Article Type : Research Article
Authors : Chakraborty AK, Halder C and Maity U
Keywords : Vibrio parahaemolyticus; Shrimp aquaculture; mdr genes; blaCARB gene; Penicillin-binding protein; PirAB toxins
The overuse of antibiotics in aquaculture eventually leads to antimicrobial resistance (AMR) in bacterial strains found in shrimp. About million MDR plasmids were sequenced from diverse bacteria and classified into bla, aph, aac, aad, tet and sul genes classes. The Vibrio parahaemolyticus was the main culprits for scrimp fish contamination and mortality. The blaPER-1, blaOXA-1, blaNDM-1, dhfr, aacA1, sul1, su12, arr3, aac3’-IId, aac6’-IIa, ANT3”, tetB, qnr1, mphA, catB3 mdr genes were sequenced from shrimp-derived V. parahaemolyticus (pVPS43, pVPH2, pVPS129) and V. alginolyticus (pVAS19, pC1394) and surprisingly were 100% homology to Escherichia, Klebsiella, Acinetobacter, Enterobacter, Shigella species plasmids suggesting horizontal gene transfer. Plasmid-mediated PirA/B toxin genes used to detect V. parahaemolyticus in shrimp and was responsible for acute hepatopancreatic necrosis disease (AHPND). Recently, AHPND-plasmids (pVa, pVA1, pVHvo, pVPE619, pVPGD2014-2 and pVPGX2015-2) were sequenced from V. parahaemolyticus as well as V. owensii and V. harveri but no mdr genes was detected. Similarly, tdh, trh and tlh virulence genes also used for diagnosis to cause membrane pore formation and located in both chromosomes and plasmids of Vibrio species. We searched NCBI database of V. parahaemolyticus genomic fragments and found very specific chromosomal mdr genes like blaCARB (carbanicillin specific beta-lactamase), PBP1B (Penicillin-binding protein) and CatC1 (chloramphenicol acetyltransferase) to design PCR primers. Further, few MDR drug efflux genes (macB, MFS, RND, emrD) and rRNA methyl transferases (RlmE, RlmM, RlmN, RsmA) were also detected to cause multi-drug resistance. BLAST search indicated that primers were very specific for V. parahaemolyticus Ch-1 or Ch-2 and had no similarities to any plasmids. In appeared toxin, virulence and mdr genes hardly located in the same V. parahaemolyticus plasmid.
We are 8000 million people in this Earth and to fed entire population is a hard task as still malnourishment prevails in West Asia, Africa and Latin America [1,2]. The fish food is rich in protein and well tolerated worldwide. Recently, mass aquaculture of Telapia, Carp, Catfish, Trout and Shrimp were taken worldwide . The shrimp was elegantly called fish-chicken and now very much popular in East India due to high demand abroad with good prise . The West Bengal low land area was being hostile for paddy cultivation due to heavy flood in rainy season and such lands were converted into shrimp ponds very quickly. The main problem of shrimp cultivation and export are: (i) healthy and nutritious fish (ii) antibiotic residue like nitrofurans in shrimp (iii) MDR bacterial contamination specifically Vibrio species and Staphylococcus aureus and (iv) presence of toxin and virulence genes in Vibrio parahaemolyticus. In truth, farming region, water source, dead fish removal frequency, antibiotic treatment and virus or bacteria contamination were all found to be significantly associated with shrimp mortality [5,6].
Shrimp population and cultures
Diseases of shrimp aquaculture
Vibrio parahaemolyticus is a marine pathogen and greatly affect shrimp aquaculture. The acute hepatopancreatic necrosis disease (AHPND) is a devastating disease that significantly affects aquaculture production of shrimp fish  (Figure 2). Photorhabdus insect-related (Pir) toxin-like genes in plasmid of V. parahaemolyticus is the causative agent of AHPND of shrimp. Lee et al elegantly showed that an AHPND-causing strain of V. parahaemolyticus contained a 70-kbp plasmid (pVA1; 70kb) with a post-segregational killing system. PirAB toxin was found in plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins, PirA and PirB. The toxin is related to Cry toxin and insecticidal Pir homologs were found in Photorhabdus and Xenorhabdus species chromosomes (FM162591.1, FN667742.1, and FO704550.1), whereas PirABvp is the only toxin to be encoded by a plasmid. Besides V. owensii, V. harveyi and V. parahaemolyticus, similar Tn903-like composite transposons were also detected in plasmid p67vangNB10 (accession no. LK021128) of V. anguillarum and also in the whole genomes of various strains of the Harveyi clade (CP006700, CP006701, CP006606 and CP000790) . Recently, V. owensii AHPND-plasmid pVHvo was sequenced (accession number: KX268305) and very identical to V. parahaemolyticus plasmids. Interestingly, both plasmids had no mdr gene. However, V. harveyi, V. alginolyticus, V. anguillarum, V. splendidus, V. salmonicida, V. vulnificus and non-AHPND causing V. parahaemolyticus that cause vibriosis.
The PirAvp corresponds to domain III of the Bacillus thuringiensis Cry toxin and PirBvp corresponds to domains I and II. The Cry toxin induces cell death by undergoing a series of processes that include receptor binding, oligomerization, and pore forming . The B. thuringiensis Cry1A toxin domain III first interacted with the GalNAc sugar on the aminopeptidase N (APN) receptor facilitating further binding of domain II to another region of the same receptor. The APN-bound Cry toxin subsequently binds to another receptor, cadherin, which facilitates the proteolytic cleavage of its domain I?1 helix. This cleavage induces the formation of Cry oligomer, which has pore-forming activity . Interestingly, such interactions trigger an alternative signal transduction pathway activating protein kinase G and adenylyl cyclase to increase cellular cAMP concentration destabilizing the cytoskeleton and ion channels on the membrane to cause cell death . It was postulated that PirAvp/PirBvp system used a similar strategy to kill host cells . V. cholerae neuraminidase (EC 220.127.116.11) releases sialic acid from higher gangliosides present on eukaryotic cells surface, exposing ganglioside GM1, which is the cholera toxin receptor and thus activates cholera toxin function .
Contaminated sea food human diseases
Human seafood-associated bacterial gastroenteritis is caused by Vibrio parahaemolyticus in many countries including United States and India . The diseases produced by Vibrio bacteria is known as vibriosis and the symptoms include watery diarrhea, vomiting, abdominal cramping, nausea, fever, and chills . The Vibrio species are halophilic bacteria that are ubiquitous in sea, coastal areas and fish ponds. Many are pathogenic to human and marine animals, and three species, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae are responsible for seafood-related human illness . The V. vulnificus and V. parahaemolyticus are naturally occurring estuarine bacteria, that causes seafood-borne mortality in USA. It was reported by the United States Centre for Disease Control and Prevention (CDC) that the incidence of Vibrio infections increased dramatically since 2001 . In August 2012, a V. parahaemolyticus outbreak involving 6 persons occurred in Maryland and the outbreak isolates were linked to the O3:K6 pandemic clone of V. parahaemolyticus that had been observed throughout the world . In July 30, 2014, ABC News reported several cases of V. vulnificus occurrence in Florida, where 32 people had contacted the bacteria and 10 had died according to the Florida Department of Health.
Drug resistance in Vibrios and MDR plasmids
In Thailand, oxytetracycline resistant Aeromonas species (4-128µg/ml) and Lactococcus species (~120µg/ml) were isolated from white leg shrimp (>25% samples) as well as black tiger shrimp (>10% samples). The TET resistance was found to be conferred by the genes tet(A), tet(C), tet(D), tet(E), tet(M) and tet(S) . Shrimp aquaculture V. parahaemolyticus isolates from Southern province of India revealed seven plasmids of 0.75, 1.2, 6, and 8 kb sizes and 3 plasmids greater than 10 kb. The bacteria were resistant to ampicillin (100), polymyxin (100), oxytetracycline (30), streptomycin (30), chloramphenicol (20-60), trimethoprim (10-60), nalidixic acid (100) but during October-January post monsoon season such resistance pattern showed inconsistent . However, both mdr genes and toxin (PirAB) genes were rarely located in same plasmids of Vibrio parahaemolyticus and such data was limited in the database. The Mexico AHPND-causing V. parahaemolyticus strain (13-306D/4 and 13-511/A1) were reported to carry tetB gene coding for tetracycline resistance gene, and V. campbellii from China was found to carry multiple antibiotic resistance genes .
Control of bacteria in shrimp ponds
In aquaculture, several strategies have already been applied to control Vibrio strains, including chemicals, probiotics, antibiotics, natural products from plants, including plant oils. The FDA approved oxacillin, florfenicol, erythromycin, oxytetracycline, sulfamerazine, and combination drugs, sulfadimethoxine and ormetoprim in fish aquaculture . The malachite green and chloramphenicol uses have reduced due to toxicities and antibiotic residue in shrimp. Similarly, floroquinolones were important drugs for human use and its use was contradicted in aquaculture due to development of drug resistance bacteria. The FDA also controls the use of nitrofurazone which may induce tumour in mammary glands [38,39]. et al showed that after 120 hrs post infection shrimp fed with a diet containing 2% of a mix with Curcuma longa and Lepidium meyenii (TuMa) and a diet containing 0.2% of vitamin C (Vit-C) showed a significantly higher survival (85%) of shrimp fishes as compared to the other treatments . The Gracilaria spp. (Gracilariaceae family) and Sargassum spp. (family Sargassaceae) have been used most for in vitro and in vivo experiments to control Vibrio species in shrimp ponds. Among the terrestrial plants, Eucalyptus camaldulensis, Psidium guajava, Rhodomyrtus tomentosa, and Syzygium cumini (Myrtaceae family) had significant activity against Vibrios [41,42]. Hannan et al. from Bangladesh screened twenty-one ethyl acetate plants extracts of which Emblica officinalis and Allium sativum were found strong inhibitory to Vibrio alginolyticus in vivo shrimp culture at 10mg/g feed . The antimicrobial peptide, vibriocin (18 KDa of molecular mass) was very effective controlling pathogenic Vibrio harveyi . The peptide acted stable in a wide range of pH, temperature, UV radiation, solvents and chemicals utilized . Chakraborty et al discovered a CU1 phyto-antibiotic that killed Mdr bacteria targeting RNA polymerase enzyme [46,47].
Other fish aquaculture
Nile tilapia (Oreochromis niloticus) cultivated in major aquaculture worldwide because the fish was easy to cultivate, adapts to a wide range of environmental conditions, grows fast with tolerant to stress and diseases . The Oreochromis species annual production reached >50MT world-wide. The reports suggested such fish was highly contaminated with MDR Aeromonas veronii [49,50]. Examples of emerging viruses in aquaculture include , , , , and . The TiLV (OM1 and OM2) virus isolated from tilapia fishes shared 94.30% and 95.52% nucleotide identity with the TiLV isolated from West Bengal, India (MF502419.1) and Israel (KJ605629.1). However, use of MYXV (Myxoma virus) and RHDV (rabbit haemorrhagic disease virus) was proposed as a potential BCA (Biological Control Agents) for (Cyprinus species) which are regarded as the most devastating invasive fish in Australia . Vibrio cholerae, V. parahaemolyticus, and V. vulnificus were identified in blue crab aquaculture . The acute hepatopancreatic necrosis disease (AHPND), also known as early mortality syndrome (EMS) was causing significant losses in shrimp production in the Southeast Asian countries due to PirAB toxins. This disease is caused by V. parahaemolyticus and affects the of infected shrimp with mortality up to 100%, in Litopenaeus vannamei and Penaus .
Materials & Methods
Growth of Vibrio species
The new chromogenic TCBS medium consists of 10 g of peptone, 10 g of sea salts mixture, 10 g of ox bile, 10 g of sodium thiosulfate, 5 g of yeast extract, 5 g of sodium citrate, 2.2 g of sodium carbonate, 2 g of lactose, 0.5 g of sodium pyruvate and 1000ml with water and PH adjusted to 8.6 and autoclaved at 15psi/15min .
Isolation of MDR bacteria and fish aquaculture
The Vibrio strains metabolize sucrose efficiently. The V. cholerae forms yellow colonies on TCBS agar, whereas other pathogenic species like V. parahaemolyticus and V. vulnificus produce green colonies in TSB agar plate . The pictures of colonies were taken in chromogenic agar plate and then confirmed by 16S rRNA sequencing.
For experiment, each group of fish (n?=?6/tank) were acclimatized in aquaria (120 × 30 × 45 cm) supplied with 120L freshwater and maintained at 35°C-370C with aeration for about 2 weeks. The fish were fed with a commercial diet (PT Central Protein, Prima) twice daily at a rate of 2% body weight. Water was 50% replaced and uneaten feed was siphoned daily. The bacteria were grown in Trypticase Soy Broth (TSB) overnight and the density of the bacterial suspension was enumerated using spread plate method on TSA. Each fish from the four groups was intraperitoneally injected with 0.1 ml bacterial suspension with a mean density of 1.0 × 107 cfu/ml. A control group was included where fish were injected with the same volume of sterile phosphate buffered saline (PBS). Clinical signs and morbidity were recorded daily for one week and the experiment was terminated when 100% morbidity or mortality occurred among the challenged groups. Newly dead or moribund shrimp were examined and tissues were inoculated onto Thiosulfate-Citrate-Bile Salt-Sucrose (TCBS) agar plate supplemented with 2% NaCl.
Identification of Vibrio parahaemolyticus
The green or bluish green colour colonies measuring about 3–5 mm was isolated from TCBS plate, and was inoculated into sterile sucrose medium supplemented with NaCl (3% w/v). Only sucrose non-fermenting colonies were streaked onto sterile tryptone soy agar slants supplemented with NaCl (3% w/v; TSAS) and maintained at room temperature for further identification. The isolates were confirmed to be V. parahaemolyticus based upon the ability to give typical biochemical reactions as listed in the USFDA (2001) viz., motile, no acid from sucrose, Gram (-), no H2S was produced on triple sugar iron agar, acetoin was not produced and grows in 3–8% NaCl but unable to grow in >10% NaCl. Each bacterium was further confirmed by RAPID Hi-Vibrio TM identification kit (KB007, HiMedia, India) and finally 16S rRNA sequencing could be performed from genomic DNA following BLAST search .
Few other sea food contaminations could be differentiated biochemically and many medium available from Himedia. The use of 6% NaCl in medium and biochemical tests for arginine dihydrolase and l-histidine decarboxylase can be useful to differentiate the growth of P. shigelloides from Vibrio species. Similarly, lysine decarboxylase and ornithine decarboxylase assays differentiate P. shigelloides from Aeromonas species and the cytochrome oxidase test differentiates P. shigelloides from other Enterobacteriaceae . Other biochemical identification tests used include indole, inositol, and glucose fermentation, production of ?-hemolysis, sensitivity to vibriostatic O/129 or a variety of commercial kits, such as API 20E, the Vitek 2 system, or the BD Phoenix
DNA extraction, PCR amplification and sequencing
Pure colonies were grown overnight in TSB medium at a concentration of 109 CFU/ml. Then 1.5 ml each culture was transferred into a microcentrifuge tube and centrifuged at 5000 rpm for 10 min. The pellet was re-suspended in 100µl Solution-I, 200µl Solution-II and 150µl Solution-III as described by Maniatis et al. 1989 . The pellet removed by centrifugation at 10,000 rpm/10min and 1ml ethanol was added. The pellet dried and suspended in TE buffer and four tubes combined into one tube and extracted with phenol-chloroform and treated with RNaseA and ethanol precipitated. The extracted gDNA was used to amplify the 16S rRNA genes using the universal primer set for prokaryotes . The PCR assay (30 ?l) contained a final concentration of 10x PCR buffer (XTPs 2mM), 0.5 mM of each primer, 2.5 U/?l of Taq DNA polymerase, 2?l of DNA sample and nuclease free water was added to achieve the total volume of PCR mixture. Then amplifications were carried out in a thermal cycler with an initial denaturation of 95°C for 3min, followed by 30 cycles of 94°C for 30 sec, 52°C for 90 sec, 72°C for 1.5min, and an additional final extension of 72°C for 7min. The expected PCR product of ~1,500bp was detected by electrophoresis in 1% agarose stained with ethidium bromide and photographed under UV light. Sequencing was done by automated dideoxy sequencer. Then the sequenced was BLAST searched to identify bacteria. The V. parahaemolyticus 16S rRNA gene (accession no. MZ015567) could be compared by Blast-2 search.
The pirAB genes PCR assay was developed for diagnosis of AHPND disease in shrimp . The Vp_PirAB-F (5’- GTG GAA ATG GTG AAC TTG CG-3’) and Vp_PirAB-R primer sequences (5’- GGC GTT GCA ATC TAA GAC AT-3’) were used for amplification of V. parahaemolyticus plasmid-derived PirAB genes (accession no. AB972427) the toxR-based PCR assay was preformed to identify V. parahaemolyticus from all the presumptive isolates. Detection of toxR gene was carried out using primer toxR-F (5?-ATA CGA GTG GTT GCT GTC ATG-3?) and toxR-R (5?-GTC TTC TGA CGC AAT CGT TG-3?) with the expected amplicon size of 368 bp (accession no. ABADIT010000001, nt. 466250-467128) . The detection of the genes tdh (Thermostable Direct Haemolysin) and trh (Thermostable direct-haemolysin Related Haemolysin) was done using the primer pairs TDHF (5-GTA AAG GTC TCT GAC TTT TGG AC-3?) and TDHR (5- TGG AAT AGA ACC TTC ATC TTC ACC-3?) for tdh and TRHF (5-TTG GCT TCG ATA TTT TCA GTA TCT-3?) and TRHR (5-CAT AAC AAA CAT ATG CCC ATT TCC G-3?) for trh . The tlh gene PCR was performed to confirm the identity of V. parahaemolyticus strains. The primers tlh-F (5' AAA GCG GAT TAT GCA GAA GCA CTG 3') and tlh-R (5' GCT ACT TTC TAG CAT TTT CTC TGC 3') were used to amplify a 450-bp fragment of the thermolabile haemolysin gene .
Plasmid-mediated mdr genes in shrimp-contaminated bacteria
Mdr genes in Vibrio parahaemolyticus plasmid pVPH1 (183730bp) was isolated from Shrimp fish (Hong Kong, 2015). The Dhfr protein is dihydro folate reductase gives trimethoprim resistance, Sul1 protein is dihydropteroate synthase, Mph protein is macrolide 2’-phosphotransferase gives aminoglycoside antibiotic resistance whereas blaPER is extended spectrum beta-lactamase that cleaves the beta-lactam ring of penicillin drugs and very much prominent in diverse bacterial species like Pseudomonas, Escherichia and Acinetobacter species (accession nos. EU022369,
Mdr genes was located in Vibrio alginolyticus plasmid pC1394 (167140bp; see, Figure 2C for plasmid structure). The bacterium was isolated from shrimp fish in China on 1st August, 2016 The Dhfr enzyme reduces 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate with NADPH as a cofactor. This is an essential step in the biosynthesis of deoxythymidine phosphate and gave resistant to trimethoprim antibiotic. The dihydropteroate synthase (Sul1) produces sulphonamide antibiotic resistance (Figure 4). The QnrA1 gene is quinolone resistant pentapeptide protein. The sul1 gene was also detected in V. cholerae strain CNRVC190247 chromosome-2 (accession number: OW443151) as well as in plasmid 3 (accession number: OW443149). But similar Blast search did not find sul1 gene in V. parahaemolyticus chromosomes (accession numbers: CP034305, CP043421, CP034295, and CP068648) and plasmids (KP324996, MH890610 and CP020036). The blaNDM-1 gene was first detected in a New Delhi patient in 2009 being a deadly mdr gene and could cleave ampicillin, oxacillin, cefotaxime as well as imipenem antibiotics (Figure 4). The blaNDM1 gene also detected in V. alginolyticus plasmids pC1394, pVb1762 and pVb2134 (accession nos. MH457126, OK146920 and OK085530) an also found in chromosome-1 of V. alginolyticus strain AUSMDU00064140 (accession no. CP110670).
The V. cholerae strain 116-17a plasmids pNDM-116-17 and pNDM-116-14 (accession nos. LN831185 and LN831184) also contained the blaNDM-1 gene to inactivate the all penicillin drugs. The blaNDM-1 gene was found in most bacterial plasmids and also in E. coli, P. mirabilis and Providencia species chromosomes (accession nos. CP053614, CP042861 and CP013483). A blaPSE4-type beta-lactamase gene located in shrimp-derived V. parahaemolyticus (protein id. NMT93259) and similar gene was found in Oyster-derived V. parahaemolyticus genomic fragments (Accession numbers: DACQME010000048, ABFJXO0000001 and AAXOFK010000086) with similar protein sequence (protein ids. HAS686330, EIZ0308401 and EGR348697) and isolated from United States in 2008, 2021 and 2013 respectively. The gene gave resistant to penicillin-G drug (see, accession no. SRKW010000001, nt. 140681-141532). However, blaTEM-1 beta-lactamase-containing plasmid pVSP43 was found in V. parahaemolyticus which was also isolated from shrimp (see, Figure 2C for plasmid structure).
The Salmonella enterica small plasmid-borne (2788bp; acc. no. KY399740) mdr genes also investigated (Figure 5). The bacterium was isolated from shrimp fish in China. The blaOXA gene cleaves oxacillin more efficiently than ampicillin. The catB3 gene acetylates chloramphenicol and acetylated drug does not able to bind ribosome to inhibit bacterial protein synthesis. The arr3 gene ribosylates rifampicin and ribosylated rifampicin does not able to bind RNA polymerase to inhibit transcription. The shrimp fish isolated V. parahaemolyticus showed resistant to as high as 5-12 antibiotics. The blaOXA-1 was found very similar to Klebsiella pneumoniae, Salmonella enterica and Shigella flexneri chromosomal genes (accession nos. DAGOGD010000073, CP034250 and ABGERN010000356) and could be used for V. parahaemolyticus diagnostic PCR . We compared the CatB3 protein of Salmonella plasmid with chromosomal catC1 protein of V. parahaemolyticus showing divergence and useful for primers design and diagnostics PCR (Table 1).
We looked V. parahaemolyticus genomic fragments and found blaCARB gene (Figure 6A) and penicillin binding protein gene (accession no. SRKW01000006, nucleotides 109851-112223; protein id. WP_0220835404) (Figure 6B). To access the heterogeneities among the conventional blaTEM-1, blaSHV-1 and blaOXA-1 enzymes as compared to blaCARB-1, we multialigneg the corresponding proteins and found strong differences that would be useful for primer design for V. parahaemolyticus (see, Figure 7A for antibiotics and corresponding genes developed naturally in bacterial plasmids and see, Figure 7B for multi-alignment data of beta-lactamases). So, blaCARB-1 was distinct and generated due to chromosomal rearrangement of V. parahaemolyticus. We also found few MDR transporters (protein ids. WP_025788558, WP_011106254) as well as MacB transporter (protein id. WP_025594350) pinpointing the multidrug resistance for penicillin (ampicillin) and macrolide (erythromycin) antibiotics in V. parahaemolyticus. Further, we detected few rRNA methyl transferasees in V. parahaemolyticus and V. Cholerae. As for example, 23S rRNA 2552Uridine 2’-O methyltransferase (RlmE; WP_015297227), 23S rRNA 1939Uridine C5-methyltransferase (WP_025789428) as well as 16S rRNA 16S rRNA 1207-Guanosine N2-methyl transferase (RsmC; WP_005479074), 16S rRNA 1518Adenine-1519Adenine-N6-di-methyltransferase (RsmA; WP_005459622) and23S rRNA 2498Cytidine 2’-O-methyltransferase (RlmM; MBE5158644). These rRNA methyltransferases may give resistance to drugs that binds ribosome (composed of 50 ribosomal proteins plus 23S, 16S and 5S RNAs) inhibiting protein synthesis of bacteria. Thus, we pinpointed the mechanism of multi-drug resistance in Vibrio species which seriously infected shrimp and other fishes in aquaculture and located in chromosome. Such mechanism appeared primitive as very few plasmids so far detected in V. parahaemolyticus (Figure 8).
Toxin genes in shrimp-contaminated bacteria
The PirA and PirB toxin genes located in many Vibrio parahaemolyticus conjugative plasmids and cause acute hepatopancreatic necrosis disease (AHPND) in shrimp [65,66]. As for examples, pirA (QHH18415) and pirB (QHH18416) located in plasmid pVPGX2015-2; pirA (QHH13410) and pirB (QHH13411) in plasmid pVPSD2016-5; pirA (AWG82359) and pirB (AWG82360) in plasmid pVpR14_74Kb; pirA (UJX11662) and pirB (UJX11663) in plasmid pVP17-1; pirA (QHH02797) and pirB (QHH02798) in plasmid pVPCZ2014-3 as well as pirA (QGT94608) and pirB (QGT94609) in plasmid pVP_Kor-D1-2 were well documented in NCBI GenBank Database. The similarly Vibrio owensii plasmid pVHvo has pirA (QGH51089) and pirB (QGH51090) proteins as well as plasmid pVa1 (accession number: CP097860) of V. parahaemolyticus (Figure 9). Vibrio campbellii strain LMB29 also has PirAB toxin genes in plasmid pVCON1 (accession number: MH890610) as well as in plasmid pVCGX1 (accession number: CP020078) but no tdh and trh virulence genes . Interestingly, no mdr genes located in pVA1, pVPE619, pVa, pVp_kor-D-1-2, pVHvo, pVPGD2014-1/2/3 and pVPGX2015-2 PirAB gene containing conjugative plasmids (accession numbers: KP324996, CP043423, CP034288, CP034293, CP034297, CP034308, AP014860 and KX268305). The Vibrio parahaemolyticus chromosomes were fully sequenced. As for examples, The PirAB genes located in Ch-1 from nt. 1276780-3063548 (accession number: CP034294, length 3358530bp). Such genome wide data suggested nt. 2500951-3163071 contained pirAB genes in accession number CP046831 and nt.2605940-2871943 in accession number CP028342 and also nt. 2605305-2871943 in accession number CP028341 of different V. parahaemolyticus strains [68,69].
Virulence genes in aquaculture fish ponds
The virulence genes tdh and trh were detected in two V. parahaemolyticus shrimp isolates from the Cochin estuary by multiplex PCR. Using 16S rRNA sequence analysis, one isolate exhibited 100 % similarity to the V. parahaemolyticus O3:K6 pandemic clone. TDH is a pore forming toxin and forms pores of ~2nm in diameter on erythrocyte membrane. These thermostable haemolysin-like proteins exert a variety of biological activities like haemolytic activity, enterotoxicity, cardiotoxicity and cytotoxicity. The trh and tdh genes share 70% homology and both proteins activate chloride channel. The TDH virulence factor is composed of four soluble monomers, in which a central pore is formed to allow the diffusion of small molecules, known as Kanagawa phenomenon (KP) (Figure 10). The Tdh+ strains of V. parahaemolyticus exhibit ?-haemolytic activity when plated on blood-agar media known as Wagatsuma agar. Purified TDH is heat stable at 100°C for 10 min . The TDH and TRH proteins cause haemolysis, enterotoxicity, cytotoxicity and cardiotoxicity in experimental animals. The TRH protein also causes haemolytic activity similar to that of TDH on blood cells (Figure 11). Moreover, TRH activates Cl ? channels and causes altered ion influx, in a manner analogous to TDH [71,72]. Thus, many disease-related reports available due to sea food contamination of Vibrio species. An Indian study reported that 178 V. parahaemolyticus strains were isolated from 13,607 diarrheal patients admitted in Infectious Diseases Hospital in Kolkata (India) since 2001-2012 . TDH and TRH proteins have overall 60% sequence similarities although perform very similar cellular tropism (Figure 12).
The tdh gene was located in chromosome-2 of V. parahaemolyticus (accession number: CP003973). It appeared that two copies of tdh gene in ch-2 (nt.1391390-1391959 and nt.1334189-1334758) with 97-99% similarities. A copy of tdh gene also found in Ch-1 of V. haemolytica (accession no. CP046785; nt. 1945873-1946442with 94% similarity). The tdh gene was also located in pFPPDNB1-3 plasmid of Photobacterium damselae strain KC-Na-NB1 with 89% similarity (accession number: CP03546). However, all V. parahaemolyticus chromosomes had no tdh or trh virulence genes as described above for chromosomes with PirAB genes (accession numbers: CP028342, CP046831 and CP034294).
The trh gene was located in Ch-2 of V. parahaemolyticus (nt. 1271013-1271582; accession number: CP066247) and also located in Ch-1 (nt.2504989-2505558; accession number: CP035701) and appeared a single copy gene found in both chromosomes. Such gene also located in plasmid pTJ187-3 (86kb) of V. parahaemolyticus strain TJ-187 with 94% similarity (nt. 3037-3606; accession number: CP068651). BLAST-2 sequence analysis showed about 73% homology between tdh and trh genes and a divergence was found at the 5’-terminal first 60 nucleotides. We also located large Nuraminidase toxin Tc-A in plasmid pva1 of V. parahaemolyticus GL601 (Figure 13).
Primer design for blaCARB-1, pbp1B and catC1 genes of V. parahaemolyticus
After the characterization of all described genes to find similarities with other bacterial genes, we came to conclusion that blaCARB-1, pbp1B and catC1 genes were very specific for Vibrio parahaemolyticus. We used NCBI Primers design software to make PCR primers for three chromosomal genes as described in (Table 1). There were ten primers selected each and we analysed the hairpin structure, dimer formation to choose one only that gave at least 100-200 V. parahaemolyticus genomic sequences with 100% similarity and 100% cover (Figure 14). The primers for pirAB, tdh, trh and tlh genes were already reported (see, Material and Methods). No other plasmid or genomic sequence had higher than 75% similarity and cover. Thus, we described many mdr genes, toxin genes and virulence genes in plasmids and chromosomes of Vibrio parahaemolyticus that caused acute destruction in shrimp aquaculture. We also found few genomic primers for identification purposes. Such article was lacking in the PubMed Database.
We clearly described the occurrence of mdr genes in Vibrio species in aqua shrimp fish culture (Figure 3 and Figure 4). The blaCARB-1 gene was distinctly located in only V. parahaemolyticus genome and had profound difference with blaTEM-1, blaSHV-1 as well as blaOXA-1 genes located in bacterial plasmids of E. coli, K. pneumoniae and A. baumannii etc. (Figure 7). Das et al have isolated many Vibrio spp. Including V. alginolyticus, V. parahaemolyticus, V. cholerae, V. mimicus, and V. fluvialis along with Aeromonas hydrophila, Aeromonas salmonicida and Salmonella enterica from the shrimp cultures on TCBS medium. The V. alginolyticus was found to be the most resistant isolate by showing multiple antibiotic resistance (MAR) index of 0.60 followed by V. mimicus (0.54) and V. parahaemolyticus (0.42) . The V. alginolyticus plasmid pVAS19 contained many mdr genes like blaPER-1, sul1, catB3, aac6’-Ia, tetB, Qnr1, dhfr and ANT3” (accession no. KX957968) .
Tendencia et al. described that most of the bacteria isolated from pond were Vibrio harveyi and resistant to at least two antimicrobials like oxolinic acid (24%) and penicillin G (19%) and rest by varying percentages to chlorotetracycline, ciprofloxacin, erythtromycin, gentamycin, neomycin, nitrofurazole, ofloxacin, oxytetracycline, polymyxin B, rifampicin, streptomycin, sulphamethezole, and sulphafurazole [76,77]. Pan J et al described that Vibrio vulnificus gram-negative bacterium found in scrimp ponds of China that were resistant or intermediate resistant to cefepime (3.03%), tetracycline (6%), aztreonam (24%), streptomycin (45%), gentamicin (94%), tobramycin (100%), and cefazolin (100%) [78,79].
Babu et al. described that in East Indian shrimp ponds mainly contaminated Enterocytozoon hepatopenaei (EHP) and V. parahaemolyticus. In this study, V. parahaemolyticus isolated from L. vannamei was sensitive to chloramphenicol and oxytetracycline but resistant to erythromycin and nalidixic acid. Interestingly, White Spot Syndrome Virus (WSSV) was also frequently observed with trace amount of Infectious Hematopoietic Necrosis Virus (IHNV) and Monodon type baculovirus . Yano et al described that isolated V. parahaemolyticus had higher affinity for non-native white-leg scrimp fish than for native black-tiger shrimp. Such bacteria were resistance to ampicillin, oxytetracycline and nalidixic acid . The bacteria like Acinetobacter, Achromobacter and Alcaligenes were also isolated from ponds, those were currently using Oxacillin antibiotic in aquaculture indicating such bacteria acquired large conjugative MDR plasmids . Such bacterial contamination and virus infections are serious threat in shrimp aquaculture. Based on baseline and unusual mortality in tilapia fishes in Bangladesh, a total loss of 875.7 million USD annually was occurred recently .
Haifa-Haryani et al. isolated many Vibrios from cultured shrimp in Peninsular Malaysia and plasmids (<10kb) were detected. These bacteria were characterized based on pyrH gene . The populations of different Vibrios were detected as follows: V. parahaemolyticus (55%), V. communis (9%), V. campbellii (8%), V. owensii (7%), V. rotiferianus (5%), V. cholerae (4%), V. alginolyticus (3%), V. brasiliensis (2%), V. natriegens (2%), V. xuii (1%), V. harveyi (1%) and V. hepatarius (0.4%). Antibiotic susceptibility profiles revealed that all isolates were resistant to penicillin G (100%), but susceptible to norfloxacin (96%). The V. haemolyticus strain V22G1 was resistant to twelve antibiotics comprising ampicillin, chloramphenicol, gentamycin, kanamycin, cefotaxime, ceftazidime, cephalothin, nitrofurantoin, sulfometioozone-trimethoprim, erythromycin, vancomycin and penicillin G with MAR index as high as 0.75. The MAR index of the isolates from the Cochin estuary ranged from 0.31 to 0.75 and that from the shrimp farm ranged from 0.19 to 0.5 (see, plasmids pVAS19 and pVPS43) (Figure 3). Mercury reductase and other mer genes located in V. parahaemolyticus plasmids .
Viral diseases were also hampered the shrimp aquaculture. In India, Penaeus monodon, black tiger shrimp was previously the foremost-cultivated shrimp species but the American white leg shrimp Litopenaeus vannamei has effectively replaced it. The White spot syndrome virus (WSSV), Hepatopancreatic parvovirus (HPV), Monodon baculovirus (MBV) and Infectious hypodermal and hematopoietic necrosis virus (IHHNV) are the other significant infectious agents of P. monodon and L. vannamei. A more recent disease of L. vannamei in India is monodon slow growth syndrome (MSGS), a component of which seems to be Laem-Singh virus (LSNV) [85,86].
The pirAB toxin genes as well as tdh, trh and tlh virulence genes primers were used to detect V. parahaemolyticus in shrimp fish. We added few chromosomal mdr genes primers for the detection of V. parahaemolyticus (table-1) . Drug sensitivity test will be performed carefully to address the spread of mdr genes in fish aquaculture. There is hope that phyto-antibiotics may be utilized to avert the multi-resistance . We have to be careful to add excessive antibiotics into aquaculture as scientists predicted that such process accelerating mdr genes spraed in the environment and as high as 10million people may die due to AMR in 2050.
Vibrio species are a group of bacteria naturally found in freshwater, estuaries and marine environments and are responsible for numerous human diseases attributed to the natural microbiota of aquatic environments and seafood. Globally, about 59.51 million people were associated with fishing or aquaculture. Fish is an important and significant source of animal protein for 4.5 billion people who rely on them. The V. parahaemolyticus, was serious food-borne pathogens and highly found in shrimp aquaculture with high molecular weight plasmids giving multi-drugs resistance. We designed chromosomal genes (blaCARB-1, pbp1B and catC1) specific primers for the detection of V. parahaemolyticus. The international aquaculture expansion and expanding global trade of shrimp have been accompanied by long distance geographical redistribution spreading many bacteria and animal viruses. The shrimp fish marketed to Europe and America from West Bengal. Thus, spread of mdr genes must be studied in fish aquaculture. We foresee new plant-based remedies for shrimp mortality control and to increase shrimp fish trade.
We thank Prof Bidhuyt Bandhopadhyay, Principal of OIST for his interest in aquaculture research. We also thank NCBI, NIH for free Database and BLAST search engine. Uttam Maity is QC Biotechnologist at Jana Brothers Seafood LLP, Digha (Email: email@example.com).
The authors declared no conflict of interest to any agency.
No animal and no human was used in this study.
No funding was obtained from any Government agency.
1. Bender W, Smith M. Feeding the future. Population today. 1997; 25: 4-5.
11. Xiao J, Liu L, Ke Y, Li X, Liu Y, Pan Y, et al. Shrimp AHPND-causing plasmids encoding the PirAB toxins as mediated by pirAB -Tn903 are prevalent in various Vibrio species. Scientific Reports. 2017; 1-11.
35. Chakraborty AK, Poira K, Saha D, Halder C, Das S. Multidrug- Resistant Bacteria with activated and diversified MDR Genes in Kolkata Water: Ganga Action Plan and Heterogeneous Phyto-Antibiotics tackling superbug spread in India. American J Drug Deli Therapeutics, 2018; 5: 2.
37. Anderson WC, Turnipseed SB, Roybal JE. Quantitative and confirmatory analyses of malachite green and leucomalachitegreen residues in fish and shrimp. USFDA Laboratory information Bulletin, LIB No. 4363. 2005.
57. Grim CJ. Aeromonas and Plesiomonas: Foodborne Infections and Intoxications. 2013; 13: 229-237.
58. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning-A laboratory manual (Cold Spring Harbor Laboratory Press, Cold spring harbour, NY, USA. 1982.
77. Chakraborty AK, Pradhan S, Das S, Maity M, Sahoo S, Poria K. Complexity of OXA Beta-Lactamases involved in Multi-Resistance. British J Bio-Medical Res. 2019; 3: 772-798.
88. Chakraborty AK. Heterogeneous phyto-antibiotics and other future therapeutics against multi-drug resistant bacteria. Advances in Biochemistry. 2019; 7: 34-50.