Scientific African 16 (2022) e01167
Contents lists available at ScienceDirect
Scientific African
journal homepage: www.elsevier.com/locate/sciaf
Isolation and multidrug drug resistance profile of Listeria
species in selected Dairy Farm’s Operational stages in Oromia
Regional State, Ethiopia
Hussein Mohammed Ahimed a, Adem Hiko a,∗, Adem Abdellah a, Yimer Muktar b,
Fanta D. Gutema c
a
College of Veterinary Medicine, Haramaya University, Ethiopia P.O. Box 138, Dire Dawa, Ethiopia
Woldia University, Ethiopia
c
College of Veterinary Medicine and Agriculture, Addis Ababa University, P.O. Box 34, Bishoftu, Ethiopia
b
a r t i c l e
i n f o
Article history:
Received 8 February 2020
Revised 1 May 2021
Accepted 19 March 2022
Editor: DR B Gyampoh
Keywords:
Dairy
Milk
Operation stage
Listeria species
Silage
a b s t r a c t
Listeriosis occurs due to consumption of contaminated food including raw milk. This study
was aimed to assess the occurrence of Listeria species along Haramaya University Dairy
Farm’s operational stages with antibiotic resistance test on the isolates in oromia-Ethiopia.
Study involves 30 lactating cows and 170 randomly collected different types of dairy environmental samples. The samples were examined for Listeria species. The isolates were
tested against eight antibiotics. Out of 200 samples, 10 (20%) were positive for Listeria
species. Listeria was isolated from cow barn, milk supply, silage feed and milk from cow
teat, milking operation and milk auditing at 30%, 26.7%, 20%, 18.6% and 10%, respectively.
Listeria species including L. monocytogenes (5.5%), L. innocua (5%), L. gray (3%), L. ivanovii
(2.5%), L. seeligeri (2%) and L. welshimeri (2%) were also isolated. High resistant L. monocytogene to tetracycline (54.5%), L. ivanovii to amoxicillin (40%), L. innocua to each of penicillin
and tetracycline (60%), L. seeligeri to tetracycline (100%), L. welsheri to penicillin (75%) and
L. gray to each of penicillin and tetracycline (66.7%) were observed. Of all the 40 Listeria
species isolates, 35%, 25%, 17.5% and 5% isolates were resistant to single, two, three and
four drugs, respectively. Detection of considerable proportion of, different species of and
antibiotic resistant Listeria isolates in the studied dairy sampling location shows there rere
risk for raw milk contamination. Thus application of basic hygiene along the studied dairy
operational stages could reduce consumer infection.
© 2022 Published by Elsevier B.V. on behalf of African Institute of Mathematical Sciences
/ Next Einstein Initiative.
This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
Abbreviations: TSYEA, Tryptic Soya Yeast Extract Agar; PALCAM, Polymixin Acriflavine Lithium Chloride Ceftazidine aesculine mannitol; CAMP, Christie–
Atkins–Munch-Peterson; CFU, Colony Forming Unit.
∗
Corresponding author.
E-mail address: Adex.2010ph@gmail.com (A. Hiko).
https://doi.org/10.1016/j.sciaf.2022.e01167
2468-2276/© 2022 Published by Elsevier B.V. on behalf of African Institute of Mathematical Sciences / Next Einstein Initiative. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
H.M. Ahimed, A. Hiko, A. Abdellah et al.
Scientific African 16 (2022) e01167
Introduction
Human infections with Listeria arises mainly from the consumption of contaminated food [1]. Listeria monocytogenes
foodborne illness rangs from mild to severe [1,2] and often fatal illness characterized by septicemia, meningitis or meningoencephalitis [2] in invasive listeriosis [3] both in humans and animals [1,2,4]. The diseases is responsible for high (91%)
hospitalization [5] with 25% mortality worldwide [6]. Various types of food were potentially contaminated and the agents
Listeria are found widely in water, soil, infected animals, human and animal feces, meat processing facilities, improperly
fermented silage and raw (unpasteurized) milk [7–9]. Listeria species including L. monocytogenes are most frequently prevalent in the milk-processing environment [10–12]. The genus Listeria comprises seven species including L. monocytogenes, L.
innocua, L. welshimeri, L. seeligeri, L. ivanovii, L. murrayi and L. grayi [11,12]. Despite advances in chilling food preservation
technologies and laboratory techniques [13], L. monocytogenes continues to be public health problem due to food contamination from various sources [10,13]. Several European countries indicates increasing rate of listeriosis [14], and occurrences
of outbreaks in the United States [15], Canada [16] and China [17].
In Ethiopia, L. monocytogenes and other Listeria species were detected and isolated of the in foods in fragments from
selected areas including Addis Ababa [18–20], Gondar [7], central highlands of Ethiopia [21], Bishoftu and Dukem [22] indicating challenges to public health. Fisseha [23] reported 80%, 40%, 15% and 10% of resistant L. monocytogens to penicillin,
nalidixic acid, tetracycline, and ampicillin, in descending order from ready-to-eat food. Similarly, Garedew et al. [7] (2015)
reported 66%, 50%, 37.5% and 16.6% of resistant L. monocytogens to penicillin, nalidixic acid, tetracycline, and chloramphenicol, respectively. Girma and Abebe [24] also reported 30.5%, 25%, 22.2% and 11.1% resistance L. monocytogenes to nalidixic
acid, tetracycline, chloramphenicol and streptomycin, respectively.
Multi-drug resistance of two in 10%, more than two in 15% [23] one or more drugs in 16.7% [7] were reported in Ethiopia.
However, there was no any structured survey to indicate the possible sources of L. monocytogenes and other Listeria species
along dairy stages in Ethiopia. This study was aimed to assess the occurrence of Listeria species along Haramaya University
Dairy Farm’s operational stages with antibiotic resistance test on the isolates in oromia-Ethiopia. This study would have
paramount contribution in prevention Listeria species along the dairy line.
Materials and methods
Description of the study area
The study was conducted in the Haramaya University Dairy Farm, located in the Haramaya District Eastern Hararghe
Zone of Oromia Regional State, at approximately 509 KMs from Addis Ababa city Ethiopia (Fig. 1). Geographically this area
lies between 90 26’N latitude and 420 3’E longitude, at an altitude of 20 0 0 meters above sea level. The area has a mean
temperature ranging from 10 to18 °C with relative humidity of 65% and an average 800 mm annual rainfall.
Description of the study farm
The studied farm has a total population of 167 including lactating cows, dry cows, heifers and calves at different age
[24,25]. The animals were feed frequently grass, hey, silage and supplement of concentrate with limited grazing. Water
was supplied at ad-libitum. Vaccination against sporadic diseases and basic biosafety were practiced. During this study
period, there are 30 cows were lactating with average milk production of the farm 600 Liters/day. The milk is supplied the
University and the surrounding community. The cows are milked twice per day (early in the morning and at afternoon) in
milking parlor of five milking machines. Sometimes by hand milking was also practiced during electricity failure. Routine
cleaning of the milking machine, bulk milk tank, milk harvesting equipment and milking parlor area were carried out after
each milking terms using cold tap water.
Study design and sampling methods
Purposive type study, which includes all of the 30 lactating dairy cows, was conducted along the Haramaya University
Dairy Farm operational stages. Sampling locations were identified from following Hiko et al. [26] and all milking stages were
considered. Thus, six dairy operational stages consisting of 14 different sampling points were identified and included.
Sample collection and sample type
Samples were collected from the animal, the product (milk) and the environment following aseptic procedure. Environmental samples were silage feed swabs, environmental samples (cow’s barn, water samples, milking machine canal swab,
teat drying towel, milking barn floor swabs, milk harvesting cylinder swab, milkers hands swab, bulk milk collectors swab
and measuring equipment swab). Per milking machine, a pooled swab sample was collected from the four canal as one swab
(i.e the 4 canal of milking machine as single swab sample). From animal, pooled milk samples from four teats of a cow were
sampled into one universal bottle. Dairy cow’s drinking water and the water used for cleaning purposes were also sampled.
2
H.M. Ahimed, A. Hiko, A. Abdellah et al.
Scientific African 16 (2022) e01167
Fig. 1. Map of study area.
From the products, sample of raw pooled milk from bulk tank at collection and at supply were aseptically collected. Thus,
about 10 ml of the raw milk and cleaning water samples were aseptically coolected. Overall 200 samples were collected.
As a transport media, sterile buffered peptone water (BPW) (Merck, Germany) and wet cotton swabs were used for each
swab samples. Then all samples were kept in ice both while collection and transported to Microbiology Laboratory, College
of Veterinary Medicine, Haramaya University, using cold ice box at 4 °C. Bacteriological examination was conducted within
24 h of collection [12].
Laboratory procedures
Isolation
A number of methods are available for the detection of Listeria species. But considering inclusive sample types, the
horizontal method for the detection and enumeration of L. monocytogenes in food and animal feed was used in this study.
Thus, ISO [12] method for the detection of L. monocytogenes, and ISO 11290-2:1998 method for enumeration were used
[11,12].
Primary enrichment
For the initial suspension, the selective primary enrichment medium (Half Fraser Broth) (Demi-Fraser) as was used according to the procedure described by ISO 11290. At a proportion of 1:10, a volume of milk and water samples was added
to Half Fraser broth while a swab samples were left in test tube containing 10 ml buffered peptone water. According to ISO
[12], the mixture was homogenized and the test portion was incubated at 30 °C for 24 h.
Secondary enrichment
A volume of 0.1 ml of the culture from primary enrichment (regardless of its color) was transferred to a tube containing
10 ml of secondary selective enrichment medium (Fraser broth) (Demi-Fraser). The sample was incubated at 37 °C for 48 h.
Plating out and identification
A loop full of the culture from the secondary enrichment was taken and streaked onto sterile PALCAM (polymixin acriflavine lithium chloride ceftazidine aesculine mannitol) agar (SIGMA-RBI) and incubated at 37 °C for 48 h to determine the
presence or absence of Listeria species based on the growth of characteristic presumed colonies.
Presumptive colonies of Listeria species in general are small, smooth, translucent, and bluish gray when viewed in normal
light, but blue green is visible by oblique light. After 48 h, the colonies become darker, with a possible greenish sheen having
about 2 mm in diameter with black halos and sunken centers [12]. For confirmation, one to five suspected colonies were
transferred on to Tryptic Soya Yeast Extract Agar (TSYEA) (Oxoid) pre dried plates incubated at 37 °C for 24 hr until growth
is satisfactory. The colonies having 1–2 mm in diameter were taken again onto TSYEA for Table 1 biochemical tests [12].
Biochemical tests for Listeria species identification
The following physiological, morphologic and biochemical test (Table 2) were used for identification and confirmation of
Listeria species. In every test, fresh colony from TSYEA was used.
3
H.M. Ahimed, A. Hiko, A. Abdellah et al.
Scientific African 16 (2022) e01167
Table 1
Biochemical reactions to identify and differentiate Listeria species.
Listeria species reactions
Test used
Catalase reaction
Gram staining
Motility
Haemolysis
Carbohydrate
utilization
CAMP reaction
L-Rhamnose
D-Xylose
D-Mannitol
S.aureus
R.equi
L. monocytogen
L. ivanovii
L. innocua
L. seeligeri
L. welshimeri
L. grayi
+
+
Motile
+
+
_
_
+
_
+
+
Motile
++
_
+
_
_
+
+
+
Motile
_
V
_
_
_
_
+
+
Motile
(+)
_
+
_
(+)
_
+
+
Motile
_
V
+
_
_
_
+
+
Motile
_
_
_
+
_
_
Note: +: positive; ++: strong positive; (+): weak positive; _: negative; V: variable [11,27].
Table 2
The Zone of inhibition in millimeter in diameter by disk diffusion method break point used to determine antimicrobial resistance profiles of Listeria isolates.
Antimicrobials and their
abbreviation
Zone of inhibition (mm) by disk diffusion method
Susceptible
Intermediate
Resistance
Amoxicillin (AML 30 μg)
Ampicillin (AMP 10 μg)
Erythromycin (ET 15 μg)
≥
≥
≥
≥
≥
≥
≥
>
≥
≥
≥
14-17
14-16
20-22
14-17
13-14
14-17
31-33
15
12-14
22-24
15-18
≤
≤
≤
≤
≤
≤
≤
≤
≤
≤
≤
Gentamycine (CN 30 μg)
Kanamycin (K 10 μg)
Penicillin (P 10 μg)
Streptomycin (S 10 μg)
Tetracycline (30 μg)
18
17
23
18
15
18
34
16
15
25
19
13
13
20
13
12
13
31
14
11
22
14
Remarks
For
For
For
For
For
For
For
For
For
For
For
Gram positives
Gram positives
L. monocytogenes
Gram positives
Gram positives
Gram positives
L. monocytogenes
Gram positives
Gram positives
L. monocytogenes
Gram positives
Source [30–32].
Gram staining: Gram staining was performed on separate colonies taken from TSYEA Listeria species was revealed as
gram-positive short rods [12].
Motility: Strait inoculation was made on motility test medium and incubated at 25 °C for 48 h. Listeria species were
motile by giving a typical umbrella like growth pattern under the sub surface [12].
Catalase reaction: A drop of 3% hydrogen peroxide (H2 O2 ) solution was used for the test. A drop of H2 O2 was added to
isolate on the glass slide, and bubble formation was considered as positive.
Hemolysis test: A Sheep blood agar plate was inoculated with isolates and incubation at 37 °C for 24 h. The isolates were
categorized based upon hemolytic reaction. L. monocytogenes show narrow, clear, light zones of ß-hemolysis. L. innocua not
shows any clear zone of hemolysis around the colony. L. seeligeri shows a weak zone of hemolysis and L. ivanovii shows a
wide clear zone of ß-hemolysis around the colonies [12].
CAMP test (Christie–Atkins–Munch-Peterson): Staphylococcus aureus obtained from reference isolates in Haramaya University Microbiology Laboratory was staked vertically on sheep blood agar. The suspected Listeria isolates were streaked
horizontally without being touching the vertical the S. aureus streaks. The culture was incubated for 24 h at 37°C. The
isolates were categorized into L. monocytogenes by an enhanced zone of hemolysis, forming an arrow head towards the S.
aureus culture; L. ivanovii by absence of formation of a wide clear “arrow head’’ hemolysis towards the S. aureus; and L.
seeligeri by the formation of a weak enhanced hemolysis around S. aureus streak. On the other hand, those non-hemolytic
were CAMP test negative and could be L. innocua, L. welshimeri, and L. gray [11,27–29].
Carbohydrate utilization tests: For carbohydrate utilization test, broth containing rhaminose, xylose and mannitol was
prepared with Bromocresol purple (Merck, Germany) as an indicator. The suspected colony was incubated at 37 °C for up to
5 days [12]. Yellow color due to acid formation indicates positive result and otherwise negative.
Antibiotic susceptibility test
Antimicrobial susceptibility test was done on all of the 40 isolates by the Kirby-Bauer disk diffusion method [30, 31]. The
tested bacterium was taken from 24 h freshly grown culture and inoculated into 5 ml Brain Heart Infusion Broth (Merck,
Germany). The inoculated broth was incubated for 4 h at 37 °C to approximately 10 6 CFU/ml at McFarland 0.5% level
of turbidity. With this culture, a bacterial lawn was spread on Mueller Hinton agar (Oxoid, UK). Antimicrobials against
disk (Oxoid, UK) which are frequently used in human and animal treatment in Ethiopia including amoxicillin (AML 30 μg),
4
H.M. Ahimed, A. Hiko, A. Abdellah et al.
Scientific African 16 (2022) e01167
Table 3
L. monocytogenes and other Listeria species along the studied dairy operational stages.
Sampling dairy operational
stages
Total samples
examined
No. (%) of samples positives
Total positives
L. monocytogenes
Other Listeria
No. (%)
χ2
p-Value
Silage and drinking water
Cow barn
Milk cow
Milking operation
Milk auditing
40
10
30
70
20
3(7.5)
1(10.0)
3(10.0)
2(2.8)
0
5(12.5)
2(20.0)
3 (10.0)
11(15.7)
2(10.0)
8(20.0)
3(30.0)
6(20.0)
13(18.6)
2(10.0)
2.80
0.73
Milk supply
30
2 (6.6)
6(20.0)
8(26.7)
Total
200
11(5.5)
29(14.5)
40(20.0)
Table 4
Distribution of Listeria species isolates long the Haramaya University Dairy Farm sampling locations.
Sampling locations
No. of samples
examined
No. (%) of
positives
Distribution of Listeria species isolates (%)
No.
L. monocytogen
L. ivanovii
L. innocua
L. seeligeri
L. welshimeri
L. gray
1
2
3
4
5
6
7
8
9
10
11
12
13
Silage feed
Drinking water
Cow barn floor
Milk from cow teat
Milkers hand
Milking machine canal
Milk harvesting cylinder
Bulk milk collector
Teat drying towel
Cleaning water
Milking parlor floor
Pooled milk at collection
Pooled milk at supply
30
10
10
30
10
10
10
10
10
10
10
20
10
8 (26.7)
0
3 (30.0)
6 (20.0)
0
2(20.0)
1(10.0)
1(10.0)
3(30.0)
2(20.0)
4(40.0)
2(10.0)
4(40.0)
3
0
1
3
0
0
0
0
0
0
2
0
1
(10.0)
0
0
0
0
0
0
1 (10.0)
0
0
0
0
1 (5.0)
2 (20.0)
2
0
0
1
0
2
0
1
2
1
0
0
1
1
0
1
0
0
0
0
0
1
0
1
0
0
1 (3.33)
0
0
1 (3.33)
0
0
0
0
0
1 (10.0)
0
0
0
1
0
1
1
0
0
0
0
0
0
1
1
0
14
Milk measuring equipment
10
4(40.0)
1 (10.0)
1 (10.0)
0
0
1 (10.0)
1 (10.0)
200
40 (20.0)
11 (5.5)
5(2.5)
10(5.0)
4(2.0)
4 (2.0)
6(3.0)
Total
(10.0)
(10.0)
(10.0)
(20.0)
(6.7)
(3.33)
(20.0)
(10.0)
(20.0)
(10.0)
(10.0)
(3.33)
(10.0)
(10.0)
(10.0)
(3.33)
(10.0)
(3.33)
(10.0)
(5.0)
Regardless of the isolated Listeria species, overall resistant isolate to tetracycline, penicillin, amoxicillin and streptomycin were 55.0%, 45.0%, 20.0% and 17.5%
in descending order (Table 5).
ampicillin (AMP 10 μg), gentamycin (CN 30 μg), erythromycin (ET 15 μg), Kanmycin (10 μg), penicillin G (P 10 μg), and
tetracycline (T 30 μg) were mounted. The culture was incubated 37 °C for 24 h.
Result was interpreted using the diameter of zone of bacterial growth inhibition (Table 2) surrounding the disc [30–32].
Data management and statistical analysis
The data obtained from this investigation was entered into Microsoft Excel 2013® and analyzed using STATA-11. The significance of association for the detection of Listeria species among explanatory variables of milk contaminant risk factors at
each milking operational stages and sampling points were expressed using percentage. Chi-square (χ 2) association was used
to determine the association between the isolates and potential risk factors (if available). Also, the significance difference
was considered at at p-value less than 0.05.
Results
Out of 200 samples collected from selected six sampling stages, 40 (20%) were positive for overall Listeria species An
overall Listeria species were detected at all sampling stages with the detection rate ranging from 10% at milk auditing to
30% at cow barn with no significant differences (χ 2 = 2.80; p-value = 0.73). Of the total samples, 11 (5.5%) were positive L.
monocytogenes. Except at milk auditing, all sampling stages were found positive L. monocytogenes at 2.8–10.0%. A total of 29
(14.5%) samples were positive for other Listeria species. The prevalence was ranged from 12.5 to 20.0% (Table 3).
Out of the total samples from 14 sampling locations, L. monocytogenes was detected from silage feed, cow barn, milk
from cow teat, milking parlor floor, pooled milk at supply and milk measuring equipment. Next to L. monocytogenes, the
predominantly detected Listeria species were L. innocua 10 (5%), L. gray 6 (3%), L. ivanovii 5 (2.5%) and L. seeligeri and L.
welsheri at 4 (2%) each in descending order (Table 4). Except for L. ivanovii, silage was found positive for all Listeria species
isolated in this study. Except for L. ivanovii and L. seeligeri, milk from cow’s teat was positive for all isolates of this study.
Likewise, milk measuring equipment was positive for all species, except for L. innocua and L. seeligeri.
5
H.M. Ahimed, A. Hiko, A. Abdellah et al.
Scientific African 16 (2022) e01167
Table 5
Antimicrobial resistance profile for Listeria species isolated from the studied dairy operation stage.
No. of tested
isolates
Responses
to the drug
Isolates and antimicrobial profiles of Listeria species
Listeria species
K
P
ET
AMP
AMX
TE
S
CN
L. monocytogenes
11
L. ivanovii
5
L. innocua
10
L. seeligeri
4
L. welsheri
4
L. gray
6
S No. (%)
I No. (%)
R No. (%)
S No. (%)
I No. (%)
R No. (%)
S No. (%)
I No. (%)
R No. (%)
S No. (%)
I No. (%)
R No. (%)
S No. (%)
I No. (%)
R No. (%)
S No. (%)
I No. (%)
R No. (%)
11(100.0)
0
0
5(100.0)
0
0
9(90.0)
1(10.0)
0
4(100.0)
0
0
3(75.0)
1(25.0)
0
5(83.3)
0
1(16.7)
10(90.9)
0
1(9.1)
2(40.0)
2(40.0)
1 (20.0)
1(10.0)
3(30.0)
6 (60.0)
0
1(25.0)
3(75.0)
1(25.0)
0
3 (75.0)
2(33.3)
0
4(66.7)
11(100.0)
0
0
4(80.0)
1(20.0)
0
10(100.0)
0
0
4(100.0)
0
0
3(75.0)
1(25.0)
0
3(50.0)
2(33.3)
1 (16.7)
8(72.7)
3(27.3)
0
4(80.0)
1(20.0)
0
8(80.0)
2(20.0)
0
4(100.0)
0
0
4(100.0)
0
0
4(66.7)
1(16.7)
1(16.7)
7(63.6)
1(9.1)
3(27.3)
2(40.0)
1(20.0)
2(40.0)
9(90.0)
1(10.0)
0
4(100.0)
0
0
1(25.0)
1(25.0)
2(50.0)
5(83.3)
0
1(16.7)
4 (36.7)
1(9.1)
6(54.5)
1(20.0)
4 (80.0)
0
3 (30.0)
1 (10.0)
6 (60.0)
0
0
4(100.0)
0
2(50.0)
2(50.0)
0
2(33.3)
4(66.7)
8 (72.7)
0
3(27.3)
5(100.0)
0
0
8 (80.0)
0
2(20.0)
3(75.0)
0
1(25.0)
4 (100.0)
0
0
4 (66.7)
1(16.66)
1(16.66)
7(63.6)
4 (36.4)
0
3(60.0)
1 (20.0)
1(20.0)
8(80.0)
1 (10.0)
1 (10)
2(50.0)
0
2(50.0)
3(75.0)
0
1(25.0)
5(83.3)
1(16.7)
0
Total
40
S. No. (%)
I. No. (%)
R. No. (%)
37(92.5)
2(5.0)
1(2.5)
16(40.0)
6(15.0)
18(45.0)
35(87.5)
4(10.0)
1(2.5)
32(80.0)
7(17.5)
1(2.5)
28(70.0)
4(10.0)
8(20.0)
8(20.0)
10(25.0)
22(55.0)
32(80.0)
1(2.5)
7(17.5)
28(70.0)
7(17.5)
5(12.5)
Note: K: kanamycin; P: penicillin; ET: erythromycin; AMP: ampicillin; AMX: amoxicillin; TE: tetracyclin; S: streptomycin; CN: gentamycine; S: susceptible;
I: intermediate; R: resistant.
Table 6
Listeria isolates showing resistance to at least one drugs of study.
Listeria species
No. tested
Resistance to at least one drug No. (%)
L.
L.
L.
L.
L.
L.
11
5
10
4
4
6
8
3
8
4
4
6
40
33 (82.5)
monocytogenes
ivanovii
innocua
seeligeri
welshimeri
gray
Total
(72.7)
(60.0)
(80.0)
(100)
(100)
(100)
Almost one or more of the isolates of the Listeria species showed resistance to one of the drugs used in this study
(Table 5). Of the 11 L. monocytogene isolates, resistant to TE (54.5%), AMX (27.3%), S (27.3%) and P (9.1%) were observed. L.
ivanovii showed resistance to TE and CN at 20% each and to AMX at 40% rate. Equal and 60% of L. innocua isolates showed
resistance to P and TE but all (100%) of the L. seeligeri were resistant to TE. Three of the four (75%) L. welsheri were resistant
to P while 50% of them were resistant to each of AMX and TE. Except for CN, 16.7% of L. gray showed resistance to the
majorities of the drug used. Resistant Isolates to each of P and TE were 66.7%.
Of the detected Listeria isolates, 33 (82.5%) expressed single to multiple drugs (MDR) resistance. As shown in Table 6, all
(100%) of the isolated L. innocua, L. seeligeri and L. welshimeri and the majorities (72.7%) of L. monocytogenes were showed
resistance to at least on drug of study.
Of all the 40 Listeria species isolates, 14 (35%), 10 (25%), 7 (17.5%) and 2 (5%) isolates were resistance to single, two, three
and four drugs, respectively. Of the L. monocytogenes, 5(45.5%), 1(9.1%) and 2(18.2%) have developed resistance to single, two
and three drugs respectively (Table 7).
Of the 33 isolates showing resistance against single to combinations of two or more drugs, for each of the isolate Listeria
were shown in Table 8.
Discussions
Listeria species along the dairy farm operational stages
Listeria species including L. monocytogenes is most frequently prevalent in the milk-processing environment including
steps, drains and floors [10]. Onsets of listeriosis in ruminants due to feeding of contaminated silage [33] were also reported. Observing Listeria spscies in all of the six operational stages and their 14 specific sampling locations indicats vast
occurance of the agent in the studied dairy farm which could be act as possible sources for milk contamination having pub6
H.M. Ahimed, A. Hiko, A. Abdellah et al.
Scientific African 16 (2022) e01167
Table 7
Single to multiple drugs resistance combination profile of the isolated Listeria species.
Resistance
profiles
Drugs
combinations
No. of
isolates
No. (%) resistant isolates of Listeria isolates
L. monocytogenes
(No. = 11)
L. ivanovii
(No. = 5)
L. inocua
(No. = 10)
L. seeligeri
(No. =4)
L. welsheri
(No. =4)
L. gray
(No. =6)
Single drug
AML
CN
P
TE
AML-TE
P-TE
P-AML
TE-S
AML-TE-S
P-TE-CN
P-TE-S
K-P-ET-TE
P-TE-S-CN
3
1
4
6
1
4
3
2
2
3
2
1
1
1
0
1
3
0
0
0
1
2
0
0
0
0
1 (33.3)
1(100)
0
0
0
0
1(33.3)
0
0
0
0
0
0
0
0
2
1
0
2
0
1
0
1
1
0
0
0
0
0
1 (16.7)
0
1 (25.0)
0
0
0
1 (33.3)
0
0
1(100)
1(33.3)
0
0
0
0
1(25.0)
1 (33.3
0
0
1(33.3)
0
0
0
0
0
1 (25.0)
1(16.7)
1 (100)
0
1 (33.3)
0
0
0
1 (50.0)
1 (100)
0
Two drugs
Three drugs
Four drugs
(33.3)
(25.0)
(50.0)
(50.0)
(100)
(50.0)
(16.7)
(50.0)
(50.0)
(33.3)
(50.0)
Note: K: kanamycin; P: penicillin; ET: erythromycin; AMP: ampicillin; AMX: amoxicillin; TE: tetracyclin; S: streptomycin; CN: gentamycine.
Table 8
Single to multiple drug resistance profiles of the isolated Listeria species.
No. (%) of MDR profiles of the isolates
Listeria species
No. of isolates
L.
L.
L.
L.
L.
L.
11
5
10
4
4
6
5(45.4)
2(40.0)
3(30.0)
1(25.0)
1(25.0)
2(33.3)
1(9.1)
1(20.0)
3(30.0)
1(25.0)
2(50.0)
2(33.3)
2 (18.2)
0
2(20.0)
1(25.0)
1(25.0)
1(16.7)
0
0
0
1(25.0)
0
1(25.0)
40
14(35.0)
10(25.0)
7(17.5)
2(5.0)
monocytogenes
ivonovii
innocua
seeligeri
welsheri
gray
Total
Single
Two
Three
Four
lic health risk. In contrary, Listeria species were not detected in samples from drinking water and milker’s hand. Van Kessel
et al. [34] reported 7.1% L. monocytogenes from dairy farm environments indicating the fecal shedding and environmental
contamination which extended to the bulk milk having difficulty to avoid. Dairy farms have been identified as a reservoir
of L. monocytogenes, and there is significant strain diversity within and across farms. The increasing of Listeria species from
the zero in drinking water and 26.7% in silage feed to the 40% in milk products indicates the possible risk of transfer of the
agent along the dairy stage. This could be due to biofilms formation of Listeria species [35] in the studied dairy farm, the
milking system and dairy environment under poor cleaning and disinfection condition acting as a consistent occurrence and
source of the bacterium for milk contamination. Van Kessel et al. [34] also provided similar suggestion by underlining the
difficulty to avoid Listeria species dairy farms have been identified. Such difficulty to avoid Listeria species from dairy farm
could also due to the biofilms formation of the agent. The present 10% Listeria species in pooled milk at collection location,
40% in each of the pooled milk at supply, and in milk measuring equipments also indicates the possible transfer and cross
contamination with Listeria species to the level of milk supply to public stations and within the operational activities in the
farm.
The present findings of 5.5% L. monocytogenes, 5% L. innocua, 3% L. gray, 2.5% L. ivanovii, and 2% each L. seeligeri and L.
welshimeri indicates the contamination of the farm line with diversified Listeria species. King et al. [36] reported that L.
monocytogenes and L. innocua share the same ecological niche. Therefore L. innocua could be used as an indicator for the
presence of L. monocytogenes. The present overall 20% isolates of Listeria species including L. monocytogenes were within the
range of the 14% from Jimma and 32.9% from Bishoftu and Dukem, Ethiopia. The present finding was still comparable with
20.88% [37], 22% [19], 24.2% [38] from Ethiopia. But, it was lower than the 60% Listeria species reported in Uganda [39]. The
present 5.5% L. monocytogenes isolation is comparable with the 5.1% [18], 5.4% [19,20] and 5.8% [22], 4.4% [40], 4.8% [21] and
8.84% [37]. The present finding was still similar with the 8.19% raw milk in Ankara [38] and7.1% from dairy operations in
U.S 34].
Listeria monocytogenes was higher 20% in milking parlor floor and 10% from each of the silage feed, raw milk from cow’s
teat, milk measuring equipment and pooled milk supply indicates these points were critical control points for corrective
action. The present finding of 10% of L. monocytogenes in raw milk is slightly lower than the 22% [19], 18.9% [21], 7.8%
[38] from varies foods in Ethiopia. The present finding was similar with 8.19% from raw milk samples in Ankara [41], 8.8%
raw cow milk samples from Ghana [42]. These could be due to poor hygienic conditions during milking, transport, milk
storage, infected cow, management practices of cattle feed (poor quality silage) leads to contamination of raw milk with L.
monocytogenes. The current 26.6% findings of Listeria species from silage feed and 10–40% in dairy farm environmental sam7
H.M. Ahimed, A. Hiko, A. Abdellah et al.
Scientific African 16 (2022) e01167
ples shows the saprophytic properties of the agent living in various area which could infect animals, contaminate animal
feed and food of animal origin. Similarly, Ueno et al. [43] reported 16.6% L. monocytogenes isolates from poor quality silage.
Since all Listeria species are potential food contaminants, the presence of any of these species on food stuffs can be considered as an indicator of their contamination and of the potential presence of L. monocytogenes. The present 20% overall and
3.3% to 10% Listeria species from cow teat indicates presence of infection of cows from the environment or an asymptomatic
cows are shedding the pathogen or acts as survival and growth of the pathogen [35] that contaminate the milking machine.
This is shown by the current 20% contaminated milking machine which could further act as source for milk contamination.
Biofilm
Antimicrobial resistance profiles of the isolates
Antimicrobial resistance profiles Listeria species this study show 82.5% of all isolates was resistant to one or more of
four drugs. The present 72.7% resistant L. monocytogenes consisting of 45% for single drug and 9% for two drugs resistance
indicated presence of and contamination the farm with resistant strain including MDR (18.2%) of three drugs. Resistant
isolates to kanamycin, erythromycin and amoxicillin was only observed only in one isolates L. gray. This might be due to
infrequent use of these drugs in veterinary sectors in Ethiopia. However, some isolates of all of the isolated Listeria species
consisting of 54.5% L. monocytogene isolates, 20% L. ivanovii, 60% L. innocua, 100% L. seeligeri, 50% L. welsheri and 66.7% L. gray
were resistant to tetracycline. This drug was due to the frequently prescription as oxytetracycline in Veterinary Medicine and
in tetracycline in human clinical cases or in other mode of actions in Ethiopia [7,23,37]. Presence resistant Listeria species
at 25% [37] and at 77.8% [40] were also reported in Ethiopia.
Next to tetracycline, higher isolates (45.0%; 20%) of the isolates consisting of L. monocytogenes (9.1%; 27.3%), L. ivanovii
(20%; 40%),L. seeligeri (75%; 0%), L. welsheri (75%; 50%)and L. gray (66.7%; 16.7%)were resistant to (penicillin; amoxicillin)
showing the risk of resistant Listeria species to beta-lacetam drugs. Such drugs are frequently used in the forms penicillin streptomycin combination in veterinary medicine in Ethiopia resulted in the development of resistance. In connection with
these drugs, 17.5% of the isolates consisting of 27.3% L. monocytogenes, 20% L. innocua, 25% L. seeligeri, and 16.7% L. gray were
resistant to streptomycin. But, Tefera [38] has reported the zero resistant isolate streptomycin in Ethiopia.
Conclusion
The present detection of L. monocytogenes from silage feed, cow barn, milk from cow teat, milking parlor floor, pooled
milk at supply and milk measuring equipment indicats these locations can act as possible sources of the agent for dairy
product contamination. In addition to this, the findings of other Listeria species including L. innocua, L. gray, L. ivanovii and
L. seeligeri and L. welsheri in different sapling location shows the occurrence and biofilms formation of Listeria species in
the studied dairy farm with risk of raw milk contamination in the entering the plant. Moreover, observing resistant isolates
to single drug to multiple of four mixed drugs indicates the the need for rationale use and monitoring of drugs along the
studied dairy farm. Therefore presence finding warrants a need for regulatory mechanism to be set and implemented chain
based hygiene application along the studied dairy operational stages.
Declaration of Competing Interest
The authors declare that they have no competing interests.
CRediT authorship contribution statement
Hussein Mohammed Ahimed: Visualization, Investigation, Writing – original draft, Writing – review & editing. Adem
Hiko: Project administration, Visualization, Data curation, Formal analysis, Investigation, Writing – original draft, Writing
– review & editing. Adem Abdellah: Formal analysis, Investigation, Writing – original draft. Yimer Muktar: Data curation,
Methodology. Fanta D. Gutema: Writing – original draft, Writing – review & editing.
Acknowledgements
This project was supported by Haramaya University Research Grant (HURG) under Thematic Research Grant (Project No.
HURG-2018-01-01-01). The Authors would like to acknowledge Haramaya University Dairy Farm management and the operating staffs for point in time support during sample collection.
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