Applied Biochemistry and Biotechnology (2022) 194:4477–4491

https://doi.org/10.1007/s12010-022-03919-3

REVIEW

Vibrio spp. and Their Vibriocin as a Vibriosis Control Measure

in Aquaculture

Hassan Sheikh1 · Akbar John2 · Najiah Musa1 · Laith A. abdulrazzak1 ·

Mulham Alfatama3 · Anis Fadhlina4

Accepted: 14 April 2022 / Published online: 22 April 2022

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022

Abstract
Vibriosis disease is a major threat to the aquaculture industry caused by Vibrio spp. that are
often resistant to antibiotics. Alternative controlling measures such as bacteriocins could
be effective due to their narrow-spectrum activity. Hence, this systematic literature review
(SLR) was carried out to review the feasibility of Vibrio spp. and their vibriocins to be
used as a vibriosis control measure in aquaculture. A literature search using the web of sci-
ence (WOS) and SCOPUS databases resulted in 42 unique articles which were reviewed.
The results showed that Vibrio spp. could be used as a probiotic to control vibriosis, but
not recommended due to their opportunistic nature and pathogenesis. Vibriocin showed
narrow-spectrum activity against Vibrio spp. including highly pathogenic strains such as
V. alginolyticus, V. harveyi, and V. parahaemolyticus. This supported this review’s hypoth-
esis of using vibriocin as a targeted vibriosis control measure. Vibrio cholerae was the
most studied and showed the highest inhibition range, inhibiting 13 different vibrio and
non-vibrio species. Various innovations were reported in the field and vibriocins can now
be produced on large scales using whole-cell culture. Vibriocins were structurally diverse,
large molecular weight, and relatively heat stable. These vibriocins mainly inhibited the
cell wall but could have other novel mechanisms. These properties could affect the extrac-
tion process as well as applications in aquaculture, hence, should be considered in future
research.

Keywords Aquaculture · Vibriosis · Bacteriocin · Vibriocin · SLR

* Hassan Sheikh
sheikhinho@gmail.com
1
Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, Kuala Nerus,
21030 Terengganu, Malaysia
2
Kulliyyah of Science, International Islamic University Malaysia, 25200 Kuantan, Pahang,
Malaysia
3
School of Pharmaceutical Technology, Faculty of Pharmacy, University Sultan Zainal Abidin,
22200 Besut, Malaysia
4
Institute of Food Security and Sustainable Agriculture, Universiti Malaysia Kelantan,
17600 Jeli, Kelantan, Malaysia

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4478 Applied Biochemistry and Biotechnology (2022) 194:4477–4491

Introduction

Vibrio species are Gram-negative bacteria widely distributed in marine environments and
contain species that are pathogenic to various aquatic organisms as well as humans [1].
These species cause vibriosis disease in numerous farmed species, cause high mortalities,
and are a major threat to the aquaculture industry [2]. Antibiotics are the first choice to
control vibriosis; however, previous studies and case reports showed a possible correlation
between virulence and antimicrobial resistance (AMR). For instance, a virulent strain of V.
harveyi showed resistance to various antibiotics such as cotrimoxazole, chloramphenicol,
streptomycin, and vibriostatic agent 0/129 [3]. Hence, there is a need for anti-vibrio agents
with low resistance risk to be used in the aquaculture industry to control vibriosis.
Bacteriocins are well-known antimicrobial agents with narrow-spectrum activity against
related species. They are protein/peptides that are ribosomally synthesized by microorgan-
isms to kill closely related species. Nisin is one of the most common bacteriocins that has
been used in more than 40 countries as a preservative due to its antibacterial properties
and is safe for human consumption [4, 5]. Similarly, bacteriocins isolated from Vibrio spp.
could provide specific activity against Vibrio spp.
Throughout the years, numerous studies attempted the detection and isolation of bac-
teriocins from Vibrio spp. Various names were also given such as vibriocin, vibriocine,
vibriocidin, vibriobactin, harveyicin, and vulnificin [6],Somova, 1965, Felsenfeld et al.,
1968; [7, 8],Ha & Whang; [9]. Hence, from this point forward, the term “vibriocin” will
be used to refer to bacteriocin produced by Vibrio spp. Hence, this systematic literature
review (SLR) was carried out to review the efficacy and suitability of Vibrio spp. and their
vibriocins to control vibriosis in aquaculture.

Data Collection: Databases and Criteria

A systematic literature review (SLR) was carried out using 5 keywords and 2 databases.
Data were retrieved up to 31 December 2021 and summarized in Fig. 1. A total of 189 arti-
cles were obtained after combining the hits from both databases and removing duplicates.

Fig. 1  Systematic literature Keywords:

review process • Vibriocin
• Bacteriocin AND vibrio
• Bacteriocin-like inhibitory substances AND vibrio
• BLIS AND vibrio

Databases: WOS and SCOPUS

Documents: n = 189 Removal of duplicates

Don’t meet selection criteria:

Articles included in
the review: n = 42
• Not in English (n = 5)
• Reviews (n = 7)
• No abstract or full paper (n = 9)
• Bacteriocins not isolated from
Vibrio species (n = 126)

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Articles were then screened and exclusion was done due to either not being in English
(n = 5), reviews (n = 7), no abstract or full paper (n = 9), or containing bacteriocins that
were not isolated from Vibrio species (n = 126). After applying the inclusion and exclusion
criteria, a total of 42 were included in this review.

Vibrio spp. as Probiotic to Control Vibriosis in Aquaculture

The reviewed articles reported that several Vibrio spp. have been successfully used as pro-
biotics in aquaculture of mollusk, crustacean, and finfish [10]. Ha and Whang [11] showed
that in all of the tested 49 V. vulnificus strains, none of these strains was resistant to chlo-
ramphenicol, tetracycline, cephalothin, or moxalactam. This indicated the relative safety of
these strains to be used as probiotics. Other studies also supported this probiotic approach.
For instance, Burks et al. [12] showed that environmental Vibrio spp. that produces bac-
teriocins were able to outcompete pathogenic V. cholerae and V. parahaemolyticus at a
starting concentration of just 300 CFU/mL. This was recommended with a condition of the
probiotic candidate not being pathogenic. Balakrishnan et al. [13] recommended vibriocin
isolated from V. parahaemolyticus to be used as an alternative for antibiotics in controlling
Vibrio infections in the mariculture industry. Thompson et al. [14] also suggested that non-
pathogenic vibrios, such as V. gazogenes, could be utilized as probiotics to protect Pacific
white shrimp, Litopenaeus vannamei, against Vibrio infections. Serrano et al. [15] isolated
Vibrio sp. V7A from the intestine of Peruvian scallop (Argopecten purpuratus). The isolate
was closely related to V. mediterranei, showed BLIS activity, and was recommended as a
probiotic. Selvendran and Babu [16] showed that inhibitory activity of Vibrio sp. MMB2
strain’s ethyl acetate extract against S. aureus, B. subtilis, V. harveyi, V. parahaemolyticus,
A. hydrophila, and P. aeruginosa was comparable with commercial antibiotics Streptomy-
cin and Chloramphenicol. Liu et al. [17] recommended the use of Vibrio sp. 33 in aquacul-
ture as a biocontrol of Vibrio splendidus pathogenic sea cucumber (Apostichopus japoni-
cus) and improved its relative survival rate by 43%.
However, several studies did not recommend the use of whole-cell Vibrio spp. in aqua-
culture due to their opportunistic nature and their well-established pathogenicity. For
instance, despite Thompson et al. [14] showing that V. alginolyticus had similar antago-
nistic activity to V. gazogenes towards indicator strains (V. harveyi, V. anguillarum, and
V. alginolyticus), the authors did not recommend it as probiotic probably due to its known
pathogenicity. Probiotics are often used in aquaculture due to their ability to produce bac-
teriocins. Hence, the use of bacteriocins produced by Vibrio spp. could be a more feasible
option in controlling vibriosis.

Vibriocin as a Vibriosis Control Measure in Aquaculture

Vibriocin‑Producing Strains

Balcazar et al. [18] suggested that bacteriocins from Vibrio spp. have great potential in
becoming biological control agents for these pathogenic species that are resistant to chem-
otherapeutic treatment. Studies as early as the 1960s [19] and as recent as 2018 [15] dis-
cussed the possibility that not all Vibrio spp. produce bacteriocins. Most of the earlier stud-
ies reported limited ability in detecting vibriocin. An early study reported that V. cholerae

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4480 Applied Biochemistry and Biotechnology (2022) 194:4477–4491

has vibriocin-producing strains such as El tor and non-producing strains such as V63/Str-
rP [20]. Another study also reported that only 1% (n = 5/410) of Vibrio cholerae El Tor
was found to produce vibriocin [21]. However, Mitra et al. [22] also studied V. cholerae
and tested 1036 strains of both agglutinable (n = 743) and inagglutinable (n = 293) and
reported that approximately 94% and 91% were able to produce bacteriocin, respectively.
Studies on other species also showed that only 2 out of 40 (5%) Beneckea harveyi strains
tested against B. harveyi KN96 were able to produce bacteriocin [8]. However, the authors
themselves believed that the percentage showed might be higher and suggested testing
against multiple indicator strains. Shehane and Sizemore [23] studied V. vulnificus, V. chol-
era, and V. parahaemolyticus and reported that only V. parahaemolyticus did not produce
bacteriocin. Other studies on unidentified Vibrio spp. also supported that very few spe-
cies produced vibriocins. For instance, McCall and Sizemore [8] reported that only 8% of
795 Vibrio spp. showed BLIS activity; moreover, subsequent experiments showed that the
active substances in these species were not bacteriocins due to very low molecular weight
and could be bacteriophages. Carraturo et al. [24] studied 45 halophilic Vibrio spp. strains
and reported that only ~ 7% (n = 3) showed BLIS activity. Burks et al. [12] carried out one
of the largest studies and tested 3456 environmental marine Vibrio isolates. The authors
reported that only 3% (n = 102) showed BLIS activity against indicator Vibrio strains.
In sum, a total of 11 identified Vibrio spp. were found to be vibriocin producers. These
species were Aliivibrio fischeri (now known as V. fischeri), V. alginolyticus, V. anguil-
larum, V. cholerae, V. fluvialis, V. gazogenes, V. harveyi, V. mediterranei 1, V. parahaemo-
lyticus, V. rotiferianus (98.5% similarity), and V. vulnificus (Table 1).

Extraction and Detection of Vibriocin: Methods and Challenges

The previous section indicated that more vibriocin producers could be identified if ideal
methods are used. Hence, this section reviews the extraction methods used throughout the
years and highlights the challenges and progress achieved in this regard. The literature
showed that 6 main methods were used. These methods were exposure to chloroform vapor
[6, 20, 22, 24–28], anaerobiosis [19], cold shock [14, 29–31], cold shock followed by chlo-
roform exposure [29, 32, 33], Mitomycin C [34–36], and whole cell [7, 8, 12, 13, 16–18,
23, 28, 37–41].
In terms of challenges encountered in detecting vibriocin, the first main issue was the
variations in vibriocin detection methods in the 1960s. Chatterjee and Maiti [42] tried to
study the discrepancy and concluded that the inconsistencies around whether Vibrio spp.
produce bacteriocins/vibriocins is due to the lack of a standard method of detecting bacte-
riocin-like inhibitory substances. However, even following the same extraction or detection
methods, the results varied. For instance, despite cold shock being the most commonly
reported method, several studies reported issues with this method. Cold shock led to a com-
plete loss of vibriocin activity in Vibrio strains [6]. Thompson et al. [14] also showed that
using the cold shock method, only 2 (V. alginolyticus and V. gazogenes) out of 8 candidate
Vibrio spp. (V. alginolyticus, V. gazogenes, V. mediterranei, V. natriegens, V. orientalis, V.
proteolyticus, V. scopthalmi, and V. tubiashii) showed antagonist activity against indicator
strains (V. harveyi, V. anguillarum, and V. alginolyticus). Smigocki and Voll [26] reported
that vibriocin isolation from 2 Vibrio spp. including one non-O1 V. cholera could not be
done following standard methods. However, numerous studies successfully used the cold
shock method to detect vibriocin activity in V. cholerae, V. harveyi, V. alginolyticus, V.
gazogenes, and Vibrio spp. [29–31]. Other researchers tried to improve detection methods

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 Table 1  Summary of vibriocin-producing Vibrio spp., their inhibition spectrum, and molecular weight of characterized bacteriocins
Producer* Inhibition spectrum Molecular weight of isolated References
bacteriocin
Gram positive Gram negative

Aliivibrio fischeri - V. ichthyoenteri, V. parahaemo- - Balcazar et al. [18]

lyticus, and V. splendidus
V. alginolyticus - V. harveyi, V. anguillarum, and
V. alginolyticus
V. anguillarum - E. coli and Vibrio spp.
V. cholerae - V. cholerae and its related strains 1.35 and 60 kDa
(V. comma/V. cholera El Tor),
Escherichia coli, Enterobacter
aerogenes, Klebsiella pneu-
moniae, E. cloaceae, Proteus
vulgaris, Shigella flexneri,
S. boydii, S. dysentariae, S.
sonnei, V. vulnificus, V. para-
haemolyticus, Plesiomonas
- Thompson et al. [14] and Tan
et al. [44]
- Zai et al. [28]
Farkas-Himsley & Seyfried, P. L.
(1962), Wahba [6], Felsenfeld
& Greer [30], Jayawardene &
Farkas-Himsley [35], Lang
et al., [25], Datta & Prescott
[20], Chakrabarty et al. [29],
Jayawardene & Farkas-Himsley
[36], Farkas-Himsley & Cheung
[34], Mitra et al. [22], Israil
Applied Biochemistry and Biotechnology (2022) 194:4477–4491

shigelloides, and unidentified et al. [33], Smigocki & Voll

Vibrio spp. [26], Somova et al. [45], Badal-
ova et al. [46], Israil et al. [32],
Basu et al. [47], Telesmanich
et al. [48], Shehane & Sizemore
[23], Liu et al. [9]
V. fluvialis - V. parahaemolyticus - Carraturo et al. [24]
V. gazogenes - V. harveyi, V. anguillarum, and - Thompson et al. [14]
V. alginolyticus
V. harveyi - V. harveyi, V. fischeri, V. gazo- 13, 20, 23, 24, 27, 28, 31, 32, McCall & Sizemore [8], Hoyt &
genes, V. parahaemolyticus, 32.6, 33, 34, 37, 38, 41, 43, 46 Sizemore [7], Zhang & Austin
and unidentified Vibrio spp. 51, 52, and 76 kDa [31], Prasad et al. [39], Cheng
et al. [38]

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4481
 Table 1  (continued)
4482

Producer* Inhibition spectrum Molecular weight of isolated References

bacteriocin
Gram positive Gram negative

13
V. mediterranei 1 - Aeromonas hydrophila, A. 63–65 kDa Carraturo et al. [24]
media, V. mediterranei 1, V.
furnissii, and V. parahaemo-
lyticus
V. parahaemolyticus - V. alcaligenes, V. fluvialis, V. 18 kDa Priya et al. [40] and Balakrishnan
harveyi, V. mimicus, V. vulnifi- et al. [13]
cus, V. natriegens, V. fischeri,
and V. proteolyticus and Vibrio
spp.
V. parahaemolyticus (97.6% - V. parahaemolyticus and V. - Balcazar et al. [18]
similarity) splendidus
V. rotiferianus (98.5% similarity) - V. alginolyticus, V. harveyi, V. - Balcazar et al. [18]
parahaemolyticus, V. ichthy-
oenteri, and V. splendidus
V. vulnificus - Serratia marcescens, Pseu- 7.5 and 9 kDa Ha & Whang [11], Shehane &
domonas aeruginosa, S. Sizemore [23]
flexneri, Salmonella typhi,
Yersinia enterocolitica, V.
vulnificus, V. cholera, and V.
parahaemolyticus
Vibrio sp. 33 - V. splendidus - Liu et al. [17]
Vibrio sp. NM 10 Bacillus spp., Coryneforms, and Pasteurella piscicida, Flavo- < 5 kDa Sugita et al. [27]
Enterococcus seriolicida bacterium spp., Pseudomonas
spp., E. coli, V. vulnificus, and
Vibrio spp.
Vibrio sp. V7A - - - Serrano et al. [15]
Applied Biochemistry and Biotechnology (2022) 194:4477–4491
 Table 1  (continued)
Producer* Inhibition spectrum Molecular weight of isolated References
bacteriocin
Gram positive Gram negative

Vibrio sp.MMB2 strain Staphylococcus aureus and B. P. aeruginosa, A. hydrophila, V. 16 & 32 kDa Selvendran & Babu [16]
subtilis harveyi, and V. parahaemo-
lyticus
Vibrio spp. - V. harveyi, V. cholera, and V. - Arijo et al. [37] and Burks et al.
parahaemolyticus [12]
*
Note: Including strains or biotypes
Applied Biochemistry and Biotechnology (2022) 194:4477–4491

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4484 Applied Biochemistry and Biotechnology (2022) 194:4477–4491

which led to advancements in purification techniques. For instance, purified vibriocin was
1000 times (62.5 × ­108 LU/mg) more active than the crude vibriocin [35].
The second issue was that excretion of vibriocin often required specific conditions or a
combination of factors. For instance, vibriocin from V. comma (V. cholerae) was detected in
small amounts and highly controlled conditions [35]. Another study also showed that vibri-
ocin from V. comma (V. cholerae) inhibited other strains only when tested under a combi-
nation of exponential phase and aerobic conditions, while it did not inhibit the growth of
cells in stationary phase and anaerobic conditions [36]. Chatterjee and Maiti [42] reported
that vibriocin from V. cholerae was produced either at a logarithmic phase when the cul-
ture reaches at least 7 × ­107 or by using complex media. Farkas-Himsley and Seyfried [19]
also found out that Streptomycin-sensitive strains of V. comma could inhibit Streptomycin-
resistant mutants of V. comma only under aerobic conditions and not anaerobic conditions.
Despite several studies reporting that either Mitomycin C [34–36] or chloroform [6, 20, 25]
can be used to induce vibriocin production, later studies reported that both methods were
unsuccessful or needed further modifications [21, 41]. Chakrabarty et al. [29] reported that
using Whaba’s methods, none of the 69 V. cholerae strains he tested against each other was
able to inhibit the growth of other strains. Bacteriocin activity was also measurable only
under highly controlled conditions of citrate–phosphate buffer (0.5 to 0.7% each) and pH
(7.5 to 7.6) using cold shock methods. Under these highly controlled conditions, 61 strains
(~ 88%) were able to produce bacteriocins. This was supported by other authors in which
when standard methods were applied, approximately 41% (n = 83) of 201 non-agglutinable
V. cholerae strains were able to produce bacteriocin [33], however, in a later study by the
same author, using Chakrabarty et al. [29] method, approximately 80% (n = 334/423) of the
tested non-group O-1 V. cholera strains were able to produce vibriocin [32].
Other factors that might affect the extraction process were studied by McCall and
Sizemore [8]. The authors mentioned that bacteriocin production is related to the
plasmid,hence, species such as B. harveyi KN96 that do not harbor any plasmid were
expected to not produce any bacteriocins. Hoyt and Sizemore [7] also reported that vibri-
ocin from B. harveyi is mediated by a plasmid. Loss of inhibitory activity was also found
to be associated with loss of the bacteriocinogenic plasmid in V. vulnificus and V. cholera
[23]. Another factor in the study field was that, unlike gram-positive species, bacteriocin
production in gram-negative bacteria such as Vibrio spp. is considered a lethal event for
the bacteria [10, 19]. However, with improvement in methods, several studies showed that
bacteriocin can be produced by Vibrio spp. without killing the cells [8, 23, 39, 41].
Loss of activity has also been reported in many studies and could indirectly contribute
to the variation in vibriocin detection mentioned previously. Bhaskaran (1970) reported
that older cultures of V. cholerae lose their bacteriocin activity. However, Chakrabarty
et al. [29] demonstrated that under optimum conditions, V. cholerae strains were able to
consistently produce bacteriocin for at least 1 year of sub-culturing. Research in the fol-
lowing years revealed more factors that lead to these variations. For instance, McCall and
Sizemore [8] mentioned that BLIS activity could be lost due to high dilution factors and/or
long durations. Mitra et al. [22] tried to improve the extraction method and found out that
the addition of ammonium chloride instead of iodoacetic acid led to consistent production
of vibriocin. Hoyt and Sizemore [7] reported that the age of the SWYE agar cell density
­(107 to 1­ 08 cells per ml) was the main factor affecting bacteriocin detections and activity
of V. harveyi. Bacteriocin production by Vibrio sp. strain NM 10 was also reported to be
significantly affected by the culture time, temperature, salinity, and pH, while optimum
conditions were described as 24-h incubation at 20 °C in a media containing 50% seawater
[27]. The temperature also affected the ability of V. anguillarum to produce vibriocin as no

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zone of inhibition was observed at 4 °C [28]. Media was also reported to yield inconsist-
ent results as vibriocins production varied between different batches when using nutrient
broth [42]. Zai et al. [28] showed that the approach of detection also significantly affects
the ability to identify BLIS activity of 50 Vibrio spp. For instance, the stab-overlay method
(exposure to chloroform) was able to detect 20 vibriocin-producing strains which made up
40% of the total number of strains tested. However, only 12 vibriocin-producing strains
(24%) were detected using the agar well diffusion method in the same study. Moreover,
Balakrishnan et al. [13] reported the highly active V. parahaemolyticus BPRIST035 vibri-
ocin was released in low concentration and need highly specialized media such as Zobell
marine broth to maintain its activity. However, the authors also demonstrated how opti-
mization of various factors could improve the activity by up to 5 folds (490 and 2351 U/
mL). These factors were temperature, agitation speed, pH, glucose concentration, and pep-
tone concentration. Ecological factors have also been associated with vibriocin production
by environmental marine Vibrios [12]. The authors reported that free-living isolates (6%)
were more active than those associated with 1-μm particles (2%), 5-μm particles (2%), and
63-μm particles (2%).
Overall, the trends of vibriocin extraction and discovery clearly showed that with the
advances in science, such as the use of various buffers and chemicals to stabilize the vibri-
ocin activity, the extraction process was significantly improved. This also led to a shift
from using plate-based methods such as exposure to chloroform, to the utilization of whole
cells in a broth. These advancements are crucial for the industry in the future to be able to
produce vibriocin continuously at a large scale.

Classification and Morphology of Vibriocin

Unlike bacteriocins produced by lactic acid bacteria which have been classified based on
genetic, structural, and chemical properties [4], very few studies tried to classify or type
vibriocins. In terms of functional typing, only earlier studies investigated this aspect such
as Mitra et al. [22] who tested 1036 strains of agglutinable and inagglutinable Vibrio chol-
erae and reported 22 different patterns of bacteriocin production/spectrum with the most
abundant group (1A) containing 259 strains (25% of all tested strains). Chakrabarty et al.
[29] also studied a total of 425 strains of V. cholerae and found 11 different inhibition pat-
terns with the most abundant group (1A) containing 259 strains (19% of all tested strains).
Israil et al. [33] reported that 83 vibriocin-producing V. cholerae were grouped in 42 dif-
ferent patterns with the most abundant group (1) containing 34 strains (~ 41% of all tested
strains). The same author studied another 423 V. cholerae non-O1 strains and reported 16
different patterns of vibriocin production which were correlated to spatial and temporal
factors [32].
In terms of morphology, earlier studies described microscope images of bacteriocins
from Vibrio spp. as bacteriophage tail-like structures (Takeya et al., 1970). Their shape
was described in more detail as a two cylindrical or tubular structure that was hollow [42].
Other studies also supported this observation and described it as contracted or extended,
while its inner cores are hollow and could be as large as 10 nm [35, 42],Lang, 1968). The
ends of these vibriocin peptides were described as a distinct neck at one end and fibers/
sheets at the other [42],Takeya et al., 1970). These extended sheets could vary between 104
[42],Takeya et al., 1970) and 300 nm [42], while widths varied between 21 [42],Takeya
et al., 1970) and 33 [42].

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4486 Applied Biochemistry and Biotechnology (2022) 194:4477–4491

These studies indicated possible structural and functional diversity of vibriocins based
on the varying inhibition spectrum obtained from all the Vibrio spp. tested. The section
also highlights the need for more studies to classify vibriocins.

Structural and Functional Characterization of Vibriocin

The previous section summarized the differences in the inhibition spectrum which indi-
cated the functional diversity of vibriocin. These functional diversities could also be due
to structural differences; hence, this section discusses the structural and functional diver-
sity of vibriocins. Characterization of vibriocins showed diversity in their building blocks,
moieties, and chemical stability. In terms of structure, the majority of the characterized
vibriocins was reported to be proteinaceous as they were inhibited by proteolytic enzymes
such as Nagarse [41], trypsin [7, 13, 18, 27, 36, 39, 41], protease [7, 18, 28], papain [7],
proteinase K [13, 18, 24, 27, 28, 39], pepsin [13, 39], and pronase E [13, 39]. However,
other studies reported vibriocins that were deactivated by α-amylase [23] and lipase [23,
39]. Deactivation by α-amylase and lipase indicated carbohydrate and lipid moieties were
part of these vibriocins, respectively. In terms of the homogeneity of the building blocks,
Balakrishnan et al. [13] mentioned that vibriocins that are inactivated by SDS are not mono
peptides. The majority of the characterized vibriocins showed mono-peptide nature when
this aspect was investigated,however, Prasad et al. [39] showed that vibriocin from V. har-
veyi VIB 571 was inactivated by SDS. Other inhibitors reported included EDTA which
inhibited a bacteriocin protease isolated from V. harveyi [38]. Solvents were also reported
as inhibitors such as acetone, benzene, chloroform, dichloromethane, DMSO, ethyl acetate,
and hexane [13].
In terms of stability, vibriocins showed significant variations which could be related to
their structure. For instance, vibriocin produced by V. parahaemolyticus was stable even
autoclaving temperature (121 °C, 15 min) and was recommended for application in food
and medical microbiology [40]. Vibriocins from Vibrio sp. MMB2 strain with a molecular
weight of 16 and 32 kDa was also stable at a temperature up to 121 °C for 15 min as well
as chloroform vapor for up to 30 min [16]. However, vibriocin with a molecular weight of
32 kDa from V. harveyi VIB 571 strain was deactivated at a temperature ≥ 60 °C for 10 min
[39]. Very sensitive BLIS with a molecular weight of < 5 kDa were extracted from Vibrio
sp. NM 10 and inhibited by temperatures as low as 30 °C; however, the molecular was very
low and might just be short peptides. These variations were also observed in another study
where three bacteriocins with a molecular weight of 1.35–9 kDa showed different stability
in terms of temperature, pH, and enzymes [23].
Only one study investigated the hemolytic nature of vibriocin extracted from V. cholerae
El tor and it was found to be non-hemolytic and agglutination-positive in chicken cells
[20]. These studies showed that very little is known regarding the structure of vibriocins
compared to bacteriocins isolated from lactic acid bacteria which are well studied and have
multiple classes. Hence, more research is needed to determine the crystal structure of vibri-
ocins and classify them. This would aid in determining which industries or products could
benefit from vibriocins.

Inhibition Spectrum and Mechanism of Vibriocins

For vibriocin to be an ideal candidate for vibriosis control in aquaculture, it is important

that they possess a relatively narrow-spectrum activity against Vibrio spp. The reviewed

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Applied Biochemistry and Biotechnology (2022) 194:4477–4491 4487

articles showed that the inhibition spectrum of vibriocin varied from the expected narrow
spectrum such as inhibiting only related strain to board spectrum such as inhibiting 78%
of the tested bacterial strains belonging to 7 genera. A detailed list of Vibrio spp. and their
inhibition spectrum are summarized in Table 1. In sum, vibriocins were able to inhibit 41
bacterial species including 36 g-negative species, 18 Vibrio spp., and only 5 g-positive spe-
cies. This correspondence to 44% (n = 18/44) of the activity observed was against related
Vibrio spp. while only 11% (n = 5/41) were against Gram-positive strains. This showed
the expected species-specific action of bacteriocins such as vibriocins. However, in terms
of genera, vibriocins were able to inhibit 12 genera namely, Aeromonas, Enterobacter,
Escherichia, Flavobacterium, Pasteurella, Plesiomonas, Proteus, Pseudomonas, Shigella,
Salmonella, Serratia, and Yersinia. This showed a degree of broad-spectrum activity which
might be improved with further optimization research such as nano-encapsulation. These
improved vibriocins could be valuable for the food and aquaculture industries.
In terms of mechanism of action, vibriocin from V. cholerae (V. comma) was shown to
inhibit the target bacteria by primarily degrading the cell membrane. It also significantly
inhibited DNA and RNA synthesis and slightly decreased protein synthesis [36]. This is
supported by the fact mentioned previously that approximately 90% of vibriocin activities
reported in the literature were against Gram-negative strains. Vibriocin was found to dam-
age the cell membrane which leads to efflux of K + as well as inhibiting the uptake of pre-
cursor metabolites crucial for DNA and RNA synthesis. Vibriocin was also reported to be
most effective in the late G2 stage [42, 43]. This could be supported by the fact that vibri-
ocin from V. comma (V. cholerae) also stopped V. cholerae cells from growing exponen-
tially, while it did not inhibit the growth of cells in the stationary phase [36]. Another study
reported that vibriocin activity depended on the availability of a certain receptor at the
cell membrane, which is shared between the Vibrio genus and other enterobacteria Israil
et al. [33]. Chloramphenicol (5 pg/ml) was shown to inhibit the activity of vibriocins which
indicated that protein synthesis is essential for vibriocin action [42]. Thompson et al. [14]
reported that BLIS activity of V. alginolyticus was only detectable against live cells, while
BLIS activity of V. gazogenes was detected regardless. This could indicate that bacterioc-
ins production could correlate quorum sensing. Burks et al. [12] tried to correlate vibriocin
production with various known factors such as genetic or ecological diversity,however,
among the active environmental Vibrio strains, no clear patterns were observed. The activ-
ity of these isolates could not be correlated consistently with these factors, hence suggest-
ing a novel mechanism of actions among active isolates and a need for more research in
this aspect.

Conclusion

This review showed that Vibrio spp. could be used as a probiotics to control vibriosis; how-
ever, it is not recommended due to their opportunistic nature and pathogenesis. The use of
vibriocin has a huge potential to be used in aquaculture due to its narrow-spectrum activ-
ity mainly against gram-negative species and specifically against related strains. Vibriocins
were effective against many Vibrio spp. including highly pathogenic strains such as V. algi-
nolyticus, V. harveyi, and V. parahaemolyticus which supports the proposed hypothesis in
this review of using vibriocin as a targeted vibriosis control measure. V. cholerae showed
the highest inhibition range, inhibiting 13 different vibrio and non-vibrio species; how-
ever, it was also the most studied species. Hence, other Vibrio spp. could also have similar

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4488 Applied Biochemistry and Biotechnology (2022) 194:4477–4491

potential if studied more. Research throughout the years revealed the factors affecting the
extraction process and vibriocins now can be produced on large scales using whole-cell
culture. Structural and functional analysis showed that vibriocins are diverse and not well
studied. The review also showed that vibriocins exist as monomers made up of amino acids
only, as well as polymers containing carbohydrate and lipids moiety. These properties are
important as they can affect the extraction process as well as applications in aquaculture.

Author Contribution H. I.: conceptualization, methodology, and original draft writing; A. J.: methodol-
ogy and original draft writing; N. M.: review and editing of final draft and funding acquisition; L. A. R.:
resources; MA: review and editing of the final draft; A. F.: validation and Formal analysis.

Funding This work was supported by Talent and Publication Enhancement Research Grant (TAPE-RG) No.
55217 from University Malaysia Terengganu. We would like to thank the Centre for Research and Innova-
tion Management (CRIM) for their assistance in gathering the resources needed in writing this review.

Data Availability All the data and resources used have been mentioned in the manuscript and supplementary
table.

Declarations
Ethical Approval Not applicable.

Consent to Participate Not applicable.

Consent to Publish Not applicable.

Competing Interests The authors declare no competing interests.

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