The comprehensive whole-genome analysis
The complete genome sequence of K. pneumoniae KPX comprises a circular chromosome (5,468,925 bp) and four plasmids (179,972 bp, 141,377 bp, 85,181 bp, and 20,247 bp) which annotated 5984 protein coding genes and identified 85 tRNA genes and 25 rRNA operons. KPX harbors multiple antimicrobial resistance determinants including aadA2, rmtB, blaCTX-M-65, blaKPC-2, blaTEM-1, blaSHV-64, blaSHV-11, qnrS1, carA, sul1, sul2, and catII, which confer resistance to aminoglycosides, β-lactams, fluoroquinolones, macrolides, sulphonamides and phenicols. The quinolone resistance-determining region (QRDR) mutations in GyrA (S83I and D87G) and ParC (S80I) were detected. The overexpression of efflux pump genes (i.e., acrA, acrB, marA, soxS, and acrAB) were determined by qRT-PCR, which was associated with tigecycline resistance in K. pneumonia. The genes, intI1, aacA4, cmlA1, qacEΔ1, tnpA, and blaCTX-M-19 were clustered in integron Class Ⅰ. The segment between the two IS26 elements was carried with aadA and sul1 genes. The virulence-related factors, aerobactin siderophore receptor and yersiniabactin encoding genes iutA and irp1/2 and YbtU/Q/T, were present. K. pneumoniae KPX was identified as sequence type (ST11) by MLST. By setting a minimum sequence homology of 95% and a minimum length of 60% in PlasmidFinder 2.0 databases, we also identified three plasmids belonging to incompatibility (Inc) group F [IncHI1B, IncFII/IncR] and ColRNAI.
The phylogenetic relationship between K. pneumoniae KPX and close ST11 K. pneumoniae strains was assessed by cgSNPs and cgMLST analysis. The analysis of cgSNPs showed that the phylogenetic tree was grouped into the same clade according to geographically related isolates (Figure 1). The most of these isolates were found from Sichuan and Hangzhou. Our results showed that SCKP020029 (NCBI Accession number: CP029384), another ST11 strain collected from a human sample in Sichuan is the most closely related strain to K. pneumoniae KPX (Figure 2).
Among the plasmids of K. pneumoniae KPX, pKPX-A carried with a set of virulence genes including iucBCD, iutA, rmpA, and rmpA2 on plasmid (179,972 bp), which belonged to an IncFIB/IncHI1B plasmid. Multiple plasmid comparisons by BRIG showed that pKPX-A plasmid was similar with two virulence plasmids, i.e. pVir_020079 (99.99% identity) and pKPN-QL24 (99.01% identity) (Figure 3). pKPX-B, the IncFII/ IncR plasmid (141,377 bp), carried the carbapenem resistance gene blaKPC-2 and shared 99.88% similarity with plasmid pKPC2_020079 from the KPC-2-producing K. pneumoniae strain SCKP020079 isolated in Sichuan, China. The resistance gene cassettes on plasmid pKPX-B was organized to IS26-rmtB- blaTEM-1-IS26, IS26-blaCTX-M-65-IS26-Tn3, and IS26-blaSHV-64-blaKPC-2-IS26. In addition, pKPX-C, IncFII-type plasmid (85,181 bp), harbored the antimicrobial resistance genes, such as sul2, qnrS1, catⅡ, and tet(R/G), which confer resistance to sulphonamide, fluoroquinolone, chloramphenicol, tetracycline, respectively.
The characteristics of drug-resistant K. pneumoniae infection subjects
Fifty strains were isolated from patients aged from 47 to 98 years old. The mean age of the patients was 78.5 ± 10.2 years, and the male to female ratio of the patients was 2.1:1. The distribution of age were shown in Table S1. The isolates were mainly collected from ICU/emergency ICU and neurosurgery department, accounting for 46.0% and 24.9%, respectively. The strains were mainly isolated from sputum (39, 78.0%), and the others were part from blood, urine, endotracheal tube suction, bronchoalveolar samples and so on.
Antimicrobial susceptibility patterns of K. pneumonia
Fifty K. pneumoniae isolates were tested for its susceptibility against 18 types of antibiotics. The overall susceptibility, intermediate and resistance were determined and the results were shown in Figure 4. Most K. pneumoniae strains showed a high percentage of resistance against Piperacillin (100%), Amoxicillin acid(100%), Ampicillin/ Sulbactam(100%), Piperacillin /Tazobactam(97.96%), cefoxitin(100%), cefoperazone/sulbactam(100%), Cefuroxime(100%), Ceftriaxone(100%), Cefotaxime(100%), Cefepime(97.87%), Ceftazidime(100%), Meropenem(100%), Imipenem(97.87%), Gentamicin(81.63%), Levofloxacin(100%), Ciprofloxacin(100%), Aztreonam(100%), but Trimethoprim/Sulphamethoxazole(47.92%). Fifty isolates were phenotypically confirmed extended-spectrum beta-lactamases ESBLs producers.
MLST analyses revealed 4 different sequence types including ST1 (n =1), ST11 (n = 47), ST15 (n = 1), and ST258 (n =1), indicating the genetic consistency of all these isolates. ST11 was the dominant clone in our study, which has been demonstrated as a predominant clone of KPC-producing K.pneumoniae in China . ST11 is a single-locus variant (tonB) of ST258. ST258 was detected in one isolate, which was reported as a dominant molecular epidemiology clone in the USA .
Figure 5 showed the representative PFGE results of the multidrug resistant isolates. Multiple DNA fragments were observed in each isolate, which had more than ten bands. The results of genetic correlation were presented as a dendrogram by SPSS software. The results revealed that 50 isolates had DNA diversity with multivariate clones. Fifty strains were mainly classified into three clones. That is, clone A had 12 isolates (24%), clone B had 9, and clone C had 7, respectively. The others had independent resource. Clonal characteristics and distributions were shown in Figure 6. Clone A, B and C strains mainly existed in ICU and EICU department, which indicated no dissemination occurred among inpatient wards. Although new DNA fingerprinting technique MLST as alternatives are widely used, the discriminatory abilities of PFGE are more robust than that of MLST.
PCR-based drug-resistant genes and virulent genes detection
According to the results of KPX strain DNA sequencing, we selected drug resistant genes- KPC-2, SHV-11, TEM-1, CTX-M, oqxB1, qnrS, sul2, sul1, int1, and virulent genes- iutA, iucABCD, rmpA2 to detect. Considering the prevalence of NDM1gene, NDM1gene has also been included. The existence of these genes were shown in Table 2. In multidrug resistant strains, 98% (49/50) isolates were found to be positive for the presence of KPC-2. All three ESBL genes (SHV-11, TEM-1 and CTX-M), as Amber class A of β-lactamases  are encoding β-lactamase enzymes which is the primary resistance mechanism in Gram-negative bacteria resulted in β-lactams degradation . All these isolates had SHV-11 out of which 49 (98.0%) isolates. 38 (76.0%) isolates carried TEM-1 gene and 38 (76.0%) of them carried CTX-M gene. 30 of the isolates carried all the three genes together. None of the isolates were PCR-positive for NDM. 66% was for Oqxb1, encoding multidrug efflux pump, which is one of the mechanisms of quinolone resistance. oqxAB genes have been prevalent among Gram-negative bacteria, especially in Enterobacteriaceae. oqxAB genes often confer cross-resistance or reduce susceptibility to multiple agents, such as chloramphenicol, trimethoprim, ciprofloxacin, enrofloxacin, norfloxacin, and tigecycline [20,21]. 70% was for qnrS, mutant in gene results in quinolone resistance. 42.0% was for class 1 integron integrase (Int1); 82.0% and 96.0% for sul1 and sul2, which is related to resistance to sulfonamide. Of antimicrobial susceptibility patterns the percentage of resistance against Trimethoprim/Sulphamethoxazole was 47.92%, which was inconsistent with sul1 and sul2 carrying rate. This can be attributed to a decline in the use of these antibiotics. But the results need to be validated in vivo. The sul1 gene is predominantly linked with class 1 integrons . Of the 41 sul1 positive isolates, 15 (36.6%) carried Int1. 88% was for iutA and 10% for iucABCD, iucABCD-iutA, encoding aerobactin, iron acquisition systems . Research has demonstrated that certain virulence traits are associated with antibiotic-resistant phenotypes . For example, carriage of the iutA gene has previously been associated with antibiotic resistance in E. coli of extraintestinal origin  and is also common among strains producing ESBLs of the CTX-M types . However, in this study we did not find this correlation. 100% was for rmpA2, capsule up-regulation genes, which are those most associated with invasive infection .
In conclusion, this study presented the hybrid genome assembly and annotation of an extensively-drug resistant K. pneumonia strain, analyzed the molecular types and antimicrobial resistance and virulence profiles of clinical isolates in Jiangsu province, China. KPC-producing K. pneumoniae isolates is widespread in Nanjing, Jiangsu. Although NDM-producing K. pneumoniae isolates are rare, nationwide surveillance is absolutely necessary to the development of strategies for prevention, diagnosis and treatment of K. pneumoniae infections.