Phage Cocktail Development against Aeromonas salmonicida subsp. salmonicida Strains Is Compromised by a Prophage.

Aquaculture is a rapidly growing food production sector. Fish farmers are experiencing increasing problems with antibiotic resistance when fighting against pathogenic bacteria such as Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis. Phage therapy may provide an alternative, but effective use must be determined. Here, we studied the inhibition of A. salmonicida subsp. salmonicida strains by five phages (HER98 [44RR2.8t.2], HER110 [65.2], SW69-9, L9-6 and Riv-10) used individually or as combinations of two to five phages. A particular combination of four phages (HER98 [44RR2.8t.2], SW69-9, Riv-10, and HER110 [65.2]) was found to be the most effective when used at an initial multiplicity of infection (MOI) of 1 against the A. salmonicida subsp. salmonicida strain 01-B526. The same phage cocktail is effective against other strains except those bearing a prophage (named Prophage 3), which is present in 2/3 of the strains from the province of Quebec. To confirm the impact of this prophage, we tested the effectiveness of the same cocktail on strains that were either cured or lysogenized with Prophage 3. While the parental strains were sensitive to the phage cocktail, the lysogenized ones were much less sensitive. These data indicate that the prophage content of A. salmonicida subsp. salmonicida can affect the efficacy of a cocktail of virulent phages for phage therapy purposes.

Isolation of a Virulent Aeromonas salmonicida subsp. masoucida Bacteriophage and Its Application in Phage Therapy in Turbot (Scophthalmus maximus).

Aeromonas salmonicida is an aquatic pathogen that can infect a variety of fish. Phage therapy has been applied to treat bacterial infections. In this study, we obtained three A. salmonicida subsp. masoucida phage isolates from sewage, and one phage (vB_AsM_ZHF) exhibited the best antibacterial effect, based on in vitro kinetics experiments. Sequencing indicated that the vB_AsM_ZHF genome is 161,887 bp (41.24% C+G content) with 237 predicted open reading frames. No antibiotic resistance or virulence genes were detected in the complete genome, which is a requirement for phage therapy safety. Intraperitoneal injection of phage vB_AsM_ZHF into turbot at 8 × 104 PFU/fish rescued turbot from A. salmonicida subsp. masoucida injection and reduced the bacterial burden by 1 order of magnitude. Injection of vB_AsM_ZHF also decreased levels of inflammatory cell infiltration in muscle tissue, cytokines interleukin-1ß (IL-1ß), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) in serum and the expression of the inflammatory factors IL-1ß, IL-6, IFN-γ, transforming growth factor ß, TNF-α, and hepcidin in the liver, spleen, and head kidney of turbot. Phage vB_AsM_ZHF demonstrated antibacterial ability in vitro and in vivo and significantly reduced mortality in turbot challenged by A. salmonicida subsp. masoucida. This study revealed that phage vB_AsM_ZHF can effectively treat the infection caused by A. salmonicida subsp. masoucida in turbot. IMPORTANCEA. salmonicida is an aquatic pathogen that can infect different fish and causes economic loss to the global aquaculture industry. Clinical strains of A. salmonicida have developed multidrug resistance, and phage therapy is being evaluated for controlling bacterial infections. Phages are biological antibacterial agents and have the potential to be therapeutic agents against multidrug-resistant bacteria. In this study, three A. salmonicida subsp. masoucida phages were isolated from sewage, and their biological behaviors were characterized. The newly isolated phage vB_AsM_ZHF could inhibit A. salmonicida subsp. masoucida infection in vitro and in vivo, suggesting that it may be an alternative strategy to antibiotics for protecting fish against multidrug-resistant A. salmonicida subsp. masoucida in the aquaculture industry.

Nine Novel Phages from a Plateau Lake in Southwest China: Insights into Aeromonas Phage Diversity.

Aeromonas species are common pathogens of fish and some of them can opportunistically cause infectious diseases in humans. The overuse of antibiotics has led to the emergence of bacterial drug-resistance. To date, only 51 complete genome sequences of Aeromonas phages are available in GenBank. Here, we report the isolation of nine Aeromonas phages from a plateau lake in China. The protein cluster, dot plot and ANI analyses were performed on all 60 currently sequenced Aeromonas phage genomes and classified into nine clusters and thirteen singletons. Among the nine isolated phages, the DNA-packaging strategy of cluster 2L372D (including 2L372D, 2L372X, 4L372D, 4L372XY) is unknown, while the other five phages use the headful (P22/Sf6) DNA-packaging strategy. Notably, the isolated phages with larger genomes conservatively encode auxiliary metabolism genes, DNA replication and metabolism genes, while in smaller phage genomes, recombination-related genes were conserved. Finally, we propose a new classification scheme for Aeromonas phages.

Phage-Encoded LuxR-Type Receptors Responsive to Host-Produced Bacterial Quorum-Sensing Autoinducers.

Quorum sensing (QS) is a process of cell-to-cell communication that bacteria use to orchestrate collective behaviors. QS relies on the cell-density-dependent production, accumulation, and receptor-mediated detection of extracellular signaling molecules called autoinducers (AIs). Gram-negative bacteria commonly use N-acyl homoserine lactones (AHLs) as their AIs, and they are detected by LuxR-type receptors. Often, LuxR-type receptors are insoluble when not bound to a cognate AI. In this report, we show that LuxR-type receptors are encoded on phage genomes, and in the cases we tested, the phage LuxR-type receptors bind to and are solubilized specifically by the AHL AI produced by the host bacterium. We do not yet know the viral activities that are controlled by these phage QS receptors; however, our observations, coupled with recent reports, suggest that their occurrence is more widespread than previously appreciated. Using receptor-mediated detection of QS AIs could enable phages to garner information concerning the population density status of their bacterial hosts. We speculate that such information can be exploited by phages to optimize the timing of execution of particular steps in viral infection.IMPORTANCE Bacteria communicate with chemical signal molecules to regulate group behaviors in a process called quorum sensing (QS). In this report, we find that genes encoding receptors for Gram-negative bacterial QS communication molecules are present on genomes of viruses that infect these bacteria. These viruses are called phages. We show that two phage-encoded receptors, like their bacterial counterparts, bind to the communication molecule produced by the host bacterium, suggesting that phages can "listen in" on their bacterial hosts. Interfering with bacterial QS and using phages to kill pathogenic bacteria represent attractive possibilities for development of new antimicrobials to combat pathogens that are resistant to traditional antibiotics. Our findings of interactions between phages and QS bacteria need consideration as new antimicrobial therapies are developed.

Complete genome sequences of Aeromonas and Pseudomonas phages as a supportive tool for development of antibacterial treatment in aquaculture.

BACKGROUND: Aquaculture is the fastest growing sector of food production worldwide. However, one of the major reasons limiting its effectiveness are infectious diseases among aquatic organisms resulting in vast economic losses. Fighting such infections with chemotherapy is normally used as a rapid and effective treatment. The rise of antibiotic resistance, however, is limiting the efficacy of antibiotics and creates environmental and human safety concerns due to their massive application in the aquatic environment. Bacteriophages are an alternative solution that could be considered in order to protect fish against pathogens while minimizing the side-effects for the environment and humans. Bacteriophages kill bacteria via different mechanisms than antibiotics, and so fit nicely into the 'novel mode of action' concept desired for all new antibacterial agents. METHODS: The bacteriophages were isolated from sewage water and characterized by RFLP, spectrum of specificity, transmission electron microscopy (TEM) and sequencing (WGS). Bioinformatics analysis of genomic data enables an in-depth characterization of phages and the choice of phages. This allows an optimised choice of phage for therapy, excluding those with toxin genes, virulence factor genes, and genes responsible for lysogeny. RESULTS: In this study, we isolated eleven new bacteriophages: seven infecting Aeromonas and four infecting Pseudomonas, which significantly increases the genomic information of Aeromonas and Pseudomonas phages. Bioinformatics analysis of genomic data, assessing the likelihood of these phages to enter the lysogenic cycle with experimental data on their specificity towards large number of bacterial field isolates representing different locations. CONCLUSIONS: From 11 newly isolated bacteriophages only 6 (25AhydR2PP, 50AhydR13PP, 60AhydR15PP, 22PfluR64PP, 67PfluR64PP, 71PfluR64PP) have a potential to be used in phage therapy due to confirmed lytic lifestyle and absence of virulence or resistance genes.

Characterization of a novel lytic myophage, phiA8-29, infecting Aeromonas strains.

Members of the bacterial genus Aeromonas are important aquatic pathogens that cause severe fish diseases. Here, we characterize a novel lytic phage, Aeromonas virus phiA8-29, isolated from the alimentary tract of a freshwater fish. Transmission electron microscopy showed that phiA8-29 has a long contractile tail and thus can be classified as a member of the family Myoviridae. The phage genome was identified as a double-stranded DNA molecule of 144,974 bp containing 185 open reading frames and nine tRNA-encoding genes. Comparative genomic analysis revealed that the phiA8-29 genome has little similarity to any of the currently sequenced Aeromonas phage genomes. Our data indicate that phiA8-29 is a novel lytic Myoviridae phage that does not belong to any of the known genera.

Complete genome analysis of bacteriophage AsXd-1, a new member of the genus Hk97virus, family Siphoviridae.

AsXd-1, a bacteriophage that infects Aeromonas salmonicida, was isolated from the wastewater of a seafood market in Shenzhen, China. The 39,014-bp genome of this phage contains 52 open reading frames (ORFs), 30 of which were found to be homologous to reference sequences that putatively encode functional phage proteins. Nine out of the remaining 22 ORFs with unknown functions were unique to AsXd-1. Gene annotation suggests that AsXd-1 has both lysogenic and lytic life cycles. Furthermore, both phylogenetic analysis based on the large subunit of terminase and genome sequence comparisons show that AsXd-1 is closely related to phages belonging to the genus Hk97virus. We thus propose AsXd-1 as a new member of the genus Hk97virus within the family Siphoviridae.

Two novel temperate bacteriophages co-existing in Aeromonas sp. ARM81 - characterization of their genomes, proteomes and DNA methyltransferases.

Aeromonas species are causative agents of a wide spectrum of diseases in animals and humans. Although these bacteria are commonly found in various environments, little is known about their phages. Thus far, only one temperate Aeromonas phage has been characterized. Whole-genome sequencing of an Aeromonas sp. strain ARM81 revealed the presence of two prophage clusters. One of them is integrated into the chromosome and the other was maintained as an extrachromosomal, linear plasmid-like prophage encoding a protelomerase. Both prophages were artificially and spontaneously inducible. We separately isolated both phages and compared their genomes with other known viruses. The novel phages show no similarity to the previously characterized Aeromonas phages and might represent new evolutionary lineages of viruses infecting Aeromonadaceae. Apart from the comparative genomic analyses of these phages, complemented with their structural and molecular characterization, a functional analysis of four DNA methyltransferases encoded by these viruses was conducted. One of the investigated N6-adenine-modifying enzymes shares sequence specificity with a Dam-like methyltransferase of its bacterial host, while another one is non-specific, as it catalyzes adenine methylation in various sequence contexts. The presented results shed new light on the diversity of Aeromonas temperate phages.

Occurrence of bacteriophages infecting Aeromonas, Enterobacter, and Klebsiella in water and association with contamination sources in Thailand.

The co-residence of bacteriophages and their bacterial hosts in humans, animals, and environmental sources directed the use of bacteriophages to track the origins of the pathogenic bacteria that can be found in contaminated water. The objective of this study was to enumerate bacteriophages of Aeromonas caviae (AecaKS148), Enterobacter sp. (EnspKS513), and Klebsiella pneumoniae (KlpnKS648) in water and evaluate their association with contamination sources (human vs. animals). Bacterial host strains were isolated from untreated wastewater in Bangkok, Thailand. A double-layer agar technique was used to detect bacteriophages. All three bacteriophages were detected in polluted canal samples, with likely contamination from human wastewater, whereas none was found in non-polluted river samples. AecaKS148 was found to be associated with human fecal sources, while EnspKS513 and KlpnKS648 seemed to be equally prevalent in both human and animal fecal sources. Both bacteriophages were also present in polluted canals that could receive contamination from other fecal sources or the environment. In conclusion, all three bacteriophages were successfully monitored in Bangkok, Thailand. This study provided an example of bacteriophages for potential use as source identifiers of pathogen contamination. The results from this study will assist in controlling sources of pathogen contamination, especially in developing countries.

The gastrointestinal phage communities of the cultivated freshwater fishes.

The phage communities in the gut of 62 cultivated freshwater fish were investigated by culture-based methods. Using three selective media, 445 pathogenic bacilli strains were isolated and used as indicators for subsequent phage isolations. Totally, 63 phages were detected and the respective host strains were identified with the comparative sequence analysis of 16S rRNA gene, including Aeromonas (29), Vibrio (1), Citrobacter (16), Serratia (4), Enterobacter (2), Proteus (3), Buttiauxella (2), Plesiomonas (2), Kluyvera (1), Morgenella (2) and Providencia (1). The diversity of Aeromonas phages was assessed by discrimination of their host strains with random amplified polymorphic DNA method. Furthermore, the isolated Aeromonas phages were characterized by host range and growth inhibition assay. The results demonstrated that there were abundant and diverse phage populations in the gut environment of the cultivated freshwater fishes. The phages could contribute to the microbiota balance in the gut ecosystem of fishes and provide reliable phage sources for future applications.

Genomic structure of the Aeromonas bacteriophage pAh6-C and its comparative genomic analysis.

pAh6-C is a virulent bacteriophage (phage), isolated from a river in Korea, that infects a multiple-antibiotic-resistant A. hydrophila strain, JUNAH. The double-stranded DNA genome of pAh6-C is composed of 53,744 bp with a G + C content of 52.83 %. The genome encodes 86 putative ORFs, four putative promoters, and seven transcriptional terminator regions. Genome sequence analysis of pAh6-C and comparative analysis with the homologous Shewanella phage Spp001 revealed that there is a high degree of similarity between pAh6-C and Spp001 in 50 of the 86 ORFs of pAh6-C. The results of this investigation indicate that pAh6-C is closely related to Spp001, especially in the genes coding for proteins involved in DNA metabolism.

Aeromonas phages encode tRNAs for their overused codons.

The GC-rich bacterial species, Aeromonas salmonicida, is parasitised by both GC-rich phages (Aeromonas phages - phiAS7 and vB_AsaM-56) and GC-poor phages (Aeromonas phages - 25, 31, 44RR2.8t, 65, Aes508, phiAS4 and phiAS5). Both the GC-rich Aeromonas phage phiAS7 and Aeromonas phage vB_AsaM-56 have nearly identical codon usage bias as their host. While all the remaining seven GC-poor Aeromonas phages differ dramatically in codon usage from their GC-rich host. Here, we investigated whether tRNA encoded in the genome of Aeromonas phages facilitate the translation of phage proteins. We found that tRNAs encoded in the phage genome correspond to synonymous codons overused in the phage genes but not in the host genes.

Isolation and characterization of broad spectrum bacteriophages lytic to Vibrio harveyi from shrimp farms of Kerala, India.

UNLABELLED: Of 33 phages isolated from various shrimp farms in Kerala, India, six were segregated to have broad spectrum lytic efficiency towards 87 isolates of Vibrio harveyi with cross-infecting potential to a few other important aquaculture pathogens. They were further tested on beneficial aquaculture micro-organisms such as probiotics and nitrifying bacterial consortia and proved to be noninfective. Morphological characterization by transmission electron microscopy (TEM) and molecular characterization by RAPD and SDS-PAGE proved them distinct and positioned under Caudovirales belonging to Myoviridae and Siphoviridae. SIGNIFICANCE AND IMPACT OF THE STUDY: In sustainable aquaculture, application of antibiotics is prohibited to manage vibriosis, including the one caused by Vibrio harveyi. In lieu of antibiotics, an eco-friendly alternative method, phage therapy, is recommended here. To facilitate the same, a set of six broad spectrum V. harveyi phages, as cocktail, has been constituted and characterized based on morphological traits and by employing molecular tools. These phages were also found to infect other aquaculture pathogens belonging to Vibrio and Aeromonas. Subsequent to in vivo trials, they can find application in shrimp hatcheries as prophylactics and therapeutics.

Isolation and partial characterization of a virulent bacteriophage IHQ1 specific for Aeromonas punctata from stream water.

Aeromonas punctata is the causative agent of septicemia, diarrhea, wound infections, meningitis, peritonitis, and infections of the joints, bones and eyes. Bacteriophages are often considered alternative agents for controlling bacterial infection and contamination. In this study, we described the isolation and preliminary characterization of bacteriophage IHQ1 (family Myoviridae) active against the Gram-negative bacterial strain A. punctata. This virulent bacteriophage was isolated from stream water sample. Genome analysis indicated that phage IHQ1 was a double-stranded DNA virus with an approximate genome size of 25-28 kb. The initial characterization of this newly isolated phage showed that it has a narrow host range and infects only A. punctata as it failed to infect seven other clinically isolated pathogenic strains, i.e., methicillin-resistant Staphylococcus aureus 6403, MRSA 17644, Acinetobacter 33408, Acinetobacter 1172, Pseudomonas aeruginosa 22250, P. aeruginosa 11219, and Escherichia coli. Proteomic pattern of phage IHQ1, generated by SDS-PAGE using purified phage particles, showed three major and three minor protein bands with molecular weights ranging from 25 to 70 kDa. The adsorption rate of phage IHQ1 to the host bacterium was also determined, which was significantly enhanced by the addition of 10 mM CaCl(2). From the single-step growth experiment, it was inferred that the latent time period of phage IHQ1 was 24 min and a burst size of 626 phages per cell. Moreover, the pH and thermal stability of phage IHQ1 were also investigated. The maximum stability of the phage was observed at optimal pH 7.0, and it was totally unstable at extreme acidic pH 3; however, it was comparatively stable at alkaline pH 11.0. At 37°C the phage showed maximum number of plaques, and the viability was almost 100%. The existence of Aeromonas bacteriophage is very promising for the eradication of this opportunistic pathogen and also for future applications such as the design of new detection and phage typing (diagnosis) methods. The specificity of the bacteriophage for A. punctata makes it an attractive candidate for phage therapy of A. punctata infections.

Pesquisa de genes codificadores de enterotoxinas e Beta-lactamases em Aeromonas jandaei e Aeromonas hydrophila provenientes de ambientes aquáticos / Enterotoxins and Beta-lactamases encoding genes investigation in Aeromonas jandaei e Aeromonas hydrophila from aquatic environments

O gênero Aeromonas está amplamente distribuído em ambientes aquáticos, e estudos recentes incluem o gênero no grupo de patógenos emergentes, devido à sua freqüente associação com infecções locais e sistêmicas em humanos. Este trabalho foi realizado com objetivo de pesquisar por meio da PCR e confirmar por meio de sequenciamento, a ocorrência dos genes de virulência act, alt, ast, e resistência cphA, bla(imp), bla(SPM-1), bla(CTX-M), bla(TEM), bla(SHV), verificando também o perfil de resistência a partir de antibiogramas, e a ocorrências de plasmídios nas cepas estudadas. A partir dos resultados observou-se que das 100 cepas selecionadas inicialmente, 87 pertenciam às espécies A. jandaei (46) e A. hydrophila (41). Dentre as quais pôde-se observar a ocorrência de act, alt,e ast, respectivamente em 70, 7 por cento (29), 97,6 por cento (40) e 26,8 por cento (11) das cepas de A. hydrophila, e em 4,4 por cento (2), 0 por cento (0) e 32,6 por cento (15) nas cepas de A. jandaei. Os genes bla(IMP), bla(VIM), bla(SPM-1), blaCTX-M, blaSHV não foram encontrados em nenhuma cepa. O gene cphA foi encontrado em 97,6 por cento (40) e 100 por cento (46) das cepas de A. hydrophila e A. jandaei, respectivamente e o gene bla(TEM) foi encontrado em 97,6 por cento (40) das cepas de A. hydrophila e em 85 por cento (39) das cepas de A. jandaei. Foi verificada a presença de plasmídios em 10/41 (24,4 por cento) das cepas de A. hydrophila e em 16/46 (34,9 por cento) das cepas de A. jandaei.

Factores de virulencia en cepas de Aeromonas aisladas de pacientes con enfermedad diarreica aguda en Cuba / Virulence factors in Aeromonas strains isolated from patients with acute diarrheas in Cuba

Objetivo: se realizó un estudio en cepas de Aeromonas aisladas de pacientes con enfermedad diarreica aguda en Cuba, para conocer la expresión fenotípica de la citotoxina y la enterotoxina como factores de virulencia. Métodos: se investigaron 46 cepas (A. hydrophila, A. veronii biovar sobria, A. caviae, A. veronii biovar veronii y Aeromonas spp.), aisladas de heces de pacientes con enfermedad diarreica aguda, en el período comprendido entre 2005 y 2006. Todas las cepas tenían identificado su patrón de susceptibilidad antimicrobiana. Se comprobó la expresión fenotípica de la citotoxina y la enterotoxina en la línea celular Vero. Resultados: el estudio demostró que 91,31 por ciento de las cepas mostraron actividad citotóxica y 43,48 por ciento actividad enterotóxica. De las cepas multirresistentes, 93,75 por ciento presentó al menos un factor de virulencia estudiado. Conclusiones: los resultados demostraron que los 2 factores de virulencia investigados estuvieron presentes en las cepas estudiadas, contribuyendo así a los múltiples esfuerzos que se realizan para conocer los mecanismos de enteropatogenicidad de este género bacteriano(AU)

Objective: A study was carried out in Aeromonas strains isolated from patients with acute diarrheas in Cuba to find out the phenotypical expression of the cytotoxin and the enterotoxin as virulence factors. Methods: Forty six strains of the genus Aeromonas (A. hydrophila, A. veronii bv sobria, A. caviae, A. veronii bv veronii and Aeromonas spp.) isolated from stool specimens taken form patients with acute diarrheal disease were studied from 2005 to 2006. All the strains had their pattern of antimicrobial susceptibility pattern identified. The phenotypic expression of the cytotoxin and the enterotoxin in the Vero cell line was checked. Results: It was demonstrated that 91,31 percent of the strains showed cytotoxic activity and 43,48 percent of them enterotoxic activity. Regarding multiresistant strains, 93,75 percent presented with at least one of the studied virulence factors. Conclusions: these results proved that the two researched virulence factors did exist in the studied strains, thus contributing to the many efforts that are being made to learn about the mechanisms of enteropathogenicity of this bacterial genus(AU)

Factores de virulencia en cepas de Aeromonas aisladas de pacientes con enfermedad diarreica aguda en Cuba / Virulence factors in Aeromonas strains isolated from patients with acute diarrheas in Cuba

Objetivo: se realizó un estudio en cepas de Aeromonas aisladas de pacientes con enfermedad diarreica aguda en Cuba, para conocer la expresión fenotípica de la citotoxina y la enterotoxina como factores de virulencia. Métodos: se investigaron 46 cepas (A. hydrophila, A. veronii biovar sobria, A. caviae, A. veronii biovar veronii y Aeromonas spp.), aisladas de heces de pacientes con enfermedad diarreica aguda, en el período comprendido entre 2005 y 2006. Todas las cepas tenían identificado su patrón de susceptibilidad antimicrobiana. Se comprobó la expresión fenotípica de la citotoxina y la enterotoxina en la línea celular Vero. Resultados: el estudio demostró que 91,31 por ciento de las cepas mostraron actividad citotóxica y 43,48 por ciento actividad enterotóxica. De las cepas multirresistentes, 93,75 por ciento presentó al menos un factor de virulencia estudiado. Conclusiones: los resultados demostraron que los 2 factores de virulencia investigados estuvieron presentes en las cepas estudiadas, contribuyendo así a los múltiples esfuerzos que se realizan para conocer los mecanismos de enteropatogenicidad de este género bacteriano.

Objective: A study was carried out in Aeromonas strains isolated from patients with acute diarrheas in Cuba to find out the phenotypical expression of the cytotoxin and the enterotoxin as virulence factors. Methods: Forty six strains of the genus Aeromonas (A. hydrophila, A. veronii bv sobria, A. caviae, A. veronii bv veronii and Aeromonas spp.) isolated from stool specimens taken form patients with acute diarrheal disease were studied from 2005 to 2006. All the strains had their pattern of antimicrobial susceptibility pattern identified. The phenotypic expression of the cytotoxin and the enterotoxin in the Vero cell line was checked. Results: It was demonstrated that 91,31 percent of the strains showed cytotoxic activity and 43,48 percent of them enterotoxic activity. Regarding multiresistant strains, 93,75 percent presented with at least one of the studied virulence factors. Conclusions: these results proved that the two researched virulence factors did exist in the studied strains, thus contributing to the many efforts that are being made to learn about the mechanisms of enteropathogenicity of this bacterial genus.

Temperate bacteriophage PhiO18P from an Aeromonas media isolate: characterization and complete genome sequence.

A group of 74 Aeromonas isolates from surface water of three ponds in Bielefeld, Germany was screened for prophage induction after UV irradiation. The phage PhiO18P was induced from the Aeromonas media isolate O18. PhiO18P belongs to the Myoviridae phage family. The complete nucleotide sequence of the double stranded DNA genome of bacteriophage PhiO18P consists of 33,985 bp. The genome has 5' protruding cohesive ends of 16 bases. On the PhiO18P genome 46 open reading frames (orfs) were identified which are organized in the modules integration and regulation, replication, head, packaging, tail and lysis. Additionally the phage DNA includes a methylase gene. Comparison of the genome architecture with those of other bacteriophages revealed significant similarities to the P2 phage family and especially to the prophages of Aeromonas salmonicida and the Vibrio cholerae phage K139.

Sampling natural viral communities from soil for culture-independent analyses.

An essential first step in investigations of viruses in soil is the evaluation of viral recovery methods suitable for subsequent culture-independent analyses. In this study, four elution buffers (10% beef extract, 250 mM glycine buffer, 10 mM sodium pyrophosphate, and 1% potassium citrate) and three enumeration techniques (plaque assay, epifluorescence microscopy [EFM], and transmission electron microscopy [TEM]) were compared to determine the best method of extracting autochthonous bacteriophages from two Delaware agricultural soils. Beef extract and glycine buffer were the most effective in eluting viable phages inoculated into soils (up to 29% recovery); however, extraction efficiency varied significantly with phage strain. Potassium citrate eluted the highest numbers of virus-like particles from both soils based on enumerations by EFM (mean, 5.3 x 10(8) g of dry soil(-1)), but specific soil-eluant combinations posed significant problems to enumeration by EFM. Observations of virus-like particles under TEM gave confidence that the particles were, in fact, phages, but TEM enumerations yielded measurements of phage abundance (mean, 1.5 x 10(8) g of dry soil(-1)) that were about five times lower. Clearly, the measurement of phage abundance in soils varies with both the extraction and enumeration methodology; thus, it is important to assess multiple extraction and enumeration approaches prior to undertaking ecological studies of phages in a particular soil.

Type III secretion genes in Aeromonas salmonicida subsp salmonicida are located on a large thermolabile virulence plasmid.

Type III secretion genes in Aeromonas salmonicida subsp. salmonicida are located on a large plasmid of approximately 140 kb. Cultivation of this organism at elevated temperatures such as 25 degrees C can, however, result in loss of this plasmid. This is accompanied by a loss of virulence for cultured fish cells.