Detection of virulence, antimicrobial resistance, and heavy metal resistance properties in Vibrio anguillarum isolated from mullet (Mugil cephalus) cultured in Korea

Abstract

In the present study, we identified and characterized 22 strains of V. anguillarum from 145 samples of mullets (Mugill cephallus) cultured in several fish farms in South Korea. They were subjected to pathogenicity tests, antimicrobial susceptibility test, and broth dilution test to detect virulence markers, antimicrobial resistance, and heavy metal resistance properties. All the isolates showed amylase and caseinase activity, followed by gelatinase (90.9%), DNase (45.5%), and hemolysis activities (α = 81.1% and β = 18.2%). The PCR assay revealed that isolates were positive for VAC, ctxAB, AtoxR, tdh, tlh, trh, Vfh, hupO, VPI, and FtoxR virulence genes at different percentages. All the isolates showed multi-drug resistance properties (MAR index ≥ 0.2), while 100% of the isolates were resistant to oxacillin, ticarcillin, streptomycin, and ciprofloxacin. Antimicrobial resistance genes, qnrS (95.5%), qnrB (86.4%), and StrAB (27.3%), were reported. In addition, 40.9% of the isolates were cadmium-tolerant, with the presence of CzcA (86.4%) heavy metal resistance gene. The results revealed potential pathogenicity associated with V. anguillarum in aquaculture and potential health risk associated with consumer health.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-023-00911-9.

Introduction  

Fisheries and aquaculture products are significant sources of affordable, nutrient-rich food and a foundation for worldwide livelihoods. Production of fisheries and aquaculture totaled 3.6 million tons of finfish, mollusks, and crustaceans in South Korea in 2018 [1]. Mugilidae fish such as red lip mullet (Chelon haematocheilus) and grey mullet (Mugil cephalus) are consumed in South Korea [2]. Production of mullets accounted for 7% of the total finfish mariculture production in South Korea in 2013 and the global production of mullets has estimated at 291,200 tonnes in 2020 [3, 4].

Vibrio anguillarum is a Gram-negative, motile, halophilic pathogenic bacteria in warm and cold water estuarine and seawater environments [5]. It is the most common causative agent of vibriosis in fish, shellfish, mollusks, and crustaceans [6]. Vibriosis is hemorrhagic septicemia, including various symptoms such as enlarged abdominal, internal and external lesions, loss of appetite, and erythema [7]. Significantly, the larval stage of fish is most exposed to disease caused by V. anguillarum, since the immune system of the fish larvae is not fully developed against bacterial infections [8]. Human infections caused by V. anguillarum are not common but can be fatal in immunocompromised patients [9].

In 2013, South Korea announced the country’s first V. anguillarum outbreak in rainbow trout (Oncorhynchus mykiss) [10]. There have been outbreaks of V. anguillarum vibriosis in fish and shellfish species in several countries, including the USA, Japan, Canada, Norway, Spain, Taiwan, Denmark, and Scotland [11]. In previous studies, Vibrio infections were reported in mullets, and most cases were caused by the V. anguillarum and V. parahaemolyticus [12, 13]. The pathogenicity of bacteria is defined by the ability to produce virulence factors expressed by virulence genes [14]. Even though several typical virulence factors have been described in V. anguillarum, they are not well characterized. They are motility, extracellular secretions associated with hemolysis and hydrolysis of proteins, lipopolysaccharides, chemotaxis, and multiple iron uptake systems responsible for causing diseases in the host [15].

Antimicrobial resistance of Vibrio spp. is another crucial factor that can enhance their pathogenicity. In accord with Pedersen et al. and Parin et al. [16, 17], V. anguillarum showed antimicrobial resistance towards various antimicrobials such as cloxacillin, ampicillin, sulfamethoxazol-trimethoprime, erythromycin, cephalothin, lincomycin, spiramycin, and colistin. In addition, industrial pollutants like heavy metals have become a considerable threat to aquaculture because heavy metals can be absorbed by marine bacteria and accumulate in the food chain [18]. Copper (Cu), cobalt (Co), cadmium (Cd), mercury (Hg), and chromium (Cr) have been detected as common metal pollutants in aquaculture [19]. Acquisition of heavy metal resistance genes in pathogenic Vibrio spp. linked with antimicrobial resistance properties and vice versa through the co-selection pathways [20].

The current study aims to evaluate the identification and occurrence of virulence markers, antimicrobial resistance, and heavy metal resistance properties of V. anguillarum isolated from mullets cultured in South Korea. We have reported phenotypic and genotypic characteristics of V. anguillarum to be aware of the potential pathogenicity associated with V. anguillarum in aquaculture and the potential health risk associated with consumers.

Materials and methods

Collection of fish samples and necropsy analysis

One hundred forty-five (145) M. cephalus fish samples with clinical symptoms of exophthalmia, hemorrhages on the mouth, operculum, and skin were collected from several fish farms in South Korea in 2020 under a routine pathological examination. Fish samples were packed separately in polythene bags and brought to the lab in a chilled condition. Necropsy analysis was done for all the fish samples.

Isolation and biochemical identification of Vibrio spp.

Bacteria were isolated from the spleen and kidney tissues of each fish by directly streaking on blood agar (MB Cell, Seoul, South Korea) supplemented with 5% (v/v) sheep blood and tryptic soy agar (TSA) (MB Cell, Seoul, South Korea) supplemented with 1.5% (w/v) NaCl and the media were incubated at 25 °C for 48 h. The most significant bacterial colonies were selected and re-inoculated to culture media for obtaining pure culture and processing for further identification. Bacterial identification was made based on standard tests using the Gram staining property, colony morphology, catalase and oxidase reaction, hemolysis, and pigment production [21]. Primary colonies were Gram-negative and curved rod shape on microscopy observation, hemolytic, white pigmented on blood agar and catalase oxidase positive. These colonies were cultured in the thiosulfate citrate bile salt sucrose (TCBS) agar (MB Cell, Seoul, South Korea) plates and incubated for 24 h at 37 °C. Yellowish or greenish colonies from TCBS media were stabbed and streaked onto triple sugar iron agar (TSI: MB Cell, Seoul, South Korea) tubes. The positive isolates with alkaline or acidic slant with the acidic butt (TSI test) were checked with an oxidase test kit (MB cell, Seoul, South Korea). Oxidase-positive isolates were subjected to a vibriostatic disc diffusion test using DD15 0129 (150 µg) discs (Oxoid, Hampshire, UK) and isolates that are sensitive to the vibriostatic test were biochemically identified as Vibrio spp.

Molecular identification of V. anguillarum

Biochemically identified Vibrio spp. were subjected to PCR assays to identify V. anguillarum isolates. Genomic DNA (gDNA) was extracted from presumptively identified Vibrio spp. isolates using the AccuPrep® gDNA extraction kit (Bioneer, South Korea). An initial PCR assay was conducted to identify samples at the Vibrio genus level by recognizing the partial recombinase A (recA) gene [22]. After that, species-level identification was done by PCR amplification of V. anguillarum-specific primer pair (forward primer van-ami8 5′-ACATCATCCATTTGTTAC-3′ and reverse primer van-ami417 5′-CCTTATCACTATCCAAATTG-3′) [23]. A total of 50 µL reaction mixture were used for the PCR assay. It consists of 250 µM of each deoxyribonucleoside triphosphate (dNTP), 10 pmol of each primer, 5 µL of 10X Taq buffer with MgCl₂, 0.5 U of Taq DNA polymerase (GeneAll, Seoul, South Korea), and distilled water up to 50 µL. The PCR thermocycling with van-ami8 and van-ami417 primer pair involved one initial cycle of denaturation at 95 °C for 10 min, followed by 25 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s, and finally, one cycle of 72 °C for 7 min (MultiGene Optimax, USA). The PCR products were electrophoresed on a 1.5% (W/V) agarose gel electrophoresis in 1X TAE buffer with RedSafe™ (Intron Biotechnology) and visualized under UV light. A 100-bp DNA ladder (Cosmo Genetech, South Korea) was used as the molecular weight marker.

Phenotypic pathogenicity test

Previously identified 22 isolates of V. anguillarum were subjected to eight phenotypic pathogenicity tests to determine their virulence factors. All isolates were maintained in the TSA (MB Cell, Seoul, South Korea) with 2% (wt/vol) NaCl for the following pathogenicity tests. TSA supplemented with 0.5% (wt/vol) skim milk was used to detect caseinase production [24]. The clear halos around the bacterial colonies were identified as positive results for caseinase production. The gelatin medium consists of gelatin (120 g/L), peptone (5 g/L), beef extract (3 g/L), and 5% peptone was used to detect gelatinase activity [25]. The inability to solidify the gelatin medium after incubation with the bacteria revealed positive results for gelatinase activity. Amylase activity was examined by adding Gram’s iodine onto the colonies grown in TSA supplemented with 0.2% (wt/vol) soluble starch. After overnight incubation at 37 °C, clear halos around the colonies were identified as positive results [26]. Isolates were streaked on DNase (MB Cell, Seoul, South Korea) test agar to detect DNase activity. 1 N HCl was added to the well-grown colonies and the positive result for the DNase activity was identified by the formation of clear opaque halos around the colonies. TSA supplemented with 1% (vol/vol) Tween 80 and TSA with 5% (vol/vol) egg yolk emulsion (MB Cell, Seoul, South Korea) were used to detect lipase and phospholipase activity [25]. The opaque halo effect around the colonies was considered positive results for phospholipase and lipase activities. Congo red uptake of the TSA media supplemented with 0.08% (wt/vol) Congo red and 5% (wt/vol) sucrose was used to detect slime production [27]. Color changes of the medium and black color colonies were identified as positive results for slime production. Hemolysin activity was determined by streaking the strains on a TSA medium (MB Cell, Seoul, South Korea) supplemented with 5% (v/v) sheep blood. Positive results for α-hemolysis and β-hemolysis were identified as greenish discoloration and a clear zone around the colonies, respectively.

Antimicrobial susceptibility test

The disc diffusion test was performed to determine the antimicrobial susceptibility of V. anguillarum. A total of 22 antimicrobial agents under the following antimicrobial groups were tested. Penicillins: ampicillin (10 μg), amoxicillin (15 mg), oxacillin (30 μg), and ticarcillin (75 μg); tetracyclines: tetracycline (30 μg) and oxytetracycline (30 μg); phenicols: chloramphenicol (30 μg); aminoglycosides: gentamycin (10 μg), streptomycin (10 μg), amikacin (30 μg), and kanamycin (30 μg); macrolides: erythromycin (15 μg); quinolones: nalidixic acid (30 μg) and ciprofloxacin (50 μg); carbapenems: imipenem (10 μg) and meropenem (10 μg); ansamycins: rifampicin (5 μg); folate pathway inhibitors: sulfamethoxazole-trimethoprim (25 μg); cephalosporins: cephalothin (30 μg), cefoxtine (30 μg), and ceftriaxone (30 μg); phosphonic acid: fosfomycin (50 μg) (Oxoid LTD, Basingstoke, Hampshire, England). Antimicrobial resistance was determined according to the Clinical and Laboratory Standards Institute guidelines and Escherichia coli ATCC 25,922 was used as a quality control agent in the test [28]. The multiple antimicrobial resistance (MAR) index was calculated by dividing the number of antimicrobials to which the isolates were resistant (a) by the total number of antimicrobials tested (b) MAR = a/b [29].

Heavy metal tolerance assay

As previously mentioned by He et al. [30], the minimal inhibitory concentrations (MIC) of the tested heavy metals against the isolates were determined using the broth dilution testing method (microdilution). Five metal chlorides, including CuCl₂, PbCl₂, CrCl₃, CdCl₂, and HgCl₂ (Samchun, Seoul, South Korea), were used to examine the Cu²⁺, Pb²⁺, Cr³⁺, Cd²⁺, and Hg²⁺ resistance properties of V. anguillarum isolates. The concentrations of Cu²⁺, Pb²⁺, Cr³⁺, and Cd²⁺ ranged from 3200 to 625 gml⁻¹, while Hg²⁺ concentrations ranged from 400 to 0.78 gml⁻¹. In our study, the isolates were considered resistant if the MIC values exceeded that of the control strain (E. coli K-12) as described in He et al. [30].

Detection of virulence, antimicrobial resistance, and heavy metal resistance genes

The prevalence of ten virulence-related genes was screened by the conventional PCR method. Primer pair nucleotide sequences and PCR conditions are listed in Table S1 [31–34]. A 20µL of PCR mixture was used to detect each virulence gene. Each sample consists of 0.2 μL of AmpOne Taq DNA polymerase (GeneAll, Seoul, South Korea), 2 μL of 10 × Taq reaction buffer, 2 μL of dNTP mix, 1 μL of each reverse and forward primer, 1 μL of template DNA, and 12.8 μL of PCR water. 1.5% (wt/vol) agarose gel was used to check the PCR amplicons by gel electrophoresis. The same PCR conditions were used to detect sixteen antimicrobial resistance genes (ARG) including extended-spectrum β lactams resistance genes (bla_TEM, bla_SHV, bla_CTX-M, and bla_IMP), tetracycline resistance genes (tetA, tet, and tetE), plasmid-mediated quinolone resistance genes (qnrA, qnrB, and qnrS), aminoglycoside resistance genes (strA-strB, aphAI-IAB, and aac(3′)-IIa) and integrons (Intl1 and gene cassette) (Table S2) [35, 36], and four heavy metal resistance genes including copA, CzcA, ChrR, and merA (Table S3) [37–39].

Results

Identification V. anguillarum by PCR

In this study, 145 infected fish samples were collected from February to April 2020 in several fish farms in South Korea. A total of 22 strains of V. anguillarum were isolated and identified using biochemical tests and PCR amplification of V. anguillarum-specific genes. The information related to all the strains are summarized in Table 1.

Table 1.

Sample collection data, antimicrobial resistance, intermediate resistant antimicrobials, and MAR index values of V. anguillarum isolated from mullets (M. cephalus)

No	Sample collection date	Location	Organ	Isolate No	Resistant antimicrobialsa	Intermediate resistant antimicrobials	MAR index
1	February 5, 2020	Hadong dachi	Spleen	Va1	AMP, OX, TIC, STR, ERY, NAL, CIP	AMOX, OTO, CMP, IPM	0.33
2	February 5, 2020	Hadong dachi	Kidney	Va2	AMP, AMOX, OX, TIC, STR, ERY, NAL, CIP, IPM	OTO	0.43
3	February 5, 2020	Hadong dachi	Kidney	Va3	AMP, OX, TIC, STR, NAL, CIP	OTO, ERY, IPM	0.29
4	February 5, 2020	Hadong dachi	Spleen	Va4	AMP, OX, TIC, STR, ERY, NAL, CIP	AMOX, OTO, IPM	0.33
5	March 13, 2020	Namhe chansan	Spleen	Va5	AMP, OX, TIC, STR, ERY, NAL, CIP, KF, FOS, IPM	AMOX, OTO, KAN,	0.48
6	March 13, 2020	Namhe chansan	Kidney	Va6	AMP, AMOX, OX, TIC, STR, AMK, ERY, NAL, CIP, IPM	OTO, GEN, FOS	0.48
7	March 13, 2020	Namhe chansan	Spleen	Va7	AMP, OX, TIC, STR, NAL, CIP, FOS, IPM	OTO, ERY, FOX,	0.38
8	March 13, 2020	Namhe chansan	Kidney	Va8	AMP, OX, TIC, STR, NAL, CIP, FOS,	AMOX, OTO, ERY, FOX, IPM	0.33
9	March 13, 2020	Namhe gamam	Spleen	Va9	AMP, AMOX, OX, TIC, STR, NAL, CIP, IPM	OTO, GEN, ERY, FOX, FOS	0.38
10	March 13, 2020	Namhe gamam	Kidney	Va10	AMP, OX, TIC, STR, ERY, NAL, CIP	AMOX, OTO, FOX	0.33
11	March 13, 2020	Namhe gamam	Spleen	Va11	AMP, OX, TIC, STR, ERY, NAL, CIP, IPM	OTO, FOX	0.38
12	March 13, 2020	Namhe gamam	Kidney	Va12	AMP, OX, TIC, STR, NAL, CIP, IPM	AMOX, OTO, AMK, ERY, KF	0.33
13	April 6, 2020	Sacheon	Spleen	Va13	OX, TIC, STR, ERY, CIP, IPM	AMP, AMOX, OTO, CMP, NAL, FOX	0.29
14	April 6, 2020	Sacheon	Kidney	Va14	AMP, AMOX, OX, TIC, STR, NAL, CIP, IPM	OTO, ERY, FOX	0.38
15	April 6, 2020	Sacheon	Spleen	Va15	AMP, AMOX, OX, TIC, STR, ERY, NAL, CIP, IPM	TET, OTO, KAN, KF	0.43
16	April 6, 2020	Sacheon	Kidney	Va16	AMP, OX, TIC, STR, ERY, NAL, CIP, IPM	OTO	0.38
17	April 21, 2020	Hadong Mibeob	Spleen	Va17	AMP, AMOX, OX, TIC, OTO, STR, NAL, CIP, IPM	CMP, ERY	0.43
18	April 21, 2020	Hadong Mibeob	Kidney	Va18	AMP, AMOX, OX, TIC, OTO, STR, NAL, CIP, IPM	CMP, ERY	0.43
19	April 21, 2020	Hadong Mibeob	Spleen	Va19	AMP, AMOX, OX, TIC, STR, NAL, CIP, IPM	OTO, ERY	0.38
20	April 21, 2020	Hadong Mibeob	Kidney	Va20	AMP, AMOX, OX, TIC, STR, NAL, CIP, IPM	OTO, ERY	0.38
21	April 21, 2020	Hadong Mibeob	Spleen	Va21	AMOX, OX, TIC, STR, NAL, CIP, IPM	AMP, OTO, ERY	0.33
22	April 21, 2020	Hadong Mibeob	Kidney	Va22	AMOX, OX, TIC, STR, NAL, CIP, IPM	AMP, OTO, ERY	0.33

^aAntimicrobials = AMP, ampicillin; AMOX, amoxicillin; OX, oxacillin; TIC, ticarcillin; TET, tetracycline; OXY, oxytetracycline; CMP, chloramphenicol; STR,streptomycin; FOS, fosfomycin; GEN, gentamycin; KAN, kanamycin; AMK, amikacin; ERY, erythromycin; NAL, nalidixic acid; CIP, ciprofloxacin; RD, rifampicin; FOX, cefoxitin; KF, cephalothin; CRO, ceftriaxone; SXT, trimethoprim-sulfamethoxazole; OT, oxytetracycline. MAR, multiple antimicrobial resistance

Determination of pathogenicity by phenotypic tests

The results of the eight-pathogenicity tests are summarized in Table 2. According to the results, all the strains were positive for the amylase and caseinase tests. Among all the tested isolates, 90.9% and 45.5% of the strains showed positive results for gelatinase and DNase activity. In the hemolysis test, 81.1% of test strains were screened as α-hemolytic strains, while 18.2% of strains were reported as β-hemolytic strains. None of the strains was positive for lipase, phospholipase, and slime production.

Table 2.

Phenotypic and genotypic pathogenicity profile of V. anguillarum

Phenotypic profile		% (No. of isolates/total no.of isolates)	Genotypicprofile	% (No. of isolates/total no.of isolates)
Amylase		100.0 (22/22)	VAC	54.5 (12/22)
Caseinase		100.0 (22/22)	ctxAB	45.5 (10/22)
Gelatinase		90.9 (20/22)	AtoxR	40.9 (9/22)
DNase		45.5 (10/22)	Tdh	31.8 (7/22)
Lipase		0	Tlh	27.3 (6/22)
Phospholipase		0	Trh	22.7 (5/22)
Slime production		0	Vfh	18.2 (4/22)
Hemolysis	α	81.8 (18/22)	hupO	13.6 (3/22)
	β	18.2 (4/22)	VPI	9.0 (2/22)
			FtoxR	9.0 (2/22)

Prevalence of virulence-related genes

Ten virulence-related genes were analyzed by the PCR, as shown in Table S1 31–34. The highest prevalence of virulence gene tested was VAC (54.5%). Other virulence-related ctxAB, AtoxR, tdh, tlh, trh, Vfh, hupO, VPI, and FtoxR genes were detected in 45.5%, 40.9%, 31.8%, 27.3%, 22.7%, 18.2%, 13.6%, 9.0%, and 9.0% respectively (Table 2).

Antimicrobial susceptibility pattern and MAR index

Table 1 shows antimicrobial resistance patterns and MAR indexes derived from disc diffusion assay. All the isolates were resistant to oxacillin, ticarcillin, streptomycin, and ciprofloxacin. Resistance to nalidixic acid, ampicillin, imipenem, amoxicillin, erythromycin, fosfomycin, oxytetracycline, cephalothin, and cefoxitin were recorded in 95.5%, 86.4%, 72.7%, 50.0%, 31.8%, 13.6%, 9.1%, 4.5%, and 4.5% of the isolates respectively. In contrast, all isolates were susceptible to rifampicin, trimethoprim-sulfamethoxazole, ceftriaxone, and meropenem.

Prevalence of antimicrobial-resistant genes

The antimicrobial resistance genes including qnrS (95.5%), qnrB (86.4%), and StrA-StrB (27.3%) were detected (Table 3).

Table 3.

Antimicrobial resistance genes of V. anguillarum

Gene	Isolates	%
qnrS	Va1, Va3, Va4, Va5, Va6, Va7, Va8, Va9, Va10, Va11, Va12, Va13, Va14, Va15, Va16, Va17, Va18, Va19, Va20, Va21, Va22	95.5
qnrB	Va3, Va4, Va5, Va6, Va8, Va9, Va10, Va11, Va12, Va13, Va14, Va15, Va16, Va17, Va18, Va19, Va20, Va21, Va22	86.4
StrAB	Va2, Va3, Va5, Va7, Va9, Va14	27.3

Heavy metal resistance

A total of 40.9% of the isolates were resistant to cadmium and 86.4% were positive for the CzcA heavy metal resistance gene. In contrast, none of the isolates showed Cu, Hg, Pb, and Cr tolerance for both phenotypic and genotypic tests (Table 4).

Table 4.

Genotypic and phenotypic results of heavy metal resistance of V. anguillarum

Isolate	Heavy-metal MIC (μg/mL)					Resisted phenotypes	Heavy-metal resistant genes
	Cu2+	Hg2+	Pb2+	Cr2+	Cd2+	-	-
E. coli K12	800	12.5	1600	800	400	-	-
Va1	50	3.125	100	800	800	Cd	CzcA
Va2	200	3.125	200	400	800	-	CzcA
Va3	100	3.125	200	400	400	-	CzcA
Va4	200	< 3.125	200	400	400	-	CzcA
Va5	200	3.125	200	400	400	-	CzcA
Va6	200	< 3.125	200	800	800	Cd	CzcA
Va7	100	< 3.125	200	400	400	-	CzcA
Va8	25	< 3.125	200	400	400	-	CzcA
Va9	100	< 3.125	200	800	400	-	CzcA
Va10	100	3.125	200	400	400	-	CzcA
Va11	100	< 3.125	200	400	800	Cd	CzcA
Va12	100	< 3.125	200	400	800	Cd	-
Va13	100	< 3.125	100	400	800	Cd	-
Va14	100	3.125	100	400	800	Cd	CzcA
Va15	100	< 3.125	100	400	800	Cd	CzcA
Va16	10	< 3.125	100	400	800	Cd	CzcA
Va17	100	3.125	200	400	400	-	CzcA
Va18	100	< 3.125	200	400	400	-	-
Va19	100	< 3.125	200	400	400	-	CzcA
Va20	100	< 3.125	100	400	400	-	CzcA
Va21	100	< 3.125	100	400	400	-	CzcA
Va22	100	6.25	200	400	800	Cd	CzcA

Discussion

Vibriosis is a considerable threat that caused significant economic losses in aquaculture. Among the pathogenic Vibrio spp., V. anguillarum is the most critical causative agent that causes vibriosis in marine fish and shellfish [12, 13]. Most vibriosis outbreaks were reported during summer and some cases were reported in late spring and early autumn in South Korea [40]. In this study, we studied 22 isolates of V. anguillarum isolated from infected mullets (M. cephalus) cultured in several fish farms located in South Korea. All isolates were subjected to phenotypical and genotypical assays to detect virulence, antimicrobial resistance, and heavy metal resistance properties.

Pathogenicity of V. anguillarum depends on the various virulence factors such as siderophore-mediated iron sequestering systems, motility of bacterial strain, and attachment [15]. In addition, the production of extracellular enzymes such as caseinase, protease, gelatinase, lipase, hemolysins, cytotoxins, dermatotoxin, metalloprotease, and hemagglutinin plays important role in pathogenesis of V. anguillarum infection [5]. In this study, V. anguillarum isolates (100%) were positive for the production of caseinase and amylase. Similar results were observed by Santos et al., Ma et al., and Younes and Gaafar [41, 42, 43]. Among all the tested isolates, 90.9% and 45.5% of the strains showed positive results for gelatinase and DNase activity. In the hemolysis test, 81.1% of test strains were screened as α-hemolytic strains, while 18.2% of strains were reported as β-hemolytic strains. None of the strains was positive for lipase, phospholipase, and slime production.

In contrast to Younes and Gaafar [43], no isolates were positive for lipase and phospholipase activity in our study. Moreover, previous studies showed that Vibrio species have the potential to produce various extracellular enzymes, including caseinase, gelatinase, hemolysins, lipase, phospholipase, amylase, and collagenase [44, 45]. Caseinolytic activity is related to producing potentially fatal toxins [44]. Gelatinase enzymes can degrade collagen, hemoglobin, and biologically active peptides in host cells. DNase is responsible for the hydrolyzation of DNA and propagation of bacterial cells while promoting their pathogenicity [46]. Alpha hemolysis activity reduces the host resistance by inhibiting phagocytosis, whereas beta hemolysis destroys the red blood cells (RBC) in hosts by depleting iron from RBC. Some isolates of V. anguillarum showed both alpha and beta hemolysis in the present study. Hemolysis activity is one of the common virulence factors presented by Vibrio spp. [47, 48].

Virulence genes indicate virulence factors that allow bacteria to infect and damage hosts. Inherited (typical) virulence and atypical virulence genes can be present in pathogenic bacteria [49]. This study contains ten virulence-associated genes, including VAC, ctxAB, AtoxR, tdh, tlh, trh, Vfh, VPI, hupO, and FtoxR which were positive as virulence markers. The highest prevalence of the tested virulence gene was VAC (54.5%). The VAC gene, specific to V. alginolyticus, is responsible for collagenase enzyme production. Previous studies reported that the VAC gene was highly presented in Vibrio spp. [50, 51].

Vibrio spp. can acquire new (atypical) virulence genes from other pathogenic bacteria or environments through horizontal gene transfer (HGT) [52]. The ctxAB gene is encoded for V. cholera-specific toxin [53]. Even though the ctxAB gene is specific to V. cholera, some previous studies and our study showed the prevalence of the ctxAB gene in other Vibrio spp. such as V. alginolyticus, V. parahaemolyticus, V. harveyi, V. diabolicus, and V. anguillarum [35, 36]. The toxR gene has an essential task in Vibrio spp. as it can be controlled the expression of other virulence genes like ctxAB in Vibrio spp. [54]. This study recorded the V. alginolyticus-specific toxR (AtoxR) gene as the third-highest virulence gene. In contrast, V. fluvialis- specific toxR (FtoxR) virulence gene presented in the lowest amount.

The tdh gene acts as an identification indicator of the pathogenicity of Vibrio spp. which is responsible for the thermostable direct hemolysin [55]. The virulence-related genes, including tdh, tlh, and trh, were reported in Vibrio spp. isolated from fish [56, 57]. In agreement with Doris et al. [58], tdh, tlh, and trh genes were found in several isolates in our study. Moreover, vfh and hupO virulence genes associated with the production of hemolysis in Vibrio spp. were detected in low percentages compared to the previous studies [59, 60].

Therapeutic substances like antimicrobials are used to treat bacterial infections in animals and humans [61]. Antimicrobial-resistant pathogenic microorganisms are formed due to the excessive and improper use of antimicrobials in aquaculture, agriculture, livestock, and human medical fields [62]. Vibrio infections in aquaculture are generally treated with antimicrobials like aminoglycosides, tetracyclines, fluoroquinolones, and third-generation cephalosporins [63].

In our study, 22 antimicrobials were screened in the antimicrobial susceptibility test. All the isolates were reported as multi-drug resistance (MAR) and scored MAR index > 0.2, which is the threshold value to identify the efficacy of antimicrobial activity [64]. In addition, oxacillin, ticarcillin, streptomycin, and ciprofloxacin were found as resistant antimicrobials in all V. anguillarum isolates. Also, more than 50% of the isolates were resistant to nalidixic acid, ampicillin and imipenem (Fig. 1).

Fig. 1.

Antimicrobial susceptibility profile of V. anguillarum isolated from mullet (Mugil cephalus) cultured in Korea. (Resistant Intermediate resistant Susceptible)

Several countries, including Japan, Turkey, China, Norway, and Egypt, have studied the antimicrobial resistance of V. anguillarum [17, 43, 64, 65]. In accordance with the previous study by Parin et al. [17], our study reported similar results that V. anguillarum isolates were resistant to ampicillin and amoxicillin. Similarly, Vaseeharan et al.[64] found that all the isolates were resistant to ampicillin and showed resistance at low rates against nalidixic acid, ciprofloxacin, erythromycin, gentamicin, and oxytetracycline. Moreover, Pedersen et al. [16] revealed that most of the O2 strains of V. anguillarum were resistant to ampicillin, cephalothin, chloramphenicol, and spiramycin.

Most gram-positive and gram-negative bacteria were susceptible to broad-spectrum antimicrobials like carbapenems (meropenem and imipenem) [65]. In our study, meropenem, rifampicin, ceftriaxone, and trimethoprim antimicrobials were effective against all the isolates of V. anguillarum. Also, most isolates were susceptible to tetracycline, chloramphenicol, gentamycin, kanamycin, and amikacin. Younes and Gaafar [43] reported that V. anguillarum isolates were susceptible to amoxicillin, oxytetracycline, erythromycin, novobiocin, chloramphenicol, and nalidixic acid. Different antimicrobial resistance qualities may exist depending on how the antimicrobials are used in a specific area [66].

ARGs are mobile genetic elements that can transfer from one pathogenic bacterium to another via vertical or HGT [67]. They are responsible for the antimicrobial resistance properties of pathogenic bacteria [68]. The ARGs including qnrS (95.5%), qnrB (86.4%), and StrAB (27.3%) were detected in our study (Table 3). Plasmid-mediated qnr (PMQR) genes confer resistance to quinolone. Three types of resistance mechanisms are employed to protect bacterial cells from quinolone [69]. They are enhanced efflux pumps by PMQR genes, quinolones hydrolysis by aac(6′)-Ib-cr protein, and protection of quinolones target sites by qnr proteins [70, 71]. In addition, some of the isolates tested positive for streptomycin resistance genes (StrAB) in our findings.

The most frequently used class of antimicrobials in aquaculture is beta-lactams. Even though several studies reported beta-lactam antimicrobial genes in Vibrio spp., none of the isolates was positive in our study [72, 73]. In addition, integrons are genetic elements that engage the antimicrobial resistance properties of pathogenic bacteria by integrating and mobilizing the gene cassettes [74]. Similar to the study done by Jeamsripong et al. [75], PCR amplicon was not detected for intl1, integron gene cassettes in our research. The non-appearance of gene cassettes in this study may be caused by early stop codons, a shortage of resistance determinants in gene cassette arrays, a lack of 3′ conserved regions, or a combination of these factors [76].

Heavy metal resistance can be triggered by anthropogenic elements like heavy metals or antimicrobial resistance genes and vice versa [77]. This phenomenon is called co-selection, which is driven by the mechanism of co-resistance [78]. In our study, V. anguillarum isolates were tested against five heavy metals (Pb, Cr, Cu, Cd, and Hg). A total of 40.9% of the isolates were resistant to cadmium and 86.4% were positive for the CzcA heavy metal resistance gene. None of the isolates showed Cu, Hg, Pb, and Cr resistance for both phenotypic and genotypic tests. However, a study by He et al.[30] reported a significant prevalence of Cu and Cr-resistant V. parahaemolyticus isolated from shrimps in China. Also, antimicrobial-resistant Vibrio spp. positive for copA, copB, copC, and nccA heavy metal resistance genes in a previous study [79]. Moreover, Ba, Cd, Co, and Cu resistance were confirmed in V. parahaemolyticus isolated from oysters in Korea [80]. In contrast, none of the V. anguillarum isolates exhibited Cu or Cr resistance in our study. Similarly, all the V. anguillarum isolates were sensitive to the heavy metals Cu, Zn, and Cd in the previous study [81]. Among 19 CzcA gene-positive isolates, only nine isolates were tolerant to cadmium phenotypically in this study. The CzcA gene, which encodes both the cadmium resistance and the cation reflux system that removes metal cations from bacterial cells, can lead to this disagreement [82]. Regardless of cadmium susceptibility, isolates were positive for the CzcA gene [83]. According to the findings of our study, only cadmium among the examined heavy metals was resistant to V. anguillarum. The excessive cadmium exposure in the mullet fish farms may be the reason for this outcome.

Conclusion

In this study, we focused on the exposure of mullets with V. anguillarum and the molecular characterization of isolated strains for the first time in South Korea. The results revealed that V. anguillarum could not be underrated as a pathogenic bacterium in aquaculture because of its virulence factors, antimicrobial resistance properties, and heavy metal resistance properties. The prevalence of virulence determinants showed the pathogenicity of the bacteria and their ability to extend the host range. Mainly, multi-drug resistance properties may impede clinical therapeutics and cause many difficulties in medicating bacterial infections. Therefore, it is necessary to prevent the over usage of antimicrobials by introducing alternative solutions (probiotics, bacteriophage, and bacteriocins), controlling the use of anthropogenic elements like heavy metals and introducing good practices in management through aquaculture to improve the quality and safety of aquaculture production and safety of the consumer’s health.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

PMK and GJH contributed to the conception and design of the study. Material preparation, data collection, and analysis were performed by PMK, SM, LADSDS, and GJH. The first draft of the manuscript was written by PMK, and all authors contributed to reviewing and editing the manuscript. All authors read and approved the final manuscript.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethical approval

Not applicable.

Conflict of interest

The authors declare no competing interests.