Concentration-dependent reduction of planktonic- and biofilm-state Vibrio alginolyticus by the bacteriophage pVa-21

There is an increasing emergence of antibiotic-resistant Vibrio alginolyticus, a zoonotic pathogen that causes mass mortality in aquatic animals as well as human infection; therefore, there is a demand for alternatives to antibiotics for treatment and prevention of infections caused by this pathogen. One possibility is through the exploitation of bacteriophages. In the present study, the bacteriophage pVa-21 belonging to Myoviridae, was isolated and characterized as a candidate biocontrol agent against V. alginolyticus. Its morphology, host range and infectivity, growth characteristics, planktonic or biofilm lytic property, stability under various conditions, and genome were investigated. Its latent period and burst size were estimated to be approximately 70 min and 58 plaque-forming units/cell, respectively. In addition, phage pVa-21 could inhibit bacterial growth both in the planktonic and biofilm state. Furthermore, phylogenetic and genome analyses revealed that the phage is closely related to ‘phiKZ-like phages’ and can be classified as a new member of the phiKZ-like phages that infect bacteria belonging to the family Vibrionaceae.


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Vibrio alginolyticus is an opportunistic pathogen frequently found in marine environments. Having a 32 wide host range, it can cause mass mortality in aquatic animals as well as human infection [1][2][3][4]. It is well 33 known that the majority of bacterial infections are caused by bacteria in biofilms [5]; therefore, biofilm 34 formation is an important feature of pathogenic microorganisms [6]. Furthermore the threat of biofilms is 35 apparent from the fact that bacterial cells in biofilms are highly tolerant to antibiotics compared with those in the 36 planktonic state [7]. Therefore, antibiotics cannot effectively inhibit bacterial proliferation within biofilms 37 resulting in regrown bacteria following failure of antibiotic treatment, which could cause serious problems [8].
38 Therefore, there is a need for effective alternatives to antibiotic treatment for managing biofilm-related bacterial 39 outbreaks.

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As viruses of bacteria, bacteriophages (phages) specifically infect and lyse the targeted bacteria.

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Recently, lytic bacteriophages have been demonstrated as potential alternatives to antibiotics in various studies 42 targeting the prophylaxis or treatment of bacterial diseases [9][10][11]. Especially in case of biofilm-related construct the growth curve, the phage lysate was inoculated in 10 ml of exponentially growing host strain 98 culture (1.5 ×10 8 CFU/ml) at an MOI of 0.001. The phage was absorbed for 15 min and then centrifuged at 99 12,000 × g for 5 min. After the supernatant was discarded, the phage-infected bacterial pellet was re-suspended 100 in 10 ml of preheated TSB and incubated at 27°C with shaking at 250 rpm. At 10-min intervals, 100-μl aliquots 101 were taken until 140 min, and the titer was immediately determined by the double-layer agar method.

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Isolation and biological properties of phage pVa-21 155 One V. alginolyticus phage was isolated from sea water samples after enrichment. The purified phage was 156 examined by TEM and classified based on the criteria proposed by Ackermann (28) Table). However, the phage did not show infectivity to the nine other bacterial strains 163 tested. Adsorption rate was assessed as described above, and the percentages of adsorbed phages in the V.
164 alginolyticus rm-8402 after 15 min was 95% (Fig 2). To identify the growth pattern and burst size of pVa-21, a 165 one-step growth curve was generated. The latent period was found to be approximately 70 min and the burst size, 166 i.e., the number of progeny released after the lysis of a single bacterial cell, was approximately 58 virions per 167 cell (Fig 3). The stability of pVa-21 was tested at various pH and temperature conditions and was estimated by 168 determining the changes in growth (based on the number of PFU). Phage pVa-21 showed relatively good 169 stability at pH 5.0, 7.0, and 9.0 after 1 h of incubation, but significant decreases in PFU counts were detected at 170 low and high pH. At pH 3.0 and 11.0, pVa-21 was extremely unstable with a nearly 100% decrease in the PFU 171 observed (S1 Fig A). The thermal stability test showed that pVa-21 was quite stable (> 90%) at 4°C, 20°C, 25°C, 172 30°C, and 35°C for 1 h, but its numbers sharply decreased at temperatures above 40°C (S1 Fig B). an OD 600 value of almost 0 after 7 h of incubation. Although the growth of these two strains was also affected by 187 the phage at an MOI of 1, the populations were sustained to some extent and did not reach a value of 0 ( Fig 4B   188 and D). Similarly, the growth of strain am-10 was affected by pVa-21 in proportion to its inoculated 189 concentration (Fig 4C). The remaining strains did not appear to be affected by phage pVa-21 showed no with further disruption to an OD value of ≤ 1 (P < 0.05) at the middle or high concentration (Fig 5). subunit sequence is shown in Fig 8A and    279 alginolyticus phage pVa-21 could safely be used as a biocontrol agent against these bacteria. However, the 280 majority of ORFs in pVa-21 did not match with the predicted function in GenBank. Thus, to ensure the safe and 281 reliable application of phages from a therapeutic perspective, further investigation of the phage genome is 282 needed to gain a deeper understanding of the roles of the encoded gene products, as they might produce novel 283 virulence factors or interact undesirably with the host genome [36].

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In conclusion, the present study highlights the importance of the initial phage concentration on its 285 antibacterial effects regardless of the growth state of the target bacteria: planktonic or biofilm. The 286 characteristics of the Vibrio phage pVa-21 and its genome are expected to broaden the phiKZ-like phage library 287 and help promote the application of bacteriophages in biofilm control to protect aquatic organisms from 288 infection. In addition, as illustrated in a recent study [37], biofilm eradication by phiKZ-like phages in multidrug 12 289 resistant bacteria suggests that the phiKZ-like phage, pVa-21, could also be used as a bio-control agent. The 290 lytic effect of pVa-21 itself and the three distinct enzymes it possesses are expected to eradicate bacteria.

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Although the precise biofilm eradication mechanism of pVa-21 remains to be elucidated, the three different lytic 292 enzymes detected are likely involved in the mechanism.