Antimicrobial resistance and genetic relationship among enterococci from siblings and non-siblings Heliconius erato phyllis caterpillars
Authors: Huff Rosana , Inhoque Pereira Rebeca , Pissetti Caroline , Mellender de Araújo Aldo , Alves d’Azevedo Pedro , Frazzon Jeverson , Guedes Frazzon Ana Paula 
Published: September 2019

Background: Studies evaluating bacterial in insects could provide information about host-microorganism-environment interactions. The gut community is recognized to have a profound effect on various physiological functions of the insects. Enterococcus is part of the gut community in humans andanimals, as well as in the guts of insects. The presence and antimicrobialresistance profileof enterococci are well studied in different animals; however, data in Heliconius erato phyllis (Lepidoptera; Nymphalidae), do not yet exist. Therefore, the aims of this study were to evaluate species distribution, antimicrobial resistance profile, virulence genes and genetic relationship amongenterococci isolated from fecal samples of siblings and non-siblings H. erato phyllis caterpillars collected from different places in South Brazil.

Methods: Three H. erato phyllis females were captured (two from a forest fragment and one from an urban area), and kept individually in open-air insectaries. Eggs were collected and caterpillars (siblings and non-siblings) were fed daily with Passiflora suberosa leaves. Fecal samples (n=12) were collected from fifth instar caterpillars, inoculated in selective medium and fifteen colonies were randomly selected from each sample. Enterococci were identified by PCR and MALDI-TOF, submitted to antimicrobial susceptibility tests by disk diffusion method, and screened for resistance and virulence genes by PCR. The genetic relationships among the strains were determined using pulsed-field gel electrophoresis (PFGE).

Results: A total of 178 enterococci were identified as: Enterococcus casseliflavus (74.15%; n=132),E. mundtii (21.34%; n=38), E. faecalis (1.12%; n=2) and Enterococcus sp. (3.37%; n=6). High rates of resistance to rifampicin (56%) and erythromycin (31%) were observed. One hundred and twenty (67.41%) out of the 178 isolates showed resistance to at least one compound and 6 (3.37%) were multidrug-resistant.None of the erythromycin-resistant strains was positive to erm(B) and msrC genes. The virulence genes esp, ace and gelE were observed in 35%, 7% and 1% of the strains, respectively. PFGE separated the enterococci into 22 patterns, being four patterns composed by strains from sibling caterpillars.

Conclusion: Enterococcus casseliflavus was the dominant species in fecal samples of fifth instar caterpillars. Resistant enterococci strains could be related to environmental pollution or linked to environmental resistome. The PFGE analysis showed related genetic relationship among some strains, suggesting that the enterococci isolated from fecal samples of fifth instar sibling caterpillars might have come from common sources, by diet (herbivory) and/or via vertical transmission (through egg surface). Further studies will be conducted to better understand the role of Enterococcus on the microbial gastrointestinal tract community of these insects, and the mechanisms involved in acquisition and maintenance of these bacteria.