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Antibiotic Resistance Pattern and Distribution of Resistance Genes in Salmonella Isolated from Chicken and Duck Eggs in Chhattisgarh, India

T. Ganjeer1, A. Patyal 1,*, S. Shakya1, C. Chandrakar1, S.K. Verma2, N.E. Gade3, C. Sannat4, V.K. Naik1, S. Parkar1
1Department of Veterinary Public Health and Epidemiology, College of Veterinary Science and Animal Husbandry, Dau Shri Vasudev Chandrakar Kamdhenu Vishwavidyalaya, Anjora, Durg-491 001, Chhattisgarh, India.
2Livestock Products Technology,  College of Veterinary Science and Animal Husbandry, Dau Shri Vasudev Chandrakar Kamdhenu Vishwavidyalaya, Durg-491 001, Chhattisgarh, India.
3Veterinary Physiology and Biochemistry, College of Veterinary Science and Animal Husbandry, Dau Shri Vasudev Chandrakar Kamdhenu Vishwavidyalaya, Durg-491 001, Chhattisgarh, India.
4Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Dau Shri Vasudev Chandrakar Kamdhenu Vishwavidyalaya, Durg-491 001, Chhattisgarh, India.

ABSTRACT

Background: Salmonella is recognized as the most prevalent bacterial cause of foodborne diseases worldwide and animal-sourced foods have been reported as a common source of Salmonella infections among humans.

Methods: The commercial chicken eggs, backyard chicken eggs, and duck eggs samples, 60 each, were processed for isolation and identification of Salmonella. All Salmonella isolates were further tested for resistance against six different antibiotics. The prevalence of virulence and antimicrobial resistance genes in the Salmonella isolates was determined by PCR.  

Result: A total of 28 Salmonella isolates were recovered with an overall prevalence of 15.6% and out of them, 11.1% and 4.4% were from eggshell and egg content, respectively. All the isolates were found sensitive to Gentamicin however maximum resistance was observed against Cefotaxime. PCR results revealed that 100% of the isolates were carrying the invA gene however stn gene was detected in 78.6% of isolates. Among presumptively identified β-lactam-resistant Salmonella isolates, 100% and 50% isolates harbored blaTEM and blaCTX-M genes, respectively whereas none of the isolates contained the blaSHV gene. All tetracycline-resistant isolates harbored the tetA gene whereas none of the isolates carried the tetB gene. 100% of fluoroquinolone-resistant isolates were carrying the gyrA gene however parC gene was present only in 60% of isolates. These results indicate that drug-resistant Salmonella spp. were prevalent in eggs sold in the study area which can pose a serious public health problem.

INTRODUCTION

Globally foodborne diseases caused 600 million foodborne diseases and 420,000 deaths in the year 2010 and the most common causes identified were bacteria such as Campylobacter spp., Salmonella enterica and Salmonella typhi (WHO, 2015; Omari et al., 2018). Foodborne salmonellosis with nontyphoidal salmonellae is among the most prevalent causes of gastrointestinal infections worldwide. It has been estimated to cause approximately 153 million cases of gastroenteritis and 57,000 deaths (Cardoso et al., 2021) and is considered as the largest burden of an enteric disease which leads to 4.07 million DALYs (Disability Adjusted Life Years) (Kirk et al., 2015).
       
In humans, salmonellosis is usually associated with the consumption of contaminated foods of animal origin especially poultry products and raw or undercooked eggs (Andino and Hanning, 2015; Karimiazar et al., 2019). Salmonella spp. is the most commonly reported bacteria associated with foodborne outbreaks caused by egg and egg products (Choi et al., 2015). In the European Union (EU), 45.6% of the salmonellosis outbreaks were reported to be associated with the consumption of eggs and egg products in the year 2018 (European Food Safety Authority (EFSA) and European Centre for Disease Prevention and Control (ECDC), 2019). Eggs may be contaminated with Salmonella spp. by two possible routes; vertical and horizontal transmission. In the vertical or transovarian route, the egg content is directly contaminated as a result of Salmonella infection of the reproductive organs, before the eggs are covered by the shell components. During horizontal transmission, the shell of eggs is contaminated with feces and environmental vectors and the bacterium penetrates through the eggshell (Zubair et al., 2017; Cardoso et al., 2021).
       
In recent times, Salmonella spp. has gained substantial attention because of its pathogenic potential and ability to harbor resistance. Salmonella spp. pathogenicity has been related to several virulence genes which help the pathogen in adhesion and invasion mechanisms inside the host. Most of these virulence genes are located on a virulence-associated plasmid (pSTV) and region of the bacterial chromosome known as chromosomal Salmonella Pathogenicity Islands (SPIs). Some genes such as invA are known to be involved in the adhesion and invasion of Salmonella into the host cell; whereas other stn genes are involved in the actual manifestation of pathogenic processes (Naik et al., 2015a). In addition, amplification of the invA gene of Salmonella has been reported as a suitable target for PCR amplification, with potential diagnostic applications and its demonstration in Salmonella isolates can be epidemiologically relevant (Malorny et al., 2003; Favier et al., 2013). Among all antibiotics, β-Lactams, cephalosporins and fluoroquinolones are the most commonly used antibiotics in the poultry industry (Fardsanei et al., 2017). Injudicious use of antibiotics in poultry has resulted in the emergence of antibiotic-resistant strains of Salmonella, which leads to increased healthcare costs and clinical treatment failure (Cui et al., 2016).
              
The Indian Government has taken various steps to promote eggs as a good source of protein and the consumption of eggs have been increased considerably (Sangeetha et al., 2019). Different states of India are also providing free eggs to school children under the mid-day meal scheme therefore it is imperative to assess the quality of eggs sold in the local markets. The present study was conducted to assess the prevalence of Salmonella in poultry eggs sold in local markets in Chhattisgarh, India and to investigate the presence of some selected antimicrobial resistance genes in the drug-resistant Salmonella isolates.

MATERIALS AND METHODS

Sample collection
 
A total of 180 fresh eggs (60 backyard chicken eggs, 60 commercial/industrial chicken eggs and 60 duck eggs) samples were collected from retail markets of the Durg, Kanker and Dhamtari districts of Chhattisgarh, India between August 2020 and July 2021. Egg samples were collected in sterile plastic bags or cardboard boxes and transported immediately to the department of Veterinary Public Health and Epidemiology, College of Veterinary Science and Animal Husbandry, DSVCKV, Durg, Chhattisgarh, India. Each egg sample was analyzed for the presence of Salmonella on the eggshell and in the egg’s internal contents.
 
Isolation and identification of Salmonella spp.
 
Isolation and identification of Salmonella spp. from egg samples was carried out according to the method described by Khan et al., (2021) with some modifications. For investigation of Salmonella contamination over the egg surface, the entire surface of the egg shell was swabbed with sterile cotton swabs soaked in buffered peptone water (BPW) (HiMedia, India). The swab was incubated in BPW at 37°C for 24 hr. For egg content contamination, eggs were sterilized by immersion in 70% alcohol for 2 min, cracked with a knife and the content was collected and homogenized. Thereafter, 25 ml of content was added to 225 ml of BPW and incubated at 37°C for 24 hr for pre enrichment. Then 1 ml of the culture was transferred to 9 ml of Tetrathionate broth (HiMedia, India) and incubated at 37°C for 24 hr for selective enrichment. The culture was streaked onto Bismuth Sulphite Agar (BSA) and Brilliant Green Agar (BGA) (HiMedia, India) and incubated at 37°C for 24-48 hr. All Salmonella isolates were subjected to various biochemical tests viz., triple sugar iron (TSI) agar, indole, methyl red (MR), Voges-Proskauer (VP), urease and citrate utilization.
       
In addition to conventional methods, Salmonella isolates were confirmed by the genus-specific polymerase chain reaction (PCR) method described by Rahn et al., (1992) for the detection of invA gene (Table 1). Template DNA of Salmonella isolates for PCR was prepared by the boiling and snap chill method (Khan et al., 2021). Salmonella genomic DNA (MBT 103, MolBioTM, HiMedia, India) and Escherichia coli isolate maintained in the departmental laboratory were used as positive and negative controls, respectively.
 
Antibiotic susceptibility testing (AST) and presumptive detection of ESBL producers
 
AST of all Salmonella isolates was conducted on Mueller-Hinton agar (MHA) (HiMedia, India) following the disc diffusion method as per the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2015). The antibiotics used were Ampicillin (10 μg), Cefotaxime (30 μg), Cephalexin (30 μg), Ciprofloxacin (5 μg), Gentamicin (10 μg) and Oxytetracycline (30 μg) (HiMedia, India). Complete inhibition zone diameter was measured and results of the AST were interpreted as resistant, intermediate and sensitive as per CLSI guidelines (CLSI, 2015). Salmonella isolate displayed resistance to more than two different classes of antimicrobials was considered as ‘Multiple Durg Resistant (MDR)’ (Weill et al., 2006). Furthermore, the Salmonella isolates found resistant to Cefotaxime disc and sensitive to Cefotaxime/Clavulanic acid (30/10 ìg) discs with more than 5 mm diameter of zone of inhibition were considered as presumptive extended spectrum β-lactamases (ESBL) producers (CLSI, 2015).
 
Molecular detection of virulence and antibiotic resistance genes
 
All Salmonella isolates were tested for the virulence associated stn gene following the protocol given by Murugkar et al., (2003) (Table 1). Salmonella isolates, which were identified as presumptive ESBL-producers and those that showed resistance to each category of antibiotics, were examined for the presence of resistance genes. Salmonella isolates phenotypically identified as ESBL-producers were tested for blaCTX-M, blaTEM and blaSHV genes and those displaying resistance to tetracycline and fluoroquinolones were tested for tetA, tetB and gyrA, parC genes, respectively (Table 1). PCR amplification was performed using Veriti® 96-Well Thermal Cycler (Applied Biosystems, Singapore). Amplified PCR products were analyzed through electrophoresis on ethidium bromide stained 1.5% (w/v) agarose gel and recorded using a Gel Documentation System (Gel DocTM XR, Biorad, USA).
 

Table 1: Primers* used for detection of virulence and antimicrobial resistance genes.

RESULTS AND DISCUSSION

Prevalence of Salmonella in eggs
 
Out of 180 egg samples tested, a total of 28 Salmonella isolates were recovered (Table 2). All the isolates were initially identified by biochemical tests and further confirmed by detecting genus-specific invA gene using PCR. The highest prevalence was observed in commercial chicken eggs (20%) followed by backyard chicken eggs (18.3%) and the least in duck eggs (8.33%). A similar prevalence rate of 20% in commercial chicken eggs was observed in a study from Tamil Nadu, India (Sangeetha et al., 2019). However, lower prevalence rates of 5.6%, 3.3% and 0% were reported from China (Li et al., 2020), Argentina (Favier et al., 2013) and Iran (Karimiazar et al., 2019), respectively. Data on the occurrence of Salmonella in backyard chicken eggs is very scarce. As compared to the present study (18.3%), lower prevalence rates of 10% and 1.66% were documented in backyard chicken eggs from West Bengal, India (Samanta et al., 2014) and Iran (Karimiazar et al., 2019), respectively. Moreover, the studies from Spain (Fenollar et al., 2019) and Egypt (Eid et al., 2015) have reported the absence of Salmonella in backyard eggs. In duck eggs, 8.33% prevalence was recorded in the present study, on the contrary, a lower prevalence rate of 1.4% was reported from England (Owen et al., 2016) and 0% prevalence rates were reported in studies from New York (Baker et al., 1985) and Malaysia (Nor Faiza et al., 2013). In the present study, prevalence of Salmonella in commercial chicken eggs was found comparatively higher than the backyard chicken eggs, it may be due to the contamination of eggs during their supply from poultry farms to wholesale and retail markets. Also poor hygiene and handling of eggs at the site of sale could be a source of contamination (Shahzad et al., 2012). The occurrence of Salmonella in backyard chicken eggs may be attributed to different factors viz. backyard chickens access to outdoors spaces, physical contact with other farm animals and birds and absence of biosecurity, vaccination, hygiene practices etc (Ferreira et al., 2020).
 

Table 2: Prevalence Salmonella in chicken and duck eggs in Chhattisgarh, India (N=180).

  
 
In this study among 28 Salmonella isolates, 11.1% and 4.4% were recovered from eggshell and egg content, respectively. However, higher prevalence rates of 34.1% and 12.7% from eggshell and egg content were reported from Pakistan (Shahzad et al., 2012). On the contrary, lower prevalence rates of 6.1% and 1.8% were also recorded in Coimbatore, South India (Suresh et al., 2006). A higher prevalence of Salmonella was observed on the egg surface in the present study which may be due to the fact that egg surface was contaminated with feces during lay in unhygienic conditions or also from infected poultry (Paul et al., 2017). Contamination of eggshell possess a high risk for the consumers because it may cross contaminate the egg contents and other foodstuffs or may directly infect the consumers (Martelli and Davies, 2012). Penetration of bacteria from the egg surface into the egg content has been already demonstrated (Gole et al., 2014). The presence of Salmonella in egg contents may be due to the ability of transovarial transmission of Salmonella from birds to eggs (Taddese et al., 2019).
 
Antibiogram profiles of Salmonella isolate
 
The results of the AST for all 28 Salmonella isolates are presented in Table 3 and Fig 1. All of the Salmonella isolates were susceptible to Gentamicin. However, a high prevalence of resistance against Cefotaxime (50%) and Ampicillin (39.3%) was observed. The degree of resistance among Salmonella isolates ranges from 3.57 to 50% was recorded against five antibiotics. Of all the isolates, 5 (17.9%) were identified as MDR. Furthermore, the Salmonella isolates also exhibited 12 different antibiotic resistance patterns (Table 3). Results further revealed that isolates recovered from egg surface showed the highest resistance against Cefotaxime (55%) and Ampicillin (40%) whereas from egg content 37.5% isolates showed resistance against Cefotaxim and Ampicillin. In the present study, the susceptibility of Salmonella isolates to Gentamicin is concurs with previous reports from Tamil Nadu, India (Sangeetha et al., 2019) and South Western Ethiopia (Taddese et al., 2019), wherein all isolates were found sensitive to Gentamicin. The high level of resistance of Salmonella isolates against Cefotaxim and Ampicillin observed in the current study is consistent with the findings from China (Wang et al., 2017) and Ethiopia (Taddese et al., 2019). Resistance to penicillins and cephalosporins by Salmonella isolates is attributable to the acquired ability of the strains to produce β-lactamase enzyme. The 25% of Salmonella isolates showed resistance against tetracycline in this study, which is highly associated with the acquisition and expression of efflux pumps that reduce toxic levels of the drug in the bacterial cells. In Salmonella, these efflux pumps are mainly encoded by the tet genes (Hur et al., 2012). Strong selective pressure due to exposure to frequently used antibiotics could be one of the main reasons behind the emergence of such antibiotic-resistant Salmonella strains (Das et al., 2021). Excessive and irrational use of antibiotics with improper dosages in poultry industries either as growth promoters or for prophylactic purposes may lead to the development of MDR strains involving genetic and biochemical mechanisms. Such resistant strains have prolonged which increases their survivability and can pass to humans through the consumption of contaminated eggs (Karimiazar et al., 2019).
 

Table 3: Antibiogram profiles of Salmonella isolates (N=28) obtained from egg samples.


 

Fig 1: Heat map of antimicrobial susceptibility profiles of Salmonella isolates to six antibiotics as indicated by a color bar (blue = Sensitive, green = Intermediate and red = Resistant).


 
Distribution of virulence and antibiotic resistance genes in Salmonella isolates
 
 In the present study, all 28 (100%) Salmonella isolates were harbored invA virulence gene. Our results are in agreement with the similar studies from Iran (Fardsanei et al., 2017), Chile (Retamal et al., 2015) and the United States (Han et al., 2013), where invA gene was detected in 100% Salmonella isolates. The invA gene sequences are unique and conserved in almost all strains of Salmonella (Naik et al., 2015b; Wajid et al., 2019). Results further revealed that 22 (78.6%) Salmonella isolates harbored stn virulence gene, which is in line with the finding from Iran (Fardsanei et al., 2017). These virulence genes appear to have influence the severity of Salmonella infections and manifest the pathogenic process in the host cell (Fardsanei et al., 2017).
              
Out of 28 Salmonella isolates, 4 (14.3%) recovered from commercial chicken eggs were phenotypically identified as presumptive ESBL producer. Similarly, 14.2% and 8% isolates were found phenotypically positive for ESBL production from South India (Pradeep et al., 2018) and Egypt  Abdel-Maskoud et al.(2015), respectively. Furthermore, among four phenotypically β-lactam-resistant Salmonella isolates, 4 (100%) and 2 (50%) isolates harbored blaTEM and blaCTX-M genes, respectively whereas none of the isolates contained blaSHV gene (Table 4). Our results are in agreement with the findings from China (Zhu et al., 2017) and Bangladesh (Parvin et al., 2020), where authors reported that among β-lactam-resistant Salmonella isolates blaTEM gene was most prevalent followed by blaCTX-M gene. Results further revealed that, all 7 (100%) tetracycline resistant Salmonella isolates contained the tetA gene and none of the isolates were found positive for tetB gene (Table 4), which is in line with the findings of some previous studies (Zamil et al., 2021; Das et al., 2021). In five fluoroquinolone resistant Salmonella isolates, gyrA and parC genes were detected in 100% and 60% isolates, respectively (Table 4), which is in agreement with the previous findings (Wajid et al., 2019). 
 

Table 4: Distribution of virulence and antimicrobial resistance genes among Salmonella isolates.

CONCLUSION

This study provides baseline data on the occurrence of MDR Salmonella spp. in poultry eggs in India. Findings showed that Salmonella spp. was prevalent in eggs from retail shops and backyard poultry farms with an overall prevalence of 15.6%. The majority of the isolates were found resistant to the routinely used antibiotics. Furthermore, this study also provided valuable information on the circulation of different virulence and antibiotic resistance genes in Salmonella spp. from eggs. The apparently healthy poultry can act as a reservoir and distributor for MDR Salmonella spp., threatens consumer’s health and can be a food safety concern for public health in the region. Therefore, the poultry sector should be provided with immediate attention by the government to maintain strict hygiene and judicious use of antibiotics. Adequate and proper cooking is recommended to kill all foodborne pathogens to reduce hazards to consumers. Along with the promotion of eggs as a complete food, consumer awareness programs/campaigns related to health risks associated with raw egg consumption may also be implemented by the national regulatory agencies.

ACKNOWLEDGEMENT

Authors are thankful to the Dean, College of Veterinary Science and Animal Husbandry, Dau Shri Vasudev Chandrakar Kamdhenu University, Anjora, Durg, Chhattisgarh (India) for providing all the necessary facilities to conduct this research.

CONFLICT OF INTEREST

None.

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