Antibiotic Resistance Analysis of Vibrio spp Isolated from Different Types of Water Sources of Bangladesh and Their Characterization

Cholera is an acute type of the diarrheal disease that torments human civilization over the centuries. The wickedness of human sufferings led physicians, and researchers to carry out extensive research on cholera and Vibrio cholerae leading to potential discoveries that opened up new areas of research or new disciplines in biomedical sciences. An attempt is made here to summarize some of these breakthroughs and outline their significance in broader perspectives. In the present study, a total of 12 samples were collected from four types of water sources for separation of Vibrio spp. Water samples are enrichment into alkaline peptone water then inoculate into culture media such as Nutrient agar, MacConkey, and Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar medium. After incubation of TCBS plates for 24h at 37°C yellow and green colonies are screened out for biochemical identification. No-sucrose fermenting Vibrio parahaemolyticus, Vibrio mimicus, and Vibrio vulnificus show green colonies and sucrose fermenting Vibrio cholerae and Vibrio alginolyticus shows yellow colonies on TCBS. To identify the Vibrio spp extracts biochemical test was carried out and typical Vibrio spp give a positive result. The majority of Vibrio spp are avirulent, but certain strains may be sporadically human pathogenic. The antibiotic resistance studies represented that among the 9 extracts were resistant against Erythromycin, Penicillin, Cephalexin, Vancomycin and 4 extracts resistant against Tetracycline. To expand present knowledge of the occurrence, ecological niche and persistence of significant human pathogenic Vibrio spp in water environments, occurrence, and laboratory studies were tested.

serve as the main water resources in rural areas used for drinking, cooking, and irrigation for agriculture in most communities that have little or no access to potable, safe water. V. cholerae is known to be normal inhabitants of surface water. Illnesses in humans occur after ingestion or direct wound contact with contaminated food or water (Cho et al., 2008;Turner et al., 2013). At least twelve Vibrio species are of clinical significance for the human, being V. cholerae, V. parahaemolyticus and V. vulnificus the three main species (Gomez-Gil et al., 2014).
Over the years, several researchers have focused on the sternness of diseases caused by V. cholerae leaving out relatively minor Vibrio species of medical interest, some of which are described as emerging pathogens able to cause mild to severe human diseases. Vibrio spp has emerged as a serious threat to human health worldwide. The growing number and severity of food poisoning incidents around the world have greatly increased public awareness about food safety (Forsythe, 2000). Many Vibrio species are pathogens to human and have been implicated in foodborne disease. Five types of diseases such as tail necrosis, shell disease, red disease, loose shell syndrome (LSS) and white gut disease (WGD) is caused by Vibrio spp in Penaeus monodon (Jayasree et al., 2006). Vibrio spp has also been implicated in mortality events in the wild (Li et al., 2006). Pathogenic Vibrio causes acute gastroenteritis characterized by diarrhea, headache, vomiting, nausea, and fever (Yang, et al., 2008 Vibrio spp is rod-shaped Gram-negative bacteria that are widespread in coastal and estuarine environments around the world. V. cholerae is a usual inhabitant of the environment and causes severe diarrheal disease contaminating thousands of people each year in developing countries. V. parahaemolyticus was first identified as a foodborne pathogen in the 1950s, and, in subsequent decades, became globally recognized as an etiological agent of diarrheal disease associated with seafood consumption (Parveen et al., 2008).
Vibrio is a Gram-negative halophilic bacterium found abundantly in marine and estuarine environments (Thompson et al., 2004;Raissy et al., 2014). Among the different species, V. parahaemolyticus has emerged as a significant pathogen for M. rosenbergii (Khuntia et al., 2008). The genus Vibrio includes some species that behave as opportunistic pathogens and can be involved both in cultured gilthead sea bream outbreaks and in cases of human disease (Austin and Zhang, 2006). Typical symptoms of the gastroenteritis may include abdominal pain, diarrhea, nausea, and fever (Tan et al., 2017).
Bacteria can have an impact on the quality of water by controlling the amount of oxygen and other elements and by causing diseases in aquatic organisms and humans. Antibiotic-resistant bacteria may represent a potential threat to human health due to direct transmission through the food chain (Duran and Marshall, 2005;Rahman et al., 2019) or by transferring the acquired antimicrobial resistance to human pathogens by mobile genetic elements (Guglielmetti et al., 2009). Vibrio spp usually, are said to be highly susceptible to most clinically used antibiotics (Mala et al., 2014;Shaw et al., 2014). The number of Vibrio spp. classified as pathogenic strains are at least 11, including V. cholerae as the main cause of diarrhea, V. parahaemolyticus as the cause of foodborne gastroenteritis (Ozer, et al., 2008). The aim of this study was to identify, characterize, and determine antibiotic resistance of Vibrio species of clinical importance in water samples and determine their pathogenicity, which will help to control Vibrioassociated gastroenteritis from Bangladesh.

Sample Collection and Cultural -
The present work was divided by separation and Identification of Vibrio spp from different types of water sources. The sample was collected from different region like as beside the Niribili area, Savar and Megna River from Daudkandi, Comilla, Bangladesh. For the separation of bacteria the enrichment, streak and spread plate approachs were followed by alkaline peptone water, Nutrient agar MacConkeyʹs agar and TCBS agar were used for the isolation purpose. UniversePG l www.universepg.com

Enrichment in Alkaline Peptone Water (APW) -
Vibrio spp grows very rapidly in APW and at 6 to 8 hours will be present in greater numbers than non-Vibrio organisms. Enrichment in APW enhances the separation of V. cholerae when few organisms are present, as in specimens from convalescent patients and asymptomatic carriers. An enrichment medium containing a selective agent may not offer any advantage over APW if it is used with short incubation time (6 to 8 hours).

Nutrient and Mac Monkeys Agar-Nutrient agar
was used for cultivating of non-fastidious microorganisms. A differential medium was also used for the separation of Coliforms and intestinal pathogens in water and biological specimens.

TCBS Agar -
TCBS is the medium of choice for the separation of V. cholerae and is widely used worldwide, it has a relatively short shelf life once prepared (3 to 5 days) unless plates are carefully protected against drying. TCBS agar is green when prepared. Overnight growth (18 to 24 hours) of V. cholerae will produce large (2 to 4 mm in diameter), slightly flattened, yellow colonies with opaque centers.

Morphological Characterization by Gram's Staining -
The most widely used staining procedure in microbiology is the gram stain. The procedure is based on the ability of microorganisms to retain the color of the stains used during the gram stain reaction.
Gram-negative bacteria are decolorized by the alcohol, losing the color of the primary stain, purple. Grampositive bacteria are not decolorized by alcohol and will remain as purple. After decolonization step and a counter stain is used to impart a pink color to the decolorized gram-negative organisms. This approach allows for maintaining their natural shape and makes it possible to observe their behavior. If an organism is motile, its activity is clearly apparent, and directional, place locomotion will be observed. This test was performed according to the approach described by Cowan to differentiate motile bacteria from non-motile one.

Antibiotic Sensitivity Test -
The list of commercially available antimicrobial disc used in this study with their concentration is given bellow After placing of the disc on the plate, the plates were inverted and incubated at 37°c for 16 to 18 hours after incubation, the diameter of the zones of complete inhibition (including the diameter of the disc) was measured and recorded in millimeters. The measurements were made with a ruler on the undersurface of the plate without opening the lid. UniversePG l www.universepg.com

Separation and study of cultural characters:
In the present study, 12 water samples were collected from different sources such as tap water, pond water, river water and sewage water for isolation and identification of Vibrio spp gram negative bacteria. All samples should be tested before enrichment and after enrichment. Before enrichment samples were inoculated into culture media such as Nutrient agar, MacConkeyʹs and TCBS agar used for separation of Vibrio spp. After enrichment into alkaline peptone water can be used to enhance the recovery of Vibrio spp where there are few organisms present. In the present study, specific biochemical tests were used for the detection of Gram-negative bacteria.

Nutrient, Mac Conkey, and TCBS Agar:
Nutrient agar represented circular, smooth and translucent colonies. MacConkey agar represented colorless colonies after prolonged incubation pink color colonies were seen. This is a highly selective medium which is used to extract and to differentiate the sucrose fermenting from non-sucrose fermenting species of Vibrio. TCBS agar represented that large 2 to 3 mm, smooth, yellow, shiny, convex and slightly flattened with opaque centers. Among the 12 samples, 2 samples did not show any growth of an organism in the media. Ten-fold dilutions of water samples were made down to 10-8, before 0.1 ml of the aliquot of each serial dilution was plated onto TCBS agar in triplicate (Letchumanan et al. 2015).   Yellow, Green 10 Green

Microscopic Examination:
Microscopic observation was performed to observe the shape and the gram reaction of the extracts. All the extracts were found to be gram-negative, curved, comma and rod shape. In Gram's staining, the morphology of extracted V. species is gram-negative, curved rodshaped bacteria with the single polar flagellum. We worked with 15 extracts of 12 samples of water after 3 times of sampling we get 15 extracts from 4 sources of water such as tap water, pond water, river water, and sewage water. Among 15 extracts, 8 extracts of 4 samples represented a zone of inhibition on blood agar media. The motile and non-motile organisms were identified by observing motility.

Sample name
Antibiotic name  We investigate the antibiotic susceptibility; Vibrio species were extracted and identified from the sample of water. Among the 15 extracts, 9 extracts represented green and 6 extracts represented yellow colony. Susceptibility of these Vibrio species was assayed by the disc diffusion approach against 10 antibiotics. Among the 9 extracts, 6 extracts represented sensitive against Chloramphenicol, 9 extracts represented highly sensitive against Sulphamethoxazole, Gentamicin, Nitrofurantoin, and 5 extracts sensitive against ciprofloxacin.
Among the 9 extracts were resistant against Erythromycin, Penicillin, Cephalexin, Vancomycin and 4 extracts resistant against Tetracycline. Vibrio species extracted in this study represented good susceptibility to most of the antimicrobial agents that are commercially available for the treatment of causing disease.

CONCLUSIONS
The present study focuses on the incidence of Vibrio spp in water bodies which are used by people in surroundings for their daily needs. For all living organisms, water is the most vital and significant matter for survival. Despite modern techniques for disinfection, sanitation, and water purification, waterborne diseases still threaten human health. Studies represented that ingestion of water contaminated with Vibrio spp like V. cholerae can create serious symptoms including diarrhea, enteritis, and even death, leading to high economic losses. These bacteria are found almost everywhere. Humans, animals, and sewage can be sources of these bacteria. Contaminated water plays a significant role in the transmission of bacteria to human body. The primary goal of this work the separation and identification of Vibrio spp from a different region of water such as tap water, pond water, river, and sewage water and was 40% 60%

Percentage of total isolates
Vibrio Cholerae Vibrio parahaemolyticus UniversePG l www.universepg.com conducted to determine the separation and identification of gram-negative Vibrio spp organisms by cultural study, staining procedure and different type of biochemical test. The purpose of the study was to extract and identify the presence of Vibrio spp in the water bodies in different areas of Bangladesh as well as determine the pattern of antibiotic resistance. Finally, more work should be done on the molecular level to confirm extracts as they are not being extracted using the cultural approach.