Paul D. Ebner

Phage Therapy To Reduce Preprocessing Salmonella Infections in Market-Weight Swine

ABSTRACT Contamination of meat products with food-borne pathogens usually results from the carcass coming in contact with the feces of an infected animal during processing. In the case of Salmonella, pigs can become colonized with the organism during transport and lairage from contaminated trailers and holding pens, resulting in increased pathogen shedding just prior to processing. Increased shedding, in turn, amplifies the likelihood of carcass contamination by magnifying the amount of bacteria that enters the processing facility. We conducted a series of experiments to test whether phage therapy could limit Salmonella infections at this crucial period. In a preliminary experiment done with small pigs (3 to 4 weeks old; 30 to 40 lb), administration of an anti-Salmonella phage cocktail at the time of inoculation with Salmonella enterica serovar Typhimurium reduced Salmonella colonization by 99.0 to 99.9% (2- to 3-log reduction) in the tonsils, ileum, and cecum. To test the efficacy of phage therapy in a production-like setting, we inoculated four market-weight pigs (in three replicates) with Salmonella enterica serovar Typhimurium and allowed the challenged pigs to contaminate a holding pen for 48 h. Sixteen naïve pigs were randomly split into two groups which received either the anti-Salmonella phage cocktail or a mock treatment. Both groups of pigs were comingled with the challenged pigs in the contaminated pen. Treatment with the anti-Salmonella phage cocktail significantly reduced cecal Salmonella concentrations (95%; P < 0.05) while also reducing (numerically) ileal Salmonella concentrations (90%; P = 0.06). Additional in vitro studies showed that the phage cocktail was also lytic against several non-Typhimurium serovars.

Effects of antibiotic regimens on the fecal shedding patterns of pigs infected with salmonella typhimurium.

An experiment was conducted to determine (i) the effects of antibiotic regimens on the shedding patterns of pigs infected with Salmonella Typhimurium and (ii) whether antibiotic resistance increases the incidence of pathogen shedding. The experiment involved 48 50-day-old pigs challenged with Salmonella Typhimurium and receiving one of four antibiotic regimens including (i) intramuscular injection of ceftiofur sodium followed by inclusion of oxytetracycline in the feed; (ii) apramycin in the feed for 14 days followed by oxytetracycline; (iii) carbadox in the feed until pigs reached 35 kg followed by oxytetracycline; (iv) no antibiotics (control). Fecal samples were collected preinoculation, 2 and 4 days postinoculation (DPI) and at weekly and biweekly intervals thereafter to determine shedding patterns. Salmonella Typhimurium isolates from 2, 4, 7, 21, 42, and 70 DPI were analyzed for antibiotic resistance. A time effect (P < 0.05) was observed, indicating that the proportion of isolates resistant to at least one antibiotic varied over time. Overall resistance was determined to be 46% at 2 DPI and increased significantly (P < .05) thereafter. Treatment x time and antibiotic x time interactions were also observed (P < 0.05) as the percentage of isolates resistant to each test antibiotic increased over time. In no case did the development of antibiotic resistance result in an increased incidence of shedding of the original inoculate. The incidence of shedding was reduced in pigs receiving the apramycin-oxytetracycline treatment, when compared to control pigs; however, no differences were observed between antibiotic treatments.

Characterization of resistance patterns and detection of apramycin resistance genes in Escherichia coli isolated from swine exposed to various environmental conditions.

Weaned pigs were separated into eight treatments including a control without exposure to apramycin; a control with exposure to apramycin; and apramycin plus either cold stress, heat stress, overcrowding, intermingling, poor sanitation, or intervention with oxytetracycline, to determine the effects of management and environmental conditions on antibiotic resistance among indigenous Escherichia coli. Pigs exposed to apramycin sulfate received that antibiotic in the feed at a concentration of 150 g/ton for 14 days. Environmental treatments were applied 5 days following initial antibiotic administration and maintained throughout the study. Fecal samples were obtained on day 0 (prior to antibiotic treatment) and on days 2, 7, 14, 28, 64, 148, and 149. E. coli were isolated and tested for resistance to apramycin using a minimum inhibitory concentration (MIC) broth microdilution method. Macrorestriction profiling, arbitrarily primed PCR, PCR targeting a gene coding for apramycin resistance, and DNA hybridization were used to characterize genetic elements of resistance. Increased (P<0.0001) resistance to apramycin was noted in E. coli from all treatment groups administered apramycin. MICs of isolates from control pigs receiving apramycin returned to pretreatment levels following removal of the antibiotic, whereas isolates from cold stress, overcrowding, and oxytetracycline groups expressed greater (P<0.05) MICs through day 64, before returning to pretreatment levels. Genetic analysis indicated that all resistant isolates carried the aac(3)IV gene sequence and this sequence was found in a variety of E. coli isotypes. Our data indicate that E. coli resistance to apramycin is increased upon exposure to various stressors.

Development of an Anti-Salmonella Phage Cocktail with Increased Host Range

Salmonella shedding in many livestock species can increase significantly after transport and lairage. Preprocessing increases in shedding can amplify the amount of Salmonella that enters the processing facility and the likelihood of end-product contamination. We previously produced an anti-Salmonella phage cocktail that reduced colonization in swine when the pigs were exposed to an environment heavily contaminated with Salmonella, similar to what might be seen in a transport trailer or processing facility holding pen. The aim of this study was to increase the efficacy of the phage treatment by (1) expanding the host-range of the cocktail and (2) developing a more cost-effective microencapsulation technique. We collected samples from wastewater treatment facilities and isolated 20 distinct phages belonging to either the Siphoviridae or Myoviridae families. From this library we identified 10 phages that together lysed a mixed culture of Salmonella enterica Typhimurium, Enteriditis, and Kentucky--three serovars commonly associated with meat and poultry products. The phages were microencapsulated using two sodium-alginate-based methods that only reduced the cocktail titer by 1.0-1.5 logs (premicroencapsulation: 10.4 log(10) PFU/mL; postmicroencapsulation method one: 9.2 log(10) PFU/mL; postmicroencapsulation method two: 8.9 log(10) PFU/mL). Microencapsulated phages remained stable at both 4°C and 22°C for up to 14 days with no appreciable drop in titer (mean titer: 8.9 log(10) PFU/mL). These data indicate that phage cocktails with wider host ranges are possible and a cost-effective microencapsulation process can protect the phages over an extended period, making simultaneous treatment of large numbers of animals with feed- or water-based delivery possible.

MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM: Development of bacteriophage treatments to reduce Escherichia coli O157:H7 contamination of beef products and produce1

Escherichia coli O157:H7 remains a foodborne pathogen of concern with infections associated with products ranging from ground beef to produce to processed foods. We previously demonstrated that phage-based technologies could reduce foodborne pathogen colonization in live animals. Here, we examined if a 3-phage cocktail could reduce E. coli O157:H7 in experimentally contaminated ground beef, spinach, and cheese. The 3 phages were chosen from our E. coli O157:H7 phage library based on their distinct origins of isolation, lytic ranges, and rapid growth (40- to 50-min life cycle). Two phages belonged to the Myoviridae family and the other phage belonged to the Siphoviridae family. The phage cocktail was added to ground beef, spinach leaves, and cheese slices contaminated with E. coli O157:H7 (10(7) cfu) at a multiplicity of infection of 1. Phage treatment reduced (P < 0.05) the concentrations of E. coli O157:H7 by 1.97 log10 cfu/mL in ground beef when stored at room temperature (24 °C) for 24 h, 0.48 log10 cfu/mL at refrigeration (4 °C), and 0.56 log10 cfu/mL in undercooked condition (internal temperature of 46 °C). Likewise, phage treatment reduced (P < 0.05) E. coli O157:H7 by 3.28, 2.88, and 2.77 log10 cfu/mL in spinach when stored at room temperature for 24, 48, and 72 h, respectively. Phage treatment, however, did not reduce E. coli O157:H7 concentrations in contaminated cheese. Additionally, 3 phage-resistant E. coli O157:H7 strains (309-PR [phage resistant] 1, 309-PR4, and 502-PR5) were isolated and characterized to test if phage resistance could limit long-term use of phages as biocontrol agents. Growth kinetics and adsorption assays indicated that phage resistance in strains 309-PR4 and 502-PR5 was mediated, at least in part, by prevention of phage adsorption. Phage resistance in strain 309-PR1 was the result of limited phage proliferation. Phage resistance was stably maintained in vitro throughout a 4-d subculture period in the absence of phage. No significant reductions in bacterial growth or cell adhesion were observed in resistant strains. Taken together, our results provide additional support for the use of phage to control E. coli O157:H7 in food products; however, the emergence of phage-resistant bacteria could limit the efficacy of phage products. Therefore, further studies are needed to develop resistance mitigation strategies to optimize phage-based technologies.

Direct Feeding of Microencapsulated Bacteriophages to Reduce Salmonella Colonization in Pigs

Salmonella shedding often increases in pigs after transportation and/or lairage. We previously showed that administering anti-Salmonella bacteriophages to pigs by gavage significantly reduced Salmonella colonization when the pigs were exposed to a Salmonella-contaminated holding pen. Here we tested whether a microencapsulated phage cocktail would remain effective if the treatment was administered to pigs in the feed. Pigs (n=21) were randomly placed into three groups: feed, gavage, and control. The feed group was direct-fed a microencapsulated phage cocktail daily for 5 days. On the fifth day, the gavage group received the same phage cocktail by gavage, whereas control pigs received a mock treatment containing no phage. All pigs were then orally challenged with Salmonella enterica serovar Typhimurium. Fecal swab samples were collected every 2 h. At 6 h postchallenge, all pigs were euthanized, and ileal and cecal contents and mesenteric lymph nodes were collected and analyzed for the challenge organism. Pigs in the feed group were less likely to shed Salmonella Typhimurium at 2 h (38.1%) and 4 h (42.9%) postchallenge than pigs in both the gavage (2 h: 71.4%; 4 h: 81.1%) and control (2 h: 71.4%; 4 h: 85.7%) groups (p<0.05). Likewise, concentrations of Salmonella Typhimurium in ileal (2.0 log(10) colony forming units [CFU]/mL [contents]) and cecal (2.7 log(10) CFU/mL) contents from feed pigs were lower than ileal (3.0 log(10) CFU/mL) and cecal (3.7 log(10) CFU/mL) contents from control pigs. High concentrations of anti-Salmonella phages were detected in ileal and cecal contents from both feed and gavage pigs (feed ileal: 1.4×10(6); feed cecal 8.5×10(6); gavage ileal 2.0×10(4); gavage cecal: 2.2×10(3)). It is concluded that direct feeding of microencapsulated phages is a practical and effective means of reducing Salmonella colonization and shedding in pigs.

Complete Genome Sequences of Two Escherichia coli O157:H7 Phages Effective in Limiting Contamination of Food Products

ABSTRACT We previously demonstrated that application of bacteriophages significantly reduced Escherichia coli O157:H7 contamination in spinach and ground beef. Here, we present the genomic sequences of two bacteriophages, vB_EcoS_FFH_1, a T5-like phage, and vB_EcoM_FFH_2, an rV5-like phage, used in those treatments.

Comparison of phenotypic traits and genetic relatedness of Salmonella enterica subspecies arizonae isolates from a colony of ridgenose rattlesnakes with osteomyelitis

Reptiles are well-known sources of human Salmonella infections; however, little is known about the ability of Salmonella to cause disease in reptiles. Thirty-seven isolates of Salmonella enterica subspecies arizonae (S. arizonae) were obtained from retrospective and prospective studies of a closed colony of ridgenose rattlesnakes (Crotalus willardi) with osteomyelitis. All isolates (N = 7) from bone lesions were of a single serotype, 56:z4,z23, and this serotype was found on only 1 occasion among 8 other serotypes isolated from 21 cloacal and intestinal samples. The remainder (N = 7) of serotype 56:z4,z23 isolates were from other extraintestinal sites, including liver, ovary, blood, and testis. S. arizonae isolates were susceptible to most antimicrobials, and plasmid profiles did not correlate with serotype or antimicrobial resistance. Isolates of the 56:z4,z23 serotype (N = 14) formed a tight cluster with 95% similarity by XbaI macrorestriction analysis. Individual isolates of serotypes, 56:z4,z23, 38:(k)-z35, and 48:i-z invaded HeLa cells but an isolate of serotype 50:r-z did not. The same individual isolates of serotype 56:z4,z23 and 48:i-z also invaded viper heart cells. The Salmonella InvA gene was detected by polymerase chain reaction (PCR) in all S. arizonae serotypes tested, including 5 serotype 56:z4,z23 isolates and individual isolates of serotypes 48:i-z and 50:r-z. A source or possible explanation for increased virulence of S. arizonae serotype 56:z4,z23 in this unique host has not been found.

The impact of orally administered phages on host immune response and surrounding microbial communities

ABSTRACT Numerous studies have shown the efficacy of phage therapy in reducing foodborne pathogen carriage in food animals. Fewer studies have focused on host reactions, especially in terms of phage-mediated acute immune responses and effects on the gut microbiome. Here we administered E. coli O157:H7 phages in low (single dose of 105 PFU) or high (single dose of 107 PFU) quantities to mice. While there were time points at which cytokine levels in different treatment groups differed from one another, all cytokine levels remained within normal ranges for mice regardless of treatment. Similarly, the patterns of these differences were not dose related, indicating that phage treatment did not result in a strong acute immune response as measured here. In separate experiments, 3-week-old pigs received a diet containing an in-feed antibiotic or daily phage treatment. After two weeks, microbial DNA of ileal, cecal, and fecal contents was characterized using 16S rRNA sequencing. There were no statistical differences in performance among the different groups. Compared to control pigs (no antibiotic, no phage), antibiotic treatment significantly altered ileal microbiome composition (P < 0.05), with Bacilli being most affected (antibiotic treated: 22%; control: 76%; FDR = 0.0572). No significant differences were observed in cecal and fecal microbiome composition between antibiotic-treated and control pigs, and there were no differences in gut microbiome composition between phage treated and control pigs in any intestinal compartment. Significant abundance differences were observed at the OTU level, with OTUs belonging to genera such as Lactobacillus and Streptococcus being over- or under-represented in either antibiotic or phage treated groups compared to control pigs. Determining whether these changes are deleterious to host, however, requires further study.

Dry-aging improves meat quality attributes of grass-fed beef loins

The objective of this study was to evaluate the effect of dry-aging on meat quality and microbiological properties of grass-fed beef loins. At 7 d postmortem, eighteen bone-in loins (M. longissimus lumborum) from 9 beef carcasses (USDA Select) were obtained. Each loin was cut in half yielding a total of 36 sections, which were assigned to three aging methods: wet-aging (WA); dry-aging (DA); and dry-aging in a water permeable bag (DW). DA resulted in greater shrink and trim loss compared to WA (P < 0.05). However, DW minimized moisture and trim loss resulting in an increase in total saleable yield up to 4%. DA samples were lowest in both aerobic/anaerobic bacteria (P < 0.05). DA steaks had significantly higher flavor and tenderness preferences compared to WA counterparts. Consumers determined DW to have greater juiciness compared with WA (P < 0.05). Our findings indicate that dry-aging could improve eating quality attributes of low marbled grass-fed beef without adversely affecting microbial characteristics.