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.

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.

Population analysis of D6-like plasmid prophage variants associated with specific IncC plasmid types in the emerging Salmonella Typhimurium ST213 genotype.

The Salmonella enterica serovar Typhimurium sequence type 213 (ST213) emerged as a predominant genotype in Mexico. It is characterized by harboring multidrug resistance (MDR) IncC plasmids (previously IncA/C) and the lack of the Salmonella virulence plasmid (pSTV). Here we show that the D6-like plasmid prophage is present in most of the ST213 strains. We used the reported nucleotide sequence of YU39 plasmid (pYU39_89) to design a PCR typing scheme for the D6-like plasmid prophages, and determined the complete nucleotide sequences for the D6-like prophages of three additional ST213 strains (YU07-18, SL26 and SO21). Two prophage variants were described: i) a complete prophage, containing homologous sequences for most of the genetic modules described in P1 and D6 phages, which most likely allow for the lytic and lysogenic lifestyles; and ii) an incomplete prophage, lacking a 15 kb region containing morphogenesis genes, suggesting that it is defective. The tail fiber gene inversion region was the most divergent one between D6 and pYU39_89 genomes, suggesting the production of a distinct set of tail fibers, which could be involved in host range preferences. A glutaminyl-tRNA synthetase gene (glnS), which could be involved in providing host cell increased fitness or plasmid maintenance functions, was found in all D6-like genomes. Population level analysis revealed a biogeographic pattern of distribution of these plasmid-phages and specific associations with variants of MDR IncC plasmids. Statistically significant associations were found between the two prophage variants (p75 or p89), the type of IncC plasmids (I or II) and geographic isolation regions (Sonora, San Luis Potosí, Michoacán and Yucatán). This work integrates results from molecular typing, genomics and epidemiology to provide a broad overview for the evolution of an emergent Salmonella genotype.

New envelope stress factors involved in σE activation and conditional lethality of rpoE mutations in Salmonella enterica.

Salmonella enterica serovar Typhimurium (S. typhimurium) can cause food- and water-borne illness with diverse clinical manifestations. One key factor for S. typhimurium pathogenesis is the alternative sigma factor σE, which is encoded by the rpoE gene and controls the transcription of genes required for outer-membrane integrity in response to alterations in the bacterial envelope. The canonical pathway for σE activation involves proteolysis of the antisigma factor RseA, which is triggered by unfolded outer-membrane porins (OMPs) and lipopolysaccharides (LPS) that have accumulated in the periplasm. This study reports new stress factors that are able to activate σE expression. We demonstrate that UVA radiation induces σE activity in a pathway that is dependent on the stringent response regulator ppGpp. Survival assays revealed that rpoE has a role in the defence against lethal UVA doses that is mediated by functions that are dependent on and independent of the alternative sigma factor RpoS. We also report that the envelope stress generated by phage infection requires a functional rpoE gene for optimal bacterial tolerance and that it is able to induce σE activity in an RseA-dependent fashion. σE activity is also induced by hypo-osmotic shock in the absence of osmoregulated periplasmic glucans (OPGs). It is known that the rpoE gene is not essential in S. typhimurium. However, we report here two cases of the conditional lethality of rpoE mutations in this micro-organism. We demonstrate that rpoE mutations are not tolerated in the absence of OPGs (at low to moderate osmolarity) or LPS O-antigen. The latter case resembles that of the prototypic Escherichia coli strain K12, which neither synthesizes a complete LPS nor tolerates null rpoE mutations.

Mutagenic and genotoxic potential of direct electric current in Escherichia coli and Salmonella thyphimurium strains.

Direct electric current has several therapeutic uses such as antibacterial and antiprotozoal action, tissues scarring and regeneration, as well as tumor treatment. This method has shown promising results in vivo and in vitro, with significant efficacy and almost no side effects. Considering lack of studies regarding direct electric current mutagenic and/or genotoxic effects, the present work evaluated both aspects by using five different bacterial experimental assays: survival of repair-deficient mutants, Salmonella-histidine reversion mutagenesis (Ames test), forward mutations to rifampicin resistance, phage reactivation, and lysogenic induction. In these experimental conditions, cells were submitted to an approach that allows evaluation of anodic, cathodic, and electro-ionic effects generated by 2 mA of direct electric current, with doses ranging from 0.36 to 3.60 Coulombs. Our results showed these doses did not induce mutagenic or genotoxic effects.

Isolation, characterization, and application of bacteriophages for Salmonella spp. biocontrol in pigs.

Foodborne illness due to Salmonella-contaminated pork products is an important public health problem, causing significant economic losses worldwide. The use of bacteriophages is a potential intervention tool that has attracted interest for the control of foodborne pathogens. The objective of this study was to detect the presence of Salmonella in commercial pig farms and to isolate specific autochthonous bacteriophages against Salmonella Typhimurium, to characterize them and to evaluate their lytic capacity against Salmonella Typhimurium in vivo and in vitro. Salmonella was isolated on 50% (4/8) of the farms, with serotype Typhimurium being the most prevalent, detected in 48.2% of samples (13/27). The isolated Salmonella Typhimurium bacteriophages belong to the Podoviridae family, were active against serotypes Abony, Enteritidis, Typhi, and Typhimurium, but not against serotypes Arizonae, Cholerasuis, Gallinarum, and Pullorum. In in vitro tests, bacteriophage at 10(7) PFU/mL and 10(9) PFU/mL significantly reduced (p<0.05) Salmonella Typhimurium counts in 1.6 and 2.5 log10 colony-forming units (CFU)/mL, respectively, after 24 h. Before the in vivo treatment with bacteriophages, Salmonella was identified in 93.3% (28/30) of the fecal samples from the pigs inoculated with 10(6) CFU/mL, and only in 56.6% (17/30) after the treatment consisting of oral administration of the pool of the bacteriophages after the fasting period, simulating a common preslaughter practice. These results indicate that the pool of bacteriophages administered was capable of reducing the colonization of Salmonella in pigs.

The Salmonella Typhi hlyE gene plays a role in invasion of cultured epithelial cells and its functional transfer to S. Typhimurium promotes deep organ infection in mice.

Comparison of genome sequences of Salmonella enterica serovars Typhi and Typhimurium reveals that S. Typhi has a small 2.3kb genomic island missing in S. Typhimurium, designated Salmonella pathogenicity island 18 (SPI-18), which includes two potential genes. One of these, hlyE, encodes a hemolysin related to the Escherichia coli K12 HlyE hemolysin. PCR assays show that SPI-18 is present in S. Typhi and in many other, but not all, serovars of S. enterica subsp. enterica belonging to the SARB collection. HlyE activity cannot be detected in S. Typhi by means of standard plate assays. Nevertheless, we were able to reveal this activity upon lysis of bacterial cells with phages, in the presence of ampicillin, and in a ompA genetic background, conditions that compromise the integrity of the bacterial envelope. Almost all serovars of the SARB collection shown to cause systemic infections in humans have SPI-18 and hlyE and express an active hemolysin revealed upon bacterial envelope destabilization. S. Typhi hlyE mutants are impaired in invasion of human epithelial cells in vitro, and its heterologous expression in S. Typhimurium improves the colonization of deep organs in mice, demonstrating that the HlyE hemolysin is a new virulence determinant.

Initial interaction of the P22 phage with the Salmonella typhimurium surface.

OBJECTIVES: The goals of these studies were to characterize the interaction of the P22 phage particle with the Salmonella cell surface and to determine the phage elements involved in this interaction by mutational analysis. BACKGROUND: The phage P22 has been characterized extensively. The gene and protein for the phage P22 tailspike, which is the phage adsorption organelle, have been intensively studied. The kinetics of the interaction of the tailspike protein with the cell surface has been studied in detail, surprisingly no mutational analysis has ever been reported that has defined these components and their interaction between themselves and the cell surface. The main and perhaps only component needed for this cell surface interaction is the tailspike protein. METHODS: Adsorption to the cell surface has been measured in the wild type phage and in mutant derivatives, isolated in this study. Phage mutants have been isolated after hydroxylamine mutagenesis. RESULTS: The adsorption of P22 to the cell surface is a temperature-independent event. Forty putative phage adsorption mutants have been isolated. A sample of them have been further analyzed. These divide the adsorption process into at least two stages. One stage contains mutants that absorb with essential wild type phage kinetics to the cell surface while the other stage with delayed adsorption kinetics. CONCLUSIONS: The interaction of the phage P22 with the Salmonella cell surface has been shown to be a complicated one which is temperature-independent and multi-stage. Mutants isolated in this study may help dissect this process even further.

Initial interaction of the P22 phage with the Salmonella typhimurium surface

OBJECTIVES: The goals of these studies were to characterize the interaction of the P22 phage particle with the Salmonella cell surface and to determine the phage elements involved in this interaction by mutational analysis. BACKGROUND: The phage P22 has been characterized extensively. The gene and protein for the phage P22 tailspike, which is the phage adsorption organelle, have been intensively studied. The kinetics of the interaction of the tailspike protein with the cell surface has been studied in detail, surprisingly no mutational analysis has ever been reported that has defined these components and their interaction between themselves and the cell surface. The main and perhaps only component needed for this cell surface interaction is the tailspike protein. METHODS: Adsorption to the cell surface has been measured in the wild type phage and in mutant derivatives, isolated in this study. Phage mutants have been isolated after hydroxylamine mutagenesis. RESULTS: The adsorption of P22 to the cell surface is a temperature-independent event. Forty putative phage adsorption mutants have been isolated. A sample of them have been further analyzed. These divide the adsorption process into at least two stages. One stage contains mutants that absorb with essential wild type phage kinetics to the cell surface while the other stage with delayed adsorption kinetics. CONCLUSIONS: The interaction of the phage P22 with the Salmonella cell surface has been shown to be a complicated one which is temperature-independent and multi-stage. Mutants isolated in this study may help dissect this process even further

A tail of protein folding.

This review describes the use of a simple genetic system that has provided important insight into the process of folding and, of its flipside, that of protein aggregation. These studies make use of the tail protein of the bacterial virus P22 which infects Salmonella typhimurium. This folding system serves as a model for a number protein structural elements and may also provide important insights into disease-related protein folding defects at a time when an increasing number of diseases are being shown to be due to protein folding alterations.

A tail of protein folding

This review describes the use of a simple genetic system that has provided important insight into the process of folding and, of its flipside, that of protein aggregation. These studies make use of the tail protein of the bacterial virus P22 which infects Salmonella typhimurium. This folding system serves as a model for a number protein structural elements and may also provide important insights into disease-related protein folding defects at a time when an increasing number of diseases are being shown to be due to protein folding alterations

Obtención, aislamiento y caracterización de mutantes auxotróficos de salmonella typhinurium / Obtain, isolation and auxotrophics mutants of salmonella typhimurium

Se comprobó el efecto inductor mutagénico de la luz uv, obteniendo mutantes auxotróficos y morfológicos de salmonella typhimurium. El aislamiento de mutantes auxotróficos mediante la técnica de réplica en placa mejora con el uso de penicilina mediante el método de Hollyday, para la identificación de los requerimientos específicos de cada mutante, se caracterizó 3 mutantes auxotróficos de 24 mutantes escogidos al azar. De 24 mutantes auxotróficos, el 30 por ciento resultó ser metronina dependientes, y 25 por ciento revirtieron la características de mutantes auxotróficos.