Indian Journal of Animal Research

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Full Research Article

Molecular Characterization and Antimicrobial Profiling of Escherichia coli isolates from Indian Camelus 

Mahender Miland Lakeshar1,*, Jyoti Chaudhary2, Sudesh Kumar2, Narasi Ram Gurjar3, Prateek Kumar4, Taruna Bhati2, B.N. Shringi2
1Department of Veterinary Microbiology, Sri Ganganagar Veterinary College, Tantia University, Sri Ganganagar-335 002, Rajasthan, India.
2Department of Veterinary Microbiology, Rajasthan University of Veterinary and Animal Sciences, Bikaner-334 001, Rajasthan, India.
3Department of Veterinary Biotechnology, Rajasthan University of Veterinary and Animal Sciences, Bikaner-334 001, Rajasthan, India.
4Department of Veterinary Livestock Production Management, Sri Ganganagar Veterinary College, Tantia University, Sri Ganganagar-335 002, Rajasthan, India.

ABSTRACT

Background: Escherichia coli is frequently associated with multiple antimicrobial resistances and a major cause of bacterial extraintestinal infections in livestock and humans. Escherichia coli resides in the lower digestive tract as harmless commensals but a subset of E. coli strains has acquired the ability due to acquisition of virulence and antibiotic genes, cause intestinal or extraintestinal diseases.

Methods: In this field-laboratory investigation during 2020-2021, samples were taken from different localities of Bikaner district and surrounding are of Rajasthan. A total no. of 70 fecal samples were collected and immediately transferred to the Department of Veterinary Microbiology. In the laboratory, the collected samples were further processed for isolation and identification of E. coli bacteria.

Result: Confirmation of E. coli was done using primary and biochemical test which are screened for hemolysin property, biofilm formation, antibiogram study and antibiotic resistance gene. All the isolates of E. coli used to show characteristic metallic sheen on EMB agar plate and excellent identification was done with VITEK 2 system. All the camel isolates shown partial hemolysis on sheep blood agarand 45.71% camel isolates were positive for biofilm formation. All these 70 E. coli isolates from camel were resistant to Penicillin (94.38%) which was followed by amoxicillin+sulbactam (85.71%), erythromycin (71.14%), cefixime+clavulanic acid (71.43%). Highest sensitivity to chloramphenicol (81.28%) followed by sulphadiazine (48.57%) and cotrimoxazole (48.28%). All these 70 isolates were screened for antibiotic resistance genes. On the basis of molecular screening of the antibiotic resistance genes, majority of the isolates carried BlaTEM gene in camel (56/70; 80%), followed by StrA (29/70; 41.40%), Sul-3 in (22/70; 31.42%), Sul-2 (18/70; 25.71%), aadA (28/70; 40%), tet(B) (22/70; 31.42%) isolates.

INTRODUCTION

The intensity of bacterial infection and antimicrobial resistance (AMR) across human and animal populations presents a considerable and growing threat to global health and economic development (Muloi et al., 2022). Escherichia coli are an important cause of disease worldwide and occur in most mammalian species, including humans and in animals. Camels are known to harbor multidrug-resistant gram-negative bacteria and could be involved in the transmission of various microorganisms to humans (Carvalho et al., 2020). In the arid zone camels having, diarrhea and other infectious diseases are considered to be the most causes of economic loss associated with poor growth, medication costs and animal death  (Bessalah et al., 2016).

Antibiotics are compounds produced by bacteria and fungi which are capable of killing, or inhibiting, competing for microbial species by mechanisms of the remarkable genetic plasticity of bacteria allows them to respond to environmental threats in wide array of action; this includes the presence of antibiotic molecules that can jeopardize their presence. All the bacteria shared the same ecological niche with antimicrobial-producing organisms have evolved ancient mechanisms to withstand the effect of the harmful antibiotic molecule. These bacteria use two major genetic strategies to adapt to the antibiotic “attack”, i) mutations in gene and  ii) acquisition of foreign DNA coding for resistance determinants (HGT) through horizontal gene transfer and resistance with in the multidrug-resistant phenotypes could also be acquired where they will be related to extra chromosomal elements acquired from other bacteria in the environment like different types of mobile DNA segments, like plasmids, transposons and integrons or adaptive like efflux pumps and other cell surface modifications created by the stress of low-level antibiotics, etc. (Munita et al., 2016).

The Camelus dromedarius (Indian camel) is an even-toed ungulate with one hump on its back and camels have been declared as the state animal of Rajasthan in 2014, as they supported 85% of the India’s camel population. Rajasthan had the highest camel population across India about 213 thousand in 2019 (20th Livestock Census 2019, Animal Husbandry Statistics).

A major problem in camel productivity is that the high mortality in camel calves in the first 3 months of life (Tibary et al., 2006). The causes of this high mortality are mainly poor management practice and infectious diseases (Kamber et al., 2001). The diseases and losses in camel population can have devastating effects on the economic success of camel production (Abbas and Omer, 2005). Diarrhea and other infectious diseases are considered to be the main causes of economic loss related to poor growth, medication costs and animal death (Mohammed et al., 2003).

MATERIALS AND METHODS

Collection of samples
 
A total of 70 fecal samples were collected from camels at different villages and also from the clinical complex of CVAS, Bikaner during 2021-22. The samples were collected using a Sterile Hi-Culture Collecting Device (Hi-Media) from rectal swabs. After that, swabs were immediately placed in cooled boxes and then transported to the Department of Veterinary Microbiology and Biotechnology, College of Veterinary and Animal Science, Bikaner (RAJUVAS), in ice-cooled containers for E. coli isolation.
 
Isolation and Identification of E. coli
 
Fecal samples were first enriched by inoculating them into nutrient broth and incubating at 37oC for 18-24 hours. Loopful inocula from the nutrient broth were plated on MacConkey agar and incubated at 37oC for 18-24 hours. Lactose-fermenting colonies, appearing as pink-colored, were selected and transferred to Eosin Methylene Blue agar (EMB) plates for further incubation at 37oC for 18-24 hours (Kumar et al., 2022). Colonies exhibiting a green metallic sheen were sub-cultured to obtain pure isolated colonies. These pure colonies were subjected to identification using biochemical tests through the VITEK-2 automated system.
 
Hemolytic properties
 
Escherichia coli isolates were propagated on blood agar base supplemented with 5% washed sheep erythrocytes. Blood agar plates were incubated at 37oC for 24 hours and colonies producing clear/partial zones of hemolysis were then recorded as hemolysin-positive. After 24 h incubation at 37oC, plates were examined for signs of β-hemolysis (clearing zones around colonies), α-hemolysis (a green-hued zone around colonies), or γ-hemolysis (no halo around colonies).
 
Slime production test activity
 
Biofilm physiology is characterized by increased tolerance to stress, biocides (including antibiotics) and host immunological defenses. Slime production in E. coli was determined by cultivation on modified Congo Red Agar plates (CRA) (Mariana et al., 2009). Cultured CRA plates were incubated at 37oC for 24 hours. The production of rough black colonies by slime-producing isolates was used to differentiate them from non-slime-producing E. coli isolates (Kagane et al., 2021).
 
Antibiogram of the E. coli isolates
 
Antibiotic susceptibility testing was done as per the disc diffusion method (Bauer et al., 1966) following the guidelines of Clinical Laboratory Standard Institute (CLSI) against 20 antibiotics of different classes.
 
Antibiotic resistance gene profiles of E. coli from fecal samples
 
PCR method was used for amplification of antibiotic resistance genes in E. coli using specific primer sequences. The primer pairs used for the PCR are given in Table 1.

Table 1: Details of the primers used for the antibiotic resistance genes of E. coli isolates (Dallenne et al., 2010).


 

RESULTS AND DISCUSSION

A total of 70 fecal samples were collected from camels in the Bikaner region for E. coli isolation. The samples were cultured on MacConkey agar and colonies from MacConkey agar were further streaked on Eosin Methylene Blue (EMB) agar for E. coli confirmation. All isolates exhibited pink colonies on MacConkey agar, with the characteristic green metallic sheen observed in the growth of cultures on EMB agar plates.
 
Biochemical identification of E. coli isolates by VITEK-2 system
 
Biochemical identification of E. coli isolates using the VITEK-2 system was conducted of 35 samples (one from every two) with pure cultures at a concentration equal to 0.5 McFarland standards. The VITEK-2 Compact system positively identified E. coli, showing excellent confidence levels with an overall probability range of 96-98% (Fig 1).

Fig 1: Biochemical test identification report of E. coli by vitek2 system.



These findings align with reports by Putra et al., (2020), which achieved 100% E. coli identification using the automatic VITEK-2 Compact method. Similar confirmation rates were observed by Voidarou et al., (2011) and Al-Marri et al., (2021). Automation of biochemical tests has significantly reduced identification time, from 5-10 hours to 3 hours for Gram-negative rods, improving reliability and efficiency with minimal manual sample preparation compared to manual miniaturized biochemical tests (Funke et al., 1998).

The VITEK-2 Compact system, known for its widespread use and automation in bacterial identification based on biochemical profiles, utilizes fluorescence and/or colorimetry. Compact plastic cards containing selective or differentiated media or reagents enable bacterial identification in a shorter time than conventional methods (Barry et al., 2003). Ueda et al., (2015), Fadlelmula et al., (2016); Putra et al., (2020) and El-Ghareeb et al., (2020) have collectively concluded that the VITEK-2 automated system is accepted, convenient and rapid for the correct identification of bacteria.

Moawad et al., (2018) applied the VITEK-2 system for phenotypical confirmation of extended-spectrum β-lactamase-producing isolates. Enterobacteriaceae were identified, with 87.5% being E. coli, 6.9% Enterobacter cloacae, 2.8% Klebsiella pneumoniae and 2.8% Citrobacter spp. Other Gram-negative and Gram-positive bacteria were also correctly identified by the VITEK-2 system.

Hara and Miller (2003) reported a correct identification rate of 93% for enteric strains, with 85.9% of gram-negative enteric strains identified at probability levels ranging from excellent to good.
 
Hemolysin property of E. coli isolates
 
In the present study 32 out of 70 camel E. coli isolates (45.71%) produces partially hemolysis on sheep blood agar (Fig 2). Red blood cell of the host organism is lysed due to the presence of hemolysin gene which in turn helps in the spread of the pathogen in the host blood (Bashar et al., 2011). The hemolytic activity of E. coli is related to the presence of hemolysin genes. Dadheech et al., (2016) and Osman et al., (2018) had 100% recovery of E. coli isolates producing β-hemolysis on blood agar.

Fig 2: Isolation of E. coli on blood agar plate.



The mechanism by which E. coli causes diarrhea does not rely on the hemolytic nature of E. coli isolates but is due to toxin produced by its strains. Roy et al., (2006) was found 45.16% E. coli isolates produced hemolysis on sheep blood agar. Similarly, Shittu et al., (2010) E. coli isolates produces (45%) both α and β-hemolysis. Hemolysin production was determined to differentiate between the virulent hemolytic isolates and the avirulent non-hemolytic isolates. According to Osman et al., (2018) 96.66% E. coli isolates producing α-hemolysin; 3.33% isolate produced β-hemolysin instead of α–hemolysin.

Hemolytic strains are more virulent than non-hemolytic strains (Vaish et al., 2016). α-Hemolysin, also known as cytotoxic necrotizing factor, is produced by invasive strains of E. coli, which sets the pace for the pathogenesis of renal disease and enhances virulence in a number of clinical infections (Herlax et al., 2010). In another cross-sectional study Singh et al., (2021), Koutsianos et al., (2021); Grakh et al. (2021) found none of the E. coli isolates as hemolytic while as Adam et al., (2022) found one isolate produce hemolysis on sheep blood agar. Al-Humam (2016) reported only (9.6%) camel E. coli isolates showed β-hemolysis on blood agar plates.
 
Slime production test of E. coli isolates
 
Biofilm formation was measured to determine the ability of isolates to colonize surfaces for environmental survival and persistence and a virulence factor. The ability to form biofilm as determined by slime production (assessed by Congo red uptake and an adherence assay in glass tubes) revealed a heterogeneity among the isolates, ranging from weak and moderate to strong biofilm formation. 32 out of 70 E. coli isolates of camel (45.71%) were positive as biofilm formation (Fig 3). Bacteria within biofilms can withstand host immune responses and are less susceptible to antimicrobials and disinfectants (Jenkins, 2018).

Fig 3: Biofilm producing E. coli on congo red plate.



Ahmad et al., (2009) reported a result of 76.92% in the growth of brick-red black-colored colonies, indicative of pathogenic E. coli. The Congo red binding ability serves as a phenotypic marker for distinguishing between E. coli strains associated with septicaemia (invasive) and those that are not. It is also an epidemiological marker useful for discriminating pathogenic strains from commensals.

In a study by Yadav et al., (2014), results indicated a Congo red binding ability of 92.86% in E. coli isolates. Similarly, Adam et al., (2022) found that all isolates, accounting for 100%, exhibited positive results in the Congo red (CR) binding test. The Congo red agar test (CRA test) is considered an essential parameter for monitoring virulence characteristics of E. coli in both human and animal communities. In line with these findings, Kagane et al., (2021) observed 100% positivity in the Congo red binding test among their E. coli isolates.
 
Antibiogram study for E. coli isolates
 
In the present investigation, all 70 E. coli isolates were subjected to antibiotic sensitivity test using 20 different antibiotics. The response of organisms was interpreted as sensitive, intermediate and resistant based on the manufacturer guidelines (Himedia). The antibiotics tested belonged to various groups i.e. β-lactam antibiotics, aminoglycosides, glycopeptides, phenicoles, quinolones, tetracyclines, sulphonamide, RNA synthesis inhibitor, polypeptides, macrolides and lincosamides. β-lactam antibiotics included penicillins, cephalosporins, monobactums and carbapenems.

In β-lactum group (cell wall sysnthesis inhibitors) all the isolates were resistant to Penicillin-G. In the present study, 94% isolates from were resistant to penicillin. E. coli isolates were more resistant to ampicillinsulbactam, amoxycillin-sulbactam 76.15% and 85.71%. E. coli isolates were least resistant with 51.42% and 54.14% resistivity to third and fourth generation cephalosporins respectively. 22.57% E. coli isolates were sensitive and 62.42%isolates showed higher resistance to gentamicin (Table 2).

Table 2: Antibiotic sensitivity for E. coli isolates from camel.



Nuesch-Inderbinen et al., (2020) reported resistance to tetracyclinefrom African camel was detected most frequently (11.7%), followed by ampicillin and streptomycin (both 10.5%) and sulfamethoxazole/trimethoprim (9.9%) and one isolatesshowed intermediate resistance to streptomycin, remaining were sensitive to amoxycillin/clavulanic acid, ciprofloxacin and kanamycin. Bessalah et al., (2016) observed that all E. coli isolates were sensitive to amikacin, chloramphenicol, ciprofloxacin, gentamicin and ceftiofur. The highest frequency of resistance was observed against tetracycline, ampicillin and streptomycin (52.8%, 37.1% and 21.4%, respectively). Resistance to sulfisoxazole and trimethoprim–sulfamethoxazole was noted in almost 20% and 18.5% of E. coli isolates, respectively. A lower percentage of resistance was identified against amoxicillin/clavulanic acid (2.8%), ceftriaxone (1.4%) and cefoxitin (2.8%).

Momtaz et al., (2013) reported higher resistance rates to gentamicin and streptomycin (62.42% and 67.32%, respectively) in E. coli isolates, which aligns with our findings. Adelaide et al., (2008) detected elevated resistance levels for tetracycline (75.9%) and cotrimoxazole (72.4%). Subedi et al., (2018) found that the maximum E. coli strains were resistant to ampicillin (98%), followed by co-trimoxazole (90%), with intermediate resistance to colistin (50%) and the highest sensitivity observed against gentamicin (84%).

The frequency of sensitivity of most susceptible antimicrobial agents observed chloramphenicol (71.14%), followed by enrofloxacin (60%), sulphadiazine (48.575%) and co-trimoxazole (48.28%) in camel E. coli isolates. Less susceptibleor intermediate antibiotics areoxytetracycline (37.49%), ciprofloxacin (31.42%) and gentamicin (22.57%). This finding is similar to Bessalah et al. (2016) who also reported chloramphenicol as most sensitive antimicrobial agent. This finding is also similar with those of previous reports on isolates associated with genital and mastitis infections of camels (Mshelia et al., 2014). Azad et al., (2017) reported 36% sensitivity to gentamicin and 100% to erythromycin. Bhave et al., (2019) revealed high degree of resistance to commonly used antimicrobials, namely tetracycline (95.89%), trimethoprim (89.04%), colistin (82.88%) and ciprofloxacin (54.11%). However, further studies would be required in order to correlate the use of antimicrobials with the fecal carriage of AMR in camels.
 
Profiling of antibiotic resistance associated genes in E. coli isolates
 
The present study was conducted to investigate detection of some genes responsible for imparting antibiotic resistance to the E. coli isolates obtained from the camel fecal samples. The outcome of this study resulted as presence of blaTEM, sul2, sul3, strA, aadA, tetA, tetB in E. coli isolates. The blaTEM gene imparts resistance against various β-lactam antibiotic like penicillin and ampicillin, Sul2 gene imparts resistance against sulfamethoxazole antibiotics, strA against streptomycin, tetA and tetB for tetracycline, aadA gene encodes for aminoglycosides adenyl transferase enzyme which imparts resistance to amino glycosides antibiotics such as streptomycin.

In the present study the blaTEM gene was detected in fifty six out of seventy (80%) camel isolates (Fig 4), for sul2 gene eighteen out of seventy (25.71%) E. coli isolates (Fig 5), Sul3 gene was obtained as twenty two out of seventy (31.42%) isolates, twenty nine out of seventy (42.85%) camel isolates for strA gene (Fig 6), twenty eight of seventy (40%) isolates carried the aadA gene (Fig 7) and twenty two out of seventy (31.42%) were found positive for tetB gene (Fig 8). tetA, tetC, tetD, tetE, gene was not found in any isolate (Table 3).

Fig 4: Amplification of blaTEM gene from camel isolates.



Fig 5: Amplification of sul2 gene from camel isolates.



Fig 6: Amplification of strAgene from camel isolates.



Fig 7: Amplification of aadA gene from camel isolates.



Fig 8: Amplification of tetBgene from camel isolates.



Table 3: Prevalence of antibiotic resistance genes in E. coli isolates.



Bhave et al. (2019) observed blaTEM (20%), blaCTX-M (40%) and blaOXA (6.66%) in E. coli isolates respectively. Abd El Tawab et al. (2016) detected blaTEM gene in the genomic DNA (100%) in all isolates and 56.25% in plasmid DNA. While blaSHV gene was detected in the genomic DNA of (37.50%) and in plasmid DNA (28.12%) isolates. Carvalho et al., (2020) and Nuesch-Inderbinen et al., (2020) isolated blaCTX-M-1 producing E. coli from camel. Saidani et al., (2019) reported blaTEM gene in 18% isolates and the occurrence of CTX-M-15- and CTX-M-1-producing Enterobacteriaceae in camel.

Resistance to sulfonamides was due to the horizontal spread of resistance genes, expressing drug-insensitive variants of the target enzymes dihydropteroate synthase and dihydrofolate reductase, for sulfonamide and trimethoprim, respectively.In present study, a low resistance was observed in camel isolates for sul2 and sul3 gene. Ngbede et al. (2021) detected Sul2 and Sul3 in 71.42% in camel E. coli isolates from Nigeria. Ben Sallem et al. (2012) isolated Escherichia coli with 100% prevalence of sul2 gene from healthy food-producing animals in Tunisia. Rawat et al. (2022) observed sulfonamide resistance gene sul3 (44%), sul2 (28%) among the poultry isolates from North India. 36.9%; sul1 (1 isolate), sul2 (4 isolates).

Lanz et al. (2003) highest tetracycline resistance phenotypes observed in the E. coli isolates were linked to the presence of the tetA gene (63.2%) and considered to be the gene commonly identified followed by tetB (34.5%) in the E. coli isolates. They are among the widest spread tet genes found in Enterobacteriaceae and their occurrence was within the range reported by other investigators.
 

CONCLUSION

The results of our study underscore the significance of multidrug-resistant E. coli in causing diarrhea and other diseases in camels, potentially impacting their health and growth rates. E. coli poses a global challenge concerning antimicrobial multidrug resistance, affecting both humans and animals. Our study revealed a high prevalence of antibiotic resistance among E. coli strains, both phenotypically and genotypically.

The identified virulence factors, hemolysin and biofilm adherence, further emphasize the pathogenic potential of E. coli. Our findings suggest that dromedary camels in Bikaner may serve as reservoirs for E. coli strains exhibiting resistance to antimicrobials commonly used in human and veterinary medicine. Additionally, our study indicates the potential presence of ESBL-producing E. coli in camels, which could pose a risk of transmission to humans through direct contact or the food chain.

Therefore, implementing improved management practices, coupled with rapid and accurate diagnostics, is crucial. Judicious use of selected drugs based on antimicrobial susceptibility testing is essential to mitigate the spread of antimicrobial resistance and minimize the potential transmission of resistant strains from camels to humans.

CONFLICT OF INTEREST

All authors declared that there is no conflict of interest.

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