The poultry industry has made a major contribution to the food sector of Pakistan, and its products are largely consumed throughout the country in order to meet important protein dietary requirements. However, there is a potential threat of bacterial infection to poultry that can result in a huge economic loss.1Salmonella is the most commonly reported cause of foodborne disease among bacterial infections.2 It is estimated that about 94 million cases of gastroenteritis due to Salmonella species occur annually worldwide, leading to 155,000 deaths every year.3 Among Salmonella species, Salmonella enteritidis is isolated predominantly from poultry and is the most frequent cause of human nontyphoidal salmonellosis.4 In recent years, S. enteritidis has been reported as a major causative agent of foodborne gastroenteritis in humans.5
The current emergence of drug resistance in S. enteritidis is a major challenge due to the nonjudicious use of antimicrobial agents in the food and livestock sector.6 Poultry, especially broiler chickens, can harbor antimicrobial-resistant strains and function as a vehicle for dissemination of these pathogens to humans.7 Standard culture and serological methods for the detection of S. enteritidis are employed as disease control measures; however, polymerase chain reaction (PCR) is a preferred diagnostic method due to its reliable sensitivity, specificity, and detection speed.8 The spvb gene, commonly involved in bacterial virulence, is routinely used for the detection of S. enteritidis.9 As far as literature mining is concerned, no data exist on the molecular detection and drug resistance pattern of S. enteritidis from the Kohat region of Pakistan.
The present study has documented for the first time molecular detection of S. enteritidis spvb gene and its associated drug resistance pattern against commonly used antibiotics. The finding of this study will be efficacious in better controlling antibiotic resistance among S. enteritidis isolates from broiler chicken samples.
2.1. Sample collection and processing
The present study was conducted at the Department of Microbiology, Kohat University of Science and Technology, Kohat, Pakistan, during the period from December 2014 to August 2015. A total of 150 different broiler chicken samples (30 samples each of heart, liver, kidney, breast tissue, and leg piece) were collected from different retail markets of three main areas of Kohat. Random samples were collected from individual chickens (1 sample from 1 chicken, which means 150 samples from 150 chickens). All the samples were processed separately and washed thoroughly with autoclaved water to avoid any cross contamination between two samples. The samples were collected in peptone water-filled sterile plastic bags and immediately transported on ice to the laboratory for inoculation on enriched medium.
2.2. Isolation and identification of Salmonella species
The culturing method to detect Salmonella species involved selective enrichment followed by plating on selective agar. One gram of poultry sample was added to 9 mL of tetrathionate broth and was incubated at 37°C for 24 hours. A loop full of broth culture from tetrathionate was streaked onto a plate of bismuth sulfite agar. Bismuth sulfite agar is the selective medium for the growth of Salmonella species. The plates were incubated at 37°C for 48 hours and checked for the growth of typical black Salmonella species colonies.
The presumptive colonies of Salmonella species were taken for further confirmation by biochemical testing, including oxidase, catalase, triple sugar iron slant reaction, motility, indole, urease, and citrate utilization tests, as described earlier.10
2.3. DNA extraction from bacterial culture
DNA was extracted by genomic DNA purification kit (Thermo Scientific, Waltham, MA, USA) as per the manufacturer’s protocol. Briefly, bacterial cells were resuspended in Tris-EDTA buffer. The sample was mixed with lysis solution and incubated at 65°C for 5 minutes. Subsequent to incubation, absolute chloroform was added and centrifuged at 12,880 g for 2–3 minutes. Following centrifugation, the upper aqueous phase was mixed with the precipitation solution and centrifuged at 10,000 rpm for 2–3 minutes. The pallet was dissolved in NaCl solution and processed for ethanol precipitation step. After incubation at –20°C for 10 minutes, the supernatant was centrifuged at 10,000 rpm for 5 minutes. The ethanol was removed and the DNA pallet was dissolved in TE buffer. The concentration and quality of DNA was checked using the Nano-drop equipment (Thermo Scientific).
2.4. Molecular detection of S. enteritidis
S. enteritidis, and S1 and S4 genes were detected using PCR (Gradient Thermal Cycler; Eppendorf, Hamburg, Germany). S. enteritidis was detected by the amplification of the spvb gene using specific primers.9 Briefly for PCR, 3 μL of DNA was added to 25 μL of the reaction mixture containing 4 μL prepared master mix (Deoxynucleotide Triphosphate (dNTPs), 10× PCR buffer, Taq polymerase, and MgCl2), and 1 μL of forward and reverse primer, while the remaining 16 μL was equalized by nuclease-free water. The prepared PCR tubes with the master mixture were placed in a gradient thermal cycler. Amplification was carried out with initial denaturation at 95°C for 5 minutes, followed by 34 cycles of denaturation (94°C for 1 minute), annealing (53°C for 1 minute), and extension (72°C for 1 minute). A final extension step was carried out at 72°C for 5 minutes. The amplified DNA product from S. enteritidis-specific PCR along with the positive control (S. enteritidis) and negative control (Escherichia coli) were analyzed with 1.3% agarose gels stained with ethidium bromide and visualized by UV transillumination.
2.5. Antimicrobial susceptibility testing of S. enteritidis
All the isolates that were identified as S. enteritidis on PCR were tested for antimicrobial susceptibility on Mueller–Hinton agar using Kirby–Bauer disk diffusion assay.11 The antibiotics tested were ampicillin (30 μg), augmentin (30 μg), ceftazidime (30 μg), cefotaxime (30 μg), ceftriaxone (30 μg), aztreonam (30 μg), tetracycline (30 μg), azithromycin (10 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), and levofloxacin (5 μg) (Oxoid, Basingstoke, UK). The results were interpreted as resistant, intermediate, and susceptible, as described by Clinical and Laboratory Standard Institute guidelines.12 Multidrug resistant (MDR) is defined as a microorganism resistant to at least one antibiotic in three or more antimicrobial categories, while extensively drug resistant (XDR) is defined as a microorganism resistant to at least one agent, but sensitive to two or equal categories. These MDR and XDR were reported as per criteria.13
A total of 150 broiler chicken samples were obtained from Kohat and processed for molecular detection, and the total number of Salmonella species identified by culture and biochemical techniques was 51 (34%). S. enteritidis was detected in 35 (23.33%) samples among the biochemically identified Salmonella species. The size of the amplified spvb gene was 250 bp (Fig. 1).
In specimen-wise distribution of S. enteritidis that included different parts (heart, kidney, liver, breast tissue, and leg piece) of poultry, a higher isolation rate of S. enteritidis was noticed in breast tissue (n=9, 30%), while the lowest detection rate was observed in heart samples (n=4; 13.3%; Table 1).
When antibiotic susceptibility was checked, most S. enteritidis isolates were resistant to ampicillin (n=29; 82.8%), tetracycline (n=28; 80%), and augmentin (n=27; 77.1%). S. enteritidis showed resistance to ciprofloxacin (42.8%) and levofloxacin (40%); however, it showed less resistance against third-generation cephalosporins (including ceftazidime, cefotaxime, and ceftriaxone; Table 2). MDR and XDR patterns were also reported among 35 S. enteritidis isolates, in which 25.7% (n=9) were non-MDR. Additionally, 54.8% (n=19) were MDR, while 20% (n=7) were the XDR isolates (Table 3).
Salmonellosis is the primary cause of foodborne diseases globally.14 A broad range of foodstuff has been associated with such diseases. However, food from animal sources, especially if poultry derived, has been implicated in periodic cases and outbreaks of human salmonellosis.15
In the present study, S. enteritidis was detected in poultry samples. Moreover, their antimicrobial susceptibility pattern was also reported. In a total of 150 broiler chicken samples, the prevalence of S. enteritidis was 23.3%, while an increased detection rate (30%) was observed in breast tissue. The results of our investigation are compatible with those of a study conducted in Faisalabad, Pakistan.4 Another study in Iran also reported 25% prevalence of S. enteritidis from broiler poultry farms.7
Resistance of Salmonella to antimicrobials is an emerging problem in developing and developed countries.16 In our study, S. enteritidis isolates were resistant to commonly used antibiotics, i.e., ampicillin (82.85%), tetracycline (80%), augmentin (77.14%), and chloramphenicol (54.2%), which is in line with the findings of Beyene et al,17 in which resistance to ampicillin, amoxicillin, and chloramphenicol were 75%, 75%, and 50%, respectively. However, our results are in contrast with the findings of the 2010 study of Akhtar et al,4 in which isolates of S. enteritidis were mostly sensitive to ampicillin, tetracycline, and chloramphenicol. The notably high rate of antimicrobial-resistant S. enteritidis strains in this study is probably due to the early introduction and subsequent widespread use of these antibiotics in human and veterinary medicine in our area.
The fluoroquinolone-class antibiotics ciprofloxacin and levofloxacin showed moderate resistance to S. enteritidis isolates (42% and 40%, respectively); however, a previous study reported that S. enteritidis isolates were sensitive to ciprofloxacin.18 Fluoroquinolone resistance among S. enteritidis isolates might designate the common use of these antibiotics.
In the current study, S. enteritidis was least resistant to third-generation cephalosporin. Lower rates of cephalosporin resistance in this study are consistent with the results of Abdel-Maksoud et al’s19 study in 2015, who reported a low prevalence of cephalosporin resistance among S. enteritidis isolates from poultry sources. Lower resistance of S. enteritidis to cephalosporin is valuable to the community as cephalosporin resistance is a noteworthy public health concern.
Overall, MDR was observed among 54.2% S. enteritidis isolates. Our findings are in line with the results of a study by Hur et al20 in 2011, in which 65.2% Salmonella isolates were multiple drug resistant. Another study from Brazil also reported 63.9% multidrug-resistant S. enteritidis isolates from chicken carcass samples.18 However, another study reported a high prevalence of MDR of 90.9% to Salmonella enterica serovars Indiana and enteritidis.21 In one recent study, 35.5% MDR Salmonella species isolates were reported.22 The increased MDR isolates can be due to the use of antimicrobial drugs in poultry food at a subtherapeutic level, which can promote antimicrobial-resistant strains.23,24
In the current study, elevated levels of S. enteritidis were detected in broiler chickens. Increased drug resistance was observed to commonly used antibiotics, which suggests an emerging problem and could negatively impact an effort to prevent and treat broiler-transmitted zoonotic S. enteritidis.
The authors wish to thank Relief International in Pakistan for providing a portion of funding for this study. The authors also thank the Department of Microbiology, Kohat University of Science and Technology, Kohat, Pakistan, for providing the facilities for carrying out this research work.
1. Lutticken D, Segers RP, Visser N. Veterinary vaccines for public health and prevention of viral and bacterial zoonotic diseases. Rev Sci Tech
2. Onyango MD, Ghebremedhin B, Waindi EN, Kakai R, Rabsch W, Tietze E, et al. Phenotypic and genotypic analysis of clinical isolates Salmonella
in western Kenya. J Infect Dev Ctries
3. Majowicz SE, Musto J, Scallan E, Angulo FJ, Kirk M, O’Brien SJ, et al. The global burden of non typhoidal Salmonella
gastroenteritis. Clin Infect Dis
4. Akhtar F, Hussain I, Khan A, Rahman SU. Prevalence and antibiogram studies of Salmonella enteritidis
isolated from human and poultry sources. Pakistan Vet J
5. Clayton DJ, Bowen AJ, Hulme AW, Buckley AM, Deacon VL. Analysis of the role of 13 major fimbrial subunits in colonization of the chicken intestines by Salmonella enteric
reveals a role for a novel locus. BMC Microbiol
6. Gyles CL. Antimicrobial resistance in selected bacteria from poultry. Anim Health Res Rev
7. Rahmani M, Peighambari SM, Svendsen CA, Cavaco LM, Agerso Y, Hendriksen RS. Molecular clonality and antimicrobial resistance in Salmonella enterica
from broilers in three Northern regions of Iran. BMC Vet Res
8. Vaneechoutte M, VanElare J. The possibilities and limitations of nucleic acid amplification technology in diagnostic microbiology. J Med Microbiol
9. Madadi MS, Hassanzadeh M, Nikbakht GHR, Bozorgmehrifard M, Shojaei H. A comparative study on the colonization of Salmonella enteritidis
hilAmutant and its parent strains in laying hens. Int J Vet Med
10. Barrow GI, Feltham RKA. 2003. Cowan and Steel's manual for the identification of medical bacteria, 3rd ed. Cambridge University Press, Cambridge.
11. Kirby WM, Yoshihara GM, Sundsted KS, Warren JH. Clinical usefulness of a single disc method for antibiotic sensitivity testing. Antibiot Annu
12. CLSI. Performance standards for antimicrobial susceptibility testing; twenty-fourth informational supplement. CLSI document M100–S24. Wayne, PA: CLSI; 2014.
13. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect
14. Eley RA., 1996. Infective bacterial food poisoning. In: Eley RA, editor., Microbial food poisoning. Chapman & Hall, London, UK, pp. 5-33.
15. Scaallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, et al. Foodborne illness acquired in the United States—major pathogen. Emerg Infect Dis
16. Schwarz S, White D., 2005. Phenicol resistance. In: White D, Alekshun M, McDemott P, editors., Frontiers in antimicrobial resistance. ASM Press, Washington, pp. 124-148.
17. Beyene G, Nair S, Asrat D, Mengistu Y, Engers H, Wain J. Multidrug resistant Salmonella
Concord is a major cause of salmonellosis in children in Ethiopia. J Infect Dev Ctries
18. Medeiros MAN, Oliveira DCN, Rodrigues DP, Freitas DRC. Prevalence and antimicrobial resistance of Salmonella
in chicken carcasses at retail in 15 Brazilian cities. Rev Panam Salud Publica
19. Abdel-Maksoud M, Abdel-Khalek R, El-Gendy A, Gamal RF, Abdelhady HM, House BL. Genetic characterisation of multidrug-resistant Salmonella enterica
serotypes isolated from poultry in Cairo, Egypt. Afr J Lab Med
20. Hur J, Kim JH, Park JH, Lee Y, Lee JH. Molecular and virulence characteristics of multi-drug resistant Salmonella enteritidis
strains isolated from poultry. Vet J
21. Lu Y, Zhao H, Sun J, Liu Y, Zhou X, Ross C, et al. Characterization of multidrug-resistant Salmonella enterica
from chickens in eastern China. PLoS One
22. Rizi KS, Peerayeh SN, Bakhshi B, Rahbar M. Prevalence of ESBLs and integrons in clinical isolates of Salmonella spp
. from four hospitals of Tehran. Int J Enteric Pathog
23. Molla W, Molla B, Alemayehu D, Muckle A, Cole L, Wilkie E. Occurrence and antimicrobial resistance of Salmonella
serovars in apparently healthy slaughtered sheep and goats of central Ethiopia. Trop Anim Health Prod
24. Zewdu E, Cornelius P. Antimicrobial resistance pattern of Salmonella
serotypes isolated from food items and workers in Addis Ababa, Ethiopia. Trop Anim Health Prod