RESUMO
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 São Paulo, Brazil. The SC counts varied from <1 to 3.4 × 10(3) PFU/100 ml in seawater (73 samples tested), from <1 to 4.7 × 10(2) PFU/g in plankton (46 samples tested), and from <1 to 2.2 × 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 São Sebastião (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 São Sebastião and Ubatuba. This is the first report of coliphage diversity in seawater, plankton, and bivalve samples collected from São 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.
Assuntos
Biodiversidade , Bivalves/virologia , Colífagos/classificação , Colífagos/isolamento & purificação , Plâncton/virologia , Água do Mar/virologia , Animais , Brasil , Colífagos/genética , Colífagos/ultraestrutura , Carga Viral , Vírion/ultraestruturaRESUMO
AIMS: To isolate, characterize and select phages as potential biocontrol agents of enterohemorrhagic and Shiga toxin-producing Escherichia coli (EHEC and STEC) in cattle. METHODS AND RESULTS: Sixteen STEC and EHEC coliphages were isolated from bovine minced meat and stool samples and characterized with respect to their host range against STEC, EHEC and other Gram-negative pathogens; their morphology by electron microscopy; the presence of the stx1, stx2 and cI genes by means of PCR; RAPD and rep-PCR profiles; plaque formation; and acid resistance. Six isolates belonged to the Myoviridae and 10 to the Podoviridae families. The phages negative for stx and cI that formed large, well-defined plaques were all isolated using EHEC O157:H7 as host. Among them, only CA911 was a myophage and, together with CA933P, had the broadest host range for STEC and EHEC; the latter phage also infected Shigella and Pseudomonas. Isolates CA911, MFA933P and MFA45D differed in particle morphology and amplification patterns by RAPD and rep-PCR and showed the highest acidity tolerance. CONCLUSIONS: Myophage CA911 and podophages CA933P, MFA933P and MFA45D were chosen as the best candidates for biocontrol of STEC and EHEC in cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: This work employs steps for a rational selection and characterization of bacteriophages as therapeutic agents. This report constitutes the first documentation of STEC and EHEC phages isolated in Argentina and proposes for the first time the use of rep-PCR as a complement of RAPD on DNA fingerprinting of phages.
Assuntos
Colífagos/isolamento & purificação , Escherichia coli Êntero-Hemorrágica/virologia , Escherichia coli Shiga Toxigênica/virologia , Animais , Argentina , Bovinos , Colífagos/genética , Colífagos/ultraestrutura , Fezes/virologia , Genes Virais , Especificidade de Hospedeiro , Carne/virologia , Reação em Cadeia da Polimerase , Fatores de Virulência/genéticaRESUMO
In the absence of urea, pressures up to 2.5 kbar promote only 10% dissociation of the whole particles of R17 bacteriophage. In the presence of concentrations of urea between 1.0 and 5.0 M, pressure promotes complete, reversible dissociation of the virus particles. At the lower urea concentrations reversible dissociation of R17 virus particles shows no dependence on protein concentration indicating a high degree of heterogeneity of the particles, but higher urea concentrations, 2.5 to 5.0 M, result in progressive restoration of the protein concentration dependence of the pressure dissociation. At still higher urea concentrations, 5.0 to 8.0 M, irreversible dissociation of virus takes place at atmospheric pressure. In contrast, the dissociation of the isolated dimers of the capsid protein was dependent on protein concentration to the extent predicted for a stochastic equilibrium, and dimers were much less stable than the whole virus both to dissociation by pressure or urea. In contradistinction, the reversible whole-virus dissociation observed at urea concentrations below 2.5 M appears to be a typical deterministic equilibrium, without appreciable dynamic exchange between whole particle and subunits during the lengthy experiments. The experiments demonstrate that the "thermodynamic individuality" of the virus particles arises in conformational differences in the assembled viruses, and that there is a direct relation between the stability of the particles and their heterogeneity.
Assuntos
Colífagos/fisiologia , Fagos RNA/fisiologia , Capsídeo/fisiologia , Colífagos/patogenicidade , Colífagos/ultraestrutura , Pressão Hidrostática , Microscopia Eletrônica , Fagos RNA/patogenicidade , Fagos RNA/ultraestrutura , Espectrometria de Fluorescência , Termodinâmica , Ureia , Vírion/fisiologiaRESUMO
Describes a comparative analysis of RNA coliphages with special emphasis on regulatory and structural features. Phylogenetic phage sequence comparison reveals kinship between different phages and facilitates deduction of their secondary structure. Complete sequences from phage fr and partial sequences from group I phages M12, R17 and f2 were determined. The nucleotide sequence from a serological intermediate JP34 was partially elucidated, Only the fr sequence proved suitable for comparison with MS2 (similarity 77 per cent). A "core" structure (3' terminal region) of phage RNA proposed a certain structural resemblance with tRNA. Comparison of the MS2 and fr nucleotide sequences revealed absence of an AUG initiator codon for the fr lysis (L) gene. However, 4 codons further downstream there is a UUG codon which proved to be the start codon for the fr lysis gene. This UUG start codon proved crucial for L-gene regulation. It is suggested that terminated but not released ribosomes mediate the L-gene activation. A general eubacterial scanning model is proposed. It seems that ribosomes can scan the mRNA in both directions and reinitiate at the first encountered restart site. This eubacterial scanning model parallels the eukaryotic translation initiation mechanism