Derra, F. A.

Felix, B.

Geyid, A.

Granier, S. A.

Hendriksen, Rene S.

Karlsmose, Susanne

Mache, A.

Monga, D. P.

Svendsen, Christina Aaby

Taye, G.

Background. Listeriosis, a bacterial disease in humans and animals, is mostly caused by ingestion of Listeria monocytogenes via contaminated food and/or water, or by a zoonotic infection. Globally, listeriosis has in general a low incidence but a high case fatality rate. Objective. The objective of this study was to investigate the occurrence, antimicrobial profiles, and genetic relatedness of L. monocytogenes from raw meat and dairy products (raw milk, cottage cheese, cream cake), collected from the capital and five neighboring towns in Ethiopia. Methods. Two hundred forty food samples were purchased from July to December 2006 from food vendors, shops, and supermarkets, using a cross-sectional study design. L. monocytogenes were isolated and subjected to molecular serotyping. The genetic relatedness and antimicrobial susceptibility patterns were investigated using pulsed-field gel electrophoresis (PFGE) and minimum inhibitory concentration determinations. Results. Of 240 food samples tested, 66 (27.5%) were positive for Listeria species. Of 59 viable isolates, 10 (4.1%) were L. monocytogenes. Nine were serotype 4b and one was 2b. Minimum inhibitory concentration determination and PFGE of the 10 L. monocytogenes isolates showed low occurrence of antimicrobial resistance among eight different PFGE types. Discussion and Conclusions. The findings in this study correspond to similar research undertaken in Ethiopia by detecting L. monocytogenes with similar prevalence rates. Public education is crucial as regards the nature of this organism and relevant prevention measures. Moreover, further research in clinical samples should be carried out to estimate the prevalence and carrier rate in humans, and future investigations on foodborne outbreaks must include L. monocytogenes

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A.; Karlsmose Pedersen, Susanne; Monga, D. P.; Mache, A.; Svendsen, Christina Aaby; Felix,B.; Granier, S. A.; Geyid, A.; Taye, G.; Hendriksen, Rene S.Published in:Foodborne Pathogens and DiseasePublication date:2013Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Derra, F. A., Karlsmose, S., Monga, D. P., Mache, A., Svendsen, C. A., Felix, B., ... Hendriksen, R. S. (2013).Occurrence of Listeria spp. in retail meat and dairy products in the area of Addis Ababa, Ethiopia. FoodbornePathogens and Disease, 10(6), 577-579.Short CommunicationOccurrence of Listeria spp. in Retail Meatand Dairy Products in the Area of Addis Ababa, EthiopiaFirehiwot Abera Derra,1 Susanne Karlsmose,2 Dharam P. Monga,3 Abebe Mache,3 Christina Aaby Svendsen,2Benjamin Fe´lix,4 Sophie A. Granier,4 Abera Geyid,1 Girum Taye,1 and Rene S. Hendriksen2AbstractBackground: Listeriosis, a bacterial disease in humans and animals, is mostly caused by ingestion of Listeriamonocytogenes via contaminated food and/or water, or by a zoonotic infection. Globally, listeriosis has in generala low incidence but a high case fatality rate.Objective: The objective of this study was to investigate the occurrence, antimicrobial profiles, and geneticrelatedness of L. monocytogenes from raw meat and dairy products (raw milk, cottage cheese, cream cake),collected from the capital and five neighboring towns in Ethiopia.Methods: Two hundred forty food samples were purchased from July to December 2006 from food vendors,shops, and supermarkets, using a cross-sectional study design. L. monocytogenes were isolated and subjected tomolecular serotyping. The genetic relatedness and antimicrobial susceptibility patterns were investigated usingpulsed-field gel electrophoresis (PFGE) and minimum inhibitory concentration determinations.Results: Of 240 food samples tested, 66 (27.5%) were positive for Listeria species. Of 59 viable isolates, 10 (4.1%)were L. monocytogenes. Nine were serotype 4b and one was 2b. Minimum inhibitory concentration determinationand PFGE of the 10 L. monocytogenes isolates showed low occurrence of antimicrobial resistance among eightdifferent PFGE types.Discussion and Conclusions: The findings in this study correspond to similar research undertaken in Ethiopia bydetecting L. monocytogenes with similar prevalence rates. Public education is crucial as regards the nature of thisorganism and relevant prevention measures. Moreover, further research in clinical samples should be carried outto estimate the prevalence and carrier rate in humans, and future investigations on foodborne outbreaks mustinclude L. monocytogenes.IntroductionListeria monocytogenes is an important cause of dis-eases in both animals and humans. The incidence of hu-man listeriosis is low (0.1–11.3 cases per million inhabitants),but it is increasing in Europe (Goulet et al., 2008). The mor-tality rate is reported to be 20–30% (Swaminathan and GernerSmidt, 2007).In Ethiopia, few findings of L. monocytogenes have beenreported, possibly due to lack of attention or resources (Mollaet al., 2004).The objective was to investigate the occurrence and preva-lence of Listeria spp., the antimicrobial resistance, and clonalrelatedness among L. monocytogenes in samples of raw meatand dairy products fromAddis Ababa and neighboring townsin Ethiopia.MethodsA cross-sectional studywas conducted in 2006 at the PublicHealth Food Microbiology Laboratory of Ethiopian Healthand Nutrition Research Institute (EHNRI), Addis Ababa,Ethiopia.A total of 240 food samples—all different samples of rawmeat, unpasteurized milk, cottage cheese, and cream cake—were purchased randomly from different retail markets,1Food Microbiology Section, Food Science and Nutrition Research Directorate, Ethiopian Health and Nutrition Research Institute, AddisAbaba, Ethiopia.2WHO Collaborating Centre for Antimicrobial Resistance in Food-borne Pathogens and EU Reference Laboratory for AntimicrobialResistance, National Food Institute, Technical University of Denmark, Kingsly Lyngby, Denmark.3Microbiology, Immunology, and Parasitology Department, Medical Faculty, Addis Ababa University, Addis Ababa, Ethiopia.4Paris-Est University, ANSES, French Agency for Food, Environmental, and Occupational Health and Safety, Laboratory for Food Safety,European Union Reference Laboratory for Listeria monocytogenes, Maisons-Alfort, France.FOODBORNE PATHOGENS AND DISEASEVolume 10, Number 6, 2013ª Mary Ann Liebert, Inc.DOI: 10.1089/fpd.2012.1361577shops, supermarkets, and food vendors in suburbs of AddisAbaba and neighboring towns from July to December 2006.Samples of 500 g or 500mL of each food category werepurchased and examined according to the standard Interna-tional Dairy Federation method (IDF, 1990).Based on biochemical testing, 66 isolates were Listeria spp.,of which 59 remained viable after prolonged storage at - 20Cin trypticase soy broth (TSB) supplemented with 10%glycerol. The isolates were then subjected to speciation bypolymerase chain reaction assay (PCR) (Huang et al., 2007),genotyped by pulsed-field gel electrophoresis (PFGE)(Graves and Swaminathan, 2001), and antimicrobial suscep-tibility testing for minimum inhibitory concentration (MIC)determination (Aarestrup et al., 2007).The isolates were identified at the species level utilizing amultiplex PCR previously described (Huang et al., 2007).Modifications to the method included one pair of oligonu-cleotide primers: 5¢-CTCCCACATTGGTGCTACTCA-3¢ and5¢-GTTGCTGCGAACTTAACTCA-3¢. Amplicon sizes of 453,373, 609, 420, 465, and 289 bp, respectively, identified each ofthe species L. monocytogenes, L. seeligeri, L. welshimeri, L. grayi,L. ivanovii, and L. innocua.The 10 L. monocytogenes isolates were subjected to L. mono-cytogenes–specific serotyping utilizing a multiplex PCR assayaccording to Ke´rouanton et al. (2010). These isolates weregenotyped by PFGE usingApaI andAscI with optimization setof 1% (Graves and Swaminathan, 2001). Comparative analysisbased on unweighted pair group method using arithmeticaverages was performed using Bionumerics software version4.6 (Applied Maths, Sint-Martens-Latem, Belgium).MICdeterminationwas performed on the 10 L.monocytogenesisolates using Mueller-Hinton broth supplemented with 5%lysed bovine blood and a dehydrated panel from TREK Diag-nostic Systems Ltd. (East Grinstead, England) with an incuba-tion period of 24h. Antimicrobials and epidemiological cut-offvalues used were based on guidelines for L. monocytogenes fromthe European Committee on Antimicrobial Susceptibility Test-ing (EUCAST) when available (i.e., for erythromycin, penicillin,tetracycline and sulfamethoxazole+ trimethoprim). For the re-maining antimicrobials—cefoxitin, chloramphenicol, cipro-floxacin, florfenicol, gentamicin, spectinomycin, streptomycin,sulfamethoxazole, tiamulin, and trimethoprim—they were uti-lized as described (Aarestrup et al., 2007).ResultsSixty-six (27.5%) of the 240 food samples were positive byconventional culture technique for Listeria spp., and 59 iso-lates viable after prolonged storage positives were raw meat62.7%, raw milk 8.5%, cottage cheese 6.8%, and cream-cakesamples 22.0%, respectively.The species-specific PCR revealed that 10 (17%) of the 59isolates were L. monocytogenes and 49 (83%) isolates belongedto the species innocua. L. monocytogeneswas isolated from four(6.8%), two (3.4%), one (1.7%), and three (5.1%) samples ofraw meat, raw milk, cottage cheese, and cream cake, respec-tively. Nine of those belonged to serotype 4b and one (isolatedfrom cream cake) to serotype 2b.MIC determination of the 10 L. monocytogenes isolatesshowed low frequency of antimicrobial resistance. Intrinsicresistance to cefoxitin was not considered relevant; only re-sistance to the following five antimicrobials was observed: FIG.1.DendrogramshowingthegenotypicrelatednessamongListeriamonocytogenesisolatesbasedonApaI/AscIpulsed-fieldgelelectrophoresis(PFGE)fingerprints.SimilarityvaluewascalculatedusingtheDicecoefficient,andtoleranceandoptimizationweresetat1%.Profilesaregroupedtogetheraccordingtotheunweightedpairgroupmethodusingarithmeticaverages.578 DERRA ET AL.ciprofloxacin (n = 3), gentamicin (n = 1), penicillin (n= 1), tia-mulin (n= 10), and sulfamethoxazole (n = 1).The 10 L. monocytogenes isolates rendered eight differentPFGE types. One cluster was identified that contained threeindistinguishable isolates, of which two originated fromcream cheese sampled at different towns and dates, and onewas isolated from rawmeat on the same day and town as oneof the cream-cake isolates (Fig. 1).DiscussionThis study as well as the previous Ethiopian study by Mollaet al. (2004) showed that Listeria species exist in the Ethiopianfood production system. Molla et al. (2004) detected all speciesof Listeria, whereas in this study only L. monocytogenes andL. innocua were found. The reason might relate to the limitednumber of collected samples. We found L. innocua to be themost predominant species (83%), which is in contrast to whathas previously been reported in theUnited States byZhang et al.(2007), who observed a prevalence rate of 65% in the same foodcategories as investigated here. The distribution of L. mono-cytogenes in indicated food types was compared with otherstudies, and similar findings were observed (Zhang et al., 2007).All 10 isolates in this study were resistant to tiamulin, aveterinary-approved antimicrobial usually described as ef-fective against L. monocytogenes for the last few decades. Aprevious study has already shown high levels of MICs totiamulin in L. monocytogenes originating from animals (Aar-estrup et al., 2007), which contributes to doubting the efficacyof tiamulin against L. monocytogenes. More worrisome is theresistance to gentamicin, which is recommended, in combi-nation with penicillins, as first-line treatment of listeriosis inhumans. A similar finding was reported in Botswana byMorobe et al. (2009).The diversity and cluster observed for the strains 22, 24,and 27 (Fig. 1) suggests a low clonal relatedness among theisolates in contrast to a normally high clonality of the species.Serotypes 4b and 2b isolated here have previously been as-sociated with human foodborne illness (Kathariou, 2002).Unfortunately, it has not been possible to compare the PFGEpatterns of serotype 4 in this study with isolates belonging toepidemic clones (Kathariou, 2002).This study highlights the presence of L. monocytogenes andL. innocua in raw food items from Ethiopia. Considering therisk of contracting listeriosis due to the practice of consumingraw food such as rawmeat and unpasteurized and fermentedflavored milk, especially in association with poor knowledgeof goodmanufacturing practice and of handling the products,we suggest that the Ethiopian authorities liaise with theWorld Health Organization to assist the implementation ofthe technology for an integrated production-to-consumptionapproach to food safety. Such an approach relies mainly onthe hazard analysis and critical control point system and goodmanufacturing practice (www.who.int/foodsafety/fs_man-agement/en/). Furthermore, we suggest that the Ethiopianauthorities provide educational information for the Ethiopianconsumers by promoting the World Health Organization’s‘‘Five Keys to Safer Food’’ (www.who.int/foodsafety/fs_management/en/).AcknowledgmentThis work was supported by the Ethiopian Health andNutrition Research Institute, Addis AbabaUniversity, UNIDO,and WHO Global Foodborne Infections Network (www.who.int/gfn).Disclosure StatementNo competing financial interests exist.ReferencesAarestrup FM, Knochel S, Hasman H. Antimicrobial suscepti-bility of Listeria monocytogenes from food products. FoodbornePathog Dis 2007;4:217–221.Goulet V, Hedberg C, Le Monnier A, De Valk H. Increasingincidence of Listeriosis in France and other European countries.Emerg Infect Dis 2008;14:734–740.Graves LM, Swaminathan B. PulseNet standardized protocol forsubtyping Listeria monocytogenes by macrorestriction andpulsed-field gel electrophoresis. Int J Food Microbiol 2001;65:55–62.Huang B, Eglezos S, Heron BA, Smith H, Graham T, Bates J,Savill J. Comparison of multiplex PCR with conventionalbiochemical methods for the identification of Listeria spp.isolates from food and clinical samples in Queensland, Aus-tralia. J Food Prot 2007;70:1874–1880.[IDF] International Dairy Federation. Detection of Listeriamonocytogenes. International Provisional IDF standard 143:1990 milk and milk products, IDF Square Vergote 41, B-1040.Brussels, Belgium, 1990.Kathariou S. Listeria monocytogenes virulence and pathogenicity,a food safety perspective. J Food Prot 2002;65:1811–1829.Ke´rouanton A, Marault M, Petit L, Grout J, Dao TT, Brisabois A.Evaluation of a multiplex PCR assay as an alternative methodfor Listeria monocytogenes serotyping. J Microbiol Methods2010;80:134–137.Molla B, Yilma R, Alemayehu D. Listeria monocytogenes and otherListeria species in retail meat and milk products in AddisAbaba, Ethiopia. J Ethiop Health Dev 2004;18:131–212.Morobe IC, Obi CL, Nyila MA, Gashe BA, Matsheka MI. Pre-valence, antimicrobial resistance profiles of Listeria mono-cytogenes from various foods in Gaborone, Botswana. African JBiotechnol 2009;8:6383–6387.Swaminathan B, Gerner-Smidt P. The epidemiology of humanlisteriosis. Microbes Infect 2007;9:1236–1243.Zhang Y, Yeh E, Hall G, Cripe J, Bhagwat AA, Meng J. Char-acterization of Listeria monocytogenes isolated from retail foods.Int J Food Microbiol 2007;113:47–53.Address correspondence to:Firehiwot Abera Derra, MScFood Microbiology SectionFood Science and Nutrition Research DirectorateEthiopian Health and Nutrition Research Institute (EHNRI)Gulele subcity of Addis Ababa, Kebele 08Addis Ababa, EthiopiaE-mail: firehiwot_a@yahoo.comLISTERIA IN ETHIOPIA 579

Occurrence of Listeria spp. in retail meat and dairy products in the area of Addis Ababa, Ethiopia.

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Occurrence of Listeria spp. in retail meat and dairy products in the area of Addis Ababa, Ethiopia.

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