Increasing protein needs, in parallel with the increase in the world’s population, has led people toward the innovative protein source of insects. Insects are one of the most promising alternative sources that can meet global protein requirements (Jantzen da Silva Lucas et al. 2020), because insects were proven in studies to be better protein and fat sources compared to other nutrient sources.
Consumption of insects, defined as entomophagy, is chosen because most insects are rich in high-quality proteins, good lipids, vitamins, minerals (like calcium, iron and zinc), fiber and chitin. Compared with traditional farm animals, farmed insects proliferate more rapidly, are more effective in converting feed into protein, require less space for reproduction and produce less greenhouse gas and ammonium emissions. However, in addition to all these positive features, entomophagy involves a range of chemical and biological hazards. Some microbial agents in the insect microbiota may be potential disease vectors in humans through consumption of insects. For this reason, it is important to research the microbiota of insects, especially those offered for human consumption (Garofalo et al. 2017; Van der Spiegel et al.2013; Makkar et al. 2014).
The intestinal microbiota of insects may contain water-derived pathogens and have the potential to be distributary agents for these pathogens. Migrating insects, especially, may act as a mechanism for the derivation and spread of diseases carried in water (Wooldridge and Wooldridge et al. 1972; Evariste et al. 2019).
Bacteria are common microorganisms in insect microbiota. The insect-bacteria interaction may be symbiotic and pathogenic. Most symbiont bacteria in the insect intestine comprise environmental samples. Literature information obtained from studies about insect intestinal assemblages reported that the Pseudomonas and Bacillus taxa are dominant, dependent to a large extent on diet (Broderick et al. 2004, Robinson et al. 2010)In this study, environmental isolates like Acinetobacter, Aeromonas, Pseudomonas, and Bacillus were densely observed. Considering the microenvironmental conditions of the insect intestine, it is a predicted outcome that environmental isolates with water and soil origin are frequently observed.
Bacteria in the Enterobacteriaceae family are known as pathogens/opportunistic pathogens and are a frequent parameter for assessment of enteric contamination in food (Barco et al. 2014; Stoops et al. 2016) The presence of these bacteria in insects indicates that the intestines cannot be cleaned or pollution of the aquatic habitat. E. coli, included in the enteric bacteria group and an element in human intestinal flora, was isolated from 2 different insect species (Laccobius syriacus Guillebeau, 1896, Hydrophilus piceus Linnaeus 1758) in this study. This bacteria, in a compatible relationship with the host organism, may cause disease in situations with emplacement in organs outside the intestines or in the intestines of another host.
Enterococcus species bacteria include species adapting at high rates to different habitats like humans, animals, insects, plants, soil, water and fermented foods. Bacteria from this family are important as they have shown antibiotic resistance in nosocomial infections since the 1970s to the present day. The inclusion of insects as a reservoir for antibiotic resistant enterococci is worrying. In our study, the E. rivorum species from this family was identified (Lebreton et al. 2014).
Two Vibrio cholerae were identified with both API 20E and molecular methods isolated from the Laccobius syriacus Guillebeau, 1896 and Helophorus brevipalpis Bedel, 1881 insect species. Vibrio are found in abundant amounts in sea water and river mouth environments. They may cause sporadic gastroenteritis and severe disease by proliferating on phytoplankton and zooplankton surfaces (Traore et al. 2014; Cabral et al. 2010). Consumption of insects from water containing sewage waste, especially, is a risk factor in terms of V. cholerae.
The Pseudomonadaceae family is commonly found in soil or water and has a role as pathogen for insects and opportunistic pathogen for humans. Additionally, it has clinical importance due to frequently developing resistance against antibiotics. It was identified in edible fresh locust and mealworm larvae samples marketed in Belgium (Stellato et al. 2015; Stoops et al. 2016). In the study, 4 Pseudomonas isolates were identified, with 1 identified at species level and 3 identified as P. putida. P. putida has both pathogenic and biotechnological importance. It is an opportunistic pathogenic microorganism in patients with suppressed immunity and was seen to colonize these individuals (Molina et al. 2016; Fernandez et al. 2015).
Aeromonas, a natural element in aquatic environments, comprised the most commonly isolated bacteria group in the study (18%). Within this group, A. hydrophila, A. caviae and A. veronii biotype sobria are defined as human pathogens. They may cause gastrointestinal and extraintestinal infectious diseases in humans. In this study, the 4 isolated A. veronii biotype sobria were frequently reported in clinical isolates and are reported to be pathogenic bacteria for traveler’s diarrhea (Al-Fatlawy and Al-Hadrawy et al. 2014). Consumption of raw shellfish was identified to be the main cause of transmission of this bacteria to humans. All species of Aeromonas, containing both mobile and immobile species, identified in this study were mobile species mostly found in freshwater, while immobile species were not encountered. A. hydrophila is a potential agent in gastroenteritis, septicemia, meningitis and wound infections, and was accepted as an opportunistic pathogen in recent years. A. hydrophila was reported to be the main cause of an epidemic occurring in people consuming oysters in Louisiana. Additionally, it was identified as a pathogenic bacterial species in fish and crab farms in China (De Silva et al.2021; Cabral JP et al. 2010) The A. rivipollensis and A. allosaccharophila species isolated in the study do not have pathogenic characteristics.
Acinetobacter was the bacteria identified most frequently after Aeromonas in this study (12%) due to being an isolate with environmental source. The clinically significant species are A. baumannii, A. nosocomialis, A. pittii and A. calcoaceticus and in this study, 3 A. pittii and 1 A. calcoaceticus were identified among pathogenic species. Attracting attention in recent years, especially as the most frequent cause of antibiotic resistance and nosocomial infections, Acinetobacter species are the most frequent causes of ventilator-associated pneumonia, blood circulation, urinary tract and intraabdominal infections in intensive care units (Esen et al. 2020).
Another isolated bacterium was Exiguobacterium sp. and species from this genus are rarely associated with human infections. Additionally, bacteremia and skin infection cases were documented. Additionally, as there is a tendency toward mistaken identification of the microorganism with routine commercial methods, deficient detection or reporting rates are high (Chen et al. 2017).
Tsukamurella inchonensis identified in Berosus spinosus (Steven, 1808) insect species was an environmental saprophyte firstly isolated from soil, arthropods, water and mud. It is an opportunistic pathogen in humans especially associated with medical devices and catheters (Safaei et al. 2018).
The most commonly used probiotic bacteria of Lactobacillus spp. is found in large scale habitats like stomach-intestinal systems and environmental media. In the study, Lactococcus lactis bacteria isolated from Laccobius syriacus Guillebeau, 1896 insect species is a microorganism accepted as having unknown virulence factors and being very safe. However, Rostagno et al. reported that these bacteria may be a source of a variety of infections (liver and brain abscess, cholangitis, peritonitis, osteomyelitis and deep neck infection) linked to consumption of raw milk and milk products by humans in the last twenty years (Rostagno et al. 2013).
Ignatzschineria larvae identified in Hydrobius fuscipes (Linnaeus 1758) insect species was first identified by isolation from larvae of Wohlfahrtia magnifica (spotted flesh fly) by Toth et al., in 2001. (Toth et al. 2007) Both pathogenic and antilarval effects of this species, which is closely related to larvae and causes bacteremia by reproducing in worm-infested wounds in humans, require further research.
Apibacter raozihei obtained from Helophorus aquaticus (Linnaeus, 1758) insect species is some facultative anaerobic bacteria. In this study, it reproduced on the 4th day in the aerobic environment and in a shorter duration (2 days) in the anaerobic environment. These bacteria were first isolated from honeybees in recent times and was defined as a microaerobic member of the bee intestine (Kwong et al. 2018). There is no literature information about pathogenicity.
Sphingobacterium sp. isolated from Helophorus aquaticus and Laccobius sulcatulus insect species has biotechnological importance in addition to being a bacterium that may be a rare infectious vector (Cai et al. 2019).
Accurate laboratory techniques have critical importance in identification of infectious syndromes linked to microorganisms with uncertain pathogenicity that are rarely isolated. 16S rRNA gene sequencing allows the opportunity to accurately identify potential pathogenic bacteria. Lysinibacillus, generally accepted as an environmental pollutant when isolated in clinical microbiology laboratories, was documented to have pathogenic potential in humans. Lysinibacillus sphaericus, identified in the study, was reported to cause 12 (2%) out of 469 bacteremia attacks in children with cancer during a 10-year duration in a pediatric cancer hospital in Italy (Wenzler et al. 2015).
Another bacterium obtained in the study was Kurthia gibsonii which is accepted as a zoonosis transmitted by sexual routes. It is commonly found in soil polluted by sewage, animal-sourced products and animal feces. This bacterium with the ability to survive in difficult living conditions was isolated from human feces in acute diarrhea, and is accepted as pathogenic in most cases due to causing gastroenteric diseases. However, the pathogenicity of this bacteria has not yet been confirmed with clinical evidence and the virulence factors are unknown (Pawar et al.2012; Cucini et al. 2020; Kövesdi et al. 2016).
In the study, 3 bacteria from Microbacterium species were identified. Microbacteria are found in soil, waste water, hospital pools and humidifiers and it is mainly known as an environmental isolate. In recent times, the frequency of reports as a potential pathogenic agent in humans, especially patients with weak immune systems, has increased (Woo et al. 2010).
Another group found densely in insect intestines and gaining importance due to spores and being pathogenic is Bacillus. In this study, 9 Bacillus species were isolated, with B. cereus isolated in 2 different insect species. The important human pathogen of B. cereus is a common cause of food-sourced gastroenteritis. Firstly, accepted as a harmless pollutant, this bacterium is known to be the etiologic agent for a variety of intestinal and extraintestinal diseases since the 1960s. (Tuipulotu et al.2020).
In spite of our study being an in vitro study characterizing cultured bacteria, bacterial density is notable. However, it is possible to identify more pathogenic microorganisms with metagenomic analyses, identifying the whole intestinal microbiome (Bektaş et al. 2021). For example, some fussy microorganisms defined with molecular methods in this study could not be isolated with traditional culture-based methods due to having specific growth requirements. For this reason, the use of methods independent of culture in research about pathogenic microorganisms in insect microbiota is very important.
The findings obtained in this study show that insect microbiota in the families studied contain many bacterial assemblages including microorganisms present in soil and water. While some of the bacteria in these microorganism assemblages, including permanent and temporary microbiota elements, protect the insect against a variety of pathogens, some may play the role of opportunistic pathogen in humans, while others have phytopathogenic properties. In the study, two bacteria (Pectobacterium cp., Bacillus mojavensis) without clear pathogenicity but accepted as phytopathogens were isolated.
Interest in insects as an alternative candidate for sustainable nutrient sources in the future will increase further in the future. Findings obtained in our study isolated both pathogenic and potentially pathogenic microorganisms and bacteria creating endospores like Bacillus sp. from insects in the Helophoridae and Hydrophilidae families. This makes it necessary to assess raw consumption of insects from these families in terms of being a risk factor for human health.
Additionally, the broad ecologic and taxonomic diversity of insects make it difficult to generalize about intestinal microbiota and there is a need for more research of this topic. The food safety of insect consumption has not been fully determined and it is thought the findings obtained in the study will provide current data about insect microbiota for the literature and contribute to future research.