The main aim of the study was to evaluate the antibiotic resistance among gram negative enteric bacteria that were isolated from different ghats of the river Khripra. For the investigation of gram negative enteric bacteria, a total of 8 sampling sites were selected, and a total of 6 gram negative bacteria were isolated. The members are E. coli, Salmonella, Enterobacter, Kliebsella and Citrobacter Along with this, Pseudomonas were also isolated, and the resistance mechanism was analysed by the antibiotic susceptibility test, also known as the disc diffusion method and the Kirby-bauer method. For the detection of multidrug resistance among the Enterobacteriaceae, the following types of beta-lactam antibiotics were used: aztreonam, ceftazidime, cefpodoxime, chloramphenicol, amikacin, ceftriaxone, ciprofloxacin, and ampicillin. Finally, after performing the disc diffusion test, we isolated a total of 21 strains of gram negative bacteria, out of which 8 strains were sensitive against these antibiotics, while 13 gram negative bacterial strains showed the mechanism of antibiotic resistance. Besides, the MAR index of all the isolates was calculated, which shows that the strains of gram negative bacteria in samples 1, 2, and 3 were sensitive except Enterobacter, which was present in sample no. 3. On analysis, we also detected the resistant strains that were present in samples 4, 5, 6, 7, and 8. After calculating the MAR index, it was found that the isolated strains from samples 4, 5, 6, 7, and 8 Together with one isolate from Sample No. 3, a MAR Index greater than 0.2 indicates a high risk source of contamination where antibiotics are commonly used.

Multidrug-Resistant Bacteria, Antibiotics, Antibiotic Resistance (AR), Antibiotic Resistance Genes (ARG), Kshripra River, MAR Index.

Coliforms are gram negative bacilli that are members of the family Enterobacteriaceae. Coliform bacteria are present in the normal flora inside the digestive tract of all warm-blooded and some cold-blooded animals. For the prevention of disease in animals, antibiotics are being used in livestock. The bacteria isolated from livestock show the mechanism of antibiotic resistance. Surface water also acts as a carrier of antibiotic-resistant bacteria. The River Missouri shows the presence of tetracycline-resistant bacteria bacteria (McDonnell et al., 2004; Gardea et al., 2016). The study done on the irrigation water system has also been polluted by multidrug-resistant bacteria, which have the possibility of entering our ecosystem directly. The strains of multidrug-resistant Enterobacter and enteric pathogens have been responsible for a main problem within a community. In various aquatic ecosystems, multiple antibiotic-resistant bacteria were observed. When this MDR bacteria causes infection inside the human being, it creates a problem during its treatment with a proper drug (Chatterjee et al., 2021). For the prevention and treatment of infection in different fields, including agriculture and poultry, antibiotic groups such as cephalosporins and fluoroquinolones are commonly used. In the last two decades, the increased and careless use of antimicrobial substances in animal husbandry, aquaculture, and food preservation has created an environment of antibiotic-resistant bacteria. Multidrug-resistant bacteria have been isolated from the main rivers of India, such as Ganga, Yamuna and Cauvery. The bacteria present in the environment receive the resistance gene by horizontal gene transfer. The mechanism of horizontal gene transfer occurs through a variety of mobile gene elements, such as plasmids, bacteriophages, genomic islands, integrative and conjugative elements, insertion sequences, transposons, integrons, and miniature inverted repeat transposable elements (Dhawde et al., 2021; Nain et al., 2021; Salikan et al., 2020; Ash et al., 2002; Purohit et al., 2020; Torkan et al., 2016; Schwartz et al., 2002; Mustafa et al., 2022; Tula et al., 2022; Besharati et al., 2018; Resende et al., 2009). It is estimated that antibiotic resistance might lead to ten million deaths annually by 2050. Thousands of deaths occur annually due to the antibiotic resistance mechanism that occurs inside the bacteria. The sewage treatment plant contains a huge population of antibiotic-resistant bacteria with antibiotic-resistant genes, which further discharge into aquatic habitats. Due to antibiotic-resistant bacteria with antibiotic-resistant genes, when they infect a human population, such types of pathogens create a negative effect on health, including treatment failure, long periods of treatment, and in chronic cases, death may occurs (Singh et al., 2020; Graham et al., 2011). The most common resistance mechanism against beta-lactam antibiotics is the production of an extended-spectrum beta-lactamase enzyme by gram-negative bacteria (Djenadi et al., 2017). The species of Pseudomonas carries antibiotic-resistant genes and also exchanges such genes with the members of the Enterobacteriaceae (Kittinger et al., 2016). The various types of water sources, such as rivers, lakes, sea, groundwater and drinking water, inside this bacteria show resistance mechanisms (Hanna et al., 2023; Ghabalo et al., 2022; Bartley et al., 2019). Antibiotics have played an important role in controlling various types of diseases that occur in animals and humans (Tadesse et al., 2012). According to the World Health Organisation, antibiotic resistance is a major threat to public health (Ogura et al., 2020; Wengenroth et al., 2021; Mustafa et al., 2022). Waste water treatment plants act as a source of antibiotic-resistant bacteria with antibiotic-resistant genes (Teshome et al., 2020). Hospital wastewater can be dangerous to people and ecosystems since it is contaminated with various types of pollutants, including radioactive, chemical, and pharmaceutical waste, as well as harmful microbes and antibiotic-resistant bacteria, along with antibiotic residue at concentrations that inhibit sensitive bacteria (Mogs et al., 2014). Using too many antibiotics and misuse of antibiotics in human and veterinary medicine are the main causes of the evolution and dissemination of antibiotic-resistant bacteria all over the world. Excretory material from animals and humans is responsible for spreading resistant enteric bacteria in aquatic habitats (Abo-State et al., 2012). According to the World Health Organisation, 80% of illnesses occur due to unsafe water (Odonkor et al., 2018; Ulfat et al., 2021). In recent years, strains of multidrug-resistant bacteria have increased fourfold (Basak et al., 2015). Per year in the United States alone, approximately 2 million infections occur due to antibiotic-resistant bacteria. Multidrug resistance among gram negative bacteria shows harmful effects (Riedel et al., 2019). Metropolitan wastewater treatment plants show the presence of antibiotic-resistant bacteria with antibiotic-resistant genes (Silva et al., 2007). There is a possibility of antibiotic-resistant bacteria being consumed by pilgrims during the mass bathing or drinking of river water. In Indian rivers, different antibiotic-resistant bacteria have been reported (Purohit et al., 2020). Bacteria can produce extracellular enzymes that can inactivate antibiotics such as penicillin, monobactam, carbapenems, and cephalosporins. These antibiotics are known as beta-lactum antibiotics. The enzyme beta-lactamase, which is normally produced by gram negative bacteria, breaks the beta-lactum ring of antibiotics, which makes the antibiotic inactive (Alam et al., 2018; Aujoulat et al., 2021; Azevedo et al., 2014). The dissemination of antibiotic resistance among bacteria is regarded as a universal hazard to human, animal, and environmental health (Moreira et al., 2014). Peudomonas aeruginosa and Enterobacteriaceae members are resistant to carbapenems and third-generation cephalosporins (Mustafa et al., 2022; Muller et al., 2018). Municipal wastewater treatment plants are antimicrobial-resistant hotspots (Mustafa et al., 2022). Bacteria genera isolated from water, including Enterobacter, and E. coli, show the mechanism of multidrug resistance (Babalola et al., 2021). The main cause of pollution inside the Halda River is the unplanned activities of various industries, including textile mills, powerplants, papermills, tanneries, etc. (Rahman et al., 2022).  Due to urbanisation, pathogenic bacteria discharge into water bodies (Afzal et al., 2021). In the past few years, the increased use of antibiotics has caused antibiotic resistance in bacteria, including coliform bacteria. Multidrug-resistant bacteria have been isolated from hospital effluent and sewage water. The presence of multiple antibiotic resistances in different water habitats has been reported in Malaysia (Lihan et al., 2017). E. coli is an important indicator organism with respect to faecal pollution in environmental water; therefore, E. coli has also been used in analysing antibiotic resistance in gram-negative bacteria (Wambugu et al., 2015). There is no proper treatment of effluents that belong to hospitals and municipalities due to this infectious agent, and antibiotic-resistant microbes are passed into rivers, which further reach into communities (Belachew et al., 2018). The rivers of Metropolitan are very prone and serve as a reservoir of antibiotic-resistant bacteria with antibiotic resistance genes. According to a report by the WHO (2018), there should be serious awareness with respect to the control of antibiotics by different international organisations in terms of pollution inside the surface water by antibiotics (Ravi et al., 2022).

A. Sampling Method

Between March 2021 and April 2022, water samples were collected. We selected eight locations on the Kshipra River to serve as sample sites. Plastic bottles with sterile screw caps were used to collect samples. After being kept at a constant 4°C in an ice box to prevent the growth of microbes, each bottle was placed in a thermal stabilising box. Within two hours, the bottles were transferred to a microbiology research laboratory for research. With the use of a pH strip and a thermometer, the temperature and pH were determined at each sample location (Wambugu et al., 2015; Teshome et al., 2020; Babalola et al., 2021; Nain et al., 2015).

B. Study Area

The Kshripra river in Ujjain is regarded as a holy river. Many pilgrims visit Ujjain in order to partake in sacred baths. It has been observed that as the water quality declines, a number of anthropogenic activities take place, including mass bathing, clothes washing, and the disposal of trash like coconuts, ashes, photos, and garland. A lot of funeral-related activities were also going on. The river rises in the northern Dhar district and flows north across the Malwa Plateau to meet the Chambal River in the Mandsaur district, which is where the MP and Rajasthan borders meet. It is a sacred river in Hinduism. The holy city of Ujjain is situated on its east bank. The Kumbh Mela (Sinhastha fair), which is held after every 12 years of period.

C. Isolation and Identification of Gram-negative Enteric Bacteria

For the isolation of gram-negative enteric bacteria streak plate method were emplyoed, one loopful of enriched media was inoculated on different culture media like Mac-Conkey Agar, Eosine Methylene Blue Agar, Nutrient Agar, Hi-chrome MM Agar, and Salmonella Shiegella Agar for the isolation of E. coli, Kliebsella, Enterobacter, Citrobacter, Salmonella, Pseudomonas. For further confirmation of Gram negative bacteria, biochemical tests IMViC were employed. Besides this, triple sugar tests, oxidase tests, catalase, gas production tests, motility tests, and gram staining techniques were also used for the identification of gram negative bacteria. After identification, the pure culture of bacteria was preserved in a nutrient agar slant for the antibiotic susceptibility test (Singh et al., 2020).

(i) Antibiotic Susceptibility Test. For the identification of multidrug-resistant bacteria, an antibiotic susceptibility test, also called the disc diffusion method, was performed on all 8 isolates. For confirmation of multidrug-resistant bacteria, Mueller-Hinton agar plates were prepared. To confirm the multidrug-resistant bacteria, a pure culture of the test bacteria was prepared, and the bacterial turbidity was set to 0.5 McFarland standards. By taking the pure culture of bacteria with the help of sterilised cotton swapped, we spread the pure culture of bacteria over the plates of Mueller Hinton agar plates. After spreading the pure culture of bacteria, we were allowed to settle down for 5–10 minutes. After this, we placed the beta lactam antibiotics at a distance of 2.5 cm with the help of a sterilised forcep. After placing the antibiotics, the plates were kept inside the incubator at 37 °C for 24 to 48 hours of incubation. After the incubation period, observe the zone of inhibition. These zones of inhibition were further compared with the CLSI standards (Clinical and Laboratory Standards Institute). The following types of beta-lactam antibiotics were used for confirmation of multidrug-resistant bacteria: aztreonam, ceftazidime, cefpodoxime, chloramphenicol, amikacin, ceftriaxone, ciprofloxacin, and ampicillin. The bacterial isolates that show resistance to three or more classes of antibiotics are termed multidrug-resistant bacteria (MDR). (Singh et al., 2020).

(ii) Calculation of the Multiple Antibiotic Resistance (MAR) Index. The multiple antibiotic resistance (MAR) index was calculated using the formula       

MAR = Number of antibiotics to which an isolate showed resistance                     

Total Number of Antibiotics (Teshome et al., 2020)  

In the study of Wambugu et al. (2015) on the Athi river water in Machakos, Kenya, they isolated a high-resistant strain of E. coli. which shows a resistant mechanism against ampicillin, cefoxitin, amoxicillin, clavulanic acid, and sulfamethoxazole, whereas the E. coli strain shows the least resistance mechanism against gentamicin, cefepime, and ceftazidime. Similar types of resistant strains of E. coli were also isolated from the Shripra River from samples 4 and 7. Besides the E. coli strain, we also studied the resistant strains of Enterobacter, Kliebsella, Salmonella, Citrobacter, and Pseudomonas, which are clearly mentioned in Table 1, 3. In the study of Belachew et al. (2018), they isolated a high level of drug-resistant strains of gram-negative bacteria from an urban river in Addid Ababa, Ethiopia. Their studies show that the strains of E. coli show a high level of resistance to ampicillin, cefalotin, cefuroxime, ceftriaxone, and cefepime. In addition to this, they also studied the resistance mechanism inside the Kliebsella bacteria, which shows resistance against ampicillin. Another strain of gram negative bacteria, Citrobacter, also shows a resistance mechanism against cefazolin and amoxicillin-clavulanic acid. However, all isolates, including E. coli, Kliebsella, and Citrobacter, were sensitive against Ceftriaxone, tetracycline, nitrofurantoin, and trimethoprim-sulfamethoxazole. Similar types of resistance strains of gram negative bacteria were also studied from the Kshripra River. The strains of gram negative bacteria were E. coli, Kliebsella, Enterobacter, Citobacter, Salmonella, and Pseudomonas, which were isolated from samples 1–8, as mentioned in Table 1, 3.  In the study of Ravi et al. (2022) on the Ghaghara River, India, it was shown that the strain of Kliebsella isolated from the Ghaghara River showed resistance mechanisms against penicillin G, cefuroxime, amoxicillin, and ampicillin. In the study of Ravi et al. (2022) on the Ghaghara River, India, it was shown that there were multiple antibiotic-resistant bacteria and provided a route to spread the multidrug-resistant pathogen in the human and animal populations through the aquatic environment. In our study on the Kshripra River with respect to analysing multidrug-resistant bacteria, we found similar findings to the study done by Ravi et al. (2022) on the Ghaghara River. In our study, the resistant strain of Kliebsella was isolated from sample no. 5, which is mentioned in Table 1. In the study of Purohit et al. (2022) on the Kshripra River, an isolated resistant strain of E. coli from river water and river sediment shows the resistance mechanism against different types of antibiotics such as ceftazidime, cefotaxime, cefapime, ampicillin, tetracycline, and co-trimoxazole. In our study on the Kshripra River, similar types of resistant strains of E. coli were also isolated from samples no -4, 7. The isolates of E. coli from sample no. 4 show a resistance mechanism with aztreonam and ceftazidime, while sample no. 7 shows a resistance mechanism with similar antibiotics as aztreonam and ceftazidime in addition to ampicillin. In the study of Besharati et al. (2018) on the Karoon River, enteric bacteria were isolated, including E. coli, Salmonella, Kliebsella, Enterobacter, and Pseudomonas aeruginosa. Multidrug resistance has been found in many bacterial isolates. The highest number of bacterial isolates were resistant to cephalexin, and the least amount of resistance was found against ciprofloxacin. Similar isolates were also isolated from the Kshripra River. The isolates were E. coli, Salmonella, Kliebsella, Enterobacter, and Pseudomonas. Out of these isolates in our study, the multidrug resistance mechanism was shown by E. coli, Kliebsella, Enterobacter, and Salmonella, as mentioned in Table  1, 3.  In the study of Afzal et al. (2021) on the swat river, they isolated a Pseudomonas resistant strain from the swat river, which shows the resistance mechanism against ampicillin. A similar result was also obtained with respect to the multidrug-resistant strain of Pseudomonas, which shows resistance against ampicillin. We obtained the resistant strain of Pseudomonas from sample no. 8, as clearly mentioned in Table 1, 3.  In the same field, another study done by Lihan et al. (2017) on recreational river water of a community resort in Baram, Sarawak, Malaysian Borneo found that Enterobacter spp. shows resistance mechanisms with nitrofurantoin, ampicillin, and piperacillin. Kliebsella spp. shows resistance mechanisms to ampicillin, piperacillin, and tobramycin. Similar types of resistance strains of Enterobacter and Kliebsella were also isolated from the Kshripra River, which is clearly mentioned in Table 1, 3. In the study of Kittinger et al. (2016) on the Danube, they isolated a resistant strain of Pseudomonas spp. The bacterial strain Pseudomonas spp. shows resistance mechanisms against meropenem, piperacillin/tazobactam, and ceftazidime. In our studies on the Kshripra River, a similar resistant strain of gram negative bacteria, Pseudomonas spp., was isolated from sample no. 8 and shows the resistance mechanism against ceftazidime, chloramphenicol, ceftriaxone and ampicillin. In the same field, another study done by Abo-State (2012) on the Rosetta branch of the Nile, Egypt, studied the Enterobacteriacae members, including Salmonella typi, E. coli, and Citrobacter freundii, which show 100% resistance against ampicillin,  methicillin, vancomycin, erythromycin, clindamycin, trimethoprim/ sulfamethoxazole and tetracycline. Also, they fail to show resistance mechanisms against norfloxacin and ofloxacin. MAR index value of Salmonella typhi shows (0.69). Citrobacter freundii shows (0.60, 0.55) and E. coli shows (0.46). Similarly, in our study, we also isolated a resistant strain of Salmonella from sample no. 6. which show resistance mechanisms against cefpodoxime, ceftazidime, ceftriaxone, ciprofloxacin, and ampicillin, and sample no. 7 Salmonella shows resistance mechanisms against aztreonam, ceftazidime, and ampicillin. Similarly, E. coli isolated from sample no. 4 shows a resistance mechanism against aztreonam, ceftazidime, and ampicillin, while E. coli isolated from sample no. 7 shows a resistance mechanism against aztreonam and ceftazidime. In our study on the Kshripra River, the resistant strain of Salmonella was isolated from sample 6, which showed a MAR index of 0.62, and Salmonella was isolated from sample 7, which showed a MAR index of 0.37. The Citrobacter strain we isolated from sample no. 6 shows a MAR index of 0.37; the Citrobacter of sample no. 7 shows a MAR index of 0.25; and the Citrobacter of sample no. 8 shows a MAR index of 0.75. Similar to E. coli, we were isolated from sample no. 4, which shows a MAR index of 0.37, and E. coli, isolated from sample no. 7, shows a MAR index of 0.25.

Fig. 1. Antibiotic Susceptibility Test for Sample-1 Isolates.

Fig. 2. Antibiotic Susceptibility Test for Sample 2 Isolates.

Fig. 3. Antibiotic Susceptibility Test for Sample 3 Isolates.

Fig. 4. Antibiotic Susceptibility Test for Sample 4 Isolates.

Fig. 5. Antibiotic Susceptibility Test for Sample 5 Isolates.

Fig. 6. Antibiotic Susceptibility Test for Sample 6 Isolates.

Fig. 7. Antibiotic Susceptibility Test for Sample 7 Isolates.

Fig. 8. Antibiotic Susceptibility Test for Sample 8 Isolates.

Fig. 9. Control for Antibiotic Susceptibility Test.

Table 1: Showing Sensitive and Resistance Mechanism as well as MAR Index by Gram Negative Bacteria.

Table 2: Total Coliform Bacterial Strains Isolated from River Kshripra Water Samples.

Table 3: Antibiotic Sensitivity Test for Detection of Multidrug Resistant Bacteria.

Graph 1: Showing the sensitive strains as well as the resistance mechanisms of Gram Negative Coliform Bacteria.

The research will help in the prevention of waterborne diseases that may occur due to contamination by multidrug-resistant bacteria. Besides, this research also gives us the idea that during the time of infection in the human population with these pathogenic Gram-negative bacteria, which types of antibiotics are more effective in treatment of multidrug-resistant bacteria. In this way, we can easily save the lives of various mankinds and make people aware that the unnecessary use of antibiotics shall be restricted.

Hinore J.S., Dass P. and Ishaque S. (2024). A Study on Analysing Gram Negative Multidrug-Resistant Enteric Bacteria from the River Kshripa, Ujjain (MP), India. Biological Forum – An International Journal, 16(2): 32-41.