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 (CB4O) and 45 bacteriophages from a municipal wastewater treatment plant (WT45O) were evaluated. In experiment 1, an in vitro crop assay was conducted with selected bacteriophage concentrations (105 to 101 pfu/mL) to determine ability to reduce Salmonella Enteritidis in the simulated crop environment. Following 2 h at 37 degrees C, CB40 or WT45O reduced Salmonella Enteritidis recovery by 1.5 or 5 log, respectively, as compared with control. However, CB40 did not affect total SE recovery after 6 h, whereas WT45O resulted in up to a 6-log reduction of Salmonella Enteritidis. In experiment 2, day-of-hatch chicks were challenged orally with 3 x 103 cfu /chick Salmonella Enteritidis and treated cloacally with 1 X 109 WT45O pfu/chick I 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 103 cfu/chick Salmonella Enteritidis and treated via oral gavage with I X 108 CB40 pfu/chick, 1.2 x 108 WT45O pfu/chick, or a combination of both, I 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.

Evaluation of Salmonella-Lytic Properties of Bacteriophages Isolated from Commercial Broiler Houses

Because of recent interest in bacteriophage therapy in poultry, information regarding the interaction of bacteriophages and potential host bacteria in the environment should be collected. The present studies were initiated with a rather typical commercial broiler integrator within the south-central United States to examine environmental Salmonella levels in two broiler complexes, attempt to isolate Salmonella-lytic bacteriophages, and elucidate a possible reason for differing apparent Salmonella prevalence. Significantly ( P<0.05) less Salmonella was isolated from houses in complex 1 ( 15/44 [ 34%] Salmonella-positive drag swabs) as compared to houses in complex 2 ( 22/24 [ 92%]). A total of seven Salmonella-lytic bacteriophages were isolated from Salmonella-positive environments, and two bacteriophages were isolated from a single Salmonella-negative house. During the initial bacteriophage isolation, individual bacteriophages did not replicate in the Salmonella host isolated from the same environment, and lysis of additional Salmonella hosts relied on high numbers of bacteriophage to be present. This suggests that the presence of these bacteriophages in the environment of a commercial broiler house had little to no effect on the presence of Salmonella. This study highlights the need to find additional bacteriophage sources, more effective isolation methods, and more innovative approaches to using bacteriophages to treat enteric disease.

Cooperative RNA Polymerase Molecules Behavior on a Stochastic Sequence-Dependent Model for Transcription Elongation

The transcription process is crucial to life and the enzyme RNA polymerase (RNAP) is the major component of the transcription machinery. The development of single-molecule techniques, such as magnetic and optical tweezers, atomic-force microscopy and single-molecule fluorescence, increased our understanding of the transcription process and complements traditional biochemical studies. Based on these studies, theoretical models have been proposed to explain and predict the kinetics of the RNAP during the polymerization, highlighting the results achieved by models based on the thermodynamic stability of the transcription elongation complex. However, experiments showed that if more than one RNAP initiates from the same promoter, the transcription behavior slightly changes and new phenomenona are observed. We proposed and implemented a theoretical model that considers collisions between RNAPs and predicts their cooperative behavior during multi-round transcription generalizing the Bai et al. stochastic sequence-dependent model. In our approach, collisions between elongating enzymes modify their transcription rate values. We performed the simulations in Mathematica® and compared the results of the single and the multiple-molecule transcription with experimental results and other theoretical models. Our multi-round approach can recover several expected behaviors, showing that the transcription process for the studied sequences can be accelerated up to 48% when collisions are allowed: the dwell times on pause sites are reduced as well as the distance that the RNAPs backtracked from backtracking sites. © 2013 Costa et al.

The role of prophage in plant-pathogenic bacteria

A diverse set of phage lineages is associated with the bacterial plant-pathogen genomes sequenced to date. Analysis of 37 genomes revealed 5,169 potential genes (approximately 4.3 Mbp) of phage origin, and at least 50 had no function assigned or are nonessential to phage biology. Some phytopathogens have transcriptionally active prophage genes under conditions that mimic plant infection, suggesting an association between plant disease and prophage transcriptional modulation. The role of prophages within genomes for cell biology varies. For pathogens such as Pectobacterium, Pseudomonas, Ralstonia, and Streptomyces, involvement of prophage in disease symptoms has been demonstrated. In Xylella and Xanthomonas, prophage activity is associated with genome rearrangements and strain differentiation. For other pathogens, prophage roles are yet to be established. This review integrates available information in a unique interface (http://propnav.esalq.usp.br) that may be assessed to improve research in prophage biology and its association with genome evolution and pathogenicity. © Copyright ©2013 by Annual Reviews. All rights reserved.

Utilização de bacteriófagos em associação com Lactobacillus no controle de Salmonella enterica subs. enterica sorotipo typhimurium antibiótico-resitente em frangos de corte (Gallus gallus domesticus - Linnaes, 1758)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Utilização de bacteriófagos ambientais no controle de biofilmes de Salmonella spp. em superfícies utilizadas na indústria de processamento e comercialização de frango de corte e derivados

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The genome sequence of the plant pathogen Xylella fastidiosa: The Xylella fastidiosa consortium of the organization for nucleotide sequencing and analysis, Sao Paulo, Brazil

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis - a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and 'two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

Separação imunomagnética associada a bacteriófago para diagnóstico de Salmonella enterica em carne de frango

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Group I Introns and Homing Endonucleases in T-even-like Bacteriophages

Homing endonucleases are rare-cutting enzymes that cleave DNA at a site near their own location, preferentially in alleles lacking the homing endonuclease gene (HEG). By cleaving HEG-less alleles the homing endonuclease can mediate the transfer of its own gene to the cleaved site via a process called homing, involving double strand break repair. Via homing, HEGs are efficiently transferred into new genomes when horizontal exchange of DNA occurs between organisms. Group I introns are intervening sequences that can catalyse their own excision from the unprocessed transcript without the need of any proteins. They are widespread, occurring both in eukaryotes and prokaryotes and in their viruses. Many group I introns encode a HEG within them that confers mobility also to the intron and mediates the combined transfer of the intron/HEG to intronless alleles via homing. Bacteriophage T4 contains three such group I introns and at least 12 freestanding HEGs in its genome. The majority of phages besides T4 do not contain any introns, and freestanding HEGs are also scarcely represented among other phages. In the first paper we looked into why group I introns are so rare in phages related to T4 in spite of the fact that they can spread between phages via homing. We have identified the first phage besides T4 that contains all three T-even introns and also shown that homing of at least one of the introns has occurred recently between some of the phages in Nature. We also show that intron homing can be highly efficient between related phages if two phages infect the same bacterium but that there also exists counteracting mechanisms that can restrict the spread of introns between phages. In the second paper we have looked at how the presence of introns can affect gene expression in the phage. We find that the efficiency of splicing can be affected by variation of translation of the upstream exon for all three introns in T4. Furthermore, we find that splicing is also compromised upon infection of stationary-phase bacteria. This is the first time that the efficiency of self-splicing of group I introns has been coupled to environmental conditions and the potential effect of this on phage viability is discussed. In the third paper we have characterised two novel freestanding homing endonucleases that in some T-even-like phages replace two of the putative HEGs in T4. We also present a new theory on why it is a selective advantage for freestanding, phage homing endonucleases to cleave both HEG-containing and HEG-less genomes.

Structural and Interaction Studies of Bacterial Polysaccharides by NMR Spectroscopy

An introduction to bacterial polysaccharides and the methods for structural determination are described in the first two parts of the thesis. In a structural elucidation of bacterial polysaccharides NMR experiments are important as is component analysis. A short description of immunochemical methods such as enzyme immunoassays is included. Two NMR techniques used for interaction studies, trNOE and STD NMR, are also discussed. The third part of the thesis discusses and summarizes the results from the included papers. The structures of the exopolysaccharides produced by two lactic acid bacteria are determined by one- and two dimensional NMR experiments. One is a heteropolysaccharide produced by Streptococcus thermophilus and the other a homopolysaccharide produced by Propionibacterium freudenreichii. The structure of an acidic polysaccharide from a marine bacterium with two serine residues in the repeating unit is also investigated. The structural and immunological relationship between two O-antigenic polysaccharides from Escherichia coli strain 180/C3 and O5 is discussed and investigated. Finally, interaction studies of an octasaccharide derived from the Salmonella enteritidis O-antigen and a bacteriophage are described which were performed with NMR experiments.

Phage therapy: A software system for phage quantification and kinetic model inference.

One of the most serious problems of the modern medicine is the growing emergence of antibiotic resistance among pathogenic bacteria. In this circumstance, different and innovative approaches for treating infections caused by multidrug-resistant bacteria are imperatively required. Bacteriophage Therapy is one among the fascinating approaches to be taken into account. This consists of the use of bacteriophages, viruses that infect bacteria, in order to defeat specific bacterial pathogens. Phage therapy is not an innovative idea, indeed, it was widely used around the world in the 1930s and 1940s, in order to treat various infection diseases, and it is still used in Eastern Europe and the former Soviet Union. Nevertheless, Western scientists mostly lost interest in further use and study of phage therapy and abandoned it after the discovery and the spread of antibiotics. The advancement of scientific knowledge of the last years, together with the encouraging results from recent animal studies using phages to treat bacterial infections, and above all the urgent need for novel and effective antimicrobials, have given a prompt for additional rigorous researches in this field. In particular, in the laboratory of synthetic biology of the department of Life Sciences at the University of Warwick, a novel approach was adopted, starting from the original concept of phage therapy, in order to study a concrete alternative to antibiotics. The innovative idea of the project consists in the development of experimental methodologies, which allow to engineer a programmable synthetic phage system using a combination of directed evolution, automation and microfluidics. The main aim is to make “the therapeutics of tomorrow individualized, specific, and self-regulated” (Jaramillo, 2015). In this context, one of the most important key points is the Bacteriophage Quantification. Therefore, in this research work, a mathematical model describing complex dynamics occurring in biological systems involving continuous growth of bacteriophages, modulated by the performance of the host organisms, was implemented as algorithms into a working software using MATLAB. The developed program is able to predict different unknown concentrations of phages much faster than the classical overnight Plaque Assay. What is more, it gives a meaning and an explanation to the obtained data, making inference about the parameter set of the model, that are representative of the bacteriophage-host interaction.

Application of in vivo induced antigen technology (IVIAT) to Bacillus anthracis.

In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets.

Direct visualization of the putative portal in the Kaposi's sarcoma-associated herpesvirus capsid by cryoelectron tomography.

Genetic and biochemical studies have suggested the existence of a bacteriophage-like, DNA-packaging/ejecting portal complex in herpesviruses capsids, but its arrangement remained unknown. Here, we report the first visualization of a unique vertex in the Kaposi's sarcoma-associated herpesvirus (KSHV) capsid by cryoelectron tomography, thus providing direct structural evidence for the existence of a portal complex in a gammaherpesvirus. This putative KSHV portal is an internally localized, umbilicated structure and lacks all of the external machineries characteristic of portals in DNA bacteriophages.

Studies of subcellular localization and complex formation of the Agrobacterium tumefaciens transport ATPase VirB11

The VirB11 ATPase is an essential component of an Agrobacterium tumefaciens type IV bacterial secretion system that transfers oncogenic nucleoprotein complexes to susceptible plant cells. This dissertation investigates the subcellular localization and homo-oligomeric state of the VirB11 ATPase in order to provide insights about the assembly of the protein as a subunit of this membrane-associated transfer system. Subcellular fractionation studies and quantitative immunoblot analysis demonstrated that $\sim$30% of VirB11 partitioned as soluble protein and $\sim$70% was tightly associated with the bacterial cytoplasmic membrane. No differences were detected in VirB11 subcellular localization and membrane association in the presence or absence of other transport system components. Mutations in virB11 affecting protein function were mapped near the amino terminus, just upstream of a region encoding a Walker 'A' nucleotide-binding site, and within the Walker 'A' motif partitioned almost exclusively with the cytoplasmic membrane, suggesting that an activity associated with nucleotide binding could modulate the affinity of VirB11 for the cytoplasmic membrane. Merodiploid analysis of VirB11 mutant and truncation derivatives provided strong evidence that VirB11 functions as a homo- or heteromultimer and that the C-terminal half of VirB11 contains a protein interaction domain. A combination of biochemical and molecular genetic approaches suggested that VirB11 and the green fluorescence protein (GFP) formed a mixed multimer as demonstrated by immunoprecipitation experiments with anti-GFP antibodies. Second, a hybrid protein composed of VirB11 fused to the N-terminal DNA-binding domain of bacteriophage $\lambda$ cI repressor conferred immunity to $\lambda$ superinfection, demonstrating that VirB11 self-association promotes dimerization of the chimeric repressor. A conserved Walker 'A' motif, though required for VirB11 function in T-complex export, was not necessary for VirB11 self-association. Sequences in both the N- and the C-terminal halves of the protein were found to contribute to self-association of the full length protein. Chemical cross-linking experiments with His$\sb6$ tagged VirB11 suggested that VirB11 probably assembles into a higher order homo-oligomeric complex. ^

Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes

The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.