Isolation and Characterization of Multiple Drug Resistant Human Enteric Pathogens from Sewage Water of Delhi

Antibiotic resistance is one of the major problems in the medical world, and the sewage waters are the primary habitats to harbour antibiotic resistance bacteria (ARB) especially multi-drug resistance (MDR) human enteric pathogens. The present study dealt with isolation, identification and characterization of human enteric pathogens showing resistance against ten different commonly prescribed antibiotics. These bacterial strains were isolated from different sewage treatment plants located in the suburb of Delhi. Initially, samples were analysed for the presence of pathogenic human enteric bacteria through morphological, biochemical and molecular analysis. Further susceptibility patterns of these isolates were studied towards clinically significant antibiotics. Doxycycline and Metronidazole were found to be most inert antibiotic as it was ineffective against all isolated enteric pathogens, whereas Meropenem was found to be most promising antibiotic. As the resistance of these microorganisms is evolving day by day, proper steps should be taken to prevent it.


INTRODUCTION
Pollution in source water is a problem in developing as well as in all developed countries. In the past few decades, uncontrolled urbanization has caused a serious pollution problem due to the disposal of sewage and industrial effluents to water bodies. Effluent wastewater treatment is the process of removing contaminants from wastewater and household sewage, both runoff (effluents) and domestic. It includes physical, chemical, and biological processes to remove physical, chemical and biological contaminants. Sewage water can be polluted by a wide variety of substances, including pathogenic microorganisms, plant nutrients, toxic chemicals, sediments, heat oil, and radioactive substances (Sharpe 2003, Ishak et al. 2011. Sewage water is one of the major sources of human enteric pathogenic bacteria. Sewage contains human faeces and therefore contains human enteric pathogens i.e. Salmonella enterica, Salmonella enteritidis, Salmonella typhimurium, Shigella dysenteriae, Enterobacter spp., Vibrio spp. These pathogens are responsible for serious gastrointestinal illness which is a significant cause of waterborne health epidemics. Gastrointestinal disease is considered the third most common cause of death in the world (Hellier & Williams 2007). For combating against these pathogens, antibiotics are generally used which has led to the evolution of multidrug-resistant strains of these pathogens. There is a great need to discover novel antibiotics due to the wide-spread emergence of resistance among pathogenic bacteria against available antibiotics .
Although the discovery of antimicrobials leads to various expectations, it has been influenced by the emergence of resistant bacterial strains against antibiotics. It has been reported that the irrigation water system also has been contaminated by these multidrug-resistant bacteria which have a chance to enter in our food chain directly. The presence of multidrug-resistant enterobacter and enteric pathogens has been regarded as a serious problem for a community (Cabrera et al. 2004, Chitnis et al. 2004, Danchaivijitr et al. 2005. A significant increase of Multiple Antibiotic Resistant (MAR) bacteria is observed in various aquatic systems. Human infections caused by such bacteria could be difficult to treat with drugs (Chandrasekaran et al. 1998, Dicuonzo et al. 2001, Lopes et al. 2005. The aim of this study was to evaluate the antibiotic resistance patterns of human enteric pathogenic bacteria which were isolated from different sewage waters from the suburb of Delhi. The study involves; (a) Collection of water samples from different sewage treatment plants (b) Isolation and identification of human enteric pathogens from sewage water samples (c) Determination of susceptibility and resistance pattern against ten different antibiotics by agar well diffusion assay (d) Interpretation of the data generated to determine the antibiotic resistance patterns of the isolated bacteria for the benefit of human welfare by increasing general awareness among the people.

(a) Collection of Water Samples
1lt capacity of bottles was used to collect samples from 10 sewage treatment plants from different locations of Delhi (Table 1). Bottles used were sterilized using gamma radiations. Samples after collection were marked with SW01 to SW10. Sample collection and transportation to the laboratory were performed aseptically. Samples analysis was done within 6 hrs of collection.

(b) Isolation and Identification of Human Enteric Pathogens
Detection of Escherichia sp: Membrane filter of 0.45µ was passed with 250mL water sample and placed in MacConkey broth. Eosin methylene blue agar and Mac Conkey agar were streaked for confirmatory identification. Characteristic pink colonies on the former and green metallic colonies on the latter are observed. Further confirmation was done by Gram's staining and HiMedia IMViC biochemical kit for E. coli as per IS: 5887(part-1)1976, Reaffirmed 2018.
Detection of Salmonella sp: Membrane filter of 0.45µ was passed with 250mL water sample and placed in Buffer peptone water incubating at 37°C for 24 hours. 10mL of Rappaport Vassiliadis medium is inoculated with 0.1mL above grown sample and incubated at 42°C for 24 hours. Further streaking is done on Brilliant green agar and Bismuth sulphide agar where the former is observed for characteristic pink colonies and the latter for black metallic sheen colonies with H 2 S. Further confirmation was done by Gram's staining and HiMedia IMViC biochemical kit for Salmonella as per IS: 5887(Part-3) 1999, Reaffirmed 2018.
Detection of Pseudomonas sp: Membrane filter of 0.45µ was passed with 250mL water sample and placed in Cetrimide broth and then incubated at 37°C for 48 hours. Cetrimide agar Plates were streaked and observed for characteristic green colonies and further confirmation was done by Gram's staining and Biochemical test as per IS: 13428:2005 (Annexure-D).
Detection of Vibrio sp: Membrane filter of 0.45µ was passed with 250mL water sample and placed in alkaline peptone water and incubated at 37°C for 24 hours. TCBS Agar is streaked and further confirmed by HiMedia IMViC biochemical kit and Gram's staining as per IS: 5887(Part-5) 1976, Reaffirmed 2018.

Detection of Shigella sp:
Membrane filter of 0.45µ was passed with 250mL water sample and placed in Nutrient broth. Deoxycholate citrate agar is streaked for further confirmation and observed for characteristic colonies such as small opaque colonies. Further confirmation was done by HiMedia IMViC biochemical kit and Gram's staining for Shigella sp as per IS: 5887(part-7)1976, Reaffirmed 2018.

(c) Molecular Identification
Further identification of bacterial isolates using 16srRNA sequencing is done. The sequence reaction using the Sanger dideoxy sequencing kit was performed following the manufacturer's instructions. Basic Local Alignment Search Tool (BLAST) algorithm is used for alignment of trimmed nucleotide sequences of different bacterial iso- lates provided by the National Centre for Biotechnology Information (NCBI). were used to check susceptibility and resistance pattern of above four bacterial isolates. All these antibiotics were obtained from a local pharmacy store and a working solution having 10µg/mL concentration of each antibiotic was used for the study.

(e) Inoculums Preparation
24hr old bacterial culture was taken for adjustment of 0.5 McFarland density in densitometer to get a bacterial population of 1.0 × 10 8 cfu/mL using saline (0.85% NaCl)

(f) Agar Well Diffusion Assay (Zone of Inhibition Evaluation)
The antibiotic assay was evaluated by agar well diffusion methods. Plates of sterile Muller Hinton Agar (MHA) with 100µL of each adjusted cultures was punched to 6mm diameter well. 100µL of each antibiotic solution were added in wells , Kaushik & Chauhan 2008.
Plates were incubated at 37 o C overnight. Zone of inhibition was observed in plates where the diameter of zones was calculated using Vernier callipers.

RESULTS AND DISCUSSION
In the present study, ten water samples were collected from ten different sewage water treatment plants located in Delhi.            The multiple alignment file was then used to create phylogram ( Fig. 1 to 5). Phylogenetic tree is a diagram that r epresents evolutionary relationships among organisms. The pattern of branching in a phylogenetic tree shows how species or other groups evolved from a series of common ancestors.
Phylogenetic analysis explores the evolutionary relationships between organisms and is a key element for microbial studies. The development of phylogenetic trees is an important stage in characterizing new pathogens and develops new methods of treating various infections. Molecular sequencing technologies and phylogenetic approaches can be used to learn more about a new pathogen outbreak. This includes finding out about which species the pathogen is related to and subsequently the likely source of transmission. 16S and 23S rRNA gene sequence data reveal a closer relationship between Salmonella and Escherichia coli than between Salmonella and Citrobacter freundii (Christensen & Olsen 1998). Escherichia fergusonii and Escherichia albertii are independent and established species (Seong et al. 2012, Farmer et al. 1985. However, these species were difficult to distinguish from E. coli by 16S rRNA gene based phylogenetic analysis. The susceptibility patterns of all isolated human enteric pathogenic strains were evaluated against ten commonly prescribed antibiotics by using an agar well diffusion assay. In this study, it is clearly seen that the bacterial isolates show non-vulnerability to different antibiotics. The antibiotic   Fig. 7: Zone of inhibition of bacterial isolates against antibiotics. resistance patterns in terms of average zones of diameter considering 4 plates for bacterial isolates against each of ten antibiotics of 10µg/mL concentration were calculated and shown in Table 2. The most vulnerable antibiotic was found to be Doxycycline and Metronidazole against which all eleven isolated human enteric pathogens shows 100% resistance (Fig. 6). The percentage of susceptibility against antibiotics was demonstrated in Table 3 Antibiotics are an essential part of combating harmful bacterial infections in vivo. During the last few decades, infectious diseases have played a significant role in the death of millions in developing countries like India. Because of the mutagenic nature of bacterial DNA, the rapid multiplication of bacterial cells, and the constant transformation of bacterial cells due to plasmid exchange and uptake, pathogenic bacteria continue to develop antimicrobial resistance, thus rendering certain antibiotics useless . It is thus become important to determine the antibiotic resistance pattern of isolated microbes as it is the part of microbial monitoring process of the water (Chatterjee et al. 2012).
The presence of multidrug resistance in human enteric pathogens is not uncommon recently, and its severity has been expanded from town to village and village to everywhere very rapidly. The rapid development of the antibiotic

Rudrangshu Chatterjee
Vol. 20, No. 2, 2021 • Nature Environment and Pollution Technology  resistant pattern of E. coli, as well as other microorganisms, might create a devastating health problem (Chauhan & Goyal 2013, Overdevest et al. 2011). Based open these study it was found that most of the isolates in the present study showed multiple tolerances to antibiotics. Since heavy metals are all similar in their toxic mechanism, multiple tolerances are common phenomena among heavy metal resistant bacteria . In sewage water, some substances have the potential to select for antibiotic resistance even though they are not antibiotics themselves. Heavy metals and biocides are two of them. Exposure to heavy metals or biocides results in the selection of bacterial strain also able to resist antibiotics. This shows that there is a close association between metal resistance and antibiotic resistance.

CONCLUSION
An alarming consequence has been occurred due to the widespread emergence of resistance among microorganisms against clinically significant antibiotics. It is clearly indicated that domestic waste and industrial waste are responsible for the development of bacterial resistance along with the risk of human health and the environment. Regular surveillance of the effluent in industrial sites is a must if the risk of disease due to such antibioticresistant organisms is to be avoided. Antibiotic resistance development among bacteria is a challenging issue that requires the improvement of next-generation treatment processes in sewage water treatment plants. The emergence of antibiotic resistance among pathogens increases the demand for novel treatment strategies. In this study, results were indicative of very high antimicrobial resistance to Doxycycline and Metronidazole among all bacterial isolates involved in our study. The uncontrolled use of this antibiotic led to the generation of multi-drug resistant strains. The fourth-generation antibiotic i.e. Meropenem is found to have significant efficacy and can be considered appropriate for the empirical treatment of the above four bacterial infections. Alternatives to antibiotics such as herbal medicines, probiotics and lytic bacteriophages can help to decrease the burden of antimicrobial resistance globally. Although the present study can lead to beneficially assist in the identification of alternate drug to control these multidrug-resistant bacterial strains.