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. monocytogenesL. 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: firstname.lastname@example.org.
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,
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
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
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
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