Current IssueEditorial BoardOnline First ArticlesArchive
Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Characterization of Antimicrobial Resistance in Escherichia coli Isolated from Goats with Enteric Disease

N
Neha Shukla1,*
0009-0001-0990-4087
R
R.C. Ghosh1
D
D.K. Jolhe1
P
Poornima Gumasta2
P
P.M. Sonkusale3
D
D.K. Giri4
P
Piyush Kumar1
1Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Dau Shri Vasudev Chandrakar Kamdhenu Vishwavidyalaya, Anjora, Durg-491 001, Chhattisgarh, India.
2Department of Veterinary Pathology, College of Veterinary and Animal Sciences, Bihar Animal Science University, Kishanganj, Patna- 800 014,  Bihar, India.
3Department of Veterinary Pathology, Nagpur Veterinary College, Maharashtra Animal and Fishery Sciences University, Nagpur- 440 001, Maharashtra, India.
4Veterinary Polytechnic, Mahasamund, Dau Shri Vasudev Chandrakar Kamdhenu Vishwavidyalaya, Anjora, Durg-491 001, Chhattisgarh, India.

ABSTRACT

Background: Overuse and misuse of antibiotics in human and farmed animals is the key risk factor for accelerating antimicrobial resistance worldwide. The study aimed at the detection and characterization of E. coli associated with enteric infections in goats of Chhattisgarh, India with its antibiogram and related pathology.

Methods: The work was carried out in the Department of Veterinary Pathology, College of Veterinary Sciences and Animal Husbandry, Durg, Chhattisgarh, from December 2021 to July 2024. A total of 274 naturally died goats were screened for enteric lesions, irrespective of age and sex. Faecal samples and swabs were collected for bacteriological examination. Intestinal tissue samples were collected for molecular, histopathological and ultrastructural study. Disc diffusion technique and Congo red agar method were used for antibiogram and biofilm assay, respectively.

Result: The prevalence of E. coli isolates in goats, died due to enteric infection was 32.45%, based on the PCR results targeting the ecp gene. The 16s rRNA gene sequencing showed 99-99.4% homology with the genome sequences available in NCBI database. The phylogenetic analysis revealed the highest similarities of E. coli isolate of this study with the isolates of Telangana (India), Iraq and China. The study revealed 83.78% of E. coli isolates as biofilm producers and 64% isolates showed multidrug resistance. Various degenerative, inflammatory and necrotic lesions were seen in the intestine. In conclusion, the emergence of antibiotic resistant E. coli in goats poses a serious threat to public health.

INTRODUCTION

In India, goat rearing is one of the prime activities of the livestock sector after cattle. Goats are often called “poor man’s cow” because of their higher productivity, survivability in adverse environments, as well as their utility as a source of income through milk, meat, skin and manure (CIRG, 2021). Chhattisgarh’s rural areas extensively rely on goat farming, which provides supplementary income to the families dependent on agriculture.
       
The incidence of diseases is one of the major barriers to the development of the goat sector. Among various diseases, enteritis is a significant problem in goats, resulting in substantial economic losses to farmers (Tsegaye et al., 2013). Although its aetiology is not well understood in all cases, it is thought to be caused by concurrent infection with several agents without inducing clinical illness in the host, among which E. coli is considered one of the most prevalent organism (Mokhbatly et al., 2022).
       
Today, antimicrobial resistance (AMR) becomes a health issue of global concern. It has been evaluated that India could witness over 2 million deaths due to AMR by the year 2050 (Prasad et al., 2024). 
       
AMR in livestock directly impacts human health and environmental ecosystems. Inappropriate antibiotic use in animals causes gut bacteria to develop the resistance. These resistant bacteria can reach to environment and food chain, reducing the efficacy of common antibiotics and contributing to multidrug resistance (Al-Khalaifah et al., 2025).
       
As long as the goats are important for human life, the bacterial infections and related antibiotic resistance pose a significant problem. Unfortunately, data on the diversity of enteric pathogens and related AMR in goats is limited and no baseline data available regarding the E. coli associated molecular and pathological study of goat mortality due to enteric diseases in Chhattisgarh. Hence, the study investigates the characterization, antibiotic resistance and pathology of E. coli linked to enteric diseases in goats of this area.

MATERIALS AND METHODS

Study area and sample collection
 
The research was carried out in the Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, DSVCKV, Anjora, Durg, Chhattisgarh.
       
Commencing from December 2021 to July 2024, a total of 274 deceased goats of several organized and unorganized goat farms of Durg, Rajnandgaon, Balod and Raipur districts of Chhattisgarh were randomly screened for the presence of prominent enteric gross lesions such as enteritis, congestion and hemorrhages in intestinal mucosa, mucosal thickening, necrotic foci on wall of intestine, ballooning of intestine, presence of necrotic debris and exudate in lumen, enlarged, oedematous and haemorrhagic mesenteric lymph nodes, during post mortem examination; of which, 114 were selected for this study.
       
Intestinal contents and swabs from the part of intestine showing lesions were collected and immediately cultured or stored at 4°C until processing. Tissue samples of the small and large intestine were stored separately at -20°C for molecular characterization. Moreover, representative tissue samples from mesenteric lymph node, small and large intestine were preserved in 10% neutral buffer formal saline solution and 2.5% glutaraldehyde solution, for histopathology and electron microscopic studies, respectively.
 
Isolation of E. coli
 
Cultural isolation of E. coli was carried out as per the standard protocol (Quinn et al., 2002). Briefly, the swabs were inoculated into nutrient broth and incubated for 4-6 h at 37°C. Thereafter, the inoculum was streaked onto MacConkey agar and incubated at 37°C for 24 h. The plates were observed for characteristic pink colonies of E. coli. Further, a single isolated pink colony was streaked on Eosin Methylene Blue (EMB) agar, incubated for 18-24 h at 37°C and observed for the development of characteristic green metallic sheen. Pure cultures of E. coli isolates were taken on Brain Heart Infusion (BHI) agar slants. These slant cultures were stored at 4-8°C for further analysis.
 
Identification of E. coli
 
The identification of E. coli was done based on cultural and biochemical characteristics (Quinn et al., 2002). Further, E. coli isolates were outsourced to National Centre for Microbial Resource (NCMR), National Center for Cell Science (NCCS), Pune, Maharashtra, India for matrix-assisted laser desorption/ ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis for confirmation of its identity.
 
Biofilm forming assay
 
For determining the virulence potential of the presumptive isolates of E. coli, biofilm forming ability was detected using Congo Red Agar (CRA) method as described by Freeman et al., (1989). Black colonies with a dry crystalline consistency indicated biofilm production in E. coli (Karthik et al., 2018).
 
Antibiotic sensitivity profile
 
The antibiotic susceptibility patterns of the E. coli isolates were determined using Kirby-Bauer disc diffusion method on Mueller-Hilton agar following the Clinical and Laboratory Standards Institute guidelines (CLSI, 2020). A total of 12 antibiotics were tested using disc (HiMedia Laboratories Pvt. Limited, Mumbai) of ampicillin (10 μg), amoxycillin clavulanic acid (20/10 μg), cefotaxime (30 μg), ciprofloxacin (5 μg), cotrimoxazole (1.25/23.75 μg), gentamicin (10 μg), tetracycline (30 μg), azithromycin (15 μg), cefixime (5 μg), streptomycin (10 μg), chloramphenicol (30 μg) and norfloxacin (10 μg). After incubation, the diameter of the zone of inhibition around discs was measured with HiAntibiotic Zone Scale-C PW297 (HiMedia Laboratories Pvt. Limited, Mumbai) and compared (CLSI, 2020).
       
According to US and European Centers for Disease Control and Prevention (CDC and ECDC), nonsusceptibility to ≥3 antimicrobial categories is taken as Multidrug-resistance (MDR) (Magiorakos et al., 2012).
       
Moreover, to determine the degree of bacterial resistance to antibiotics, the multiple antibiotic resistance index (MARi) was calculated (Krumperman, 1983). This index was assessed by dividing the numbers of antibiotics to which the organism were resistant to (a), by the numbers of antibiotics tested (b) and MARi greater than 0.2 is considered a health risk (Temikotan and Daniels, 2022).
 
Molecular characterization
 
The Escherichia coli common pilus (ecp) gene was targeted for molecular characterization.
       
The genomic DNA from E. coli isolates were extracted by using HiPurA® Multi sample DNA Purification kit (HiMedia Laboratories Pvt. Limited, India) as per the manufacturer’s guidelines.
       
The PCR reaction was carried out in a total volume of 20 µL containing 3 µL of extracted DNA, 1µL of each primer ECP F (5’TGG TAA TTA CCG ACG AAA ACG GC 3’) and ECP R (5’ACG CGT GGT TAC AGTC TTC CG 3’), 10 µL 2X PCR TaqMixture (Himedia) and 5 μl nuclease free water. Amplification was carried out in a thermal cycler using an initial denaturation step at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 45 sec, annealing step at 60°C for 45 sec, elongation at 72°C for 90 sec and final extension at 72°C for 10 min. The reaction mixture was run by 1.5% agarose gel electrophoresis alongside a 100 base pair marker (Himedia). The amplified products (amplicon size 500 bp) were viewed using the gel documentation system (Bio Rad, UK) (Avelino et al., 2010).
 
Nucleotide sequencing
 
Partial nucleotide sequencing of 16s rRNA gene of one representative E. coli isolate was carried out by NCMR, NCCS, Pune, Maharashtra. The obtained sequences were aligned using the pairwise alignment tool BioEdit. Based on the BLAST results, sequences were further compared with other nucleotide sequences of E. coli in GenBank database. In addition, CLUSTAL W feature of MEGA XI software was used for phylogenetic tree analysis employing neighbor-joining algorithm with 1000 bootstrap replicates (Tamura et al., 2021).
       
The final sequences were submitted in NCBI: https://www.ncbi.nlm.nih.gov, in order to get the accession number.
 
Pathomorphological study
 
For pathomorphological studies, formal saline fixed tissue samples were subjected to routine processing, paraffin embedding, sectioning at 4-5 μm thickness followed by staining with haematoxylin and eosin (Gridley, 1960).
 
Ultrastructural study
 
Representative tissue samples (n=10) of intestine were outsourced to Palamur Biosciences Laboratory, Telangana to explore the ultrastructural alterations using scanning electron microscope.
 
Statistical analysis
 
The data was statistically analysed using IBM SPSS statistics 24 software. P-values of correlations less than 0.05 were considered statistically significant.

RESULTS AND DISCUSSION

Prevalence
 
In the current study, the prevalence of E. coli in naturally dead goats with enteric lesions was recorded as 32.45% (37/114). Although E. coli strains are said to be harmless commensals in the intestine of animals, but this finding indicates that most of these bacterial strains were pathogenic and associated with enteric disease in goats which also corroborates with preceding reports (Singh et al., 2018; Sharma et al., 2020; Gupta et al., 2024). These workers isolated E. coli from diarrheal goats and demonstrated the bacterium as an enteric pathogen. Begum et al., (2016) recorded the 92% prevalence of E. coli in diarrhoeic goats while 84.76% prevalence of E. coli were recorded by Mishra et al., (2020), in goats suffering from diarrhoea, in their study. However, in several studies, relatively lower prevalence were reported as 18.82% and 23.5% by Kamal et al., (2018) and Azmat et al., (2024), respectively.
 
Identification of E. coli
 
The cultural and biochemical characteristics of E. coli isolates, observed in our study, are in accordance with the previous literature (Ahmed et al., 2010; Njoroge et al., 2013; Chatzopoulos et al., 2016), where they showed characteristic colonies on MacConkey and EMB agar (Fig 1). Biochemically, the bacteria were indole, methyl red positive and Voges-Proskauer (VP), citrate, oxidase and urease negative (Fig 2). Moreover, bacteria were able to ferment glucose, lactose, maltose, dextrose, sucrose and mannitol.

Fig 1: Escherichia coli isolate producing characteristic green metallic sheen on eosine methylene blue agar.



Fig 2: Escherichia coli isolate producing characteristic IMViC pattern + + - -.


       
Further, in MALDI-TOF MS analysis, the bacteria showed best matching with Escherichia coli DH5alpha of MALDI-TOF MS biotyper database with score value of 2.457 followed by the second-best matching with Escherichia coli ATCC 25922 with score value 2.338 (Fig 3). Roncarati et al., (2021) used this technique in their study and confirmed the identity of E. coli isolates.

Fig 3: MALDI TOF MS spectra of Escherichia coli isolate.


 
Biofilm forming assay
 
Biofilm formation serves as an important virulence factor for bacteria that provides protective environment for their survival and decreases the susceptibility of bacteria to the host immune response and antimicrobial agents (Zhao et al., 2023). The study revealed 83.78% isolates of E. coli were biofilm producer while 16.21% were non biofilm former.
       
Similar to our study, Dadawala et al., (2010) used Congo red (CR) assay for the assessment of biofilm production in E. coli and observed that out of 14 isolates, 12 (85.71%) were producing black colonies.
 
Antibiotic sensitivity profile
 
Antibiotic sensitivity assay revealed that none of the antimicrobials had 100% sensitivity and resistance towards E. coli isolates in the study area. The isolates showed the highest resistance to tetracycline (78.37%), followed by ampicillin (72.97%), amoxiclav (amoxicillin/clavulanic acid) (48.64%), co-trimoxazole (45.94%) and cefotaxime (40.54%). However, relatively lower resistance was observed against ciprofloxacin (32.43%), cefixime (24.32%), gentamicin (21.62%), norfloxacin (16.21%), azithromycin (16.21%), streptomycin (13.51%) and chloramphenicol (08.10%). In the present study, 64% of the isolates were identified as multidrug-resistant (MDR) based on a Multiple Antibiotic Resistance (MAR) index.
       
Moreover, it was observed that antimicrobial resistance was statistically significant (p<0.05) with biofilm production. Our results are in close conformity with earlier reports, where Zare et al., (2014), observed 65% E. coli isolates with multiple antibiotic resistance (MAR), in their study. Hariharan et al. (2004) recorded high resistance of E. coli to tetracycline in different animals while Adefarakan et al. (2014) reported higher number of resistant E. coli isolates to co-trimoxazole, nitrofurantoin and tetracycline in goat faeces. The present study finding agrees with the reports by Singh et al., (2017); Haulisah et al. (2021) and Kumar et al., (2022), who demonstrated the presence of multiple antibiotic resistance in more than 60% E. coli strains associated with clinical diarrhoea in goats. Also, Katongole et al. (2020) reported the statistically significant association between MDR and biofilm production in uropathogenic E. coli.
       
Long-term exposure of bacteria to subtherapeutic antibiotic doses is the cause of the increment in antibiotic resistance (Samreen et al., 2021). It may also originate in farms from contaminated water with subtherapeutic antibiotic concentrations. Antibiotic-resistant bacteria can transfer their resistance genes to DNA and plasmid of bacteria, during their multiplication, causing the number of resistant bacteria to increase (Xu et al., 2022). Moreover, antibiotic resistance in animals may also attributed to biofilm forming ability of bacteria. The proximity of cells within a biofilm can facilitate a plasmid exchange and hence enhance the spread of antimicrobial resistance (Watnick and Kotler, 2000).
 
Molecular characterization
 
The results of PCR amplification in the present study showed that all the E. coli isolates (n=37), were positive for ecp gene, producing an amplicon of 500 bp (Fig 4). This finding is in close agreement with the earlier reports where E. coli isolates were confirmed using ecp gene amplification (Avelino et al., 2010; Deshmukh et al., 2023; Munhoz et al., 2023). Munhoz et al., (2023) described that E. coli common pilus (ecp) fimbrae present in several diarrheagenic E. coli (DEC) associated pathotypes and plays an important role in host cell adherence and biofilm formation.

Fig 4: Gel image of amplification of ecp gene of Escherichia coli isolates producing an amplicon size of 500 bp.


 
Nucleotide sequencing
 
The partial nucleotide sequencing of 16S rRNA gene of E. coli isolate (NE1 DSVCKV/DURG/CG/INDIA) revealed final consensus of 1008 bp sequence. The Basic Local Alignment Search Tool (BLAST) analysis discovered 99.00-99.40% homology with other nucleotide sequences of E. coli available in the reference database of GenBank. The accession number for the E. coli isolate of the present study was provided by NCBI GenBank as PQ334875.
       
Phylogenetic analysis showed highest similarities of E. coli isolate (PQ334875) of this study with the E. coli isolates of Telangana, India (MK716402.1), Iraq (LC796919.1) and China (MK621216.1) (Fig 5).

Fig 5: Cladogram of Escherichia coli (NE1 DSVCKV/DURG/CG/INDIA) isolate.


 
Gross and histopathological examination
 
During the necropsy procedure, the majority of the goats were found emaciated, dehydrated, with rough hair coat and pale conjunctiva. The perianal region and tail-base were soiled with diarrhoeic faeces. The gross lesions observed were enlargement, congestion and multiple necrotic foci on liver, enlarged and oedematous mesenteric lymph nodes and severe ballooning of the small intestine with hyperaemic serosa, as well as congestion and haemorrhage in mucosa (Fig 6). The microscopic examination revealed cellular swelling and vacuolation, congestion and infiltration of inflammatory cells with distended sinusoids in liver (Fig 7) and infiltration of mononuclear cells over a homogenous meshwork of fibrin threads in the medulla of mesenteric lymph nodes. Moreover, desquamation of mucosal epithelium was accompanied by infiltration of neutrophils, lymphocytes in the lamina propria, villous atrophy, stunting or complete loss of villi in jejunum (Fig 8). The above lesions associated with E. coli infection in goats corroborate with the findings of Kumar et al., (2015) and Gupta et al., (2023).
 

Fig 6: Jejunum of Escherichia coli positive goat showing congestion.



Fig 7: Photomicrograph of liver from Escherichia coli infected goat showing cellular swelling characterized by swollen hepatocytes with eosinophilic cytoplasm and vacuolation (H and E×1000).



Fig 8: Photomicrograph of jejunum showing erosion of mucosal epithelium with infiltration of polymorphonuclear cells in lamina propria, haemorrhagic crypts and accumulation of sloughed off epithelial debris with inflammatory cells in lumen (H and E × 100).



Ultrastructural studies
 
Ultrastructural lesions were characterized by fusion, atrophy and loss of villi accompanied with enlarged crypt orifices in jejunal mucosa as well as covering of mucosa of small intestine with necrotic and fibrinous layer (Fig 9). The above mentioned changes corroborate with the findings of Neog et al., (2011), who studied the Rota virus and E. coli related pathology in diarrhoeic pigs.

Fig 9: Ultrastructural changes in small intestine showing necrotic and fibrinous layer completely covering the mucosa (SEM ×500).

CONCLUSION

This study shows that E. coli is one of the major cause of enteric infection in goats, which may result in fatal infections. Moreover, goats may serve as carrier for multidrug-resistant bacteria, raising concerns about their potential introduction into environment and eventually to the food chain posing threat to other animals and human food safety; hence, the irrational use of antibiotics in animals should be discouraged.

ACKNOWLEDGEMENT

The study was supported by College of Veterinary Science and Animal Husbandry, DSVCKV, Anjora, Durg, (C.G.).
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

CONFLICT OF INTEREST

The authors declare no conflicts of interest regarding the publication of this article.

REFERENCES


  1. Adefarakan, T.A., Oluduro, A.O., David, O.M., Ajayi, A.O., Ariyo, A.B. and Fashina, C.D. (2014). Prevalence of antibiotic resistance and molecular characterization of Escherichia coli from faeces of apparently healthy rams and goats in Ile-Ife, Southwest, Nigeria. Ife Journal of Science. 16(3): 447-460.

  2. Ahmed, A., Egwu, G.O., Garba, H.S. and Magaji, A.A. (2010). Prevalence of bacterial pathogens and serotyping of E. coli isolates from diarrhoeic lambs in Sokoto state, Nigeria. Sokoto Journal of Veterinary Sciences. 8(1): 42-45.

  3. Al-Khalaifah, H., Rahman, M. H., Al-Surrayai, T., Al-Dhumair, A. and Al-Hasan, M. (2025). A one-health perspective of antimicrobial resistance (AMR): Human, animals and environmental health. Life. 15(10): 1598.  

  4. Avelino, F., Saldana, Z., Islam, S., Monteiro-Neto, V., Dall’Agnol, M., Eslava, C.A. and Giron, J.A. (2010). The majority of enteroaggregative Escherichia coli strains produce the E. coli common pilus when adhering to cultured epithelial cells. International Journal of Medical Microbiology. 300(7): 440-448.

  5. Azmat, M., Khan, J.A., Ijaz, M., Ashraf, K. and Naseer, O. (2024). Molecular epidemiology of enteric pathogens associated with diarrheal disease in goat kids. Pakistan Journal of Zoology. pp 1-8. 

  6. Begum, F., Islam, M.M., Sohidullah, M., Kabir, S.M.L., Islam, M. and Rahman, M.T. (2016). Molecular identification and antibiogram profiles of Escherichia coli isolated from apparently healthy and diarrheic goats. Bangladesh Journal of Veterinary Medicine. 14(2): 203-208.

  7. Chatzopoulos, D.C., Sarrou, S., Vasileiou, N.G.C., Ioannidi, K.S., Peteinaki, E., Valiakos, G., Tsokana, C.N., Papadopoulos, E., Spyrou, V., Mavrogianni, V.S. and Giannakopoulos, A. (2016). Dissemination of intestinal pathogens between lambs and puppies in sheep farms. Small Ruminant Research. 141: 5-10.

  8. CIRG. (2021). Annual Report 2021. Central Institute for Research on Goats, ICAR. pp 05.

  9. Clinical and Laboratory Standards Institute. (2020). Performance Standards for Antimicrobial Susceptibility Testing CLSI Supplement M100. 30th edn., Clinical and Laboratory Standards Institute, Wayne, PA, USA.

  10. Dadawala, A.I., Chauhan, H.C., Chandel, B.S., Ranaware, P., Patel, S.S, Singh, K., Rathod, P.H., Shah, N.M. and Kher, H.N. (2010). Assessment of Escherichia coli isolates for  in vitro biofilm production. Veterinary World. 3(8): 364-366.

  11. Deshmukh, O., Suryawanshi, R., Kurkure, N., Kaore, M., Badar, S., Shinde, O., Awandkar, S. and Gaikwad, N. (2023). Molecular characterization of multidrug-resistant avian pathogenic Escherichia coli isolated from poultry and poultry products. Indian Journal of Animal Sciences.  93(5): 431-436.

  12. Freeman, D.J., Falkiner, F.R. and Keane, C.T. (1989). New method for detecting slime production by coagulase negative staphylococci. Journal of Clinical Pathology. 42(8): 872- 874.

  13. Gridley, M.F. (1960). Manual of Histological and Special Staining Technique. McGraw-hill book company, New York, Toronto, London. pp 202.

  14. Gupta, R.K., Niyogi, D., Joshi, R.K., Yadav, V., Jaiswal, V., Shrivastava, D.P., Singh, S.V., Jaiswal, S., Singh, J. and Alam, K. (2024). Prevalence and phylogenetic analysis of E. coli in diarrhoeic goats in eastern plain zone of Uttar Pradesh. Indian Journal of Animal Research. 1-8. doi: 10.18805/IJAR.B-5179.

  15. Gupta, R.K., Niyogi, D., Joshi, R.K., Yadav, V., Singh, S., Singh, J.P., Jaiswal, S. and Mishra, A. (2023). Pathomorphological alterations in intestine of goats infected with E. coli and rotavirus in eastern plain zone of Uttar Pradesh. Journal of Experimental Zoology India. 1(2): 26.

  16. Hariharan, H., Mada, C., Poole, D. and Page, R. (2004). Antibiotic resistance among enterotoxigenic Escherichia coli from piglets and calves with diarrhea. Canadian Veterinary Journal. 45: 605-606.

  17. Haulisah, N.A., Hassan, L., Bejo, S.K., Jajere, S.M. and Ahmad, N.I. (2021). High levels of antibiotic resistance in isolates from diseased livestock. Frontiers in Veterinary Science. 8: 652351. 

  18. Kamal, M.R., Fakhruzzaman, M., Akter, M.R. and Haque, M.A. (2018). Isolation, identification and antibiogram profiles of enterovirulent Escherichia coli from diarrhoeic goat in some selected areas of Rangpur district of Bangladesh. Asian Journal of Medical and Biological Research. 4(1): 36-43. 

  19. Karthik, R., Ambica, R. and Nagarathnamma, T. (2018). Study of biofilm production and antimicrobial susceptibility pattern in clinical isolates of Proteus Species at a tertiary care hospital. International Journal of Current Microbiology and Applied Sciences. 7(1): 574-586. 

  20. Katongole, P., Nalubega, F., Florence, N.C., Asiimwe, B. and Andia, I. (2020). Biofilm formation, antimicrobial susceptibility and virulence genes of uropathogenic Escherichia coli isolated from clinical isolates in Uganda. BMC Infectious Diseases. 20: 453.

  21. Krumperman, P.H. (1983). Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Applied and Environmental Microbiology. 46(1): 165-170.

  22. Kumar, N., Kumar, M. and Kaur, A. (2022). Escherichia Coli antimicrobial resistance profile obtained from productive livestock. Journal of Research in Medical and Dental Science. 10(S1): 6-10.

  23. Kumar, S., Jakhar, K.K., Nehra, V. and Pal, M. (2015). Pathomorphological and microbiological studies in sheep with special emphasis on gastrointestinal tract disorders. Veterinary World. 8(8): 1015-1020.

  24. Magiorakos, A.P., Srinivasan, A., Carey, R.B., Carmeli, Y., Falagas, M.E., Giske, C.G., Harbarth, S., Hindler, J.F., Kahlmeter, G., Olsson-Liljequist, B.J. and Paterson, D.L. (2012). Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clinical Microbiology and Infection. 8(3): 268-281.

  25. Mishra, A.K., Singh, D.D., Kumar, N., Kumarsen, G., Paul, S. and Kumar, A. (2020). Role of bacterial and parasitic pathogens in occurrence of neonatal diarrhoea in goat-kids. Indian Journal of Animal Research. 10(3): 389-395. doi: 10.30954/ 2277-940X.03.2020.9.

  26. Mokhbatly, A.A., Elsheikh, N., Ghazy, E.W., Elgamal, A.M., Hegazy, Y.M. and Assar, D.H. (2022). Prevalence of Shiga toxin- producing Escherichia coli and Salmonellae and some associated hematologic and biochemical profile alterations in lambs. Veterinary Research Forum: An International Quarterly Journal. 13(2): 155-162.

  27. Munhoz, D.D., Richards, A.C., Santos, F.F., Mulvey, M.A. and Piazza, R.M.F. (2023). E. coli common pili promote the fitness and virulence of a hybrid aEPEC/ExPEC strain within diverse host environments. Gut Microbes. 15(1):  2190308. 

  28. Neog, B.K., Barman, N.N., Bora, D.P., Dey, S.C. and Chakraborty, A. (2011). Experimental infection of pigs with group A rotavirus and enterotoxigenic Escherichia coli in India: Gross, histopathological and immunopathological study.  Veterinaria Italiana. 47(2): 117-128.

  29. Njoroge, S., Muigai, A.W.T., Njiruh, P.N. and Kariuki, S. (2013). Molecular characterisation and antimicrobial resistance patterns of Escherichia coli isolates from goats slaughtered in parts of Kenya. East African Medical Journal. 90(3): 72-83.

  30. Prasad, D.K., Shukla, R. and Ahammad, S.Z. (2024). Antibiotic resistance in the Ganga River: Investigation of antibiotic- resistant bacteria and antibiotic resistance genes and public health risk assessment. Journal of Environmental Chemical Engineering. 12(6): 114931.

  31. Quinn, P.J., Markey, B.K., Carter, M.E., Donnelly, W.J., Leonard, F.C. and Maguire, D. (2002). Veterinary Microbiology and Microbial Disease. Blackwell Science Ltd. pp106-118. 

  32. Roncarati, G., Foschi, C., Ambretti, S. and Re, M.C. (2021). Rapid identification and detection of β-lactamase-producing enterobacteriaceae from positive blood cultures by MALDI-TOF/MS. Journal of Global Antimicrobial Resistance. 24: 270-274.

  33. Samreen, Ahmad, I., Malak, H.A. and Abulreesh, H.H. (2021). Environmental antimicrobial resistance and its drivers: A potential threat to public health. Journal of Global Antimicrobial Resistance. 27: 101-111.

  34. Sharma, S.K., Manat, N. and Joshi, M. (2020). Prevalence of colibacillosis in goat kids in Udaipur district of Rajasthan. Indian Journal of Veterinary Sciences and Biotechnology. 16(2): 98-100.

  35. Singh, B.R., Kumar, V., Sinha, D.K., Bhardwaj, M., Saraf, A. and Vadhana, P. (2017). Antimicrobial resistance profile of enteropathogens isolated from diarrhea patients: Herbal antimicrobials, a ray of hope. Annals of Pharmacology and Pharmaceutics. 2(13): 1068.

  36. Singh, D.D., Pawaiya, R.S., Gururaj, K., Gangwar, N.K., Mishra, A.K. andani, D., Singh, M.K., Bhushan, S. and Kumar, A. (2018). Molecular detection of Clostridium perfringens toxinotypes, enteropathogenic Escherichia coli, rotavirus and coronavirus in diarrheic fecal samples of neonatal goat kids. Veterinarski Arhiv. 88: 1-20.

  37. Tamura, K., Stecher, G. and Kumar, S. (2021). MEGA11: Molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution. 38(7): 3022-3027.

  38. Temikotan, T. and Daniels, A.O. (2022). Intestinal flora of selected Agricultural animals and their antibiotic resistance and plasmid profiles. IOSR Journal of Pharmacy and Biological Sciences. 17(6): 56-64.

  39. Tsegaye, D., Belay, B. and Haile, A. (2013). Prevalence of major goat diseases and mortality of goat in Daro-Labu district of West Hararghe, Eastern Ethiopia. Journal of Scientific and Innovative Research. 2(3): 665-672.

  40. Watnick, P. and Kotler, R. (2000). A biofilm, a city of microbes. Journal of Bacteriology. 182: 2675-79.

  41. Xu, C., Kong, L., Gao, H., Cheng, X. and Wang, X. (2022). A review of current bacterial resistance to antibiotics in food animals. Frontiers in Microbiology. 13(5): 822689.

  42. Zare, P., Ghorbani, C.H., Jaberi, S., Razzaghi, S., Mirzaei, M. and Mafuni, K. (2014). Occurrence and antimicrobial resistance of Salmonella spp. and Escherichia coli isolates in apparently healthy slaughtered cattle, sheep and goats in East Azarbaijan province. International Journal of Enteric Pathogens. 2(1): e15451.

  43. Zhao, A., Sun, J. and Liu, Y. (2023). Understanding bacterial biofilms: From definition to treatment strategies. Frontiers in Cellular and Infection Microbiology. 13: 1137947. 
Published In
Indian Journal of Animal Research
Article Metrics
Metrics Icon
0
Views
0
Citations
Reviewed By
Share Article
Download Article
In this Article
    Contact us
    Follow us

    Full Research Article

    Characterization of Antimicrobial Resistance in Escherichia coli Isolated from Goats with Enteric Disease

    N
    Neha Shukla1,*
    0009-0001-0990-4087
    R
    R.C. Ghosh1
    D
    D.K. Jolhe1
    P
    Poornima Gumasta2
    P
    P.M. Sonkusale3
    D
    D.K. Giri4
    P
    Piyush Kumar1
    1Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Dau Shri Vasudev Chandrakar Kamdhenu Vishwavidyalaya, Anjora, Durg-491 001, Chhattisgarh, India.
    2Department of Veterinary Pathology, College of Veterinary and Animal Sciences, Bihar Animal Science University, Kishanganj, Patna- 800 014,  Bihar, India.
    3Department of Veterinary Pathology, Nagpur Veterinary College, Maharashtra Animal and Fishery Sciences University, Nagpur- 440 001, Maharashtra, India.
    4Veterinary Polytechnic, Mahasamund, Dau Shri Vasudev Chandrakar Kamdhenu Vishwavidyalaya, Anjora, Durg-491 001, Chhattisgarh, India.

    ABSTRACT

    Background: Overuse and misuse of antibiotics in human and farmed animals is the key risk factor for accelerating antimicrobial resistance worldwide. The study aimed at the detection and characterization of E. coli associated with enteric infections in goats of Chhattisgarh, India with its antibiogram and related pathology.

    Methods: The work was carried out in the Department of Veterinary Pathology, College of Veterinary Sciences and Animal Husbandry, Durg, Chhattisgarh, from December 2021 to July 2024. A total of 274 naturally died goats were screened for enteric lesions, irrespective of age and sex. Faecal samples and swabs were collected for bacteriological examination. Intestinal tissue samples were collected for molecular, histopathological and ultrastructural study. Disc diffusion technique and Congo red agar method were used for antibiogram and biofilm assay, respectively.

    Result: The prevalence of E. coli isolates in goats, died due to enteric infection was 32.45%, based on the PCR results targeting the ecp gene. The 16s rRNA gene sequencing showed 99-99.4% homology with the genome sequences available in NCBI database. The phylogenetic analysis revealed the highest similarities of E. coli isolate of this study with the isolates of Telangana (India), Iraq and China. The study revealed 83.78% of E. coli isolates as biofilm producers and 64% isolates showed multidrug resistance. Various degenerative, inflammatory and necrotic lesions were seen in the intestine. In conclusion, the emergence of antibiotic resistant E. coli in goats poses a serious threat to public health.

    INTRODUCTION

    In India, goat rearing is one of the prime activities of the livestock sector after cattle. Goats are often called “poor man’s cow” because of their higher productivity, survivability in adverse environments, as well as their utility as a source of income through milk, meat, skin and manure (CIRG, 2021). Chhattisgarh’s rural areas extensively rely on goat farming, which provides supplementary income to the families dependent on agriculture.
           
    The incidence of diseases is one of the major barriers to the development of the goat sector. Among various diseases, enteritis is a significant problem in goats, resulting in substantial economic losses to farmers (Tsegaye et al., 2013). Although its aetiology is not well understood in all cases, it is thought to be caused by concurrent infection with several agents without inducing clinical illness in the host, among which E. coli is considered one of the most prevalent organism (Mokhbatly et al., 2022).
           
    Today, antimicrobial resistance (AMR) becomes a health issue of global concern. It has been evaluated that India could witness over 2 million deaths due to AMR by the year 2050 (Prasad et al., 2024). 
           
    AMR in livestock directly impacts human health and environmental ecosystems. Inappropriate antibiotic use in animals causes gut bacteria to develop the resistance. These resistant bacteria can reach to environment and food chain, reducing the efficacy of common antibiotics and contributing to multidrug resistance (Al-Khalaifah et al., 2025).
           
    As long as the goats are important for human life, the bacterial infections and related antibiotic resistance pose a significant problem. Unfortunately, data on the diversity of enteric pathogens and related AMR in goats is limited and no baseline data available regarding the E. coli associated molecular and pathological study of goat mortality due to enteric diseases in Chhattisgarh. Hence, the study investigates the characterization, antibiotic resistance and pathology of E. coli linked to enteric diseases in goats of this area.

    MATERIALS AND METHODS

    Study area and sample collection
     
    The research was carried out in the Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, DSVCKV, Anjora, Durg, Chhattisgarh.
           
    Commencing from December 2021 to July 2024, a total of 274 deceased goats of several organized and unorganized goat farms of Durg, Rajnandgaon, Balod and Raipur districts of Chhattisgarh were randomly screened for the presence of prominent enteric gross lesions such as enteritis, congestion and hemorrhages in intestinal mucosa, mucosal thickening, necrotic foci on wall of intestine, ballooning of intestine, presence of necrotic debris and exudate in lumen, enlarged, oedematous and haemorrhagic mesenteric lymph nodes, during post mortem examination; of which, 114 were selected for this study.
           
    Intestinal contents and swabs from the part of intestine showing lesions were collected and immediately cultured or stored at 4°C until processing. Tissue samples of the small and large intestine were stored separately at -20°C for molecular characterization. Moreover, representative tissue samples from mesenteric lymph node, small and large intestine were preserved in 10% neutral buffer formal saline solution and 2.5% glutaraldehyde solution, for histopathology and electron microscopic studies, respectively.
     
    Isolation of E. coli
     
    Cultural isolation of E. coli was carried out as per the standard protocol (Quinn et al., 2002). Briefly, the swabs were inoculated into nutrient broth and incubated for 4-6 h at 37°C. Thereafter, the inoculum was streaked onto MacConkey agar and incubated at 37°C for 24 h. The plates were observed for characteristic pink colonies of E. coli. Further, a single isolated pink colony was streaked on Eosin Methylene Blue (EMB) agar, incubated for 18-24 h at 37°C and observed for the development of characteristic green metallic sheen. Pure cultures of E. coli isolates were taken on Brain Heart Infusion (BHI) agar slants. These slant cultures were stored at 4-8°C for further analysis.
     
    Identification of E. coli
     
    The identification of E. coli was done based on cultural and biochemical characteristics (Quinn et al., 2002). Further, E. coli isolates were outsourced to National Centre for Microbial Resource (NCMR), National Center for Cell Science (NCCS), Pune, Maharashtra, India for matrix-assisted laser desorption/ ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis for confirmation of its identity.
     
    Biofilm forming assay
     
    For determining the virulence potential of the presumptive isolates of E. coli, biofilm forming ability was detected using Congo Red Agar (CRA) method as described by Freeman et al., (1989). Black colonies with a dry crystalline consistency indicated biofilm production in E. coli (Karthik et al., 2018).
     
    Antibiotic sensitivity profile
     
    The antibiotic susceptibility patterns of the E. coli isolates were determined using Kirby-Bauer disc diffusion method on Mueller-Hilton agar following the Clinical and Laboratory Standards Institute guidelines (CLSI, 2020). A total of 12 antibiotics were tested using disc (HiMedia Laboratories Pvt. Limited, Mumbai) of ampicillin (10 μg), amoxycillin clavulanic acid (20/10 μg), cefotaxime (30 μg), ciprofloxacin (5 μg), cotrimoxazole (1.25/23.75 μg), gentamicin (10 μg), tetracycline (30 μg), azithromycin (15 μg), cefixime (5 μg), streptomycin (10 μg), chloramphenicol (30 μg) and norfloxacin (10 μg). After incubation, the diameter of the zone of inhibition around discs was measured with HiAntibiotic Zone Scale-C PW297 (HiMedia Laboratories Pvt. Limited, Mumbai) and compared (CLSI, 2020).
           
    According to US and European Centers for Disease Control and Prevention (CDC and ECDC), nonsusceptibility to ≥3 antimicrobial categories is taken as Multidrug-resistance (MDR) (Magiorakos et al., 2012).
           
    Moreover, to determine the degree of bacterial resistance to antibiotics, the multiple antibiotic resistance index (MARi) was calculated (Krumperman, 1983). This index was assessed by dividing the numbers of antibiotics to which the organism were resistant to (a), by the numbers of antibiotics tested (b) and MARi greater than 0.2 is considered a health risk (Temikotan and Daniels, 2022).
     
    Molecular characterization
     
    The Escherichia coli common pilus (ecp) gene was targeted for molecular characterization.
           
    The genomic DNA from E. coli isolates were extracted by using HiPurA® Multi sample DNA Purification kit (HiMedia Laboratories Pvt. Limited, India) as per the manufacturer’s guidelines.
           
    The PCR reaction was carried out in a total volume of 20 µL containing 3 µL of extracted DNA, 1µL of each primer ECP F (5’TGG TAA TTA CCG ACG AAA ACG GC 3’) and ECP R (5’ACG CGT GGT TAC AGTC TTC CG 3’), 10 µL 2X PCR TaqMixture (Himedia) and 5 μl nuclease free water. Amplification was carried out in a thermal cycler using an initial denaturation step at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 45 sec, annealing step at 60°C for 45 sec, elongation at 72°C for 90 sec and final extension at 72°C for 10 min. The reaction mixture was run by 1.5% agarose gel electrophoresis alongside a 100 base pair marker (Himedia). The amplified products (amplicon size 500 bp) were viewed using the gel documentation system (Bio Rad, UK) (Avelino et al., 2010).
     
    Nucleotide sequencing
     
    Partial nucleotide sequencing of 16s rRNA gene of one representative E. coli isolate was carried out by NCMR, NCCS, Pune, Maharashtra. The obtained sequences were aligned using the pairwise alignment tool BioEdit. Based on the BLAST results, sequences were further compared with other nucleotide sequences of E. coli in GenBank database. In addition, CLUSTAL W feature of MEGA XI software was used for phylogenetic tree analysis employing neighbor-joining algorithm with 1000 bootstrap replicates (Tamura et al., 2021).
           
    The final sequences were submitted in NCBI: https://www.ncbi.nlm.nih.gov, in order to get the accession number.
     
    Pathomorphological study
     
    For pathomorphological studies, formal saline fixed tissue samples were subjected to routine processing, paraffin embedding, sectioning at 4-5 μm thickness followed by staining with haematoxylin and eosin (Gridley, 1960).
     
    Ultrastructural study
     
    Representative tissue samples (n=10) of intestine were outsourced to Palamur Biosciences Laboratory, Telangana to explore the ultrastructural alterations using scanning electron microscope.
     
    Statistical analysis
     
    The data was statistically analysed using IBM SPSS statistics 24 software. P-values of correlations less than 0.05 were considered statistically significant.

    RESULTS AND DISCUSSION

    Prevalence
     
    In the current study, the prevalence of E. coli in naturally dead goats with enteric lesions was recorded as 32.45% (37/114). Although E. coli strains are said to be harmless commensals in the intestine of animals, but this finding indicates that most of these bacterial strains were pathogenic and associated with enteric disease in goats which also corroborates with preceding reports (Singh et al., 2018; Sharma et al., 2020; Gupta et al., 2024). These workers isolated E. coli from diarrheal goats and demonstrated the bacterium as an enteric pathogen. Begum et al., (2016) recorded the 92% prevalence of E. coli in diarrhoeic goats while 84.76% prevalence of E. coli were recorded by Mishra et al., (2020), in goats suffering from diarrhoea, in their study. However, in several studies, relatively lower prevalence were reported as 18.82% and 23.5% by Kamal et al., (2018) and Azmat et al., (2024), respectively.
     
    Identification of E. coli
     
    The cultural and biochemical characteristics of E. coli isolates, observed in our study, are in accordance with the previous literature (Ahmed et al., 2010; Njoroge et al., 2013; Chatzopoulos et al., 2016), where they showed characteristic colonies on MacConkey and EMB agar (Fig 1). Biochemically, the bacteria were indole, methyl red positive and Voges-Proskauer (VP), citrate, oxidase and urease negative (Fig 2). Moreover, bacteria were able to ferment glucose, lactose, maltose, dextrose, sucrose and mannitol.

    Fig 1: Escherichia coli isolate producing characteristic green metallic sheen on eosine methylene blue agar.



    Fig 2: Escherichia coli isolate producing characteristic IMViC pattern + + - -.


           
    Further, in MALDI-TOF MS analysis, the bacteria showed best matching with Escherichia coli DH5alpha of MALDI-TOF MS biotyper database with score value of 2.457 followed by the second-best matching with Escherichia coli ATCC 25922 with score value 2.338 (Fig 3). Roncarati et al., (2021) used this technique in their study and confirmed the identity of E. coli isolates.

    Fig 3: MALDI TOF MS spectra of Escherichia coli isolate.


     
    Biofilm forming assay
     
    Biofilm formation serves as an important virulence factor for bacteria that provides protective environment for their survival and decreases the susceptibility of bacteria to the host immune response and antimicrobial agents (Zhao et al., 2023). The study revealed 83.78% isolates of E. coli were biofilm producer while 16.21% were non biofilm former.
           
    Similar to our study, Dadawala et al., (2010) used Congo red (CR) assay for the assessment of biofilm production in E. coli and observed that out of 14 isolates, 12 (85.71%) were producing black colonies.
     
    Antibiotic sensitivity profile
     
    Antibiotic sensitivity assay revealed that none of the antimicrobials had 100% sensitivity and resistance towards E. coli isolates in the study area. The isolates showed the highest resistance to tetracycline (78.37%), followed by ampicillin (72.97%), amoxiclav (amoxicillin/clavulanic acid) (48.64%), co-trimoxazole (45.94%) and cefotaxime (40.54%). However, relatively lower resistance was observed against ciprofloxacin (32.43%), cefixime (24.32%), gentamicin (21.62%), norfloxacin (16.21%), azithromycin (16.21%), streptomycin (13.51%) and chloramphenicol (08.10%). In the present study, 64% of the isolates were identified as multidrug-resistant (MDR) based on a Multiple Antibiotic Resistance (MAR) index.
           
    Moreover, it was observed that antimicrobial resistance was statistically significant (p<0.05) with biofilm production. Our results are in close conformity with earlier reports, where Zare et al., (2014), observed 65% E. coli isolates with multiple antibiotic resistance (MAR), in their study. Hariharan et al. (2004) recorded high resistance of E. coli to tetracycline in different animals while Adefarakan et al. (2014) reported higher number of resistant E. coli isolates to co-trimoxazole, nitrofurantoin and tetracycline in goat faeces. The present study finding agrees with the reports by Singh et al., (2017); Haulisah et al. (2021) and Kumar et al., (2022), who demonstrated the presence of multiple antibiotic resistance in more than 60% E. coli strains associated with clinical diarrhoea in goats. Also, Katongole et al. (2020) reported the statistically significant association between MDR and biofilm production in uropathogenic E. coli.
           
    Long-term exposure of bacteria to subtherapeutic antibiotic doses is the cause of the increment in antibiotic resistance (Samreen et al., 2021). It may also originate in farms from contaminated water with subtherapeutic antibiotic concentrations. Antibiotic-resistant bacteria can transfer their resistance genes to DNA and plasmid of bacteria, during their multiplication, causing the number of resistant bacteria to increase (Xu et al., 2022). Moreover, antibiotic resistance in animals may also attributed to biofilm forming ability of bacteria. The proximity of cells within a biofilm can facilitate a plasmid exchange and hence enhance the spread of antimicrobial resistance (Watnick and Kotler, 2000).
     
    Molecular characterization
     
    The results of PCR amplification in the present study showed that all the E. coli isolates (n=37), were positive for ecp gene, producing an amplicon of 500 bp (Fig 4). This finding is in close agreement with the earlier reports where E. coli isolates were confirmed using ecp gene amplification (Avelino et al., 2010; Deshmukh et al., 2023; Munhoz et al., 2023). Munhoz et al., (2023) described that E. coli common pilus (ecp) fimbrae present in several diarrheagenic E. coli (DEC) associated pathotypes and plays an important role in host cell adherence and biofilm formation.

    Fig 4: Gel image of amplification of ecp gene of Escherichia coli isolates producing an amplicon size of 500 bp.


     
    Nucleotide sequencing
     
    The partial nucleotide sequencing of 16S rRNA gene of E. coli isolate (NE1 DSVCKV/DURG/CG/INDIA) revealed final consensus of 1008 bp sequence. The Basic Local Alignment Search Tool (BLAST) analysis discovered 99.00-99.40% homology with other nucleotide sequences of E. coli available in the reference database of GenBank. The accession number for the E. coli isolate of the present study was provided by NCBI GenBank as PQ334875.
           
    Phylogenetic analysis showed highest similarities of E. coli isolate (PQ334875) of this study with the E. coli isolates of Telangana, India (MK716402.1), Iraq (LC796919.1) and China (MK621216.1) (Fig 5).

    Fig 5: Cladogram of Escherichia coli (NE1 DSVCKV/DURG/CG/INDIA) isolate.


     
    Gross and histopathological examination
     
    During the necropsy procedure, the majority of the goats were found emaciated, dehydrated, with rough hair coat and pale conjunctiva. The perianal region and tail-base were soiled with diarrhoeic faeces. The gross lesions observed were enlargement, congestion and multiple necrotic foci on liver, enlarged and oedematous mesenteric lymph nodes and severe ballooning of the small intestine with hyperaemic serosa, as well as congestion and haemorrhage in mucosa (Fig 6). The microscopic examination revealed cellular swelling and vacuolation, congestion and infiltration of inflammatory cells with distended sinusoids in liver (Fig 7) and infiltration of mononuclear cells over a homogenous meshwork of fibrin threads in the medulla of mesenteric lymph nodes. Moreover, desquamation of mucosal epithelium was accompanied by infiltration of neutrophils, lymphocytes in the lamina propria, villous atrophy, stunting or complete loss of villi in jejunum (Fig 8). The above lesions associated with E. coli infection in goats corroborate with the findings of Kumar et al., (2015) and Gupta et al., (2023).
     

    Fig 6: Jejunum of Escherichia coli positive goat showing congestion.



    Fig 7: Photomicrograph of liver from Escherichia coli infected goat showing cellular swelling characterized by swollen hepatocytes with eosinophilic cytoplasm and vacuolation (H and E×1000).



    Fig 8: Photomicrograph of jejunum showing erosion of mucosal epithelium with infiltration of polymorphonuclear cells in lamina propria, haemorrhagic crypts and accumulation of sloughed off epithelial debris with inflammatory cells in lumen (H and E × 100).



    Ultrastructural studies
     
    Ultrastructural lesions were characterized by fusion, atrophy and loss of villi accompanied with enlarged crypt orifices in jejunal mucosa as well as covering of mucosa of small intestine with necrotic and fibrinous layer (Fig 9). The above mentioned changes corroborate with the findings of Neog et al., (2011), who studied the Rota virus and E. coli related pathology in diarrhoeic pigs.

    Fig 9: Ultrastructural changes in small intestine showing necrotic and fibrinous layer completely covering the mucosa (SEM ×500).

    CONCLUSION

    This study shows that E. coli is one of the major cause of enteric infection in goats, which may result in fatal infections. Moreover, goats may serve as carrier for multidrug-resistant bacteria, raising concerns about their potential introduction into environment and eventually to the food chain posing threat to other animals and human food safety; hence, the irrational use of antibiotics in animals should be discouraged.

    ACKNOWLEDGEMENT

    The study was supported by College of Veterinary Science and Animal Husbandry, DSVCKV, Anjora, Durg, (C.G.).
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

    CONFLICT OF INTEREST

    The authors declare no conflicts of interest regarding the publication of this article.

    REFERENCES


    1. Adefarakan, T.A., Oluduro, A.O., David, O.M., Ajayi, A.O., Ariyo, A.B. and Fashina, C.D. (2014). Prevalence of antibiotic resistance and molecular characterization of Escherichia coli from faeces of apparently healthy rams and goats in Ile-Ife, Southwest, Nigeria. Ife Journal of Science. 16(3): 447-460.

    2. Ahmed, A., Egwu, G.O., Garba, H.S. and Magaji, A.A. (2010). Prevalence of bacterial pathogens and serotyping of E. coli isolates from diarrhoeic lambs in Sokoto state, Nigeria. Sokoto Journal of Veterinary Sciences. 8(1): 42-45.

    3. Al-Khalaifah, H., Rahman, M. H., Al-Surrayai, T., Al-Dhumair, A. and Al-Hasan, M. (2025). A one-health perspective of antimicrobial resistance (AMR): Human, animals and environmental health. Life. 15(10): 1598.  

    4. Avelino, F., Saldana, Z., Islam, S., Monteiro-Neto, V., Dall’Agnol, M., Eslava, C.A. and Giron, J.A. (2010). The majority of enteroaggregative Escherichia coli strains produce the E. coli common pilus when adhering to cultured epithelial cells. International Journal of Medical Microbiology. 300(7): 440-448.

    5. Azmat, M., Khan, J.A., Ijaz, M., Ashraf, K. and Naseer, O. (2024). Molecular epidemiology of enteric pathogens associated with diarrheal disease in goat kids. Pakistan Journal of Zoology. pp 1-8. 

    6. Begum, F., Islam, M.M., Sohidullah, M., Kabir, S.M.L., Islam, M. and Rahman, M.T. (2016). Molecular identification and antibiogram profiles of Escherichia coli isolated from apparently healthy and diarrheic goats. Bangladesh Journal of Veterinary Medicine. 14(2): 203-208.

    7. Chatzopoulos, D.C., Sarrou, S., Vasileiou, N.G.C., Ioannidi, K.S., Peteinaki, E., Valiakos, G., Tsokana, C.N., Papadopoulos, E., Spyrou, V., Mavrogianni, V.S. and Giannakopoulos, A. (2016). Dissemination of intestinal pathogens between lambs and puppies in sheep farms. Small Ruminant Research. 141: 5-10.

    8. CIRG. (2021). Annual Report 2021. Central Institute for Research on Goats, ICAR. pp 05.

    9. Clinical and Laboratory Standards Institute. (2020). Performance Standards for Antimicrobial Susceptibility Testing CLSI Supplement M100. 30th edn., Clinical and Laboratory Standards Institute, Wayne, PA, USA.

    10. Dadawala, A.I., Chauhan, H.C., Chandel, B.S., Ranaware, P., Patel, S.S, Singh, K., Rathod, P.H., Shah, N.M. and Kher, H.N. (2010). Assessment of Escherichia coli isolates for  in vitro biofilm production. Veterinary World. 3(8): 364-366.

    11. Deshmukh, O., Suryawanshi, R., Kurkure, N., Kaore, M., Badar, S., Shinde, O., Awandkar, S. and Gaikwad, N. (2023). Molecular characterization of multidrug-resistant avian pathogenic Escherichia coli isolated from poultry and poultry products. Indian Journal of Animal Sciences.  93(5): 431-436.

    12. Freeman, D.J., Falkiner, F.R. and Keane, C.T. (1989). New method for detecting slime production by coagulase negative staphylococci. Journal of Clinical Pathology. 42(8): 872- 874.

    13. Gridley, M.F. (1960). Manual of Histological and Special Staining Technique. McGraw-hill book company, New York, Toronto, London. pp 202.

    14. Gupta, R.K., Niyogi, D., Joshi, R.K., Yadav, V., Jaiswal, V., Shrivastava, D.P., Singh, S.V., Jaiswal, S., Singh, J. and Alam, K. (2024). Prevalence and phylogenetic analysis of E. coli in diarrhoeic goats in eastern plain zone of Uttar Pradesh. Indian Journal of Animal Research. 1-8. doi: 10.18805/IJAR.B-5179.

    15. Gupta, R.K., Niyogi, D., Joshi, R.K., Yadav, V., Singh, S., Singh, J.P., Jaiswal, S. and Mishra, A. (2023). Pathomorphological alterations in intestine of goats infected with E. coli and rotavirus in eastern plain zone of Uttar Pradesh. Journal of Experimental Zoology India. 1(2): 26.

    16. Hariharan, H., Mada, C., Poole, D. and Page, R. (2004). Antibiotic resistance among enterotoxigenic Escherichia coli from piglets and calves with diarrhea. Canadian Veterinary Journal. 45: 605-606.

    17. Haulisah, N.A., Hassan, L., Bejo, S.K., Jajere, S.M. and Ahmad, N.I. (2021). High levels of antibiotic resistance in isolates from diseased livestock. Frontiers in Veterinary Science. 8: 652351. 

    18. Kamal, M.R., Fakhruzzaman, M., Akter, M.R. and Haque, M.A. (2018). Isolation, identification and antibiogram profiles of enterovirulent Escherichia coli from diarrhoeic goat in some selected areas of Rangpur district of Bangladesh. Asian Journal of Medical and Biological Research. 4(1): 36-43. 

    19. Karthik, R., Ambica, R. and Nagarathnamma, T. (2018). Study of biofilm production and antimicrobial susceptibility pattern in clinical isolates of Proteus Species at a tertiary care hospital. International Journal of Current Microbiology and Applied Sciences. 7(1): 574-586. 

    20. Katongole, P., Nalubega, F., Florence, N.C., Asiimwe, B. and Andia, I. (2020). Biofilm formation, antimicrobial susceptibility and virulence genes of uropathogenic Escherichia coli isolated from clinical isolates in Uganda. BMC Infectious Diseases. 20: 453.

    21. Krumperman, P.H. (1983). Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Applied and Environmental Microbiology. 46(1): 165-170.

    22. Kumar, N., Kumar, M. and Kaur, A. (2022). Escherichia Coli antimicrobial resistance profile obtained from productive livestock. Journal of Research in Medical and Dental Science. 10(S1): 6-10.

    23. Kumar, S., Jakhar, K.K., Nehra, V. and Pal, M. (2015). Pathomorphological and microbiological studies in sheep with special emphasis on gastrointestinal tract disorders. Veterinary World. 8(8): 1015-1020.

    24. Magiorakos, A.P., Srinivasan, A., Carey, R.B., Carmeli, Y., Falagas, M.E., Giske, C.G., Harbarth, S., Hindler, J.F., Kahlmeter, G., Olsson-Liljequist, B.J. and Paterson, D.L. (2012). Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clinical Microbiology and Infection. 8(3): 268-281.

    25. Mishra, A.K., Singh, D.D., Kumar, N., Kumarsen, G., Paul, S. and Kumar, A. (2020). Role of bacterial and parasitic pathogens in occurrence of neonatal diarrhoea in goat-kids. Indian Journal of Animal Research. 10(3): 389-395. doi: 10.30954/ 2277-940X.03.2020.9.

    26. Mokhbatly, A.A., Elsheikh, N., Ghazy, E.W., Elgamal, A.M., Hegazy, Y.M. and Assar, D.H. (2022). Prevalence of Shiga toxin- producing Escherichia coli and Salmonellae and some associated hematologic and biochemical profile alterations in lambs. Veterinary Research Forum: An International Quarterly Journal. 13(2): 155-162.

    27. Munhoz, D.D., Richards, A.C., Santos, F.F., Mulvey, M.A. and Piazza, R.M.F. (2023). E. coli common pili promote the fitness and virulence of a hybrid aEPEC/ExPEC strain within diverse host environments. Gut Microbes. 15(1):  2190308. 

    28. Neog, B.K., Barman, N.N., Bora, D.P., Dey, S.C. and Chakraborty, A. (2011). Experimental infection of pigs with group A rotavirus and enterotoxigenic Escherichia coli in India: Gross, histopathological and immunopathological study.  Veterinaria Italiana. 47(2): 117-128.

    29. Njoroge, S., Muigai, A.W.T., Njiruh, P.N. and Kariuki, S. (2013). Molecular characterisation and antimicrobial resistance patterns of Escherichia coli isolates from goats slaughtered in parts of Kenya. East African Medical Journal. 90(3): 72-83.

    30. Prasad, D.K., Shukla, R. and Ahammad, S.Z. (2024). Antibiotic resistance in the Ganga River: Investigation of antibiotic- resistant bacteria and antibiotic resistance genes and public health risk assessment. Journal of Environmental Chemical Engineering. 12(6): 114931.

    31. Quinn, P.J., Markey, B.K., Carter, M.E., Donnelly, W.J., Leonard, F.C. and Maguire, D. (2002). Veterinary Microbiology and Microbial Disease. Blackwell Science Ltd. pp106-118. 

    32. Roncarati, G., Foschi, C., Ambretti, S. and Re, M.C. (2021). Rapid identification and detection of β-lactamase-producing enterobacteriaceae from positive blood cultures by MALDI-TOF/MS. Journal of Global Antimicrobial Resistance. 24: 270-274.

    33. Samreen, Ahmad, I., Malak, H.A. and Abulreesh, H.H. (2021). Environmental antimicrobial resistance and its drivers: A potential threat to public health. Journal of Global Antimicrobial Resistance. 27: 101-111.

    34. Sharma, S.K., Manat, N. and Joshi, M. (2020). Prevalence of colibacillosis in goat kids in Udaipur district of Rajasthan. Indian Journal of Veterinary Sciences and Biotechnology. 16(2): 98-100.

    35. Singh, B.R., Kumar, V., Sinha, D.K., Bhardwaj, M., Saraf, A. and Vadhana, P. (2017). Antimicrobial resistance profile of enteropathogens isolated from diarrhea patients: Herbal antimicrobials, a ray of hope. Annals of Pharmacology and Pharmaceutics. 2(13): 1068.

    36. Singh, D.D., Pawaiya, R.S., Gururaj, K., Gangwar, N.K., Mishra, A.K. andani, D., Singh, M.K., Bhushan, S. and Kumar, A. (2018). Molecular detection of Clostridium perfringens toxinotypes, enteropathogenic Escherichia coli, rotavirus and coronavirus in diarrheic fecal samples of neonatal goat kids. Veterinarski Arhiv. 88: 1-20.

    37. Tamura, K., Stecher, G. and Kumar, S. (2021). MEGA11: Molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution. 38(7): 3022-3027.

    38. Temikotan, T. and Daniels, A.O. (2022). Intestinal flora of selected Agricultural animals and their antibiotic resistance and plasmid profiles. IOSR Journal of Pharmacy and Biological Sciences. 17(6): 56-64.

    39. Tsegaye, D., Belay, B. and Haile, A. (2013). Prevalence of major goat diseases and mortality of goat in Daro-Labu district of West Hararghe, Eastern Ethiopia. Journal of Scientific and Innovative Research. 2(3): 665-672.

    40. Watnick, P. and Kotler, R. (2000). A biofilm, a city of microbes. Journal of Bacteriology. 182: 2675-79.

    41. Xu, C., Kong, L., Gao, H., Cheng, X. and Wang, X. (2022). A review of current bacterial resistance to antibiotics in food animals. Frontiers in Microbiology. 13(5): 822689.

    42. Zare, P., Ghorbani, C.H., Jaberi, S., Razzaghi, S., Mirzaei, M. and Mafuni, K. (2014). Occurrence and antimicrobial resistance of Salmonella spp. and Escherichia coli isolates in apparently healthy slaughtered cattle, sheep and goats in East Azarbaijan province. International Journal of Enteric Pathogens. 2(1): e15451.

    43. Zhao, A., Sun, J. and Liu, Y. (2023). Understanding bacterial biofilms: From definition to treatment strategies. Frontiers in Cellular and Infection Microbiology. 13: 1137947. 
    In this Article
      Contact us
      Follow us
      Published In
      Indian Journal of Animal Research

      Editorial Board

      View all (0)