2.1. Sample collection A total of 25 samples, five samples from each of five dairy beverage items locally named as Labang, Lassi, Borhani, Strawberry milkshake, and Pistachio nut sherbet were collected randomly from five different nooks and corners of Dhaka University campus and adjacent areas. Samples were collected from canteen, restaurant, fast food shops, and street vendor. The producers used simple technology such as measuring the required ingredients proportionately into a mixing container and mixing accordingly to prepare the final products. The ingredients were mostly measured by eye estimation.
In this study, we collected five samples, prepared in different batches, of a particular beverage items from a particular selling point/shop which was randomly selected from a total of 21 identified selling points/shops in the study area. Samples were taken in sterile containers and were kept in ice-boxes and transported to the laboratory for further examination. Labang, Lassi, and Borhani contain fermented dairy products whereas Strawberry milkshake and Pistachio nut sherbet were nonfermented beverages. It was a general observation that dairy beverages were prepared in unhygienic environment with raw materials such as milk, yogurt, water, ice, syrup, sugar, mint, salts, etc. Samples were collected and analyzed during June to August 2018.
2.2. Sample processing Samples were taken out from the sterilized container and placed in a sterilized petri-dish by pipetting out 10 mL. One milliliter of liquid sample was taken and transferred into sterilized cotton plugged test tubes containing 9 mL of 0.1% peptone water. Then, they were mixed thoroughly by shaking 20 times. This time the solution was allowed to stand for 5–10 min. Thus, the initial dilution of homogenate was prepared and from this homogenization, further serial dilutions were prepared. The samples were diluted at 10 fold dilution up to 10−6 according to American Public Health Association (APHA) sample dilution guidelines (American Public Health Association [APHA], 1992).
2.3. Bacteriological study For bacterial isolation, the spread plate method was followed in this experiment. To identify the isolated bacteria; cultural, morphological and biochemical characteristics were studied using Bergey’s Manual of Determinative Bacteriology, 9th Edition (Holt, Krieg, Sneath, Staley, & Williams, 1994). Different types of non-selective and selective agar such as Plate Count Agar (PCA) for viable count, MacConkey (MC) agar for gram-negative enteric bacteria, Salmonella-Shigella (SS) agar for different species of Salmonella and Shigella, Eosine-Methylene Blue (EMB) agar for coliform bacteria, and Thiosulphate Citrate Bile Sucrose (TCBS) agar selective for Vibrio were used for isolation. Cooked Meat Media was used for Clostridium and Listeria, and Potato Dextrose Agar (PDA) was used for observing fungal growth. From each tube of serially diluted sample suspension, 50 µL was transferred to the petri-dishes and tubes which were prepared with PCA, MacConkey, EMB, SS, TCBS, and PDA and were incubated for 24–48 h at 37°C. The bacterial colonies grew on different types of medium were collected and maintained in nutrient slant agar for further analysis. Morphological, cultural, gram staining and some biochemical tests such as Kliger’s Iron Agar (KIA) test, Motility test, Indole test, Urea (MIU) test, Catalase and Oxidase tests were performed for the identification of bacterial isolates.
2.4. Antibiogram Total 34 isolates were found among which 27 were gram-negative bacteria and seven were grampositive. Gram-negative isolates of bacteria from five samples were selected for antibiotic susceptibility test. Six common antibiotics such as Ampicillin (AMP10), Colistin (CT10), Ciprofloxacin (CIP5), Levofloxacin (LE5), Ceftriaxon (CRO30), Gentamycin (GEN10) were used for each type of bacteria to observe the sensitivity and resistance toward antibiotics. Finally, Multiple Antibiotic Resistance (MAR) index was calculated as the ratio of number of antibiotics to which the isolate showed resistance to total number of antibiotics to which the isolate was exposed (Krumperman, 1983). The MAR index is a good tool for health risk assessment which identifies if isolates are from a region of high or low antibiotic use. A MAR index with 0.2 indicates a “high-risk” source of contamination.