Filamentous Bacteriophages of Vibrio parahaemolyticus as a Possible Clue to Genetic Transmission

We have previously reported the isolation and characterization of two filamentous bacteriophages of Vibrio parahaemolyticus, designated Vf12 and Vf33. In this study, to understand the potential of these phages as tools for genetic transmission, we investigated the gene structures of replicative-form (RF) DNAs of their genomes and the distribution of these DNAs on chromosomal and extrachromosomal DNAs. The 7,965-bp nucleotide sequences of Vf12 and Vf33 were determined. An analysis of the overall gene structures revealed that Vf12 and Vf33 had conserved regions and distinctive regions. The gene organization of their conserved regions was similar to that of CTX phage of Vibrio cholerae and coliphage Ff of Escherichia coli, while their distinctive regions were characteristic of Vf12 and Vf33 phage genomes. Southern blot hybridization testing revealed that the filamentous phage genomes integrated into chromosomal DNA of V. parahaemolyticus at the distinctive region of the phage genome and were also distributed on some plasmids of V. parahaemolyticus and total cellular DNAs of one Vibrio damsela and one nonagglutinable Vibrio strain tested. These results strongly suggest the possibilities of genetic interaction among the bacteriophage Vf12 and Vf33 genomes and chromosomal and plasmid-borne DNAs of V. parahaemolyticus strains and of genetic transmission among strains through these filamentous phages.     

A Filamentous Phage of Vibrio parahaemolyticus O3:K6 Isolated in Laos

A filamentous phage, ‘lvpf5’, of Vibrio parahaemolyticus O3:K6 strain LVP5 was isolated and characterized. The host range was not restricted to serotype O3:K6, but 7 of 99 V. parahaemolyticus strains with a variety of serotypes were susceptible to the phage. The phage was inactivated by heating at 80 C for 10 min and by treating with chloroform. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phage exhibited a 3.8 kDa protein. The amino-terminal amino acid sequence of the coat protein was determined as AEGGAADPFEAIDLLGVATL. The phage genome consisted of a single-stranded DNA molecule. The activity of the phages was inhibited by anti-Na2 pili antibody.     

Characterization of a new Bacillus megaterium bacteriophage,MJ-1, from tropical soil

A new Bacillus megaterium bacteriophage is characterized. It is a tailed phage with regular polyhedral head belonging to Bradley's group B. Head and tail dimensions are 56.4 and 300 nm, respectively. Lysis was restricted to strains of B. megaterium. No antigenic relationship with pumilus phage FP-1 or subtilis phage FS-1 was observed. The phage is sensitive to 60°C and moderately sensitive to chloroform. The nucleic acid is double-stranded linear DNA with a G-C mole % of 38.8 and a mol wt of (53±3)×10⁶.     

Occurrence of Vibrio parahaemolyticus and Its Specific Phages from Shrimp Ponds in East Coast of India

Vibrio parahaemolyticus is a natural microflora of marine and coastal water bodies and associated with mortality of larval shrimp in penaeid shrimp in ponds. Bacteriophages occur virtually in all places where their hosts exist. In this study, total distribution of V. parahaemolyticus and its phages were examined in shrimp ponds, seawater, estuary, animal surface, and tissues. Total vibrio count in sediments of two ponds was found to be 2.6 × 10³ and 5.6 × 10³ cfu/g. Incidence of V. parahaemolyticus in the ponds was close, while it was markedly higher in the animal surface and tissue samples. Biochemically identified eight strains of V. parahaemolyticus (V1, V3–V6, V9, V11, and V12) were taken for further infection studies with bacteriophage. Totally five bacteriophages capable of infecting V. parahaemolyticus MTCC-451 strain were isolated from all the samples. One of the isolated bacteriophage Vp1 from estuary was able to lyse all the isolated V. parahaemolyticus strains we used. Therefore, the morphology of Vp1 was estimated in TEM. Vp1 phage head measuring approximately about 50–60 nm diameter with icosahedral outline and a contractile tails of diameter 7 nm and length 100 nm and it was identified as Myoviridae. Therefore, the phages have the potential application in destroying bacterial pathogens.     

Rapid detection of Vibrio parahaemolyticus by PCR targeted to the histone-like nucleoid structure (H-NS) gene and its genetic characterization

Aims: The aim of this study was to explore a new PCR target gene for Vibrio parahaemolyticus, based on the histone‐like nucleoid structure (H‐NS) gene. Methods and Results: Primers for the H‐NS gene were designed for specificity to V. parahaemolyticus and incorporated into a PCR assay. The PCR assay was able to specifically detect all of the 82 V. parahaemolyticus strains tested, but did not result in amplification in the 47 other Vibrio spp. and nonVibrio spp. strains. The detection limit of the PCR assay was 0·14 pg purified genomic DNA and 1·8 × 10⁵ CFU g^?1 spiked oyster samples from V. parahaemolyticus RIMD2210633. Furthermore, a multiplex PCR assay targeting the hns, tdh and trh genes was successfully developed to detect virulent V. parahaemolyticus strains. Conclusions: The H‐NS‐based PCR assay developed in this study was sensitive and specific, with great potential for field detection of V. parahaemolyticus in seawater or seafood samples. Significance and Impact of the Study: The H‐NS gene was validated as a new specific marker gene in PCR assays for accurate detection and identification of V. parahaemolyticus, which has the potential to be applied in diagnostics and taxonomic studies.     

Lysogeny among mycobacteria

Our investigations to detect naturally lysogenic strains of mycobacteria were limited to 1 strain ofMycobacterium smegmatis, 4 strains ofMycobacterium borstelense var.niacinogenes, and to 5 strains ofMycobacterium marinum (Syn:Mycobacterium balnei), all together 10 strains. They were chosen because as a sign of lysis they secrete a large quantity of cytoplasmatic components (nucleic acids proteins, amino acids etc.) into the fluid medium (for instance phosphate buffer), in which they are suspended. In a first series of experiments culture filtrates were tested on 84 strains of slowly and rapidly growingMycobacterium species as indicator strains. Using this method free phage particles were only found in the culture filtrate of 1 strain,Mycobacterium smegmatis SN 46, isolated from a patient with achalasia. Phage particles could not be found in the filtrates of the other 9 probably lysogenic strains. In a second series of experiments more closely related indicator strains were used. The 10 probably lysogenic strains were cultured in bovine serum or antiphage-antiserum containing medium and single selected colony cultures a small part of which showed sensitivity to the filtrates. The released and adapted phages, designated as B₂₄, B₃₀, B₃₂, B₃₃, B₃₄ and B₃₅ have a very narrow host range. The plaques are very small and turbid. On electron micrographs the temperate phages B₂₄, B₃₀ and B₃₅ exhibit the typical head-tail morphology. The head of the temperateborstelense var.niacinogenes phage B₃₀ is 45 nm in diameter, the tength of tail is about, 120nm. The average dimensions of the long head ofsmegmatis phage B₂₄ are 40 × 80 nm, the tail is about 160 nm long. The balnei phage B₃₅ is very similar morphologically to phage B₃₀. The head is about 50 nm in diameter, the length of tail about 160 nm. The phage sensitive variants are not “carrier” strains. Their phage sensitivity is not a stable property. After several culture passages in serum-free medium the variants regain their phage immunity completely and release phages like the lysogenic parent strains. The sensitive variants must therefore be considered to be also lysogenic. TheMycobacterium borstelense var.niacinogenes phages are serologically very related. Dedicated to Academician Ivan Málek on the occasion of his 60th birthday     

A new group of cosmopolitan bacteriophages induce a carrier state in the pandemic strain of Vibrio parahaemolyticus

A clonal population of pathogenic Vibrio parahaemolyticus O3 : K6 serovar has spread in coastal waters, causing outbreaks worldwide since 1996. Bacteriophage infection is one of the main factors affecting bacterial strain concentration in the ocean. We studied the occurrence and properties of phages infecting this V. parahaemolyticus pandemic strain in coastal waters. Analysing 143 samples, phages were found in 13. All isolates clustered in a closely related group of podophages with at least 90% nucleotide sequence identity in three essential genes, despite distant geographical origins. These bacteriophages were able to multiply on the V. parahaemolyticus pandemic strain, but the impact on host concentration and subsequent growth was negligible. Infected bacteria continued producing the phage but were not lysogenized. The phage genome of prototype strain VP93 is 43 931 nucleotides and contains 337 bp direct terminal repeats at both ends. VP93 is the first non‐Pseudomonas phage related to the ΦKMV‐like subgroup of the T7 supergroup. The lack of a major effect on host growth suggests that these phages exert little control on the propagation of the pandemic strain in the environment. This form of phage growth can be modelled if phage‐sensitive and ‐resistant cells that convert to each other with a high frequency are present in clonal cultures of pandemic V. parahaemolyticus.     

Morphological Features of a Filamentous Phage of Vibrio cholerae O139 Bengal

A filamentous phage was isolated from carrier strain AI-1841 of Vibrio cholerae O139 Bengal and thus was termed fs phage. The phage was measured to be approximately 1 μm in length and 6 nm in width. One end of the phage was slightly tapered and had a fibrous appendage. The plaques developed on strain AI-4450 of V. cholerae O139 were small and turbid. The phage grew in strain AI-4450 and reached a size of 10⁸ to 10⁹ pfu/ml at 5 hr after infection without inducing any lysis of the host bacteria. The group of phages attached on rod-shaped materials like fimbriae of this bacteria, with their fibrous appendages at the pointed end, were often found in the phage-infected culture. The anti-fimbrial serum effectively inhibited the infection of fs phage to the host strain AI-4450. We thus concluded that the phage can be adsorbed on fimbriae with a fibrous appendage on the pointed end of the phage filament.     

Isolation and Characterization of Bacteriophages from Inland Saline Aquaculture Environments to Control Vibrio parahaemolyticus Contamination in Shrimp

Among the various bacterial pathogens associated with the aquaculture environment, Vibrio parahaemolyticus the important one from shrimp and human health aspects. Though having been around for several decades, phage-based control of bacterial pathogens (phage therapy) has recently re-emerged as an attractive alternative due to the availability of modern phage characterization tools and the global emergence of antibiotic-resistant bacteria. In the present study, a total of 12 V. parahaemolyticus specific phages were isolated from 264 water samples collected from inland saline shrimp culture farms. During the host range analysis against standard/field isolates of V. parahaemolyticus and other bacterial species, lytic activity was observed against 2.3–45.5% of tested V. parahaemolyticus isolates. No lytic activity was observed against other bacterial species. For genomic characterization, high-quality phage nucleic acid with concentrations ranging from 7.66 to 220 ng/µl was isolated from 9 phages. After digestion treatments with DNase, RNase and S1 nuclease, the nature of phage nucleic acid was determined as ssDNA and dsDNA for 7 and 2 phages respectively. During transmission electron microscopy analysis of phage V5, it was found to have a filamentous shape making it a member of the family Inoviridae. During efficacy study of phage against V. parahaemolyticus in shrimp, 78.1% reduction in bacterial counts was observed within 1 h of phage application. These results indicate the potential of phage therapy for the control of V. parahaemoyticus in shrimp.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-021-00934-6.     

Characterization of filamentous bacteriophage PE226 infecting Ralstonia solanacearum strains

Aims: The aim of this study was to isolate and characterize new bacteriophages that infect a wide range of plant pathogenic Ralstonia solanacearum strains. Methods and Results: Fifteen bacteriophages were isolated from pepper, tomato and tobacco plant rhizospheres infected with R. solanacearum. A host specificity analysis of the isolated phages using nine strains of R. solanacearum indicated great phage diversity in a single soil. Two phages, PE226 and TM227, showed clear plaques on all nine bacterial hosts tested and were virtually identical in morphology and genome. PE226, an Inovirus, is a long, flexible, filamentous phage carrying a circular (+) sense single‐strand DNA genome of 5475 nucleotides. DNA sequences of PE226 exhibited nine open reading frames (ORF) that were not highly similar to those of other phages infecting R. solanacearum. The genome organization of PE226 was partially similar to that of p12J of Ralstonia pickettii. One ORF of PE226 showed identity to the zot gene encoding zonula occludens toxin of Vibrio cholera. Orf7 of PE226 was also present in the genome of R. solanacearum strain SL341. However, SL341, a highly virulent strain in tomato, was still sensitive to phage PE226. Conclusions: A new, flexible, filamentous phage PE226 infected wide range of R. solanacearum strains and carried unique circular single‐strand DNA genome with an ORF encoding Zot‐like protein. Significance and Impact of the Study: PE226 may be a new type of temperate phage, based on its lytic nature on a wide range of hosts and the presence of a zot homologue in a host bacterial genome.     

Molecular characterization of Vibrio parahaemolyticus of similar serovars isolated from sewage and clinical cases of diarrhoea in Calcutta,India

Vibrio parahaemolyticus is a seafood-borne halophilic pathogen that causes acute gastroenteritis in humans. During the course of an investigation on the incidence of V. parahaemolyticus in sewage water samples of Calcutta, India, we isolated eight (26.7%) strains of V. parahaemolyticus from 30 samples. Among these strains, five (62.5%) carried the thermostable direct hemolysin (tdh) gene, a major virulence marker of V. parahaemolyticus. Two strains belonged to serovar O5:K3 and the remaining three to O5:KUT, which is common among clinical strains of V. parahaemolyticus isolated from hospitalized patients of Calcutta with acute diarrhoea. The tdh positive sewage strains of V. parahaemolyticus were compared by randomly amplified polymorphic DNA (RAPD)-PCR and pulsed-field gel electrophoresis (PFGE) with strains of similar serovars selected from our culture collection to determine the genetic relatedness. Our results showed that except for sharing the similar serovar, sewage and clinical strains of V. parahaemolyticus were genetically different. In addition, toxRS-targeted group-specific (GS) PCR and open reading frame 8 (ORF-8) PCR showed that the sewage strains did not belong to the pandemic genotype. Since the sewage in Calcutta is directly used for cultivation of vegetables and for pisciculture, the presence of tdh positive V. parahaemolyticus in the sewage highlights the need for constant monitoring of the environment.     

Air Breathing and Gill Ventilation Frequencies in Juvenile Tarpon, Megalops atlanticus: Responses to Changes in Dissolved Oxygen, Temperature, Hydrogen Sulfide, and PH

This study quantified the air-breathing frequency (ABf in breaths h^–1) and gill ventilation frequency (Vf in ventilations min^–1) of tarpon Megalops atlanticusas a function of PO₂, temperature, pH, and sulphide concentration. Ten tarpon held at normoxia at 22–33°C without access to atmospheric oxygen survived for eight days, and seven survived for 14 days (at which point the experiment was terminated) suggesting that the species is a facultative, rather than an obligate, air breather. At temperatures of 29°C and below ABf was highest and Vf was lowest at low oxygen partial pressures. Tarpon appear to switch from aquatic respiration to air breathing at PO₂levels of roughly 40 torr. The gills were the primary organ for oxygen uptake in normoxia, and the air-breathing organ the primary mechanism for oxygen uptake in hypoxia. At 33°C, both ABf and Vf were elevated but highly variable, regardless of PO₂. There were no mortalities in tarpon exposed to total H₂S concentrations of 0–232µM (0–150.9µM H₂S); however, high sulfide concentrations resulted in very high ABf and Vf near zero. Vf was reduced when pH was acidic. We conclude that air breathing provides an effective means of coping with the environmental conditions that characterize the eutrophic ponds and sloughs that juvenile tarpon typically inhabit.     

The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Vibrio parahaemolyticus

Aims: The current study was aimed to develop a loop‐mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay for rapid and specific detection of Vibrio parahaemolyticus. Methods and Results: Biotinylated LAMP amplicons were produced by a set of four designed primers that recognized specifically the V. parahaemolyticus thermolabile haemolysin (tlh) gene followed by hybridization with an FITC‐labelled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP–LFD method accurately identified 28 isolates of V. parahaemolyticus but did not detect 24 non‐parahaemolyticus Vibrio isolates and 35 non‐Vibrio bacterial isolates. The sensitivity of LAMP–LFD for V. parahaemolyticus detection in pure cultures was 120 CFU ml^?1. In the case of spiked shrimp samples without enrichment, the detection limit for V. parahaemolyticus was 1·8 × 10³ CFU g^?1 or equivalent to 3 CFU per reaction while that of conventional PCR was 30 CFU per reaction. Conclusions: The established LAMP–LFD assay targeting tlh gene was specific, rapid and sensitive for identification of V. parahaemolyticus. Significance and Impact of the Study: The developed LAMP–LFD assay provided a valuable tool for detection of V. parahaemolyticus and can be used effectively for identification of V. parahaemolyticus in contaminated food sample.     

Bacteriophages drive strain diversification in a marine Flavobacterium: implications for phage resistance and physiological properties

Genetic, structural and physiological differences between strains of the marine bacterium Cellulophaga baltica MM#3 (Flavobacteriaceae) developing in response to the activity of two virulent bacteriophages, ΦS_M and ΦS_T, was investigated during 3 weeks incubation in chemostat cultures. A distinct strain succession towards increased phage resistance and a diversification of the metabolic properties was observed. During the incubation the bacterial population diversified from a single strain, which was sensitive to 24 tested Cellulophaga phages, into a multistrain and multiresistant population, where the dominant strains had lost susceptibility to up to 22 of the tested phages. By the end of the experiment the cultures reached a quasi steady state dominated by ΦS_T‐resistant and ΦS_M + ΦS_T‐resistant strains coexisting with small populations of phage‐sensitive strains sustaining both phages at densities of > 10⁶ plaque forming units (pfu) ml^?1. Loss of susceptibility to phage infection was associated with a reduction in the strains' ability to metabolize various carbon sources as demonstrated by BIOLOG assays. This suggested a cost of resistance in terms of reduced physiological capacity. However, there was no direct correlation between the degree of resistance and the loss of metabolic properties, suggesting either the occurrence of compensatory mutations in successful strains or that the cost of resistance in some strains was associated with properties not resolved by the BIOLOG assay. The study represents the first direct demonstration of phage‐driven generation of functional diversity within a marine bacterial host population with significant implications for both phage susceptibility and physiological properties. We propose, therefore, that phage‐mediated selection for resistant strains contributes significantly to the extensive microdiversity observed within specific bacterial species in marine environments.     

Phages C-2 and J: IncC and IncJ plasmid-dependent phages, respectively

Phages C-2 and J were isolated from sewage. Phage C-2 was filamentous and formed plaques on Salmonella typhimurium strains carrying various C plasmids. It also plated on Proteus mirabilis and Serratia marcescens strains carrying particular C plasmids, but failed to form plaques on lines of Escherichia coli K12 strains harbouring most of these plasmids, although in all cases, phage multiplication on the strains was demonstrated. No phage increase occurred in any strain which lacked a C plasmid or contained plasmids of other incompatibility groups. The phage was sensitive to chloroform and, unlike other filamentous bacterial viruses, adsorbed to shafts of conjugative pili. It had a disc-like structure at the end which attached to the pilus. Phage C-2 had a buoyant density of 1 . 30 g cm-3 and a single-stranded circular DNA genome of 3 . 0 MDal. Phage J had an hexagonal head with an inter-apical distance of 40 nm and a short noncontractile tail. It was resistant to chloroform and diethyl ether. The phage formed plaques or propagated on E. coli strains harbouring some IncC plasmids and all IncJ and IncD plasmids tested. The phage did not form plaques but propagated on P. mirabilis and Ser. marcescens strains carrying these plasmids. It did not plate or propagate on S. typhimurium strains harbouring the plasmids. The plaques were very hazy and variable in size. The phage attached sparsely, at a site which appeared to be located at the base of the tail, to sides of conjugative pili.     

φ20, A Temperate Bacteriophage Isolated from Bacillus anthracis Exists as a Plasmidial Prophage

This study describes the isolation of temperate B. anthracis phages, from 4 out of 20 B. anthracis strains screened, by use of the inducing agents mitomycin C and UV light. Phage φ20 isolated from B. anthracis Sterne 34F₂ (pXO1⁺ pXO2⁻) was shown to have double-stranded DNA of size 48756 bp and a restriction site map showing nine sites for enzymes BamHI, BglII, and SstI is included. The φ20 genome was found to exist as a plasmidial prophage and the phage itself to have a polyhedral head of diameter 65 nm and tail 217 nm long and 15 nm wide.     

Morphology of pili determined by the N incompatibility group plasmid N3 and interaction with bacteriophages PR4 and IKe

The IncN plasmid N3 was transferred to bald strains of Salmonella typhimurium LT2 and Escherichia coli K-12. In both cases, transconjugants were found to carry short pili, which were designated N pili. They were very easily detached from cells and readily broken into short pieces. N pili were found to be straight inflexible rods 9.5 nm thick and sharply pointed at the distal end. The N-specific filamentous bacteriophage IKe and the lipid-containing phage PR4 both adsorbed to the pointed tips when mixed with cell-free suspensions of N pili.     

Mutants at conserved positions in gene IV, a gene required for assembly and secretion of filamentous phages

The filamentous phage protein pIV is required for assembly and secretion of the virus and possesses regions homologous to those found in a number of Gram-negative bacterial proteins that are essential components of a widely distributed extracellular protein-export system. These proteins form multimers that may constitute an outer membrane channel that allows phage/protein egress. Three sets of f1 gene IV mutants were isolated at positions that are absolutely (G355 and P375) or largely (F381) conserved amongst the 16 currently known family members. The G355 mutants were non-functional, interfered with assembly of plV⁺ phage, and made Escherichia coli highly sensitive to deoxycholate. The P375 mutants were non-functional and defective in multimerization. Many of the F381 mutants retained substantial function, and even those in which charged residues had been introduced supported some phage assembly. Some inferences about the roles of these conserved amino acids are made from the mutant phenotypes.     

Detection of Vibrio parahaemolyticus in Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes

We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenic Vibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters. V. parahaemolyticus has been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype of V. parahameolyticus O3:K6. Total V. parahaemolyticus was targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 10⁴ CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFU V. parahaemolyticus per g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3 × 10⁹ CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive for tlh and 4/33 samples that were positive for tdh. This assay specifically and sensitively detected total and pathogenic V. parahaemolyticus and is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.