Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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The Occurrence, Antibiotic Susceptibility Pattern and Multiple Drug Resistance Index Studies of E. coli from Fresh Meats in Marathwada Region of Maharashtra

Rahul Suryawanshi1,*, Ashok Bhosale2, Onkar Deshmukh1, Aishwarya Jogdand1, Onkar Shinde1, Niraj Hatwar1, Hrishikesh Kamat1
1Department of Veterinary Public Health, College of Veterinary and Animal Sciences, Maharashtra Animal and Fishery Sciences University, Udgir-413 517, Latur, Maharashtra, India.
2Department of Veterinary Microbiology, College of Veterinary and Animal Sciences, Maharashtra Animal and Fishery Sciences University, Udgir-413 517, Latur, Maharashtra, India.

Background: Foodborne diseases are a huge social burden in both developed and underdeveloped countries. Escherichia coli is a natural inhabitant of the human and warm-blooded animal digestive tracts. It is considered as an indicator organism for direct or indirect faecal contamination of raw meats. The presence of antibiotic-resistant microorganisms in meat accounts for a public health risk.

Methods: In current study, all 39 characterized E. coli isolates recovered on screening 405 fresh meat samples collected from different butcher shops in the Udgir city, Marathwada region of Maharashtra were also tested for Congo red dye binding assay and haemolysis assay. Antimicrobial resistance profiling of recovered isolates was carried out using the disc diffusion method.

Result: Overall occurrence of E. coli in fresh meats was noted to the tune of 9.63% and the corresponding group wise occurrence in chicken and chevon samples were 12% and 7.32%, respectively. All E. coli tested positive in the Congo red dye binding assay and haemolysis assay, suggesting virulent nature of isolated organisms. On screening for antimicrobial resistance, these isolates were found resistant to mainly â-lactams (94.87%), lincosamides (84.62%), 4th Generation cephalosporins (82.05% each), DHFR Inhibitors (64.10%), glycopeptides (51.28%). The multidrug resistant isolates showed resistance to a minimum of 4 and maximum of 12 antibiotics with MAR index ranging from 0.27 to 0.8 and 9 resistance patterns.

The indiscriminate usage and misuse of different antibiotics have led to the emergence of resistance, which has a negative impact on the efficacy of bacterial infection treatment and prevention (Deshmukh et al., 2023). Escherichia coli has become one of the microorganisms that are frequently resistant to antimicrobials due to its ubiquity in humans and animals, as well as its roles as a pathogenic and commensal bacterium (Zhao et al., 2012). According to several investigations, infections with drug-resistant E. coli in humans have been caused by strains from animals and those infectious agents had the same mobile resistance genes in different species of bacteria originating from diverse animal sources (von Baum and Marre, 2005; Hammerum and Heuer, 2009; Johnson et al., 2009). Although antimicrobial-resistant E. coli strains have been found from a variety of foods, a large number of resistant strains have been isolated from common retail meats and poultry (Schroeder et al., 2004).
In response to the increasing public concern over food safety, the current study focused on the precise identification, characterization and antimicrobial resistance of bacterial food-borne pathogens. Its objective was to isolate, identify and confirm pathogenic E. coli from fresh meat samples obtained from meat shops in Udgir, Maharashtra’s Marathwada region.
Samples collection and transportation
A total of 405 fresh meat samples, including 200 samples of chicken and 205 samples of chevon were collected at random from different butcher shops in the Udgir city, Maharashtra. All raw meat samples were collected in sterile sample containers (HiMedia, India). All samples were labeled and immediately transported to the laboratory of Department of Veterinary Public Health and Epidemiology, College of Veterinary and Animal Sciences, Udgir by maintaining cold chain for isolation and identification of E. coli.
Isolation, identification and in vitro pathogenicity assessment of E. coli
The cultural isolation and identification of E. coli was carried out according to the conventional procedure given by Feng et al., (2002). Pure cultures of recovered E. coli isolates were grown on Brain Heart Infusion (BHI) agar slants. These slant cultures were kept at 4-8°C for biochemical characterization and in vitro pathogenicity assays. Gram staining of presumptive E. coli isolates was performed as per Preston and Morel (1962) and pink Gram-negative rods were identified under a binocular microscope (Olympus, Japan). The biochemical characterization was carried out using the technique outlined by Quinn et al., (1994). IMViC (Indole, Methyl Red, Voges Proskauer and Citrate) tests were employed for determining E. coli.
The in vitro pathogenicity assays viz. Congo red dye binding and haemolysis assays (on 5% defibrinated sheep blood agar) were used to determine the virulence potential of the presumptive isolates of E. coli. The Congo red dye binding assay was performed as per the procedure given by Berkhoff and Vinal (1986). In case of haemolysis assay, the freshly grown E. coli isolates were streaked and cultured on 5% Sheep blood agar for 24-48 hours at 37°C following the procedure described by (Beutinet_al1989). The zone of haemolysis production was recorded.
Antimicrobial susceptibility testing
Antimicrobial resistance profiling of recovered isolates was carried out using the disc diffusion method, as described by Bauer et al., (1966). Using an antibiotic zone scale, zones of inhibition were measured after 18 hours and again after 48 hours of incubation. The isolates were classified as sensitive, intermediate sensitive or resistant based on the diameter of the zone of growth inhibition (CLSI, 2020). The isolates were evaluated against a panel of 15 unique antibiotics belonging to different classes and which are routinely used in human and animal treatment as shown in Table 1.

Table 1: Antimicrobial susceptibility pattern of E. coli.

Determination of the multiple drug resistance (MAR) index
The MAR index (b) was calculated using the method described by Krumperman (1983), which divides the number of antibiotics used in the investigation by the number of antibiotics an isolate is resistant to (a). The calculating formula is as follows: MAR index equals a/b.
Occurrence of E. coli isolates
In this investigation, on screening a total of 405 raw fresh meat samples collected from several butcher shops in the Udgir city, Maharashtra altogether 39 samples were found positive for E. coli suggesting the overall occurrence of E. coli to the tune of 9.63%. Chicken and chevon samples exhibited 12% (24/200) and 7.32% (15/205) occurrences of target organism, respectively.
In vitro pathogenicity assessment
All 39 presumed E. coli isolates tested positive in the Congo red dye binding experiment and haemolysis assay used to assess the pathogenicity of bacteria. The positive isolates developed intense brick red colonies on Congo red agar and showed haemolysis in haemolysis assay.
Antibiogram profiling of recovered E. coli isolates
The results showed that the majority of the isolates had multidrug resistance characteristics (Table 3). Two individual isolates recovered from raw chicken meat sample demonstrated resistance against 12 and 11 antibiotics respectively. while, two other isolates recovered from chicken samples and 3 isolates recovered from chevon samples showed resistance against 10 antibiotics (Table 3). Similar trends were also seen in the current study’s observation of sample group-wise antibiotic resistance of E. coli isolates. As shown in Table 2, a significant percentage of E. coli isolates obtained from chicken and chevon exhibited antibiotic resistance to several drugs. The majority of these isolates were found to be resistant to antibiotic classes based on the data obtained are β-lactams (94.87%), lincosamides (84.62%), 4th Generation cephalosporins (82.05% each), DHFR Inhibitors (64.10%), glycopeptides (51.28%) followed by fluoroquinolones, quinolones, 2nd and 3rd generation cephalosporins (48.72% each) (Table 1).

Table 2: Sample group wise antibiotic resistance of E. coli isolates.


Table 3: Multiple drug resistance (MAR) index of E. coli isolates from different type of samples.

Multiple drug resistance (MAR) index
The multi-drug resistant (MDR) isolates in the present study showed resistance to a minimum of 4 and a maximum of 12 antibiotics. Thus, 9 resistance patterns were observed ranging from 4 to 12 antibiotics with maximum multiple antibiotic resistance (MAR) index of 0.8 and a minimum MAR index of 0.27 in MDR isolates as depicted in Table 3. A MAR greater than 0.2 indicates that places with high usage of antibiotics are the source of contamination (Davis and Brown, 2016).
Escherichia coli organism is also regarded as an indicator of faecal contamination in food and water. It is a commonly found commensal bacterium in humans and the environment (Rahman et al., 2017). In this study, the overall occurrence of E. coli was observed to the tune of 9.63% in raw fresh meat samples collected from different butcher shops in the Udgir city, Maharashtra. These findings can be corroborated with Suryawanshi et al., (2023) and Deshmukh et al., (2023) who made similar observations, reporting an overall prevalence of E. coli of 9.17% and 12%, respectively, in raw fresh meats collected from same geographic area.
In current investigation, chicken and chevon samples revealed 12% (24/200) and 7.32% (15/205) occurrence of target organism. Suryawanshi et al., (2023) screened 425 samples of fresh meats comprising chicken, mutton, chevon and carabeef. Researchers found that the occurrence of E. coli was 12.50% (15/120) in chicken and 9.52% (10/105) in chevon, which is consistent with the results of the present study. However, these results contradict with the findings reported by Bhoomika et al., (2016) and Mawia et al., (2012). Bhoomika et al., (2016) reported 66.32% (65/98) and 46.34% (38/82) occurrence of E. coli in chicken meat and chevon samples collected in Chhattisgarh, India. While, Mawia et al., (2012) also obtained 47 (28.14%) E. coli isolates comprising 22 (25.88%) from chevon samples and 25 (30.49%) from chicken on screening 167 meat samples collected from local markets of Jammu, India. These values of higher prevalence of E. coli from chicken and chevon samples reported can be attributed to the variation of sampling methods, detection protocols, poor sanitary practices adopted during handling of meat with the use of microbiologically contaminated water and difference of area of sample collection.
Berkhoff and Vinal (1986) and Roy et al., (2006) confirmed the recovery of 100% Congo red dye-binding isolates in their investigations. Based on their findings, researchers suggested using the Congo red binding assay as a phenotypic marker to discriminate between pathogenic and non-pathogenic isolates. In current investigation, all 39 E. coli isolates were tested positive in the Congo red dye binding experiment and also showed haemolysis in haemolysis assay, suggesting their invasive character.
Microbiologists and public health veterinarians are urged all over the world to survey the antibiotic resistances of key foodborne pathogens in order to deliver epidemiological data to the professionals in charge of public health in order to make recommendations on the appropriate use of antibiotics. For current experiment, the most frequently used antibiotics in both human and animal health were chosen to be tested against recovered E. coli isolates. The investigation’s findings showed that the majority of the isolates had a pattern of multidrug resistance. All 39 isolates recovered were found to be resistant to at least four of the antibiotics that were tested against them. Occurrence of two isolates from chicken showing resistance against 12 and 11 antibiotics respectively was also noted. While, 06 isolates including 03 from chicken and remaining 03 from chevon, displayed resistance against 10 different antibiotics. In our investigation, a very high percentage of antibiotic resistance was noted against amoxycillin-clavulanic acid (94.87%), lincomycin (84.62%), cefepime (82.05%), trimethoprim (64.10%), vancomycin (51.28%), ofloxacin, nalidixic acid, cefotaxime, cefaclor and tetracycline (48.72% each) followed by other antibiotics with resistance levels under 40%. Similar high multidrug resistance pattern was also reported by other researchers like Deshmukh et al., (2023), Adzitey (2015) and Uzeh et al., (2021). The results of present study are consistent with that of observed by Deshmukh et al., (2023) who screened E. coli isolates recovered from poultry farms environment, chicken meat retailers shop, raw chickens from Udgir city of Maharashtra, wherein they reported high percentage of antibiotic resistance against lincomycin and tetracycline (85.29%), nalidixic acid and vancomycin (82.35% each), ofloxacin (67.64%), amoxycillin-clavulanic acid (61.76%), cefepime (41.17%), trimethoprim (38.23%) and cefaclor (47.05%) with similar MDR pattern of each isolate showing resistance to at least four antibiotics. Adzitey (2015) screened 45 E. coli isolates from Beef and its related samples of Ghana and observed a very high resistance to amoxycillin-clavulanic acid (86.67%), trimethoprim (82.22%) and vancomycin (88.89%). Similarly, Uzeh et al., (2021) studied E. coli isolates from raw meats and revealed MDR in 22% of the isolates, as well as resistance against ampicillin (57%), tetracycline (45%) and sulfamethoxazole-trimethoprim (21%).
Despite the lack of antibiotic usage histories to link with susceptibility data, findings of this study might be interpreted as reflecting, at least in part, the selective pressures exerted by antimicrobial use in food animal production and processing contexts. It is hypothesised that increasing levels of resistance in food animals are attributable in part to modern production practises, in which antibiotics for disease prevention and control are provided through water as well as feed. Resistance to expanded-spectrum cephalosporins, as observed in current experiment, is of particular concern because these antimicrobials are used as first-line therapy for a variety of Gram-negative infections, especially systemic and paediatric salmonellosis.
In this study, it was noted that a noteworthy percentage of E. coli isolates recovered from chicken as well as chevon samples exhibiting virtually identical trend of MDR pattern to set of antibiotics It may be the result of a meat-selling strategy used by retailers in India, in which the same shops sell both chicken and chevon meat, based on the preferences of the customers., It additionally introduces the chance that E. coli bacteria may appear as a secondary contamination while cutting chicken and chevon meats with the same contaminated cutting boards or knives and/or washing water. Insufficiently cleaned chopping boards and post-processing meat handling equipment have been linked to cross-contamination, according to Suryawanshi et al., (2023). Uzeh et al., (2021) also speculated the possibility of contamination of raw meats with antibiotic resistant pathogenic organisms at unhygienic slaughter as well as sale points.
Finding regarding MDR isolates in the present study showing resistance to a minimum of 4 and a maximum of 12 antibiotics with minimum MAR index of 0.27 and maximum MAR index of 0.8 and 9 resistance patterns are comparable with Adzitey (2015), who observed MAR index ranging from 0.11to 0.78 shown by E. coli isolates recovered from meats. Multidrug resistant E. coli was also discovered by Hassanien et al., (2016), Adzitey et al., (2020), Abass et al., (2020), Jaja et al., (2020), Mir et al., (2022) Ahmed et al., (2023), with MAR indices ranging from 0.14-1, 0.13-1, 0.22-0.78, 0.2–0.5, 0.45-0.81 and 0.32-0.95, respectively.
In conclusion, the current analysis shows a high occurrence of E. coli in retail meats, showing that faecal contamination at slaughter and processing is prevalent and emphasizing the significance of consumer awareness about proper food handling and cooking. Retail meats are an exposure site close to the customer, so it’s important to keep track of the occurrence of antimicrobial resistance among commensal and pathogenic microorganisms that are found in such products in order to identify emerging resistance issues in the food supply chain.
Authors are grateful to the Associate Dean, COVAS, Udgir for providing facilities to carry out this research work.
Authors declare that there is no conflict of interest.

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