Antimicrobial susceptibility testing and genotyping of Mycobacterium avium isolates of two tertiary tuberculosis designated hospital, China

Infection, Genetics and Evolution 36 (2015) 141–146

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Antimicrobial susceptibility testing and genotyping of Mycobacterium avium isolates of two tertiary tuberculosis designated hospital, China Guomei Wei a,1, Mingxiang Huang b,1, Guirong Wang a, Fengmin Huo a, Lingling Dong a, Yunxu Li a, Hairong Huang a,⁎ a National Clinical Laboratory on Tuberculosis, Beijing Key laboratory for Drug-resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, China b Fuzhou Pulmonary Hospital, Fujian Province, China

a r t i c l e

i n f o

Article history: Received 15 July 2015 Received in revised form 14 September 2015 Accepted 15 September 2015 Available online xxxx Keywords: Mycobacterium avium Antimicrobial susceptibility Genotype

a b s t r a c t Background: : Mycobacterium avium is frequently isolated from clinical samples, while the bacteriological features of M. avium clinical isolates from China have never been well defined. Methods: : A total of 50 M. avium isolates were recruited from two tertiary tuberculosis designated hospitals, one located in Beijing whereas another in Fujian Province, which are northern and southern parts of China, respectively. Subspecies identification was conducted by sequencing the variable 3′ end of the hsp65 gene. The susceptibility against 15 antimicrobial agents, widely administered for the treatment of non-tuberculosis mycobacteria (NTM) infections, was tested by broth microdilution assay. Variable number of tandem repeats (VNTR) assay was also performed using the 16-loci genotyping method. Results: : All of the 50 M. avium isolates were identified as M. avium subsp. hominissuis. The drug susceptibility test revealed that clarithromycin (98%, 49/50) and moxifloxacin (86%, 43/50) had the best antimicrobial activities in vitro against the M. avium isolates. The overall Hunter–Gaston Discriminatory Index (HGDI) value for the VNTR typing was 0.95. However, the genotyping method yielded much greater discriminative power for isolates of northern China than that of southern China (1.00 V.S. 0.86, P b 0.05). Conclusion: : M. avium subsp. hominissuis is the dominate subspecies among M. avium clinical isolates in China. The 16-loci VNTR genotyping method is more discriminative in Beijing than in Fujian Province. The bacteriological features of M. avium isolates from different regions of China demonstrated dramatic variations, and stressed the importance of building up knowledge from the local isolates. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The non-tuberculosis mycobacteria (NTM), a complex of various species including opportunistic pathogens for a variety of animal hosts, are ubiquitously present in various environmental sources such as water, soil and biofilms (Glassroth, 2008; Griffith, et al., 2007). Mycobacterium avium complex (MAC) constitutes the pathogens that most commonly isolated from respiratory samples and is responsible for most of the human-associated NTM infections in some countries (O'Brien et al., 1987; Griffith, et al., 2007). As a member of MAC, M. avium is an important pathogen that causes infections in the respiratory tract, lymph node, and, occasionally in the soft tissue of the immunocompetent individuals (Good, 1985; Prince et al., 1989). Moreover, secondary infections with M. avium have gained increased attention over the decades due to longevity of the immunocompromised population and the administration of immunosuppressive chemotherapeutics to cancer ⁎ Corresponding author at: Beimachang Rd 97, Beijing 101149, China. E-mail address: [email protected] (H. Huang). 1 These authors contributed equally to this article.

http://dx.doi.org/10.1016/j.meegid.2015.09.015 1567-1348/© 2015 Elsevier B.V. All rights reserved.

patients (Miguez-Burbano et al., 2006). Newer molecular identification methods have further subdivided M. avium into four major subspecies: M. avium subsp. avium which is well recognized as the causative agent of avian tuberculosis, M. avium subsp. paratuberculosis has consistent association with Crohn's disease, M. avium subsp. silvaticum also causes tuberculosis in birds, and M. avium subsp. hominissuis which is the opportunistic pathogen for both animals and humans (Tell et al., 2001; Harris and Barletta, 2001; Kaevska et al., 2011). Although with distinct pathogenic characteristics and host preference, all the subspecies are considered to be involved in zoonoses. MAC infection is difficult and frustrating to be treated, especially in patients with AIDS, since it is highly resistant to standard antituberculosis agents. Furthermore, there is no routine reference method recommended by the American Thoracic Society/Infectious Diseases Society of America (ATS/IDSA) for susceptibility testing of most of the drugs being administered for MAC treatment. Macrolides are the only antimicrobial agents for which the correlation between in vitro susceptibility tests and in vivo clinical response has been observed. For other antimicrobials, such correlation has never been concluded either because of the existing controversial data, or most possibly, because

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no system evaluation has been performed (Miguez-Burbano et al., 2006; Griffith et al., 2007). Variable number of tandem repeats (VNTR) technology has been widely used for genotyping of Mycobacterium tuberculosis and recently it has also been applied to NTM as well but for a few species only. Researchers from Japan used M. avium tandem repeat (MATR) loci (MATR-VNTR) for genotyping the M. avium isolates and demonstrated that the 15-loci MATR-VNTR possess high discriminative capacity (Moriyama et al., 2006). In this assay, we tested the susceptibility of 50 clinical M. avium isolates, collected from two tertiary tuberculosis designated hospitals in China, against 15 widely used antibiotics for NTM infection treatment, and genotyped the strains by VNTR. The rifampicin resistance determination region (RRDR) of rpoB gene and the ribosomal 16S rRNA (rrs) gene were sequenced to interpret the relationship between specific single nucleotide polymorphisms (SNPs) and drug resistance of M. avium isolates. The systemic bacteriological research on M. avium clinical isolates from China has never been reported before. 2. Materials and methods 2.1. Isolation and identification of M. avium The criteria for isolates recruitment: All the M. avium strains recovered from sputum samples of pneumonia patients visiting Beijing (BJ) Chest Hospital (located in northern China) or Fuzhou Pulmonary Hospital of Fujian (FJ) Province (located in southern China) during the given period of times were recruited; only one isolate was recruited for each patient. Traditional species identification was performed with paranitrobenzoic acid (PNB) containing media to distinguish NTM from M. tuberculosis complex. Sequencing of multiple genes, including 16S rRNA, rpoB and 16S–23S rRNA internal transcribed spacer (ITS) was performed to identify the strains into species level. Consequently, a total of 50 strains were identified as M. avium and included in this study. Among them, 20 isolates were collected in the past 5 years in the Beijing Chest Hospital, where NTM accounted to 2.49% of mycobacterial isolates and M. avium constituted about 5.26% of all the NTM isolates (Wang et al., 2014); in contrast, 30 isolates were collected in the past one year in the Fuzhou Pulmonary Hospital, where NTM accounted to approximately 10.2% of mycobacterial isolates and M. avium accounted for 21.3% of all the NTM isolates (Huang et al., 2014). For subspecies identification, a 1059-bp fragment of the variable 3′ end of the hsp65 gene (from positions 574 to the 6th base downstream the terminator codon) was amplified and sequenced. Primers used for amplification included MAChsp65F_574 (5′-CGGTTCGACAAGGGTTAC

AT-3′) and MAChsp65R (5′-ACTGACTCAGAAGTCCATG-3′), as reported by Turenne (Turenne et al., 2006). hsp65 codes were assigned according to Turenne's nomenclature (Turenne et al., 2006). 2.2. Antimicrobial susceptibility testing (DST) Antimicrobial susceptibility testing was performed at the National Tuberculosis Clinical Laboratory in Beijing Chest Hospital for clarithromycin, moxifloxacin, linezolid, rifampicin, amikacin, ethambutol, capreomycin, levofloxacin, cefoxitin, ofloxacin, antoflolxacin, rifapentine, streptomycin, cycloserine, ciprofloxacin. All the mentioned drugs were purchased from Sigma-Aldrich (USA) except antoflolxacin that was a gift from Anhui Huanqiu Pharmaceutical Co. Ltd (Hefei, China). Stock solutions were prepared in accordance with manufacturer instructions and kept in aliquots at −80 °C. From the stocks, working solutions were prepared in distilled water. The minimal inhibitory concentration (MIC) for 15 antimicrobial agents was determined by broth microdilution method in cationadjusted Mueller–Hinton broth supplemented by 10% OADC according to the guidelines by the Clinical and Laboratory Standards Institute (CLSI) (Clinical and Laboratory Standards Institute, 2011). The breakpoints of the following antimicrobial agents were referred to the CLSI recommendations: clarithromycin ≥ 32 μg/ml; moxifloxacin ≥ 4 μg/ml; linezolid ≥ 32 μg/ml. The breakpoints for some other antimicrobial agents were deciphered from previous literatures: rifampicn ≥ 32 μg/ml (Obata et al., 2006), ethambutol N8 μg/ml (Kobashi et al., 2006), amikacin ≥ 32 μg/ml (Brown-Elliott et al., 2013) and capreomycin ≥ 16 lg/ml (Heifets, 1988). Otherwise, the drug concentrations that inhibited 50% and 90% of the tested strains of M. avium were expressed as MIC50 and MIC90, respectively. The M. avium reference strain (ATCC 25291) was used as control. 2.3. PCR amplification and DNA sequencing of rpoB and rrs genes The rpoB gene fragment, including the region homologous to the 81-bp RRDR of M. tuberculosis were amplified by primers MA-F2 and MA-R2 as described elsewhere (Obata et al., 2006). For the ribosomal 16S rRNA endocing rrs gene, reported as amikacin resistance related gene, primers were used as previously described (Alangaden et al., 1998). PCR mixtures were subjected to 30 cycles of denaturation at 94 °C for 15 s, primer annealing at 60 °C for 30s, and DNA elongation at 72 °C for 1 min. The PCR products were sequenced at RuiBo Biotech Company (Beijing, China). All the sequencing results were compared to the GenBank database. The same amino acid sequence numbering system, used for E. coli, was also applied in this study.

Table 1 Susceptibilities of 50 clinical isolates of M. avium against 15 antimicrobial agents. Antimicrobial agents

Clarithromycin Moxifloxacin Linezolid Rifampicin Amikacin Ethambutol Capreomycin Levofloxacin Cefoxitin Ofloxacin Antofolxacin Rifapentine Streptomycin Cycloserine Ciprofloxacin

No. of strain with different MIC (=μg/ml) ≤0.5

1

2

24 16 3 14 6

2 19 6 11 1

1 8 13 6 4

9 5 3 3 6

5

1 7

1 7

2 19

1 28 1

2 1 4 5

9 13 6 7

8

15

13

4

% resistant strains 8

16 2 1 2

9 7 3 10

11 1 2 1 16 1 4 4 1 17 15 4 4

6

4

32

≥64 1

7 2 9 6

5 3 9 6 5 1

6 6

5 5

7 2 2

8 8 1

16 12 1 41 28 1 49 2 2 5 8 40 1

MIC50

MIC90

1 1 4 1 8 ≥64 ≥64 4 ≥64 8 8 1 8 ≥64 2

8 4 ≥64 ≥64 32 ≥64 ≥64 16 ≥64 32 32 8 ≥64 ≥64 8

2 14 42 30 20 98 84 – – – – – – – –

MIC50 and MIC90, concentration of the antimicrobial agent required for inhibiting 50% of the strains; The breakpoint of antimicrobial agents were recommended by the CLSI: clarithromycin, ≥32 μg/ml; moxifloxacin, ≥4 μg/ml; linezolid, ≥32 μg/ml. For other antimicrobial agents, the breakpoints were taken as recommended in previous literatures: rifampicin ≥32 μg/ml (Obata et al., 2006), ethambutol N8ug/ml (Kobashi et al., 2006), amikacin ≥32 μg/ml (Brown-Elliott et al., 2013), capreomycin ≥16 μg/ml (Heifets, 1988).

143

32 ⩾64 ⩾64 ⩾64 ⩾64

Ciprofloxacin Cycloserine Levofloxacin

Cefoxitin

All of the 50 M. avium isolates were identified as M. avium subsp. hominissuis based on the sequencing analysis of the 3′ end of the hsp65 gene. Five strains (5/50, 10%) from Beijing belonged to hsp65 sequevar code 1, while the remaining 45 strains (45/50, 90%) belonged to hsp65 sequevar code 2. The M. avium ATCC 25291 belonged to code 4. No other hsp65 sequevar was found.

16 16 1 2 32

Streptomycin

3.1. Subspecies identification of M. avium isolates

⩽0.5 2 8 ⩾64 ⩽0.5

Rfapentine Ofloxacin

3. Results

2 4 16 2 8

Antofloxacin

Statistical analyses were performed using SPSS v12.0 (SPSS Inc.). Chi-square and Crosstab analyses were used to test whether the differences between two statistical data were significant, and P value less than 0.05 were defined as significant.

4 4 16 1 8

2.5. Statistical analysis

⩾64 ⩾64 ⩾64 ⩾64 ⩾64

The primers and method reported by Kikuchi et al. (Kikuchi et al., 2009) were used for VNTR typing analysis. The estimated size of PCR products for different alleles was deduced from the allele size range and from the basic unit length as previously reported (Selander et al., 1986). The allelic diversity of each VNTR locus was calculated using Selander's formula and the discriminatory power of VNTR using Hunter and Gaston's formula (Hunter &Gaston, 1988). The VNTR data was analyzed using BioNumerics (Version5.0, Applied Maths, SintMartens-Latem, Belgium) software. Cluster analysis was performed and a dendrogram was generated in BioNumerics using the UPGMA coefficient.

2 2 4 ⩽0.5 4

2.4. VNTR analysis

1 1 8 2 4

G. Wei et al. / Infection, Genetics and Evolution 36 (2015) 141–146

Resistance (%)

1 2 9 7 5 20 15

5.00 10.00 45.00 35.00 25.00 100.00 75.00

0 5 12 8 5 29 27

0.00 16.67 40.00 26.67 16.67 96.67 90.00

Capreomycin Ethambutol

⩾64 ⩾64 ⩾64 ⩾64 ⩾64 8 16 32 8 2

Amikacin Rifapicin

⩽0.5 ⩾64 ⩾64 ⩾64 ⩽0.5 1 2 2 ⩾64 ⩽0.5

Linezolid Moxifloxacin

No. of resistant strains

⩽0.5 ⩽0.5 2 ⩽0.5 1

Resistance (%)

Clarithromycin

Fujian (n = 30)

No. of resistant strains

⩽0.5 8 ⩽0.5 8 4

Clarithromycin Moxifloxacin Linezolid Rifampicin Amikacin Ethambutol Capreomycin

Beijing (n = 20)

Isolates

Drug

BJ-390 BJ-254 BJ-56 BJ-155 BJ-65

Table 2 A comparison of resistant strains between two tertiary hospitals.

Table 3 Susceptibility profile of 5 M. avium isolates belonging to code 1.

The results of the drug susceptibility tests for the 50 M. avium clinical isolates are shown in Table 1. Clarithromycin (98%, 49/50) and moxifloxacin (86%, 43/50) demonstrated the best in vitro antimicrobial activities against the M. avium isolates. Secondly, 80%(40/50), 70%(35/50) and 58%(29/50) of the strains were susceptible to amikacin, rifampicin and linezolid, respectively. However, except one strain, all others were resistant to ethambutol. In addition, the antimicrobial activities of other antibiotics were revealed according to the MIC50 and MIC90 values. The MIC50 and MIC90 values of the three injectable anti-tuberculosis drugs were in the following order: amikacin ≤ streptomycin b capreomycin, whereas the MIC50 and MIC90 values of the five different Fluoroquinolones were in the order as moxifloxacin b ciprofloxacin b levofloxacin b antoflolxacin ≤ ofloxacin. The comparison of the isolates' origins revealed that isolates from Beijing had higher rifampicin resistant rate than those from Fujian Province, but there was no significant statistic difference (P = 0.5, Table 2). Five isolates, belonging to hsp65 sequevar code 1, did not show any specific drug susceptibility feature compared with that of other isolates (Table 3).

⩾64 ⩾64 8 16 2

3.2. Antimicrobial susceptibility profiles of M. avium isolates

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Table 4 SNPs detected within the DRDR of rpoB gene among 5 rifampicin resistant M. avium isolates. Strain

MIC (μg/ml)

Codon (amino acid substitution, position) containing a mutation at position Gly-507

Leu-511

Ser-512

Gln-513⁎

Ser-522

Gly-523

Leu-524

Arg-528

Arg-529

ATCC 25291 FJ-2 FJ-738 FJ-140 FJ-486 BJ-169 (Glu)

≤0.5 ≥64 ≥64 32 32 ≥64 _

GGC GGG _ _ _ _ _

CTG _ _ _ CTC _ _

TCC TCG TCG TCG TCG _ _

CAG _ _ _ _ GAG _

TCG TCC _ TCC _

GGG _ GGT _ GGT

CTC _ _ _ CTG

CGC CGT CGT CGT CGT

CGC _ CGT _ CGT

Same nucleotide as in the ATCC 25291 strain. ⁎ Amino acid substitution.

3.3. SNPs in RRDR of rpoB gene amd rrs gene

3.4. VNTR genotyping and clustering analysis

All of the 35 rifampicin susceptible M. avium isolates and the reference strain (ATCC25291) had wile-type (WT) sequences in the 81-bp core region. Among the 15 rifampicin resistant M. avium isolates, 10 (77.7%) had WT sequences and 5 (33.3%) possessed SNPs in the core region, but only one such SNP caused amino acid change (Gln513Glu). The SNPs are listed in Table 4. None of the 10 amikacin resistant isolates contained mutation in the rrs gene. However, 7 out of 10 (70%) amikacin resistant isolates were found to be cross-resistant to capreomycin.

VNTR differentiated the 50 isolates into five clusters and acquired a total of 27 unique patterns (fig. 1). Among the 23 clustered strains, 22 isolates were from Fujian Province, and only 1 was from Beijing. The 16-loci used for VNTR analysis gave a overall discriminatory power of 0.95, but it was 1.00 and 0.86 for Beijing and Fujian isolates respectively. The allelic differences between M. avium isolates from the two regions were also significant. Five VNTR loci (MATR−3,− 5,−6,−7, and −8) had a high diversity index (h ≥ 0.5) while 1 locus (MATR-12) showed low diversity index (h ≤ 0.10). On the other side, VNTR grouped the

Fig. 1. Cluster analysis of 50 M. avium clinical isolates and ATCC25291 based on VNTR profiles, generated in Bionumerics 6.1.

G. Wei et al. / Infection, Genetics and Evolution 36 (2015) 141–146

Fujian isolates into five clusters and exhibited 8 unique patterns; only 1 VNTR loci (MATR13) had a high diversity index (h ≥ 0.5) whereas 6 VNTR loci (MATR − 2, − 3, − 6, − 9, − 11, − 12 and − 14) did not show any allelic diversity (Table 5). 3.5. Association between drug resistance profiles and mycobacterial genotypes Among the 23 clustered isolates, the percentages of resistance to rifampicin was 8.7%, while was 48.1% for the 27 unclustered isolates. Statistical analysis revealed that rifampicin resistance was commonly more significant among the unclustered strains than the clustered strains (χ2 = 9.206,P = 0.004). On the other hand, resistance against moxifloxacin (χ2 = 0.347,P = 0.430), linezolid (χ2 = 0.144, P = 0.778) and amikacin (χ2 = 0.005,P = 0.736) was not independently associated with cluster (Table 6). 4. Discussion Non-tuberculosis mycobacteria (NTM) infection had been neglected in the past in China mainly because it was considered less important in contrast with tuberculosis. With the advancements in identification methods and increased awareness of those organisms, NTM-associated diseases have gained increasing attentions in the recent years. However, the knowledge on either bacteriology or clinical medicine of NTM infection remains far from being comprehensive. M. avium complex (MAC) is shown to be the most frequently isolated NTM according to

Table 5 A comparison of MATR-VNTR allelic distribution among M. avium isolated from two different geographic regions of China. Locus

Sourcea

MATR-1

BJ FJ BJ FJ BJ FJ BJ FJ BJ FJ BJ FJ BJ FJ BJ FJ BJ FJ BJ FJ BJ FJ BJ FJ BJ FJ BJ FJ BJ FJ BJ FJ

No. of isolates with the specified MATR allele 0

MATR-2c c

MATR-3

MATR-4 MATR-5 MATR-6c MATR-7 MATR-8 MATR-9c MATR-10 MATR-11

c

MATR-12c MATR-13 MATR-14 MATR-15 MATR-16

c

15 30

4 1

1

2

3 10 1

16 20 4

6 30 14 21 1

4

2

1 8 9 28 4

1

10 21 7 21 17 30 12 18 17 30 19 30 17 19 17 30 14 28 4 7

7 1 2

11

3

4

5

6

Allelic diversity (h)b 7

8

1

9 1 4

1

1

6

1 1 1

30

4 9 1 1 1

1 1 3

2

2 7

2

1 7 11 1

1 8 5 1

2

1 3 1 16 23

5

2

0.30 0.43 0.36 – 0.75 – 0.44 0.42 0.57 0.10 0.59 – 0.59 0.42 0.74 0.40 0.23 – 0.17 0.49 0.23 – 0.05 – 0.23 0.52 0.22 – 0.42 0.09 0.28 0.34

a BJ represents strains isolated from north China (n = 20), FJ represents strains isolated from south China (n = 30). b Calculated as described by Selander et al. (Selander et al., 1986). c The locus did not show any allelic diversity for strains isolated from north China.

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Table 6 Drug susceptibilities of 23 clustered and 27 unclutered strains of M. avium. Drug

Strain class

Sensitive strain

Resistant strain

χ2

P

Rifampicin

Clustered Unclustered Clustered Unclustered Clustered Unclustered Clustered Unclustered

21 14 21 22 14 15 19 21

2 13 2 5 9 12 4 6

9.206

0.004

0.347

0.430

0.144

0.778

0.005

0.736

Moxifloxacin Linezolid Amikacin

the limited data published from China (Wang et al., 2010). Apart from some epidemiological information, comprehensive studies on bacteriological features of M. avium clinical isolates are absent in China. In this study, the constitution of different subspecies of M. avium was analyzed and the results categorized all of them as M. avium subsp. honimiussis. This outcome highlighted that China and its surroundings are dominated by this subspecies of M. avium or its characteristics of human preference. The lack of other subspecies is also possible due to inadequate culture methods used to recover such animal pathogens (Tran and Han, 2014). M. avium subsp. hominissuis has also been reported as the dominated subspecies in some other countries as well. Shin et al. identified 77 clinical isolates collected from different countries and found that all of them were M. avium subsp. hominissuis (Shin et al., 2010). Quynh et al. tested a collection of 257 human clinical M. avium strains from the United States and noted 238 (92.6%) being M. avium subsp. hominissuis (Tran and Han, 2014). The drug susceptibility test of our assay provided new information regarding the candidate antimicrobial agents against M. avium. The isolates did not show uniform susceptibility against any of the tested drugs, which indicated that DST is necessary before establishing a regimen. Consistent with previously studies, clarithromycin showed the best in vitro activity against M. avium isolates among the 15 tested antimicrobial agents (Miguez-Burbano et al., 2006). Several other investigators have validated the in vitro activity of quinolones, such as moxifloxacin and ciprofloxacin, against M. avium (Kohno et al., 2007; Tomioka et al., 2000). Our results are in general agreement with theirs, i.e. moxifloxacin showed better in vitro activity against M. avium isolates than other four fluoroquinolones. Comparison of the MICs for three injectable anti-tuberculosis drugs for M. avium isolates showed that amikacin and streptomycin had lower MIC50 and MIC90 values than capreomycin. This finding is also in agreement with previous reports about the stronger activities of streptomycin and kanamycin than capreomycin (Heifets, 1988; Tomioka et al., 2000). In addition to macrolide and aminoglycoside, rifampicin and ethambutol are also included in the macrolide-containing multidrug treatment regimens for MAC disease as recommended by ATS/IDSA (Griffith et al., 2007). In our study, 70% of M. avium were susceptible to rifampicin, while 98% of M. avium were resistant to ethambutol. The substantial discrepancy for the reported drug susceptibility profiles of M. avium isolates might reflect the strain difference in different countries. Molecular approach has been reported to be beneficial while screening drugs for MAC treatment (Obata et al., 2006; Alangaden et al., 1998). However, we only found 1 out of 15 rifampicin resistant isolates which harbored mis-sense mutation within RRDR of rpoB gene, whereas no rrs mutations were found for the 10 amikacin resistant isolates. Our outcomes demonstrated that M. avium can have altogether different drug resistance mechanisms in contrast with M. tuberculosis dose. Although the silent mutations we found in the RRDR are less likely to have relation with rifampicin resistance, but it is very interesting that they were only detected among the rifampicin resistant strains. We presume those sequence polymorphisms might relate with the evolution of drug resistant strains for M. avium, however more information is needed to prove this hypothesis.

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In agreement with the previous report (Kikuchi et al., 2009), our study showed that the 16-loci VNTR analysis yielded a total Hunter– Gaston discriminatory index (HGDI) of 0.95. In this study, 23 isolates were clustered into 5 genotypes thus the clustering rate remained 46%. It is noteworthy that 22 out of the 23 clustered strains were from Fuzhou Pulmonary Hospital, hence the clustering rate in this hospital was 73.3% whereas the HGDI was 0.86. The extremely high clustering rate may reflect contamination during culture, but the drug resistant patterns of the strains in same cluster were extremely very variable, which disproves this hypothesis. Therefore, our VNTR outcomes indicate that some M. avium genotypes with similar genotypes are predominated in the surroundings of Fujian Province and the 16-loci VNTR system had low resolution among Fujian isolates. In our assay, we found that rifampicin resistance was more common among the unclustered strains than the clustered strains. According to other report, human disease-associated M. avium isolates are more resistant to rifampicin than the isolates from natural sources (Saito et al., 1989). On the other hand, cluster indicates a high transmissibility, which is a hallmark of virulence. Those inconsistent outcomes highlighted the necessity of clinically significant analysis for M. avium strains isolated from different regions. This study has several limitations. First, due to small number of the recruited strains and different sampling period between these two hospitals, bias may exist for this assay. Second, since it was mainly a study on pooled strains, we could not develop the clinical relevance with patients in this assay. In conclusion, our study demonstrated that M. hominissuis is the dominate subspecies among M. avium clinical isolates in China, and clarithromycin and moxifloxacin presented strong antimicrobial activities against the M. avium isolates. Additionally, we observed that the 16-loci VNTR genotyping method is more discriminative in Beijing than in Fujian Province. Compared with isolates from Fujian, M. avium was sparsely isolated but less clustered in Beijing area. The bacteriological features of isolates from different regions of China demonstrated dramatic variations, besides reflecting the large size of Chinese territories, it also stressed the importance of building up knowledge from the local isolates. Acknowledgment All the mycobacterial strains used in this project were acquired from the “Beijing Bio-Bank of clinical resources on Tuberculosis” (D131100005313012). The work was supported by the research funding of Infectious Diseases Special Project from Ministry of Health of China (2012ZX10003002-009). and Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (ZYLX201304). References Alangaden, G.J., Kreiswirth, B.N., Aouad, A., Khetarpal, M., Igno, F.R., Moghazeh, S.L., Manavathu, E.K., Lerner, S.A., 1998. Mechanism of resistance to amikacin and kanamycin in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 42, 1295–1297. Brown-Elliott, B.A., Iakhiaeva, E., Griffith, D.E., Woods, G.L., Stout, J.E., Wolfe, C.R., Turenne, C.Y., Wallace, R.J., 2013. In vitro activity of amikacin against isolates of Mycobacterium avium complex with proposed MIC breakpoints and finding of a 16S rRNA gene mutation in treated isolates. J. Clin. Microbiol. 51, 3389–3394. Clinical and Laboratory Standards Institute, 2011. Susceptibility Testing for Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes:Approved Standard M24-A. Clinical and Laboratory Standards Institute, Wayne, PA. Glassroth, J., 2008. Pulmonary disease due to nontuberculous mycobacteria. Chest 133, 243–251.

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