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Table of Contents
Year : 2021  |  Volume : 18  |  Issue : 2  |  Page : 80-84

Isolation and characterization of microbial population associated with industrial waste effluent and their antibiotic sensitive pattern

1 Division of Microbiology, ICMR-National Institute of Occupational Health, Ahmedabad, Gujarat, India
2 Division of Toxicology, ICMR-National Institute of Occupational Health, Ahmedabad, Gujarat, India

Date of Submission16-Mar-2021
Date of Decision12-Apr-2021
Date of Acceptance16-Apr-2021
Date of Web Publication28-May-2021

Correspondence Address:
Mahesh Chandra Sahu
Division of Toxicology, ICMR-National Institute of Occupational Health, Meghani Nagar, Ahmedabad - 380 016
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/am.am_23_21

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Background: Both organic and inorganic chemicals are deposited in industrial effluents, which contain radioactive, metals, antibiotics, and carcinogenic substances. These effluents are directly or indirectly affecting the daily life of human. Through food chains, it migrates to human health and cased different diseases with different drug-resistant strains. Materials and Methods: Fifty-five industrial waste effluent samples were collected from waste effluent sites serially diluted and documented CFU from individual effluents and subcultured for isolated colony on nutrient agar plate, and then, grown bacteria were identified with culture morphology and biochemical tests. Disc-diffusion method was used for antibiotic sensitivity pattern of isolated bacteria. Results: From 55 industrial waste samples, total 13 different types of bacterial strains were found from industrial waste effluent. Among all isolated bacteria, Pseudomonas sp., Staphylococcus epidermidis, Pseudomonas aeruginosa, Bacillus sp., Acinetobacter sp., Staphylococcus aureus, Micrococcus sp., Citrobacter sp., Shigella sp., and Escherichia coli. Moreover, also 2 Aspergillus sp. and 2 unidentified fungus were revealed from this study. Antibiotic sensitivity pattern revealed that all the organisms show 100% resistant to amoxiclav, 71% resistant to ampicillin, 43% resistant to oxacillin antibiotics, 23% resistant to streptomycin, and 15% resistant to both gentamycin and tetracycline antibiotics. Furthermore, we revealed 31% methicillin-resistant Staphylococcus aureus from industrial waste, whereas rest 69% revealed methicillin-susceptible Staphylococcus aureus strain. All the Gram-positive strains are shown highly resistant against beta-lactam group antibiotics. Conclusion: Our findings raise potential public health concerns for industrial waste effluent. Workers and individuals exposed to reclaimed wastewater. Because of increasing use of reclaimed wastewater, further study is needed to evaluate the risk of exposure to antibiotic-resistant bacteria in treated wastewater.

Keywords: Antibiotic sensitivity pattern, bacteria, CFU, industrial waste effluent, methicillin-resistant Staphylococcus aureus

How to cite this article:
Shukla A, Sahu MC. Isolation and characterization of microbial population associated with industrial waste effluent and their antibiotic sensitive pattern. Apollo Med 2021;18:80-4

How to cite this URL:
Shukla A, Sahu MC. Isolation and characterization of microbial population associated with industrial waste effluent and their antibiotic sensitive pattern. Apollo Med [serial online] 2021 [cited 2021 Sep 19];18:80-4. Available from: https://www.apollomedicine.org/text.asp?2021/18/2/80/317185

  Introduction Top

Industrial waste is the waste produced by industrial activity which includes any material that is delivered useless or waste materials during a manufacturing process and reactions. It can be in the form of solid, liquid, or gaseous form either it can be hazardous or nonhazardous. Hazardous waste may be toxic, poisonous, inflammable, ignitable, corrosive, reactive, or may be radioactive. It may pollute or contaminate the air, soil, and water source. Heavy metal waste, chemicals, hazards, and sewage released into sources of water directly affect to marine living organism community and the health of those people who depend on the waters as drinking water sources. Water pollution can have destructive and dangerous effects on the human body with the main ones being infections from bacteria, parasites, and toxic chemicals. Waste effluents are always contaminated with different biological living organisms such as bacteria, viruses, protozoa, fungi, flatworms, or roundworms.[1] Among them, pathogenic bacteria can cause the most serious epidemiological risk to the human beings. It can cause various diseases in humans.

Staphylococcus aureus is one of the most important clinical pathogens globally, with the development of antibiotic resistance, specifically, methicillin-resistant Staphylococcus aureus (MRSA), having imposed heavy burdens on the health-care system.[2]

The global antibiotic resistance problem is likely to be the use and misuse of antibiotics in humans and animals and somewhere harmful. However, the role of the environment revealed the source of novel resistance genes and route for the transmission of both resistant bacteria and resistance genes.[3],[4],[5] With extensive use of antibiotics, pathogens, which were earlier sensitive to antibiotics, now started developing resistance mechanism to many classes of antibiotics. Many plasmids carrying resistance genes are transferred by the process of conjugation. Conjugation is a explicative process in which both donor and recipient cells have a copy of the plasmid after the process.[6]

The occurrence of antibiotics and other chemicals and pharmaceuticals in the environment have become an increasing public concern as recent environmental monitoring activities reveal the presence of much number of pharmaceuticals in soil and water. Many pharmaceutical companies discharge pharmaceutical waste to environments. It causes the main source of antibiotic-resistant microorganism in the environment. Antibiotic resistance in the environment increased the emergence of resistance among pathogenic bacteria.[7]

The objective of the present study was to isolate and screen antibacterial metabolite-producing bacteria from industrial waste effluent samples in Ankleshwar GIDC. The outcome of this finding may be important to give direction for researchers and for future treatment of multidrug-resistant human pathogens.

  Materials and Methods Top

Collection of sample

Industrial liquid waste effluent was collected from different industrial area of GIDC of Ankleshwar (21°61´and 73°02´) at Bharuch district of Gujarat state of India. All the samples were placed in separate sterile screw cap vials (10 ml) and stored in a refrigerator at 4°C till use.

Isolation of bacteria from waste effluent

Serial dilution techniques were used for the isolation of bacteria. In this technique, sample suspension was prepared by adding mixed with waste. Liquid effluent 1 ml of liquid waste effluent pick with the sterile pipette and transfer into the dilution tube which feel up 9 ml with distilled water, mark different dilution (10 − 1 to 10 − 7). 1 ml from stock was transferred into the 10 − 1 dilution tube. Then again, 1 ml of stock from 10 to 1 dilution transfer into the 10 − 2 dilution tube then again same sequentially make dilution 10 −3, 10 −4, 10 −5, 10 −6, and 10 −7. From each dilution tube, take 0.5 ml dilution fluid was transfer into N-agar plate under aseptic condition, spread it gently with spreader, and incubate it overnight 37°C. Labeling should be done. Another day observes the results of overnight incubation. The CFU were determined with counting the number of colonies on nutrient agar plate by pour plate method.

Isolation of fungi from industrial liquid waste effluents

Samples were spread on Czapek-Dox agar medium and incubate at 25°C for 5 days. After 5 days of incubation, the mycoflora was identified on basis of morphology and characterization. Moreover, for subculture, fungal spores were spread in Czapek agar medium with surgical needle and grow at 25°C.

Isolation of bacteria from soil samples

Solid soil sample was first weight 100 mg and the add to sterile N–broth and incubate overnight 37°C and streak on blood agar media and MacConkey agar media. Differential colony from both blood agar plate and MacConkey plate was taken by sterile loop and subculture on N-agar plate. Then incubate at 37°C, for 24 h in incubator. The morphological characterstics of bacterial colonies (size, shape, elevation, opacity, margin, pigmentation etc.) were determined for identification of bacteria.

Identification of unknown bacterial strain

Identification of unknown bacterial species was done by microscopy. The purified colonies were subjected to gram staining and characterized using biochemical tests and consulting the pertinent literature.[8],[9],[10]

Antibiotic sensitivity test

The sensitivity to different antibiotics was assessed using the disc-diffusion method on Mueller-Hinton Agar. Disk diffusion is considered as one of the traditional techniques to test antimicrobial susceptibility of microorganisms, and yet, it is one of the most commonly used tests in routine clinical application. It is convenient for testing the majority of the microorganisms including bacterial pathogens. Six antibiotics were selected as representative of commonly used antibiotic classes. This method consists of placing discs of absorbent paper containing the antibiotic of interest (all purchased from hi media), ampicillin (10 mg), amoxicillin (30 mg), vancomycin (30 mg), oxacillin (1 mg), gentamycin (10 mg), tetracycline (30 mg), chloramphenicol (30 mg), streptomycin (10 mg), cefepime (30 mg), imipenem (10 mg), cefotaxime (30 mg), and ceftazidime (30 mg) on a plate inoculated with the bacteria collected from all four sites of sampling. Plates were incubated 20 h at 37°C to allow growth of the bacteria and time for the antibiotics to diffuse into the agar. Strains were classified as resistant to the antibiotic tested when no inhibition zone was observed around the disk, or as sensitive, when a clear zone of inhibition was seen.

  Results and Discussion Top

Effluents discharged by industries constitute one of the major causes of environmental pollution and a significant public health hazard. This study was based on the microbial population found in industrial waste effluent and their antibiotic sensitivity pattern from collected waste effluent sample by different industries. The result showed heavy microbial population in industrial waste effluent. We have documented CFU from individual effluents and subcultured for isolated colony on nutrient agar plate, and then, grown bacteria were identified with culture morphology and biochemical tests.

Adewoye and Lateef, 2004, obtained a bacterial count of 2.15 × 10[5] cfu/ml from a pharmaceutical effluent in Nigeria, while in our study, we obtained bacterial counts in the order of 1 × 10[2] cfu/ml to 4.70 × 10[9] cfu/ml from industrial waste effluent from different pharma and chemical industries of the Ankleshwar (Gujarat) region [Table 1], which is contradictory to his finding (Adewoye and Lateef, 2004).[7]
Table 1: Total bacterial count

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In this study, a total 13 different types of bacterial strains were found from industrial waste effluent. Among all isolated bacteria, Pseudomonas sp. was found highest (20%), followed by Staphylococcus epidermidis (18%), Pseudomonas aeruginosa (10%), Gram-positive bacilli (8%), Acinetobacter sp. (6%), S. aureus (5%), Micrococcus sp. and Citrobacter sp. (3%), Shigella sp. (2%), and E. coli (1%) [Figure 1]. Furthermore, 2 Aspergillus sp. [Figure 2] and 2 unidentified fungi were revealed from this study [Table 2]. Whereas, from another study of Ali and Naseem, it was revealed that most predominant organism isolated from industrial waste, Pseudomonas which is similar to our study. Isolates were identified as genus of Flavobacterium, Cupriavidus, Enterobacter, Pseudomonas, Yersinia, Proteus, Klebsiella, Serratia, and Acinetobacter. The other Gram-negative coccus was identified as genus Bordetella. Six strains of Gram-positive cocci were identified as genus of Staphylococcus, Micrococcus, Trichococcus, Deinococcus, Syntrophospora, and Vagococcus. Gram-negative Bacillus bacteria constituted the majority of species in the industrial effluents. According to Ali and Naseem, the majority of the isolated Gram-negative bacteria belonged to the genus Pseudomonas, while two of isolate belonged to genus Bacillus. The presence of Gram-positive bacteria has been reported from some worker and Gram-positive bacteria found from each effluent belonged to the genus Micrococcus. These bacterial strains are no validation in the microdiversity of industrial effluents of industries.[11]
Figure 1: Bacterial culture on nutrient agar

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>Figure 2: Characterization of Aspergillus sp. with culture morphology and microscopic image

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Table 2: Total number of isolates

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An antibiotic sensitivity test was conducted on bacterial isolates to ascertain level of resistance. The result of antibiotic sensitivity test was interrupted and is presented as the resistance of bacterial isolates to the antibiotics [Table 3].
Table 3: Frequency of isolation of methicillin-resistant Staphylococcus aureus and methicillin-susceptible Staphylococcus aureus from industrial waste

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According to one study, Acinetobacter sp. were resistant to ceftazidime which is similar to our study in which Acinetobacter sp. were resistant to ceftazidime.[12] According to finding of one study, Enterococci were resistant against ampicillin and vancomycin which coincide with our study as we also found enterococcus sp. to be resistant against ampicillin and vancomycin.[13] As revealed by Pazhani et al. 2008, Shigella sp. were resistance to streptomycin which is similar to our study as most of Shigella sp. were resistant to streptomycin.[14]

According to one study, resistance of S. aureus to antibiotics varied considerably with the highest resistance recorded to ampicillin and penicillin (96.7%), rifampicin and clindamycin (80%), oxacillin (73.3%), chloramphenicol (83.3%), and tetracycline (56.7%).[15] From our study, out of 5 class of antibiotics of Gram-positive antibiotic, staphylococcus strains were founded mostly resistant to beta-lactam group of antibiotics. S. aureus shows 80% resistance to both ampicillin and amoxiclav antibiotic and some of also shown resistance again chloramphenicol antibiotic. S. epidermidis shows highest resistance against all group of antibiotics. It was resistant to whole beta-lactam antibiotic group and some of them show resistance against streptomycin and tetracycline antibiotic.

According to one study, revealed that Enterococcus faecium was resistant to vancomycin, S. aureus was resistant to oxacillin which is similar to our study as we found Enterococcus sp. resistant to vancomycin and S. aureus resistant to oxacillin (Dweba et al., 2018).[16] We were found 4 strain methicillin resistant out of 15 staphylococcus strain. There were 31% MRSA type staphylococcus strain founded and 69% methicillin-susceptible Staphylococcus aureus type strain reported [Table 3] from industrial waste effluent.

Our research indicates that all these types of bacteria occur even in industrial effluents. Thus, the pathogenic bacteria and nonpathogenic appear to have a very wide range of antibiotic-resistant pattern.

Out of 5 class of antibiotics, staphylococcus strains were founded mostly resistant to beta-lactam group of antibiotics. S. aureus shows 80% resistance to both ampicillin and amoxiclav antibiotic and some of also shown resistance again chloramphenicol antibiotic. S. epidermidis shows highest resistance against all group of antibiotics. It was resistant to whole beta-lactam antibiotic group and some of them show resistance against streptomycin and tetracycline antibiotic. Enterococcus sp. and Bacillus sp. are shown resistant against vancomycin (glycopeptides antibiotics). Enterococcus sp. are shown resistant against beta-lactam antibiotics and tetracycline resistance also found.

Here, Pseudomonas sp. were shown more resistant against cephalosporin antibiotics (10% to cefepime and 15% to cefotaxime), and some of them also found carbapenem group antibiotic (imipenem) resistant. Acinetobacter sp. are shown resistance again cephalosporin antibiotic (33% resistant to cefotaxime and 17% resistance to ceftazidime). Most of the Shigella sp. are found resistant to whole aminoglycoside antibiotics (50% resistant to both gentamycin and streptomycin) [Table 4].
Table 4: Resistance of the bacterial strain to individual antibiotic

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Tetracyclines and chloramphenicol were active against all the bacterial isolates, while ampicillin and amoxiclav were not active against any of bacterial isolates. The total resistance of the bacteria to the antibiotics ranged from 0% for tetracyclines and chloramphenicol to 100% for ampicillin and amoxiclav as shown in [Table 4]. The cumulative effectiveness of antibiotics as obtained in this study is Tetracyclines > Chloramphenicol > Gentamicin > Cefepime = Imipenem > Streptomycin = Cefotaxime =Ceftazidime > Vancomycin = Oxacillin > Ampicillin= Amoxiclav.

Antibiotic prescriptions in hospitals are given without clear evidence of infection or adequate medical indication. Broad-spectrum antibiotics are sometimes given in place of narrow-spectrum drugs as a substitute for culture and sensitivity testing, with the consequential risk being super infections and the selection of drug-resistant mutants.[17] In developing countries, drugs are available to the public, and thus, people may practice self-administration of antibiotics and further increase the prevalence of drug-resistant strains. The long-standing practice of using low doses of antibiotics for a long period of time for growth promotion in animals is a strong contributor to the development of antibiotic-resistant bacteria in the environment. The consumption of antibiotic is enormous, and it has been estimated that the antibiotic market consumption worldwide lies between 100,000 and 200,000 tons.[18]

  Conclusion Top

Industrial waste contains different microorganisms. Furthermore, with this study, it is concluded that several drug-resistant organisms are found in industrial waste effluents. Which spreads the MDR bacterial diseases. Hence, industrial waste should be treated and purified before sending outside. Every pharmaceutical industry necessary to discharge drug free waste in the final treated effluent. So that microbial strain becomes less resistant against antibiotics.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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Bengtsson-Palme J, Boulund F, Fick J, Kristiansson E, Larsson DG. Shotgun metagenomics reveals a wide array of antibiotic resistance genes and mobile elements in a polluted lake in India. Front Microbiol 2014;5:648.  Back to cited text no. 5
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Holt WE, Shen-Tu B, Haines J, Jackson J. On the Determination of Self-Consistent Strain Rate Fields Within Zones of Distributed Continental Deformation. Washington DC: American Geophysical Union; 2000. p. 121.  Back to cited text no. 8
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Adewoye SO, Lateef A. Assessment of the microbiological quality of clariasgariepinus exposed to an industrial effluent in Nigeria. Environmentalist 2004;24:249-54.  Back to cited text no. 12
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Pazhani GP, Niyogi SK, Singh AK, Sen B, Taneja N, Kundu M, et al. Molecular characterization of multidrug-resistant Shigella species isolated from epidemic and endemic cases of shigellosis in India. J Med Microbiol 2008;57:856-63.  Back to cited text no. 14
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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3], [Table 4]


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