Atypical and dual biotypes variant of virulent SA-NAG-Vibrio cholerae: an evidence of emerging/evolving patho-significant strain in municipal domestic water sources

The recent cholera spread, new cases, and fatality continue to arouse concern in public health systems; however, interventions on control is at its peak yet statistics show continuous report. This study characterized atypical and patho-significant environmental Vibrio cholerae retrieved from ground/surface/domestic water in rural-urban-sub-urban locations of Amathole District municipality and Chris Hani District municipality, Eastern Cape Province, South Africa. Domestic/surface water was sampled and 759 presumptive V. cholerae isolates were retrieved using standard microbiological methods. Virulence phenotypic test: toxin co-regulated pili (tcp), choleragen red, protease production, lecithinase production, and lipase test were conducted. Serotyping using polyvalent antisera (Bengal and Ogawa/Inaba/Hikojima) and molecular typing: 16SrRNA, OmpW, serogroup (Vc-O1/O139), biotype (tcpAClas/El Tor, HlyAClas/El Tor, rstRClas/El Tor, RS1, rtxA, rtxC), and virulence (ctxA, ctxB, zot, ace, cep, prt, toxR, hlyA) genes were targeted. Result of 16SrRNA typing confirmed 508 (66.9%) while OmpW detected/confirmed 61 (12.01%) V. cholerae strains. Phenotypic-biotyping scheme showed positive test to polymyxin B (68.9%), Voges proskauer (6.6%), and Bengal serology (11.5%). Whereas Vc-O1/O139 was negative, yet two of the isolates harbored the cholera toxin with a gene-type ctxB and hlyAClas: 2/61, revealing atypical/unusual/dual biotype phenotypic/genotypic features. Other potential atypical genotypes detected include rstR: 7/61, Cep: 15/61, ace: 20/61, hlyAElTor: 53/61, rtxA: 30/61, rtxC: 11/61, and prtV: 15/61 respectively. Although additional patho-significant/virulent genotypes associated with epidemic/sporadic cholera cases were detected, an advanced, bioinformatics, and post-molecular evaluation is necessary. Such stride possesses potential to adequately minimize future cholera cases associated with dynamic/atypical environmental V. cholerae strains.


Introduction
Cholera, a disease that is associated with dirt, gutter, unhealthy contact with contaminated material and other unhygienic practices has been affirmed to be associated with the pathogen Vibrio cholerae. This organism Igere et al. Annals of Microbiology (2022) 72:3 has been classified according to the Bergeys manual of identificative bacteriology among the family Vibrionaceae with more than twelve human and animal pathogenic strains identified. Over the years, there have been reports of sustained implication of the pathogen in acute watery diarrhea, wound infections, otitis media, neonatal meningitis, septicemia, sporadic, and cholera cases (Lu et al. 2014;Marin et al. 2013;Talkington et al. 2011). World Health Organization (WHO) and other investigators in vibriology have affirmed the annual burden of cholera cases to range from 1.3 million to 4.0 million with annual mortality rate ranging from 21,000 to 143,000 worldwide (Ali et al. 2015;WHO facts 2018). Due to their high clinical relevance, they are subdivided into serogroups, serotypes, phage types, and biotype (Momba and Azab El-Liethy 2017). Serogrouping are based on their extracellular polysaccharide into O1, O139, and somatic antigen non-agglutinating V. cholerae (SA-NAG-Vibrio cholerae or non-O1/non-O139) (Igbinosa et al. 2009). Biotyping is based on their first place and time of isolation which include Classical, El Tor, Bengal, Calcutta (Mandal et al. 2011). However, two biotypes have been globally distributed and implicated in outbreak reported cases (El Tor and Classical V. cholera). Evident in the early studies was the observation of an El Tor biotype which was first isolated from camp quarantined pilgrims of the El Tor City, located at Sinai Pennisula, Egypt in 1905. According to Zaw et al. (2016) and Mandal et al. (2011), biotyping cholera Vibrio members possess a major epidemiological relevance in the classification scheme of the family Vibrionaceae especially as the pathogen has shown in history, a remarkable disappearance and re-establishment as well as dominance since 1992 (Mandal et al. 2011;Zaw et al. 2016).
Reports from various Vibrio studies have revealed that there exist some new genetic variants of dual biotype with virulent genes suggesting an ongoing evolution as reported by various investigators (Faruque et al. 2007;Goel et al. 2008;Grim et al. 2010;Nair et al. 2006;Zaw et al. 2016). Further results from the study of Ansaruzzaman et al. (2004) in Mozambique, after the cholera outbreak episode in Eastern African countries revealed the presence of an atypical variant of V. cholerae with dual biotype and resistant to phage type IV. These reports were affirmed in the studies of Faruque et al. (2007) and Das et al. (2007), who observed two tandem copies of prophage with similar sequence type as CTXΦ clas in the pathogen small chromosome. Other investigators of Vibrio have also reported the occurrence of dual phenotype and genotype among pathogenic V. cholerae (Ceccarelli et al. 2011;Lee et al. 2009;Nair et al. 2006;Nguyen et al. 2009;Safa et al. 2008;Zaw et al. 2018). Such features are positive and/or negative to chicken red cell agglutination test, Voges Proskauer test, sensitive, and/or resistance to both polymyxin B and phage V but resistance to phage IV (Safa et al. 2010).
Such strains also anchor their genomic composition on two functional but distinct gene clusters, namely, the satellite phage (RS1) and CTX-phi (CTXΦ) phage Heidelberg et al. 2000). The classical biotype is shown to possess only the CTXΦ, whereas the El Tor biotype possesses both the satellite phage (RS1) and CTXΦ where RS1 is located adjacent to CTXΦ as depicted in Fig. 1A , B below (Castillo et al. 2018;Heidelberg et al. 2000;Safa et al. 2010). Present as gene content of CTXΦ are other two distinct clusters referred to as RS2 and core. The RS2 is also a satellite phage-like particle as the RS1; however, it is similar in genetic content as the RS1 except that it lacks the anti-repressor protein which promotes transcription of CTXΦ during the generation and gene expression of infectious particles (rstC) (Banerjee et al. 2014;Safa et al. 2010;Waldor et al. 1997). The core consists of accessory genes needed for secretion and assembling of infectious particles such as CT (ctxA, ctxB), psh, cep, g IIICTX , Ace, and Zot (Fig. 1A). The satellite phage (RS1) consists of rstA gene which is responsible for replication of infectious particles, rstB, which is necessary for integration of viral particles while rstR and rstC are necessary for regulation of site specific recombination and gene expression of viral particle, although the rstC is an anti-repressing protein (Rezaie et al. 2017;Banerjee et al. 2014).
It is important to note that the ctx B gene is present in both the wild-type CTX-phi and the atypical genome. Although the ctx B gene differs in its amino acid sequences, the CTX-phi gene of the altered/atypical has a notable genome similarity with the Classical CTX-phi genes except for the difference which occurred at rstR. The difference in ctxB genotypes is associated with the global distribution and the wave of evolution over a period of time Banerjee et al. 2014;Safa et al. 2010;Heidelberg et al. 2000). The gene types found in atypical or altered strains of the pathogen are mainly of RS1 and CTXΦ (RS2 and core) origins which include rstA, rstR, rstB, rstC, and CTXΦ (cep, ace, zot, rstA, rstB, rstR, psh, g II-ICTX , ctxA, ctxB). Other patho-significant determinants of clinical relevance are rstR cla , rstR El Tor , rtxA, rtxC, and other virulent characteristics (Plaza et al. 2018;Kim et al. 2014;Lee et al. 2009;Nguyen et al. 2009;Safa et al. 2006Safa et al. , 2008Faruque et al. 2007;Das et al. 2007). The inclusion of the RTX toxin is evident as earlier reported by Heidelberg et al. (2000) in a comparative genomics study which shows that located at the far end of the CTXΦ is a region encoding an RTX toxin (rtxA), and its activator (rtxC) with nucleotide composition suggesting the RTX region was horizontally acquired along with other sensor histidine kinase/ response regulator. It also suggests that the regulators affect the expression of some closely related RTX transcriptional units Plaza et al. 2018;Kim et al. 2014). Atypical strains are rarely reported among environmental SA-NAG-V. cholerae strains. In the current study, we confirm atypical/unusual trait/ genes both phenotypically and genotypically among environmental SA-NAG-V. cholerae or non-O1/Non-O139 V. cholerae as evidence of emerging/evolving patho-significant strain in municipal domestic water sources.

Study area and sample collection
One liter of domestic water sources was collected onto sterile Nalgene bottles from each of the twenty sampling points which include the waste water treatment plants, receiving water shed, rivers, irrigation canals, dam, and tap water with geographical coordinates as follows: S 31° 54.548′, E 026° 50.715′, S 31° 54.548′, E 026° 50.715′, S 31° 54.548′, E 026° 50.715′, S 31° 54.548′, E 026° 50.715′. These samples were collected from Amathole District municipality and Chris Hani District municipality of Eastern Cape Province, South Africa. The sampled treatment plant receives municipal wastewater from household/domestic usage and release effluents after treatment into receiving water shed, rivers, dams, and irrigation canal. Such released water is used by the rural population for swine husbandry, irrigation of farm lands, drinking, household/domestic activities, and recreational activities. Most irrigated farm lands with sampled water cultivate and harvest large variety of vegetables both local and commercial farm lands in sub-urban and peri-urban area of study municipalities (Queens Town, Alice). Water samples were collected from treated final effluent before discharge, point of discharge for receiving body of water, 500 m downstream, and 500 m upstream of the receiving water discharge point. Samples were collected monthly between February 2018 and August 2018. Specified water samples were collected onto 1 L screw capped glass Nalgene containers, pre-cleaned/washed using non-ionic detergents and rinsed/bathe in smooth running tap water. Additional and further pre-cleaning includes treatment with 5% concentrate of hydrogen chloride acid and finally with deionized water. Prior to collection of water specimen at various sampling points, 1.7 mL volume of sterile 1% sodium thiosulphate solution was added asceptically to specimen bottles. At the collection site/location, midstream water sampling was done by dipping each sterile-secured sample bottle to approximately 20-30 cm below water surface, projecting the open-mouth of glass bottle or container against water flow direction. Collected samples were thereafter transported in cooler boxes containing ice-packs to the Applied and Environmental Microbiology Research Group (AEMREG) laboratory at the University of Fort Hare, Alice, for respective physicochemical and microbial analyses within 6 to 8 h after collection.

Physicochemical analysis
The various sample site geographical coordinates were taken, the distance from analysis laboratory taken, the flow of water into municipality observed, and the wastewater treatment plant capacity design taken. The physicochemical parameters was measured using the following: HANNA HI 96711 free/residual and total chlorine meter for chlorine estimation after pre-preparation with the Merck lovibond DPD-1 tablets; HANNA is a multiple parameter waterproof meter made in Romania Europe (www. hanna inst. com). Other machines including HACH HQ40D an optimum density meter; HACH Model CO150 conductivity meter; HACH DR 900 a multiple parameter meter HACH Company/Hach Lange GmbH, 2013 Model 210A, Orion Laboratories and Life Science membrane filtration system Model No: DOA -P730 -BN, Patr No: 13158 were used to analyze the various physiological properties of the collected water samples on/out site. The study encompasses sampling within three seasons of study area, (Winter, Summer, and Autumn).

Bacterial strains isolation
Following the previous methods as described by various investigators with few modifications, filtered water samples (vacuum pump/pressure pump: model No. DOA-P730-BN) (www. lasec. co. za) using 0.45-μm nitrocellulose paper (Merck, Germany, www. merck-chemi cals. com) were plated onto pre-prepared Thiosulphate Citrate Bile Salts-Sucrose agar (TCBSA) in triplicate and incubated at 37 °C for 24 h Igbinosa et al. 2009;Pfeffer and Oliver 2003;Rivera et al. 2013;Uddin et al. 2018). Presumptive yellow and green V. cholerae colonies were sub-cultured onto nutrient agar to ascertain the purity subsequently. Three to five colonies per-plate were stored in aliquot on glycerol stock.
Further cultural morphology, colonial morphology, and biochemical characteristics of representative strains were observed/recorded as suspected or presumptive colonies which were subculture onto TCBSA and subsequently onto nutrient agar (NA) to ascertain/confirm purity. These characteristic tests include Gram reaction, oxidase test, motility test, Voges Proskauer test (VP test), and D-mannitol catabolism, while positive strains were selected for confirmation as V. cholera/non-Vibrio strains using serotyping, molecular typing scheme, biotyping serogrouping, and pathotyping as specified by investigators (Beshiru and Igbinosa 2018).

Vibrio numerical density/count
The method as described by Huq et al. (2012) and Rivera et al. (2013) was employed for isolation with few modifications. Each of the samples was separated into plankton free and plankton associated. Plankton-free samples were filtered after a tenfold dilution treatment using 0.45-μm nitrocellulose filter membrane (Merck filter membrane Germany, www. merck-chemi cals. com), filtered membrane were plated in triplicate onto thiosulphate citrate bile salts-sucrose agar (TCBSA) and incubated at 37 °C for 24 h (Pfeffer and Oliver 2003;Baumann et al. 1984). Plankton-associated samples were pre-treated by centrifuging at 5000 rpm for 1 min and 1 ml of concentrated homogenates inoculated onto 9-ml sterile Alkaline peptone water (APW). The prepared plankton associated culture was incubated for 6-8 h and subcultured directly onto thiosulfate citrate bile salt-sucrose agar (TCBSA) and incubating at 37 °C for 24 h Alam et al. 2006;WHO 2004;Oliver and Kapper 1997).

Presumptive identification, biochemical reaction, and in vitro virulence test
Pure colonies of presumptive V. cholerae isolates were tested using Gram reaction, oxidase reaction, motility and D-mannitol catabolism, Voges-Proskauer, and resistance to polymyxin B. An in vitro presumptive virulence test of isolates was determined using the string test, protease production (protein degrading enzyme test), lipase production (Ryan test or lipid degrading enzyme test), lecithinase production (lecithin degrading enzyme test), and cholera red production (Rivera et al. 2013;Huq et al. 2012;Alam et al. 2006;WHO 2004). Although these determinative tests may not be sufficient for virulence specific identity, they are used as preliminary/presumptive pathotyping procedure for both clinical/environmental V. cholerae members. Isolates that reveal positive/ negative reactions to these aforementioned multiple tests were selected presumptively for gene-based confirmation as virulent non-agglutinating O1/O139 V. cholerae members.

Storage of V. cholerae strains
Yellow and green single colony from a fresh culture were purified and stored in aliquot {containing 50% glycerol in 1 ml of sterile Luria Bertani broth on sterile 2 ml graduated skirted-Cryo-preservation tubes (Starlabs, Milton Keynes, UK)}. The tubes were vortexed briefly to ensure thorough mixing of contents and immediately stored frozen at −80 °C.

Optimum resuscitation broth (ORB)
The optimum resuscitation broth {nutrient broth (Oxoid CM0733)} was used for preparation of isolate dilutions during water samples collection and isolation of V. cholerae. It was prepared following appropriately the steps on manufacturer's instruction manuals. A 9.5 g quantity of nutrient broth powder was dissolved in 1 liter of distilled water and sterilized by autoclaving at 15 psi, 121 °C for 15 min. Aliquot was stored aseptically at room temperature prior usage.

Phenotypic biotyping of strains
There are two basic biotypes of V. cholerae somatic serogroups, which are Classical and El Tor biotypes. Although a third biotype known as the Bengal biotype appeared as emerging member, it has remained in the Asian continent. The phenotypic biotyping test for isolates was applied using polymyxin B susceptibility, hemolysin test, and Voges-Proskauer test (Forbes et al. 2007).

Polymyxin B (PB) sensitivity
A modified technique as previously described by Han and Khie (1963) was applied. Mueller-Hinton agar plates were inoculated with standardized saline culture of test organism (4 h at 35 °C) to obtain confluent growth and then 50 unit PB antibiotic disk was placed on the surface of medium, was inverted, and was incubated overnight at 37 °C. Classical biotype V. cholerae shows a 12-15 mm zone of inhibition, whereas El Tor Biotype shows complete resistance.

Voges-Proskauer test
A prepared methyl red and VP reagent was added to pure 24 h culture of the presumptive isolates. A deep red coloration of medium as the reagent is added to the culture indicates a positive result.

Bengal biotype using serology
The MAS-AGGL-M15001 Bengal antisera were purchased from the Davis diagnostics {(Pty) Ltd., 141 Oak Avenue, Ferndale, Randburg, 2194, Gauteng, South Africa (www. davie sdiag nosti cs. co. za)}. A single colony from a pure 18-24 h growth culture plate was smeared on a grease-free slide containing two drops of normal saline.
Two drops of Bengal antisera was added and homogenized following manufacturer instructions. Positive report was recorded on observation of agglutination.

Choleragen red
Alkaline peptone water (APW) [VM 768100 704, Merck KGaA, 64271 Darmstadt, Germany (www. merck-chemi cals. com)] was prepared according to manufacturer's instruction. Pure 24 h culture of isolates was inoculated onto prepared broth and incubated for 24 h. Few drops of concentrated H 2 SO 4 were added to the 18-24 h broth culture growth of APW. Positive results were considered when the pale yellow color of APW is turned to pink or pale red.

DNA extraction
The boiling method as previously described by Maugeri et al. (2006) was applied. A single colony of the overnight pure culture of presumptive isolate was harvested and/or suspended in ice cold nuclease free 0.5 molar TE {Tris ethylene diamine tetracetate (EDTA)} in a 1.5-mL microfuge tube. The tube was heated to boiling using Lasec heating block (www. lasec. co. za) for 10 min and cooled centrifuged at 15,000 rpm for 8 min in Eppendorf centrifuge (Hermle Microrifuge; Labortechnik GmbH Germany). Supernatant of the above preparation was use as bacterial lysate or DNA template during the PCR amplification/detection experiments.

PCR amplification
The amplification was carried out in a 200-μl microfuge tube. Fifty picomolar to 1 micromolar DNA extract was used for the PCR in a final volume of 25 μl, a GoTaq ℗ G2 green master mix supplied in 2X Green GoTaq ℗ G2 pH: 8.5 reaction solution containing, dNTPs {400 μM each of dATP, dGTP, dCTP, and dTTP}, 3 mM MgCl 2, and GoTaq ℗ G2 DNA polymerase at optimal concentration for efficient PCR amplification as specified by Promega Corporation USA (www. prome ga. com). Primer concentration of 0.5 μM with the PCR machine (Bio-Rad T100 TM thermal cycler) was used. The thermocycling condition were 4 min at 94 °C followed by 35 cycles of 94 °C for 1 min, 53.0 °C for 1 min, and 72.0 °C for 1 min and a final extension step at 72.0 °C for 8 min. Other virulent genes cycling conditions are as shown in Table S1 below.

Tris acetate-EDTA (TAE) or Tris Borate-EDTA (TBE) buffer for gel electrophoresis
A TAE stock solution was prepared by adding 242 g of Trizma base, 5.7 ml glacial acetic acid and 100 ml 0.5M EDTA (pH: 8.0) into a 1 L of deionized water to make a 1 L 50× TAE buffer (pH 8.0). The working solution consists 20 ml of the stock solution to 980 ml of deionized water (DW) to prepare a 1 L of 1 × TAE buffer.

Gel electrophoresis
A small-sized gel was prepared by adding 1.5 g of agarose (Sigma Aldrich, UK) powder and 100 ml 1 × TAE buffer. The mixture was dissolved in a 250-ml conical flask, boiled on a microwave for approximately 3 min using high heat with intermittent mixing, allowed to cool to about 50 °C before adding 2 μl per 50 ml of ethidium bromide. The gel was casted, allowed to polymerize, transferred onto an electrophoresis tank filled with 1 × TAE buffer to mark. PCR products or amplicons from a thermal cycling process were mixed with loading buffer (bromophenol blue, 0.01% w/v, glycerol, 50% v/v, EDTA, 0.025 M pH 8.0) at the ratio of 4:1 (sample:buffer) before loading into the comb wells. The DNA ladder or molecular marker was then loaded without DNA-loading dye. Thereafter, samples were electrophoresed at an appropriate voltage of 100 V for 50 min until the dye front was 2 cm away from the end of the gel. The power was switched off and the electrophoresis tank was disconnected from the power pack. The gel was carefully removed from the casting tray and photograph using the Gel Doc imaging system (Bio Rad, USA).

Statistical analysis
Pearson correlation was employed to describe and determine level of significances between Vibrio colonial mean cell density/count and the recorded physicochemical parameters in relation to the study sites. This is done to determine the statistical significance of the physicochemical parameters to the proliferation tendency of such emerging strains and/or the Vibrio-colonial density. Such generated data were transferred onto Excel spread sheets to develop tables while other generated data were described using figures.

Results
The environmental SA-NAG V. cholerae strains retrieved was phenotyped and genotyped using microbiological culture dependent methods and PCR. A total of 61 (12.01%) isolates were PCR confirmed after the detection of 508 (66.9%) genus specific isolates from 759 presumptive isolates during the study as shown in Fig. 2 and Table 2. The phenotypic character of isolates also further showed that some of them were producer of the cholera toxin as depicted by the choleragen red test (Fig. 3, Table 2). The serological test reveals some of them as Bengal (7/61) serotype, whereas 68.9% (42) of isolates showed susceptibility to polymyxin B test, while 6.6% (4) were Voges-Proskauer test positive, protease test [203; (26.8%)] ( Fig. 4; Table 2).
The PCR detection of the CTX-phi, RS 1, RTX cluster, HLY-A cluster, and the rstR region of the Classical and El Tor biotype revealed the presence of various genes of emerging relevance. Table S1 shows the various sets of oligonucleotide-primer sequences ranging from genus specific 16SrRNA primer (Yong et al. 2006) to specie specific OmpW gene (Nandi et al. 2000) which confirms the various isolates as V. cholerae. The ctxA gene was not detected during the study whereas ctxB and other toxigenicity associated genes such as RTX, cep, and ace were detected among isolates. All the detected gene markers are located within the CTX-phi, satellite phage, RTX cluster, and the hemolysin A cluster of the classical and El Tor strains of V. cholerae (Safa et al. 2010;Goel and Jiang 2010;Heidelberg et al. 2000;Waldor and Mekalanos 1996). The detection of the RTX toxin (repeat toxin) cluster reveals virulence determinants while rtxC gene detection reconfirm that these isolates where of the El Tor biotype as previously reported by Chow et al. (2001). The PCR detection of El Tor and the Classical hemolysin gene in 3.27% (2/61) of the isolates analyzed also confirms the occurrence of emerging and unusual genotype among the strains. Further studies are currently ongoing on these atypical V. cholerae members in our laboratory and environment which include whole genome sequence analysis, bioinformatics, and phylogenetic study of these members.

Discussion
New reports of V. cholerae strains continue to surface from diverse region of the world as some reports are associated with the environmental strains. Other reports queried emergence of new strains with high virulent nature necessitating public health interest. The sampled domestic water physico-chemical indices show statistical significance with Vibrio-colonial density/counts (Table 1). This reveals the impact of physicochemical parameters on the proliferating emerging strains. The strains show positive reaction to diverse phenotypic virulent determinants including protease, oxidase, choleragen red test (Figs. 3, 4), and other atypical phenotypes of pathogenic V. cholerae as depicted in Table 2 above. The study revealed that 68.9% (42/61) of isolates were susceptible to polymyxin B test, whereas 6.6% (4/61) of isolates were Voges proskauer test positive, while 11.48% (7/61) were positive to the Bengal serological test (Table 2). This implies that the SA-NAG members are emerging with such dual/atypical biotype. This result is similar to the reports of numerous investigators who have observed the dual phenotype   (Zaw et al. 2018;Safa et al. 2005Safa et al. , 2006Safa et al. , 2010Nguyen et al. 2009;Chun et al. 2009;Das et al. 2007;Faruque et al. 2007). Observing variability in susceptibility to polymyxin B by the SA-NAG strains reveals the atypical biotype. It is important to note that the polymyxin sensitivity or resistance has been applied for several decades as an epidemiological bio-marker in the biotyping scheme for V. cholerae. This occurrence and other phenotypic virulent indices (Figs. 5,6,7,and Table 2) has aroused the need to source track the spread/distribution of the patho-biotype exhibiting emergent trait.
The study also reveals a remarkable occurrence of atypical/unusual genes which are found both in the Classical biotype and the El Tor biotype as previously reviewed by Safa and his colleagues (Safa et al. 2010). The observed genotype/phenotypes were located around the RS1 and CTXɸ regions of either the small and/or large chromosomes (Heidelberg et al. 2000;Safa et al. 2010). According to Faruque and his group, it was reported that these strains with atypical or altered and mixed biotype may have risen from primordial El Tor strains that lost their CTXɸ El from their large chromosome with a subsequent tandem location of CTXɸ Cla in the small chromosome (Faruque et al. 2007). Nguyen et al. (2009) also reported that these variant biotype strains may possess their CTXɸ Clas and CTXɸ El Tor either in tandem array or located within two different chromosomes.
It is important to note that previous vibriology investigators studied the O1 and O139 pathogenic strains of V. cholerae, it appears that the environmental strains (SA-NAG-O1/O139) are currently evolving with related dynamics which calls for prompt survey and/or control measure to avert future difficult to control strain.
Other atypical markers were also observed among our isolates which were similar to those reported recently in the study of Jena et al. (2018) and Samanta et al. (2018), revealing that the occurrence of two biotypes in V. cholerae O1 strain, exhibits diverse pattern of susceptibility to polymyxin B. The result of the PCR detection (Figs. 5, 6, 7) as represented in Table 3 showed that fifteen of the confirmed SA-NAG V. cholerae isolates harbored ace (24.6%) gene, while fourteen harbored cep (23%) gene. These genes were harbored in the CTXɸ region of the pathogen chromosomes which reveals the presence of the nucleic acid based genotype. In an earlier study of Chun et al. (2009), he referred to strains with the above genotype with the term "a shift or drift between two distinct phyletic lineages" which is seen to be occurring in the environmental SA-NAG strains, and indicates potential evolution/emergence of new strains with related pathogenic determinants. Estuarine environment has been shown as V. cholerae autochthonous habitat; however, the observation of such atypical or dual biotype strains among environmental V. cholerae shows evolving tendency which pose high risk to human and environmental health. Such risk and public health concerns are specifically directed to the urban and sub-urban individuals who use the studied water as their domestic water source. The need for adequate application of hygienic practice and supply of safe water cannot be over emphasized. This observation is also a pointer to the potential occurrence of a transitional representative of genetically identical strains, which possess high virulent potential as the pathogenic strain. It also implies that there is a possibility of an ongoing shift/drift of the environmental strains due to acquisition of new genes over time. Very indicative in the study was the observation of pathogenic emerging indices in somatic antigen non-agglutinating V. cholerae strains (SA-NAG-Vc-1/139) which were previously known nonpathogenic V. cholerae members.
Among other observed genotypes is an RTX toxigenic gene detected in the RTX cluster which is reported to encode cytotoxic reaction. Tables 3 and 4 show the numbers of isolates with related reports. It is reported to be physically linked to the CTX-phi in the V. cholerae genome (Heidelberg et al. 2000;Faruque and Mekalanos 2003) and associated with other residual features in attenuated cholera vaccines. About 49.2% of the isolates possess rtxA genotype whereas 18% possess the rtxC genotypes. These observations were also previously reported in the comparative genomics study of Heidelberg et al. (2000) and the characterization of RTX by Chow et al. (2001). It is said that the detection of RTX cluster is associated with actin depolymerisation when cross-linked with Hep-2. Such depolymerisation results villi effacement, hemorrhagic colitis, and bloody diarrhea in infection cases (Chow et al. 2001). This indicates that in any infection case associated with the atypical environmental strains of SA-NAG V. cholerae, bloody diarrhea and other symptoms cannot be removed from such cases. The study of Goel and Jiang (2010) also reported a similar occurrence of RTX (rtxA and rtxC) virulence and cytotoxin genes with such potential clinical symptoms.
Also revealed from this study was the detection of cep gene (23%) which is associated with the expression of CTX-phi coat protein among strains. This report is similar to the previous annual report of NICED (2004) which indicated that the detection of cep gene is of high and paramount clinical/epidemiological relevance. Cep gene detection may be used instead of ctxA gene detection while tracking the CTX-phi genes in V. cholerae isolates in both outbreak associated location and the environment.  The genotyping analysis of hlyA gene revealed that all isolated SA-NAG-Vc-1/139 strains possessed the El-Tor hemolysin gene which is affirmed to be frequently observed among non-O1/non-O139 V. cholerae (SA-NAG-Vc 1/139) with the exception of two which possess both the Classical and El Tor gene type (Table 4 and Fig. 6). These genotype is said to be unusual among members of the V. cholerae family and rare among the non-O1/non-O139 V. cholerae strains. The detection of hly A atypical genotype among the non-O1/non-O139 cholera Vibrio members indicates that strains are changing its nature within the environment over time. A previous study by Chatterjee et al. (2009) and Rivera et al. (2001) had reported the presence of the El Tor hemolysin gene (hlyA-El Tor) among non-O1 and non-O139 V. cholerae members; however, the dual phenotype was not observed in their study. Our position remains that the detection of the atypical V. cholerae has opened a new area of interest about the potential pathogen in our environmental and domestic water sources.

Conclusion
A notable report from this study is the observation of biotype gene content as well as other associated genes which are located within the small and large chromosome of the cholera Vibrio. It shows that present in the environment are emerging and evolving strains of SA-NAG V. cholerae which pose threat to the public in the urban and sub-urban area from which these isolates were retrieved. This is used to describe the potential evidence of virulence among environment associated V. cholerae (SA-NAG-Vc-1/139) isolated during the study. This study is also necessary as it provide reports of new area of research among V. cholerae with specific interest in relevant autochthonous diagnostic scheme, autochthony dependent vaccine design/application and need for other therapeutics. In avoidance of any doubt, more biotype variants with other virulent indices and phenotypic characters may arise in the future which pose futuristic threat to public health. These emerging trend, change, or occurrence necessitate an urgent routine survey with a view to avoiding any looming outbreak. The need for research based and clinical management preparedness for such evolving and emerging strains remains or offers prime hope for possible future management of outbreaks associated with variant and/or altered biotype non-agglutinating V. cholerae O1/O139 in various environmental nexus.
Additional file 1: Table S1. The various primer sets, annealing temperature and expected amplicon size