Occurrence, antimicrobial resistance pattern and molecular characterization of Listeria monocytogenes isolated from bovine’s milk and meat in Mekelle City, Ethiopia

doi:10.21203/rs.3.rs-3082175/v1

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Occurrence, antimicrobial resistance pattern and molecular characterization of Listeria monocytogenes isolated from bovine’s milk and meat in Mekelle City, Ethiopia

https://doi.org/10.21203/rs.3.rs-3082175/v1

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Background

Listeria monocytogenes is an opportunistic and emerging foodborne zoonotic pathogen that encompasses a diversity of strains with varied virulence and can cause serious human and animal infections worldwide. It has low incidence but high hospitalization and case fatality rates. The aims of this study were to assess the prevalence of L. monocytogenes and its serotypes, and determine antimicrobial resistance pattern of isolates in Mekelle City, Ethiopia.

Methods

A cross-sectional study was conducted from December 2016 to June 2017 in Mekelle City, Ethiopia and a total of 768 (n = 384 of milk and n = 384 meat) samples of bovine origin were collected using a purposive random sampling technique. Isolation and identification of L. monocytogenes were done according to standard and recommended bacteriological procedures. Genome-based confirmation of each isolate was performed at species and serovar levels by targeting Iap, Imo0737, ORF2819, and ORF2110 genes using specific primers. In vitro antimicrobial susceptibility testing was performed using agar plate antibiotic disk diffusion method. Descriptive statistics, Chi-square test (χ²), and logistic regression were applied.

Results

The overall prevalence of L. monocytogenes was 26 (3.39%). Prevalence rates of L. monocytogenes were 4.17% and 2.6% in meat and milk samples, respectively. There was a statistically significant difference (p < 0.05) in the prevalence rates of the organism in meat samples collected from abattoir (1.67%), butcher shops (8.33%), and restaurants (8.33%). Serovars that were identified belonged to 1/2b and 4b. Large proportions of isolates were highly susceptible to ampicillin (88.46%) and vancomycin (84.62%). However, the isolates had shown the highest level of resistance against nalidixic acid (96.15%). The highest intermediate resistance rate was observed for amoxicillin (57.69%). Moreover, 42.31% of the isolates developed resistance to more than two antimicrobial agents.

Conclusions

Hence, both its occurrence and development of a multi-drug resistance indicated that a coordinated effort is imperative to reduce or eliminate the risk posed by this pathogen in food chains and on controlled and careful use of antimicrobials both in veterinary and human treatment regimes.

Antimicrobial susceptibility testing

Bacteriological procedures

Bovine

Gene

L. monocytogenes

Milk

Meat

prevalence

Foodborne diseases remain a major public health problem across the globe. The problem is severe in developing countries due to difficulties in securing optimal hygienic food handling practices and with the increase in consumption of raw products of animal origin. Hence, food safety and particularly the safety of products of animal origin is an increasingly significant issue concerning human health (Grace 2015; Havelaar et al. 2013; WHO 2000). There are over 200 known microbial, chemical or physical agents that can cause illness when ingested (Acheson 1999). Out of the agents associated with the current worldwide increase in cases of food-borne diseases, L. monocytogenes is a concern especially in the developing world (WHO 2002).

Listeria spp. are Gram-positive, non-spore-forming, non-capsular, short, regular rods, motile with a characteristic tumbling movement, and a low Guanine + Cytosine (G + C) content. The six species of Listeria are L. monocytogenes, L. ivanovii, L. seeligeri, L. innocua, L. welshimeri, and L. grayi. However, only L. monocytogenes and L. ivanovii are considered virulent. Human cases of L. ivanovii infection are rare (Cummins et al. 1994; Lovett 1990; Seeliger and Jones 1986), whereas L. monocytogenes is an important foodborne pathogen (Swaminathan 2001). L. monocytogenes is the causative agent of listeriosis, a food-borne disease that affects mainly at-risk populations such as the elderly, pregnant women, newborns, and immunocompromised individuals (Buchanan 2017). Listeriosis manifests itself clinically in ruminants as encephalitis, neo-natal mortality (abortion), and septicemia (Quinn et al. 2002). Even though its incidence is low, ranging from 0.1 to 1 case per 100 000 population, listeriosis is responsible for 19% of known causes of foodborne disease-related death in the United States and 17% in France (Scallan et al. 2011; Vaillant et al. 2005).

L. monocytogenes comprises 13 serotypes with different virulence potential namely: 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7 based on somatic (O factor) and flagella (H factor) antigens (Elliot and Elmer 2007; Jadhav et al. 2012). However, more than 95% of human listeriosis is commonly associated with isolates that belong to serotypes 1/2a, 1/2b, and 4b, and of these serotype 4b has been related to the most recent outbreaks of listeriosis, and serotypes 1/2a and 4b are commonly reported in animals (Datta et al. 2013; Doumith et al. 2004a; Doumith et al. 2004b; Liu et al. 2006; Mateus et al. 2013; Kasper et al. 2009; Roberts et al. 2006).

L. monocytogenes can be found in a wide variety of raw and processed foods including dairy products, raw meat, vegetables, and fishery products and all these food products have been associated with Listeria contamination and with outbreaks or sporadic cases of listeriosis (Bell and Kyriakides 2005; Dhama et al. 2013; Elliot et al. 2007; Schlech 2000; Yücel et al. 2005). For instance, in raw milk and the dairy environment, the source of L. monocytogenes contamination is mainly poor silage and bedding (Sanaa et al. 1993; Wagner et al. 2005). L. monocytogenes is transmitted from man to man or from animal to man mostly through ingestion of the organism with contaminated raw milk, dairy products, and raw meat. Hence, these food products have been connected to several outbreaks of listeriosis (Kasalica et al. 2011).

The central characteristics of L. monocytogenes contributing to food-borne transmission are the ability to grow as low as -0.4°C, resist heat, salt, nitrite, acidity, endure osmotic stress, and survive mild preservation treatment measures commonly used to control the growth of organisms in food (Walker et al. 1990). Despite the poor surveillance programs and lack of epidemiological data to establish a comprehensive incidence rate of human listeriosis in Africa, some studies reported the occurrence of L. monocytogenes in foods from African countries (Bouayad and Hamdi 2012; Derra et al. 2013; El-Shenawy et al. 2011; Matle et al. 2016). Moreover, strains of L. monocytogenes recovered from foods, the environment, and sporadic cases of human listeriosis had shown the development of resistance to one or more antibiotics (Charpentier et al. 1995; Franco Abuin et al. 1994; Hadorn et al. 1993). In Ethiopia, the actual epidemiological data about the prevalence as well as antimicrobial susceptibility pattern of L. monocytogenes is lacking both in the veterinary and public health sectors. Moreover, there is no published research work done on the epidemiology and antimicrobial susceptibility patterns of L. monocytogenes and its strain in foods of bovine origin, particularly in raw milk and meat in the current study area. Hence, this study was stipulated to assess the prevalence of L. monocytogenes and its serotypes, and determine the antimicrobial susceptibility of L. monocytogenes from raw milk and raw meat samples of bovine origin collected from different sources in Mekelle City, Ethiopia.

Study area

Mekelle City is the capital city of Tigray Regional State, which is located at 39° 29` E and 13°-30` N at an average altitude of 2000 m. a. s. l. and 783Km far from Addis Ababa, Ethiopia. The climate of the study area conforms to that of the Ethiopian Highlands. The mean annual rainfall is 619mm and is bimodal with a short rainy season occurring from March to May and another from middle September to February. The annual minimum and maximum temperatures are 11.8°C and 29.9°C, respectively (BoPED 2011). It is administratively divided into 7 sub-cities and is subdivided into 33 Kebelles (Fig. 1).

Study design and study population

A cross-sectional study was conducted from December 2016 to June 2017 in Mekelle City, Ethiopia. The study populations comprised of purposively selected milking dairy cows and slaughtered cattle found in Mekelle City. Raw milk and meat samples were collected from randomly selected dairy farms, milk shops, and cafeterias for bovine milk; and abattoir, butcher shops, and restaurants for beef. The samples were examined to assess the prevalence of L. monocytogenes and its serotypes and to determine its antimicrobial sensitivity pattern by following the standard protocols.

Sample size and sample collection

Using a 95% confidence interval and 5% sampling error, the sample size was determined using the formula recommended by Thrusfield (2005). Since there was no previously published data in the study area, the expected prevalence of L. monocytogenes was taken as 50%.

\(n=\frac{{{Z^2}P\exp (1 - P\exp )}}{{{d^2}}}\) \(n=\frac{{{{1.96}^2}P\exp (1 - P\exp )}}{{{d^2}}}\)

Where, n = sample size, z = statistic for a level of confidence

d = required absolute precision, P_exp = expected prevalence

Accordingly, a minimum sample size of 384 was calculated but to increase the precision of the estimation, the sample size was doubled to a total of 768 which was distributed to an equal proportion of both raw milk and meat samples. Hence, a total of 768 samples, comprised of milk (n = 384) and meat (n = 384), were collected using a purposive random sampling technique. The samples were collected based on the willingness of the owners until the required sample size was achieved. Thirty (30) ml of the bovine milk samples were collected from dairy farms (n = 166), milk shops (n = 120), and cafeterias (n = 98). Similarly, 25g of bovine meat samples were collected from the abattoir (n = 240), butcher shops (n = 84), and restaurants (n = 60) using sterile plastic bags. All samples were labeled and transported with icebox to Molecular Biotechnology Laboratory of College of Veterinary Sciences, Mekelle University at the date of collection and stored at 4°C; and analyzed within 24 hrs.

Enrichment, culturing, and identification

The procedure described by the International Organization for Standardization (ISO 11290-1 2004) was used for milk samples for the isolation and identification of L. monocytogenes. While the techniques recommended by the International Standards Organization (ISO 11290-1 2010; ISO 11290-1 1996) and the French Association for Standardization (AFNOR 1993) with minor modifications were employed for meat samples.

The raw milk sample was thoroughly mixed and inoculated into Listeria Enrichment Broth (LEB) at 1:9 ratios. After 48 hrs of incubation at 30^○C, the culture from LEB suspensions was inoculated into Secondary Fraser Enrichment Broth (SFEB) and incubated at 35^○C for 24 hrs. Similarly, for the meat samples, 1:9 proportion of sample to LEB was placed in sterile Stomacher plastic bags and homogenized for 1 min in a laboratory stomacher (Lab Blender 400, Seward Medical, London, England). Then, the resulting suspension was incubated for 48 hrs. After 48 hrs of incubation, 0.1ml of the culture was transferred to previously prepared SFEB and then incubated for 24 hrs (Kiiyukia 2003).

After 24 hrs of incubation, aliquots of positive SFEB medium with black/dark brown or dark green color were taken and streaked onto Listeria Oxford Agar Plates containing selective media with manufacturer's supplements, and the plates were incubated at 35^○C for 24–48 hrs. Then, the growth of Listeria species on the Listeria Oxford Agar Plate was examined (Additional file 1: Fig. S1).

Suspected Listeria colonies were sub-cultured onto a Tryptose Soya Agar plate (TSA) and incubated at 37^○C for 18–24 hrs. Those suspected Listeria colonies were characterized by using Gram’s staining (Additional file 1: Fig. S2), oxidase test, catalase test (Additional file 1: Fig. S3), motility test (Additional file 1: Fig. S4) characteristics of hemolysis (Kiiyukia 2003) (Additional file 1: Fig. S5 A), carbohydrate utilization (xylose, rhamnose (Additional file 1: Fig. S6), and Christie Atkins Munch Peterson (CAMP) test (Additional file 1: Fig. S5 B) following standard methods (AFNOR 1993; ISO 11290-1 1996; Kiiyukia 2003). All culture media and selective supplements were from Oxoid (Basingstoke, UK).

Genomic DNA extraction and PCR amplification

The DNA extraction was performed using the phenol-chloroform method for all the isolates from samples of milk and meat according to Sambrook and Russel (2001) and the quality and quantity of the extracted DNA were estimated using agarose gel electrophoresis and ultraviolet (uv) spectrophotometer, respectively.

Following preliminary bacteriological identification and genomic DNA extraction of L. monocytogenes isolates, genome-based confirmation of each isolate was performed. The PCR condition that was used in this study has been explained previously by Nayak et al. (2010) and the sequence of primers by Bubert et al. (1999) for L. monocytogenes and Bobade et al. (2016) for its serovars. A standard strain of L. monocytogenes ATCC 7644 and nuclease-free ultrapure water were used as positive and negative controls, respectively. The primers used for the PCR reaction were F-3'-TTA TAC GCG ACC GAA GCC AAC-5' and R-3'-CAA ACT GCT AAC ACA GCT ACT-5' for amplification of Iap gene of L. monocytogenes (660 bp); F-5'-AGG GCT TCA AGG ACT TAC CC-3' and R-5'-ACG ATT TCT GCT TGC CAT TC-3' for amplification of Imo0737 gene of L. monocytogenes serovar 1/2a (691 bp); F-5'-AGC AAA ATG CCA AAA CTC GT-3' and R-5'-CAT CAC TAA AGC CTC CCA TTG-3' for amplification of ORF2819 gene of L. monocytogenes serovar1/2b (471 bp); and F-5'-AGT GGA CAA TTG ATT GGT GAA-3' and R-5'-CAT CCA TCC CTT ACT TTG GAC-3' for amplification of ORF2110 gene of L. monocytogenes serovar 4b (597 bp).

Amplification was carried out with thermal cycling conditions of an initial denaturation at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 45s, annealing at 50°C for 1 min, and extension at 72°C for 1 min, and with a final extension at 72°C for 10 min for detection of L. monocytogenes. But the detection of serovars was optimized with cycling conditions of an initial denaturation at 94ºC for 5 minutes followed by 35 cycles of denaturation at 94ºC for 30s, annealing at 54ºC for 15s, and extension at 72º C for 75s, with a final extension at 72ºC for 10 min. Finally, the PCR products along with DNA molecular weight marker (3B Black Bio Biotech) were separated by running on a 2% (w/v) agarose gel containing 0.3mg/ml ethidium bromide. Electrophoresis was conducted in a horizontal equipment system for 120 min at 90 V using 1X TAE buffer. The amplicons were visualized under a gel documentation system and their molecular weights were estimated.

Antimicrobial susceptibility testing

All the phenotypically and molecularly characterized isolates of L. monocytogenes were tested for antibiotic susceptibility patterns. The method applied for the in vitro antimicrobial susceptibility testing of L. monocytogenes isolates was the agar plate antibiotic disk diffusion method using Kirby-Bauer technique (Bauer et al. 1966). The following thirteen antimicrobial disks (belong to eight classes of antimicrobials) (Himedia Laboratory Pvt Limited, Mumbai, India) with their concentrations given in parentheses were used in the antibiogram testing: Penicillin class antimicrobials (amoxicillin (25µg), ampicillin (10µg), cloxacillin (5µg), methicillin (30µg), and penicillin G (10µg)); Fluoroquinolones class antimicrobial (ciprofloxacin (5µg)); Lincomycin class antimicrobial (clindamycin (10µg)); Macrolide class antimicrobial (erythromycin (15µg)); Aminoglycoside class antimicrobials (gentamycin (10µg) and streptomycin (10µg)); Quinolone class antimicrobial (nalidixic acid (30µg)); Tetracyclineclass antimicrobial (tetracycline (30µg)); and Glycopeptides class antimicrobial (vancomycin (30µg)). The selection of these antimicrobials was based on the availability and frequent use of these antimicrobials in the study area both in veterinary and human medicine. Standard strains of L. monocytogenes ATCC 7644 and S. aureus ATCC 25923 were used as positive and negative controls, respectively. The results were interpreted as Susceptible (S), Intermediate (I), and Resistant (R) categories based on the critical points recommended by the Clinical and Laboratory Standards Institute (Additional file 1: Fig. S7) (CLSI 2014).

Data management and processing

The data collected from the results of the laboratory investigation were entered into Microsoft Excel Spreadsheet (Microsoft Crop., Redmond, WA, USA) and transferred to STATA Version 11.1 for statistical analysis. Descriptive statistics, Chi-square (χ²) test, and logistic regression were applied to see the associations of the food type and corresponding source as risk factors with that of the occurrence of the positive result; the degree of association was determined using the Odds ratio (OR) and 95% confidence interval (CI). For all tests, a p-value less than 0.05 was considered statistically significant.

Prevalence of L. monocytogenes

The prevalence of L. monocytogenes, from the total 768 samples, was found to be 26(3.39%). The sample type prevalence of L. monocytogenes was found to be16 (4.17%) and 10 (2.6%) in meat and milk samples, respectively. The prevalence of L. monocytogenes in meat samples collected from an abattoir, butcher shops, and restaurants were 1.67%, 8.33%, and 8.33%, respectively. The result was significantly higher (p < 0.05) in butcher shops and restaurants than an abattoir. Whereas, out of 384 milk samples collected from dairy farms, milk shops, and cafeterias, the prevalence was 1.81%, 2.50%, and 4.08%, respectively. However, there was no statistically significant difference (p > 0.05) in the prevalence of L. monocytogenes among the various sources of milk samples (Table 1).

Table 1

Prevalence of *L. monocytogenes* in the collected samples
Sample type	Place of collection	No. of examined	No. of positive samples (%)	95% CI	p-value
Milk	Farms	166	3(1.81)	0.0 − 1.00	0 .531464
	Shops	120	3(2.50)	0.27–7.02
	Cafeterias	98	4(4.08)	0.51–10.55
	Total	384	10 (2.6)
Meat	Abattoir	240	4(1.67)	0.27–4.90	0.005659
	Butchers	84	7(8.33)^a	1.24–19.62
	Restaurants	60	5(8.33)	0.84–17.87
	Total	384	16 (4.17)
Grand Total		768	26(3.39)		0.0417

PCR confirmation of L. monocytogenes isolates and serotyping

All the phenotypically characterized L. monocytogenes isolates were also molecularly confirmed as L. monocytogenes using L. monocytogenes species-specific primer, which targets Iap gene as it is depicted below in (Additional file 1: Fig. S8).

Moreover, the molecularly confirmed L. monocytogenes isolates were further identified at serovar levels using serovar-specific primers with the help of multiplex PCR. The serovars that were identified belonged to 1/2b and 4b as shown below in (Additional file 1: Fig. S9).

Antimicrobial susceptibility pattern

The antibiogram profile results indicated as large proportions of the isolates of this study were found to be highly susceptible to ampicillin (88.46%) and vancomycin (84.62%). However, the isolates had shown the highest level of resistance against nalidixic acid (96.15%). The highest intermediate was observed to amoxicillin (57.69%) (Table 2). Moreover, 34.62% and 42.31% of the isolates developed resistance to two and more than two drugs, respectively, as is shown in Fig. 2.

Table 2

*In vitro* antimicrobial susceptibility pattern of *L. monocytogenes* isolates
Antimicrobial Agents	Interpretations
Antimicrobial Agents	No. of Susceptible (%)	No. of Intermediate (%)	No. of Resistance (%)
Penicillin G	13(50.00)	10(38.46)	3(11.54)
Amoxicillin	9(34.62)	15(57.69)	2(7.69)
Ampicillin	23 (88.46)	3(11.54)	0(0)
Vancomycin	22(84.62)	4(15.38)	0(0)
Ciprofloxacillin	20(76.92)	6(23.08)	0(0)
Streptomycin	11(42.31)	12(46.15)	4(15.38)
Nalidixic Acid	0(0)	1(3.85)	25(96.15)
Gentamycin	21(80.77)	5(19.23)	0(0)
Methicillin	19(73.07)	7(26.92)	0(0)
Erythromycin	15(57.69)	9(34.62)	2(3.85)
Clindamycin	19(73.07)	6(14.93)	0(0)
Cloxacillin	17(65.38)	7(26.92)	2(7.69)
Tetracycline	5(19.23)	7(26.92)	14(53.85)

The overall prevalence of L. monocytogenes from both sample types was found to be 26 (3.39%), which was characterized and confirmed both phenotypically and molecularly. The current finding is in line with the findings of Kramarenko et al. (2013) (2.6%), Reda et al. (2016) (2.94%), Ndahi et al. (2014) (4%), Walker et al. (1990) (4.1%), Morobe et al. (2009) (4.3%), Mengesha et al. (2009) (4.8%), Molla et al. (2004) (5.1%), Leong et al. (2014) (5.3%), Gebretsadik et al. (2011) (5.4%), and Garedew et al. (2015) (6.25%). However, it is lower than the findings of Wang et al. (2013) (12.4%), Khen et al. (2015) (17%), Zafar et al. (2020) (26.66%), and Rahimi et al. (2010) (32.7%). But it is higher than the report of Nayak et al. (2015) (1.5%).

The meat sample-based prevalence of L. monocytogenes was found to be 4.17%. This is in agreement with the findings of Gebretsadik et al. (2011) (2.6%), Nastasijevic et al. (2017) (3.3%), Ndahi et al. (2013) (3.92%), and Garedew et al. (2015) (6.66%). However, it is lower than the reports of Reda et al. (2016) (8%), Shen et al. (2013) (9.6%), Wang et al. (2013) (10.3%), Teixeira et al. (2019) (12%), Khen et al. (2015) (14%), Awadallah and Suelam (2014) (15%), Indrawattana et al. (2011) (15.4%), Kramarenko et al. (2013) (18.7%), and Demaîtrea et al. (2020) (45.6%). But it is higher than the finding of Nayak et al. (2015) (0%). Likewise, the milk sample-based prevalence of L. monocytogenes was found to be 2.6%. The current report is in agreement with the reports of Rawool et al. (2007) (0.41%), Rahimi et al. (2010) (1.1%), Garedew et al. (2015) (4.0%), Muhammed et al. (2013) (4%), Nayak et al. (2015) (4.0%), Hamdi et al. (2007) (4.29%), Kevenk and Gulel (2015) (5%), and Kalorey et al. (2008) (5.1%). But it is lower than the reports of Seyoum et al. (2015) (5.6%), Obaidat and Stringer (2019) (7.54%), Gebretsadik et al. (2011) (13%), Morobe et al. (2009) (5.3%), Reda et al. (2016) (8%), James et al. (2018) (8.8%), Kramarenko et al. (2013) (18.1%), Jamali et al. (2013) (21.7%), and Zafar et al. (2020) (40%). In general, the variation both in the overall and sample-wise prevalence rates of L. monocytogenes might be due to differences in sample types of foods of bovine origin, sources of the samples, processing plants, approaches of sample collection, sample size, methodological approaches, isolation and identification techniques, prevalence calculation/interpretation, geographical locations, hygienic conditions, handling and transportation of samples, and contamination rates from utensils and personnel. The serovars that were identified in the current study belonged to 1/2b and 4b. Demaîtrea et al. (2020) and Kevenk and Gulel (2015) were also reported these serovars in addition to some others. In general, the most common causes of human listeriosis among the 13 serotypes of L. monocytogenes are 1/2a, 1/2b, and 4b, and of these, serotypes 4b has been related to the most recent outbreaks of listeriosis, and serotypes 1/2a and 4b are commonly reported in animals (Datta et al. 2013; Doumith et al. 2004a; Doumith et al. 2004b; Kasalica et al. 2011; Kasper et al. 2009; Liu et al. 2006; Mateus et al. 2013; Roberts et al. 2006). The presence of molecular serogroup 1/2b and 4b isolates (potential serotype 4b) in food of bovine origin may pose a great health threat since L. monocytogenes 4b has caused numerous human listeriosis outbreaks (Ward et al. 2010).

In the present study, the antimicrobial susceptibility results indicated as large proportions of the isolates were found to be highly susceptible to ampicillin (88.46%) and vancomycin (84.62%). However, the isolates had shown the highest level of resistance against nalidixic acid (96.15%). The highest intermediate was observed in amoxicillin (57.69%). This is in agreement with the reports of Garedew et al. (2015) who reported a high degree of resistance against nalidixic acid (50%) and tetracycline (37.5%) but the highest level of sensitivity to vancomycin (100%); Khen et al. (2015) who reported 97% of susceptibility to ampicillin and vancomycin, Shen et al. (2013) who reported 100% of susceptibility vancomycin, Zafar et al. (2020) who reported 100% of susceptibility to ampicillin and vancomycin, James et al. (2018) who reported 100% of susceptibility to ampicillin, gentamycin, and vancomycin and 24.2% of resistance against tetracycline, Kevenk and Gulel (2015) who reported a high level of sensitivity ampicillin (78.9%), penicillin G (77%), erythromycin (71.2%), and vancomycin (67.3%), Rahimi et al. (2010) who reported 100% of susceptibility to vancomycin and 96.4% of resistant against nalidixic acid. This is contradicted by the findings of Welekidan et al. (2019) who reported a high degree of resistance against amoxicillin (50%) and vancomycin (50%), Jamali et al. (2013) who reported a high degree of resistance against clindamycin (100%), ampicillin (95.7%), erythromycin (95.7%), penicillin (91.3%), tetracyclin (82.6%), streptomycin (78.3%), and vancomycin (69.7%), Garedew et al. (2015) who reported a high degree of resistance against penicillin (66.7%) and the highest level of sensitivity to amoxicillin (100%), cloxacillin (100%), and Gentamicin (100%). It is similar to the findings of Wang et al. (2013) who reported 13.6% of intermediate response to ciprofloxacin. Moreover, 23.10%, 34.62%, and 42.31% of the isolates showed resistance to one, two, and more than two drugs, respectively. This is lower than the findings of Obaidat and Stringer (2019) who reported 96.9% of multidrug resistance. But, it is higher than the report of Garedew et al. (2015) (16.7%), and Escolar et al. (2017) who reported a 2% of resistance to two antibiotics and 2% resistance to three antibiotics, Kevenk and Gulel (2015) who reported that 15.3% of the isolates were resistant to only one drug and 36.5% were resistant to multiple drugs. But it is lower than the reports of Wang et al. (2013) who reported 72.3% of multiple drug resistance. In general, L. monocytogenes is usually susceptible to a wide range of antimicrobials. Nevertheless, the evolution of bacterial resistance towards antibiotics has been accelerated considerably by the selective pressure exerted by the over-prescription of drugs in clinical settings and their heavy use as promoters in animals husbandry (Charpentier et al. 1995). Moreover, the genes responsible for antibiotic resistance could be transferred through movable genetic elements such as conjugative transposons, mobilizable plasmids, and self-transferable plasmids to other foodborne bacteria in the gastrointestinal tract. In Listeria spp., efflux pumps have also been reported as the resistant mechanism (Lungu et al. 2011). Hence, the use of antimicrobials in veterinary medicine is the main cause of the development of AMR foodborne bacterial pathogens including L. monocytogenes, as AMR pathogens can easily be transported from animal to human via food consumption (Conter et al. 2009).

In conclusion, the current study revealed the occurrence of L. monocytogenes with different pathogenic serovars (1/2b and 4b) in raw meat and milk samples of bovine origin collected from different sites in the study area and the development of multidrug resistance to the tested antimicrobials. Hence, regardless of its prevalence rate, foods of bovine origin can serve as a potential vehicle for transmitting L. monocytogenes and can be a risk of infection to consumers of raw or undercooked milk and meat and are of significant concern for public health and give a warning signal for the possible occurrence of a foodborne outbreak in humans in the study area. It has an important public health implication when antimicrobial-resistant L. monocytogenes is present in raw foods, especially in developing countries like Ethiopia where there is a very common practice of consumption of raw meat and milk products and widespread and uncontrolled use of antibiotics. As a result, both its occurrence and development of a multi-drug resistance should receive particular attention and is an alert for the concerned bodies. Thus, a coordinated effort is imperative to reduce or eliminate the risk posed by this pathogen at various stages in the food chains and on the controlled and careful use of antimicrobials both in veterinary and human treatment regimes. Moreover, awareness creation should be made on foodborne diseases caused by L. monocytogenes with due consideration to the safe handling and consumption of foods of bovine origin and selection and safe use of antimicrobials. Finally, nationwide molecular epidemiology, phenotypic and molecular characterization, in-depth typing, and drug-resistant gene identification of L. monocytogenes and its serotypes should be undertaken.

ATCC American Type Culture Collection

CI Confidence interval

DNA Deoxyribonucleic acid

L. monocytogenes Listeria monocytogenes

LEB Listeria Enrichment Broth

OR Odds ratio

PCR Polymerase Chain Reaction

S. aureus Staphylococcus aureus

SFEB Secondary Fraser Enrichment Broth

TAE Tris-acetate-EDTA

TSA Tryptose Soya Agar plate

UK United Kingdom

USA United States of America

χ² Chi-square

Ethics approval and consent to participate

This study was reviewed and approved by the Research Ethics Committee of the College of Veterinary Sciences, Mekelle University. Owners, managers, and workers of the different sites were informed of the procedures and significance of the study. Each data and analysis result were kept confidential and communicated to concerned bodies. Any participants who were not volunteers were not forced to be included.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was funded by Norwegian Agency for Development Cooperation (NORAD) and One Health Central and Eastern Africa (OHCEA) project funds of College of Veterinary Sciences, Mekelle University. However, the APC was not funded. The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Authors’ Contributions

All the authors, TH, GG, YT, MA, and NA, were contributing in conceptualization, designing, funding acquisition, carrying out, data curation, analyzing and interpreting, writing, and revising the manuscript. All authors have read and agreed to the published version of the manuscript.

Acknowledgements

The authors would like to acknowledge the owners, managers, and workers of the different dairy farms, abattoir, butcher shops, restaurants, milk whole sellers, and cafeterias of the study site for their keen interest and cooperation during the collection of the samples, and to the College of Veterinary Sciences staff members who directly or indirectly helped us during the research period.

A preprint has previously been published (Kidanu et al. 2021).

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Occurrence, antimicrobial resistance pattern and molecular characterization of Listeria monocytogenes isolated from bovine’s milk and meat in Mekelle City, Ethiopia

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