Cyperus esculentus L, also known as nutsedge, earth nuts, earth almond, rush nuts, and chufas, is a member of the grass family Cyperaceae 1 . The genus name Cyperus is from Cypeirus, an ancient Greek name, while the species name esculentus is of Latin origin meaning edible 2 3 . The nuts are a minor crop grown in the temperate and tropical zones of the world 4 . They have become naturalized in Ghana, Nigeria and Sierra Leone 5 . There are two types of tiger nuts (Figure 1a); the light brown type (or yellowish) and the black type (or dark brown) and they vary in size from about 1-3 cm in length to 1-2 cm in diameter 6 .

Tiger nuts are consumed for their nutritional or medicinal values. Typically 100 g of the nuts contain 386 kcal (1635 kJ) of energy (7% of which are of proteins, 26% fats/oils, 31% starch and 21% glucose), and they contain an appreciable level of fibre (12% soluble and 14% non-soluble). They are also known to be rich in minerals such as sodium, calcium, iron, zinc, phosphorus, potassium, magnesium, copper and manganese 7 . In some parts of the world, they are medicinally known to have aphrodisiac, carminative, diuretic, emmenagogue and stimulating properties and also used in the preparation of tonics 8 . In Ayurvedic medicine, they are used for the treatment of flatulence, indigestion, colic, diarrhoea, dysentery, debility and excessive thirst 8 9 .

In Ghana, the nuts are known and eaten mainly for their aphrodisiac property though the other uses are known to some people. They are sold packaged in plain polythene bags in markets and on the road side (Figure 1b). These nuts are sometimes processed into porridge (locally known as “atagwe” milk) and consumed domestically or sold to the public (Figure 1c). The majority of people buying the nuts purchases them whilst in a vehicle and consume without washing because they assumed sellers have washed already. Studies so far in some parts of the world have found some contaminants other than parasites. For example, in a small market survey of the beverage “Horchata” derived from tiger nuts frequently consumed in Southern Europe, aflatoxin B₁ was detected 10 . In Nigeria, Adebanjo in 1993 found aflatoxins present at toxicologically unsafe levels on tiger nuts 11 . Besides aflatoxins, Aspergillus flavus have also been reported to contaminate tiger nuts 12 . Other contaminants such as stones, animal droppings and some other extraneous materials have been found to be associated with tiger nuts 13 . In most parts of Africa, the passion for eating outweighs other considerations such as food safety; this drive has led to considerable rise in food-borne intoxications and contamination 14 . The current study therefore, aimed at identifying common parasitic and bacterial contaminants on the crop, as it is sold and eaten by Ghanaians.

Parasitological investigations found life form of four different parasites from the tiger nuts collected (Table 1). The parasites identified were in the following proportions; Cryptosporidium parvum oocysts (12/40, 30.0%), Ancylostoma duodenale ova (10/40, 25.0%), Strongyloides stercoralis larvae (9/40, 22.5%) and Cyclospora cayetanensis oocysts (9/40, 22.5%). Cyclospora cayetanensis was found to be present on nuts from three (3) of the five (5) street vendors studied, namely, UTC, Shangrila and Madina. Cryptosporidium parvum, the most predominant parasite found was present on nuts from two locations; UTC and Shangrila (Table 1). Interestingly, all parasites found were associated with nuts bought from street vendors (40/40, P-Value <0.001). Fifty percent (50% ) of the street vendors admitted washing their nuts in several changes of clean tap water before bagging for sale whereas 15% stated that they washed with salt water. The percentage of vendors that do not treat the nuts before selling was 25%. The remaining 10% could not tell how the nuts were treated before bagging because they buy already bagged nuts at a lower price from other vendors and sell to make profit. Meanwhile, there was no association between vendor washing the nuts and the number of parasites or bacterial colonies obtained (χ2 = 0.042, DF = 1, P-Value = 0.840).

*No parasite was found in the tiger nuts bought from market places (Agbogbloshie, Kaneshie and Makola).

Overall, five bacterial species were isolated. Bacterial isolates were found to be associated with nuts bought from both street vendors and market place. The predominant isolates were grouped into coliforms, non-lactose fermenter (NLF) and Staphylococcus spp, and all showed varied colony forming units (Table 2). Different percentage of occurrences amongst all location was recorded. Among the coliforms were Klebsiella oxytoca (3/22, 13.6%), Enterobacter cloacae (7/22, 31.8%) and Enterobacter spp (3/22, 13.6%). NLF observed was Proteus vulgaris (1/22, 4.6%) whiles Staphylococcus spp recorded 36.4% (8/22).

*Only sterile normal saline was cultured. NC means, no colonies observed and cfu/g means, colony forming unit per gram.

Some locations recorded more isolates than others (Figure 2). Inhibition zones have been presented in Figure 2, showing the isolates that were sensitive or resistance to different abtibiotics. These included the resistance of Staphylococcus spp from Kaneshie to ampicillin and resistance of Staphylococcus spp from Airport, Agbogloshie, Makola and Madina, to tetracycline (Figure 2a). Enterobacter spp from Agbogbloshi and Airport, and Klebsiella oxytoca from Shangrila were also resistant to ampicillin. Also, Enterobacter spp from Airport and Madina, and K. oxytoca from Mokola were all resistant to cotrimoxazole. Enterobacter spp from Madina and K. oxytoca from Mokola were resistant to chloramphenicol. K. oxytoca from Mokola and Circle, and Enterobacter spp from Madina were all resistant to tetracycline (Figure 2b). Enterobacter Cloacae from Kaneshie was resistant to ampicillin. One notable pattern was the multiple resistance of Proteus vulgaris from Circle to ampicillin, cotrimoxazole, chloramphenicol and tetracycline (Figure 2c).

Antibiotic sensitivity patterns of the bacteria isolated from the tiger nuts are reported in Table 3. In the present study Staphylococcus spp was highly sensitive to all the antibiotics tested, except Tetracycline. K. oxytoca was highly sensitive to ciprofloxacin, gentamicin, piperacillin, amikacin, levofloxacin and ofloxacin, relatively sensitive to cefotaxime and ampicillin, but resistant to cotrimoxazole and tetracycline. P. vulgaris was highly sensitive to 7 of the antibiotics (ciprofloxacin, ceftizoxime, piperacillin, amikacin, levofloxacin, ofloxacin and gentamicin), but was resistant to 5 antibiotics, including ampicillin, cotrimoxazole, chloramphenicol, tetracycline and cefotaxime. Enterobacter spp. displayed sensitivity to all antibiotics with the exception of ampicillin and cefotaxime. E. cloacae showed sensitivity to all the antibiotics.

n = number of bacteria isolates. Total number of location = 8.

In this study, we investigated bacterial and parasitic contamination of Cyperus esculentus L sold on the Ghanaian market and street. A wide range of both bacterial and parasitic contaminants were detected. A number of factors, such as direct contact of nuts with the soil, unhygienic handling, and the use of contaminated water for irrigation, could have accounted for the contamination observed. These factors make the nuts prone to parasitic and bacterial agents that have long been associated with fresh vegetables and fruits 22 23 24 25 26 . An indirect factor that could have also contributed to the contamination observed in the current study is urbanization. The increase in urbanization 27 28 29 has resulted in high food demand and a similar pressure on social amenities like clean water. Access to clean potable water is a challenge in the Accra metropolis. As a result, vendors who require larger amounts of water to wash food items do so, not under running water, but in a bowl. This practice is likely to have resulted in the contaminants found on the nuts.

Five bacterial agents, some with serious health implications, were found in the current investigation. Studies have shown that individuals with recurrent infections and those with structural abnormalities of the urinary tract have an increased frequency of infection caused by Proteus vulgaris and other bacteria such as Klebsiella spp, Enterobacter, Pseudomonas, enterococci and staphylococci 30 31 . Hence isolation of some of these bacteria on the tiger nuts is a health concern. Although P. vulgaris recorded low prevalence rate (4.6%), its presence is reliable proof of faecal contamination of the tiger nuts which could be as a result of irrigation with contaminated water sources and direct soil contact. This practice is common in developing countries, with Ghana not being an exception. P. vulgaris and Staphylococcus spp. have been implicated in food spoilage and food-borne diseases, hence, they should be of interest when food hygiene is being considered 22 . This supports the assertion that, the intake of contaminated food can cause diarrhoeal-associated illnesses with bacteria being one of the major causes 32 . Yeboah-Manu et al., reported high levels of bacterial contamination among sellers in Accra, which included contaminants such as Staphylococcus spp., Enterobacter cloacae and Proteus spp 33 , therefore, the hygiene and packaging methods of these tiger nut must be monitored. Bacterial counts on tiger nuts showed different bacterial loads. All the samples bought from market places recorded unacceptable levels of coliforms, which are indicators of food hygiene. This result is expected since the market women do not seem to wash the nuts before selling it to the vendors. Although most of the vendors interviewed (including those from Korle-Bu and Circle) admitted they washed their products with several changes of tap water before bagging and selling, this level of contamination is not surprising because, chlorinated water in distribution here in Accra has been found to be contaminated 34 . Even sachet water, which is regarded as being more purified, has been identified to contain bacteria counts ranging from 2.8 × 10³ to 5.9 × 10⁵ cfu/mL 34 . The high microbial loads of the tiger nut sampled from the study locations, which were unacceptable, supports the finding of Nyarko et al. 35 . In that study, they found high levels of microbial loads on non-sterilized tiger nuts 35 . Vendors from Shangrila and Airport residential area recorded low bacterial levels. This could be because, the aforementioned locations are regarded as residence for the elite class in the city. The vendors, therefore, wash the nuts to try and make them presentable, and in the process reduce bacterial contaminants, before bagging.

From the outcome of the sensitivity testing, it is noteworthy that, Staphylococcus spp, K. oxytoca and P. vulgaris were all resistant to tetracycline. Two out of these bacteria (K. oxytoca and P. vulgaris), together with Enterobacter spp., were resistant to ampicillin whiles K. oxytoca and P. vulgaris were resistant to cotrimoxazole (Table 3). Proteus vulgaris showed multiple resistance to ampicillin, cotrimoxazole, chloramphenicol and tetracycline. These antibiotics, especially tetracycline and cotrimoxazole, have been on the Ghanaian market for a long time and therefore might have been exposed to frequent use or abuse. This might account for the levels of resistance observed. The resistance recorded in the current study supports the finding of other researchers who also worked on isolates from food items 36 37 . These researchers therefore suggested that the incidence of resistant bacteria (especially enterobacterial pathogens) is a worldwide phenomenon and a major public health threat 36 37 . All the bacterial isolates were sensitive to Ciprofloxacin, Piperacillin, Gentamicin, Amikacin, Levofloxacin and Ofloxacin. This finding suggests that, medical officers can have a numbers of options to choose from, when treating bacterial infection acquired from the consumption of contaminated tiger nuts sold on the Ghanaian market.

The parasitic contaminants found on the nuts are associated with gastrointestinal infections 38 . To the best of our knowledge, this is the first study to report parasitic contamination of tiger nuts sold in a Ghanaian city. The most prevalent parasite, Cryptosporidium parvum (30.0%), is known to contaminate both food and water, and have sometimes caused small and large outbreak of acute watery diarrhoea 39 40 . Occurrence of this parasite on the nuts supports the suggestion that contaminated leafy vegetables and fruits can be connected to Cryptosporidium parvum transmission 41 42 . Although Cryptosporidium parvum is regarded as a minimally invasive mucosal pathogen, infection can be associated with diarrhoea and marked mucosal inflammation 43 . Some cases of pulmonary and tracheal cryptosporidiosis, coughing and fever can also be seen 44 . Mostly, the diarrhoea is self-limiting; however, immuno-compromised individuals can develop uncontrollable forms leading to severe dehydration that is potentially fatal 45 . Parasites such as Ancylostoma duodenale and Strongyloides stercoralis are common species of soil-transmitted helminthes, which can be related to infection associated with tiger nut consumption considering the way it is cultivated. Infection of these parasitic agents can cause significant nutritional deficiencies, delayed physical and cognitive development during childhood, and reduced productivity in adults 46 47 . In spite of Cyclospora cayetanensis recording low prevalence (22.5%), its occurrence should not be overlooked, since it has been recognized as an emerging food and water-borne pathogen that causes protracted diarrhoea in humans 48 . The clinical presentation is somewhat different in areas with varying levels of endemicity. Nevertheless, younger children have more severe clinical symptoms 48 . Coinfection of Cyclospora cayetanensis with Cryptosporidium parvum and other parasites has been described for both immunocompetent and immunocompromised individuals to cause more severe presentations 49 , and this is noteworthy since both parasites were found to be present on the tiger nuts. In spite of the good nutritional and medicinal values of tiger nuts, consumption of untreated nuts has the potential of increasing transmission of both intestinal parasites and pathogenic bacteria that could lead to a rise in water and food-borne infections if not properly processed. Washing with salt water promises to be an effective decontamination procedure. Another method that is also recommended is surface sterilization. This method of decontamination is in conformity with the study conducted by Yousuf et al., in which different chemicals including calcium hypochlorite solution and oxalic acid in a form of lemon juice were used, as decontaminants of shrimp and prawn, and yielded significant reduction in bacterial contamination 50 . In view of this, investigation on agricultural practices of farmers and vendors that tend to expose the nuts to contaminants need to be undertaken periodically to monitor transmission of bacterial and parasitic diseases.

Buying and eating fruits and vegetables from street vendors without further washing could result in infection with pathogenic parasites and bacteria. Pre-treated fruits or vegetables being sold on both streets and markets in Ghana could be a potential source of transmission of food borne diseases. There is therefore a need for regular monitoring of the quality of such food items sold. There is also the need for consumers to pay critical attention to proper washing and surface sterilization of the tiger nuts before consumption.

NLF: Non-lactose fermenter; CFU: Colony-forming unit; K. oxytoca: Klebsiella oxytoca; E. cloacae: Enterobacter cloacae; Enterobacter spp: Enterobacter specie; P. vulgaris: Proteus vulgaris; Staphylococcus spp: Staphylococcus species; C. parvum: Cryptosporidium parvum; NCLS: North Country Library System; hrs: hours; mm: millimeters.

The authors declare that they have no competing interests.

PFA-K conceived and designed the experiments: KOD participated in collection and analyses of the data and drafting of the manuscript. ASO contributed in interpretation of the data. PBT-Q and RHA Jointly developed the structure and arguments for the manuscript. SM and KA-O made critical revisions and approved final version. All authors read and approved the final manuscript.