Marine Plastics from Norwegian West Coast Carry Potentially Virulent Fish Pathogens and Opportunistic Human Pathogens Harboring New Variants of Antibiotic Resistance Genes

To our best knowledge this is the first study characterizing fish pathogens isolated from marine plastics from the West coast of Norway for their potential for pathogenicity using whole genome sequencing. Marine plastic polymers identified as polyethylene, polyethylene/ethylene vinyl acetate copolymer and polypropylene, yielded a total of 37 bacterial isolates dominated by Pseudomonas spp. (70%). Six isolates representing either fish pathogens or opportunistic human pathogens were selected for whole genome sequencing (WGS). These included four isolates belonging to Aeromonas spp., one Acinetobacter beijerinckii isolate and one Morganella morganii isolate. Three Aeromonas salmonicida isolates were potentially virulent and carried virulence factors involved in attachment, type II and type VI secretion systems as well as toxins such as aerA/act, ahh1, ast, hlyA, rtxA and toxA. A. salmonicida and Acinetobacter beijerinckii carried new variants of antibiotic resistance genes (ARGs) such as β-lactamases and chloramphenicol acetyltransferase (catB), whereas Morganella morganii carried several clinically relevant ARGs. Our study shows that marine plastics carry not only potentially virulent fish pathogens but also multidrug resistant opportunistic human pathogens like M. morganii and may serve as vectors for transport of these pathogens in the marine environment.

Pollution with plastics and accumulation of plastics in the oceans has a big impact on the marine ecosystem [8][9][10]. Ingestion of plastic by marine organisms can disturb their energy balance, affect behavior and sometimes block the intestinal tract leading to sublethal effect or, in the worst-case, cause death [11,12]. Small microplastic (<10 µm) and nanoplastic particles appear to have an increased plastic particle toxicity to aquatic life [13]. Plastic can also be toxic due to the breakdown products as well as due to the presence of chemicals absorbed or released from plastics, such as plasticizers [14][15][16]. Although some polymers are biodegradable, albeit at a very slow rate, others are not [1] and thus plastics stay in the environment for a long time.
Plastic provides a hydrophobic surface for the attachment of microbes and hence different microbes colonize plastics in the marine environment [5,17,18]. Therefore, marine plastics may function as vectors for the transport of bacteria, including fish pathogens, in the oceans [19]. Colonization of plastics by bacteria can also facilitate horizontal gene transfer of antibiotic resistance genes (ARGs), owing to biofilm formation [20]. Accordingly, a study has shown presence of multidrug resistant bacteria on plastics from marine environments [5].
Plastic pollution has been reported in the marine environment in Norway [21]. To date, Norway is a country with a low burden of antibiotic resistance in clinics and in the environment [22]. Although this is true, there is lack of knowledge about microbiota associated with plastic found in the marine environment in Norway as well as associated ARGs. The aim of our study was to investigate the presence of fish pathogens and antibiotic resistant bacteria on marine plastics collected from the West coast of Norway. We isolated fish pathogens and opportunistic human pathogens from plastic surfaces, analyzed whole genome sequences of these isolates and report their potential for pathogenicity as well as new variants of ARGs.

Sample Collection
Macroplastic samples (n = 7), with different sizes ranging from one centimeter to 15 cm, were collected from the intertidal zone at beaches in Øygarden, Vestland county, Norway. The coordinates for the sampling areas are 60 • 29 57.6 N 4 • 55 05.3 E and 60 • 29 54.5 N 4 • 54 52.6 E, respectively. The samples were collected in either sterile 50 mL tubes or sterile plastic bags. These samples were then stored at 4 • C and transported back to the lab for analysis.

Isolation and Identification of Bacterial Strains
Plastic samples were carefully washed with sterile phosphate buffer saline (PBS) before making suspensions. Suspensions were made by adding 10 mL of sterile PBS to each plastic sample in 50 mL sterile tubes. Four sterile glass beads (4 mm diameter) were added to the tubes, followed by vortexing for 90 s at maximum speed. Serial dilutions of the suspension were prepared in sterile PBS and spread on Mueller-Hinton agar plates and MacConkey agar plates, both media containing either ampicillin (100 µg/mL), cefotaxime (10 µg/mL) or no antibiotics. Mueller-Hinton agar plates were incubated at 25 • C for 24-48 h, while MacConkey agar plates were incubated at 37 • C for 24-48 h. A total of 37 colonies were picked and purified by restreaking on Mueller-Hinton agar with ampicillin. Fish pathogens like Aeromonas spp. and Vibrio spp. are intrinsically resistant to penicillins, thus, ampicillin facilitated selection of fish pathogens [23,24]. All isolates were stored as glycerol stocks at −80 • C for further use. For identification, isolates were grown on Mueller-Hinton agar with ampicillin at 30 • C for 24 h. The strains were typed using a Bruker MALDI Biotyper at Veterinaer instituttet (Bergen, Norway) using the MALDI Biotyper database.

Genomic DNA Extraction and Illumina Sequencing
Genomic DNA was extracted from isolates cultivated overnight on Mueller-Hinton agar with ampicillin using QlAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. NanoDrop 1000 and Qubit 2.0 (Thermo Scientific, Waltham, MA, USA) were used to quantify the extracted DNA. Extracted DNA was sent for sequencing to the Norwegian Sequencing Centre (Oslo University Hospital, Ullevål, Oslo, Norway). Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA, USA) was used for preparing sequencing libraries. Sequencing was performed on an Illumina MiSeq platform (Illumina, San Diego, CA, USA), using 2 × 300 bp chemistry.

Phylogenetic Analysis
Amino acid sequences for CphA, RtxA and AerA were extracted from the genome sequences of Aeromonas spp. Additional sequences for respective proteins were downloaded from GenBank (www.ncbi.nlm.nih.gov/genbank). The sequences were aligned using clustalX version 2.1 [30]. The phylogenetic tree was generated by the neighbor joining method using MEGA-X with bootstrap analysis (1000 replicates) [31].

Antibiotic Susceptibility Testing
Isolates were grown on Mueller-Hinton agar with ampicillin at 30 • C overnight and were used for making suspensions for determination of minimum inhibitory concentration (MIC). MICs were determined for cefotaxime (CT), tetracycline (TC), ciprofloxacin (CI), ampicillin (AM), meropenem (MP), streptomycin (SM), trimethoprim (TR), gentamicin (GM), imipenem (IP) and chloramphenicol (CL) using E-test according to manufacturer's protocol (bioMérieux, Paris, France). Escherichia coli strain CCUG 17,620 was used as a control strain for E-test quality check.

Identifying Plastic Polymer Type
The polymer type for plastic samples was identified by attenuated total reflection infrared spectroscopy (Cary670 FTIR spectrometer, Agilent Technologies) equipped with a monolithic diamond crystal unit (GladiATR™, PIKE Technologies, Madison, WI, USA). Spectra were collected over the wavenumber range 440-4000 cm −1 . The samples were analyzed by 32 co-added scans, with a resolution of 8 cm −1 . The spectra were compared to spectra of known standards using a library of environmental relevant synthetic and natural polymers [32], and commercial libraries for polymers (Bio-Rad Sadtler) in the KnowitAll Informatics System software (BioRad Laboratories, Hercules, CA, USA). Each polymer type was determined by a combination of the best-fitted spectra and the expertise of the operator on interpreting polymer IR spectra.

Identification of the Plastic Polymers and Bacterial Strains
The plastic polymers were identified as polyethylene, polyethylene/ethylene vinyl acetate copolymer or polypropylene ( Figure 1). A total of 37 isolates were obtained from the samples (Table S1). Only two isolates were obtained from polypropylene, while the rest were found on polyethylene or polyethylene/ethylene vinyl acetate copolymer. Pseudomonas spp. dominated the isolates (26 of 37). Four isolates representing fish pathogens Aeromonas spp. and two isolates representing opportunistic human pathogens (Morganella morganii and Acinetobacter beijerinckii, respectively) were chosen for characterization with whole genome sequencing (WGS). Genome sequence assembly statistics and GenBank accession number for the genome sequences of the isolates are represented in Table S2. Sequencing coverage ranged from 55× to 110×.

Antibiotic Resistance and ARGs
All three A. salmonicida isolates were resistant to ampicillin. M. morganii and Ac. beijerinckii were multidrug resistant, with resistance against at least three different classes of antibiotics (Table 1). Class C β-lactamases and chloramphenicol acetyltransferase (catB) were detected in all six isolates. Class B2 metallo-β-lactamase cphA was detected in three A. salmonicida isolates, with a new variant of cphA (≤ 98% identity) present in two of the three A. salmonicida isolates (Table 2 and Figure S1). Along with cphA variants all Aeromonas isolates carried qnrA gene. Isolate 2HC4 (Ac. beijerinckii) carried new variants of four different ARGs including a class A and a class C β-lactamase, aminoglycoside acetyltransferase and chloramphenicol acetyltransferase. M. morganii carried several resistance genes with 100% homology to previously described ARGs. The list of ARGs detected in the genome sequences of these isolates is presented in Table 2.

Antibiotic Resistance and ARGs
All three A. salmonicida isolates were resistant to ampicillin. M. morganii and Ac. beijerinckii were multidrug resistant, with resistance against at least three different classes of antibiotics (Table 1). Class C β-lactamases and chloramphenicol acetyltransferase (catB) were detected in all six isolates. Class B2 metallo-β-lactamase cphA was detected in three A. salmonicida isolates, with a new variant of cphA (≤98% identity) present in two of the three A. salmonicida isolates (Table 2 and Figure S1). Along with cphA variants all Aeromonas isolates carried qnrA gene. Isolate 2HC4 (Ac. beijerinckii) carried new variants of four different ARGs including a class A and a class C β-lactamase, aminoglycoside acetyltransferase and chloramphenicol acetyltransferase. M. morganii carried several resistance genes with 100% homology to previously described ARGs. The list of ARGs detected in the genome sequences of these isolates is presented in Table 2.

Virulence Factors
All three A. salmonicida isolates had genes for virulence factors involved in attachment, type II (T2SS) and type VI (T6SS) secretion systems as well as toxins such as aerA/act, ahh1, ast, hlyA, rtxA, rtxB, rtxC, rtxD, rtxE, rtxH and toxA. This suggests that they have potential for causing infection. M. morganii carried genes involved in acid resistance, genes involved in iron uptake, and type six secretion system proteins. A phylogenetic tree of RtxA and AerA proteins from A. salmonicida isolates and closely related proteins from other species is presented in Figure 2. A list of virulence genes detected in all sequenced isolates is presented Table S3.   (T2SS) and type VI (T6SS) secretion systems as well as toxins such as aerA/act, ahh1, ast, hlyA, rtxA, rtxB, rtxC, rtxD, rtxE, rtxH and toxA. This suggests that they have potential for causing infection. M. morganii carried genes involved in acid resistance, genes involved in iron uptake, and type six secretion system proteins. A phylogenetic tree of RtxA and AerA proteins from A. salmonicida isolates and closely related proteins from other species is presented in Figure 2. A list of virulence genes detected in all sequenced isolates is presented table S3.

Discussion
To our best knowledge, this is the first study analyzing genome sequences of multidrug resistant bacteria, including fish pathogens, associated with plastic found in the marine environment in Norway. Using 16S rDNA clone libraries, Viršek et al. [33] suggested that microplastics might serve as a vector for the transport of pathogenic bacteria, especially fish pathogen A. salmonicida. Our study strengthens the notion that fish pathogens as well as other opportunistic human pathogens are present on marine plastics. We further show the presence of virulence genes and new variants of antibiotic resistance genes in A. salmonicida isolated from these plastics.
Most Aeromonas spp. are opportunistic pathogens and are widespread in the marine environments [34,35]. Aeromonas spp. are known to cause infection in both humans and animals [36]. A. salmonicida is one of the most important fish pathogens [37], causing disease in healthy wild and cultured stocks of salmon and other fish species [36]. All A. salmonicida isolates in this study carried virulence factors like the rtxA toxin. Repeats in toxins (RTX) rtxA is a cytotoxin that is an important virulence factor for many fish pathogens, including Vibrio spp. and Aeromonas spp. It disrupts host cells membranes, aiding pathogenesis [38,39]. Along with rtxA, other cytotoxins like aerolysin aerA [40], extracellular hemolysin ahh1, heat-stable cytotonic enterotoxin ast, hemolysin III and hylA were detected in these isolates [41]. A study suggests that the presence of both aerA and ahh1 represents the most cytotoxic genotype in A. hydrophila [41]. In addition, Aeromonas isolates also carried Exotoxin-A (toxA), which is a major virulence factor in Pseudomonas aeruginosa [42]. Two A. salmonicida isolates (2HA2 and 2MA4) carried genes involved in immune evasion (capsular polysaccharide), phagocytosis prevention (rmlC, wbfU, wbfY) and endotoxin from Bordetella. In addition to these, several others virulence factors like adhesion factors, T2SS [43,44] and T6SS [45,46] were detected. The presence of virulence factors in the genome does not necessarily guarantee expression of these virulence factors and further in-depth studies are needed to establish the virulence of these isolates. Nevertheless, the presence of these virulence genes indicates that these isolates have potential for causing infections. This emphasizes the risk posed by plastics as vectors for the transport of potentially virulent fish pathogens in the marine environment, especially in Norway where aquaculture is one of the major activities.
All three isolates of A. salmonicida in our study carried resistance genes like ampC, bla OXA , cphA, qnrA and catB. New variants of cphA (Class B2 metallo-β-lactamase) were present in the two isolates (5HA1 and 2MA4) of A. salmonicida whereas new variants of FOX/MOX family class C β-lactamase (96.3% to 98.4% identity) were detected in all three isolates. Marine bacteria have been shown to be a source of ARGs found in the clinics [47][48][49]. A recent study by Ebmeyer et al. [50] showed that Aeromonas spp. are the origin of several clinically important β-lactamases like CMY-1/MOX-family AmpC β-lactamases MOX-1, MOX-2 and MOX-9. Similarly, another study from Ebmeyer et al. [51] showed that FOX AmpC β-lactamases originated in A. allosaccharophila. They proposed that the mobilization and fixation of these genes may be recent and may have happened during the antibiotic era. Plastics provide a hydrophobic surface for the attachment of microbes promoting colonization and biofilm formation [52]. In accordance, an increased frequency of resistance plasmid transfer in bacteria associated with microplastics was observed [20]. Plastics also absorb a range of pollutants including antibiotic and heavy metals that are known to be drivers of antibiotic resistance [5,[53][54][55]. This may create selection pressure, aiding transfer and/or mobilization of ARGs in the surface associated microbiota on marine plastics [56]. In order to better understand the role of marine plastics in mobilization and selection of ARGs, more research is thus warranted.
We detected opportunistic human pathogens like M. morganii [57][58][59], Ac. beijerinckii [60] and A. popoffii [61] on plastic surfaces. M. morganii previously belonged to family Enterobacteriaceae, which also consists of classical human pathogens like Escherichia coli, Salmonella typhi and Klebsiella pneumoniae. It was recently reclassified to be included in its own family Morganellaceae [62]. Although reclassified, M. morganii is an important emerging opportunistic human pathogen causing a variety of infections ranging from wound infections, urinary tract infections to meningitis [57][58][59]. M. morganii isolate (2MA1) carried virulence genes involved in acid resistance (ureB, ureG), genes involved in iron uptake, and T6SS proteins. Further, this isolate was resistant to tetracycline, chloramphenicol and β-lactams as well as carried several clinically important resistance genes like tetD, aac3 and catB [59]. The presence of multi-drug resistant human associated bacteria carrying multiple ARGs suggests that plastic may serve as vectors for transport of not only fish pathogens but also opportunistic human pathogens in the marine environment. Our results are in accordance with a recent study that showed presence of potentially pathogenic bacteria on plastic debris from Guanabara Bay in Brazil [63].
Aquaculture is both historically and economically important for Norway. Norway is considered the world's second largest exporter of seafood after China and delivers fish to more than 100 countries [64,65]. In 2018, more than 1.35 million ton of fish, mostly salmon (Salmo salar) (1.28 million ton) and rainbow trout (Oncorhynchus mykiss) (68,345 ton) were farmed in Norway, with a first-hand value of 67.8 billion NOK (8.34 billion USD) [66,67]. The presence and spread of potentially virulent fish pathogens on marine plastics, could have a major impact on aquaculture in Norway. Hence, more research on understanding of the role of plastic surface-associated microbial communities and their biogeochemical functions in the marine environment is needed, especially in Norway.

Conclusions
Our study demonstrates the presence of fish pathogens and human associated bacteria on marine plastics from Norway. We show the potential for pathogenicity of A. salmonicida isolates obtained from marine plastics, with presence of genes encoding toxins, hemolysins and adhesion factors in their genome sequences, as well as describe new variants of ARGs carried by plastic associated bacteria. Our study strengthens the notion that plastic debris may serve as vectors for transport for fish pathogen as well as other opportunistic human pathogens in the marine environment. Marine plastics colonized by potentially virulent fish pathogens may impact aquaculture. Hence, in-depth follow-up studies for better understanding the role of plastic in the spread of antibiotic resistant pathogens in the marine environment are needed.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2607/8/8/1200/s1, Figure S1: Phylogenetic tree for cphA β-lactamases based on the amino acid sequence. The proteins highlighted with circles are new variants found in A. salmonicida isolates in this study. Accession numbers are presented for sequences downloaded from GenBank. PFM1 (QDC33502.1) representing subclass B2 metallo-beta-lactamase; PFM-1 from Pseudomonas fluorescens is used as an out group for construction of phylogenetic tree. Numbers at nodes are bootstrap values (%) based on 1000 resampled datasets; only values >50% are given. Table S1: Identification and isolation source of the isolates obtained in the study. Table S2: Genome sequence assembly statistics for the whole genome sequences of the six isolates. Table S3  Acknowledgments: The authors acknowledge Didrik Hjertaker Grevskott from Institute of Marine Research (IMR) for help with sample collection. We acknowledge Ørjan Bjorøy from IMR for providing technical support and Julia Storesund from IMR for discussions regarding bioinformatics analysis. The authors also acknowledge Monica Sanden, Cecilie Smith Svanevik and Bjørn Tore Lunestad from IMR for supporting the work. The sequencing service was provided by the Norwegian Sequencing Centre (www.sequencing.uio.no), a national technology platform hosted by the University of Oslo and supported by the "Functional Genomics" and "Infrastructure" programs of the Research Council of Norway and the Southeastern Regional Health Authorities.

Conflicts of Interest:
The authors have no conflict of interest.