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

Abstract

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 Information

The online version contains supplementary material available at 10.1007/s12088-021-00934-6.

Introduction

Among the various aquaculture species, shrimp (Litopenaeus vannamei) is the important one. High global demand and export potential have led to the culture intensification and horizontal expansion of shrimp farming [1, 2]. Due to its ability to survive and grow in low salinity, inland saline areas (previously considered as wastelands) with salinity as low as 5 ppt have emerged as attractive destinations for vannamei shrimp farming [3, 4]. However, frequent disease outbreaks in shrimp farming not only leads to economic losses for the farmers but may also lead to serious health consequences for the consumers, as many of these aquatic pathogens may also cause foodborne infections [5]. Among the various bacterial pathogens associated with shrimp farming, Vibrio parahaemolyticus has often been found as the causative agent of acute gastroenteritis in humans resulting from consumption of poor quality, undercooked or raw seafood [6]. V. parahaemolyticus also causes a devastating shrimp early mortality syndrome (EMS) also known as acute hepatopancreatic necrosis disease (AHPND) [7]. Previous studies have reported the presence of V. parahaemolyticus in low salinity inland saline areas [8, 9]. As shrimp culture in inland saline areas is expanding at a rapid rate, the risk of V. parahaemolyticus related infections cannot be neglected. Due to the global emergence of antibiotic-resistant bacteria and the presence of antibiotic residues in food products destined for human consumption, phage therapy has recently re-emerged as an attractive alternative for targeted control of bacterial pathogens without affecting the non-target natural microbiota. Before therapeutic applications, extensive host range, morphological and genomic characterization of phages is of utmost important [10]. Thus, the present study was carried out to isolate and characterize V. parahaemolyticus specific phages from inland saline shrimp culture environments. After characterization, efficacy of a selected phage against V. parahaemolyticus in shrimp was also investigated.

Materials and Methods

Phage Enrichment and Isolation

For phage enrichment and isolation, water samples were collected from inland saline shrimp culture farms located in the states of Punjab and Haryana. Two isolates of V. parahaemolyticus were used as hosts for phage enrichment from these water samples. The first isolate of V. parahaemolyticus (designated as Vp7PE) was obtained from the Department of Aquatic Animal Health, College of Fisheries, Mangalore, whereas the second isolate (designated as Vp25) was obtained from a water sample collected from Inland saline shrimp farm located in Punjab state. Both the isolates were subjected to species-level identification by biochemical testing and PCR as per previously described protocols [11]. The basic protocols for phage enrichment, isolation and soft agar overlay assay have been described elsewhere [12, 13]. The detailed protocols for all the steps have also been provided in the supplementary section.

Evaluation of Phage Host Range

To evaluate the host range spectrum of isolated phages, their lytic activity against various isolates of V. parahaemolyticus and other related bacteria was studied (Supplementary Table 1). The lytic activity was tested by lawn and plaque assays against log phase (OD₆₀₀ 0.5–0.7) cultures of bacteria as per the above-described methodology. The presence of a zone of lysis (in the case of lawn assay) and plaques (during plaque assay) was taken as the sign of lytic activity of phage against specific bacteria.

Genomic Characterization of Phages

For genomic characterization, phage nucleic acid was isolated by Guanidine hydrochloride and Proteinase K mediated lysis method followed by silica column purification. The quantitative and qualitative QC of purified phage nucleic acid was performed by Qubit fluorometric (ThermoFisher, USA) and agarose gel electrophoresis methods, respectively. For determining the RNA/ssDNA/dsDNA nature of phage nucleic acid, it was subjected to separate treatments with DNase I (MP Biomedicals, USA), RNase A (GeNei, India) and S1 nuclease (Promega, USA) followed by agarose gel electrophoresis as per previously described protocols [14].

Phage Morphological Characterization

The morphology of fixed and stained phage preparations was observed with Hitachi H-7650 Transmission Election Microscope (TEM) at Electron Microscopy & Nanoscience Laboratory (Punjab Agricultural University, Ludhiana) as per previously described protocols [13]. As per the criteria described by the International Committee on Taxonomy of Viruses (ICTV) (http://www.ictvonline.org/) and Novik et al.[15], phage morphology, as well as taxonomy, was determined. Phage morphometric measurements were performed by ImageJ v1.52a software [16].

Evaluation of Phage Efficacy Against V. parahaemolyticus in Shrimp

After isolation, host range evaluation, genomic and morphological characterization, the efficacy of a selected phage V5 was evaluated against V. parahaemolyticus contamination in raw deveined shrimps. For bacterial inoculation, 50 g of raw deveined shrimps were sprayed with one millilitre of log-phase V. parahaemolyticus culture to achieve the final counts of 1 × 10⁴ cfu/g of shrimp followed by incubation at room temperature for 1 h. After incubation, the shrimp samples were sprayed with one millilitre of phage V5 to achieve the final phage counts of 10⁶ pfu/g at the theoretical multiplicity of infection (MOI) of one hundred. Shrimps with V. parahaemolyticus but without phage and shrimps without V. parahaemolyticus but with phage were used as positive and negative controls respectively. To determine the lytic effect of phage, the V. parahaemolyticus counts at 0 h (immediately after addition of phage cocktail), 1 h and 3 h were determined by spread plate method on thiosulfate-citrate-bile salts-sucrose TCBS agar. Student’s t test was to determine any significant difference in the V. parahaemolyticus counts between phage treated shrimps and positive control.

Results and Discussion

Phage Isolation and Characterization

In the present study, a total of 12 V. parahaemolyticus specific phages were isolated from 264 water samples. Among these, 5 phages (AMN2, FT2, FT3, KD1 and V1 were isolated against V. parahaemolyticus host isolate Vp7PE, whereas 7 phages (AMN1, AMN3, PL1, V2, V4, V5 and V6 were isolated against V. parahaemolyticus Vp25 isolate (Fig. 1). It is well known that phages are highly host-specific with the host range limited to one or a few strains of the same bacterial species [17]. To isolate the phages with varying host range, two different V. parahaemolyticus hosts were used in the present study.

Fig. 1.

Lytic activity of various phages against V. parahaemolyticus Vp25 (a–c) and V. parahaemolyticus Vp7PE hosts (d, e). Representative plaque assay images for phage V5 (f) and AMN1 (g) have also been shown. Negative control is the filtered extract of the overnight grown host bacterium without any phage

During the host range analysis, lytic activity was observed against 2.3 to 45.5% of tested V. parahaemolyticus isolates. With lytic activity against 45.5% of tested V. parahaemolyticus isolates, maximum host range was determined for phage V5 and AMN1, whereas phages AMN2, FT2 and V1 showed the lowest host range with lytic activity only against 2.3% of V. parahaemolyticus isolates. No lytic activity was observed against other bacterial species. (Supplementary Table 1). Phages isolated against V. parahaemolyticus Vp25 showed higher lytic activity against V. parahaemolyticus isolates obtained from the same inland saline shrimp culture habitats. On the other hand, the lytic activity of phages isolated against V. parahaemolyticus Vp7PE was generally limited to only the host strain with almost no activity against other V. parahaemolyticus isolates (Supplementary Table 1). The receptor diversities between these two V. parahaemolyticus isolates might also have been responsible for the distinct host range of their respective phages.

Ideally for therapeutic applications, a phage should be able to infect and lyse all available strains of targeted bacterial species. However, such kinds of broad range phages are generally not available in nature. Thus, one approach to increase the host range during phage therapy is to use the mixture of genetically diverse phages (phage cocktail) with varying host ranges [18]. In the present study, no combination of phages (phage cocktail) was able to cover the host range spectrum of more than 50% of total isolates tested, suggesting the need for further phage isolation and characterization from inland saline shrimp culture environments.

For the determination of phage genome type and approximate genome size, nucleic acid from 9 phages was isolated. The concentrations of phage nucleic acid ranged from 7.66 to 220 ng/µl (Table 1). The significant variation in nucleic acid concentrations for various phages could be due to variations in genome size, genome type (dsDNA/ssDNA/RNA), burst size and generation time [19, 20]. All the phage nucleic acid samples showed very good intact bands on agarose gels with minimal degradation and RNA contamination (Fig. 2). Among these 9 phages, 7 contained ssDNA genome, whereas two others had dsDNA as their genetic material (Table 1). As per the ICTV database, phages with dsDNA are the dominant ones with almost 89% prevalence. On the other hand, ssDNA phages constitute only 11% of the ICTV phage database [21]. Thus, phages with ssDNA genomes in our study contribute significantly to relatively very fewer numbers of ssDNA phages characterized to date. Based on agarose gel electrophoresis results, the genome sizes of ssDNA phages were estimated to be 5—6 kb, whereas genomes of dsDNA phages were determined to be around 20–21 kb (Fig. 2). As per previous studies, the linear genomes of dsDNA phages range from 16 to 500 kb, whereas ssDNA phages contain circular genomes varying from 4.5 to 12.4 kb [21].

Table 1.

Genome types and nucleic acid concentrations of V. parahaemolyticus phages

S. no	Phage	Genome type	DNA conc. (ng/µl)	Total amount (µg) (in 50 µl vol.)
1	AMN1	ssDNA	119a	6.0
2	AMN3	ssDNA	220a	11.0
3	PL1	ssDNA	8.22a	0.4
4	V2	ssDNA	49.6a	2.5
5	V4	ssDNA	108a	5.4
6	V5	ssDNA	54.6a	2.7
7	V6	ssDNA	64.4a	3.2
8	FT2	dsDNA	7.66b	0.4
9	V1	dsDNA	15.7b	0.8

^aNucleic acid concentration was determined by Qubit ssDNA assay kit

^bNucleic acid concentration was determined by Qubit dsDNA broad range assay kit

Fig. 2.

Agarose gel QC of phage genomic DNA samples. Lane M: Lambda DNA/EcoRI plus HindIII marker (ThermoFisher), Lane 1: Phage nucleic acid. The sizes of marker bands preceding and succeeding the phage nucleic acid have also been shown

During the TEM analysis, the phage V5 was found to have filamentous shaped morphology making it a member of the family Inoviridae (Fig. 3). The diameter and length of the V5 phage was 9.10 ± 2.15 nm (mean ± SD) and 1,194 ± 40.46 nm, respectively. Members of the family Inoviridae have a thin, filamentous capsid consisting of thousands of subunits of major capsid proteins enclosing the ssDNA circular genome within it. For ssDNA phages, the reported capsid diameter is around 6 nm, whereas the length may vary between 700 and 3700 nm [21].

Fig. 3.

Transmission electron micrograph of phage V5 after staining with 2% phosphotungstic acid

Efficacy of Phage Against V. parahaemolyticus Contamination in Shrimp

In the present study, the efficacy of phage V5 against V. parahaemolyticus infection in shrimp was tested by inoculating the raw deveined shrimps with fixed counts of V. parahaemolyticus followed by phage application, incubation at room temperature and determination of V. parahaemolyticus at regular intervals. After one hour, average V. parahaemolyticus counts in control and phage treated groups were found to be 5.33 × 10⁴ cfu/g and 1.17 × 10⁴ cfu/g, respectively signifying the reduction of 78.1% (0.66 log₁₀ units) in Vibrio parahaemolyticus counts due to phage applications within one hour (t test, p < 0.05). During one to 3 h, no further reduction in V. parahaemolyticus counts was observed, and bacterial counts instead increased to 1.77 × 10⁴ cfu/g in treatment groups. However, these counts were still significantly lower than the 5.57 × 10⁴ cfu/g in control groups, signifying the reduction of 68.3% (0.50 log₁₀ units) in V. parahaemolyticus counts due to phage application (t test, p < 0.05) (Fig. 4). Throughout the study, no V. parahaemolyticus was detected in the negative controls. In a previous study, a five phage cocktail was able to reduce the Shigella from 0.2 to 1.6 log units in various food products (lettuce, salmon, chicken, corned beef, yoghurt and melon) within five minutes of application [22]. The efficacy of phage treatment in food products against V. parahaemolyticus has also been tested. During the application of pVp-1 phage in the oyster, V. parahaemolyticus counts reduced by approximately 2 and 4 log units after 2 and 12 h of application, respectively [23]. In another study, the application of phage OMN on the oyster surface for 48 and 72 h resulted in inactivation of 90% and 99% V. parahaemolyticus, respectively [24]. Though the application of phage V5 resulted in a significant reduction in V. parahaemolyticus counts in the present study, the quantum of reduction was relatively less in comparison to the previous studies. This could be due to the infection mechanism and lifecycle of filamentous V5 phage used in the present study. Filamentous phages with ssDNA genome do not lyse their hosts during virion release, but instead, continuously shed viral particles causing ‘stable’ or ‘chronic’ infections [25]. In these cases, mortality in host bacterium occurs due to stress and exhaustion resulting from overexploitation of the host`s biosynthesis machinery by phage [21]. This could explain the relatively low extent of V. parahaemolyticus reduction by V5 phage in the present study. Still, these results indicate the potential of phage therapy for control of V. parahaemoyticus in shrimp.

Fig. 4.

Efficacy of phage treatment against V. parahaemolyticus contamination in shrimp. V. parahaemolyticus counts in control and treatment groups have been shown at different time intervals. Different superscripts (a, b) on the graph represent the significant difference in V. parahaemolyticus counts (t test, p < 0.05)

In conclusion, the present study, for the first time, resulted in isolation and characterization of V. parahaemolyticus specific phages from the inland saline shrimp culture environment. In contrast to more prevalent dsDNA phages in nature, the majority of phages in the present study were found to have ssDNA genome, and these could of significant biotechnological significance. The host range analysis results indicated the need for isolation and characterization of more phage from inland saline areas for potential therapeutic applications. More studies are also needed to be carried on the efficacy of phages against V. parahaemolyticus in shrimp under different storage conditions.

Supplementary Information

Below is the link to the electronic supplementary material.

Authors’ Contributions

AT conceived the study, analyzed the data and drafted the manuscript. SD collected the samples and performed the experiments. AS contributed to phage efficacy studies. NBT and NKS contributed to phage genomic characterization. All authors contributed to manuscript correction.

Declarations

Conflict of interest

The authors declare that they no conflict of interest.

Ethical Approval

No special permission was required for this study.