ORIGINAL ARTICLE


https://doi.org/10.5005/jp-journals-10068-3027
Journal of Gastrointestinal Infections
Volume 9 | Issue 1 | Year 2019

A Study on the Physicochemical and Bacteriological Analysis of Sewage Water from Coastal Karnataka, India


Anusha Hoskere Chandrashekhara1, Sohan R Bangera2, Vignesh Shetty3, Suganthi M Devadas4, Poornima Bhagavath5, Mamatha Ballal6

1–4,6Department of Microbiology, Enteric Diseases Division, Kasturba Medical College, Manipal Academy of Higher Education, Manipal, Karnataka, India
5Department of Chemistry, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka, India

Corresponding Author: Mamatha Ballal, Department of Microbiology, Enteric Diseases Division, Kasturba Medical College, Manipal Academy of Higher Education, Manipal, Karnataka, India, Phone: +91 9901466320, e-mail: mamatha.ballal08@gmail.com

How to cite this article Chandrashekhara AH, Bangera SR, Shetty V, et al. A Study on the Physicochemical and Bacteriological Analysis of Sewage Water from Coastal Karnataka, India. J Gastrointest Infect 2019;9(1):10–14.

Source of support: Nil

Conflict of interest: None

ABSTRACT

Background and objectives: Water, the elixir of life, gets polluted by fecal contaminants, household wastes, and industrial effluents into sewage water and acts as a vehicle in the transmission of human diseases associated with microorganisms. The present study was carried out to isolate and identify the pathogenic microorganisms along with their antimicrobial resistance pattern and also to chemically analyze the sewage water sources of Udupi district.

Materials and methods: The 12 sewage water samples were collected from different localities and subjected to bacterial isolation, identification, and antimicrobial susceptibility testing (AST). The chemical analysis of pH value, total hardness, and amount of chloride present in the samples was determined.

Results: A total of 26 organisms were isolated which consisted of waterborne pathogens, Vibrio alginolyticus (V. alginolyticus) (n = 2) and Aeromonas sobria (A. sobria) (n = 2) along with the environmental pathogens (n = 22). A. sobria showed resistance to cefotaxime and the other isolates were susceptible to the antimicrobial drug tested. The pH ranged from 5.7 to 7.5. Ten samples were considered as hard, and in two samples, the chloride content was above normal.

Conclusion: Contamination of groundwater with the sewage water poses a threat to human health. Hence, treating the sewage water in an appropriate sewage treatment plant (STP) is recommended and the need of the hour.

Keywords: Antimicrobial resistance, Bacterial isolation, Chloride, Hardness of water, pH value, Sewage water.

INTRODUCTION

Water is an important factor essential for life but the quality of water can be compromised by the presence of infectious agents, toxic chemicals, and radiological hazards. Water acts as a vehicle in the transmission of human diseases associated with microorganisms. The waterborne diseases cause major health issues in most parts of the globe, owing to about 1.7 billion cases of diarrhea per year. Due to these waterborne diseases, children under the age of 5 years are the severely affected leading to increased morbidity and mortality.1 The sewage water consists of physical, chemical, and biological wastes which can be separated from water by various methods. The microbial flora of sewage water mainly comprises of bacteria, viruses, protozoa, algae, fungi, and helminths.2 Among the bacteria that contaminate water sources, Escherichia coli (E. coli) is frequently seen in sewage and waste waters, since it is the normal flora of the human and animal gut and can easily contaminate water bodies.3 The sewage water comprises of certain human toxic heavy metals to which these bacteria are resistant, such as chromium, cobalt, and nickel.4 The hardness of water is caused by divalent metal ions like calcium and magnesium. The chloride ion is an inorganic anion which is usually present in sewage and wastewater. The pH of water is to measure the intensity of acidic or basic character obtained from sewage or other water sources. Drug resistance has been acquired by some of these sewage bacteria in the due course of time. The transfer of antibiotic resistance genes is very frequently seen in sewage and lake waters due to pooling of antimicrobial resistant microorganisms. The transfer of genetic component responsible for drug resistance in Gram-negative bacteria is mainly through conjugation process. Some of the physicochemical parameters also lead to transfer of resistant genes among the microorganisms.5 The collection of wastewater is usually done through underground sewage pipes to one or more centralized STPs, where the sewage water is treated.6 The spread of drug resistance is increasing in the developing countries due to inadequate usage of antibiotics. The wastewater treatment plants are connected to household and hospitals where more antibiotics are used and chances of bacteria acquiring drug resistance are high. Once these resistant organisms come in contact with wastewater treatment plants, they transfer the resistance to the endogenous microbial community.7 The present study was carried out to isolate and identify the pathogenic microorganisms along with their antimicrobial resistance pattern and also to chemically analyze the sewage water sources of Udupi district, Karnataka.

MATERIALS AND METHODS

Bacteriological Analysis

Sample Collection

The 12 sewage water samples were collected from different parts of Udupi district. Sampling was done in sterilized 100 mL bottle containing 0.1 mL of freshly added 1.8% aqueous solution of sodium thiosulfate. The water was collected from a depth of 30 cm with its mouth facing the water current.8 The samples were transported to the laboratory in ice and stored in −80°C until used for further studies. Samples were stored in the −80°C at Enteric Diseases Division, Central Research Lab, Kasturba Medical College, Manipal, until further processing.

Isolation of Bacteria

The samples were thawed at room temperature. Dilutions of 1:10 and 1:100 were performed. 10 μL of undiluted, 1:10, and 1:100 diluted water samples were inoculated into 5% sheep blood agar and MacConkey agar by spread plate method. The culture plates were incubated at 37°C for 18–24 hours. The water samples were also inoculated into alkaline peptone water, selenite F broth, and glucose broth and incubated at 37°C for 4 hours, 18 hours, and 24 hours, respectively, and subcultured into thiosulfate citrate bile salt sucrose agar (TCBS), Hektoen enteric agar (HEA), 5% sheep blood agar, and MacConkey agar, which were further incubated for 24 hours at 37°C to look for the presence of enteric pathogens. Colonies were identified by Gram staining, and a battery of biochemical tests.9 Further confirmation and speciation was done by MALDI-TOF (Biomerieux).

Antimicrobial Susceptibility Testing

The AST was performed for the isolates by the Kirby–Bauer disc diffusion method according to clinical and laboratory standards institute (CLSI) guidelines.10 The antibiotics used for members of Enterobacteriaceae family were amikacin, ampicillin, amoxicillin–clavulanic acid, ceftriaxone, ciprofloxacin, and cotrimoxazole; for Pseudomonadaceae family were ceftazidime, gentamicin, piperacillin, imipenem, amikacin, and ciprofloxacin; for Vibrionaceae family were tetracycline, ampicillin, cotrimoxazole, and ciprofloxacin; and for Aeromonas species, cefotaxime was included. The plates were incubated at 37°C for 24 hours. The zone of inhibition was measured and interpreted according to CLSI guidelines.11

Chemical Analysis

Determination of pH

The pH or the hydrogen ion concentration was determined to check the intensity of acidic or basic property of water by the pH meter.12

Determination of Total Hardness

The hardness of the sewage water sample was determined by titration method.12

Determination of Chloride

The amount of chloride content present in sewage water sample was determined by titration method.12

RESULTS

Of the 12 sewage samples collected, a total of 26 bacterial pathogens were isolated as shown in Figure 1. The waterborne pathogens, A. sobria (7.69%, n = 2), and V. alginolyticus (7.69%, n = 2) were isolated among the other environmental pathogens. The other organisms predominantly seen were E. coli (15.3%, n = 4), Klebsiella pneumoniae, Pseudomonas species, and Acinetobacter johnsonii as shown in Table 1.

A. sobria showed resistance to cefotaxime. Out of four E. coli isolates, one of the E. coli isolate was resistant to ampicillin and cotrimoxazole, whereas the other one showed resistance to amoxicillin–clavulanic acid. An isolate of A. lwoffii was resistant to piperacillin. The other isolates were susceptible to the antimicrobial agents tested as shown in Table 1.

The chemical analysis of pH determination showed a variable range of pH 5.7–7.5 that was interpreted as acidic, basic, and neutral. Of 12 sewage samples, 50% (6/12) were neutral, 41.6% (5/12) were acidic, and one sample showed basic pH. The total hardness of water was tested and ten samples were found to be hard (>200 ppm). The amount of chloride content present in water was detected and two samples were found to be above the normal range as shown in Table 2.

DISCUSSION

The wastewater consists of feces and urine of both human beings and animals that carry many pathogenic organisms. Some of the human pathogenic bacteria usually seen in sewage water are Vibrio, Aeromonas, E. coli, Shigella, Salmonella, Pseudomonas, and fecal streptococci due to fecal contamination. These organisms cause illness like cholera, typhoid, diarrhea, and dysentery.13

Fig. 1: Bacterial pathogens isolated from sewage samples

Table 1: Sample site, organisms isolated, and their antimicrobial resistance pattern
Sampling siteOrganismAntimicrobial resistance
1RiverEscherichia coliAmoxicillin–clavulanic acid
Acinetobacter johnsonii
2Lake near highwayVibrio alginolyticus
3Open sewer lineEscherichia coli
Klebsiella pneumoniae
Pseudomonas aeruginosa
4Open sewer lineCitrobacter braakii
Acinetobacter lwoffiiPiperacillin
5Open sewage in the city near highwayEnterobacter cloacae
Klebsiella pneumoniae
Providencia alcalifaciens
6LakeStenotrophomonas maltophila
7Sewage outlet near highwayVibrio alginolyticus
8Sewage water outletAcinetobacter johnsonii
Enterobacter cloacae
9Sewage outletAerobic Spore Bearing bacilli
10Sewage water outlet connectedAeromonas sobriaCefotaxime
Pseudomonas mandocina
Acinetobacter johnsonii
11Sewage water connected to public toiletAeromonas sobriaCefotaxime
Escherichia coli
Klebsiella pneumoniaeAmoxicillin–clavulanic acid
Pseudomonas spp.
12Sewage treatment plantEscherichia coliAmpicillin and cotrimoxazole
Klebsiella pneumoniaeAmoxicillin–clavulanic acid
Pseudomonas aeruginosa
Stenotrophomonas maltophila
Table 2: Physicochemical values of the sewage water samples
Sample no.pH valueInterpretationTotal hardness (ppm)InterpretationChloride (ppm)Interpretation
16.8Neutral     265Hard  74.5Acceptable
26.1Acidic16,270Hard  59.6Acceptable
37.1Neutral     720Hard178.8Acceptable
47.1Neutral     260Hard253.3Unacceptable
57.2Neutral     265Hard193.7Acceptable
66.3Acidic     265Hard178.8Acceptable
76.4Acidic     686Hard  89.4Acceptable
85.7Acidic     420Hard305.4Unacceptable
96.1Acidic14,940Hard  29.8Acceptable
107.5Basic       79.5Soft149Acceptable
116.9Neutral     360Hard149Acceptable
126.8Neutral     180Soft149Acceptable

According to WHO: (a) pH value: 6.5–9.5, (b) total hardness: 200 ppm, and (c) chloride: 250 ppm23

Our study showed that among 12 sewage water samples collected, the total bacterial isolates were 26 in number. In our study, the pathogenic A. sobria (7.6%, n = 2) was isolated from the sewage outlets, one near the beach and the other near public toilet. Aeromonas species is usually present in aquatic and waste water, raw sewage, sewage effluents, sewage-contaminated waters, and activated sludge14 and is tolerant to high temperatures and basic pH and survives during summer.15 They cause gastrointestinal infection, wound infection, and also septicemia. Holmberg et al.16 reported the ability of Aeromonas species in untreated well water to cause gastrointestinal infection. The presence of Aeromonas species in aquatic environments is an alarming threat to public health as they are resistant to chlorination and can survive in aquatic systems, thereby acquiring virulence properties linked to human disease.

The other rare isolate was V. alginolyticus (7.6%, n = 2) and was isolated from two different sewage samples (one from lake and the other from a sewage outlet). The presence of V. alginolyticus from the lake suggests its habitat.17V. alginolyticus is a halophilic marine organism, which enters the human body and causes enteric diseases and extraintestinal infections such as otitis media, wound infections, sepsis, conjunctivitis, and tissue necrosis. It may be isolated from the stool specimen of a healthy person due to ingestion of contaminated raw seafood. It also causes infection in people who recently come in contact with sea water.18

The other pathogens isolated in our study were similar to a study by Moges et al.11 These pathogens cause gastrointestinal infection, urinary tract infection, cholecystitis, bacteremia, cholangitis, wound infection, and skin infections.19 There was no isolation of Gram-positive organisms in our study.

The bacterial isolates were tested for drug resistance. A. sobria showed resistance to cefotaxime. Studies have shown a diverse pattern of resistance for Aeromonas species and the urban wastewater effluents are thought to contribute to the antibiotic resistance seen in environmental Aeromonas.14 In our study, one of the E. coli isolate was resistant to amoxicillin–clavulanic acid and the other one was resistant to ampicillin and cotrimoxazole, respectively, which was similar to a study done in Austria for E. coli isolates from sewage and sludge.20 The environmental Acinetobacter species are generally susceptible to antimicrobial drugs when compared to the clinical isolates. In our study, an isolate of A. lwoffii was resistant to piperacillin. This is in contrast to the study reported by Agrawal et al.21 There was no resistance seen for ciprofloxacin by any of the Gram-negative bacteria in our study, contradicting a study by Moges et al.11 in Northwest Ethiopia, where resistance to ciprofloxacin was 12%. The chemical analysis of pH determination, total hardness, and chloride determination was performed for all the 12 sewage water samples. These tests are essential to evaluate the reliable indices of the toxic impact of effluents in the aquatic environment.

In a developing country, there are a very few wastewater treatment plants employed for treatment of sewage disposal and it is often observed that people below socioeconomic status have their dwelling near these sewage outlets. The untreated waste gets directly added to the water bodies such as rivers, lakes, ponds, and reservoirs, etc., polluting the fresh water of these regions and thereby making it unfit for consumption and usage. This also acts as a reservoir for the drug resistant pathogenic organisms, posing a global threat to mankind and health. The groundwater sources get contaminated by storage tank leakage, industrial waste, and sewer and septic leakage.21 Most of the antibiotics are incompletely metabolized by the patients after administration, and hence, they enter municipal sewage through patients’ excreta. These drugs affect the biological process in STPs and may remain in the aquatic environment, causing an increase in the spread of drug resistance.22,23

Hence, the microbiological quality and safety of water systems is important and has to be maintained to prevent the high risks to human health. Furthermore, epidemiological studies in testing of water bodies should be undertaken to assess the potential risks to public health.

CONCLUSION

In the present study, sewage water samples were chemically analyzed and the pathogenic microorganisms were identified along with their antimicrobial resistance pattern. Contamination of drinking water or groundwater by the sewage water may cause a threat to human health. Resistance to antibiotics may be due to patients’ excreta from the nearby hospitals which can lead to survival of bacteria and may spread to other water sources due to lack of treatment of sewage water. Hence, treating the sewage water in an appropriate STP is recommended and is the need of the hour.

ACKNOWLEDGMENTS

The authors are thankful to Manipal University; Dean, Kasturba Medical College, Manipal; Enteric Disease Division, Central Research Lab, Kasturba Medical College, Manipal; Department of Chemistry, Manipal Institute of Technology, Manipal for providing all the facility and support to carry out this work.

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