First Report and Characterization of a Plasmid-Encoded blaSFO-1 in a Multi-Drug-Resistant Aeromonas hydrophila Clinical Isolate

Antibiotic resistance remains one of the most pressing public health issues facing the world today. At the forefront of this battle lies the ever-increasing identification of extended-spectrum beta-lactamases and carbapenemases within human pathogens, conferring resistance towards broad-spectrum and last-resort antimicrobials. This study was prompted due to the identification of a pathogenic Aeromonas hydrophila isolate (strain MAH-4) collected from abdominal fluid, which presented a robust resistance pattern against second-, third-, and fourth-generation cephalosporins, ertapenem, ciprofloxacin, gentamicin, levofloxacin and moxifloxacin, and beta lactam/beta-lactamase inhibitor combinations. Whole genome sequencing was performed and identified a 328 kb plasmid (pMAH4) encoding 10 antibiotic resistance genes, including blaSFO-1, blaTEM-1, and blaOXA-1 of A. hydrophia MAH-4. This is the first report of beta-lactamase SFO-1 within a clinical strain of Aeromonas. Due to the remarkable sequence identity of pMAH4 to plasmids associated with Enterobacterales genera like Klebsiella and the extensive capabilities of Aeromonas for horizontal gene transfer, our identification of a clinical isolate encoding SFO-1 on a plasmid suggests antibiotic resistance gene mobility between Enterobacterales and non-Enterobacterales species.


Introduction
Antimicrobial resistance (AMR) remains one of the world's top public health emergencies, claiming roughly 1.27 million lives in 2019 [1].With discoveries of new AMR far outpacing discoveries of new antibiotics, this situation is only becoming more dire.Projections currently estimate that AMR bacterial-caused mortalities will reach 10 million by the year 2050 [1].Despite overwhelming evidence and repeated warnings surrounding this public health crisis, global economic development and increasing access to medical treatments have continued to drive an increase in antimicrobial consumption [2].Alarmingly, broad-spectrum and last-resort antibiotics are becoming more frequently employed than ever before [2].Consequently, as these compounds' usage increases, so does the identification of bacterial strains resistant to them [3].This often leads to longer hospital stays, higher healthcare costs, and increased mortality, particularly within areas with developing economies [4].Within bacterial pathogens most responsible for human mortalities, extended-spectrum beta-lactamases (ESBLs) and carbapenemases rank among the top acquired antibiotic resistance genes (ARGs) [1].AMR among Enterobacterales members Escherichia coli and Klebsiella pneumoniae alone were solely responsible for 23.4% and 19.9% of deaths attributed to AMR pathogens in high-and low-income regions, respectively [1].Therefore, the Center for Disease Control and Prevention has deemed members Microorganisms 2024, 12, 494 2 of 14 of carbapenem-resistant and ESBL-producing Enterobacterales as urgent and serious threats to human health [5].
Some of the most pervasive ESBLs currently being identified within clinical pathogens originate from common broad-spectrum beta-lactamases, such as TEM, SHV, OXA, and CTX-M [6], with the CTX-M type demonstrating the greatest prevalence worldwide [7].Due to their commonality, as well as the high degree of genetic diversity within the CTX-M family, their dissemination throughout the world has often been referred to as the "CTX-M pandemic" [8].While these beta-lactamases and ESBLs constitute the majority of ARGs within common pathogens, other concerning ESBLs are being identified more frequently than they were in years past [7].SFO-1, regulated by the AmpR regulator, is a rarer class A ESBL with the capacity to hydrolyze numerous beta-lactams other than cephamycins and carbapenems.Initially identified in 1988 from a self-transferable plasmid within a clinical strain of Enterobacter cloacae [9], SFO-1 has since been found within clinical isolates of various species of Enterobacterales such as Klebsiella spp., Escherichia coli, and Enterobacter cloacae complex [7].The first SFO-1 epidemic was identified in Spain and was caused by Enterobacter cloacae between 2006-2009 [10].The SFO-1 outbreak was significantly correlated with administration of beta-lactam antibiotics, chronic renal failure, tracheostomy, and prior hospitalization, further emphasizing the impact of antibiotic overconsumption [10].Then, between 2011-2015, SFO-1-encoding strains of E. hormaechei evolved into epidemic clones (ST93, ST114, and ST418) among community-acquired strains found in four provinces in China [7].
Apart from minimizing antibiotic overconsumption, the search for a solution to the growing AMR issue must also consider the horizontal spread of ARGs between various biome resistomes [11].To confront challenges on multiple fronts like what is seen within the issue of AMR, approaches such as One Health use the expertise from several disciplines to identify solutions to complex issues facing the world [5].However, a challenge in using this tactic against AMR resides in identifying a proper indicator species.One potential indicator which has been proposed is Aeromonas [3].This genus of bacteria resides ubiquitously in aquatic environments across the globe, including fresh and brackish waters, and has been correlated with a variety of diseases among cold-and warm-blooded animals [12].Aeromonas-related diseases within humans often afflict children or those with compromised immune systems.Predominantly, these diseases include gastrointestinal infections like gastroenteritis or soft-tissue infections such as cellulitis, abscesses, and necrotizing fasciitis [13,14].Furthermore, aeromonads readily participate in both intra-and interspecies horizontal gene transfer (HGT) [15], allowing for monitoring of ARGs within an environment [16].Aquaculture and wastewater treatment facilities, both common habitats for Aeromonas [3,17], have been identified as hot-spots of ARG transmission [18].Therefore, it has been proposed that aeromonads may provide a useful tool in tracking antimicrobial resistance patterns around the world [3,19].
Here, we report and characterize the first clinical case of plasmid-encoded bla SFO-1 in a multi-drug-resistant isolate of Aeromonas hydrophila to further understand the driving factors of resistance, especially among ESBLs and carbapenemases.

Bacterial Isolation and Identification
This study was approved on 18 December 2019 by the Medical College of Wisconsin and the Froedtert Institutional Review Board (PRO# 00036609).Abdominal fluid was surgically collected from a patient and submitted to the hospital's diagnostic laboratory in a sterile syringe.The sample was submitted and worked up as an aerobic/anaerobic culture.Confirmation of the isolates was performed using a Bruker MALDI-TOF analyzer (Bruker Daltonics, Bremen, Germany) with subsequent identification comparing the protein spectrum of the isolate to the Bruker MTB Compass Library (Revision H, 10,833 MSP).Glycerol frozen stocks were made for future analysis.

Antimicrobial Susceptibility
A fresh subculture of the clinical isolate was used to perform antimicrobial susceptibility testing using a BD Phoenix M50 analyzer and BD EpiCenter System V7.21 (Becton, Dickinson and Company, Sparks, MD, USA).The BD EpiCenter System uses current CLSI guidelines and customized rules designated by the laboratory to determine the susceptibility interpretations from the MIC results given from the BD Phoenix.A Kirby disk diffusion assay was used to also analyze antimicrobial susceptibility to the following antibiotics: aztreonam (30 mcg), cefotaxime (30 mcg), chloramphenicol (30 mcg), ciprofloxacin (5 mcg), meropenem (10 mcg), tetracycline (30 mcg), sulfamethoxazole-trimethoprim (23.75/1.25 mcg).Antimicrobial susceptibility was determined using values provided in the Clinical and Laboratory Standards Institute M45-A for Aeromonas spp.[20].

Isolation of Genome DNA and Whole Genome Sequencing
An overnight culture of A. hydrophila MAH-4 was pelleted and genomic DNA was obtained using a Zymo DNA Miniprep (Zymo Research, Irvine, CA, USA).Long reads and short paired-end reads (2 × 151 bps) were obtained by the Oxford Nanopore (Oxford Nanopore, Oxford, UK) platform and Illumina NextSeq 2000 (Illumina, Inc., San Diego, CA, USA) platform, respectively, for whole genome sequencing (WGS).Quality control and adapter trimming were performed using bcl-convert and porechop followed by hybrid assembly of the reads using Unicycler [21].The genome was submitted to NCBI Accession no.CP143514 and CP143515 (pMAH4).Annotations of the assemblies were performed using BV-BRC 3.33.16[22].Phylogenetic reconstruction of MAH-4 with other representative Aeromonas species was carried out using 100 different conserved and concatenated genes and was run in BV-BRC 3.33.16(Supplemental Table S1) [22].Further confirmation of species was performed by determining an average nucleotide identity (ANI) in comparison to reference strain Aeromonas hydrophila subspecies hydrophila ATCC 7966 using JSpeciesWS version 4.1.1[23].

Bioinformatics
We conducted a blastn analysis [24] against the NCBI nr nucleotide database to identify associations, i.e., contiguous regions of high sequence identity, of the pMAH4 DNA sequence with other bacterial genomes (accessed on 18 January 2024).Seven bacterial isolate genomes with very high identity (>99.9%) and ≥18% coverage of pMAH4 were selected for further analysis and visualization.Additionally, the blastn analysis identified seven Aeromonas spp.genomes in NCBI that had high identity (>95%) to a portion (>5%) of pMAH4.The matching sequence region from the 14 selected genomes was added to the program Blast Ring Image Generator (BRIG; [25]) for sequence identity visualization and mapping.Within BRIG, pMAH4 was set as the "reference sequence" and the other 14 genomes as "query sequences".Nucleotide sequences were used in the pairwise blastn query between all 14 sequences and pMAH4, and BLAST settings were left as default.The final BRIG image was exported, and final image annotations were modified in Adobe Illustrator 2024 (Adobe Inc., San Jose, CA, USA).
Various online bioinformatic tools were used to further characterize pMAH4.Identification of antibiotic resistance genes (ARGs) was conducted in both Proksee CARD [26,27] and ResFinder 4.4.2[28] from the Center for Genomic Epidemiology.The ID threshold was set at 90% and at 80% for minimum length.BLAST ® was further used to analyze ARGs on the chromosome because it has a larger library of Aeromonas sequences.Identification and location of mobile genetic elements (MGEs) were performed using MobileElementFinder [29], ISFinder [30], and Proksee mobileOG-db [31].PHASTER was used to identify similarity to bacteriophage DNA [32][33][34].Comparison of tcb operon nucleotide similarity among members of the Aeromonadaceae/Succinivibrionaceae group (taxid: 135,624) was performed with BLAST ® [35].

Whole Genome Sequence Analysis
Due to the intense antimicrobial resistance patterns, we wanted to understand the molecular mechanisms driving these resistance phenotypes.Therefore, whole genome sequencing of strain MAH-4 was performed with both long and short reads resulting in two closed contig loops, a chromosomal genome of 5,110,450 bp with a 327,540 bp plasmid (pMAH4).The chromosome was 60.78% GC content with an estimated 4,803 CDS (Figure 1).Phylogenetic analysis using 100 different genes comprising 115,401 concatenated nucleotides placed strain MAH-4 within a clade of A. hydrophila species (Figure 2).The MAH-4 chromosome also had 96.61% average nucleotide identity (ANI) with A. hydrophila subspecies ATCC 7966 which is above the 95% cutoff value for grouping genomes within the same species.From the genome-based phylogeny and ANI results, we concluded that strain MAH-4 is an Aeromonas hydrophila species.A. hydrophila has been commonly associated with a myriad of clinical diseases ranging from gastroenteritis and colitis [36] to wound infections and life-threatening necrotizing fasciitis [36].The evolution of many clinically relevant Aeromonas species has included the acquisition of multiple beta-lactamases resulting in intrinsic resistance to many beta-lactam antibiotics.Often, the chromosome of these organisms carries Ambler class B2 metallo-b-lactamase, Ambler class D penicillinase, and Ambler class C cephalosporinases [37].A. hydrophila MAH-4 carries all of these beta-lactamases: bla cphA2 , bla OXA-12 , and bla ampC (Table 2).Additionally, A. hydrophila MAH-4 encodes multiple genes associated with the ATP-binding cassette family (ABC), the resistance-nodulation-cell division (RND) family, the small multi-drug-resistance (SMR) family, the multi-drug and toxic compound extrusion family (MATE), and tri-and tetrapartite multi-drug efflux pumps.These pumps may enhance or confer resistance patterns observed beyond the presence of ARGs [38].
cleotides placed strain MAH-4 within a clade of A. hydrophila species (Figure 2).The MAH-4 chromosome also had 96.61% average nucleotide identity (ANI) with A. hydrophila subspecies ATCC 7966 which is above the 95% cutoff value for grouping genomes within the same species.From the genome-based phylogeny and ANI results, we concluded that strain MAH-4 is an Aeromonas hydrophila species.A. hydrophila has been commonly associated with a myriad of clinical diseases ranging from gastroenteritis and colitis [36] to wound infections and life-threatening necrotizing fasciitis [36].The evolution of many clinically relevant Aeromonas species has included the acquisition of multiple beta-lactamases resulting in intrinsic resistance to many beta-lactam antibiotics.Often, the chromosome of these organisms carries Ambler class B2 metallo-b-lactamase, Ambler class D penicillinase, and Ambler class C cephalosporinases [37].A. hydrophila MAH-4 carries all of these beta-lactamases: blacphA2, blaOXA-12, and blaampC (Table 2).Additionally, A. hydrophila MAH-4 encodes multiple genes associated with the ATP-binding cassette family (ABC), the resistance-nodulation-cell division (RND) family, the small multi-drug-resistance (SMR) family, the multi-drug and toxic compound extrusion family (MATE), and tri-and tetrapartite multi-drug efflux pumps.These pumps may enhance or confer resistance patterns observed beyond the presence of ARGs [38].The fourth ring represents antimicrobial resistance CDS followed by virulence CDS on the fifth ring.
The purple ring presents the GC content throughout the chromosome followed by the GC skew in the innermost ring.

Characterization of pMAH4
To further our understanding of the transmission of antimicrobial resistance, we characterized the genetic signatures associated with resistance of A. hydrophila MAH-4.The 327,540 bp plasmid (pMAH4) had a 52.89%G+C content but could not be assigned an incompatibility group using PlasmidFinder [39], although a traX gene contained 100%

Characterization of pMAH4
To further our understanding of the transmission of antimicrobial resistance, we characterized the genetic signatures associated with resistance of A. hydrophila MAH-4.The 327,540 bp plasmid (pMAH4) had a 52.89%G+C content but could not be assigned an incompatibility group using PlasmidFinder [39], although a traX gene contained 100% identity to TraX of the IncF plasmid (Figure 3).Ten different ARGs intermixed with numerous MGE were encoded on this plasmid, creating a mosaic evident of multiple mobilization events.Beyond the intrinsic resistance provided by the chromosomally encoded beta-lactamases, A. hydrophila pMAH4 encoded bla SFO-1 , bla TEM-1 , and bla OXA-1 (Figure 3 and Table 2) .This is the first report of bla SFO-1 from a clinical strain of Aeromonas.
Microorganisms 2024, 12, x FOR PEER REVIEW 7 of 14 identity to TraX of the IncF plasmid (Figure 3).Ten different ARGs intermixed with numerous MGE were encoded on this plasmid, creating a mosaic evident of multiple mobilization events.Beyond the intrinsic resistance provided by the chromosomally encoded beta-lactamases, A. hydrophila pMAH4 encoded blaSFO-1, blaTEM-1, and blaOXA-1 (Figure 3 and Table 2).This is the first report of blaSFO-1 from a clinical strain of Aeromonas.SFO-1 was first identified in an Enterobacter cloacae isolate in Japan [9], which shared strong amino acid similarity to chromosomal-encoded AmpA in Serratia fonticola.Typically, SFO-1 is identified in members of the Enterobacterales and is located on plasmids [7,10].However, to the authors' knowledge, this is the first clinical case of an Aeromonas species or non-Enterobacterales encoding SFO-1.Immediately adjacent to the blaSFO-1 was the transcriptional regulator ampR (Figure 1).SFO-1 production provides hydrolysis to oxyimino-cephalosporins like cefotaxime, although this activity is inhibited in the presence of imipenem or clavulanic acid [9].BLASTp only identified one isolate among Aeromonas species for SFO-1 in the NCBI nt database and it was Aeromonas hydrophila AFG_SD03_1510_Ahy_093 from stray dog fecal matter in Afghanistan [40].Outside of this one isolate, all other SFO-1-encoding organisms listed in the NCBI Identical Protein Groups (n = 515) were from Enterobacterales members.Although, it is possible that the frequency of SFO in clinical isolates may be underreported due to misidentification as CTX-M in lateral flow assays [41].
Upstream of the blaSFO-1, the aminoglycosides phosphotransferase aph( 6)-Id and aph(3′)-lb are found as part of a Tn3 family including the transposase (TnpA) and resolvase (TnpR).Additionally, the presence of a composite transposon Tn6082 and insertion sequence ISAhy2 upstream of blaSFO-1-ampR followed by ISCfr1 and Tn3 transposon downstream suggests that numerous recombination events shaped the content of pMAH4.Downstream of the SFO-1-ampR is a class 1 composite transposon flanked by IS6100 elements rich in ARGs between 30,939-53,611 bp: IS6100-mphR(A)-mrx-mphA-IS26-aac(3)-IId SFO-1 was first identified in an Enterobacter cloacae isolate in Japan [9], which shared strong amino acid similarity to chromosomal-encoded AmpA in Serratia fonticola.Typically, SFO-1 is identified in members of the Enterobacterales and is located on plasmids [7,10].However, to the authors' knowledge, this is the first clinical case of an Aeromonas species or non-Enterobacterales encoding SFO-1.Immediately adjacent to the bla SFO-1 was the transcriptional regulator ampR (Figure 1).SFO-1 production provides hydrolysis to oxyiminocephalosporins like cefotaxime, although this activity is inhibited in the presence of imipenem or clavulanic acid [9].BLASTp only identified one isolate among Aeromonas species for SFO-1 in the NCBI nt database and it was Aeromonas hydrophila AFG_SD03_1510_Ahy_093 from stray dog fecal matter in Afghanistan [40].Outside of this one isolate, all other SFO-1-encoding organisms listed in the NCBI Identical Protein Groups (n = 515) were from Enterobacterales members.Although, it is possible that the frequency of SFO in clinical isolates may be underreported due to misidentification as CTX-M in lateral flow assays [41].
Two of the earliest identified ESBLs associated with plasmids were identified on pMAH4: TEM-1 and OXA-1.The ESBL TEM-1 has been around since the 1960s and provides resistance to first-generation cephalosporins, while OXA-1 enables hydrolysis of fourth-generation cephalosporins, like cefepime [8].The presence of an aminoglycoside Nacetyltransferase (aac(6 ′ )-lb-cr) is commonly found in class 1 integrons and associated with IS26 elements similar to pMAH4 [43].This ARG provides resistance to aminoglycosides and moderate resistance due to fluoroquinolone acetylation [44] and has been found among ciprofloxacin-resistant Aeromonas hydrophila clinical isolates as well as within environmental samples surrounding aquaculture and hospitals [45].Lastly, catB3 and sul1 were found furthest downstream of the composite transposon.Chloramphenicol resistance is associated with the inducible production of chloramphenicol O-acetyltransferase (catB3) and has been identified in many species of Aeromonas, including on both a plasmid and chromosome of A. hydrophila [26].The last ARG, a sulfonamide-resistant dihydropteroate synthase (sul1), is commonly found among Gram-negative bacteria, including Aeromonas species where it is more common to be encoded on the chromosome than the plasmid [26].However, it is a common ARG among the Aeromonas mobilome and in some cases has been identified on plasmids and associated with transposons, and class 1 integrons, as well as clustered within rich ARG regions [46].The absence of resistance to SXT in the presence of sul1 might be associated with a decreased expression considering there is a seven-nucleotide overlap of qacE delta 1 [47].
Although a large portion of pMAH4 contains unknown open reading frames (ORFs), a mercury resistance operon (MerRTPEDAFPTR) encoding genes to detoxify the heavy metal mercury is present.MerT, P, F, and E are associated with transport, whereby MerD serves as a transcriptional repressor.This operon has been identified in both Gram-positive and Gram-negative bacteria.Considering the ubiquitous presence of Aeromonas in aquatic environments and common mercury contaminants in anthropogenic-impacted waters [8], the acquisition of a mercury resistance operon is not surprising.Additionally, conjugative transfer of plasmids increases in the presence of low levels of mercury contamination [48] and this operon has already been identified in clinical Aeromonas strains showing varying resistance to mercury [49].

Evolution of pMAH4
Comparing pMAH4 to most other plasmids using BLAST provided some insights into its potential origin.The majority (75%) of pMAH4 was made up of DNA with very few predicted ORFs and an absence of high sequence identity (>50%) to other sequences in the NCBI nucleotide database (Figure 4 and Table S2).The lone exception was a very high identity match to an A. caviae isolate acquired from a hospital patient's urine in November 2022 from the province of Guangdong in China.This isolate's genome contained a plasmid pAC1520 with 95.88% nucleotide identity to pMAH4 within this region, but the remaining ~25% (75 kb) of the pMAH4 plasmid did not match pAC1520.This region of pMAH4 was rich in ARGs and MGEs and contained segments with a very high sequence identity (often >99.9%) to plasmids from members of Enterobacterales, predominantly from the genera Klebsiella and Enterobacter (Figure 4 and Table S2).These isolates were acquired from human samples from at least four different provinces in China from 2016-2021 and shared a very high sequence identity (99.9-100%) to the ARG-containing region on pMAH4 including bla SFO-1 (Figure 4 and Table S2) .
identified.Lower socio-economic countries are commonly associated with increased levels of AMR through all sectors [3], which are also correlated frequently with antimicrobial overuse and an absence or deficiency of wastewater treatment facilities [3,57].It is imperative to develop global policies regulating antimicrobial use and for higher socio-economic countries to increase support for infrastructure, like wastewater systems, in countries with lower gross domestic income.Among Aeromonas spp., there were seven genomes that contained a very high sequence identity (99.9-100%) to the plasmid region containing the ten ARGs (Figure 4 and Table S2).Only one genome, Aeromonas hydrophila AFG_SD03_1510_Ahy_093, harbored bla SFO-1 .This strain of A. hydrophila was acquired from stray dog feces in Afghanistan in 2015 [40].Although it was the only Aeromonas isolate encoding a bla SFO-1 , it was missing the majority of the other ARGs encoded downstream of bla SFO-1 in pMAH4: mphA-aac(3)-IId-bla TEM-1 -aac(6 ′ )-lb-cr-bla OXA-1 -catB3-qacEdelta1-sul1.Only one other Aeromonas isolate had high sequence identity to this ARG-rich region, Aeromonas caviae SCLZ552.This isolate was acquired from wastewater in the Sichuan province in China in 2019.One other isolate, Aeromonas caviae WP3-S18-ESBL-02, from Tokyo, Japan, had high plasmid sequence identity to the ARG-rich region in pMAH4, but it was broken into several smaller matching regions.This A. caviae isolate was acquired in 2018 from wastewater effluent as well.Aeromonas spp.are common municipal and hospital wastewater residents and also comprise a high percentage of ESBL-producing bacteria in final treated wastewater effluents, even post-disinfection [50].Aeromonas spp., discharged in final effluent, have been identified with the following carbapenemases: blaKPC-2, blaVIM-2, blaOXA-48, blaIMP-13, blaGES, and blaMOX genes [51].Their presence in treated effluent emphasizes the potential role Aeromonas can serve as a vehicle for transferring dangerous ARGs across environmental sectors [3] as well as an opportunist for exchanging ARGs both intra-and interspecies.
Other Aeromonas isolates with a high degree of nucleotide similarity to pMAH4, though without bla SFO-1 , were from clinical strains.These isolates spanned three different Chinese provinces from 2019-2022 ranging from urine (A.caviae AC1520) to bile (A.caviae FAHZZU2447).One study performed between 2012-2016 by the NIH Clinical Center in the USA focused on identifying potential reservoirs of carbapenemaseproducing bacteria around hospital environments to help understand the cause of increased carbapenem-resistant hospital-acquired infections [52].The study identified six bla KPCencoding Aeromonas isolates from within wastewater manholes associated with hospitals.In this study, one patient did have an Aeromonas strain containing a bla KPC-2 on a 143.4 kb plasmid (pASP-135 from A. hydrophila AHNIH1) though it was unrelated to the hospital environmental isolates [52].Many of these isolates encoded a chromosomal trb operon, which is associated with type IV secretion systems [53]. A. hydrophila MAH-4 also encodes a trb operon (trbK-VirD4-trbBCDEJKLFGI) in its chromosome.This region shared the highest nucleotide identity (≥99.98%) with the trb operon in 244 kb and 49 kb bla GES -encoding plasmids from A. caviae Aero21 and KAM329, respectively (Figure S1). A. caviae Aero21 was from hospital wastewater in Brazil [54] and KAM329 from an unknown source in Japan.The next most similar Aeromonas isolates had ≥97.65% sequence identity within the trb operon and were found on chromosomes from isolates (Aeromonas sp.ASNIH1, ASNIH5, and ASNIH7) in the bla KPC study mentioned above (Figure S1).Aeromonas sp.ASNIH5 and ASNIH7 were acquired from wastewater manholes outside the hospital associated with the patient infected with Aeromonas ASNIH1 in 2015.Together, this suggests a correlation of trb operon genes and carbapenemases among Aeromonas species.
Considering the diverse environments Aeromonas is known to colonize, the complex mobilome associated with this genus is ever evolving.It is evident that Aeromonas engages in interspecies horizontal gene transfer [55], including with members of Enterobacterales [56].Additionally, we found MGEs scattered throughout pMAH4, including an IS6100 composite transposon between 30,877-53,665 bp (Figure 3 and Table S3).Within this composite transposon is a type 1 integrase as well as multiple ARGs.The presence of IS26 and ISCfr1 inside this composite transposon likely reflects numerous mobilization events that resulted in this multi-drug-resistant plasmid.Additionally, ISPst3, ISAs1, and ISAhy2 are all within 5000 bp of each other (Table S3), reaffirming a high frequency of recombination and HGT events in this isolate.
The presence of SFO-1 from Aeromonas isolates collected from humans, wastewater, and dogs further supports addressing antimicrobial resistance from a One Health approach.In previous work, we used Aeromonas as a global indicator species for analyzing antimicrobial resistance over a twenty-year period.We identified similar resistance levels across human, agricultural, and environmental sectors for the majority of the 21 antimicrobials investigated; the few exceptions were mainly for last-resort antimicrobials like cefepime and aztreonam [3].Among these antibiotics, wastewater contains significantly higher resistance profiles than all other sectors, which stresses that wastewater may be an origin or source for increased HGT and ARG acquisition.The findings of SFO-1 in wastewater globally further highlight the importance of characterizing AMR from multiple sectors so that the ultimate drivers of resistance mobilization and proliferation may be identified.Lower socio-economic countries are commonly associated with increased levels of AMR through all sectors [3], which are also correlated frequently with antimicrobial overuse and an absence or deficiency of wastewater treatment facilities [3,57].It is imperative to develop global policies regulating antimicrobial use and for higher socio-economic countries to increase support for infrastructure, like wastewater systems, in countries with lower gross domestic income.

Conclusions
We report the first clinical isolate of an Aeromonas species encoding bla SFO-1 from a multi-drug-resistant strain in the USA.Isolate MAH-4 contained numerous ESBLs both chromosomally and on a 327 kb plasmid.Genomic evidence strongly indicates interspecies HGT between Klebsiella and Aeromonas.This conclusion is further strengthened when taking into account the common presence and relatively high abundance of both genera in wastewater.Wastewater may be an accelerator of AMR as it often harbors compounds like heavy metals and subinhibitory concentrations of antimicrobials, both of which have been shown to increase genetic exchange.Although wastewater presents a difficult challenge in the fight against AMR, it also provides an opportunity via wastewater-based epidemiology to monitor rising resistance patterns and ARGs across disparate areas so that resistance risks can be identified before they become clinical problems.

Figure 1 .
Figure 1.Chromosome map of Aeromonas hydrophila MAH-4.The coding sequences (CDS) of the forward strand are on the outermost ring followed by the CDS of the reverse strand.The colors of the CDS represent the various subsystems: blue-metabolism; green-protein processing; orange-energy; purple-stress response; defense, and virulence; red-membrane transport; pink-DNA processing; brown-cellular processes; gray-RNA processing; olive-cell envelope; artic blue-miscellaneous; turquoise-regulation and cell signaling.The third ring from the outside represents RNA genes.

Figure 1 .
Figure 1.Chromosome map of Aeromonas hydrophila MAH-4.The coding sequences (CDS) of the forward strand are on the outermost ring followed by the CDS of the reverse strand.The colors of the CDS represent the various subsystems: blue-metabolism; green-protein processing; orangeenergy; purple-stress response; defense, and virulence; red-membrane transport; pink-DNA processing; brown-cellular processes; gray-RNA processing; olive-cell envelope; artic blue-miscellaneous; turquoise-regulation and cell signaling.The third ring from the outside represents RNA genes.The fourth ring represents antimicrobial resistance CDS followed by virulence CDS on the fifth ring.The purple ring presents the GC content throughout the chromosome followed by the GC skew in the innermost ring.

Figure 2 .
Figure 2. Phylogenetic tree reconstruction derived from conserved concatenated genes of clinical isolate Aeromonas hydrophila MAH-4 and other representative Aeromonas species.

Figure 2 .
Figure 2. Phylogenetic tree reconstruction derived from conserved concatenated genes of clinical isolate Aeromonas hydrophila MAH-4 and other representative Aeromonas species.

Figure 3 .
Figure 3. Mobile genetic element (MGE) and antimicrobial resistance gene (ARG) characterization of pMAH4.Blue arrows represent predicted ARGs with purple arrows representing MGEs of the 327,540 bp plasmid from Aeromonas hydrophila MAH-4.SnapGene v. 7.1.1 was used to create the figure.

Figure 3 .
Figure 3. Mobile genetic element (MGE) and antimicrobial resistance gene (ARG) characterization of pMAH4.Blue arrows represent predicted ARGs with purple arrows representing MGEs of the 327,540 bp plasmid from Aeromonas hydrophila MAH-4.SnapGene v. 7.1.1 was used to create the figure.

Table 1 .
Phenotypes and genotypes of antimicrobial resistance of Aeromonas hydrophila MAH-4.

Table 2 .
Similarity and location of ARGs from A. hydrophila MAH-4.

Table 2 .
Similarity and location of ARGs from A. hydrophila MAH-4.