Isolation and Molecular Detection of Antimicrobial Resistance Genes in Escherichia coli and Staphylococcus Species from Joint Ill Cases of Calves in Puducherry 

Joint ill is a septicemic polyarthritis condition, primarily affects young calves and neonates in the age group of one week to one month. It is considered to be one of the major cause of calf mortality and leads to significant economic loss to the livestock farming (Bagga et al., 2009). Severe inflammation of the affected joints as a result of infection from the umbilicus and its associated structures causes critical lameness in young farm animals (Abdullah et al., 2015). Prevalence of arthritis in cattle is about 8.34% in India (Rao et al., 2020). Localization of bacteria within the joints to cause an infectious arthritis. Bacteria enter the bloodstream from the gut and upper respiratory tract in calves born in poor sanitary conditions with delayed or inadequate colostrums intake (Lorenz et al., 2011).
       
Joint illness, also known as neonatal arthritis, is a result of unsanitary environmental conditions in the calving shed and improper umbilical cord asepsis (Radostits et al., 2007). The clinical signs include varying degrees of lameness, fever, inappetence, synovial effusion, lameness, hot and swollen joints with open wounds, limited range of joint motion, pain on manipulation and flexion of the affected joints and varying levels of hypersensitivity.
       
The most common cause of joint illness is bacteria, which includes Escherichia coli, Corynebacterium sp, Staphylococcus aureus and Pseudomonas aeruginosa. These bacteria commonly inhabits the environment, food and warm blooded animals lower gut, skin and urinary tract etc (Tamilarasu et al., 2020). For successful treatment of joint ill, early diagnosis is essential. The choice of antibiotic will depend on the causative bacterial pathogen (Robson, 2003).
       
Antimicrobial resistance occurs when microorganisms develop resistance to antimicrobial drugs such as antibiotics over time. Antibiotic resistant bacteria (superbugs) are of emerging problem now a days both in human and veterinary medicine (Chaodhary et al., 2017, Venkatvasan et al., 2020, Devanathan et al., 2024). The widespread use of antibiotics has resulted in the emergence and spread of resistance genes which leads to an imbalance in the system as well as in environment’s microbiota (Thakur et al., 2018, Indhuja et al., 2021). Inappropriate antibiotic usage and delayed treatment results in the formation of irreversible lesions in the joint tissue of calves.
       
Antibiotics commonly used for treating joint infections include penicillin and tetracycline. The mechanism of antimicrobial resistance (AMR) in Staphylococcus aureus is particularly notable, as it has become a prominent superbug resistant to β-lactam antibiotics, including methicillin, resulting in methicillin-resistant Staphylococcus aureus (MRSA) (Dookie et al., 2016). The resistance to methicillin is attributed to the presence of Penicillin-binding protein 2a (PBP 2a), which has a low affinity for β-lactam antibiotics and is encoded by the mecA gene (Peacock and Paterson, 2015).
       
Tetracycline resistance in various bacterial species arises from multiple factors, including the acquisition of mobile genetic elements, mutations in ribosomal binding sites and chromosomal mutations that enhance the expression of intrinsic resistance mechanisms. Increased antibiotic resistance in animals may have great impact on human health especially in an environment where animals and humans share the same ecosystem. (Pavelquesi et al., 2021).
       
Although there are various studies on detection of bacteria causing joint illness in calves (Naik et al., 2011, Goodarzi et al., 2015, Jalal et al., 2016), there is no reports on antibiotic resistant determinants among the bacterial pathogens causing joint ill condition of calves in India. Hence, the present study was aimed to isolate and identify the bacterial pathogens from a joint ill cases in calves at Union Territory of Puducherry (India) and to detect the presence of antibiotic resistant genes in them.

Source of samples
 
A total of 31 pus swabs were collected aseptically from joint ill cases presented to the Veterinary Clinical Complex (VCC), Rajiv Gandhi Institute of Veterinary Education and Research (RIVER), Puducherry, India, with the history of painful and swollen joints with pus accumulation and the samples were collected during the period of January 2023 to August 2023 (8 months). The samples were transported to the Department of Veterinary Microbiology, RIVER, Puducherry under cold chain for the bacterial isolation and further microbiological analysis.
 
Isolation and identification of the bacteria
 
The samples were inoculated onto Luria broth and incubated at 37°C for 16 to 18 hours. The inoculum were streaked onto Muller Hinton agar. Further isolation was done by sub culturing onto MacConkey’s agar, Eosin-methylene blue agar and Mannitol salt agar. The isolated colonies in the culture medium were subjected to Gram’s staining and conventional biochemical methods includes catalase, oxidase, IMViC (Indole, Methyl red, Voges-Proskauer and Citrate), urease and sugar fermentation tests for species-level identification as per Cowan and Steel (1974). The biochemically confirmed E. coli and S. aureus isolates were further subjected to molecular detection using polymerase chain reaction (PCR).
 
Molecular detection of E. coli and S. aureus
 
The template DNA was extracted from colonies by boiling and snap chilling method as described by Zhang et al., (2015). The PCR was carried out with the primers targeting alr genes specific for E. coli with the product size of 366 bp (Yokoigawa et al., 1999) and nuc gene specific for Staphylococcus aureus with the product size of 270 bp (Brakstad et al., 1992) as described in Table 1. For both the genes, PCR assay was optimized with 12.5 μl reaction mixture containing 2.5 μl of DNA template, 6.25 μl of 2 X master mix (Taq amplicon red Mix, Emerald), 1 μl each of forward and reverse primers (10 pmol/μl) and the rest of the volume is made by adding nuclease free water for 31 reactions. The cycling conditions were as follows: initial denaturation at 95°C for 6 min; 35 cycles of 95°C for 1 min, 55°C for 45 sec and 72°C for 45 sec and a final elongation step at 72°C for 5 min. The PCR amplification was carried in an automated thermal cycler (Eppendorf Master Cycle, Germany). The PCR products were analyzed by 1.5% agarose gel electrophoresis, visualized under UV transilluminator.


 
Molecular detection of antimicrobial resistance gene
 
All the PCR positive E. coli and S. aureus isolates were further screened for the presence of AMR gene by PCR using the primers targeting tet and mec A respectively (Table 1). For both genes, the PCR assay was optimized using a 12.5 μl reaction mixture, as described earlier for  PCR reaction mixture preparation for E. coli and S. aureus. The amplification process with the annealing temperature of 53°C for 1 min for tet (Olobatoke and Mulugeta, 2015) and 50°C for 1 min for mec A (Oliveira and de Lencastre, 2002) was followed. The PCR amplification was carried in an automated thermal cycler (Eppendorf Master Cycle, Germany). The PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized under UV transilluminator. The PCR products which yielded 700 bp and 162 bp was considered positive for tet and mec A gene of E. coli and S. aureus respectively.
 
Antibiotic sensitivity test (ABST)
 
All the isolates were subjected to antibiotic sensitivity tests using 8 antibiotic agents by the disc diffusion method (Bauer et al., 1966). The inoculum was prepared from a single bacterial colony picked up with sterile loop from at least 4-5 well isolated similar type colonies and inoculated into 5 ml sterile Luria broth and then the inoculated broth was incubated at 37°C until a slight visible turbidity appeared usually within 2-4 hours, so as to correspond with McFarland tubes. The incubated broth was swabbed and lawn cultured over the entire surface of the MH agar plate three times, with the plate rotated approximately 60° each time to ensure even distribution of the inoculum. A final sweep of the swab was made around the agar rim. The antimicrobial agents used were Enrofloxacin (EX, 5 μg), Tetracycline (TE, 30 μg) Cefotaxime (30 μg), Co- Trimoxazole (COT, 25 μg), Amoxyclav (AMX, 5 μg), Gentamicin (GEN, 10 μg), Pencillin-G (10 μg) and Ceftriaxone (CTR, 30 μg) were used in this study. The inoculated plates were incubated at 37°C for 24 hrs. The interpretation of zone of inhibition was read as per Clinical Laboratory Standard Institute guidelines (CLSI, 2022. The reference strain S. aureus MTCC 87 and E. coli ATCC 25922 maintained in the Department of Veterinary Microbiology, RIVER, was used as positive control for genus and species specific PCR assay.

Joint ill usually occurs less than 1 year of age in calves, as a result of inflammation due to infection of the tissue of the umbilicus after parturition in dirty environment (Blowey and Weaver, 2011). The tissue of umbilicus get infected by bacterial contamination soon after parturition in unhygienic areas. Among 31 samples, 17 lactose fermenting colonies in MacConkey’s agar and 14 mannitol fermenting colonies in mannitol salt agar (Fig 1 and 2) were obtained. The isolated organisms were identified as E. coli (54.84%) and S. aureus (45.16%) respectively by conventional biochemical tests. The results of morphological, cultural and biochemical tests were in agreement with the reports of Nuss (2012), Goodarzi et al., (2015), Waseem et al., (2016), Ruban et al., (2018) and Debbarma et al., (2020).




       
All the 17 (54.84%) isolates were further confirmed as E. coli using PCR assay by targeting alr gene with the amplicon size of 366 bp and 14 (45.16%) isolates were confirmed as S. aureus by amplifying the nuc gene with the product size of 270 bp. The study conducted by Waseem et al., (2016) reported 9/20 (45%) S. aureus isolates from synovial fluid samples from kids in Uttarpradesh. Lower incidence of E. coli (2, 5%) and S. aureus (12, 30%) isolates were detected from the synovial fluid of calf in Iran were reported by Goodarzi et al., (2015). Jalal et al., (2016) found the 2.47% prevalence rate of joint ill in calves at Bangladesh and Wudu et al., (2008) detected 6% prevalence rate in Ethiopia which were lower than the report in our study. It might be due to differences in the geographical area. Anderson and Rings (2008) reported that most umbilical infections are caused by Actinomyces pyogenes and Escherichia coli being the second most commonly isolated bacteria and is believed to be the most likely cause of systemic infection and septic polyarthritis.
       
According to this study, the occurrence of joint ill was higher in crossbred female calves (57.14%) than male calves (42.86%) which are supported by the research findings of Ramanathan (2007), Goodarzi et al., (2015), Jalal et al., (2016), Dogan et al., (2016) and Ibrahim (2019). Septic arthritis was frequently reported as neonatal polyarthritis in young calves (Blowey and Weaver (2011). An abnormal birth circumstance was also may be a reason for increased susceptibility to neonates to septic arthritis in calves (Firth, 1983). In our study, 24 animals were less than 1 year of age (77.42%) and 7 animals were more than 1 year of age (22.58%). Similar findings were also reported by Jackson (1999) in first 8 weeks of age. Rohde et al., (2000) observed higher incidence in calves of below 1 year age (70.49%) than adults (29.51%). Whereas Merkens et al., (1984) reported that the incidence was not age dependent. The failure of passive transfer of immunity lead to high risk of developing calf hood infections in neonatal calves (Wittum et al., 1985, Blowey and Weaver, 2011).
       
All physiological parameters were within the normal range, with the exception of pyrexia, which was present in all cases upon presentation. Following treatment, body temperature returned to normal levels. The elevated body temperature observed served as a clinical indicator of systemic involvement in the context of arthritis which is also reported by Mee et al., (2008).
       
Among the 17 E. coli isolates, 4/17 (23.53%) isolates were harboring tet gene and among 14 S. aureus isolates, 4 isolates (28.57%) found to harbour mecA gene by PCR (Fig 3 and 4). Even though only 4 isolates found to harbour tet gene responsible for that particular drug resistance, phenotypic disk diffusion method shows 50% of the E. coli isolates were resistant to tetracycline on the contrary to the study by Jalal et al., (2016), Paul et al., (2010) and Sunder et al., (2021) who reported that the E. coli isolates were sensitive to tetracycline and doxycycline. MRSA has emerged as a significant animal health problem in veterinary medicine and resistant is mediated by mecA gene that encodes the production of a modified penicillin-binding protein. In the present study also, a high prevalence (28.57%) of MRSA was noticed which was in accordance with Jalal et al., (2016) and Nuss (2012).




       
Antibiogram patterns obtained from joint ill cases in this study showed resistant to most commonly used antibiotics (Fig 5). All the isolates were highly sensitive with Enrofloxacin (100%) and Gentamicin (77%), but were resistant to Amoxyclav (70%) and tetracycline (50%). Enrofloxacin and Gentamicin was detected as the most effective antibiotic which was in agreement with the previous reports of Goodarzi et al., (2015).

The present study showed that E. coli and S. aureus is the frequently isolated bacteria from joint ill cases in calves in Puducherry region. Enrofloxacin and Gentamicin were found to be the most effective antibiotics and the isolates showed resistant to antibiotics such as Amoxyclav, Ceftriaxone and tetracycline. The incidence of tetracycline and methicillin-resistance genes might probably be the result of indiscriminate use of antibiotics in large animal practice in Puducherry region. Further studies on antibiotic resistance genes in E. coli and S. aureus may help to understand the prevalence of such AMR infection and to formulate control strategies against them.

The authors are thankful to the Dean, Rajiv Gandhi Institute of Veterinary Education and research, Puducherry, India for providing all the necessary funds and facilities to carry out this study.

The authors declare that they have no conflict of interest.