Purification and Host Specificity of Predatory Halobacteriovorax Isolates from Seawater

Abstract

c Halobacteriovorax (formerly Bacteriovorax) is a small predatory bacterium found in the marine environment and modulates bacterial pathogens in shellfish. Four strains of Halobacteriovorax originally isolated in Vibrio parahaemolyticus O3:K6 host cells were separated from their prey by an enrichment-filtration-dilution technique for specificity testing in other bacteria. This technique was essential, since 0.45-m filtration alone was unable to remove infectious Vibrio minicells, as determined by scanning electron microscopy and cultural methods. Purified Halobacteriovorax strains were screened for predation against other V. parahaemolyticus strains and against Vibrio vulnificus, Vibrio alginolyticus, Escherichia coli O157:H7, and Salmonella enterica serovar Typhimurium DT104, all potential threats to seafood safety. They showed high host specificity and were predatory only against strains of V. parahaemolyticus. In addition, strains of Halobacteriovorax that were predatory for E. coli O157:H7 and S. Typhimurium DT104 were isolated from a tidal river at 5 ppt salinity. In a modified plaque assay agar, they killed their respective prey over a broad range of salinities (5 to 30 ppt). Plaques became smaller as the salinity levels rose, suggesting that the lower salinities were optimal for the predators' replication. These species also showed broader host specificity, infectious against each other's original hosts as well as against V. parahaemolyticus strains. In summary, this study characterized strains of Halobacteriovorax which may be considered for use in the development of broad-based biocontrol technologies to enhance the safety of commercially marketed shellfish and other foods. Bdellovibrio and like organisms (BALO) are Gram-negative, aerobic bacteria which are predatory toward other Gram-negative bacteria and consist of freshwater/terrestrial and marine forms. Bdellovibrio and like organisms are divided into four genera, Bdellovibrio, Bacteriolyticum, Peredibacter, and Bacteriovorax, which was recently renamed Halobacteriovorax (1). These organisms enter into a susceptible host bacterium and reside within the periplasmic space where they utilize the cytoplasmic nutrients of the host to support growth and replication. The replicative form, known as a bdelloplast, elongates within the host and septates into progeny cells as the host is lysed, releasing cells capable of attacking more prey. Halobacteriovorax organisms are the marine forms, which are small, polar flagellated, highly motile, intracellular predators in the class Deltaproteobacteria family Halobacteriovoraceae (1).Bdellovibrio and like organisms, particularly the nonhalophilic Bdellovibrio bacteriovorus, have been proposed for use in reducing the levels of phytopathogens associated with bacterial blights in soybeans and rice (2, 3) and reducing the levels of Salmonella enterica serovar Typhimurium on freshwater fish fillets (4). Other uses involve the treatment of various infections in animals, including bovine keratoconjunctivitis caused by Mora...

Halobacteriovorax isolated from marine water of the Adriatic sea, Italy, as an effective predator of Vibrio parahaemolyticus, non-O1/O139 V. cholerae, V. vulnificus

“…Despite the low level of prey used in the experiment, HBXCO1 strain was still able to contain its growth, showing predatory activity already in the first 24 h. Moreover, HBXCO1 showed prey specificity preferentially for Vibrio sp., not being able to attack other bacteria, frequently present in marine environment as potential threats to seafood safety, (Salmonella and Aeromonas), or as contaminants (E. coli and Pseudomonas). Selective predation by BALOs has been documented before (Pineiro et al, 2004;Li et al 2011;Richards et al 2016). This prey specificity probably also reflects the nature of the primary prey that was used to obtain HBXCO1 which was V. parahaemolyticus.…”

“…; Richards et al . ). This prey specificity probably also reflects the nature of the primary prey that was used to obtain HBXCO1 which was V. parahaemolyticus .…”

“…Briefly, 1 μ l of filtered HBXCO1 undiluted enrichment, 1 ml of prey culture at an OD 600 of 0·2 for Vibrio and 0·9 for non‐ Vibrio preys (approximately 10 8 PFU per ml), were added to 7·5 ml of sterilized artificial seawater (30 ppt salinity) and 7·5 ml of Pp 20 top agar (Richards et al . , ). For each prey specificity assay, a positive control, represented by V. parahaemolyticus NCTC 10885 and HBXCO1, and a negative control, which consisted of the prey alone, were performed.…”

“…Prey specificity and predator efficiency of HBXCO1 were determined by monitoring its abilities to form clear lytic halos on a lawn of Vibrio and non-Vibrio preys, using a double layer agar plating technique. For non-Vibrio preys, the plaque assay procedure for Halobacteriovorax in V. parahaemolyticus was modified to incorporate one-quarter-strength LB broth (added as a powder before autoclaving) with normalstrength Pp20 agar (modified Pp20 agar) to provide additional nutrients to support growth at the higher salinities (Richards et al 2016). Briefly, 1 ll of filtered HBXCO1 undiluted enrichment, 1 ml of prey culture at an OD 600 of 0Á2 for Vibrio and 0Á9 for non-Vibrio preys (approximately 10 8 PFU per ml), were added to 7Á5 ml of sterilized artificial seawater (30 ppt salinity) and 7Á5 ml of Pp 20 top agar (Richards et al 2012(Richards et al , 2016.…”

“…The potential application of marine BALOs in safeguarding seafood safety has started to be investigated. Recent studies report that marine BALOs are capable of significantly reducing Vibrio parahaemolyticus and Vibrio vulnificus levels in both, seawater and oysters, at the laboratory scale Li et al 2011;Richards et al 2012Richards et al , 2016Williams et al 2015). In Italy, bivalve contamination by pathogenic vibrios is an increasing problem in the last decades, and toxigenic V. parahaemolyticus, non-O1/O139 Vibrio cholerae and V. vulnificus have been isolated in Mytilus galloprovincialis (Normanno et al 2006;Ottaviani et al 2009Ottaviani et al , 2010aOttaviani et al , 2013Beneduce et al 2010).…”