Method for detecting pan-resistant K. pneumoniae, method for screening antibiotics, and recording media / database.

Abstract

This method includes a step of performing a whole genome analysis of the bacteria contained in the sample; and a step of comparing the base sequence of the obtained whole genome with the base sequence of SEQ ID NO: 1 or 2, and determining whether the bacteria in the sample are pan-resistant K. pneumoniae based on the degree of match.

Description

Technical Field

[0001] The present invention relates to a method for detecting pan-resistant K. pneumoniae, a method for screening antibacterial drugs, and a recording medium / database.

Background Art

[0002] In recent years, it has been reported that multidrug-resistant (MDR) bacteria such as carbapenemase- and extended-spectrum β-lactamase (ESBL)-producing bacteria are very prevalent in specific regions of the world (Non-Patent Documents 1 and 2). In particular, Gram-negative bacteria that produce Klebsiella pneumoniae carbapenem (KPC) have become a major problem worldwide (Non-Patent Documents 3 and 4), and travelers from endemic areas are at risk of infection with MDR bacteria. Infections with carbapenem-resistant enterobacteria are clinically serious problems because they are difficult to treat with conventional antibacterial agents.

[0003] In Japan, it has been reported that less than 1% of hospital-associated enterobacterial infections are caused by carbapenem-resistant bacteria (Non-Patent Document 6). Usually, resistance to carbapenem is mediated by imipenemase (IMP)-type metallo-β-lactamase or overexpression of AmpC cephalosporinase in combination with porin mutations. Carbapenemases including KPC, New Delhi metallo-β-lactamase (NDM), and oxacillinase-48 (OXA-48) are becoming common, but their detection remains sporadic (Non-Patent Documents 4 and 7).

[0004] In Asia, KPC isolates are limited to East Asia including Southeast Asia (Non-Patent Documents 3 and 7). The first case of KPC-producing Klebsiella pneumoniae reported in Japan (in 2014) was isolated from a patient who had originally been hospitalized in Brazil (Non-Patent Document 8). KPC-producing Klebsiella pneumoniae is often MDR and poses serious problems in terms of clinical treatment and infection control.

[0005] Furthermore, pathogenic bacteria known as ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter sp.) often acquire drug resistance. Depending on the number of drugs to which the bacteria develop resistance, they are classified as MDR, broad drug resistance (XDR), and ultimately pandrug-resistant (PDR). Pathogens that have acquired pandrug-resistant (PDR) are known to be unaffected by any available antimicrobial agents (Non-patent Literature 9, 10). However, since the whole genome sequences of PDR-acquired pathogens are rarely available, there are currently no reports on the genetic structure and systems of drug resistance. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Jacoby GA, Munoz-Price LS. The New β-Lactamases. N. Engl. J. Med. 2005; 352:380-391. [Non-Patent Document 2] Wellington EM, Boxall AB, Cross P, et al. The role of the natural environment in the emergence of antibiotic resistance in Gram-negative bacteria. Lancet Infect. Dis. 2013;13:155-165. [Non-Patent Document 3] Munoz-Price LS, Poirel L, Bonomo RA, et al. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect. Dis. 2013; 13:785-796. [Non-Patent Document 4] Nordmann P, Poirel L. The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae worldwide. Clin. Microbiol. Infect. 2014; 20:821-830. [Non-Patent Document 5] van der Bij AK, Pitout JDD. The role of international travel in the worldwide spread of multiresistant Enterobacteriaceae. J. Antimicrob. Chemother. 2012; 67:2090-2100. [Non-Patent Document 6] Infectious Disease Surveillance Center NI of ID. Carbapenem-resistant Enterobacteriaceae Infection, Japan. Infect. Agents Surveill. Rep. 2014; 35:281. [Non-Patent Document 7] Lee C-R, Lee JH, Park KS, Kim YB, Jeong BC, Lee SH. Global Dissemination of Carbapenemase-Producing Klebsiella pneumoniae: Epidemiology, Genetic Context, Treatment Options, and Detection Methods. Front. Microbiol. 2016; 7:895. [Non-Patent Document 8] Saito R, Takahashi R, Sawabe E, et al. First report of KPC-2 Carbapenemase-producing Klebsiella pneumoniae in Japan. Antimicrob. Agents Chemother. 2014; 58:2961-3. [Non-Patent Document 9] Falagas ME, Bliziotis IA. Pandrug-resistant Gram-negative bacteria: the dawn of the postantibiotic era? Int. J. Antimicrob. Agents 2007; 29:630-636. [Non-Patent Document 10] Magiorakos A-P, Srinivasan A, Carey RB, et al. Multidrug-resistant, extensively drugresistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 2012; 18:268-281. [Non-Patent Document 11] Jolley KA, Maiden MCJ. BIGSdb: Scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics 2010; 11:595.

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Summary of the Invention

Problems to be Solved by the Invention

[0007] As one of three different carbapenem-resistant Gram-negative bacteria from patients transferred from a hospital in Jakarta, Indonesia, the inventors obtained a novel PDR K. pneumoniae ST11 isolate and performed whole-genome analysis of this strain.

[0008] The present invention has been made in view of the above circumstances, and an object thereof is to provide a novel PDR K. pneumoniae, a method for detecting pan-resistant K. pneumoniae, a method for screening antibacterial drugs, and a recording medium / database using the base sequence information obtained by this whole-genome analysis.

Means for Solving the Problems

[0009] The present invention is a method for detecting pan-resistant K. pneumoniae, comprising: a step of performing whole-genome analysis of bacteria contained in a sample; and a step of comparing the base sequence contained in the obtained whole-genome with the base sequence of SEQ ID NO: 1 or 2, and determining whether the bacteria in the sample are pan-resistant K. pneumoniae based on the coincidence rate and is characterized by including the above.

[0010] The antimicrobial screening method is characterized by including a step of supplying a sample containing candidate antimicrobial substances to pan-resistant K. pneumoniae that has chromosomal DNA consisting of the base sequence of SEQ ID NO: 1 and plasmid DNA consisting of the base sequence of SEQ ID NO: 2.

[0011] The storage medium or database of the present invention is characterized by storing the base sequence information of sequence number 1 or 2. [Effects of the Invention]

[0012] The method for detecting pan-resistant K. pneumoniae according to the present invention can accurately determine whether or not the bacteria in a sample are pan-resistant K. pneumoniae. The antimicrobial screening method according to the present invention can reliably screen for antimicrobial agents that are effective not only against pan-resistant K. pneumoniae but also against other bacteria. The storage medium or database according to the present invention enables the use of various analyses, research, and commercial applications based on the nucleotide sequence information of pan-resistant K. pneumoniae. [Brief explanation of the drawing]

[0013] [Figure 1] This figure shows a phylogenetic tree of the K. pneumoniae chromosome. It is a phylogenetic tree of the complete K. pneumoniae chromosome sequence constructed from the entire genome, and single nucleotide polymorphisms are shown using PathoBacTyper. [Figure 2]This figure shows the phylogenetic analysis of K. pneumoniae ST11 isolates. (a) A phylogenetic tree of 48 K. pneumoniae ST11 isolates constructed by whole-genome single nucleotide polymorphism analysis using PathoBacTyper is shown. Black circles indicate strains reported to carry pathogenic plasmids. (b) A figure showing a comparative analysis of plasmids carried by K. pneumoniae ST11 isolates. Plasmid sequences were compared using Easyfig. The top, middle, and bottom sequences represent pKP1034, p69-2, and pTK1401, respectively. [Figure 3] This figure shows a comparative genomic analysis of K. pneumoniae ST11 isolates KP69 and TK1401. The chromosome sequences of KP69 and TK1401 were compared using the BLAST Ring Image Generator (BRIG). [Figure 4] This figure shows a comparative genomic analysis of K. pneumoniae ST11 isolates KP69 and TK1401. The plasmid sequences of KP69 and TK1401 were compared using BRIG. [Figure 5] This figure shows a comparative analysis of the genomes of K. pneumoniae ST11 isolates. (a) shows a comparative analysis of the chromosomes of K. pneumoniae ST11 isolates JM45, HS11286, KP69, and TK1401 (performed using progressiveMauve). The locations of the prophage and ltrA (group II intron reverse transcriptase) genes are indicated by arrows. (b) This figure shows a comparative analysis of plasmids contained in K. pneumoniae ST11 isolates. Plasmid sequences were compared using Easyfig. The top, middle, and bottom sequences represent pKP1034, pTK1401, and pKSH203-KPC, respectively. [Figure 6] This figure shows the phylogenetic tree of the complete plasmid sequence of K. pneumoniae. Single nucleotide polymorphisms are shown using parsnp. [Modes for carrying out the invention]

[0014] The inventors performed whole-genome sequencing on K. pneumoniae isolated from a Japanese patient with a history of treatment in Indonesia, and determined the base sequence of 5.5 Mb of chromosomal DNA (sequence listing: SEQ ID NO: 1) and the base sequence of 128 kb of plasmid DNA (sequence listing: SEQ ID NO: 2).

[0015] Furthermore, this K. pneumoniae possesses 16 drug resistance genes for 9 classes of antibiotics, and has been confirmed to be pan-resistant (PDR), meaning that commercially available antibiotics are ineffective against it.

[0016] One embodiment of the present invention's method for detecting pan-resistant K. pneumoniae will be described.

[0017] The present invention provides a method for detecting pan-resistant K. pneumoniae, comprising the steps of (1) performing whole genome analysis of the bacteria contained in the sample, and (2) A step of comparing the base sequence contained in the whole genome obtained in step (1) with the base sequence of Sequence ID No. 1 or 2, and determining whether or not the bacteria in the sample is pan-resistant K. pneumoniae based on the agreement rate. Includes.

[0018] In step (1), for example, genomic DNA is isolated from a sample taken from a patient or the environment using a known method, and the bacterial genome is analyzed using a known method (e.g., Illumina's MiSeq, Genome Analyzer series, HiSeq series, Lifetechnologies' ion proton, ion PGM, SoLid series, Roche's GS series, PacBio's PacBio RS series, etc.). This provides the base sequence information of the entire genome of the bacteria contained in the sample.

[0019] In step (2), the base sequence of the whole genome obtained in step (1) is compared with the base sequence of sequence number 1 or 2, and it is determined whether or not the bacteria in the sample are pan-resistant K. pneumoniae based on the agreement rate.

[0020] The whole genome sequence information entered into the computer can be processed using commercially available software to compare it with the sequence of Sequence ID No. 1 or 2 (including calculating the agreement rate). For example, if the sequence agreement rate is 99.9% or higher, it can be determined to be pan-resistant K. pneumoniae. In addition, when making this determination, the inventors can focus on the 16 drug resistance genes that pan-resistant K. pneumoniae possesses.

[0021] Furthermore, the present invention also provides a storage medium or database in which the nucleotide sequence information of Sequence ID No. 1 or 2 is stored. The storage medium is not particularly limited, but examples include magnetic storage media such as floppy disks, hard disk storage media and magnetic chips; optical storage media such as CD-ROMs; and electrical storage media such as RAM and ROMs. The database can also be exemplified in the form of a computer-based system having the nucleotide sequence information of Sequence ID No. 1 or 2 stored in a data storage means.

[0022] Next, the antimicrobial screening method of the present invention will be described.

[0023] The present invention provides a method for screening antimicrobial agents, which includes the step of supplying a sample containing a candidate antimicrobial agent to a pan-resistant K. pneumoniae having chromosomal DNA consisting of the base sequence of SEQ ID NO: 1 and plasmid DNA consisting of the base sequence of SEQ ID NO: 2.

[0024] The pan-resistant K. pneumoniae discovered by the present inventors possesses chromosomal DNA consisting of the nucleotide sequence of Sequence ID No. 1 and plasmid DNA consisting of the nucleotide sequence of Sequence ID No. 2, and contains 16 drug resistance genes to 9 classes of antibacterial agents, thus rendering commercially available antibacterial agents ineffective. Therefore, if a sample containing a candidate antibacterial agent expected to be effective against pan-resistant K. pneumoniae is given to pan-resistant K. pneumoniae, and a decrease in the number of surviving pan-resistant K. pneumoniae is confirmed, for example, the antibacterial agent can be evaluated as effective not only against pan-resistant K. pneumoniae but also against other bacteria. In this way, by utilizing the pan-resistant K. pneumoniae discovered by the present inventors, it is possible to screen for active ingredients of new antibacterial agents.

[0025] Furthermore, the nucleotide sequence information (SEQ ID NOs: 1, 2) or representative fragments obtained from the genome analysis of K. pneumoniae can be used by those skilled in the art for various analyses, studies, and other purposes.

[0026] For example, it is conceivable to use primers and probes discovered by the inventors to detect pan-resistant K. pneumoniae based on the nucleotide sequence information shown in Sequence IDs 1 and 2 in the design of these primers and probes. For instance, these can be used to establish methods for specifically detecting genomic DNA, such as PCR (Polymerase Chain Reaction) using primer sets, Southern hybridization using probes, and FISH (Fluorescent in situ hybridization).

[0027] The pan-resistant K. pneumoniae detection method, antimicrobial agent screening method, recording medium, and database of the present invention are not limited to the embodiments described above. [Examples]

[0028] The present invention will be described in more detail below with reference to examples, but the present invention is not limited in any way to these examples.

[0029] <1> Acquisition of isolated shares The K. pneumoniae isolate TK1401 was previously collected from a patient's stool samples, tracheal samples (including aspirates and sputum), and pharyngeal and wound swabs as part of a surveillance screening program for infection prevention. The patient was a 74-year-old Japanese male with a history of hospitalization in a hospital in Jakarta, Indonesia.

[0030] <2> Culture and isolation of bacterial strains Screening samples were cultured in CHROM Agar mSuperCARBA (Kanto Chemical Co., Ltd., Tokyo, Japan) and stored in glycerol at -80°C. Frozen stocks of isolates were streaked onto Lysogeny broth (LB) (Miller) agar (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated at 37°C. They were grown in LB (Miller) (Sigma-Aldrich, St Louis, MO, USA) at 37°C, and single colonies were cultured in liquid in Bio-shaker BR-40LF (Tytec Co., Ltd., Tokyo, Japan). Genomic DNA was then isolated using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany).

[0031] <3> Antimicrobial susceptibility testing The tests were performed using a MicroScan WalkAway device (Beckman Coulter, Brea, CA, USA). The minimum inhibitory concentration (MIC) of colistin was confirmed using the E-test (Biomérieux, Marci Étoile, France) and the broth microdilution method. Antimicrobial susceptibility was defined by the breakpoints described in the Clinical and Laboratory Standards Institute (CLSI) guidelines (M100-S24).

[0032] <4> Genome analysis Genomic DNA was isolated from K. pneumoniae TK1401 cells using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany). The whole genome sequence was determined by single-molecule real-time sequencing (SMRT) using PacBio RSII (Pacific Biosciences, Menlo Park, CA, USA). The sequence consisted of 210,166 reads and 12,144 bp N 50 A 1,896 Mb sequence consisting of read lengths was obtained. Hierarchical Genome Assembly Process version 3 was used to assemble the two circular contigs. The complete sequences of the chromosome and plasmid contained 5,488,304 bp with a 57.4% GC content and 128,638 bp with a 54.4% GC content, respectively (SEQ ID NOs: 1 and 2). Using the DDBJ Fast Annotation and Submission Tool beta, 173 protein-coding regions were identified in the plasmid and 5,156 protein-coding regions in the chromosome, confirming the presence of 9 copies of 5S, 8 copies of 16S and 23S rRNA, and 86 tRNAs.

[0033] Genome sequences were analyzed using the bacterial isolation genome sequence database (BIGSdb; http: / / bigsdb.pasteur.fr / klebsiella / ) (Non-Patent Literature 11), the comprehensive antibiotic resistance database (CARD; http: / / arpcard.mcmaster.ca) (Non-Patent Literature 12), and compared using the Phase Search Tool Enhanced Release (PHASTER; http: / / phaster.ca) (Non-Patent Literature 13), Easyfig v2.2.2 (Non-Patent Literature 14), BLAST Ring Image Generator (BRIG) (Non-Patent Literature 15), and progressiveMauve (Non-Patent Literature 16). Genome similarity was analyzed using NCBI BLASTN 2.10.0 (Non-Patent Literature 17). Phylogenetic analysis was performed using whole-genome single nucleotide polymorphism (wgSNP) analysis with PathoBacTyper (http: / / hast.nhri.org.tw / PathoBacTyper / ) and Harvest v1.1.2, and the phylogenetic tree was represented using FigTree v1.4.3. The genome sequence is registered in DDBJ (BioProject number PRJDB8036).

[0034] <5> result (Antibacterial agent susceptibility test) The results of the antimicrobial susceptibility test are shown in Table 1.

[0035] [Table 1]

[0036] The isolated strain TK1401 was unresponsive to penicillin, cephalosporins, carbapenems, monobactams, aminoglycosides, fluoroquinolones, polymyxins, tetracyclines, glycylcyclines, phenicol, trimethoprim-sulfamethoxazole, or fosfomycin. These results suggest that strain TK1401 possesses the PDR phenotype. This is believed to be the first reported case in Japan of a patient carrying PDR (pan-resistant) K. pneumoniae.

[0037] Genome sequencing of this K. pneumoniae isolate revealed a 129kb plasmid and a 5.5Mb chromosome belonging to genomic MLST (gMLST) ST11. The plasmid incompatibility group was the hybrid incompatibility group IncR-F33:A-:B-. This incompatibility plasmid is a non-transferable plasmid (Non-patent Literature 18). Furthermore, conjugation between K. pneumoniae isolate TK1401 and Escherichia coli JE53 was not confirmed.

[0038] (Genome analysis) The complete genome sequence of K. pneumoniae isolate TK1401 is shown as sequence number 1 for chromosomal DNA and sequence number 2 for plasmid DNA in the sequence listing.

[0039] The chromosome of K. pneumoniae isolate TK1401 was classified into single nucleotide polymorphisms (SNPs) ranging from 38 to 4,351 in the top 100 hits. It was a population of ST11 isolates identified in Asia (Figure 1). Figure 2(a) shows a phylogenetic tree constructed from wgSNPs comparing the genome sequence of K. pneumoniae ST11 strain TK1401 with that of other K. pneumoniae ST11 strains, primarily from China. Genomic capsular typing using BIGSdb wzc and wzi sequences revealed that K. pneumoniae ST11 isolates mainly form populations with K47 type (wzc47, wzi209), K64 type (wzc64, wzi64), and other capsule synthesis (cps) genes. The chromosomes of K. pneumoniae isolate TK1401 were most similar to KPC-2-producing K. pneumoniae isolate KP69 (GenBank accession number CP025456) isolated at Fudan University and Huashan Hospital of Shanghai Medical University in Shanghai, China (99% search range, 99% identity). Genomic analysis of KP69 using CARD revealed that KP69 is not a pan-resistant genotype (Table 2). Tigecycline and polymyxin resistance mutations were identified in the ramR and mgrB genes of isolate TK1401, but not in those of KP69.

[0040] [Table 2]

[0041] The genome sequence of K. pneumoniae ST11 strain TK1401 was compared with the genome sequences of K. pneumoniae ST11 strains (JM45, HS11286, and KP69) originating from China (Figure 1). For comparison, ST11 isolates JM45 (GenBank accession number CP006656) and HS11286 (GenBank accession number CP003200) were used in addition to KP69. Overall, the genome structure was conserved. Differences were found in SNPs, insertions and deletions of mobile genetic elements (prophages, integrative co-factors (ICE), transposons (Tn)), and insertion sequences.

[0042] HS11286 possesses two ICEs (ICEKpnHS11286-1 and ICEKpnHS11286-2) (Non-Patent Literature 19). ICEKpnHS11286-2 was conserved in JM45, HS11286, KP69, and TK1401. In contrast, ICEKpnHS11286-1, recently classified as ICEKp3, was conserved in HS11286, KP69, and TK1401 (Figure 1). These results suggest that TK1401 belongs to the ST11 strain that possesses both of these ICEs, which may contribute to the pathogenicity of the ST11 strain.

[0043] A comparison of the genomes of pan-resistant TK1401 and non-pan-resistant KP69 revealed gene deletions in TK1401 (Figures 3-5). The deletion region upstream of the ISKpn25 transposase contains genes encoding DinI, LtrA (group II intron reverse transcriptase), DNA adenine methylase, DNA replication endonuclease, and eight virtual proteins and three prophage-related proteins. KP69 and TK1401 have eight and seven ltrA genes, respectively.

[0044] Most proteins, including DNA adenine methylases and DNA replication endonucleases, are thought to be involved in prophage function. Since DinI regulates recombination, loss of DinI may upregulate recombination. One hypothetical protein, CYD98_21555, is similar to TraR, which belongs to the DskA_TraR superfamily. TraR is known as a comprehensive transcription regulator (Non-Patent Literature 21, 22). Furthermore, to find the IncR-F33:A-:B- plasmid containing seven resistance genes—fosA3, blaKPC-2, blaCTXM-65, blaSHV-12, blaTEM-1, rmtB, and catA2—we searched the nucleotide collection (nr / nt) using NCBI BLASTN 2.10.0. For the rmtB, blaKPC-2, blaSHV-12, fosA3, and catA2 sequences, we used three GenBank data (accession numbers KP893385, CP034324, and LR59607) as genetic markers. All three strains possessing each plasmid belonged to ST11. These data indicate that genetic markers (rmtB, blaKPC-2, blaSHV-12, fosA3, and catA2) are helpful in identifying multidrug-resistant IncR-F33:A-:B plasmids (particularly those containing seven resistance genes: fosA3, blaKPC-2, blaCTX-M-65, blaSHV-12, blaTEM-1, rmtB, and catA2K) and in understanding the evolution of K. pneumoniae ST11. Since the fosA3 gene has been detected in K. pneumoniae ST11 containing wzi209 (K47) and wzi64 (K67) type cps genes (Non-Patent Literature 18), it is suggested that the fosA3 plasmid may be evolving in K47 and K67 K. pneumoniae ST11. Using Easyfig, pKP1034, pTK1401, and pKSH203-KPC were compared (Figure 5(b)). This comparison confirmed that pKP1034-like plasmids and recombination rearrangements may be caused by transposition factors (IS and Tn). Plasmid pTK1401, identified from the TK1401 genome sequence, was phylogenetically analyzed in 41 of the top 97 BLAST hits of K. pneumoniae isolates (Figure 6). It was most similar to plasmid p69-2 from the KPC-2-producing K. pneumoniae isolate KP69 (GenBank accession number CP025458, 99% search range, 99% identity). pKP69-2 was very similar to IncR-F33:A-:B- plasmid pKP1034 (GenBank accession number KP893385) from the infectious KPC-2-producing K. pneumoniae isolate KP1034, which contains fosA3, blaKPC-2, blaCTX-M-65, blaSHV-12, and rmtB.

[0045] Next, we used Easyfig to compare the KPC-2 encoding plasmids pKP1034, p69-2, and pTK1401 (Figure 2(b)). This comparison suggests that the pKP1034-like plasmid may have been rearranged by recombination to form p69-2. Further recombination of the p69-2-like plasmid may have formed pTK1401.

[0046] The IncR-F33:A-:B- plasmid, containing fosA3, blaKPC-2, blaCTX-M-65, blaSHV-12, and rmtB, formed a population with pKP1034, pKP69-2, pKPGD4, pKPC-CR-HvKP4, and pTK1401. These plasmids were identified from K. pneumoniae ST11 possessing the KL47-type cps gene. pKPC-CRHvKP4 was isolated and sequenced from a highly virulent KPC-2-producing K. pneumoniae ST11 strain possessing the pathogenic plasmid pVir-CR-HvKP4 (Non-Patent Literature 23). In this example, pTK1401 was identified by genomic analysis using BIGSdb from pan-resistant K. pneumoniae ST11 possessing the KL47-type cps gene and lacking the pathogenic plasmid. Furthermore, the major pathogenicity genes were conserved in the KL47 ST11 isolate (Table 3).

[0047] [Table 3]

[0048] The K. pneumoniae isolate TK1401 is related to the highly pathogenic carbapenem-resistant K. pneumoniae (hvpk-5 GenBank accession number NJPJ00000000 and TVGHCRE225 GenBank accession number CP023722) recently identified in China and Taiwan, but lacks the pathogenic plasmid (Non-patent documents 23, 24).

[0049] Table 1 lists the resistance genes contained in the plasmids and chromosomes of the TK1401 isolate. Mutations in the quinolone resistance determinant regions of the chromosomal gyrA(D87G) and parC(S80I) genes are associated with resistance to fluoroquinolones (Non-Patent Literature 28). Mutations in the ramR(T162I) transcriptional repressor gene confer resistance to tigecycline (Non-Patent Literature 27). Overexpression of the AcrAB efflux pump may contribute to resistance to multiple drugs, including chloramphenicol, fluoroquinolones, tetracyclines, and glycylcyclines. Colistin resistance in TK1401 has been confirmed to be associated with a frameshift mutation in mgrB (FS_aa8). Furthermore, as shown in Table 1, this plasmid contains the carbapenemase gene blaKPC-2, the ESBL gene blaCTX-M-65, and the β-lactamase genes blaTEM-1B, blasHV-12, and blaSHV-11. These five β-lactamases are thought to provide resistance to all β-lactams listed in Table 1. In addition to β-lactamases, chromosomal genes encoding outer membrane porins (ompK35, ompK36, and ompK37), which are associated with membrane permeability and drug resistance, were detected. TK1401 has a frameshift mutation in ompK35 (FS_aa29), which differs from the ompK35 (FS_aa134) found in K. pneumoniae ST11 strain HS11286, which produces KPC (Non-Patent Literature 25). The ompK36 gene of TK1401 is identical to the ompK36 mutant (GenBank accession number JX310552) possessed by KPC-producing K. pneumoniae ST11 strain HS092187 isolated in China, and is associated with carbapenem resistance (Non-Patent Literature 26). The ompK37 gene of TK1401 is identical to that of HS11286 and NDM-producing K. pneumoniae ST11 isolate JIE2713 (GenBank accession number KC534871) (Non-Patent Literature 26). All ompK genes in TK1401 are conserved in KP69, suggesting that KP69 may be the ancestral strain. In addition, colistin resistance in pan-resistant K. pneumoniae has been found to be associated with frameshift mutations in mgrB(FS_aa8). While colistin-resistant and KPC-2-producing ST11 strains have been reported in Asia, the emergence of pan-resistant K. pneumoniae ST11 strains possessing both phenotypes poses a threat to both clinical treatment and infection control.

[0050] As described above, by utilizing the whole genome sequence of K. pneumoniae isolate TK1401 analyzed by the present inventors, it is possible to compare the base sequence of the whole genome of the bacteria contained in the sample with the base sequence of Sequence ID No. 1 or 2, and determine whether the bacteria in the sample are pan-resistant K. pneumoniae based on the agreement rate. Furthermore, by supplying a sample containing candidate antimicrobial agents to the pan-resistant K. pneumoniae discovered by the present inventors, it is possible to screen for effective antimicrobial agents.

[0051] Tables 4-7 show the strains and plasmids used in the phylogenetic analysis of K. pneumoniae. [Table 4] [Table 5] [Table 6] [Table 7]

Claims

[Claim 1] A method for detecting pan-resistant K. pneumoniae, The process of performing whole genome analysis of bacteria contained in the sample: and A step in which the nucleotide sequence contained in the obtained whole genome is compared with the nucleotide sequence of Sequence ID No. 1 or 2, and the degree of agreement is used to determine whether the bacteria in the sample are pan-resistant K. pneumoniae. A method for detecting pan-resistant K. pneumoniae, characterized by including the following:

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