IDENTIFICATION AND CHARACTERIZATION OF HONEY BEE (Apis mellifera) GUT BACTERIAL FLORA IN KHYBER PAKHTUNKHWA, PAKISTAN

Abstract

Honey bee (Apis mellifera) populations are declining and one of the factors involved may be gut flora such as pathogenic bacteria because comprised honey bee immune systems endure various infectious pathogens. Gut microbiota has been recognized to play a key role in honey bee (Apis mellifera) physiology. Therefore, the present study was designed to identify and characterize the gut bacterial flora of the honey bee in north-west Pakistan. However, a comprehensive survey of honey bee bacterial disease (AFB) was carried out in the study areas before subjecting the bee guts for bacterial identification. American foulbrood (AFB) disease is a deleterious bacterial disease worldwide, caused by the spore forming bacterium Paenibacillus larvae that affects honey bee larvae and causes significant decrease in honey bee populations. Following symptomatical and bacteriological approaches combined with 16S rDNA sequencing, an assessment has been made to evaluate the presence of AFB disease in North-West Pakistan, as no record for bee-associated bacterial disease from Pakistan was available. A total of 1276 samples from 1520 bee colonies (15 apiaries) were collected, of which 476 samples (37.30%) were found with symptoms of AFB. Biochemical and 16S rDNA analysis indicated that all of these farms have Paenibacillus larvae infection. It was concluded that the prevalence of AFB bacterial disease is at such an extent in these regions of Pakistan that it will devastate the apicultural industries to a large scale. A total of 150 aerobic and facultative anaerobic bacteria were characterized from the guts of 45 worker bees using biochemical assays and 16S rDNA sequencing followed by bioinformatics analysis. Cell morphology of the selected isolates was studied and characteristics such as form, color, margin, texture, and elevation were examined and reported. The gut isolates were classified into different bacterial phyla Firmicutes, Proteobacteria and Actinobacteria. High numbers of bacterial spp. observed were from the phylum Firmicutes followed by Proteobacteria. Most of the isolates belonged to the genera and families of Staphylococcus, Bacillus, Enterococcus, Ochrobactrum, Sphingomonas, Ralstonia, Enterobacteriaceae, Corynebacterium and Micrococcineae. Many of these bacteria were tolerant to acidic environments and fermented sugars, hence considered to be beneficial gut inhabitants and involved in the maintenance of a healthy microbiota. However, several opportunistic commensals that proliferate in the hive environment including members of the Staphylococcus haemolyticus group and Sphingomonas paucimobilis also were identified. Additionally, in order to study the cultivable gut bacteria of honey bees collected from Kohat district, a total of 219 bacteria were isolated and characterized by bacteriological parameters. The potential pathogenic bacterial isolates were identified based on 16S ribosomal DNA sequencing. Combined microbiological practices revealed the presence of the following bacterial genera in honey bee alimentary canals; Bacillus, Enterococcus, Escherichia, Micrococcus, Morganella, Ochrobactrum Pseudomonas, Salmonella, Shigella, Sphingomonas and Staphylococcus. Two pathogenic bacteria Salmonella enterica and Shigella sonnei, which cause diseases affecting human beings and other animals, were characterized. This is the first report on bee gut microbiota from north-west Pakistan. A total of 15 selected isolates from various time points were exposed to antibiotic susceptibility assays. Most of them did not demonstrate high resistance or increasing resistance in tested antibiotics. However, a relative increasing resistance was observed in few bacterial isolates. Among all bacterial isolates used in this essay KK9d (Bacillus sp.) was found to be the most resistant against the various antibiotic tested. The most susceptible isolates were BN8G1 (Bacillus subtilis) followed by KK8bA/KK10 (Paenibacillus larvae) and KK8bB/KT1E (Klebsiella pneumonia). In recent years challenging bacterial resistance to common antibiotics has been detected. One of the main causes of this growing concern is biofilm formation. Biofilm is a process by which bacterial cells are held together by an extracellular matrix to form sessile communities. These communities provide protection to the individual from external physical and chemical stresses. Therefore, preventing or eradicating biofilm formation of the bacterium is a medically important challenge. In the current study a total of 76 bacterial isolates were subjected to biofilm formation tests, nine isolates were found to be strong biofilm formers. Initial biofilm formation was examined on glass cover slides showing three strong, moderate and weak biofilm formations by different honey bee gut bacterial isolates. Scanning electron micrographs confirmed the biochemical data of biofilm formation and biofilm disruption. Additionally, using microscopy, bacterial adhesion trends mirrored the previous quantitative data as shown in the present work. Thus, a number of actively dispersed bacterial cells were seen using scanning electron microscope images compared with the control. Furthermore, after 24 h of biofilm growth on the glass coverslips in six-well plates, the biofilm structures were rinsed and imaged by atom force microscope without any chemical treatment. Many bacterial cells can be seen entrenched together in a biofilm matrix not treated with any type of honey dilutions. On the other hand bacterial cells matrixes are dissolved due to 13% Acacia honey and furrows in bacterial cells appeared after honey treatment with different concentrations. Finally, a comparative efficacy of plant extracts, including Neem (Azadirachta indica) and Barbaka (Vitex trifolia) against gut bacteria of the honey bee A. mellifera (Hymenoptera: Apidae) were assessed. The in vitro activities of the plant extracts were determined using standard methods against five bee gut bacterial isolates, including the well-known bee pathogenic bacteria Paenibacillus larvae. The obtained results from the phytochemical screening of Barbaka and Neem extracts showed inhibitory zones with diameters of 23 mm with 60 mg/mL against P. larvae and 14 mm with 60 mg/mL against Escherichia coli, respectively. None of the extracts proved to be effective against Salmonella enterica and the Neem extract showed intermediate activity against Bacillus subtilis and Staphylococcus hominis. The last step of the current study concluded that Barbaka and Neem extracts have antibacterial activity and are reasonably environmentally safe. However, more trials have to be conducted, in order to validate these results.