BioNanoScience

https://doi.org/10.1007/s12668-024-01375-3

RESEARCH

A Study on the Antipathogenic Effects of Nanoemulsion Against

V. parahaemolyticus in Shrimp Aquaculture: Antibacterial
and Antibiofilm Activities
Jahangir Ahmed1 · Karthikeyan Ramalingam1

Accepted: 24 March 2024

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2024

Abstract
Antimicrobial resistance is a significant concern in aquaculture, prompting the exploration of nanoemulsions (NEs) with
a non-resistant mode of action as a safer and more effective alternative to antibiotics. In Asian countries, the culture of
Penaeus vannamei has faced growing concerns as V. parahaemolyticus (Vp) has been increasingly linked to shrimp diseases,
leading to huge economic losses. In response, stable ozonated oil-in-water NEs (NE-25, 26, 28, 29) were produced using a
microfluidization method and characterized by their polydispersity index and average droplet size (NE-25, 26, 28, 29), which
were found to 0.108, 0.251, 0.223, 0.173, and 229.7nm, 190.2nm, 201.8nm, and 145.8nm, respectively. The therapeutic
potential of NEs is tested against pathogenic strains having tdh and toxR virulence gene, which were isolated from diseased
shrimp (VP-S7 and VP-S14), these were found to be more resistant compared to commercial V. parahaemolyticus (MTCC-
451) strain. Our NEs confirmatory antibacterial tests include bacteriostatic, bactericidal, antibiofilm, adherence, live/dead
assays, and oxygen consumption rate (OCR) studies. All tested nanoemulsions showed effectiveness against planktonic and
biofilm stages of V. parahaemolyticus; NE-25 showed strong adherence inhibition, with results of VP-S7 (49.55%), VP-S14
(53.47%), and MTCC-451 (68.76%). The biofilm inhibition of NE-25 (55.97%) is comparable to inhibition with antibiotics
gentamicin (58.73%). The NE OCR results (1 × 107 cell/ml) compared to antibiotics treated and untreated, the fold reduc-
tion for NE-28 (0.2) is highly significant when compared with commercial antibiotics (0.6) and the growth control (1.0).
Therefore, using NEs, which possess high antibacterial properties, is a promising alternative for treating V. parahaemolyticus
compared to current antibiotics.

Keywords Biocidal alternative · Disease prevention · Nanotechnology · Oxygen consumption rate · Penaeus vannamei ·
Vibrio pathogens

1 Introduction shrimp. In 2018, 9.4 million tons of crustaceans were pro-

In Asian countries, aquaculture is the most important and
rapidly developing industry, dramatically increasing the
demand for expensive shrimp goods on the global market
[1]. Commercial shrimp production, which was dominated
by two species of penaeid shrimp in 2015, observed that
95% of the shrimp come from either the Penaeus monodon
(giant tiger shrimp) or the exotic P. vannamei (white-leg)
* Karthikeyan Ramalingam
karthikeyan.sls@crescent.education
1 lions of dollars worldwide yearly [6–8]. These Vibrionaceae
School of Life Sciences, B.S. Abdur Rahman Crescent
Institute of Science and Technology, Chennai, Tamil Nadu,
India
duced through aquaculture worldwide, producing USD 69.3
billion [2, 3]. Vibriosis is one of the most significant issues
caused by a group of bacteria faced by the crustacean indus-
try. Early mortality syndrome (EMS) occurs within 35 days
after stocking P. monodon in grow-out ponds, resulting in
severe mortality [4]. Vibriosis in India linked history back to
Vibrio spp., causing shell disease, white gut, and loose shell
syndrome in the coastal area [5]. In addition, common vibrio
illnesses caused by Vibrio spp. (Vibrio harveyi, V. campbel-
lii, and V. parahaemolyticus), luminous vibriosis (Vibrio fis-
cheri, V. harveyi, V. cholerae) produced in shrimp farms as
mortality led to 50–100% loss to the shrimp industry, bil-
family facultative anaerobic bacteria frequently cause total
mortality in prawn hatcheries and grow-out ponds [7].

Vol.:(0123456789)

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Following a disease outbreak and mass mortality on 2 Material and Methods

India’s east coast from October to November 2013, cultures
of V. parahaemolyticus were obtained from the haemolymph 2.1 Bacterial strains and Growth Conditions
of moribund shrimp. Independently, V. parahaemolyticus
strains cause mass mortalities of cultured shrimp by activat- The strain commercial V. parahaemolyticus (MTCC-451)
ing virulent genes like a transmembrane transcriptional regu- was obtained from the Microbial Type Culture Collection
lator gene (toxR), thermostable direct haemolysin (tdh), and (MTCC), Chandigarh, India. A shrimp disease-causing strain
Thermolabile haemolysin gene (tlh) expression of virulence (VP-S7 and VP-S14) was acquired from the Central Institute
factors [9–11]. Among bacterial lethal infections since 2009, of Brackishwater Aquaculture in Chennai (Tamil Naidu),
acute hepatopancreatic necrosis disease (AHPND), caused by India [28]. The sunflower (SF), castor oil (CA), and olive
Photorhabdus insect-related toxins (PirA/B), is a lethal illness oil (OL) are purchased from the local market in Tambaram,
that has risen in Southeast Asian nations [12, 13]. In such a Chennai, Tamil Naidu, India. The surfactant Polysorbate 60
case, various chemicals and antibiotics were used to quickly (Tween-60), polyethylene glycol dodecyl ether (Brij-30), and
control the bacterial infection. Antibiotic overuse in the aqua- co-surfactant cetylpyridinium chloride, agar, and tryptose
culture industry has resulted in momentous economic loss soya broth (TSB) were purchased from HI-media and Sisco
and the establishment of antibiotic-resistant bacterial strains Research Laboratories (SRL) Mumbai, India. The bacterial
[14, 15]. Therefore, finding novel treatment routes is a pre- cultures were preserved in TSB with 50% (v/v) glycerol
requisite for standard antibiotics treatment and guarantees at − 80°C. Inoculated stock mother cultures were cultivated
more sustainable growth for the aquaculture industry [16]. at 37°C in sterile, fresh broths containing 1.5% NaCl. Fresh
The NE’s physicochemical properties, including the inoculums were utilized in every experiment; the bacterium
oil phase, aqueous phase, and emulsifiers, determine how cultured optical density was measured at 600nm, which
NEs form and stabilize [17]. Because of their effective interprets a bacterial concentration of about ­107 CFU/mL.
encapsulation, solubilization, targeted distribution, and
bioavailability, food-grade NEs are increasingly focused in the
feed industry. The nanosized NEs are regarded as one of the
2.2 Production and Preparation of Nanoemulsions
crucial carriers for the maintenance release of phytocompounds
According to earlier studies, four nanoemulsions (NEs) were
[18, 19]. Typically, emulsion-based delivery safeguard from
synthesized and processed as oils in water (O/W) combina-
harmful environmental factors, improve product stability
tions [29]. Nanoemulsion composition was used for one set
during storage, and preserve their functional activity [20].
of combinations such as castor oil (25%) and olive oil (25%)
When lipophilic compounds are encapsulated within NEs,
with Polysorbate 60 (Tween-60–2.5%) as surfactant and
such as Docosahexaenoic acid [21], fish oil [22], beta-carotene
cetylpyridinium chloride (CPC-1%) as co-surfactant. The
[23], curcumin [24], etc., they have a more significant ability
sunflower oil (20%) used in another set of combinations,
to boost bioavailability. Due to their increased bioactivity and
such as NE-28 (Epigallocatechin-gallate-EGCG, 0.4%) and
widespread use in antibacterial, disinfectants, and antiseptics,
NE-29 (Taxifolin-TF, 0.5%) with Brij-30 (3%) as well as
NEs can also be utilized as an antimicrobial agent [25, 26].
CPC (0.5%) was used.
The anti-biofilm and anti-adherence feature of the NEs
The Microfluidizer-LM10 was used to emulsify all NEs
recommends that they may also help develop anticarcinogenic
(Microfluidics Int. Corporation, USA). The solutions were
agents [27].
mixed thoroughly using a magnetic stirrer, until samples
In vitro screening of NEs was performed parallel to
were mixed well with the oil, and the remaining sterile
understand the effect of the administrated dose on three
water ­(DH2O) was added gently to create a homogeneous,
vibrio pathogens’ growth. Our study focuses on developing
transparent solution. Finally, the solution was emulsified at
antibacterial (ozone-enriched oil used) NEs from phytocom-
20,000psi lb/in2, three times at room temperature (one set),
pounds such as epigallocatechin-gallate, and taxifolin, with
whereas another set was produced in the presence of ice
sunflower oil in this set and the next set of olive oil and
(phytocompounds). The Schematic representation is shown
castor oil. The antibacterial NEs synthesized using high-
in Fig. 1. Using a magnetic stirrer, the samples were mixed
pressure micro-fluidization (20,000 psi) techniques. The
well with the oil, and the remaining sterile water solution
oil combinations are said to be tested as a unique class of
was added gently to create a homogeneous, transparent
NEs, because of better antibacterial potential confirmed by
solution. The prepared nanoemulsion was further character-
MIC followed by MBC for further evaluation studies. The
ized by dynamic light scattering for size and zeta-potential
antibiofilm, anti-adherence, and live/dead studies were done
analysis.
by slanted glass coverslip (SGC) methods, and the oxygen
consumption rate was analyzed independently.

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Fig. 1  Schematic representation
of the synthesis of ozonated
oil nanoemulsion, charac-
terization (DLS), and antibac-
terial efficacy tests against V.
parahaemolyticus strains from
diseased shrimp (P. vannamei)
culture
2.3 Fourier Transform Infrared Spectroscopy
of Synthesized Nanoemulsions
The oils (castor, olive, and sunflower) nanoemulsions were
prepared, and FTIR analysis was used to examine whether
the active ingredients involved in the optimized nanoemul-
sion composition were compatible. The prepared nanoemul-
sion’s FTIR spectra were performed on a JASCO (JASCO
INFRARED SPECTRUM) FTIR-spectrophotometer. The
nanoemulsion’s FTIR spectra were scanned at A-399–4000
­cm−1 [30].
2.4 MIC and MBC Against V. Parahaemolyticus
Pathogens
The bacteriostatic and bactericidal approach used the micro-
dilution method in a microtiter (96-well) plate to assess the
antibacterial properties of produced nanoemulsions against
V. parahaemolyticus pathogenic strains. Briefly, 100µl of
(2 ×) TS-broth and 100µl of NEs from each group were
added to the initial well and orderly diluted up to the 12th
well. In the next step, 5µl of overnight active bacteria with
­107 CFU/ml of the mixed solution was added to each well,
except the negative (media) control. As a negative control,
sterile ultrapure water ­(UPH2O) containing medium without
bacteria and positive control (gentamicin) media with bacte-
ria in an identical volume were maintained, respectively. A
microtiter plate containing four distinct NE-treated V. para-
haemolyticus strains and an untreated control was incubated
at 37° for 24 h. The greatest dilutions that exhibit no bac-
terial growth were used to calculate the MIC, with the no
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visible turbidity acting as a growth inhibitor NEs well of the
microbes [31]. Following the incubation time, the concentra-
tions of bactericidal nanoemulsions were also determined in
the poor turbid and clear wells. To provide more detail, each
well was chosen, a 5µl solution was extracted, and doublet
spots of the suspension were placed on T-SB agar plates.
These plates were then incubated for 24 h at 37°C [29]. The
maximum suspension of NEs that resulted in a (99.9%) bac-
terial cell count, or the endpoint of the effective reductions
of the microbial population, was noted as the bactericidal
concentration in terms of the solution.
2.5 Slanted Coverslip Method for Biofilm
Quantification
For the slanted glass coverslip (SGC) method, a coverslip
(18 × 18 mm) was used to develop a biofilm. Because the
coverslip (18 mm × 18 mm) bulges roughly one-third of its
length above the level of the medium, the formation of a
biofilm could begin at the level of the medium on the upper
surface. After that, a consistent biofilm spread across the
entire coverslip developed both below (into the medium)
and above this level of the coverslip. To evaluate the biofilm
yield, overnight bacterial cultures containing ­107 CFU/ml
of different strains of V. parahaemolyticus (MTCC-451),
shrimp isolated (VP-S7 and VP-S14), were added to 2.5 ml
of media (T-SB) in 12-well microtiter plates with inserted
slanted glass coverslips. Every 12 h, the media containing
the suspended bacterial cells were removed, and an equiva-
lent volume of fresh sterile medium was added [32, 33].
After 72 h, the grown biofilm was treated with NEs in an

equal volume covering the biofilm completely in an aseptic
condition for a fixed period. After incubation, the glass cov-
erslips and the well plate with the biofilm-developed bacteria
were placed for 15 min in 2.5 ml of methanol in each well.
After, the well plate was emptied and left to dry naturally,
with and without glass coverslips. Each well was stained for
5 min with a 0.1% (w/v) crystal violet solution. The plates
were thoroughly cleaned of extra stains using wash bottles
in a controlled setting. Using glacial acetic acid, 33% (v/v),
the dye bound to adherent cells was removed from the well
plate with and without glass coverslips. Finally, the optical
densities of the resultant solutions (590nm) were measured
[34]. Under each experimental condition, three different tri-
als were conducted. The mean optical density of the negative
control was subtracted from the readings of all other samples
in each case.
2.6 Adherence Assay by Slanted Coverslip Method
The adherence inhibition properties of V. parahaemolyti-
cus pathogens grown on the slanted glass coverslip (SGC-
18 × 18mm) were performed according to earlier studies [33],
with little modifications. On selected bacteria, the bacte-
rial adherent behavior was conducted: V. parahaemolyticus
(MTCC-451) pathogens and two shrimp isolated (VP-S7 and
VP-S14). Based on predetermined results of MBC, NE-25,
NE-26, NE-28, and NE-29 were used for the anti-adherence
assay. In 12-well plates with slanted glass coverslips put at
their appropriate Sub-MBC dilutions for the MTCC and
shrimp isolate strains, overnight-grown bacterial cultures
containing ­107 cells/ml, were introduced. Slanted glass cov-
erslips were used to maintain a total volume of 2.5 ml in each
well, which included positive and negative (sterilized water)
controls [34]. For 1h, the plates were incubated at 37°C. Fol-
lowing the fixation procedure is the removing of the residual
medium once the incubation period is complete and using 5
ml of 100% methanol per well for 15 min. Following medium
emptying and air drying, adhering layers remained on the fixa-
tion at the tilted glass coverslip surface. The well was stained
for 5 min with 3ml/well of 0.1% (w/v) crystal violet. The wells
were uniformly washed using a water bottle to eliminate the
extra stains. After air drying, the coverslip was taken out and
transferred to new wells (microtiter 12-well plates), the num-
ber of labelled adhering cells was quantified, and 3mL of 33%
(v/v) glacial acetic acid was added to each well [35]. Each
experiment was performed in three independent sets for each
repeat to conclude the results. The bound bacteria’s solution
was measured using a multilabel-detector (Perkin Elmer) at
590nm. To subtract the reading from all experiment sets, the
­UPH2O (negative control) was used as a blank.
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2.7 Live/Dead Assay
Slanted glass coverslip (SGC) was used to build the bio-
films for 72 h, stained with the LIVE/DEAD-BacLight,
and then treated with ozone-enriched oil-based nanoemul-
sions (OZNEs). A multilabel-detector VICTOR X3 (Perkin
Elmer) was used to measure the fluorescence intensity [36].
The staining solution containing the components SYTO-9
was mixed in (0.1µl, SYTO-9 can enter all cells and is used
for assessing total cell counts), quantities from stock solu-
tions in each well and maintained in an aseptic condition
for a further 1 h [37]. The untreated bacterial cells in the
untreated wells were designated as controls and counted to
remove the absorbance readings of lives and dead cells. For
each experimental condition, three separate experiments
were conducted. In each case, the mean optical density of
the negative control was subtracted from all other sample
results recorded.
2.8 Oxygen Consumption Assay
The measurement of oxygen consumption rates (OCR) was
measured using a Clarke electrode-based oxygraph instru-
ment (Hansatech, Norfolk, UK) following the manufactur-
er’s instructions. The cells in the exponential growth phase
were subjected to centrifugation at 8000 rpm for 2–3 min
and then resuspended in fresh TS-broth with or without the
test compounds, such as NE-25, 26, 28, and 29, and antibi-
otics like gentamicin at a concentration of approximately
1 × ­107 cells. The obtained results were analyzed using ­O2
view software version 2.0, and the rates were expressed as
nanomoles of O ­ 2 consumed per 1 × ­107 cells/ml/min, based
on the findings of previous studies [38].
3 Statistical Analysis
To determine the significance level within each row, statisti-
cal analyses were reviewed using Dunnett’s multiple com-
parisons test., simple effects within the rows, and compara-
ble columns. The two-way and one-way ANOVA method
was used to analyze variance for experimental data, and
statistical significance was considered as P values < 0.05.
4 Results and Discussions
4.1 Nanoemulsion Production and Preparation
After passing the solution through Microf luidizer-
LM10, the visual appearance of the emulsions changed
BioNanoScience
noticeably, making them physically transparent and leav-
ing no turbidity behind. Under high vacuum pressure, the
combined phase (oil and water) solutions were treated in
the intake of a microfluidizer to produce emulsions with
fine nanodroplets as mentioned in Fig. 2.
4.2 Nanoemulsion Size and Zeta Potential
Determination
The nanoemulsions were emulsified with the micro-
fluidizer and tested for size and zeta potential (DLS-
Malvern Zetasizer Nano ZSP instrument) measurement.
Each NE employed for a dynamic light scattering (DLS)
instrument for zeta potential analysis (mV) as NE-25,
NE-26, NE-28, and NE-29 are 41.8mV, 49.1mV, 40.8mV,
and 49.1mV and PDI values of 0.108, 0.251, 0.223,
and 0.173. Dynamic light scattering size distribution
histogram predicted that the mean diameter size of
nanoemulsions would be calculated as an average of
the sizes [39]. The NE-25, NE-26, NE-28, and NE-29
showed distinct single peaks and particles of an average
size of 229.7nm, 190.2nm, 201.8nm, and 145.8nm,
shown in Fig. 2a–d. The produced nanoemulsions
quality was determined by the unique peak discovered
by DLS analysis [40]. The formulations suggested that
the successful preparation of the nanoemulsion at the
nanometres scale is shown by high-intensity dispersion
in smaller droplet size ranges.
Fig. 2  An analysis of synthe-
sized nanoemulsions’ mean
droplet size distribution was
carried out using a 25, b 26, c
28, and d 29 nanoemulsions. To
create the four nanoemulsions,
three emulsifications at 20,000
lb/in2 were performed using a
microfluidizer LM-10. DLS was
used to analyze the average size
distributions of all the nanoe-
mulsions
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4.3 Fourier Transform Infrared Spectroscopy
Analysis
The nanoemulsion of edible oils, including castor, olive,
and sunflower oils exhibits peaks in the FTIR spectrum. The
corresponding groups analyzed O–H stretch, C-H stretch,
C = O stretch, C = C stretch, C-O stretch, and C–Br stretch at
3336–3359, 2855–2926, 1460–1461, 1742–1744, 1638–1639,
and 586–626 cm − 1, represented all NEs (NE-25, 26, 28, 29)
(Fig. 2a–d). The O–H stretch of all NEs has a broad and strong
peak in the 3200–3555 cm − 1, indicating NE involvement
with the oil’s functional groups. The shift in peak towards
the lower wavenumber is due to the increased mass of the
molecule, indicating that the vibration frequency decreases as
the mass of a molecule increases [41]. The C-H stretch is also
observed in the range of 3333–3267 cm − 1, with strong and
sharp peaks, and in the case of all NEs, this stretch is observed
at an increased wavenumber of 2921 cm − 1. The FTIR spectra
of the loaded nanoemulsion reveal all the significant active
ingredient peaks, indicating no incompatibility between the
active and inactive ingredients. Therefore, it can be concluded
that all the characteristic peaks of studied NEs are related to
functional groups of edible oils involved in nanoformulations
[42, 43] mentioned in Fig. 3a–d.
4.4 MIC and MBC Determination
The antibacterial activities of the developed nanoemul-
sions were assessed using MIC and MBC. The lowest

Fig. 3  Fourier Transform
Infrared Spectra were obtained
for formulated nanoemulsions
containing castor oil (NE-25),
olive oil (NE-26), and sunflower
oil, with incorporated phyto-
compounds NE-28 (Epigallo-
catechin-gallate) and NE-29
(Taxifolin-TF)
concentration, at which no apparent growth of the test
pathogens was seen, was recorded as the MIC. The anti-
bacterial activity of all (four) developed nanoemulsions
was evaluated using the micro-dilution method [44] against
V. parahaemolyticus commercial (MTCC-451) and a path-
ogenic diseased shrimp strain (VP-S7 and VP-S14). All
synthesized (NE-25, 26 28, 29) NE efficacy was to exhibit
noteworthy antibacterial activity as determined by bacte-
riostatic and bactericidal investigations; however, NE-25
showed effectual and significant catalytic activity at high
dilution ranges against V. parahaemolyticus (MTCC-451,
VP-S14) (MIC-2:4 to 2:4096 and MBC- 1:2 to 1:2048) and
VP-S7 (MIC-2:4 to 2:1024; MBC- 1:2 to 1:512), shown in
Fig. 4  The bacteriostatic and bactericidal solutions were determined
at various dilutions against pathogenic strains of V. parahaemolyti-
cus. The different oil-based nanoemulsions efficacy tested against the
commercial strain of V. parahaemolyticus as well as diseased shrimp
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BioNanoScience
Fig. 4 a–c, strains of V. parahaemolyticus pathogens in the
bacteriostatic and bactericidal assay. The fraction dilution
(D) (1:10, 1:100, and 1:1000 D) of NEs also showed suc-
cessful inhibition against shrimp pathogenic strains and
commercial strains tested in Fig. 5a–c. Individually, castor
oil, olive, and sunflower (20%) were tested, and no killing
effects were noted against all the pathogens. The presence
of castor, olive, and sunflower oils can capsulate active
ingredients, improve antibacterial effects, and function as
nanoscale catalysis in nanodroplets. The potential use of
NEs incorporate bioactive components, as a practical and
effective method either to increase their physical stability
or to decrease their possible harmful sensory effects [45].
strains: a VP-MTCC-451; b VP-S7; c VP-S14); NE-25-castor oil
nanoemulsion; NE-26 olive oil nanoemulsion; NE-28, 29-sunflower
oil nanoemulsion
BioNanoScience

Fig. 5  Three fractional dilutions were utilized to check (D-1:10, VP-S14), our NEs showed successful inhibitions at various dilutions,
1:100, 1:1000) minimum inhibitory and bactericidal concentrations comparable to antibiotics (gentamicin): NE-25-(castor oil nanoe-
against three V. parahaemolyticus. Based on the a V. parahaemo- mulsion); NE-26 (olive oil nanoemulsion); NE-28, 29 (sunflower oil
lyticus (MTCC-451) and b, c diseased shrimp strains (VP-S7 and nanoemulsion)

4.5 Adherence Inhibition of Nanoemulsions
Four nanoemulsions were produced and tested by ant-
adherence assays, based on the results obtained from MBIC
dose inhibitory potential on strains of V. parahaemolyticus
from shrimp aquaculture. The adherence efficacy of nanoe-
mulsions on three strains of V. parahaemolyticus (MTC-
451) and two from shrimp (VP-S7; VP-S14), developing
cells to the glass surfaces of slanted glass coverslips, was
investigated. The outcomes of our results stated here the
adherence potential of NE-25 (68.76%) and gentamicin as
(69.70%) inhibition reported against the V. parahaemolyti-
cus (MTCC-451) when treated with 62.5ug/ml. The shrimp
obtained strains’ (VP-S7; VP-S14) adherence was reported
at (49.55 and 53.48%), and with antibiotics at (53.50;
54.39%) in Fig. 6a. The various dilutions (10, 100, 1000D)
were also utilized to know adherence inhibitions on all the
strains of V. parahaemolyticus grown here, which are shown
in Fig. 6b–d. Amongst all strains, the NE (NE-25)-treated
maximum elimination was found in MTCC-451, compared
to shrimp strains.
The clinically isolated strains may possess a higher
degree of resistance due to virulence factors (tlh, toxR) pres-
ently reported and the influential activity of these genes.
The VP-14 genome was discovered to carry all 39 virulence
factors listed in the Virulence Factors Database as T3SS1
(responsible for the cytotoxicity of host cells) [9, 28]. The
nanoemulsion is positively charge-bearing particles due to

Fig. 6  Adherence potential

of four nanoemulsions tested
against the V. parahaemo-
lyticus strains. The oil-based
nanoemulsions in effectiveness
tested against the commercial
strain (MTCC-451) as well as
diseased shrimp strains: a V.
parahaemolyticus (VP-S7 &
VP-S14). Additionally, nanoe-
mulsions dilutions were used
1:10, 1:100, and 1:1000 D to
test adherence inhibition poten-
tial against b V. parahaemolyti-
cus (MTCC-451), and shrimp
strains c, d VP-S7 and VP-S14,
respectively. NE (nanoemul-
sion); PC (positive control); GC
(growth control); D (Dilution);
VP (V. parahaemolyticus); S
(shrimp strains 7 and 14)

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 BioNanoScience

cetylpyridinium chloride (CPC). The addition of CPC (cati-
onic halogen) into the nanoemulsion mixture can enhance
antibacterial potential [46]. Additionally, nanoemulsions
having nano-droplet size can penetrate the biofilm matrix
and interact with bacteria embedded within it. This can dis-
rupt the matrix and weaken the biofilm’s structural integrity,
decreasing adherence to the glass surface [47]. Extracellular
curli fibrils, heteropolymeric filaments of major, and minor
subunits produced by many bacteria, including V. para-
haemolyticus, have been linked to adhesion, biofilm forma-
tion, and virulence. However, the additional components of
planktonic form a complex extracellular matrix that supports
and protects the bacterial community [48]. It is important
to note that the efficacy of nanoemulsions in reducing bac-
terial biofilm adherence on glass surfaces can be affected
by various factors, including the specific biofilm-forming
bacteria, the composition and concentration of the nanoe-
mulsion, contact time, and application method. Resistance
to currently available antibiotics and their spread into the
food chain and environment necessitates the development of
safe and effective alternative methods against this bacterium.
4.6 Nanoemulsions Effect on Biofilm
Quantifications
Antibiotic-resistant gene associations in bacteria promote
the formation of a matrix through the production of vari-
ous surface components and virulence factors [49, 50]. The
effect of edible oils NE, including antibiotics, on the biofilm
of V. parahaemolyticus, was quantitatively evaluated by the
following treatments. Quantitative analysis of the biofilm
assay showed biofilm inhibition in the presence of castor
and olive oil nanoemulsion (NE-25, 26), phytocompound
sunflower oil nanoemulsions as (NE-28-Epigallocatchin gal-
late 0.5%, and NE-29-Taxifolin 0.4%), and nanoemulsions
inhibitions observed dose-dependent manner. Minimum
biofilm inhibition concentration (MBIC) values for NE-25
and 26 are 62.5ug/ml, and NE-28 and 29 were found to be
125 μg/ml, respectively. Therefore, results showed higher
inhibition in NE-25 (55.96%) and less inhibition in NE-29
(44.36%), including gentamicin (58.28%) against V. para-
haemolyticus (MTCC-451). The shrimp obtained pathogenic
strains with higher inhibition in NE-25 (51.74%), less inhi-
bition in NE-29 (44.04%), and gentamicin (54.91%). The
VP-S14 maximum biofilm inhibition (52.73%), lowest in
NE-28 (41.64%), and with gentamicin (54.28%), respec-
tively shown in Fig. 7a. Furthermore, the biofilm was also
quantified at various dilutions (D) against V. parahaemo-
lyticus (MTCC-451) as well as shrimp obtained strains
(CIBA-VP-S7; VP-S14) (1:10; 1:50; 1:1000D) showed in
shown in Fig. 7b–d; and in each dilution, inhibition was
also quantified. The study found that increasing the dilution
of nanoemulsion resulted in a decrease in its effectiveness
in inhibiting the biofilm formation against V. parahaemo-
lyticus, thus suggesting that NE-25 has a stronger ability
to inhibit biofilm formation at higher concentrations. The
biofilm of V. parahaemolyticus was treated with NEs and
stained with crystal violet as quantitative data was esti-
mated. These results demonstrated that a novel castor, olive
oil, and phytocompounds loaded poorly with water-soluble

Fig. 7  Percentage of biofilm

formation determined against
three strains of V. parahaemo-
lyticus. The different oil-based
nanoemulsions efficacy tested
against commercial as well as
the shrimp isolated pathogenic
strains: a V. parahaemolyticus
(MTCC-451) and (VP-S7 and
VP-S14). Further, dilutions (D)
as 1:50, 1:100, 1:1000, b rep-
resented V. parahaemolyticus
(MTCC-451), and c, d diseased
shrimp strains. NE-25-(castor
oil nanoemulsion); NE-26 (olive
oil nanoemulsion); NE-28 &
29 (sunflower oil NEs with
phytocompounds). S1-(VP-
MTCC-451), S-shrimp strains
(S7 & 14); D1 (1:10 dilution),
D2 (1:100 dilution), D3 (1:1000
dilution)

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BioNanoScience

nanoemulsion could improve this agent’s aqueous solubility
and antimicrobial activity against V. parahaemolyticus in
vitro. So nanoemulsion inhibits effect on biofilm formation
and enhances damage to the structure of biofilm. Similarly,
earlier studies reported nanoemulsion biofilm effectuality
against the Streptococcus mutans [29]. The MIC test is used
to determine planktonic bacteria’s antimicrobial sensitivity.
It is the lowest antibiotic concentration required to halt bac-
terial growth under certain conditions. Unlike planktonic
bacteria cultivated in liquid media, bacterial biofilms can
withstand antibiotic dosages up to 1000 times the MIC. The
presence of extracellular polysaccharides, the spatial organi-
zation of the community, and the physiological changes
caused by biofilm formation all help bacteria survive. As a
result, the MIC is not proportional to the minimal biofilm
elimination concentration (MBEC), as the reason biofilm
changes is specific to environmental conditions [51].
4.7 Quantifications of Dead Cell Populations
The biofilm that had grown was treated with NE-25 to NE-29
followed treatment; the cells were stained with LIVE/DEAD
BacLight TM Bacterial Viability Kit (Molecular Probes Inc.)
After the treatment, the well was rinsed and washed once
with PBS (1 ×) solutions. We utilized the minimum biofilm
inhibition concentration (MBIC) values for NE-25 and 26,
62.5ug/ml, and NE-28 and 29 were found to be 125 μg/ml,
to quantify the dead cell numbers, respectively. The results
determined that NE-25 (48.68%) showed the dead popula-
tions against V. parahaemolyticus (MTCC-451) and standard
antibiotics gentamicin-54.06%. The diseased shrimp strains
VP-S7 and VP-S14 at (43.19 and 44.92%), whereas anti-
biotics at (48.28 and 52.09%) are shown in Fig. 8a respec-
tively. The dilutions (1:10, 1:100, and 1:1000 D) results also
assessed the reduction efficiency in dead cells against V. par-
ahaemolyticus strains tested here, and successful inhibitions
were noted, as shown in Fig. 8b–d. The separate mechanisms
of action of nanoemulsions (EGCG; TF) and CPC may be
operating to reduce biofilm formation on the glass coverslip.
The positively charged emulsion may remain attached to the
biofilm for a longer time than individual phytocompounds
do and, therefore, can prevent the formation of furthermore
biofilm. Additionally, after treatment, the fractional cell was
stained with SYTO 9 and quantified by the multimode reader
[27]. We used SYTO 9 to enter into cells continuously while
entering quantified cells by fluorescence lost within 0.1ns
[52]. The SYTO9 is a hydrophobic cell-permeant nucleic
acid dye that exhibits a significant fluorescence enhancement
after penetrating the cell’s intact membrane and binding to
the nucleic acids. The SYTO9 can be permeable as (i) it can
enter both the living and dead cells. (ii) It penetrates dead
cells more than live cells due to their impaired membrane.
(iii) It has greater penetration frequently to dead cells than
live cells due to their larger membrane pores [37].
4.8 Oxygen Consumption Rate Determination
The permeability and efflux of drugs are significantly influ-
enced by the bacterial membrane, which is highly depend-
ent on the active membrane potential. Previous research

Fig. 8  Assessment of percent-

age of dead cells of pathogens
treated with ozonated oil
nanoemulsions and gentamicin
(PC) by employing already
determined concentrations as
on pre-formed biofilms. The
nanoemulsion potential tested
against commercially purchased
V. parahaemolyticus (MTCC-
451) as well as diseased shrimp
strains (VP-S7 and VP-S14)
a) combine graph of all three
strains and dilutions employed
as 1:10, 1:100, 1:1000D. The b
V. parahaemolyticus (MTCC-
451); c, d shrimp isolated
VP-S7 and VP-S14 strains—D,
dilution; PC, positive control-
(gentamicin); NE, nanoemul-
sion

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 BioNanoScience

Fig. 9  Nanoemulsion OCR

effect against V. parahaemolyti-
cus a, b (MTCC-451); c, d (VP-
S7); and e, f (VP-S14) culture
were grown for 6-8h and treated
with the desired concentrations
of NEs and PC (gentamicin),
by maintaining 10.7cells/ml
with OD at 600nm. The graph
shows OCR affected; signifying
cell death compared to growth
control. The average values
from each experiment were
plotted after being repeated
three times. The values were
presented as means ± SD, with
n = 3, compared to untreated
cells, *P < 0.05; ***P < 0.001.
MTCC, microbial type of
culture collection; Ab, antibiot-
ics; NE, nanoemulsion; OCR,
oxygen consumption rate; VP,
V. parahaemolyticus; S, shrimp
strains-7 And 14; growth
control (Vp-MTCC, VP-S7, and
VP-S14)

by Kosuru et al., [53] tested the oxygen consumption rate
against P. aeruginosa using gallic acid and tannic acid. In
this study, nanoemulsions (NE-25, 26), including phyto-
compounds (NE28-EGCG and NE29-TF) used by Ahmed
et al., [54] at optimized concentrations of 125μg/ml, were
employed as a therapeutic treatment against V. parahaemo-
lyticus strains. The current antibiotic at a concentration of
175μg/ml impacted the OCR in a commercial strain of VP
(MTCC-451). It resulted in a reduction of 0.58-fold (Fig. 9a),
indicating a lower rate than the growth control (1.0). The
increase in the nanomoles of oxygen/ml/min was recorded
(Fig. 9b). Meanwhile, NE-25, NE-28, and NE-29 exhibited
reductions of 0.38-, 0.16-, and 0.31-fold. Interestingly, these
values are 0.2-, 0.42-, and 0.27-fold lower than antibiotics
(0.58), as shown in Fig. 9a. Consequently, the reduction in
the OCR observed in the cells was significantly increased
when measured in terms of nanomoles of oxygen/ml/min
(Fig. 9b). The results were observed with NE-25, and NE-29
were tested against VP-S7. These NEs exhibited reductions
of 0.23 and 0.20, which were 0.26- and 0.29-fold lower than
the reduction caused by antibiotics, which measured at 0.49
(Fig. 9c). Furthermore, the impact of OCR on cell death
was evident as there was an increase in the measurement of
nanomoles of oxygen/milliliter/minute (Fig. 9d). In the case
of VP-S14, NE-25, and NE-26 demonstrated reductions of
0.12 and 0.22, and antibiotics resulted in a reduction of 0.62.
These values indicate that NE-25 and NE-26 were 0.5- and
0.40-fold lesser than the antibiotics, as depicted in Fig. 9e.
Moreover, the impact of OCR on cell death was noticeable,
as there was an increase in the measurement of nanomoles
of oxygen/milliliter/minute, as recorded in Fig. 9f. Hence,
all tested NEs against V. parahaemolyticus from diseased
shrimp exhibited a notable reduction in cell count at a con-
centration of 125μg/ml. Additionally, there was an observed
increase in the measurement of nanomoles of oxygen/mil-
liliter/minute compared to the concentration of antibiotics
at 175μg/ml and the growth control (0-μg/ml). The NEs
with high-pressure nanodroplet particles, when hitting with
pathogen surfaces, generate a bombing effect and release
a substantial amount of energy that disrupts and kills the
pathogens [55].
5 Conclusion
A recent study produced four nanoemulsions using a high-
pressure homogenization technique. The nanoemulsion’s
zeta potential, droplet sizes, and PDI values indicate the

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BioNanoScience
uniformity and stability of the formulations. To assess
the impact of ozone-enriched nanoemulsions (OZNEs) on
V. parahaemolyticus strains from diseased shrimp were
employed, each exhibiting varying degrees of sensitivity to
gentamicin. Remarkably, the NE-25 showed strong adher-
ence inhibition, with results of VP-S7 (49.55%), VP-S14
(53.47%), and MTCC-451 (68.76%). In contrast, the bio-
film inhibition assays revealed that NE-25 and NE-26 at a
concentration of 62.5 μg/ml, as well as NE-28 and NE-29
at a concentration of 125 μg/ml, exhibited independent
efficacy against all strains of V. parahaemolyticus. We
utilized the minimum biofilm inhibition concentration
(MBIC) values for NE-25 and 26 (62.5μg/ml) and NE-28
and 29 (125 μg/ml) to quantify the dead cell. The results
determined that NE-25 (48.68%) showed the dead cells of
V. parahaemolyticus (MTCC-451) and current antibiotics
gentamicin-54.06%. The diseased shrimp strains VP-S7 and
VP-S14 were at 43.19 and 44.92%), whereas current antibi-
otics were at 48.28 and 52.09%. For the OCR assays, opti-
mized concentrations of NEs, including phytocompounds
NEs (125 μg/ml) and antibiotics gentamicin-175 μg/ml,
were utilized for treatments. The better fold reduction for
Vp-MTCC strain, NE-25 and NE-28 (0.42; 0.2), indicat-
ing lower than the growth control (1.0), is highly signifi-
cant when compared with the antibiotics (0.58). Similarly,
against VP-S7, the reduction was noted as NE-29 was 0.29-
fold lower than the reduction caused by antibiotics, which
measured at 0.49, whereas VP-S14, NE-25, and NE-26 dem-
onstrated reductions as 0.12 and 0.22, compared to antibi-
otics 0.62 and growth control (1.0). Hence, all tested NEs
against V. parahaemolyticus from diseased shrimp exhib-
ited a notable reduction in cell count at a concentration of
125μg/ml. Additionally, an increase in the measurement of
nanomoles of oxygen/milliliter/minute was observed, com-
pared to the concentration of antibiotics at 175μg/ml and the
growth control (0-μg/ml). Hence, the results from all tests
affirm that NEs demonstrate comparable and, in some cases,
even superior antibacterial effectiveness compared to cur-
rent antibiotics. Therefore, NEs are an extremely promising
substitute in combatting V. parahaemolyticus strains, which
cause diseases in shrimp aquaculture.
Acknowledgements We acknowledge B. S. Abdur Rahman Crescent
Institute of Science and Technology for providing the facility to per-
form this research; and Dr. Sneha Unnikrishnan for data curation, for-
mal analysis, and investigation. We thank Dr. Soumen Bera and Mr
Md. Aashique for their help in doing OCR studies using the Clarke
electrode-polygraph instrument.
Author Contribution Jahangir Ahmed: All experimental work, data
curation, formal analysis, investigation, methodology, and writing of
the original draft. Dr. Karthikeyan Ramalingam: conceptualization,
supervision; validation; writing, review, and editing.
Funding This work was supported by the Indian Council of Medical
Research [Project ID: 2020–4964].
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Data Availability The datasets generated during and/or analyzed dur-
ing the current study are available from the corresponding author upon
reasonable request.
Code Availability Not applicable.
Declarations
Competing interests The authors declare no competing interests.
Research Involving Humans and Animals Statement None.
Informed Consent Not applicable.
Ethics Approval Not applicable.
Consent to Participate Not applicable.
Consent for Publication All authors read and agreed to the manuscript
for publication.
Conflict of interest None.
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