Oct 19, 2007 - (Linda). M. avium subsp. paratuberculosis C b3 (C5). Human. USA ..... We are grateful to Gerard Cangelosi (University of Washington),.
JOURNAL OF BACTERIOLOGY, Apr. 2008, p. 2479–2487 0021-9193/08/$08.00⫹0 doi:10.1128/JB.01691-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 190, No. 7
Mycobacterium avium subsp. paratuberculosis and M. avium subsp. avium Are Independently Evolved Pathogenic Clones of a Much Broader Group of M. avium Organisms䌤† Christine Y. Turenne,1 Desmond M. Collins,2 David C. Alexander,1 and Marcel A. Behr1* McGill University Health Centre, Montreal, Quebec, Canada H3G 1A4,1 and AgResearch, NCBID Wallaceville, Upper Hutt, New Zealand2 Received 19 October 2007/Accepted 23 January 2008
Mycobacterium avium comprises organisms that share the same species designation despite considerable genomic and phenotypic variability. To determine the degree and nature of variability between subspecies and strains of M. avium, we used multilocus sequencing analysis, studying 56 genetically diverse strains of M. avium that included all described subspecies. In total, 8,064 bp of sequence from 10 gene loci were studied, with 205 (2.5%) representing variable positions. The majority (149/205) of these variations were found among M. avium subsp. hominissuis organisms. Recombination was also evident in this subspecies. In contrast, there was comparatively little variability and no evidence of recombination within the pathogenic subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. silvaticum. Phylogenetic analysis showed that M. avium subsp. avium and M. avium subsp. silvaticum strains clustered together on one branch, while a distinct branch defined M. avium subsp. paratuberculosis organisms. Despite the independent origin of these pathogenic subspecies, an analysis of their rates of nonsynonymous (dN) to synonymous (dS) substitutions showed increased dN/dS ratios for both: 0.67 for M. avium subsp. paratuberculosis and 0.50 for M. avium subsp. avium/M. avium subsp. silvaticum, while the value was 0.08 for M. avium subsp. hominissuis organisms. In conclusion, M. avium subsp. hominissuis represents a diverse group of organisms from which two pathogenic clones (M. avium subsp. paratuberculosis and M. avium subsp. avium/M. avium subsp. silvaticum) have evolved independently. while IS901 has served to distinguish between subsets of M. avium differing in host range, virulence, and capacity to interfere with Mycobacterium bovis BCG protection (6, 15). Similarly, LSPs also enable typing at the subspecies and strain levels (29). While an association between insertion sequences, select LSPs, and select subspecies has proven highly useful in the context of diagnostics, their strict correlation precludes them from enhancing the understanding of the genetic diversity across M. avium as a whole. Multilocus sequence analysis (MLSA) provides an additional modality to document and quantify genetic variability. MLSA exploits sequence level variation in housekeeping genes that are conserved across a broad range of organisms (17). The availability of two genome sequences for M. avium provides the opportunity to select variable genes and design appropriate primers to amplify and sequence genes across M. avium subsets. Unlike the Mycobacterium tuberculosis complex, where limited genetic variability has restricted the utility of MLSA, M. avium genes such as hsp65 exhibit sufficient variability for classification purposes (33). Therefore, we hypothesized that the study of a set of housekeeping genes across a panel of M. avium strains would provide insights into the relationships between M. avium organisms and the degree of diversity both within and between subspecies.
Mycobacterium avium comprises four named subspecies: M. avium subsp. avium, M. avium subsp. silvaticum, M. avium subsp. paratuberculosis, and M. avium subsp. hominissuis (22, 32, 34). Despite their taxonomic relationship, these subspecies represent phenotypically diverse organisms, ranging from environmental bacteria that cause opportunistic infections of swine and immunocompromised people to professional pathogens of birds and ruminants. The availability of complete genome sequences for two M. avium strains (M. avium subsp. paratuberculosis K-10 and M. avium subsp. hominissuis 104) has provided an unprecedented opportunity to determine the basis of this phenotypic variability (16, 30, 39). These sequences have helped to define how M. avium is distinct from other mycobacteria and revealed that M. avium subspecies exhibit extensive genomic differences. Previous studies of M. avium diversity have targeted an assortment of variable genetic elements, including insertion sequences (3) and large sequence polymorphisms (LSPs) (28, 30, 39). The presence (or absence) of specific insertion elements can be used to type an organism to the subspecies level (3), while variations in copy number can facilitate strain tracking. In particular, IS900 is routinely used as a marker for the diagnosis and epidemiology of M. avium subsp. paratuberculosis, * Corresponding author. Mailing address: Division of Infectious Diseases and Medical Microbiology, A5-156, Montreal General Hospital, 1650 Cedar Avenue, Montreal, QC H3G 1A4, Canada. Phone: (514) 934-1934, ext. 42815. Fax: (514) 934-8423. E-mail: marcel.behr @mcgill.ca. † Supplemental material for this article may be found at http://jb .asm.org/. 䌤 Published ahead of print on 1 February 2008.
MATERIALS AND METHODS Bacterial strains. A collection of 56 strains of M. avium organisms was assembled for this study (Table 1). Three strains of Mycobacterium intracellulare (ATCC 13950T, FCC 1804, and 90331) were also included to provide an outgroup for phylogenetic analysis. From M. intracellulare and M. avium subsp. hominissuis, we selected isolates that have previously been shown to have different hsp65 alleles. To further ensure that a genetically diverse range of M.
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TABLE 1. List of strains used, their known characteristics, and their molecular classification as per previously published markers Strain IDa
Strain 104 ATCC 700898 (MAC 101) FCC 268 9700626 MB063764 9700613 ATCC 49601 76102 98838 102 9501281 108 9801021 DV 9800848 28132 MB068331 MB064490 9501268 WAg208 (1464bd) 2000–333 2000–325 HMC36 822 T
ATCC 25291
IWGMT 19 (14141–1395; ATCC 35716; TMC 715) V11 ATCC 35713 ATCC 35718 R13 ATCC 15769 IWGMT 17 (B-92) ATCC 49884 (6409) 9800851 9801574 TMC1613 K-10 (ATCC BAA-968) 989
T
RFLPb
Host
Source
IS900
IS901
LSPA17
LSPA8
LSPA20
M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. hominissuis M. avium subsp. avium M. avium subsp. avium
Multiband (⬎20)
Human
USA
⫺
⫺
⫹
⫹
⫹
Multiband (⬎20)
Human
USA
⫺
⫺
⫹
⫹
⫹
Multiband (⬎20)
Unknown
Australia
⫺
⫺
⫺
⫹
⫹
Multiband
Porcine
Netherlands
⫺
⫺
⫹
⫹
⫹
Multiband (⬎20)
Human
Canada
⫺
⫺
⫺
⫹
⫹
Multiband
Porcine
Netherlands
⫺
⫺
⫹
⫹
⫹
Multiband (6)
Human
USA
⫺
⫺
⫺
⫹
⫹
Multiband (⬎15)
Human
Canada
⫺
⫺
⫹
⫹
⫹
M. avium subsp. avium M. avium subsp. avium M. avium subsp. avium M. avium subsp. avium M. avium subsp. avium M. avium subsp. avium M. avium subsp. silvaticum M. avium subsp. silvaticum M. avium subsp. silvaticum M. avium subsp. paratuberculosis C M. avium subsp. paratuberculosis C M. avium subsp. paratuberculosis C
Multiband (⬎15)
Human
Canada
⫺
⫺
⫹
⫹
⫹
Multiband (8)
Human
USA
⫺
⫺
⫺
⫹
⫹
Multiband
Porcine
Brazil
⫺
⫺
⫺
⫹
⫹
Multiband (⬎15)
Human
USA
⫺
⫺
⫹
⫹
⫹
Cigarette
Netherlands
⫺
⫺
⫹
⫹
⫹
Multiband (⬎15)
Human
Canada
⫺
⫺
⫺
⫹
⫹
Multiband
Dog
Belgium
⫺
⫺
⫺
⫹
⫹
Multiband (⬎15)
Human
Canada
⫺
⫺
⫺
⫹
⫹
ND
Human
Canada
⫺
⫺
⫺
⫹
⫹
ND
Human
Canada
⫺
⫺
⫺
⫹
⫹
Type 2
Human
Argentina
⫺
⫺
⫺
⫹
⫹
Cigarette
c
Cervine
New Zealand
⫺
⫺
⫹
⫹
⫹
Type 6
c
Human
USA
⫺
⫺
⫺
⫹
⫹
Type 5
c
Human
USA
⫺
⫺
⫺
⫹
⫹
ND
Human
USA
⫺
⫺
⫹
⫹
⫹
ND
African buffalo
South Africa
⫺
⫺
⫹
⫹
⫹
Three-band bird type
Chicken
⫹
⫺
⫹
⫹
Bovine
Reference (serotype 2) Reference (serotype 2)
⫺
Three-band bird type
⫺
⫹
⫺
⫹
⫹
Three-band bird type
Porcine
Netherlands
⫺
⫹
⫺
⫹
⫹
Three-band bird type
Chicken
⫺
⫹
⫺
⫹
⫹
Three-band bird type
Human
⫺
⫹
⫺
⫹
⫹
Three-band bird type
Crane
⫺
⫹
⫺
⫹
⫹
Three-band bird type
Chicken
⫺
⫹
⫺
⫹
⫹
Three-band bird type
Unknown
⫺
⫹
⫺
⫹
⫹
Three-band M. avium subsp. silvaticum type Three-band M. avium subsp. silvaticum type Three-band M. avium subsp. silvaticum type b1 (C6)
Wood pigeon
Reference (serotype 3) Reference (serotype 3) Netherlands (serotype 3) Reference (serotype 1) Reference (serotype 1) France
⫺
⫹
⫺
⫹
⫹
Penguin
Belgium
⫺
⫹
⫺
⫹
⫹
Wood pigeon
Belgium
⫺
⫹
⫺
⫹
⫹
Bovine
Reference
⫹
⫺
⫹
⫺
⫹
b2 (C1)
Bovine
USA
⫹
⫺
⫹
⫺
⫹
b2 (C1)
Bovine
New Zealand
⫹
⫺
⫹
⫺
⫹
ND
Continued on following page
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TABLE 1—Continued Strain
Strain ID
04–4531 5979 7428 316F 119 ATCC 19698 ATCC 43015 (Linda) 87/8880 6601 7296 6756 6758 P133/79 6759 85/14 86–45 LN20 P465
T
a
M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis M. avium subsp. paratuberculosis
RFLP
b
Host
Source
IS901
LSPA17
LSPA8
LSPA20
b2 (C1)
Bovine
Canada
⫹
⫺
⫹
⫺
⫹
b2 (C1)
Cervine
New Zealand
⫹
⫺
⫹
⫺
⫹
b4 (C2)
Bovine
New Zealand
⫹
⫺
⫹
⫺
⫹
b7 (C7)
Vaccine
⫹
⫺
⫹
⫺
⫹
b9 (CU1)
Bovine
United Kingdom Canada
⫹
⫺
⫹
⫺
⫹
b3 (C5)
Bovine
⫹
⫺
⫹
⫺
⫹
b3 (C5)
Human
USA; reference USA
⫹
⫺
⫹
⫺
⫹
b5 (C3)
Bovine
Australia
⫹
⫺
⫹
⫺
⫹
b6 (C4)
Bovine
Australia
⫹
⫺
⫹
⫺
⫹
b8 (C8)
Cervine
New Zealand
⫹
⫺
⫹
⫺
⫹
s1 (S1; type I)
Ovine
New Zealand
⫹
⫺
⫹
⫺
⫺
s1 (S1; type I)
Ovine
New Zealand
⫹
⫺
⫹
⫺
⫺
s3 (S2; type I; pigmented) s5 (type I)
Ovine
⫹
⫺
⫹
⫺
⫺
Ovine
Faeroe Islands New Zealand
⫹
⫺
⫹
⫺
⫺
s6 (type III)
Ovine
Canada
⫹
⫺
⫹
⫺
⫺
s2 (I1; type III)
Ovine
Canada
⫹
⫺
⫹
⫺
⫺
s2 (I1; type III)
Porcine
Canada
⫹
⫺
⫹
⫺
⫺
s4 (I2; type III)
Ovine
Iceland
⫹
⫺
⫹
⫺
⫺
C C C C C C C C C C S S S
IS900
S S S S S
a
The identity (ID) of strains was confirmed by established genetic methods as described in Materials and Methods. C, cattle type; S, sheep type. IS1245 RFLP result for non-M. avium subsp. paratuberculosis isolates, with the number of bands indicated in parentheses, or IS900 RFLP result for M. avium subsp. paratuberculosis isolates, with published profile designations indicated in parentheses, as per references 4, 5, and 25. I, intermediate; ND, not done. c IS1245 RFLP profiles of “cigarette” type, type 5 and type 6 are characterized by a low-copy, weak, two-band pattern (22). b
avium subsp. hominissuis was sampled, we included isolates with the typical multiband IS1245 RFLP profile as well as strains with low-copy-number IS1245 patterns distinct from the three-band M. avium subsp. avium and M. avium subsp. silvaticum patterns (22). In the case of M. avium subsp. paratuberculosis, which presents with restricted hsp65 variability, isolates were selected on the basis of previously published polymorphisms by insertion sequence-based restriction fragment length polymorphism (RFLP) and included “type III/intermediate” strains and a pigmented strain. For M. avium subsp. avium and M. avium subsp. silvaticum, we selected the type strain of each organism as well as members representing serotypes 1, 2, and 3 associated with avian strains. To ensure the subspecies designations of all strains prior to MLSA, for each we performed insertion sequence typing, hsp65 sequencing, and testing for LSP regions that have previously been reported to be absent from specific subspecies. Strains of M. avium subsp. avium (n ⫽ 8) and M. avium subsp. silvaticum (n ⫽ 3) were characterized by the presence of the IS901 element and a common hsp65 sequence and the absence of LSPA17. M. avium subsp. paratuberculosis strains were characterized by the presence of the IS900 element and the absence of LSPA8 (29). Bovine (n ⫽ 13) and ovine (n ⫽ 8) subtypes were distinguished by their hsp65 sequences and LSPA20 profiles, such that LSPA20 was absent from ovine strains (29). Strains that were IS900 and IS901 negative were called M. avium subsp. hominissuis (n ⫽ 24). These also possessed hsp65 sequences distinct from the M. avium subsp. paratuberculosis and M. avium subsp. avium/M. avium subsp. silvaticum alleles. MLSA. MLSA targets were selected in two ways. We first selected enzymes demonstrating the most intraspecies variability in M. avium by multilocus enzyme electrophoresis (37, 40). Enzyme-encoding genes were then identified by crossreferencing the protein name and Enzyme Commission numbers against the annotated M. avium strain K-10 and 104 genome sequences. Gene sequences were compared, and those demonstrating sufficient genetic variability (ⱕ99.1% DNA identity, akin to that found in the 5⬘ hsp65 [33]) were used for MLSA. These were aspB, gnd1, lipT, pepB, and gnd and a putative esterase, annotated as
a hypothetical protein in M. avium subsp. paratuberculosis, that we designated “est.” Second, additional genes were selected from previously published reports on single nucleotide polymorphisms (SNPs) of mycobacteria (18, 41) that when verified in the bigenomic comparison of K-10 versus 104 revealed a 98 to 99% sequence identity. These were recF, sodA, and groEL1. The genome locus tag for each gene in strains K-10 and 104 is indicated in Table S1 the supplemental material. For all genes selected, primers were designed in Primer3 (http://frodo.wi.mit .edu/cgi-bin/primer3/primer3_www.cgi) to amplify an approximately 1-kb region per gene and encompass the majority of SNPs that distinguish K-10 and 104. Primers targeting groEL1 amplified a larger region that includes almost the complete gene. For an initial group of strains, sequences were determined in both forward and reverse directions. This method permitted the identification of regions of greatest variability and information content. The regions selected for analysis across the complete panel, along with primer sequences, preferred sequencing primers, and region of analysis, are detailed in Table S1 in the supplemental material. DNA preparation and PCR. DNA lysates for RFLP and PCR were prepared as previously described (36). In occasional instances where ambiguous results were obtained, such as in the case of mixed cultures or cross-contamination, samples for which live cultures were available were reprocessed from a single colony and the identity of the strain was reconfirmed. If a lysate was suspected as mixed but no live culture was conveniently available, it was excluded from the panel (data not shown). The amplification of MLSA genes was performed in a 25-l final reaction mixture volume consisting of approximately 25 ng of DNA template, 2.5 mM MgCl2, 1⫻ PCR buffer (Invitrogen, Carlsbad, CA), 5% acetamide, 0.2 mM deoxynucleoside triphosphates, 0.5 M of each primer, and 1 U of Taq DNA polymerase (Fermentas). PCR was performed with an Applied Biosystems Gene Amp PCR system 2700 using the following conditions: 94°C for 3 min; 35 cycles with intervals of 94°C (45 s), 55°C (45 s), and 72°C (1 min); 72°C
2482
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FIG. 1. (A) Phylogenetic representation of each ST determined for this study and generated in SplitsTree4. Strains identified as M. avium subsp. paratuberculosis of cattle type (MAP-C) and sheep type (MAP-S) are highlighted, as are strains of M. avium subsp. avium (MAA) and M. avium subsp. silvaticum (MAS). (B) Expanded subtree of the M. avium subsp. paratuberculosis branch and expanded subtree of the M. avium subsp. avium and subsp. silvaticum branch. Scale bar, 1 nucleotide difference.
for 10 min; and holding at 4°C. The amplification of groEL1 was performed at an annealing temperature of 60°C to eliminate the appearance of nonspecific bands. The amplification of M. intracellulare strains was performed at a lower annealing temperature (53°C), which enabled 6 of the 10 MLSA targets to be successfully amplified. PCR product (5 l) was visualized on a 1.5% agarose gel containing ethidium bromide. The remaining PCR product was submitted to a core sequencing facility (McGill University and Genome Quebec Innovation Centre) for sequencing on a 3730XL DNA analyzer system. Phylogenetic analysis. Following dideoxy nucleotide sequencing, sequence editing and multiple alignments were performed in MEGA, version 4.0 (31). Sequences from the nine MLSA loci were combined with 5⬘ hsp65 sequence data and concatenated using START2 (11), generating an 8,064-bp semantide. Phylogenetic analysis was performed in two manners by using SplitsTree, version 4.8 (10). First, data for all 10 genes were used to generate an unrooted tree of M.
avium organisms. Second, genes that were successfully amplified in M. intracellulare strains were included with the M. avium strains in a separate analysis to generate a rooted tree, with M. intracellulare serving as the out-group. In both cases, the split network phylogeny was computed by NeighborNet analysis in SplitsTree4. Distances were determined in MEGA 4.0 using the neighbor-joining (p-distance) method by pairwise analysis to include gaps and with 1,000 bootstrap replicates. Expanded subtrees in Fig. 1B were visualized in MEGA 4.0 using the neighbor-joining (number of differences) method. Recombination events within M. avium were evaluated using two programs: DnaSP 4.10 (27) and the recombination detection program RDP3 Beta 22, a recent upgrade from RDP2 (19). Since the MLSA loci were separate from each other in the genome and too few SNPs existed within a single locus to identify specific recombination breakpoints, we tested for evidence of recombination in the concatenated sequence. To test for a larger recombination event that would
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TABLE 2. MSLA-defined sequence types and allele distribution Sequence type
Allelic profile
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 20 21 22 23 24 25 26 27 30 31 32 33 34 35 36
1-1-1-1-1-1-1-1-1-1 2-1-1-2-1-1-1-1-1-1 2-1-1-2-2-1-1-1-1-2 2-1-1-3-1-1-2-11-1-1 2-2-2-3-3-2-2-2-1-3 2-3-2-4-1-3-2-1-2-3 2-1-1-4-1-8-1-1-2-1 2-3-8-4-1-3-2-1-2-3 2-3-2-3-3-9-2-11-2-7 2-1-1-5-2-4-1-1-3-2 2-1-1-2-2-4-1-1-3-4 2-1-1-3-2-4-1-1-3-2 3-8-7-8-9-10-5-2-15-9 4-1-8-4-10-3-1-1-1-3 4-7-1-4-3-9-2-1-7-3 5-2-2-4-7-9-2-11-2-7 5-2-2-8-7-9-2-2-8-7 5-1-2-3-1-9-4-1-9-1 6-1-1-6-4-5-1-3-4-5 6-1-1-7-4-5-1-3-4-5 6-1-1-7-4-5-1-10-4-5 7-1-1-7-5-5-1-3-4-5 7-1-1-7-4-5-1-3-4-5 7-1-1-7-4-5-1-9-4-8 8-1-3-7-6-6-1-4-4-5 8-1-4-7-6-6-1-4-4-5 9-4-5-9-8-7-3-5-5-6 9-4-5-9-8-7-3-6-5-6 9-4-6-9-8-7-3-5-5-6 10-5-6-9-8-7-3-7-6-6 11-5-6-9-8-7-3-8-6-6 11-6-6-9-8-7-3-7-6-6 12-5-6-9-8-7-3-7-6-6
Subspecies
M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M.
avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium avium
subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp.
Strain(s) presenting with ST
hominissuis hominissuis hominissuis hominissuis hominissuis hominissuis hominissuis hominissuis hominissuis hominissuis hominissuis hominissuis hominissuis hominissuis hominissuis hominissuis hominissuis hominissuis avium avium avium avium avium avium silvaticum silvaticum paratuberculosis paratuberculosis paratuberculosis paratuberculosis paratuberculosis paratuberculosis paratuberculosis
manifest in the allele for the sequenced genes, we considered that such an event would consist of one or more complete alleles in the concatenated sequence. To look for recombination events within a particular group of organisms (M. avium subsp. hominissuis, M. avium subsp. paratuberculosis, and M. avium subsp. avium/M. avium subsp. silvaticum), we used the DnaSP program, analyzing for recombinant alleles in each of these three groups of organisms. As a second approach, RDP3 was used to estimate the number of recombination events by applying the algorithms RDP and GENECONV (24), evaluating all sequence types (STs) together or each of the three groups delineated as per DnaSP. To determine the nature of the genetic variability, we used MEGA4 to calculate taxon-specific ratios of nonsynonymous (dN) to synonymous (dS) polymorphisms (dN/dS) (26). Results were based on the pairwise analysis of the 33 sequence types and computed for each group. Analyses were conducted using the Nei-Gojobori method with the Jukes-Cantor correction (23, 31). Nucleotide sequence accession numbers. The sequences representing each allele of every MLSA target, with the exception of that of hsp65, which was previously published (33), were deposited in the National Center for Biotechnology Information database under GenBank accession numbers EU409971 to EU410052.
RESULTS MLSA. The number of different sequence alleles found for each gene varied between 5 (gnd1) and 12 (recF) alleles (see Table S2 in the supplemental material). With some exceptions, the majority of alleles (86/92) were exclusive to a single subspecies (see Table S2 in the supplemental material). For each strain, sequence data from the 10 MLSA gene loci, distributed around the M. avium genomes, were concatenated to form an 8,064-bp in-frame semantide. In total, 205 polymorphic sites
C C C S S S S
104, ATCC 700898 FCC 268, 9700626 MB063764 9700613 ATCC 49601 76102, 98838 102, 9501281 108 9801021 DV, 9800848 28132, MB068331 MB064490 9501268 WAg208 2000–333 2000–325 HMC36 822 ATCC 25291T IWGMT 19 V11 ATCC 35713, ATCC 35718, R13 ATCC 15769 IWGMT 17 ATCC 49884T 9800851, 9801574 TMC1613, K-10, 989, 04–4531, 5979, 7428, 316F 119 ATCC 19698T, ATCC 43015, 87/8880, 6601, 7296 6756, 6758, P133/79, 6759 85/14 86–45, LN20 P465
were found (2.5%), consisting of 204 SNPs and one codon deletion. When combined, these variable alleles permitted us to separate tested strains into a total of 33 STs (Table 2). Importantly, each ST comprised only one subspecies of M. avium; M. avium subsp. hominissuis had 18 STs, M. avium subsp. avium had 6 STs, M. avium subsp. silvaticum had 2 STs, and M. avium subsp. paratuberculosis had 7 STs. Sequence variability was greatest among M. avium subsp. hominissuis isolates, where 149 of the 205 variable sites were detected. In these strains, genetic recombination events were evident, as strains were found to carry alleles from various other strains in a number of combinations, resulting in a weblike topology due to incongruent individual gene phylogenies (Table 2; see Fig. S1 and S2 in the supplemental material). Supporting this result, when we used DnaSP, the minimum number of recombinations within M. avium subsp. hominissuis alone was estimated to be 21, whereas the numbers of recombination signals were estimated as 9 (RDP method) and 13 (GENECONV method) for RDP3. In contrast, sequence variability was much lower among the other subspecies, where no recombination was observed irrespective of the method used. There were seven polymorphisms that distinguished the IS901positive “avian strains” from the other M. avium strains and only four SNPs that distinguished M. avium subsp. silvaticum from M. avium subsp. avium. A similar pattern was observed for the IS900 subset, where there were SNPs that distinguished
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J. BACTERIOL. TABLE 3. SNP markers for specific lineages of interesta
Group
M. avium subsp. paratuberculosis C (all) M. avium subsp. paratuberculosis S (all) M. avium subsp. paratuberculosis S, intermediate/type III Variable SNPs in M. avium subsp. paratuberculosis M. avium subsp. avium/M. avium subsp. silvaticum (all) M. avium subsp. silvaticum Variable SNPs within M. avium subsp. avium/M. avium subsp. silvaticum
recF
782
b
sodA
aspB
gnd1
lipT
pepB
b
397
“est”
183
hsp65
groELI b
1269, 1296 1435b
299b 840
521b 927
292b, 296b 247–249b (deletion)
865b (M. avium subsp. avium serotype 2)
340b 447b (M. avium subsp. silvaticum)
899b
1209 603 293b (M. avium subsp. avium serotype 3)
54, 347b, 451b
899b 138 426, 479b
645
843
128b
a Base pair position from annotated start codon in the M. avium subsp. paratuberculosis K-10 genome. Not included in this table are SNPs that distinguish M. avium subsp. paratuberculosis as a whole (n ⫽ 27) or distinguishing markers of various M. avium subsp. hominissuis strains. b Synonymous change.
M. avium subsp. paratuberculosis as a group and a set of SNPs that further distinguished the M. avium subsp. paratuberculosis subgroups. M. avium subsp. paratuberculosis isolates were divided into seven STs, with MLSA reliably distinguishing cow and sheep subtypes of M. avium subsp. paratuberculosis and also distinguishing type III isolates by a single SNP in recF. Most notably, there were more SNPs (n ⫽ 27) that distinguished M. avium subsp. paratuberculosis from M. avium subsp. hominissuis and avian organisms than there were among M. avium subsp. paratuberculosis organisms (n ⫽ 12). A list of SNPs that were strictly associated with subspecies and strains tested is provided in Table 3. Phylogenetic analysis. In addition to uncovering SNPs that permit subspecies and strain differentiation, MLSA also provides data toward phylogenetic analysis. By inputting data from all 10 genes studied across M. avium organisms, the unrooted phylogeny depicted in Fig. 1 was obtained. In keeping with the diversity and recombination noted within the M. avium subsp. hominissuis subset, these organisms present as a complex web in Fig. 1A. In contrast, the host-associated lineages are present as two independent branches that are distinct from the M. avium subsp. hominissuis group. The longer branch is composed exclusively of M. avium subsp. paratuberculosis strains, subdivided into cow and sheep strains (magnified in Fig. 1B). The second branch includes M. avium subsp. avium and M. avium subsp. silvaticum isolates, each responsible for disease in birds (magnified in Fig. 1B). For 6 of the 10 genes (recF, sodA, aspB, gnd1, pepB, and hsp65), we were able to successfully amplify and sequence the homologues from M. intracellulare isolates. Using data for only these six genes, the rooted phylogeny shown in Fig. 2 is obtained. Here, one observes that the distance between M. intracellulare and M. avium is much greater than the distances within M. avium. The mean similarity between M. intracellulare and M. avium is 91%, whereas it is ⬎99% within each group. Consistent with the topology in Fig. 1, the M. avium subsp. hominissuis group presents the most diversity, and again, the host-associated groups present as independent lineages. Addi-
tionally, the rooted phylogeny links M. intracellulare with a putative ancestor within the M. avium subsp. hominissuis group and also indicates that both M. avium subsp. paratuberculosis and M. avium subsp. avium/M. avium subsp. silvaticum arose from M. avium subsp. hominissuis. dN/dS ratio. Because the phylogenetic representation pointed to M. avium subsp. paratuberculosis and M. avium subsp. avium/M. avium subsp. silvaticum as distinct from M. avium subsp. hominissuis, we hypothesized that SNPs along these branches would differ in nature from those among M. avium subsp. hominissuis organisms. To test this possibility, the dN/dS ratio was determined for each of the following groups: M. avium subsp. hominissuis (18 STs), M. avium subsp. avium and M. avium subsp. silvaticum (8 STs), and M. avium subsp. paratuberculosis (7 STs). For M. avium subsp. hominissuis organisms, the dN/dS was 0.08, compatible with observations of bacterial genomes presumed to be under stabilizing selection (26). In contrast, the dN/dS for the pathogenic subspecies was increased to levels comparable to that observed for M. tuberculosis (⬃0.6) (9, 26); the dN/dS ratios were 0.67 for M. avium subsp. paratuberculosis and 0.50 for M. avium subsp. avium/M. avium subsp. silvaticum. DISCUSSION Traditional mycobacterial species designations are based on phenotypic features, including host range, pathological presentation, growth characteristics, and nutrient requirements. The validity of many species designations is supported by genetic analysis. However, DNA-based typing methods have also revealed the limitations of such phenotype-based taxonomies. Long considered distinct species, it is now recognized that ‘M. avium,’ ‘M. paratuberculosis,’ and ‘M. silvaticum’ share identical 16S rRNA gene sequences (32). As such, these organisms, along with ‘M. hominissuis’ strains, are currently described as subspecies of M. avium. The phylogenetic framework we have developed highlights both the similarities and differences among these M. avium strains.
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FIG. 2. Phylogenetic analysis of M. avium, based on six genes (4,626 bp), using M. intracellulare as an out-group. (A) Distance between the two species M. avium and M. intracellulare, emphasizing relative closeness of members of the same species. (B) Expansion of M. avium and M. intracellulare clusters demonstrating comparable topology with the unrooted phylogeny shown in Fig. 1. MI-1, M. intracellulare ATCC 13950T; MI-2, M. intracellulare FCC 1804; MI-3, M. intracellulare 90331.
While the 10 genes studied showed conservation across all strains tested, with ⬎98% sequence identity, sufficient differences were uncovered to provide valuable information about the evolutionary relationship of M. avium subspecies. By MLSA, the greatest variability was observed within the M. avium subsp. hominissuis subspecies. From this heterogeneous network of M. avium subsp. hominissuis strains emerged two distinct groups, one consisting exclusively of M. avium subsp. paratuberculosis strains and the other consisting of the avian subspecies M. avium subsp. avium and M. avium subsp. silvaticum. Relative to M. avium subsp. hominissuis organisms, the nature of genetic diversity within the pathogenic subspecies was altered, as the proportion of SNPs that was nonsynonymous was increased. This points to M. avium subsp. paratuberculosis and M. avium subsp. avium/M. avium subsp. silvaticum as groups that are more recently evolved and/or under differential selection than is M. avium subsp. hominissuis. A similar pattern of reduced and differential selection has been observed for other pathogenic clones, such as Yersinia pestis (1), Salmonella enterica subsp. enterica serovar Typhi (12), and Bordetella pertussis (7). However, while there is considerably less genetic variability within these pathogenic subsets compared to that in M. avium as a whole, there is nonetheless sufficient variability for subtyping into genetic variants, such as the sheep and cow subtypes of M. avium subsp. paratuberculosis. In fact, the variability within M. avium subsp. paratuberculosis organisms alone is on the same order as that observed between M. tuberculosis and virulent M. bovis. For the nine genes tested that present high homology in the M. tuberculosis complex, only six variable
positions can be found between M. tuberculosis H37Rv and M. bovis AF2122/97, while nine SNPs were found across the same sequences in both M. avium subsp. paratuberculosis and M. avium subsp. avium/M. avium subsp. silvaticum organisms. Unlike the case for pathogenic subspecies, MLSA data demonstrated considerable heterogeneity among M. avium subsp. hominissuis strains, including evidence of genetic exchange (see Fig. S1 and S2 in the supplemental material). These findings concur with evidence of recombination as demonstrated on a small scale by the sequencing of genes of the glycopeptidolipid cluster in four M. avium strains (14). Though novel, these results are perhaps not surprising given the diverse environmental habitats in which M. avium subsp. hominissuis organisms reside. Moreover, the opportunities for genetic exchange with other species or with other M. avium subsp. hominissuis organisms are likely considerable in microbial communities, such as water and peat-rich soil, where M. avium subsp. hominissuis organisms figure prominently (2, 8, 20, 21, 35). An interesting observation was that the avian subspecies M. avium subsp. avium and M. avium subsp. silvaticum emerged as a lineage independent from M. avium subsp. paratuberculosis. As these organisms have yet to be the subject of a wholegenome sequence study, their genetic situation has been less certain. It has been observed that these IS901-positive strains contain certain genomic regions that vary between M. avium subsp. paratuberculosis K-10 and M. avium subsp. hominissuis 104, tempting us to contemplate that avian strains might occupy an intermediate position between the sequenced strains. The present MLSA data argue instead that avian strains form
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a branch distinct from M. avium subsp. paratuberculosis, shared by the closely related M. avium subsp. avium and M. avium subsp. silvaticum. In fact, the number of SNPs that separate M. avium subsp. silvaticum from M. avium subsp. avium (n ⫽ 4) was similar to the number of SNPs within M. avium subsp. avium (n ⫽ 6). Further information on these pathogens, including the determination of whether these organisms harbor unique genomic elements not found in other M. avium subsets, would be uncovered by determining the whole-genome sequence of a member of this M. avium subsp. avium/M. avium subsp. silvaticum lineage. While considerable attempts were made to include a representative panel of strains, we consider it likely that the selection of isolates studied may underrepresent the diversity that exists within M. avium subsp. hominissuis. We attribute this bias to the fact that organisms available from most culture collections are highly selected for those that cause disease in human hosts. Therefore, we consider it worthwhile to apply the MLSA strategy described herein to well-defined collections of environmental isolates (13) and animal isolates, to better document the degree of variability within M. avium subsp. hominissuis. Nonetheless, with the limited number of animal isolates tested, we did observe that strains of M. avium subsp. hominissuis did not distribute into any evident pattern where human and nonhuman isolates were distinct. Rather, one pig and one dog strain shared identical sequence types with two human strains, while other animal strains (two pig, one deer, and one African buffalo) were also genetically distributed within the complex web that encompassed human strains of M. avium subsp. hominissuis. Moreover, while there are documented associations between host provenance and microbe genotype, as in the case of ovine and bovine strains of M. avium subsp. paratuberculosis, exceptions to these associations are not uncommon. Our panel included two strains of M. avium subsp. avium that were not from birds (one human and one porcine) and an M. avium subsp. paratuberculosis strain of the ovine subtype isolated from a pig. The spillover of host-associated pathogens into alternative hosts is well documented (3, 38) and emphasizes the importance of genotypically defining isolates in studies that investigate various aspects of host-pathogen relationships. Another limitation of our study may stem from the choice of genes applied to the MLSA study. We suspect that the use of different targets will uncover different SNPs in other genes, including different markers of strains and subspecies. Moreover, by using more genes to uncover more SNPs, we suspect that greater information might be obtained on branches with seemingly limited variability. For instance, our data suggest that there may be more variability within ovine strains of M. avium subsp. paratuberculosis compared to that within bovine strains; however, more sequence data would be required to formally test this hypothesis. Our MLSA-derived phylogeny indicates that the species M. avium may consist of many subspecies, but for practical purposes, one can divide them between a genetically variable pool of organisms, collectively known as M. avium subsp. hominissuis, and the two pathogenic lineages, M. avium subsp. paratuberculosis and M. avium subsp. avium/M. avium subsp. silvaticum. These divisions concur with the use of variable LSP regions and also with the presence/absence of subspecies-as-
J. BACTERIOL.
sociated insertion elements; therefore, it would appear that each of these modalities can be used for classification purposes. Whether M. avium subsp. hominissuis organisms need to be divided into further groupings will depend in part on the results of functional studies of these organisms to determine whether certain M. avium subsp. hominissuis lineages possess unique characteristics that merit consideration as a defined subspecies. ACKNOWLEDGMENTS C.Y.T. is supported by a Lloyd-Carr-Harris McGill Major Fellowship and an F.C. Harrison scholarship from the McGill Department of Microbiology and Immunology. M.A.B. is a Chercheur Bousier Senior of the Fonds de la Recherche en Sante´ du Que´bec (FRSQ) and a William Dawson Scholar of McGill University. Work on M. avium genetics and genomics is funded by a grant from the Natural Science and Engineering Research Council (grant number no. GEN2282399). We are grateful to Gerard Cangelosi (University of Washington), Anita Michel (Onderstepoort Veterinary Institute, South Africa), Petra de Haas (RIVM, The Netherlands), Debby Cousins (Australian Reference Laboratory for Bovine Tuberculosis, South Perth, Australia), and Louise Thibert (Laboratoire de Sante´ Publique du Que´bec, Canada) for the provision of strains or DNA samples. We also thank Speranza Masala for assistance with RFLP and Paul Harrison for discussion on population genetics. REFERENCES 1. Achtman, M., G. Morelli, P. Zhu, T. Wirth, I. Diehl, B. Kusecek, A. J. Vogler, D. M. Wagner, C. J. Allender, W. R. Easterday, V. Chenal-Francisque, P. Worsham, N. R. Thomson, J. Parkhill, L. E. Lindler, E. Carniel, and P. Keim. 2004. Microevolution and history of the plague bacillus, Yersinia pestis. Proc. Natl. Acad. Sci. USA 101:17837–17842. 2. Angenent, L. T., S. T. Kelley, A. St. Amand, N. R. Pace, and M. T. Hernandez. 2005. Molecular identification of potential pathogens in water and air of a hospital therapy pool. Proc. Natl. Acad. Sci. USA 102:4860–4865. 3. Bartos, M., P. Hlozek, P. Svastova, L. Dvorska, T. Bull, L. Matlova, I. Parmova, I. Kuhn, J. Stubbs, M. Moravkova, J. Kintr, V. Beran, I. Melicharek, M. Ocepek, and I. Pavlik. 2006. Identification of members of Mycobacterium avium species by Accu-Probes, serotyping, and single IS900, IS901, IS1245 and IS901-flanking region PCR with internal standards. J. Microbiol. Methods 64:333–345. 4. Collins, D. M., D. M. Gabric, and G. W. de Lisle. 1990. Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization. J. Clin. Microbiol. 28:1591–1596. 5. de Lisle, G. W., D. M. Collins, and H. F. Huchzermeyer. 1992. Characterization of ovine strains of Mycobacterium paratuberculosis by restriction endonuclease analysis and DNA hybridization. Onderstepoort. J. Vet. Res. 59:163–165. 6. de Lisle, G. W., B. J. Wards, B. M. Buddle, and D. M. Collins. 2005. The efficacy of live tuberculosis vaccines after presensitization with Mycobacterium avium. Tuberculosis 85:73–79. 7. Diavatopoulos, D. A., C. A. Cummings, L. M. Schouls, M. M. Brinig, D. A. Relman, and F. R. Mooi. 2005. Bordetella pertussis, the causative agent of whooping cough, evolved from a distinct, human-associated lineage of B. bronchiseptica. PLoS Pathog. 1:e45. 8. Falkinham, J. O., III, C. D. Norton, and M. W. LeChevallier. 2001. Factors influencing numbers of Mycobacterium avium, Mycobacterium intracellulare, and other mycobacteria in drinking water distribution systems. Appl. Environ. Microbiol. 67:1225–1231. 9. Gutacker, M. M., J. C. Smoot, C. A. Migliaccio, S. M. Ricklefs, S. Hua, D. V. Cousins, E. A. Graviss, E. Shashkina, B. N. Kreiswirth, and J. M. Musser. 2002. Genome-wide analysis of synonymous single nucleotide polymorphisms in Mycobacterium tuberculosis complex organisms: resolution of genetic relationships among closely related microbial strains. Genetics 162: 1533–1543. 10. Huson, D. H., and T. H. Kloepper. 2005. Computing recombination networks from binary sequences. Bioinformatics 21(Suppl. 2):ii159–ii165. 11. Jolley, K. A., E. J. Feil, M. S. Chan, and M. C. Maiden. 2001. Sequence type analysis and recombinational tests (START). Bioinformatics 17:1230–1231. 12. Kidgell, C., U. Reichard, J. Wain, B. Linz, M. Torpdahl, G. Dougan, and M. Achtman. 2002. Salmonella typhi, the causative agent of typhoid fever, is approximately 50,000 years old. Infect. Genet. Evol. 2:39–45. 13. Kirschner, R. A., Jr., B. C. Parker, and J. O. Falkinham III. 1992. Epidemiology of infection by nontuberculous mycobacteria. Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in acid,
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