Encapsulation Strategies of Bacteriophage (Felix O1) for Oral Therapeutic Application.

Due to emerging antibiotic-resistant strains among the pathogens, a variety of strategies, including therapeutic application of bacteriophages, have been suggested as a possible alternative to antibiotics in food animal production. As pathogen-specific biocontrol agents, bacteriophages are being studied intensively. Primarily their applications in the food industry and animal production have been recognized in the USA and Europe, for pathogens including Salmonella, Campylobacter, Escherichia coli, and Listeria. However, the viability of orally administered phage may rapidly reduce under the harsh acidic conditions of the stomach, presence of enzymes and bile. It is evident that bacteriophages, intended for phage therapy by oral administration, require efficient protection from the acidic environment of the stomach and should remain active in the animal's gastrointestinal tract where pathogen colonizes. Encapsulation of phages by spray drying or extrusion methods can protect phages from the simulated hostile gut conditions and help controlled release of phages to the digestive system when appropriate formulation strategy is implemented.

Temporal distribution of encapsulated bacteriophages during passage through the chick gastrointestinal tract.

Encapsulation of bacteriophages ("phage") protects phage against environmental deactivation and provides a product that is easy to handle for storage and application with animal feed as an antibiotic alternative. The objective of this study was to evaluate an orally administered, encapsulated phage for efficient phage release in the gastrointestinal tract (GIT) of young chicks receiving feed. An optimized formulation that consisted of 0.8% low molecular weight (MW) alginate, 2% ultra-low molecular weight alginate and 3% whey protein completely released the encapsulated phage within 60 min under simulated intestinal conditions. This product was given to broiler chicks to determine passage time and distribution of the viable phage within the GIT. Following a single oral dose of 109 plaque-forming unit (PFU)/chick, the major portion (peak concentration) of the encapsulated phage passed through the chick's GIT and was detected in the feces within 4 h, with low levels being continuously excreted for up to 24 h. In comparison, the passage of free phage through the GIT occurred faster as indicated by a peak concentration in feces after 1.5 h. In assessing the temporal phage distribution, both encapsulated and free phage treatments showed no apparent difference, both having low levels of 102 to 106 PFU/g of contents along the entire GIT after 1, 2 and 4 h. These low concentrations recovered in vivo led us to examine various exposure conditions (with feed, fecal material, and buffer solutions) that were suspected to have affected phage viability/infectivity during oral delivery, sample recovery, and enumeration by plaque assay. Results showed that the exposure conditions examined did not significantly reduce phage viability and could not account for the observed low phage levels following oral administration in chicks that are on feed. In conclusion, an oral encapsulated phage dose can take more than 4 h to completely move through the GIT of young chicks. Thus, repeated or higher doses may be necessary to attain higher phage concentrations in the GIT.

Supersize me: Cronobacter sakazakii phage GAP32.

Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB_CsaM_GAP32 (GAP32), which possesses the second largest sequenced phage genome (358,663bp). A total of 571 genes including 545 protein coding sequences and 26 tRNAs were identified, thus more genes than in the smallest bacterium, Mycoplasma genitalium G37. BLASTP and HHpred searches, together with proteomic analyses reveal that only 23.9% of the putative proteins have defined functions. Some of the unique features of this phage include: a chromosome condensation protein, two copies of the large subunit terminase, a predicted signal-arrest-release lysin; and an RpoD-like protein, which is possibly involved in the switch from immediate early to delayed early transcription. Its closest relatives are all extremely large myoviruses, namely coliphage PBECO4 and Klebsiella phage vB_KleM-RaK2, with whom it shares approximately 44% homologous proteins. Since the homologs are not evenly distributed, we propose that these three phages belong to a new subfamily.

Efficiency of bacteriophage therapy against Cronobacter sakazakii in Galleria mellonella (greater wax moth) larvae.

Cronobacter sakazakii, an opportunistic pathogen found in milk-based powdered infant formulae, has been linked to meningitis in infants, with high fatality rates. A set of phages from various environments were purified and tested in vitro against strains of C. sakazakii. Based on host range and lytic activity, the T4-like phage vB_CsaM_GAP161, which belongs to the family Myoviridae, was selected for evaluation of its efficacy against C. sakazakii. Galleria mellonella larvae were used as a whole-animal model for pre-clinical testing of phage efficiency. Twenty-one Cronobacter strains were evaluated for lethality in G. mellonella larvae. Different strains of C. sakazakii caused 0 to 98% mortality. C. sakazakii 3253, with an LD50 dose of ~2.0×10(5) CFU/larva (24 h, 37 °C) was selected for this study. Larvae infected with a dose of 5×LD50 were treated with phage GAP161 (MOI=8) at various time intervals. The mortality rates were as high as 100% in the groups injected with bacteria only, compared to 16.6% in the group infected with bacteria and treated with phage. Phage GAP161 showed the best protective activity against C. sakazakii when the larvae were treated prior to or immediately after infection. The results obtained with heat-inactivated phage proved that the survival of the larvae is not due to host immune stimulation. These results suggest that phage GAP161 is potentially a useful control agent against C. sakazakii. In addition, G. mellonella may be a useful whole-animal model for pre-screening phages for efficacy and safety prior to clinical evaluation in mammalian models.

Lactobacillus zeae protects Caenorhabditis elegans from enterotoxigenic Escherichia coli-caused death by inhibiting enterotoxin gene expression of the pathogen.

BACKGROUND: The nematode Caenorhabditis elegans has become increasingly used for screening antimicrobials and probiotics for pathogen control. It also provides a useful tool for studying microbe-host interactions. This study has established a C. elegans life-span assay to preselect probiotic bacteria for controlling K88(+) enterotoxigenic Escherichia coli (ETEC), a pathogen causing pig diarrhea, and has determined a potential mechanism underlying the protection provided by Lactobacillus. METHODOLOGY/PRINCIPAL FINDINGS: Life-span of C. elegans was used to measure the response of worms to ETEC infection and protection provided by lactic acid-producing bacteria (LAB). Among 13 LAB isolates that varied in their ability to protect C. elegans from death induced by ETEC strain JG280, Lactobacillus zeae LB1 offered the highest level of protection (86%). The treatment with Lactobacillus did not reduce ETEC JG280 colonization in the nematode intestine. Feeding E. coli strain JFF4 (K88(+) but lacking enterotoxin genes of estA, estB, and elt) did not cause death of worms. There was a significant increase in gene expression of estA, estB, and elt during ETEC JG280 infection, which was remarkably inhibited by isolate LB1. The clone with either estA or estB expressed in E. coli DH5α was as effective as ETEC JG280 in killing the nematode. However, the elt clone killed only approximately 40% of worms. The killing by the clones could also be prevented by isolate LB1. The same isolate only partially inhibited the gene expression of enterotoxins in both ETEC JG280 and E. coli DH5α in-vitro. CONCLUSIONS/SIGNIFICANCE: The established life-span assay can be used for studies of probiotics to control ETEC (for effective selection and mechanistic studies). Heat-stable enterotoxins appeared to be the main factors responsible for the death of C. elegans. Inhibition of ETEC enterotoxin production, rather than interference of its intestinal colonization, appears to be the mechanism of protection offered by Lactobacillus.

Efficacy of bacteriophage LISTEX™P100 combined with chemical antimicrobials in reducing Listeria monocytogenes in cooked turkey and roast beef.

The aim of this study was to verify the effectiveness of the commercially available anti-Listeria phage preparation LISTEX(™)P100 in reducing Listeria monocytogenes on ready-to-eat (RTE) roast beef and cooked turkey in the presence or absence of the chemical antimicrobials potassium lactate (PL) and sodium diacetate (SD). Sliced RTE meat cores at 4 and 10 °C were inoculated with cold-adapted L. monocytogenes to result in a surface contamination level of 10(3)CFU/cm(2). LISTEX(TM)P100 was applied at 10(7) PFU/cm(2) and samples taken at regular time intervals during the RTE product's shelf life to enumerate viable L. monocytogenes. LISTEX(™)P100 was effective during incubation at 4 °C with initial reductions of L. monocytogenes of 2.1 log10 CFU/cm(2) and 1.7 log10 CFU/cm(2), respectively, for cooked turkey and roast beef without chemical antimicrobials (there was no significant difference to the initial L. monocytogenes reductions in the presence of LISTEX(TM)P100 for cooked turkey containing PL and roast beef containing SD-PL). In the samples containing no chemical antimicrobials, the presence of phage resulted in lower L. monocytogenes numbers, relative to the untreated control, of about 2 log CFU/cm(2) over a 28-day storage period at 4 °C. An initial L. monocytogenes cell reduction of 1.5 log10 CFU/cm(2) and 1.7 log10 CFU/cm(2), respectively, for cooked turkey and roast beef containing no chemical antimicrobials was achieved by the phage at 10 °C (abusive temperature). At this temperature, the L. monocytogenes cell numbers of samples treated with LISTEX™ P100 remained below those of the untreated control only during the first 14 days of the experiment for roast beef samples with and without antimicrobials. On day 28, the L. monocytogenes numbers on samples containing chemical antimicrobials and treated with LISTEX(TM)P100 stored at 4 and 10 °C were 4.5 log10 CFU/cm(2) and 7.5 log10 CFU/cm(2), respectively, for cooked turkey, and 1.2 log10 CFU/cm(2) and 7.2 log10 CFU/cm(2), respectively, for roast beef. In both cooked turkey samples with and without chemical antimicrobials stored at 10 °C, the phage-treated samples had significantly lower numbers of L. monocytogenes when compared to the untreated controls throughout the 28-day storage period (P<0.0001). For roast beef and cooked turkey containing chemical antimicrobials treated with LISTEX(TM)P100 and stored at 4 °C, no more than a 2 log CFU/cm(2) increase of L. monocytogenes was observed throughout the stated shelf life of the product. This study shows that LISTEX(™)P100 causes an initial reduction of L. monocytogenes numbers and can serve as an additional hurdle to enhance the safety of RTE meats when used in combination with chemical antimicrobials.

The Genome of Cronobacter sakazakii Bacteriophage vB_CsaP_GAP227 Suggests a New Genus within the Autographivirinae.

The genome of Cronobacter sakazakii podovirus vB_CsaP_GAP227 was fully sequenced. The DNA of this lytic phage consists of 41,796 bp and has a G+C content of 55.7%. Forty-nine open reading frames and no tRNAs were identified. This phage is related to Yersinia phages ϕR8-01 and ϕ80-18 and Aeromonas phage phiAS7.

Complete genome sequence of Cronobacter sakazakii bacteriophage vB_CsaM_GAP161.

Cronobacter sakazakii is an opportunistic pathogen that causes infant meningitis and is often associated with milk-based infant formula. We have fully sequenced the genome of a newly isolated lytic C. sakazakii myovirus, vB_CsaM_GAP161, briefly named GAP161. It consists of 178,193 bp and has a G+C content of 44.5%. A total of 277 genes, including 275 open reading frames and two tRNA-encoding genes, were identified. This phage is closely related to coliphages RB16 and RB43 and Klebsiella pneumoniae phage KP15.

Genome sequence of Cronobacter sakazakii myovirus vB_CsaM_GAP31.

Cronobacter sakazakii is a pathogen that predominantly infects immunocompromised individuals, especially infants, where it causes meningitis. The genome of lytic C. sakazakii myovirus vB_CsaM_GAP31 has been fully sequenced. It consists of 147,940 bp and has a G+C content of 46.3%. A total of 295 genes, including 269 open reading frames and 26 tRNA genes, were identified. This phage is related to Salmonella phage PVP-SE1 and coliphages vB_EcoM-FV3 and rV5.

Use of Caenorhabditis elegans for preselecting Lactobacillus isolates to control Salmonella Typhimurium.

Host-specific probiotics have been used to control enteric pathogens, including foodborne pathogens, in food animal production. However, evaluation of the efficacy of these probiotics requires costly in vivo assays in the target animal. The nematode Caenorhabditis elegans has been used for prescreening of antimicrobial agents and for studies of host-pathogen interactions. In the present study, 17 Lactobacillus isolates from chicken and pig intestines were tested with C. elegans, and the ability of these isolates to prevent death from Salmonella infection was variable. Two Lactobacillus isolates (S64, which gave full protection, and CL11, which gave no protection) were further studied. Both isolates exhibited a similar colonization profile in the C. elegans intestine. Although different culture fractions of CL11 were not protective, both live and heat-killed S64 cells provided full or partial protection of C. elegans from death caused by Salmonella infection. In contrast, different culture fractions from both isolates had similar effects on the colonization of the nematode intestine by Salmonella Typhimurium DT104. Our preliminary results from a pig performance trial revealed a correlation between the degree of protection in the C. elegans survival assay and the performance of 35-day-old weaned piglets that were treated with the same Lactobacillus isolates, suggesting that C. elegans can be used as a laboratory animal model for preselecting probiotics for control of Salmonella infections.

Microencapsulation of bacteriophage felix O1 into chitosan-alginate microspheres for oral delivery.

This paper reports the development of microencapsulated bacteriophage Felix O1 for oral delivery using a chitosan-alginate-CaCl(2) system. In vitro studies were used to determine the effects of simulated gastric fluid (SGF) and bile salts on the viability of free and encapsulated phage. Free phage Felix O1 was found to be extremely sensitive to acidic environments and was not detectable after a 5-min exposure to pHs below 3.7. In contrast, the number of microencapsulated phage decreased by 0.67 log units only, even at pH 2.4, for the same period of incubation. The viable count of microencapsulated phage decreased only 2.58 log units during a 1-h exposure to SGF with pepsin at pH 2.4. After 3 h of incubation in 1 and 2% bile solutions, the free phage count decreased by 1.29 and 1.67 log units, respectively, while the viability of encapsulated phage was fully maintained. Encapsulated phage was completely released from the microspheres upon exposure to simulated intestinal fluid (pH 6.8) within 6 h. The encapsulated phage in wet microspheres retained full viability when stored at 4 degrees C for the duration of the testing period (6 weeks). With the use of trehalose as a stabilizing agent, the microencapsulated phage in dried form had a 12.6% survival rate after storage for 6 weeks. The current encapsulation technique enables a large proportion of bacteriophage Felix O1 to remain bioactive in a simulated gastrointestinal tract environment, which indicates that these microspheres may facilitate delivery of therapeutic phage to the gut.

Characterization of bacterial populations recovered from the teat canals of lactating dairy and beef cattle by 16S rRNA gene sequence analysis.

Bovine mastitis is of major concern to the dairy industry worldwide. The bovine teat canal is the primary route through which pathogens enter the mammary gland. The microflora of the teat canals of dairy and beef cattle was investigated by analysis of 16S rRNA gene sequences. The goal was to examine the global difference between dairy cattle, which are sanitized on a regular basis, and beef cattle, which receive little sanitary management. A diverse population of 16S rRNA gene sequences was recovered from both the dairy and the beef herd samples, with diversity higher in the beef sample. Analysis revealed the presence of 90 operational taxonomic units (OTUs) among 156 sequences, with 45 OTUs in the dairy sample and 53 OTUs in the beef sample. Only eight OTUs were common to both samples. Members of the classes Clostridia and Bacilli dominated both samples, followed by Actinobacteria and Proteobacteria. The dairy sample contained a cluster (20/80 clones) of Staphylococcaceae members, seven of which were identifiable as coagulase-negative Staphylococcus species. The beef sample was dominated by members of the genus Clostridia, many of which have not been previously cultured. The results suggest that the microorganisms present in the bovine teat canal are more diverse than previously described.

Diversity and phylogenetic analysis of bacteria in the mucosa of chicken ceca and comparison with bacteria in the cecal lumen.

We reported the first attempt to describe mucosa-associated bacterial populations in the chicken ceca by molecular analysis of 16S rRNA genes. Bacteria in the mucosa were highly diverse but mainly Gram-positive with low G+C. Fusobacterium prausnitzii and butyrate-producing bacteria comprised the largest groups among 116 cloned sequences. Twenty five percent of the clones had less than 95% homology to database sequences. Many sequences were related to those of uncultured bacteria identified in human feces or the bovine rumen. Terminal restriction fragment length polymorphism (T-RFLP) analysis revealed some differences between bacterial populations present in the mucosa and lumen of ceca. Greater resolution of bacterial population was obtained using a culture-independent approach rather than a culture-based approach.

Molecular analysis of bacterial populations in the ileum of broiler chickens and comparison with bacteria in the cecum.

Bacterial populations in the ileum of broiler chickens were analyzed by molecular analysis of 16S rRNA genes and compared to those in the cecum. Bacteria found in the ileal mucosa were mainly Gram-positive with low G+C content. There were 15 molecular species among 51 cloned sequences. More than 70% of the cloned sequences were related to lactobacilli and Enterococcus cecorum. Two sequences had 95% or less homology to existing database sequences. Terminal restriction fragment length polymorphism (T-RFLP) analysis revealed differences among bacterial populations present in the mucosa and lumen of the ileum. Comparative studies by T-RFLP and sequence analyses of 16S rRNA genes indicated a less diverse bacterial population in the ileum (mucosa and lumen) than in the cecum. Lactobacilli, E. cecorum, and butyrate-producing bacteria related (including both identified and unidentified species) sequences were the three major groups detected in ilea and ceca. Although butyrate-producing bacteria may have good potential in the development of novel probiotics for poultry, verifying the presence of the bacteria in the chicken gut is required to warrant further investigation.