Multilocus Sequence Typing of Mycobacterium xenopi 1

David C. Alexander1,8, Theodore K. Marras2,3,4 Jennifer H. Ma1, Samia Mirza1,2, Daniel 2

Liu1, Julianne V. Kus1,2, Hafid Soualhine5, Vincent Escuyer6, David Warshauer7, Sarah 3

K. Brode3,4, David J. Farrell1, and Frances B. Jamieson1,2 * 4

5

1 Public Health Ontario Laboratories, Public Health Ontario, Toronto, ON, Canada 6

2 University of Toronto, Department of Laboratory Medicine and Pathobiology, Toronto, 7

ON, Canada 8

3 University of Toronto, Department of Medicine, Toronto, ON, Canada 9

4 University Health Network and Mount Sinai Hospital, Department of Medicine, 10

Toronto, ON, Canada 11

5 Laboratoire de Santé Publique du Québec, Institut National de Santé Publique du 12

Québec, Sainte–Anne–de–Bellevue, QC, Canada 13

6 Wadsworth Center, New York State Department of Health, Albany, NY, USA 14

7 Wisconsin State Laboratory of Hygiene, Madison, WI, USA 15

8 Saskatchewan Disease Control Laboratory, Saskatchewan Ministry of Health, Regina, 16

SK, Canada 17

18

19

20

21

* Corresponding author: Frances.Jamieson@oahpp.ca 22

23

24

JCM Accepts, published online ahead of print on 10 September 2014J. Clin. Microbiol. doi:10.1128/JCM.01601-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Abstract 25

Mycobacterium xenopi (Mxe) is an opportunistic mycobacterial pathogen of increasing 26

clinical importance. Surveillance of Mxe is hampered by the absence of tools for 27

genotyping and molecular epidemiology. In this study, we describe the development and 28

evaluation of an effective multilocus sequence typing strategy for Mxe. 29

30

Introduction 31

Mycobacterium xenopi (Mxe) is an opportunistic mycobacterial pathogen of increasing 32

clinical importance. Mxe disease is most common among patients with underlying 33

medical conditions (e.g. chronic obstructive pulmonary disease, bronchiectasis, 34

immunodeficiencies) (1-4). Cases usually present as tuberculosis–like pulmonary disease, 35

but extrapulmonary (e.g. bone, joint, and muscle) infections have also been reported (5). 36

37

In North America, Mxe disease is not reportable, so the actual incidence of infection is 38

unknown. However, in a recent global survey of nontuberculous mycobacteria (NTM), 39

Mxe accounted for 8% of all NTM species isolated from pulmonary samples (6). 40

Although rarely seen in Asia, Australia, or South America, Mxe is common in North 41

America and Europe. Isolation frequency is highest in central Europe (e.g. Germany, 42

Italy, Switzerland, Hungary, Croatia, Poland and the Czech Republic), the English 43

Channel region (e.g. southeastern England, northern France, Belgium), and the Great 44

Lakes region of Canada and the United States (6, 7). In Hungary, Mxe is the dominant 45

NTM species and accounts for almost half of all NTM isolated from pulmonary samples 46

(6). In Ontario, Canada, Mxe prevalence is second only to that of the M. avium complex 47

(8). Moreover, population–based studies indicate that, in Ontario, the prevalence of Mxe 48

pulmonary disease has increased from 7.4 cases/100,000 persons during 1998–2002 to 49

9.5 cases/100,000 persons during 2006–2010 (9). 50

51

Surveillance of Mxe is hampered by the absence of effective tools for molecular 52

epidemiology. Restriction fragment length polymorphism (RFLP) methods have been 53

described but are not in routine use, and no public IS1081 or IS1395 RFLP databases 54

exist (10-12). Moreover, RFLP is time consuming, labor–intensive and, like other gel 55

electrophoresis–based typing methods, suffers from poor inter–lab reproducibility and 56

portability. In contrast, multilocus sequence typing (MLST) is readily portable and 57

reproducible. MLST is a DNA sequencing–based method that exploits nucleotide 58

variations present in predefined sets of genetic loci (13, 14). The traditional approach 59

involves Sanger sequencing of PCR amplicons, but MLST is compatible with whole 60

genome sequencing technologies, and thus, is future–proof. MLST schemes have been 61

developed for dozens of bacterial species and some pathogenic fungi. Although not 62

widely used for Mycobacterium species, a MLST scheme for the M. abscessus complex 63

has recently been described (15, 16). In this study, we describe the development and 64

evaluation of an effective MLST strategy for Mxe. 65

66

Materials and Methods 67

Bacterial Strains. A collection of 106 Mxe isolates was assembled for this study. 68

Clinical isolates (n=104) were provided by the Public Health Ontario Laboratories 69

(PHOL; n = 37), the Laboratoire National de Santé Publique du Québec (LSPQ; n = 22), 70

the Wisconsin State Laboratory of Hygiene (WSLH; n = 27), and the Wadsworth Center, 71

New York State Department of Health (WCNY; n = 18). The clinical strains include 72

respiratory isolates cultured from specimens submitted to the source laboratories for 73

routine mycobacterial testing as well as referral isolates sent to the laboratories for 74

identification. Two independent aliquots of the Mxe type strain ATCC 19250T, provided 75

by the LSPQ and WSLH were also examined. All Mxe isolates were submitted to the 76

PHOL for analysis. 77

78

DNA Extraction. Mxe isolates obtained from the PHOL, LSPQ, WSLH and WCNY 79

were subcultured on Lowenstein Jensen slants (Bio–media Unlimited Ltd., Toronto, ON, 80

Canada) and incubated at 40ºC until sufficient growth was observed (4 to 6 weeks). DNA 81

was extracted using a modified boiling lysis method (17). Briefly, a loopful of culture 82

was resuspended in 200 µL buffer (10 mM Tris, 1 mM EDTA, pH 8), vortexed to 83

disperse any clumps, and then heated at 95ºC for 20 minutes. Samples were centrifuged 84

to pellet cellular debris and the DNA–containing supernatant was transferred to a fresh 85

tube and stored at –20ºC until required for downstream applications. 86

87

PCR. Six genes (atpD, fusA1, glnA1, pheT, secA1, and topA) plus the 16S–23S rRNA 88

internal transcribed spacer (ITS) region were used for MLST of Mxe. Primer pairs for 89

each MLST target are listed in Table 1. PCR was performed with the HotStarTaq Plus 90

Master Mix Kit (Qiagen, Louisville, KY, USA). Each 20 µL amplification reaction 91

contained 10 μL of 2× HotStartMM master mix, 2 μL of Q solution, 2 μL of 10× CL 92

buffer, 3 μL of nuclease free water, 1 µL (10 nmol) of each primer, plus 1 μL of crude 93

DNA lysate. PCR was performed for 35 cycles using the following conditions: 94

denaturation (94°C for 45 sec), annealing (57°C for 45 sec), and extension (72°C for 1 95

minute), with a final extension cycle at 72°C for 10 minutes. Agarose gel electrophoresis 96

was used to confirm amplification of each MLST target and estimate amplicon 97

concentration. 98

99

DNA Sequencing. The BigDye Terminator v3.1 Cycle Sequencing Kit (Life 100

Technologies Inc., Burlington, ON, Canada) was used for DNA sequencing of amplicons 101

for MLST. Labelling reactions were performed following the manufacturer’s instructions 102

(Life Technologies). For each MLST target, bidirectional sequencing was performed. 103

Unincorporated dyes were removed by ethanol/EDTA precipitation and the labeled 104

amplicons were resuspended in 10 µL of nuclease–free water. DNA sequencing was 105

performed via capillary electrophoresis with an Applied Biosystems 3730xl DNA 106

Analyzer. Editing, analysis, and alignment of DNA sequences were performed with 107

MEGA 5.2 (18). 108

109

Results 110

MLST. DNA sequencing results for the MLST targets are summarized in Table 2 and 111

Supplementary Table 1. Individual genes were found to have two (atpD), three (fusA1, 112

topA), four (glnA1, pheT) or five (secA1) allelic variants. All single nucleotide 113

polymorphisms (SNPs) associated with the atpD, fusA1, glnA1 and topA1 alleles were 114

synonymous. However, the pheT ST–2 , pheT ST–3, secA1 ST–3 and secA1 ST–5 115

variants contained non–synonymous SNPs. The six locus MLST strategy (MLST–6) 116

divided the 106 Mxe isolates into 15 distinct sequence types (ST). The MLST profile 117

shared by the two aliquots of ATCC19250T was designated ST–1. Most STs were only 118

observed once or twice, but three STs were prevalent among the clinical isolates: ST–2 119

(n= 48; 46.1%), ST–5 (n=23; 22.1%) and ST–9 (n=20; 19.2%). 120

Previous studies have documented the existence of three distinct Mxe 16S–23S rRNA 121

ITS alleles (i.e. Mxe–A, Mxe–B, Mxe–C) (19). As such, the ITS region was selected as a 122

seventh MLST target. However, for some isolates, sequencing of this target was 123

confounded by the presence of multiple, non–identical ITS sequences. Whereas genomes 124

from the majority of ‘slow growing’ mycobacterial species feature a single rRNA operon, 125

the Mxe genome contains two. Because the ITS PCR did not use operon–specific primers, 126

both ITS loci were amplified and sequenced together. Thus, homozygous strains (e.g. ITS 127

A/A) generated ‘clean’ sequences but heterozygous strains (e.g. ITS A/B) generated 128

‘mixed’ electropherograms. By comparing the mixed electropherograms to published ITS 129

sequences, it was possible to identify characteristic mixed base patterns for heterozygous 130

allele combinations (e.g. ITS A/B, ITS A/C, ITS B/C) and assign ITS profiles to all but 131

nine isolates. The seven loci MLST strategy (MLST–7; MLST–6 plus ITS) divided the 132

remaining 97 Mxe isolates into 21 STs (Table 2). In most cases, strains that clustered by 133

six loci MLST also clustered by ITS. For example, all isolates in the ST–9 group shared 134

the same ITS A/A profile. In contrast, ST–2 was subdivided into four ITS clusters (A/A = 135

3, A/B = 26, B/B = 4, B/C = 9), ST–5 was subdivided into three clusters (B/B = 1, B/C = 136

16, C/C = 4), and the ST–13 pair included one A/B and one B/B isolate. 137

138

MLST of Consecutive Isolates from Individual Patients. Table 3 includes MLST–6 139

results for a subset of PHOL strains: 17 consecutive isolates recovered from 7 patients. 140

For five patients, only Mxe isolates of identical ST were recovered. However, the isolates 141

from Patient F were distinct (ST–11 in 2011 and ST–5 in 2012) and the four isolates 142

recovered from Patient G included one of ST–2 and three of ST–9. 143

144

Geographic Distribution of STs. The geographic distribution of MLST–6 defined STs is 145

summarized in Figure 1 and Supplementary Figure 1. Most STs were only observed once 146

or, in the case of the ST–3 and ST–13 pairs, were isolated by a single source laboratory. 147

ST–9 strains were isolated at both the PHOL and LSPQ, and ST–5 strains were isolated 148

at three facilities, but only ST–2 strains were isolated by all four source laboratories. 149

150

Discussion 151

Despite increasing recognition of Mxe as an important human pathogen, little is known 152

about the genetic diversity of this species. MLST is a sequencing–based method that has 153

been used for genotyping of many bacterial species. Traditionally, MLST strategies target 154

‘housekeeping’ or ‘core’ genes, loci that are conserved within the species of interest. For 155

typing to be effective, these targets must also be heterogeneous, such that distinct isolates 156

exhibit distinct sequence polymorphisms. To develop an MLST strategy for Mxe, we 157

initially targeted 19 housekeeping genes: alaS, atpD, dnaK ffh, ftsK, fum, fusA1, gap, 158

gcvB, glnA1, gyrB, hsp65, infB, leuS, pheT, tpi, rpoB, secA1, and topA. Preliminary 159

sequencing results, derived from a panel of 21 Mxe isolates, revealed that 13 of these loci 160

were invariant, and thus, unsuitable for MLST (20). 161

The MLST–6 approach used in this study targets six polymorphic Mxe loci: atpD, fusA1, 162

glnA1, pheT, secA1, and topA. The MLST–7 strategy includes one additional target, the 163

Mxe ITS region. The two approaches were used to genotype a collection of 106 Mxe 164

isolates provided by four mycobacteriology laboratories (Table 2, Figure 1, 165

Supplementary Figure 1). The MLST–6 strategy divided the collection into 15 STs. The 166

MLST–7 strategy provided greater resolution (21 STs) even though genotyping could not 167

be completed for nine isolates. Despite the potential utility of the MLST–7 strategy, 168

technical challenges, especially the requirement for interpretation of ‘mixed’ ITS 169

electropherograms, make the current MLST–7 approach less suitable for routine use than 170

MLST–6. However, if the Sanger sequencing method used here was replaced by a next 171

generation approach, it would be possible to resolve distinct ITS loci and type additional 172

targets. Tools to facilitate such analysis, sometimes called core genome multilocus 173

sequence typing (cgMLST), have recently been described for M. tuberculosis (21). 174

175

For patients with multiple positive cultures or multiple episodes of disease, MLST can be 176

used to distinguish cases of relapse from those of reinfection. MLST–6 analysis of 17 177

consecutive isolates obtained from 7 patients is summarized in Table 3. In six of the 178

seven cases, multiple isolates of identical ST were obtained. These STs persisted over a 179

period of months (Patients C, D, G) to two (Patient A) and three (Patients B, E) years. 180

The two isolates from Patient F were of different STs and isolated in different years, 181

which is consistent with a case of reinfection. However, in the case of Patient G, isolates 182

of ST–2 and ST–9 were obtained within a few months of one another. Since subsequent 183

isolates were also of ST–2, the situation may be indicative of strain replacement. Or, it 184

may represent a polyclonal infection, a phenomenon that is common among patients with 185

infections due to other nontuberculous mycobacteria, such as M. avium (22, 23). 186

187

MLST analysis demonstrates that Mxe isolates are genotypically heterogeneous. Nine of 188

the MLST–6 defined STs, and twelve of the MLST–7 defined STs, were only observed 189

once (Table 2, Figure 1). Because Mxe is an ‘environmental’ mycobacterial species, it is 190

possible that this genotypic diversity is related to geographic distribution. Although ST–2 191

strains were isolated by all four source laboratories, the distribution of other STs was 192

restricted. For example, ST–9 strains were only isolated by the Canadian laboratories, 193

while both ST–3 isolates were collected by WSLH. Most of the PHOL isolates were 194

obtained from patients living in the Greater Toronto Area (GTA) of southern Ontario, but 195

the genotypically distinct ST–15 strain was isolated from a patient in central Ontario. 196

Analysis of the PHOL isolates suggests a geographic trend, with distribution of the ST–5, 197

ST–9 and ST–2 strains restricted to the east/central, north/central, and west regions of the 198

GTA, respectively. Additional isolates and environmental sampling are required to 199

confirm this observation, but Mxe is typically associated with aquatic ecosystems of both 200

natural and anthropogenic origin (24, 25). The geographic distribution of STs in the GTA 201

may overlap with patient exposure to specific engineered (e.g. domestic water reservoirs, 202

distribution systems, hot tubs) or natural (e.g. lakes, rivers, aquifers) water sources. 203

204

The ST–1 profile assigned to Mxe type strain ATCC 19250T was not shared by any of the 205

clinical isolates. Most likely, this is due to the fact that the type strain is from a different 206

place and time (i.e. England, c1958) (26). However, Mxe ATCC 19250T was isolated 207

from the skin of an amphibian (Xenopus laevis), not the respiratory tract of a mammal. 208

As such, the ST differences may be indicative of differences in the pathogenic potential 209

and host preferences of genotypically distinct Mxe populations. Among other 210

mycobacterial species, including the M. tuberculosis complex and M. avium complex, 211

lineage–specific differences in pathogenicity and host range are well documented(27-29). 212

213

Despite its prevalence and increasing clinical importance, little is known about the 214

genetic diversity and molecular epidemiology of Mxe. Gel electrophoresis–based 215

methods are widely considered the ‘gold standard’ for mycobacterial typing and RFLP 216

can been used to differentiate Mxe isolates. Unfortunately, for many laboratories, RFLP 217

is not a practical option for routine typing. In contrast, in an era where labor is expensive 218

but sequencing is cheap, MLST may be a faster and more affordable alternative. With the 219

development of an effective MLST scheme, it becomes possible to routinely examine the 220

distribution, evolution, and population structure of Mxe. Future studies may provide 221

insight into the geographic preferences of specific STs, or uncover associations between 222

Mxe genotype and disease phenotype, antibiotic susceptibility, and pathogenic potential. 223

224

Acknowledgements 225 226 The authors would like to acknowledge the skill and dedication of the laboratory staff 227 from PHOL, LSPQ, WLSH, and WCNY responsible for the initial isolation and 228 cultivation of the M. xenopi isolates used in the study. 229 230

231

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2 3 4 5 6 7 8 9 10 11 12 13 14 15

Sequence Type (MLST-6)

# I

sola

tes

Figure 1. Geographic Distribution of Mycobacterium xenopi MLST-6 Sequence Types.

Isolates from PHOL (black), LSPQ (grey), WSLH (hatched), and WCNY (white) are shown.

Table 1. Oligonucleotide Primers for MLST Analysis of Mycobacterium xenopi

Target Primer 1 Primer 2 Size (bp) Function atpD 5'-CGGTGTTCTCCAAGGGCATC-3' 5'-TTGTCGGTGTTGTCGGCCTG-3' 499 ATP synthase (subunit β) fusA1 5'-GCATCATGGCGCACATCGAC-3' 5'-TTCATCTCGACCAGGTCGACGACG-3' 505 Elongation factor G glnA1 5'-ATTCTGCACCATGCGCCTTC-3' 5'-ACACGTCGAAGTACAGCGCG-3' 511 Glutamine synthetase pheT 5'-TACAGCTGGCTGCGCGAAGT-3' 5'-ATGGCCAGGTGGAACACCACGT-3' 494 Phenylalanyl-tRNA synthetase (subunit β) secA1 5'-CGAGCACCTCTACGAGATGG-3' 5'-TTAGCGCTTCTTGGCCGATT-3' 502 Preprotein translocase topA 5'- AAAACCCGCTCGCCCCAAAC-3' 5'- GTGCCTTGCGGGTCGTCTTCTT-3' 515 DNA topoisomerase I ITS 5’-TTGTACACACCGCCCGTCA-3’ 5’-TCTCGATGCCAAGGCATCCACC-3’ 445 16S-23S rRNA Internal Transcribed Spacer

Table 2. MLST Analysis of Mycobacterium xenopi

MLST-6 atpD fusA1 glnA1 pheT secA1 topA ITS # Isolates†

ST-1 1 1 1 1 1 1 AA 2

ST-2 1 1 1 1 2 1 variable1 48

ST-3 1 1 1 1 3 1 AB 2

ST-4 1 1 1 1 1 2 CC 1

ST-5 1 1 1 1 2 2 variable2 23

ST-6 1 1 2 1 2 1 BC 1

ST-7 1 1 3 1 2 2 CC 1

ST-8 1 1 3 1 2 3 CC 1

ST-9 1 1 2 2 2 1 AA3 20

ST-10 1 1 4 3 4 1 AC 1

ST-11 1 1 2 2 2 2 AA 1

ST-12 1 1 4 4 5 3 CC 1

ST-13 1 2 1 1 2 2 variable4 2

ST-14 1 3 1 1 2 1 AB 1

ST-15 2 1 2 2 2 1 AA 1

† Duplicate isolates (i.e. genotypically identical isolates from individual patients; see Table 3) were only counted once.

1 ST-2 isolates were associated with multiple ITS patterns: AA (n=3), AB (n=26), BB (n=4), BC (n=9), non-typable (n=6)

2 ST-5 isolates were associated with multiple ITS patterns: BB (n=1), BC (n=16), CC (n=4), non-typable (n=2)

3 The ITS of one ST-9 isolate was non-typable

4 ST-13 isolates were associated with two ITS patterns: AB (n=1) and BB (n=1)

Table 3: MLST-6 Analysis of Consecutive Isolates from Individual Patients

Patient Isolate Date MLST-6

A 1 10/2010 ST-5 2 11/2011 ST-5

B 1 08/2010 ST-15 2 08/2012 ST-15

C 1 09/2010 ST-5 2 10/2010 ST-5

D 1 09/2012 ST-2 2 11/2012 ST-2

E 1 2009 ST-5 2 10/2010 ST-5 3 01/2011 ST-5

F 1 10/2010 ST-11 2 01/2012 ST-5

G

1 02/2012 ST-2 2 04/2010 ST-9 3 08/2012 ST-9 4 10/2012 ST-9