Prevalence and Antimicrobial Resistance of Staphylococcus aureus Isolated from Retail Ready-to-eat Foods in Nigeria
The occurrence and antibiotic resistance of Staphylococcus aureus in 120 samples of ready-to-eat foods obtained from retail outlets and street vendors was studied. About 17.2% of the isolates were detected in suya, 13.4% in moin-moin, 29.7% in meat sausages and 32.6% in fish sausages. Rinse and wash water from food preparation centers had counts>103 cfu mL-1. The antibiotic resistance of the isolates was tested using eleven different antibiotics including ampicillin, streptomycin, tetracycline, chloramphenicol and ciproflox. The isolates were resistant to two or more antibiotics and seven different resistance patterns were recorded. Resistance to streptomycin and tetracycline was found for 60.0 and 59.2%, respectively for the isolates. Lower levels of resistance were found for erythromycin (0.83%) and rifampicin (1.6%). Ten strains of the isolates showed evidence of β-lactamase activity. The results indicate that ready-to-eat food samples are frequently contaminated with S. aureus and could be potential vehicle of resistant Staphylococcus foodborne intoxications.
Staphylococcus aureus continues to be a major cause of community-acquired and health-care related infections throughout the world. Animals, human food and the inanimate environment can provide a favorable environment for the transmission of S. aureus (Bertolatti et al., 2003; Lateef 2004).
Staphylococcus aureus as an indicator of contamination of processed foods could come from the skin, mouth, or nose of handlers (Acco et al., 2003). Colonization of humans with S. aureus occurs in the anterior nares and other body sites with a carriage rate of 19-40% reported for a normal population (Noble et al., 1967; Acco et al., 2003). It has been isolated from foods such as rice, spices, meat and dairy products (Owhe-Ureghe et al., 1993; Sokari, 1991). In developing countries, street vending of foods is common because it offers inexpensive foods at convenient locations. In contrast to their potential benefits, concerns over the safety and quality of these foods have been raised, because the vendors lack appreciation of basic food safety issues (Bryan et al., 1988; Ekanem 1998; Umoh and Odaba 1999). A number of data confirm the fact that S. aureus causes many outbreaks of food poisoning resulting from hand contact (Bryant et al., 1988). As reported by Sokari (1991), 86% of meat products, 13% of cowpea-based food products and 55% of fish products from part of eastern Nigeria were contaminated with S. aureus.
Antimicrobial resistance associated with food and water has been a global concern
(Kumar et al., 2005). It is now widely accepted that there is an association
between the use of antimicrobial agents and the occurrence of resistance. Antimicrobials
exert a selective pressure on microorganisms that acts as a driving force in
the development of antibiotic resistance and therefore their use is considered
a key issue in epidemiological studies (McGeers, 1998). Moreover, there remains
the possibility that resistance may be transmitted from antibiotic resistant
bacteria to the susceptible ones (Kessie et al., 1998). Multidrug resistant
pathogens travel not only locally but also globally, with newly introduced pathogens
spreading rapidly in susceptible hosts.
Antimicrobial resistant bacteria in foods threaten the efficacy of human drugs if antimicrobial resistance genes become incorporated into human bacterial populations (Smith et al., 2002). Surveillance of antimicrobial resistance is essential for providing information on the interventions, especially because the prevalence of resistance may vary widely between and within countries and over time (WHO, 2001).
The purpose of this study was to investigate the prevalence of Staphylococcus aureus in some ready-to-eat foods collected from different vendors in Umuahia Nigeria and to characterize the isolates by antimicrobial susceptibility. This will help to determine the potential hazards and possible health implications to humans that may be associated with the consumption of these foods.
MATERIALS AND METHODS
Description of Food Samples
Suya, thin slices of condiment-coated roasted beef on skewers, were purchased
at random from hawkers at traditional suya spots in Umuahia. Moin-moin (cowpea
pudding) prepared by placing slurries, contained in small aluminium plates or
wrapped in leaves in water and boiling until the slurries set as firm gels,
were purchased from retail hawkers in the market. Fish and meat sausage rolls,
fried fish and meat products were purchased from retail hawkers and from supermarkets.
Three independent replicate surveys of each vendor were collected. Water was
scooped from dish washing and hand washing basins using sterile beakers. All
the samples were transported to the laboratory of the Department of Microbiology
and analysed within 1h of collection or refrigerated at 5°C before being
analysed, but never longer than 2 days.
Soaking 10 g samples separately in 90 mL of sterile distilled water and
shaking them for 30 min at room temperature rehydrated the food samples. Serial
dilutions of all the samples were made in sterile distilled water. The dilutions
(0.1 mL) were spread on Mannitol Salt Agar (Oxoid Ltd, Basingstoke, UK) and
incubated for 24-48 h at 37°C. Typical Staph aureus colonies on MSA
were transferred to nutrient agar slants and incubated at 37°C. Only coagulase
positive strains (Collins et al., 1990) were tested further in accordance
with standard procedures (Speck, 1984).
Characterization of Isolates
All isolates of staphylococci were inoculated on to blood agar plates, which
were incubated aerobically at 37°C overnight to identify mucoid and pigmented
strains. Randomly selected Staph aureus isolates were subjected to the
following tests; catalase activity (Harrigan and McCance , 1976), growth in
nutrient agar supplemented with 7.5% (w/w) and 10% (w/w) NaCl and growth in
nutrient agar at 10° and 45°C (Baird-Parker, 1979). Nitrate reduction
in nitrate broth and coagulase activity by the tube assay (Baird-Parker, 1979).
Further characterization included production of acid from glucose, arabinose,
mannitol, mannose, lactose, sucrose and xylose (Devriese et al., 1985),
Antimicrobial Susceptibility Testing
The susceptibility of Staph aureus isolates to antimicrobial agents
was tested by the disk diffusion method using Mueller-Hinton agar (Oxoid, Basingstoke
England) and antibiotic disks (Abtek Biologicals Ltd.) according to the guidelines
of the National Committee for Clinical Laboratory standards (NCCLS, 2001). The
antibiotics used were Ciproflox (Cpx), Norfloxacin (Nfx), Gentamicin (Gen) Streptomycin
(Str), Rifampicin (Rfp), Erythromycin (Ery), Chloramphenicol (Chl.), Cloxacillin
(Clo), Cotrimoxazole (Cot), Amoxycillin (Amx) and Tetracycline (Tet). The commercial
antibiotic disks were placed on the agar plates previously seeded with 18 h-broth
cultures of the test organisms. The plates were incubated at 37°C for 48
h. The inhibition zones were measured, scored as sensitive, intermediate susceptibility
and resistant according to the NCCLS recommendations. Staphylococcus aureus
ATCC 12600 was used as a reference strain.
Determination of Minimum Inhibitory Concentrations (MIC)
The MIC of a commonly used antibiotic, amoxycillin was determined using
the paper disc method (Oloke, 2000). Sterile paper discs were dipped into different
concentrations of amoxycillin. Sterile paper discs dipped into sterile water
used for the dilution of the antibiotic were used as control. Each soaked disc
was then layered on Mueller-Hinton agar plates already seeded with an 18 h-broth
culture of the Staph aureus strains in duplicate. Each plate was incubated
at 37°C for 24 h and then examined for zones of inhibition. The lowest concentration
of the antibiotic, which inhibited growth, was taken as the MIC.
Assay for β -Lactamase Production
β-lactamase production was assayed using the method of Ahmad and Yadav
(1979). The cultures of the test organism was spot inoculated on to starch agar
and then incubated overnight at 37°C. The plates were then flooded with
freshly prepared phosphate-buffered saline containing potassium iodide, iodine
and penicillin. β-lactamase converts penicillin to penicilloic acid, which
reduces iodine to iodide monitored via decolorization of the starch iodine complex.
The presence of clear colorless zones around the bacterial growth is an indication
of β-lactamase production. All the bacterial isolates were tested for the
production of β-lactamases.
Three replicates were used for all the parameters tested and the mean of
three values reported. Results were subjected to a one-way analysis of variance
to compare mean values.
RESULTS AND DISCUSSION
The enumeration of S. aureus in food products is employed generally as a sanitation index (Adams and Moss, 1996). The presence of these organisms in food beyond certain critical limit is interpreted as indicating that the food in question has been exposed to condition that might introduce or allow proliferation of pathogenic microorganisms (Mukhopadhyay et al., 2002). The results obtained by the plating analysis of the different food products and related samples are shown in Table 1. Average staphylococcal counts noted were 1.8 and 2.1x103 cfu g-1, respectively and detected in about 32.6% of fish sausages and 29.7% of meat sausages. Presence of S. aureus was observed in 17.2% of suya and 13.4% of moin-moin samples. The mean log10 (±SD) counts of this microorganism per g of the different food products were not statistically different (p>0.05). Mean staphylococcal counts for wash water from food preparatory centers exceeded those of food samples in all cases (Table 1). These findings agree with previous results of contamination of ready-to-eat foods, which ranged from 13 to 86% (Sokari 1991; Mosupye and von Holy, 2000). Higher prevalence and mean counts of S. aureus have however, been reported in other ready-to-eat products (Adesiyun and Balsbirsingh 1996; Umoh and Odaba, 1999).
S. aureus is an important food-poisoning organism because of its cosmopolitan
distribution in nature. Its presence in ready-to-eat foods may be traced back
to the environment or human sources (Newsome 1988; Neihart et al., 1988;
|| Prevalence of S. aureus in ready-to-eat foods*
|*Values represent the means of triplicate determinations
The high incidence of S. aureus in meat and fish products may be a
reflection of, repeated hand contact with these foods at the point of sale (Sokari
1991). In addition, temperatures of 26-38°C are often encountered in the
retail points from which the samples were purchased; hence, growth of this organism
may have been favored. Outbreaks of staphylococcal food-poisoning have been
reported to occur as the result of contamination of precooked food, often through
unsanitary handling and holding food at temperatures that allow the growth and
toxin production (Newsome, 1988; Bergdoll, 1989; Synder and Poland, 1991). Whatever
the cause, appropriate control and good hygienic display of foods in protected
cabinets will minimize potential hazards. The observed high counts of Staph
aureus in rinsing water and wash water samples from food restaurants may
be attributed partly to post hand contamination from the variety of customers
who patronize these eating-houses. Additionally this may result from the washing
of cookery, cutlery and other utensils (Sokari, 1991). In traditional Nigeria
restaurants, it is common to observe customers eating with bare hands. The sanitary
conditions of the environment of these restaurants may also lead to contamination
of foods. The microbial contamination of ready-to-eat foods could be closely
related to the method of preparation and handling. As expected, fried fish and
meat products had lower contamination levels probably due to the combined treatment
of frying followed by garnishing with onions and pepper. Heat stressed microorganisms
that survived frying was probably capable of growing if sample were not conserved
under appropriate temperature conditions. International microbiological standards
recommend limits of bacterial contamination in the range of 10-102
cfu g-1 for total aerobic plate counts (ICMSF, 1986). About 33% of
the total food samples investigated was contaminated with S. aureus at
102 cfu g-1 levels. In comparison, although contamination
of ready-to-eat foods with S. aureus is not common in the UK, according
to studies by Elson et al. (2004), 75% of ready-to-eat cold sliced meats
and pate from catering and retail premises were of satisfactory/acceptable microbiological
quality and about 25% were unsatisfactory/unacceptable.
Since large numbers, typically >106 cfu g-1, are required for the production of enough toxin to cause illness, contamination is necessary but is not alone sufficient for an outbreak to occur (Adams and Moss, 1996). In particular, holding the product for sale at temperature and time that allow the organism to grow to hazardous levels could be risky. It is an acknowledged fact that unsold samples are usually presented for sale the next day perhaps with gentle heat treatment. Such unwholesome practices could result in higher levels of toxins due to build-up on subsequent days. S. aureus strains demonstrate elevated thermal resistance, which precludes inactivation by current culinary heating techniques (Synder and Poland, 1991).
The antibiotic sensitivity test and assay for the production of β-lactamase
were conducted to ascertain the source of the multi drug resistance phenomenon
frequently encountered among bacterial isolates in Nigeria. Notwithstanding
the comparably low counts of S. aureus in the ready-to-eat food products
examined, the fact that some of the isolated strains exhibited antibiotic resistance
indicates that the products could pose a public health risk to consumers.
|| Antibiotic Resistance In S.aureus Strains Isolated from Ready-
|| Resistance of S.aureus strains of different origins to a
variety of antibiotics
|| Antibiotic resistance profiles of S. aureus strains
isolated from ready-to-eat foods
|| MIC values of amoxycillin against strains of Staphylococcus
Table 2 summarizes the resistance patterns of all the S.
aureus strains to 11 antimicrobial agents. The percentage resistance of
the isolates for the antibiotics varied from 60% for streptomycin to 0.83% for
erythromycin. Most of the resistant strains for streptomycin were obtained from
moin-moin. Fried fish and meat samples yielded low levels of antimicrobial
resistant strains. High resistance was also found for tetracycline (59.2%) and
gentamicin (49.2%). Lower levels of resistance were observed among the strains
for chloramphenicol, norfloxacin and ciproflox. 65.8% of S. aureus strains
were multiresistant (resistance to two or more antibiotics) (Table
3). Most of the isolates fell into one of seven major resistance groups.
The predominant pattern was resistance to streptomycin, gentamicin and tetracycline
(Table 4). Even though the food samples were obtained from
hawkers and retail outlets at different locations, the isolates showed similar
antibiotic resistance patterns. The MIC of Amoxycillin against the strains of
S. aureus was determined (Table 5). The MIC (μg
mL-1) ranged between 0.25 to ≥256 μg mL-1. Out
of the 18 strains of S. aureus tested, 10 were found to be positive for
β-lactamase production. The production of β-lactamase by bacteria
with high resistance to β-lactam antibiotics has been reported (Raham Khan
and Malik, 2001; Lateef, 2004).
The present data has indicated that infection of humans by transmission through food products contaminated with antibiotic-resistant strains is plausible. The antibiotic-resistant isolates may have originally come from humans, considering that the level of carriage among human S. aureus isolates is over 40% in Nigeria (Paul et al., 1982; Lamikanra et al., 1985). The level of resistance to antimicrobial drugs is a reflection of the indiscriminate misuse and abuse of antibiotics in the environment (Umoh et al., 1990; Chigbu and Ezeronye, 2003).
There is need to educate the hawkers and retailers of ready-to-eat foods on the hazards of contamination. Such control measures as displaying foods in glass cabinets, washing hands at regular intervals, not allowing customers to pick up foods with bare hands and selling off all items on the same day are necessary to prevent hazardous conditions from developing or occurring.
The antibiotic resistance patterns of ready-to-eat food strains of Staphylococcus aureus observed in this study suggests a greater risk in the form of transfer of resistance to other forms of bacteria since they can form commensal flora through the food chain. A comprehensive surveillance is required to determine the presence and distribution in foods, animals and agriculture in Nigeria.
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