Isolation of bacteriophages specific to bovine mastitis-causing bacteria and characterization of their lytic activity in pasteurized milk.

Background and Aim: Bovine mastitis is one of the most serious issues in dairy production. It is caused by contagious and coliform pathogens such as Staphylococcus spp., Escherichia coli, and Klebsiella pneumoniae. In addition, the emergence of drug-resistant bacteria raises urgent concerns in the field of drug treatment, thus requiring the exploration of alternative treatments. Bacteriophage therapy has been shown to be a promising alternative approach for the control of antibiotic-resistant pathogens. In this study, we aimed to isolate phages specific to contagious mastitis and coliform mastitis, characterize the isolated phages, and examine their ability to lyse bacteria in pasteurized milk samples. Materials and Methods: The Staphylococcus phage vB_Sau-RP15 isolated from raw milk in our previous study was used in this study. Other three phages, vB_Eco-RN12i1, vB_Kpn-RN14i1, and vB_Ssc-RN20i3, were isolated from wastewater using E. coli 5823, K. pneumoniae 194, and Staphylococcus sciuri MM01 as hosts, respectively. The host range and efficiency of plating (EOP) were determined following phage isolation. Moreover, the lysis activities of these phages against their hosts were investigated in pasteurized milk using a multiplicity of infections (MOIs) of 10 and 100 at 37°C. Phages were applied using individual and combination phages. Results: According to the EOP results, all phages showed high specificity to their respective hosts. They are tailed phages with distinct morphologies. Individual phage treatments in spiked pasteurized milk with their respective bacterial hosts significantly reduced the bacterial counts in both MOI conditions during the first 2 h of the treatment (approximately 1-8 log reduction compared to the control). Although these phages specifically infected only their hosts, the phage cocktail resulted in a better result compared to the use of individual phage. However, bacterial regrowth was observed in all experiments, which may be related to the development of phage-insensitive mutants. Conclusion: Our findings suggest that the application of phages could be used to treat bovine mastitis. Phage cocktail is suitable to promote the efficacy of phage treatment in pasteurized milk. However, when considering the use of phages in dairy cows, certain phage properties in raw milk and in vivo and ex vivo should be highlighted to ensure their effectiveness as biocontrol agents for bovine mastitis treatment.

A novel Staphylococcus phage, vB_Sau-RP15, and its application in contaminated milk.

The vB_Sau-RP15 phage, selected for its potential use as a phage treatment in milk, was isolated from raw milk using Staphylococcusaureus NP01 as the host. The host range test revealed that the phage was able to lyse 12 strains of Staph. aureus from raw milk. This phage was stable at 4-37°C and pH 6-9 for at least 1 h. The adsorption rate was ~78% within the first 3 min. A low frequency of phage-insensitive mutant induction (4.6 × 10-6) was observed. Genomic analyses revealed that the vB_Sau-RP15 represented a novel species in the genus Silviavirus. Even though no virulence or antibiotic resistance genes were detected, the phage genome carried lysogenic-associated genes. Phage treatments (108 PFU per ml) in pasteurized milk contaminated with low (104 CFU per ml) and high (107 CFU per ml) concentrations of Staph. aureus confirmed the proficiency of the phage in the diminishing of the number of bacterial cells at 4°C and ambient temperature. A Staphylococcus phage, vB_Sau-RP15, could be a promising agent for controlling Staph. aureus contamination in milk.

In vivo assessment of bacteriophages specific to multidrug resistant Escherichia coli on fecal bacterial counts and microbiome in nursery pigs.

Escherichia coli is the most common cause of economic loss in swine industry. Nowadays, bacteriophages have been proven as good candidates for controlling bacterial infections. In this study, 6 phages were isolated and selected based on their high efficacy against 11 stains of E. coli isolated from diarrheal pigs. Six groups of weaned piglets were assigned (control, bacterial control (BC), two phage control (PC) and two phage treatment (PT) groups). Two titers (2 × 109 PFU/animal and 2 × 1010 PFU/animal) of phage cocktails consisting of these phages were tested in the PC and PT groups via oral gavage at 24, 48, and 72 h against an E. coli cocktail (2 × 109 CFU/animal) that was given to the piglets at 0, 12, 24, and 48 h of the trial. A significant reduction of fecal E. coli counts was observed in both PT groups from day 1 to 7 following the final phage dosage when compared to those of the BC group. Microbiomes in feces obtained 24 h after the final phage administration revealed phage therapy with both dosages could restore the gut's bacterial composition. Moreover, the given phage cocktails resulted in a significantly higher average daily gain of piglets during the first few weeks in both PC groups and the PT group receiving a higher phage dosage. These findings suggest that bacteriophages might be a potential alternative to antibiotics in the treatment of pathogens. In addition, they could also be utilized to improve pig growth performance.

Characterization and complete genome analysis of a novel Escherichia phage, vB_EcoM-RPN242.

The novel Escherichia phage vB_EcoM-RPN242 was isolated using a strain of Escherichia coli originating from a diarrheic piglet as a host. The phage was able to form plaques on the E. coli lawn at 15-45 °C. Moreover, it was stable over a wide pH (4-10) and temperature (4-70 °C) range. The vB_EcoM-RPN242 genome was found to be a linear, double-stranded DNA consisting of 154,840 base pairs. There were 195 protein-encoding genes and two tRNAs detected in the genome; however, no genes associated with virulence, toxins or antimicrobial resistance were found. According to overall nucleotide sequence comparisons, vB_EcoM-RPN242 possibly represents a new species in the genus Agtrevirus.

Bacteriophage efficacy in controlling swine enteric colibacillosis pathogens: An in vitro study.

Background and Aim: Swine enteric colibacillosis caused by Escherichia coli is a major problem in the swine industry, causing diarrhea among swine and resulting in substantial financial losses. However, efforts to counter this disease are impeded by the increase in antimicrobial resistance (AMR) worldwide, so intensive research is being conducted to identify alternative treatments. This study isolated, characterized, and evaluated the efficacy of bacteriophages to control pathogens causative of swine enteric colibacillosis. Materials and Methods: Five sewage samples were collected from different areas of a swine farm in Suphanburi province, Thailand and the bacteriophages were enriched and isolated, followed by purification by the agar overlay method using E. coli RENR as the host strain. The selected phages were characterized by evaluating their morphology, while their specificity was verified by the host range test. The efficiency of plating and multiplicity of infection (MOI) were also determined. Results: Four selected phages, namely, vB_Eco-RPNE4i3, vB_Eco-RPNE6i4, vB_Eco-RPNE7i1, and vB_Eco-RPNE8i3, demonstrated different patterns of host range and phage efficiency. They significantly decreased E. coli concentration at the tested MOIs (0.01-100) from 1 h onward. However, bacterial regrowth was observed in all phage treatments. Conclusion: This study shows the potential of using phages as an alternative treatment for swine enteric colibacillosis. The obtained results demonstrated that the selected phages had a therapeutic effect against pathogens causative of swine enteric colibacillosis. Therefore, phages could be applied as an alternative treatment to control specific bacterial strains and reduce AMR arising from the overuse of antibiotics.

Selection and characterization of bacteriophages specific to Salmonella Choleraesuis in swine.

Background and Aim: Salmonella Choleraesuis is the most common serotype that causes salmonellosis in swine. Recently, the use of bacteriophages as a potential biocontrol strategy has increased. Therefore, this study aimed to isolate and characterize bacteriophages specific to S. Choleraesuis associated with swine infection and to evaluate the efficacy of individual phages and a phage cocktail against S. Choleraesuis strains in simulated intestinal fluid (SIF). Materials and Methods: Three strains of S. Choleraesuis isolated from pig intestines served as host strains for phage isolation. The other 10 Salmonella serovars were also used for the phage host range test. The antibiotic susceptibility of the bacterial strains was investigated. Water samples from natural sources and drain liquid from slaughterhouses were collected for phage isolation. The isolated phages were characterized by determining the efficiency of plating against all Salmonella strains and the stability at a temperature range (4°C-65°C) and at low pH (2.5-4.0) in simulated gastric fluids (SGFs). Furthermore, morphology and genomic restriction analyses were performed for phage classification phages. Finally, S. Choleraesuis reduction in the SIF by the selected individual phages and a phage cocktail was investigated. Results: The antibiotic susceptibility results revealed that most Salmonella strains were sensitive to all tested drugs. Salmonella Choleraesuis KPS615 was multidrug-resistant, showing resistance to three antibiotics. Nine phages were isolated. Most of them could infect four Salmonella strains. Phages vB_SCh-RP5i3B and vB_SCh-RP61i4 showed high efficiency in infecting S. Choleraesuis and Salmonella Rissen. The phages were stable for 1 h at 4°C-45°C. However, their viability decreased when the temperature increased to 65°C. In addition, most phages remained viable at a low pH (pH 2.5-4.0) for 2 h in SGF. The efficiency of phage treatment against S. Choleraesuis in SIF showed that individual phages and a phage cocktail with three phages effectively reduced S. Choleraesuis in SIF. However, the phage cocktails were more effective than the individual phages. Conclusion: These results suggest that the newly isolated phages could be promising biocontrol agents against S. Choleraesuis infection in pigs and could be orally administered. However, further in vivo studies should be conducted.

Yeast diversity and novel yeast D1/D2 sequences from corn phylloplane obtained by a culture-independent approach.

Culture-independent techniques have recently been used for evaluation of microbial diversity in the environment since it addresses the problem of unculturable microorganisms. In this study, the diversity of epiphytic yeasts from corn (Zea mays Linn.) phylloplanes in Thailand was investigated using this technique and sequence-based analysis of the D1/D2 domains of the large subunit ribosomal DNA sequences. Thirty-seven samples of corn leaf were collected randomly from 10 provinces. The DNA was extracted from leaf washing samples and the D1/D2 domains were amplified. The PCR products were cloned and then screened by colony PCR. A total of 1049 clones were obtained from 37 clone libraries. From this total, 329 clones (213 sequences) were closely related to yeast strains in the GenBank database, and they were clustered into 77 operational taxonomic units (OTUs) with a similarity threshold of 99 %. The majority of sequences (98.5 %) were classified into the phylum Basidiomycota. Sixteen known yeast species were identified. Interestingly, more than 65 % of the D1/D2 sequences obtained by this technique were suggested to be sequences from new yeast taxa. The predominant yeast sequences detected belonged to the order Ustilaginales with relative frequency of 68.0 %. The most common known yeast species detected on the leaf samples were Pseudozyma hubeiensis pro tem. and Moesziomyces antarcticus with frequency of occurrence of 24.3 and 21.6 %, respectively.

The assessment of epiphytic yeast diversity in sugarcane phyllosphere in Thailand by culture-independent method.

The diversity of epiphytic yeasts from sugarcane (Saccharum officinarum Linn.) phyllospheres in Thailand was investigated by culture-independent method based on the analysis of the D1/D2 domains of the large subunit rRNA gene sequences. Forty-five samples of sugarcane leaf were collected randomly from ten provinces in Thailand. A total of 1342 clones were obtained from 45 clone libraries. 426 clones (31.7 %) were closely related to yeast strains in the GenBank database, and they were clustered into 31 operational taxonomic units (OTUs) with a similarity threshold of 99 %. All OTU sequences were classified in phylum Basidiomycota which were closely related to 11 yeast species in seven genera including Cryptococcus flavus, Hannaella coprosmaensis, Rhodotorula taiwanensis, Jaminaea angkoreiensis, Malassezia restricta, Pseudozyma antarctica, Pseudozyma aphidis, Pseudozyma hubeiensis, Pseudozyma prolifica, Pseudozyma shanxiensis, and Sporobolomyces vermiculatus. The most predominant yeasts detected belonged to Ustilaginales with 89.4 % relative frequency and the prevalent yeast genus was Pseudozyma. However, the majority were unable to be identified as known yeast species and these sequences may represent the sequences of new yeast taxa. In addition, The OTU that closely related to P. prolifica was commonly detected in sugarcane phyllosphere.

Assessment of endophytic yeast diversity in rice leaves by a culture-independent approach.

Endophytic microorganisms inhabit internal plant tissues in the host plant without causing any symptoms or negative effects. Although the diversity of endophytes has been evaluated by both culture-dependent and culture-independent methods, less information is available on yeast communities. Therefore, in this study a culture-independent method was used to examine endophytic yeasts associated with rice leaves based on the large subunit of ribosomal DNA using a semi-nested PCR technique. Sequence analysis indicated that the colonization frequency and the relative species frequency (RF) of endophytic yeast phylotypes were 0.41 and 0.06, respectively, and the majority of the yeast phylotypes were basidiomycetous yeasts. The phylotypes were designated as five known species (Cryptococcus victoriae, Debaryomyces hansenii, Debaryomyces vindobonensis, Meyerozyma guilliermondii and Pseudozyma antarctica), together with seventeen phylotypes closest to Candida metapsilosis, Cryp. foliicola, Cryp. laurentii, Pseudozyma abaconensis, Pseudozyma aphidis and Trichosporon asahii, among which some could be novel species. The most prevalent phylotypes were those closest to Cryp. foliicola (47.5 % RF) followed by D. hansenii (22.8 % RF) and P. antarctica (16.8 % RF). The presence of the phylotypes related to species known for their potential applications as biocontrol agents and plant growth promoting hormone producers suggests that they may have valuable applications. In addition, our findings revealed the occurrence of novel phylotypes at high frequency, which should encourage extensive studies to discover novel yeast species and to understand their roles in the rice leaves.

Assessment of epiphytic yeast diversity in rice (Oryza sativa) phyllosphere in Thailand by a culture-independent approach.

The epiphytic yeast diversity in rice phyllosphere in Thailand was investigated by a culture-independent technique based on the RFLP pattern and the sequence of the D1/D2 domain of the large subunit rRNA gene. Forty-four samples of rice leaf were collected randomly from six provinces. The DNA was extracted from leaf washing samples and the D1/D2 domain was amplified using PCR technique. The PCR products were cloned and then screened by colony PCR. Of total 1121 clones, 451 clones (40.2 %) revealed the D1/D2 domain sequences closely related to sequences of yeasts in GenBank, and they were clustered into 45 operational taxonomic units (OTUs) at 99 % homology. Of total yeast related clones, 329 clones (72.9 %) were identified as nine known yeast species, which consisted of 314 clones (8 OTUs) in the phylum Basidiomycota including Bullera japonica, Pseudozyma antarctica, Pseudozyma aphidis, Sporobolomyces blumeae, Sporobolomyces carnicolor and Sporobolomyces oryzicola and 15 clones (6 OTUs) in the phylum Ascomycota including Metschnikowia koreensis, Meyerozyma guilliermondii and Wickerhamomyces anomalus. The D1/D2 sequences (122 clones) that could not be identified as known yeast species were closest to 3 and 14 species in Ascomycota and Basidiomycota, respectively, some of which may be new yeast species. The most predominant species detected was P. antarctica (42.6 %) followed by B. japonica (25.9 %) with 63.6 and 22.7 % frequency of occurrence, respectively. The results of OTU richness of each sampling location revealed that climate condition and sampling location could affect epiphytic yeast diversity in rice phyllosphere.

Multicomponent synthetic polymers with viral-mimetic chemistry for nucleic acid delivery.

The ability to deliver genetic material for therapy remains an unsolved challenge in medicine. Natural gene carriers, such as viruses, have evolved sophisticated mechanisms and modular biopolymer architectures to overcome these hurdles. Here we describe synthetic multicomponent materials for gene delivery, designed with features that mimic virus modular components and which transfect specific cell lines with high efficacy. The hierarchical nature of the synthetic carriers allows the incorporation of membrane-disrupting peptides, nucleic acid binding components, a protective coat layer, and an outer targeting ligand all in a single nanoparticle, but with functionality such that each is utilized in a specific sequence during the gene delivery process. The experimentally facile assembly suggests these materials could form a generic class of carrier systems that could be customized for many different therapeutic settings.

Combination dual responsive polypeptide vectors for enhanced gene delivery.

Variation in amino acid sequences on a disulfide-linked polypeptide backbone generates differing pK(a) vectors for DNA delivery, which release nucleic acids under reducing conditions and transfect cells with greater efficacy than non-reducible or non-variable pK(a) analogues.