Phylogenetic of ERIC-DNA Fingerprinting and New Sequencing of Aeromonas Species and V. Cholerae DNA

Corresponding Author: Hawraa Natiq Kabroot ALFatlawy Departmetn Biology-Genetic Microbiology, College of Sciences, University of Kuf, Iraq Email: hawraanatiq@gmail.com Abstract: The current study included 44 isolate of A. hydrophila, A. sobria and V. cholerae and other bacteria isolated from stool samples and environmental samples (Kufa river water and hospital environmental samples). ERIC DNA Fingerprinting with ERIC primers pairs generated distinct amplification bands ranging in size from (87 bp to 8000 kb). The 44 isolates produced 93 different patterns by ERIC DNA fingerprinting. The fingerprinting patterns of the isolates were constructed using cluster analysis the UPGMA (group method) using average linkages Number of different bands (Similarity coefficient), 1% tolerance. The PCR method of gene (16SrDNA and 16SrRNA) were the best methods for diagnosis, which has led to isolate and diagnosis of A. hydrophila A. sobria and V. cholerae are distributed as clinical isolates of A. hydrophila A. sobria were diarrhea samples. While, the environmental isolates were isolate of V. cholerae from Kufa river water. Sequencing technology is used to diagnosis of A. hydrophila, A. sobria and V. cholerae isolates were examined by (16SrDNA, 16SrRNA) genes. Recorded the new isolates in Nucleotide/Blast and recorded as the first sequencing in Gene-Bank/NCBI, DDBJ and ENA (INSDC). Each sequence have Accession number (No.: Gene bank: LC194875 Aeromonas sobria-HNK1, Gene bank: LC194876 Aeromonas hydrophila-HNK2, Gene bank: LC194877 Vibrio cholerae-HNK3) this is the first study in Iraq for discovery of new isolates by new sequences. The frequency of A. hydrophila, A. sobria and V. cholerae isolates in Najaf were higher among clinical and environmental isolates. The ERIC band pattern is an adequate tool for epidemiological investigations of A. hydrophila, A. sobria and V. cholerae isolates.


Introduction
The Gram-negative bacilli Aeromonas hydrophila and Aeromonas sobria are species of the genus Aeromonas, which belongs to the family Aeromonadaceae that received increasing attention opportunistic pathogen s because of its association with both dysenteric diarrheal and extra intestinal infections in human disease especially in children and persons with impaired immune system (Naharro et al., 2009;Uche and Johnkennedy, 2014). Aeromonas bacteria are linked to two types of gastroenteritis, the first type is a disease similar to cholera which causes rice-watery diarrhea and the other type of disease is dysenteric gastroenteritis that causes loose stools filled with blood and mucus, while dysenteric gastroenteritis is the most severe out of the two types and distributed of A. hydrophila is widely in fresh and salt water also frequently found in chlorinated and nonchlorinated drinking water (Galindo and Chopra, 2007).
And other hand, Cholera is an acute diarrheal infection caused by ingestion of food or water contaminated with the bacterium Vibrio cholera that belongs to genus vibrio, family Vibrionaceae (WHO, 2016).
Genetic diversity studies on Aeromonas spp. have received a little attention in Iraq. The present study is the first study in Najaf/Iraq and carried out to achieve the following objectives 1. Isolation of A. hydrophila, A. sobria and V. cholerae isolates from diarrheal samples and environmental samples and identification

Samples Collection
Diarrheal samples were 272 from patients suffering of diarrhea infection in AL-Main Health Laboratory of Najaf governorate during the period (April 2016 to November 2016). While, 34 environmental samples were involve three different of environmental regions (Kufa river water). A. hydrophila isolates were diagnosed by four methods as (Culture, biochemical tests, Vitek@2GN/ID cards system and Polymerase Chain Reaction (PCR) methods). Most characteristics of A. hydrophila, A. sobria and V. cholerae bacteria were examine dofgram's stain bacteria, the microscopic properties (Jawetz et al., 2016). Culturally, colonies characteristics of isolates were recorded on the specific media for primary identification of A. hydrophila A. sobria and V. cholerae (Collee et al., 1996) and ThioSulphate Citrate Bile Salt Sucrose Agar (TCBS) (Henry, 1996), Alkaline Peptone Water Medium (APW) (Gomez-Gil and Roque, 2006) and Aeromonas Isolation Medium Base (Moyer, 1987).

VITEK@2 GN ID Card System
The identified Aeromonas ssp and V. choleraeisolates were confirmed with the automated VITEK@2 compact system by using GN ID cards. The GN ID card is based on established biochemical (64 reaction) methods and newly developed substrates, measuring various metabolic activities (BioMérieux Company/http://www.bioMérieux.com),

Genomic DNA Extraction Bacteria Chromosomal DNA (Promega/USA)
Extraction Chromosomal DNA is 44 isolates of (34 A. hydrophila 2 A. sobria, 3 V. cholerae, 1 E. coli O 157, 1 E. coli, 1 Pseudomonas aeruginosa, 1 Enterobacter complex and 1 Acinetobacter manniuii) bacteria. The wizard genomic DNA purification kit is designed for extraction of DNA from bacteria. Extraction of genomic DNA was performed as follow: • Added 1 mL of an overnight culture to a 1.5 mL microcentrifuge tube • Used centrifuge the growth at 14,000×g for 2 min to pellet the cells and remove the supernatant

PCR Amplification
PCR technique has been amplify genes of 16SrDNA, 16SrRNA with ERIC1, ERIC2 primers with genomic DNA of all isolates A. hydrophila A. sobria and V. cholerae and other bacteria. The wizard genomic DNA purification kit is designed for extraction of DNA A. hydrophila A. sobria and V. cholerae and other bacteria. Gel electrophoresis was used for detection of DNA by UV transilluminator (Sambrook and Russell, 2013). The PCR assay was performed to detect the (16S rRNA, 16SrDNA) gene for confirmation the identification of A. hydrophila A. sobria and V. cholerae and other bacteria. These primers synthesized by Alpha DNA company, Canada Program of PCR.
PCR products and the DNA marker are resolved by electrophoresis on (1.5%) agarose gel shown in Table 1.

Clonal Analysis by ERIC DNA Fingerprinting A: ERIC-PCR Assay of A. Hydrophila and A. Sobria, V. Cholerae and Other Bacteria
According to Rathinasamy et al. (2014), the assessing genotypic distinctions in Aeromonas hydrophila, Aeromonas sobria, Vibrio cholera and E. coli O157 isolates, direct PCR examination was carried out to these isolates. One set of primers were used specifying. The reaction was carried out by using a 50 µL mixture including (25) µL GoTaq® Green Polymerase Mix, (5) µL for each ERIC 1 and ERIC2 separately, (8) µL of genomic DNA and the volume was completed with nuclease free water.
The PCR was performed with a Biometra, professional thermal cycler under the following conditions.
The PCR products were loaded with gel electrophoresis after mixing with loading dye. The electrophoresis done with Biometra gel electrophoreses in 1.5% agarose gels and photographed on gel documentation system.

B: Bioinformatics' Analysis for ERIC DNA Fingerprinting
Computer analysis of ERIC DNA fingerprinting was carried out using Bio Numerics Gel-Compar II version 7.6. Applied Maths. Similarity between fingerprints were calculated with the Dice coefficient. Cluster analysis was performed using the unweighted pair-group method.

Gene Sequencing and Analysis
Sequenceing of diagnosis genes for Aeromonas hydrophila, Aeromonas sobria and Vibrio cholera (16SrRNA, 16SrDNA genes) were performed by Macrogen's sequencing service, Sequencing Technology in Korea http://dna.macrogen.com. Sequence Analysis Software Programs; blast online program (www.ncbi.nlm.nih.gov). Basic Local Alignment Search Tool (BLAST) program is available at the National Center Biotechnology Information (NCBI) online at http://blast.ncbi.nlm.nih.gov/Blast.cgi for Aeromonas hydrophila and Aeromonas sobria and Vibrio cholera isolates in this study.

Bioinformatics
Bioinformatic and biostatistical service technology has been advancing the field of medical and ecological research and diagnostics in Next-Generation Sequencing (NGS). Bioinformatic specialized in life science and setting new standards for high level services of data analysis including high expertise in next-generationsequencing (Ghatak et al., 2016).

Results
Identification of Aeromonas spp. and V. Cholerae.
During the study period were collected272clinical samples from diarrhea cases and, 34 environmental samples from Kufa river water and hospital environmental samples Identification of A. hydrophila, A. sobria and V. cholerae isolates is depended on initial identification the colonial morphology, microscopically and biochemical tests. The colonies of A. hydrophila A. sobria and V. cholerae are grown on culture media once revealed the typical characteristics, on blood agar A. hydrophila, A. sobria and V. cholerae produces smooth, convex, rounded and β-hemolytic colonies and pale white to grey color, but colonies were green with black center Aeromonas isolates on Aeromonas media and V. cholerae on Chromo agar is light blue colonies as show in (Fig. 1). Biochemically tests were positive results for indole tests, oxidase test and simmone citrate test.

VITEK@2 GN ID Card System
The identification was contained biochemical tests. The results demonstrate that isolates of A. hydrophila A. sobria and V. cholerae isolates were confirmed with ID message confidence level ranging excellent (Probability percentage from 94 to 99.7%). The 44 isolates of (34 A. hydrophila 2 A. sobria, 3 V. cholerae, 1 E. coli O 157, 1 E. coli, 1 Pseudomonas aeruginosa, 1 Enterobacter complex and 1Acinetobacter manniuii,) of isolates on MacConkey agar.

Molecular Identification
PCR technique has been amplify genes of 16SrDNA and 16SrRNAgenes with genomic DNA of all isolates A. hydrophila, A. sobria and V. cholerae. The results of all isolates diagnosis by PCR technique for detection both 16SrDNA and 16SrRNA clarify isolates of A. hydrophila, A. sobria and V. cholera producers carrying 16SrDNA and 16SrRNA genes, as show in (Fig. 2).
Dendrogram of phylogenetic analysis revealed the diversity of all isolates in the Najaf. The percentage level of similarity clearly showed that the isolates examined by species were divided into (8) distinct cluster numbers, in addition to (4) single isolates, that clustered at a similarity level of (93%). Cluster I was the largest characterized by domination of phylogenetic group and specifically subgroup.

Sequencing and Analysis of 16SrDNA and 16SrRNA Gene Sequences
A. hydrophila, A. sobria and V. cholerae isolates were examined by sequencing technology to diagnosis of isolates and record it by (16SrDNA, 16SrRNA) genes. All isolates were success in processing of a good running of sequencing by a Company DNA-Macrogen/Korea. The results were indicated to the first Iraqi isolates after compared in Gene Bank/ BLAST is available at the NCBI online, using Nucleotide/Blast and recorded as the first sequencing in Gene-Bank/NCBI, DDBJ and ENA (INSDC).
16SrDNA and 16SrRNA genes were successfully amplified using specific PCR primers for A. hydrophila, A. sobria and V. cholerae isolates which observed of results, which showed PCR amplification for 16SrDNA and 16SrRNA genes, which have a specific products (1500 and 300) bp respectively. Results analysis of the sequencing were revealed discovering new strains and recorded for each gene of isolates; A. hydrophila, A. sobria also V. cholerae isolates were recorded as the first sequencing in Gene-Bank by Accession numbers in Gene-Bank/NCBI, DDBJ and ENA (INSDC): Accession numbers: Gene bank/NCBI/Nucleotide Gene bank: LC194875Aeromonas sobria-HNK1 Gene bank: LC194876Aeromonas hydrophila-HNK2 Gene bank: LC194877Vibrio cholerae-HNK3 Gene bank/NCBI, DDBJ: ENA (INSDC) USA, these Accession numbers as showed in index (1) and (2).

Discussion
Identification of A. hydrophila, A. sobria and V. cholerae depending on the morphology, biochemical tests, VITEK@2GN ID system and molecular identification. The colonies are green with black center Aeromonas isolates on Aeromon as media (Carriero et al., 2016). PCR technique has been amplify genes of 16SrDNA and 16SrRNAgenes with genomic DNA of A. hydrophila, A. sobria and V. cholera and other bacteria. The current results of all isolates diagnosis by PCR technique for detection both 16SrDNA and 16SrRNA clarify of A. hydrophila, A. sobria and V. cholera and other bacteria, producers carrying 16SrDNA and 16SrRNA genes. Singh et al. (2012) who noted that the ribosomal mainly 16SrRNA gene has be a stable and specific for the identification of A. hydrophila, A. sobria bacteria. The present results are agree with (Al-Fatlawy and Al-Ammar, 2013;Boustanshenas et al., 2016).
ERIC Sequence greater heterogeneity among the clinical and environmental isolates of A. hydrophila, A. sobria and V. cholerae and other have been demonstrated by ERIC DNA Fingerprinting. The isolates revealed a clear structure on this basis, conclusion that the isolates of Aeromonas and other bacteria having genetically heterogeneous (Rathinasamy et al., 2014).
Our results demonstrated that DNA Fingerprinting with ERIC primers amplification bands ranging in size (87 bp to 8000 kb). The 44 isolates produced 93 different patterns by ERIC fingerprinting (Fig. 4). The fingerprinting patterns of the isolates were constructed using cluster analysis the UPGMA (group method) using average linkages Number of different bands (Similarity coefficient), 1% tolerance.
Dendrogram of phylogenetic analysis revealed the diversity of all isolates in the Najaf. The percentage level of similarity clearly showed that the isolates examined by species were divided into (8) distinct cluster numbers, in addition to (4) single isolates, that clustered at a similarity level of (93%). Cluster I was the largest characterized by domination of phylogenetic group and specifically subgroup.
A. hydrophila, A. sobria and V. cholerae isolates were examined by sequencing technology to diagnosis of isolates and record it by (16SrDNA, 16SrRNA) genes. All isolates were success in processing of a good running of sequencing by a Company DNA-Macrogen/Korea. The results were the first Iraqi isolates after compared in Gene Bank/BLAST is available in NCBI/Nucleotide/Blast and recorded as the first sequences in Gene-Bank/NCBI, DDBJ and ENA (INSDC).
16SrDNA, 16SrRNA and hlyA genes were successfully amplified using specific PCR primers for A. hydrophila, A. sobria and V. cholerae isolates which observed of results which showed PCR amplification for 16SrDNA and 16SrRNA genes, which have a specific products (1500 and 300) bp respectively, as index (3).
Results analysis of the sequencing were revealed for each gene of isolates; A. hydrophila, A. sobria also V. cholerae isolates and recorded as the first Iraqi sequencing in Gene-Bank by Accession numbers in Gene-Bank/NCBI, DDBJ and ENA (INSDC).
All these sequences Recording of Iraqi sequences in NCBI-Gene-Bank and DDBJ of INSDC A. hydrophila, A. sobria and V. cholerae isolates sequences were isolated from clinical specimens and hospitals environment in NAJAF city and each sequence have Accession number (No. Gene bank: LC194875 Aeromonas sobria-HNK1, LC194876 Aeromonas hydrophila-HNK2 and LC194877 Vibrio cholerae-HNK3) Gene bank/NCBI, DDBJ and ENA (INSDC) USA as showed in appendix (1), (2) and (3 ).
16SrDNA, 16SrRNAgenes sequence submitted to Gene bank. The results of these sequences were analyzed and examined by professional staff in Gene bank/NCBI, DDBJ and ENA (INSDC). All these sequences accepted in Gene bank and each sequence take accession number.
These results recorded and published in the International Nucleotide Sequence Database Collaboration (INSDC).
Local isolates in Najaf do not studying by sequences technology in past, therefore, the current study is discovering new isolates by contamination between patients and environments so that demonstrated these isolates in Najaf.

Conclusion
• The frequency of A. hydrophila, A. sobria and V.
cholerae isolates in Najaf were higher among clinical and environmental isolates • Identification by VITEK@2GN card system and molecular technique is necessary for detection of pathogenic bacteria between clinical and environmental samples • The ERIC-DNA Fingerprinting band pattern is an adequate tool for epidemiological investigations of A. hydrophila, A. sobria and V. cholerae isolates