Vibrio harveyi phages: Isolation, characterization and evaluation of their potential as phage therapeutics on Vibrio harveyi in shrimp hatcheries

Aquaculture is a global industry providing food and employment thereby contributing to the economy. For the sustenance of aquaculture, disease management is a major requirement. Among the bacterial pathogens Vibrio harveyi remains to be the major one especially in shrimp culture systems. Rapid and mass mortality of shrimp larvae due to Vibrio harveyi infection is well known, and the pathogen causes serious economic losses in grow out systems as well. It suggests that a well defined management strategy has to be built up to protect the crop from Vibrio harveyi infection in aquaculture systems. Antibiotics have been the choice for quite some times which led to residues in meat and development of multidrug resistant bacteria which invited ban on their application. In this context several alternate options have been thought off such as probiotics, immunostimulants and vaccines. Phage therapy is yet another option. Phages being natural parasites of bacteria and are abundant in aquatic environments their application to control bacterial pathogens in aquaculture has commendable potential in lieu of antibiotics. For that matter the therapeutic effect of phages has been proven in several antibiotic resistant pathogens inclusive of Vibrio harveyi.

Relative survival of Escherichia coli and Salmonella typhimurium in a tropical estuary

Microcosm studies have been carried out to find out the relative survival of Escherichia coli and Salmonella typhimurium in a tropical estuary. Survival has been assessed in relation to the important self-purifying parameters such as biotic factors contained in the estuarine water, toxicity due to the dissolved organic and antibiotic substances in the water and the sunlight. The results revealed that sunlight is the most important inactivating factor on the survival of E. coli and S. typhimurium in the estuarine water. While the biological factors contained in the estuarine water such as protozoans and bacteriophages also exerted considerable inactivation of these organisms, the composition of the water with all its dissolved organic and inorganic substances was not damaging to the test organisms. Results also indicated better survival capacity of E. coli cells under all test conditions when compared to S. typhimurium

Fagoterapia como alternativa ao uso dos antibióticos convencionais

Nas últimas décadas, a investigação de antibióticos com novos mecanismos de acção, tem vindo a ser motivada pela contínua emergência de estirpes bacterianas multirresistentes. No entanto, nos últimos anos esse desenvolvimento tem vindo a abrandar, o que representa um grave problema de saúde pública. Antes da era dos antibióticos a fagoterapia representava a terapêutica de primeira linha no tratamento de infecções bacterianas. Como a ausência de recursos impossibilitava a compreensão dos mecanismos de acção moleculares do fago, a fagoterapia era apenas sustentada pelo conhecimento empírico. A ausência de conhecimento associada ao início da era dos antibióticos foram condições suficientes para que a terapêutica fágica fosse posta de parte, à excepção de alguns países da Europa do Leste. De acordo com a literatura disponibilizada por estes países, vários têm sido os casos de sucesso no tratamento de infecções bacterianas, incluindo infecções causadas por estirpes multirresistentes aos antibióticos convencionais. No entanto, contrariamente aos ensaios clínicos, a maioria destes estudos omite informação crítica que impossibilita a interpretação dos respectivos resultados. Actualmente, as novas ferramentas oferecidas pelos avanços biotecnológicos possibilitam não só a compreensão do mecanismo de infecção bacteriana como também permitem compreender melhor a interacção entre os bacteriófagos e o organismo humano. Como tal, no futuro, a fagoterapia pode ser considerada uma alternativa efectiva para solucionar os casos críticos de multirresistência bacteriana aos antibióticos convencionais.

Comparative genomics of Brachyspira pilosicoli strains: genome rearrangements, reductions and correlation of genetic compliment with phenotypic diversity.

Background. The anaerobic spirochaete Brachyspira pilosicoli causes enteric disease in avian, porcine and human hosts, amongst others. To date, the only available genome sequence of B. pilosicoli is that of strain 95/1000, a porcine isolate. In the first intra-species genome comparison within the Brachyspira genus, we report the whole genome sequence of B. pilosicoli B2904, an avian isolate, the incomplete genome sequence of B. pilosicoli WesB, a human isolate, and the comparisons with B. pilosicoli 95/1000. We also draw on incomplete genome sequences from three other Brachyspira species. Finally we report the first application of the high-throughput Biolog phenotype screening tool on the B. pilosicoli strains for detailed comparisons between genotype and phenotype. Results. Feature and sequence genome comparisons revealed a high degree of similarity between the three B. pilosicoli strains, although the genomes of B2904 and WesB were larger than that of 95/1000 (~2,765, 2.890 and 2.596 Mb, respectively). Genome rearrangements were observed which correlated largely with the positions of mobile genetic elements. Through comparison of the B2904 and WesB genomes with the 95/1000 genome, features that we propose are non-essential due to their absence from 95/1000 include a peptidase, glycine reductase complex components and transposases. Novel bacteriophages were detected in the newly-sequenced genomes, which appeared to have involvement in intra- and inter-species horizontal gene transfer. Phenotypic differences predicted from genome analysis, such as the lack of genes for glucuronate catabolism in 95/1000, were confirmed by phenotyping. Conclusions. The availability of multiple B. pilosicoli genome sequences has allowed us to demonstrate the substantial genomic variation that exists between these strains, and provides an insight into genetic events that are shaping the species. In addition, phenotype screening allowed determination of how genotypic differences translated to phenotype. Further application of such comparisons will improve understanding of the metabolic capabilities of Brachyspira species.

The role of prophage-like elements in the diversity of Salmonella enterica serovars

The Salmonella enterica serovar Typhi CT18 (S. Typhi) chromosome harbours seven distinct prophage-like elements, some of which may encode functional bacteriophages. In silico analyses were used to investigate these regions in S. Typhi CT18, and ultimately compare these integrated bacteriophages against 40 other Salmonella isolates using DNA microarray technology. S. Typhi CT18 contains prophages that show similarity to the lambda, Mu, P2 and P4 bacteriophage families. When compared to other S. Typhi isolates, these elements were generally conserved, supporting a clonal origin of this serovar. However, distinct variation was detected within a broad range of Salmonella serovars; many of the prophage regions are predicted to be specific to S. Typhi. Some of the P2 family prophage analysed have the potential to carry non-essential "cargo" genes within the hyper-variable tail region, an observation that suggests that these bacteriophage may confer a level of specialisation on their host. Lysogenic bacteriophages therefore play a crucial role in the generation of genetic diversity within S. enterica. (C) 2004 Elsevier Ltd. All rights reserved.

Viruses in soils: morphological diversity and abundance in the rhizosphere

Soil viruses are potentially of great importance as they may influence the ecology and evolution of soil biological communities through both an ability to transfer genes from host to host and as a potential cause of microbial mortality. Despite this importance, the area of soil virology is understudied. Here, we report the isolation and preliminary characterisation of viruses from soils in the Dundee area of Scotland. Different virus morphotypes including tailed, polyhedral (spherical), rod shaped, filamentous and bacilliform particles were detected in the soil samples. An apparent predominance of small spherical and filamentous bacteriophages was observed, whereas tailed bacteriophages were significantly less abundant. In this report, we also present observations and characterisation of viruses from different soil functional domains surrounding wheat roots: rhizosheath, rhizosphere and bulk soil. In spite of the differences in abundance of bacterial communities in these domains, no significant variations in viral population structure in terms of morphology and abundance were found. Typically, there were approximately 1.1–1.2 × 109 virions g−1 dry weight, implicating remarkable differences in virus-to-bacteria ratios in domains close to roots, rhizosphere and rhizosheath (approximately 0.27) and in bulk soil (approximately 4.68).

Within-host evolution of Staphylococcus aureus during asymptomatic carriage

Background Staphylococcus aureus is a major cause of healthcare associated mortality, but like many important bacterial pathogens, it is a common constituent of the normal human body flora. Around a third of healthy adults are carriers. Recent evidence suggests that evolution of S. aureus during nasal carriage may be associated with progression to invasive disease. However, a more detailed understanding of within-host evolution under natural conditions is required to appreciate the evolutionary and mechanistic reasons why commensal bacteria such as S. aureus cause disease. Therefore we examined in detail the evolutionary dynamics of normal, asymptomatic carriage. Sequencing a total of 131 genomes across 13 singly colonized hosts using the Illumina platform, we investigated diversity, selection, population dynamics and transmission during the short-term evolution of S. aureus. Principal Findings We characterized the processes by which the raw material for evolution is generated: micro-mutation (point mutation and small insertions/deletions), macro-mutation (large insertions/deletions) and the loss or acquisition of mobile elements (plasmids and bacteriophages). Through an analysis of synonymous, non-synonymous and intergenic mutations we discovered a fitness landscape dominated by purifying selection, with rare examples of adaptive change in genes encoding surface-anchored proteins and an enterotoxin. We found evidence for dramatic, hundred-fold fluctuations in the size of the within-host population over time, which we related to the cycle of colonization and clearance. Using a newly-developed population genetics approach to detect recent transmission among hosts, we revealed evidence for recent transmission between some of our subjects, including a husband and wife both carrying populations of methicillin-resistant S. aureus (MRSA). Significance This investigation begins to paint a picture of the within-host evolution of an important bacterial pathogen during its prevailing natural state, asymptomatic carriage. These results also have wider significance as a benchmark for future systematic studies of evolution during invasive S. aureus disease.

Bacterial motility confers fitness advantage in the presence of phages

Dispersal provides the opportunity to escape harm and colonize new patches, enabling populations to expand and persist. However, the benefits of dispersal associated with escaping harm will be dependent on the structure of the environment and the likelihood of escape. Here, we empirically investigate how the spatial distribution of a parasite influences the evolution of host dispersal. Bacteriophages are a strong and common threat for bacteria in natural environments and offer a good system with which to explore parasite-mediated selection on host dispersal. We used two transposon mutants of the opportunistic bacteria, Pseudomonas aeruginosa, which varied in their motility (a disperser and a nondisperser), and the lytic bacteriophage ФKZ. The phage was distributed either in the central point of colony inoculation only, thus offering an escape route for the dispersing bacteria; or, present throughout the agar, where benefits of dispersal might be lost. Surprisingly, we found dispersal to be equally advantageous under both phage conditions relative to when phages were absent. A general explanation is that dispersal decreased the spatial structuring of host population, reducing opportunities for parasite transmission, but other more idiosyncratic mechanisms may also have contributed. This study highlights the crucial role the parasites can play on the evolution of dispersal and, more specifically, that bacteriophages, which are ubiquitous, are likely to select for bacterial motility.

Klebsiella pneumoniae subsp. pneumoniae–bacteriophage combination from the caecal effluent of a healthy woman

A sample of caecal effluent was obtained from a female patient who had undergone a routine colonoscopic examination. Bacteria were isolated anaerobically from the sample, and screened against the remaining filtered caecal effluent in an attempt to isolate bacteriophages (phages). A lytic phage, named KLPN1, was isolated on a strain identified as Klebsiella pneumoniae subsp. pneumoniae (capsular type K2, rmpA+). This Siphoviridae phage presents a rosette-like tail tip and exhibits depolymerase activity, as demonstrated by the formation of plaque-surrounding haloes that increased in size over the course of incubation. When screened against a panel of clinical isolates of K. pneumoniae subsp. pneumoniae, phage KLPN1 was shown to infect and lyse capsular type K2 strains, though it did not exhibit depolymerase activity on such hosts. The genome of KLPN1 was determined to be 49,037 bp (50.53 %GC) in length, encompassing 73 predicted ORFs, of which 23 represented genes associated with structure, host recognition, packaging, DNA replication and cell lysis. On the basis of sequence analyses, phages KLPN1 (GenBank: KR262148) and 1513 (a member of the family Siphoviridae, GenBank: KP658157) were found to be two new members of the genus “Kp36likevirus”.

Diversity of Somatic Coliphages in Coastal Regions with Different Levels of Anthropogenic Activity in Sao Paulo State, Brazil

Bacteriophages are the most abundant and genetically diverse viruses on Earth, with complex ecology in both quantitative and qualitative terms. Somatic coliphages (SC) have been reported to be good indicators of fecal pollution in seawater. This study focused on determining the concentration of SC and their diversity by electron microscopy of seawater, plankton, and bivalve samples collected at three coastal regions in Sao Paulo, Brazil. The SC counts varied from < 1 to 3.4 x 103 PFU/100 ml in seawater (73 samples tested), from < 1 to 4.7 x 10(2) PFU/g in plankton (46 samples tested), and from < 1 to 2.2 x 10(1) PFU/g in bivalves (11 samples tested). In seawater samples, a relationship between the thermotolerant coliforms and Escherichia coli and SC was observed at the three regions (P = 0.0001) according to the anthropogenic activities present at each region. However, SC were found in plankton samples from three regions: Baixada Santista (17/20), Canal de Sao Sebastiao (6/14), and Ubatuba (3/12). In seawater samples collected from Baixada Santista, four morphotypes were observed: A1 (4.5%), B1 (50%), C1 (36.4%), and D1 (9.1%). One coliphage, Siphoviridae type T1, had the longest tail: between 939 and 995 nm. In plankton samples, Siphoviridae (65.8%), Podoviridae (15.8%), Microviridae (15.8%), and Myoviridae (2.6%) were found. In bivalves, only the morphotype B1 was observed. These SC were associated with enteric hosts: enterobacteria, E. coli, Proteus, Salmonella, and Yersinia. Baixada Santista is an area containing a high level of fecal pollution compared to those in the Canal de Sao Sebastiao and Ubatuba. This is the first report of coliphage diversity in seawater, plankton, and bivalve samples collected from Sao Paulo coastal regions. A better characterization of SC diversity in coastal environments will help with the management and evaluation of the microbiological risks for recreation, seafood cultivation, and consumption.

Análise proteômica de Chromobacterium violaceum: acúmulo estacionário e diferencial após exposição à luz UVC

Chromobacterium violaceum is a free-living bacillus, Gram-negative commonly found in water and sand of tropical and subtropical regions. One of its main characteristic it's the ability to produce the purple pigment named violacein, that shows countless biological activities. In 2003, the genome of this organism was totally sequenced and revealed important informations about the physiology of this bacteria. However, few post-genomics studies had been accomplished. This work evaluated the protein profile of C. violaceum cultivated in LB medium at 28ºC that allowed the identification and characterization of proteins related to a possible secretion system that wasn't identified and characterized yet in C. violaceum, to the quorum sensing system, to regulatory process of transcription and translation, stress adaptation and biotechnological potential. Moreover, the response of the bacteria to UVC radiation was evaluated. The comparison of the protein profile, analyzed through 2-D electrophoresis, of the control group versus the treatment group allowed the identification of 52 proteins that arose after stress induction. The obtained results enable the elaboration of a stress response pathway in C. violaceum generated by the UVC light. This pathway, that seems to be a general stress response, involves the expression of proteins related to cellular division, purine and pirimidine metabolism, heat chock or chaperones, energy supply, regulation of biofilm formation, transport, regulation of lytic cycle of bacteriophages, besides proteins that show undefined function. Despite the response present similarities with the classic SOS response of E. coli, we still cannot assert that C. violaceum shows a SOS-like response, mainly due to the absence of characterization of a LexA-like protein in this organism

Structural basis of nuclear import of flap endonuclease 1 (FEN1)

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

Tethered DNA scaffolds on optical sensor platforms for detection of hipO gene from Campylobacter jejuni

DNA biosensors have gained increased attention over traditional diagnostic methods due to their fast and responsive operation and cost-effective design. The specificity of DNA biosensors relies on single-stranded oligonucleotide probes immobilized to a transduction platform. Here, we report the development of biosensors to detect the hippuricase gene (hipO) from Campylobacter jejuni using direct covalent coupling of thiol- and biotin-labeled single-stranded DNA (ssDNA) on both surface plasmon resonance (SPR) and diffraction optics technology (DOT, dotLab) transduction platforms. This is the first known report of the dotLab to detect targeted DNA. Application of 6-mercapto-1-hexanol as a spacer thiol for SPR gold surface created a self-assembled monolayer that removed unbound ssDNA and minimized non-specific detection. The detection limit of SPR sensors was shown to be 2.5 nM DNA while dotLab sensors demonstrated a slightly decreased detection limit of 5.0 nM (0.005 μM). It was possible to reuse the SPR sensor due to the negligible changes in sensor sensitivity (∼9.7 × 10 -7 ΔRU) and minimal damage to immobilized probes following use, whereas dotLab sensors could not be reused. Results indicated feasibility of optical biosensors for rapid and sensitive detection of the hipO gene of Campylobacter jejuni using specific ssDNA as a probe. © 2011 Elsevier B.V.

Clonagem e expressão de fragmentos funcionais(scFv) de anticorpo contra o Parvovírus canino-2 com a técnica da Phage display

Pós-graduação em Medicina Veterinária - FMVZ

Bacteriophage- induced reduction in Salmonella Enteritidis counts in the crop of broiler chickens undergoing preslaughter feed withdrawal

Salmonella food poisoning is a public health problem. Feed withdrawal from broiler chickens before slaughter can favor the multiplication of Salmonella in the cecum and crop of contaminated animals and subsequently lead to contamination of carcasses in the processing plant. In the present study, a cocktail of lytic bacteriophages isolated from sewage water was orally administered to 45-d-old broiler chickens 1 h after they received an oral dose of 107 cfu/mL Salmonella enterica subspecies enterica serotype Enteritidis. Immediately after phage administration and 30 min, 1, 3, 6, and 12 h thereafter, groups of chicken were killed. Ceca and crops were analyzed for the presence of Salmonella. At 3 h posttreatment, there were 103 cfu/g and 101 cfu/g of cecal and crop suspension, respectively. At 6 h after treatment, the number of Salmonella was 103 cfu/g in the cecal suspension, but below the detection limit in the crops. our results suggest that bacteriophage therapy may be able to reduce the contamination of chicken carcasses by reducing the preslaughter load of Salmonella in the birds.