Assessment of bacterial community and characterization of Pseudomonas aeruginosa in Diptera flies from livestock kraals and residential dumping sites in Potchefstroom

Abstract

The gut microbiota of Diptera flies is poorly understood, especially those that coexist with rural community farms and human waste sites. Pseudomonas aeruginosa pose challenges in both clinical and environmental contexts due to its capacity to colonize natural ecosystems and resistance to antibiotics. This study characterized P. aeruginosa from illegal residential dumping sites in urban Ikageng and livestock kraals in rural Matlwang village of Potchefstroom, South Africa. Diptera flies were collected in January-February 2022 and were identified using morphological features and by amplification and sequencing of the Cytochrome Oxidase 1 (CO1) gene. Meta-taxonomic approach was utilized to investigate the gut bacterial diversity. Furthermore, P. aeruginosa was characterized using classical microbiological test, species specific PCR assay targeting the housekeeping gyraseB gene, and confirmed by 16S rRNA gene sequencing. Antimicrobial resistance patterns were investigated on the isolates using disc diffusion assay (DDA) and antibiotic resistant genes (ARGs) by PCR assay. Additionally, six virulence genes (exoS, plcN, plcH, toxA, lasB and algD) were screened among the isolates. The prevalent Diptera flies in Matlwang livestock kraal were identified as Hemipyrellia spp. (n = 435/1422, 31%), followed by Tabanus spp. (n = 341/1422; 24%) and Chrysomya spp. (n = 176/1422; 12%). Ikageng illegal residential dumping site was dominated by Hemipyrellia spp. (n = 2461/2793; 88%), followed by Sarcophagidae spp. (n = 200/2793; 7.2%), Chrysomya spp. (n = 53/2793; 1.9 %), and Synthesiomyia spp. (n = 46/2793; 1.6 %). Tabanus spp. was only found in Matlwang livestock kraal sites. Meta-taxonomic approach showed that the gut-fly consisted of 92.13% and 90.7% Proteobacteria phyla in Matlwang livestock kraals and Ikageng residential dumping, respectively. Pseudomonas species were more prevalent at the Ikageng residential dumping site (98.97%) than in the Matlwang livestock kraal (62.41%). Rickettsia spp. were prevalent in Matlwang livestock kraal with a relative abundance of 79%. Culture-based and molecular assays showed that P. aeruginosa is prevalent in Matlwang (n = 36/48; 75%) and Ikageng (n = 23/48; 48%). The most commonly detected virulent gene (96.6%) among the isolates was exoS, followed by plcN and algD genes (83.1%), lasB (81.4%), toxA (76.3%), and plcH (47.5%). All isolates were resistant to Metronidazole, Sulphamethoxazole, Cefazolin and Amoxicillin and susceptible to Neomycin (n = 26/59; 44.07%) and Gentamicin (n = 29/59; 49.1%) based on DDA. The sulphamethoxazole, sulI gene (n = 52/59; 88.1%) was the most detected ARG found in the isolates, followed by acc(3)-IV (n = 36/59; 80.6%) and rdxA (n = 21/59; 35.6%) genes that confirms resistance to gentamicin and metronidazole, respectively. The absence of the genes for Amoxicillin (Pbp1A) and Neomycin (aph (2'')-Ib) in all isolates suggests that they can be utilised to treat P. aeruginosa infections. The P. aeruginosa was present in both areas; however, it was more common in residential dumping sites, while Rickettsia spp. was prominent in livestock kraals. This study has highlighted the significance of the bacteria carried by Diptera flies that are of public health and veterinary importance. The results of this study will assist in informing management decisions on the prevention of bacterial pathogen transmission.