JP2020115793A - Genotyping methods of klebsiella pneumoniae and related strains and primer sets therefor - Google Patents

Abstract

To provide methods for identifying Klebsiella pneumoniae and related strains suitable for actual industrial (medical site) use, and primer sets therefor.SOLUTION: Disclosed is a method for identifying Klebsiella pneumoniae and related strains, the method comprising: detecting in any order the presence or absence of: (1) at least one genomic islet-constituting ORF and (2) at least one ORF constituting hyper-variable ORF in genomic islands, on the chromosomal gene sequence of Klebsiella pneumoniae and related strains; and determining a strain with the combinations of the presence and absence of these ORFs.SELECTED DRAWING: None

Description

The present invention relates to a clone identification method and/or strain identification method for Klebsiella pneumoniae and closely related bacterial species, and a primer set used therefor.

Klebsiella pneumoniae is a pathogenic bacterium that has become a problem as one of the bacteria causing respiratory infections such as urinary tract infections and pneumonia. In particular, in recent years, strains showing drug resistance, such as substrate-specific extended β-lactamase (ESBL)-producing Klebsiella pneumoniae that has acquired third-generation cephalosporin resistance, and carbapenem-resistant Klebsiella pneumoniae, are increasing in number. Increasing number of cases of infection is regarded as a problem.
In medical institutions in Europe and the United States, the increase and spread of Klebsiella pneumoniae, which produces carbapenemase, which is a carbapenem-degrading enzyme, and belongs to Sequence Type 258 (ST258), is a serious problem. However, the characteristics of Klebsiella pneumoniae strains isolated in Japan are different from those in Europe and the United States, and the STs of the isolates are rarely biased toward a specific ST such as ST258, and are classified into a wide variety of STs. In addition, drug-resistant strains have not been as widespread as in Europe and the United States, and according to the Ministry of Health, Labor and Welfare's Nosocomial Infection Control Surveillance Business, the third-generation cephalosporosis among the Klebsiella pneumoniae isolated in major hospitals in Japan as of 2017. The proportion of phosphorus-resistant strains is 8.9%, and the proportion of carbapenem-resistant strains is 1% or less. In Japan, it is necessary to maintain this situation.

The classification of Klebsiella pneumoniae has been recently reviewed, and Klebsiella variicola and Klebsiella quasipneumoniae exist as closely related bacterial species. These two bacterial species are also included in the scope of application of the present invention.

When nosocomial infection is suspected, it is necessary to determine whether the strains isolated from different patients are genetically identical in order to identify the infection route. For this purpose, the characteristics of the genome possessed by the strain are detected to identify the strain, which is called typing.
As a method of typing Klebsiella pneumoniae, conventionally, pulse field gel electrophoresis has been widely used. The pulse field gel electrophoresis method is a method for determining the genotype of a strain as its electrophoretic pattern by utilizing the fact that there are polymorphisms in the positions and cleavage lengths of restriction enzyme cleavage sites that exist in the chromosomal DNA of bacteria. Although it takes more than 3 days to carry out, it is a standard method for identifying strains of Klebsiella pneumoniae because of its high reproducibility.

However, since the pulse field gel electrophoresis method needs to be determined by comparing complicated band patterns, it tends to have subjective factors. In addition, even in the case of strains that co-migrated at the same time, a slight difference may occur in the band pattern. In that case, a skilled person is required to judge whether or not the genotypes are the same. Therefore, it was very difficult to determine whether or not genotypes of multiple isolates were equivalent by a method of comparing the results of pulse field gel electrophoresis obtained at different times or in different laboratories. ..

On the other hand, Patent Document 1 discloses a genotyping method for Staphylococcus aureus and a primer set used therefor, and Patent Document 2 describes a genotyping method for Pseudomonas aeruginosa and a primer set used therefor. The genotyping method for Acinetobacter spp. and the primer set used therefor are shown in 3, Patent Reference 4 shows the genotyping method for Escherichia coli and the primer set used therefor, and Patent Reference 5 shows Clostridium difficile. Genotyping method of and the primer set used for this are described

JP, 2011-120528, A JP, 2013-146244, A JP, 2014-207895, A JP, 2016-49109, A JP, 2019-4811, A

Klebsiella pneumoniae is a Gram-negative bacterium belonging to the family Enterobacteriaceae, and is one of the common bacteria that constitutes the normal bacterial flora of the intestinal tract of healthy individuals. However, it is a pathogenic bacterium that not only causes urinary tract infections in healthy subjects but also often causes pneumonia in immunocompromised persons and elderly persons who have a reduced ability to protect against infection by bacteria. In addition, the number of reports of severe liver onset abscess and sepsis caused by this bacterium is increasing, and the clinical significance is high. In addition, multiple clones that have acquired drug resistance have emerged, and infection control in the medical field has become an issue. Therefore, it is important to identify the infection route at the time of outbreak of the outbreak and to grasp the scale of infection in order to implement countermeasures against drug-resistant bacteria and effective infection control. Is required. In cases where nosocomial infection is suspected, multilocus sequence typing (MLST) or pulse field gel (PFGE) electrophoresis is often used to confirm clonal transmission of bacteria. The MLST method requires determining the nucleotide sequence of at least 7 positions on the chromosome, which increases the cost. The pulse field gel electrophoresis method requires a long time to carry out because it requires analysis by a special electrophoresis apparatus after enzymatic treatment of chromosomal DNA. In addition, since it is necessary to compare complicated band patterns, dedicated software is required to compare the strains electrophoresed at different dates and times, which is difficult to carry out in the medical field.

In view of the above situation, the present invention develops a method for rapidly and easily identifying strains of Klebsiella pneumoniae and closely related strains, and more suitable for actual industrial (medical field) use, for identifying strains of Klebsiella pneumoniae. The aim is to provide a way for. Another object of the present invention is to provide a primer set used in the method for identifying the above strain.

MLST method, which is the standard method for Klebsiella pneumoniae typing, has low ability to discriminate strains, but in the case of closely related clones in which the MLST type (sequence type, ST type) of Klebsiella pneumoniae is the same when the whole genome data is compared and examined, The above diversity is shown to be low. Therefore, it is difficult to improve the strain discriminating ability by simply detecting the foreign gene island, and it is necessary to search for a hypervariable detection site in which the strains are greatly different. While paying attention to this point as well, the inventors of the present invention have made extensive studies to solve the above problems, compared and examined the nucleotide sequence data of a large number of Klebsiella pneumoniae chromosomes that are being accumulated in the database, and performed clinical isolation. As a result of investigating the ORF retention patterns in the strains, we succeeded in discovering the ORF effective for discrimination at the PFGE level and the strain level. Specifically, the present inventors determined whether or not (1) an ORF that constitutes a genomic islet on a chromosomal gene sequence and (2) a hyper variable ORF that exists in a genomic island on a chromosomal gene sequence is arbitrary. It was found that the genotyping of Klebsiella pneumoniae and related strains can be performed more effectively by sequentially detecting and performing genotyping by combining these ORFs.

That is, according to the present invention, on the chromosomal gene sequences of Klebsiella pneumoniae and closely related fungal species, (1) ORFs constituting a genomic islet and (2) ORFs constituting a hyper variable ORF existing in a genomic island. There is provided a method for identifying strains of Klebsiella pneumoniae and related strains, which comprises the steps of detecting the presence or absence of A. cerevisiae in any order and performing genotyping based on the combination of the presence or absence of these ORFs.

The ORFs listed here mean the open reading frame of a gene, and indicate a region of approximately 99 bp or more sandwiched between a start codon and a stop codon, and include those that are translated into proteins and those that are not translated.

In the method for identifying strains of Klebsiella pneumoniae and closely related bacterial strains of the present invention, the ORF constituting the genomic islet of (1) above is KPHS_23160, KPHS_25730, KPHS_14300, A7321_12080, KPHS_05620, A7321_12740, KPHS_27850, KPHS_19280, PMK1_19280, PMK1_19280, PMK1_19,280 ABY63_05860, KPNIH10_15035, PMK1_03738, KPHS_04610, KPHS_05440, KPHS_10130, KPHS_12340, KPHS_12360, KPHS_14360, KPHS_17140, KPHS_22680, KPHS_27840, KPHS_28160, KPHS_30110, KPHS_33080, KPHS_35440, KPHS_44950, KPHS_45270, BB751_16465, BB751_24340, KPNIH10_06180, KPNIH10_06335, KPNIH10_06350, KPNIH10_10880, KPNIH10_13265, A7321_01305, A7321_14925, KPHS_22950, KPHS_34550, KPHS_35720, KPHS_49450, KPHS_51010, A7321_00110, A7321_01475, especially A7321_03820, A7321_09350, A7321_09350, A7321_15655, KPNIH29_02070, KPNIH29_2970 Is an ORF of KPHS_23160, KPHS_25730, KPHS_14300, A7321_12080, KPHS_05620, A7321_12740, KPHS_27850, KPHS_19280, PMK1_04123, ABY63_05860, KPNIH10_15035 and 16 kinds of the 16K of ORFs present in 16 of the 16 kinds of the 16K of ORFs of 16 kinds of 16 in the above-mentioned 16 of KBYS_27850, KPNIH10_15035 and PMK1_03738. , Kpn2146_5469, KPHS_34600, KPHS_12700, KPHS_01250, KPHS_10080, KPHS_12370, KPHS_25140, KPHS_30440, KPHS_34840, KPHS_34850, K At least one selected from the group consisting of PHS_35110, KPHS_35580, N559_1651, Kpn2146_6120, KPNJ1_02432, KPNJ2_02710 and KPR0928_21110 (more preferably at least 3 kinds, particularly preferably AXK16_16575, Kpn2146_5469, KPHS_34600 and KPHS_34700 and KPHS_12700). preferable.

The method for identifying the strain of the present invention is as follows: SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ IDs 5 and 6, SEQ IDs 7 and 8, SEQ IDs 9 and 10, and SEQ IDs 11 and 12. , SEQ ID NOS: 13 and 14, SEQ ID NOS: 15 and 16, SEQ ID NOS: 17 and 18, SEQ ID NOS: 19 and 20, SEQ ID NOS: 21 and 22, and combinations of 12 sets of nucleotide sequences shown in SEQ ID NOS: 23 and 24 (however, The above-mentioned base sequence may have the addition, substitution, deletion and/or insertion of 2 or less bases), and the detection of the ORF of (1) above by PCR using 12 sets of primers. The combination of the four sets of base sequences shown in SEQ ID NOs: 101 and 102, SEQ ID NOS: 103 and 104, SEQ ID NOS: 105 and 106, and SEQ ID NOS: 107 and 108 (however, the base sequence is 2 bases or less) (Which may have an addition, a substitution, a deletion and/or an insertion) of 4), and the ORF of (2) above is detected by the PCR method.

As described above, the primer may have addition, substitution, deletion and/or insertion of 2 bases or less, but from the viewpoint of highly accurate genotyping, the primer is It is preferred that there are no additions, substitutions, deletions and/or insertions.

When the multiplex PCR method is adopted as the PCR method, the cost of genotype classification can be suppressed.

When the detection results of the presence or absence of ORF in (1) and (2) above are replaced with 1 (present) and 0 (absent), respectively, and coded in a binary system, the detection results are digitized for easy viewing.
By further converting the binary coded result into a decimal code, the number of digits of the result is reduced, the result can be more easily discriminated, and the result obtained at another date and time, in another laboratory, etc. It becomes possible to make an accurate comparison with.

Further according to the present invention, SEQ ID NOS: 1 and 2, SEQ ID NOS: 3 and 4, SEQ ID NOS: 5 and 6, SEQ ID NOS: 7 and 8, SEQ ID NOS: 9 and 10, SEQ ID NOS: 11 and 12, SEQ ID NOS: 13 and 14, sequence Combinations of 12 sets of base sequences shown in Nos. 15 and 16, SEQ ID Nos. 17 and 18, SEQ ID Nos. 19 and 20, SEQ ID Nos. 21 and 22, and SEQ ID Nos. 23 and 24 (however, the above-mentioned base sequence is 2 bases or less. Which may have addition, substitution, deletion and/or insertion of bases), SEQ ID NOS: 101 and 102, SEQ ID NOS: 103 and 104, SEQ ID NOS: 105 and 106, SEQ ID NO: 107 and 6 sets of primers consisting of the combination of 4 sets of base sequences shown in 108 (however, the base sequence may have addition, substitution, deletion and/or insertion of 2 bases or less) A primer set for classifying genotypes of Klebsiella pneumoniae and closely related bacterial species is provided.

As described above, the primer may have addition, substitution, deletion and/or insertion of 2 bases or less, but from the viewpoint of highly accurate genotyping, the primer is It is preferred that there are no additions, substitutions, deletions and/or insertions.

According to the present invention, strain identification of Klebsiella pneumoniae and closely related strains can be performed easily, quickly, objectively and with high precision without requiring special equipment or skill of an operator. And, the occurrence of nosocomial infections of closely related bacterial species can be promptly detected, and the spread of nosocomial infections can be achieved.

FIG. 1 shows an electrophoretic photograph of Example 1. In FIG. 1, “PC” is an abbreviation for positive control, and the numerical value such as “12.. .” indicates the number of the isolate. "M" indicates a 50 bp ladder. FIG. 2 shows an electrophoretic photograph of Example 2. In FIG. 2, “PC” is an abbreviation for positive control, and the numerical value such as “12.. .” indicates the number of the isolate. "M" indicates a 50 bp ladder.

Hereinafter, the present invention will be specifically described.
The method for identifying strains of Klebsiella pneumoniae and closely related bacterial strains according to the present invention includes (1) ORFs constituting genomic islets on chromosomal gene sequences of Klebsiella pneumoniae and closely related bacterial species, and (2) chromosomal gene Possession of different ORFs derived from these two types, including the step of detecting the presence or absence of hyper variable ORFs present in the genomic island on the sequence in any order and performing genotypic classification based on the combination of the presence or absence of these ORFs. The method is characterized by having a step of performing genotype-based classification according to the combination of the presence or absence of. Hereinafter, these ORFs will be described.

(Genetic characteristics of Klebsiella pneumoniae)
Pulse field gel electrophoresis and multilocus sequence typing (MLST) analysis are used to systematically classify the genetic background of Klebsiella pneumoniae.

In MLST analysis, the nucleotide sequences of seven housekeeping genes (adk, atpA, dxr, glyA, recA, soda, tpi) were determined, and the sequence type (ST) was determined based on the polymorphism to identify clones. I do. In addition, clonality may be determined as a clone complex (CC) that is a collection of closely related ST populations. In addition, it was shown in Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter, Escherichia coli, and Clostridium difficile that it was effective to detect the possession pattern of genomic islet to estimate ST type or CC, but pneumonia Until now, it was unclear whether the same method was effective for bacilli.

Klebsiella pneumoniae is one of the indigenous bacteria of the human intestinal tract and has a symbiotic relationship with humans for a long time, so that there are those with various genetic backgrounds. The findings of the genetic background (including the results of detection of various ORFs) in clinically isolated actual wild strains are available as fragmentary information in individual research facilities as a literature, but by themselves, It is not sufficient to develop a strain identification method for Klebsiella pneumoniae by detecting ORF.

Therefore, the present inventor collected clinically isolated Klebsiella pneumoniae, conducted MLST analysis, and investigated their genetic background. As a result, it was found that Klebsiella pneumoniae clinically isolated in Japan have strains classified into various ST types, and for the identification of clones that caused nosocomial infection, strain identification was sufficient to identify ST types. There is a need to build a competent test method.

[(1) ORFs that make up the genomic islet]
In order to distinguish the ST type from the above, it was found that it is useful to detect ORF that constitutes the genomic islet in Klebsiella pneumoniae (see Table 1 below). A genomic islet is a portion of a bacterial chromosomal gene sequence that differs in size by about 5 kbp or less when compared to each other.This is scattered throughout the chromosomal gene sequence, and the survival probability of the individual. It is thought that it has been left behind in the process of evolution without affecting the.

Table 1 shows the results of an examination of the genomic islet possession status in clinical isolates. KPHS_23160 to PMK1_02643 are identification symbols of individual genomic islets.

Table 1 shows the results of an investigation of the relationship between ST type of clinical isolates and (1) ORFs constituting the genomic islet. 1 indicates detection, 0 indicates not detected. As can be seen from Table 1, the strains of Klebsiella pneumoniae with different ST types have different patterns of genomic islet (KPHS_23160 to PMK1_02643).

It has been shown that various clones are clinically isolated in Klebsiella pneumoniae, and in the analysis of collective infections, the strain discrimination ability can be obtained mainly by typing by detection of genomic islet.

The combination of KPHS and numbers (KPHS_01250 etc.) means the ORF name registered in HS11286 (Accession No. CP003200),
The combination of A7321 and numbers (A7321_00110 etc.) means the ORF name registered in W14 (Accession No. CP015753),
It means the ORF name registered in the combination of PMK1 and number (PMK1_02643 is PMK1 etc.) (Accession No. CP008929),
The combination of ABY63 and numbers (ABY63_05860 etc.) means the ORF name registered in CAV1392 (Accession No. CP011578),
The combination of KPNIH10 and numbers (KPNIH10_06180 etc.) means the ORF name registered in KPNIH10 (Accession No. CP007727),
The combination of BB751 and numbers (BB751_16465 etc.) means the ORF name registered in Kp_Goe_149832 (Accession No. CP018695),
The combination of KPNIH29 and numbers (KPNIH29_25465 etc.) means the ORF name registered in KPNIH29 (Accession No. CP009863),
The combination of AM275 and numbers (AM275_13120 etc.) means the ORF name registered in UCLAOXA232KP_Pt0 (Accession No. CP012560).
For example, KPHS — 01250 ORF is not only present in the HS11286 strain, but is also detected in, for example, strain No. 4, and may be present in strains derived therefrom and in Klebsiella pneumoniae of a different strain.

In addition, ORF KPHS_23160 can be detected by PCR by using the primers of SEQ ID NO: 1 (forward) and 2 (reverse) in the sequence listing,
ORF KPHS_25730 can be detected by PCR using the primers of SEQ ID NOs: 3 (forward) and 4 (reverse) in the sequence listing,
ORF KPHS_14300 can be detected by PCR using the primers of SEQ ID NOs: 5 (forward) and 6 (reverse) in the sequence listing,
ORF A7321_12080 can be detected by PCR by using the primers of SEQ ID NO: 7 (forward) and 8 (reverse) in the sequence listing,
ORF KPHS_05620 can be detected by PCR using the primers of SEQ ID NOs: 9 (forward) and 10 (reverse) in the sequence listing,
ORF A7321_12740 can be detected by PCR using the primers of SEQ ID NOs: 11 (forward) and 12 (reverse) in the sequence listing,
ORF KPHS_27850 can be detected by PCR using the primers of SEQ ID NO: 13 (forward) and 14 (reverse) in the sequence listing,
ORF KPHS_19280 can be detected by PCR using the primers of SEQ ID NOs: 15 (forward) and 16 (reverse) in the sequence listing,
ORF PMK1_04123 can be detected by PCR by using the primers of SEQ ID NOs: 17 (forward) and 18 (reverse) in the sequence listing,
ORF ABY63_05860 can be detected by PCR using the primers of SEQ ID NOs: 19 (forward) and 20 (reverse) in the sequence listing,
ORF KPNIH10_15035 can be detected by PCR by using the primers of SEQ ID NOs: 21 (forward) and 22 (reverse) in the sequence listing,
ORF PMK1_03738 can be detected by PCR by using the primers of SEQ ID NOs: 23 (forward) and 24 (reverse) in the sequence listing,
ORF KPHS_04610 can be detected by PCR using the primers of SEQ ID NOs: 25 (forward) and 26 (reverse) in the sequence listing,
ORF KPHS_05440 can be detected by PCR by using the primers of SEQ ID NOs: 27 (forward) and 28 (reverse) in the sequence listing,
ORF KPHS — 10130 can be detected by PCR using the primers of SEQ ID NOs: 29 (forward) and 30 (reverse) in the sequence listing,
ORF KPHS_12340 can be detected by PCR using the primers of SEQ ID NO: 31 (forward) and 32 (reverse) in the sequence listing,
ORF KPHS_12360 can be detected by PCR using the primers of SEQ ID NO: 33 (forward) and 34 (reverse) in the sequence listing,
ORF KPHS_14360 can be detected by PCR using the primers of SEQ ID NOs: 35 (forward) and 36 (reverse) in the sequence listing,
ORF KPHS_17140 can be detected by PCR using the primers of SEQ ID NOs: 37 (forward) and 38 (reverse) in the sequence listing,
ORF KPHS_22680 can be detected by PCR using the primers of SEQ ID NOs: 39 (forward) and 40 (reverse) in the sequence listing,
ORF KPHS_27840 can be detected by PCR by using the primers of SEQ ID NOs: 41 (forward) and 42 (reverse) in the sequence listing,
ORF KPHS_28160 can be detected by PCR by using the primers of SEQ ID NOs: 43 (forward) and 44 (reverse) in the sequence listing,
ORF KPHS_30110 can be detected by PCR by using the primers of SEQ ID NOs: 45 (forward) and 46 (reverse) in the sequence listing,
ORF KPHS_33080 can be detected by PCR using the primers of SEQ ID NOs: 47 (forward) and 48 (reverse) in the sequence listing,
ORF KPHS_35440 can be detected by PCR using the primers of SEQ ID NOs: 49 (forward) and 50 (reverse) in the sequence listing,
ORF KPHS_44950 can be detected by PCR by using the primers of SEQ ID NOs: 51 (forward) and 52 (reverse) in the sequence listing,
ORF KPHS_45270 can be detected by PCR by using the primers of SEQ ID NOs: 53 (forward) and 54 (reverse) in the sequence listing,
ORF BB751_16465 can be detected by PCR by using the primers of SEQ ID NOs: 55 (forward) and 56 (reverse) in the sequence listing,
ORF BB751_24340 can be detected by PCR by using the primers of SEQ ID NOs: 57 (forward) and 58 (reverse) in the sequence listing,
ORF KPNIH10_06180 can be detected by PCR using the primers of SEQ ID NOs: 59 (forward) and 60 (reverse) in the sequence listing,
ORF KPNIH10_06335 can be detected by PCR by using the primers of SEQ ID NO: 61 (forward) and 62 (reverse) in the sequence listing,
ORF KPNIH10_06350 can be detected by PCR by using the primers of SEQ ID NO: 63 (forward) and 64 (reverse) in the sequence listing,
ORF KPNIH10_10880 can be detected by PCR using the primers of SEQ ID NO: 65 (forward) and 66 (reverse) in the sequence listing,
ORF KPNIH10_13265 can be detected by PCR using the primers of SEQ ID NO: 67 (forward) and 68 (reverse) in the sequence listing,
ORF A7321_01305 can be detected by PCR using the primers of SEQ ID NOs: 69 (forward) and 70 (reverse) in the sequence listing,
ORF A7321_14925 can be detected by PCR by using the primers of SEQ ID NOs: 71 (forward) and 72 (reverse) in the sequence listing,
ORF KPHS_22950 can be detected by PCR using the primers of SEQ ID NOs: 73 (forward) and 74 (reverse) in the sequence listing,
ORF KPHS_34550 can be detected by PCR by using the primers of SEQ ID NOs: 75 (forward) and 76 (reverse) in the sequence listing,
ORF KPHS_35720 can be detected by PCR using the primers of SEQ ID NOs: 77 (forward) and 78 (reverse) in the sequence listing,
ORF KPHS_49450 can be detected by PCR using the primers of SEQ ID NOs: 79 (forward) and 80 (reverse) in the sequence listing,
ORF KPHS_51010 can be detected by PCR by using the primers of SEQ ID NOs: 81 (forward) and 82 (reverse) in the sequence listing,
ORF A7321_00110 can be detected by PCR using the primers of SEQ ID NOs: 83 (forward) and 84 (reverse) in the sequence listing,
ORF A7321_01475 can be detected by PCR using the primers of SEQ ID NOs: 85 (forward) and 86 (reverse) in the sequence listing,
ORF A7321_03820 can be detected by PCR using the primers of SEQ ID NOs: 87 (forward) and 88 (reverse) in the sequence listing,
ORF A7321_09350 can be detected by PCR using the primers of SEQ ID NOs: 89 (forward) and 90 (reverse) in the sequence listing,
ORF A7321_15655 can be detected by PCR using the primers of SEQ ID NOs: 91 (forward) and 92 (reverse) in the sequence listing,
ORF KPNIH29_02070 can be detected by PCR by using the primers of SEQ ID NOs: 93 (forward) and 94 (reverse) in the sequence listing,
ORF KPNIH29_25465 can be detected by PCR using the primers of SEQ ID NOs: 95 (forward) and 96 (reverse) in the sequence listing,
ORF AM275_13120 can be detected by PCR using the primers of SEQ ID NOs: 97 (forward) and 98 (reverse) in the sequence listing,
ORF PMK1 — 02643 can be detected by PCR by using the primers of SEQ ID NOs: 99 (forward) and 100 (reverse) in the sequence listing.
By detecting the above ORF, ST type can be easily distinguished.

Further, when these are detected using the conditions of PCR and electrophoresis described later, the target DNA fragment is easily amplified, and the extra band is less likely to appear.

In the above, the detection of ORF(1) of genomic islet by PCR method has been mainly described, but ORF(1) of genomic islet can be detected by other methods, for example, by hybridization. is there.

[(2) ORFs that make up the genomic island]
It is known that among the Klebsiella pneumoniae, there are clones having a relatively high frequency of isolation from clinical specimens. Since frequently isolated clones are often isolated between nosocomial infection cases that are geographically and temporally unrelated, a strain identification method having a higher identification ability than clone identification is required.

In recent years, the genome has been decoded, and a gene cluster consisting of 5 to 100 foreign genes is found in Klebsiella pneumoniae. This cluster is called a genomic island. Genomic islands include lysogenic phages, pathogenic islands, and resistant islands, as well as those of unknown function. Each genomic island has a size of approximately 5 kbp or more, and is composed of 5 to 100 ORFs. There are cases where ORFs are combined in a mosaic pattern on the genomic island and there is diversity in the ORF composition within the genomic island, and there are cases in which the ORF composition is almost the same even if the strains are different and no diversity is observed.

However, various strains of Klebsiella pneumoniae exist. The genotype survey results (including the results of detection of various ORFs) in actual wild strains can be used as individual fragmentary information as a nucleotide sequence database that carries literature or genomic information, but the minimum ORF is sufficient It is not sufficient to develop a method for identifying Klebsiella pneumoniae strains by detection.

Therefore, the present inventor designed a primer for detecting ORFs of various genomic islands by utilizing the nucleotide sequence information of Klebsiella pneumoniae registered in GenBank and the draft genome data of clinical isolates, and designed various genetic backgrounds. We investigated the ORF composition of K. pneumoniae that possesses the genomic island region.

As a result of repeated scrutiny of all nucleotide sequence data in strains registered in the genome database and repeated experiments to confirm ORF possession by clinical isolates, it is effective to detect several ORFs in the genomic island region in order to identify Klebsiella pneumoniae strains. It became clear.

As a result, AXK16_16575, Kpn2146_5469, KPHS_34600, KPHS_12700, KPHS_01250, KPHS_10080, KPHS_12370, KPHS_25140, KPHS_30440, KPHS_34840, KPHS_34850, KPHS_351K, KPHS_351K, KPHS_35K, KPHS_351K, KPHS_351K, KPHS_351K, KPHS_351K, KPHS_351K The present inventor has found that it is possible to discriminate the difference in the target background with high accuracy (see Table 2 below). By detecting the above ORFs, improved strain discrimination ability was achieved in most clones. By detecting all the ORFs that compose these genomic islands, the maximum strain discrimination ability is realized.

The combination of AXK16 and numbers (AXK16_16575 etc.) means the ORF name registered in KP38731 (Accession No. CP014294),
The combination of Kpn2146 and numbers (Kpn2146_6120 etc.) means the ORF name registered in ATCC_BAA-2146 (Accession No. CP006659),
The combination of KPHS and numbers (KPHS_34600 etc.) means the ORF name registered in HS11286 (Accession No. CP003200),
The combination of N559 and numbers (N559_1651 etc.) means the ORF name registered in GCA_000445405 (Accession No. CP006656),
The combination of KPNJ1 and numbers (KPNJ1_02432 etc.) means the ORF name registered in 30660-NJST258_1 (Accession No. CP006923),
The combination of KPNJ2 and numbers (KPNJ2_02710 etc.) means the ORF name registered in 30684-NJST258_2 (Accession No. CP006918),
The combination of KPR0928 and numbers (KPR0928_21110 etc.) means the ORF name registered in KPR0928 (Accession No. CP008831).
For example, the AXK16_16575 ORF does not exist only in the KP38731 strain and is also detected in, for example, the strain number 5, so that it may be present in the strain derived therefrom or in the Klebsiella pneumoniae of a different strain.

Table 2 shows the results of a survey on the genomic island possession status in clinical isolates. It was clarified that the clinical isolates also showed a strain-specific ORF retention pattern. In addition, AXK16_16575 to KPR0298_21110 in Table 2 below are identification symbols of genes including individual ORFs.

In addition, ORF AXK16_16575 can be detected by PCR by using the primers of SEQ ID NOs: 101 (forward) and 102 (reverse) in the sequence listing,
ORF Kpn2146_5469 can be detected by PCR using the primers of SEQ ID NOs: 103 (forward) and 104 (reverse) in the sequence listing,
ORF KPHS_34600 can be detected by PCR by using the primers of SEQ ID NOs: 105 (forward) and 106 (reverse) in the sequence listing,
ORF KPHS_12700 can be detected by PCR by using the primers of SEQ ID NOs: 107 (forward) and 108 (reverse) in the sequence listing,
ORF KPHS — 01250 can be detected by PCR using the primers of SEQ ID NOs: 109 (forward) and 110 (reverse) in the sequence listing,
ORF KPHS_10080 can be detected by PCR by using the primers of SEQ ID NOs: 111 (forward) and 112 (reverse) in the sequence listing,
ORF KPHS_12370 can be detected by PCR by using the primers of SEQ ID NOs: 113 (forward) and 114 (reverse) in the sequence listing,
ORF KPHS_25140 can be detected by PCR using the primers of SEQ ID NOs: 115 (forward) and 116 (reverse) in the sequence listing,
ORF KPHS_30440 can be detected by PCR by using the primers of SEQ ID NOs: 117 (forward) and 118 (reverse) in the sequence listing,
ORF KPHS_34840 can be detected by PCR by using the primers of SEQ ID NOs: 119 (forward) and 120 (reverse) in the sequence listing,
ORF KPHS_34850 can be detected by PCR using the primers of SEQ ID NOs: 121 (forward) and 122 (reverse) in the sequence listing,
ORF KPHS_35110 can be detected by PCR using the primers of SEQ ID NOs: 123 (forward) and 124 (reverse) in the sequence listing,
ORF KPHS_35580 can be detected by PCR using the primers of SEQ ID NOs: 125 (forward) and 126 (reverse) in the sequence listing,
ORF N559_1651 can be detected by PCR by using the primers of SEQ ID NOs: 127 (forward) and 128 (reverse) in the sequence listing,
ORF Kpn2146_6120 can be detected by PCR by using the primers of SEQ ID NOs: 129 (forward) and 130 (reverse) in the sequence listing,
ORF KPNJ1_02432 can be detected by PCR by using the primers of SEQ ID NOs: 131 (forward) and 132 (reverse) in the sequence listing,
ORF KPNJ2_02710 can be detected by PCR by using the primers of SEQ ID NOs: 133 (forward) and 134 (reverse) in the sequence listing,
ORF KPR0928_21110 can be detected by PCR by using the primers of SEQ ID NOs: 135 (forward) and 136 (reverse) in the sequence listing.

In addition, when these are detected using the conditions of PCR and electrophoresis described later, DNA is easily amplified and extra bands are less likely to appear.

In the above, the detection of the ORF (2) forming the genomic island by the PCR method has been mainly described, but the ORF (2) forming the genomic island can also be detected by other methods, for example, hybridization. Can be detected by.

[ORF detection means]
The presence or absence of the ORF (1) constituting the genomic islet and the ORF (2) constituting the genomic island on the chromosomal gene sequence of Klebsiella pneumoniae described above can be detected in any order. Specifically, these ORFs may be detected in any order for each ORF, or may be collectively detected in any order for a plurality of ORFs, or all ORFs may be detected simultaneously. Good. Means for detecting ORF include PCR and hybridization.

<PCR>
When detecting each ORF separately, for example, using a set of primers (forward primer and reverse primer) corresponding to each ORF to be detected, in an independent system, together with the Klebsiella pneumoniae DNA extraction sample, , Real-time PCR reaction, etc. are performed, and the generated signal fluorescence of PCR amplification product is detected in real time.

In addition, when multiple ORFs are to be detected collectively, multiple sets of primers (a set of forward primer and reverse primer) corresponding to each ORF are mixed and put in the same reaction system to carry out PCR reaction. Multiplex PCR can be performed. In this case, by setting the position of the primer so that the length of the PCR product can be distinguished from the others as an independent clear band after electrophoresis, the presence or absence of the PCR amplification product corresponding to each ORF It can be confirmed by the presence or absence of a band obtained by electrophoresis of the latter solution by electrophoresis, for example, agarose gel electrophoresis, capillary electrophoresis, or the like.

When detecting all ORFs at the same time, for example, microarray is applied to detect the ORFs. In microarray detection, a probe that hybridizes with each ORF is created in each well of the microarray, and the DNA purified by column extraction or phenol-chloroform extraction from the culture is hybridized to unreacted DNA. After washing, the target ORF can be detected by labeling with a fluorescent dye that binds to double-stranded DNA and measuring with a machine that captures fluorescence.

The ORF can also be detected on a computer by analyzing the entire genome base sequence using a next-generation sequencer.
Of these, multiplex PCR is preferable because it can efficiently detect a plurality of ORFs collectively by a simple method, but other analyzers that can obtain the same results as those can also be used.

(Design of primer)
The ORFs that compose the Klebsiella pneumoniae have different PCR amplification efficiencies depending on the site.To design primers suitable for multiplex PCR, actually use them for PCR and multiplex PCR, and confirm that they function and the amplification efficiency. However, it is necessary to proceed with the design. Furthermore, with respect to the ORF (1) that constitutes the above-mentioned genomic islet and the ORF (2) that constitutes the genomic island, since ORFs having a nucleotide sequence homology of about 90% are found in clinical isolates and databases, these homologues are similar. By designing a primer in a region with few mutations so that ORF can also be detected, the primer can be used universally for most Klebsiella pneumoniae that is often clinically isolated and is less susceptible to mutation. If the effect of mutation may be unavoidable, set another primer set that produces a PCR product of the same length and correct it by mixing multiple primer sets that can detect the same ORF. You can also

In designing a primer that detects the ORF (1) that constitutes the above-mentioned genomic islet and the ORF (2) that constitutes the genomic island, when the primer itself is bent, the complementary portions are bound to form a double chain. Or different primers do not bind to each other at their complementary portions to form a dimer or more conjugate.

Further, considering the detection by multiplex PCR, it is preferable to make the GC ratio of the primer about 50% and devise to match the Tm value. Moreover, it is desirable to adjust the size of the PCR amplification product to be about 50 bp to 2000 bp, preferably 70 bp to 600 bp so that the amplification efficiency and the separation in a short time during electrophoresis can be performed. In addition, in ORF detected in the same reaction system, it is essential to design primers so that the sizes of PCR amplification products are not the same.

By setting the conditions as described above, it is possible to obtain a primer and a primer set that can obtain a highly reproducible amplification result in multiplex PCR. Such a primer can be designed by commercially available software or software that can be freely obtained and used through the web, but by actually performing PCR and confirming the amplification, it becomes a practical primer.

Specifically, as described in the ORF items (1) and (2) above,
(1) Twelve pairs of primers as primers for ORFs constituting a genomic islet,
(2) 4 pairs of primers as primers for hyper variable ORF existing in genomic island,
The present inventor designed a total of 16 sets of primers (SEQ ID NOS: 1 to 24, 101 to 108 in the sequence listing).

When performing multiplex PCR using the above-mentioned primer set, as shown in Table 3 below as a primer mixture, a reaction system in which 6 primer sets are combined and mixed, and shown in Table 4 below as a primer mixture. As described above, it is desirable to perform PCR in a reaction system in which 10 primer sets are combined and mixed. The clone identification and the strain identification of Klebsiella pneumoniae may be performed simultaneously or separately.

The specific primers shown above are sequences optimized for amplifying the target ORF, and even if these primers have changes such as addition, substitution, deletion and/or insertion of about 2 bases, The function as a primer is not lost, and the target ORF can be amplified by PCR. Addition means that a base is added to the 5'-side or 3'-side end of the primer shown in the table, and insertion means between the base and the base inside the primer, not at the above-mentioned end. Say that a base is added to. However, when 2 or less bases are added or substituted at the 3'end of the base sequence, the base at the 3'end of the base sequence after addition or substitution is preferably complementary to the target ORF.

From the viewpoint of exhibiting high performance as a primer, the change is preferably addition, substitution, deletion, or insertion of 2 bases or less, addition of 2 bases or less or 5'side or 3'side of 2 bases or less. Is more preferable, and a deletion of 1 base or less at the end of 5'side or 3'side is particularly preferable. Generally, the modification on the 5'side is unlikely to lose the function as a primer, and the modification on the 3'side tends to lose the function of the primer.

The primer set shown in Tables 3 and 4 may be reacted with a detection primer targeting, for example, a pathogenic factor marker or a drug resistance gene.

<Hybridization>
When the ORF is detected by hybridization, first, DNA purified from a culture bacterium (Klebsiella pneumoniae) by column extraction or phenol-chloroform extraction is alkali-denatured and fixed on a nylon membrane, a cellulose membrane or a microplate. A probe that hybridizes with each ORF detected in the present invention is prepared. The probe may be artificially synthesized DNA or may be prepared by PCR using the primer described above. Label the probe with biotin, digoxigenin, a fluorescent dye, or the like. The probe DNA is hybridized with the bacterium-extracted DNA, and depending on the label of the probe, enzyme addition, color development, luminescence, etc. are performed to capture a signal. This is done for each detected ORF.

[ORF Detection Species Identification and Clone Identification and Strain Identification Method Implementation Method]
Hereinafter, a multiplex PCR is performed, and among the ORFs of the above (1) to (2), the case of detecting a preferred ORF as the above-mentioned detection target is taken as an example, and the clone identification method of Klebsiella pneumoniae of the present invention and the procedure for strain identification Will be explained.

(Identification of bacterial species)
Klebsiella pneumoniae is isolated from a sample derived from a patient such as stool using a medium capable of separating Klebsiella pneumoniae such as DHL medium. Then, it is identified as Klebsiella pneumoniae by morphological confirmation by Gram staining, confirmation of biochemical properties, mass spectrometry and the like.

The strain identified as Klebsiella pneumoniae is cultured overnight at 37° C. using a liquid medium capable of growing Klebsiella pneumoniae or an agar medium (ordinary agar medium, tryptosoy agar medium, etc.).

(DNA extraction)
From the cultured cells, heat extraction, phenol extraction, DNA extraction with a commercial DNA extraction kit, or suspension of the cells in distilled water or Tris-EDTA buffer and heating to obtain the DNA Extract and use as a sample (template) for PCR reaction.

(PCR reaction)
12 ORFs (1) constituting the genomic islets and 4 ORFs (2) constituting the genomic islands shown in Tables 3 and 4 above are detected by multiplex PCR. Specifically, the number of sets of primers (forward primer and reverse primer) corresponding to the ORF group to be detected is 6 sets (for ORF (1)) and 10 sets (6 sets for ORF (1) and ORF). (4 sets for (2)) divided into 2 groups, and each set is put in the same reaction tube and the PCR reaction is performed collectively. The bacterial species identification, the clone identification, and the bacterial strain identification may be performed simultaneously or separately.

(Electrophoresis)
The PCR amplification product is electrophoresed using a gel such as agarose gel under conditions such that a DNA fragment of about 50 bp to 600 bp is sufficiently separated. The migration distance may be about 5 to 6 cm, and in the case of mini gel, 100 V, about 50 minutes can be sufficiently separated. After electrophoresis, stain with ethidium bromide or cyber green and take a picture. It is also possible to separate and image PCR products using various capillary-type electrophoresis devices such as the Agilent 2100 Bioanalyzer or Shimadzu MultiNA.

(Result judgment)
A maximum of 11 or 10 bands will appear. In the genotyping method of the present invention, the band size is known in advance, so it is determined whether or not there is a band of the desired size in each strain and reaction system. In some cases, nonspecific bands other than the target size may appear, but bands considered nonspecific are ignored. A binary code is created by setting 1 if a band of a desired size (a band corresponding to each ORF and a band corresponding to a positive control) is amplified, and setting 0 when amplification is not observed.

This code may be created for each ORF of (1) to (2), or all of them may be created as a single code, or ORFs of (1) to (2) and Binary toxin (cdtA), toxinA (tcdA), toxinB (tcdB) may be created collectively. When this method for identifying bacterial strains and clones is used in the actual medical field, if all ORFs (1) to (2) are created as a single code, the number of digits will increase. Since it is difficult to see, it is preferable to combine several ORF results into one code.

Here, as described above, the ORF retention pattern of (1) has a high correlation with the ST type and is useful for estimating the ST type. Strains can be identified by comparing the ORF retention patterns of (2).

Therefore, the ORF detection result of (1) is preferably created as one code, and the ORF detection result of (2) is preferably created as one code.

Then, for example, the binary code created in this manner is converted into, for example, a decimal system to make it a genotype code in order to reduce the number of digits and make it easier to see. An example of the above code creation is shown in Table 5 below. The example in Table 5 is for isolate number 1 in FIG.

First, the presence or absence of each ORF in Klebsiella pneumoniae to be tested is detected by PCR using a suitable primer and electrophoresis, and it is encoded by the binary method. Then, this code is converted into a decimal system to obtain a genotype code (code).

In the example shown in Table 5, since codes 1 and 2 are a summary of the ORF detection results of (1), when genotype codes were created for multiple Klebsiella pneumoniae, the ST type of Klebsiella pneumoniae was determined. It becomes the corresponding numerical value. From this value, it can be judged whether the clone is a highly pathogenic clone or a drug resistant clone. Code 3 is unique to the strain and can be calculated into 16 different genotypes. The ORF of the code 3 is selected so that the strain discriminating ability in most Klebsiella pneumoniae is high.

Specifically, for example, the presence or absence of 16 ORFs (KPHS_23160 to KPHS_12700) in 16 strains of Klebsiella pneumoniae clinical isolates are binary coded and decimal coded as described above, It is as shown in Table 6.

In Table 6, the top row is the strain number, and the second row from the top is the Sequence type (ST) type by MLST analysis.
By comparing the genotype codes obtained in this way, it is possible to easily and objectively identify the strains isolated at different times or at different facilities.

Table 7 shows Code 1, Code 2 and Code 3 predicted from the Klebsiella pneumoniae, Klebsiella quasipneumoniae and Klebsiella variicola whole genome sequences on the genome database. The presence or absence of the ORF shown in Table 6 was determined by searching the sequence of each ORF by BLASTn, and Codes 1 to 3 were calculated according to the method shown in Table 5.

The slv added to the parentheses of the ST type is an abbreviation for single locus variant, indicating that it is closely related to the original ST type.

Furthermore, most of the ORFs to be detected in the present invention are ORFs that are not related to the pathogenicity of Klebsiella pneumoniae. Genes that contain ORFs that have specific functions, such as those associated with pathogenicity, increase the probability that Klebsiella pneumoniae will grow in the host, or their gene products will target the immune system of the host, and conversely increase the survival probability. May be lowered. In other words, it is considered that most of Klebsiella pneumoniae possesses such a gene, or, on the contrary, hardly possesses such a gene. In the present invention, genotyping is performed by selecting an appropriate ORF so that such a bias is not applied.
Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

Example 1
The 22 strains of Klebsiella pneumoniae clinically isolated at a hospital were genotyped by ORF detection according to the following procedure.

(Culture of Klebsiella pneumoniae)
Twenty-two strains identified as Klebsiella pneumoniae were cultured for 2 days at 37°C in Brucella HK agar.

(DNA extraction)
DNA was extracted from the cultured bacteria using a commercially available column DNA extraction kit, and the 20-fold solution was used as the template DNA.

(Preparation of PCR reaction solution)
Roche FastStart DNA polymerase was used according to the instructions. At that time, the reaction liquid volume was 20 μl. The composition of the reaction solution (20 μl) is 10×FastStart Taq Buffer (2 μl), 10 mM dNTP Mix (0.4 μl), FastStart Taq DNA polymerase (0.16 μl), distilled water (15.24 μl) and primer mixture (0.2 μl). To this, 2 µl of the above DNA heat-extracted sample (1/10 volume of the reaction solution) was added.

Two reaction tubes, that is, a reaction tube in which 6 sets of primers are combined (reaction system 1) and a reaction tube in which 10 sets of primers are combined (reaction system 2) are used, and 16 ORFs are multiplexed. PCR was performed. The combinations of primers and the final concentrations thereof are shown in Table 8 (Reaction system 1) and Table 9 (Reaction system 2) below.

Reaction system 1

Reaction system 2

(PCR reaction)
Set a mixture of extracted DNA and reaction solution in a thermal cycler (GeneAtlas G02, manufactured by ASTEC), treat at 95°C for 10 minutes first, and then at 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 2 minutes The cycle was repeated 30 times. After completion of the cycle reaction, the PCR product was stored at 15°C.

(Electrophoresis)
A mini gel was prepared using agarose gel (4% NuSieve 3:1 in TBE buffer), and 3 μl of the PCR product was electrophoresed at 100 V for 60 minutes. The migration distance was about 5 cm. After electrophoresis, it was stained with ethidium bromide and photographed.

(Result judgment)
A maximum of 6 bands appeared in reaction system 1 and a maximum of 10 bands in reaction system 2 per reaction. It was determined whether or not there was a band of the desired size in each strain and reaction system. If the band of the desired size was amplified, then 1 was set, and if no amplification was observed, 0 was set. Created a binary code. Furthermore, the binary code was converted into a decimal code to obtain a genotype code.
The results are shown in FIG. 1 and Table 10.

In Table 6, the ST type is found from the code 1 and the code 2 as described above, but it can be seen that the strain of the same ST type is further identified by the code 3.

Example 2
Hereinafter, an example in which the primer sequence is changed by about 2 bases will be described more specifically.
As in the previous example, 16 strains of Klebsiella pneumoniae clinically isolated at a hospital were genotyped by ORF detection by the following procedure.

(Culture of Klebsiella pneumoniae)
Twenty-two strains identified as Klebsiella pneumoniae were cultured for 2 days at 37°C in Brucella HK agar.

(DNA extraction)
DNA was extracted from the cultured bacteria using a commercially available column DNA extraction kit, and the 20-fold solution was used as the template DNA.

(Preparation of PCR reaction solution)
Roche FastStart DNA polymerase was used according to the instructions. At that time, the reaction liquid volume was 20 μl. The composition of the reaction solution (20 μl) is 10×FastStart Taq Buffer (2 μl), 10 mM dNTP Mix (0.4 μl), FastStart Taq DNA polymerase (0.16 μl), distilled water (15.24 μl) and primer mixture (0.2 μl). To this, 2 µl of the above DNA heat-extracted sample (1/10 volume of the reaction solution) was added.

Two reaction tubes, that is, a reaction tube in which 6 sets of primers are combined (reaction system 1) and a reaction tube in which 10 sets of primers are combined (reaction system 2) are used, and 16 ORFs are multiplexed. PCR was performed. The combinations of primers and their final concentrations are shown in Table 11 (reaction system 1) and Table 12 (reaction system 2) below.

Reaction system 1

Reaction system 2

Incidentally, SEQ ID NO: 137 is obtained by substituting 1 base for SEQ ID NO: 1,
SEQ ID NO:138 is obtained by adding 2 bases to SEQ ID NO:2,
SEQ ID NO:139 is a deletion of 2 bases from SEQ ID NO:3,
SEQ ID NO: 140 is obtained by adding 2 bases to SEQ ID NO: 4,
SEQ ID NO: 141 is obtained by replacing 2 bases with respect to SEQ ID NO: 5,
SEQ ID NO: 142 is obtained by substituting 1 base for SEQ ID NO: 6,
SEQ ID NO: 143 is obtained by inserting 1 base into SEQ ID NO: 7,
SEQ ID NO: 144 is obtained by adding 2 bases to SEQ ID NO: 8,
SEQ ID NO: 145 is obtained by replacing 2 bases with respect to SEQ ID NO: 9,
SEQ ID NO: 146 is obtained by adding 2 bases to SEQ ID NO: 10,
SEQ ID NO: 147 is obtained by substituting 1 base for SEQ ID NO: 11,
SEQ ID NO: 148 is obtained by inserting 1 base into SEQ ID NO: 12,
SEQ ID NO: 149 is obtained by adding 2 bases to SEQ ID NO: 13,
SEQ ID NO: 150 is obtained by adding 2 bases to SEQ ID NO: 14,
SEQ ID NO: 151 is obtained by replacing 2 bases with respect to SEQ ID NO: 15,
SEQ ID NO: 152 is obtained by inserting 2 bases into SEQ ID NO: 16,
SEQ ID NO: 153 is obtained by substituting 1 base for SEQ ID NO: 17,
SEQ ID NO: 154 is obtained by adding 2 bases to SEQ ID NO: 18,
SEQ ID NO: 155 is obtained by substituting 1 base for SEQ ID NO: 19,
SEQ ID NO: 156 is obtained by adding 2 bases to SEQ ID NO: 20,
SEQ ID NO: 157 is obtained by replacing 2 bases with respect to SEQ ID NO: 21,
SEQ ID NO:158 is obtained by inserting 2 bases into SEQ ID NO:22,
SEQ ID NO: 159 is obtained by inserting 2 bases into SEQ ID NO: 23,
SEQ ID NO: 160 is a deletion of 2 bases from SEQ ID NO: 24,
SEQ ID NO: 161 is obtained by replacing 2 bases with respect to SEQ ID NO: 101,
SEQ ID NO: 162 is obtained by substituting 1 base for SEQ ID NO: 102,
SEQ ID NO: 163 is obtained by replacing 2 bases with respect to SEQ ID NO: 103,
SEQ ID NO: 164 is obtained by adding 2 bases to SEQ ID NO: 104,
SEQ ID NO: 165 is obtained by adding 2 bases to SEQ ID NO: 105,
SEQ ID NO:166 is obtained by adding 2 bases to SEQ ID NO:106,
SEQ ID NO: 167 is obtained by replacing 2 bases with respect to SEQ ID NO: 107,
SEQ ID NO: 168 is obtained by inserting 1 base into SEQ ID NO: 108.

(PCR reaction)
Set a mixture of extracted DNA and the reaction solution in a thermal cycler (GeneAtlas G02, manufactured by ASTEC), treat at 95°C for 10 minutes first, and then at 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 2 minutes. The cycle was repeated 30 times. After completion of the cycle reaction, the PCR product was stored at 15°C.

(Electrophoresis)
A mini gel was prepared using agarose gel (4% NuSieve 3:1 in TBE buffer), and 2 μl of the PCR product was electrophoresed at 100 V for 50 minutes. The migration distance was about 5 cm. After electrophoresis, it was stained with ethidium bromide and photographed.

(Result judgment)
A maximum of 10 bands appeared per reaction. It was determined whether or not there was a band of the desired size in each strain and reaction system. If the band of the desired size was amplified, then 1 was set, and if no amplification was observed, 0 was set. Created a binary code. Furthermore, the binary code was converted into a decimal code to obtain a genotype code.

In Table 6, the ST type is found from the code 1 and the code 2 as described above, but it can be seen that the strain of the same ST type is further identified by the code 3.
The results are shown in Figure 2. Results similar to those in FIG. 1 and Table 10 were obtained.

According to the present invention, it is possible to easily and quickly identify strains of Klebsiella pneumoniae and related strains that are important pathogenic bacteria as a cause of nosocomial infection. The present invention is particularly useful for preventing nosocomial infections of Klebsiella pneumoniae and closely related bacterial species, preventing the spread of nosocomial infections, and the like.

The present invention is not limited to the description of the embodiments and examples of the invention. Various modifications are also included in the present invention within the scope that can be easily conceived by those skilled in the art without departing from the scope of the claims. The contents of papers, published patent publications, patent publications, and the like, which are specified in the present specification, are incorporated by reference in their entirety.

SEQ ID NOs: 1 to 168: Description of artificial sequence: primer

Claims (12)

On the chromosomal gene sequences of Klebsiella pneumoniae and related species,
(1) ORFs that make up a genomic islet, and (2) ORFs that make up a hyper variable ORF that exists in a genomic island,
A method for identifying strains of Klebsiella pneumoniae and related strains, which comprises the steps of detecting the presence or absence of any of these in any order and performing genotyping by combining the presence or absence of these ORFs. Wherein (1) the ORF constituting the genomic islet it is KPHS_23160, KPHS_25730, KPHS_14300, A7321_12080, KPHS_05620, A7321_12740, KPHS_27850, KPHS_19280, PMK1_04123, ABY63_05860, KPNIH10_15035, PMK1_03738, KPHS_04610, KPHS_05440, KPHS_10130, KPHS_12340, KPHS_12360, KPHS_14360, KPHS_17140, KPHS_22680, KPHS_27840, KPHS_28160, KPHS_30110, KPHS_33080, KPHS_35440, KPHS_44950, KPHS_45270, BB751_16465, BB751_24340, KPNIH10_06180, KPNIH10_06335, KPNIH10_06350, KPNIH10_10880, KPNIH10_13265, A7321_01305, A7321_14925, KPHS_22950, KPHS_34550, KPHS_35720, KPHS_49450, KPHS_51010, A7321_00110, A7321_01475, A7321_03820, It is at least one ORF selected from the group consisting of A7321_09350, A7321_15655, KPNIH29_02070, KPNIH29_25465, AM275_13120 and PMK1_02643, and hypervariable ORFs present in the above-mentioned (2) genomic island are AXK16_16575, Kpn2146_5600, KPHS, KPH2PH_S600, KPH2S_5469, KPHS. , KPHS_10080, KPHS_12370, KPHS_25140, KPHS_30440, KPHS_34840, KPHS_34850, KPHS_35110, KPHS_35580, N559_1651, Kpn2146_6120, KPNJ1_02432, KPNJ2_02710 and at least one of the group 1 of KPRJ928_21710. Wherein (1) genomic island in the ORF of KPHS_23160, KPHS_25730, KPHS_14300, A7321_12080, KPHS_05620, A7321_12740, KPHS_27850, KPHS_19280, PMK1_04123, ABY63_05860, KPNIH10_15035, PMK1_03738, KPHS_04610, KPHS_05440, KPHS_10130, KPHS_12340, KPHS_12360, KPHS_14360, KPHS_17140, KPHS_22680, KPHS_27840, KPHS_28160, KPHS_30110, KPHS_33080, KPHS_35440, KPHS_44950, KPHS_45270, BB751_16465, BB751_24340, KPNIH10_06180, KPNIH10_06335, KPNIH10_06350, KPNIH10_10880, KPNIH10_13265, A7321_01305, A7321_14925, KPHS_22950, KPHS_34550, KPHS_35720, KPHS_49450, KPHS_51010, A7321_00110, A7321_01475, A7321_03820, A7321_09350, A7321_15655, KPNIH29_02070, KPNIH29_25465, AM275_13120 and at least four ORF selected from the group consisting of PMK1_02643, hyper variable of ORF present in genomic island in (2) is AXK16_16575, Kpn2146_5469, KPHS_34600, KPHS_12700, KPHS_01250, KPHS_10080 , KPHS_12370, KPHS_25140, KPHS_30440, KPHS_34840, KPHS_34850, KPHS_35110, KPHS_35580, N559_1651, Kpn2146_6120, KPNJ1_02432, KPNJ2_02710, and at least 4 kinds of methods selected from the group consisting of 1 or more of F selected from the group consisting of at least 4 kinds of ORs selected from the group consisting of F or more. The genomic island in the ORF of (1) above is KPHS_23160, KPHS_25730, KPHS_14300, A7321_12080, KPHS_05620, A7321_12740, KPHS_27850, KPHS_19280, PMK1_04123, ABY63_05860, KPNIH10_15035 in the island and PMK1_03738 of species 12 and PMK1_03738 of PMK1_03F. The method according to claim 1, wherein the existing hyper variable ORFs are four kinds of ORFs of AXK16_16575, Kpn2146_5469, KPHS_34600 and KPHS_12700. Sequence number 1 and 2, sequence number 3 and 4, sequence number 5 and 6, sequence number 7 and 8, sequence number 9 and 10, sequence number 11 and 12, sequence number 13 and 14, sequence number 15 and 16 of sequence listing , SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 20 and 21, SEQ ID NOs: 21 and 22, and 12 combinations of base sequences shown in SEQ ID NOs: 23 and 24 (provided that the base sequence is 2 The ORF of (1) above is detected by PCR using 12 sets of primers consisting of addition, substitution, deletion and/or insertion of bases up to and including bases. And 102, SEQ ID NOS: 103 and 104, SEQ ID NOS: 105 and 106, and combinations of the four sets of nucleotide sequences shown in SEQ ID NOS: 107 and 108 (provided that the above-mentioned nucleotide sequences are additions, substitutions, deletions of 2 or less nucleotides). The ORF of (1) and (2) above is detected by a PCR method using 4 sets of primers consisting of a deletion and/or an insertion). The method described in the section. The method according to claim 5, wherein the base sequence has no base addition, substitution, deletion and/or insertion. The method according to claim 5 or 6, wherein the PCR method is a multiplex PCR method. The method according to any one of claims 1 to 7, wherein the detection results of the presence or absence of ORF in (1) and (2) are replaced by 1 (present) and 0 (absent), respectively, to perform binary coding. .. The method according to claim 8, wherein the binary coded result is further decimal coded. The method according to any one of claims 1 to 9, wherein the closely related bacterial species are Klebsiella quasipneumoniae and Klebsiella variicola. Sequence number 1 and 2, sequence number 3 and 4, sequence number 5 and 6, sequence number 7 and 8, sequence number 9 and 10, sequence number 11 and 12, sequence number 13 and 14, sequence number 15 and 16 of sequence listing , SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 20 and 21, SEQ ID NOs: 21 and 22, and 12 combinations of base sequences shown in SEQ ID NOs: 23 and 24 (provided that the base sequence is 2 12 pairs of primers consisting of addition, substitution, deletion and/or insertion of bases not more than the bases), SEQ ID NOS: 101 and 102, SEQ ID NOS: 103 and 104, SEQ ID NOS: 105 and 106, sequence 4 consisting of a combination of 4 sets of base sequences shown in Nos. 107 and 108 (however, the above base sequence may have addition, substitution, deletion and/or insertion of 2 bases or less) A primer set for identifying strains of Klebsiella pneumoniae and closely related strains, including a set of primers. The primer set according to claim 11, wherein the closely related bacterial species are Klebsiella quasipneumoniae and Klebsiella variicola.

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