Prevalence , assessment , and antimicrobial resistance patterns of Salmonella from raw chicken eggs in Haramaya , Ethiopia

Introduction: The presence of antimicrobial-resistant Salmonella in poultry and poultry products, including eggs, is a global public health concern. This study aimed to estimate the levels and patterns of antimicrobial resistance of Salmonella from chicken eggs and assess consumers’ raw egg consumption and farmers’ handling practices. Methodology: A total of 300 egg samples were collected from Haramaya open market (n = 150) and Haramaya University poultry farm (n = 150) in Ethiopia. Questionnaires were administered to egg sellers and buyers. A sterile cotton swab was used to sample the surface of eggs. The shells were sterilized and the egg content sampled. Isolation was done using the conventional methods for the detection of Salmonella, following the standard guidelines from ISO 6579. Sensitivity to 12 selected antibiotics was tested following the procedure of the Clinical and Laboratory Standards Institute. Results: A level of 5.3% was observed among eggs shells from the open market and 0% among egg shells from the poultry farm, for an overall level of 2.7%. There was a significant difference (p = 0.004) between the prevalence of Salmonella spp. in sample site and sample type. Of the antimicrobials tested, Salmonella isolates were all resistant to erythromycin and clindamycin. Isolates were sensitive to ciprofloxacin (100%) and chloramphenicol (87.5%). All isolates were resistant to multiple antibiotics. One-third of the consumers were found to have eaten raw eggs for perceived medicinal values. Conclusion: To minimize the potential contamination of eggs by pathogens, the eggs should be properly handled, transported, and stored.


Introduction
Poultry meat, eggs, and egg products are among the most nutritious foods on earth and are an important part of the human diet.However, they are perishable, just like other meat, fish and other food items, and consumption of improperly handled poultry and egg products is closely associated with foodborne illnesses, such as salmonellosis [1].
The Food Standards Agency (FSA) [2] of the United Kingdom has drawn attention to the risk associated with eating raw and lightly cooked eggs and issued public health advice on the safe handling and use of eggs.It is estimated that, in the United States, Salmonella transmission through contaminated shelled eggs or egg products results in 700,000 cases of salmonellosis and costs $1.1 billion annually [3].One study in Ethiopia found that from a total of 400 chicken eggs examined for Salmonella, 46 (11.5%) were positive; 25 (6.3%) and 27 (6.8%) of isolates were found on egg shells and in egg contents, respectively [4].
Overuse of antimicrobials in food animals can generate genomic selective pressures to enable microbes to adapt and acquire resistance [5].Ultimately, increases in bacterial antimicrobial resistance pose a considerable threat to public health, especially for vulnerable populations including young children, the elderly, and immunocompromised individuals [6].Concentrated animal feeding operations (CAFOs) in agricultural practices have evolved to accommodate food consumption rates with increased agricultural output at the risk of introducing antimicrobial-resistant pathogens into the environment.Several studies have suggested that characteristics of agricultural environmental settings, including animal crowding, CAFO hygiene, temperature, ventilation control, and stress can influence antimicrobial resistance and pathogen risk [7].There are reports of high levels of resistance in Salmonella isolates from 1231 countries such as Taiwan [8], India [9], the Netherlands [10], France [11], Canada [12], and Ethiopia [13][14][15][16].
The presence of resistant organisms in poultry and poultry products for consumption is a safety concern to the population [17].In Ethiopia, the extent of Salmonella contamination of eggs sold at local markets and farms, and the antimicrobial profile of the Salmonella isolates has not been adequately studied; there is very limited information available, and none from eastern Hararghe.This study aimed to estimate the levels and determine antimicrobial resistance patterns of Salmonella isolated from chicken eggs collected from Haramaya University (HU) poultry farm and the nearby local market and to assess the consumption and handing (storage) practices of farmers and consumers of raw chicken eggs.

Study design, sample collection and transportation
Sampling of chicken eggs from HU poultry farm and local chicken eggs directly from the farmers in Haramaya district was undertaken from November 2012 to April 2013.The sample size required for this study was determined based on a study on the prevalence of Salmonella among chicken table eggs in Kombolcha, northern Ethiopia, by Minte et al. [4].The estimate was desired to be with 5% absolute precision and 95% confidence [18].Accordingly, a total of 300 eggs (150 eggs from HU poultry farm and 150 eggs from Haramaya district open market) were collected directly from the farmers.Ten eggs from the market (one egg from one egg seller) and 10 eggs from HU caged white leghorn birds were collected once per week using a simple random sampling technique.Eggs were collected individually in sterile plastic bags and transported on ice in an ice box for analysis in the microbiology laboratory of the College of Veterinary Medicine at HU within 2-4 hours of collection.

Sample processing
The sterile plastic bags containing sampled eggs were opened with scissors and the samples were processed immediately.A swab technique was used to sample the shell surface of the intact eggs.Sterile cotton swabs dipped into sterile buffered peptone water (BPW) were used to swab the entire surface area of the egg shell.The swabs were then incubated separately in a test tube that contained 10 mL BPW (Oxoid Ltd, Hampshire, UK; Lab M Ltd., Quest Park, UK).The same eggs from which the shell samples were collected were used for interior (egg content) sampling.The egg's surface was sterilized by immersing the egg in 70% alcohol for 2 minutes; the egg was then air dried in a sterile chamber for 10 minutes, and then cracked with a sterile scalpel blade.The egg contents were homogenized in stomacher bags containing 225 mL sterile BPW for 1 minute in a stomacher.The isolation was conducted using the conventional methods for the detection of Salmonella, following the standard guidelines from ISO 6579.Confirmation was done by using biochemical tests such as triple sugar iron (TSI) agar, urea agar, citrate utilization test, L-lysine decarboxylation medium, and indole test according to ISO 6579 [19] (Table 1-S).

Questionnaire survey
A questionnaire was used to collect information from 75 farmers and consumers, who were chosen randomly, to identify conditions of handling (storage) practice, transportation, and preparation and utilization patterns of chicken eggs in the study area.

Data management and analysis
Data management was carried out using Microsoft Office Excel 2007, which was used to generate descriptive statistics such as percentage and proportion.The Chi-square (χ 2 ) test was used to determine the difference in levels between study groups.A p value of less than 0.05 was considered statistically significant.All statistical analyses were performed using SPSS software package, version 15.0 (IBM, Armonk, USA).

Prevalence of Salmonella spp. in raw chicken egg shell and egg contents
Out of the 300 eggs examined for bacteriological status, only 8 samples yielded Salmonella.All of the isolates were from egg shell samples collected from Haramaya local market.The other 150 eggs collected from the HU poultry farm were negative for Salmonella spp.(Table 1).
The levels of Salmonella varied among the sampling sites and types.The level of Salmonella in the local market was significantly higher than the level of Salmonella in the poultry farm (p = 0.007).The level of Salmonella in egg shells from the open market was also significantly higher than the level of Salmonella in egg content from both the open market and poultry farm (p = 0.007).Similarly, the level of Salmonella in egg shells from the open market was significantly higher than the level of Salmonella in egg shells from the poultry farm (p = 0.007).The overall level of Salmonella in egg shells was significantly higher than the overall level of Salmonella in egg contents (Table 1).

Farmers' and consumers' chicken egg use and handling (storage) practices
The questionnaire administered to farmers and egg consumers indicated that 28% of the respondents had a preference for raw egg consumption, while 72% disliked eating raw eggs.The habit of washing eggs before consumption was also investigated, and the results showed that 90.7% did not have the habit of washing eggs, and the other 9.3% had this habit only when the eggs become extensively dirty.Egg-keeping practices of respondents showed that 57.3% used open containers such as baskets, cartons, and trays, while 42.7% of the respondents kept eggs together with different cereal crops and coffee (Table 2).

Discussion
In Ethiopia, Minte et al. [4] reported that the prevalence of Salmonella from egg shells and egg contents was 6.3% and 6.8%, respectively, with a total of 11.5%.The number of Salmonella isolates observed from market egg shells was 11 (5.5%), and from farm egg shells was 14 (7.0%).The overall level of Salmonella spp.and the finding in egg shells of the previous study was higher than that in the present study, and the result in the egg content was different from that in the current study in that no Salmonella was isolated 1233 from egg contents.However, the report [4] on market egg shells (5.5%) was in line with the prevalence found in the current study (5.3%).A study conducted by Wiriya et al. [21] reported a prevalence of 5.2% from egg shells, whereas none of the egg content samples were Salmonella positive from different poultry housing systems, which is in agreement with a study carried out in Bangladesh by Kohinur et al. [5], who found 4 isolates (0.4%) out of 1,000 poultry eggs and 3 isolates (0.27%) out of 1,100 domestic eggs.A survey of retail eggs in the United Kingdom found an overall prevalence of Salmonella contamination of 0.34% [22], and a survey of 5,000 samples in Northern Ireland and the Republic of Ireland found only 2 positive samples (0.04%) [23].
Overall Salmonella levels on eggs in this study (2.7%) (Table 1) were in agreement with the finding of Salmonella spp.recovered from table eggs in Denmark, with a prevalence of 2.6% [24].Salmonella on egg shells from the local market in this study (5.3%) was also in agreement with the work of Adesiyun et al. [25], who reported 5.4% Salmonella prevalence on egg shells in Trinidad.Suresh et al. [26] from India also reported that the prevalence of Salmonella on egg shells was 6.1%.However, in the United Kingdom, the prevalence of Salmonella on egg shells was 0.38% [27].Furthermore, 0.29% Salmonella egg shell prevalence was reported from the United Kingdom [2].This Salmonella egg shell prevalence was lower than that of the present finding and could be due to egg shell surface contamination with feces, dust, and hands of egg collectors.
In this study, the negative results of the egg contents for the presence of Salmonella were, in general, in agreement with the literature, but there was a high difference in the number of egg samples used.In the study reported by UK Food Standards Agency in 2003, none of the 4,753 pooled egg contents of retail samples were Salmonella positive.Similarly, in a study conducted by a research group in Belgium, none of the 490 table eggs from 49 different producers were Salmonella positive [28].In another study conducted in England and Wales, a total of 12,615 eggs were collected from catering premises from September 2002 to November 2004.Salmonella was detected in 5.5% of eggs imported from Spain, in 6.3% of eggs of unknown origin, and in 1.1% of eggs imported from the UK.Salmonella was not detected in eggs from other countries (France, Germany, Portugal, USA: 0%) [29,30].1234 Based on the results of the questionnaire, one-third of the consumers practiced eating raw eggs for medicinal values; this could have some negative effects on health.Around half of the respondents used open containers and kept eggs together with other cereals or coffee husks.This situation might have led to a warmer micro-environment conducive to multiplication of a small number of Salmonella isolates acquired from the environment or from handlers during egg collection.A study conducted in Ethiopia by Minte et al. [4] indicated that 6.2% of egg consumers preferred to consume raw eggs, while 93.8% did not.
In a study carried out by Ayalu et al. in eastern Ethiopia [14], the sensitivity of the Salmonella isolates was 0.0% to ampicillin and amoxicillin, 14.2% to tetracycline, 28.6% to chloramphenicol, and 92.8% to gentamicin.In a report by Sibhat et al. [16], the highest level of resistance (66.7%) was seen in S. Newport to streptomycin.Zewdu and Cornelius [31] assessed antimicrobial resistance of 98 isolates of Salmonella serovars recovered from food in Addis Ababa, Ethiopia, and revealed that 32 Salmonella isolates were resistant to one or more of the 24 antimicrobials tested.The most common resistance was to streptomycin (75%), ampicillin (59.4%), tetracycline (46.9%), and spectinomycin (40.6%), which is in agreement with the present study.Miko et al. [32] determined antimicrobial resistance of Salmonella serovars isolated from German foodstuff; they showed that the most prevalent resistance among the multidrug-resistant Salmonella serovars isolated from foods were to streptomycin (94%), sulfamethoxazole (92%), tetracycline (81%), ampicillin (73%), spectinomycin (72%), chloramphenicol (48%), and trimethoprim (27%).The result regarding nitrofurantoin with 50% resistance is in agreement with that of Tassios et al. [33] in Brazil, who reported 57.82% resistance among Salmonella isolates.These drugs are among the most commonly used drugs in Ethiopia both in humans and animals.
A study conducted by Kohinur et al. [5] reported that 100% strains isolated from poultry eggs were found to be sensitive to ciprofloxacin and chloramphenicol.On the other hand, different strains have shown resistance to ampicillin, tetracycline, and kanamycin.Chen et al. [34] analyzed 133 isolates of Salmonella serovars and found that 73 (82%) of these Salmonella serovars were resistant to at least one antimicrobial agent.Resistance was common to tetracycline (68%) and ampicillin (29%).The problem is probably associated with the widespread use of these antimicrobials in humans and animals for treatment of enteric infections.

Conclusions
The study showed that Salmonella contamination of eggs in Haramaya area is low, with an overall prevalence of 2.7%.However, people consume raw and cracked eggs in the area, indicating a lack of awareness of zoonosis.The presence of Salmonella contamination in local chicken eggs is of public health concern, as these are the most widely available and used egg types.Therefore, the public should be made aware of risks associated with consumption of raw chicken eggs and raw eggs cracked during storage and transportation.

Table 1 .
Prevalence of Salmonella from raw chicken eggs of local market and poultry farm.

Table 2 .
Handling and consumption practice of eggs among farmers and consumers from Haramaya.
a Carton, box, basket, tray; b Barley, sorghum, maize, wheat and together with coffee.

Table 4 .
Resistance patterns exhibited by Salmonella isolates against 12 antimicrobial agents.

Table 2 -
S. Inhibition zone diameter size interpretive standards for Enterobacteriaceae (for selected antimicrobial disks appropriate for Salmonella spp.).

Table 3 -
S. Summary of disk diffusion zone diameter of isolated Salmonella spp.for selected antimicrobials in millimeters.