Aquaculture is experiencing rapid growth compared to other food production sectors and playing a vital role in ensuring food security [1–4]. Despite its benefits, the expansion of aquaculture is negatively impacting aquatic ecosystems [5]. This has led to the widespread adoption of environmentally sustainable systems such as aquaponic systems around the world [6, 7]. In aquaponic systems, vegetables are grown alongside tilapia in a closed recirculation system where water is not discarded [8], and nutrients from fish tanks are used as a source of plant nutrition [6]. According to [9], food safety is compromised when vegetables are grown in the presence of fish farming effluent.

Human illnesses and deaths caused by the consumption of food contaminated with pathogenic bacteria are a constant threat to public health worldwide [10, 11]. Escherichia coli and Vibrio cholerae are considered highly pathogenic to humans and have been identified as parts of the microbiota of tilapias [12–14]. Lettuce is the most common raw food associated with foodborne illnesses globally [15], and is also the most commonly cultivated in aquaponic systems [8]. Tilapia, which is the most commonly cultured fish in aquaponic systems [11], can harbor a diverse microbiota [12–14], some of which are pathogenic to humans and can be transmission through the consumption of uncooked food [16, 17].

In recent years, the use of aquaponic systems for food production has raised concerns about the potential contamination of vegetables by microorganisms [18]. Several researchers [16, 19–23], have recommended conducting further studies on the potential contamination of vegetables grown in aquaponic systems, highlighting the multiple pathways through which pathogenic bacteria can be introduced. These researchers have emphasized the need for research to understand how and when microbial contamination occurs in aquaponic systems, driven by concerns over food safety. They also proposed investigating preventive strategies to reduce the risk of contamination of vegetables grown in aquaponic systems.

Due to the risks of microbiological contamination in aquaponic systems, it is recommended to adopt preventive methods to control diseases [21]. The use of probiotics may be a safe alternative [24] as they do not pose a risk to the health of cultured fish and vegetables or humans [21]. In aquaponic systems, probiotics can be introduced either through feed supplementation or direct inoculation into the water, and they can be employed either as single-species preparation or as a combination of multiple species [25]. Bacillus spp. is one of the most commonly used probiotic bacteria in aquaculture [26]. Specifically, B. subtilis and B. licheniformis have been shown to improve growth performance and reduce pathogens in Nile tilapia [27]. According to [28], B. subtilis can be mixed with malic acid to reduce pathogens in the gut of Nile tilapia, and B. licheniformis can be combined with other probiotics and give good results in the host.

This systematic review aimed to comprehensively outline the existing research on lettuce contamination by pathogenic bacteria in aquaponic systems, specifically E. coli and V. cholerae. Several studies [29–37] have reported the presence of these pathogens in tilapia intestines and rearing water. According to these studies, V. cholerae is a significant human pathogen that can proliferate in aquatic environments and spread to the human intestine. This bacterium can colonize the tilapia intestine, and survive for at least 15 days. E. coli is one of the most prevalent microorganisms in food, with various strains that differ in their pathogenicity on the host. Outbreaks caused by these two pathogens are often associated with the consumption of uncooked food, which can become contaminated through waterborne feces. The internalization of these pathogens in lettuce leaves can put public health at risk. Therefore, their presence and prevention in aquaponic systems should be investigated to ensure food security. We also aimed to elucidate the efficacy of Bacillus strains in controlling these pathogens in aquaponic systems, as these probiotics have been extensively used in aquaculture and have shown the potential to control pathogenic bacteria.

This systematic review followed the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) Statement 2015, PRISMA 2020 Checklist (S1 Checklist), and the protocol (S1 Protocol) was registered on Open Science Framework (OSF) with the associated project: osf.io/va3nk and registration Doi: https://doi.org/10.17605/OSF.IO/K64EN.

This systematic review aimed to address two key research questions: first, to explore existing studies concerning lettuce contamination by pathogenic bacteria in aquaponic systems, and second, to assess the effectiveness of B. subtilis and B. licheniformis as probiotics in controlling E. coli and V. cholerae within such systems. To answer these questions, we critically evaluated five of the most relevant studies on the topic.

Overall, it appears that in aquaponic environments, the use of nutrient-rich water from fish tanks to grow plants increases the risk of contamination of edible parts of vegetables by pathogenic microorganisms. If the vegetables are consumed raw, a significant number of pathogenic bacteria can survive on the produce posing a public health hazard [42, 43].

In fish-rearing systems, factors such as the discharge of organic matter, environmental conditions, and untreated water can influence microbial metabolic processes and promote microbial proliferation [44–46]. According to [46], temperatures between 30–35°C, low levels of dissolved oxygen (<5mg/l), higher levels of NH3 (1-5mg/l), and pH (8.0) are associated with pathogens outbreaks in fish farms. The growth of E. coli in aquatic environments is influenced by various environmental factors, including temperature, pH, dissolved oxygen, nutrient availability, and humidity. This pathogen can survive for an extended period, up to 91 days, in aquatic environments at normal temperatures [47]. A study carried out by [48] reported that E. coli O177:H7 is resistant to temperature variation and can survive for 12 weeks in water at 25°C.

The isolation of pathogenic bacteria from foods is alarming because their presence poses a food safety risk, with potential implications for public health [49]. The presence of 100 CFU/100ml of coliforms in effluents, can significantly increase public health risk [42]. For instance, [50], reported the presence of 6.94 log10 CFU/g and 3.25 log10 CFU/g of aerobic mesophilic bacteria and coliforms, respectively in lettuce samples from organic farming systems. Similarly, [44], reported 7.57± 0.57 log10 CFU/g of aerobic mesophilic bacteria and below 3.38 log10 CFU/g of total coliforms in lettuce samples.

[51] investigated the microbial quality of lettuce leaves finding 5.3 ± 0.7 log CFU/g of aerobic mesophilic bacteria; 5.2±0.8 CFU/g of Enterobacteriaceae, and 4.3±0.7 CFU/g, of coliforms. [52] reported 2.87±0.76 x 10⁹ CFU/g of human pathogens bacteria in lettuce samples from different marketplaces. The number of Colony Forming Units per gram in lettuce samples was 6.35±0.69log10 CFU/g for aerobic mesophilic bacteria, 5.82±1.01log10 CFU/g for Psychotropic microorganisms, and 5.16±1.01 CFU/g for Enterobacteriaceae [53]. In the study of (49), the CFU/g of aerobic mesophilic bacteria and total coliforms were 4.82±2.55 log10 CFU/g and 3.50±1.00 CFU/g, respectively.

The presence of E. coli at around 10⁵ CFU/g or 1–3.8 CFU/g as well as coliforms at ≤4log 10 CFU/g or 3–6.4 CFU/g in food is a signal of public health risk [42, 54]. According to [55], bacterial count above 10³ CFU/g in food, exceeds the permissible limit recommended by the World Health Organization (WHO) and the International Commission on Microbiological Specification for Food (CMSF) standards.

The analysis of the reviewed studies revealed that Enterobacteriaceae, coliforms, and Shiga-toxin-producing E. coli play a significant role in the microbiological contamination of lettuce leaves in aquaponic systems. The Enterobacteriaceae, E. coli, and other microorganisms mentioned in the studies, such as Aerobic mesophilic count, psychrotrophic microorganisms, Yeast and mold, lactic acid bacteria, A. hydrophila, and Pseudomonas spp were found in lettuce grown in the organic farming system and also were isolated from tilapia faces [53, 56]. These pathogenic bacteria have been found in the effluent water, the root zone [57], and fish tanks [58]. E. coli can internalize in plants grown in hydroponic systems through the roots and move up to the edible parts of the plants. This internalization increases when roots are exposed to high densities of pathogens [59]. According to [60], enteric pathogens can survive outside their animal host and attach to plants by forming of biofilms, and internalization into plant tissue. The presence of those pathogens, specifically Shiga toxin-producing E. coli, in lettuce leaves is a public health concern [61]. Studies have shown that all samples from organic systems contain E. coli and coliforms [22, 62, 63].

The included studies did not report the presence of V. cholerae in aquaponic systems, likely, because none of them specifically investigated V. cholerae. However, studies targeting V. cholerae in tilapia and rearing water reported the isolation of this pathogen. In Taiwan, Bangladesh, and Egypt, [64–66], isolated V. cholerae from tilapia and from freshwater ponds in which tilapia was raised. Some researchers challenged tilapia with V. cholerae in closed systems and they reported colonization of tilapia by different strains of V. cholerae and its presence in rearing water. In Tanzania, [35] challenging tilapia with four strains of V. cholerae (V. cholerae O1 Classical biotype, V. cholerae O1, EI Tor biotype, V. cholerae O1 Δtox T, and V. cholerae no-O1), by inoculating it in the water. On days 1, 2, 3, 7, and 14, they observed that all strains of V. cholerae had colonized tilapia and these strains were also detected in aquarium water. In Egypt, [67, 68], conducted a study by injecting V. cholerae intraperitoneally into the tilapia reared in closed systems, and they reported that tilapia were colonized by V. cholerae after 10 days.

Regarding the source of contamination, the analysis of the included studies showed that water and fish feces are the main sources of contamination in aquaponic systems. Researchers have identified a link between aquaponic systems and the water source concerning microbial contamination [69]. Feces and water are common sources of pathogenic microorganisms in fresh produce [70]. In aquaponic systems, pathogens enter when proper hygiene practices are not applied [61]. Those pathogens can survive for extended periods in water [71]. Pathogenic microorganisms such as Coliforms, Yeast and Mold, E. coli, Pseudomonas, A. hydrophila, and Enterobacteriaceae have been detected in water samples from tilapia rearing system [20, 22, 43, 56]. If the water used in aquaponic systems is not treated, the microbial count of Enterobacteriaceae, E. coli, and total bacteria increases. The prevalence of these pathogens is influenced by favorable water temperatures [61]. According to [72], microbial density increases when the water temperature reaches approximately 21°C.

The included studies did not report the use of B. subtilis and B. licheniformis as probiotics to control E. coli and V. cholerae, however, these two Bacillus promote lettuce growth in aquaponic systems by regulating the microbiome [73] and improving weight gained, specific growth rate, feed conversion ratio, and thrombocyte counts in the bloodstream of Nile tilapia [27, 74].

These two probiotics have also been shown to reduce the Proteobacteria Phylum (to which E. coli belongs) in Nile tilapia cultured in aquaponic systems [27]. Their administration can enhance the immune systems of Nile tilapia by boosting their defense mechanisms [75]. B. subtilis has been shown to control vibriosis, Aeromonas hydrophila, and Streptococcus aureus in reared fish [76–78] and protects tilapia against bacterial infection in the ponds [79]. It can also inhibit the proliferation of pathogenic species of vibrio in Letopenaeus Vannamei, Penaeus monodon, and tilapia through exclusive competition mechanism in aquaculture [80, 81].

In the present review, we identified notable patterns of bias among the included studies. For example, 50% of the studies exhibited a high risk of bias in two domains: random sequence generation and allocation concealment. Additionally, 25% of the studies showed a high risk of bias in the domains of incomplete outcome data and selective reporting. The randomization and allocation methods were often unclear, raising concerns about the reliability of their findings. Moreover, the method used for detecting pathogenic bacteria in the study design labeled as another bias in this systematic review, was conventional, and probably failed to detect microorganisms that cannot thrive under such conditions. In one study, incomplete outcome data resulted from not reporting the source of contamination, casting doubt on the accuracy of its results and affecting our systematic review findings. Many of the studies analyzed, used both conventional and molecular methods for detect microorganisms in aquaponic systems. Overall, the high risk of bias across the studies reviewed stemmed from methodological disparities. A common limitation was the lack of a contamination analysis specifically targeting the lettuce leaves, the edible part of the plant. These methodological variations significantly impacted the outcomes of our systematic review.

This systematic review revealed that roots and leaves of lettuce grown in aquaponic systems can be contaminated by pathogenic bacteria. The most common bacteria contaminating lettuce leaves in aquaponic systems include Enterobacteriaceae, Coliforms, and Shiga Toxin-Producing E. coli. In aquaponic systems, Shiga-toxin producing E. coli can internalize into lettuce leaves, and potentially infect humans when raw lettuce is consumed. V. cholerae is the causative agent of cholera, and commonly inhabits freshwater environments, with fish serving as its reservoir. Therefore, its presence in aquaponic systems must be monitored to ensure the safety of harvested vegetables. To improve pathogen identification and provide comprehensive insights into the microbial landscape in aquaponic systems, conventional methods should be combined with molecular techniques. Since water and fish feces are the main sources of pathogenic bacteria in these systems, further research is needed to explore the role of tilapia acting as a contamination source of vegetables grown in aquaponic systems and investigate the effectiveness of Bacillus strains in preventing human pathogenic bacteria.