Honey bee gut an unexpected niche of human pathogen

1 Introduction

A wide range of pathogens as viruses, bacteria and parasites may affect honey bee colony, some are more harmful that leads to colony collapse (Potts et al., 2010; Evans and Schwarz, 2011). The honey bee populations are going to decline in various parts of the world and these could be due to adverse effect of multiple honey bee pathogens. Further, increasing prevalence of parasites and pathogens are among the most significant threats to managed bee colonies. (O’Neal et al., 2018; Cox-Foster et al., 2007). Like two known bacteria (Melissococcus plutonius and Paenibacillus larvae) that effect honey bees brood not adults and causes significant losses to beekeepers around the world. Many aspects of their transmissions, virulence and adult host mortality are poorly documented and still remain obscure (Genersch, 2010).

Contaminated water not only acts a reservoir of pathogenic bacteria (Khalil et al., 1994) but access to polluted water affect the health of insects especially honey bee (Staveley et al., 2014). The usage of reclaimed water sugar solution as a drinking water had negative impacts on the average deaths of the honeybee colonies. Reclaimed water also alters the shape of mid gut of bees (Hananeh, et al., 2014). Water resources are polluted having various animal and plants pathogenic bacteria. Several pathogens, in feces of animals are transported to plants by non-potable irrigation water, fertilizer and insects (Tyler and Triplett, 2008). As honey bee spread plant bacterial pathogens including Erwinia amylovora (fire blight pathogen) and Pseudomonas syringae while pollinating various plants (Pattemore et al., 2014)

A range of techniques has been used so far to characterize the gut bacterial flora of honey bee like culture dependent method and phenotypic screenings. Although biochemical characterization is very helpful in bacterial identification but Sanger-based and next generation DNA sequencing techniques are useful in identification of distinctive set of bacteria present in honey bees gut (Li et al., 2012; Cox-Foster et al., 2007; Engel et al., 2012). So this study aimed to investigate the bacterial communities from the digestive tract of managed honey bee workers, captured in North West districts of Khyber Pakhtunkhwa Pakistan, using bacteriological and molecular techniques. These findings will improve current knowledge on the composition and structure of bee gut microbiota and provide the framework for understanding their contribution to honey bee health and potential application in disease control.

2 Material and methods

3 Results discussions

A total of 219 bacterial isolates from gut of 30 worker honeybees were obtained and the presence of 11 genera were confidently identified (Fig. 1). 23% Salmonella and 18% Shigella were characterized up to species level by using bacteriological parameters combined with 16s rDNA gene sequencing. BLAST searching revealed that 87% of the obtained sequences were identical to Salmonella enterica and 84% of Shigella sonnei (Table 1). Generally, gut bacterial diversity of honey bee collected from three zones were almost similar but the Enterococcus spp in zone 1 and zone 2 and Pseudomonas spp in zone 3 were prevalent (Fig. 2). Five bacterial genera, Bacilli, Staphylococci, Escherichia, Pseudomonas and Micrococci (Fig. 1) appeared recurrently and may contribute the major flora of bee gut. Colonies PCR of the selected pathogenic bacterial isolates were amplified, except few colonies of Salmonella and Shigella (Fig. 3). The isolates KHT03 and KHT30 produced low quality PCR product (Fig. 3) which were not subjected to BLAST analysis.

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Fig. 1

Diversity of bacteria harboring in the guts of worker bees in Kohat.

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Fig. 2

Guts bacterial isolates of honeybee collected from different regiones in Kohat.

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Fig. 3

Showing bands of 16s rDNA gene amplification, two lanes (KHT02 and KHT23) visualizing Shigella sonnei DNA fragments while three lanes (KHT21, KHT17 and KHT1C) separating Salmonella enterica DNA fragments, 100 bp DNA ladder (L).

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Indeed, honey bee populations all over the world are at high risk of decline due to unidentified reasons however a high load of parasites and microbial pathogens especially bacteria strongly connected with the disappearing of bee population (Olofsson and Vásquez, 2008; Gilliam, 1997; Di Prisco et al., 2013; Core et al., 2012). Additionally, pathogens are not passive microbes when they enter the arthropod vector but actively influence vector gene expression that can manipulate the local environment (Abraham et al., 2017)

The present study demonstrated 219 bacterial isolates of 11 bacterial genera, identified (Fig. 1) from worker honey bee gut, collected from the different bee yards in Kohat. The worker bee harbored some well-known pathogenic or potentially pathogenic bacteria species, including Shigella sonnei and Salmonella enterica. These bacteria are known for various diseases such as diarrheal illnesses, nausea, vomiting, and fever in human being (Greeley, 2013; Karlsson et al., 2013; Holman et al., 2014). The Diptera flies as a carrier of pathogenic bacteria like Salmonella enterica have previously been reported (Holt et al., 2007), however the presence of the human pathogen in bee gut are quit alarming. Although it has been reported that honey bee spread plant bacterial pathogens including Erwinia amylovora and Pseudomonas syringae while pollinating various plants (Pattemore et al., 2014).).

Enterococcus spp in the guts of Apis nigrocincta based on an identity BLAST search of its COI region also revealing the richness of the group in the gut Apis spp. (Lombogia et al; 2020). Recently, bacterial isolates (Enterococcus spp.) were isolated from Egyptian honey bee’s intestinal tract and were expected that honey bee has the potential to be a source of new bacteria. (Elzeini et al; 2020). Pseudomonas spp. Enterococcus spp. were also recovered from ventriculum of Apis mellifera ligustica in Italy (Kačániová et al; 2020). Consequently, prevalence of the Enterococcus spp Enterococcus spp in bee guts collected from Zone 1–3 are in accordance of the recent published reports. However, differences and richness may be due to the type strains, procedures, and condition of cultivation. Similarly, Pseudomonas spp were also most abundant bacteria followed by Bacillus sp in drinking water of the study areas (Hussain et al; 2013).

The high incidence of bacteria presents in bee’s gut is a public health risk, as the synanthropic behavior of bees may conducive to disseminate through a wide variety of routes (Menasria et al., 2014). Worker honey bees forage in different sites where sugar food is prepared, processed, stored and thus it may increase likelihood of the risk of bacterial transmission. The lack of sufficient food is partially a management issue in bee keeping practices (Mattila and Otis, 2007). Beekeepers usually feed sugar solution during starvation but quality and diversity of sugar sources can affect number of bees (Pernal and Currie, 2001). Much like the human gut microbiota, many bee gut bacteria are specific to the bee gut and can be directly transmitted between individuals through social interaction. (Zheng et al., 2018). Apart from bee sociability, the main risk factor of transmission is water as in the study area it is highly contaminated with human pathogenic bacteria (Hussain et al., 2013). It is suggested that these pathogenic bacteria in bee gut are transferred from foraging sites or sugar supplements through contaminated water.

Moreover, it has been reported that sugar feeding like HFCS (high fructose corn syrup and SS (Sucrose syrup) decrease life span of worker bees (Sammataro and Weiss, 2013) and also colony collapse disorder (CCD) is an alarming colony mortality, correlated with the deleterious effects of sugar feeding (Van Engelsdorp et al., 2007; VanEngelsdorp and Meixner, 2010). But it is observed in the current study that beekeepers always use feeder continuously without sterilization for sugar feeding during starvation to honey bees. It is obvious that the feed or the water may be contaminated with pathogens like bacteria and may be ultimately resulted in bee’s population declining and also a source of transmission. However, there is a need of research to correlate bacteria present in bee gut to honey bee morbidity and mortality and to evaluate the sugar solutions used for bee feeding, hive tools, feeder and water as source of transmission of these bacteria.

Presence of human pathogenic bacteria in bee gut may be the main cause of declining of bee population. The same were proved while injecting various pathogenic bacteria (Salmonella enterica, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa and Serratia marcescen) to honey bees Apis mellifera. Moreover, the infected model bee with S. aureus also act as a vector to other hive mate and 50% of them died within 24 hrs (Ishii et al., 2014). Similar studies were carried out on Drosophilla melanogestar infected with P. aeruginosa and same results were obtained (Linder et al., 2008). Serratia marcescens, an opportunistic pathogen of many plants and animals, including humans, is a virulent opportunistic pathogen of honey bees, which could contribute to bee decline (Raymann et al., 2018). This indicates that human pathogenic bacteria may be the main cause of honey bee mortality and bee population declining.

4 Conclusion

It is concluded that honey bee gut is an alternate habitat for human pathogenic bacteria as high load of these bacteria recovered from the alimentary canal of honey bees. Furthermore, Salmonella and Shigella may play major role in mortality of honey bee in the study area.