Carbapenem-resistant Citrobacter freundii harboring blaKPC−2 and blaNDM−1: a study on their transferability and potential dissemination via generating a transferrable hybrid plasmid mediated by IS6100

Introduction The increase in clinical Enterobacteriaceae with dual carbapenemase has become a serious healthcare concern. It is essential to characterize the transferability and potential dissemination of blaKPC−2- and blaNDM−1-coharboring carbapenem-resistant Citrobacter freundii (CRCF). Methods Four blaKPC−2- and blaNDM−1-coharboring CRCF strains were collected from our surveillance of the prevalence of carbapenem-resistant Enterobacteriaceae. The isolates were assessed using species identification, antimicrobial susceptibility testing, conjugation assays, whole-genome sequencing, plasmid stability, and fitness costs. Clonality, genome, plasmidome, and phylogeny were analyzed to reveal potential dissemination. Results Three ST523 blaKPC−2- and blaNDM−1-coharboring CRCF strains, collected from the same hospital within 1 month, exhibited high homology (both identity and coverage >99%), implying clonal dissemination and a small-scale outbreak. Moreover, the blaKPC−2 and blaNDM−1 genes were coharbored on an IncR plasmid, probably generated by a blaKPC−2-harboring plasmid acquiring blaNDM−1, in these three strains. Importantly, the IncR plasmid may form a transferable hybrid plasmid, mediated by IS6100 via transposition, with another IncFII plasmid included in the same C. freundii strain. Furthermore, the blaKPC−2 and blaNDM−1 of the fourth CRCF strain are located on two different non-transferable plasmids lacking complete transfer elements. Additionally, throughout the course of the 10-day continuous passage, the genetic surroundings of blaNDM−1 in four CRCF strains were gradually excised from their plasmids after the 8th day, whereas they maintained 100% retention for blaKPC−2. Genome and plasmidome analyses revealed that blaKPC−2- or blaNDM−1-harboring C. freundii were divergent, and these plasmids have high homology to plasmids of other Enterobacteriaceae. Conclusion Clonal dissemination of ST523 blaKPC−2- and blaNDM−1-coharboring CRCF strains was detected, and we first reported blaKPC−2 and blaNDM−1 concomitantly located on one plasmid, which could be transferred with mediation by IS6100 via transposition. Continued surveillance should urgently be implemented.


Introduction:
The increase in clinical Enterobacteriaceae with dual carbapenemase has become a serious healthcare concern.It is essential to characterize the transferability and potential dissemination of bla KPC− -and bla NDM− -coharboring carbapenem-resistant Citrobacter freundii (CRCF).
Methods: Four bla KPC− -and bla NDM− -coharboring CRCF strains were collected from our surveillance of the prevalence of carbapenem-resistant Enterobacteriaceae.The isolates were assessed using species identification, antimicrobial susceptibility testing, conjugation assays, whole-genome sequencing, plasmid stability, and fitness costs.Clonality, genome, plasmidome, and phylogeny were analyzed to reveal potential dissemination.
Results: Three ST bla KPC− -and bla NDM− -coharboring CRCF strains, collected from the same hospital within month, exhibited high homology (both identity and coverage > %), implying clonal dissemination and a smallscale outbreak.Moreover, the bla KPC− and bla NDM− genes were coharbored on an IncR plasmid, probably generated by a bla KPC− -harboring plasmid acquiring bla NDM− , in these three strains.Importantly, the IncR plasmid may form a transferable hybrid plasmid, mediated by IS via transposition, with another IncFII plasmid included in the same C. freundii strain.Furthermore, the bla KPC− and bla NDM− of the fourth CRCF strain are located on two di erent non-transferable plasmids lacking complete transfer elements.Additionally, throughout the course of the -day continuous passage, the genetic surroundings of bla NDM− in four CRCF strains were gradually excised from their plasmids after the th day, whereas they maintained % retention for bla KPC− .Genome and plasmidome analyses revealed that bla KPC− -or

Introduction
Citrobacter freundii, a Gram-negative and facultative anaerobic bacillus, is an opportunistic pathogen and causes diverse infections, including those of the urinary tract, respiratory tract, and bloodstream (Liu et al., 2018).Carbapenemase-producing C. freundii (CPCF), carrying bla KPC encoding a class A serine βlactamase KPC or bla NDM encoding a class B metallo-β-lactamase NDM, has been increasingly reported in recent years and resulted in a small-scale, sporadic outbreak (Hammerum et al., 2016;Bartsch et al., 2017;Jimenez et al., 2017;Bitar et al., 2019;Babiker et al., 2020).Both KPC-2 and NDM-1, the subclasses of KPC and NDM, respectively, show a broad spectrum of hydrolytic activity against penicillins, cephalosporins, and carbapenems.The former can also hydrolyze monobactams and be inhibited by most β-lactamase inhibitors, such as avibactam and relebactam.By comparison, the latter cannot hydrolyze monobactams and is unresponsive to the above β-lactamase inhibitors (Bush, 2013;Bush and Bradford, 2019).Importantly, bla KPC−2 -and bla NDM−1coproducing CPCF strains have been recovered from patient specimens and clinical waste in China, leading to limited options for antibacterial treatment (Feng et al., 2015;Wu et al., 2016;Ouyang et al., 2018;Li et al., 2022).Nevertheless, each of those studies only described a single CRCF strain carrying bla KPC−2 and bla NDM−1 on two separate plasmids.
Herein, we identified four bla KPC−2 -and bla NDM−1coharboring CRCF strains isolated from urine specimens and renal perfusion fluid of four patients in China.We first identified bla KPC−2 and bla NDM−1 concomitantly located on one IncR plasmid of three CRCF strains and also compared their genetic environments in conjunction with data available on NCBI, revealing its potential dissemination mechanism.

Materials and methods
Identification of bla KPC--and bla NDM--coharboring CRCF strains and their clinical data Four bla KPC−2 -and bla NDM−1 -coharboring CRCF strains were collected during our surveillance of the prevalence of carbapenemresistant Enterobacteriaceae isolates.Species identification was determined using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik, Bremen, Germany), as described previously (Lu et al., 2021).

Plasmid conjugation assays
Plasmid conjugation experiments were conducted for four CRCF strains, as described previously (Zhang et al., 2023).Azideresistant E. coli J53 and amikacin-resistant K. pneumoniae KP54 were used as the recipient strains.In brief, the four CRCF strains and recipient KP54 were adjusted to a McFarland standard of 0.5 and mixed at a ratio of 1:3, and a 0.1-milliliter aliquot of mixture was transferred into LB broth without antibiotics.After an 18h incubation at 37 • C, 200-ml cultures were streaked onto China blue agar (CBA, addition of rosolic acid as the pH indicator) plates containing both amikacin (16 mg/L) and meropenem (1 mg/L) to screen the bla KPC−2− or bla NDM−1 -carrying transconjugants.Similarly, KP54_CF2075K-N and recipient E. coli J53 were mixed, and transconjugants carrying bla KPC−2 or bla NDM−1 were also selected on CBA plates containing both azide (150 mg/L) and meropenem (1 mg/L).The above transconjugants were confirmed by antimicrobial susceptibility testing (AST), polymerase chain reaction (PCR), and pulsed-field gel electrophoresis (PFGE), respectively (Zhang et al., 2023).
Plasmid stability and fitness cost of CF , CF , CF , CF , and the transconjugants The plasmid stability and fitness cost were assessed as previously described but with slight modifications (Gao et al., 2020).The stability of CF2075, CF2084, CF2085, CF26019, and the transconjugants was evaluated by a passage experiment.In brief, the aforementioned strains were grown in LB broth and transferred at a 24-h interval for 10 consecutive days (approximately 200 generations), at a 1:1000 dilution, into fresh LB broth.The cultures at the 2nd, 4th, 6th, 8th, and 10th days were serially diluted and streaked onto the antibiotic-free LB agar.Approximately 50 colonies were randomly selected to identify the retention of bla KPC−2 and bla NDM−1 using PCR.All the above experiments were conducted in triplicate on different days.
Fitness cost was evaluated by growth curves.In brief, CF2085, CF2085 NDM (obtained in the above passage assay), CF26019, CF26019 NDM, the recipient KP54, and its transconjugants were cultured and shaken at 200 rpm overnight at 37 • C in 10 mL LB broth.The overnight cultures were diluted and incubated at 37 • C for 25 h to measure the optical density values (OD 600 ).The experiment was repeated two times.The growth curves were estimated using Tukey's multiple comparison tests with a one-way analysis of variance (ANOVA).
Plasmidome analysis of bla KPC--or bla NDM--carrying plasmids of C. freundii To better unravel the plasmidome of bla KPC−2 -or bla NDM−1harboring plasmids in C. freundii, we searched the RefSeq database on NCBI and obtained the intact plasmids harboring bla KPC−2 (42) or bla NDM−1 (21) in C. freundii worldwide as of 20 November 2022.Blasting was performed with BLASTn and illustrated with the R ggplot2 package.

Statistical analyses
Data analyses were performed using GraphPad Prism 8.2.1.One-way analysis of variance was used for assessing significant differences, with a P-value of <0.05 being considered statistically significant.

Clinical data for CRCF strains
From 11 September 2020 to 12 October 2020, three bla KPC−2and bla NDM−1 -coharboring CRCF strains (CF2075, CF2084, and CF2085) were recovered from urine specimens of three inpatients with congenital hydronephrosis post-operation or urinary infection (aged 8 months to 9 years) at the same urological ward of a tertiary hospital in Henan, China (Table 1).The CRCF strain CF26019 was isolated from the renal perfusion fluid of Patient 4 with chronic kidney disease at another tertiary hospital in Beijing, China, after kidney transplantation (Table 1).The four patients all finally recovered from urological diseases.

Transferability, stability, and fitness cost of bla KPC--and bla NDM--coharboring CRCF strains
The transferability of bla KPC−2 and bla NDM−1 was evaluated by conjugation assays.The results revealed that bla KPC−2 and bla NDM−1 of CF2075, CF2084, and CF2085 could be concomitantly transferred to the recipient K. pneumoniae KP54, a clinical strain isolated from urine samples, and the transconjugants were designated as KP54_CF2075K-N, KP54_CF2084K-N, and KP54_CF2085K-N, respectively (Supplementary Figure S1B).Further conjugation experiments revealed that bla KPC−2 and bla NDM−1 of KP54_CF2075K-N could also be simultaneously conjugated into recipient E. coli J53, designated as E. coli J53_K-N (Supplementary Figure S1C).The above transconjugants were verified by resistance phenotype using PCR and PFGE.However, no transconjugant was obtained after co-culturing CF26019 with KP54, even though we repeated the conjugation assay dozens of times.
Unexpectedly, S1-PFGE showed that all the transconjugants, KP54_CF2075K-N, KP54_CF2084K-N, and KP54_CF2085K-N, contained a novel fusion plasmid co-carrying bla KPC−2 and bla NDM−1 , named pCfr_tK-N, which is different from any plasmids of CF2075, CF2084, and CF2085 in size.However, the size of the pCfr_tK-N is almost equal to the sum of the size of pCF2075-1 and pCF2075-2; that is to say, pCfr_tK-N was probably generated by the recombination of pCF2075-1 and pCF2075-2.To unravel the forming mechanism of pCfr_tK-N, WGS was performed.Sequence analysis revealed that pCfr_tK-N was indeed the recombination result of pCF2075-1 and pCF2075-2 (Figures 3A, B), and the junctions of these two plasmids were two insertion sequence IS6100s.Of note, two 8-base-pair direct repeat (DR, ATGCTCAG) sequences were adjacent to the left inverted repeat sequence (IRL) of one IS6100 and the right inverted repeat sequence (IRR) of another IS6100, which implied that the recombination was mediated by IS6100 (Figure 3C).The plasmid stability of four CRCF strains and transconjugants was evaluated by passage experiment (Supplementary Figure S1E).Excisions of bla NDM−1 strains were obtained from CF2085 and CF26019 on 8th day, named CF2085 NDM and CF26019 NDM, respectively.However, S1-PFGE and the sequence analysis showed that bla NDM−1 -harboring genetic surroundings were excised from the plasmids of CF2085 and CF26019, which were inconsistent with the loss of bla NDM−1 usually caused by the removal of plasmids harboring bla NDM−1 in most cases (Figure 2B and Supplementary Figure S1B).A similar phenomenon was also observed in CF2075 and CF2084 on the 10th day of the passage experiment.However, the transconjugants (KP54_CF2075K-N, KP54_CF2084K-N, KP54_CF2085K-N, and E. coli J53_K-N) maintained 100% retention during the process of 10-day continuous passage, showing that the hybrid plasmids harboring bla KPC−2 and bla NDM−1 could be stably inherited.In summary, bla KPC−2 -and bla NDM−1 -coharboring CRCF strains and transconjugants can retain the stable inheritance of bla KPC−2 -and bla NDM−1 -co-carrying plasmids (>90% retention in the 10-day passage) that might slow the clearance of resistance genes and facilitate clonal dissemination.Growth rates were measured to assess the impact of the acquisition of bla KPC−2 -and bla NDM−1 -coharboring plasmids on biological fitness cost.A significant growth difference (P < 0.0001) was shown between CF2085 and CF2085 NDM, KP54 and KP54_CF2075K-N, and E. coli J53 and E. coli J53_K-N, whereas no significant difference (P > 0.5) was observed in the growth rates between CF26019 and CF26019 NDM.Taken together, the bla KPC−2 -and bla NDM−1 -coharboring plasmids exhibited punishment of fitness in CF2075, CF2084, CF2085, and their transconjugants.in this study, were analyzed via phylogenetic tree (Figure 4).These 42 C. freundii strains were recovered from urine, sediment, rectal swab, and blood and also from wastewater in the hospital environment and were gathered in Spain, the Czech Republic, China, and USA.Heterogeneous STs were identified in bla KPC−2 -harboring C. freundii, including ST8, ST18, ST22, ST65, ST118, ST257, ST259, ST523, and ST632.The C. freundii strains belonging to the same STs might carry both closely related plasmids (such as pCF2075-1, pCF2084-1, and pCF2085-1 or CP037739.1 and CP054297.1),largely implying a clonal spread, and distantly related ones (CP011608.1 and CP011656.1).Plasmid incompatibility groups revealed that the above closely related plasmids had the same replicon, including IncP6, IncR, and IncN.Furthermore, bla KPC−2 -harboring plasmids showed ≤52% and ≤59% coverage compared with the bla KPC−2 -and bla NDM−1 -co-carrying plasmids pCF2075-1 and pK254-KPC_NDM, respectively.Notably, most bla KPC−2harboring plasmids (43/46, 93.5%) exhibited ≥82.0%coverage at 100% identity to the plasmids contained in non-C.freundii Enterobacteriaceae.Similarly, bla KPC−2 -harboring plasmids (34/46, 74.0%) also exhibited ≥82.0%coverage to the plasmids of C. freundii.
The bla KPC−2 -harboring plasmids in both P10159 and SCLZS47 were identical, although not grouped into the same cluster, and CF2075, CF2084, and CF2085 also contained the same plasmids.Therefore, four different bla KPC−2 -harboring plasmids were identified in the seven CRCF strains.These different plasmids, other than the type of pCF2075-1, have a high coverage of similar sequence (bi-directional ≥90%) in Enterobacteriaceae.For bla NDM−1 -harboring plasmids, three different plasmid types were identified.It is likely that the coverage of similar sequences among these plasmids was also distantly related (4.0-16.0%).Only the type of pP10159-1 (MF072961.1)had similar plasmids (bi-directional coverage and identity ≥99.9%) reported before in Enterobacteriaceae, and others were novel ones.Furthermore, P10159 and SCLZS47 were isolated from Chongqing and Sichuan in China, which are geographically close, and had identical bla KPC−2 -and bla NDM−1 -harboring plasmids, but the chromosomes of P10159 had 81.0%coverage of similar sequence at 95.94% to SCLZS47 and 80.0% the other way around, hinting at a horizontal gene transfer (HGT) by plasmids.

Discussion
CRCF coharboring bla KPC−2 and bla NDM−1 was first reported in 2015 (Feng et al., 2015) and has emerged continually in recent years, especially in China (Feng et al., 2015;Wu et al., 2016;Ouyang et al., 2018;Li et al., 2022).However, comprehensive analyses for the CRCF strains were lacking, and the potential dissemination mechanism has remained unclear.
In the current study, four bla KPC−2 -and bla NDM−1coharboring CRCF strains were identified.Three of them (CF2075, CF2084, and CF2085), recovered from urine samples within 1 month in the same ward, indicated a clonal outbreak according to PFGE, WGS, and phylogenetic tree analyses.WGS revealed a bla KPC−2 -and bla NDM−1 -coharboring plasmid in these three CRCF strains, which was first reported here.The clonal outbreak of coharboring dual carbapenemase genes will pose a severe threat to    of the above plasmid and chromosome were highly identical, facilitating the generation of TE and further spread.However, the bla KPC−2 -carrying genetic surroundings of them were Tn1721/Tn6296-based transposons, which were usually generated in an ancestor transposon by inserting another transposon, and lacked an intact inverted repeat sequence (IR) at both sides of the core bla KPC−2 region, resulting in difficult transfer.Taken together, it is reasonable to hypothesize that a bla KPC−2 -and bla NDM−1 -coharboring plasmid or chromosome might be derived from bla KPC−2 -harboring plasmid or chromosome progenitors that acquired bla NDM−1 by HGT, such as TUs and transposons.AST revealed that aztreonam-avibactam was probably an option for bla KPC−2 -and bla NDM−1 -coharboring CRCF strains.
To evaluate the transferability of bla KPC−2 and bla NDM−1 , a series of transconjugants were obtained by conjugation assays and showed consistency of resistance phenotype and genotype.However, S1-PFGE showed that pCF2075-1 was not transferred alone to the recipient, and further analysis revealed that during conjugation, the fusion plasmid pCfr_tK-N was generated and could be transferred to the recipient strains KP54 and E. coli J53, respectively.Sequence analysis indicated that pCfr_tK-N was a recombinant of pCF2075-1 and pCF2075-2 mediated by IS6100, and this intermolecular transposition was first reported.Similar recombination, mediated by IS26, ISKpn14, ISKpn74, and IS903B, was also reported in conjugations among different resistant or virulent plasmids in recent years, which could accelerate the dissemination of resistance and virulence genes (Wang et al., 2022a,b;Yang et al., 2022).The pCF2075-1 being transferred by generating recombinants might be explained by the fact that the self-transferrable plasmids usually contain four modules: origin of transfer site oriT, relaxase gene, gene encoding type IV coupling protein (T4CP), and gene cluster for bacterial type IV secretion system (T4SS).Putative transferability analysis revealed that pCF2075-1 only contained oriT and relaxase gene, which prevented it from being transferred alone.However, pCF2075-2 contained all four complete modules.Therefore, pCF2075-1 is required to be integrated into pCF2075-2 to be transferrable.Furthermore, the stability and fitness cost of the CRCF strains and transconjugants were evaluated.These exhibited higher retention for bla KPC−2 and bla NDM−1 (>90.0%after 10 days) but required biological cost.
Plasmidome is extremely essential for understanding its origin and taking measures to prevent its propagation.In total, 46 bla KPC−2 -harboring plasmids and 25 bla NDM−1 -harboring plasmids retrieved from NCBI were systematically analyzed.Analysis revealed that C. freundii strains with bla KPC−2 -or bla NDM−1 -harboring plasmids shared the same STs, such as ST18 and ST257, that might generate more bla KPC−2 -and bla NDM−1 -coharboring CRCF strains in future.Importantly, most bla KPC−2 -or bla NDM−1 -harboring plasmids in C. freundii had high homology to the plasmids of other Enterobacteriaceae, hinting at the high transferability of bla KPC−2 -or bla NDM−1 -harboring plasmids.Moreover, the types of core bla KPC−2 and bla NDM−1 regions were relatively conserved, although bla KPC−2 or bla NDM−1 genetic surroundings exhibited multiple types by different ISs, indicating that ISs play a significant role in driving resistance genes' transfer.
We next comprehensively elaborated on the evolutionary relationships of seven bla KPC−2 -and bla NDM−1 -coharboring CRCF strains.These were grouped into three clusters.Notably, P10159 and SCLZS47 had the same bla KPC−2 -and bla NDM−1 -harboring plasmids, although they did not belong to the same cluster.The discordance of homology of bla KPC−2 -or bla NDM−1 -harboring plasmids to the genome evolution indicated that these have a strong ability to transfer and adapt to different hosts.As mentioned above, bla KPC−2 -or bla NDM−1 -harboring plasmids in C. freundii belong to diverse STs.Therefore, the formation of bla KPC−2and bla NDM−1 -coharboring CRCF strains probably occurs in two ways: bla KPC−2 -harboring C. freundii acquires bla NDM−1harboring plasmids, or bla NDM−1 -harboring C. freundii acquires bla KPC−2 -harboring plasmids.
In conclusion, we identified four bla KPC−2 -and bla NDM−1coharboring CRCF strains and reported, for the first time, bla KPC−2 and bla NDM−1 concomitantly located on one plasmid.Notably, the plasmid was integrated into another plasmid to generate an uncommon fusion plasmid, mediated by IS6100 via transposition, which could be transferred into a different genus in Enterobacteriaceae.Genome and plasmidome analyses revealed that bla KPC−2 -or bla NDM−1 -harboring C. freundii were divergent, and these plasmids have high homology to plasmids of other Enterobacteriaceae.

FIGURE
FIGURE Origin and formation of pCF -. (A) Linear sequence alignment analysis on plasmid pA -KPC (MT .), pCF -, pM A-NDMgene cluster (KX .), and pB B (CP .). (B) Proposed formation mechanism of pCF -as follows.Step : pA -KPC evolves to generate a cointegrate by losing insignificant segments (arc dotted line) via similar recombination or transposition.Step : ISCR , IS , and Tn mediate the production of transposable elements (TEs), and the cointegrate is integrated into the hybrid plasmid pCF -by these TEs.

A
FIGURE Linear alignment of bla KPC− -or bla NDM− -bearing plasmid.(A) Linear sequence analysis of bla KPC− -and bla NDM− -bearing chromosome and plasmid on CHS chromosome (CP .), pCF -, and pK -KPC_NDM (OM .). (B) Linear comparison of pCF -between CF and CF NDM.CF NDM was derived from excision of bla NDM− core structure for CF in conjugation.(C) Linear alignment of pCF structure on CF and CF NDM.CF NDM was derived from excision of bla NDM− core structure for CF in conjugation.

FIGURE
FIGURE Emergence and recombination mechanism of hybrid plasmid pCfr_tK-N generated during conjugation.(A) Linear sequence comparison of hybrid plasmid pCfr_tK-N with pCF -and pCF -. (B) Circular alignment analysis of pCF -, pCF -, and hybrid plasmid pCfr_tK-N.Alignments were generated using the BLAST Ring Image Generator (BRIG).(C) Proposed model for the formation of the fusion plasmid pCfr_tK-N mediated by IS transposition.

FIGURE
FIGUREPlasmidome analysis of bla KPC− -bearing plasmid in C. freundii.(A) bla KPC− genetic surrounding was analyzed in bla KPC− -bearing plasmid of C. freundii strains, including strains from NCBI and from this study.(B) The maximum-likelihood phylogenetic tree was built by PhyML from complete plasmids' sequence alignment generated by ma t.The tree was visualized and annotated using Interactive Tree Of Life (iTOL, https://itol.embl.de).Class-di erent types of bla KPC− genetic surrounding on the left.Alignment-alignment to pCF -and pK -KPC_NDM.Coverage-coverage to plasmids in non-Citrobacter sp., Enterobacteriaceae, and Citrobacter sp.

FIGURE
FIGURE Plasmidome analysis of bla NDM− -bearing plasmid in C. freundii.(A) bla NDM− genetic surrounding was analyzed in bla NDM− -bearing plasmid C. freundii strains, including strains from NCBI and from this study.(B) The maximum-likelihood phylogenetic tree was built by PhyML from complete plasmids' sequence alignment generated by ma t.The Interactive Tree Of Life (https://itol.embl.de)was used for visualization.Class-di erent types of bla NDM− genetic surrounding on the left.Alignment-alignment to pCF -and pK -KPC_NDM.Coverage-coverage to plasmids in non-Citrobacter sp., Enterobacteriaceae, and Citrobacter sp.

FIGURE
FIGUREMaximum-likelihood phylogenetic tree of seven bla KPC− -and bla NDM− -coharboring CRCF strains was built by RaxML.The tree is annotated based on ST, Inc_pKPC (incompatibility group of bla KPC− -bearing plasmids), Inc_pNDM (incompatibility group of bla NDM− -bearing plasmids), transferability, species, year, geography, and similarity among bla KPC− -bearing plasmids (blue heatmap) and bla NDM− -bearing plasmids (purple heatmap) in C. freundii.The similarity is defined as the coverage of homology regions for query plasmid (row-wise) and subject plasmid (column-wise).
a Time span: Interval between admission and specimen collection.