Genotyping method for enterobacter bacteria and primer set for use therein

Abstract

To provide an ECC strain identification method suitable for practical industrial (medical field) applications, and a primer set for use therein.SOLUTION: The present invention provides a method for the identification of species and / or strain of test bacteria. This method includes the steps for detecting the presence or absence of one or more open reading frames (ORFs) on the chromosomes of the test bacteria: (1) Enterobacter hormaechei-specific ORFs, (2) Enterobacter asburiae-specific ORFs, (3) Enterobacter sp. close to E. asburiae-specific ORFs, (4) ORFs that constitute genome islands suitable for ST type estimation on the chromosome of E. hormaechei, and ORFs that constitute genome eyelets suitable for ST type estimation, (5) ORFs that constitute genome islands suitable for ST type estimation on the chromosome of E. asburiae, (6) ORFs that constitute genome islands suitable for strain identification on the chromosome of E. hormaechei and ORFs that constitute genome eyelets suitable for strain identification, and performing genotype classification based on the combination of ORF presence or absence.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for identifying the species and strain of bacteria of the genus Enterobacter, particularly Enterobacter cloacae complex (hereinafter also referred to as ECC). [Background technology]

[0002] ECC is widely distributed in the environment and is a normal inhabitant of the human intestine. It is generally not pathogenic to humans, but it can cause opportunistic infections in immunocompromised patients. ECC is a collective term for bacterial species genetically closely related to Enterobacter cloacae (hereinafter referred to as E. cloacae), and the five representative species are E. cloacae, Enterobacter hormaechei (hereinafter referred to as E. hormaechei), Enterobacter asburiae (hereinafter referred to as E. asburiae), Enterobacter kobei (hereinafter referred to as E. kobei), and Enterobacter ludwigii (hereinafter referred to as E. ludwigii).

[0003] In recent years, ECC resistant to almost all β-lactam antibiotics, including carbapenem antibiotics, which are said to be the last line of defense in the treatment of infectious diseases, have been reported. Among ECC resistant to carbapenem antibiotics, ECC that produce carbapenemase, a carbapenem-degrading enzyme, are often resistant to antibiotics other than β-lactam antibiotics, making infections caused by such ECC difficult to treat. In addition, since the gene for the carbapenem-degrading enzyme is encoded by plasmid DNA, it is known that drug resistance can be transmitted across bacterial species. The resistance rate of E. cloacae to carbapenem antibiotics in Japan is low at about 1% as of 2020, and this state needs to be maintained in the future.

[0004] For these reasons, it is important for medical institutions to detect the causative bacteria early in order to control infection and take measures against hospital-acquired infections. When hospital-acquired infection is suspected, in order to identify the route of infection, it is necessary to determine whether the strains of bacteria isolated from different patients and from the environment, such as shared medical equipment and facilities, are identical and whether they have resistance genes to carbapenem antibiotics.

[0005] One method for identifying bacterial species is mass spectrometry, which determines the bacterial species from the molecular weight information pattern of protein components derived from the bacteria. This method identifies the bacterial species by ionizing the bacterial body or a sample obtained from the bacterial body, separating the ions according to mass, and comparing the peak pattern of the mass spectrum obtained from mass spectrometry with an existing database, and has been widely used in recent years because it can provide results quickly.

[0006] Bacterial strains are often identified by detecting the genomic characteristics that the strains possess, using methods such as multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). MLST is a method for identifying strains using sequence types (ST), which are quantified variations in the base sequences of seven housekeeping genes. However, this method is costly because it requires determining the base sequences of seven genes. In addition, it cannot identify closely related clones with the same ST type. PFGE is a method to determine the genotype of a bacterial strain by cutting the genomic DNA of the bacteria with a restriction enzyme and determining the genotype of the strain based on the electrophoretic pattern of the fragments. It has high discrimination ability and reproducibility. However, it takes time to obtain the results, about 3 to 4 days at the earliest after the isolation and identification of the strain, and the work is complicated and requires the skilled operator.

[0007] On the other hand, there have been reports on genotyping methods for various bacteria other than Enterobacter. For example, Patent Document 1 describes a genotyping method for Staphylococcus aureus and a primer set used therefor, Patent Document 2 describes a genotyping method for Pseudomonas aeruginosa and a primer set used therefor, Patent Document 3 describes a genotyping method for Acinetobacter and a primer set used therefor, Patent Document 4 describes a genotyping method for Escherichia coli and a primer set used therefor, Patent Document 5 describes a genotyping method for Clostridium difficile and a primer set used therefor, and Patent Document 6 describes a genotyping method for Klebsiella pneumoniae and closely related bacterial species and a primer set used therefor. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2011-120528 A [Patent Document 2] JP 2013-146244 A [Patent Document 3] JP 2014-207895 A [Patent Document 4] JP 2016-49109 A [Patent Document 5] JP 2019-4811 A [Patent Document 6] JP 2020-115793 A Summary of the Invention [Problem to be solved by the invention]

[0009] ECC is a normal intestinal flora, but it can cause opportunistic infections in immunocompromised patients. In addition, ECC that are resistant to almost all β-lactam antibiotics, including carbapenem antibiotics, which are said to be the last line of defense in the treatment of infectious diseases, have been found, making infection control in medical settings a challenge. Therefore, in order to implement measures against drug-resistant bacteria and effective infection control, it is important to identify the source and route of infection, and for this purpose, molecular epidemiological analysis of isolated strains is required. In cases where hospital infection is suspected, mass spectrometry is often used to identify the bacterial species. However, with mass spectrometry, many of the bacterial species contained in ECC are simply identified as ECC or E. cloacae without detailed identification of the bacterial species, and it can be said that it is not possible to accurately identify bacterial species that are very closely related genetically. During the development process of the present invention, the species of ECC was identified using the average nucleotide identity (hereinafter referred to as ANI) method, which is a species identification method using the whole genome sequence of the strain, and it was revealed that many of the ECCs were E. hormaechei. For example, the present inventors have now found that of 618 strains registered as E. cloacae as of May 7, 2018 on GeneBank, a public database provided by the National Center for Biotechnology Information (hereinafter referred to as NCBI), only 57 were E. cloacae and 413 were E. hormaechei. Therefore, the results of species identification based on the ANI method performed by the present inventors have suggested for the first time that many of the strains identified as E. cloacae on GeneBank at present are E. hormaechei. In other words, although most ECC strains are E. hormaechei, many of the bacterial species contained in ECC by mass spectrometry are determined to be ECC without detailed species determination or E. cloacae, so it is not possible to determine whether they are E. hormaechei or not. In addition, the present inventors suspected that there is a new species in ECC that is closely related to E. asburiae, although the species name has not been registered, but this has not been clarified until now. Pulsed-field gel electrophoresis (PFGE) is often used to identify bacterial strains, but the PFGE method is cumbersome and requires the skilled operator. Furthermore, when it is necessary to determine the presence or absence of carbapenem antibiotic resistance genes at the same time as identifying the bacterial strain, traditionally, identification of the bacterial species, identification of the bacterial strain, and detection of the resistance genes were performed using separate methods, which was time-consuming and required a great deal of effort.

[0010] In view of the above-mentioned circumstances, an object of the present invention is to provide a method that enables simple, rapid, objective and highly sensitive identification of ECC bacterial species and / or strains. [Means for solving the problem]

[0011] As a result of intensive research to achieve the above object, the present inventors compared the complete genome sequence (chromosome sequence and plasmid sequence) data of ECC that is being accumulated in the NCBI database with the sequence data obtained by whole genome analysis of clinical isolates, investigated the pattern of open reading frames (hereinafter referred to as ORFs) present in clinical isolates, and succeeded in discovering ORFs that are effective for identification at the bacterial species level, ST type typing level, and strain level. Specifically, the present inventors have found that it is possible to more effectively identify ECC species and strains by detecting, in any order, one or more of: (1) E. hormaechei-specific ORFs on a chromosome; (2) E. asburiae-specific ORFs on a chromosome; (3) Enterobacter species close to Enterobacter asburiae (hereinafter referred to as Enterobacter sp. close to E. asburiae)-specific ORFs on a chromosome; (4) ORFs constituting a genomic island useful for ST type estimation on the E. hormaechei chromosome and ORFs constituting a genomic islet useful for ST type estimation; (5) ORFs constituting a genomic island useful for ST type estimation on the E. asburiae chromosome; and (6) ORFs constituting a genomic island useful for strain discrimination on the E. hormaechei chromosome and ORFs constituting a genomic islet useful for strain discrimination, and then performing genotyping based on the presence or absence of these ORFs in combination. Furthermore, (7) we found that by further detecting the genes of the blaIMP-1 group, which are carbapenem-degrading enzymes, it is possible to determine the presence or absence of resistance genes to carbapenem antibiotics.

[0012] That is, according to the present invention, there is provided a method for species identification and / or strain identification of a test bacterium, particularly a bacterium of the genus Enterobacter, and particularly preferably ECC, comprising the steps of detecting, in any order, one or more of (1) E. hormaechei-specific ORFs on the chromosome, (2) E. asburiae-specific ORFs on the chromosome, (3) Enterobacter sp. close to E. asburiae-specific ORFs on the chromosome, (4) ORFs constituting a genomic island useful for ST type estimation on the E. hormaechei chromosome and ORFs constituting a genomic islet useful for ST type estimation, (5) ORFs constituting a genomic island useful for ST type estimation on the E. asburiae chromosome, and (6) ORFs constituting a genomic island useful for strain discrimination on the E. hormaechei chromosome and ORFs constituting a genomic islet useful for strain discrimination, and classifying the test bacterium by genotype based on the combination of the presence or absence of these ORFs. In the method of the present invention, the presence or absence of a resistance gene to carbapenem antibiotics can be determined by detecting the presence or absence of (7) the blaIMP-1 group gene, which is a carbapenem-degrading enzyme, on the plasmid in any order.

[0013] In the method for identifying bacterial species of the present invention, it is preferable that the ORF (1) is LI66_04605 (SEQ ID NO: 106), the ORF (2) is NF29_17240 (SEQ ID NO: 107), and the ORF (3) is ECNIH4_02210 (SEQ ID NO: 108), thereby enabling the identification of the bacterial species E. hormaechei, E. asburiae, and Enterobacter sp. close to E. asburiae, respectively. In the method for estimating the ST type of E. hormaechei and E. asburiae, the ORFs constituting the genomic island on the E. hormaechei chromosome in (4) above are six types, AM432_16960 (SEQ ID NO: 109), AM432_02885 (SEQ ID NO: 110), AM432_06075 (SEQ ID NO: 112), AM432_12345 (SEQ ID NO: 115), AM451_02655 (SEQ ID NO: 116), and AM432_08555 (SEQ ID NO: 118), and the ORF constituting the genomic islet is one type, AM432_17020 (SEQ ID NO: 114), and the E. It is preferable that the ORFs constituting the genomic island on the C. asburiae chromosome are the three types ACJ69_15600 (SEQ ID NO: 111), NF29_16315 (SEQ ID NO: 113), and AB190_16475 (SEQ ID NO: 117). In the method for identifying E. hormaechei strains (6), it is preferable that the ORFs constituting the genomic island on the chromosome of E. hormaechei are nine types, namely LI64_04005 (sequence number 119), LI66_19120 (sequence number 123), AM409_17200 (sequence number 124), LI63_021875 (sequence number 125), LI63_018475 (sequence number 127), AM383_04310 (sequence number 128), LI62_04085 (sequence number 129), LI62_09620 (sequence number 120), and LI66_10685 (sequence number 121), and the ORFs constituting the genomic islet are two types, namely LI66_08680 (sequence number 122) and LI66_19065 (sequence number 126). In the method for determining the presence or absence of a resistance gene to a carbapenem antibacterial agent, the carbapenem degrading enzyme gene in (7) above is preferably the blaIMP-1 group.

[0014] Furthermore, the method of the present invention for identifying bacterial species, differentiating bacterial strains, and determining the presence or absence of a resistance gene to carbapenem antibacterial agents comprises detecting the ORF of (1) above by PCR using primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 3 and 4 in the sequence listing (however, the nucleotide sequences may have additions, substitutions, deletions, and / or insertions of 2 or less nucleotides), and detecting the ORF of (1) above by PCR using primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 5 and 6 (however, the nucleotide sequences may have additions, substitutions, deletions, and / or insertions of 2 or less nucleotides). The ORF of (2) above is detected by PCR using primers consisting of the combination of the nucleotide sequences shown in SEQ ID NOs: 7 and 8 (however, the above nucleotide sequences may have addition, substitution, deletion and / or insertion of 2 or less nucleotides), and the ORF of (3) above is detected by PCR using 7 combinations of the nucleotide sequences shown in SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, and SEQ ID NOs: 27 and 28 (however, the above nucleotide sequences may have addition, substitution, deletion and / or insertion of 2 or less nucleotides). The ORF in (4) above was detected by PCR using seven pairs of primers consisting of three combinations of base sequences shown in SEQ ID NOs: 13 and 14, SEQ ID NOs: 17 and 18, and SEQ ID NOs: 25 and 26 (however, the above base sequences may have addition, substitution, deletion, and / or insertion of two or less bases), and the ORF in (5) above was detected by PCR using three pairs of primers consisting of three combinations of base sequences shown in SEQ ID NOs: 31 and 32, SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, and SEQ ID NOs: 41 and 42. 2. Using 11 pairs of primers consisting of 11 pairs of combinations of base sequences shown in SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, SEQ ID NOs: 47 and 48, SEQ ID NOs: 49 and 50, and SEQ ID NOs: 51 and 52 (however, the above base sequences may have addition, substitution, deletion and / or insertion of 2 or less bases), the ORF of (6) above is detected by PCR, and using primers consisting of a combination of base sequences shown in SEQ ID NOs: 29 and 30 in the Sequence Listing (however, the above base sequences may have addition, substitution, deletion and / or insertion of 2 or less bases),This can be carried out by detecting the gene (7) above by PCR.

[0015] As described above, the above primers may have additions, substitutions, deletions and / or insertions of two or less bases, but from the viewpoint of highly accurate genotype classification, it is preferable that the primers do not have additions, substitutions, deletions and / or insertions of bases.

[0016] When a multiplex PCR method is used as the PCR method, the cost of genotyping can be reduced.

[0017] If the detection results for the presence or absence of the ORFs and genes in (4), (5), (6), and (7) are converted to binary code by replacing them with 1 (presence) and 0 (absence), respectively, the detection results can be quantified and made easier to read. By further converting the binary coded results into decimal coding, the number of digits in the results is reduced, making the results easier to interpret and enabling accurate comparison with results obtained on other dates, in other laboratories, etc.

[0018] Furthermore, according to the present invention, there are 11 pairs of primers consisting of combinations of 11 pairs of base sequences shown in 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: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, SEQ ID NOs: 27 and 28, and SEQ ID NOs: 29 and 30 (however, the above-mentioned base sequences may have addition, substitution, deletion and / or insertion of 2 or less bases), SEQ ID NOs: 31 and 32, SEQ ID NOs: 33 and 34, and SEQ ID NOs: 35. and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, SEQ ID NOs: 47 and 48, SEQ ID NOs: 49 and 50, and SEQ ID NOs: 51 and 52 (however, the above-mentioned base sequences may have addition, substitution, deletion, and / or insertion of two or less bases).

[0019] As described above, the above primers may have additions, substitutions, deletions and / or insertions of two or less bases, but from the viewpoint of highly accurate genotype classification, it is preferable that the primers do not have additions, substitutions, deletions and / or insertions of bases.

[0020] That is, the present invention relates to the following. [1] A method for species identification and / or strain identification of a test bacterium, comprising: On the chromosome of the test bacteria, (1) an E. hormaechei-specific open reading frame (ORF), (2) E. asburiae-specific ORF, (3) Enterobacter sp. close to E. asburiae-specific ORF (4) ORFs constituting a genomic island useful for ST type estimation on the E. hormaechei chromosome and ORFs constituting a genomic islet useful for ST type estimation, (5) ORFs constituting genomic islands on the chromosome of E. asburiae that are useful for ST typing, (6) ORFs constituting genomic islands and genomic islets useful for strain identification on the E. hormaechei chromosome and classifying the gene by genotype based on a combination of the presence or absence of the ORF. [2] The method of [1], detecting the presence or absence of ORFs (1), (4) and / or (6). [3] Method [1] or [2] to detect the presence or absence of ORFs (1), (2), and (3). [4] Detect the presence or absence of the ORFs in (2) and (5) using any of the methods described in [1] to [3]. [5] The ORF of (1) is LI66_04605 (sequence number 106), the ORF of (2) is NF29_17240 (sequence number 107), the ORF of (3) is ECNIH4_02210 (sequence number 108), the ORFs constituting the genomic island on the E. hormaechei chromosome of (4) are six types, AM432_16960 (sequence number 109), AM432_02885 (sequence number 110), AM432_06075 (sequence number 112), AM432_12345 (sequence number 115), AM451_02655 (sequence number 116), and AM432_08555 (sequence number 118), and the ORF constituting the genomic islet is one type, AM432_17020 (sequence number 114), and the ORF of (5) is E. (6) the ORFs constituting the genomic island on the E. asburiae chromosome are ACJ69_15600 (SEQ ID NO: 111), NF29_16315 (SEQ ID NO: 113), and AB190_16475 (SEQ ID NO: 117), and / or The methods of [1] to [4], wherein the ORFs constituting the genomic island on the chromosome of the ST78 strain are nine types, namely LI64_04005 (sequence number 119), LI66_19120 (sequence number 123), AM409_17200 (sequence number 124), LI63_021875 (sequence number 125), LI63_018475 (sequence number 127), AM383_04310 (sequence number 128), LI62_04085 (sequence number 129), LI62_09620 (sequence number 120), and LI66_10685 (sequence number 121), and the ORFs constituting the genomic island are two types, namely LI66_08680 (sequence number 122) and LI66_19065 (sequence number 126). [6] detecting the ORF of (1) by PCR using primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 3 and 4 (however, the nucleotide sequences may have additions, substitutions, deletions and / or insertions of 2 or less nucleotides); detecting the ORF of (2) by PCR using primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 5 and 6 (however, the nucleotide sequences may have additions, substitutions, deletions and / or insertions of 2 or less nucleotides); (3) detecting the ORF by PCR using primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 7 and 8 (wherein the nucleotide sequences may have additions, substitutions, deletions and / or insertions of 2 or less nucleotides); (4) detects the ORF by PCR using seven pairs of primers consisting of seven combinations of base sequences shown in SEQ ID NOs: 9 and 10, 11 and 12, 15 and 16, 19 and 20, 21 and 22, 23 and 24, and 27 and 28 (however, the above base sequences may have addition, substitution, deletion, and / or insertion of two or less bases); (5) detecting the ORF by PCR using three pairs of primers consisting of three combinations of base sequences shown in SEQ ID NOs: 13 and 14, SEQ ID NOs: 17 and 18, and SEQ ID NOs: 25 and 26 (however, the above base sequences may have additions, substitutions, deletions, and / or insertions of two or less bases); and / or (6) ORF is detected by PCR using 11 pairs of primers consisting of 11 combinations of base sequences shown in SEQ ID NOs: 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, and 51 and 52 (however, the above base sequences may have addition, substitution, deletion, and / or insertion of 2 or less bases); Method [5] [7] The method of [6], wherein the base sequence does not have any additions, substitutions, deletions and / or insertions of bases. [8] (7) Genes of the blaIMP-1 group, which are carbapenem degrading enzymes The methods of [1]-[7] further comprising detecting the presence or absence of. [9] The method of [8], comprising detecting the gene of (7) by PCR using primers consisting of a combination of the base sequences shown in SEQ ID NOs: 29 and 30 (wherein the base sequences may have additions, substitutions, deletions and / or insertions of 2 or less bases).

[10] The method of [9], wherein the base sequence does not have any additions, substitutions, deletions and / or insertions of bases.

[11] The methods of [6], [7], [9] and

[10] , in which the PCR method is a multiplex PCR method.

[12] A method described in [1]-

[11] in which the results of detection of the presence or absence of ORFs (4), (5), and / or (6) are binary coded by replacing them with 1 (presence) and 0 (absence), respectively.

[13] A method described in [8]~

[12] in which the results of the detection of the presence or absence of a gene (7) are converted to binary code by replacing them with 1 (presence) and 0 (absence), respectively.

[14] The method of

[13] in which the binary-coded result is further coded into decimal.

[15] The method of [1]-

[14] in which the test bacteria are E. cloacae and closely related species.

[16] Eleven pairs of primers consisting of combinations of eleven pairs of nucleotide sequences shown in SEQ ID NOs: 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 15 and 16, 19 and 20, 21 and 22, 23 and 24, 27 and 28, and 29 and 30 (however, the above nucleotide sequences may have additions, substitutions, deletions, and / or insertions of two or less nucleotides), as well as SEQ ID NOs: 31 and 32, 33 and 34, and SEQ ID NO: 35. and 36, SEQ ID NO: 37 and 38, SEQ ID NO: 39 and 40, SEQ ID NO: 41 and 42, SEQ ID NO: 43 and 44, SEQ ID NO: 45 and 46, SEQ ID NO: 47 and 48, SEQ ID NO: 49 and 50, and SEQ ID NO: 51 and 52 (however, the above-mentioned base sequences may have addition, substitution, deletion, and / or insertion of two or less bases), a primer set for use in genotyping ECC and determining the presence or absence of resistance to carbapenem antibacterial agents.

[17] A method of isolating subjects who possess bacterial species and / or strains resistant to carbapenem antibiotics from subjects who do not, based on information on the bacterial species and / or strains identified and / or differentiated by the methods of [1] to

[15] . Effect of the Invention

[0021] According to the present invention, the species and strain of a test bacterium, and / or the presence or absence of a resistance gene to carbapenem antibacterial drugs can be identified simply, quickly, objectively, and highly sensitively by an ECC identification method more suitable for practical industrial (medical) use, that is, without requiring special equipment or operator skill. According to one aspect of the present invention, when the test bacterium is E. hormaechei, E. asburiae, or Enterobacter sp. close to E. asburiae, it can be identified as these species, when it is E. hormaechei, its ST type can be estimated, when it is E. asburiae, its ST type can be estimated, and / or when it is E. hormaechei, its strain can be identified. Furthermore, according to the present invention, a primer set for use in the above method can be provided. Furthermore, according to the present invention, nosocomial infection of drug-resistant ECC can be detected quickly, and the spread of nosocomial infection can be prevented by isolating a patient carrying the ECC. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1A shows an electrophoretic photograph of Example 1. In FIG. 1A, "P" indicates a positive control, and numbers such as "123..." indicate the numbers of the isolated strains. Also, "M" indicates a 50 bp ladder marker. FIG. 1B shows an electrophoretic photograph of Example 2. In FIG. 1B, "P" indicates a positive control, and numbers such as "123..." indicate the numbers of the isolated strains. Also, "M" indicates a 50 bp ladder marker. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention will be specifically described below. The method of the present invention for identifying the species and strain of a test bacterium and / or determining the presence or absence of a resistance gene to carbapenem antibacterial agents comprises determining the presence or absence of (1) an E. hormaechei-specific ORF on a chromosome, (2) an E. asburiae-specific ORF on a chromosome, (3) an Enterobacter sp. close to E. asburiae-specific ORF on a chromosome, (4) an ORF constituting a genomic island useful for ST type estimation on the E. hormaechei chromosome and an ORF constituting a genomic islet useful for ST type estimation, (5) an ORF constituting a genomic island useful for ST type estimation on the E. asburiae chromosome, and / or (6) an ORF constituting a genomic island useful for strain identification on the E. hormaechei chromosome and an ORF constituting a genomic islet useful for strain identification, by arbitrarily detecting the presence or absence of (7) a carbapenem-degrading enzyme blaIMP-1 on a plasmid of the test bacterium. The present invention is characterized by comprising a step of detecting the presence or absence of genes of group in any order, and classifying the strain by genotype and / or detecting carbapenem resistance genes based on the combination of the presence or absence of these ORFs and the detection of genes. These ORFs and the blaIMP-1 group genes are described below.

[0024] [(1)~(3) Bacterial species-specific ORF] The five representative species that make up ECC are E. cloacae, E. hormaechei, E. asburiae, E. kobei, and E. ludwigii. When the complete genome sequences (chromosome sequences and plasmid sequences) of strains registered as the same species in the NCBI database were compared, there were strains that showed different patterns of sequences common to strains of the same species, and strains that showed similar patterns to other species. Since it was suspected that the species name may have been incorrectly named at the database registration stage, the ANI (Average Nucleotide Identity) method was used to determine the correct species. This analysis revealed that the majority of ECC strains registered in the database are E. hormaechei. Furthermore, it was found that there is a species that the inventor calls Enterobacter sp. close to E. asburiae, which is not registered as a species name but is closely related to E. asburiae. Here, Enterobacter sp. close to E. asburiae refers to a strain that is closely related to E. asburiae, with an ANI value of around 91 with E. asburiae, among strains that are not identified as any species of the Enterobacter genus by the ANI method. In addition, clinically isolated ECC strains were collected and whole genome analysis was performed. It was found that the majority of ECC clinically isolated in Japan was E. hormaechei, followed by E. asburiae and Enterobacter sp. close to E. asburiae in that order. Mass spectrometry, which is often used to identify bacterial species in clinical testing, is difficult to use to identify ECC species, which could lead to delays and confusion in infection control measures. Therefore, based on the accurate bacterial species determined and the chromosomal sequence information of ECC obtained from the database, we selected and determined species-specific ORFs that are conserved within each bacterial species and cannot be found in other bacterial species.

[0025] (E. hormaechei specific ORF) Genome information for E. hormaechei available in public databases includes 4928STDY7071111 (Accession Numbers CABHJV000000000, CABHJV010000001:CABHJV010000054), 921_ECLO (Accession Numbers JUNO00000000, JUNO01000001:JUNO01000140), AS012434 (Accession Numbers VLMG00000000, VLMG01000001:VLMG01000339), EC32 (Accession Numbers SORQ00000000, SORQ01000001:SORQ01000089), H2F4R (Accession Numbers JACGFV000000000, JACGFV010000001:JACGFV010000545). The genome information of E. hormaechei was compared with other ECCs to select ORFs found specifically in E. hormaechei. PCR primers capable of amplifying the selected ORFs were designed, and E. hormaechei-specific ORFs were selected using clinical isolates that had been accurately identified by the ANI method (LI66_04605). E. hormaechei-specific ORFs can be easily detected by PCR using primers of SEQ ID NO: 3 (forward) and SEQ ID NO: 4 (reverse) in the sequence listing. Note that in order to detect the presence or absence of a specific ORF, it is not necessarily necessary to detect the full length of the ORF, and it is sufficient to determine the presence or absence of the ORF.

[0026] (E. asburiae specific ORF) Genome information for E. asburiae available in public databases includes 1009_ECLO (Accession Number: JWGM00000000, JWGM01000001:JWGM01000139), AGK0506 (Accession Number JAKWIV000000000, JAKWIV010000001:JAKWIV010000166), L1 (Accession Number AWXI00000000, AWXI01000001:AWXI01000195), e800 (Accession Number FKGO00000000, FKGO01000001:FKGO01000133), nEC133 (Accession Number JADHKU000000000, JADHKU010000001:JADHKU010000036). The genome information of E. asburiae was compared with other ECCs, and ORFs contained in many E. asburiae species were selected. PCR primers capable of amplifying the selected ORFs were designed, and E. asburiae-specific ORFs useful for species identification of E. asburiae were selected using clinical isolates for which accurate species identification by the ANI method had been completed (NF29_17240 (SEQ ID NO: 107)). E. asburiae-specific ORFs can be easily detected by PCR using primers of SEQ ID NO: 5 (forward) and SEQ ID NO: 6 (reverse) in the sequence listing. Note that in order to detect the presence or absence of a specific ORF, it is not necessarily necessary to detect the full length of the ORF, and it is sufficient to determine the presence or absence of the ORF.

[0027] (Enterobacter sp. close to E. asburiae-specific ORF) Enterobacter sp. close to E. asburiae is an ECC that is relatively frequently isolated from patients, although the species name has not been determined. Genome information of Enterobacter sp. close to E. asburiae available in public databases includes 145e9 (Accession Number QMCN00000000, QMCN01000001:QMCN01000148), AGK0550 (Accession Number JAKWIQ000000000, JAKWIQ010000001:JAKWIQ010000295), MGH178 (Accession Number NGRP00000000.1, NGRP01000001:NGRP01000005), TUM11131 (Accession Number BEGI00000000, BEGI01000001:BEGI01000138), Y07 (Accession Number JAASKF000000000, JAASKF010000001:JAASKF010000019). The genome information of Enterobacter sp. close to E. asburiae was compared with other ECCs, and Enterobacter sp. close to E. asburiae-specific ORFs that can identify Enterobacter sp. close to E. asburiae were selected. PCR primers capable of amplifying the selected ORFs were designed, and Enterobacter sp. close to E. asburiae-specific ORFs useful for identifying Enterobacter sp. close to E. asburiae were selected using clinical isolates for which accurate species identification by the ANI method had been completed (ECNIH4_02210 (SEQ ID NO: 108)). Enterobacter sp. close to E. asburiae-specific ORFs can be easily detected by PCR using primers of SEQ ID NO: 7 (forward) and SEQ ID NO: 8 (reverse) in the sequence listing. In order to detect the presence or absence of a particular ORF, it is not necessarily necessary to detect the entire length of the ORF, but it is sufficient to be able to determine the presence or absence of the ORF.

[0028] [(4), (5), (6) ORFs constituting a genomic island and / or ORFs constituting a genomic islet] Multilocus sequence typing (MLST) analysis is used to systematically classify the genetic background of ECC. In MLST analysis, the base sequences of seven housekeeping genes (dnaA, fusA, gyrB, leuS, pyrG, rplB, rpoB) are determined, and sequence types (STs) are determined, making it possible to distinguish strains with the same genetic background, which is essential for epidemiological studies. It has been shown that detecting the pattern of genome islets is effective for estimating ST types in Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter spp., Escherichia coli, Clostridium difficile, and Klebsiella pneumoniae, but it was unclear whether a similar method would be effective for Enterobacter spp.

[0029] ECCs have various genetic backgrounds. Therefore, the present inventors collected clinically isolated ECCs and performed whole genome analysis to investigate their genetic backgrounds. As a result, it was found that many of E. hormaechei, which accounts for the majority of ECCs clinically isolated in Japan, were classified as ST78 type, and other strains classified as ST113, ST133, and ST171 were detected, while E. asburiae strains classified as ST24, ST25, ST252, and ST484 were detected. Therefore, in order to roughly classify isolated strains, it is necessary to realize a strain discrimination ability that can distinguish these ST types.

[0030] (ORFs constituting a genomic island useful for ST typing on the E. hormaechei chromosome and ORFs constituting a genomic island useful for ST typing) It was found that the detection of the ORFs that compose the genomic islet alone is not sufficient to distinguish the ST types of E. hormaechei. A genomic islet is a part of the chromosomal gene sequence of bacteria that differs in size by about 5 kbp or less when comparing the chromosomal gene sequences of bacteria. This part is scattered throughout the chromosomal gene sequence, does not affect the survival probability of individuals, and is thought to have been left behind during the evolutionary process.

[0031] The present inventors also investigated the discrimination of ST types by detecting the ORFs constituting the genomic island, but the above ST types could not be discriminated by only detecting the ORFs constituting the genomic island. A genomic island is a gene cluster consisting of foreign genes. In recent years, genome deciphering has progressed, and gene clusters consisting of 5 to 100 foreign genes have been found in ECC. Genomic islands include lysogenic phages, pathogenic islands, resistance islands, and those with unknown functions. Each genomic island is approximately 5 kbp in size or larger. It has been found that detecting the pattern of genomic island possession is effective for discriminating strains in Acinetobacter, Clostridium difficile, and Klebsiella pneumoniae.

[0032] In ECC, ORFs are combined in a mosaic pattern in genomic islands, and in some cases, the ORF composition within the genomic island is diverse, while in other cases, the ORF composition is almost the same even though the ST type is different, and no diversity is observed.

[0033] Therefore, the present inventors attempted to classify the ST types by detecting the ORFs constituting the genomic island and the ORFs constituting the genomic islet in combination. Using the nucleotide sequence information of E. hormaechei registered in GenBank and the draft genome data of clinical isolates, we designed primers to detect the ORFs of various genomic islands and genomic islets, and investigated the ORF composition of the genomic island and genomic islet regions of E. hormaechei strains with diverse genetic backgrounds.

[0034] After careful examination of the entire genome data registered in the genome database and repeated experiments to confirm the presence of ORFs in clinical isolates, it became clear that detecting several combinations of ORFs constituting genomic islands and ORFs constituting genomic islets is effective in distinguishing ST types of E. hormaechei.

[0035] The results of investigating the presence of each ORF are shown in Table 1. [Table 1]

[0036] Table 1 shows the results of an investigation into the ST types of clinical isolates and (4) the relationship between the ORFs constituting the genomic island and the ORFs constituting the genomic islet. As can be seen from Table 1, strains of E. hormaechei with different ST types have different patterns of ORFs constituting the genomic island (AM432_08555, AM432_16960, AM432_12345, AM432_02885, AM451_02655, AM432_06075) and ORF constituting the genomic islet (AM432_17020).

[0037] The combination of AM432 and a number (AM432_08555, etc.) refers to the ORF name registered in AR_0053 (Accession No. CP021776.1), and the combination of AM451 and a number (AM451_02655) refers to the ORF name registered in AR_0072 (Accession No. CP026850.1). For example, AM432_08555 is not present only in the AR_0053 strain, but has also been detected in, for example, strain number 1 and may be present in strains derived from it or in E. hormaechei of a different lineage.

[0038] The gene AM432_16960 can be detected by PCR using primers of SEQ ID NO: 9 (forward) and 10 (reverse) in the sequence listing. The gene AM432_02885 can be detected by PCR using primers of SEQ ID NO: 11 (forward) and 12 (reverse) in the sequence listing; The gene AM432_06075 can be detected by PCR using primers of SEQ ID NO: 15 (forward) and 16 (reverse) in the sequence listing; The gene AM432_17020 can be detected by PCR using primers of SEQ ID NO: 19 (forward) and 20 (reverse) in the sequence listing; The gene AM432_12345 can be detected by PCR using primers of SEQ ID NO: 21 (forward) and 22 (reverse) in the sequence listing; The gene AM451_02655 can be detected by PCR using primers of SEQ ID NO: 23 (forward) and 24 (reverse) in the sequence listing; The gene AM432_08555 can be detected by PCR using primers of SEQ ID NO: 27 (forward) and 28 (reverse) in the sequence listing. By detecting the above ORFs, ST types can be easily distinguished. In order to detect the presence or absence of a particular ORF, it is not necessarily necessary to detect the entire length of the ORF, but it is sufficient to be able to determine the presence or absence of the ORF.

[0039] Furthermore, when these are detected using PCR and electrophoresis, which will be described later, the DNA is easily amplified and extra bands are unlikely to appear. In the above, we have mainly explained the detection of ORF(4), which varies depending on the ST type on the E. hormaechei chromosome, by PCR. However, ORF(4), which varies depending on the ST type on the E. hormaechei chromosome, can also be detected by other methods, such as hybridization.

[0040] (ORFs constituting a genomic island useful for ST typing on the E. asburiae chromosome) In order to distinguish the ST types of E. asburiae, we carried out a similar study to that for E. hormaechei and found that detecting a combination of several ORFs that make up the genomic island is effective for distinguishing ST types. The results of the investigation into the presence of each ORF are shown in Table 2. [Table 2]

[0041] Table 2 shows the results of an investigation into the relationship between the ST types of clinical isolates and (5) the ORFs that make up the genomic island. As can be seen from Table 2, the patterns of ORFs (ACJ69_15600 to AB190_16475) that make up the genomic island are different among the ST24, ST25, ST252, and ST484 types of E. asburiae.

[0042] The combination of ACJ69 and a number (ACJ69_15600) means the ORF name registered in ATCC 35953 (Accession No. CP011863.1). The combination of NF29 and a number (NF29_16315) means the ORF name registered in colR / S (Accession No. CP010512.1). The combination of AB190 and a number (AB190_16475) means the ORF name registered in CAV1043 (Accession No. CP011591.1). For example, ACJ69_15600 is not present only in the ATCC 35953 strain, but has also been detected in strain number 17, for example, and may also be present in strains derived from it or in different lineages of E. asburiae.

[0043] In addition, the gene ACJ69_15600 can be detected by PCR using primers of SEQ ID NO: 13 (forward) and 14 (reverse) in the sequence listing. The gene NF29_1631 can be detected by PCR using primers of SEQ ID NO: 17 (forward) and 18 (reverse) in the sequence listing; The gene AB190_16475 can be detected by PCR using primers of SEQ ID NO: 25 (forward) and 26 (reverse) in the sequence listing. By detecting the above ORFs, ST types can be easily distinguished. In order to detect the presence or absence of a particular ORF, it is not necessarily necessary to detect the entire length of the ORF, but it is sufficient to be able to determine the presence or absence of the ORF.

[0044] Furthermore, when these are detected using PCR and electrophoresis, which will be described later, the DNA is easily amplified and extra bands are unlikely to appear. In the above, we have mainly explained the detection of ORF(5), which varies depending on the ST type on the E. asburiae chromosome, by PCR. However, ORF(5), which varies depending on the ST type on the E. asburiae chromosome, can also be detected by other methods, such as hybridization.

[0045] (ORFs constituting a genomic island useful for strain identification on the chromosome of E. hormaechei and ORFs constituting a genomic islet useful for strain identification) Among E. hormaechei clinically isolated in Japan, many were classified as ST78 type. However, many ST78 types with diverse ORF structures have been detected among the epidemic clones in Japan, and therefore a strain identification method with higher discrimination ability than ST type estimation is required.

[0046] Therefore, the inventors used the base sequence information obtained from the database to design primers to detect ORFs that showed diversity, and investigated the structure of the ORFs possessed by ST78 E. hormaechei strains with diverse genetic backgrounds.

[0047] As a result of detailed examination of the chromosomal sequences of strains registered in the database and repeated experiments to confirm the presence of ORFs in clinical isolates, it became clear that detection of several combinations of ORFs constituting genomic islands and ORFs constituting genomic islets is effective for identifying E. hormaechei strains, especially ST78 E. hormaechei strains.

[0048] The present inventors have found that, for specific ORFs, the ORFs constituting the genomic island (LI66_10685, LI66_19120, AM383_04310, AM409_17200, LI62_04085, LI62_09620, LI64_04005, LI63_018475, LI63_021875) and the ORFs constituting the genomic island (LI66_19065, LI66_08680) can be detected to classify E. hormaechei strains, particularly ST78 E. hormaechei strains, with high accuracy (see Table 3 below). By detecting the above ORFs, high strain discrimination ability can be obtained for clones with high clinical isolation frequency in Japan. By detecting all of these ORFs, maximum strain discrimination ability can be achieved. These ORFs were determined by examining their size, ease of primer construction, and ease of amplification using clinical isolates. [Table 3]

[0049] In addition, the ORF LI64_04005 can be detected by PCR using primers of SEQ ID NO: 31 (forward) and 32 (reverse) in the sequence listing. ORF LI66_08680 can be detected by PCR using primers of SEQ ID NO: 33 (forward) and 34 (reverse) in the sequence listing. ORF LI66_19120 can be detected by PCR using primers of SEQ ID NO: 35 (forward) and 36 (reverse) in the sequence listing. The ORF AM409_17200 can be detected by PCR using primers of SEQ ID NO: 37 (forward) and 38 (reverse) in the sequence listing. ORF LI63_021875 can be detected by PCR using primers of SEQ ID NO: 39 (forward) and 40 (reverse) in the sequence listing. ORF LI66_19065 can be detected by PCR using primers of SEQ ID NO: 41 (forward) and 42 (reverse) in the sequence listing. ORF LI63_018475 can be detected by PCR using primers of SEQ ID NO: 43 (forward) and 44 (reverse) in the sequence listing. The ORF AM383_04310 can be detected by PCR using primers of SEQ ID NO: 45 (forward) and 46 (reverse) in the sequence listing. ORF LI62_04085 can be detected by PCR using primers of SEQ ID NO: 47 (forward) and 48 (reverse) in the sequence listing. ORF LI62_09620 can be detected by PCR using primers of SEQ ID NO: 49 (forward) and 50 (reverse) in the sequence listing. ORF LI66_10685 can be detected by PCR using primers of SEQ ID NO: 51 (forward) and SEQ ID NO: 52 (reverse) in the sequence listing. In order to detect the presence or absence of a particular ORF, it is not necessarily necessary to detect the entire length of the ORF, but it is sufficient to be able to determine the presence or absence of the ORF.

[0050] Furthermore, when these are detected using PCR and electrophoresis, which will be described later, the DNA is easily amplified and extra bands are unlikely to appear. In the above, we have mainly explained how to detect ORF(6), which is present in different strains on the chromosome of E. hormaechei, by PCR. However, ORF(6), which is present in different strains on the chromosome of E. hormaechei, can also be detected by other methods, such as hybridization.

[0051] [(7) Genes of the blaIMP-1 group, which are carbapenem degrading enzymes] Carbapenemase-producing Enterobacteriaceae bacteria (hereafter referred to as CPE) are drug-resistant bacteria that have become a problem in recent years, and when examining CPE by bacterial species, it is said that the majority of CPE are ECC species. In addition, since most carbapenemase genes are present on plasmids, the carbapenemase genes can be transmitted to other bacteria by conjugative transfer of plasmids. Therefore, when CPE is detected, it is necessary to take special care and take measures against infection. In Japan, most CPE possess the IMP-1 group as a carbapenemase gene. For these reasons, it is important to detect and monitor ECC that possess the IMP-1 group as a carbapenemase gene.

[0052] Therefore, the present inventors have made it possible to determine the presence or absence of carbapenem antibiotic resistance genes by using gene sequence information obtained from a database and detecting the IMP-1 group gene blaIMP-1 group, which confers resistance to carbapenem antibiotics.

[0053] The blaIMP-1 group gene can be detected by PCR using primers of SEQ ID NO: 29 (forward) and 30 (reverse) in the sequence listing.

[0054] Furthermore, when these are detected using PCR and electrophoresis, which will be described later, the DNA is easily amplified and extra bands are unlikely to appear. Although the above mainly describes the use of PCR to detect the carbapenem-degrading enzyme blaIMP-1 group gene (7), the carbapenem-degrading enzyme blaIMP-1 group gene (7) can also be detected by other methods, such as hybridization.

[0055] [Methods for detecting ORFs and / or genes] The above-mentioned E. hormaechei-specific ORF (1), E. asburiae-specific ORF (2), Enterobacter sp. close to E. asburiae-specific ORF (3), ORFs constituting a genomic island useful for ST type estimation on the E. hormaechei chromosome and ORFs constituting a genomic islet useful for ST type estimation on the E. hormaechei chromosome (4), ORFs constituting a genomic island useful for ST type estimation on the E. asburiae chromosome (5), and / or ORFs constituting a genomic island useful for strain discrimination on the E. hormaechei chromosome and ORFs constituting a genomic islet useful for strain discrimination on the E. hormaechei chromosome (6), and optionally the gene of the blaIMP-1 group, which is a carbapenem degrading enzyme on a plasmid (7), can be detected in any order. Specifically, these ORFs and genes may be detected in any order for each ORF and gene, or multiple ORFs and / or genes may be detected together in any order, or all ORFs and genes may be detected simultaneously. Means for detecting ORFs include PCR and hybridization.

[0056] <pcr> When detecting each ORF and gene separately, for example, a pair of primers (forward and reverse primers) corresponding to each ORF and gene to be detected is used to perform a PCR reaction or the like in an independent system using an ECC DNA extraction sample as a specimen, the reaction solution after the PCR reaction is separated by amplified size by electromigration or the like, and the DNA is stained with ethidium bromide or the like for detection. Alternatively, a fluorescent dye or fluorescent probe is added to the reaction solution in advance to perform a real-time PCR reaction, and the fluorescent signal generated with DNA amplification is detected in real time.

[0057] In addition, when detecting multiple ORFs and / or genes collectively, multiple sets of primers (pairs of forward and reverse primers) corresponding to the multiple ORFs and / or genes respectively can be mixed and placed in the same reaction system to perform a PCR reaction, thereby performing multiplex PCR. In this case, the positions of the primers are set so that the length of the PCR product can be distinguished from others as an independent and clear band after electrophoresis, and the presence or absence of a PCR product corresponding to each ORF and / or gene can be confirmed by the presence or absence of a band obtained by electrophoresis of the solution after the reaction, for example, agarose gel electrophoresis, capillary electrophoresis, etc.

[0058] When all ORFs are to be detected simultaneously, for example, a microarray is used to detect the ORFs. In detection using a microarray, a probe that hybridizes with each ORF is prepared in each well of the microarray, and DNA purified from cultured bacteria by column extraction or phenol-chloroform extraction is hybridized with the probe. After washing off unreacted DNA, the probe is labeled with a fluorescent dye that binds to double-stranded DNA, and the target ORF can be detected by measuring the fluorescence using a device. In addition, the whole genome sequence can be analyzed using a next-generation sequencer, and the above ORFs can be detected on a computer. Among these, multiplex PCR is preferred because it allows efficient simultaneous detection of multiple ORFs using a simple method, but other analytical devices that can obtain equivalent results can also be used.

[0059] (Primer design) The PCR amplification efficiency of the ORFs constituting the ECC genome varies depending on the site. To design primers suitable for multiplex PCR, it is necessary to actually use PCR and multiplex PCR. It is necessary to proceed with the design while confirming the functionality and amplification efficiency. Furthermore, for the ORFs constituting the genomic island useful for ST type estimation on the E. hormaechei chromosome and the ORFs constituting the genomic islet useful for ST type estimation (4), the ORFs constituting the genomic island useful for ST type estimation on the E. asburiae chromosome (5), and the ORFs constituting the genomic island useful for strain discrimination on the E. hormaechei chromosome and the ORFs constituting the genomic islet useful for strain discrimination (6), ORFs with approximately 90% homology in base sequence are found in clinical isolates and databases, so by designing primers in the parts with few mutations so that these homologous ORFs can also be detected, primers that can be used universally for most ECCs that are often clinically isolated and are not easily affected by mutations will be obtained. In designing primers to detect the above-mentioned ORFs (1)(2)(3) for species identification, the ORFs (4) on the E. hormaechei chromosome that differ depending on each ST type, the ORFs constituting the genomic island for ST type estimation on the E. asburiae chromosome (5), and the ORFs constituting the genomic island for strain identification on the E. hormaechei chromosome and the ORFs constituting the genomic islet for strain identification (6), the primers should be arranged so that the primers themselves do not bend and bind to each other at their complementary portions to form a double strand, and different primers do not bind to each other at their complementary portions to form a dimer or higher complexes. Furthermore, in consideration of detection by multiplex PCR, it is preferable to set the GC ratio of the primers to about 50% and to devise a method to match the Tm value. Also, it is desirable to adjust the size of the PCR amplification product to about 50 bp to 2000 bp, preferably 70 bp to 600 bp, so that it can be separated in a short time during electrophoresis and has high amplification efficiency. It is essential to design primers so that the sizes of the PCR amplification products are not the same for ORFs detected in the same reaction system. By setting the above conditions, it is possible to obtain primers and primer sets that provide highly reproducible amplification results in multiplex PCR. Such primers can be designed using commercially available software or software that is freely available and usable via the web, but practical primers can be determined by actually performing PCR and confirming amplification.

[0060] Specifically, as described above in items (1) to (7), (1) a pair of primers for the E. hormaechei specific ORF on the chromosome; (2) one pair of primers for the E. asburiae -specific ORF on the chromosome; (3) a pair of primers for the Enterobacter sp. close to E. asburiae specific ORF on the chromosome; (4) Seven pairs of primers for ORFs constituting a genomic island useful for ST type estimation on the E. hormaechei chromosome and ORFs constituting a genomic islet useful for ST type estimation. (5) Three pairs of primers for the ORFs constituting a genomic island useful for ST typing on the E. asburiae chromosome: (6) Eleven pairs of primers for ORFs constituting a genomic island on the chromosome of E. hormaechei, which are useful for strain discrimination, and ORFs constituting a genomic islet, which are useful for strain discrimination, were used. (7) A pair of primers for the genes of the blaIMP-1 group, which are carbapenem degrading enzymes on the plasmid; The present inventors designed a total of 25 pairs of primers (SEQ ID NOs: 3 to 52 in the sequence listing).

[0061] When multiplex PCR is performed using the above primer sets, it is preferable to perform PCR in a reaction system in which 15 pairs of primers are combined and mixed, including 11 pairs of primer sets, primers for detecting LI66_04665 as an ECC marker (SEQ ID NOs: 1 and 2 in the sequence listing) and primers for identifying the species (SEQ ID NOs: 3 and 4, 5 and 6, and 7 and 8 in the sequence listing), as shown as a primer mixture in Table 4 below, and 12 pairs of primers are combined and mixed, including 11 pairs of primer sets, primers for detecting LI66_04605 as an E. hormaechei marker (SEQ ID NOs: 3 and 4 in the sequence listing), as shown as a primer mixture in Table 5 below. Note that ECC species identification, strain identification, and / or the presence or absence of a resistance gene to carbapenem antibiotics may be performed simultaneously or separately.

[0062] [Table 4]

[0063] [Table 5]

[0064] The specific primers shown above are sequences optimized for amplifying the target ORF or gene, and even if these primers are modified by addition, substitution, deletion and / or insertion of about two bases, they do not lose their function as primers and can amplify the target ORF by PCR. Addition refers to the addition of a base to the 5' or 3' end of the primer shown in the table, and insertion refers to the addition of a base between bases inside the primer, not to the end. However, when the 3' end of the base sequence has an addition or substitution of two or less bases, it is preferable that the base at the 3' end of the base sequence after addition or substitution is complementary to the target ORF.

[0065] From the viewpoint of exerting high performance as a primer, the modification is preferably an addition, substitution, deletion, or insertion of 2 bases or less, more preferably an addition of 2 bases or less or a deletion of 2 bases or less at the 5' or 3' end, and particularly preferably an addition of 1 base or less complementary to the target ORF or a deletion of 1 base or less at the 5' or 3' end. In general, modifications at the 5' end are unlikely to cause the primer to lose its function, while modifications at the 3' end tend to cause the primer to lose its function.

[0066] <Hybridization> When detecting ORFs by hybridization, first, DNA purified by column extraction or phenol-chloroform extraction from cultured bacteria (ECC) is denatured with alkali and fixed on a nylon membrane, a cellulose membrane, or a microplate. A probe that hybridizes with each ORF to be detected in the present invention is created. The probe may be artificially synthesized DNA or may be created by PCR using the primers described above. The probe is labeled with biotin, digoxigenin, fluorescent dye, or the like. The bacterial extract DNA is hybridized with the probe, and the signal is captured by adding an enzyme, coloring, or emitting light depending on the label of the probe. This is performed for each detection ORF.

[0067] [Methods for bacterial species identification, strain identification, and / or determination of the presence or absence of resistance to carbapenem antibiotics] The procedure for performing multiplex PCR to detect the above ORFs and genes (1) to (7) and for identifying the bacterial species and strain of the ECC of the present invention and determining the presence or absence of a resistance gene to a carbapenem antibacterial agent will be described below.

[0068] (Identification of fungal species) ECC-like bacteria are isolated from patient-derived specimens such as blood, cerebrospinal fluid, sputum, and throat swabs using a medium capable of isolating ECC, such as blood agar medium (for general bacterial isolation) or MacConkey medium. They are then identified as ECC by morphological confirmation using Gram staining, biochemical properties, mass spectrometry, etc. Strains identified as ECC are cultured overnight at 37°C in a liquid medium capable of growing ECC or in an agar medium (such as nutrient agar medium or tryptosoy agar medium). (DNA extraction) DNA is extracted from the cultured cells by heat extraction, phenol extraction, DNA extraction using a commercially available DNA extraction kit, or by heat extraction in which the cells are suspended in distilled water or Tris-EDTA buffer and heated, and used as a sample (template) for PCR reaction. (PCR reaction) In multiplex PCR, 3 ORFs for species identification, 7 ORFs whose state varies depending on each ST type on the E. hormaechei chromosome, 3 ORFs whose state varies depending on each ST type on the E. asburiae chromosome, and 11 ORFs whose state varies depending on each strain on the E. hormaechei chromosome, as well as LI66_04665 (SEQ ID NO: 105) as an ECC common marker and bla IMP-1 group, which is a resistance gene against carbapenem antibiotics, are detected. Specifically, the number of primer pairs (forward primers and reverse primers) corresponding to the ORF group to be detected are divided into two groups, 15 pairs and 12 pairs, and each group is placed in the same reaction tube and PCR reaction is performed all at once. Note that species identification, clone identification, and strain identification may be performed simultaneously or separately. (Electrophoresis) The PCR amplified products are electrophoresed using gels such as agarose gels under conditions that allow sufficient separation of DNA fragments of approximately 50 bp to 600 bp. The migration distance is approximately 5 to 6 cm, and in the case of minigels, sufficient separation is possible at 100 V for about 50 minutes. After electrophoresis, the samples are stained with ethidium bromide or Cyber ​​Green and photographed. It is also possible to separate and image the PCR products using various capillary electrophoresis devices such as the QIAGEN QIAxcel Connect and the Agilent 2100 Bioanalyzer.

[0069] (Result judgment) A maximum of 10 or 12 bands will appear. In the genotyping method of the present invention, the band sizes are known in advance, so it is determined whether or not there are bands of the desired size in each strain and reaction system. In some cases, non-specific bands other than the desired size may appear, but these non-specific bands are ignored. A binary code is created with 1 if bands of the desired size (bands corresponding to each ORF or gene and bands corresponding to the positive control) are amplified, and 0 if no amplification is observed.

[0070] This code may be created for each ORF or gene of (1) to (7), or may be created as a single code for all of them, or may be created as a single code for the ORFs or genes of (1) to (7), etc. When the method of the present invention for identifying bacterial species, distinguishing bacterial strains, and / or determining the presence or absence of a resistance gene to a carbapenem antibacterial agent is used in actual medical practice, it is preferable to create the results of several ORFs or genes together as a single code, since creating a single code for the ORFs or genes of (1) to (7) together would result in a large number of digits that would be difficult to read.

[0071] As mentioned above, the ORFs (1) to (3) are related to the identification of bacterial species, the pattern of (4) is highly correlated with the ST type of E. hormaechei, and the pattern of (5) is highly correlated with the ST type of E. asburiae, so (4) and (5) are useful for estimating the ST type. Comparing the patterns of the ORFs (6) enables the identification of E. hormaechei strains. In addition, the presence or absence of resistance genes to carbapenem antibiotics can be determined by detecting the gene (7).

[0072] Therefore, it is preferable to create a single code for the detection results of the ORFs or genes in (4) and (7), it is preferable to create a single code for the detection results of the ORFs or genes in (5) and (7), and it is preferable to create a single code for the detection results of the ORFs in (6).

[0073] The binary code thus created is then converted to a decimal code, for example, to reduce the number of digits and make it easier to read, and used as the genotype code. An example of the above-mentioned code creation is shown in Table 6 below. The example in Table 6 is for isolate number 1 in Figure 1A. [Table 6]

[0074] First, the presence or absence of each ORF or gene in the ECC to be tested is detected by PCR and electrophoresis using appropriate primers, and coded in binary form, which is then converted to a decimal form to obtain the genotype code (Code).

[0075] In the example shown in Table 6, code 1 is a compilation of the detection results of ORFs (4) and (7), so when genotype codes are created for multiple E. hormaechei strains, the code 1 is a number that corresponds to the ST type of E. hormaechei. This number can be used to determine whether the strain is a pandemic clone or not. Code 2 is a strain-specific code, and can be mathematically divided into 2048 genotypes. The ORFs in code 2 were selected to provide high strain discrimination ability for most E. hormaechei strains.

[0076] Specifically, for example, the presence or absence of 21 ORFs (AM432_16960 to LI66_10685) and blaIMP-1 in 14 ECC clinical isolates was binary-coded and then decimal-coded in the same manner as above, as shown in Table 7 below.

[0077] [Table 7]

[0078] In Table 7, the top line shows the strain number, and the second line from the top shows the Sequence type (ST) determined by MLST analysis. By comparing the genotype codes obtained in this manner, it is possible to easily and objectively identify strains isolated at different times or in different facilities.

[0079] Furthermore, most of the ORFs targeted for detection in the present invention are ORFs that are not related to the pathogenicity, etc., of ECC. Genes with specific functions, such as those related to pathogenicity, can increase the probability of the ECC proliferating in the host, or their gene products can become targets of the host's immune system, thereby decreasing the probability of survival. In other words, it is believed that many ECCs have such genes, or conversely, that very few ECCs have them. In the present invention, appropriate ORFs are selected to avoid such bias when performing genotyping. The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples. EXAMPLES

[0080] Example 1 The ECC14 strains clinically isolated from various medical institutions were genotyped by ORF detection using the following procedure.

[0081] (ECC strain culture) Fourteen strains identified as ECC were cultured overnight at 37°C in tryptic soy liquid medium.

[0082] (DNA extraction) DNA was extracted from 10 μL of the culture medium using a commercially available DNA extraction reagent, Cica Geneus DNA Extraction Reagent (Kanto Chemical), and used as a sample (template) for the PCR reaction.

[0083] (Preparation of PCR reaction solution) The enzyme used was AptaTaq DNA Master Mix from Roche. The reaction volume was 20 μL, and the composition was 4 μL of 5×AptaTaq Master Mix, 4 μL of 10 mmol / L urea solution, and 4 μL of primer mixture prepared in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). 8 μL of the above template was added to this.

[0084] Multiplex PCR was performed for 27 ORFs using two reaction tubes, one with 15 primer combinations (reaction system 1) and the other with 12 primer combinations (reaction system 2). The primer combinations and their final concentrations are shown in Table 8 (reaction system 1) and Table 9 (reaction system 2) below.

[0085] [Table 8-1] [Table 8-2]

[0086] [Table 9]

[0087] (PCR reaction) The mixture of template and reaction solution was placed in a thermal cycler (Veriti 96, Applied Biosystems) and a cycle of 94°C for 15 seconds and 60°C for 1 minute was repeated 15 times, followed by 10 cycles of 94°C for 15 seconds and 60°C for 3 minutes.

[0088] (Electrophoresis) A minigel was made using agarose gel (4% Agarose KANTO HC in TBE buffer), and 5 μL of the PCR product was electrophoresed at 100 V for 50 minutes. The migration distance was about 5 cm. After electrophoresis, the gel was stained with ethidium bromide and photographed.

[0089] (Result judgment) A maximum of eight bands appeared per reaction in reaction system 1, and a maximum of nine bands appeared in reaction system 2. Whether or not a band of the desired size was present in each strain and reaction system was determined, and a binary code similar to that in Table 6 above was created, with a value of 1 if a band of the desired size was amplified and a value of 0 if no amplification was observed. The binary code was then converted to a decimal system to create a genotype code. The results are shown in Figure 1A and Table 7.

[0090] In Table 7, as mentioned above, code 1 identifies the ST type, but it can be seen that code 2 further distinguishes strains of the same ST type.

[0091] Example 2 Below, an example in which about two bases are changed in the primer sequence will be described more specifically. As in the previous examples, ECC14 strains clinically isolated from various medical institutions were subjected to genotyping by ORF detection according to the following procedure.

[0092] (ECC strain culture) Fourteen strains identified as ECC were cultured overnight at 37°C in tryptic soy liquid medium.

[0093] (DNA extraction) DNA was extracted from 10 μL of the culture medium using a commercially available DNA extraction reagent, Cica Geneus DNA Extraction Reagent (Kanto Chemical), and used as a sample (template) for the PCR reaction.

[0094] (Preparation of PCR reaction solution) The enzyme used was AptaTaq DNA Master Mix from Roche. The reaction volume was 20 μL, and the composition was 4 μL of 5×AptaTaq Master Mix, 4 μL of 10 mmol / L urea solution, and 4 μL of primer mixture prepared with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) as the solvent. 8 μL of the above template was added to this.

[0095] Multiplex PCR was performed for 27 ORFs using two reaction tubes, one with 15 primer combinations (reaction system 1) and the other with 12 primer combinations (reaction system 2). The primer combinations and their final concentrations are shown in Table 10 (reaction system 1) and Table 11 (reaction system 2) below.

[0096] [Table 10-1] [Table 10-2]

[0097] [Table 11]

[0098] SEQ ID NO:53 is a sequence in which one base has been added to SEQ ID NO:1. SEQ ID NO:54 is SEQ ID NO:2 with two additional bases. SEQ ID NO:55 is SEQ ID NO:3 with two bases deleted, SEQ ID NO:56 is SEQ ID NO:4 with two additional bases. SEQ ID NO:57 is SEQ ID NO:5 with one additional base added. SEQ ID NO:58 is a sequence having two base substitutions compared to SEQ ID NO:6, SEQ ID NO:59 is a sequence obtained by inserting one base into SEQ ID NO:7. SEQ ID NO:60 is SEQ ID NO:8 with one additional base. SEQ ID NO:61 is SEQ ID NO:9 with two additional bases. SEQ ID NO:62 is a sequence in which two bases have been inserted into SEQ ID NO:10. SEQ ID NO:63 is a sequence having two base substitutions compared to SEQ ID NO:11, SEQ ID NO:64 is SEQ ID NO:12 with one additional base. SEQ ID NO:65 is a sequence having one base substitution in comparison with SEQ ID NO:13, SEQ ID NO:66 is SEQ ID NO:14 with two bases deleted, SEQ ID NO:67 is SEQ ID NO:15 with two additional bases. SEQ ID NO:68 is SEQ ID NO:16 with two bases deleted, SEQ ID NO:69 is SEQ ID NO:17 with two additional bases. SEQ ID NO: 70 is a sequence in which two bases have been inserted relative to SEQ ID NO: 18, SEQ ID NO: 71 is SEQ ID NO: 19 with two additional bases. SEQ ID NO: 72 is a sequence in which two bases have been substituted relative to SEQ ID NO: 20, SEQ ID NO: 73 is SEQ ID NO: 21 with one additional base. SEQ ID NO: 74 is SEQ ID NO: 22 with two bases deleted, SEQ ID NO: 75 is SEQ ID NO: 23 with one additional base added. SEQ ID NO: 76 is SEQ ID NO: 24 with two additional bases. SEQ ID NO: 77 is SEQ ID NO: 25 with two bases deleted, SEQ ID NO:78 is a sequence in which two bases have been substituted relative to SEQ ID NO:26, SEQ ID NO: 79 is SEQ ID NO: 27 with one additional base added. SEQ ID NO:80 is SEQ ID NO:28 with one base deleted, SEQ ID NO:81 is SEQ ID NO:29 with two additional bases. SEQ ID NO:82 is the same as SEQ ID NO:30 except that one base has been deleted.

[0099] SEQ ID NO:83 is SEQ ID NO:31 with two additional bases. SEQ ID NO:84 is a sequence in which two bases have been substituted relative to SEQ ID NO:32, SEQ ID NO:85 is SEQ ID NO:33 with two additional bases. SEQ ID NO:86 is a sequence in which two bases have been substituted relative to SEQ ID NO:34, SEQ ID NO:87 is SEQ ID NO:35 with two bases deleted, SEQ ID NO:88 is SEQ ID NO:36 with one additional base added. SEQ ID NO:89 is SEQ ID NO:37 with two additional bases. SEQ ID NO: 90 is a sequence in which two bases have been inserted relative to SEQ ID NO: 38, SEQ ID NO: 91 is a sequence obtained by deleting two bases from SEQ ID NO: 39, SEQ ID NO: 92 is SEQ ID NO: 40 with two additional bases. SEQ ID NO: 93 is SEQ ID NO: 41 with two additional bases. SEQ ID NO: 94 is a sequence obtained by inserting two bases into SEQ ID NO: 42. SEQ ID NO:95 is SEQ ID NO:43 with two bases deleted, SEQ ID NO:96 is a sequence having two base substitutions compared to SEQ ID NO:44, SEQ ID NO: 97 is SEQ ID NO: 45 with one additional base added. SEQ ID NO:98 is a sequence in which two bases have been substituted relative to SEQ ID NO:46, SEQ ID NO: 99 is SEQ ID NO: 47 with two bases deleted, SEQ ID NO: 100 is SEQ ID NO: 48 with two additional bases. SEQ ID NO: 101 is SEQ ID NO: 49 with two additional bases. SEQ ID NO: 102 is a sequence in which two bases have been substituted relative to SEQ ID NO: 50, SEQ ID NO: 103 is SEQ ID NO: 51 with two additional bases. SEQ ID NO:104 is SEQ ID NO:52 with one base deleted.

[0100] (PCR reaction) The mixture of template and reaction solution was placed in a thermal cycler (Veriti 96, Applied Biosystems) and a cycle of 94°C for 15 seconds and 60°C for 1 minute was repeated 15 times, followed by 10 cycles of 94°C for 15 seconds and 60°C for 3 minutes.

[0101] (Electrophoresis) A minigel was made using agarose gel (4% Agarose KANTO HC in TBE buffer), and 5 μL of the PCR product was electrophoresed at 100 V for 50 minutes. The migration distance was approximately 5 cm. After electrophoresis, the gel was stained with ethidium bromide and photographed.

[0102] (Result judgment) A maximum of eight bands appeared per reaction in reaction system 1, and a maximum of nine bands appeared in reaction system 2. Whether or not a band of the desired size was present in each strain and reaction system was determined, and a binary code similar to that in Table 6 above was created, with a value of 1 if a band of the desired size was amplified and a value of 0 if no amplification was observed. The binary code was then converted to a decimal system to create a genotype code. The results are shown in Figure 1B. Similar results to Figure 1A and Table 7 were obtained.< / pcr>

Claims

1. A method for species identification and / or strain identification of a test bacterium, comprising: On the chromosome of the test bacteria, (1) Enterobacter hormaechei-specific open reading frame (ORF), (2) Enterobacter asburiae-specific ORF, (3) Enterobacter sp. close to E. asburiae-specific ORF (4) ORFs constituting a genomic island useful for ST type estimation on the E. hormaechei chromosome and ORFs constituting a genomic islet useful for ST type estimation, (5) ORFs constituting genomic islands useful for ST type estimation on the chromosome of E. asburiae, (6) ORFs constituting genomic islands and genomic islets useful for strain identification on the E. hormaechei chromosome and classifying the gene by genotype based on the combination of the presence or absence of the ORF.

2. (7) Genes encoding the blaIMP-1 group of carbapenem-degrading enzymes The method of claim 1 further comprising detecting the presence or absence of

3. (a) detecting the presence or absence of ORFs (1), (4) and / or (6); (b) detecting the presence or absence of the ORFs (1), (2), and (3); and / or (c) detecting the presence or absence of the ORFs (2) and (5); The method according to claim 1 or 2.

4. The ORF of (1) is LI66_04605 (SEQ ID NO: 106), the ORF of (2) is NF29_17240 (SEQ ID NO: 107), the ORF of (3) is ECNIH4_02210 (SEQ ID NO: 108), the ORFs constituting the genomic island on the E. hormaechei chromosome of (4) are six types: AM432_16960 (SEQ ID NO: 109), AM432_02885 (SEQ ID NO: 110), AM432_06075 (SEQ ID NO: 112), AM432_12345 (SEQ ID NO: 115), AM451_02655 (SEQ ID NO: 116), and AM432_08555 (SEQ ID NO: 118), and the ORF constituting the genomic islet is one type: AM432_17020 (SEQ ID NO: 114), and the ORF of (5) is E. (6) the ORFs constituting the genomic island on the E. asburiae chromosome are ACJ69_15600 (SEQ ID NO: 111), NF29_16315 (SEQ ID NO: 113), and AB190_16475 (SEQ ID NO: 117); and / or (6) E. hormaechei The method described in claim 1 or 2, wherein the ORFs constituting the genomic island on the chromosome of the ST78 strain are nine types: LI64_04005 (SEQ ID NO: 119), LI66_19120 (SEQ ID NO: 123), AM409_17200 (SEQ ID NO: 124), LI63_021875 (SEQ ID NO: 125), LI63_018475 (SEQ ID NO: 127), AM383_04310 (SEQ ID NO: 128), LI62_04085 (SEQ ID NO: 129), LI62_09620 (SEQ ID NO: 120), and LI66_10685 (SEQ ID NO: 121), and the ORFs constituting the genomic islet are two types: LI66_08680 (SEQ ID NO: 122) and LI66_19065 (SEQ ID NO: 126).

5. (1) detecting the ORF by PCR using primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 3 and 4 (wherein the nucleotide sequences may have additions, substitutions, deletions, and / or insertions of two or less nucleotides); (2) detecting the ORF by PCR using primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 5 and 6 (note that the nucleotide sequences may have additions, substitutions, deletions, and / or insertions of two or less nucleotides); (3) detecting the ORF by PCR using primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 7 and 8 (wherein the nucleotide sequences may have additions, substitutions, deletions, and / or insertions of two or less nucleotides); (4) ORF is detected by PCR using seven pairs of primers consisting of seven combinations of base sequences shown in SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, and SEQ ID NOs: 27 and 28 (however, the above base sequences may have addition, substitution, deletion, and / or insertion of two or less bases), (5) ORF is detected by PCR using three pairs of primers consisting of three combinations of base sequences shown in SEQ ID NOs: 13 and 14, SEQ ID NOs: 17 and 18, and SEQ ID NOs: 25 and 26 (however, the above base sequences may have addition, substitution, deletion, and / or insertion of two or less bases); (6) ORF is detected by PCR using 11 pairs of primers consisting of 11 pairs of combinations of base sequences shown in SEQ ID NOs: 31 and 32, SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, SEQ ID NOs: 47 and 48, SEQ ID NOs: 49 and 50, and SEQ ID NOs: 51 and 52 (however, the above base sequences may have addition, substitution, deletion, and / or insertion of 2 or less bases), and / or The gene (7) is detected by PCR using primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 29 and 30 (however, the nucleotide sequences may have additions, substitutions, deletions, and / or insertions of two or less nucleotides). The method of claim 4.

6. The method of claim 5, wherein the base sequence does not have any additions, substitutions, deletions and / or insertions of bases.

7. The method according to claim 5, wherein the PCR method is a multiplex PCR method.

8. The method of claim 1, wherein the results of detecting the presence or absence of ORFs (4), (5), and / or (6) are binary coded by replacing them with 1 (presence) and 0 (absence), respectively.

9. Detection results for the presence or absence of ORFs (4), (5), and / or (6). and / or (7) Detection result of the presence or absence of the gene 3. The method of claim 2, wherein the values ​​are binary coded by replacing the values ​​with 1 (presence) and 0 (absence), respectively.

10. 10. The method according to claim 8, wherein the binary coded result is further coded into a decimal code.

11. 3. The method according to claim 1, wherein the test bacteria are E. cloacae and related species.

12. Eleven pairs of primers consisting of combinations of eleven pairs of base sequences shown in 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: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, SEQ ID NOs: 27 and 28, and SEQ ID NOs: 29 and 30 (however, the above-mentioned base sequences may have additions, substitutions, deletions, and / or insertions of two or less bases), as well as SEQ ID NOs: 31 and 32, SEQ ID NOs: 33 and 34, and SEQ ID NO: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, SEQ ID NOs: 47 and 48, SEQ ID NOs: 49 and 50, and SEQ ID NOs: 51 and 52 (however, the above-mentioned base sequences may have additions, substitutions, deletions, and / or insertions of two or less bases), for use in genotyping ECC and determining the presence or absence of resistance to carbapenem antibiotics.

13. A method for isolating subjects carrying bacterial species and / or strains resistant to carbapenem antibiotics from subjects not carrying such species and / or strains, based on information on the bacterial species and / or strains identified and / or differentiated by the method of claim 1 or 2.

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