Recovery of Pasteurization-Resistant Vagococcus lutrae from Raw Seafoods Using a Two-Step Enrichment, Its Presumptive Prevalence, and Novel Classification Phenotypes

Abstract

V. lutrae is an emerging human pathogen attributed to increasing hospitalization cases in humans; however, its biology and epidemiology are under-explored. The present study explored V. lutrae recovery, prevalence, and biology. A two-step enrichment method (i.e., step 1, nourishment; step 2, heat, 80 °C, 20 min) and thiosulfate–citrate–bile salts–sucrose (TCBS) agar were employed for recovering V. lutrae in raw seafood. Bacterial colonies were streaked for purification before 16S rRNA bacterial identification. Confirmed V. lutrae isolates were analyzed for their culture-challenged turbidity and virulence. Of 41 bacterial isolates, 9 confirmed V. lutrae, including regular (33%; nourished 24 h) and heat-resistant (67%; nourished 48 h plus heating) isolates, were exclusively from yellow colonies (i.e., TCBS) and were exclusively recovered from nourished shrimp (78%) and crab (22%) only. The culture and virulence biology revealed that they could diversely tolerate salinity (i.e., 0–17.5% additional NaCl), pasteurization (63 °C, 8 h), oxygen availability, and antibiotic sensitivity (i.e., erythromycin, gentamicin, and vancomycin). Further, this pathogen exhibited no visible hemolytic and alkalization activities. Emerging foodborne pathogens could readily evade the established food safety regime. The present study reveals systematic investigation and diverse phenotypes of V. lutrae to enhance its detection and contribute to public health initiatives.

1. Introduction

V. Lutrae is a Gram-positive, non-spore-forming [1,2], facultative anaerobic, motile [3], catalase-negative cocci [2]. This bacterial genus consists of nine species, including the human pathogen V. fluvialis, and they were separately chronologically determined between 1989 and 2014. [2,4]. Since the discovery of V. lutrae in 1999 in a dead marine animal’s organs, such as the blood, liver, lungs, and spleen, due to a road accident [3,5], it has been recurringly (i.e., three cases between 1999 and 2023) causing severe diseases in elderly patients with immunocompromised complications [3,6,7]. This could very likely be attributed to its difficult differentiation phenotypically and physiologically from other bacterial genera, including lactic acid bacteria and clinical Enterococcus, thereby complicating diagnostic identification and leading to false pathogen confirmation and case reports [2,8].

V. lutrae infections in humans can manifest as severe skin lesions [3], bacteremia [6], and other potential complications, including dehydration [3,6], depression [3,6], fever [6], increased heartbeat [6], and sugar level [3] and blood pressure [6] fluctuations. The reported cases have been presumably connected with processed ready-to-eat food (i.e., canned seafood), but this predetermination and the extremophilic property of the pathogen have not been experimentally confirmed [3,6]. Attributed to its physiological and phenotypical similarities to human symbiont bacteria, including Lactococcus and Enterococcus, its accurate diagnosis could be compromised, thereby leading to rare human cases (i.e., three) documented between 2015 and 2023 [3,6,7].

V. lutrae has been widely found in marine ecosystems (i.e., otter, fish contaminated water) [5,8], food/non-food animals, insects, soil, and contaminated food, including dairy products; however, these associated carriers have not been experimentally validated to date [6].

Maldi-tof [3,6,7] and 16S rRNA [3,5,6,8] of bacterial cultures in blood samples are the routinely employed detection methods for V. lutrae. Both general purpose non-enrichment-blood-supplemented Columbia medium [5] and non-enrichment-supplemented bile esculin azide (BEA) medium [8] have been used to non-selectively and selectively differentially (i.e., determined by metabolism-based colony color) cultivate V. lutrae, respectively. Recently, Meza et al. [9] and Tiong et al. [10] used the modified two-step enrichment method and thiosulfate–citrate–bile salts–sucrose agar (TCBS) cultivation to analyze seafood for viable-but-nonculturable (VBNC) Vibrio species contamination. The study accidentally recovered V. lutrae contaminant, confirmed by 16S rRNA bacterial identification (not documented), thereby leading to the exploration of various biological aspects of V. lutrae, including the effect of heat on its biology (in this study).

Various antibiotics, including amoxicillin, ceftriaxone, gentamicin, erythromycin, rifampicin, clindamycin, doxycycline, vancomycin [3], and piperacillin/tazobactam [6], were clinically confirmed to be effective against infectious V. lutrae isolates. Specifically, amoxicillin [3] and piperacillin/tazobactam [6] were administered to patients [3,6].

The present study explored the human emerging pathogen V. lutrae’s nourished recovery, presumptive prevalence, and culture and clinical characterizations. Using the two-step enrichment method of Meza et al. [9], through which the same lab previously selectively differentially recovered on TCBS agar regular and heat-resistant Vibrio species [9,10] and other non-specific bacteria, including V. lutrae (not documented), which otherwise could be nonculturable, various environmental and wet-market raw seafood types were analyzed for the presumptive availability of V. lutrae. After 16S rRNA bacterial identification of bacterial isolates, confirmed V. lutrae isolates were subject to biochemical (i.e., culture and virulence biology) and physical (i.e., culture and thermal biology) characterizations for assessing their growth biology, food safety, and clinical relevancy, including saline-growth dependence, pasteurization-resistance (63 °C, 8 h), pasteurization-dependent re-resuscitation of lab dormant cells (63 °C, 90 min/8 h), oxygen-growth dependence, virulence, and antibiotic sensitivity profiles.

2. Materials and Methods

2.1. Seafood Collection

Raw seafood organisms were acquired from various sources around Pensacola Island, Florida, including estuarine environments (i.e., Santa Rosa Island, lagoon, oyster; and Gulf Island National beach, crab) and a wet market (i.e., the Joe Patti seafood market, shrimp) (Figure 1,

Table 1. List of raw seafood used in this study.

Seafood Type	Source	Sample #	Storage Condition
Crab	Beach	2	4 °C
Oyster	Lagoon	3	4 °C
Shrimp	Wet market	3	4 °C

). Seafood samples were individually packaged and transported in cold conditions (0–2 °C) to the research laboratory cold storage (~4 °C) before experimental analyses (Figure 1). Environmental conditions during sample acquisition were recorded as pH8.5 and temperature 30.3 °C.

2.2. Two-Step Enrichment, Selective/Differential Isolation, and Colony Purification

The 2-step enrichment method of Meza et al. [9] and a Vibrio species selective/differential agar (i.e., TCBS) (9) were adopted to selectively cultivate regular (i.e., involved the step 1 APW nourishment only) and heat-resistant (i.e., involved both the steps 1 BPW nourishment and 2 heat processing) Vibrio species found in seafood samples (i.e., crab, oyster, and shrimp) (Table 1). Attributed to V. cholerae comparable metabolism (i.e., 0% additional NaCl to TCBS agar, pH7.4) and colony phenotypes (i.e., yellow color colony on TCBS agar) (Figure 2), V. lutrae was accidentally recovered in this process (not documented). Briefly, enrichment broth (i.e., alkaline peptone water, APW) (pH 7.4, 8, or 8.6) [11,12,13] containing peptone (5%, w/v) and NaCl (0%, 3%, or 8% additional NaCl) was inoculated with homogenized (Stomacher^® 400 Circulator lab blender, Seward, Weber Scientific, Hamilton, NJ, USA) seafood solution (i.e., 1/10 dilution), incubated for specific times (i.e., 0–72 h), and nourishment dilutions were inoculated on TCBS agar (Difco, Detroit, MI, USA) (i.e., three replications per sample) or heat-treated (80 °C, 20 min) before inoculated onto the agar (i.e., three replicates per sample), followed by parafilm wrapping (i.e., to retain agar moisture) and incubation at 35 °C for one week or until bacterial colonies emerged. Bacterial colonies (i.e., 2–5 colonies) in green and yellow colors were streaked on sterile Brain Heart Infusion (BHI, Difco, Detroit, MI, USA) agar plates for purification.

2.3. Bacterial Culture and Storage Conditions

Presumptive pure colony isolates were separately cultured in fresh sterile BHI broth (pH7.4) for 16–48 h before being subject to storage conditions (BHI supplemented with 20% glycerol, −70 °C). Prior to experimentation, thawed bacterial cultures were inoculated into fresh sterile BHI broth (i.e., inoculant dilution 1/100) (pH7.4), incubated at 35 °C for 16 h, and followed by another subculture in fresh sterile BHI broth. BHI culture agar plates were used for bacterial colony formation and enumeration.

2.4. DNA Extraction

Bacterial genomic DNA was extracted by subjecting fresh bacterial cultures to bead-collision [10]. Briefly, bacterial subculture pellets were repeatedly (2×) washed by sequential resuspension in sterile deionized water (DI H₂O) and centrifugation (12K RPM, 2 min) (VWR, Suwanee, GA, USA). After final centrifugation and discard of supernatants, Tris buffer (10 mM, pH7.4) suspensions (0.1 mL) containing bacterial cells and sterile glass beads (5 µm) were subject to physical lysis via pulsing vortex collision (SPW Industrial, Laguna Hills, CA, USA), and the unleased cytoplasmic DNA supernatants were collected following centrifugation (12K RPM, 2 min) and stored at −20 °C until experimental analyses. Bacterial DNA quality was analyzed at 260 nm/280 nm in a UV spectrophotometer (Thermo Scientific, South San Francisco, CA, USA) and gene-specific PCR (i.e., 16S rRNA gene primers).

2.5. PCR Conditions and 16S rRNA Bacterial Identification

The GoTaq Flexi DNA Polymerase’s instructions (Promega, Madison, WI, USA) were followed to prepare 16S rRNA PCR reactions. Briefly, 5× PCR buffer (Promega), 0.4 µM of gene-specific primers (16S 515F, GTGCCAGCMGCCGCGGTAA; 16S 1391R, GACGGGCGGTGTGTRCA) [14] (IDT, Coralville, IA, USA), 1.5 mM MgCl₂ (Promega), 0.2 mM deoxynucleoside triphosphate mix (dNTPs, Fisher Scientific, Fair Lawn, NJ, USA), <0.5 µg/50 µL template DNA, and 1.25 U of GoTaq polymerase (Promega) were premixed and subject to PCR cycle step 1, 1 cycle of 1 min denaturation at 95 °C; step 2, 40 cycles of sequential 45 s denaturation, 40 s annealing, and DNA extension at 95 °C, 56 °C, and 72 °C, respectively; step 3, 1 cycle of extended DNA extension for 10 min at 70 °C before infinite holding at 4 °C, in a GeneAmpPCR System 9700 Thermal Cycler (Applied Biosystems, Thermo Scientific, South San Francisco, CA, USA). Agarose gel (1.6% agarose + 1× Tris-borate-EDTA buffer) containing GelStarTM Nucleic Acid Gel Stain solution (ratio 5 µL stain solution: 50 mL gel solution, Lonza Walkersville Inc., Walkersville, MD, USA) was used to analyze PCR amplicons with a UV transilluminator (Ultra-Lum UV Transilluminator Muvb-20, ULTRA-LUM INC., Carson, CA, USA) before subject to 1-directional Sanger sequencing using 515F primer (GTGCCAGCMGCCGCGGTAA) [14], chromatogram editing (i.e., MegaX), and the National Center for Biotechnology Information (NCBI) blastn bacterial identification (i.e., default choose search set and program selection). The identity of bacteria with 100% nucleotide query coverage, 0 E value, and 100% percent identity was dictated for each edited nucleotide sequence search.

2.6. V. lutrae Characterization

2.6.1. Culture Biology

Saline-Challenged Growth Assay

Bacterial subcultures (i.e., VHT6, 10⁴ cfu/mL; VHT7, 10³ cfu/mL) were inoculated into sterile BHI broth containing specific concentrations of additional NaCl (VWR, Suwanee, GA, USA), including 0% (i.e., regular BHI; contains 0.5% indigenous NaCl) and 2.5–20% (i.e., experimentally modified BHI) NaCl, incubated at 35 °C for 24–48 h, and the growth was assessed by turbidimetry (i.e., OD₆₀₀ absorbance reading measured before and after incubation) followed by plating bacterial dilutions on regular BHI (i.e., no additional NaCl) agar, with which cell viability was confirmed with colony formation. Initial, reference bacterial turbidity (i.e., blank turbidity) was recorded for each inoculated NaCl-supplemented culture solution (i.e., 0–20% NaCl) prior to incubation at 35 °C. Bacterial countable range beyond 30 and 300 colony forming units (i.e., <30 or >300 CFU) was called the colony detection limit.

Pasteurization-Challenged Growth Assay

Heat-resistant V. lutrae isolates (i.e., 80 °C, 20 min) were evaluated for their resistance to the USDA-recommended pasteurization/cooking temperature for seafood [15] according to Tiong et al. [10]. Briefly, thawed cultures were cultivated (i.e., inoculant dilution 1/100) in screw cap tubes containing sterile culture broth. Culture tubes with turbidity (viable/non-dormant heat-resistant isolates) were subcultured in fresh sterile culture broth (i.e., inoculant dilution 1/100), incubated at pasteurization conditions (i.e., 63 °C, 8 h), followed by culture incubation (i.e., 35 °C, 16 h), viable cell plating, incubating with an anaerobic jar containing paladin, gas pak (AnaeroGenTM 2.5 L, Thermo Scientific, South San Francisco, CA, USA), and viable colonies were streaked for purification before being subjected to culture storage conditions and 16S rRNA bacterial identity confirmation.

Thermal Resuscitation of V. lutrae Lab Dormant Strains

Thermal resuscitation of lab dormant cultures was evaluated for both regular and heat-resistant V. lutrae (in this study) in accordance with Tiong et al. (10) with modifications. Briefly, fresh BHI broth inoculated with thawed V. lutrae cultures (i.e., inoculant dilution 1/100) was incubated at 35 °C for 20 h, pasteurized at 63 °C for 90 min or 8 h, and subject to viable cultivation at 35 °C for 20 h before bacterial viability was assessed by growth turbidity (i.e., indicative of successful resuscitation) and viable colony formation on BHI agar, and the identity was determined with 16S rRNA gene sequencing.

Oxygen-Required Growth Assay

Oxygen exposure of bacterial culture on inoculated agar plates (i.e., BHI) was modulated by direct anaerobic incubation in anaerobic jars containing palladium and gas pak (AnaeroGenTM 2.5 L, Thermo Scientific, South San Francisco, CA, USA) (i.e., zero O₂ availability) or by parafilm wrapping (i.e., minute O₂ availability) vs. no-parafilm wrapping (i.e., full O₂ availability) prior to culture incubating (i.e., 35 °C, 48 h). Bacteria with varied O₂-availability dependencies, including strict aerobe, microaerophile, and facultative anaerobe, were dictated with visible colonies formed on the inoculated agar plates. Additionally, bacterial growth behavior (i.e., non-planktonic cells aggregated in the tube bottom) in BHI broth (i.e., 35 °C, 20 h) was recorded.

2.6.2. Clinical Biology

Hemolysis Assay

Bacterial hemolytic activity was assessed on BHI agar plates containing sheep erythrocytes (5%, v/v) (Alsever, pooled, Carolina Biological Supply Company, Burlington, NC, USA) or human erythrocytes (i.e., acquired from >50-year-old male donor; 1.5%, v/v) (Human Red Blood Cells, 3.2% NaCit, Pooled, Male; BIOIVT, Hicksville, NY, USA) following patch inoculation or streak-inoculation using a sterile inoculating loop, with or without parafilm-wrapped, and incubation at 35 °C until visible growth formed. The hemolytic activity of alpha (α), beta (β), and gamma (γ) was assigned to partial, complete, and no hemolysis, respectively.

Urease Assay

According to Meza et al. [9], pelleted cells (i.e., 1 mL fresh subcultures, 35 °C, 16 h) (16K RPM, 2 min) were resuspended with 1 mL sterile buffered peptone water (i.e., 0.1%, w/v; 10⁸–10⁹ CFU/mL final cell concentration) containing a commercial urea pellet (Urease Test Tablets Key Scientific, Hardy diagnostics, Santa Maria, CA, USA), followed by incubation at 35 °C until a pink color, indicative of urease-positive reactions, appeared (i.e., 1–24 h).

Antibiotic Disc Diffusion Assay

Antibiotic discs, including Amoxicillin/clavulanic acid (AMC, 20/10 µg or AMC, 30 µg), ceftriaxone (CEF, 30 µg), clindamycin (CLI, 2 µg), doxycycline (DOX, 30 µg), erythromycin (ERY, 15 µg), gentamicin (GEN, 10 µg), rifampin (RIF, 5 µg), and vancomycin (VAN, 30 µg), were acquired commercially (BD BBLTM Sensi-DiscTM Antimicrobial Susceptibility Test Discs, Fisher Scientific, Waltham, MA, USA), applied, and evaluated according to Meza et al. [9]. Briefly, antibiotic discs were placed onto agar media inoculated with bacterial lawns of fresh subcultures (i.e., 16 h), and the plates were incubated at 35 °C until inhibition zones formed (i.e., 24–48 h). Bacterial sensitivity profiles were recorded and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) M100-S21 (M2): Disk Diffusion Supplemental Tables, Performance Standards for Antimicrobial Susceptibility Testing, provided for V. lutrae closely related genera, including Enterococci [6] (i.e., DOX, ERY, RIF, VAN) and a Gram-positive bacterium, Staphylococcus spp. (i.e., AMC, CEF, CLI, GEN), in the manufacturer products.

2.7. Turbidimetric Assay

Bacterial growth turbidity was measured with an automated spectrophotometer (Spectronic Genesys 2, Thermo Scientific, South San Francisco, CA, USA). Bacterial solution (i.e., 0.5 mL each) was transferred into disposable cuvettes (BrandTechTM BrandTM Plastic cuvettes, Fisher Scientific, Waltham, MA, USA), and optical density at 600 nm (i.e., OD₆₀₀) was measured. Blank reading was made using fresh sterile culture solution (i.e., BHI broth).

2.8. Statistical Analysis

The two-sample T-test was used to determine the significance of varied salinity growth and antibiotic profiles among two V. lutrae strains (i.e., salinity, VHT6 vs. VHT7; antibiotic, VHT84 vs. VHT85). The profile significant variation was called at p < 0.05.

3. Results

3.1. Bacteria Enrichment, Selection, and Isolation

Using the 2-step enrichment method, V. lutrae was recovered from raw seafood samples (Table 1 and

Table 2. Strains of V. lutrae (√VL) and non-V. lutrae (VP, JC, SE, VA, MN, VC, and VM) recovered in this study.

Strain ID 1	16S ID 3	Colony Color	Nourishment Conditions 4			Seafood Type
			NaCl, pH	Time (h)	Step #
VHT1	VP	G	M	48	2	Oyster
VHT2	VP	G	M	48	2	Oyster
VHT5	VL	Y	m	48	2	Crab
VHT6	√VL	Y	m	48	2	Crab
VHT7	√VL	Y	m	48	2	Shrimp
VHT9	JC	G	E	48	2	Shrimp
VHT10	JC	G	E	48	2	Shrimp
VHT11	JC	G	E	48	2	Shrimp
VHT12	JC	G	E	48	2	Shrimp
VHT13	JC	G	E	48	2	Shrimp
VHT14	VP	G	M	48	2	Oyster
VHT15	VP	G	M	48	2	Oyster
VHT16	VP	G	M	48	2	Oyster
VHT17	VP	G	M	24	1	Oyster
VHT18	VP	G	M	24	1	Oyster
VHT20	VP	G	M	24	1	Oyster
VHT21	VP	G	M	24	1	Oyster
VHT22	VP	G	E	72	1	Oyster
VHT23	SE	G	E	72	1	Oyster
VHT25	VP	G	E	72	1	Oyster
VHT26	VP	G	E	72	1	Oyster
VHT27	√VL	Y	m	48	2	Shrimp
VHT28	√VL	Y	m	48	2	Shrimp
VHT29	√VL	Y	m	48	2	Shrimp
VHT31	VA	Y	m	8	2	Oyster
VHT32	VA	Y	m	8	2	Oyster
VHT33	VA	Y	m	8	2	Oyster
VHT37	MN	Y	m	48	1	Oyster
VHT38	MN	Y	m	48	1	Oyster
VHT39	VC	Y	m	48	1	Oyster
VHT40	VC	Y	m	48	1	Oyster
VHT41	√VL	Y	m	24	1	Shrimp
VHT42	VM	Y	m	24	1	Shrimp
VHT43	√VL	Y	m	24	1	Shrimp
VHT44	√VL	Y	m	24	1	Shrimp
VHT45	VM	Y	m	24	1	Shrimp
VHT46	VA	Y	m	12	1	Crab
VHT47	VA	Y	m	12	1	Crab
VHT48	VA	Y	m	12	1	Crab
VHT49	VA	Y	m	12	1	Crab
VHT50	VA	Y	m	12	1	Crab
VHT82 2	√VL	NA	NA	NA	NA	VHT27
VHT83 2	√VL	NA	NA	NA	NA	VHT28
VHT84 2	√VL	NA	NA	NA	NA	VHT29
VHT85 2	√VL	NA	NA	NA	NA	VHT44

¹ These bacterial isolates are confirmed V. lutrae (√) by 16S rRNA bacterial identification. ² These 16S rRNA confirmed V. lutrae derivatives are survivors of pasteurization conditions (i.e., 63 °C, 8 h). ³ JC, Jeotgalicoccus sp.; MN, Morganella sp.; SE, Staphylococcus epidermidis; VA, Vibrio anguillarum; VC, Vibrio cholerae; VM, Vibrio metschnikov; VP, Vibrio parahaemolyticus. ⁴ mild (m), 0% additional NaCl, pH7.4; moderate (M), 6% additional NaCl, pH8; extreme (E), 8% additional NaCl, pH8.6. The two-step enrichment method step 1 (i.e., APW nourishment) was applied to enrich/activate presumptive V. lutrae contaminant in seafood samples for 0–72 h prior to TCBS cultivation of the pathogen. The two-step enrichment method steps 1 (i.e., nourishment, 0–72 h) and 2 (i.e., heat) were sequentially applied before heat-dependent/resistant bacteria in seafood were cultivated on selective, differential TCBS agar. NA, not applicable.

). Of 41 bacterial isolates (i.e., including V. lutrae) differentially acquired from seafood samples after one-step (i.e., 12 h, 24 h, 48 h, and 72 h) (

Table 3. Bacterial colony-forming profile of enriched seafood on TCBS agar at the post step 1 of the 2-step enrichment method ¹.

Enrichment Time (h) 2	Seafood Type and Enrichment Only Conditions 3
	Crab			Oyster			Shrimp
	Mild	Moderate	Extreme	Mild	Moderate	Extreme	Mild	Moderate	Extreme
0	X	X	X	X	X	X	X	X	X
8	X	X	X	X	X	X	X	X	X
12	√, Y	X	X	X	X	X	X	X	X
24	X	X	X	X	√, G	X	√, Y+	X	X
48	X	X	X	√, Y	X	X	X	X	X
72	X	X	X	X	X	√, G	X	X	X

¹ One-step enrichment employs step 1 of the two-step enrichment method of Meza et al. [9], which involves enrichment incubation (35 °C, 0–72 h) of homogenized seafood in APW, plating of enrichment dilutions on TCBS agar and incubation (35 °C) of inoculated TCBS agar until colony formation or 72 h. The data represent non-retail seafood only (i.e., environmental and wet market seafood) analyzed (in this study). ² Specific time-nourished samples were inoculated on TCBS agar for viable, culturable bacteria isolation. ³ mild (m), 0% additional NaCl, pH7.4; moderate (M), 6% additional NaCl, pH8; extreme (E), 8% additional NaCl, pH8.6; √, bacterial growth (colony) was visible; Y (yellow) or G (green), bacterial colony color on the agar; +, V. lutrae positive, identified via 16S rRNA gene sequencing.

) and two-step (i.e., 8 h, 48 h) (

Table 4. Bacterial colony-forming profile of enriched seafood on TCBS agar at the post steps 1 and 2 of the 2-step enrichment method ¹ followed by overnight cold storage.

Enrichment Time (h) 2	Seafood Type and Enrichment Conditions 3 + Heat
	Crab			Oyster			Shrimp
	Mild	Moderate	Extreme	Mild	Moderate	Extreme	Mild	Moderate	Extreme
0	X	X	X	X	X	X	X	X	X
8	X	X	X	√, Y	X	X	X	X	X
12	X	X	X	X	X	X	X	X	X
24	X	X	X	X	X	X	X	X	X
48	√, Y+	X	X	X	√, G	X	√, Y+	X	√, G
72	X	X	X	X	X	X	X	X	X

¹ Two-step enrichment sequentially employs steps 1 and 2 of the two-step enrichment method of Meza et al. [9], which involves enrichment incubation (35 °C, 0–72 h) of homogenized seafood in APW, heating of enriched seafood homogenate, plating of the heated homogenate on TCBS agar, and incubation (35 °C) of inoculated TCBS agar until colony formation or 72 h. ² Specific time-nourished samples were followed by heat processing before being inoculated on TCBS agar for heat-resistant bacteria isolation. ³ mild (m), 0% additional NaCl, pH7.4; moderate (M), 6% additional NaCl, pH8; extreme (E), 8% additional NaCl, pH8.6; √, bacterial growth (colony) was visible; Y (yellow) or G (green), bacterial colony color on the agar; +, V. lutrae positive, identified via 16S rRNA gene sequencing.

) enrichments (i.e., one-step, nourishment; two-step, nourishment and heat processing), 22 and 19 were regular and heat-resistant bacteria (Table 2), respectively, in which (i.e., of the 41 isolates) 18 and 23 were green and yellow colonies, respectively, on TCBS agar.

3.2. V. lutrae Presumptive Prevalence and Distribution

Of these isolates, 9 V. lutrae (i.e., 22% of 41 total bacterial isolates) were exclusively detected in yellow colony isolates (i.e., 39% of 23 yellow isolates) acquired after mild enrichment conditions (i.e., 3% NaCl, pH7.4, 24 h or 48 h enrichment time) (Table 2, Table 3 and Table 4), including the regular (i.e., post 24 h nourishment time; 33% of nine V. lutrae isolates) (Table 2 and Table 3) and heat-resistant strains (i.e., after 48 h enrichment time; 67% of nine V. lutrae isolates) (Table 2 and Table 4). Of all V. lutrae isolates (i.e., nine), two and seven were exclusively acquired from crab and shrimp samples, respectively (Table 2, Table 3 and Table 4), with the regular and heat-resistant isolates exclusively recovered in shrimp (Table 2 and Table 3) and both crab and shrimp (Table 2 and Table 4) samples, respectively.

3.3. V. lutrae Culture Biology

Select V. lutrae isolates were analyzed for physical and biochemical characterizations.

3.3.1. Growth Response to Salinity

Biochemically, select heat-resistant V. lutrae isolates (i.e., VHT6 and VHT7) were evaluated for their NaCl-challenged viability. Both VHT6 and VHT7 differentially exhibited significantly decreasing visible turbidity (p < 0.05) as NaCl concentration increased (Figure 3); 0% and 12.5–20% additional NaCl completely unleashed and abolished V. lutrae viability, respectively, as demonstrated in their turbidity readings (i.e., relative to inoculated BHI turbidity before culture incubation). Subsequently, NaCl-challenged cells were assessed for viable plate counts (i.e., colony forming unit, CFU). Both VHT6 and VHT7 exhibited a negative correlation (i.e., visible turbidity vs. CFU below detection or zero visible turbidity vs. CFU) between their turbidimetric reading and viable count after 48 h-incubation with 0–7.5 and 12.5 NaCl concentrations (Figure 3 and Figure 4). Both were visibly viable at 10% and 12.5% NaCl, as demonstrated by their significantly differential visible colony counts (i.e., VHT6 vs. VHT7) (p < 0.05) formed on BHI agar plates (Figure 4). Comparatively, VHT6 NaCl resilience outperformed VHT7 (i.e., VHT6 CFU vs. VHT7 CFU below detection) at increasing NaCl concentrations (i.e., 15% and 17.5%), but its viability was completely abolished (i.e., CFU below detection) at 20% NaCl alongside VHT7 (Figure 4).

3.3.2. Pasteurization Sensitivity Profile of V. lutrae

Select V. lutrae heat-resistant isolates (in this study) were evaluated for their resistance to prolonged pasteurization (i.e., physical characterization) as dictated by their growth turbidity at post-incubation (i.e., 35 °C, 16 h). All tested isolates (VHT27, VHT28, and VHT29) remained viable at post-pasteurization conditions (i.e., 63 °C, 8 h), (

Table 5. Pasteurization sensitivity of heat-resistant V. lutrae isolated in this study.

Strain ID	Heat-Resistant 1	Pateurization-Resistant 2	Re-ID 3
VHT27	v	v	VHT82
VHT28	v	v	VHT83
VHT29	v	v	VHT84

¹ V. lutrae isolates acquired by the two-step enrichment method steps 1 and 2 of Meza et al. [9]. ² Fresh viable broth cultures (i.e., 16 h) were treated at 63 °C for 8 h and followed by 35 °C incubation until the formation of visible growth turbidity (i.e., 20 h) before pasteurization sensitivity was evaluated. ³ Pasteurization-resistant colonies of heat-resistant VHT27, VHT28, and VHT29 (Figure 6) were analyzed by 16S rRNA gene sequencing and confirmed V. lutrae strains were renamed as VHT82, VHT83, and VHT84, respectively.

) as evidenced by their growth turbidity following culture incubation (i.e., 35 °C, 16 h) (Figure 5) and 16S rRNA V. lutrae confirmation of their colonies (Figure 6).

3.3.3. Dormant/VBNC Cells Resuscitated Using Pasteurization Temperature

V. lutrae dormant revertants (i.e., culturable V. lutrae converted into VBNC while adapted to the lab storage and culture conditions) were thermally analyzed for their ability to return to their culturable state. Of thirteen VBNC V. lutrae lab strains (i.e., nine isolated in this study and four pasteurization-resistant derivatives), two and one of each of regular and heat-resistant V. lutrae, respectively, were resuscitated following 63 °C pasteurization at specific incubation times (i.e., 90 min, VHT82; 8 h, VHT44) (

Table 6. Thermal resuscitation of VBNC V. lutrae lab strains isolated in this study.

Strain ID 1	8 h Thermal 2	1.5 h Thermal 2	Heat-Resistant 3	Regular 4
VHT5	-	-	v	NA
VHT6	-	-	v	NA
VHT7	-	ND	v	NA
VHT27	ND	ND	v	NA
VHT28	ND	-	v	NA
VHT29	ND	-	v	NA
VHT41	-	-	NA	v
VHT43	-	-	NA	v
VHT44	+	-	NA	v
VHT82	ND	+	VHT27 der.	NA
VHT83	ND	-	VHT28 der.	NA
VHT84	ND	-	VHT29 der.	NA
VHT85	ND	-	NA	VHT44 der.

¹ Non-culturable thawed (i.e., after being subject to the lab storage conditions, −70 °C in BHI supplemented with 20% glycerol) V. lutrae (i.e., VBNC) isolates at 35 °C incubation for >16 h. ² Non-culturable V. lutrae (i.e., VBNC; after >16 h incubation at 35 °C) at post-pasteurization conditions, including 8 h or 1.5 h incubation at 63 °C before being subject to culture incubation conditions (i.e., 35 °C, 16 h) and visible growth turbidity determination. “-”, no visible growth turbidity; “+”, visible turbidity was detected. ³ V. lutrae isolates were acquired by the two-step enrichment method steps 1 and 2 of Meza et al. [9]. ⁴ V. lutrae isolates were acquired by the two-step enrichment method step 1 of Meza et al. [9]. ND, not determined; NA, not applicable.

), as evidenced by bacterial growth turbidity (i.e., 35 °C, 16 h) and confirmed V. lutrae identity by 16S rRNA colony sequencing (Figure 7).

3.3.4. Growth Response to Oxygen Availability

Subsequently, select V. lutrae strains, including heat-resistant (i.e., VHT28 and VHT29) and pasteurization-resistant (i.e., VHT82-VHT85) strains (

Table 7. Oxygen-dependent growth profile of select V. lutrae isolates.

O2 Availability 1	V. lutrae Strains 2 in This Study
	VHT28	VHT29	VHT82	VHT83	VHT84	VHT85
Layer (full)	ND	ND	v	v	x	v
Wrap (minute)	x	v	ND	x	v	v
Anaerobic jar (zero)	ND	ND	v	ND	ND	ND
Classification	Strict aerobe	Microaerobe	Facultative anaerobe	Strict aerobe	Microaerobe	Facultative anaerobe

¹ Inoculated agar plates were directly incubated anaerobically (i.e., anaerobic jar, zero O₂ availability) or wrapped with (i.e., wrap, minute O₂ availability) or without (i.e., layer/surface, full O₂ availability) parafilm. ² VHT28 and VHT29, heat-resistant V. lutrae; VHT82, VHT83, VHT84, and VHT85, pasteurization-resistant V. lutrae derived from heat-resistant V. lutrae VHT27, VHT28, VHT29, VHT44, respectively. ND, not determined.

), were analyzed for their oxygen-dependent viability (i.e., CFU availability). Examined V. lutrae strains exhibited diverse oxygen dependencies, including strict aerobe (i.e., VHT83), facultative anaerobes (i.e., VHT82 and VHT85), and microaerophile (i.e., VHT84) (Table 7), as evidenced in the formation of CFU on agar plates (i.e., select V. lutrae strains examined, VHT28 and VHT29) (Figure 8). The oxygen-dependence of VHT28 (i.e., strict aerobe) and VHT29 (i.e., microaerobe) was comparably retained with their derivative, pasteurized strains (i.e., pasteurization-resistant strains), VHT83 and VHT84, respectively (Table 7, Figure 8). Further, VHT27, VHT28, and VHT29 exhibited comparable non-planktonic growth behavior in culture broth media (Figure 9).

3.4. V. lutrae Clinical Biology

3.4.1. Hemolysis Assay

Select V. lutrae strains were assessed for hemolytic activity. Both heat-resistant (i.e., VHT5 and VHT6) and pasteurization-resistant (i.e., VHT84 and VHT85) V. lutrae inoculated and differentially incubated BHI blood agar plates (i.e., VHT5 and VHT6, non-parafilm-wrapped; VHT84 and VHT85, parafilm wrapped) exhibited visible bacterial growth with no visible hemolysis (Figure S1).

3.4.2. Urease Assay

The urease activity profile of V. lutrae was assessed to determine their ability to modulate biological pH. Of all test strains (i.e., thirteen), only three strains, including heat-resistant (i.e., VHT7 and VHT27) and pasteurization-resistant (i.e., VHT82) V. lutrae, were culturable in broth media. All exhibited negative urease activity (Figure S2) relative to the test negative (non-pink color) and positive (i.e., pink color) controls.

3.4.3. Antibiotic Sensitivity Profile

Select V. lutrae strains were evaluated to dictate the variation and multi-antibiotic resistance index (MAR) of the bacterium. Pasteurization-resistant V. lutrae, including VHT84 and VHT85, exhibited a high degree of similarity in antibiotic sensitivity profile (i.e., determined by inhibition size, mm) to most of the antibiotics examined (Figure 10) except gentamycin, with which VHT84 was interpreted as slightly gentamicin resistant (i.e., intermediate) as opposed to VHT85 (i.e., resistant) (Figure 10,

Table 8. Antibiotic interpreted sensitivity ¹ and MAR index of select V. lutrae strains.

Antibiotic	VHT84				VHT85
	Avg Inhibition size (mm) ± S.D.	S	I	R	Avg Inhibition size (mm) ± S.D.	S	I	R
AMC30	40 ± 0.82	v			40 ± 1.25	v
CEF30	40 ± 1.25	v			39 ± 0.82	v
CLI2	38 ± 1.25	v			39 ± 0.82	v
DOX30	40 ± 0.47	v			40 ± 0.82	v
ERY15	38 ± 0.47	v			40 ± 0.47	v
GEN10	13 ± 0.82		v		8 ± 0.47			v
RIF5	41 ± 0.94	v			41 ± 0.47	v
VAN30	29 ± 0.82	v			39 ± 0.82	v
MAR index		0				0.125

¹ Adapted in part from the inhibition size interpretive chart of CLSI Document M100-S21 (M2) provided by the antibiotic manufacturer. S, susceptible; I, intermediate; R, resistant.

). VHT84 and VHT85 differentially exhibited a MAR index of 0 and 0.125, respectively (Table 8).

4. Discussion

4.1. Selective and Differential Culture Recovery of Environmental and Wet-Market V. lutrae

The two-step enrichment method of Meza et al. [9] revealed the presence of V. lutrae in environmental and wet-market seafood tested (in this study) (Table 1 and Table 2), thereby suggesting, for the first time, that V. lutrae could be VBNC (Table 3 and Table 4) and that they could not be recovered with the currently available non-enrichment-supplemented cultivation techniques [5,8] (i.e., 0 nourishment time in this study did not recover any V. lutrae) (Table 3). Additionally, this method could resuscitate VBNC regular (i.e., recovered using the step 1 of the two-step enrichment method) (Table 3) and heat-resistant (i.e., recovered using both steps 1 and 2 of the two-step enrichment method) (Table 4) groups of V. lutrae (Table 3 and Table 4), and the heating step (i.e., 80 °C, 20 min) could improve V. lutrae recovery selectivity among other seafood non-Vagococcus contaminants (Table 2). Its VBNC and heat-resistant features could be driven by gene transfers from the environmentally enriched VBNC bacteria and heat-resistant spore-forming/non-spore-forming bacteria community [16] and global warming [17], respectively. V. lutrae exhibits exclusive yellow colonies on TCBS (i.e., 0% additional NaCl, pH7.4) (Figure 2, Table 2, Table 3 and Table 4), hence suggesting, for the first time, the differentiability of Vibrio species selective and differential TCBS agar against V. lutrae and that this agar could be a comparable alternative medium for V. lutrae selective isolation and differentiation from many non-Vibrio and other Vibrio species (i.e., green colonies of V. parahaemolyticus and V. vulnificus) (Table 2, Table 3 and Table 4) bacteria, as opposed to currently available Columbia [5] and BEA media [8]. It is worth noting that V. lutrae did not exhibit visible growth in Luria Bertani (i.e., LB) and Tryptic Soy broth (i.e., TSB), which led to the exclusive use of BHI media throughout this study.

4.2. Presumptive Prevalence of V. lutrae in Raw Seafood

Of the three types of seafood examined (i.e., crab, oyster, and shrimp) (Table 1), V. lutrae was exclusively and differentially associated with shrimp (i.e., 78% of nine V. lutrae isolates) and crab (i.e., 22% of nine V. lutrae isolates) (Table 2, Table 3 and Table 4), experimentally confirming, for the first time, the presumptive association of another Vagococcus species (i.e., V. lutrae) with environmental and wet-market raw seafood, including crab [18] and shrimp [19]. V. lutrae’s presumptive exclusive prevalence in non-oyster seafood examined concurs with prior reports of oyster’s exclusive symbiont specificity with V. parahaemolyticus [9,10].

4.3. V. lutrae Culture Biology

4.3.1. Saline Dependence

NaCl availability could modulate the viability of Gram-positive bacteria [20], including V. lutrae. This study demonstrated V. lutrae non-parallel growth profiles between turbidimetric values and viable plate count numbers (i.e., full O₂ availability) analyses (Figure 3 and Figure 4), thereby suggesting the need for modified culture conditions (i.e., osmotically adjusted media) for plate-enumerating NaCl-osmotic-stress adapted cells [20]. The negative correlation between the NaCl-challenged (i.e., 0–7.5%, 12.5–17.5% NaCl) viability (i.e., optical density) (Figure 3) vs. viable count (i.e., agar plate, non-parafilm wrapped) (Figure 4) of V. lutrae could be attributed to their diverse oxygen dependencies as richly reported on other bacteria [21] and NaCl-modulated metabolism preferences [22]. The examined strains’ diverse turbidimetric values could not be attributed to the diverse inoculant sizes as the final turbidimetric reading of the cell viability was measured at after 48 h cultivation (35 °C), conferring sufficient time to achieve maximum cell numbers, thereby suggesting the availability of diverse V. lutrae strains in NaCl metabolism (Figure 3), including halophiles, which could pose a food safety threat in saline food. For the first time, a means of classifying V. lutrae isolates is revealed. Additionally, the findings suggest the need to pair the study of saline-challenged growth in broth with agar plating to ensure result validity from non-viable cell turbidimetric readings. The fact that 10% additional NaCl could improve V. lutrae growth (Figure 3 and Figure 4), both liquid and agar, could be restrictive in a similar study (i.e., including V. lutrae hemolysis assay) due to NaCl-posed osmotic stresses [23].

4.3.2. Pasteurization Resistance

Select heat-resistant strains of V. lutrae (in this study) possessed pasteurization resistance (i.e., 63 °C, 8 h) (Table 5), thereby shedding light on previously documented assumptions of the presence of human infectious extremophile V. lutrae (i.e., the presence of thermal-processing resistant strain of V. lutrae) and their availability in a processed food products [3,6]. This corroborates the availability of pasteurization-resistant bacterial pathogens (i.e., V. parahaemolyticus) in environmental and wet-market raw seafood [9].

4.3.3. Pasteurization Re-Resuscitation of Select Dormant Cells

Select lab dormant strains of V. lutrae (i.e., VHT44, regular; VHT82, pasteurization-resistant) were successfully resuscitated following pasteurization intervention (i.e., VHT44, 63 °C, 8 h; VHT82, 63 °C, 90 min) (Table 6, Figure 7). Tiong et al. [10] reported the continuous dormancy of a Gram-negative, non-spore-forming bacterium following these thermal resuscitation conditions (i.e., 63 °C, 8 h), thereby suggesting for the first time that dormant, non-spore-forming lab bacterial cultures, including regular and related heat-resistant bacteria, could be resuscitated at lower thermal conditions than the VBNC recovery conditions (i.e., 80 °C, 20 min) (in this study). These cultivation tests (i.e., pasteurization resistance and re-resuscitation) demonstrated additional V. lutrae phenotypes that could differ among strains, including pasteurization resistance and pasteurization dependent resuscitation, thereby disclosing the prevalence of thermally variant V. lutrae (i.e., pasteurization resistance) and their potential cause of food poisoning in thermally processed seafood products, including canned seafood suspected of causing V. lutrae infections [3,6].

4.3.4. Oxygen Dependency

Further work (in this study) demonstrated that the pathogen exhibited differential oxygen sensitivity responses, as demonstrated by their ability to form colonies on diverse types of incubation (i.e., non-parafilm wrapped, parafilm-wrapped, anaerobically incubated) of inoculated plates (Table 7, Figure 8), including strict aerobic, microaerobic, and facultative anaerobic. This confirms the availability of V. lutrae with diverse O₂-dependencies (Figure 3 and Figure 4) and suggests, for the first time, another bacterial phenotype that could be used to classify V. lutrae isolates. Additionally, this V. lutrae phenotype (i.e., varied O₂ dependencies) (i.e., VHT28, its pasteurized der., VHT83; VHT29, its pasteurized der., VHT84) could withstand thermal treatment (Table 7, Figure 8B). The inconsistent growth profiles demonstrated for facultative anaerobes (i.e., VHT27/VHT82), strict aerobes (i.e., VHT28/VHT83), and microaerobes (i.e., VHT29/VHT84) in agar media (i.e., plate) (Figure 8) and broth media (i.e., all cultures pelleted in the least oxygenated liquid bottom) (Figure 9) [24] reveal, for the first time, that the V. lutrae O₂ dependency could be conclusively dictated by agar plating alone and with modifications (i.e., parafilm-wrapped, anaerobically incubated). Additionally, the varied colony profiles (i.e., size) exhibited as the result of different incubation types employed in V. lutrae recovery (i.e., from samples, parafilm-wrapped only to retain agar moisture) (Figure 2) and cultivation (i.e., lab stored strains; non-parafilm wrapped, parafilm wrapped, and anaerobically incubated) (Figure 8) could be attributed to the availability of O₂-bypassing mechanisms, including environmentally or TCBS available unknown or undetermined growth factors, respectively, that are not originally available in the BHI cultivation agar media used in this study. Comparatively, full O₂ exposure of inoculated agar plate incubation could effectively culture most V. lutrae lab strains as opposed to parafilm-wrapped or anaerobic incubation (Table 7, Figure 8), thereby restraining the use of the latter time-consuming incubation preparations (i.e., parafilm-wrapped and anaerobic incubation).

4.4. Clinical Biology

4.4.1. Hemolysis Profile

A prior report documented V. lutrae bacteremia in humans [6]. For the first time, negative hemolysis (i.e., gamma hemolysis type) was determined in all groups of V. lutrae isolates (in this study), including the regular, heat-resistant, and pasteurization-resistant strains (Figure S1), thereby further characterizing the bacterium’s invasiveness (i.e., blood infection) but non-hemolytic bacteremia (i.e., in human bloodstream) as opposed to the exclusive bacteremia (i.e., previous work did not explore the bacterium hemolytic activity) revealed in the report by Altintas et al. [6]. This non-hemolytic bacteremia phenotype is typical in Streptococcal species [25,26], hence validating V. lutrae’s findings (in this study). Lawson et al. [5] reported the use of horse erythrocytes in culture media to cultivate V. lutrae. It is worth noting that this study demonstrated, for the first time, V. lutrae diverse growth densities on sheep blood-supplemented BHI agar (i.e., poor growth) relative to human blood-containing BHI agar (Figure S1).

4.4.2. Urease Profile

The urease-negative activity of V. lutrae (Figure S2) examined revealed its immunity from a urease-carrying gene that could be available in its community bacteria, including urease-positive V. parahaemolyticus strains (i.e., uh gene) [9,27], that was co-recovered using the modified 2-step enrichment method of Meza et al. [9]. The urease gene of V. parahaemolyticus usually co-exist with the thermostable-direct-related hemolysin gene (i.e., trh) [27], and its activity is positively correlated with the hemolysis activity [28], thereby explaining the non-hemolytic (Figure 5) and non-urease (Figure S2) activity of V. lutrae.

4.4.3. Antibiotic Profile

Diverse antibiotic sensitivity profiles (Figure 10, Table 8) with significantly diverse levels of sensitivity (i.e., per the inhibition size, mm, of erythromycin and vancomycin against V. lutrae) (Figure 10) and MAR indexes (Table 8) of V. lutrae were detected in this study, revealing, for the first time, the availability of antibiotic-sensitively diverse (Figure 10) and gentamicin-resistant (i.e., VHT85) (Table 8) V. lutrae phenotypes. This is in contrast to prior reports (i.e., they were sensitive to antibiotics tested) by Garcia et al. and Altintas et al. [3,6], and revealed that antibiotic sensitivity profiling could be employed to determine the pathogen variability.

5. Conclusions

Microbial food safety is an ongoing threat to the food industry. It requires continuous investigative exploration and intervention attributed to the emergence and evolving features of microbial pathogens, including V. lutrae. The present study utilized the two-step enrichment method to successfully recover total V. lutrae on TCBS agar, including the novel VBNC regular and heat-resistant groups of V. lutrae. These V. lutrae groups are not recoverable with the currently available non-enrichment-supplemented investigative cultivation techniques [5,8]. Together, the present findings suggest, for the first time, that they are positively associated with environmental and wet-market raw seafood (i.e., foodborne) and that they could be selectively differentially (i.e., yellow color colony) recovered on TCBS agar. Additionally, this study discovered diverse biological (i.e., salinity-, pasteurization-, and O₂-challenged viability) and antibiotic profiles of the isolates acquired from raw seafood, thereby revealing, for the first time, a systematic investigation of VBNC V. lutrae that enables pathogen recovery (i.e., using the two-step enrichment method in this study) and biological and virulence classifications (i.e., salinity-, pasteurization-, O₂-, antibiotic-challenged viability).