Keywords

Antimicrobials; Debre-Birhan; Detection; Isolation; Listeria monocytogene; Raw milk; Susceptibility

Introductions

The genus Listeria is a group of closely related Gram-positive, facultative anaerobic, non-spore forming, rod shaped, and motile bacteria. Genus Listeria comprises ten species, i.e., L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. seeligeri, L. grayi, L. murrayi, L. marthii, L. fleischmannii, and L. weihenstephanensis, [1]. Listeria monocytogenes is the principal pathogen in humans and animals. Listeria ivanovii is a pathogen of animals but is occasionally implicated in human disease. The other Listeria species are generally considered as non-virulent [2].

Listeria monocytogenes, the causative agent of Listeriosis is ubiquitous in the environment and has been recognized as animal pathogen since the 1920s but in the past two decades, it has been implicated in several outbreaks of food-borne illness in humans [3,4]. Contamination of silage leads to infection of farm animals resulting in possible human infection by way of the food chain [5,6]. Food-borne transmission constitutes the main acquisition route of Listeriosis [7].

In humans, food-borne L. monocytogenes causes large outbreaks of Listeriosis, with a mortality rate of 9% to 44% [8]. Certain sections of the population are predisposed to the development of Listeriosis due to presence of existing chronic illness, suppression of the immune system, pregnancy, or extreme youth or age (under 1 year or over 60 years) [5]. This presents a significant public health problem because in such population, Listeriosis is fatal in up to 30% of cases [9] which may increase up to 75% in high risk groups such as pregnant women, neonates and immune-compromised persons [10].

L. monocytogenes the leading cause of death among bacterial pathogens acquired primarily through the consumption of contaminated foods [11]. Transmission is generally through eating contaminated food, particularly dairy products made from unpasteurized milk and ready-to-eat meat and fish products [12]. The incidence of L. monocytogenes in soft and semi-soft cheese varied from 0.50% to 46.00% [3,13,14].

Raw milk can be contaminated from the environment or by direct excretion into the milk; therefore, consumption of raw milk is associated with increased risk factors. L. monocytogenes has been largely studied in the past decades because of its importance as a food-borne human pathogen [15,16]. Ongoing efforts are needed to further reduce the incidence of listeriosis, due to the manifestation of its high mortality rate [17].

Listeria species detection and identification in foods traditionally involve culture methods based on selective pre-enrichment, enrichment and plating. This is followed by the characterization of Listeria species using colony morphology, sugar fermentation and haemolytic properties [18,19]. In the last years there has been a notable development of new culture media for the improved detection of L. monocytogenes in foods, and efficient methods based on antibodies or molecular techniques have also been developed [18,19].

In developing countries most of the time, there have been few or no reports on L. monocytogenes. This is true because no one has given it attentions or awareness on the occurrence of L. monocytogenes in food [20]. In Ethiopia, there is inadequate published information regarding to the prevalence and antimicrobial susceptibility patterns of Listeria species in both Veterinary and Public health sectors [21]. Therefore, the present study was undertaken to isolate Listeria species from raw milk and to determine the antimicrobial resistance profile.

Materials and Methods

Study area

The study was conducted in Debre-birhan town, North Shoa, Ethiopia. The Town is located 130 km away from the capital city (Addis Ababa) at 9041’ N latitude and 39032’ E longitude and the elevation is ranged from 2700 to 2800 meter above sea level. The area receives annual rain fall ranges from 814 to 1080 mm with about 70% rain falling between June and September. The annual average temperature of the town ranges between 40C in the coldest month (August) to 26°C in the hottest month (April) which is categorized under “dega” or tropical climate weather condition [22].

Study design

A cross-sectional study was conducted at Debre-Birhan town, to determine the prevalence of L. monocytogenes and other Listeria species, and also the antimicrobial susceptibility profile of L. monocytogenes.

Sample size determination

Since there was no previous study in the area on raw milks from dairy producers, sample size was estimated by taking 50% expected prevalence with 95% confidence interval and 5% desired absolute precision [23]. Accordingly a total of 384 dairy producers were included in this study. In addition, 23 venders were also sampled for the detections of L. monocytogenes.

Sampling method and collection

Study samples were selected using simple random sampling technique and 384 dairy producers that deliver their milk to 13 milk collection centers were sampled. All dairy producers encountered in this study were smallholder dairy farms having one to five, and some are more than five lactating dairy cows. Each raw milk samples were collected in sterile snap-cap milk (sterile plastic containers). 30 ml of raw milk were collected aseptically in sterile plastic containers according to Jorgensen [24], from milk containers of the dairy producers at the sites of milk collection centers. 23 milk samples from bulk milk container of vendor’s were also taken. The collected samples were identified by date of collection, code of the sample and their source. All milk samples were transported immediately by using icebox to Debre-brihan agricultural research center microbiology laboratory and stored at 4°C until analysis.

Isolation and identifications of Listeria species

Bacteriological examination was done to isolated L. monocytogenes and other species of Listeria from raw milk samples. For the isolations and identifications of Listeria species in food samples, the techniques recommended by the International Standards Organization (ISO 11290-1, 1996) [25] and the French Association for Standardization [26], were employed. Oxford agar was used for selective plating and identification of Listeria species.

Primary and secondary selective enrichment

Twenty five milliliters of each sample from the composite mixture was measured and transferred into 225 ml of previously prepared half Fraser broth followed by mixing the suspended sample for two minutes using a laboratory blender at 2400 rev/ min. Finally the samples were incubated at 300C for 24 hrs. After 24 hrs of incubation, the inoculated half Fraser suspension was mixed by swirling the Erlenmeyer flask and 0.1 ml was drawn and inoculated into 10 ml of Fraser broth and then incubated for 48 hrs at 350C. Growth in the Frazer broth was very apparent, with colour change from the initial golden yellow to dark green or black; unlike in the half Fraser which has no showed colour change.

Isolation and identification

As a continuation of the procedure from the selective enrichment broth, a positive Fraser broth media with black /dark brown or dark green color was inoculated by taking a loop-full of the suspension into selective media Oxford agar (OXA) and the plate were incubated at 370C for 48 hrs. Growth of 1-2 mm diameter black or black green colony with a black halo and black sunken center was taken as positive for Listeria species. The colonies of Listeria were identified as per Falana [27], on Oxford agar colonies appeared brown black or greenish black with a depressed center and a surrounding black halo. When the colonies were grown and well isolated on the selective media, about 3-5 colonies from each medium was picked and inoculated into pre dried plates of Tryptone Soya Yeast Extract Agar and incubated for 18 to 24 hrs at 370C to obtain pure culture. Any grey-whitish colony was suspected to be positive for Listeria. Typical colonies (1 mm to 2 mm in diameter, convex, colorless and opaque), were used for further biochemical tests. Gram staining, Catalase test, Motility test, and CAMP tests were also carried out. For the carbohydrate utilization test, the isolated colonies from TSYEA were transferred into test tubes that contain Xylose, Rhamnose and Mannitol and incubated at 37°C for up to 5 day. A positive reaction (acid formation) was indicated by yellow color production and occurred mostly within 24 h to 48 h (Table 1) [28].

Table 1: Differentiations of main Listeria species [28].

Antimicrobial susceptibility test

Antimicrobial susceptibility test were performed for L. monocytogenes isolates by using Muller Hinton Agar. The common conventional antimicrobial drugs (cephalotin, Nalidixic acid, chloramphenicol, kanamycin, steptomycin, tetracycline, vancomycin, gentamicin and Ampcillin) were tested. The method applied for antimicrobial testing was agar plate antibiotic disk diffusion method by adjusting to 0.5 McFarland turbidity standards [29,30]. About 2-3 pure colonies of the isolates were taken from the Tryptone Soya Yeast Extract Agar and suspended in Muller Hinton broth and then, incubated at 370C for 1-2 hrs. The suspension was checked for the development of slight turbidity. It was inoculated to Mueller-Hinton agar plate by using a sterile cotton swab. Plates were seeded uniformly by rubbing the swab against the entire agar surface. After the inoculums were dried, antibiotics impregnated disc were firmly placed on the surface of the inoculated plates. The plates were incubating at 370C for 24 hrs. After incubation the zones of inhibition around each disc were measured and the results were interpretate as sensitive, intermediate and resistant using a standard zone interpretative chart [31].

Quality control

The correct performance of all stages of the analysis, including enrichment, screening tests, plating and all confirmatory tests were verified through the use of L. monocytogenes (ATCC 11911) reference strains. Staphylococcus aureus (S. aureus) (American type culture collection (ATCC) 25923), L. monocytogenes (ATCC 11911) kindly obtained from the Ethiopian Public Health Institute (EPHI), Addis Ababa, Ethiopia.

Data management and analysis

Microsoft Excel was utilized for data entry and storage and analysis were done by using statistical package for social science (SPSS 2007, version 16). Descriptive statistics were used to describe and process the data. Comparisons between detection rates of groups were analysed by using Chi square (χ2). For all tests, p-value less than 0.05 were considered to be significant.

Result

A total of 407 samples of raw bovine milk, out of which 384 from Dairy producers and 23 from Vendors were sampled for isolation and identifications of Listeria species by bacteriological examination, 85 (20.88%) proved positive for Listeria. The identification results showed that raw bulk milk from vendors was highly contaminated with Listeria species with the detection rates of 30.4% and raw milk from dairy producers were 20.3%. Statistically there was no significant difference (P>0.05) with Listeria isolation among dairy producers and vendor at the study area (Table 2).

Table 2: Isolation and detections of Listeria species in raw milk from Dairy producers and Vendors.

In this study, both pathogenic and nonpathogenic Listeria species were isolated from raw milk samples of dairy producers and vendors in the study area. The detections of L. monocytogenes in both dairy producers and vendors were 8.6% and 13% respectively (Table 3).

Table 3: The prevalence of L. monocytogenes from different sources of samples.

As depicted in the above Table 3, the level of identification of L. monocytogenes in vendor was 1.6 times (OR=1.6, CI=0.45- 5.65) higher compared to milk samples originated from dairy producers. But it was not statistically significant (p=0.75).

The bacteriological analysis indicated that L. monocytogenes and L. innocau were found with higher prevalence followed by L. seeligeri. The frequency of isolation of Listeria species from raw milks of the dairy producers and vendor are summarized as depicted below in (Table 4).

Table 4: Numbers of Listeria species isolated from raw milks of dairy producers and Vendors.

Antimicrobial susceptibility test

Of the total 36 L. monocytogenes species subjected for antimicrobial susceptibility test, 11(30.5%) exhibited resistance for Nalidixic acid, 9(25%), 8(22.2%) and 4(11.1%) of L. monocytogenes were resistance to Tetracycline, Chloramphenicol and Streptomycin’s respectively. All of L. monocytogenes isolated in this study were sensitive to Cephalotin, Kanamycin, Vancomycin and Ampcillin (Table 5).

Table 5: Antimicrobial resistance profiles of Listeria monocytogenes from milk samples.

Of these L. monocytogenes exhibited resistance for one or more antimicrobials, one (2.7%) L. monocytogenes were resistances to four antimicrobials (Streptomycin, Chloramphenicol, Tetracycline and Naldixic acid), Nine (25%) L. monocytogenes were resistance to two antimicrobials and Ten (27.8%) of L. monocytogenes were resistance to one antimicrobial.

Discussion

In this finding raw milk samples were analyzed for the presence of L. monocytogenes and other Listeria species. The overall isolations of Listeria species were 20.88% from 407 raw milk samples examined. The present finding was comparable with other reports in Ethiopia; Addis Ababa 22 % and Gondar 25% from raw milk samples [21,32], respectively. In others countries like Uganda, 60% of bulk raw milk were positive for Listeria species [33], which is with higher prevalence compared to the present studies (20.88%). Such differences are might be due to poor hygiene and sanitation activities in the milk production, processing and supplying chains, since the origins of contaminations of milk with Listeria are mainly faeces [34]. In Ethiopia relatively low prevalence’s of Listeria were reported in Jimma town 14 % and in Addis Ababa 8.3% from raw milk samples [35,36], respectively. These different isolations might be due to either by the difference of samples sizes or hygienic practicing and /or sample collecting origin. The isolated species of Listeria in the present study was L. monocytogenes 36(8.8%), L. innoccau 28(6.88%), L. seeligeri 14(3.4%), L. grayi (0.74%), L. welshimeri (0.49%) and L. murrayi (0.49%) indicated as diminishing orders.

In this study almost from both (dairy producers and vendors) sampling origin the presence of L. monocytogenes in raw milk, higher bacterium isolation were observed in vendors. This might be due to supply chain contamination from dairy producer to the retailer transportations or might be the retailers’ handling exposed to contaminations. Because of L. monocytogenes are found everywhere; in water, soil, vegetables and in healthy persons without showing symptom [2]. One of the main reasons for lower detection of Listeria in the dairy producers might be due to early supplying of the milk to collection centers where there are few chances of contamination from handlers and the environment [36].

Money researchers have been reported the occurrence of Listeria species in raw milks, according to Yakubu [37], report, out of 192 raw milk samples 76 (39.58%) were positive for Listeria species. Out of which 39 (20.3%) were L. innocua, 14 (7.3%) L. ivanovii, 17(8.9%) L. monocytogenes, 4(2.1%) L. welshimeri and 2(1%) L. seeligeri. Another studies conducted in Algeria by Hamdi [38], from raw milk samples, the isolations of L. monocytogenes were 7.7%. The detections of L. monocytogenes from raw milk was 6.5% in Ireland and 5.5% in Finland [39,40], respectively. A total of 240 milk samples from farm bulk milk tanks collected in Tehran province, Iran, 54 (22.5%) were positive for Listeria species out of which L. monocytogenes 13(5.4%), L. welshimeri 6(2.5%) L. innocau 33 (13.8%) and L. seeligeri 2 (0.8%) [41]. From these reports of Listeria species isolation, L. monocytogene were comparable with the present findings. Whereas other more recent reports in the isolations of Listeria species from unpasteurized raw milk were 13.46% and L. monocytogenes 4.81% [42], relatively lower detection compared to the present studies. Similarly D’costa [43], study showed that the isolations of L. monocytogenes was 4.82% in raw milk samples from different markets of Mumbai city, this study result was lower compared to the present findings.

Antimicrobial resistance profiles of L. monocytogenes were exhibited in this study. Of these 58.3% of isolated L. monocytogenes were resistances at least for one or more antimicrobials tested. One (2.7%), Nine (25%) and Ten (27%) of L. monocytogenes were resistance to four antimicrobial (Streptomycin, Chloramphenicol, tetracycline and Naldixic acid), two antimicrobial and one antimicrobials respectively. Further analysis of antimicrobial susceptibility tests result showed that 25% of the isolated L. monocytogenes were resistance to tetracycline which might be ascribed to high level utilization of this antibiotic due to its relatively cheaper price and easily accessible. Tetracycline is one of the most frequently prescribed drugs in the current study area for most of infectious diseases in veterinary medicine that could be mentioned as one of the reasons for the development of such higher resistance profile. Recent study conducted by saha [44] L. monocytogenes were 100% sensitivity to penicillin, tetracycline and gentamicin. In our finding, majority of L. monocytogenes isolated were susceptible to Cephalotin and Vankomycin which was in line with the recent study in Gondar, Ethiopia [21].

Resistance of L. monocytogenes to nalidixic acid, tetracycline and chloramphenicol were observed in the present study which is similar with studies conducted by [3,43,44]. The presence of antimicrobial resistant L. monocytogenes in raw food products has an important public health implication especially in developing countries where there is a wide spread and uncontrolled use of antimicrobials [45]. The problem can be higher in Ethiopia since consumption of raw meat, raw milk and raw milk products are very common and large number of high-risk population are found in the country. The increased antimicrobial administration to animals can cause the development of antimicrobial resistance L. monocytogenes [46]. Multidrug resistant L. monocytogene isolated from foods and human Listeriosis has been previously reported by Safdar and Marian [47-49]. The overall frequencies and patterns of resistance can vary remarkably from one country to another and also one reports to other even within a country.

Conclusion and Recommendations

In this study the results of bacteriological assessment showed that milk from dairy producers and vendors are positive for Listeria. In this finding both pathogenic L. monocytogenes and nonpathogenic Listeria species were isolated. The presence of L. monocytogenes in food can poses a threat to human life and can cause outbreaks of morbidity and mortality. Isolations of L. monocytogenes in raw milk and its antimicrobial resistant is an indication of public health hazards to the consumer, particularly to the high risk group such as young, elder, pregnant women and immune-compromised individuals. Raw milk intended for human consumption must be subjected for pasteurization or heat treatment at equivalent to pasteurization temperature. Further studies on the risk factors, molecular characterization and serotyping of the bacteria should be carried out. Awareness creation on food safety and implementation of regulations about the use of antimicrobials in humans and animals should be strongly practiced.

Acknowledgement

We would like to thank Debre-Birhan Agricultural Research Center for laboratory facility support to smoothly accomplishment of this work. Although we want to acknowledge Ethiopian public health institute (EPHI) and Aklilu lemma institute of pathobiology, Addis Ababa University for providing us control strain (S. aureus and L. monocytogens) and chemicals respectively.

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