pathogenic biogroups or serogroups, and none carried genetic markers for virulence. However, some strains were enterotoxigenic in infant mice, while others were associated with prolonged carriage in adult mice.