Evaluating the Antagonistic Activity of Lactic Acid Bacteria in Cadaverine Production by Vibrio Strains during Co-Culture

: Vibrio cholerae and Vibrio parahaemolyticus are common pathogens linked to human gastroenteritis, particularly in seafood like shrimp. This study investigated the impact of lactic acid bacteria on V. cholerae and V. parahaemolyticus regarding the production of cadaverine, a concerning compound. V. cholerae NCCP 13589 and V. parahaemolyticus ATCC 27969 were significant producers of amines in experiments conducted using white-leg shrimp ( Litopenaeus vannamei ) and lysine decarboxylase broth. Notably, the Lactiplantibacillus plantarum NCIMB 6105 and Leuconostoc mesenteroides ATCC 10830 lactic acid bacteria strains demonstrated a pronounced antagonistic effect on the production of biogenic amines by these food-borne pathogenic bacteria. The presence of lactic acid bacteria led to a substantial reduction in cadaverine production in the lysine decarboxylase broth and shrimp extract. The co-culture of two lactobacilli species reduced the cadaverine production in V. cholerae and V. parahaemolyticus by approximately 77 and 80%, respectively. Consequently, the favorable influence of lactic acid bacteria in curbing cadaverine production by food-borne pathogens presents clear advantages for the food industry. Thus, effectively managing these pathogens could prove pivotal in controlling the biogenic amine levels in shrimp.


Introduction
Biogenic amines (BAs) are a group of low-molecular-weight organic bases produced and broken down as components of cellular metabolic activity in microorganisms, plants, and animals [1].They are associated with diverse cellular processes, including cell growth, differentiation, and receptor function [2].The significance of biogenic amines is particularly notable owing to their possible toxicity when accumulated at high concentrations, particularly during histamine consumption in fish and related seafood products [3].
Most biogenic amines (including cadaverine) are characterized by an unpleasant odor, toxicity, and carcinogenic properties.Exposure to these compounds can lead to a range of adverse health effects, including dizziness, fainting, headache, heart palpitations, mucosal burns, and eye irritation.Although putrescine and cadaverine are far less toxic, their interactions with amine oxidases may slow down metabolism and increase histamine toxicity [4].Putrescine and cadaverine, which are commonly found in decomposed fish and shellfish, may generate introsopyrrolidine and nitrosopiperidine N-nitroso compounds, respectively [5].Putrescine and cadaverine are commonly found in various shrimp products, whereas the histamine levels tend to be lower than those in shellfish products [4].
Vibrio cholerae and Vibrio parahaemolyticus are halophilic, rod-shaped, Gram-negative bacteria that are ubiquitously found in marine environments.They are globally recognized as the primary triggers of foodborne disease outbreaks associated with seafood consumption [6].These bacteria are commonly found in aquaculture [7].Vibrio species such as V. cholerae and V. parahaemolyticus are significant contributors to acute gastroenteritis outbreaks in humans worldwide, often linked to the ingestion of raw or inadequately cooked seafood, such as shrimp.An increased demand for such products is particularly prevalent in Asian countries and the United States [8,9].
Pathogenic microorganisms such as Vibrio spp.cause one of the diseases that frequently affect shrimp (especially Litopenaeus vannamei) aquaculture [10].Vibrio sp. is one of the most common bacteria in the aquaculture industry, where it can cause widespread mortality in shrimp aquaculture ponds [11].This presents a potential public health hazard and underscores the necessity for measures to reduce the bacterial presence in shrimp [12].The Vibrio and Flavobacterium genera isolated from fish can produce cadaverine and putrescine [13].Bacteria capable of amine formation, identified in fish and shrimp, were categorized as putrescine-and cadaverine-forming, with no detection of histamine-forming bacteria [13].An analysis of the genome sequences of several Vibrio strains revealed genes encoding carboxyspermedine decarboxylase, diaminopimelate decarboxylase, ornithine decarboxylase, and lysine decarboxylase [14].
Probiotics were initially studied as a countermeasure against antibiotic resistance [15].They can curb the growth of V. parahaemolyticus in in vitro co-cultures [16] and in vivo mouse models [17].Lpb.plantarum strains isolated from shrimp intestines exhibit inhibitory effects against a wide range of Gram-positive and Gram-negative pathogenic bacteria, including Vibrio harveyi and Aeromonas hydrophila [18].Leuconostoc mesenteroides is a lactic acid bacterium isolated from the intestines of tilapia fish and inhibits the pathogenic bacteria V. harveyi and Mycobacterium marinum [19].These examples illustrate the main potential of lactic acid bacteria in counteracting pathogens before categorizing them as probiotics or beneficial organisms.
Current studies lack clarity on how lactic acid bacteria interact with foodborne pathogenic bacteria, specifically in terms of cadaverine and other amines' production in living organisms.It is crucial to investigate specific strains relevant to the food industry given that biogenic amine formation seems to vary depending on the bacterial strain rather than on the species.Thus, this study aimed to elucidate the effects of lactic acid bacteria on cadaverine production by foodborne pathogenic bacteria.

Strains and Growth Conditions
Seven lactic acid bacteria strains were selected to screen for activity against cadaverineproducing Vibrio strains: Lpb.plantarum NCIMB 6105, Le. mesenteroides ATCC 10830, Bacillus cereus ATCC 13061 (used as a negative control), V. parahaemolyticus ATCC 27969, Lactobacillus brevis ATCC 8287, Lactobacillus curvatus ATCC 25601, Lactobacillus sakei subsp.sakei ATCC 15521, Lactococcus lactis subsp.lactis ATCC 19435, and Weissella confusa ATCC 10881.V. cholerae NCCP 13589 was acquired from the National Culture Collection for Pathogens (Cheongju, Republic of Korea).Lpb.plantarum NCIMB 6105 was purchased from the National Collection of Industrial Food and Marine Bacteria (Aberdeen, UK).The remaining bacterial strains were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Cultivation of Vibrio Strains
White-leg shrimp (Litopenaeus vannamei) were procured from a local supermarket in Ansung, Republic of Korea.A 100 g portion of shrimp was mixed with 500 mL of sterile water and ground into a paste.The shrimp paste was filtered two to three times using a cotton cloth to eliminate impurities and was further clarified by centrifugation (5000× g for 5 min at 4 • C).The resulting supernatant was filtered using a 0.22 µm MCE membrane filter (Millipore, Billerica, MA, USA).
In a separate process, lactic acid bacteria and Vibrio strains were individually propagated in their respective broths and incubated at 37 • C for 24 h.Then, 1 mL of each culture was centrifuged at 5000× g for 10 min at 4 • C to collect the cell pellets.The collected cells were washed twice with phosphate buffer (PB, pH 7.2) and subsequently resuspended in 1 mL of prepared shrimp extract (ShE).These suspensions were stored at −20 • C until further use.

Screening of Lactic Acid Bacteria against Cadaverine-Producing Vibrio Strains
The antimicrobial activity of lactic acid bacteria was screened using a modified colony overlay method based on the protocol outlined in [20].Briefly, 5 µL of an overnight lactic acid bacteria culture was spotted onto a fresh MRS agar plate and incubated at 37 • C for 24 h.Subsequently, the Petri plates (diameter of 90 mm) were overlaid with 5 mL of thiosulfate-citrate-bile salts-sucrose (Difco) soft agar (containing 0.5% agar) pre-mixed with 0.5 mL Vibrio culture.The presence of an inhibition zone (a clear circular area with a diameter of 10 mm or more, measured with a ruler), extending horizontally from the edge of the lactic acid bacteria spot after 24 h incubation at 37 • C, was regarded as an indicator of positive inhibition.

Co-Culturing of Vibrio spp. and Lactic Acid Bacteria
This study aimed to investigate the antagonistic effects of lactic acid bacteria on V. cholerae NCCP 13589 and V. parahaemolyticus ATCC 27969 using co-culture experiments.Lactic acid bacteria and Vibrio strains were cultured separately in broth media and incubated at 37 • C for 24 h.One mL of each incubated culture was centrifuged at 5000× g for 10 min at 4 • C to collect the cell pellets.The cells were washed twice with PB and re-suspended in 1 mL of decarboxylase medium.Each resuspended culture was inoculated into 20 mL of lysine decarboxylase broth media individually and in co-culture and incubated at 37 • C under anaerobic conditions for 48 h.The pH of the medium was adjusted to 6.5, and anaerobic conditions were maintained using an anaerobic chamber.
The culture conditions were based on the optimal growth temperature and time according to the handling information provided by the NCCP and ATCC for the individual strains.Additionally, to create conditions that maximized the cadaverine production by the Vibrio strains, the pH of the medium was adjusted to 6.5.
One mL aliquots of monoculture and co-culture suspensions were promptly collected to analyze the cadaverine production and assess the cell growth of the Vibrio strains after 4, 12, 24, and 48 h of the decarboxylation reaction period.These collected suspensions were centrifuged at 5000× g for 10 min at 4 • C. The resulting supernatant and cell pellet were used for subsequent experiments.

Quantitative Analysis of Cadaverine Using High-Performance Liquid Chromatography (HPLC)
The quantification of cadaverine by HPLC involved the centrifugation of samples of broth culture media at 26,000× g for 8 min at 4 • C, followed by filtration through a membrane (regenerated cellulose, 0.45 µm pore size) and derivatization with O-phthaldialdehyde.Samples were injected into an HPLC system equipped with a Waters 600s controller (Waters, Milford, MA, USA), Waters 474 scanning fluorescence detector (Waters, Milford, MA, USA), and Clarity Lite (Petrzilkova, Prague, Czech Republic).Fluorescence was detected at excitation and emission wavelengths of 330 and 440 nm, respectively.The molecules were separated by gradient elution using Solvent A (100 mM acetate buffer, pH 5.8) and Solvent B (100% acetonitrile) on a Nova-Pak C18 column (Waters, Milford, MA, USA) (4 µm, 3.9 mm × 150 mm).The gradient elution was as follows: 0-10 min, 40% B (isocratic); 10-11 min, 40-60% B (linear gradient); and 11-22 min, 60-65% B linear gradient.The flow rate was set at 1 mL/min, and the column temperature was maintained at 35 • C. The retention time of cadaverine was 17 min.The quantification of cadaverine-producing Vibrio strains and lactic acid bacteria cells was performed using RT-qPCR.After the decarboxylation reaction, the liquid cultures were subjected to centrifugation at 5000× g for 10 min at 4 • C. Subsequently, DNA was extracted from the harvested cells using a Qiagen DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA), following the manufacturer's guidelines.The extracted DNA samples were used as templates for RT-qPCR analysis using an Applied Biosystems StepOnePlus Real-Time PCR System and the obtained data were analyzed using the StepOnePlus Software v2.0 (Applied Biosystems, Foster City, CA, USA).For the qPCR reaction, a reaction mixture of 20 µL was prepared, consisting of 10 µL BIOFACT™ 2X Real-Time PCR Master Mix (for SYBR Green I; BIOFACT, Daejeon, Republic of Korea), 1 µL of 10 pmol/µL of each primer, 3 µL of DNA template, and 5 µL of PCR-grade distilled water.The thermal cycling conditions included a polymerase pre-activation step at 95 • C for 15 min, followed by 40 cycles of denaturation at 95 • C for 15 s, annealing at 60 • C for 20 s, and extension at 72 • C for 30 s. Melting curve analysis was performed to verify the specificity of the qPCR products.The Ct values were automatically determined using this instrument.No template controls were included for the target genes in the PCR runs to account for potential contamination.All samples were analyzed in triplicate.
Standard curves were established for each strain based on the cycle threshold (Ct) values of serial dilutions of a 24 h enrichment culture.Briefly, DNA was extracted using a Qiagen DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA).Subsequently, the Ct values were plotted against the logarithms of the cell numbers to generate standard curves.Viable cell counts were determined by plating 0.1 mL of a 10 −7 dilution of each culture onto LB agar for Vibrio strains and MRS agar for lactic acid bacteria strains in triplicate.The resulting colonies were counted after incubation at 37 • C for 24 h [21,22].
Standard curves were constructed to quantify the growth of V. cholerae NCCP 13589, V. parahaemolyticus ATCC 27969, Lpb.plantarum NCIMB 6105, and Le.mesenteroides ATCC 10830 using species-specific primers.For cadaverine-producing V. cholerae, a primer pair (Vc_lys_F and Vc_lys_R) was designed to amplify a 180 bp region of its ldc gene, which served as the amplification target.For V. parahaemolyticus ATCC 27969, a specific primer pair (irgB_F and irgB_R) was used to amplify a 369 bp region of the iron-regulated virulence regulatory gene [23].For Lpb. plantarum NCIMB 6105, a species-specific primer pair (LplF and LplR) was used to amplify a 313 bp region of the cadmium-manganese transport ATPase gene (AF136521) [24].Similarly, for Le.mesenteroides ATCC 10830, a specific primer pair (LmeF and LmeR) was used to amplify a 358 bp region of the alcohol acetaldehyde dehydrogenase gene (AY804189) [24].The primer sequences used for RT-qPCR analysis are listed in Table 1.

Screening of Lactic Acid Bacteria against Cadaverine-Producing Vibrio Strains
The principle of competitive exclusion, whereby beneficial probiotic strains overpower and replace harmful pathogens, is a crucial and beneficial phenomenon that has been widely recorded across various vertebrate groups, including humans, poultry, and pigs [25].The antimicrobial activity of the lactic acid bacteria against Vibrio strains was screened using a colony overlay method (Table 2).All seven lactic acid bacteria strains tested were active against the cadaverine-producing Vibrio strains, except for Lc.lactis subsp.lactis ATCC 19435 and W. confusa ATCC 10881, which showed no consistent activity.Seven of the tested lactic acid bacteria strains produced antimicrobial substances that were active against at least one Vibrio strain.Five lactic acid bacteria strains (Lb.brevis ATCC 8287, Lb. curvatus ATCC 25601, Lpb.plantarum NCIMB 6105, Lb. sakei subsp.sakei ATCC 15521, and Le.mesenteroides ATCC 10830) were active against both Vibrio strains.Among the seven lactic acid bacteria strains tested, Lpb.plantarum NCIMB 6105 and Le.mesenteroides ATCC 10830 showed high antibacterial activity against V. cholerae NCCP 13589 and V. parahaemolyticus ATCC 27969.Lpb.plantarum NCIMB 6105 strongly inhibited V. parahaemolyticus ATCC 27969 compared with V. cholerae NCCP 13589.Le. mesenteroides ATCC 10830 strongly inhibited V. cholerae NCCP 13589 compared with V. parahaemolyticus ATCC 27969.Lpb.plantarum NCIMB 6105 and Le.mesenteroides ATCC 10830 were selected for their strong antimicrobial effects against V. cholerae NCCP 13589 and V. parahaemolyticus ATCC 27969 and were tested in co-culture experiments with cadaverine-producing-Vibrio strains.

Cadaverine Production and Bacterial Cell Growth in Lysine Decarboxylase Broth
The effects of the lactic acid bacteria and Vibrio strains on cadaverine production when co-cultured in lysine decarboxylase broth (LDB) are shown in Figure 1a,b.After 24 h of incubation, V. cholerae NCCP 13589 and V. parahaemolyticus ATCC 27969 individually produced cadaverine amounts of 569.27 ± 28.67 and 570.33 ± 4.53 µg/spent culture mL, respectively.

Cadaverine Production and Bacterial Cell Growth in Lysine Decarboxylase Broth
The effects of the lactic acid bacteria and Vibrio strains on cadaverine produ when co-cultured in lysine decarboxylase broth (LDB) are shown in Figure 1a Interestingly, the cadaverine production decreased to 42.37 ± 2.62 (92.98%) and ± 15.93 µg/spent culture mL (95.61%) when V. cholerae was co-cultured with Lpb. plan NCIMB 6105 and Le.mesenteroides ATCC 10830, respectively.Similarly, the cadav production levels fell to 116.21 ± 3.32 (79.65%) and 132.11 ± 7.97 µg/spent cultur (76.84%) when V. parahaemolyticus ATCC 27969 was co-cultured with Lpb. plan NCIMB 6105 and Le.mesenteroides ATCC 10830, respectively.There was a significa crease in cadaverine production by Vibrio strains capable of producing this compou the presence of lactic acid bacteria.
V. cholerae NCCP 13589 and V. parahaemolyticus ATCC 27969 growth significant creased (p < 0.05) when co-cultured with lactic acid bacterial strains compared to m cultures.
The initial bacterial cell counts for the different lactic acid bacteria strains ra from 4.55 to 4.48 log CFU/mL, with V. cholerae NCCP 13589 having the highest cell c After 24 h, the lactic acid bacteria strains showed a cell growth range of 5.64 to 5.9 Interestingly, the cadaverine production decreased to 42.37 ± 2.62 (92.98%) and 25.24 ± 15.93 µg/spent culture mL (95.61%) when V. cholerae was co-cultured with Lpb.plantarum NCIMB 6105 and Le.mesenteroides ATCC 10830, respectively.Similarly, the cadaverine production levels fell to 116.21 ± 3.32 (79.65%) and 132.11 ± 7.97 µg/spent culture mL (76.84%) when V. parahaemolyticus ATCC 27969 was co-cultured with Lpb.plantarum NCIMB 6105 and Le.mesenteroides ATCC 10830, respectively.There was a significant decrease in cadaverine production by Vibrio strains capable of producing this compound in the presence of lactic acid bacteria.
V. cholerae NCCP 13589 and V. parahaemolyticus ATCC 27969 growth significantly decreased (p < 0.05) when co-cultured with lactic acid bacterial strains compared to monocultures.
The initial bacterial cell counts for the different lactic acid bacteria strains ranged from 4.55 to 4.48 log CFU/mL, with V. cholerae NCCP 13589 having the highest cell count.After 24 h, the lactic acid bacteria strains showed a cell growth range of 5.64 to 5.90 log CFU/mL.Co-culture with Lpb.plantarum NCIMB 6105 and Le.mesenteroides ATCC 10830 significantly reduced the growth of V. cholerae NCCP 13589 and V. parahaemolyticus ATCC 27969.V. cholerae NCCP 13589 exhibited cell growth reductions of 2.13 and 2.93 log CFU/mL, respectively, while V. parahaemolyticus ATCC 27969 exhibited cell growth reductions of 1.36 and 0.86 log CFU/mL, respectively.These findings demonstrate the inhibitory effect of these lactic acid bacteria strains against pathogens (Figure 2a,b).
The co-culture of lactic acid bacterial strains with Vibrio strains resulted in a s cant (p < 0.05) decrease in the growth of V. cholerae NCCP 13589 and V. parahaemo ATCC 27969 compared with their growth in the monoculture.After 24 h of incubati cholerae NCCP 13589 exhibited cell growth of 3.37 log CFU/mL in the monoculture.ever, its cell growth decreased to 1.11 and 0.74 log CFU/mL when co-cultured wit plantarum NCIMB 6105 or Le.mesenteroides ATCC 10830, respectively.Similarly, V Various types of microorganisms or strains generate distinct amino acid decarboxylases that subsequently result in the production of different BA [26].There was a significant reduction in V. parahaemolyticus growth (5.9 log CFU/g) following treatment with 150 mg/mL lactic acid and 300 mg/mL citric acid.These findings highlight the efficacy of lactic acid in combination V. parahaemolyticus [27].
The interaction between pathogenic bacteria and lactic acid bacteria in a co-culture involves nutrients and the production of inhibitory substances.These interactions can significantly influence the growth conditions for both groups of microorganisms, especially int the context of pH variations.Under favorable conditions for pathogenic bacteria, such as those with a neutral to slightly alkaline pH, the activity of constitutive decarboxylases like LdcC in Escherichia coli is optimal.LdcC, which has an optimum pH of 7.6, can continuously produce biogenic amines like cadaverine, contributing to the pathogenicity and survival of the bacteria in such environments [28].Under these conditions, the inhibitory effects of lactic acid bacteria are diminished as the production of organic acids by lactic acid bacteria is not sufficient to significantly lower the pH to levels that would inhibit the pathogens.Additionally, neutral pH conditions do not favor the production of acidic compounds like bacteriocins and other organic acids that are crucial for lactic acid bacteria's antagonistic activity [29].Consequently, pathogenic bacteria can thrive and potentially cause spoilage or disease.
Conversely, when the conditions favor the growth of lactic acid bacteria, such as in environments where the pH is acidic, the production of organic acids like lactic acid significantly lowers the pH, creating a hostile environment for pathogenic bacteria.Lactic acid bacteria can produce a range of inhibitory substances, including bacteriocins, hydrogen peroxide, and enzymes that contribute to the inhibition of competing microorganisms [29].The decrease in pH due to organic acid production can permeate bacterial membranes, leading to their damage and inhibiting a broad range of pathogens [30].
However, the relationship between the pH and decarboxylase activity introduces an additional layer of complexity: while a lower pH inhibits the activity of constitutive decarboxylases, it can induce the production of inducible decarboxylase like CadA, which is active at an optimum pH of 5.5 [28].This inducible enzyme allows pathogenic bacteria to counteract the acidic conditions by producing biogenic amines that neutralize the pH to some extent.Nevertheless, the overall inhibitory environment created by lactic acid bacteria often outweighs the defense mechanisms of pathogens, leading to the reduced growth and viability of pathogenic microorganisms [31].
The co-culture of lactic acid bacterial strains with Vibrio strains resulted in a significant (p < 0.05) decrease in the growth of V. cholerae NCCP 13589 and V. parahaemolyticus ATCC 27969 compared with their growth in the monoculture.After 24 h of incubation, V. cholerae NCCP 13589 exhibited cell growth of 3.37 log CFU/mL in the monoculture.However, its cell growth decreased to 1.11 and 0.74 log CFU/mL when co-cultured with Lpb.plantarum NCIMB 6105 or Le.mesenteroides ATCC 10830, respectively.Similarly, V. parahaemolyticus ATCC 27969 showed cell growth of 2.33 log CFU/mL in the monoculture (Figure 2c,d).However, its growth was reduced to 0.46 and 0.23 log CFU/mL when co-cultured with Lpb.plantarum NCIMB 6105 or Le.mesenteroides ATCC 10830, respectively.This demonstrates that these lactic acid bacterial strains significantly inhibited the growth of V. cholerae NCCP 13589 and V. parahaemolyticus ATCC 27969 in a co-culture environment.The biogenic amine content demonstrated considerable variability across various shrimp paste samples, which could be attributed to the diverse microbial compositions of each sample [32].Various microorganisms or strains generate distinct amino acid decarboxylases that produce different BAs [27].Additionally, they indicate that the use of lactic acid wash solutions could provide a cost-effective, natural, and efficient method to manage V. parahaemolyticus in the oyster industry, particularly in sterilized shucked oysters [26].

Conclusions
This study compared cadaverine-producing Vibrio strains under two different conditions (LDB and ShE media) using lactic acid bacteria and co-cultures.Certain lactic acid bacteria strains possess beneficial properties that positively affect vertebrates and invertebrates.However, the competitive interactions between probiotics and pathogens in shrimp remain largely unexplored.In this study, we demonstrated that Lpb.plantarum and Le.mesenteroides competitively reduced the amount of cadaverine produced by V. cholerae and V. parahaemolyticus in LDB and ShE under co-culture conditions.We also confirmed the decrease in the number of Vibrio strains when co-cultured with lactic acid bacteria in LDB and ShE media, confirming the inhibitory effect of the lactic acid bacteria strains on the growth of V. cholerae and V. parahaemolyticus.The utilization of lactic acid bacteria strains in shrimp presents an interesting approach to address the challenges associated with eliminating V. cholerae and V. parahaemolyticus from shrimp prior to commercialization.Moreover, the combination of beneficial shrimp extracts and lactic acid bacteria provides a natural and attractive solution for the industry, potentially offering additional protection against the growth of V. cholerae and V. parahaemolyticus in shrimp fermentation and processed products.Further studies are needed to determine the optimal conditions for lactic acid bacteria inoculation in processed shrimp products.

2. 6 .
Quantification of Bacterial Cell Growth by Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR)

Table 2 .
Antimicrobial activity of lactic acid bacteria selected against Vibrio strains.