First report of multidrug-resistant carbapenemase-producing Aeromonas caviae co-harboring mcr-3.43 and mcr-7.2

ABSTRACT Hospital sewage serves as a crucial reservoir for antibiotic resistance genes. As colistin and carbapenems are the last-resort antibiotics, the emergence of their resistance genes has become a significant concern in clinical settings. In this study, we found that two novel mcr alleles (mcr-3.43 and mcr-7.2) with two carbapenemase genes (blaNDM-1 and blaKPC-2) were encoded in a single Aeromonas caviae strain isolated from hospital sewage. Our phylogenetic analysis revealed that the mcr-3.43 gene clustered with mcr-3.17 (with 95.55% amino acid identity), while the mcr-7.2 gene clustered with mcr-7.1 (with 68.68% amino acid identity). BLAST search against GenBank showed that mcr-7.2 was exclusively detected in Aeromonas spp. Mobile genetic elements were not found in the genetic context of mcr-7.2, suggesting that the dissemination of mcr-7.2 in Aeromonas spp. may be dependent on vertical transfer or recombination. The blaNDM-1 was adjacent to a recombinase gene and flanked by two IS91 elements, indicating a potential mobilization mechanism mediated by recombination and/or ISs. The blaKPC-2 gene was located on an IncU plasmid and adjacent to an ISKpn6. In summary, our study provides evidence for Aeromonas spp. as one of the potential reservoirs of colistin and carbapenem resistance genes. IMPORTANCE The study discovered two novel mcr genes (mcr-3.43 and mcr-7.2) and two carbapenemase genes (blaNDM-1 and blaKPC-2) in a single Aeromonas caviae strain retrieved from hospital sewage. Using phylogenetic analysis and comparative data evaluation, the study revealed the genetic relatedness and dissemination potential of the detected resistance genes. With the exclusive discovery that mcr-7.2 is only present in Aeromonas spp. and the lack of mobile genetic elements in its genetic context, there is a strong indication of limited dissemination. The identification of these four resistance genes in a single strain of Aeromonas provided valuable insights into their potential presence in this genus. This study revealed that hospital sewage functions as a significant reservoir for antibiotic resistance genes, including colistin and carbapenem resistance genes.

To detect the presence of novel mcr genes in G77, we performed WGS.The sequenc ing short and long reads were hybridly assembled to obtain no-gap contigs, resulting in a chromosome of 4,8313,750 bp and five contigs with sizes ranging in size from 5,079 bp to 110,200 bp.The contigs were confirmed to be complete by PCR and Sanger sequencing.The isolate was further confirmed as an A. caviae strain by KmerFinder (https://cge.food.dtu.dk/services/KmerFinder/). In silico mining revealed that A. caviae G77 carried 31 ARGs (Table 1), including homologs of two colistin resistance genes mcr-3.17(95.55% amino acid identity) and mcr-7.1 (68.89% amino acid identity), and the carbapenemase genes bla NDM-1 and bla KPC-2 .The mcr genes and bla NDM-1 were located on the chromosome, while bla KPC-2 was detected on a 12,135-bp contig.Replicon typing by Mob_Suite detected an IncU replicon on the contig, suggesting that the contig may belong to a plasmid.Two novel KPC-2-producing multidrug-resistant IncU plasmids (pEC2341-KPC, pEC2547-KPC) from E. coli in China were reported in 2021 (25).The blast analysis showed that the coverage of pEC2341-KPC, pEC2547-KPC are both 20.82%, compared with the KPC-2-producing IncU plasmid in this study with a threshold at 80% identity.We searched the National Center for Biotechnology Information (NCBI) genome database and found the co-existence of bla KPC-2 and mcr-3 in three Aeromo nas genomes (assembly accession numbers GCA_003925855.2, GCA_009831085.1, and GCA_014169675.1), and of bla NDM-1 and mcr-7 in one genome (assembly accession number GCA_017280155.1).However, genomes containing all four genes were not available.We tested 22 mcr-carrying isolates for the bla KPC-2 and bla NDM-1 genes by PCR and Sanger sequencing; the results showed that four other isolates besides G77 also carry bla KPC-2 and bla NDM-1 .This is the first time that these four genes have been identified in a single Aeromonas strain.
The mcr-7.2 gene was found to be chromosomally located in strain G77.We observed that the mcr-7.2segment was flanked by functional genes rather than mobile elements, as depicted in Fig. 2B.Through comparative Blast analysis, we discovered a significant similarity between the mcr-7.2segment in G77 and three other A. caviae strains: KAM376 (GenBank accession number AP024402.1),1605-27183 (GenBank accession number CP047983.1),and KAM471 (GenBank accession number AP026370.1).Upstream of the mcr-7-like gene, we identified the presence of the aspS and yebC genes.Downstream, a cluster of genes consisting of nagC-nagA-2-nagB-nagE was observed, along with the inclusion of a hexosaminidase gene.
The genetic arrangement of bla NDM-1 on the G77 chromosome exhibited specific mobile elements located both upstream and downstream.Upstream, we observed the presence of IS91, ΔISAba125, and ΔIS91 elements, while a complete IS91 element was identified downstream.The precise genetic context of bla NDM-1 on the G77 chromosome was represented as "IS91-hp-ΔISAba125-ΔIS91-ΔIS91-ΔISAba125-bla NDM-1ble MBL -PRAI-DsbD-IS91-sul1-qceE", which closely resembles the arrangement observed in E. coli 13ZX 28 (GenBank accession number MN101850.1).Furthermore, the down stream region of sul1-qceE contained an IntI1-recombinase-Tn3 segment, similar to that observed in Providencia rettgeri P138 (GenBank accession no.MZ670000.1).However, compared to the genetic context of bla NDM-1 in P. rettgeri P138, the "groES-groEL" segment appeared to be truncated in G77 and K. pneumoniae KP67 (GenBank acces sion no.CP101561.1)(Fig. 3A).It is noteworthy that unlike G77, where bla NDM-1 was integrated into the chromosome, bla NDM-1 in 13Z × 28, P138, and KP67 was located on plasmids.Based on the genetic context of bla NDM-1 in G77, we can infer that its origin likely involved plasmid-mediated transmission, facilitated by the integration of IS91 into the chromosome.Inverse PCR confirmed that no circular intermediate is generated, indicating that the bla NDM-1 is not acquired by translocatable unit cointegration (27).These findings provide valuable insights into the mechanisms underlying the dissemina tion of bla NDM-1 and its potential impact on antimicrobial resistance.
The bla KPC-2 gene was identified on an IncU plasmid within A. caviae G77.Upstream of the bla KPC-2 -bla TEM segment in G77, we discovered the presence of a mobile element called ISKpn6, exhibiting sequence similarities with E. cloacae 30860 (GenBank accession number MN477223.1),K. pneumoniae A1706 (GenBank accession number MH909350.1),and K. pneumoniae A1705 (GenBank accession number MH909348.1).However, in contrast to these segments, the region downstream of the bla KPC-2 -bla TEM segment in G77 lacked the ISKpn27 mobile element.Instead, it was followed by the sequence "APH (6)-Ic-Rep-hp-Mobc."This distinct organization of bla KPC-2 and bla TEM , coupled with the presence or absence of specific mobile elements, underscores the potential genetic diversity associated with carbapenem resistance genes among different bacterial strains (Fig. 3B).A comprehensive understanding of these genetic variations is critical to unraveling the dynamics of antibiotic resistance dissemination and formulating effective countermeasures.

Expression of mcr-and carbapenemase-encoding genes under colistin and meropenem stress in A. caviae G77
Induction assays were performed using different concentrations of colistin and meropenem to evaluate the expression of mcr-3.43,mcr-7.2,bla KPC-2 , and bla NDM-1 in A. caviae G77.Compared to the untreated control group, mcr-3.43showed upregulated expression in the colistin 0.5 (P < 0.05) and 1 mg/L (P < 0.01) groups and downregulated expression in the colistin 8 mg/L group (P < 0.05).In contrast, compared with the untreated control group, the transcript level of the mcr-7.2gene was induced when bacteria were inoculated with 0.5 and 1 mg/L of colistin while repressed when colistin was ≥2 mg/L (P < 0.05).In contrast, the expression of bla KPC-2 and bla NDM-1 shows no significant difference with less than twofold change when treated with a series of meropenem (Fig. 4).The divergence of MCR enzymes exists in the context of antibiotic resistance, as demonstrated by the colistin killing assay (Fig. 5).To test the activity of mcr-3.43 and mcr-7.2, the coding regions of mcr-3.43,mcr-7.2,mcr-9.1, and mcr-1.1 (positive control) were cloned into pBAD vector and expressed in E. coli Top10.The strains were incubated with twofold colistin dilutions (from 0 to 16 mg/L).mcr-3.43expression conferred colistin resistance up to 8 mg/L, but the resistance was weaker than that of mcr-1.Meanwhile, mcr-7.2expression conferred colistin resistance at 4 mg/L, and mcr-9.1 conferred resistance only at 1 mg/L.The data suggest that the activity of mcr-3.73 against colistin is higher than that of mcr-7.2, and it may play a more critical role in colistin resistance.

Plasmid transformability and stability
Conjugation assays showed that pKPC2 could not be successfully transferred into EC600 successfully by conjugation, albeit multiple attempts were made.
To determine the stability of the bla KPC-2 plasmid, a plasmid stability experiment was performed.The retention rate of bla KPC-2 in the G77 isolate was 100% after 10-day passages, suggesting that pKPC2 is stable in G77.In addition, the plasmid is a low-copynumber plasmid, with 1.02 ± 0.08 copy/CFU at day 0 and 1.06 ± 0.07 copy/CFU at day 10, with no significance.

DISCUSSION
The genus Aeromonas encompasses a wide range of aquatic environments and is associated with both intestinal and extra-intestinal infections in humans and animals (6).However, the emergence of multidrug-resistant organisms, particularly carbapenemresistant organisms (CRO) (28), represents a formidable global challenge.Colistin, a last-resort antimicrobial agent, remains one of the limited treatment options for severe CRO infections.Unfortunately, the alarming increase in CRO strains with acquired colistin resistance has now become a worldwide phenomenon, posing a significant threat to the efficacy of colistin therapy (29).The IS-mediated mcr colistin resistance genes are of particular concern, which are capable of horizontal transfer between different species and spreading across continents.This represents a specific and significant threat to public health and clinical management on a global scale (28).In the ongoing battle against antimicrobial resistance, continuous surveillance and monitoring play a pivotal role in upstanding the mechanisms of resistance transmission and formulating effective strategies to combat this escalating problem (30).In this way, we can protect public health and ensure the continued effectiveness of antimicrobial treatments for present and future generations.
This study identified a colistin-resistant strain of A. caviae, designated as G77, which carries multiple antibiotic resistance genes (ARGs).Through comprehensive wholegenome sequencing, we revealed the presence of novel genes, including mcr-3.43,mcr-7.2,bla NDM-1 , and bla KPC-2 , within the G77 strain.Notably, bla KPC-2 was specifically located on an IncU plasmid.Remarkably, this is the first documented case of all four genes coexisting within a single Aeromonas strain.Of particular interest, MCR-3.43 shares the highest amino acid identity (95.55%) with MCR-3.17, while MCR-7.2 shares the highest amino acid identity (68.68%) with MCR-7.1.These findings provide valuable insights into the emergence of multidrug-resistant strains within the Aeromonas genus and highlight the potential for widespread dissemination of antibiotic resistance within this group.These important discoveries contribute to the understanding of the genetic dynamics underlying multidrug resistance in Aeromonas species.Such knowledge is critical to addressing the global challenge of antibiotic resistance and developing effective strategies to mitigate its impact.
A comparative analysis investigated the relationship between the newly identified MCRs and the known MCR variants (MCR-1 to MCR-10).The analysis reveals that MCR-3.43 and MCR-3.17show only a few amino acid variations (26 aa), while MCR-7.2 and MCR-7.1 exhibit a substantial number of amino acid variations (166 aa).MCR-3.43 forms a distinct cluster alongside MCR-3.17 and shares a high amino acid identity of ≥90.91% with the known MCR-3 variants.Consequently, the newly discovered protein is designated as MCR-3.43.Furthermore, phylogenetic analysis demonstrated that MCR-7.2 also forms a separate cluster with MCR-7.1, exhibiting a moderate amino acid identity of 68.89%.Notably, MCR-7.2 and MCR-7.1 stand out from other MCRs in the phylogenetic tree.These findings provide valuable insights into the genetic relationships and variations among different MCR variants, enhancing our understanding of their diversity and evolution.
We discovered that mcr-3.43 is located adjacent to ISAhy2 and shares several elements similar to Tn6518, suggesting its potential mobility.In contrast, mcr-7.2 is surrounded by a few functional genes and is situated on the chromosome.Interestingly, the identical genetic context of mcr-7.2found in A. caviae suggests a limited dissemination of this gene thus far.Moving on to the G77 strain, the bla NDM-1 segment is on the chromosome, flanked by two IS91 elements.Conversely, bla KPC-2 is positioned on an IncU plasmid adjacent to a complete ISKpn6.These observations suggest that IS elements facilitate the transmission of the two carbapenemase genes.Understanding the genetic arrange ments and mobile elements associated with resistance genes is crucial for comprehend ing their dissemination and devising effective strategies to combat antibiotic resistance.These findings contribute significantly to our knowledge of the genetic characteristics and potential transmission mechanisms of resistance genes in A. caviae.
In conclusion, our study highlights that A. caviae is an essential reservoir of colistinresistant genes and carbapenemase genes in hospital wastewater, which could be a risk for the wide dissemination of these genes in the future.Developing robust surveillance strategies and effective countermeasures is urgent for the prevention of such reservoir dissemination.

Limitation of the study
This study is limited by its reliance on a single strain of A. caviae isolated from hospital sewage.Findings need to be completed in terms of both diversity and prevalence of resistance genes in Aeromonas spp.We recommend further research, including broader sampling and functional analysis, to promote a more comprehensive grasp of colistin and carbapenem resistance in Aeromonas spp.and other relevant pathogens.
The complete genome sequence of G77 has been deposited in GenBank under accession no.JAVHYK000000000.

Functional evaluation of mcr-3.43 and mcr-7.2
The coding regions of mcr-3.43 and mcr-7.2 were amplified from G77 and ligated with pBAD vector and expressed in E. coli Top10.E. coli Top10 harboring mcr-1.1 and empty pBAD vector previously constructed by our team (31) were employed as the positive and negative controls, respectively.The colistin killing assay was performed as previously described (36).In brief, different strains were incubated at 37°C to an optical density (OD 600 ) value of 0.3 to 0.4 and were then diluted 1:100 in LB broth, respectively.Fifty microliters of the diluted cultures was mixed with 50 µL of colistin dissolved in a phosphate-buffered saline (PBS) solution and was placed in a 96-well plate to ensure the final colistin concentrations of 0, 1, 2, 4, 8, and 16 mg/L.After 1 h of incubation at 37°C with aeration, the cultures were serially diluted in PBS, and 5 µL each was plated onto LB agar plates.

Antibiotic induction, bacterial RNA Isolation, and quantitative real-time PCR (RT-qPCR)
For induction assays, G77 was grown in LB without antibiotic until OD 600 = 0.5.Overnight culture of the bacteria was diluted in fresh LB broth (1:1,000, vol/vol) with final colistin or meropenem concentrations of 0.5, 1, 2, 4, and 8 mg/L.The antibiotic-free medium was used as control.The culture was incubated at 37°C for 30 min and harvested by centrifugation at 4°C.RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions, followed by genomic DNA elimina tion.cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara Bio USA).gDNA removal was confirmed using PCR.RNA size, integrity, and total amount were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, MA, USA).
The primers used for RT-qPCR were designed using Primer Premier 6.0 and are listed in Table S1.The amplification efficiency of all primer pairs was tested using standard dilution procedures.RT-qPCR analysis was conducted on an Applied Biosystems ViiA 7 sequence detection system with SYBR green fluorescence dye.The 16S rRNA gene was used as a reference control for normalization.The relative differences in gene expression were calculated as a fold change using the formula 2 −ΔΔCT (Livak and Schmittgen, 2001).Expression changes > twofold with P < 0.05 were considered statistically significant.

Stability and transferability of the bla KPC-2 -carrying plasmid
The plasmid stability was confirmed as previously described with minor modifications (37).Briefly, single colonies were enriched in 5 mL of LB broth at 37°C for 24 h.The suspension was then inoculated with 1:1,000 dilution in antibiotic-free LB broth.The freshly inoculated culture constituted time point one.Isolates were cultured at 37°C in a shaking bath (200 rpm) and serially passaged for 10 days.Cultures were diluted and plated onto antibiotic-free LB plates every 24 h.A total of 50 resulting colonies were randomly selected for PCR assays to determine the proportion of mcr or carbapene mase-positive bacteria in each population.Plasmids were considered stable when the retention rates were still over 80% at the end of the experiment.Plasmid copy number was estimated by qPCR according to the previous study (31).
Conjugation experiments were performed using the rifampicin-resistant E. coli EC600 recipient strain.Both donor and recipient strains were cultured in exponential phase and then mixed at a 1:1 ratio.The transconjugants were selected on agar plates supple mented with 4 mg/L of meropenem plus 100 mg/L of rifampicin.PCR was performed to confirm the presence of bla KPC-2 in transconjugants.Positive transconjugants were validated using Vitek MS to confirm the species.

FIG 1
FIG 1The phylogenetic tree of MCR.The phylogenetic tree was constructed by IQ-TREE 2's LG + I + G4 model.The cluster of MCR-1, MCR-2, MCR-5, and MCR-6 is collapsed.The column of alleles is the protein ID of MCR.The column of length is the MCR's amino acid length.The column of identity is the sequences' identity compared with MCR-3.43 using the blastp.The column of Accession number is the protein's accession number of MCR.The column of the host is the host genome of MCR.

FIG 2
FIG 2 The genetic context of (A) mcr-3.43 and (B) mcr-7.2.Different-colored arrows indicate different open reading frames (ORFs), while the direction of the arrow indicates its transcription direction.Light gray shadows represent homologous areas.

FIG 3
FIG 3 The genetic context of (A) bla NDM-1 and (B) bla KPC-2 .Different-colored arrows indicate different open reading frames (ORFs), while the direction of the arrow indicate its transcription direction.Light gray shadows represent homologous areas.

FIG 4
FIG 4 Gene expression with and without antibiotic exposure (colistin and meropenem).Fold changes in mRNA levels were determined by RT-qPCR.The data have been normalized to values for reference rpoD gene.The experimental groups included LB supplemented with 0.5, 1, 2, 4, and 8 mg/L of colistin or meropenem, and antibiotic-free LB was used as control.The data are shown as the means ± SD of the results from three individual assays.Statistical significance was determined by Student's t-test relative to the CI for competition between experimental groups versus control; *P < 0.05; **P < 0.01; ***P < 0.001.

FIG 5
FIG 5 Growth viability of E. coli harboring different versions of MCR family of enzymes.The isolates were grown on the LBA plates supplied with varied levels of colistin.Designation: Vec, pBAD33.

TABLE 1
Plasmid replicons and antimicrobial resistance genes detected in G77 a a Nucleotide blast with minimum identity thresholds of 80% and 70% and minimum coverage thresholds of 90% and 90%, for PlasmidFinder and ResFinder, respectively.