White plague (WP)-like diseases of tropical corals are implicated in reef decline worldwide, although their etiological cause is generally unknown. Studies thus far have focused on bacterial or eukaryotic pathogens as the source of these diseases; no studies have examined the role of viruses. Using a combination of transmission electron microscopy (TEM) and 454 pyrosequencing, we compared 24 viral metagenomes generated from Montastraea annularis corals showing signs of WP-like disease and/or bleaching, control conspecific corals, and adjacent seawater. TEM was used for visual inspection of diseased coral tissue. No bacteria were visually identified within diseased coral tissues, but viral particles and sequence similarities to eukaryotic circular Rep-encoding single-stranded DNA viruses and their associated satellites (SCSDVs) were abundant in WP diseased tissues. In contrast, sequence similarities to SCSDVs were not found in any healthy coral tissues, suggesting SCSDVs might have a role in WP disease. Furthermore, Herpesviridae gene signatures dominated healthy tissues, corroborating reports that herpes-like viruses infect all corals. Nucleocytoplasmic large DNA virus (NCLDV) sequences, similar to those recently identified in cultures of Symbiodinium (the algal symbionts of corals), were most common in bleached corals. This finding further implicates that these NCLDV viruses may have a role in bleaching, as suggested in previous studies. This study determined that a specific group of viruses is associated with diseased Caribbean corals and highlights the potential for viral disease in regional coral reef decline.
Antibiotics offer an efficient means for managing diseases caused by bacterial pathogens. However, antibiotics are typically broad spectrum and they can indiscriminately kill beneficial microbes in body habitats such as the gut, deleteriously affecting the commensal gut microbiota. In addition, many bacteria have developed or are developing resistance to antibiotics, which complicates treatment and creates significant challenges in clinical medicine. Therefore, there is a real and urgent medical need to develop alternative antimicrobial approaches that will kill specific problem-causing bacteria without disturbing a normal, and often beneficial, gut microbiota. One such potential alternative approach is the use of lytic bacteriophages for managing bacterial infections, including those caused by multidrug-resistant pathogens. In the present study, we comparatively analysed the efficacy of a bacteriophage cocktail targeting Escherichia coli with that of a broad-spectrum antibiotic (ciprofloxacin) using an in vitro model of the small intestine. The parameters examined included (i) the impact on a specific, pre-chosen targeted E. coli strain, and (ii) the impact on a selected non-targeted bacterial population, which was chosen to represent a defined microbial consortium typical of a healthy small intestine. During these studies, we also examined stability of bacteriophages against various pH and bile concentrations commonly found in the intestinal tract of humans. The bacteriophage cocktail was slightly more stable in the simulated duodenum conditions compared to the simulated ileum (0.12 vs. 0.58 log decrease in phage titers, respectively). It was equally effective as ciprofloxacin in reducing E. coli in the simulated gut conditions (2–3 log reduction), but had much milder (none) impact on the commensal, non-targeted bacteria compared to the antibiotic.
Multiple studies have explored microbial shifts in diseased or stressed corals; however, little is known about bacteriophage interactions with microbes in this context. This study characterized microbial 16S rRNA amplicons and phage metagenomes associated with Montastraea annularis corals during a concurrent white plague disease outbreak and bleaching event. Phage consortia differed between bleached and diseased tissues. Phages in the family Inoviridae were elevated in diseased or healthy tissues compared with bleached portions of diseased tissues. Microbial communities also differed between diseased and bleached corals. Bacteria in the orders Rhodobacterales and Campylobacterales were increased while Kiloniellales was decreased in diseased compared with other tissues. A network of phage-bacteria interactions was constructed of all phage strains and 11 bacterial genera that differed across health states. Phage-bacteria interactions varied in specificity: phages interacted with one to eight bacterial hosts while bacteria interacted with up to 59 phages. Six phages were identified that interacted exclusively with Rhodobacterales and Campylobacterales. These results suggest that phages have a role in controlling stress-associated bacteria, and that networks can be utilized to select potential phages for mitigating detrimental bacterial growth in phage therapy applications.
ShigaShield™ is a phage preparation composed of five lytic bacteriophages that specifically target pathogenic Shigella species found in contaminated waters and foods. In this study, we examined the efficacy of various doses (9x105-9x107 PFU/g) of ShigaShield™ in removing experimentally added Shigella on deli meat, smoked salmon, pre-cooked chicken, lettuce, melon and yogurt. The highest dose (2x107 or 9x107 PFU/g) of ShigaShield™ applied to each food type resulted in at least 1 log (90%) reduction of Shigella in all the food types. There was significant (P<0.01) reduction in the Shigella levels in all phage treated foods compared to controls, except for the lowest phage dose (9x105 PFU/g) on melon where reduction was only ca. 45% (0.25 log). The genomes of each component phage in the cocktail were fully sequenced and analyzed, and they were found not to contain any “undesirable genes” including those listed in the US Code for Federal Regulations (40 CFR Ch1). Our data suggest that ShigaShield™ (and similar phage preparations with potent lytic activity against Shigella spp.) may offer a safe and effective approach for reducing the levels of Shigella in various foods that may be contaminated with the bacterium.
Contamination of pet food with Salmonella is a serious public health concern, and several disease outbreaks have recently occurred due to human exposure to Salmonella tainted pet food. The problem is especially challenging for raw pet foods (which include raw meats, seafood, fruits, and vegetables). These foods are becoming increasingly popular because of their nutritional qualities, but they are also more difficult to maintain Salmonella-free because they lack heat-treatment. Among various methods examined to improve the safety of pet foods (including raw pet food), one intriguing approach is to use bacteriophages to specifically kill Salmonella serotypes. At least 2 phage preparations (SalmoFresh Ò and Salmonelex TM ) targeting Salmonella are already FDA cleared for commercial applications to improve the safety of human foods. However, similar preparations are not yet available for pet food applications. Here, we report the results of evaluating one such preparation (SalmoLyse Ò ) in reducing Salmonella levels in various raw pet food ingredients (chicken, tuna, turkey, cantaloupe, and lettuce). Application of SalmoLyse Ò in low (ca. 2-4£10 6 PFU/g) and standard (ca. 9£10 6 PFU/g) concentrations significantly (P < 0.01) reduced (by 60-92%) Salmonella contamination in all raw foods examined compared to control treatments. When SalmoLyse Ò -treated (ca. 2£10 7 PFU/g) dry pet food was fed to cats and dogs, it did not trigger any deleterious side effects in the pets. Our data suggest that the bacteriophage cocktail lytic for Salmonella can significantly and safely reduce Salmonella contamination in various raw pet food ingredients.
Vibrio coralliilyticus and Vibrio tubiashii are pathogens responsible for high larval oyster mortalities in shellfish hatcheries. Bacteriophage therapy was evaluated to determine its potential to remediate these mortalities. Sixteen phages against V. coralliilyticus and V. tubiashii were isolated and characterized from Hawaiian seawater. Fourteen isolates were members of the Myoviridae family and two were Siphoviridae. In proof-of-principle trials, a cocktail of five phages reduced mortalities of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) by up to 91% at 6 days post-challenge with lethal doses of V. coralliilyticus. Larvae survival depended on the oyster species, the quantity of phages and vibrios applied, and the species and strain of Vibrio. A later-generation cocktail, designated VCP300, was formulated with three lytic phages subsequently named Vibrio phage vB_VcorM-GR7B; vB_VcorM-GR11A, and vB_VcorM-GR28A (abbreviated 7B, 11A and 28A). Together, these three phages displayed host specificity toward eight V. coralliilyticus strains and a V. tubiashii strain. Larval C. gigas mortalities from V. coralliilyticus strains RE98 and OCN008 were significantly reduced by > 90% (P < 0.0001) over 6 days with phage treatment compared to untreated controls. Genomic sequencing of phages 7B, 11A and 28A revealed 207,758; 194,800; and 154,046 bp, linear DNA genomes, respectively, with the latter showing 92% similarity to V. coralliilyticus phage YC, a strain from the Great Barrier Reef, Australia. Phage 7B and 11A genomes showed little similarity to phages in the NCBI database. This study demonstrates the promising potential for phage therapy to reduce larval oyster mortalities in oyster hatcheries. IMPORTANCE Shellfish hatcheries encounter episodic outbreaks of larval oyster mortalities, jeopardizing the economic stability of the hatcheries and commercial shellfish industry. Shellfish pathogens, like Vibrio coralliilyticus and Vibrio tubiashii, have been recognized as major contributors of larval oyster mortalities in U.S. East and West Coast hatcheries for many years. This study isolated, identified, and characterized bacteriophages against these Vibrio species, and demonstrated their ability to reduce mortalities from V. coralliilyticus in larval Pacific oysters and both V. coralliilyticus and V. tubiashii in larval Eastern oysters. Phage therapy offers a promising approach in stimulating hatchery production to ensure the well-being of hatcheries and the commercial oyster trade.
The importance of domestic Alternaria exposure to allergic disease in urban communities is underrecognized relative to cockroach, dust mite, and mouse exposures. 1,2 Concentrations of Alternaria are higher in outdoor than in indoor air; however, dampness, leaks, and resident behaviors can influence fungal penetrance and secondary growth indoors. 3 Moreover, given the amount of time children spend indoors, domestic exposure may contribute more than outdoor exposure to asthma morbidity. Byproducts from fossil fuel combustion are common in urban air and are the most well-established anthropogenic environmental adjuvants of allergic sensitization. 4 Black carbon and elemental carbon (EC), indicators of combustion exposure, vary across cities such as New York (NYC) because of vehicle and residential heating sources. 5,6 Among NYC children, we previously demonstrated an interaction between exposure to combustion byproducts and cockroach allergen on cockroach sensitization 7 and an association between black carbon measured inside homes and fractional exhaled nitric oxide (FENO), a marker of airway inflammation. 5 Therefore, combustion byproducts might enhance the effect of fungal exposure on allergic disease outcomes among NYC children. We hypothesized that, among NYC children, (1) sensitization to Alternaria alternata would be associated with increased asthma symptoms, (2) domestic exposure to A alternata would be associated with increased FENO, and (3) the association with FENO would be modified by neighborhood EC concentrations. The NYC Neighborhood Asthma and Allergy Study is an asthma case-control study. Seven-to eight-year-old children were recruited through a health insurance provider primarily serving middle-income families. 5 Study details and demographic characteristics (see Table E1 in this article's Online Repository at www.jacionline.org) for children included (n 5 270) are available in this article's Online Repository at www.jacionline. org. FENO and serum IgE to inhalant allergens, including A alternata and mixed fungal species (Mx2, Phadia, Uppsala, Sweden), were measured. 5 A alternata was measured by quantitative PCR from sieved settled dust samples collected from the child's bedroom floor. 8 Children's neighborhood annual airborne EC was estimated using published data. 6 A alternata was detected in 85% of homes, ranging from less than 10 to 33,158 spore equivalents/mg dust collected (geometric mean 5 57 spores equivalents/mg), which was approximately 60% higher than was measured in a study of US homes. 8 In bivariate analyses, A alternata concentrations were associated with neighborhood asthma prevalence, housing type, carpeting, and household and neighborhood income (all P < .001; see Tables E2 and E3 in this article's Online Repository at www.jacionline.org). Higher A alternata concentrations were observed with wet mopping (P 5 .020) and inversely correlated with neighborhood EC (P 5 .029). In multivariable analyses, only the presence of carpet remained independently associated with A alternata (P 5...
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