Microbiological quality of fresh lettuce sold at Lilongwe market, Malawi: Does purchasing time matter?

Fifteen (15) samples of fresh lettuce were bought from Lilongwe city market, Malawi in the morning, afternoon and evening and were examined for microbiological quality. Counts ranged from 1.2×10 5 to 6.9×10 5 cfu/g for total Enterobacteriaceae and 1.5×10 4 to 1.1×10 6 mpn/g for total coliforms. Higher total Enterobacteriaceae and total coliform counts in the afternoon samples and presence of fecal coliforms in all evening samples suggested that comparatively safer lettuce was sold in the morning. Poor handling practices at the market and temperature abuse could be responsible for contamination and microbial proliferation during retail of the lettuce.


INTRODUCTION
Fruits and vegetables are important sources of vitamins, minerals and antioxidants in human diets.Vegetables contribute large quantities of vitamin C, but the vitamin's high water solubility and sensitivity to heat makes it susceptible to loss during thermal preparation (Howard et al., 1999;Murcia et al., 2000;Dewanto et al., 2002;Gupta et al., 2008).Lettuce (Lactuca sativa) on the other hand, is typically eaten cold and raw, in salads, and many other dishes (Brackett et al., 2001).Lettuce stored at 0°C can retain up to 46% ascorbic acid after 200 h of storage, hence lettuce has a potential of high retention of the vitamin and other nutrients at consumption (Moreira et al., 2006).
However, consumption of lettuce with minimal processing increases its risk as a vehicle for microbio-logical infection.Bacteria pathogens associated with minimally processed vegetables include Listeria monocytogenes, Escherichia coli and Salmonella (Francis et al., 1999a;Manani et al., 2006;Seo et al., 2010;Tian et al., 2011) among others.These pathogens have been linked to outbreaks caused by consumption of minimally processed vegetables.Other pathogens linked to consumption of vegetables include viruses (Norwalk-like, hepa-titis A), and parasites (Cryptosporidium, Cyclospora) (Abadias et al., 2008;Seo et al., 2010).Foodborne outbreaks linked to lettuce have also been reported (Ho et al., 1986;Francis et al., 1999;Francis and O'Beirne, 2001;Srebernich et al., 2012).
Like most leafy green vegetables available in the market, lettuce can get contaminated at every stage of the production process to the consumer.Amoah et al. (2006) reported fecal and helminth contamination of lettuce, cabbage and spring onions in urban markets in Ghana.In their study, fecal coliform counts exceeded the International Commission on Microbiological Specification for Food (ICSMF) recommended levels of 10 4 (Amoah et al., 2006).Exposure to microbial contamination and microbial growth can occur at various stages of harvest, Doku et al., 2010;Berger et al., 2010;Seo et al., 2010).During distribution and retail, time and temperature abuse, lack of hygienic handling and storage practices are important factors (Brackett, 1990).In most markets in storage, processing and production (Tian et al., 2011).Contamination during production occurs due to contact with soil or manure, irrigation water or due to birds or animal fecal matter (Brackett et al., 2001; Amponsah-*Corresponding author E-mail: mananitinna@gmail.com. African countries including Malawi, vegetables are sold without refrigeration in open stalls throughout the day and water is sprinkled on the vegetables (market refreshing water) to prevent wilting.Refreshing water can be a source of produce contamination.Amponsah-Doku et al. (2010), reported thermotolerant coliform levels of 9.0 × 10 3 to 4.3 × 10 10 in market refreshing water which were higher as compared to counts on the market lettuce.
There is limited information on vegetable production and marketing and general vegetable research in Malawi.According to Mtukuso (2008), the vegetable research and development policy highlighted in the Agricultural Research Master Plan (1995) aimed at overcoming technical constraints of production and post-harvest losses.No plans were made to address consumer safety issues.Yet it has been stated that a Malawian is a vegetarian by default, due to the nation's low per capita intake of animal products (Mtukuso, 2008).About 80% of vegetable production in Malawi is mainly by smallholder farmers, who sell their produce to local markets (Mwandira, 2008).Various vegetables are produced in large quantities during winter under irrigation and during summer rains.Malawi agricultural production estimates do not provide or attempt to collect data to estimate production in the country (Daudi and Mwenda, 2008).However, Mwandira (2008), stated that the local demand for horticultural crops was projected at 15,000 tonnes of assorted horticultural crops (including fruits, Irish potatoes, beans and leafy vegetables like cabbage, lettuce and indigenous vegetables) per year for the urban population of roughly 1,500,000 inhabitants.There is little organized marketing of horticultural products and there are at least four levels of middlemen before the vegetables reach the consumers (Daudi and Mwenda, 2008).The bulk of horticultural marketing takes place in informal market sector; which is characterized by poor quality produce, lack of grading, and little or no packaging.This sector is dominated by vendor marketing groups (Mwandira, 2008).
Studies on microbiological quality and safety of fresh market produce have not been conducted in Malawi.Therefore, this study forms a basis for justification for the need of microbiological examination of market sold fresh vegetables.Thus, the study was conducted to assess the microbiological quality of fresh lettuce sold in Lilongwe city market, in Malawi.In addition, the study also determined whether there is a difference in microbial quality at different purchasing times (morning, afternoon and evening) in order to determine the purchasing time with reduced risk of infection.

Sample collection and treatment
A total of 15 lettuce samples were purchased from different vendors randomly selected from Lilongwe city market.The samples were put in sterile Ziploc bags in a cooler box with ice during transportation and were refrigerated upon reaching the laboratory.Five samples were purchased in the morning, afternoon and evening between 6:00-7:00 am, 12:00-13:00 pm and 17:00-18:00pm, respectively.The samples were analyzed immediately upon reaching the laboratory (within 24 h of purchase) for total Enterobacteriaceae, total coliforms and fecal coliforms.

Microbiological analysis
From each sample, 50 g was transferred aseptically into a sterile stomacher bag before adding 450 ml sterile diluent containing 0.1% buffered peptone water (Mast DM494D, Mast Group Ltd, Merseyside,UK) and homogenising in a VWR Star-blender TM LB400 (VWR, France) for 120 s.The mixtures were serially diluted to 10 -7 .

Total Enterobacteriaceae count
Violet red bile glucose agar (VRBGA, Merck, Darmstadt, Germany) was used to grow colonies of Enterobacteriaceae using the spread plate technique.Each serial dilution was plated in duplicate Petri dishes.The Petri dishes were incubated at 37°C for 24 h and all colonies were counted after the 24 h.

Total coliforms
Three tube most-probable-number (MPN) method using lauryl tryptose broth was used (Manani et al., 2006).Three 1 ml aliquots from each dilution (10 -3 to 10 -7 ) were aseptically transferred to three tubes containing 9 ml sterile lauryl tryptose broth (Oxoid Ltd, Basingstoke, Hampshire, England) and inverted Durham's tubes.Incubation was done at 30°C for 24 h.Tubes showing gas and growth were considered as positive for total coliforms.Results were interpreted using probability tables for estimating values of MPN for three tubes (Harrigan, 1998).

Fecal coliforms
Presence of presumptive fecal coliforms were detected by aseptically transferring 1 ml of inocula from each dilution showing positive results for total coliforms to tubes containing 9 ml of sterile lauryl tryptose broth and inverted Durham's tubes.Direct isolation of fecal coliforms from a solid sample requires prior enrichment in presumptive medium like lauryl tryptose broth, for optimum recovery of fecal coliforms (United States Environmental Protection Agency, 2010).In this case, the incubation for total coliforms was the preenrichment.Incubation was done at 44°C for 24 h.Tubes showing growth and gases were considered positive for fecal coliforms (Manani et al., 2006).

Data entry and analysis
Analysis of variance (ANOVA) at 0.05 significance level was performed in SPSS 15.0 (SPSS Inc., Chicago, Illinois., USA).When significant differences were found, least significance difference (LSD) test was used to compare the means.

RESULTS AND DISCUSSION
Enterobacteriaceae include many of the more familiar pathogens such as Salmonella and Escherichia coli which are linked to most cases of food borne illnesses.They are often used in food microbiology as indicator organisms (Washington State Department of health, 2009).Figure 1 shows the mean values of total Enterobacteriaceae counts.The lowest count was observed in the evening (3.64×10 5 cfu/g) then in the morning (3.90×10 5 cfu/g) while the highest was observed in the afternoon (5.14×10 5 cfu/g).However, there were no significant differences (p>0.05) between the observations at the different times.Abadias et al. (2008) reported an Enterobacteriaceae count range of 1.0-7.1 log cfu/g in fresh-cut lettuce.
According to guidelines for microbial quality of various ready-to-eat foods, the acceptable levels of Enterobacteriaceae range from 10²-10 4 cfu/g (Gilbert et al., 2000).Samples in this study had counts ranging from 1.2×10 5 to 6.9×10 5 cfu/g, exceeding the acceptable levels.Enterobacteriaceae are not only pathogens and commensals of the mammalian gastrointestinal tract, but they are also found in abundance in almost any moist environment.Some, like E. coli, are always considered to be of fecal origin, and exist only transiently in other environments.Others are natural environmental strains and can be found among the flora associated with growing vegetables (Osterblad et al., 1999).Since Enterobacteriacea includes pathogens, therefore levels exceeding standards should raise food safety concerns especially for vegetables eaten raw like lettuce.
Fecal and total coliform bacteria are indicators of potential fecal pollution and water-borne pathogenic threats to human health.The presence of coliforms indicates that pathogens like E. coli, Salmonella and Shigella species could be present in a food (Washington State Department of health, 2009).Figure 2 shows total coliform counts at the different sampling times.Counts were highest in the afternoon than the other times and there were significant differences between the afternoon and the evening mean counts (p<0.05).
Coliform counts reported in this study were within the reported ranges by others.Amponsah-Doku et al. ( 2010) reported coliform counts between 6.0 × 10 1 -2.3 × 10 8 mpn/g in market sold lettuce; Seo et al. ( 2010) reported a total coliform population range of 2.2 to 7.5 log cfu/g, while Amoah et al. ( 2006) reported a higher total coliform count range of 9.3 x 10 5 to 1.5 x 10 11 mpn/g in market sold lettuce in Ghana.According to the guidelines for the microbial quality of ready to eat foods for leafy green vegetables, more than 10 4 cfu/g of total coliforms are unacceptable (Gilbert et al., 2000).All the samples in this study exceeded the set limits which mean they had an increased risk of harboring pathogenic organisms.The highest counts in the afternoon suggested increased risk of infection from consumption of lettuce purchased in the afternoons.
High Enterobactericeae and total coliform counts observed in the afternoon reflect the effect of timetemperature abuse, as the afternoons tend to have warmer temperatures encouraging rapid proliferation of microorganisms.Storage temperature has been recognized as the most important factor affecting the growth of microorganisms in ready-to-use (RTU) vegetables (Francis and O'Beirne, 2001;Tian et al., 2011).Francis and O'Beirne (2001) showed that pathogen survival and growth was higher at 8°C than at 4°C storage.Tian et al. (2011) also found similar results in which four pathogens increased in population during 7 days of storage at 15°C, while there was no significant increase in population of the same pathogens when minimally processed vegetables including lettuce were stored at 4°C.In Malawian markets, vegetables are normally sold on open stalls without refrigeration hence they are subjected to abusive temperatures which encourage microbial proliferation throughout the day.In addition, vegetables tend to wither during retail due to exposure to direct sunlight and air in the open stalls.Therefore, refreshing water is sprinkled a lot more in the afternoon than the other times to maintain freshness.The refreshing water could be responsible for contaminating the produce further, with microorganisms from the water and from the hands of the vegetable vendors.
Fecal coliforms were detected in all the five samples in the evening, in three samples in the afternoon and in two samples in the morning.Amoah et al. (2006) reported a range of 4.0 x 10 3 to 9.3 x10 8 mpn/g fecal coliforms in lettuce in Ghana.The presence of fecal coliforms indicated possible fecal contamination or E. coli contamination.Possible sources of fecal contamination could be use of polluted irrigation water and manure applied to the crops.Another contamination source could be marketrelated handling, especially where provision for better sanitary facilities such as clean water for crop washing and refreshing is lacking (Amoah et al., 2006).Another area of concern is the hygienic practices of the vegetable vendors.Vendors spend the whole day at the market and may handle their produce after visiting the toilet without properly washing their hands hence increasing the risk of contamination with fecal matter.The risk can build up during retail due to microbial proliferation during the day.
More evening samples had fecal coliforms than the afternoon and morning samples.Increase in fecal coliform contamination with time could be attributed to unhygienic handling and use of refreshing water continuously during the day.In a study by Amponsah-Doku et al. (2010), thermotolerant coliform counts between 9.0 × 10 3 to 4.3 × 10 10 mpn/g were reported in refreshing water.While Amoah et al. (2006) observed use of a single bucket of water the whole day to refresh produce by all the sellers, which made the vegetables dirtier.Even with the use of portable water, studies have shown that effective washing and decontamination of ready-to-eat fruits and vegetables is difficult.Effect of washing in reducing the number of bacteria present is small with reductions of 10 1 units (Beuchat, 1998).In conclusion, higher total Enterobacteriaceae and total coliform counts in the afternoon samples suggested higher risk of infection from lettuce sold in the afternoon.However, the presence of fecal coliforms in all five evening samples suggested that comparatively safer lettuce was sold in the morning.Therefore, it would be advisable if consumers purchased the produce in the morning and refrigerated it for later use.Presence of fecal coliforms in all the evening samples indicated questionable water quality and market handling practices.Although the study was limited, it raised consumer safety issues that need collective efforts to address.

Figure 1 .
Figure 1.Total Enterobacteriaceae counts in lettuce at the different sampling times.

Figure 2 .
Figure 2. Mean total coliform counts in lettuce at the different sampling times.