Introduction

Cholera remains a perpetual health problem in the world by causing significant morbidity and mortality throughout human history. This diarrheal disease results in large volumes of watery stool lead to rapid dehydration and deaths 50-70% of patients without medicine (Faruque, Albert, & Mekalanos, 1998). The disease caused by mucosal pathogenic bacteria called Vibrio cholerae is a major public health problem in developing countries. Although V. cholerae is acquired from environmental sources, a small portion only of environmental bacteria can cause cholera (Lutz, 2013). V. cholera is classified into 206 serogroups based on variations in the heat-stable somatic O antigens, only serogroups O1 and O139 are associated with cholera (Barzamini, Moghbeli, & Vibrio, 2014).

In the aquatic environment, several kinds of living organisms were infected with V. cholera, including plants, protozoa, bivalves, birds, the exoskeleton of chitin. These organisms regarded as an environmental reservoir for the bacterium (Vezzulli, 2010). The virulence factor of Vibrio cholerae known as cholera toxin (CT), initiates a pathway in epithelial cells that leads to the severe diarrhea characteristic of cholera known as rice-water stool (RWS) (Morris, 2011).

The first stage of infection, bacteria rapidly replicate and increase expression of key virulence factors. The middle stage of infection is characterized by upregulation of the chemotaxis and flagellar genes. This allows V. cholerae to exit the luminal fluid of the small intestine. Finally, in the late stage of infection, chemotaxis becomes repressed as V. cholerae is shed in the RWS (Nguyen, L.M.S., & Camilli, 2017). Cholera enterotoxin was encoded by ctxAB gene, which responsible of severe diarrhea, consist of two subunits one A subunit (ctxA) that provides the activity of intracellular, and five B subunit (ctx B) that binds with the cell receptor (Pal, 2014). The ctx AB gene existing in all strains of V. cholerae and retain on the 6.9kb of CTX prophage that is combined within a chromosome (Maysa & Al-Shukri, 2017). The infection by V. cholerae dose, not a systemic infection, therefore antibiotics are not essential to determination cholera symptoms and cannot be used as a treatment for the disease; however, it is important to combine oral rehydration therapy with antibiotic treatment. Cholera treatment should be used as an oral solution containing glucose, sodium chloride, potassium chloride and trisodium citrate with antibiotics tetracycline and quinolones to reduce the symptoms of cholera (Kitaoka, Miyata, & Unterweger, 2011). The problems of V. cholerae are become drug-resistant to antibiotics by having several mechanisms including efflux pumps, chromosomal mutations, developing genetic resistance via the exchange of conjugative plasmids, conjugative transposons, integrons or self-transmissible chromosomally integrating SXT elements (Garg et al., 2001).

The study aimed at determining the accuracy method for detection of V. cholerae in water via the following steps:

• Isolation of V. cholerae from Kufa River

• The conventional method for identification of these bacteria by using selective media and biochemical tests

• Molecular method for identification of these bacteria by using PCR

Determining the prevalence and antibiotic resistance pattern

Materials and Methods

Traditional methods for isolating and characterization V. cholerae from water samples were presented by (Colwell, Kaper, & Joseph, 1977). who modified protocols originally intended for clinical samples (Colwell et al., 1977).

Study of location

The study was carried out in Kufa River/ Al-Najaf Al-Ashraf/Iraq, different locations were randomly selected from four regions which are Albohadari, Hawatem/Albodheres, Hawatem/ Albohaijasim, Hawatem/ Albotrad.

From September 2017 to August 2018, one hundred samples were collected and selected comprising of 30 water master for drinking, 20 Sewage drainage pipes, 20 from the River containing aquatic plant, 15 River centers, 15 River ranges.

Samples collection

One hundred milliliter of water were taken from each of the 100-water samples in a sterile container containing Carry-Blair transport media, sealed, labelled and transported immediately to the Biology Department laboratory of the Science Faculty/ Kufa University for further analysis.

Sample processing

Culturing with traditional methods for detection of V. cholera were continuing to be improved with the result. The process involves each water sample was filtered by 0.45 µm Millipore filter then the membranes were transferred to slants with a cup containing 2ml of alkaline peptone water and incubated for 5-8 hours at 37ᵒC. After enrichment, aliquots are subcultured onto a selective medium- TCBS (Thiosulphate Citrate Bile Salt Sucrose) agar and incubated for 24h at 37ᵒC. The colonies were taken characterized by yellow, smooth, slightly flattened with opaque centers and translucent peripheries considered as Vibrio species and were sub-cultured on enrichment media such as blood agar for biochemical tests.

Biochemical Tests

All presumptive isolates were confirmed by using a series of biochemical tests including Oxidase test, Simmon Citrate test, DNase test, Indole test, kligler Iron Agar (KIA) test, MacConkey agar test and motility.

Serological Tests

Confirmed V. cholera strains were then screening for slide agglutination test by using commercially antisera polyvalent and monovalent O1 and O139 for determine strain serotype.

Preservation of Bacterial Isolates

For long-time preservation, stab the organism into the trypticase soya agar and incubated at 35ᵒC. Close tubes with screw-cup and dip the cap into molten paraffin wax to seal, store at room temperature and transfer after one year (Vandepitte, Jozef, Engbaek, Kraesten, & Rohner, 2003).

Antibiotic Susceptibility

Muller Hinton agar and broth support the growth of V. cholera are suitable for detection of antibiotic susceptibility. Thirteen standardized disc diffusion was used for determining the sensitivity of V. cholera isolates according to Clinical and Laboratory Standards Institute (CLSI) guidelines for the following antibiotics: Amikacin (Ak, 30mcg), Ceftriaxone (Ci, 30mcg), Ceftazidime (Ca, 30mcg), Gentamycin (G, 10mcg), Tetracycline (T, 30mcg), Streptomycin (S, 10mcg), Tobramycin (Tb, 10mcg), Cephotaxime (Ce, 30mcg), Nalidixic Acid (Na, 30mcg), Norfloxacin (Nx, 10mcg), Cephalothin (Ch, 30mcg), Rifampicin (R, 5mcg), Cefixime (Cfx, 5mcg).

Extraction of Genomic DNA

The genomic DNA was extracted from a pure culture of V. cholerae cells by the phenol/chloroform method as described previously (Kochl, Niederstatter, & Parson, 2005).

Polymerase Chain Reaction (PCR) Assay

In this study, we used specific primer F: 5-CAAATGATGATAAGTTATATCGG- 3´and R: 5´-GACCAGACAATATAGTTTGACC- 3´ (CUSABIO/China), which designed according to ctxA gene sequences. This primer was detection and amplifying 241 base pairs of the ctxA gene (Barzamini et al., 2014). The primer was provided in a lyophilized form by dissolved in TE buffer to give a final concentration of 100 pmol/µL, ninety microliters of stock solution were mixed with 100µl TE buffer to an obtained working solution. PCR mixture was set up in a total volume of 30µl included 15µl of PCR premix (Solgent/Korea), 2µl of primer and 5µl of extracted DNA, the rest volume was completed 6µl of sterile deionized distilled water. PCR reaction tubes were centrifuged briefly to mix and bring the contents to the bottom of the tubes and placed into thermocycler PCR instrument where DNA was amplified as indicating in below Table 1.

Results and Discussion

We take 100 ml of water sample because a small amount of water sample is not enough for contain enough bacterial cells for detection. Thus, it is important to examined appropriate volumes of water by using several methods to determine the presence of V. cholerae in a given sample (Huq, Haley, & Taviani, 2012).

All isolates were tested, and the positive ones were selected. From one hundred water samples were detected, fifty-six samples with colonies characterized by yellow, smooth, flattened with two to four mm in diameter, opaque centers and translucent peripheries on TCBS agar medium which had pH above of 7 and motile and positive for Simmon citrate, Indole, Oxidase, DNase and Alkaline/Acid for KIA and growth on MacConkey agar characteristics as Vibrio species (Table 2), but only 15 isolates confirmed as V. cholera. These fifteen isolate showed agglutination with polyvalent and monovalent O1 antisera, and two strains represented Ogawa from other strains that showed Inaba (Table 3).

The susceptibility of V. cholerae isolates to antibiotics. Each V. cholerae strain was subjected to antibiotic susceptibility test to a battery of antibiotics (Table 4). The fifteen isolates exhibited an identical response to each antibiotic examined. They were sensitive to Ceftriaxone (Ci), Ceftazidime (Ca), Tobramycin (Tb), Cephotaxime (Ce), Gentamycin (G), Nalidixic Acid (Na), Rifampicin (R) and Tetracycline (T). On the other hand, ten strains included polyvalent Inaba were resistant to Amikacin (Ak), four strains to Streptomycin (S), one strain numbered 91 was resistant to Cefixime (Cfx), one strain to Norfloxacin (Nx), one strain to Cephalothin (Ch).

The bacterial DNA were isolated from the pure V. cholerae, then electrophoresed on 1.7% gel aga­rose, and one band was observed.

The assay of polymerase chain reaction (PCR) was carried out on the DNA extracted from the V. cholerae, and the negative control sample with primers de­signed for ctxA and 241bp band was observed. The results of PCR between strains of V. cholerae isolated from different regions of Al-Najaf Al-Ashraf listed in the previous table shown in (Figure 1), only the genome of V. cholerae is positive.

A thiosulfate citrate bile salts sucrose (TCBS) agar, highly selective medium, removes most nontarget bacteria in experimental samples but is not enough for isolation of V. cholerae from environmental samples because many bacteria existing in natural water sources can growth and produce yellow colonies on TCBS agar whose appearance is like that colony of V. cholerae. Additionally, the series of biochemical tests commonly used to identify V. cholerae (Choopun, 2002). The conventional methods including culturing, serotyping and biochemical testing which used to identify V. cholerae are consumed the time and laborious, while molecular techniques such as PCR are suitable for detection of bacterial culture because of rapid, specificity, and high accuracy (Barzamini et al., 2014). PCR was effectively for detecting of toxigenic V. cholerae directly in seawater and samples. This is providing evidence for an environmental reservoir for pathogenic bacteria (Lipp, 2003). The detection of V. cholerae O1 using PCR method was described by Koch et al. (1993) in food samples, they amplification specific sequences ctxAB gene within the cholera toxin by seeded oysters, crabmeat, shrimp, and lettuce with V. cholerae and washed with alkaline peptone water (6-8h), they resulted only Vibrio stains contain ctxAB gene (Koch, 1993). Shirai et al. were used specific primers for detection of ctx operon of V. cholerae by PCR isolated from rice water stool samples of patients with cholerae. They give rise to that the strains of V. cholerae were identical with those obtained (Shirai, Nishibuchi, & Ramamurthy, 1991). Vibrio that confirmed and identified as Vibrio spp. By using PCR from Vibrio strains isolated from seawater was demonstrated by (Kim, 2015). Other researchers used cultural and PCR methods for the presence of V. cholerae in a total of 245 samples, including 35 samples from water. The results showed 80 samples (19 water) were positive by the PCR method, while the samples were positive by cultural method, only 59 samples (12 water). The study proved the excellent tool for detection of V. cholerae in the water environment is PCR (Maheshwari & Krishnaiah, 2011).

(Alishahi, 2013) were isolate V. cholerae from patients by using multiplex PCR assay with three separate primers for ctxA, toxR, and ompU genes, which is important in pathogenicity and toxigenicity. They found 72 samples identified as V. cholerae carried toxR, and ompU genes but only 61 isolates carried ctxA gene. They indicate that pathogenicity of V. cholerae does not depend on the existence of the cholerae toxin encoded by ctx gene (Alishahi, 2013).

In this study, specific primers were designed for ctxA gene screening of V. cholerae by using PCR, similar to the study by (Barzamini et al., 2014), who using specific primers for ctxA gene of cholera toxin-producing V. cholerae in water and wastewater by PCR assay.

Contaminated water in developing countries plays an important role for transmission of pathogenic bacteria to the human and causes cholera, that regarded as serious problems in healthy countries. Therefore, the rapid detection of V. cholerae in water its important for early diagnostic and prevent the transmission of these bacteria.

Most strains of V. cholerae in this study were resistant to at least two antibiotics of a different group, but there are several antibiotics were still more potent against V. cholerae.

Resistance to antibiotics usually varies from one place to another, V. cholerae becomes to antibiotics by several mechanisms including efflux pumps, chromosomal mutations and genetic resistance. The resistance genes can be able to transfer to commensals bacteria or enteric pathogens in the human gut by sharing traits (Dengo-Baloi, 2017).

Antimicrobial treatment is important for a treat of patients with cholera, although rehydration therapy is the support of therapy. The antibiotics doxycycline and tetracycline were the drugs of choice for the treatment of cholera for long of a time, but the resistance by V. cholerae against antibiotics was demonstrated by many studies (Chillarge, 2015). (Gupta, 2016) demonstrated V. cholerae resistant to ampicillin and cotrimoxazole, but all isolates were sensitive to tetracycline, doxycycline, levofloxacin and azithromycin (Gupta, 2016).

Conclusion

Water is the main source of V. cholerae can transfer to the human, which causes fatal disease. Therefore we should examine the water by isolation these bacteria and detection by several methods to know the accuracy and faster method for identification. Tow methods used in this study for detection of V. cholerae, conventional and molecular method, the PCR technique is faster and accuracy.

Antibiotics are used in combination with rehydration therapy for the treatment of cholerae because antibiotics are widely used for the treatment of cholerae. Therefore, the number of pathogenic V. cholerae strains becomes resistant to one or more of antibiotics. This study used several antibiotics to be determining the sensitivity of V. cholerae to these antibiotics.