Desvendando Staphylococcus nepalensis: avaliação da larva Tenebrio molitor como modelo de patogenicidade e caracterização de plasmídeos carreadores de resistência antimicrobiana

Abstract

Staphylococcus nepalensis is a coagulase-negative Staphylococcus (CoNS) initially identified as part of goat’s normal microbiota. However, it is now known to be found in different locations and hosts, including some cases of opportunistic infections in humans. Similar to other CoNS, S. nepalensis may serve as a reservoir for antimicrobial resistance (AMR) genes, potentially impacting more pathogenic species like Staphylococcus aureus. The aim of this project is to characterize the S. nepalensis species and its plasmids: i) evaluating virulence and horizontal gene transfer in an in vivo model and ii) establishing an efficient protocol for plasmid extraction to sequence and characterize the plasmids obtained. First, we established Tenebrio molitor larvae as an in vivo model to evaluate the virulence of Staphylococcus spp. Our findings demonstrated that S. aureus, Staphylococcus saprophyticus and S. nepalensis killed T. molitor in a dose-dependent manner and stimulates the cellular immune response of larvae. Notably, when compared at the same concentration, only the S. nepalensis 136b strain was able to kill more than 50% of the larvae at doses lower than 105 CFU (Colony forming unit at 72h post-infection. We also utilized T. molitor larvae as a model for plasmid transfer. For this, larvae were inoculated with the donnor strain S. aureus RN7242 (106 CFU), which carries the gentamicin resistance plasmid pGO1, and the recipient strain S. aureus RN2677 105 CFU. We successfully obtained transconjugant colonies with a frequency of 1.6 × 10−5 per donor cell, demonstrating the suitability of T. molitor as a host for staphylococcal horizontal gene transfer. Next, we tested different plasmid extraction methods to ensure higher-quality samples for sequencing. To evaluate the efficiency of plasmid recovery, we compared two approaches using the S. nepalensis 136b strain: i) total DNA extraction and ii) plasmid-enriched DNA extraction. The plasmid-enriched extraction using Qiagen’s miniprep kit, with an additional lysozyme step, improved assembly results, allowing the recovery of four complete plasmids. Three mobilizable plasmids were identified carrying the resistance genes aadk, cat, and tetK, which confer resistance to streptomycin, chloramphenicol, and tetracycline, respectively. Comparative and phylogenetic analyses revealed high sequence similarity between these plasmids and mobile genetic elements found in various pathogenic and environmental bacteria, including S. aureus, S. epidermidis, Enterococcus sp., and Pseudomonas aeruginosa, indicating plasmid circulation across different genera. Additionally, a novel plasmid was identified, showing low similarity to any sequence deposited in databases, suggesting the existence of uncharacterized plasmid lineages in commensal Staphylococcus. This study highlights the importance of monitoring commensal species such as S. nepalensis, which can act as reservoirs of resistance genes, promoting their dissemination and posing a potential risk for opportunistic infections.