Bacteriophages Isolated From Turkeys Infecting Diverse Salmonella Serovars.

Salmonella is one of the leading causes of foodborne illnesses worldwide. The rapid emergence of multidrug-resistant Salmonella strains has increased global concern for salmonellosis. Recent studies have shown that bacteriophages (phages) are novel and the most promising antibacterial agents for biocontrol in foods because phages specifically kill target bacteria without affecting other bacteria, do not alter organoleptic properties or nutritional quality of foods, and are safe and environmentally friendly. Due to the vast variation in Salmonella serotypes, large numbers of different and highly virulent Salmonella phages with broad host ranges are needed. This study isolated 14 Salmonella phages from turkey fecal and cecal samples. Six phages (Φ205, Φ206, Φ207, ΦEnt, ΦMont, and Φ13314) were selected for characterization. These phages were from all three families in the Caudovirales order. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that each phage had a unique structural protein profile. Each phage had a distinct host range. Φ207 and ΦEnt are both siphophages. They shared eight hosts, including seven different Salmonella serovars and one Shigella sonnei strain. These two phages showed different restriction banding patterns generated through EcoRI or HindIII digestion, but shared three bands from EcoRI digestion. ΦEnt displayed the broadest and very unusual host range infecting 11 Salmonella strains from nine serovars and three Shigella strains from two species, and thus was further characterized. The one-step growth curve revealed that ΦEnt had a short latent period (10 min) and relatively large burst size (100 PFU/infected cell). ΦEnt and its host showed better thermal stabilities in tryptic soy broth than in saline at 63 or 72°C. In the model food system (cucumber juice or beef broth), ΦEnt infection [regardless of the multiplicity of infections (MOIs) of 1, 10, and 100] resulted in more than 5-log10 reduction in Salmonella concentration within 4 or 5 h. Such high lytic activity combined with its remarkably broad and unusual host range and good thermal stability suggested that ΦEnt is a novel Salmonella phage with great potential to be used as an effective biocontrol agent against diverse Salmonella serovars in foods.

Bacteriophages Infecting Gram-Negative Bacteria in a Commercial Cucumber Fermentation.

Cucumber fermentations are one of the most important vegetable fermentations in the United States. The fermentation is usually driven by lactic acid bacteria (LAB) indigenous to fresh cucumbers. But LAB are greatly outnumbered by many Gram-negative bacteria on fresh cucumbers, which may influence the growth of LAB and the incidence of bloater defect (hollow cavities formed inside fermented cucumbers) leading to serious economic loss to the pickle industry. Rapid elimination of Gram-negative bacteria is crucial to the dominance of LAB and the reduction of bloater defect in the fermentation. Various factors can affect the viability of Gram-negative bacteria in cucumber fermentation. Bacteriophages (phages) may be one of such factors. This study explored the abundance, diversity, and functional role of phages infecting Gram-negative bacteria in a commercial cucumber fermentation. Cover brine samples were taken from a commercial fermentation tank over a 30-day period. On day 1 and day 3 of the fermentation, 39 Gram-negative bacteria and 26 independent phages were isolated. Nearly 67% of Gram-negative bacterial isolates were susceptible to phage infection. Phage hosts include Enterobacter, Citrobacter, Escherichia, Pantoea, Serratia, Leclercia, Providencia, and Pseudomonas species. About 88% of the isolated phages infected the members in the family Enterobacteriaceae and 58% of phages infected Enterobacter species. Eight phages with unique host ranges were characterized. These phages belong to the Myoviridae, Siphoviridae, or Podoviridae family and showed distinct protein profiles and DNA fingerprints. The infectivity of a phage against Enterobacter cancerogenus was evaluated in cucumber juice as a model system. The phage infection at the multiplicity of infection 1 or 100 resulted in a 5-log reduction in cell concentration within 3 h and rapidly eliminated its host. This study revealed the abundance and variety of phages infecting Gram-negative bacteria, particularly Enterobacteriaceae, in the commercial cucumber fermentation, suggesting that phages may play an important role in the elimination of Gram-negative bacteria, thereby facilitating the dominance of LAB and minimizing bloater defect. To our knowledge, this is the first report on the ecology of phages infecting Gram-negative bacteria in commercial cucumber fermentations.

Assessment of the non-lactic acid bacteria microbiota in fresh cucumbers and commercially fermented cucumber pickles brined with 6% NaCl.

Limited documentation of the cucumber fermentation microbiome has impeded the understanding of the role of microbes on the quality of finished products. We characterized the microbiome of fresh and fermented cucumber samples using culture dependent and independent techniques, with an emphasis on the non-lactic acid bacteria (non-LAB) population. Insubstantial microbiome variations were observed among fresh cucumber types with Rhizobium (31.04%), Pseudomonas (14.08%), Pantoea (9.25%), Stenotrophomonas (6.83%), and Acinetobacter (6.5%) prevailing. The relative abundance of LAB remained below 0.4% and 4.0% on fresh cucumbers and day 3 of the fermentations brined with 6% sodium chloride, respectively. Fermentation cover brine samples collected on day 1 harbored Pseudomonas, Pantoea, Stenotrophomonas, Acinetobacter, Comamonas, Wautersiella, Microbacterium, Flavobacterium, Ochrobactrum and the Enterobacteriaceae, Citrobacter, Enterobacter and Kluyvera. Plate counts for presumptive Klebsiella and Pseudomonas from fermentation cover brine samples reached 2.80 ±â€¯0.36 and 2.78 ±â€¯0.83 log of CFU/mL, respectively, in 30% and 60% of the nine tanks scrutinized with selective media. Both genera were found in cover brine samples with pH values at 4.04 ±â€¯0.15. We aim at elucidating whether the low relative abundance of non-LAB in commercial cucumber fermentations, in particular Pseudomonas and Enterobacteriaceae, impacts the quality of fermented cucumbers.

Antibacterial Activities of Wasabi against Escherichia coli O157:H7 and Staphylococcus aureus.

Escherichia coli O157:H7 and Staphylococcus aureus are two of the major pathogens frequently involved in foodborne outbreaks. Control of these pathogens in foods is essential to food safety. It is of great interest in the use of natural antimicrobial compounds present in edible plants to control foodborne pathogens as consumers prefer more natural "green" foods. Allyl isothiocyanate (AITC) is an antimicrobial compound naturally present in wasabi (Japanese horseradish) and several other edible plants. Although the antibacterial effects of pure AITC and wasabi extract (essential oil) against several bacteria have been reported, the antibacterial property of natural wasabi has not been well studied. This study investigated the antibacterial activities of wasabi as well as AITC against E. coli O157:H7 and S. aureus. Chemical analysis showed that AITC is the major isothiocyanate in wasabi. The AITC concentration in the wasabi powder used in this study was 5.91 ± 0.59 mg/g. The minimum inhibitory concentration (MIC) of wasabi against E. coli O157:H7 or S. aureus was 1% (or 10 mg/ml). Wasabi at 4% displayed higher bactericidal activity against S. aureus than against E. coli O157:H7. The MIC of AITC against either pathogen was between 10 and 100 µg/ml. AITC at 500 µg/ml was bactericidal against both pathogens while AITC at 1000 µg/ml eliminated E. coli O157:H7 much faster than S. aureus. The results from this study showed that wasabi has strong antibacterial property and has high potential to effectively control E. coli O157:H7 and S. aureus in foods. The antibacterial property along with its natural green color, unique flavor, and advantage to safeguard foods at the point of ingestion makes wasabi a promising natural edible antibacterial plant. The results from this study may be of significant interest to the food industry as they develop new and safe foods. These results may also stimulate more research to evaluate the antibacterial effect of wasabi against other foodborne pathogens and to explore other edible plants for their antimicrobial properties. To our knowledge, this is the first report on the antibacterial activity of wasabi in its natural form of consumption against E. coli O157:H7 and S. aureus.

Escherichia coli O157:H7 bacteriophage Φ241 isolated from an industrial cucumber fermentation at high acidity and salinity.

A novel phage, Φ241, specific for Escherichia coli O157:H7 was isolated from an industrial cucumber fermentation where both acidity (pH ≤ 3.7) and salinity (≥5% NaCl) were high. The phage belongs to the Myoviridae family. Its latent period was 15 min and average burst size was 53 phage particles per infected cell. The phage was able to lyse 48 E. coli O157:H7 strains, but none of the 18 non-O157 strains (including E. coli O104:H7) or the 2 O antigen-negative mutants of O157:H7 strain, 43895Δper (also lacking H7 antigen) and F12 (still expressing H7 antigen). However, the phage was able to lyse a per-complemented strain (43895ΔperComp) which expresses O157 antigen. These results indicated that phage Φ241 is specific for O157 antigen, and E. coli strains lacking O157 antigen were resistant to the phage infection, regardless of the presence or absence of H7 antigen. SDS-PAGE profile revealed at least 13 structural proteins of the phage. The phage DNA was resistant to many commonly used restriction endonucleases, suggesting the presence of modified nucleotides in the phage genome. At the multiplicity of infection of 10, 3, or 0.3, the phage caused a rapid cell lysis within 1 or 2 h, resulting in 3.5- or 4.5-log-unit reduction in cell concentration. The high lytic activity, specificity and tolerance to low pH and high salinity make phage Φ241 a potentially ideal biocontrol agent of E. coli O157:H7 in various foods. To our knowledge, this is the first report on E. coli O157:H7 phage isolated from high acidity and salinity environment.

DNA fingerprinting of lactic acid bacteria in sauerkraut fermentations.

Previous studies using traditional biochemical identification methods to study the ecology of commercial sauerkraut fermentations revealed that four species of lactic acid bacteria, Leuconostoc mesenteroides, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis, were the primary microorganisms in these fermentations. In this study, 686 isolates were collected from four commercial fermentations and analyzed by DNA fingerprinting. The results indicate that the species of lactic acid bacteria present in sauerkraut fermentations are more diverse than previously reported and include Leuconostoc citreum, Leuconostoc argentinum, Lactobacillus paraplantarum, Lactobacillus coryniformis, and Weissella sp. The newly identified species Leuconostoc fallax was also found. Unexpectedly, only two isolates of P. pentosaceus and 15 isolates of L. brevis were recovered during this study. A better understanding of the microbiota may aid in the development of low-salt fermentations, which may have altered microflora and altered sensory characteristics.

Cross-subtype T-cell immune responses induced by a human immunodeficiency virus type 1 group m consensus env immunogen.

The genetic diversity among globally circulating human immunodeficiency virus type 1 (HIV-1) strains is a serious challenge for HIV-1 vaccine design. We have generated a synthetic group M consensus env gene (CON6) for induction of cross-subtype immune responses and report here a comparative study of T-cell responses to this and natural strain env immunogens in a murine model. Three different strains of mice were immunized with CON6 as well as subtype A, B, or C env immunogens, using a DNA prime-recombinant vaccinia virus boost strategy. T-cell epitopes were mapped by gamma interferon enzyme-linked immunospot analysis using five overlapping Env peptide sets from heterologous subtype A, B, and C viruses. The CON6-derived vaccine was immunogenic and induced a greater number of T-cell epitope responses than any single wild-type subtype A, B, and C env immunogen and similar T-cell responses to a polyvalent vaccine. The responses were comparable to within-clade responses but significantly more than between-clade responses. The magnitude of the T-cell responses induced by CON6 (measured by individual epitope peptides) was also greater than the magnitude of responses induced by individual wild-type env immunogens. Though the limited major histocompatibility complex repertoire in inbred mice does not necessarily predict responses in nonhuman primates and humans, these results suggest that synthetic centralized env immunogens represent a promising approach for HIV-1 vaccine design that merits further characterization.

Antigenicity and immunogenicity of a synthetic human immunodeficiency virus type 1 group m consensus envelope glycoprotein.

Genetic variation of human immunodeficiency virus (HIV-1) represents a major obstacle for AIDS vaccine development. To decrease the genetic distances between candidate immunogens and field virus strains, we have designed and synthesized an artificial group M consensus env gene (CON6 gene) to be equidistant from contemporary HIV-1 subtypes and recombinants. This novel envelope gene expresses a glycoprotein that binds soluble CD4, utilizes CCR5 but not CXCR4 as a coreceptor, and mediates HIV-1 entry. Key linear, conformational, and glycan-dependent monoclonal antibody epitopes are preserved in CON6, and the glycoprotein is recognized equally well by sera from individuals infected with different HIV-1 subtypes. When used as a DNA vaccine followed by a recombinant vaccinia virus boost in BALB/c mice, CON6 env gp120 and gp140CF elicited gamma interferon-producing T-cell responses that recognized epitopes within overlapping peptide pools from three HIV-1 Env proteins, CON6, MN (subtype B), and Chn19 (subtype C). Sera from guinea pigs immunized with recombinant CON6 Env gp120 and gp140CF glycoproteins weakly neutralized selected HIV-1 primary isolates. Thus, the computer-generated "consensus" env genes are capable of expressing envelope glycoproteins that retain the structural, functional, and immunogenic properties of wild-type HIV-1 envelopes.