Genomic Insights Into the Pathogenicity of a Novel Biofilm-Forming Enterococcus sp. Bacteria (Enterococcus lacertideformus) Identified in Reptiles

Abstract

Whole genome analysis of a novel species of enterococci, Enterococcus lacertideformus, causing multi-systemic and invariably fatal disease in critically endangered Christmas Island reptiles was undertaken to determine the genetic elements and potential mechanisms conferring its pathogenic nature, biofilm-forming capabilities, immune recognition avoidance, and inability to grow in vitro. Comparative genomic analyses with related and clinically significant enterococci were further undertaken to infer the evolutionary history of the bacterium and identify genes both novel and absent. The genome had a G + C content of 35.1%, consisted of a circular chromosome, no plasmids, and was 2,419,934 bp in length (2,321 genes, 47 tRNAs, and 13 rRNAs). Multi-locus sequence typing (MLST), and single nucleotide polymorphism (SNP) analysis of multiple E. lacertideformus samples revealed they were effectively indistinguishable from one another and highly clonal. E. lacertideformus was found to be located within the Enterococcus faecium species clade and was closely related to Enterococcus villorum F1129D based on 16S rDNA and MLST house-keeping gene analysis. Antimicrobial resistance (DfreE, EfrB, tetM, bcrRABD, and sat4) and virulence genes (Fss3 and ClpP), and genes conferring tolerance to metals and biocides (n = 9) were identified. The detection of relatively few genes encoding antimicrobial resistance and virulence indicates that this bacterium may have had no exposure to recently developed and clinically significant antibiotics. Genes potentially imparting beneficial functional properties were identified, including prophages, insertion elements, integrative conjugative elements, and genomic islands. Functional CRISPR-Cas arrays, and a defective prophage region were identified in the genome. The study also revealed many genomic loci unique to E. lacertideformus which contained genes enriched in cell wall/membrane/envelop biogenesis, and carbohydrate metabolism and transport functionality. This finding and the detection of putative enterococcal biofilm determinants (EfaAfs, srtC, and scm) may underpin the novel biofilm phenotype observed for this bacterium. Comparative analysis of E. lacertideformus with phylogenetically related and clinically significant enterococci (E. villorum F1129D, Enterococcus hirae R17, E. faecium AUS0085, and Enterococcus faecalis OG1RF) revealed an absence of genes (n = 54) in E. lacertideformus, that encode metabolic functionality, which potentially hinders nutrient acquisition and/or utilization by the bacterium and precludes growth in vitro. These data provide genetic insights into the previously determined phenotype and pathogenic nature of the bacterium.

Genomic Insights of Enterococcus faecium UC7251, a Multi-Drug Resistant Strain From Ready-to-Eat Food, Highlight the Risk of Antimicrobial Resistance in the Food Chain

“…Moreover, unique genes epsM and epsL coding for putative acetyltransferase and sugar transferase, respectively, were detected. They are involved in the production of the exopolysaccharide (EPS) component of the extracellular matrix during biofilm formation (Agius et al, 2021 ). Gene cbh_2 , choloylglycine hydrolyse, catalyzes the de-conjugation of bile acids (Chand et al, 2018 ).…”

Experimental infection of Asian house geckos with Enterococcus lacertideformus demonstrates multiple disease transmission routes and the in-vivo efficacy of antibiotics

“…The treatments administered during the trial were chosen based on one or more of the following criteria: efficacy against Gram positive bacteria, ability to penetrate biofilms, therapeutic index, previous use in reptiles, capacity to administer via the oral route, and susceptibility to E. lacertideformus and other species of enterococci. A screen of the E. lacertideformus genome for antimicrobial resistance genes in a previous study revealed a resistance profile only to the antibiotics trimethoprim, tetracycline, streptothricin, and bacitracin 7 .…”

“…Therefore, understanding the modes of transmission, clinical course of infection, and susceptibility of E. lacertideformus to antimicrobial treatments are critically important to inform disease control and management. Based on a recent metagenomics study 7 , it appears that E. lacertideformus has limited antibiotic resistance and is likely susceptible to fluoroquinolones, macrolides, broad-spectrum penicillins, and rifamycins, antimicrobials that are often effective against other enterococci and have the added advantage of high penetrating capacity of biofilms 8 , 9 . These antibiotics have also been shown to be safe when administered to reptiles and can be given orally, which is a significant advantage when treating reptiles as small as two grams.…”

Multisystemic Enterococcosis in Brown Anoles (Anolis sagrei) from Florida, USA

“…We performed PCR amplification and Sanger sequencing of an approximately 1,400 base pair (bp) portion of the 16S SSU gene (GenBank accession number ON017515) on mass material from the index case using previously described methods (Schuurman et al 2004). Molecular analysis revealed 100% sequence identity to Enterococcus lacertideformus, a bacterium first observed in 2014 as the cause of similar lesions in multiple lizard species from Christmas Island, an Australian territory in the Indian Ocean (Rose et al 2017;Agius et al 2021). For further comparison, primers were designed to amplify the ITS region of the Enterococcus genome (Enter-o10F: 5 0 -TCAAAGATGCTGGGTTCCCC-3 0 and Entero553R: 5 0 -TGGGGTGAAGTCG-TAACAAGG-3 0 ) from mass material of 3/6 anoles, resulting in a 482-bp amplicon after primer editing that also exhibited 100% sequence identity to E. lacertideformus (Gen-Bank ON032371).…”