Study Area
Bay of Bengal lies between latitude 5° N to 22° N and longitude between 80° E to 94° E. The Bay is semi-enclosed and surrounded by India, Bangladesh, Myanmar, the Andaman, and the Nicobar Islands. The Bay receives a huge amount of freshwater influx (1.6 × 1012 m3 per year) along with sediment from the Brahmaputra, Cauvery, Ganges, Godavari, Irrawaddy, Krishna, and Pennar rivers (Subramanian, 1993; Varkey et al., 1996). The population living in this region is one-third of the world. Industries in this region are a major part of the growth, development and economy of the countries. Visakhapatnam – Guntur industrial region falls under the 8 major industrial regions of India with many industries related to maritime, petroleum chemicals, pharmaceuticals, etc. The presence of a major port and several minor ports add to the industrial activities of this region. In this study, the coastline from Pydibhimabaram to Tuni in the district of Visakhapatnam was chosen. This region covers ten major sampling locations including treated effluent discharge points from industries and harbours (Fig. 1). The details of the study sites, industries, type of discharge and the number of stations sampled from each station are listed in Table 1. Except for 12 major discharge points, nearby stations varying in numbers from 9 to 24 surrounding the discharge points are also sampled. A total of 147 stations were sampled as part of this study. All these stations are between 0.5 to 6.6 km from the coast. For easier understanding, the stations are broadly divided into three groups; north stations (NS), middle stations (MS) and south stations (SS) based on their geographic location and time of collection. The north stations (NS) included PBM and TPM, the middle stations (MS) included CHP, VSH, GPH and VSP and the south stations (SS) included TVM, PDM, NKP and TUN.
Sample collection and analyses
Samples were collected during two time periods. During the 1st phase of sampling (8th to 11th Dec 2018), Water samples from all the stations from CHP, VSH, GPH, and VSP were collected. In the 2nd phase of sampling (24th Dec 2018 to 1st Jan 2019) water samples from the rest of the stations were collected. The second phase of sampling was carried out so that water samples can be collected from the stations that included industrial discharge points during their respective discharge period. Niskin bottle (10 litres) was used for a sample collection from 147 stations along the coast of the study area. Both surface (below 0.5 m from the surface) and bottom (above 1 m from the bottom) water samples were collected from each station and fixed/preserved accordingly for downstream analyses.
Physicochemical parameters
Vertical profiles of pressure, temperature (°C), and salinity (ppt) were measured using a portable conductivity, temperature, and depth (CTD) profiler (SBE 19 plus; Sea-Bird Electronics, USA). First, bubble-free water is collected for dissolved oxygen (DO) and immediately fixed with Winkler’s reagents which were later analyzed following the Winkler titration method of Carritt and Carpenter (1966). Water samples were collected in cleaned plastic bottles for nutrient analysis and fixed by adding mercury chloride (for ammonia, nitrate, nitrite, and phosphate). The concentrations of nutrients were measured following standard spectrophotometric procedures (Grasshoff et al., 1983). A 2-L water sample was filtered through a GF/F filter (0.5 μm pore size, Whatman). Chlorophyll-a on the filter was first extracted with 90% acetone at 4 °C in the dark for 12 h and then spectrofluorometrically analyzed. Samples collected in acid-washed glass bottles were analyzed for pH by potentiometric method (Dickson et al., 2007) using 835 Titrando (Metrohm, Switzerland). For total suspended matter (TSM), 1-2 l of water samples were filtered through 0.22 µm filter paper (MCE, 47 mm, Millipore) and they were dried and weighed and calculated in grams per litre.
Bacteriological parameters
About 100 ml of sample was taken into a pre-sterilized screw-capped bottle for bacterial analysis from both surface and bottom water from the Niskin water sampler’s nozzle. All samples were collected after rinsing the sterile collected bottle with precautions required for microbiological analysis while wearing gloves. Samples were kept in an ice box for transportation to the lab where it is analyzed soon after or kept at 4 °C whenever necessary to stunt bacterial growth (APHA, 2017; Ramaiah et al., 2004). The bacteriological examinations were carried out following Ramaiah et al. (2004) and Prasad et al. (2015) for the enumeration of heterotrophic, indicator, and few pathogenic bacteria. A 100 µl of the sample was taken and spread on various plates and incubated at 25 °C for 24-48 hours. Sample dilution (10 times) was performed whenever the plate became overcrowded and replated. All the samples were plated in replicates. The number of colonies was then counted using a colony counter. The specific colonies (unique to the organism of interest) on the respective agar media (HIMEDIA) were quantified and the results were expressed in colony-forming units (CFU ml−1) after averaging out from the replicate plates according to Nagvenkar and Ramaiah (2009). Different selective, isolation and differential media were used for the specific bacterial groups. Heterotrophic bacterial counts were determined on R2A agar, enumeration of coliforms on MacConkey agar, Aeromonas hydrophila on Aeromonas Isolation agar, Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia, Proteus mirabilis, Staphylococcus aureus on Universal differential medium, Pseudomonas sp. like Pseudomonas aeruginosa on Cetrimide agar, Salmonella spp. and Shigella spp. on Xylose-Lysine-Deoxycholate (XLD) agar, and Vibrio spp. like Vibrio cholerae, Vibrio fluvialis, Vibrio parahaemolyticus and Vibrio vulnificus on thiosulphate citrate bile salts sucrose (TCBS) agar.
Antibiogram
From respective agar plates, presumptive pathogenic bacterial colonies were isolated by the streak plate method. A total of 46 isolates from respective agar plates were transferred to 1 ml of Mueller Hinton broth and left to grow overnight. The concentrations of the cultures were adjusted to 0.5 McFarland (OD of 0.1 at 600 nm) before plating. Mueller Hinton agar plates were prepared and fresh cultures from the tubes were spread evenly on them by using sterilized cotton swabs covering the whole surface. A total of 21 antibiotic discs used for testing sensitivity/resistance of Enterobacteriaceae isolates as per CLSI guidelines, are cefuroxime (CXM), cefoxitin (CX), ceftazidime (CAZ), ceftriaxone (CTR), cefoperazone (CPZ), cefotaxime (CTX), cefepime (CPM), piperacillin (PI), piperacillin/tazobactam (PIT), ampicillin/sulbactam (A/S), nalidixic acid (NA), norfloxacin (NX), ciprofloxacin (CIP), gentamicin (GEN), amikacin (AK), imipenem (IPM), meropenem (MRP), azithromycin (AZM), tetracycline (TE), aztreonam (AT), and chloramphenicol (C). These antibiotic discs were placed on MH agar plates and left overnight to grow. The next day, the diameter of the inhibition zones was measured using a scale specifically for this purpose. Among them, 11 antibiotics are considered for Pseudomonas aeruginosa isolates as per CLSI guidelines in M100 (CLSI, 2017).
The resulting inhibition zones were compared with the inhibition zone diameter range given in the current CLSI (clinical and laboratory standards institute) guidelines that determine the inhibition zone diameter breakpoint where the bacterial strain is resistant, intermediate or susceptible to the given antibiotic which follows Kirby-Bauer disc diffusion method (Bauer et al., 1996). For the Enterobacteriaceae family, 21 and Pseudomonas, 11 antibiotics were taken for testing. The antibacterial resistance (AR) index for these samples based on the isolates was determined.
AR index = y/nx
Where, y = Number of resistant microbes in the sample
n = population size,
x = total number of antibiotics used.
For isolates resistant to 3 or more antibiotics multiple antibiotic resistance (MAR) index was calculated.
MAR index = a/b
Where a = No. of antibiotics, the isolate is resistant to, b = No. of antibiotics, the isolate is exposed to/tested against.
Statistical analyses
To compare the means of the parameters and whether they are varying significantly spatially, pair-wise statistical analyses were performed between NS, MS and SS combining both values in surface and sub-surface water samples. For physicochemical parameters, a parametric Tukey’s HSD (honestly significant difference) test was performed. For bacteriological parameters, non-parametric Dunn’s post hoc test is carried out. Principal component analysis (PCA) was performed to infer the effect of physical (Temperature, Salinity, PAR, and Fluorescence) and chemical parameters (Dissolved oxygen, Chlorophyll-a, Total suspended matter, pH, Ammonium, Nitrate, Nitrite, and Phosphate) on bacteriological parameters (Heterotrophic bacterial count, total coliforms, Escherichia coli, Enterococcus faecalis, Klebsiella pneumonia, Aeromonas hydrophila, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella, Shigella, Vibrio cholera, and Vibrio parahaemolyticus) for surface samples and bottom samples separately. All the statistical analyses were performed using PAST 4.03 software.