Sarab Hussein Khaleel1, Ibtisam H. Al-Azawia2 and
Ali Hussein Khlebos3

1Department of Anatomy, College of Medicine. University of Al- Qadisiyah, Diwaniyah, Iraq.
2Department of Microbiology, College of Medicine. University of Al- Qadisiyah, Diwaniyah, Iraq.
3Iraq Ministry Healthy, Al-Dewany Teaching Hospital, Laboratory Department, Diwaniyah, Iraq.

Abstract

Cholera is one of the most important epidemic diseases globally. It is causing of morbidity and mortality in the world. Severe watery diarrhea causes a composite process involving several component that help them reach the lining of the small intestine, form colonies and generate bacterial toxins.This survey was carried out to discover the genetic patterns of clinical isolates according to the presence or absence of toxic genes for the city of Diwaniyah in southern Iraq. Sixty isolates were isolated from patients with cholera. The isolates included the center of the city and the surrounding rural areas. Biochemical and serological diagnosis. All the isolates were V. cholera  serogroup O 1 of the serotyping  Ogawa in  biotype El Tor  . Genetic testing was carried out using PCR technique and base on the presence or absence of toxin genes. Three genotypes were identified for the region.

Keywords: Vibrio, Cholera, biochemical and surgical diagnosis.

Introduction

One of the epidemic disease is cholera. Acute watery diarrhea disease is causing  common in Iraq . there are several region of Africa , Latin America and south of Asia where seasonal outbreaks happen (Neelam,et al.2012)And  It was public health problem ( Shears ,2001). cholera belong to the family Vibrionaceae which have 104 species (DSMZ. Leibniz) . The strains of genus V. cholera classified to serogroups according to epitopic different for lipopolysacharide for surface of cell(Yamai et al. 1997) The serogroup O 1 and O 139 among 210 that causal agent for cholera  (Revera,et al.2012).

There are two  distinct biotypes  for O1 are classical and E1 Tor, every one of biotype has three serotypes are Ogawa , Inaba and Hokojima.  (Nair, 2001).

Globally , the V. cholera have seven pandemics . Indian was originated for the first six pandemics of cholera via classical type for v. cholera O1.The seventh pandemic was E1 Tor that spread to the world which originated from Sulawesi in Indonesia .  ( Carlos and Eduardo, 1996)

Iraq is confronting tremendous calamities of obliteration of the foundations with lack of sterilized water of drinking particularly in poor areas and exile camp that aide for the presence of contaminate water with cholera. (Al-Abbasi,et all.2005)

In Iraq, 1999 reported the first occurrence of O 139 in Baghdad although few cases.(Al-Abbassi , et al.2005).After war of  Iraq in 2003 with program of the communicable disease reported cholera epidemics in many region of Basrah(Valencino et al., 2003). Biotype El Tor of serogroup  O1 include serotype Inaba isolated from 4,667 cases in Iraq when outbreak epidemic cholera in 2007.( Khwaif et al. 2010 ) . As well as , Ogawa serotype reported in this epidemic (Saleh, et al.2011)

All governorates in Iraq isolate  Vibrio cholera O1 serotype Inaba with few cases only of Ogawa serotype form 2013 to 2015. (Al-Abbassi et al.2005)

The  enterotoxin (CT) of cholera which codes by ctxAB gene is causing the severity of disease (Joseph,et al.2015). Strains O1 and O139 of V. cholera   possess the ctxAB gene on the 6.9-kb CTX prophage integrated within chromosome which responsible for encoding enterotoxins  . These Enterotoxins in V. cholera cause diarrhea . And  ace gene coding for accessory cholera enterotoxin as well as zot coding for zonula occludens toxin.

The aim of present study to determined genotypes of clinical isolates of V. cholera according to presence and absence of enterotoxin genes are ompW , ctxA , ace , zot by using PCR technique for outbreak and sporadic cholera in Diwaniyah city.

Martial and methods

Specimen collection
In all, 60 isolates of V. cholera O1 were obtained in 2016 from patients having acute watery diarrhea .The samples were collected from Al Diwaniyah hospital in Diwaniyah city of Iraq.

These isolates were identified  V.cholera O1 after culture and isolation (following World Health Organization 2014 guidelines) on TCBS agar (BIOMARK, Laboratories, India) ‘media via usual biochemical tests that included those for the fermentation of sugars in triple sugar iron agar medium, H2S and gas, indole formation, mannitol motility, oxidase, urease production , catalase production , nitrate reductase . Then isolates were confirmed by serology with commercially available antisera (Denka Seiken, Gosen-shi, Nilgata, Japan). Culture (18–24 h old) for these  strains with specific density (0.5–0.63 McFarland standard, measured with colorimeter supplied with the system) and inoculate into sterile normal saline (supplied by BioMerieux, Lyon, France) then load on to Vitek 2 Compact system has software version 4.01 (BioMerieux, Durham, NC) following manufacturer’s instructions for identification using GN (BioMerieux) cards. The isolates were also sent to the Central Health Laboratory  of Cholera and Enteric Diseases, Bagdad , which be the WHO Collaborating Centre for research on diarrheal diseases  to identity for these isolates.

PCR test
Genomic DNA for V.coleara extracted carried out by geniod kit (Geniad ).Then amplification the target DNA by PCR in a thermal cycler (Techne TC-3000. USA) was performed in a reaction mixture tube which add to it 1µl for each” primer “(forward and reverse),5 µl from gDNA extract and add dionized water to complete the final volume 20 µl.”

The primers were used for cholera toxin in this study(ompW) (ctxA), (ace), (zot) . All primers weres provided by (Bioneer company . Korea) showing in (Table 1) . The program of thermocycler  was as follows: preincubation at 95 C for 2 min , 30 cycles for 30 sec. at 95C for denaturation, 1 min at 64.8C for annealing, 30 sec. at ( 57.8 ,62.9 and 59.9 for ompW, ctxA and zot respectively)C for elongation  and incubation at 72C for 5 min for final elongation. This protocol for each ompW ,ctxA and zot genes. While the protocol for ace gene was touchdown  PCR protocol . It was performed on 45 cycles of predenaturation at 95°C for 2 min, followed by the 15 cycles with successive annealing temperature decrements that 0.5°C changed from 63°C to 56.5°C in every cycle versus time increments (30 sec in the first 15 cycles and 30 sec in the last 20 cycles). The reaction was denatured at 95°C for 30 min, followed by annealing at this temperature algorithm and polymerization at 72°C for 40 sec. Subsequently, the 20 cycles of denaturation at 95°C for 30 sec, annealing at 56°C for 30 sec, and elongation at 72°C for 40 sec were employed. The last primer extension step was done at 72°C for 5 min. Amplicons were made electrophoresis in agarose 1.5% gel . (Appligene, Illkirch, France) then stained with ethidium bromide (5mg/ml) for visualized

Tables 1. Primers used in PCR*

Gene Sequence of nucleotides (5’-3’)

 

PCR product
Size (bp
ompW- F

ompW – R

 

5’- CACCAAGAAGGTGACTTTATTGTG-3’

5’- GAACTTATAACCACCCGCG3’

588
ctxA – F

ctx – R

 

5’- CGGGCAG ATTCTAGACCTCCTG-3’

5’- CGATGATCTTGGAGCATTCC CAC-3’

564
ace – F

ace – R

 

5’- TAAGGATGTGCTTATGATGGACACCC-3’

5’- CGTGATGAATAAAGATACTCATAGG-3’

316
zot – A

zot – R

 

5’- TCGCTTAACGATGGCGCGTTTT-3’

5’- AACCCCGTTTCACTTCTACCCA-3’

947

 

Results

Table 2. Results of vibrio choleara genes

Genes No.teseted No. positive Positive %
ompW gene 60 60 100
ctxA gene 60 60 100
Ace gene 60 36 60
Zot gene 60 57 95

 

Table 3. Genotypes of the Isolates

Grouping of genoype CtxA Ace Zot No.isolates %
Genotype 1 + + + 36 60%
Genotype 2 + + 21 35%
Genotype 3 + + 3 5%
Total 60 100%

 

Biotyping and serotyping
A sixty isolates of  V. cholerae were examined by serogroup, serotyping and biotyping . All isolates  were  diagnosed    V. cholera  serogroup  O1 of  serotyping  Ogawa in  biotypeb El Tor.

Genotyping
Genotypically,  the description  of   isolates were shown in table(2). All V. cholera  O1 isolates were positive for the species specific gene ompW. The gene of toxin ctxA was found in all isolates. And  ace gene showed 36(60% ) of the isolate  while 24(40%) was negative for this gene .Fifty seven  of isolates (95%) was positive for Zot gene and 3 (5%) was negative . For study genotypes for isolates grouped isolate to three isolate based on presence and absence toxins of cholera  table – 3.

Fig. 1.  Agarose (1.5%) gel electrophoresis of  ompW gene with 588 bp in lanes (1-20 ). M was DNA marker

Fig. 2. Agarose (1.5%) gel electrophoresis of ctxA gene with 564 bp in lanes (1-20 ).  M was DNA marker

Fig. 3. Agarose (1.5%) gel electrophoresis of  ace gene with 316 bp in lanes (5,7,9,10,11,12,15,16,17,18and 19).  M was DNA marker

Fig. 4. Agarose (1.5%) gel electrophoresis of zot gene with 947 bp in lanes (1-20 except lane 6 ).  M was DNA marker

Discussion

The larger part of  V. cholerae strain in nature are thought  its innocuous estuarine microbes. Nonetheless, particular strains seem to be advanced that reason sickness for people by successfully colonize in small digestive system and discharging a powerful enterotoxin . Four genes in chromosomal DNA codes for destructiveness related factors in V.  cholerae (OmpW, ctxA, ace and zot) were incorporated into this investigation.

It found all isolate positive for Omp gene .So that indicate all samples were V cholera . The strains isolated from the common source are important for epidemiology, through which the genetic relationship of the isolates is determined and determines the source of transmission, spread and  origin of cholera infection.

(6). In previous studies demonstrated that  omp W gene reoresented as inner control  for testing  V. cholera ( 10, 12 ,17 ,16 ).our results agreement with Menezes et all  concerned that  identification of  v.choleara via  ompW gene give result 100% (Menezes et al. 2014).

“The genetic elements of CTX has a compound transposon with 4.5 kb central core region (ctx AB ,zot , ace ,orfU, and cep)which encodes for both CT and functions for virion and flanked with one or more copies of a 2.7Kb repetitive sequences that responsible for regulation ,replication and integration of ctxφ “(Rivera et al.2001). we tested three genes in this region (ctxA , ace ,and zot) in our isolates.

Enterotoxin  gene ctx A  found in all isolates . This gene is caustive agent to sever diarrhea . All our isolates are Toxogenic strains because  possessing ctx  elements (Julianai et al., 2000; Chein et al., 2004).  And ctxA gene located in this element (Waldor et al., 1996) .This result agree with parrick et al.,2012 and  Riveraa et al.,2001that their result found ctx A gene was 100% in your toxigenic isolates . Ratnam et al. show 98% for their isolation of v.cholera was positive for ctx gene in their clinical isolate (Ratnam et al. 2015) .  However Menezes et al. Show negative results when tested environmental samples via ctxAB gene .other studies not found positive tested for ctxA gene in their isolates.(13) While THEOPHILO et al found one positive isolate for virulence factor ctx A gene in their environmental strains of non O1 and O139(34).

Accessory cholera enterotoxin (ace) gene and (zot) gene show high percentage which was 60% and 95%  in all our isolates .In contrast GRIMONT et al found lower percentage for virulence cassette genes (GRIMONT et al.2002).The zot toxin is increases permeability of the gut via intercellular tight  junction  or (zonula  occludens) (fasno et al.,1999).

However, in all our result show 60%  of  V.Cholera has three genes ctx, ace and zot in genotype 1.In genotype 2 show presence ctx ,zot genes and absence ace gene was 35% for all isolates of v.cholera .However Rivera et all. found that ctxA and zot genes in their toxigenic isolates was 100%.(Revira et al. 2001). Third  genotype show presence ctx ,ace genes and absence zot gene was 5% for all isolates of v.cholera that result agree with many studies (Colwell, et al.,1995 ;  Ghosh et al., 1997; karasawa, et   al. , 1993).

Cholera is still a major epidemic disease that must be investigated and deepened. The continuous monitoring of cholera strains should be carried out through genomic research and the investigation of new strains .Despite the development of medical services. Precautions must be taken to prevent infection. Therefore , the clones were determined their genotype according to virulence factors and  resistance to antibiotic .

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