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African Journal of Biotechnology Vol. 8 (22), pp. 6383-6387, 16 November, 2009 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 2009 Academic Journals Prevalence, antimicrobial resistance profiles of Listeria
monocytognes from various foods in Gaborone,
Botswana
Morobe, I. C.1, 3, Obi, C. L.2*, Nyila, M. A.1, Gashe, B. A.3 and Matsheka, M. I.3
1School of Agriculture and Life Sciences, Department of Life and consumer Sciences, University of South Africa, 2Academic and Research Directorate, Walter Sisulu University, Nelson Mandela Drive Mthata, Eastern Cape, South 3Department of Biological Sciences, University of Botswana, Mabutu Drive, Private Bag UB00704 Gaborone, Botswana. Listeria monocytogenes is known to cause epidemic and sporadic cases of listeriosis. The present
study investigated the occurrence, antibiograms and molecular serotypes of the organism in various
retail outlets in Gaborone, Botswana. Food samples were obtained randomly from selected
supermarkets and street vendors in 5 geographical areas of Gaborone from May, 2007 to September
2007. L. monocytogenes was isolated and positively identified by using morphological and biochemical
tests. From a total of 1324 food samples tested 57(4.3 %) were positive for L. monocytogenes. Out of
the 57 isolates of L. monocytogenes 7 (12.3%), 3 (5.3%), 0 (0%), 27 (47.4%) and 20 (35.1%) were isolated
from cheese, raw milk, meat (biltong), frozen cabbage and salad (coleslaw), respectively. From the 5
geographical areas selected for sampling in this study, Gaborone South recorded the highest number
19 (33.3%) of L. monocytogenes isolates while Gaborone West recorded the least, 7 (12.3%). The
findings in this study reveal the presence of L. monocytogenes serotypes 1/2a and 1/2b in ready to eat
food and highlight the need for education and training programmes in food safety in Gaborone,
Botswana.

Key words: Listeria monocytogenes, antibiotic resistance, molecular serotyping, food, Botswana.


INTRODUCTION
Listeria monocytogenes causes a very serious il ness try due to its ability to grow in gas or vacuum-packaged known as listeriosis. Individuals who are particularly products at refrigeration temperatures (Duffy et al., 1994), susceptible to this condition are those who are low water activity (Nolan et al., 1992) as wel as low pH immunocompromised (as in HIV/AIDS infection), preg- (Buchanan et al., 1993) and al these measures are nant women, newborn babies, and the elderly (Farber important in the control of food pathogens. and Peterkin, 1991; McLauchlin et al., 2004). Although L. monocytogenes is also problematic due to its resis- the incidence of listerosis is low, what is significant is that tance to antibiotics. The first multiresistant strain of L. very high fatalities ranging from 20 to 30% have been monocytogenes was isolated in France in 1988 (Poyart - Salmeron et al., 1990), thereafter L. monocytogenes L. monocytogenes is widely distributed in nature and strains resistant to one or more antibiotics have since has been isolated from a wide array of food products. been isolated (Franco Albuin et al., 1994; Charpentier et The organism is considered hazardous in the food indus- It has been shown by various studies that listeriosis is a food-mediated il ness (Embarek, 1994; Slutsker and Schuchat, 1999). A wide range of foods such as salads, *Corresponding author. E-mail: lobi@wsu.ac.za. seafoods, meat, and dairy have been implicated in liste- riosis (Bel and Kyriakides, 1998; Schlech, 2000), which Table 1. Incidence of Listeria monocytogenes in various food
fol ows the oral ingestion of the contaminated food (Finlay, 2001). Studies have shown that the number of L. monocytogenes cel s can rise fol owing refrigeration from % of positive
fewer than 100 cel s per gram, and this is the dose that is Food product
No. of isolates
isolates
general y accepted for healthy people (Huss et al., 2000; Serotyping has been used extensively to characterize L. monocytogenes (Wieldmann, 2002; Wagner and Al erberger, 2003). Thirteen L. monocytogenes serotypes (serovars) have been characterized in this species by using specific and standardized sera (Seeliger and Langer, 1979). Although most clinical isolates belong to serovars 1/2a, 1/2b, and 4b are the majority of strains were sub-cultured on nutrient agar. A Gram stain was performed on suspected colonies and Gram positive short rods colonies were which have caused large outbreaks were serovar 4b sub-cultured onto tryptose soy agar (Merck, Darmstadt, Germany) (Kathariou, 2000), and serovar 1/2a (Jacquet et al., 2002;
slants and these were maintained at 4°C. Fol owing this, the Zhang and Knabel, 2005). Serovar identification by catalase test was performed on al the isolates and catalase serological tests has remained popular. However, positive isolates were identified to the species level by API Listeria numerous molecular biology methods such as multiplex (Oxoid). Isolates that were confirmed as L. monocytogenes were PCR (Doumith et al., 2004) have come to the fore in the preserved in a solution containing 80% tryptose soy broth (Oxoid) and 20% glycerol at -80°C for use in the steps that fol owed. characterization of L. monocytogenes serotypes. Even though a recent study (Manani et al., 2006) reported the occurrence of L. monocytogenes in frozen vegetables in Antibiotic susceptibility testing
this country, there is little data on the occurrence of this pathogen in foods in Botswana. This investigation was Isolates that were confirmed as L. monocytogenes were inoculated carried out to unravel the prevalence and scope of on Muel er-Hinton broth (Oxoid, Basingstoke, Hampshire, England). The flasks were incubated at 37˚C on a Gal enkamp shaker (200 antimicrobial resistance profiles of L. monocytogenes rpm) for 24 h. The turbidity of the actively growing broth culture was serovars isolated from various retail foods in Gaborone, adjusted with sterile saline to obtain turbidity optical y comparable to that of the 0.5 McFarland standard. One mil iliter of the cel suspension was then transferred onto the surface of Muel er-Hinton agar (Oxoid, Basingstoke,Hampshire, England) and then spread evenly. The susceptibilities of al isolates to different antimicrobial MATERIALS AND METHODS
agents were tested by the disk-agar method as standardized by the National Committee for Clinical Laboratory Standards (NCCLS, Sampling
1998). The fol owing panel of antimicrobial disks and concentrations were used; chloramphenicol (25 g), erythromycin (5 g), fusidic Samples were obtained randomly from selected supermarkets and acid (10 g), methicil in (10 g), novobicin (5 g), penicil in G (1 U), street vendors in 5 geographical areas of Gaborone (east, west, streptomycin (10 g), tetracycline (25 g) (Mast Diagnostics, north, south, and central). Samples col ected were; raw vegetables Merseyside, UK) as wel as ampcil in (25 g), cephalothin (30 g), (cabbage), salads, raw milk, cheese and meat (biltong). In this sulphamethaxozole/trimethoprim (25 g), gentamicin (30 g), and study, 250 - 300 samples per product were obtained. The samples nitrofurantoin (10 g) (Oxoid). They were obtained from the South were put in separate properly labeled sterile specimen bags and African Bureau of Standards (Pretoria, South Africa) L. monocyto- placed into a cooler box containing ice packs. Gloves were worn to genes ATCC 19115 was used as the reference strain. avoid cross-contamination between samples from different super- markets and street vendors. Aseptic technique was fol owed to avoid contamination during transport of the samples from the Prevalence of L. monocytogenes
Enrichment, culturing, morphological and biochemical
identification
L. monocytogenes was found in al food products except biltong. Table 1 shows that among the food products, the On arrival at the laboratory, the samples were transferred to pro- perly labeled stomacher bags and then homogenised with the organism was frequently isolated from frozen cabbage Stomacher (Seward 400, Tekmar, and Cincinnati Ohio, USA) set at (10.11%), while raw milk recorded the least number of medium speed. The homogenised samples were enriched by isolates (1.08%). L. monocytogenes was not isolated at placing 25 g of the sample into 225 ml enrichment broth (Mast Diagnostcs, Merseyside, UK) and incubated at 30˚C for 48 h on The geographical areas from which L. monocytogenes Innova 4000 Newbrunswick Scientific incubator shaker. A loop ful was sampled seemed to affect the incidence of the of culture was sub-cultured on Modified Listeria Selective Agar (Oxoid, Basingstoke, UK) supplemented with Listeria Selective organism (Table 2). Of the geographical zones sampled Enrichment Supplement (Oxoid) and then incubated at 37ºC for 24 in this study, Gaborone South recorded the most number h. Dark brown colonies with black zones characteristic of Listeria (33.33%) whilst Gaborone West recorded the least Table 2. Prevalence of L. monocytogenes in different localities in
cheese was less than the 8.2% reported in the United Kingdom (Greenwood et al., 1991) and the 6.4% reported in Germany (Rudolph and Scherer, 2001). In a previous No. of positive
% of positive
study in Iran (Moshtaghi and Mohamadpour, 2007), the Geographical area
isolates
isolates
incidence of L. monocytogenes in raw milk was found to be 1.6%, and is similar to the 1.08% in the present study. The prevalence of the organism in cheese is hazardous to consumers because cheese is a ready to eat (RTE) food. Although raw milk is heat treated (pasteurization) before consumption, where the raw milk is processed to dairy products before pasteurization it poses a health risk Of public health concern is the occurrence of the (12.28%) with Gaborone East and North recording microorganism in frozen cabbage (10.11%) and salads (7.41%). A recent study (Little et al., 2007) found a lower proportion (6.0%) of salads to be contaminated with L. Antimicrobial
susceptibility
L.
monocytogenes while another study conducted in the monocytogenes
United States of America (Prazak et al. 2002) found an even lower number (2.34%) of cabbage samples to be Antimicrobial susceptibility testing was performed on al contaminated. The high incidence of the microorganism the 57 confirmed L. monocytogenes isolates. Of these in salads and cabbage are alarming because the salads isolates, 31 (54.39%) were found to be resistant to one or (coleslaw) are ready to eat and the cabbage is more antibiotic. Resistant rates to penicil in G, sometimes eaten raw when it is mixed with other sulphamethaxozole/trimethoprim, chloramphenicol, and vegetables to make salads. According to Little et al. tetracycline were encountered in 42.11, 29.82, 28.30, and (2007), the occurrence of L. monocytogenes in pre- 22.81%, respectively (Table 3). Antibiotic resistance was packaged mixed salads could result from the original not encountered for fusidic acid, erythromycin, methicil in, contamination of raw material, cross contamination ampicil in and cephalothin. In al , 15 different resistance during processing, packaging or at retail. In chil ed foods patterns were observed. Of the food products tested, such as coleslaw and frozen cabbage, temperature is the frozen cabbage and salads recorded the highest diversity principal control ing factor for their safety. However, L. of resistance patterns. From the resistance patterns, only monocytogenes has been shown to thrive under one pattern (penicil in G and tetracycline) was common refrigeration temperatures (Duffy et al., 1994). among al the food products that tested positive for L. Among the five geographical areas sampled in the monocytogenes (Table 4). Otherwise, the rest of the present study, the highest prevalence rate was recorded resistant patterns were unique or peculiar to the different in Gaborone south (33.33%) whereas Gaborone west recorded the least (12.28%). It is important to note that Gaborone South is characterized by overcrowding and
DISCUSSION
the inhabitants are general y of low economic and educational status. An interesting point to note from this In the present study contamination rates of food products study is that there were no significant differences in the with L. monocytogenes were 2.75, 1.08, 0.00, 10.11, and prevalence of L. monocytogenes in retail supermarkets 7.41% for cheese, raw milk, biltong, frozen cabbage, and and street vendors. A previous study in South Africa salads (coleslaw), respectively. L. monocytogenes is (Lues et al., 2006) found the microbiological quality of known to contaminate milk and milk products such as foods served by street vendors to be within acceptable cheese because of the complex nature of these products safety limits. In the same study the occurrence of specific (Cimmons, 2001). The non-occurrence of L. monocyto- microorganisms was thought to be indicative of a degree genes in biltong is significant in public health because the of ignorance on the part of the food handlers towards product is a ready-to-eat (RTE) and dry meat. Most reports on the occurrence of this food borne pathogen in This study found resistance to penicil in G, meats have tended to concentrate on raw meat, meat sulphamethaxozole/trimethoprim, chloramphenicol, and mixed with salads and poultry. Low water activity had tetracycline to be 42.11, 29.82, 28.30 and 22.81%, been shown to profoundly limit the growth and multipli- respectively. However, no isolate was resistant to fusidic cation of the pathogen in some conditions (Vermeulen et acid, erythromycin, methicil in, ampicil in and cephalothin. al., 2007). Biltong from this country is exported to the EU In contrast to the present study, Dhanashree et al. (2003) markets and the absence of the organism may be due to found no strain that was resistant to chloramphenicol in a the zero tolerance limits in the processing of the meat. study in India as did Facinel i et al. (1991) in a survey of The incidence rate (2.75%) of L. monocytogenes in Italian meat and dairy products. Tetracycline resistance Table 3. Susceptibility of L. monocytogenes to 13 antimicrobial agents.
Antibiotic
Sensitive (%)
Intermediate (%)
Resistant (%)
Table 4. Resistance patterns of L. monocytogenes isolates from food sources.
Food source
No. of isolates Resistant pattern
No. of resistant strains
% Resistant

The antibiotics are Chloramphenicol (C), Gentamycin (CN), Novomycin (NO), Ntrofurantoin (NI), Penicil in G (PG),
Streptomycin (S) ,Sulphamethoxazole/Trimethoprim (SXT), Tetracyclin (T). has been the most frequently observed phenotype al., 1990) multi-resistant strains have been extensively among L. monocytogenes strains (Charpentier et al., 1995). Tetracycline resistance in this study was noted to The findings clearly highlight the occurrence of L. be much higher than the 8.4% reported USA (Zhang et monocytogenes (serotypes 1/2a and 1/2b) among al., 2007). Tetracycline resistance is thought to originate retailers and street vendors in the 5 geographical areas from the use of antibiotic in animal production (Schroeder of Gaborone. The presence of this human pathogen in et al., 2002). Antibiogram profiles point to the high diver- ready-to-eat foods should be considered as having signi- sity of the microorganism in the food products tested. ficant public health implications, particularly among the Since the first isolation of a multiresistant strain of L. immunocompromised and HIV/AIDS persons who are at monocytogenes in France in 1998 (Poyart -Salmeron et It was evident that L. monocytogenes was resistant to Huss HH, Jorgenssen LV, Vogel BF (2000). Control options for Listeria chloramphenicol, penicil in G, sulphamethaxazole/ monocytogenes in seafood. Int. J. Food Microbiol. 62: 267-274. trimethoprim and tetracycline, suggesting that the Jacquet C, Gouin E, Jeannel D, Cossart P, Rocourt J (2002). Expres- sion of ActA, Ami, InlB, and listeriolysin O in Listeria monocytogenes incorrect use of these antimicrobial agents for therapeutic of human and food origin. Appl. Environ. Microbiol. 68: 616-622. purposes in animals and humans may lead to the Kathariou S (2000). Pathogenesis determinants of Listeria monocyto- genes, In Cary JW, Linz JE, Bhatnagar D, Lancaster PA (ed.), We recommend education of stakeholders such as Technomics Publishing Co., Inc., Microbial Foodborne Dis. pp. 295- product suppliers, supermarket management, cleaning Little CL, Taylor FC, Sagoo SK, Gil espie LA, Grant K, McLauchlin J staff and hygiene specialists on the biology of food borne (2007). Prevalence and level of Listeria monocytogenes and other infections and the intricate need to maintain the cold Listeria species in retail pre-packaged mixed vegetable salads in the chain order to prevent food borne outbreaks. Lues JF, Rasephei MR, Venter P, Theron MM (2006). Assessing food safety and associated food handling practices in street food vending. Int. J. Environ. Health Res. 16: 319-28. ACKNOWLEDGEMENTS
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