Unveiling Mobilizable Multiresistance Clusters in Marine Bacteria ^†

Abstract

The occurrence and spread of antibiotic resistance have become a pressing global health concern. Understanding the genetic elements that facilitate the dissemination of antibiotic resistance genes (ARGs) in marine environments is crucial for effective microbial surveillance and management strategies. This study aimed to reveal the presence of mobilizable multiresistance clusters, consisting of ARGs associated with mobile genetic elements (MGEs), in marine bacterial communities. Water samples were collected from two beaches in Jeju, South Korea, and screened to identify multi-drug resistant bacteria. A total of 20 bacterial isolates were selected for whole genome sequencing, and through comprehensive genomic analysis, we identified and characterized nine such clusters primarily composed of betalactams, aminoglycosides, and tetracycline ARGs associated with MGEs like IS6, IS9, and Tn3. Additionally, an extensive analysis of 900 marine bacterial genomes from the National Center for Biotechnology Information (NCBI) database was conducted to gain a broader perspective. Our results provide valuable insights into the prevalence and diversity of mobilizable multiresistance clusters in marine bacterial communities.

1. Introduction

Antibiotic resistance is a global concern in the realm of human health. The emergence of community-acquired infections caused by resistant bacteria has amplified interest in natural environments [1]. These natural environments encompass a diverse range of ecosystems, including animals, soils, glaciers, and marine habitats, all of which serve as pivotal reservoirs for antibiotic resistance genes (ARGs). Despite the typically dilute nature of marine waters, they have evolved into significant reservoirs for antibiotic resistance. This phenomenon is primarily driven by escalating anthropogenic activities, which facilitate the introduction of antibiotic-resistant bacteria and residual antibiotics into marine ecosystems. Consequently, it is imperative to acquire a comprehensive understanding of the dissemination patterns of antibiotic resistance among marine bacteria. This knowledge is fundamental for the implementation of effective antibiotic control measures and the development of strategies to address this pressing issue [1].

Mobile genetic elements (MGEs), such as insertion sequences and transposons, possess the remarkable capacity to capture ARGs from bacterial chromosomes and subsequently transfer them horizontally, either through plasmids or phages, to other bacterial species. In this study, our objective is to illuminate the prevalence of ARGs within marine bacteria and their genomic colocation with MGEs. This research promises to yield valuable insights into the potential for ARG dissemination among marine bacteria, which, in turn, may exacerbate the spread of antibiotic resistance within marine ecosystems.

2. Materials and Methods

Water samples were collected in triplicate from Jungmun beach, located in Jeju, South Korea. Culturable bacteria were isolated from these samples using Marine agar (BD Difco, Difco Laboratory, Detroit, MI, USA) by incubating at 30 °C. Subsequently, isolates were sub-cultured on trypticase soy agar (BD Difco) with 3% additional salt content at 30 °C. A total of 100 distinct colonies were selected for plasmid carriage determination based on their resistance to 14 different antibiotics, as determined through a disk diffusion test. Isolates exhibiting resistance to three or more distinct antibiotics were then screened for plasmids using the alkaline lysis method. This process identified 60 plasmid-carrying isolates whose plasmid sizes were estimated using gel electrophoresis. Twenty isolates were selected from this group based on the distinct sizes of their plasmids for further analysis.

DNA from these 20 isolates was extracted using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany), and the DNA quantity was assessed using Qubit^TM (Thermo Fisher Scientific, Waltham, MA, USA). Short-read sequencing was conducted using Illumina Hiseq 2500 (Macrogen, Seoul, Republic of Korea), while long-read sequencing employed MinION (Oxford nanopore technology). Raw reads from MinION were subjected to quality trimming using fastp [2] and Filtlong (https://github.com/rrwick/Filtlong/, accessed on 23 January 2022), respectively. Assembly was performed using Unicycler [3], and taxonomic identification was carried out via the RDP classifier [4]. Further, functional analysis for antibiotic resistance genes was executed using AMRFinder [5], and mobile genetic elements (MGEs), including plasmids, prophages, transposons, and integrases were identified through plasmidFinder [6], phaster [7], and Isescan [8].

To complement our findings, we retrieved 900 marine bacterial genomes from the National Center for Biotechnology Information (NCBI) for additional analysis (Figure 1). The assembly and analysis of these genomes followed the same procedures mentioned above.

3. Results and Discussion

Taxonomic analysis revealed that the isolated bacteria belonged to various species, including Vibrio cholera (6), Vibrio alginolyticus (4), Vibrio parahaemolyticus (3), Phaeobacter inhibens (3), Aeromonas salmonicida (2), and Edwardsiella anguillarum (2). Antibiotic susceptibility testing (AST) showed increased resistance to beta-lactam, aminoglycoside, and tetracycline antibiotics, particularly within the Vibrio species, aligning with genotypically identified ARGs as shown in

Table 1. The alignment of phenotypic resistance with the corresponding genotypic resistance in MDR marine bacteria.

Bacterial Taxonomy	Antibiotic Class	AST	ARG	Plasmids	Prophages
Vibrio cholerae	Betalactam	Ampicillin Imipenem Meropenem Ceftazidime	blaTEM-1 blaS1 YEM-1	IncFII_1 (2) IncH12A-1 (2) Unnamed plasmids (3)	PHAGE_Vibrio_K139 PHAGE_Vibrio_VP882 PHAGE_Klebsi_ST16 PHAGE_Escher_520873 (2)
	Aminoglycoside	Kanamycin	cpxA
	Tetracycline	Oxytetracycline	tetA
Vibrio alginolyticus	Betalactam	Ampicillin Imipenem Aztreonam	blaOXA50 MexR	p3442-IMI-2c Unnamed plasmids (3)	PHAGE_Vibrio_K139 PHAGE_Vibrio_VfO3K6 (2) PHAGE_Vibrio_VCY (2) PHAGE_Erwini_vB_EhrS (2)
	Aminoglycoside	Kanamycin	AAC(6’)-34
	Tetracycline	Oxytetracycline	tetA, tetB
	Fluoroquinolones	Ciprofloxacin	oqxA, oqxB
	Phenicol	Chloramphenicol	Cat
Vibrio parahaemolyticus	Betalactam	Ampicillin Imipenem Aztreonam	blaXCARB2 MexR	Unnamed plasmids (3)	PHAGE_Vibrio_K139 PHAGE_Klebsi_ST16 PHAGE_Klebsi_ST17 PHAGE_Staphy_Spbeta
	Aminoglycoside	Kanamycin	AAC(6’)-34
	Tetracycline	Oxytetracycline	tetB
	Polymyxin B	Colistin	PmrC
Phaeobacter inhibens	Betalactam	Ampicillin	blaTEM-1	IncFII_1 Unnamed plasmids (4)	PHAGE_Entero_mEp237 PHAGE_Entero_mEp235
	Fluoroquinolones	Levofloxacin	gyrA, gyrB
	Phenociol	Florfenicol	floR
Aeromonas salmonicida	Aminoglycoside	Kanamycin	cpxA	IncFII_1 Unnamed plasmids (2)	PHAGE_Entero_mEp237 (2) PHAGE_Klebsi_ST17 PHAGE_Salmon_118970_sal3 (2)
	Tetracycline	Oxytetracycline	tetB
Edwardsiella anguillarum	Betalactam	Ampicillin	blaTEM-1	Unnamed plasmids (2)	PHAGE_Entero_mEp235
	Polymyxin B	Colistin	eptA

.

These antibiotics are extensively used in livestock farms for treatment and prophylactic purposes [9]. This suggests that the antibiotic residues from neighboring livestock farms may intensify the selective pressure on marine bacteria, leading to heightened resistance to these antibiotics. Moreover, multi-drug resistant (MDR) Vibrio species are significant pathogens for aquatic life and humans. Therefore, the resistance observed in these isolates may pose an increased risk to both human and animal health [10].

The concordance observed between phenotypic and genotypic resistance implies the potential utility of whole genome sequencing for resistance surveillance across diverse environments [11]. Genomic analysis unveiled the presence of 25 intact prophages in the MDR isolates, with PHAGE_Vibrio_K139 (n = 3) and PHAGE_Entero_mEp237 (3) being the most prevalent. The majority of these prophages were classified within the Siphoviridae family. (Figure 2). These findings inspired us to further investigate the functional roles of these prophages and their potential influence on the prevalence of antibiotic resistance within marine bacterial species.

Furthermore, our analysis unveiled the presence of 24 plasmid sequences encoded within these bacterial genomes. These plasmids exhibited diversity, with the majority categorized as Unnamed plasmids (17), while others belonged to the IncFII_1 (4), IncH12A-1 (2), and p3442-IMI-2c (1) categories (see Table 1). The co-occurrence of plasmids and prophages within these genomes is a noteworthy observation, as it implies a potential interplay between mobile genetic elements and their role in shaping the genetic landscape of these marine bacterial species [12].

We identified a total of nine distinct associations between ARGs and MGEs within both prophage-like sequences (Figure 3) and plasmid contigs (Figure 4). Notably, MGEs such as IS6, IS5, and IS110 exhibited predominant associations with ARGs. These specific MGEs are recognized for their capacity to facilitate the transposition of associated genes within genomes, subsequently transferring them to other horizontally gene transfer (HGT) vehicles, including plasmids and prophages [13].

Significantly, we have identified two prophage-like sequences, Vibrio phage VP882 and Vibrio phage VFO3k6, each encoding betalactam (blaTEM-1) and aminoglycoside (aph(3″)-Ib) genes, respectively, within various marine bacterial species (Figure 5). Notably, these genes exhibited 100% identical sequences across these diverse species. This highlights transduction as a key driver in the widespread distribution of ARGs by these prophages, showcasing their remarkable conservation [14].

4. Conclusions

Our study sheds light on the intricate dynamics of antibiotic resistance in marine bacterial communities, revealing the significant role of plasmids and prophages in ARGs’ spread. Future research should delve deeper into the functional roles of these MGEs.