Characterization and efficacy of Salmonella phage cocktail PHA46 in the control of Salmonella Newport and Typhimurium internalized into cherry tomatoes.

Non-typhoid Salmonella enterica causes salmonellosis illness, and this bacterium can contaminate food throughout the production chain, including those that are consumed as raw products. Salmonella enterica can adhere to and internalize into fresh produce such as cherry tomatoes. It has been reported that lytic bacteriophages (phages) can be used as a biocontrol agent in the agricultural field, being an alternative for the control of Salmonella in red meat, fish, lettuce, and cabbage. The aim of this study was to characterize the two phages present in the PHA46 cocktail to determine their morphology, genome, host range, and resistance to different temperatures and pHs values; and later evaluate their lytic activity to reduce the adherence to and internalization of Salmonella enterica serovars Newport and Typhimurium into cherry tomatoes. In addition, in this work, we also explored the effect of the PHA46 cocktail on the virulence of S. Newport-45 and S. Typhimurium SL1344, recovered from the interior of cherry tomatoes, on the lifespan of the animal model Caenorhabditis elegans. The nematode C. elegans, recently has been used to test the virulence of Salmonella and it is easy to maintain and work with in the laboratory. The results revealed that the morphology obtained by Transmission Electron Microscopy of two phages from the PHA46 cocktail correspond to a myovirus, the analyses of their genomes sequences did not report virulence or antimicrobial resistance genes. The PHA46 sample is specific for 33 different serovars from different Salmonella strains and shows stability at 7 °C and pH 6. Also, the PHA46 cocktail was effective in reducing the adherence of S. Newport-45 and S. Typhimurium SL1344 to cherry tomatoes, at an average of 0.9 log10, respectively. Regarding internalized bacteria, the reduction was at an average of 1.2 log10, of the serovars mentioned above. The lifespan experiments in C. elegans showed by itself, that the PHA46 cocktail was harmless to the nematode, and the virulence from both Salmonella strains grown in vitro is diminished in the presence of the PHA46 cocktail. In conclusion, these results showed that the PHA46 cocktail could be a good candidate to be used as a biocontrol agent against Salmonella enterica.

Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5.

Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 µg ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 µg ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 µg ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 µg ml-1) and P. aeruginosa P2307 (65.00 µg ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.

Long-Term Interactions of Salmonella Enteritidis With a Lytic Phage for 21 Days in High Nutrients Media.

Salmonella spp. is a relevant foodborne pathogen with worldwide distribution. To mitigate Salmonella infections, bacteriophages represent an alternative to antimicrobials and chemicals in food animals and food in general. Bacteriophages (phages) are viruses that infect bacteria, which interact constantly with their host. Importantly, the study of these interactions is crucial for the use of phages as a mitigation strategy. In this study, experimental coevolution of Salmonella Enteritidis (S. Enteritidis) and a lytic phage was conducted in tryptic soy broth for 21 days. Transfer to fresh media was conducted daily and every 24 hours, 2 mL of the sample was collected to quantify Salmonella OD600 and phage titter. Additionally, time-shift experiments were conducted on 20 colonies selected on days 1, 12, and 21 to evaluate the evolution of resistance to past (day 1), present (day 12), and future (day 21) phage populations. The behavior of the dynamics was modeled and simulated with mathematical mass-action models. Bacteria and phage from days 1 and 21 were sequenced to determine the emergence of mutations. We found that S. Enteritidis grew for 21 days in the presence and absence of the phage and developed resistance to the phage from day 1. Also, the phage was also able to survive in the media for 21 days, however, the phage titer decreased in approx. 3 logs PFU/mL. The stability of the lytic phage population was consistent with the leaky resistance model. The time-shift experiments showed resistance to phages from day 1 of at least 85% to the past, present, and future phages. Sequencing of S. Enteritidis showed mutations in genes involved in lipopolysaccharide biosynthesis genes rfbP and rfbN at day 21. The phage showed mutations in the tail phage proteins responsible for recognizing the cell surface receptors. These results suggest that interactions between bacteria and phage in a rich resource media generate a rapid resistance to the infective phage but a fraction of the population remains susceptible. Interactions between Salmonella and lytic phages are an important component for the rational use of phages to control this important foodborne pathogen.

The gut microbiota of chickens in a commercial farm treated with a Salmonella phage cocktail.

The microbiota in broiler chicken intestines affects the animals' health, metabolism, and immunity both positively and negatively. Accordingly, it has a significant impact on animal productivity. Phages, host-specific parasites of bacterial cells, are a promising antimicrobial alternative that selectively target pathogens without disturbing the microbiota. The purpose of this study is to further characterize the commensal microbial community at production scale in broiler chickens treated with a Salmonella phage treatment. We evaluated the cecal microbiota of broilers reared in a commercial farming system where a phage cocktail against Salmonella, SalmoFree was supplied to animals. To do so, two field trials were conducted, incorporating three doses of phages in the broilers' drinking water. Our results showed that the core microbiome (taxa that were present in more than 50% of samples) contained species that are key to microbiota adaptation in the last stage of the production cycle. Among these, there are some important degraders of complex polysaccharides and producers of short chain fatty acids (SCFA) such as Eisenbergiella and Lachnoclostridium. The phage cocktail did not affect the normal development of the microbiota's structure. The addition of the phage cocktail resulted in a significant reduction in Campylobacter and an increase in Butyricimonas, Helicobacter and Rikenellaceae, which are common inhabitants in chicken gut with known negative and positive effects on their health and metabolism. Altogether, we consider that these results contribute valuable information to the implementation of large-scale phage therapy technologies.

Efficacy of Salmonella Bacteriophage S1 Delivered and Released by Alginate Beads in a Chicken Model of Infection.

Modern bacteriophage encapsulation methods based on polymers such as alginate have been developed recently for their use in phage therapy for veterinary purposes. In birds, it has been proven that using this delivery system allows the release of the bacteriophage in the small intestine, the site of infection by Salmonella spp. This work designed an approach for phage therapy using encapsulation by ionotropic gelation of the lytic bacteriophage S1 for Salmonella enterica in 2% w/v alginate beads using 2% w/v calcium chloride as crosslinking agent. This formulation resulted in beads with an average size of 3.73 ± 0.04 mm and an encapsulation efficiency of 70%. In vitro, the beads protected the bacteriophages from pH 3 and released them at higher pH. To confirm that this would protect the bacteriophages from gastrointestinal pH changes, we tested the phage infectivity in vivo assay. Using a model chicken (Gallus gallus domesticus) infected with Salmonella Enteritidis, we confirmed that after 3 h of the beads delivery, infective phages were present in the chicken's duodenal and caecal sections. This study demonstrates that our phage formulation is an effective system for release and delivery of bacteriophage S1 against Salmonella Enteritidis with potential use in the poultry sector.

Salmonella enterica Serovar Enteritidis Control in Poultry Litter Mediated by Lytic Bacteriophage Isolated from Swine Manure.

We report the use of bacteriophages for control of Salmonella Enteritidis in poultry production. Phage was isolated by the double-agar plate assay from agricultural waste samples, and one isolate, named SM1, was selected and propagated for application in poultry litter. Two experimental protocols were tested: single treatment and repeated treatment (re-application of phage SM1 after 6 h and 12 h). Each treatment cycle involved 25 g of poultry litter placed in plastic boxes and contaminated with 105 Colony Forming Units mL-1 (CFU mL-1) of S. Enteritidis, in independent duplicates. The contaminated litter was treated with 106 Plaque Forming Units mL-1 (PFU mL-1) of SM1 phage by dripping. Repeated application of phage SM1 reduced Salmonella counts by over 99.9%; the phage persisted in poultry litter for over 35 days. This study illustrates the application of SM1 treatment as a promising technology for bacterial control in production matrices that could allow safe and sustainable use of agricultural waste products as biofertilizers.

Two Phages of the Genera Felixounavirus Subjected to 12 Hour Challenge on Salmonella Infantis Showed Distinct Genotypic and Phenotypic Changes.

Salmonella Infantis is considered in recent years an emerging Salmonella serovar, as it has been associated with several outbreaks and multidrug resistance phenotypes. Phages appear as a possible alternative strategy to control Salmonella Infantis (SI). The aims of this work were to characterize two phages of the Felixounavirus genus, isolated using the same strain of SI, and to expose them to interact in challenge assays to identify genetic and phenotypic changes generated from these interactions. These two phages have a shared nucleotide identity of 97% and are differentiated by their host range: one phage has a wide host range (lysing 14 serovars), and the other has a narrow host range (lysing 6 serovars). During the 12 h challenge we compared: (1) optical density of SI, (2) proportion of SI survivors from phage-infected cultures, and (3) phage titer. Isolates obtained through the assays were evaluated by efficiency of plating (EOP) and by host-range characterization. Genomic modifications were characterized by evaluation of single nucleotide polymorphisms (SNPs). The optical density (600 nm) of phage-infected SI decreased, as compared to the uninfected control, by an average of 0.7 for SI infected with the wide-host-range (WHR) phage and by 0.3 for SI infected with the narrow-host-range (NHR) phage. WHR phage reached higher phage titer (7 × 1011 PFU/mL), and a lower proportion of SI survivor was obtained from the challenge assay. In SI that interacted with phages, we identified SNPs in two genes (rfaK and rfaB), which are both involved in lipopolysaccharide (LPS) polymerization. Therefore, mutations that could impact potential phage receptors on the host surface were selected by lytic phage exposure. This work demonstrates that the interaction of Salmonella phages (WHR and NHR) with SI for 12 h in vitro leads to emergence of new phenotypic and genotypic traits in both phage and host. This information is crucial for the rational design of phage-based control strategies.

Host Resistance, Genomics and Population Dynamics in a Salmonella Enteritidis and Phage System.

Bacteriophages represent an alternative solution to control bacterial infections. When interacting, bacteria and phage can evolve, and this relationship is described as antagonistic coevolution, a pattern that does not fit all models. In this work, the model consisted of a microcosm of Salmonella enterica serovar Enteritidis and φSan23 phage. Samples were taken for 12 days every 48 h. Bacteria and phage samples were collected; and isolated bacteria from each time point were challenged against phages from previous, contemporary, and subsequent time points. The phage plaque tests, with the genomics analyses, showed a mutational asymmetry dynamic in favor of the bacteria instead of antagonistic coevolution. This is important for future phage-therapy applications, so we decided to explore the population dynamics of Salmonella under different conditions: pressure of one phage, a combination of phages, and phages plus an antibiotic. The data from cultures with single and multiple phages, and antibiotics, were used to create a mathematical model exploring population and resistance dynamics of Salmonella under these treatments, suggesting a nonlethal, growth-inhibiting antibiotic may decrease resistance to phage-therapy cocktails. These data provide a deep insight into bacterial dynamics under different conditions and serve as additional criteria to select phages and antibiotics for phage-therapy.

Backyard Farms Represent a Source of Wide Host Range Salmonella Phages That Lysed the Most Common Salmonella Serovars.

The genus Salmonella has more than 2,600 serovars, and this trait is important when considering interventions for Salmonella control. Bacteriophages that are used for biocontrol must have an exclusively lytic cycle and the ability to lyse several Salmonella serovars under a wide range of environmental conditions. Salmonella phages were isolated and characterized from 34 backyard production systems (BPSs) with a history of Salmonella infections. BPSs were visited once, and cloacal or fecal samples were processed for phage isolation. Four hosts, Salmonella serovars Enteritidis, Heidelberg, Infantis, and Typhimurium, were used for phage isolation. The host range of the phages was later characterized with a panel of 23 Salmonella serovars (serovar diversity set) and 31 isolates obtained from the same farms (native set). Genetic relatedness for 10 phages with a wide host range was characterized by restriction fragment length polymorphism, and phages clustered based on the host range. We purified 63 phages, and 36 phage isolates were obtained on Salmonella Enteritidis, 16 on Salmonella Heidelberg, and 11 on Salmonella Infantis. Phages were classified in three clusters: (i) phages with a wide host range (cluster I), (ii) phages that lysed the most susceptible Salmonella serovars (serogroup D) and other isolates (cluster II), and (iii) phages that lysed only isolates of serogroup D (cluster III). The most susceptible Salmonella serovars were Enteritidis, Javiana, and Dublin. Seven of 34 farms yielded phages with a wide host range, and these phages had low levels of genetic relatedness. Our study showed an adaptation of the phages in the sampled BPSs to serogroup D Salmonella isolates and indicated that isolation of Salmonella phages with wide host range differs by farm. A better understanding of the factors driving the Salmonella phage host range could be useful when designing risk-based sampling strategies to obtain phages with a wide lytic host range for biocontrol purposes.

Salmonella phages and prophages: genomics, taxonomy, and applied aspects.

Since this book was originally published in 2007 there has been a significant increase in the number of Salmonella bacteriophages, particularly lytic virus, and Salmonella strains which have been fully sequenced. In addition, new insights into phage taxonomy have resulted in new phage genera, some of which have been recognized by the International Committee of Taxonomy of Viruses (ICTV). The properties of each of these genera are discussed, along with the role of phage as agents of genetic exchange, as therapeutic agents, and their involvement in phage typing.

Análise de critérios para utilização na seleção de bacteriófagos líticos com potencial para utilização médica: dados preliminares / Criteria for selection of lytic bacteriophage for use in medical field (setting): preliminary data / Análisis de los criterios para la selección de bacteriófagos líticos con potencial para uso médico: datos preliminares

A fagoterapia é um método de controle de infecções conhecido há mais de cem anos, porém deixou-se de ser utilizada devido à praticidade do uso de antibióticos. No entanto, com o aumento mundial da resistência bacteriana, a fagoterapia ressurge como opção terapêutica, porém os padrões de seleção de amostras utilizados são empíricos. O presente artigo descreve o comportamento in vitro de 62 amostras de bacteriófagos líticos isolados de esgoto hospitalar humano usando uma amostra de Salmonella Enteritidis de origem avícola. No estudo, foram testados quatro perfis distintos comumente utilizados nas pesquisas com bacteriófagos e observou-se que há uma variância comportamental considerável em que atividade lítica, tamanho de halo em gel difusão e índice de proliferação são fatores independentes de virulência, porém amostras prolíferas apresentam maior espectro de atividade lítica.

This article analyzed, in vitro, the behavior of 62 bacteriophages samples lytic for Salmonella Enteritidis. The lytic potential was assessed by analyzing following parameters: proliferation, halo size in gel diffusion, superficial halo size and virulence degree. Proliferation degree of samples was determined by quantifying the amount of plaque forming unit (PFU) using gel diffusion technique. To determine UFP, minor dilution with measurable halos was considered. Lytic spectrum in the gel column was measured with an acrylic precision ruler. For to superficial halo size and virulence degree, the "spot-on-lawn" technique was employed. We observed that lytic activity spectrum, halo size in gel diffusion and proliferation are factors independent of virulence, but prolific samples shown higher spectrum of lytic activity. Prolific samples that present a high degree of virulence demonstrate in vitro a good potential for medical application.

La fagoterapia es un método de control de la infección conocido por más de cien años, sin embargo, su uso se dejó de aplicar debido a la practicidad de los antibióticos. Con el aumento mundial de la resistencia bacteriana, la fagoterapia resurge como una opción terapéutica, pero los patrones de selección de muestras utilizadas son empíricas. En este trabajo, se describe el comportamiento in vitro de 62 muestras de los bacteriófagos líticos aislados de aguas residuales del hospital humano utilizando una muestra de Salmonella Enteritidis original de productos avícolas. En el estúdio, se probaron cuatro diferentes perfiles comúnmente utilizados en la investigación en bacteriófagos y se observó que existe una variación considerable en donde el espectro de comportamiento de actividad lítica, tamaño de halo en gel difusión y el índice de proliferación fueron factores independientes de la virulencia, pero las muestras prolíficos tienen un mayor espectro de acción.

Avaliação da suscetibilidade antimicrobiana e do perfil de macrorrestrição do DNA de isolados humanos de Salmonella serovar Typhimurium fagotipo 193, no período de 1970 a 2008 / Assessment of antimicrobial susceptibility and macrorestriction DNA of human isolates of Salmonella serovar typhimurium phage type 193 in the period 1970 to 2008

Para analisar cepas de Salmonella ser. Typhimurium isoladas de processos entéricos e extraintestinais humanos ocorridos no período de 1970 a 2008 de diferentes regiões do país foram selecionadas, com base nos registros contidos no banco de dados do Laboratório de Enterobactérias do IOC/FIOCRUZ, RJ, amostras do fagotipo prevalente 193, visando precipuamente o reconhecimento de clones epidêmicos. Foram selecionadas 553 cepas de Salmonella ser. Typhimurium fagotipo 193 representadas por 91, 65, 70 e 327 amostras referentes as décadas de 70, 80, 90 e ao período de 2000 a 2008, respectivamente. Na análise global da sensibilidade destas cepas, 52% apresentaram um ou mais marcadores de resistência a antibióticos incluídos no perfil ACSSuT. Este perfil de resistência completo foi verificado em 20,9% dos isolados, sendo os 21,9% restantes, sensíveis a todas as drogas testadas, especialmente no período de 2000 a 2008, representadas por 121 amostras (37,0%) em relação as 327 culturas dessa época. O maior percentual de resistência foi observado nas amostras da década de 70 (99%) sendo o perfil ACSSuT detectado em 35,2% dos isolados, ressaltando-se que todas as amostras foram isoladas de processos gastroentéricos ocorridos na cidade de São Paulo. Ao longo das quatro décadas de estudo, descreve-se um ponto de ruptura entre a prevalência de resistência e a suscetibilidade na transição entre as décadas de 80 e 90. Embora o número de isolados de Salmonalla ser. Typhimurium fagotipo 193 tenha aumentado no último período considerado, o percentual de mono e multirresistência aos antimicrobianos se situou em nível elevado (63,0%). A análise do polimorfismo obtido após macrorrestrição com a enzima Xbal revelou que cepas isoladas na década de 90 apresentaram elevado percentual de similaridade (>-85%) com cepas isoladas recentemente (período de 2000-2008), sendo agrupadas nos mesmos "subclusters". Por outro lado, as cepas da década de 70 inserem-se em "subclusters" independentes...

To analyze strains of Salmonella ser. Typhimurium isolated from human cases of enteric and extraintestinal occurred during the period 1970 to 2008 of different regions of Brazil were selected, based on records in the database from Enterobacteria Laboratory of IOC/FIOCRUZ, RJ, samples prevalent phage type 193 in order to recognition of epidemic clones. We selected 553 strains of Salmonella ser. Typhimurium phage type 193 represented by 91, 65, 70 and 327 samples concerning the 1970s, 1980s, 1990s and the period from 2000 to 2008, respectively. In a global analysis of the sensitivity of these strains, 52% had one or more antibiotic resistance markers included in the profile ACSSuT. This resistance profile was found complete in 20.9% of isolates and the remaining 21.9%, sensitive to all drugs tested, especially in the period 2000 to 2008, represented by 121 samples (37.0%) compared the 327 cultures of that time. The highest percentage of resistance was observed in the samples of the 70 (99%) being the profile ACSSuT detected in 35.2% of isolates, emphasizing that all strains were isolated from gastrointestinal processes occurring in São Paulo city. Over the four decades of study, we describe a breaking point between the prevalence of resistance and susceptibility in the transition between the 1980s and 1990s. Although the number of isolates of Salmonella ser. Typhimurium phage type 193 has increased in the last period, the percentage of mono-and-multidrug resistance to antimicrobial agents stood at high level (63.0%). The analysis of polymorphism obtained after macrorestriction with the enzyme Xbal showed that isolates in the 1990s showed a high percentage of similarity (>-85%) with strains isolated recently (2000-2008) and are grouped in the same subclusters. Moreover, the strains of the 1970s fall into subclusters independent, although the percentage of similarity between such subsclusters and the other is >- 70%, the same was observed for the strains...

Aerosol spray treatment with bacteriophages and competitive exclusion reduces Salmonella enteritidis infection in chickens.

A combination of three different Salmonella-specific bacteriophages (BPs) and one competitive exclusion (CE) product were used to reduce Salmonella Enteritidis (SE) colonization in experimentally infected chickens. Equal numbers of 7-day-old chickens were used in each of three groups: a CE group (treated with CE), a BP group (treated with BP), and a CE-plus-BP group (treated with both products). The CE product was administered via coarse spray at 1 day of age and the cocktail of three BPs was given via spray at 6 days of age using a multiplicity of infection of 10(3) plaque-forming units. All the experimental groups, except a healthy control group, were challenged orally with 2.95 x 10(5) colony-forming units (CFU)/ml of an SE strain at 7 days of age. Seven days postchallenge, the chickens were euthanatized for individual SE detection, quantitative bacteriology, and phage isolation from ceca and an internal organ pool. The qualitative bacteriology demonstrated that the use of the CE product diminished the incidence of SE to 75.7% and the mixture of BPs reduced it to 80%; when CE plus BP were used, the incidence dropped significantly to 38.7% (P < 0.0001), as compared with the infection control group (100%). A significant difference in the incidence was observed between the CE and the CE-plus-BP groups, and the BP and the CE-plus-BP groups (P = 0.0027 and P = 0.0010, respectively). The mean SE cecal count diminished with the use of CE plus BP (1.6 x 10(2) CFU/g, P = 0.0003) compared with the control group (1.56 x 10(5) CFU/g), the CE group (4.23 x 10(3) CFU/g), and the BP group (9.48 x 10(3) CFU/g). On the basis of the present study, it may be concluded that the use of both types of biocontrollers can be an effective method for reducing SE colonization in commercial chickens, but further basic and applied research is needed.

Bacteriophage treatment reduces Salmonella colonization of infected chickens.

Three different lyric bacteriophages (BPs) were isolated from the sewage system of commercial chicken flocks and used to reduce Salmonella Enteritidis (SE) colonization from experimental chickens. Ten-day-old chickens were challenged with 9.6 x 10(5) colony-forming units (CFU)/ml of a SE strain and treated by coarse spray or drinking water with a cocktail of the three phages at a multiplicity of infection (MO1) of 10(3) plaque-forming units (PFU) 24 hr prior to SE challenge. Chickens were euthanatized at day 20 of age for individual SE detection, quantitative bacteriology, and phage isolation from the intestine and from a pool of organs. SE detection was performed by both bacteriologic culture and genome detection by polymerase chain reaction (PCR). Qualitative bacteriology showed that aerosol-spray delivery of BPs significantly reduced the incidence of SE infection in the chicken group (P = 0.0084) to 72.7% as compared with the control group (100%). In addition, SE counts showed that phage delivery both by coarse spray and drinking water reduced the intestinal SE colonization (P < 0.01; P < 0.05, respectively). BPs were isolated at 10 days postinfection from the intestine and from pools of organs from BP-treated chickens. We conclude that the phage treatment, either by aerosol spray or drinking water, may be a plausible alternative to antibiotics for the reduction of Salmonella infection in poultry.

Ability of bacteriophages isolated from different sources to reduce Salmonella enterica serovar enteritidis in vitro and in vivo.

Salmonella enterica serovar Enteritidis-lysing bacteriophages isolated from poultry or human sewage sources were used to reduce Salmonella Enteritidis in vitro and in experimentally infected chicks. Cocktails of 4 different bacteriophages obtained from commercial broiler houses (CB4Ø) and 45 bacteriophages from a municipal wastewater treatment plant (WT45Ø) were evaluated. In experiment 1, an in vitro crop assay was conducted with selected bacteriophage concentrations (10(5) to 10(9) pfu/mL) to determine ability to reduce Salmonella Enteritidis in the simulated crop environment. Following 2 h at 37 degrees C, CB4Ø or WT45Ø reduced Salmonella Enteritidis recovery by 1.5 or 5 log, respectively, as compared with control. However, CB4Ø did not affect total SE recovery after 6 h, whereas WT45Ø resulted in up to a 6-log reduction of Salmonella Enteritidis. In experiment 2, day-of-hatch chicks were challenged orally with 3 x 10(3) cfu/chick Salmonella Enteritidis and treated cloacally with 1 x 10(9) WT45Ø pfu/chick 1 h postchallenge. One hour later, chicks were treated or not with a commercially available probiotic (Floramax-B11). Both treatments significantly reduced Salmonella Enteritidis recovery from cecal tonsils at 24 h following vent lip application as compared with controls, but no additive effect was observed with the combination of bacteriophages and probiotic. In experiment 3, day-of-hatch chicks were challenged orally with 9 x 10(3) cfu/chick Salmonella Enteritidis and treated via oral gavage with 1 x 10(8) CB4Ø pfu/chick, 1.2 x 10(8) WT45Ø pfu/chick, or a combination of both, 1 h postchallenge. All treatments significantly reduced Salmonella Enteritidis recovered from cecal tonsils at 24 h as compared with untreated controls, but no significant differences were observed at 48 h following treatment. These data suggest that some bacteriophages can be efficacious in reducing SE colonization in poultry during a short period, but with the bacteriophages and methods presently tested, persistent reductions were not observed.

Identification of the Salmonella phage epsilon 34 tailspike gene.

To understand the interaction between lipopolysaccharide (LPS) and proteins in molecular detail, a molecular genetic approach has been employed, using phage as a model system. The phage epsilon(34) is a Salmonella phage whose tailspike protein (TSP) uses the host LPS as its initial host cell receptor. Previous studies indicated that there was a similarity between the well-studied tail protein of Salmonella phage P22 and the epsilon(34). This study reports the identification of the gene for the epsilon(34) TSP as well as its initial characterization. In addition, some aspects of the structure of the epsilon(34) TSP have been deduced.

Oral treatment with bacteriophages reduces the concentration of Salmonella Enteritidis PT4 in caecal contents of broilers.

Bacteriophages isolated from free-range chickens were tested as a therapeutic agent for reducing the concentration of Salmonella enterica serovar Enteritidis phage type 4 (S. Enteritidis PT4) in caeca of broilers. One-day-old broilers infected with S. Enteritidis PT4 by a seeder bird method were orally treated on the seventh day of age with a mixture of 10(11) plaque-forming units of each of three bacteriophages. Five days after treatment the bacteriophage-treated group showed a reduction of 3.5 orders of magnitude on colony-forming units of S. Enteritidis PT4 per gram of caecal content. Samples collected at 10, 15, 20 and 25 days after treatment revealed that treated birds still had lower colony-forming units of S. Enteritidis PT4 per gram of caecal content. These data gave us compelling evidence that a mixture of bacteriophages may be efficacious in reducing S. Enteritidis PT4 concentration in broilers' caeca and therefore reducing contamination of poultry products by this food-borne pathogen.

Conservation of the N-terminus of some phage tail proteins.

To study the interaction between lipopolysaccharide and protein, a comparative approach was employed using seven Salmonella enterica serovar Typhimurium typing phages as the protein model systems. This interaction has been studied in detail in the Salmonella enterica serovar Typhimurium phage P22 system and involves only the viral tailspike protein. Similarity between these phages and phage P22 was monitored in this Report by assaying restriction endonuclease digestions, capsid size, reactivity to the P22 tailspike protein monoclonal antibody, mAb92, which reacts with the N-terminus of the P22 tail protein and the ability to produce a PCR fragment using primers made to the ends of the P22 tailspike gene. The data indicate that tailspike similarity exists between most of these phages and a scheme reclassifying them is presented and that the N-terminus of the P22 tailspike protein may be a motif for many phage systems and may serve as a aid in the taxonomy of phages. The data suggest a classification scheme in which the N-terminus of some tailspike proteins (head-binding region in some tail proteins) may play a critical element role in the classification of Salmonella viruses.

Phage types of Salmonella enteritidis isolated from clinical and food samples, and from broiler carcasses in southern Brazil.

272 isolates of Salmonella Enteritidis (111 isolated from frozen broiler chicken carcasses, 126 from human food and other biological materials involved in food poisoning outbreaks and 35 from different poultry materials) were selected for phage typing. From these, 111 were phage typed, 57.65% being classified as phage type 4, 32.43% as phage type 4a, 3.60% as phage type 6a and 0.90% as phage type 7, whereas 5.40% samples were not phage typeable. The predominance of phage type 4 is in agreement with the results published worldwide, and reinforces the need for studies related to the epidemiological meaning of these findings.