Mechanism Of Horizontal Gene Transfer Research Articles

Effect of chemotherapeutic agents on natural transformation frequency in Acinetobacter baylyi

Natural transformation is the ability of a bacterial cell to take up extracellular DNA which is subsequently available for recombination into the chromosome (or maintenance as an extrachromosomal element). Like other mechanisms of horizontal gene transfer, natural transformation is a significant driver for the dissemination of antimicrobial resistance. Recent studies have shown that many pharmaceutical compounds such as antidepressants and anti-inflammatory drugs can upregulate transformation frequency in the model species Acinetobacter baylyi. Chemotherapeutic compounds have been shown to increase the abundance of antimicrobial resistance genes and increase colonization rates of potentially pathogenic bacteria in patient gastrointestinal tracts, indicating an increased risk of infection and providing a pool of pathogenicity or resistance genes for transformable commensal bacteria. We here test for the effect of six cancer chemotherapeutic compounds on A. baylyi natural transformation frequency, finding two compounds, docetaxel and daunorubicin, to significantly decrease transformation frequency, and daunorubicin to also decrease growth rate significantly. Enhancing our understanding of the effect of chemotherapeutic compounds on the frequency of natural transformation could aid in preventing the horizontal spread of antimicrobial resistance genes.

Comparison of integron mediated antimicrobial resistance in clinical isolates of Escherichia coli from urinary and bacteremic sources

BackgroundAntimicrobial resistance (AMR) is a global threat driven mainly by horizontal gene transfer (HGT) mechanisms through mobile genetic elements (MGEs) including integrons. The variable region (VR) of an integron can acquire or excise gene cassettes (GCs) that confer resistance to antibiotics based on the selection pressure. Escherichia coli plays a significant role in the genetic transfer of resistance determinants to other Gram-negative bacteria. Current study is aimed to detect and compare integron-mediated resistance in clinical isolates of E. coli. Unique isolates of E. coli from urine or blood cultures were studied for their antimicrobial resistance patterns and integrons were detected using polymerase chain reaction assays followed by Sanger sequencing of GCs.ResultsDuring the study period, a total of 470 E. coli isolates were obtained, 361 (76.8%) from urinary and 109 (23.1%) from bacteremic sources. Class 1 integrons were detected in 66 (18.2%) and 26 (23.8%) isolates respectively. Urinary isolates of E. coli harbouring Class 1 integrons demonstrated significantly higher rates of resistance (p < 0.05) for most antibiotics (12/16, 75%) compared to integron negative isolates. Although not statistically significant, similar differences were observed in bacteremic isolates. Among the urinary isolates, 27 (40.9%) had a VR, in which the most common GC array detected was DfrA17-AadA5 (n = 14), followed by DfrA5 (n = 4) and DfrA12 (n = 3). Among bacteremic isolates, only 4 (15.3%) had a VR, all of which were carrying DfrA17. The detected GC array correlated with the respective isolates’ phenotypic resistance patterns.ConclusionWe found a strong correlation between integron positivity and trimethoprim resistance among E. coli from urinary sources. Although higher rates of resistance were observed in bacteremic isolates, they mostly carried empty integrons.

Strong suppression of silver nanoparticles on antibiotic resistome in anammox process

This study comprehensively deciphered the effect of silver nanoparticles (AgNPs) on anammox flocculent sludge, including nitrogen removal performance, microbial community structure, functional enzyme abundance, antibiotic resistance gene (ARGs) dissemination, and horizontal gene transfer (HGT) mechanisms. After long-term exposure to 0–2.5 mg/L AgNPs for 200 cycles, anammox performance significantly decreased (P < 0.05), while the relative abundances of dominant Ca. Kuenenia and anammox-related enzymes (hzsA, nirK) increased compared to the control (P < 0.05). For antibiotic resistome, ARG abundance hardly changed with 0–0.5 mg/L AgNPs but decreased by approximately 90% with 1.5–2.5 mg/L AgNPs. More importantly, AgNPs effectively inhibited MGE-mediated HGT of ARGs. Additionally, structural equation model (SEM) disclosed the underlying relationship between AgNPs, the antibiotic resistome, and the microbial community. Overall, AgNPs suppressed the anammox-driven nitrogen cycle, regulated the microbial community, and prevented the spread of ARGs in anammox flocs. This study provides a theoretical baseline for an advanced understanding of the ecological roles of nanoparticles and resistance elements in engineered ecosystems.

Wall teichoic acid substitution with glucose governs phage susceptibility of Staphylococcus epidermidis.

The species- and clone-specific susceptibility of Staphylococcus cells for bacteriophages is governed by the structures and glycosylation patterns of wall teichoic acid (WTA) glycopolymers. The glycosylation-dependent phage-WTA interactions in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) have remained unknown. We report a new S. epidermidis WTA glycosyltransferase TagE whose deletion confers resistance to siphoviruses such as ΦE72 but enables binding of otherwise unbound podoviruses. S. epidermidis glycerolphosphate WTA was found to be modified with glucose in a tagE-dependent manner. TagE is encoded together with the enzymes PgcA and GtaB providing uridine diphosphate-activated glucose. ΦE72 transduced several other CoNS species encoding TagE homologs, suggesting that WTA glycosylation via TagE is a frequent trait among CoNS that permits interspecies horizontal gene transfer. Our study unravels a crucial mechanism of phage-Staphylococcus interaction and horizontal gene transfer, and it will help in the design of anti-staphylococcal phage therapies.IMPORTANCEPhages are highly specific for certain bacterial hosts, and some can transduce DNA even across species boundaries. How phages recognize cognate host cells remains incompletely understood. Phages infecting members of the genus Staphylococcus bind to wall teichoic acid (WTA) glycopolymers with highly variable structures and glycosylation patterns. How WTA is glycosylated in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) species has remained unknown. We describe that S. epidermidis glycosylates its WTA backbone with glucose, and we identify a cluster of three genes responsible for glucose activation and transfer to WTA. Their inactivation strongly alters phage susceptibility patterns, yielding resistance to siphoviruses but susceptibility to podoviruses. Many different CoNS species with related glycosylation genes can exchange DNA via siphovirus ΦE72, suggesting that glucose-modified WTA is crucial for interspecies horizontal gene transfer. Our finding will help to develop antibacterial phage therapies and unravel routes of genetic exchange.

Phages in different habitats and their ability to carry antibiotic resistance genes

As the most abundant organisms on Earth, phages play a key role in the evolution of bacterial antibiotic resistance. Although previous studies have demonstrated the molecular mechanisms of horizontal gene transfer mediated by mobile genetic elements, our understanding of the intertwined relationships between antibiotic resistance genes (ARGs) and phages is limited. In this study, we analysed 2781 metagenomic samples to reveal the composition and species interactions of phage communities in different habitats as well as their capacity to carry ARGs with health risks. The composition of phage communities varies in different habitats and mainly depends on environmental conditions. Terrestrial habitats display more complex and robust interactions between phages than aquatic and human-associated habitats, resulting in the highest biodiversity of phages. Several types of phages in certain taxa (4.95–7.67%, mainly belonging to Caudoviricetes) have the capacity to carry specific ARGs and display a high potential risk to human health, especially in human-associated habitats. Overall, our results provide insights into the assembly mechanisms of phage communities and their effects on the dissemination of antibiotic resistance.

Role of membrane vesicles in the transmission of vancomycin resistance in Enterococcus faecium

Clonal transmission and horizontal gene transfer (HGT) contribute to the spread of vancomycin-resistant enterococci (VRE) in global healthcare. Our study investigated vesiduction, a HGT mechanism via membrane vesicles (MVs), for vanA and vanB genes that determine vancomycin resistance. We isolated MVs for VRE of different sequence types (STs) and analysed them by nanoparticle tracking analysis. Selected MV samples were subjected to DNA sequence analysis. In resistance transfer experiments, vancomycin-susceptible enterococci were exposed to MVs and bacterial supernatants of VRE. Compared to bacteria grown in lysogeny broth (MVs/LB), cultivation under vancomycin stress (MVs/VAN) resulted in increased particle concentrations of up to 139-fold (ST80). As a key finding, we could show that VRE isolates of ST80 and ST117 produced remarkably more vesicles at subinhibitory antibiotic concentrations (approx. 9.2 × 1011 particles/ml for ST80 and 2.4 × 1011 particles/ml for ST117) than enterococci of other STs (range between 1.8 × 1010 and 5.3 × 1010 particles/ml). In those MV samples, the respective resistance genes vanA and vanB were completely verifiable using sequence analysis. Nevertheless, no vancomycin resistance transfer via MVs to vancomycin-susceptible Enterococcus faecium was phenotypically detectable. However, our results outline the potential of future research on ST-specific MV properties, promising new insights into VRE mechanisms.

NO3− as an electron acceptor elevates antibiotic resistance gene and human bacterial pathogen risks in managed aquifer recharge (MAR): A comparison with O2

Managed aquifer recharge (MAR) stands out as a promising strategy for ensuring water resource sustainability. This study delves into the comparative impact of nitrate (NO3−) and oxygen (O2) as electron acceptors in MAR on water quality and safety. Notably, NO3−, acting as an electron acceptor, has the potential to enrich denitrifying bacteria, serving as hosts for antibiotic resistance genes (ARGs) and enriching human bacterial pathogens (HBPs) compared to O2. However, a direct comparison between NO3− and O2 remains unexplored. This study assessed risks in MAR effluent induced by NO3− and O2, alongside the presence of the typical refractory antibiotic sulfamethoxazole. Key findings reveal that NO3− as an electron acceptor resulted in a 2 times reduction in dissolved organic carbon content compared to O2, primarily due to a decrease in soluble microbial product production. Furthermore, NO3− significantly enriched denitrifying bacteria, the primary hosts of major ARGs, by 747%, resulting in a 66% increase in the overall abundance of ARGs in the effluent of NO3− MAR compared to O2. This escalation was predominantly attributed to horizontal gene transfer mechanisms, as evidenced by a notable 78% increase in the relative abundance of mobile ARGs, alongside a minor 27% rise in chromosomal ARGs. Additionally, the numerous denitrifying bacteria enriched under NO3− influence also belong to the HBP category, resulting in a significant 114% increase in the abundance of all HBPs. The co-occurrence of ARGs and HBPs was also observed to intensify under NO3− influence. Thus, NO3− as an electron acceptor in MAR elevates ARG and HBP risks compared to O2, potentially compromising groundwater quality and safety.

Genomic Analysis of a Community-Acquired Methicillin-Resistant Staphylococcus aureus Sequence Type 1 Associated with Caprine Mastitis.

This study aimed to investigate the genomic and epidemiological features of a methicillin-resistant Staphylococcus aureus sequence type 1 (MRSA ST1) strain associated with caprine subclinical mastitis. An S. aureus strain was isolated from goat's milk with subclinical mastitis in Paraiba, Northeastern Brazil, by means of aseptic procedures and tested for antimicrobial susceptibility using the disk-diffusion method. Whole genome sequencing was performed using the Illumina MiSeq platform. After genome assembly and annotation, in silico analyses, including multilocus sequence typing (MLST), antimicrobial resistance and stress-response genes, virulence factors, and plasmids detection were performed. A comparative SNP-based phylogenetic analysis was performed using publicly available MRSA genomes. The strain showed phenotypic resistance to cefoxitin, penicillin, and tetracycline and was identified as sequence type 1 (ST1) and spa type 128 (t128). It harbored the SCCmec type IVa (2B), as well as the lukF-PV and lukS-PV genes. The strain was phylogenetically related to six community-acquired MRSA isolates (CA-MRSA) strains associated with human clinical disease in North America, Europe, and Australia. This is the first report of a CA-MRSA strain associated with milk in the Americas. The structural and epidemiologic features reported in the MRSA ST1 carrying a mecA-SCCmec type IVa suggest highly complex mechanisms of horizontal gene transfer in MRSA. The SNP-based phylogenetic analysis suggests a zooanthroponotic transmission, i.e., a strain of human origin.

Structure of the Portal Complex from Staphylococcus aureus Pathogenicity Island 1 Transducing Particles In Situ and In Isolation

Staphylococcus aureus is an important human pathogen, and the prevalence of antibiotic resistance is a major public health concern. The evolution of pathogenicity and resistance in S. aureus often involves acquisition of mobile genetic elements (MGEs). Bacteriophages play an especially important role, since transduction represents the main mechanism for horizontal gene transfer. S. aureus pathogenicity islands (SaPIs), including SaPI1, are MGEs that carry genes encoding virulence factors, and are mobilized at high frequency through interactions with specific “helper” bacteriophages, such as 80α, leading to packaging of the SaPI genomes into virions made from structural proteins supplied by the helper. Among these structural proteins is the portal protein, which forms a ring-like portal at a fivefold vertex of the capsid, through which the DNA is packaged during virion assembly and ejected upon infection of the host. We have used high-resolution cryo-electron microscopy to determine structures of the S. aureus bacteriophage 80α portal itself, produced by overexpression, and in situ in the empty and full SaPI1 virions, and show how the portal interacts with the capsid. These structures provide a basis for understanding portal and capsid assembly and the conformational changes that occur upon DNA packaging and ejection.

Systematic investigation of recipient cell genetic requirements reveals important surface receptors for conjugative transfer of IncI2 plasmids

Bacterial conjugation is a major horizontal gene transfer mechanism. While the functions encoded by many conjugative plasmids have been intensively studied, the contribution of recipient chromosome-encoded genes remains largely unknown. Here, we analyzed the genetic requirement of recipient cells for conjugation of IncI2 plasmid TP114, which was recently shown to transfer at high rates in the gut microbiota. We performed transfer assays with ~4,000 single-gene deletion mutants of Escherichia coli. When conjugation occurs on a solid medium, we observed that recipient genes impairing transfer rates were not associated with a specific cellular function. Conversely, transfer assays performed in broth were largely dependent on the lipopolysaccharide biosynthesis pathway. We further identified specific structures in lipopolysaccharides used as recipient cell surface receptors by PilV adhesins associated with the type IVb accessory pilus of TP114. Our strategy is applicable to study other mobile genetic elements and understand important host cell factors for their dissemination.

Occurrence and Role of Bacterial Biofilms in Different Systems

Bacterial biofilms are complex communities of bacteria that adhere to surfaces, including living tissues, and form a protective matrix of extracellular polymeric substances (EPS). Biofilms are widespread and play crucial roles in various processes, such as nutrient cycling, bioremediation, and biofouling. They have significant implications for public health. Biofilms provide an ideal environment for bacteria to exchange genetic material, including resistance genes, via horizontal gene transfer mechanisms such as conjugation, transformation, and transduction. Moreover, biofilms can protect bacteria from antibiotics and host immune responses, enabling them to persist and cause chronic infections. The EPS matrix, which can act as a physical barrier, limits the penetration of antibiotics into the biofilm, and the slow-growing or dormant cells within the biofilm are less susceptible to antibiotics than their planktonic counterparts. The significance of bacterial biofilms in the development of antibiotic resistance has prompted research efforts to understand their formation and mechanisms of resistance. Novel strategies to prevent or disrupt biofilm formation are also being explored, including the development of antibiofilm agents and biofilm-disrupting enzymes. Understanding the role of biofilms in the spread of antibiotic resistance is crucial for the development of effective treatment and prevention strategies to combat the chronic infections associated with biofilm-producing bacteria.

Restriction-methylation systems regulate transformation in Acinetobacter baumannii.

Antimicrobial resistance in Acinetobacter baumannii is a major concern. Natural transformation remains understudied as a horizontal gene transfer (HGT) mechanism for the spread of resistance genes. Recent work (Vesel et al.) reveals a profound impact of the state of donor DNA methylation with strong implications for HGT of resistance determinants in this worrisome pathogen.

Molecular characterization of methicilin-resistant Staphylococcus aureus strain BM85 isolated from a Vietnamese patient with bloodstream infection

Methicillin-resistant Staphylococcus aureus (MRSA) is a threat to global health due to its resistance to β-lactam antibiotics and many other antibiotic classes developed via both intrinsic and acquired mechanisms. In this study, molecular characteristics related to antibiotic resistance of MRSA strain BM85 were investigated by whole-genome sequencing of a sample isolated from a patient with bloodstream infection at Bach Mai Hospital, Vietnam. Antibiotic susceptibility testing was also performed to determine the correlation between the presence of antibiotic-resistant genes and resistance phenotypes. Genomic analyses showed that the MRSA strain BM85 belonged to the major community-acquired (CA)-MRSA lineage ST59 originating from Taiwan. The strain harbored Staphylococcal Cassette Chromosome mec (SCCmec) type Vb (5C2&amp;5) and Panton-Valentine leukocidin (PVL). Additionally, MRSA strain BM85 also possessed various antibiotic-resistant genes including tet38, tetK, blaZ, mecA, aph(3')-IIIa, aacA-aphD, and ermB which were located on mobile genetic elements MESPM1, Tn553-Tn4001 transposon, and a plasmid carrying the tetK gene, which was responsible for tetracycline resistance. The genotypic-resistant results were concordant with the phenotypic-resistant profile in which MRSA strain BM85 was resistant to penicillin, cefoxitin, gentamicin, kanamycin, tobramycin, erythromycin, and tetracycline. The sequencing data for the MRSA strain BM85 was deposited in GenBank, NCBI under accession number: BioProject PRJNA857185. In conclusion, the acquisition of the foreign genetic elements associated with antibiotic resistances through horizontal gene transfer mechanisms was the key driver of multidrug resistance in the multidrug-resistant MRSA strain BM85.

Inhibition of Plasmid Conjugation in Escherichia coli by Targeting rbsB Gene Using CRISPRi System.

Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic-resistant genes (ARGs) among human pathogens. The spread of ARGs can be halted or diminished by interfering with the conjugation process. In this study, we explored the possibility of using an rbsB gene as a single target to inhibit plasmid-mediated horizontal gene transfer in Escherichia coli by CRISPR interference (CRISPRi) system. Three single-guide RNAs (sgRNAs) were designed to target the rbsB gene. The transcriptional levels of the rbsB gene, the conjugation-related genes, and the conjugation efficiency in the CRISPRi strain were tested. We further explored the effect of the repressed expression of the rbsB gene on the quorum sensing (QS) system and biofilm formation. The results showed that the constructed CRISPRi system was effective in repressing the transcriptional level of the rbsB gene at a rate of 66.4%. The repressed expression of the rbsB gene resulted in the reduced conjugation rate of RP4 plasmid by 88.7%, which significantly inhibited the expression of the conjugation-related genes (trbBp, trfAp, traF and traJ) and increased the global regulator genes (korA, korB and trbA). The repressed rbsB gene expression reduced the depletion of autoinducer 2 signals (AI-2) by 12.8% and biofilm formation by a rate of 68.2%. The results of this study indicated the rbsB gene could be used as a universal target for the inhibition of conjugation. The constructed conjugative CRISPRi system has the potential to be used in ARG high-risk areas.

Detecting patterns of accessory genome coevolution in Staphylococcus aureus using data from thousands of genomes

Bacterial genomes exhibit widespread horizontal gene transfer, resulting in highly variable genome content that complicates the inference of genetic interactions. In this study, we develop a method for detecting coevolving genes from large datasets of bacterial genomes based on pairwise comparisons of closely related individuals, analogous to a pedigree study in eukaryotic populations. We apply our method to pairs of genes from the Staphylococcus aureus accessory genome of over 75,000 annotated gene families using a database of over 40,000 whole genomes. We find many pairs of genes that appear to be gained or lost in a coordinated manner, as well as pairs where the gain of one gene is associated with the loss of the other. These pairs form networks of rapidly coevolving genes, primarily consisting of genes involved in virulence, mechanisms of horizontal gene transfer, and antibiotic resistance, particularly the SCCmec complex. While we focus on gene gain and loss, our method can also detect genes that tend to acquire substitutions in tandem, or genotype-phenotype or phenotype-phenotype coevolution. Finally, we present the R package DeCoTUR that allows for the computation of our method.

The complex regulation of competence in Staphylococcus aureus under microaerobic conditions

To perform natural transformation, one of the three main Horizontal Gene Transfer mechanisms, bacteria need to enter a physiological differentiated state called genetic competence. Interestingly, new bacteria displaying such aptitude are often discovered, and one of the latest is the human pathogen Staphylococcus aureus.Here, we show an optimized protocol, based on planktonic cells cultures, leading to a large percentage of the population activating the development of competence and a significant improvement of S. aureus natural transformation efficiencies. Taking advantage of these conditions, we perform transcriptomics analyses to characterize the regulon of each central competence regulator. SigH and ComK1 are both found essential for activating natural transformation genes but also important for activation or repression of peripheral functions. Even though ComK2 is not found important for the control of transformation genes, its regulon shows an important overlap with that of SigH and ComK1. Finally, we propose that microaerobic conditions, sensed by the SrrAB two-component system, are key to activate competence in S. aureus.

Catch me if you can: capturing microbial community transformation by extracellular DNA using Hi-C sequencing

The transformation of environmental microorganisms by extracellular DNA is an overlooked mechanism of horizontal gene transfer and evolution. It initiates the acquisition of exogenous genes and propagates antimicrobial resistance alongside vertical and conjugative transfers. We combined mixed-culture biotechnology and Hi-C sequencing to elucidate the transformation of wastewater microorganisms with a synthetic plasmid encoding GFP and kanamycin resistance genes, in the mixed culture of chemostats exposed to kanamycin at concentrations representing wastewater, gut and polluted environments (0.01–2.5–50–100 mg L−1). We found that the phylogenetically distant Gram-negative Runella (102 Hi-C links), Bosea (35), Gemmobacter (33) and Zoogloea (24) spp., and Gram-positive Microbacterium sp. (90) were transformed by the foreign plasmid, under high antibiotic exposure (50 mg L−1). In addition, the antibiotic pressure shifted the origin of aminoglycoside resistance genes from genomic DNA to mobile genetic elements on plasmids accumulating in microorganisms. These results reveal the power of Hi-C sequencing to catch and surveil the transfer of xenogenetic elements inside microbiomes.

Guidelines for the estimation and reporting of plasmid conjugation rates

Conjugation is a central characteristic of plasmid biology and an important mechanism of horizontal gene transfer in bacteria. However, there is little consensus on how to accurately estimate and report plasmid conjugation rates, in part due to the wide range of available methods. Given the similarity between approaches, we propose general reporting guidelines for plasmid conjugation experiments. These constitute best practices based on recent literature about plasmid conjugation and methods to measure conjugation rates. In addition to the general guidelines, we discuss common theoretical assumptions underlying existing methods to estimate conjugation rates and provide recommendations on how to avoid violating these assumptions. We hope this will aid the implementation and evaluation of conjugation rate measurements, and initiate a broader discussion regarding the practice of quantifying plasmid conjugation rates.

THE PREFERENCE PRIORITY OF Bacillus subtilis IN UPTAKING FREE DNA DURING THE NATURAL TRANSFORMATION

Although genetic material is vertically transferred between generations via sexual or asexual reproduction, similarities in some chromosome and gene parts of unrelated organisms provide important clues for another way of transfer. The mobility of genetic information among different organisms, known as horizontal gene transfer (HGT) has immediate or delayed effects on the recipient host. One of the most notable mechanisms of HGT is natural transformation (NT), a process in which cells take free DNA from the extracellular environment and incorporate it into their chromosomes by homologous recombination. NT is widely conserved in many bacterial species as it can promote to spread of resistance genes. Although it is known that many organisms rely on HGT, there is limited information about how they decide which particular genetic material to horizontally transfer. Here, I have investigated the preference priority among different gene sources presented under certain stress conditions for Bacillus subtilis possessing NT ability. To test this, two DNA specimens (E and C) with different sequence contents of the same length were presented to B. subtilis under different stress environments (BK, BC, BE and BCE). The hypothesis was evaluated according to the analysis of the results of colonial formations on selective plates (pE, pC and pCE). The obtained data presented a strong positive correlation that the bacteria have preference priority during NT depending on a stimulator. The tendency of the bacteria to uptake useful DNA fragments in a specific environment can be suggested. For instance, the majority of colonies grow on pE plates rather than the pC and pCE when the transformation media includes erythromycin (Eryt) as an inducer. Although the data significantly overlaps with the idea claiming that the bacteria have a preference priority to uptake free DNAs during NT, further investigations are needed to support the present data and for better understanding of the phenomenon.

Metagenomic profiling of antibiotic resistance genes in Red Sea brine pools.

Antibiotic resistance (AR) is an alarming global health concern, causing an annual death rate of more than 35,000 deaths in the US. AR is a natural phenomenon, reported in several pristine environments. In this study, we report AR in pristine Red Sea deep brine pools. Antimicrobial resistance genes (ARGs) were detected for several drug classes with tetracycline and macrolide resistance being the most abundant. As expected, ARGs abundance increased in accordance with the level of human impact with pristine Red Sea samples having the lowest mean ARG level followed by estuary samples, while activated sludge samples showed a significantly higher ARG level. ARG hierarchical clustering grouped drug classes for which resistance was detected in Atlantis II Deep brine pool independent of the rest of the samples. ARG abundance was significantly lower in the Discovery Deep brine pool. A correlation between integrons and ARGs abundance in brine pristine samples could be detected, while insertion sequences and plasmids showed a correlation with ARGs abundance in human-impacted samples not seen in brine pristine samples. This suggests different roles of distinct mobile genetic elements (MGEs) in ARG distribution in pristine versus human-impacted sites. Additionally, we showed the presence of mobile antibiotic resistance genes in the Atlantis II brine pool as evidenced by the co-existence of integrases and plasmid replication proteins on the same contigs harboring predicted multidrug-resistant efflux pumps. This study addresses the role of non-pathogenic environmental bacteria as a silent reservoir for ARGs, and the possible horizontal gene transfer mechanism mediating ARG acquisition.