Prevalence and antibiogram of Salmonella species isolated from poultry products in Ebonyi State, Nigeria

Objective: This study evaluated the occurrence and antimicrobial susceptibility profile of Salmonella species isolated from various poultry products including chicken meat, poultry eggs, poultry bird’s drinking water, and poultry feed. Materials and methods: A total of 79 samples comprising of chicken meat (n=20), egg shell (n=15), poultry egg contents (n=18), drinking water (n=14), and poultry feed (n=12) were bacteriologically and microscopically analyzed for the isolation of Salmonella species. Results: Overall, this study reported a high prevalence of Salmonella species (62%) from various poultry products especially in poultry (chicken) meat and poultry egg contents where the percentage occurrence of Salmonella species was 100% and 20.4% respectively. The antibiogram conducted on the Salmonella species isolated from the various poultry samples reveal that all the isolates were multi-drug resistant to more than 50% of the tested antibiotics especially to tetracycline, gentamicin, tobramycin, nitrofurantoin and imipenem. However, most of the Salmonella species were also found to be highly susceptible to ceftriaxone, cefotaxime, ertapenem and ceftazidime. It was also observed in this study that the highest level of resistance to the tested antibiotics was recorded in Salmonella species isolated from poultry meat samples. Conclusion: Salmonellosis due to the consumption of contaminated or infected poultry products could pose serious public health problem to the general public if allowed. Thus, poultry farms and other poultry product outlets should be operated under sanitized conditions that ward-off the incidence of foodborne pathogens such as Salmonella. The use of antibiotics as growth promoting agents and prophylaxis in the production of poultry birds in this region should be discouraged – since such practices allowed drug-resistant bacteria to emerge and spread in the community.


INTRODUCTION
The food chain especially those that are of poultry origin could be a serious public health concern if they serve as route for the emergence and dissemination of resistant bacteria including Salmonella species in the community. According to Marcus et al. (2007), poultry remains an important vehicle of Salmonella transmission to humans, and most bacterial infections due to Salmonella usually occur via the consumption of contaminated meat. Bacterial infections caused by Salmonella species is amongst the most frequent foodborne gastrointestinal infection or disease that occur in the community; and this foodborne pathogen (Salmonella) is easily transmitted via poultry products including poultry eggs, meat and water (Hennessy et al., 2004;Kimura et al., 2004). Salmonella species is notorious in invading the epithelial cells of the gastrointestinal tract (GIT) during its invasion of a host to cause foodborne illness; and other pathogenic strains of Salmonella also cause other bacterial-related infections such as typhoid and paratyphoid fever. When poultry products including eggs and meat and other agricultural products are poorly handled and treated, they could serve as route via which foodborne infection spread in the community (Aarestrup, 2005). Salmonella sp. is predominantly associated with poultry products and other products of animal origin, and they are mainly responsible for the zoonotic transmission of salmonellosis in the community (Crump et al., 2008). In some cultures, people eat raw or uncooked eggs. This practice could predispose the people to foodborne infections such as salmonellosis. In most developing countries, the processes involved in the production of poultry birds and their products for human consumption may be unhygienic, and this is a critical public health challenge because of the direct transfer of resistant human pathogens including Salmonella species through the food chain. The emergence and spread of antibiotic resistance in foodborne bacteria including Salmonella species is well noted; and these organisms portend serious public health challenges since they cause a plethora of communityacquired infections such as salmonellosis (Threlfall et al., 2000;Glynn et al., 2004).
Bacterial resistance to some commonly available antimicrobial agents continues to remain a significant public health concern worldwide; and this phenomenon limits the choice of antibiotic therapy in some bacterialrelated infections or diseases. Several studies have shown that the continued use of antibiotics in animal rearing and other agricultural activities including the rearing and production of poultry birds as a major driving force behind the development and dissemination of antimicrobial-resistant Salmonella in the community (Mathew et al., 2002;van den Bogaard et al, 2000;Rajashekara et al., 2000;Devasia et al., 2005). The undue use of antimicrobial agents as growth promoting agents and prophylactic agents in the production of food producing animals such as poultry birds and livestock is a major contributing factor to the widespread emergence and dissemination of drug resistant bacteria in the community (van den Bogaard et al., 2000;Threlfall et al., 2000). Thus, this study evaluated the incidence and antimicrobial susceptibility profile of Salmonella species isolated from some poultry products in Abakaliki metropolis, Nigeria.

MATERIALS AND METHODS
Collection and processing of samples: Ethical approval was sought for and obtained prior to the commencement of this research project. A total of 79 environmental samples including samples from poultry bird (chicken) meat, poultry egg content, poultry bird's drinking water, egg shell, and poultry feed were used for this study. The samples were aseptically collected into clean zip lock bags using a clean spatula and swab sticks; and each of the sample containers were labeled and transported to the microbiology laboratory unit of Ebonyi State University, Abakaliki for further microbiological analysis.
Bacteriological analysis: Sterile swab sticks was used to swab the surface of the collected samples, and these were inoculated in 5 mL nutrient broth (Oxoid, UK). The drinking water of the poultry birds (1 mL each) was inoculated in 5 mL nutrient broth. All the inoculated samples were incubated at 35 o C for 18-24 h. After incubation, all the tubes showing turbidity after incubation were bacteriologically cultured on xyloselysine deoxycholate (XLD) agar and Salmonella-Shigella agar (SSA) (Oxoid, UK) for the selective isolation of Salmonella species. The culture plates were incubated at 35 o C for 18-24 h (Cheesbrough, 2000;Khan et al., 2005). And each of the plates was macroscopically examined for the presence of colonies that resemble Salmonella species. Suspected bacterial colonies on the culture media plates were aseptically subcultured onto freshly prepared SSA and XLD agar plates for the isolation of pure cultures of Salmonella species. And the isolated organisms were purified on nutrient agar plates for further bacteriological studies.

Identification of bacteria:
Suspected bacterial isolates on the respective culture media plates were identified using standard microbiological identification techniques including motility test, indole, triple sugar iron test, H2S production test, sugar fermentation test, citrate utilization test, Voges-Proskauer test, and Methyl-red test (Cheesbrough, 2000). . Briefly, the test isolate (adjusted to 0.5 McFarland turbidity standards) were aseptically swabbed on the surface of Mueller-Hinton (MH) agar plates, and the antibiotic disks were aseptically inserted into the MH agar plates using sterile forceps. Inoculated plates were incubated at 35 o C for 18-24 h, and the inhibition zone diameter (IZD) were recorded and reported as per the CLSI standards based on the organism susceptibility and resistance profile.

RESULTS
A total of 79 poultry samples including chicken meat, poultry egg contents, poultry bird's drinking water, egg shell and poultry feed were bacteriologically examined in this study for the isolation of Salmonella species. Table 1 showed the percentage occurrence of Salmonella isolated from the test samples. It was observed in this study that a total of 49 (62%) isolates of Salmonella species were recovered from the various poultry samples analyzed. The least number of Salmonella species isolated was recovered from poultry feed. However, higher number of Salmonella species was isolated from chicken meat; and this was followed by poultry egg content, egg shell and drinking water used for poultry production. Table 2 showed the antimicrobial susceptibility profile of the Salmonella species isolated from poultry feed. The Salmonella species isolated from poultry feed samples showed varying levels of susceptibility and resistance to the tested antibiotics. The inhibition zone diameter of the Salmonella species to the tested antibiotics was in the range of 6-26 mm ( Table 2). The isolates were least susceptible to penicillin, tetracycline, amoxycillinclavulanic acid, nitrofurantoin and amoxycillin but they showed better susceptibility to the other tested antibiotics especially to cefotaxime, gentamicin, ceftazidime, imipenem and ertapenem. Table 3 showed the antimicro-bial susceptibility profile of the Salmonella species isolated from the drinking water of the poultry birds. The Salmonella species from the drinking water of poultry birds had inhibition zone diameter that ranged from 6-28 mm. Table 4 showed the inhibition zone diameter of the Salmonella species isolated from egg shell. The inhibition zone diameter of Salmonella species isolated from egg content is shown in Table 5. The Salmonella species isolated from the egg shell samples were very susceptible to the tested antibiotics especially to ceftriaxone, imipenem, ertapenem, ofloxacin, cefotaxime, gentamicin and ceftazidime. However, the Salmonella species isolated from the egg shell samples were least susceptible to penicillin, amoxycillin, nitrofurantoin, and tobramycin. Table 6 show the inhibition zone diameter of the Salmonella species isolated from poultry meat. The Salmonella species isolated from poultry meat were most susceptible to ceftriaxone, cefotaxime, ertapenem and ceftazidime. However, the isolates were highly resistant to tetracycline, nalidixic acid, penicillin, gentamicin, tobramycin, nitrofurantoin, imipenem, sulphameth-oxazoletrimethoprim, amoxycillin-clavulanic acid and amoxycillin.

DISCUSSION
Salmonella species are common bacterial pathogens implicated in most foodborne infections. The organism causes salmonellosis and it is also the leading cause of gastroenteritis in humans. Salmonellosis can be transmitted through the food chain especially those of poultry origin when proper hygiene and infection control practices are not imbibed. This study evaluated the prevalence and antimicrobial susceptibility profile of Salmonella species isolated from various poultry products including chicken meat, poultry eggs, poultry bird's drinking water, and poultry feed. A total of 49 isolates of Salmonella species were isolated from the various poultry product samples. Only 4 (8.2%) samples were found to be positive for Salmonella species isolated from 12 poultry feed samples. And this was the least isolation of Salmonella species from the test samples. All the chicken meat samples were positive for the isolation of Salmonella species. Out of the 18 samples of poultry egg content analyzed in this study, Salmonella species was isolated from only 10 (20.4%) samples of egg content. However, a total of 8 (16.3%) samples and 7 (14.3%) samples were positive for Salmonella species isolation from the egg shell samples and poultry drinking water respectively. The overall prevalence of Salmonella species (62%) in this study is high. The prevalence of Salmonella species in       , 2000). A recent report in Bangladesh reported a higher prevalence of Salmonella isolation from poultry products (Mahmud et al., 2015). Elsewhere in Imo State, southeast Nigeria, Salmonella species have been reported to be prevalent in poultry products as observable in this study (Okoli et al., 2006). Egg contents of poultry birds have been reported to be low in Salmonella incidence due to the protected barrier of the shell and the antibacterial components of the albumen as was reported by (Mahmud et al., 2015). And this usually account for the low isolation of Salmonella species from the egg content of poultry birds. But in this study, the isolation of Salmonella species from the egg content samples was high (20.4%) compared to previous reports which show that Salmonella isolation from egg shell content is usually low (Mahmud et al., 2015). Mahmud et al. (2015) reported 3% occurrence rate Table 6. Inhibition zone diameter of Salmonella species isolates from poultry meat. CN TE  CAZ  AML  F  AMC  P  CRO  TOB SXT  ETP  NA IPM  OFX  CTX  1  Salmonella sp.  10  06  19  06  10  10  06  12  06  06  20  06  15  06  15  2  Salmonella sp.  10  06  19  06  09  10  06  19  06  06  19  06  14  06  19  3  Salmonella sp.  14  06  21  06  14  08  06  23  07  06  20  14  15  19  23  4  Salmonella sp.  09  06  18  06  11  06  06  19  07  06  16  09  13  10  21  5  Salmonella sp.  14  06  22  06  09  09  06  24  06  06  21  06  15  11  of Salmonella species from 103 poultry egg samples analyzed in a similar study in Bangladesh. Studies have shown that the penetration of the egg shell by pathogenic bacteria such as Salmonella is attributed to fecal contamination of the egg shell (Schoeni et al., 1995;Detha and Datta, 2016). The antibiotic susceptibility test conducted on the Salmonella species isolated from the various poultry samples reveal that all the isolates were multiple resistant to more than 50% of the tested antibiotics. The Salmonella species showed reduced susceptibility to tetracycline, nalidixic acid, penicillin, gentamicin, tobramycin, nitrofurantoin, imipenem, sulphamethoxazole-trimethoprim, amoxy-cillin-clavulanic acid and amoxycillin. However, most of the Salmonella species were also found to be highly susceptible to ceftriaxone, cefotaxime, ertapenem and ceftazidime. It was also observed in this study that the highest level of resistance to the tested antibiotics was recorded in Salmonella species isolated from poultry meat samples; and the resistance of these isolates was high to tetracycline, amoxycillin, penicillin, tobramycin, sulphamethoxazoletrimethoprim, ofloxacin and nalidixic acid in which the test Salmonella species had lower inhibition zone diameter. The level of antimicrobial resistance recorded in the isolated Salmonella species is similar to the study of Okoli et al. (2006) who showed that Salmonella species from poultry origin is multiple resistant to some commonly available antibiotics. The resistance of the Salmonella species isolated in this study to some commonly available antibiotics also substantiates previous findings that . The consumption of animal-based foods especially those that are of poultry origin have usually been attributed to the incidence of some foodborne infections such as salmonellosis which is caused by Salmonella species in humans. Salmonellosis due to the consumption of contaminated or infected poultry products could pose serious public health problem to the general public if allowed.

CONCLUSION
This study reported a high prevalence of Salmonella species (62%) from various poultry products especially in poultry meat and poultry egg contents. And the Salmonella species were multiple resistant to some conventional antibiotics especially to gentamicin, ofloxacin, tetracycline, nitrofurantoin, amoxycillin-clavulanic acid, penicillin, sulphamethoxazole-trimethoprim and amoxycillin. We therefore recommend that those involved in the rearing and production of poultry birds and their products ensure that their premises and other materials used in their enterprise are maintained in a sanitized state that excludes feacal contamination of poultry products. The continuous monitoring of poultry farms and other outlets where poultry products are sold to the public by public health personnel is very vital to the containment of salmonellosis outbreak in this region.

CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.