1. Introduction
Antibiotics represent a distinct class of therapeutic agents in the fight against infectious diseases [
1]. They are produced by microorganisms and kill pathogenic microbes by targeting specific microbial components [
1]. However, the emergence of antibiotic resistance poses a serious threat to the effectiveness of antibiotics, leading to a worldwide public health crisis that cannot be understated [
2,
3,
4]. According to the World Health Organization’s (WHO’s) 2014 report, the world is at the brink of a post-antibiotic era, characterized by inoperable infections, an increased mortality rate, and escalating healthcare costs [
5]. In 2019, an estimated 4.95 million deaths were attributed to antibiotic resistance, with 1.27 million cases directly caused by infections resulting from this phenomenon [
4]. If effective preventive measures are not implemented immediately, the worldwide death toll from antibiotic resistance is projected to potentially surge to 10 million annually by 2050 [
6]. Human activities, such as the improper use of antibiotics, inappropriate disposal of unused medicines, as well as intensive agricultural practices, have exacerbated this natural phenomenon. The increasing concern over the rise of antibiotic-resistant bacteria in clinical settings has presently prompted efforts to identify the environmental reservoirs of antibiotic resistance genes (ARGs), as these reservoirs serve as crucial intermediaries and important routes for the spread of antibiotic resistance [
7,
8,
9]. Lakes, rivers, hot springs, and coastline areas, including soils, can act both as natural reservoirs of antibiotic resistance and pathways for the dissemination of clinically relevant ARGs [
10].
While the research has primarily focused on antibiotic resistance in thermophilic and mesophilic bacteria, both pathogenic and non-pathogenic extremophilic bacteria have received less attention, despite their extensive use in biotechnological and industrial sectors. For instance, moderate thermophiles, which cause diseases, such as meningitis, endocarditis, and septicemia, have been found to be resistant to various antibiotics, including erythromycin, tetracycline, sulfamethoxazole, tobramycin, and netilmicin [
11]. Similarly, isolates of
Arthrobacter sp. and
Hafnia sp. from hot springs have been found resistant to antibiotics [
12]. However, the role of poly-extremophilic environments, which accommodates multiple extremities of temperature, salinity, and heavy metal concentrations, in the distribution of ARGs and their antibiotic resistance profiles remains underexplored.
In addition to the biological factors, the widespread distribution of heavy metals in the environment is another consequential aspect of industrialization and urbanization that has had harmful impacts on human health and ecological risks [
13]. Growing evidence suggests that metal contamination in natural environments significantly contributes to the persistence and proliferation of antibiotic resistance [
14,
15,
16,
17,
18]. The historical exposure of bacteria to metals has made them a major source of environmental contamination. It is believed that co-selection is one of the primary mechanisms through which heavy metals contribute to the increased ARG levels in bacterial-rich environments [
19,
20,
21,
22,
23]. For instance, soil bacterial resistomes have been found to expand under the selective pressure of Cu exposure [
24]. Similarly, Hu and co-authors [
25] reported an increase in the frequencies and abundances of ARGs in agricultural soils with elevated nickel levels. Recent studies using metagenomics and metagenome-assembled genomes have further demonstrated the extensive co-occurrence of ARGs and MRGs in bacterial hosts found within activated sludge and urban rivers [
26,
27]. This co-occurrence might suggest a significant environmental risk in areas with high levels of heavy metals. The close linkages between these resistance genes suggest the potential for co-transferability and co-expression mechanisms. This study was conducted in the Danakil Depression, specifically Lake Afdera and the Assale salt plain, where salt mining is a routine activity. The unregulated extractions of salt in these areas can lead to human contamination, as salt miners often lack the proper sanitation facilities. Moreover, Lake Afdera is utilized for both bathing and laundry, enhancing the potential for contamination. Consequently, it is imperative to ensure the health-related safety of these ecologically important reservoirs from further anthropogenic interventions. This is crucial not only because they can potentially serve as reservoirs for the spread of ARGs, but also due to their general exposure to elevated levels of heavy metals. The primary goal of this study is to determine the abundance of ARGs and MRGs in Lake Afdera and the Assale salt plain in the Danakil Depression. The study also aims to explore the potential correlations between environmentally toxic heavy metals and their associated ARGs. For this purpose, shotgun metagenome sequences are mined against databases, such as the comprehensive antibiotic resistance database (CARD) and BacMetScan V.1.0., available from the BacMet AntiBacterial Biocide and MRG databases to identify potential ARGs and MRGs, respectively.
3. Discussion
Over the course of the antibiotic era, human health has shown remarkable improvements, leading some scientists to claim the end of infectious diseases [
28]. However, the dramatic rise in antibiotic resistance among microorganisms has emerged as a growing global concern for human health [
5]. Although environmental microbes, which are not disease-causing and are closely linked with antibiotic synthesis, have been largely overlooked, they are presently recognized to play a vital role in the evolution of antibiotic resistance. It is believed that pathogens are naturally vulnerable to antibiotics and do not inherently possess ARGs, but acquire them from environmental microbes. At present, the mechanism through which ARGs evolve and spread in environmental microorganisms remains unclear. Notably, every habitat, even habitats isolated for centuries or millennia, contains bacteria resistant to antibiotics and genes linked to resistance [
29,
30,
31].
The research on antibiotic resistance has primarily focused on bacteria from thermophilic, mesophilic, and psychrophilic environments [
32]. However, no attention has been given to the unique poly-extreme environments of the Danakil Depression, in the northern Afar region of Ethiopia. To address this gap, this study was designed to investigate the distribution of ARGs and MRGs in the extreme settings of Dallol and Lake Afdera. Previous studies have shown that the Danakil Depression is heavily enriched with various heavy metals, contributing to the remarkable color variations observed in the region’s hot springs [
33,
34,
35]. The combination of high metal concentrations and extreme physico-chemical parameters (e.g., temperature, pH, and salinity) has led to the creation of a unique multi-extreme environment in the Danakil Depression. A taxonomic analysis revealed the dominance of Pseudomonadota in both extreme environments, suggesting a resemblance in the bacterial community structures between the two locations. While the abundance percentage varied, the major phyla, Pseudomonadota, Actinomycetota, and Bacilliota, were consistent, except for Bacteroidota, which was found exclusively in Lake Afdera and not in the Assale salt plain. This miniscule community composition difference might stem from variations in the physicochemical factors and distant geographical locations [
32]. The low taxonomic diversity in Lake Afdera and the Assale salt plain aligned with the previous findings in other extreme hot springs, such as Sikkim, a northeastern state in India [
36]. Additionally, similarities in community structure can also be observed when compared to the Rehai geothermal field located in China [
37].
Our metagenomic analysis detected several putative ARGs in Lake Afdera, while the Assale salt plain had less (
Supplementary Table S1). Of the identified ARGs, half were considered housekeeping genes and similarity searches using CARD returned no matches. These genes included
ACC-1,
OXA-50,
vatD,
MexC,
APH3-Ia,
adeH,
OpmB,
ACT-29,
vanHB,
LRA-19,
AAC6-Ib-cr,
SMB-1,
LRA-13,
TEM-71,
chrB,
AER-1,
IND-5,
tetX,
tetO,
rmtB,
APH2-IIa,
MexD,
tet32,
emrB,
mexN,
OpmH,
lnuA,
MuxB,
mexM,
srmB,
Sed-1,
tetH,
dfrA10,
Erm38,
OpmH,
arr-4,
ErmR,
EXO-1,
mexM, and
tetA46. An annotation procedure via RAST revealed their multi-functionality properties. For instance, the
emrB gene encodes both DNA gyrase subunit A (EC 5.99.1.3) and DNA gyrase subunit B (EC 5.99.1.3), which are engaged in cell division and DNA replication. Another example is the
ChrB gene, which encodes a chromate transport protein. Interestingly, these genes, along with metal-dependent hydrolases of the beta-lactamase superfamily I proteins and multidrug resistance efflux pumps, were present in the RAST MAGs of Bacilliota (
Supplementary Table S14). However, strains, like
Bacillus cereus and
Lysinibacillus fusiformis, despite carrying these genes, showed susceptibility to antibiotics (unpublished data). This suggests that these genes may serve alternative metabolic roles, making them potentially inactive or hypothetical in antibiotic resistance. Numerous studies on
Bacillus species and their isolates have consistently produced findings that indicate their susceptibility to various tested antibiotics. For instance, a study conducted by Coonrod and co-authors demonstrated that 49
Bacillus isolates were susceptible when tested against six different classes of antibiotics [
38]. Investigations in Jordanian hot springs and other extreme environments, such as remote cave microbiomes, Antarctic marine waters, and pristine mountain rivers, have also shown the susceptibility of
Bacillus strains to antibiotics [
39]. The presence of resistance genes for β-lactam antibiotics is not surprising, given the involvement of β-lactamases in various common bacterial functions, like cell wall biosynthesis, signaling molecules, the detoxification of metabolites, and other processes [
40].
The limited abundance of a few phyla, Actinomycetota and Bacilliota, which are not the key hosts of ARGs, combined with the observed low diversity, suggests diminished competition among microbial communities for acquiring ARGs in the current ecosystem. Moreover, the metagenomic investigation of the Assale salt plain identified only a few ARGs, predominantly related to Gram-negative bacteria (
Supplementary Table S10). This scarcity of ARGs might be intrinsic to the environment or result from contamination, potentially from salt soil microflora or human skin flora introduced during salt mining practices. Previous research by Miller et al. emphasized the link between human activities and antibiotic resistance in remote sampling stations, such as Palmer in the Antarctic [
41]. Given the uncertainty surrounding this issue, we examined the abundance of heavy metals and MRGs, and also the correlations that possibly existed between ARGs and MRGs. Several heavy metals were detected, including Cu, Fe, Zn, Cd, Ni, and Pb. The samples from the Danakil Depression, especially those from Lake Afdera, which originated from a depth of 160 m, were influenced by the surrounding rocks, leading to the wide distribution of metallic pollutants. Notably, Cu and Zn were abundant in the study sites and were directly linked to the development of heavy metal tolerance in the environmental microflora since the dawn of time [
42]. In line with this, metal tolerance was assessed and the potential co-occurrence of heavy metal resistance with antibiotic resistance was explored. While metals, like copper and zinc, are essential nutrients supporting various physiological and cellular functions in microorganisms, they become toxic at high concentrations [
43]. This necessitates the development of resistance mechanisms by microorganisms in response to prolonged exposure [
44]. Accordingly, MRGs associated with copper, cobalt zinc, cadmium mercury, nickel, lead, arsenic, and chromium were identified in this study. Corroborating these findings, MRGs were also found in a separate study conducted on a functional metagenomic analysis [
32]. Previous research has documented a significant increase in metal tolerance, particularly for copper, among several bacterial isolates, including, but not limited to,
E. coli [
45], and
S. enterica [
46],
G. thermoleovorans, and
G. thermantarcticus [
47].
In the environmental samples, heavy metals were observed alongside antibiotic-resistant microbial communities [
48]. More than half of the total microbial resistance genes detected in our study were linked to commonly consumed antibiotics, such as betalactam (32.1%), aminoglycoside (11.1%), and tetracycline (11.1%), as well as heavy metals, like Cu, Hg, and Cd. Similar findings were reported in previous abundance-based surveys [
49,
50]. This pattern suggests a potential correlation between the abundance profiles of ARGs and MRGs across different microbial phyla. Pseudomonadota (82.6%) emerged as the dominant phylum carrying ARGs and MRGs, followed by Bacilliota (7.6%), Actinomycetota (4.3%), and Bacteroidota (1.1%), indicating a potential risk of resistance gene dissemination. This distribution suggests a correlation between microbial communities, particularly those associated with humans, and the abundance of ARGs and MRGs (
Figure 6). The long evolutionary periods, influenced by significant anthropogenic activities, like unhygienic salt mining practices, might facilitate the dissemination of genes, providing microorganisms with enhanced protection against harsh environmental conditions [
51,
52].
The observed abundance profiles of ARGs and MRGs within various genome phyla, combined with the selection pressure from a metallic-rich environment, raise our interest. Additionally, the recently reported abundance correlation in clinically important genera (e.g.,
Escherichia,
Shigella, and
Klebsiella) [
53] prompted us to investigate the possible co-occurrence between ARGs and MRGs. The Spearman’s correlation analysis (
r2 ≥ 0.8,
p < 0.05) revealed significant associations between specific heavy metals (Cd and Zn) and ARGs (
Table 4 and
Table 5). These findings are consistent with previous studies that also report similar correlations, particularly between Zn and ARGs originating from sulfonamides [
53] and tetracycline [
48], though the trends are diversified [
54,
55]. Additionally, significant correlations were observed between ARGs and MRGs (
Table 6), as well as between specific ARGs, MRGs, and heavy metals from the network analysis (
Figure 7). This supports the hypothesis of the co-occurrence of MRGs and ARGs in the resident microbes in the sampling sites, a phenomenon increasingly reported in the literature, particularly in intestinal microbes subjected to antibiotic and metal pressures [
56,
57,
58]. Further, this co-occurrence was observed in experiments conducted by Pal and colleagues [
50], suggesting a novel co-selection potential, and was also observed in full-scale biogas reactors [
59]. Some other previous studies have also reported the selection of ARGs by various metals [
48,
60], including those found in animal manure [
61] and copper tailing dam areas [
55].
The co-selection of MRGs with ARGs is hypothesized to be primary pathway for the spread and persistence of antibiotic resistance in different environments [
62,
63]. This link can occur through shared functions, the co-regulation of gene expression, and physical co-localization of resistance genes (with MRGs and ARGs located in the same mobile element) [
64]. Efflux pumps, like the multidrug efflux pump
mdrL in
Listeria monocytogenes, can lead to cross resistance by expelling both antibiotics and heavy metals [
65]. In the cases of co-regulation, the gene expressions of both ARGs and MRGs were influenced by a common factor. For example, in
Pseudomonas aeruginosa, the characterization of efflux pumps (
CzcCBA), which are responsible for resistance to Zn and Cd, revealed that their expressions were regulated by two genes,
CzcS and
CzcR. These genes also controlled the expression of the
OprD porin, which was responsible for resistance against carbapenems (a class of β-lactam antibiotics) [
60]. Additionally, the co-localization of MRGs and ARGs on mobile genetic elements played significant role in co-selection, leading to the dissemination of ARGs as a consequence of heavy metal contamination. This suggests that ARGs can be maintained in the environment through the co-selection with MRGs in polluted areas, where the level of heavy metal contamination is remarkably higher than that of antibiotics pollution [
66]. Overall, the results of the present study imply the heavy metal-induced selection of ARGs in multi-metal polluted extreme environments, raising concerns for human and animal health due to frequent exposure to the sampling area. Further studies are necessary to investigate the potential public health risks associated with these co-selection mechanisms and their role in promoting resistance in bacterial communities.