Genome-centric metagenomes unveiling the hidden resistome in an anchialine cave

Abstract

Background: Antibiotic resistance is a critical global concern, posing significant challenges to human health and medical treatments. Studying antibiotic resistance genes (ARGs) is essential not only in clinical settings but also in diverse environmental contexts. However, ARGs in unique environments such as anchialine caves, which connect both fresh and marine water, remain largely unexplored despite their intriguing ecological characteristics.

Results: We present the first study that comprehensively explores the occurrence and distribution of ARGs and mobile genetic elements (MGEs) within an anchialine cave. Utilizing metagenomic sequencing we uncovered a wide array of ARGs with the bacitracin resistance gene, bacA and multidrug resistance genes, being the most dominant. The cave's microbial community and associated resistome were significantly influenced by the salinity gradient. The discovery of novel β-lactamase variants revealed the cave's potential as a reservoir for previously undetected resistance genes. ARGs in the cave demonstrated horizontal transfer potential via plasmids, unveiling ecological implications.

Conclusions: These findings highlight the need for further exploration of the resistome in unique environments like anchialine caves. The interconnected dynamics of ARGs and MGEs within anchialine caves offer valuable insights into potential reservoirs and mechanisms of antibiotic resistance in natural ecosystems. This study not only advances our fundamental understanding but also highlights the need for a comprehensive approach to address antibiotic resistance in diverse ecological settings.

Keywords: Anchialine cave; Antibiotic resistance gene; Metagenomics; Mobile genetic element; Resistome; Subterranean estuary.

Fig. 1

Study area. Location map of Croatia, Šibenik, and the Sarcophagus Cave in Čapljina. Water samples were collected in August 2021 from the anchialine cave at six different depths (SC1-SC6), ranging from 0 to 12 m, along the salinity gradient. The cave’s entrance is inaccessible to most, and skilled divers are needed to collect the samples. The topology of the anchialine cave was adapted from the plan of Vedran Jalžić

Fig. 2

Relative abundances of antimicrobial resistance classes and genes per sample based on the reads analysis. The composition of samples according to A antimicrobial resistance classes and B top 25 most abundant ARGs. The top 25 most abundant ARG were filtered out, and based on their total sum, their relative abundance per depth was calculated

Fig. 3

Taxonomical composition on the phylum level of metagenome-assembled genomes (MAGs) which carry antibiotic-resistance genes (ARGs) at a specific depth (SC1-SC6)

Fig. 4

The association between antibiotic resistance gene (ARG) resistance classes (according to CARD terminology) and taxonomic composition of the microbial community at the phylum level based on the ARG-carrying MAGs. Phyla of MAGs are displayed on the outer circos layer. Pseudomonadota are presented as classes (Gammaproteobacteria, Alphaproteobacteria and Magnetococcia). Archaea are presented as phyla Iainarchaeota, Nanoarchaeota, and Thermoplasmatota. MDR multidrug–resistance; confers resistance to at least three different drug classes)

Fig. 5

Heatmap of antibiotic resistance genes (ARGs) for six sampling sites (SC1-SC6). The abundance of ARGs was quantified and normalized based on the abundance of a particular ARG-containing MAG at a specific sampling depth. Rows were clustered using the “complete” method with the pheatmap function (presented in Fig. S6A)

Fig. 6

Distribution and taxonomic composition of mobile genetic elements (MGEs) in the anchialine cave. A Heatmap of MGE categories for six sampling sites (SC1–SC6). The abundance of MGE categories was quantified and normalized based on their abundance in a particular MAG at a specific sampling depth. Rows were clustered using the “complete” method with the pheatmap function. B The bar plots represent the relative abundance of bacterial phyla corresponding to the top 10 most abundant MGEs

Fig. 7

β-lactamases and MAGs phylogenetic trees. A β-lactamases phylogenetic tree based on maximum–likelihood (1000 ultrafast bootstrap approximation replicates) contains novel anchialine β-lactamase sequences and representative sequences from the Comprehensive Antibiotic Resistance Database (CARD). B Metagenome—assembled genomes from this study were aligned with the representative genomes and a phylogenetic tree was constructed using the Genome Taxonomy Database Toolkit (GTDB-Tk). β-lactamase sequences and MAGs detected in this study are coloured in blue