Bacteriological Quality and Antimicrobial Susceptibility Patterns Among Raw Milk Producers and Vendors in Gomole District, Borena Zone, Southern Ethiopia.

Background and Aim: Milk consumption plays a great role in the nutrition of consumers and the income of producers as well as vendors, but their bacteriological quality causes loss of those benefits. Hence, the aim of this study was to determine the bacteriological quality, associated factors and antimicrobial susceptibility pattern among raw milk of producers and vendors in Gomole district, Borena zone, South Ethiopia, from March 1 to April 30, 2019.
Methods: A cross-sectional study was conducted on 130 purposively selected study participants. Pretested structured questionnaires and observation checklists were used to collect data. Then, 15 to 20 mL of milk samples were collected from producers and vendors for laboratory analysis. Standard plate count agar and eosin methylene blue agar were used for total bacterial count and total coliform count, respectively. Bacterial isolation from poor-quality milk was performed with biochemical tests and antimicrobial susceptibility tests using Kirby Bauer's disk diffusion method. After completeness checking, the data were analyzed by Statistical Package for the Social Sciences version 21. Chi-square (χ 2) was used to analyze association factors, and a p value <0.05 was considered statistically significant.
Results: The overall means ± standard deviation of the total bacterial count (TBC) and total coliform count (TCC) were 7.75 ± 0.882 and 6.69 ± 1.545 log10 CFU/mL, respectively. The mean TBC was significantly different between producers' and vendors' milks t = 2.1 (P < 0.001). The proportions of raw milk TBC and TCC of poor quality were 90% and 80%, respectively. Poor hand washing practices before milking, water source and cleanliness of milk containers were associated with poor milk quality. The isolated bacteria were E. coli (30.8%), S. aureus (17.9%) and Salmonella spp. Out of the bacterial isolates, 80.4% were extensive drug resistant, 14.3% were multidrug resistant, and 5.4% were resistant against all antimicrobials used in this study.
Conclusion: Ensuring proper hygienic practices during milking, storage and transportation to reduce milk contamination.

Introduction

Milk is an animal-derived food product that provides essential nutrients such as protein, energy, vitamins and minerals that promote the growth and maintenance of body tissue.1 Biologically active compounds such as casein and whey proteins in milk have been found to be important for physiological and biochemical functions that have crucial impacts on human metabolism and health. Immunoglobulin in milk is important to protect newborns against a number of diseases.2

In Ethiopia, milk and milk products have a high value in feeding pastoral communities. Especially in the lowlands where livestock keeping is the main occupation, all groups of society consume milk.3 Fresh milk is sold without pasteurization to the public either directly from small producers, via informal markets or through dairy farmers. These practices create milk contamination during production, handling and transportation and processing that acts as a vehicle for the transmission of milk-borne pathogens to humans.4

Bacterial contamination of milk is the major cause of food-borne diseases, which pose a serious threat to the health of millions of people worldwide. The magnitude of foodborne disease, death or other complications associated with milk contamination increases day-to-day and imposes a substantial burden on health care systems.5 A major public health challenge is the increase in foodborne illness, which causes the death of millions of people. Including Salmonella, food poisoning is one of the most common and widely distributed diseases in the world, estimated to cause 1.3 billion cases of gastroenteritis and three million deaths worldwide.6 Staphylococcal food poisoning (SFP) is among the most prevalent causes of gastroenteritis worldwide.7 In addition, E. coli O157:H7, the most important foodborne pathogen, causes diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans.8

In Africa, foodborne diseases are responsible for 33–90% of deaths in children and represent a serious problem for the continent.9 Nevertheless, in developing countries such as Ethiopia, milk is a significant source of foodborne diseases and other infectious diseases. This happens where milk production and various dairy products take place under unsanitary conditions and poor production practices.10

Multidrug resistance (MDR) has increased worldwide and is considered a public health threat. Several recent investigations reported the emergence of multidrug-resistant bacterial pathogens from different origins, including humans, birds, cattle, and food, which increases the need for routine application of antimicrobial susceptibility testing to detect the antibiotic of choice as well as the screening of emerging MDR strains.11–20 This is worsening the clinical scenario and is currently one of the greatest medical challenges. It is also the reason for low cure rates, loss of human life and animal and milk products.21,22

In Ethiopia, bacteriological quality control of raw milk and milk products is not usually conducted on a routine basis.23 This situation results in the higher poor bacteriological quality of raw milk of pastoral communities in South Ethiopia.24 Moreover, the raw milk producers and vendors in the Gomole district supply their milk directly to consumers without appropriate measures taken. No previous study has investigated bacteriological quality, factors contributing to the deterioration of raw milk quality and appropriate drugs for the treatment of individuals infected from milk-borne illness in Gomole district pastoral communities. Therefore, this study was carried out to determine the bacteriological quality, associated factors and antimicrobial susceptibility pattern among raw milk producers and vendors in Gomole district, Southern Ethiopia.

Methods

Study Area and Period

The study was conducted in Gomole district located in the Borena zone of Oromia Regional state from March 1 to April 30, 2019. The district is located 525 km from Addis Ababa along the Addis Ababa - Moyale highway. The Gomole district has 67,798 total human populations, and more than 90% of the populations are pastoralists. The district climate is arid and semiarid, and the annual mean daily temperature varies from 17 °C to 30 °C with a bimodal rain season. In 2018, animal statistics showed that there were 37,000 cattle, 14,890 camels and 108,222 goats in the district. Raw milk is the main product exported to neighboring districts and crosses the border to Kenya.

Study Design and Population

A community-based cross-sectional study was conducted on purposively selected raw milk producers and vendors in Gomole district.

Inclusion and Exclusion Criteria

Raw milk producers and vendors in four randomly selected kebeles of Gomole district during the study period were included in the study, while milk imported from outside of the study area and their producers or vendors were not included in the study.

Sample Size Determination

The sample size was calculated by Epi Info Version 7.2.1.0 using the double population proportion formula,25 with the following assumptions: power of the study=80%, 95% confidence level, the ratio of unexposed:exposed is 1:1 including 15% contingency. The final sample size was 132.

Sampling Technique

Out of 14 kebeles (locations) in Gomole district, initially, four kebeles were selected using a simple random method. Then, producers and vendors in Kebeles were proportionally allocated. Finally, 132 study participants (97 producers and 35 vendors) were selected using a purposive sampling approach (Figure 1).

Figure 1

This figure shows schematic presentation of sampling techniques. In this figure N is the total number of producers and vendors in the selected kebeles, where Sp= the study sample size of producers in the selected kebeles; Sv=the study sample size of vendors from selected kebeles; and n= the total sample size.

Data and Sample Collections

Data were collected through face-to-face interviews with the study participants by pretested structured questionnaires. The questionnaire contains sociodemographic characteristics such as age and sex and other factors affecting the quality of raw milk; cleaning practice of udder, source and type of water used for cleaning purposes, preservation method, hand washing practices before milking, types of milk containers and others. In addition, direct observation to assess participant personal hygiene, condition around milk and cleanliness of using containers was also noted in the checklists (S1 file). Then, approximately 15–20 milliliter (mL) raw milk samples were aseptically collected from producers’ and vendors’ containers placed into sterile screw cups. Consequently, the samples were labeled, placed in a 4°C ice box and transported to the laboratory within two hours for bacterial quality determination and antimicrobial susceptibility testing.

Laboratory Analysis

Total Bacteria and Coliform Enumeration

The total coliform count was determined by sterile standard plate count agar eosin methylene blue agar (EMBA). One milliliter of raw milk sample was added to a sterile test tube containing 9 mL of sterile peptone water. After thoroughly mixing, the suspension was serially diluted up to 10−7. From all diluted tubes, 1 mL was taken and spread on sterile 15–20 mL standard plate count agar (Oxoid, Basingstoke, UK) and eosin methylene blue agar (EMB) media (Oxoid, Basingstoke, UK). The inoculated petri dishes were incubated at 35°C for 24 hours.26 The plates with colonies ranging from 25–250 colony forming units per milliliter (CFU/mL) of sample were selected for determination of TBC.27 TBC was determined as the total number of CFU per milliliter of milk sample, which was calculated using the formula. CFU/mL=Average number of colonies counted/Dilution factor ×Volume plated. Total bacterial counts were judged according to East African standard milk samples and were considered good quality if the bacterial load was < 2×106 CFU/mL and poor if the bacterial load was > 2×106 (>6.3 log10) CFU/mL.26 Regarding coliforms after incubation, green metallic sheen dark centered nucleated colonies appeared on the plates counted as total coliforms.27 The results were interpreted as East African standard raw milk containing less than 50,000 (<4.7 log10) CFU/mL TCC, which is acceptable.28

Bacterial Isolation and Identification

Escherichia coli were isolated from coliform-contaminated eosin methylene blue agar (EMBA) plates, and one or two typical greenish metallic sheen suspected colonies were subjected to Gram staining. The presence of Gram-negative rod isolates was confirmed by biochemical tests.29 For Salmonella spp. identification, 1 mL of milk was preenriched in 9 mL of lactose broth at 37°C for 24 hours. Then, 1 mL of preenrichment sample was inoculated into 10 mL of selenite F broth and incubated at 37°C for 24 hours. A loop full of selective enrichment was streaked on xylose-lysine decarboxylase (XLD) (Oxoid, Basingstoke, UK) and incubated at 37°C for 24 hours. All suspected nonlactose-fermenting Salmonella colonies were picked from plate agar, streaked onto nutrient agar plates and then incubated at 37°C for 24 hours. From plate agar, pure isolate single colonies were picked for biochemical tests.24 Staphylococcus aureus was isolated by placing 0.1 mL aliquots from dilutions of poor quality graded samples into properly labeled mannitol salt agar (MSA) plates. The plates were spread and incubated at 37°C for 24 to 48 hours. For confirmation, one or two yellowish suspected colonies were transferred from MSA plates into nutrient broth (NB) tubes and incubated at 35°C for 48 hours. Following the incubation period, a loop full of NB was streaked on the nutrient agar plates and incubated at 35°C for 48 hours. The pure isolate colonies were subjected to Gram staining, catalase tests and coagulase tests. Those forming clumping by the coagulase test were considered positive.30

Antimicrobial Susceptibility Test

The antimicrobial susceptibility test (AST) of the bacterial isolate was performed by the Kirby-Bauer disk diffusion technique as modified by the Clinical and Laboratory Standard Institute (CLSI). From a pure culture, 3–5 selected colonies of bacteria were taken and transferred to a tube containing 4–5 mL sterile normal saline and mixed gently to make a homogenous turbid suspension adjusted to a McFarland 0.5 standard. A sterile cotton swab was dipped into the standardized suspension of bacteria and then uniformly streaked over the entire surface of Mueller-Hinton agar (Oxoid Ltd., UK) as per the guidelines of the Clinical and Laboratory Standards Institute.31 Then, paper discs impregnated with a fixed concentration of the antimicrobial discs obtained from Himedia (Mumbai, India) were used in the study. The criteria used to select five antimicrobial agents, ampicillin, chloramphenicol, ciprofloxacin, tetracycline and gentamicin, were based on the availability and frequency of prescription for the management of bacterial infections in animals as well as for humans in Ethiopia.32 Antimicrobial discs were placed on the agar surface and incubated in an inverted position at 37°C for 24 hours. After incubation for 24 hours, clear zones of inhibition were produced by the bacterial growth and diffusion of the antibiotics, which were measured in millimeters using a ruler and interpreted as susceptible, intermediate and resistant.31

Quality Control

Qualities of the obtained data were ensured by following a standard procedure in each step of the work. The questionnaire for the study was adopted from prior studies and developed according to the local context. The questionnaire was pretested for completeness and appropriateness to the local context on 5% of producers and vendors in Fichwa district. Based on the results of the pretest, some questions were modified. Every day, questionnaires were checked in the field after data collection by data collectors, supervisors and principal investigators. The calibrated equipment was used for the measurement of reagents and other materials before employing the process. The quality of the reagents, antibiotic disks and disinfectant solutions was ensured following the manufacturer’s direction, and the expiration date was checked. Before dispensing, the antimicrobial disks were kept at room temperature for one hour. The culture media were prepared and sterilized based on the manufacturer’s instructions, after which the sterility test of the media was checked by incubating 3–5% of the batch at 35–37°C overnight. American Type Culture Collection (ATCC) reference strains, such as E. coli ATCC 25922, S. aureus ATCC 25923 and S. typhimurium ATCC 14028, were used for the quality control of culture.

Data Processing and Analysis

The total bacterial and coliform count results were interpreted as direct and converted into Log10 CFU/mL by a logarithm calculator. The data were analyzed by SPSS version 21 for descriptive statistics, frequency, percentage and chi-square (χ2) for association tested between dependent and independent variables, and a t-test was used for mean comparison. For all analyses, a 95% confidence level and p value <0.05 were considered statistically significant. The information is presented using tables and graphs.

Results

Questionnaire and Observational Survey Results

Out of a total, 130 study participants were enrolled in this study, with a response rate of 98.5%. Among the study participants, 95 (73.1%) were producers, and the remaining 35 (26.9%) were vendors. The age of the study participants ranged from 19 years to 67 years with a mean and standard deviation of 38.1 ±11.4. In terms of sex, the majority 108 (83.1%) of study participants were female. Regarding education status, 114 (87.7%) were unable to read and write, and only 5 (3.8%) attended primary school. On the other hand, among the study participants, only 21 (16.2%) trained on milk handling, and 101 (77.7%) did not know the diseases transmitted through contaminated raw milk (Table 1). Out of the study participants interviewed, the majority 106 (81.5%) of them used plastic containers, more than half of them 69 (53.1%) used motorcycles as a transportation means, and 43.1% transported milk for less than two hours (Table 2).

Table 1

Sociodemographic Characteristics and Knowledge of Raw Milk Producers and Vendors in Gomole District, Borena Zone, South Ethiopia, 2019

Factors	Category	Producers	Vendors	Total
		(n=95)	(n=35)	(n=130)
		No (%)	No (%)	No (%)
Sex of participants	Male	18(19)	4(11.4)	22(16.9)
	Female	77(81)	31(88.6)	108(83.1)
Age group	19–34 years	39(41.1)	15(42.9)	54(41.5)
	35–50 years	40(42.1)	15(42.9)	55(42.3)
	51–67 years	16(16.8)	5(14.2)	21(16.2)
Educational status	Cannot read and write	84(88.4)	30(85.7)	114(87.7)
	Can read and write	7(7.4)	4(11.4)	11(8.5)
	Primary school	4(4.2)	1(2.9)	5(3.8)
Train on milk handling	Yes	13(13.7)	8(22.9)	21(16.2)
	No	82(86.3)	27(77.1)	109(83.8)
Know milk borne diseases	Yes	21(22.1)	8(22.9)	29(22.3)
	No	74(77.9)	27(77.1)	101(77.7)

Table 2

Hygienic Practices, Environment, Transportation and Milk Container Characteristics Among Raw Milk Producers and Vendors in Gomole District, Borena Zone, South Ethiopia, 2019

Factors	Category	Producers	Vendors	Total
		(n=95)	(n=35)	(n=130)
		No (%)	No (%)	No (%)
Types of milk equipment	Plastic	72(75.8)	34(97)	106(81.5)
	Aluminum/Steel	3(3.2)	–	3(2.3)
	Wood	12(12.6)	–	12(9.2)
	Traditional pot	8(8.4)	1(3)	9(7)
Source of water for cleaning	Tap water	63(66.3)	19(54.3)	82(63.1)
	Well	18(18.9)	11(31.4)	29(22.3)
	Spring	13(13.7)	4(11.4)	17(13.1)
	Pond	1(1.5)	1(2.9)	2(1.5)
Type of water used to wash milk containers	Cold water	20(21)	5(14.3)	25(19.2)
	Cold water +soap	–	1(2.9)	1(0.8)
	Hot water only	66(69.5)	23(65.7)	89(68.5)
	Hot water + soap	9(9.5)	6(17.1)	15(11.5)
Mode of transportation	On foot	35(36.8)	11(31.4)	46(35.4)
	By motorcycle	52(54.7)	17(48.6)	69(53.1)
	By animal	3(3.2)	–	3(2.3)
	By vehicle	5(5.3)	7(20)	12(9.2)
Transportation hours	Less than 1 hour	32(33.7)	11(31.4)	43(33.1)
	Less than 2 hours	46(48.4)	10(28.6)	56(43.1)
	3 hours and above	17(17.9)	14(40)	31(23.8)
Mix milk from different sources	Yes	80(84.2)	34(97.1)	114(87.7)
	No	15(15.8)	1(2.9)	16(12.3)
Milk Preserving method	By smoking	94(98.9)	35(100)	129(99.2)
	By fridge	1(1.1)	–	1(0.8)
Clean barn practice	Daily	6(6.3)	–	6(6.3)
	Once a week	32(33.7)	–	32(33.7)
	Once a month	57(60)	–	57(60)
Hand wash before milking	Yes	10(10.5)	–	10(10.5)
	No	85(89.5)	–	85(89.5)
Udder wash practice	Yes	1(1.1)	–	1(1.1)
	No	94(98.9)	–	94(98.9)

Regarding observations of hygienic practices, approximately 88.4% of participants used containers with poor cleanliness, and 54.7% observed good personal hygiene. Approximately 38.5% of the milk was kept under cool conditions (Table 3).

Table 3

Observational Findings Among Raw Milk Producers and Vendors in Gomole District, Borena Zone, South Ethiopia, 2019

Factors	Category	Producers (n=95)	Vendors (n=35)	Total (n=130)
		No (%)	No (%)	No (%)
Personal hygiene	Looks poor	52(54.7)	10(28.6)	62(47.7)
	Looks good	43(45.3)	25(71.4)	68(52.3)
Cleanliness of milk container	Looks poor	84(88.4)	27(77.1)	111(85.4)
	Looks good	11(11.6)	8(22.9)	19(14.6)
Conditions around milk	Clean	13(13.7)	4(11.4)	17(13.1)
	Dusty	16(16.8)	5(14.3)	21(16.2)
	Cool conditions	41(41.2)	9(25.7)	50(38.5)
	Hot conditions	25(26.3)	17(48.6)	42(32.3)

Total Bacteria and Coliform Counts

The overall total bacterial count (TBC) mean was 7.75±0.882 log10 (5.6×107) CFU/mL, while the total coliform count (TCC) mean was 6.69±1.545 log10 (4.89×106) CFU/mL. The mean of TBC was significantly different between producers’ and vendors’ milks t=2.1 (P<0.001); however, no TCC mean significantly differed t= 1.4 (P >0.062) (Table 4).

Table 4

Total Bacteria and Coliform Counts Among Raw Milk of Producers and Vendors in Gomole District, Borena Zone, South Ethiopia, 2019

Bacterial Count	Producers’ Milk	Vendors’ Milk	Overall Milks	t-test
log10 CFU/mL	Mean ± SD	Mean ± SD	Mean ± SD	t value 95% CI	P value
TBC	7.57±0.83	8.25±0.838	7.75±0.882	2.1(1.00, 3.5)	0.001
TCC	6.54±1.53	7.11±1.54	6.69±1.545	1.4(0.028, 1.17)	0.062

Abbreviations: TBC, total bacteria count; TCC, total coliform count; SD, standard deviation; CI, confidence interval.

Raw Milk Quality and Bacterial Isolates

The overall TBC and TCC poor-quality milks were 117 (90%) and 104 (80%), respectively (Figure 2). The proportion of poor quality was higher among milk collected from vendors, but the difference was not statistically significant (P>0.05). Bacteria isolated from poor-quality milk were Salmonella spp. (2.6%), S. aureus (17.9%), and Escherichia coli (30.8%). However, the high prevalence of S. aureus, Salmonella spp. and E. coli isolated from vendors’ raw milk was not statistically significant (p>0.05) (Table 5).

Figure 2

This figure shows poor quality proportion of total bacteria count (TBC) and total coliforms count (TCC) among producers’ milk, vendors’ milk and both milks.

Table 5

Proportion of Bacteriological Quality and Bacterial Isolates Among Raw Milk Producers and Vendors in Gomole District, Borena Zone, South Ethiopia, 2019

Poor Quality	Producers’ Milk	Vendors’ Milk	Overall Milks	p value
	No (%)	No (%)	No (%)
TBC	84(88.4)	33(94.3)	117(90)	0.323*
S. aureus	14(16.7)	7(21.2)	21(17.9)	0.566
Salmonella spp.	1(1.2)	2(6.1)	3(2.6)	0.135*
TCC	75(78.9)	29(82.8)	104(80)	0.621
E. coli	23(30.7)	9(31)	32(30.8)	0.971

Note: *Fisher’s exact value.

Abbreviations: TBC, total bacteria count; TCC, total coliform count.

Factors Associated with Raw Milk Bacteriological Quality

In the present study, the source of water for cleaning, handwashing practices before milking and cleanliness of milk containers were significantly associated with poor bacteriological quality of raw milk (p<0.05) (Table 6).

Table 6

Factors Associated with the Bacteriological Quality of Raw Milk from Producers and Vendors in Gomole District, Borena Zone, South Ethiopia, 2019

Factors	Category	Poor Quality (No. %)	Good Quality (No. %)	χ2	P value	df
Gender	Male	21(95.4)	1(4.6)	0.875	0.349*	1
	Female	96(88.9)	12(11.1)
Age group	19–34	48(88.9)	6(11.1)	0.765	0.682*	2
	35–50	49(89.1)	6(10.9)
	51–67	20(95.2)	1(4.8)
Educational status	Not read and write	104(91.2)	10(8.8)	1.560	0.213*	2
	Can read and write	9(81.8)	2(18.2)
	Primary grade 1–8	4(80)	1(20)
Source of water	Tape water	70(85.4)	12(14.6)	5.299	0.021*	1
	Nontape water	47(97.9)	1(2.1)
Transportation hours	<1 hour	36(83.7)	7(16.3)	3.299	0.199*	2
	<2 hours	53(94.6)	3(5.4)
	3<hours	28(90.3)	3(9.7)
Mix milk from different sources	Yes	102(89.5)	12(10.5)	0.285	0.593*	1
	No	15(93.8)	1(6.2)
Train on milk handling	Yes	18(85.7)	3(14.3)	0.511	0.475*	1
	No	99(89)	10(11)
Know disease transmit by contaminated milk	Yes	24(82.8)	5(17.2)	2.175	0.140	1
	No	93(92.1)	8(7.9)
Hand wash before milking	Yes	4(40)	6(60)	25.59	0.001*	1
	No	80(94.1)	5(5.9)
Personal hygiene	Looks poor	57(91.9)	5(8.1)	0.493	0.482	1
	Looks good	60(88.2)	8(11.8)
Cleanliness of milk containers	Looks poor	110(99.1)	1(0.9)	69.86	0.001*	1

Notes: *Fischer’s exact value; good quality milk ≤6.3 log10; poor quality milk>6.3 log10 CFU/mL.

Abbreviations: χ2, Chi-square; df, degrees of freedom.

Antimicrobial Susceptibility and Resistance Profile of Bacterial Isolates

The bacterial isolates were mostly susceptible to ciprofloxacin. All isolated bacteria showed resistance against ampicillin, but higher resistance was observed against E. coli (81.2%). Salmonella spp. was the only bacterial isolate that showed high resistance against all antimicrobials used in this study (Table 7). The overall multidrug resistance (MDR) and pandrug resistance (all tested antimicrobial resistant) of all bacterial isolates were 14.3% and 5.4%, respectively (Table 8).

Table 7

Antimicrobial Susceptibility Pattern of Bacterial Isolates from Raw Milk

Antimicrobials	E. coli (n=32)		S. aureus (n=21)		Salmonella spp. (n=3)
	S (%)	R (%)	S (%)	R (%)	S (%)	R (%)
Ampicillin	6(18.8)	26(81.2)	8(38.1)	13(61.9)	1(33.3)	2(66.7)
Ciprofloxacin	32(100)	–	21(100)	–	2(66.7)	1(33.3)
Chloramphenicol	32(100)	–	15(71.4)	6(28.6)	1(33.3)	2(66.7)
Gentamicin	32(100)	–	19(90.5)	2(9.5)	2(66.7)	1(33.3)
Tetracycline	32(100)	–	16(76.2)	5(23.8)	1(33.3)	2(66.7)

Abbreviations: S, Susceptible; I, intermediate; R, resistance.

Table 8

Antimicrobial Resistance Profile of Bacterial Isolates

Bacteria Strains	No. of Isolates	Extensive Drug Resistant (XDR)	Multidrug Resistant (MDR)	Pandrug Resistant (PDR)
		No. (%)	No. (%)	No. (%)
E. coli (n=32)	32	26(81.2)	–	–
S. aureus (n=21)	21	16(76.2)	6(28.6)	–
Salmonella spp.	3	3(100)	2(66.7)	1(33.3)
Total	56	45(80.4)	8(14.3)	3(5.4)

Notes: XDR ≤2 drugs resistant. MDR ≥3 drug resistant.

Abbreviation: PDR, all drug resistant.

Discussion

The present study revealed that the total bacterial count (TBC) overall mean was 5.6×107 (7.75±0.8824 log10) CFU/mL. This finding is in line with a study conducted in Borena Yabello (8.149 ± 0.043 log10), South Ethiopia.33 However, this finding was higher than that of a study conducted in Iran (1.03×106 ±0.0022 CFU/mL),34 Dire Dawa (6.76 Log10 CFU/mL).35 In addition, the overall total coliform count (TCC) mean of raw milk was 6.69±1.545 log10 (4.89×106) CFU/mL. This result is comparable with the study in Borena Yabello (6.323 ± 0.028 log10),33 Hawassa (6.52 log10 CFU/mL),25 and Eastern Ethiopia (7.32±0.07 log10).36 However, this finding is higher than that of Tanzania (2.8x104 CFU/mL),37 Kenya (4.00±0.66 log10 CFU/mL),38 and Mersa Ethiopia (5.15 log10 CFU/mL).39 The differences in studies might be attributed to variations in the surrounding temperature, geographical area, seasonal variations, fecal contamination cleanliness of milk containers, hygienic practices during milking, and transportation.10

The overall proportion of TBC poor-quality milk was 90%. This finding is similar to a study conducted in Tanzania (90%)37 but higher than a study from Adigrat Ethiopia (60%)40 and lower than a study from Borena Abaya, Ethiopia (99%).24 A higher TBC can reduce milk quality, shelf life and the nutritional quality of milk and threatens the health of consumers due to toxic metabolites produced by different organisms growing in it.30

Ensercheia coli isolated was 30.8%, which was higher than that in a study conducted in Zambia (13%),41 Tanzania (3.5%),37 Mekele, Ethiopia (17.6%),42 Hawassa, Ethiopia (8.8%),43 and Borena Abaya South Ethiopia (12.9%).24 However, lower than Iran (69%),34 Arusha Tanzania (90.67%),44 two district Tanzania (3.5%),37 Isiolo Kenya (38.2%),38 and Mekele Ethiopia (44.4%).45 The number of Staphylococcus aureus isolates was 21 (17.9%), which was lower than that in studies conducted in Iran (41.66%),34 Zambia (22%),41 Ethiopia Hawassa (35.2%),43 Mekele (27.5%),42 and Mekele (26.7%).45 In contrast, the prevalence was higher than that in a study in Tanzania (0.9%)37 and Borena Abaya, Ethiopia (7.29%).24 In addition to E. coli and S. aureus, Salmonella spp. were the third most common isolates, with a 2.6% prevalence, which was lower than that in a study in Arusha Tanzania (37.4%)44 and Borena Abaya District, South Ethiopia (11.5%).24 The difference in prevalence might vary with the geographic region of the area samples collected, sample size and high proportion of those bacteria in milk related to the level of poor hygienic practices.

In this study, factors such as hand wash practice before milking, source of water for cleaning and cleanliness of milk containers showed statistically significant (p<0.05) associations with poor bacterial quality of raw milk. This finding agrees with studies conducted in Hawassa, Ethiopia,43 Yabelo Borena, Ethiopia33 and Kampala Uganda.46

Antimicrobial susceptibility tests showed that E. coli isolates were 100% susceptible to gentamicin, ciprofloxacin and chloramphenicol. This finding is similar to studies conducted in Adigrat, Ethiopia,40 Tanzania47 and Egypt48 but higher sensitivity than studies in Bangladesh49 and Jigjiga Ethiopia.32 However, E. coli was highly resistant to ampicillin (81.2%). This study is similar to other studies.40,47,49 Staphylococcus aureus was highly sensitive to ciprofloxacin, gentamicin, tetracycline and chloramphenicol (100%, 90.5%, 76.2% and 71.4%, respectively), which was comparable to a study conducted in Adigrat, Ethiopia,40 Tanzania,47 Egypt,48 and Bangladesh.49 In contrast, S. aureus was highly resistant to ampicillin (61.9%), which was similar to a study conducted in Bangladesh49 and Egypt48 and lower than a study from Adigrat Ethiopia.40

In terms of Salmonella spp. isolates, 33.3% were sensitive to chloramphenicol and tetracycline, and 66.7% were sensitive to gentamicin and ciprofloxacin, which is lower than the results of studies conducted in Bangladesh,49 Tanzania47 and Jigjiga Ethiopia.32 In the present study, Salmonella spp. showed 66.7% resistance to ampicillin, which is comparable to a study conducted in Bangladesh49 and lower than that in Tanzania47 but higher than that in Jigjiga, Ethiopia.32 This high level of resistance of E. coli, S. aureus and Salmonella spp. to ampicillin in this study might be a result of suboptimal, prolonged, indiscriminate and use of inappropriate antibiotics for the treatment of diseases and excessive use of antimicrobial therapeutic and prophylactic treatment.

The present study obtained 14.3% overall multidrug resistance. The finding of the study was lower than that of a study conducted in Jigjiga (55.2%).32 S. aureus isolates were found to be 4.8% multidrug resistant (≥3 drugs). This finding is lower than that of a study conducted in Egypt, which found S. aureus 34.8% and E. coli 94.9%.48 Although MDR S. aureus has a low prevalence in raw milk samples from the study area, it is hurdling to develop a healthy and safe living environment for humans. On the other hand, Salmonella spp. isolates demonstrated a high level of multidrug resistance (66.7%), especially among commonly used drugs such as ampicillin, chloramphenicol and tetracycline.

Limitations of the Study

This study did not identify the virulence genes and antimicrobial resistance genes of isolated bacteria due to the inability of resources. The correlation between phenotypic and genotypic MDR was not assessed.

Conclusion and Recommendations

The findings of this study showed that both producers’ and vendors’ raw milk was highly contaminated with total bacteria and coliforms. The overall bacteria isolated from raw milk with poor bacteriological quality were 32 (30.8%) Escherichia coli, 21 (17.9%) Staphylococcus aureus and 3 (2.6%) Salmonella spp. Antimicrobial-resistant bacteria isolated from milk may be a great challenge for the healthcare system. Based on the findings, milk producers and vendors should practice cleaning the milk container properly and washing their hand before milking. Local, regional and national governments should establish diagnostic centers for the identification, antimicrobial resistance surveillance and prevention of zoonotic transmission of pathogens.