Bacteriological Quality of Raw Meat, Antibiotic Susceptibility Pattern of Bacterial Isolation and Associated Risk Factors Among Butcher Houses of Adama Town, Oromia, Ethiopia, 2020

Foodborne diseases continue to be an important public health problem globally and most of the food safety hazards are caused by foods of animal sources. It is also reported that contaminated raw meat has been identied as one of the main sources of foodborne illnesses. In Ethiopia, the widespread habit of raw meat consumption, lack of compliance with standard quality and sanitation protocols is a potential cause for foodborne illnesses. The purpose of this study was to assess bacteriological quality, antibiotic susceptibility pattern of bacterial isolation of raw meat and associated factors among butcher houses of Adama town, Ethiopia, 2019. Three-fourth (¾ th ) of collected raw meat was unacceptable bacterial load of Total Aerobic Plate count based on Gulf Standard. The average contamination was (5.89±0.86) log Colony Forming Unit per gram for total aerobic plate count. Raw meat collected from meat handlers who trained on meat hygiene (AOR=5.8, 95%CI:(1.99-17.34), collecting money (AOR= 0.14,95%CI (0.04-0.43) were associated with bacteriological quality of raw meat. Whereas, proportion of meat samples that were positive for Salmonella and shigella were (9.8% and 2.67%) respectively. The resistance of Salmonella was most frequently observed to Ampicillin (100%), Amoxicillin/ Clavunilic (54.5%), Tetracycline (36.3%) Trimethoprim-sulfamethoxazole (18.2%). Shigella expressed resistance to Ampicillin (50%) and 100% sensitive to the rest antibiotics used. Bacterial logarithmic mean values were beyond the acceptable standard indication of poor hygiene, making it a potential source of food-borne infection. Therefore, cm -2 (35). In a cross-sectional study on 50 beef samples from the slaughter unit in Aizawland and revealed the total aerobic plate count of 6.13±0.09 log10cfu/g and 12% positive for E. coli(40).In 68 raw meat samples from butcher shops collected in Iran, reported total aerobic plate count that varied from 10 3 to 2.6×10 6 CFU/g, Coliforms varied from <10 1 to 2.4×10 4 CFU/g, E. coli varied from <10 1 to 3×10 2 CFU/g and S. aureus 5×10 2 CFU/g(41). A study conducted on S. aureus, E. coli and Salmonella from raw meat at abattoirs and butcher shops in different areas of the Lahore city,in Pakistan 51% of samples had Aerobic plate count more than acceptable value (42).Another studies from Asia, eastern Nepal show 84%,68%34% Coliforms,S.aureu and Salmonella species were isolated respectively from raw meat sold in retail shops(43).A study conducted on raw meat Sold in Sylhet Sadar, in Bangladesh Tota Aerobic count of the samples ranged between 2.5 × 10 5 to 2.25 × 10 5 cfu/g and 28% were above acceptable value(44). A survey conducted on bacteriological quality and safety of raw beef from selected outlets in Windhoek,Namibia the overall prevalence of total plate count on the beef samples was 98.9%.Of these 98.9%, 26% samples were satisfactory,49 % samples were within acceptable level and 25% exceeded the acceptable level(12).In a study to investigate the microbiological quality and safety of beef from East Java Province, Indonesia 32.5% of the samples were contaminated with E. coli and 20.0% with S. aureus. The mean values of TAPC and S. aureus were 4.15 logCFU/g and1.39 logCFU/g respectively.About 29.66% of meat samples were found to exceed the maximum limit of TAPC based on the Food Safety and Standards regulation of India(28). In a study on the bacteriological quality of raw meat in Laa metropolis,Nigeria shows mean Aerobic plate of ampicillin of and chloramphenicol and levels of to cefotaxime (0%), gentamicin (3.2%), and kanamycin (4.3%) (26). A in the Wa Municipality of Ghana on Prevalence of Salmonella species isolated from raw meat and liver of cattle resistant to teicoplanin (96.77%) but susceptible to chloramphenicol (100%), ciprooxacin (100%), tetracycline (100%), suphamethoxazole/ trimethoprim (100%), amoxicillin/clavulanic acid (93.55%), ceftriaxone (93.55%) and gentamicin (83.87%)(66).Another study in Addis Ababa, 25% of Salmonella species were found resistant to ampicillin. 9% of Salmonella species and 2% of E. coli O157:H7 isolates were found to be resistant to ceftriaxone(15). A study in Addis Ababa, Gullele sub-city E. coli isolates were observed to be the most resistant to penicillin (60%) followed by Amoxicillin (40%) and Ampicillin (40%) of the isolates were resistance for chloramphenicol. All isolates of Salmonella (100%) were resistant to penicillin and Vancomycin. And 66.67% of the isolates resistant to Ampicillin. isolates Ciprooxacin(67).A on microbial ora and food borne pathogens on raw meat revealed Shigella was 100% resistant to co-trimoxazole and tetracycline whereas from the two Salmonella isolates one was susceptible against all 12 tested antimicrobials, while the other to all the 11 except cephalexin(68). A cross-sectional study done on prevalence of Salmonella in raw meat in dukem town Ethiopia, All the isolates (100%) were sensitive to Kanamycin where as 94.4%, 88.9% and 83.3% of the isolates were found to be sensitive to Susoxazole, Tetracycline and Nalidixic acid, respectively(69). In Ethiopia, there are a number of reports on foods including meat to have high incidence of bacteria(15),nonetheless, there are limited reports on the health implication of food borne diseases from raw meat that are displayed in open-air for sales.Thus,the objective of this study is to assess bacteriological quality of raw meat in order to assess the risk to public health, specically via the enumeration of microbiological process hygiene criteria. of meat evidence of medical certicate. This study conrms that there exist personnel medical health requirements in Ethiopia there is very little attention given to their implementation and enforcement in a food enterprise like butcher shops. Therefore, there is a high possibility of the meat handlers contaminating meat with microorganisms(79).Handling of meat and money with the same unwashed hands is one sources of meat contamination. Results of this study revealed sixty four(57.1%)(95% CI:48.2,66.1) of the meat handlers handled money (papers/coins) which may result into cross contamination of meat with microbes. Similar study in Mekelle, Ethiopia 91.7% of the meat handlers collect money while serving meat(78).According to compliance study based on gulf standard raw meat in this classies 85/112(75.89%) (95% CI: 67.9,83.9) of meat have unacceptable bacteriological quality(80).Comparable ndings were also obtained in meat retail shops of Meknes City, Morocco, reported a total aerobic plate count of 67% in beef produced and marketed with unacceptable quality(81).It is higher than in study done in Sylhet Sadar, Bangladesh in which 28% of meat were un acceptable quality(44).However, it is lower than study done in bahir in which all samples or hundred percent unacceptable(11). According to food and agricultural organization Total aerobic plate counts exceeding 5.0 log10 on fresh meat are not acceptable and alarm signals on meat hygiene(16). erent

A cross-sectional study conducted in the Federal Capital Territory of Nigeria showed knowledge and practice were in uenced by previous training, whereas food handlers who had worked for long years had better practices of food hygiene (57). A study conducted in Mexico, Latin America on the prevalence and antibiotic susceptibility pattern of Salmonella on raw meat showed high levels of resistance to ampicillin (66.7% of isolates), tetracycline (61.3%), and chloramphenicol (64.5%) and low levels of resistance to cefotaxime (0%), gentamicin (3.2%), and kanamycin (4.3%) (26). A study conducted in the Wa Municipality of Ghana on Prevalence of Salmonella species isolated from raw meat and liver of cattle highly resistant to teicoplanin (96.77%) but susceptible to chloramphenicol (100%), cipro oxacin (100%), tetracycline (100%), suphamethoxazole/ trimethoprim (100%), amoxicillin/clavulanic acid (93.55%), ceftriaxone (93.55%) and gentamicin (83.87%) (66).Another study in Addis Ababa, Ethiopia shows about 25% of Salmonella species were found resistant to ampicillin. Besides, 9% of Salmonella species and 2% of E. coli O157:H7 isolates were found to be resistant to ceftriaxone (15). A study done in Addis Ababa, Gullele sub-city E. coli isolates were observed to be the most resistant to penicillin (60%) followed by Amoxicillin (40%) and Ampicillin (40%) and none of the isolates were resistance for chloramphenicol. All isolates of Salmonella (100%) were resistant to penicillin and Vancomycin. And also 66.67% of the isolates were resistant to Ampicillin. None of the isolates were resistant to Cipro oxacin (67).A study conducted in Jimma,Ethiopia on microbial ora and food borne pathogens on raw meat revealed Shigella was 100% resistant to co-trimoxazole and tetracycline whereas from the two Salmonella isolates one was susceptible against all 12 tested antimicrobials, while the other to all the 11 except cephalexin (68). A cross-sectional study done on prevalence of Salmonella in raw meat in dukem town Ethiopia, All the isolates (100%) were sensitive to Kanamycin where as 94.4%, 88.9% and 83.3% of the isolates were found to be sensitive to Su soxazole, Tetracycline and Nalidixic acid, respectively (69). In Ethiopia, there are a number of reports on foods including meat to have high incidence of bacteria (15),nonetheless, there are limited reports on the health implication of food borne diseases from raw meat that are displayed in open-air for sales.Thus,the objective of this study is to assess bacteriological quality of raw meat in order to assess the risk to public health, speci cally via the enumeration of microbiological process hygiene criteria.

Results:
A total of 112 study participants were involved, making a response rate of 112/119(94%).The mean age of the participants was (32.83±8.31) years (table  1).  (Table 3).  Bacteriological quality of the raw meat: Raw meat samples collected form butcher shops during the study period 85/112 (75.89%) have unacceptable bacteriological quality based on gulf standard. The Enumeration of the TAPC ranged between 3.70log10cfu/g to 7.43log10cfu/g with an average count of 5.89log10cfu/g. Enumeration of TCC ranged 2.73log10cfu/g -5.76log10cfu/g with an average of 4.27log10cfu/g, whereas FCC and TSAC had mean of 3.16cfu/g and 3.02cfu/g respectively. In bivariate analysis, training of meat handlers, practices such as wearing white coat, head cover, collecting money and washing using sanitizer were signi cantly associated (p-value less than 0.25) with overall bacteriological quality of raw meat and moved to multivariable logistic regression model. However in multivariable logistic regression model training and collecting money while handling meat were signi cantly associated (p-value less than 0.05) with bacteriological quality of raw meat in the butcher shops. (Table 6).     (80).Comparable ndings were also obtained in meat retail shops of Meknes City, Morocco, reported a total aerobic plate count of 67% in beef produced and marketed with unacceptable quality (81).It is higher than in study done in Sylhet Sadar, Bangladesh in which 28% of meat were un acceptable quality (44).However, it is lower than study done in bahir in which all samples or hundred percent unacceptable (11). According to food and agricultural organization Total aerobic plate counts exceeding 5.0 log10 on fresh meat are not acceptable and alarm signals on meat hygiene (16).
The average TAPC was 5.89log CFU/g (95% CI: 5.7,6.1).Finding of this result is higher than East Java,Indonesia where mean of TAPC was 4.158 CFU/g and Chennai city, India(4.78log10) (28,82).However it is less than Addis Ababa,Ethiopia (6.44 log CFU/g ) (31). The variations of bacterial load observed in different studies might be due to lack of good processing, handling practices,sampling and sanitary standard operating procedures of meat handlers.Raw meat collected from butchers who trained on meat safety hygiene was 5.8 times more likely to be acceptable than those who did not receive training(AOR=5. 8,1.99-17.34).This is because training of food handlers about the basic concept and requirements of personal hygiene and its environment plays an important part in safeguarding the safety of products to consumers (83).Regarding collecting money,in the current study,raw meat which were collected from butcher SHOPS in which meat handlers handle money while selling meat was 86% less likely to be acceptable than their counter part(AOR=0.14,0.04-0.43).According to WHO/FAO report, handling of foods with bare hands result in cross contamination and high microbial load. Furthermore, WHO recommends food handlers should be educated, encouraged or supervised to stop their business promptly if at any time, they suffer from diarrhea, vomiting, fever, sore throat or have visibly skin lesions.Eventhough it is not independent predictor in this study, fty percent of meat handlers had practice of working while they were ill. With regard to contamination by Total coliform, the average is 4.27 logCFU/g (95% CI: 4.1,4.4). This value is higher than that of commercial beef meat in Tanzania (4.13log CFU/g) and in India (2.07 log CFU/g) but lower than that found in La a metropolis, Nigeria(4.19) (45). Variations in total coliform counts among studies may be due to differences in storage conditions and season in which samples were collected. The average contamination of meat by Feacal coliform is 2.77log CFU/g, (95% CI: 5.7,6.1) it The result is lower than that of retail beef meat in Algeria (3.41 log CFU/g and higher than in beef meat of Namibi(1.70 log10CFU/g) (84).
The data in the present study indicate that 81(72.3%) of samples collected in the town showed contamination with faecal coliforms. Presence of faecal coliforms suggests faecal contamination which is normally associated with poor hygiene and faulty sloughtering.It also suggests the possibility of nding enteric pathogens such as salmonelll,shigella and others (81). The average contamination of meat by Staphylococcus aureus is 3.14logCFU/g, (95% CI: 2.9,3.3).This value is higher than that of commercial beef meat in Chennai city, India (2.07log10) (28).However, it is Lower than study done in Bahir dar, Ethiopia (11).The highest number of S.aureus on meat indicates the presence of cross-contamination, which usually related to human skin, hair, hand and discharge from nose, and clothing (61). Concerning the prevalence of pathogenic bacteria Salmonella was detected in 11(9.8%) of analyzed samples. This nding reveled that there was a considerable rate of contamination in the butcher SHOPS of Adama town, which potentially poses a risk of causing foodassociated illness. The prevalence reported in the current study is higher than other reports such as in United State of America (6%).However, the result of this study was much lower than that found in Senegal (87%) and Bahir Dar (70%) (11,85).This difference possibly arises from the source of animals, types of samples, and sampling technique. With regard to the antimicrobial susceptibility pro les of Salmonella isolates revealed a higher rate of resistance against Ampicillin9(81.8%) and Amoxicillin6(54.5%).These ndings is in agreement with (86),where salmonella was 100% resistant to Ampicillin. An intermediate resistance of 2(18.2%) was also found for Ampicillin. On the other hand, Interestingly all of the isolates were 100% susceptible to gentamycine,cipro oxacin,tetracycline and ceftraxione.This is in line with the study conducted in Ghana (66).In addition 7(63.6%) and 9(81.8%) exhibited susceptibility to Tetracycline and Trimethoprim-sulfamethoxazole respectively. This result is also in aligning with study done in Gondar, Ethiopia. However, resistance to Ampicillin is much higher than in Bahir dar(23.8%) (11,25).
In other way the study revealed an overall Shigella prevalence of 2.67% which is higher than study done in Jimma,Ehiopia but, lower than in Karachi,Pakistan and Gondar (7,68,79), however, similarly lower rate of isolation was reported from Ebony,Nigeria (1). The antimicrobial susceptibility pro les of the isolates revealed resistance against ampicillin 1(50%), but, all the isolates were sensitive to Amoxicillin, Tetracycline, and Trimethoprim-Sulfamethoxazole, Erythromycin gentamycin, Cipro oxalin and ceftraxione. However, higher rate of resistance against ampicillin was observed in Gondar (79).Differences in the geographical location of the isolates or the emergency of drug resistant strains could partially explain this discrepancy.

Conclusion
Compliance study based on gulf standard 75.9% of raw meat collected from butcher shops have unacceptable bacteriological quality. Under observation more than half of meat handlers in the butcher shops handle money with their bare hands while processing of meat and serving of custom. The following areas are need big attention and concern for future suggestion to: Adama town ablators enterprise should get training on how to cope with good handling practices on the use of proper clothing such as hand gloves, head covers, clean white coat and dedicated cashier to collect money. Adama health bureau should regular be inspected butcher shops in the town. Owners of butcher shops should be dedicated cashier in order to collect money and consumers refrain from eating raw meat appropriately to avoid intoxication and infection due to microbes. Further investigation should be carried out to isolate and characterize the bacterial load of raw meat along with meat production chain.

Methods
The study designed to assess bacteriological quality, its associated factors and antibiotic susceptibility pattern of the isolated raw meat from butcher shops of Adama Town from October 1 to December 2019, Oromia Ethiopia.Study design was Cross-Sectional study design conducted from October 1 to December 30,2019.Source population was all the butcher shops in Adama town. Study population was all butcher shops in which cattle meat were sold. Study unit was butcher shops in which sample were actually collected. Inclusion criteria was Butcher shops which used to sell meat of cattle originally and exclusion criteria was Butcher shops in which goat ' s and sheep's meat sold; Butcher shops which closed and shifted their task during the study period and nonvolunteer. All 119 butcher shops which were working during study period were included in the study and simple random sampling method was employed to select meat handlers for interview.

Data collection tools
A pre-tested structured questionnaire initially developed in English and then translated in to local language translation expert and then translated back to English by another person to check its consistency. The questionnaire structured into three distinct parts including demographic information such as respondents' sex, age, years of experience, medical checkup and attending meat safety training. The second section of the questionnaire is about meat safety knowledge.
Questions on knowledge referred to mainly about their personal hygiene, cross contamination and temperature. It contains 15 close ended questions and each question has three optional answers ("Yes", "No" and "I do not know").The response was analyzed as categorical variables (right or wrong answer). A score of one was given to right answer and zero to the wrong and I do not know answer. The last section dealt with meat hygiene practices. The question comprises the issues of personal hygiene, hand washing practices, practices against food borne diseases and cross contamination. This section had 17 questions with two possible responses: "yes", and "no". Each correct practice reported scored one point (51).

Observational check list
Observational check list was developed after reviewing relevant literatures to assess the butcher shops' hygienic status and practice. The check list incorporated personal hygiene of meat handlers and hygienic conditions of the butcher shops' premises.

Data collection procedure
Face-to-face interview and the general sanitary condition of the butcher shops as well as the workers were observed. After nishing of questionnaires one hundred gram of raw meat sample was collected for laboratory investigation.

Data quality assurance
The data collectors were selected based on their educational background (two environmental health) and the selected data collectors were trained on the purpose and objective; bene t of the study, individual's right, informed consent and techniques of the interview for one day. Daily check-up of data completeness were made by the principal investigator.
The questionnaire was pre-tested on 5% of butcher shops in Olanciti town neighboring town 25km to Adama town before the study. The structured questionnaire was then rephrased in the light of the responses.

Statistical analysis
Before analysis, data were checked for completeness, consistencies and entered into computer using Epi info version 7.2.3.1software. Then the data was exported to SPSS version 25, coded, categorized, sorted and cleaned to facilitate analysis. Descriptive statistics was computed for the study variables and frequency distribution tables were used to describe most of the ndings. All bacterial counts were normalized to CFU/g and converted into Log10 values. Mean and standard deviation were also computed. Variables with p-value less than 0.25 in binary logistic regression analysis were entered to binary multiple logistic regression using Enter methods to determine factors independently associated with bacteriological quality of raw meat. Odds ratio with their 95% con dence intervals were computed to identify the presence of association and statistical signi cance were declared if p value is < 0.05.All other assumptions of the analysis like normality of variables were checked. Odd Ratio was considered to assess the strength of association between dependent and independent variables.
Laboratory work: One hundred gram of pooled raw meat cuts from leg area, limb area and ank area of hanging display for sale were collected from butcher SHOPS in a sterile zipped plastic bag in an icebox (70).The samples were collected in the morning (9:00am-10:00am), After labelling properly, they were keptin an ice box between 2-4ºC and were immediately transported to Adama public health research and referral laboratory centre in Adama town Oromia, Ethiopia.
The samples were analysed immediately upon arrival in the laboratory. From 100g of grinded and homogenized meat 25g was weighted and placed in 225 ml sterile 0.1% buffered peptone water. The grinded meat and diluent were thoroughly vortexed on a platform shaker for 5 minutes to wash off and dislodge any microbe that may be resident on the surface of the meat. The mixture was considered to be a 10 -1 dilution. The mixture (1ml) were transferred to a tube containing 9 ml of normal saline diluent to make 10 -2 dilution. Further dilutions were made by transferring 1 ml of the succeeding dilutions to the tubes containing 9 ml diluent up to 10 -6 . After preparation, bacteriological analyses of the samples were performed to assess the selected microbial attributes such as Total aerobic plate count, Total coliform count,fecal coliform count and Total staphylococcus aureous count (TSC) in cattle meat by using Plate Count (PC) agar, MacConkey (MC) agar and Manitol salt agar (28).All the media used were from Hardy diagnostic, America.

Determining Total aerobic plate count and Judging Meat Quality
For the enumeration of total aerobic bacteria in raw meat samples conventional standard plate Count method was used. Tenfold serial dilution up to 10 -6 was made from the homogenized sample. One mL from each serial dilutions(10 -3 , 10 -4 , 10 -5 and10 -6 ) of the test sample was pipetted into sterile Petri dishes and then molten, cooled nutrient agar was added and incubated for 24h at 37°C.Plates with colonies lying between 30-300 were counted using colony counter (TT20,Techmel, USA) and the average count was calculated and expressed as logCFU/gm (11,71,72). After determining TAPC by counting each visible colony of bacteria, the Quality of each raw meat samples were judged based on Guideline levels for determining microbial Quality of ready-to eat food(Gulf Standards).Meat samples of TAPC <5log10CFU/gm were acceptable and > 5log10CFU/gm were unacceptable (28,73).

Enumeration of Total coliforms and fecal coliforms:
For the TCC and FCC 0.1ml of each of dilution from 10 -1 ,10 -2 and 10 -3 was transferred and spread on triplicate on MacConkey agar. Then plates were incubated at 37 °C and 44ºC for 24 hours for TCC and for FCC counts respectively. Enumeration of the TCC and FC (typical pink colonies resulting from the fermentation of lactose) (74). For Staphylococci aures count, mannitol Salt Agar (MSA, HARDY DIAGNOSTIC) was surface plated with 0.1ml of the homogenate from duplicates of 10 -1 & 10 -2 .The inoculum was evenly spread on the surface of the agar and allowed to dry for 15 min at room temperature. The plates were inverted and incubated for 24 to 48h at 37ºC.Typical colonies of Staphylococci aures(golden yellow colonies shining and convex) after 24 hours' incubation were isolated, puri ed and tested for catalase and coagulase positive as a con rmatory test (75).

Isolation of Salmonella and Shigella
For the isolation of Salmonella and shigella samples were pre-enriched in buffered peptone water (incubated aerobically at 37 0 c for 24), followed by secondary enrichment in selenite cysteine broth(incubated aerobically at 37 0 c for 24 ) and plated on to XLD incubated aerobically at 37 0 c for 24 ,The suspected colonies were sub-cultured on the blood agar and incubated at 37 °C for 24hr. Further identi cation was made with triple sugar iron agar (TSI),urea broth, lysine iron agar (LIA),citrate broth and then incubated for 24 to 48 hours at 37 0 C.All biochemical test reagents were obtained from Hardy diagnostic, America (11). Antimicrobial susceptibility tests were performed using the modi ed Kirby-Bauer disk diffusion technique (76).Bacterial suspension turbidity was adjusted to 0.5McFarland standard.
Culture Media quality control Quality of culture media was maintained after checking its expiration date and preparation according to manufacturer instruction by sterilizing at 121 °C (15 lbs. sp) for 15 minutes. Sterility of culture media were also checked using strains kept for quality checking at APHRRLC.To exclude lab contaminants and check whether media and diluent completely sterilized, a representative number of a plate with media and broth without the test sample were incubated at 37 °C for 48hours. If any growth observed on control media, this batch will be discarded and another media will be replaced. Gram staining reagents were also checked for their expiry dates of each reagent, their storage condition, and checked with known quality control organisms (ATCC, American type culture collection Organism) before performing study samples.S.aureus ATCC 25923,E.coli ATCC 25922, Shigella exineri ATCC 12022 and Salmonella typhimurium ATCC 14028 were used as quality control reference strains.