Patent Grant
8110359
1. Field of the Invention
The present invention relates to a method of identifying a serotype of Klebsiella pneumoniae, in particular to a method using specific polymerase chain reaction (PCR) primer sets designed according to a fragment of a capsular polysaccharide synthesis (cps) region to identify a K57 or a NTUH-N1 serotype.
2. Background of the Invention
Klebsiella pneumoniae belongs to the family of Enterobacteriaceae; which is a Gram-negative, facultative anaerobic, rod shaped bacterium with polysaccharide capsules. It's an opportunistic infectious pathogen. Strains of Klebsiella pneumoniae exist in the respiratory or digestive tract of healthy people. Infection occurs most commonly in people with low immunity that usually causes serious infection, and may result in pneumonia, septicemia, urinary tract infections, or wound infections and the like.
Pyogenic liver abscess caused by Klebsiella pneumoniae, which was always combined with septicemia, was found with unique clinical characteristics over the past two decades. This is a global emerging disease, particularly common in Taiwan. Different from the traditional disease caused by Klebsiella pneumoniae, people acquired this disease led an active daily life prior to hospitalization. Most patients do not have intra-abdominal infections or biliary tract diseases but may have peritonitis or septic shock, or may further be combined with bacteremia, septic endophthalmitis, meningitis, and so on.
The known virulence factors of Klebsiella pneumoniae include capsular polysaccharides, lipopolysaccharide, adhesions, membrane and extracellular protein. Among them, capsular polysaccharides are the major virulence factor and the base for serotype classification. There are at least 77 serotypes for Klebsiella pneumoniae according to serum epidemiology. The virulence of each strain varied with serotype. For example, serotype K1 or K2 has higher severity of infection (more virulent) than others, which cause pyogenic liver abscess easily, followed by combination with meningitis and endophthalmitis. The identification of serotypes was carried out by immune response using the immune sera (antibodies) to react with the extracted capsules of Klebsiella pneumoniae from the patients to determine the precipitation line with the reference strains of all serotypes. However, drawbacks of this method include low specificity, low sensitivity, and high expenses of serum.
Pyogenic liver abscess induced by infection of Klebsiella pneumoniae is susceptible to first generation antibiotics cephalosporin and gentamicin. However, mortality as high as 10-20% for primary liver abscess and as high as 30-40% among those with meningitis has been reported with effective doses of antibiotics. Due to the serious and invasive Klebsiella pneumoniae disease in epidemiology, it is important to develop a Klebsiella pneumoniae serotype detection method to facilitate the early diagnosis and rapid treatment in order to lower the mortality and provide insight to the prevention and treatment of diseases associated with Klebsiella pneumoniae.
The objective of the present invention is to provide a method of identifying a serotype of Klebsiella pneumoniae using specific PCR primer sets. The primer sets are designed according to the sequence of capsular polysaccharide synthesis (cps) region. The method comprises: (1) extracting DNA from a sample of Klebsiella pneumoniae for use as a DNA template; (2) performing PCR using the DNA template from Step (1), and primer sets of PCR primers comprising a primer set of nucleotide sequence #8282-#19243 of SEQ ID NO:44 and a primer set of nucleotide sequence #6039-#19131 of SEQ ID NO:45; and (3) identifying a K57 serotype of Klebsiella pneumoniae if PCR products are found from the primer set of SEQ ID NO:44, or identifying a NTUH-N1 serotype of Klebsiella pneumoniae if PCR products are found from the primer set of SEQ ID NO:45.
Another objective of the present invention is to provide a method of identifying a serotype of Klebsiella pneumoniae based on immune response. The method comprises: (1) obtaining capsular extracts from a sample of Klebsiella pneumoniae; (2) reacting the capsular extract of Step (1) with anti-NTUH-N1 antiserum; and (3) identifying a NTUH-N1 serotype of Klebsiella pneumoniae if positive result is revealed.
Yet another objective of the present invention is to provide an isolated and biologically pure Klebsiella pneumoniae strain, comprising an identifiable characteristics of Klebsiella pneumoniae A1517 stored in Bioresource Collection and Research Center (Food Industry Research and Development Institute, Hsinchu, Taiwan) with an accession number of BCRC No. 910412 as well as the identifiable characteristics of serotype NTUH-N1. The strain can cause pyogenic liver abscess and septicemia, which contains the nucleotide sequence of SEQ ID NO:45 and can be used to prepare anti-NTUH-N1 antiserum.
Still yet another objective of the present invention is to provide a kit for detecting a serotype of Klebsiella pneumoniae, comprising a primer set of nucleotide sequence #8282-#19243 of SEQ ID NO:44 or a primer set of nucleotide sequence #6039-#19131 of SEQ ID NO:45.
Still yet another objective of the present invention is to provide an isolated gene of capsular polysaccharide synthesis (cps) from Klebsiella pneumoniae, comprising SEQ ID NO:44 or SEQ ID NO:45, and a complementary sequence of SEQ ID NO:44 or SEQ ID NO:45. The Klebsiella pneumoniae can cause pyogenic liver abscess and septicemia clinically. The isolated gene can be used in rapid molecular diagnosis to determine if the patients are infected by serotype K57 or NTUH-N1 of Klebsiella pneumoniae, and to have early diagnosis and treatment for decreasing the mortality.
These methods and the kit comprise the primer set of nucleotide sequence #8282-#19243 of SEQ ID NO:44 is selected from the group consisting of a first primer set (SEQ ID NO:21 and SEQ ID NO:22) and a second primer set (SEQ ID NO:23 and SEQ ID NO:24); while the primer set of nucleotide sequence #6039-#19131 of SEQ ID NO:45 is selected from the group consisting of a third primer set (SEQ ID NO:25 and SEQ ID NO:26), a forth primer set (SEQ ID NO:27 and SEQ ID NO:28), a fifth primer set (SEQ ID NO:29 and SEQ ID NO:30), a sixth primer set (SEQ ID NO:31 and SEQ ID NO:27), a seventh primer set (SEQ ID NO:32 and SEQ ID NO:33), and a eighth primer set (SEQ ID NO:34 and SEQ ID NO:35).
The present invention is further explained in the following embodiment illustration and examples. Those examples below should not, however, be considered to limit the scope of the invention, it is contemplated that modifications will readily occur to those skilled in the art, which modifications will be within the spirit of the invention and the scope of the appended claims.
The present invention used the strains listed in Table 1 to establish a method of identifying a serotype of Klebsiella pneumoniae in the field samples. Klebsiella pneumoniae strain A1517 was screened and isolated by the present invention, which was stored in Bioresource Collection and Research Center (Food Industry Research and Development Institute, Hsinchu, Taiwan) with an accession number of BCRC 910412 on Nov. 20, 2008. The nucleotide sequences of capsular polysaccharide synthesis (cps) region from A1142, A1517, and A7754 strains were determined. The specific primers were designed to identify a serotype of Klebsiella pneumoniae according to these sequences to perform PCR. Classic immune responses or other serotype detection method with high sensitivity were used to confirm the accuracy of the serotype detection method in the present invention. Strain A1517 was therefore identified to have a novel serotype NTUH-N1, and strain A1142 as well as strain A7754 were determined to belong to serotype K57.
1. Bacterial Strains and Preparation of Plasmids
All the bacterial strains used in the present invention were listed in Table 1, which include forty-two clinical isolates of Klebsiella pneumoniae obtained from patients admitted to the National Taiwan University Hospital (NTUH) with pyogenic liver abscess with or without septic complications, such as meningitis and the cps genotypes of A1142, A7754, and A1517; twenty-one non-blood isolates from nonseptic patients at the NTUH; thirteen strains from patients at En Chu Kong Hospital (ECKH; Sansia, Taiwan); thirty four strains obtained from patients at Far Eastern Memorial Hospital (FEMH; Banciao, Taiwan); twenty four strains purchased from the American Type Culture Collection (ATCC), including strain MGH78578; eighty strains from Canada (Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada); and one strain from Finland (Department of Clinical Microbiology, Kuopio University Hospital, Finland). Klebsiella pneumoniae and Escherichia coli were cultured in Luria-Bertani (LB) medium supplemented with appropriate antibiotics, including ampicillin (100 μg/ml) or kanamycin (50 μg/ml).
Klebsiella pneumoniae strains
Escherichia coli
2. Serum resistance test for strains A1142, A7754, and A1517
The serotypes of strains A1142, A7754, and A1517 isolated from patients were identified using serum resistance test. These bacterial strains were mixed with human sera from healthy volunteers. After incubation with serum for 3 h, the CFU counts in A1142, A7754, and A1517 decreased respectively to about 1%, 20%, and 4% of those in the initial inoculum. Therefore, the three strains were all serum sensitive and were less virulent than serotype K1 and serotype K2.
3. Nucleotide Sequencing of the cps Region
The nucleotide sequences of capsular polysaccharide synthesis (cps) region obtained from PCR amplification (
Primers for conserved sequences flanking the cps region were designed according to the sequences of the cps genomic regions of Klebsiella pneumoniae. PCR amplifications were performed and the nucleotide sequences were determined (
PCR amplification was carried out with primers CPS-F and CPS-R2 (SEQ ID NO:1 & SEQ ID NO:2) for A1142 and A7754 bacterial strains. In addition, 5′-end of cps region was amplied with primers pre-galF-F and 9534 cps 5′R (SEQ ID NO:13 & SEQ ID NO:14), and 3′-end of cps region was amplied with primers 13-730F and post gnd R (SEQ ID NO:15 & SEQ ID NO:16) to get a complete cps region of A1142 and A7754 bacterial strains. The nucleotide sequence of the complete cps region of A1142 was listed in SEQ ID NO:44 with an accession number of AB334776 in GenBank database.
Another PCR amplification was carried out with primers wzi-2 and gnd-R (SEQ ID NO:4 & SEQ ID NO:8) for A1517 bacterial strain. In addition, 5′-end of cps region was amplied with primers yegH and 1517 conserve 5′R (SEQ ID NO:17 & SEQ ID NO:18), and 3′-end of cps region was amplied with primers 1517 conserve 3° F. and ugd (SEQ ID NO:19 & SEQ ID NO:20) to get a complete cps region of A1517 bacterial strain. The nucleotide sequence of the complete cps region of A1517 was listed in SEQ ID NO:45 with an accession number of AB334777 in GenBank database.
The PCR reaction contained the abovementioned reactants with the cycling program of: 96° C. for 3 min, followed by 30 temperature cycles of 96° C. for 30 s, 52° C. for 15 s, and 72° C. for 2 to 5 min. A final elongation step of 10 min at 72° C. was added. The sequence of PCR products were determined and combined with the sequence of the middle cps region to obtain a complete cps region of 10-20 kb DNA fragments (from galF to gnd).
Referring to
On the other hand, sequence analysis of amplified 10-20 kb fragments of cps region of A1142 and A7754 with primer pairs CPS-F and CPS-R2 showed similar sequence. This indicated that both strains have the same serotype. In addition, the A1142 and a reference strain which belong to K57 have a very similar cps variable region (44 bases difference among 4323 bases), while A1517 has a quite unique sequence among any known serotype in cps region. It is suggested that this cps region in the present invention was responsible for capsular polysaccharide synthesis according to the figure and the predicted function of ORFs.
4. Serotyping of A1142, A7754 and A1517
K1 and K2 specific primers were used in PCR to detect the cps genotype of A1142, A7754 and A1517. Detection of serotype K1-K6 with Klebsiella antisera (SEIKEN) at the same time showed no result on these three strains. Therefore, they did not belong to serotype K1-K6.
Specific primer pairs were designed according to the nucleotide sequences of cps regions of these three strains since they did not belong to the major serotypes K1 or K2, the pyogenic liver abscess associated Klebsiella pneumoniae strains. PCR was performed by using reference strains with 77 known serotypes as templates. The PCR reaction contained: 1 μl template DNA to a solution containing 2 μl of 10× buffer, 2.5 U of Taq polymerase, 1 mM of each deoxynucleoside triphosphates (dNTPs), and primers at final concentrations of 0.4 mM each in a total volume of 20 μl. The cycling program consisted of one denaturation step of 3 min at 96° C. and 30 cycles of 30 s at 96° C., 30 s at 53° C., and 30 s at 72° C. Other serotype detection methods, as described below, were also used to confirm the PCR results.
(1) Identification of Serotypes A1142 and A7754
The designed specific primers for cps region of A1142 or A7754: a first primer set: 9471F and 9897R (SEQ ID NO:21 & SEQ ID NO:22), and a second primer set: 1142×F and 1142XR (SEQ ID NO:23 & SEQ ID NO:24) were used in cps PCR genotyping with the 77 known serotypes. The annealing sites for these two primer pairs are shown in
Further PCR screening was performed with primers 9471F and 9897R among total of 173 strains, which contained 21 NTUH nonblood isolates, 13 ECKH strains, 34 FEMH strains, 24 ATCC strains, 80 Canada strains, and 1 Finland strain. Only 2 of the 13 ECKH strains (designated E7 and E12) and the Finland strain have positive results.
Immunoblot serotyping was further performed to confirm that A1142 belonged to cps genotype K57 in the present invention. Ten microliters of each capsular extract was vacuum spotted onto a nitrocellulose membrane. The membrane was blotted and blocked, followed by hybridization with K57 serotype-specific antiserum. The results are shown in
The cps PCR-restriction fragment length polymorphism (RFLP) method was carried out in the K57 reference strain, A1142, and A7754. PCR amplifications were amplified with primers wzi2 and rCPS2 (SEQ ID NO:4 & SEQ ID NO:43). The amplified products were digested with HincII, followed by electrophoresis (
(2) Identification of Serotypes A1517
Six sets of primers, including a third primer set: 1517×F and 1517XR (SEQ ID NO:25 & SEQ ID NO:26), a forth primer set: 1517YF and 1517YR (SEQ ID NO:27 & SEQ ID NO:28), a fifth primer set: 12R STAR and 12 STAR (SEQ ID NO:29 & SEQ ID NO:30), a sixth primer set: 1517YF and 15R-2 (SEQ ID NO:31 & SEQ ID NO:27), a seventh primer set: 7R STAR and 7 STAR (SEQ ID NO:32 & SEQ ID NO:33), and a eighth primer set: 3-2 and 12R-2 (SEQ ID NO:34 & SEQ ID NO:35) were used in A1517 cps PCR genotyping. The annealing sites were shown in
Further PCR screening was performed with primers 12R STAR and 12 STAR among the abovementioned 173 strains. Only one of the Canada strains (designated Canada 05-14) showed a positive result, which suggested that they belonged to same cps genotype of A1517.
Immunoblot serotyping was further performed to confirm the result of A1517 serotyping in the present invention (with the abovementioned procedures, except the hybridization antiserum was replaced with anti-A1517 antiserum). The results are shown in
The cps PCR-restriction fragment length polymorphism (RFLP) method was also carried out in the A1517 and Canada 05-14 strains. PCR amplifications were amplified with primers CPS-1 and rCPS. The amplified products were digested with HincII, followed by electrophoresis (
To characterize the K57 determinant gene of the cps region in A 1142, an unmarked deletion mutant of the wzy gene located in the cps variable region of A1142 (K57) was generated in the present invention. The function of the wzy gene was studied, which was associated with capsule synthesis using a string test. A cps deletion strain of A1142 was generated by using a pKO3-Km plasmid constructed in the invention. First, primers 9471F (952 bp upstream of wzy, SEQ ID NO:21) and R5 (824 bp downstream of wzy, SEQ ID NO:36) were designed to amplify the target gene wzy and its flanking region. The amplified products were cloned to a pGEM-T Easy vector, followed by inverse PCR to delete the entire wzy gene with the intact flanking regions remained. The flanking regions of wzy were digested by restriction enzymes and ligated into pKO3-Km vector. Plasmid pKO3-Km_wzy was transformed into A1142, and a wzy gene deleted strain was generated after serial selection. For trans-Complementation test, the intact wzy gene and its ribosomal binding site were cloned to a modified pGEM-T Easy vector, which was transformed into the wzy mutant of A1142.
The result showed that the wzy mutant of A1142 lost mucoviscosity with a string test, indicating attenuated capsule synthesis. This mutant strain also showed negative results after immunoblot serotyping with anti-K57 antiserum. In trans-complementation test, the positive result was restored after transformation of a wzy-carrying pGEM-T Easy-Km vector into the mutant strain. Therefore, wzy was the essential gene for capsular synthesis of serotype K57. And the gene cluster of the cps region was responsible for the capsular synthesis.
Countercurrent immunoelectrophoresis and double immunodiffusion are commonly used for identifying the serotypes of Klebsiella pneumoniae. However, these two methods had problems in correct serotyping. Initially, double immunodiffusion was used to confirm if the serotype of A1142 belonged to K57. Sample to be assayed formed an ambiguous precipitation line in contrast to that for the K57 reference strain. Modified immunoblot serotyping method presently employed showed an increased sensitivity and reduced the consumption of antiserum. The result of immunoblot analysis with anti-A1517 antiserum also showed several cross-reactions. DNA sequences were different among A1517 and these known serotypes. Therefore, cps PCR genotyping would be a more sensitive and specific way for serotype identification.
On the other hand, a molecular serotyping method such as cps PCR-RFLP analysis, has a higher discriminatory power than classical serotyping according to recent studies. However, cps PCR-RFLP pattern variations were found among strains of serotype K57. This was also shown in the abovementioned example. The cps PCR-RFLP pattern of reference strain K57 was quite different from those of A1142 and A7754 though sequences of these strains in the cps variable region were very similar (
From the description and results of the abovementioned examples, the present invention provided a more sensitive and specific method for serotyping, a PCR-based cps genotyping for capsular type, to solve the problems of insufficient specificity and sensitivity in conventional immune method. This PCR-based cps genotyping method not only solves the problems of insufficient specificity and sensitivity caused by conventional immune method, but can be applied in clinical diagnosis with the advantages of rapidity and low cost. In addition, the rate of unidentifiable strains can also be reduced by this method.
| Number | Date | Country | Kind |
|---|---|---|---|
| 97146314 A | Nov 2008 | TW | national |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20110065111 | Sampath et al. | Mar 2011 | A1 |
| Number | Date | Country | |
|---|---|---|---|
| 20100136534 A1 | Jun 2010 | US |
