Genetically manipulated phages with improved pH resistance for oral administration in veterinary medicine

Orally administered phages to control zoonotic pathogens face important challenges, mainly related to the hostile conditions found in the gastrointestinal tract (GIT). These include temperature, salinity and primarily pH, which is exceptionally low in certain compartments. Phage survival under these conditions can be jeopardized and undermine treatment. Strategies like encapsulation have been attempted with relative success, but are typically complex and require several optimization steps. Here we report a simple and efficient alternative, consisting in the genetic engineering of phages to display lipids on their surfaces. Escherichia coli phage T7 was used as a model and the E. coli PhoE signal peptide was genetically fused to its major capsid protein (10A), enabling phospholipid attachment to the phage capsid. The presence of phospholipids on the mutant phages was confirmed by High Performance Thin Layer Chromatography, Dynamic Light Scattering and phospholipase assays. The stability of phages was analysed in simulated GIT conditions, demonstrating improved stability of the mutant phages with survival rates 102107 pfu.mL1 higher than wild-type phages. Our work demonstrates that phage engineering can be a good strategy to improve phage tolerance to GIT conditions, having promising application for oral administration in veterinary medicine.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and under the scope of the Project PTDC/BBB-BSS/6471/2014 (POCI-01-0145-FEDER-016678). Franklin L. Nobrega and Ana Rita Costa acknowledge FCT for grants SFRH/BD/86462/2012 and SFRH/BPD/94648/2013, respectively. Melvin F. Siliakus acknowledges funding from the Biobased Ecologically Balanced Sustainable Industrial Chemistry (BE-BASIC) foundation. Electron microscopy work was performed at the Wageningen Electron Microscopy Centre (WEMC) of Wageningen University

Orally Administered P22 Phage Tailspike Protein Reduces Salmonella Colonization in Chickens: Prospects of a Novel Therapy against Bacterial Infections

One of the major causes of morbidity and mortality in man and economically important animals is bacterial infections of the gastrointestinal (GI) tract. The emergence of difficult-to-treat infections, primarily caused by antibiotic resistant bacteria, demands for alternatives to antibiotic therapy. Currently, one of the emerging therapeutic alternatives is the use of lytic bacteriophages. In an effort to exploit the target specificity and therapeutic potential of bacteriophages, we examined the utility of bacteriophage tailspike proteins (Tsps). Among the best-characterized Tsps is that from the Podoviridae P22 bacteriophage, which recognizes the lipopolysaccharides of Salmonella enterica serovar Typhimurium. In this study, we utilized a truncated, functionally equivalent version of the P22 tailspike protein, P22sTsp, as a prototype to demonstrate the therapeutic potential of Tsps in the GI tract of chickens. Bacterial agglutination assays showed that P22sTsp was capable of agglutinating S. Typhimurium at levels similar to antibodies and incubating the Tsp with chicken GI fluids showed no proteolytic activity against the Tsp. Testing P22sTsp against the three major GI proteases showed that P22sTsp was resistant to trypsin and partially to chymotrypsin, but sensitive to pepsin. However, in formulated form for oral administration, P22sTsp was resistant to all three proteases. When administered orally to chickens, P22sTsp significantly reduced Salmonella colonization in the gut and its further penetration into internal organs. In in vitro assays, P22sTsp effectively retarded Salmonella motility, a factor implicated in bacterial colonization and invasion, suggesting that the in vivo decolonization ability of P22sTsp may, at least in part, be due to its ability to interfere with motility… Our findings show promise in terms of opening novel Tsp-based oral therapeutic approaches against bacterial infections in production animals and potentially in humans

Occurrence of Salmonella sp in laying hens

This study was carried out to investigate the presence of Salmonella sp in flocks of white laying hens. In different farms, the transport boxes of twelve flocks were inspected at arrival for the presence of Salmonella. Four positive (A, B, L and M) and one negative (I) flocks were monitored at each four weeks using bacteriological examination of cecal fresh feces up to 52 weeks. Birds were also evaluated at 52 weeks, when 500 eggs were taken randomly, and at 76 weeks, after forced molt. Salmonella enterica serovar Enteritidis and S. enterica rough strain were isolated from the transport boxes of the four positive flocks (flocks A, B, L and M). Salmonella sp was not isolated from the transport boxes or from the feces after 76 weeks-old in flock I. Salmonella sp was isolated in the 1st, 11th, 34th, 42nd and 76th weeks from flock A; in the 1st, 4th, 11th and 76th weeks from flock B; in the first week and in the 17th to 52nd weeks from flock L; and in the 1st and 76th weeks from flock M. S. Enteritidis, S. enterica rough strain and Salmonella enterica serovar Infantis were isolated from the four positive flocks. Besides, Salmonella enterica serovar Javiana was isolated from flocks B and L, and Salmonella enterica serovar Mbandaka was isolated from flock L. Eggs produced by flock A and by flock L were contaminated with S. Enteritidis and S. enterica rough strain. According to these results, Salmonella-infected flocks may produce contaminated eggs

Occurrence of Salmonella sp in laying hens

This study was carried out to investigate the presence of Salmonella sp in flocks of white laying hens. In different farms, the transport boxes of twelve flocks were inspected at arrival for the presence of Salmonella. Four positive (A, B, L and M) and one negative (I) flocks were monitored at each four weeks using bacteriological examination of cecal fresh feces up to 52 weeks. Birds were also evaluated at 52 weeks, when 500 eggs were taken randomly, and at 76 weeks, after forced molt. Salmonella enterica serovar Enteritidis and S. enterica rough strain were isolated from the transport boxes of the four positive flocks (flocks A, B, L and M). Salmonella sp was not isolated from the transport boxes or from the feces after 76 weeks-old in flock I. Salmonella sp was isolated in the 1st, 11th, 34th, 42nd and 76th weeks from flock A; in the 1st, 4th, 11th and 76th weeks from flock B; in the first week and in the 17th to 52nd weeks from flock L; and in the 1st and 76th weeks from flock M. S. Enteritidis, S. enterica rough strain and Salmonella enterica serovar Infantis were isolated from the four positive flocks. Besides, Salmonella enterica serovar Javiana was isolated from flocks B and L, and Salmonella enterica serovar Mbandaka was isolated from flock L. Eggs produced by flock A and by flock L were contaminated with S. Enteritidis and S. enterica rough strain. According to these results, Salmonella-infected flocks may produce contaminated eggs.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Experimental infection by Salmonella enterica subsp enterica serovar kottbus in day-old broiler chickens

The strain used in this work was a Salmonella enterica subsp enterica serovar Kottbus (6,8:e,h:1,5) isolated from imported day-old ducklings in Laboratório Nacional Agropecuário (LANAGRO/SP) of the Ministry of Agriculture of Brazil (MAPA). In view of the lack of information available about this Salmonella isolate and also because it was detected in day-old imported birds, this study was carried out to investigate the dissemination of S. Kottbus among newly hatched chicks. The birds were placed in three groups: one group of 20 birds received 0.1 mL of S. Kottbus culture containing 1.2 x 10(8) CFU/mL, the second group of 20 birds was inoculated with 1.2 x 10(5) CFU/mL and the third group of 10 birds was untreated (control group). Results were similar for both infected groups. The bacterium was recovered from cloacal swabs collected from the first day following the experimental infection until the end of the trial (42 days post-inoculation). At 15 and 42 days post-inoculation (dpi), half of the birds of each group were killed for bacteriological examination of cecal contents, liver and spleen. At 15 dpi, viable cell counts of S. Kottbus were obtained in all kinds of samples. At 42 dpi, Salmonella was present in the liver and spleen of few birds, but in large amounts in the cecal contents of almost all birds