Rapid identification and differentiation of the species of the Mycobacterium chelonae／abscessus complex by hsp65 and rpoB PCR－RFLP

LI Yan－bing，ZHANG Yuan－yuan，HUANG Ming－xiang，ZHAO Xiu－qin，ZHANG Li－shui ，LIU Wen－en，WAN Kang－lin

（1．Xiangya Hospital，Central South University，Changsha 410008，China；2．National Institute for Communicable Disease Control and Prevention，Chinese Center for Disease Control and Prevention／State Key Laboratory for Infections Disease Prevention and Control，Beijing 102206，China；3．Fuzhou Pulmonary Hospital／Clinical Teaching Hospital of Fujian Medical University，F(xiàn)uzhou 350008，China）

Rapid identification and differentiation of the species of the Mycobacterium chelonae／abscessus complex by hsp65 and rpoB PCR－RFLP

LI Yan－bing1，2，ZHANG Yuan－yuan1，HUANG Ming－xiang3，ZHAO Xiu－qin1，ZHANG Li－shui3，LIU Wen－en2，WAN Kang－lin1

（1．Xiangya Hospital，Central South University，Changsha 410008，China；2．National Institute for Communicable Disease Control and Prevention，Chinese Center for Disease Control and Prevention／State Key Laboratory for Infections Disease Prevention and Control，Beijing 102206，China；3．Fuzhou Pulmonary Hospital／Clinical Teaching Hospital of Fujian Medical University，F(xiàn)uzhou 350008，China）

The Mycobacterium chelonae／abscessus（M．chelonae／abscessus）complex belongs to the rapidly growing genus Mycobacterium （RGM）．It is one of the most important pathogenic members of Mycobacterium leading to nosocomial infections and outbreaks．It includes members of M．chelonae，M．immunogenum，M．a(chǎn)bscessus，M．massiliense，and M．bolletii．In order to investigate the epidemiological characteristics of the M．chelonae／abscessus complex in China and to conduct the molecular methods for species identification of M．chelonae／abscessus，we collected clinical M．chelonae／abscessus complex strains identified by phenotypic tests．Members were verified by sequencing of 16S rRNA．Species and subspecies were identified by hsp65 and rpoB PCR－RFLP methods．In total，27 clinical specimens were identified as Mycobacterium chelonae／abscessus complex by phenotypic tests．16s rRNA gene sequence analysis of all 27 clinical samples shared over 99．7%similarity with M．chelonae and M．a(chǎn)bscessus．Species identification with hsp65 PCR－RFLP and rpo B PCR－RFLP revealed that 18 specimens were M．a(chǎn)bscessus and 4 were M．a(chǎn)bsecces．The remaining 5 samples displayed a pattern that failed to match any previously reported pattern．Thus，this might represent a novel species that is part of the Mycobacterium chelonae／abscessus complex．We identified that a majority of the chronic lung infection in China is caused by the M．chelonae／abscessus complex．Specifically，the M．a(chǎn)bscessus species might be the most infectious，while other species in the complex can still cause infection．Interestingly，there may be a novel or previously unidentified species that is a part of the complex．Finally，we show that species identification can be carried out more accurately by combined use of hsp65 and rpoB PCR－RFLP．

non－tuberculosis mycobacterium，Mycobacterium chelonae／abscessus group，PCR－RFLP

Nontuberculous mycobacteria （NTM）has been reported to cause chronic respiratory diseases and nosocomial infections—a trend that has increased in recent years．The Mycobacterium chelonae／abscessus （M．chelonae／abscessus）complex，which belongs to the rapidly growing Mycobacterium（RGM）family，is one of the most important pathogenic Mycobacterium．Infection by this complex can lead to contaminated skin wounds，post－surgical soft tissue infections［1－2］，nosocomial infections［3］，or outbreaks［4－6］．The M．chelonae／abscessus complex contains at least five species，including M．chelonae，M．a(chǎn)bscessus，M．immunogenum［7］，M．massiliense［8］，and M．bolletii［9］．M．a(chǎn)bscessus，in particular，is associated with hypersensitivity chronic pneumonia and other respiratory illnes－ses，including bronchitis，fibrillation，and occupational lung disease［10－11］．Increasing incidences of nosocomial infections and occupational respiratory illnesses caused by the M．chelonae／abscessus complex and the need for species－specific differences in patient management［12］have made it clinically important to rapidly and unambiguously identify individual species of this complex．Although the species are phenotypically similar to one another，they may differ in their sensitivity to antibiotics．Although it has been reported that the M．chelonae／abscessus complex is naturally resistant to many antibiotics，amikacin and clindamycin have been proposed to be effective drugs for the treatment of infection［13］．However，more recent reports［8］suggest that doxycycline is more effective than amikacin and clindamycin for the treatment of M．massiliense infection．A recent study by Won－Jung Koh concluded that treatment response rates to combined antibiotic therapies，including regimens with clarithromycin，were much higher in patients with M．massiliense lung disease than in those with M．a(chǎn)bscessus lung disease［14］．A long period of time was needed for the treatment of M．chelonae／abscessus complex infection．This is especially important considering that the likelihood of resistance increases over time．Thus，there are important clinical considerations for species－specific differences in the M．chelonae／abscessus complex that are centered on the choice of appropriate therapies．

Compared with traditional methods，molecular－based methods for species identification were more effective．PCR－based methods have proved to be useful for the rapid diagnosis of Mycobacterium infection．Amplification and sequencing of the 16S rRNA gene of mycobacterial species were performed for many years for taxonomic and phylogenetic studies．This was generally considered the"reference method"for detecting species differentiation of Mycobacteria［15－16］．16S rRNA gene based phylogenetic analysis has had a major influence on our analysis of taxonomic relationships among NTM．However，since M．chelonae and M．a(chǎn)bscessus share more than 99%similarity in terms of 16S rRNA gene sequence，they can only be classified as part of the M．chelonae／abscessus complex and cannot be distinguished from one another．

PCR－restriction fragment length polymorphism （PCR－RFLP）analysis is a useful tool for species identification of Mycobacterium since it yields species－specific patterns by digesting PCR products with a specific restriction endonuclease．Variation analysis of the partial fragment of 65－k Da heat shock protein （hsp65）［17－18］（over 441 bp）or rpo B encoding theβsubunit of RNA polymerase（over 360 bp）by PCR－RFLP has been widely used for identification of Mycobacterium［19］．Genes for hsp65 and rpo B exist in almost all species of bacteria．They are both evolutionarily conserved to the same extent as 16S rRNA，but they display more polymorphism in their nucleotide sequences．In general，both require multiple enzymes in various combinations in a two－step restriction digestion process to differentiate particular species．

In this study，isolates previously identified as NTM were confirmed to be part of the M．chelonae／abscessus complex by amplifying and sequencing the 16S rRNA gene（over 1 500 bp）．PCRRFLP of both hsp65 and rpoB were used for species or subspecies distinction of the M．chelonae／abscessus complex．Finally，three patterns of hsp65／rpoB PCR－RFLP were identified from all M．chelonae／abscessus complex strains that were collected．

Material and methods

Bacterium strains

A total of 178 clinical strains were isolated from patients suspected to have tuberculosis（TB）in three local hospitals in China，including Fuzhou Pulmonary Hospital of Fujian Province，Chaoyang Center for Disease Control and Prevention，Beijing，and the General Hospital of the People＇s Liberation Army of China．Patients were screened from 2005 to 2010 in local laboratories．Repeating tests of L－J and PNB／TCH media were performed in our laboratory following standard procedures［20］．Twenty－eight Mycobacterium tuberculosis （M．tuberculosis）isolates were rejected from our study，and 150 isolates were verified as NTM strains．Conventional biochemical tests were performed for the 150 NTM isolates，and 27 isolates were identified as M．a(chǎn)bscessus strains，which were used in this study．M．a(chǎn)bscessus ATCC19977，M．massiliense CIP108297 and M．chelonae ATCC35752 were stored at－70℃in our laboratory．They were resuscitated when needed and used as reference strains．

Phenotypic tests

Phenotypic tests were performed according to standard procedures［20－21］．Such tests included analysis of growth rate，pigment production，growth at 42℃，and on MacConkey agar，arylsulfatase activity，nitrate reduction activity，and tolerance to 5%NaCl．

Genomic DNA extraction and amplification of 16S rRNA，hsp65 and rpoB

Genomic DNA from 27 clinical isolates and reference strains were extracted from 1 mg wet bacteria by a genomic DNA extraction kit（TIANGEN BIOTECH，BEIJING）according to the man－ufacturer＇s instructions．The concentration of nucleic acid extracted was determined by a spectrophotometer（Nanodrop Technologies，Inc，Wilmington，DE，USA）．The full length 16S rRNA gene sequence（1 500 bp）was amplified with primers 16S rRNA P1（5′－AGAGTTTGATCCTGGCT CAG－3′）and 16S rRNA P2 （5′－AAGGAGGTGATCCAGCCGCA－3′）．The total volume of each reaction was 50μl，including 25μl 2×Taq PCR Master Mix （TIANGEN BIOTECH，BEIJING），2μl of each primer（10μM／μl），5μl DNA solution（30μg／ml），and up to 50μl with distilled water．The amplification reactions were performed as follows：10 min at 94℃followed by 30 cycles of 94℃for 1 min，63℃for 45 s，and 72℃for 1 min，and a final extension step at 72℃ for 10 min．hsp65（441 bp）and rpo B （360 bp）were amplified as previously described［17，19］．

Sequencing of 16S rRNA and phylogenetic relationship and gene diversity analysis

To sequence the 1 500 bp 16S rRNA gene，in addition to the primers for PCR，the following primers were set and used： p1 forward （5′－ACGCTCGGACCCTACCTATT－3′）； p2 reverse（5′－CTCGTGTCGTGAGATGTTGG－3′）；p2．W1R1（5′－GCGTTGCATCGAATTAAT－3′）；p2．W1R2（5′－CCACCTTCCGAGTTGA－3′）．The sequences were aligned with BioEdit（BioEdit software，version 7．0．5）．This was followed by cluster analysis with ClustalW program．A phylogenetic tree of the 16S rRNA gene was drawn with the assistance of the Neighbor－Joining method of Molecular Evolutionary Genetics Analysis（MEGA software，version 5）．The number of bootstrap trails was chosen to be 1 000．Additionally，by using MEGA software，gene diversities were analyzed by comparing the sequences of each gene target within clinical isolates and within the reference strains．

hsp65 PCR－RFLP and rpoB PCR－RFLP

For PCR－restriction analysis of hsp65，the amplified products were digested with Bst EⅡand HaeⅢ as previously described［17］．Briefly，10μl of the PCR amplification product，1μl enzyme BstEⅡ （10 U／μl）or HaeⅢ （10 U／μl），2μl of restriction buffer（10×），and 2μl of distilled water were added to a fresh tube．The mixtures were in－cubated for at least 2 hours at 60℃for Bst EⅡand at 37℃for HaeⅢ．Restriction fragments were separated by electrophoresis in 4%Metaphor agarose gel（Rockland ME USA）．RFLP patterns were determined after calculating the molecular weights of the band fragments by Ec3 Imaging System（UVP，America）．M．a(chǎn)bscessus ATCC19977 were used as the control．For PCR－restriction analysis of rpo B，the amplified products were digested with MspⅠand HaeⅢas previously described［19］．The mixture was incubated for at least 2 hours at 37℃for MspⅠand HaeⅢ．Restriction fragments were separated and RFLP patterns were determined as described above．

Results

Phenotypic characteristics analysis

Twenty－seven clinical isolates （CIs）shared similar phenotypic characteristics with each other and with the strains M．a(chǎn)bscessus，M．massiliense，and M．chelonae（Table 1）．They were characterized as acid－fast rods that grew into colonies by 3－7 days on L－J solid culture medium at 37℃．Additionally，they were without pigment production and exhibited 3－day arylsulfatase activities．The isolates that were most similar to the M．a(chǎn)bscessus strain，for example FJ05160，grew on MacConkey agar medium without crystal violet and L－J slants containing 5%NaCl at 30℃．The isolates that were the most similar to M．chelonae were unable to tolerate the presence of 5%NaCl．

Phylogenetic relationship analysis based on the sequence of 16S rRNA

The 16S rRNA gene sequence of 27 CIs shared more than 99．7%similarity with M．chelonae and M．a(chǎn)bscessus．Thus，they were indistinguishable from one another and identified as a M．chelonae／abscessus complex．We drew a phylogenetic tree based on sequence analysis of the 16S rRNA gene．The tree shows the relationships between the 27 CIs and the strains of selected M．a(chǎn)bscessus that were related to rapidly growing Mycobacterium．The phylogenetic tree illustrated that all 27 isolates fell into a similar group with M．chelonae，M．a(chǎn)bscessus，M．massiliense，and M．bolletii（Figure 1）．

Tab．1 Comparison on biochemical characteristics of clinical isolates and closely related mycobacterial species type strains

Type strains were selected based on their relatedness to the rapidly growing mycobacterium M．a(chǎn)bscessus． These included M．chelonae，M．a(chǎn)bscessus， M． massiliense， M． bolletii，M．immunogenum， and M． salmoniphilum．M．tuberculosis H37Ra and H37Rv were included as outlier groups．The 16s rRNA gene sequence of all type strains was obtained from the published database in GenBank．CI1 to CI27 of 27 clinical isolates were numbered for this study（Table 1）．

Gene diversity analysis

Sequence analysis of the 16s rRNA gene showed only minimal diversity between the 27 CIs and M．a(chǎn)bscessus ATCC19977．The few changes that were detected included an A insert at 752 bp of CI5 and CI10，a G insert at 760 bp of CI12，and a C→T substitution at 1 003 bp position of CIs 5，6，7，10，and 11（Figure 2）．

Pattern of hsp65 PCR－RFLP

We compared the patterns of hsp65 PCRRFLP from the 27 CIs with patterns from published data．All analyses identified the CIs as members of the M．a(chǎn)bscessus strain，including 23 isolates that were mostly identical to M．a(chǎn)bscessus．In fact，they shared the same hsp65 PCR－RFLP patterns of 235 bp and 210 bp（Bst EⅡ）／145 bp，70 bp，60 bp，and 55 bp（HaeⅢ）with the published hsp65 PCR－RFLP pattern of M．a(chǎn)bscessus ATCC19977［17］．The other 4 isolates shared the same patterns of 235 bp and 210 bp（BstEⅡ）／200 bp，70 bp，60 bp，and 50 bp（HaeⅢ）with the published hsp65 PCR－RFLP pattern of the M．a(chǎn)bscessus group （M． abscessus ATCC14472，M．Massiliense CIP108297， and M．bolletii CIP108541）［17］．The hsp65 PCR－RFLP patterns of 27 CIs are shown in Figure 3．

Fig．2 Compared gene diversity in the 1 500 bp 16S rRNA gene of some CIs with that of M．a(chǎn)bscessus ATCC19977

Fig．3 PCR－RFLP patterns of hsp65 gene digested by BstEⅡ（A）and HaeⅢ （B）M：Size standards of 20 bp DNA ladders；M．a(chǎn)b：M．a(chǎn)bscessus ATCC 19977；Line 1－27：Clinical isolates；Line 1，12，24 and 25 shared the same pattern which was reported as the pattern of M．a(chǎn)bscessus ATCC14472， M． Massiliense CIP108297 and M．bolletii CIP108541［17］；Line 2－11，13－23，26 and 27 shared the same pattern of M．a(chǎn)bscessus ATCC19977．

Pattern of rpoB PCR－RFLP

Patterns of rpo B PCR－RFLP were obtained by using enzymes MspⅠand HaeⅢfor clinical isolates．We compared the pattern of the 27 CIs to those of published data［19］．Twenty－two clinical isolates displayed a pattern of 100 bp，95 bp，and 80 bp （Msp Ⅰ）／130 bp，100 bp，and 90 bp（HaeⅢ）．This pattern matched those of M．chelonae and M．a(chǎn)bscessus，which were previously published．We also identified 5 strains that displayed a novel rpoB PCR－RFLP pattern of 200 bp，100 bp，and 80 bp （Msp Ⅰ）／200 bp and 135 bp（HaeⅢ）．These included CI5 （FJ05242）in line 5，CI6（FJ05517）in line 6，CI7（FJ05520）in line 7，CI10（FJ05534）in line 10，and CI11（FJ05541）in line 11（Figure 4）．All 5 failed to match previously published patterns and，thus，separated themselves from other clinical M．chelonae／abscessus complex strains that display the pattern 100 bp，95 bp，and 80 bp （Msp Ⅰ）／130 bp，100 bp，and 90 bp （Hae Ⅲ）．Patterns of rpo B PCR－RFLP are shown in Figure 4．

Fig．4 PCR－RFLP patterns of the rpoB gene digested by MspⅡ （A）and HaeⅢ （B）M：Size standards of 20 bp DNA ladders；M．a(chǎn)b：M．a(chǎn)bscessus ATCC 19977；Line 1－27：Clinical isolates；Line 1－4，8，9，12－27 shared the same pattern of M．chelonae or M．a(chǎn)bscessus；Line 5，6，7，10 and 11 shared a pattern of 200 bp，100 bp and 80 bp（Msp I）／200 bp and 135 bp（Hae III），failing to match any pattern reported before．

Discussion

Of 178 isolates identified as NTM by local laboratories were verified by phenotypic tests in our own hands．According to our analysis，28 were identified as M．tuberculosis strains，and 150 were identified as NTM．Twenty－seven of the 150 NTM shared phenotypic characteristics of the M．chelonae／abscessus complex，including the ability to grow in both L－J and PNB／TCH culture mediums and the ability to grow to mature colonies in less than 7 days．Additionally，they did not produce pigment and displayed arylsulfatase activity by the third day．All strains were further validated by genetic tests．We sequenced the 16S rRNA gene（over 1 500 bp）from all strains．We constructed a phylogenetic tree based on the 16S rRNA gene sequence of the 27 CIs，which indicated that all 27 isolates fell into one group and only little diversity existed among the isolates．One important aspect was that we identified a C→T base substitution at position 1 003 in the 16S rRNA gene sequence of 5 isolates，including CI5，CI6，CI7，CI10，and CI11（FJ05242， FJ05517， FJ05520， FJ05534，and FJ05541，respectively）．Thus，the 27 isolates were classified as part of the M．chelonae／abscessus complex by both phenotypic and genetic analyses．In order to distinguish the M．chelonae／abscessus complex isolates at the species or subspecies levels，hsp65 PCR－RFLP and rpoB PCR－RFLP meth－ods were used in our study．In total，we identified three distinct patterns of hsp65 and rpo B PCRRFLPs in all collected M．chelonae／abscessus complex strains． Twenty－three of the 27 isolates shared the same hsp65 PCR－RFLP pattern of 235 bp and 210 bp （Bst EⅡ）／145 bp，70 bp，60 bp，and 55 bp（HaeⅢ）with a previously published hsp65 PCR－RFLP pattern［13］of M． abscessus ATCC19977．Four isolates shared the same pattern of 235 bp and 210 bp （BstE Ⅱ ）／200 bp，70 bp，60 bp，and 50 bp（HaeⅢ）with a previously published hsp65 PCR－RFLP pattern from the M．a(chǎn)bscessus group［13］（M．a(chǎn)bscessus ATCC14472，M．Massiliense CIP108297，and M．bolletii CIP108541）．Interestingly，5 of the 23 isolates displayed a unique，previously unidentified hsp65 PCR－RFLP pattern of 200 bp，100 bp，and 80 bp（MspⅠ）／200 bp and 135 bp（HaeⅢ）．This failed to match any known patterns of the M．chelonae／abscessus complex，including those obtained from M．chelonae，M．immunogenum，M．a(chǎn)bscessus，M．massiliense，and M．bolletii．These five were also unique when compared to another 18 clinical M．a(chǎn)bscessus strains with the pattern 100 bp，95 bp，and 80 bp（MspⅠ）／130 bp，100 bp，and 90 bp （Hae Ⅲ ）． In total， of the 27 M．chelonae／abscessus complex strains，18 isolates were identified as M．a(chǎn)bscessus，four isolates were identified to be part of the M．a(chǎn)bscessus group but the exact species could not be identified，and 5 isolates differed from all type strains of the M．chelonae／abscessus complex．These 5 isolates were the ones that possessed the C→T substitution at 1 003 bp position in the 16S rRNA gene．These five may represent a novel species but more work needs to be done to definitively prove this．

For detection and identification of Mycobacterium at the species level，PCR－based molecular methods tend to be more efficient compared to conventional biochemical methods．The hsp65 gene and the rpoB gene have been primarily used to differentiate species in the M．chelonae／abscessus complex，but each target has its own limits in differentiating closely related Mycobacteria．Gene diversity may exist in the restriction loci of either hsp65 or rpo B．From our study，23 of the 27 M．chelonae／abscessus complex isolates shared the same pattern．Among the 23 isolates，5 isolates separated from another 18 isolates by displaying different patterns of rpo B PCR－RFLP．Significant diversity may exist within restriction loci for both enzymes digesting the rpo B gene in the five isolates．hsp65 PCR－RFLP was considered to be reliable in species identification of Mycobacterium［17］and been used routinely for Mycobacterium species identification in the laboratory［18］．However，in our study，when we identified the M．a(chǎn)bscessus isolates at the species level by PCR－RFLP and sequencing of the hsp65 gene，22．2% （6／27）of the results were inaccurate．Instead of using hsp65 PCR－RFLP on its own，greater accuracy can be ensured by combining hsp65 PCR－RFLP with rpo B PCR－RFLP．

Based on species identification in this study，we demonstrate that most of the chronic lung infection caused by the M．chelonae／abscessus complex in China is due specifically to the M．a(chǎn)bscessus species．Other species within the M．chelonae／abscessus complex may also cause infection，and，interestingly，there may be a novel species within the complex that has yet to be identified．If only hsp65 PCR－RFLP or rpo B PCR－RFLP is used individually，some isolates among the M．chelonae／abscessus complex may not be properly identified at the species level．Instead，to ensure proper identification，combined hsp65 PCR－RFLP and rpo B PCR－RFLP should be used．Alternatively，one could use other methods that have demonstrated greater identification ability，such as the motile target gene sequencing method［23］．

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hsp65 and rpoB PCR－RFLP用于龜／膿腫分枝桿菌復(fù)合群種的快速鑒定＊

李艷冰1，2，張媛媛2，黃明翔3，趙秀芹2，張麗水3，萬(wàn)康林2，劉文恩1

目的探討和評(píng)價(jià)hsp65 and rpoB PCR－RFLP用于龜／膿腫分枝桿菌復(fù)合群種的快速鑒定。方法收集經(jīng)PNB／TCH鑒別培養(yǎng)基表型鑒定和16s rRNA基因測(cè)序鑒定為龜／膿腫分枝桿菌復(fù)合群的臨床分離菌株，用hsp65 and rpoB PCR－RFLP進(jìn)行種／亞種鑒定。結(jié)果經(jīng)表型鑒定為非結(jié)核分枝桿菌的27株臨床菌株，16s rRNA基因測(cè)序分析與龜／膿腫分枝桿菌的同源性達(dá)到99．7%。經(jīng)hsp65 PCR－RFLP and rpoB PCR－RFLP鑒定18株為膿腫分枝桿菌（M．a(chǎn)bscessus），4株為潰瘍分枝桿菌（M．a(chǎn)bsecces），另5株表現(xiàn)為獨(dú)特的指紋特征，可能是一個(gè)新的亞種。結(jié)論能夠快速進(jìn)行龜／膿腫分枝桿菌復(fù)合群種／亞種的鑒定。

非結(jié)核分枝桿菌；龜／膿腫分枝桿菌復(fù)合群；PCR－RFLP

R378．91

A

1002－2694（2012）07－0645－08

劉文恩，Email：liuwenen＠hotmail．com；萬(wàn)康林，Email：wankanglin＠icdc．cn李艷冰、張媛媛和黃明翔在本文中為同等貢獻(xiàn)。

1．中南大學(xué)湘雅醫(yī)院，長(zhǎng)沙 410008；2．中國(guó)疾病預(yù)防控制中心傳染病預(yù)防控制所／傳染病預(yù)防控制國(guó)家重點(diǎn)實(shí)驗(yàn)室，北京 102206；3．福州肺科醫(yī)院／福建醫(yī)科大學(xué)臨床教學(xué)醫(yī)院，福州350008

10．3969／cjz．j．issn．1002－2694．2012．07．001

This research was supported by the projects 2008ZX100／03－010 and 2011ZX10004－001 of China Mega－Project for Infectious Disease．

Wan Kang－lin，Email：wankanglin＠icdc．cn；Liu Wen－en，Email：liuwenen＠hotmail．com

Li Yan－bing，Zhang Yuan－yuan and Huang Ming－xiang contributed equally to this study．

Fig．1 Phylogenetic tree based on the 16S rRNA gene sequence of 27 CIs（CI1－27）and type strains drawn by the neighbor－joining method

＊國(guó)家重大傳染病專(zhuān)項(xiàng)課題（2008ZX100／03－010和2011ZX10004－001）

2012－05－09；Revision accepted：2012－06－12