Prevalence and Antimicrobial Resistance of Salmonella in Poultry Products in Central Ethiopia

Salmonellosis is a bacterial infection caused by salmonella, a member of the Enterobacteriaceae family. From December 2021 to May 2021, a cross-sectional study was carried out to isolate Salmonella from poultry farms in the towns of Bishoftu and Adama and to determine the antimicrobial susceptibility frequency of the isolates. A total of 384 samples were tested for the presence of Salmonella, including 259 feces, 56 eggs, and 69 types of meat, using the ISO, 2002 standard procedures. The raw data were organized, coded, and entered into an Excel spreadsheet before being analyzed with STATA via descriptive analysis with chi-square. From 384 collected samples, 62 (16.15%) isolates were obtained, with 9.9%, 3.65, and 2.6% found in feces, eggs, and meat, respectively. Statistically, there was a significant difference between breeds (p value = 0.036). Bovines had the highest prevalence (32.83%), while Saso had the lowest (30.81%). The variation within each sample type, housing condition, and age group was not statistically significant (p value >0.05). Antimicrobial resistance was found in 29 (96.77%) of the isolates. Ampicillin and sulphamethoxazole-trimethoprim were effective against all isolates. Salmonella was isolated from various locations, sample types, ages, and breeds, indicating a wider distribution. Salmonellosis detection isolates suggested that it could be an emerging poultry and public health issue. As a result, future research should concentrate on isolating and identifying salmonella from poultry in backyard systems and comparing it to that of an intensive farm, as well as molecular characterization for serotyping and genetic studies, as well as genes responsible for salmonella pathogenicity and drug resistance.


Introduction
Foodborne pathogens are a major source of economic and health problems around the world [1]. Salmonella is one of the leading causes of foodborne illness worldwide, with 3.7billion-dollar annual economic loss [2]. It is the leading cause of acute gastroenteritis in several countries and continues to be a major public health concern globally, particularly in developing countries [3]. Although diseases caused by this pathogen have been linked to a wide range of food sources, poultry in particular has been identifed as the single most common source of human salmonellosis [4].
Animal-derived foods, particularly poultry and poultry products, are frequently implicated in sporadic cases and outbreaks of human salmonellosis [5]. Poultry and poultry products are a common source of foodborne illness and consistently rank among the top animal sources of Salmonella that enter the human food supply. He also mentioned that humans are exposed to this problem when they consume raw or undercooked food, particularly poultry and egg products [6].
Salmonella is a type of bacteria that is heterogeneous, short bacilli, 0.7-1.5 × 2.5 m, Gram-negative, aerobic or facultative anaerobic, oxidase negative, catalase positive, indole and Voges Proskauer (VP) negative, methyl red and Simmons citrate positive, H 2 S producing, and urea negative. Except for Salmonella pullorum and Salmonella gallinarum, which are nonmotile, they ferment sugars with gas production, are nonsporogenic, and typically have peritrichous fagella [7]. Around 7.0 is the ideal pH for multiplication; pH values of 9.0 or lower are bactericidal. Te ideal temperature range is between 35 and 37°C, with a minimum of 5°C and a maximum of 47°C. Salmonella cannot survive concentrations of salt higher than 9%. It shares a close relationship with the genus Escherichia and can be found on every continent in both warm-blooded and cold-blooded animals as well as in nonliving environments [8].
Except for Salmonella serotype Typhi, which only produces acid, the majority of salmonellae catabolize a variety of carbohydrates including glucose, mannitol, and maltose into acid and gas. In contrast, lactose, sucrose, and saline are not fermented by the majority of salmonellae [9]. Salmonella falls under the domain of bacteria. Proteobacteria as a phylum, Gammaproteobacteria as a class, enterobacteriales as an order, Enterobacteriaceae as a family, and Salmonella as a genus [10].
Currently, the Centers for Disease Control and Prevention (CDC) employs the Salmonella nomenclatural system recommended by the World Health Organization (WHO) Collaborating Center [11]. Based on diferences in their 16S rRNA sequence analysis, the genus Salmonella is divided into two species, Salmonella enterica and Salmonella bongori. Salmonella enterica is classifed into six subspecies such as arizonae, diarizonae, enterica, houtenae, indica, and salamae [12].
Currently, about 2541 Salmonella serotypes have been identifed [12]. Tese serotypes can be distinguished by the type of somatic (O) and fagellar (H) antigens present. In addition, Salmonella typhi and a few other Salmonella serovars, including Salmonella Dublin, have a capsular polysaccharide virulence antigen [13].
Many animal species, particularly chickens, pigeons, and reptiles, are possible reservoirs for this bacterium [14]. Salmonella is typically transmitted among poultry through a fecal-oral route, most often through the consumption of contaminated food or water. Human transmission can occur via a variety of routes. Consumption of contaminated food products (milk, eggs, and meats), direct contact with animals and their environments, and cross-contamination through direct contact of foods with contaminated surfaces such as stainless steel, hanging material, knives, and buckets where milk is collected are all important mechanisms for pathogens to contaminate food products [15]. Salmonella infections range from gastrointestinal infections characterized by infammation of intestinal epithelia, diarrhea, and vomiting to typhoid fever, a potentially fatal infection whose severity is determined by the host immune status and the pathogenicity of the bacterium [16]. Young and immunocompromised patients are more vulnerable to dangerous complications, which are typically treated with fuoroquinolones and extended-spectrum cephalosporins, which are commonly used in veterinary medicine [17].
Ethiopia has approximately 56.87 million chickens, the majority (95%) of which are kept in low-input low-output village chicken production systems [18]. Salmonella was found in large numbers and was widely distributed in Ethiopia, according to research. Te number of Salmonella outbreaks in humans in the country has increased significantly in recent years. Much more is now known about the scope of foodborne illness and how severe it can be, both in terms of acute illness and long-term consequences. Various percentages of Salmonella isolates were found in Ethiopian towns, according to studies. Furthermore, a high proportion of S. typhi isolates are resistant to antimicrobial agents [19].
Salmonella serotypes isolated from animal foods have multidrug resistance frequency, according to studies from various countries [20]. Meat and poultry products have also been implicated in the spread of antimicrobial-resistant zoonotic bacterial pathogens [21]. Several studies on the prevalence and antimicrobial resistance of Salmonella in processed poultry, poultry products, and poultry processing plants have been conducted in other countries, including Agada et al. [22], Orji et al. [23] from Nigeria, Kagambega et al. [24] from Burkina Faso, Al-Abadi and Al-Mayah [25] from Iraq, and Khan et al. [26] from Pakistan.
Regardless, there are more published and unpublished papers in Ethiopia on Salmonella from poultry, dairy cattle, abattoirs of large and small ruminants, and other feed items. Abunna et al. [27], Asefa et al. [28], and unpublished: Tadesse [29]. However, little is known about Salmonella isolation from poultry products in Ethiopia. As a result, increased and long-term surveillance of the most risk factors is required, as is the isolation of Salmonella from poultry and poultry products. Tus, the objective of the current study was to detect salmonella from poultry products like meat, feces, and eggs, and to analyze the efectiveness of antimicrobial resistance for selected drugs in the study area.

Study Area.
From December 2021 to May 2021, the cross-sectional study was carried out in Bishoftu and Adama. 45 kilometers southeast of Addis Ababa, the capital city of Ethiopia. Bishoftu had a long rainy season from June to October and a short rainy season from March to May, the region has a bimodal rainfall pattern and is located at an altitude of 1850 meters above sea level. Te region experiences 875 mm of annual rainfall and average high and low temperatures of 26 degrees Celsius and 14 degrees Celsius, respectively. Adama is 99 kilometers to the southeast of Addis Ababa. It is situated at an elevation of 1,712 meters above sea level at 8.54°N 39.27°E. It has a yearly average minimum and maximum temperatures of 18 and 32 degrees Celsius, respectively, and receives 600 to 1,150 mm of rainfall on average [30].

Study Animal.
Te study animals were poultry from the intensive poultry production systems of the private and government farms in Bishoftu and Adama. Each poultry farm received letters requesting their support before sample collection. Unafected by age, sex, or color, the randomly selected chicken was evaluated. Owners and workers on the farm were provided information regarding them.

Study Design.
A cross-sectional study was carried out to isolate Salmonella from poultry farms between December 2021 and May 2021. Te sampling was done using a straightforward random sampling method, and each poultry farm was given a diferent set of sampling days.

Sample Size Determination and Sampling Techniques.
Te straightforward random sampling technique was used, giving chickens from intensive farms in Bishoftu and Adama an equal chance of being included in the sample. Te necessary sample size for this study was meticulously calculated [31]. Since no research has been done in this area before, a sample size of 50% prevalence has been chosen. A 95% confdence interval and a desired absolute precision of 5% were also factors in the sample size calculation.
Tus, the total sample was 384.  [33]. Te testing of suspect food items and animal feces samples discovered through foodborne disease surveillance programs is intended to be guided by this protocol. Tis protocol is only meant to be applied to animal waste and food samples. According to this theory, Salmonella must be detected in the following four steps: pre-enrichment in nonselective liquid media, enrichment in selective liquid media, and selective plating on selective solid agar. Suspect isolates were found and verifed through screening against fve biochemical tests [7].

Antimicrobial Resistance. Antimicrobial Sensitive Test
(Disk Difusion or Disk Plate Technique). It is the most commonly used method to determine qualitative antibiotic sensitivity tests "in vitro." It is almost a qualitative way of determining antibiotic sensitivity tests based on difusion. Te most common test medium is called Mueller-Hinton Agar (MHA). Small discs containing diferent antibiotics, or impregnated paper disks, are dropped in diferent zones of the culture on Muller Hinton Agar medium. Since the agar plate is a nutrient-rich environment in which bacteria can grow, the antibiotic will difuse in the area surrounding each tablet, and a disc of bacterial lysis will become visible. Te area around the disc where there is no growth of bacteria is called the zone of inhibition. Te area around the disc where there is no growth of bacteria is called the zone of inhibition [34].

Data Management and Analysis.
Te study's raw data were coded, organized, and entered into an Excel spreadsheet using Microsoft ® Ofce Excel 2010. Ten, using STATA, a descriptive analysis using chi-square statistics was performed on the data. Te analyses' fndings will be broken down into proportional descriptions. Te percentage was calculated based on the number of samples that tested positive for Salmonella out of the total samples tested as well as the ratio of antimicrobial-resistant isolates to positive samples.  Table 1).

Frequency of Antimicrobial Resistance Distribution.
Te 30 positive Salmonella isolates were selected and screened for antimicrobial susceptibility tests against fve antimicrobials such as sulphamethoxazole-trimethoprim, gentamycin, streptomycin, ampicillin, and ciprofoxacin. 29 (96.77%) were resistant to one or more of the antimicrobials. Only one isolate from fecal samples was sensitive to the entire fve selected antimicrobial drugs. All isolates were susceptible to ampicillin and sulphamethoxazoletrimethoprim. Although all isolates were supposedly susceptible to ciprofoxacin, 4 (13.3%) isolates were intermediately susceptible. In addition, all isolates were supposedly susceptible to gentamycin but 5 (16.7%) isolates were intermediately susceptible. 26

Discussion
Te current study evaluated the antibiogram frequency and isolation and identifcation of salmonella from poultry products. Salmonella was present in the sample collected overall at a rate of 16.14%. Te study found that some of the prevalence of Salmonella isolation was consistent with that reported in Ethiopia and other nations. At 15.5% [28] from chicken table eggs by bacteriological methods in Ethiopia, 15.12% [27] from poultry cloacal swabs, fresh feces, litter samples, chicken feed samples, poultry drinking water, and chicken handlers by bacteriological methods in Ethiopia, and 12.5% [23] from chicken handlers in Nigeria.
Higher prevalence than the current fndings has also been reported in Ethiopia and other countries, including 41.9% [35] from fecal samples by the bacteriological method in Ethiopia and 35.7% [36] and 55% [24] in Burkina Faso. Tis discrepancy may be caused by the protocol followed and variations in chicken management practices between nations and chicken farms.
In Ethiopia and other nations with a lower prevalence than that of the current study, similar studies to isolate Salmonella from various poultry products were carried out. A few instances include the isolation of Salmonella enterica serovar Gallinarum at a rate of 2.6% [37] from local chicken in Tanzania, the isolation of Salmonella at a rate of 2.5% [38] from clean eggs in Ethiopia, the isolation of Gallinarum and S. pullorum at a rate of 0.8% [39] from cloacal. Te use of primary and pre-enrichment media, the use of a large amount of sample, which increased the likelihood of Salmonella recovery from poultry products as described by ISO, and the pooling of samples in the present study, which was based on Wallace et al. [40] and ISO [33].
Tese diferences (higher or lower prevalence) from the current fnding could, in general, be attributed to diferences in the isolation technique, sample type and quantity, geographic location, bird breeds, and work quality.
All of the Salmonella isolates used in the current study were derived from feces, meat, and egg samples. As fecal 38 (61.29%), egg 14 (22.58%), and meat 10 (16.13%) were positive out of the total sample, there is no statistically signifcant diference in the distribution of isolates between diferent sample types [2 � 3.4380, p value � 0.179]. Te current percentage of fecal isolates (61.29%; 38/62) difered from the results of Orji et al. [23] in Nigeria (12.5%) and Kagambega et al. [24] in Burkina Faso (55%). 10.33% (16/62) of the salmonella isolates in the current study came from meat samples. Te current study did not support the fndings of Abunna et al. [27]; and Davies and Hinton [41]; which found no salmonella.
Salmonella prevalence was found to be 2.5% in similar studies to isolate Salmonella from clean eggs conducted in Ethiopia [38]. 14 (or 25%) of the 56 egg samples had results that did not agree with those of the current study. Te geographic region, sample size, sampling technique, and breeds of poultry from which the eggs were taken could all be contributing factors to the diference.
Sulphamethoxazole-trimethoprim, gentamycin, streptomycin, ampicillin, and ciprofoxacin were the fve antimicrobial drugs tested for antimicrobial susceptibility on the thirty chosen Salmonella isolates. Ampicillin and sulphamethoxazole-trimethoprim were efective against all isolates. Although all isolates should have been able to respond to Ciprofoxacin and Gentamycin, only 4 (13.3%) and 5 (16.7%) isolates showed intermediate susceptibility. Te outcome was consistent with those reported by [42], Al-Ledeni et al. [43], Maria [44] from America, Tabo et al. [45] in Chad, and Carramiñana et al. [46] from Spain, who all came to the same conclusion that frst-line medications like chloramphenicol, gentamycin, and sulfamethoxazoletrimethoprim as well as currently Te fndings of Adesiji  [50] from India, and Antunes et al. [51] from Portugal were in disagreement with the sensitivity results of this study, but they difered with resistant patterns in which tested Salmonellae were highly resistant to ciprofoxacin, gentamycin, and sulfamethoxazole Disagreement may result from various isolate strains as well as poor processing quality. 26 salmonella isolates (86.7%) were streptomycin resistant. Te results of the current study were consistent with those of studies by Cardoso et al. [48], Agada et al. [22] from Nigeria, Abunna et al. [27] from Ethiopia, and Davies and Hinton [41], which found that the majority of Enterobacteriaceae family members, including Salmonella, are resistant to antibiotics like aminoglycosides like streptomycin and betalactams. Tus, the most prevalent single resistance was streptomycin (86.7%). Tese could be brought on by the widespread use of streptomycin in conjunction with penicillin (pin strip), as well as the drug's accessibility from all veterinary medications in Ethiopia, afordability from a neighborhood pharmacy, and frequency of use, which increases salmonella exposure to medications that encourage the emergence of resistance.
Only three isolates (10%) were resistant to gentamycin, streptomycin, and ciprofoxacin, three antimicrobial drugs. Furthermore, 5 isolates (16.67%) were resistant to two antimicrobial drugs. Te rest only 21 (70%) of the isolates had streptomycin resistance. Tis fnding disagreed with those of studies by Abunna et al. [27]; which found that 19 of 30 (63.33%) resistance isolates were resistant to four to seven diferent antimicrobials, Payne et al. [52] on broiler farms, where 96% of the isolates were resistant to more than one antimicrobial agent (s), and Singh et al. [53] on resistance isolated from chicken eggs, poultry farms, and markets, which found that two isolates were resistant to out of 27 multiresistant isolates, fve were resistant to fve diferent antimicrobials, according to Jahan et al. [54]. Tis discrepancy might be caused by the drugs' unavailability, high cost, or afordability, which may prevent both humans and animals from being exposed to them. Antimicrobial resistance against drugs such as ciprofoxacin, sulphamethoxazole-trimethoprim, and gentamycin was less common in Ethiopia because these medications were more expensive for veterinary use.

Conclusion and Recommendations
Te current study demonstrated that Salmonella is prevalent overall at a rate of 16.14% in the study area. One of the leading causes of morbidity and mortality in poultry, salmonellosis is still a distressing public health concern on a global scale. Te Salmonella strains' genetic makeup enables them to adapt to a variety of environments, including hosts that are both animal and nonanimal. Tis makes getting rid of the bacteria more challenging. Te majority of isolated Salmonella was found to be sensitive to the majority of the tested antibiotics. Most resistant isolates were only susceptible to one or two antimicrobial medications. Streptomycin resistance was present in the majority of salmonella isolates, which may be related to the drug's widespread use in Ethiopia. However, their use for treating typhoid and salmonellosis in humans and animals should continue with justifcation, and treatment is necessary if illness persists, especially in immune-compromised people (HIV/AIDS). Te recommendations that were made were as follows in light of the aforementioned result and conclusion: (i) Te public health sectors should create awareness through various pieces of training, workshops, and seminars to inform health stakeholders (ii) People should be made aware of the need to refrain from practicing serving raw eggs to children or using them as traditional medicine (iii) Further studies should concentrate on molecular characterization for serotyping and genetic studies and genes responsible for Salmonella's pathogenicity and drug resistance

Data Availability
All the datasets generated or analyzed during this study are included in this manuscript.

Ethical Approval
Ethical consent was perused from Wolaita Sodo University, Research Review Committee to collect research and conduct the research, and the committee approved this research work.