Resistome and Genome Analysis of an Extensively Drug-Resistant Klebsiella michiganensis KMIB106: Characterization of a Novel KPC Plasmid pB106-1 and a Novel Cointegrate Plasmid pB106-IMP Harboring blaIMP-4 and blaSHV-12

Klebsiella michiganensis is a recently emerging human pathogen causing nosocomial infections. This study aimed to characterize the complete genome sequence of a clinical Klebsiella michiganensis strain KMIB106 which exhibited extensive drug-resistance. The whole genome of the strain was sequenced using PacBio RS III systems and Illumina Nextseq 500. Annotation, transposable elements and resistance gene identification were analyzed by RAST, prokka and Plasmid Finder, respectively. According to the results, KMIB106 was resistant to multiple antimicrobials, including carbapenems, but it remained susceptible to aztreonam. The genome of KMIB106 consisted of a single chromosome and three predicted plasmids. Importantly, a novel KPC plasmid pB106-1 was found to carry the array of resistance genes in a highly different order in its variable regions, including mphA, msrE, mphE, ARR-3, addA16, sul1, dfrA27, tetD and fosA3. Plasmid pB106-2 is a typical IncFII plasmid with no resistant gene. Plasmid pB106-IMP consists of the IncN and IncX3 backbones, and two resistance genes, blaIMP-4 and blaSHV-12, were identified. Our study for the first time reported an extensively drug-resistant Klebsiella michiganensis strain recovered from a child with a respiratory infection in Southern China, which carries three mega plasmids, with pB106-1 firstly identified to carry an array of resistance genes in a distinctive order, and pB106-IMP identified as a novel IncN-IncX3 cointegrate plasmid harboring two resistance genes blaIMP-4 and blaSHV-12.


Introduction
Klebsiella michiganensis was first identified in Michigan from a toothbrush holder in 2012 and was initially identified as Klebsiella oxytopca due to their highly similar 16S rRNA sequences (approximately 99% identity) until it was then recognized as a new species by DNA-DNA hybridization.Since then, K. michiganensis has been recognized as a potential emerging human pathogen responsible for nosocomial infections.Commonly isolated from blood and rectal swabs, this bacterium often infects immune compromised patients, with associated infection cases being increasingly reported in recent years.In addition, the antimicrobial resistance in K. michiganensis has become a major concern in the therapeutic treatment of relevant infections, especially for K. michiganensis exhibiting multidrug resistance (MDR) [1][2][3].Also, the carriage of various antibiotic resistance genes has been found to be associated with the multidrug resistance phenotype [1][2][3].In particular, over the past decades, carbapenemase-producing Klebsiella spp.and their carriage of various carbapenem resistance genes have been frequently identified, which has become a leading challenge to clinicians [2,4].For K. michiganensis, strains harboring two to three carbapenem resistance genes have been previously reported in South Africa and China [5,6].
Carbapenemase is able to hydrolyze most of the β-lactam antibiotics [7].As an Ambler class B metallo-β-lactamase, imipenemase (IMP) confers resistance to all the β-lactam antibiotics except for the monocyclic β-lactam antibiotic and is not susceptible to β-lactamases inhibitors [8].Up to now, a total of 62 IMP variants have been identified (https://bitbucket.org/genomicepidemiology/resfinder_db/src/master/beta-lactam.fsa; accessed on 20 October 2020).Among them, IMP-4 is the prevalent imipenemase in China [9], which was first detected in Acinetobacter spp. in Hong Kong in 2001 and ever since has been spread worldwide [10].The carriage of the bla IMP-4 gene provides the host strain with high minimum inhibitory concentration (MIC) values for almost all β-lactams.Wide dissemination of IMP is often due to various transferable plasmids [11][12][13][14], and IMP genes are frequently reported to be carried in combination with other resistance genes.As a consequence, the coexistence of IMP and other resistance genes in a plasmid confers higher resistance to antibiotics, raising a significant challenge for clinical treatment [15].
Plasmids are DNA genetic elements in bacteria that can replicate themselves.Typical plasmids are composed of a backbone and several variable regions, which usually carries different species and different numbers of mobilizable elements including transposons, integrons and insertion sequences (ISs).Moreover, these mobilizable elements usually carry antibiotic resistance genes; therefore, horizontal gene transfer mediated by plasmids makes a significant contribution to the dissemination of antibiotic resistance genes between different bacterial species [16].
In this study, an extensively drug-resistant K. michiganensis strain recovered from a child with a respiratory infection from a teaching hospital in Southern China was subjected to genomic sequencing and further bioinformative analysis.For the first time, we have identified three mega plasmids, with pB106-1 first identified to carry an array of resistance genes in a distinctive order which provides important evidence on the evolution of antimicrobial resistance in K. michiganensis

Isolation of the K. michiganensis KMIB106 Strain
The K. michiganensis strain KMIB106 was isolated from a 7-year-old girl in the First Affiliated Hospital of Guangzhou Medical University (FAHGMU) in Southern China.The child was hospitalized with a diagnosis of severe pneumonia in April 2019, who then suffered from fever with a maximum temperature of 41 • C and paroxysmal continuous cough.During the treatment, 3 different types of bacterial strains were isolated from independent sputum samples, including Acinetobacter baumannii, K. michiganensis and Elizabethkingia meningoseptica.

Bacterial Identification and Antimicrobial Susceptibility Testing
For all bacterial strains, biochemical identification to species level and the susceptibility test of 20 common antibiotics were performed using the Vitek2 TM Automated Susceptibility System [17], including ceftazidime, cefepime, cefuroxime sodium, ceftriaxone, cefuroxime axetil, cefoxitin, piperacillin/tazobactam, amoxicillin/clavulanic acid, ticacillin/clavulanic acid, compound sulfamethoxazole, minocycline, doxycycline, imipenem, ertapenem, colistin, aztreonam, amikacin, tobramycin, ciprofloxacin and levofloxacin.The MICs were interpreted according to the guidelines recommended by the Clinical and Laboratory Standards Institute (CLSI).

Genome Sequencing and De Novo Assembly and Correction
Bacterial genome sequencing was performed by PacBio RS III systems and Illumina Nextseq 500.For sequencing by PacBio RS III, genomic DNA was extracted using the CTAB method, followed by a concentration and quality test.Then, genomic DNA was fragmented with G-tubes and end-repaired to prepare SMRTbell DNA template libraries (with fragment size of >10 Kb selected by the Blue Pippin system).After the average fragment size was estimated on a Bioanalyzer 2100, SMRT sequencing was performed on the Pacific Biosciences Sequel.For sequencing by Illumina Nextseq 500, qualified genomic DNA was sonicated randomly, then end-repaired, A-tailed, and adaptor-ligated using NEBNext ® MLtra™ DNA Library Prep Kit for Illumina (NEB, TX, USA).DNA fragments with a length of 300-400 bp were enriched by PCR.PCR products were purified using the AMPure XP system (Beckman Coulter, Brea, CA, USA).The libraries were analyzed for size distribution by a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and quantified using real-time PCR.Genome sequencing was performed on the Illumina Novaseq 6000 sequencer using the pair-end technology.Continuous long reads obtained from SMRT sequencing runs were used for further de novo assembly using Falcon (version 0.3.0)[18].Continuous long reads obtained from ONT sequencing were used for de novo assembly using Flye (version 2.8.1-b1676) [19].Raw data from the Illumina platform were filtered using FASTP (version 0.20.0)[20] by the following standards: (1) removing reads with ≥10% unidentified nucleotides (N); (2) removing reads with ≥50% bases having Phred quality scores ≤ 20; (3) removing reads aligned to the barcode adapter.After filtering, the clean reads were used to correct the genome sequences to improve the quality of the assembly and determine the final genome sequences using Pilon (version 1.23) [21].

Genome Annotation and Analysis
The ORFs (open reading frames) were predicted using the NCBI prokaryotic genome annotation pipeline.For noncoding RNAs, rRNAs, tRNAs and sRNAs, predictions were carried out using rRNAmmer (version 1.2), tRNAscan (version 1.3.1)and cmscan (version 1.1.2),respectively.Gene islands and transposons were predicted using IslandPath-DIMOB (version 1.0.0) and Transposon PSI (version 20100822).Interspersed repeat elements were identified by Repeat Masker (version 4.0.5).The acquired genome sequences were further annotated with RAST and prokka tools [22,23].The replicon types of plasmids were identified with Plasmid Finder-1.3[24] and the resistance genes of plasmids were also predicted with ResFinder [25].The genome of KMIB106 was searched for in CRISPR regions over the public databases using CRISPRfinder [26].

Genomic Analysis of Bacterial Chromosome
The average genome sequencing depth was more than 100X in coverage, including 1.2 G and 1.7 G data acquired from PacBio and Illumina platforms, respectively.The genome of the K. michiganensis strain KMIB106 was assembled into a single linear chromosome and three circular plasmids (Figure S1).The chromosome consisted of 5,907,184 bp with an overall G+C content of 55.48%.Among the 5667 predicted CDs from the chromosome, a total of 86 tRNA and 25 rRNA were identified, with 2 CRISPR-cas sequences further identified by the prokka and the CRISPRfinder server.

Correlation between Antimicrobial Resistance Phenotype and Genes Profile
The correlation between antimicrobial resistance phenotype and resistance genes profile was further analyzed in the K. michiganensis strain KMIB106.As shown in Table 2, the antimicrobial resistance phenotype was in accordance with the carriage of relevant resistance gene.For each antimicrobial agent group to which the K. michiganensis strain KMIB106 was resistant, the strain carried the relevant resistance gene.Remarkably, high MIC values were obtained for Beta-lactams and compound sulfamethoxazole, for which KMIB106 harbors 3 bla genes and sul1 with dfrA27, respectively.

Genomic Analysis of Plasmid pB106-2
Belonging to IncFII incompatibility groups, the size of plasmid pB106 bp, with the average G+C content at 51.74% and the total number of predict The plasmid pB106-2 exhibited similarity (94% coverage, 99% identity) wit plasmid (unnamed1, accession number: CP033823.1)from the Klebsiella Figure 1.The comparison between variable regions of plasmid pB106-1 and plasmid pKB18-31; genes are denoted by arrows, resistance genes are highlighted in red, other genes are shown in green and the gray area represents that the similarity between these sequences is more than 99%.

Genomic Analysis of Plasmid pB106-2
Belonging to IncFII incompatibility groups, the size of plasmid pB106-2 was 189,346 bp, with the average G+C content at 51.74% and the total number of predicted CDs at 228.The plasmid pB106-2 exhibited similarity (94% coverage, 99% identity) with an unnamed plasmid (unnamed1, accession number: CP033823.1)from the Klebsiella sp.FDAARGOS_511 strain.The plasmid pB106-2 contained two replicons, IncFII and IncFIB, but with none of the antimicrobial resistance gene.

Genomic Analysis of Plasmid pB106-IMP
The size of plasmid pB106-IMP was 85,326 bp, with the average G+C content at 48.80% and the total number of predicted CDs at 132.The backbone of plasmid pB106-IMP included sequences responsible for plasmid maintenance (stbC and stbA genes), antirestriction function (adrA, adrR and adrK genes), UV protection (mucA and mucB genes) and conjugative transfer (traM, trbA and trbK genes).
It is noteworthy that two different replication genes were found in the backbone regions of plasmid pB106-IMP, including the IncN-type replication gene repE and the IncX3-type replication gene repB.Remarkably, approximately 43.37% of the sequences were similar to a series of IncX3-type plasmids (identity > 99%, coverage 100%) and 51.92% of the sequences were similar to a series of IncN-type plasmids (identity 100%, coverage > 99%), with both regions accounting for more than 95% of the whole sequences of pB106-IMP.In addition, these two regions were linked by a composite transposon, and this composite transposon was bracketed by two identical IS26, with a ESBLs gene bla SHV-12 inserted.Another resistance gene bla IMP-4 was located on the IS-mediated transposition unit, organized as IS26-∆IntI1-bla IMP-4 -ItrA-mobC-IS6100.A 54-bp segment of the integrase gene was deleted and the IS26 was located at the 5'-end of In823 integrons.A similar structure was reported in the bla IMP-4 -carrying plasmid pIMP-HZ1 (the GenBank accession number: KU886034.1)originating from the K. pneumoniae strain in China [28,29].The sequence of bla IMP-4 was 100% identical to those frequently detected in the broad host range of IncN-type plasmids [28].The most common genetic environments of bla IMP-4 were complex class 1 integrons (In809 and In823).In809 is a genetic structure with bla IMP-4 as the core (bla IMP-4 -qacG2-aacA4-catB3-∆qacE) and IS26 nearby.In823 was first reported in plasmid pIMP-HZ1 from the K. pneumoniae strain [28,29], in which bla IMP-4 was captured as a cassette by the class 1 integrons.A type II intron, Kl.pn.I3, was located downstream of bla IMP-4 and a qnrS1 (quinolone resistance gene) region was located upstream of In823.However, in our study, in In823, the upstream of truncated IntI1 was IS26 and a genetic structure of bla SHV-12 (IS26-bla SHV-12 -ygbI-IS26) was found at the same variable region in plasmid pB106-IMP [30].As a consequence, we hypothesized that the formation of the variable region in plasmid pB106-IMP occurs due to the recombination event mediated by the transposition formed by two copies of IS26 in the transposon.

Discussion
In this study, the whole genomic sequences of the K. michiganensis strain KMIB106 were analyzed, including a chromosome and three mega plasmids.For the profile of resistance genes, only two resistance genes, oqxB and bla OXY-4-1 , were located on the chromosome.As previously found, bacteria carrying the resistance gene bla OXY-4-1 showed natural resistance to amino and carboxy penicillins.In comparison with the chromosome, plasmid pB106-1 and pB106-IMP were found to carry a large number of resistance genes which are associated with resistance to cephalosporins, β-Lactamase inhibitors, carbapenems, tetracycline antibiotics and compound sulfamethoxazole.Coincidentally, the K. michiganensis strain KMIB106 exhibited antimicrobial resistance to such antibiotics.Thus, the large proportion of the resistance genes that are likely relevant to the resistance phenotypes in KMIB106 were located on plasmids, which strongly suggested such mega plasmids with various resistance genes are the crucial factor responsible for the multidrug resistance of the host strain.Thus, horizontal gene transfer mediated by mobile genetic elements, such as plasmids, transposons and integrons, plays a major role in the dissemination of antibiotic resistance genes, as well as the ultimate adaptation of the host strain to the antibiotic pressure.
In general, each plasmid has one specific replicon gene and a plasmid might be driven away by other plasmids if they carry an incompatible replicon.However, recently, a large number of mega plasmids containing two or even more functional replicons have been reported.Importantly, the carriage of multiple replicons in one single plasmid may allow these plasmids to contain more than one incompatible replicon, and thus expand the host range and further facilitate the dissemination of these plasmids.Concerning bla IMP-4 and bla shv-12 , the plasmids containing bla IMP-4 often belong to several replicon types, including IncN, IncHI2, IncF, IncL/M and IncI1 [31][32][33], and the plasmid replicon types associated with the widespread dissemination of bla shv-12 include IncX3, IncA/C, IncF, IncHI2, IncI1α/γ, IncL/M, IncN, IncP, IncR, and IncK [34].In our study, the plasmid pB106-IMP can be divided into three regions, including the IncN section, the IncX3 section and the 4029 bp segment carried bla SHV-12 .The IncN section was similar to an IncN plasmid pIMP-HZ1, containing several conjugative transfer genes which implies the potential capability of horizontal transmission by conjugation for plasmid pB106-IMP.The IncX3 section of plasmid pB106-IMP was identical to an IncX3 plasmid p128379-NDM (the GenBank accession number: MF344560.1)which carries both bla NDM-1 and bla SHV-12 genes.In comparison, the 4029 bp bla SHV-12 segment in plasmid pB106-IMP linking the IncN and IncX3 section was likely derived from the plasmid p128379-NDM (100% coverage, 99.96% identity).Furthermore, there were three identical IS26 fragments in plasmid pB106-IMP which were located at the junction of the IncX3 section; the 4029 bp segment carried bla SHV-12 and IncN section, respectively.As a consequence, it could be inferred that the cointegrate plasmid pB106-IMP was formed by fusion of an IncX3 plasmid and an IncN plasmid, with the IS26 structure located between these two sections (Figure 2, Figure 3).Notably, this is the first report of an IncN and IncX3 cointegrate plasmid.As a novel IncN-IncX3 cointegrate plasmid, pB106-IMP is potentially capable of facilitating the dissemination of bla shv-12 and bla IMP-4 among Klebsiella spp.
ferred that the cointegrate plasmid pB106-IMP was formed by fusion of an IncX3 pla and an IncN plasmid, with the IS26 structure located between these two sections (F 2, Figure 3).Notably, this is the first report of an IncN and IncX3 cointegrate plasmi a novel IncN-IncX3 cointegrate plasmid, pB106-IMP is potentially capable of facilit the dissemination of blashv-12 and blaIMP-4 among Klebsiella spp.segment carried blaSHV-12 and IncN section, respectively.As a consequence, it could b ferred that the cointegrate plasmid pB106-IMP was formed by fusion of an IncX3 plas and an IncN plasmid, with the IS26 structure located between these two sections (Fi 2, Figure 3).Notably, this is the first report of an IncN and IncX3 cointegrate plasmid a novel IncN-IncX3 cointegrate plasmid, pB106-IMP is potentially capable of facilita the dissemination of blashv-12 and blaIMP-4 among Klebsiella spp.ISs are simple autonomous mobile elements that mediate recombination events within genomes and further cause structural rearrangements.For a genetic structure composed of terminal inverted repeats and one ORF-encoded transposase, the terminal inverted repeats function as the specific recognition sites for the transposase, and the transposase further conducts the strand cleavage and transfer reactions.Various ISs are reported to play critical roles in the formation of these cointegrate plasmids [35].IS26, as an important member of the IS6 family, contains an 820 bp nucleotide sequence, with two inverted repeats and only one gene tnp26 encoding 234 amino acids.IS26 has been reported to play an important role in genetic mobility in plasmids and chromosomes among Enterobacteriaceae, which reorganize plasmids by replicative transposition or targeted conservative [36].

Conclusions
This study, for the first time, described a novel IncN-IncX3 cointegrate plasmid pB106-IMP.It contained two replicons and two carbapenem genes bla IMP-4 and bla SHV-12 , giving its nature host KMIB106 carbapenem-resistance.Our work first expanded the diversity of the plasmids incompatibility group in Enterobacterales.Revealing the mechanisms underlying the formation of this novel cointegrate plasmid will help to find new ways to inhibit dissemination of resistance genes through this type of plasmid.

Figure 2 .
Figure 2. Whole sequence of pB106-IMP and comparison of p128379-NDM, pIMP-HZ1.Pla pB106-IMP was used as a reference to compare with the other two plasmids.The black cycl sents the GC content of pB106-IMP.The outer two rings comprise the coding sequenc pB106-IMP and key features are present in different colors: resistance genes are in red, rep genes are in purple, other genes are in grey and mobile elements are in green.

Figure 2 .
Figure 2. Whole sequence of pB106-IMP and comparison of p128379-NDM, pIMP-HZ1.Plasmid pB106-IMP was used as a reference to compare with the other two plasmids.The black cycle presents the GC content of pB106-IMP.The outer two rings comprise the coding sequences of pB106-IMP and key features are present in different colors: resistance genes are in red, replicon genes are in purple, other genes are in grey and mobile elements are in green.

Figure 2 .
Figure 2. Whole sequence of pB106-IMP and comparison of p128379-NDM, pIMP-HZ1.Pla pB106-IMP was used as a reference to compare with the other two plasmids.The black cycle sents the GC content of pB106-IMP.The outer two rings comprise the coding sequenc pB106-IMP and key features are present in different colors: resistance genes are in red, rep genes are in purple, other genes are in grey and mobile elements are in green.

Figure 3 .
Figure 3. Proposed fusion mechanism of the cointegrate plasmid pB106-IMP mediated by IS26.IS26s are shown as green squares (the white arrow in green square indicates the orientation of IS26), resistance genes are shown as red arrows and other genes are shown as grey arrows, pink circle represents the IncN plasmid and pink circle represents the IncX3 plasmid.
"/" indicates that the interpretive standard does not exist in Clinical and Laboratory Standards Institute.R, resistant; S, susceptible; MIC, minimum inhibitory concentration.

Table 2 .
Resistant determinants identified by genomic for KMIB106.