Multidrug-Resistant Mycobacterium tuberculosis of the Latin American Mediterranean Lineage, Wrongly Identified as Mycobacterium pinnipedii (Spoligotype International Type 863 [SIT863]), Causing Active Tuberculosis in South Brazil

Abstract

We recently detected the spoligotype patterns of strains of Mycobacterium pinnipedii, a species of the Mycobacterium tuberculosis complex, in sputum samples from nine cases with pulmonary tuberculosis residing in Porto Alegre, South Brazil. Because this species is rarely encountered in humans, we further characterized these nine isolates by additional genotyping techniques, including 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing, verification of the loci TbD1, RD9, pks15/1, RD(Rio), and fbpC, the insertion of IS6110 at a site specific to the M. tuberculosis Latin American Mediterranean (LAM) lineage, and whole-genome sequencing. The combined analysis of these markers revealed that the isolates are in fact M. tuberculosis and more specifically belong to the LAM genotype. Most of these isolates (n8) were shown to be multidrug resistant (MDR), which prompted us to perform partial sequencing of the rpoA, rpoB, rpoC, katG, and inhA genes. Seven isolates (77.8%) carried the S315T mutation in katG, and one of these (11%) also presented the C((-17)T single-nucleotide polymorphism (SNP) in inhA. Interestingly, six of the MDR isolates also presented an undescribed insertion of 12 nucleotides (CCA GAA CAA CCC) in codon 516 of rpoB. No putative compensatory mutation was found in either rpoA or rpoC. This is the first report of an M. tuberculosis LAM family strain with a convergent M. pinnipedii spoligotype. These spoligotypes are observed in genotype databases at a modest frequency, highlighting that care must be taken when identifying isolates in the M. tuberculosis complex on the basis of single genetic markers.

Full text

Multidrug-Resistant Mycobacterium tuberculosis of the Latin American
Mediterranean Lineage, Wrongly Identified as Mycobacterium
pinnipedii (Spoligotype International Type 863 [SIT863]), Causing
Active Tuberculosis in South Brazil
Elis R. Dalla Costa,
a
Sidra E. G. Vasconcelos,
b
Leonardo S. Esteves,
a,c
Harrison M. Gomes,
b
Lia L. Gomes,
b
Pedro Almeida da Silva,
d
João Perdigão,
e
Isabel Portugal,
e
Miguel Viveiros,
f
Ruth McNerney,
g
Arnab Pain,
h
Taane G. Clark,
g,i
Nalin Rastogi,
j
Gisela Unis,
k
Maria Lucia R. Rossetti,
a,c
Philip Noel Suffys
b,l
Fundação Estadual de Produção e Pesquisa em Saúde (FEPPS), Porto Alegre, Brazil
a
; Laboratório de Biologia Molecular Aplicada a Micobactérias, Instituto Oswaldo Cruz,
Fiocruz, Rio de Janeiro, Brazil
b
; Universidade Luterana do Brasil (ULBRA/RS), Porto Alegre, Brazil
c
; Núcleo de Pesquisa em Microbiologia Médica, Faculdade de Medicina,
Universidade Federal do Rio Grande, Rio Grande do Sul, Brazil
d
; Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia da Universidade de
Lisboa, Lisbon, Portugal
e
; Grupo de Micobactérias, Unidade de Microbiologia Médica, Global Health and Tropical Medicine (GHMT), Instituto de Higiene e Medicina
Tropical (IHMT), Universidade Nova de Lisboa (UNL), Lisbon, Portugal
f
; Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London,
United Kingdom
g
; Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi
Arabia
h
; Faculty of Epidemiology and Population Health, London School of Hygiene & Tropical Medicine, London, United Kingdom
i
; Unité de la Tuberculose et des
Mycobactéries, Institut Pasteur de Guadeloupe, Les Abymes, Guadeloupe, France
j
; Hospital Sanatório Partenon (HSP), Porto Alegre, Brazil
k
; Unit of Mycobacteriology at
the Institute of Tropical Medicine, Antwerp, Belgium
l
We recently detected the spoligotype patterns of strains of Mycobacterium pinnipedii, a species of the Mycobacterium tuberculo-
sis complex, in sputum samples from nine cases with pulmonary tuberculosis residing in Porto Alegre, South Brazil. Because this
species is rarely encountered in humans, we further characterized these nine isolates by additional genotyping techniques, in-
cluding 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing, verification
of the loci TbD1, RD9, pks15/1,RD
Rio
, and fbpC, the insertion of IS6110 at a site specific to the M. tuberculosis Latin American
Mediterranean (LAM) lineage, and whole-genome sequencing. The combined analysis of these markers revealed that the isolates
are in fact M. tuberculosis and more specifically belong to the LAM genotype. Most of these isolates (nⴝ8) were shown to be
multidrug resistant (MDR), which prompted us to perform partial sequencing of the rpoA,rpoB,rpoC,katG, and inhA genes.
Seven isolates (77.8%) carried the S315T mutation in katG, and one of these (11%) also presented the C(ⴚ17)T single-nucleotide
polymorphism (SNP) in inhA. Interestingly, six of the MDR isolates also presented an undescribed insertion of 12 nucleotides
(CCA GAA CAA CCC) in codon 516 of rpoB. No putative compensatory mutation was found in either rpoA or rpoC. This is the
first report of an M. tuberculosis LAM family strain with a convergent M. pinnipedii spoligotype. These spoligotypes are ob-
served in genotype databases at a modest frequency, highlighting that care must be taken when identifying isolates in the M. tu-
berculosis complex on the basis of single genetic markers.
Tuberculosis (TB) is a disease caused by organisms belonging to
the Mycobacterium tuberculosis complex (MTBC), which are
known to infect humans and domestic and wild animals. The
MTBC complex includes M. tuberculosis,M. africanum,M. mi-
croti,M. bovis,M. bovis bacillus Calmette-Guérin (BCG), M. ca-
prae,M. pinnipedii,M. orygis,M. mungi, and M. suricattae (1–8).
M. tuberculosis is the predominant cause of human TB worldwide,
but M. africanum and M. bovis remain important agents of human
disease in certain geographical regions (9). The MTBC species
share identical 16S rRNA sequences, and recent studies have im-
proved our knowledge on the genetic diversity, host range, epide-
miological aspects, and differences in pathogenicity and virulence
among the species of the complex (10,11). Based on the various
genotyping techniques, like spoligotyping (12), restriction frag-
ment length polymorphism (RFLP) (13), mycobacterial inter-
spersed repetitive-unit–variable-number tandem-repeat (MIRU-
VNTR) typing (14), and whole-genome sequencing (15), M.
tuberculosis strains have been subdivided into lineages and fami-
lies. The most geographically widespread family worldwide (spo-
ligotyping) is the Latin American Mediterranean (LAM) lineage,
which is part of the heterogeneous Euro-American lineage, one of
seven M. tuberculosis lineages (16). The LAM family strains are
widespread across all five continents, with a marked incidence in
Received 24 July 2015 Returned for modification 1 September 2015
Accepted 16 September 2015
Accepted manuscript posted online 23 September 2015
Citation Dalla Costa ER, Vasconcelos SEG, Esteves LS, Gomes HM, Gomes LL,
Almeida da Silva P, Perdigão J, Portugal I, Viveiros M, McNerney R, Pain A, Clark TG,
Rastogi N, Unis G, Rossetti MLR, Suffys PN. 2015. Multidrug-resistant
Mycobacterium tuberculosis of the Latin American Mediterranean lineage, wrongly
identified as Mycobacterium pinnipedii (spoligotype international type 863
[SIT863]), causing active tuberculosis in South Brazil. J Clin Microbiol
53:3805–3811. doi:10.1128/JCM.02012-15.
Editor: G. A. Land
Address correspondence to Elis R. Dalla Costa, dallacostaer@gmail.com, or
Philip Noel Suffys, psuffys@gmail.com.
E.R.D.C. and S.E.G.V., and M.L.R.R. and P.N.S., contributed equally to this article.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/JCM.02012-15.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
December 2015 Volume 53 Number 12 jcm.asm.org 3805Journal of Clinical Microbiology

South America, and they account for 17% of all strains in the
SITVITWEB database (17).
In a previous work, nine drug-resistant isolates sourced from
Porto Alegre (Rio Grande do Sul, Brazil) presented with the spo-
ligotype international type 863 (SIT863), a strain type previously
described by Perizzolo et al. (18). This spoligotype, according to
the SITVITWEB database, is representative of M. pinnipedii and,
more specifically, the PINI2 clade (17). This species was originally
described as the etiological agent of TB in seals and sea lions and in
some terrestrial mammals, while a single report related putative
transmission to a zoo caretaker but without evolution to active
disease (6,19–21). There is a paucity of information concerning
M. pinnipedii as a cause of human disease and on its pathogenicity/
virulence, drug resistance, and epidemiology. The nine strains
with the PINI2 spoligopattern were therefore submitted to more
extensive genetic characterization and evaluation of their drug
susceptibility patterns.
MATERIALS AND METHODS
Mycobacterium strains. Nine MTBC isolates were derived from sputum
samples from individuals who were diagnosed in 2006 (n⫽1) and 2010
(n⫽8) with pulmonary TB as part of routine diagnosis at the Hospital
Sanatório Partenon, the reference center for drug-resistant TB in Porto
Alegre, the capital of Rio Grande do Sul, South Brazil. The sputum sam-
ples from these patients were processed for acid-fast bacilli microscopy
detection on Ziehl-Neelsen-stained slides and cultured in Lowenstein-
Jensen medium. The isolates were submitted to standard bacteriological
and biochemical tests for differentiation of species within the MTBC and
nontuberculous mycobacteria, including biochemical testing for niacin,
para-nitrobenzoic acid, and tiofeno-2-carboxylic acid hydrazine (22). In
addition, they were submitted to drug susceptibility testing using the pro-
portion method on Lowenstein-Jensen solid medium (22,23).
Nucleic acid extraction and genotyping controls. For genotyping,
nucleic acids were extracted as described by van Soolingen et al. (24). All
PCR-based genotyping reactions included negative controls (ultrapure
water) and positive controls, composed of DNA from M. pinnipedii
(strains 76 and 8; kindly provided from the collection of the National
Institute for Public Health and the Environment-RIVM) (25), M. tuber-
culosis H37Rv (ATCC 27294), and two M. tuberculosis isolates of the LAM
genotype from Brazil, as defined by spoligotyping and 24-locus MIRU-
VNTR typing (26).
Genotyping. All PCRs were performed on a Veriti thermal cycler (Ap-
plied Biosystems, Foster City, CA). Spoligotyping was performed using
the commercially available kit from Ocimum Biosolutions (Hyderabad,
India), as described by Kamerbeek et al. (12). For 24-locus MIRU-VNTR
typing, amplification of loci was performed by using a commercial typing
kit (Genoscreen, Lille, France) and automated MIRU-VNTR analysis,
performed as previously described (27). The fragment sizes of the ampli-
cons were analyzed on an ABI3730 DNA sequence analyzer (Applied Bio-
systems), and the number of copies of each locus was determined by
automated assignment using the GeneMapper 4.0 software (Applied Bio-
systems). In the case of doubtful results, the sizes of the repeats were
double checked by size estimation compared to a DNA ladder (50 and 100
bp) and the positive control (H37Rv) on agarose gel and by comparing to
a reference table, as described previously (14).
Comparison of genotypes with spoligotype and MIRU-VNTR
databases. The spoligopatterns of the strains were compared with the
SITVITWEB database (http://www.pasteur-guadeloupe.fr:8081/SITVIT
_ONLINE) for defining the spoligotype international type (SIT) and distri-
bution frequency on a global level (17). The spoligotype and 24-locus
MIRU-VNTR profiles were also compared with the MIRU-VNTRplus da-
tabase (http://www.miru-vntrplus.org/MIRU/index.faces#)(28). The defini-
tion of lineages was based on 24-locus MIRU-VNTR typing using best-
match analysis and tree-based identification using the categorical index.
Detection of a LAM-specific IS6110 insertion. Briefly, the LAM fam-
ily was confirmed based on the verification of the presence or not of an
IS6110 element at position 932204 of the H37Rv genome (GenBank ac-
cession no. NC_000962.2), as described by Sampson et al. (29).
Verification of RD
Rio
status. Verification of RD
Rio
status was per-
formed as described by Lazzarini et al. (30) using a multiplex PCR proto-
col. The amplified products of 1,175 bp or 530 bp in the presence or
absence of the deletion, respectively, were analyzed in a 1.5% agarose gel.
Characterization of the fbpC
103
SNP. Characterization of the fbpC
103
SNP was performed as described by Vasconcellos et al. (31). The amplified
products of 519 bp were analyzed on 2% agarose gels after staining with
ethidium bromide. Partial sequencing was performed using the BigDye
Terminator kit (PE Applied Biosystems) on an ABI 3730 DNA analyzer
(Programa de Desenvolvimento Technológico em Insumos para Saúde
[PDTIS] DNA sequencing platform at Fiocruz [http://www.dbbm.fiocruz
.br/PDTIS_Genomica/]), and the results were analyzed with SeqScape
software version 3.0 (Applied Biosystems, CA, USA), as previously de-
scribed (32,33).
Detection of the insertion in pks15/1 and of RD9 and TbD1. Six- or
7-bp insertions were detected by partial sequencing of the pks15/1 locus.
The initial amplifications were performed as described by Huard et al.
(33). The sequencing was performed using sequencing kits and an ana-
lyzer, as described above; the results were interpreted as previously de-
scribed (32,33). For analysis of the RD9 and TbD1 loci, we used 3-primer
combinations (site specific), as described by Vasconcellos et al. (31). The
3-primer PCRs were each designed to amplify a product of one size when
the target locus is intact or to produce a different band size when a known
long-sequence polymorphism is present.
Sequencing of rpoA,rpoB,rpoC,katG, and inhA.For rpoB,katG, and
inhA, the PCR-based sequencing was performed as described by Ra-
masoota et al. (34), de Oliveira et al. (35), and Dalla Costa et al. (36), while
for rpoA and rpoC, the conditions were as described by de Vos et al.
(37). Sequencing and analysis of rpoA,rpoB, and rpoC genes was per-
formed at Fiocruz-RJ, as described above, while the katG and inhA
genes were sequenced and analyzed at the sequencing platform at Cen-
tro de Desenvolvimento Científico e Tecnológico (CDCT)-Fundação
Estadual de Produção e Pesquisa em Saúde (FEPPS).
Whole-genome sequencing. Three isolates (M. tuberculosis RG74,
RG112, and RG621257) were submitted to paired-end sequencing (105
bp) using the Illumina HiSeq 2500 platform at the King Abdullah Univer-
sity of Science and Technology (KAUST), Saudi Arabia. Raw read
data were mapped to the reference genome of M. tuberculosis H37Rv
(GenBank accession no. NC_000962.3) using the Burrows-Wheeler
Aligner tool, and variants were called using the SAMtools package (38,
39). The obtained mean fold coverages were 249.51, 251.44, and 221.94
for RG74, RG112, and RG621257, respectively. Comma-separated files
containing all detected variants are available in Tables S1 to S3 in the
supplemental material. A script was written to extract the nucleotide cov-
erage at each reference genomic position, and an R script was developed to
classify each strain according to the recent M. tuberculosis SNP barcode
typing system (15) (see Files S1 and S2 in the supplemental material).
Nucleotide sequence accession number. All sequencing data have
been submitted to the European Nucleotide Archive (http://www.ebi.ac
.uk/ena/) under study accession no. PRJEB10715.
RESULTS
Bacteriological identification. The Ziehl-Neelsen-stained micro-
scopic slides showed the presence of acid-fast bacilli, while the
Lowenstein-Jensen cultures presented rough colonies without
pigmentation after 3 to 4 weeks of incubation at 37°C. The isolates
scored positive for niacin and para-nitrobenzoic acid but were
nitratase negative, which is characteristic of organisms of the
MTBC.
Conventional and genetic testing of drug susceptibility. The
proportion method for drug susceptibility testing on Lowenstein-
Dalla Costa et al.
3806 jcm.asm.org December 2015 Volume 53 Number 12Journal of Clinical Microbiology

Jensen solid medium demonstrated that eight isolates were mul-
tidrug resistant (MDR), while one isolate was resistant to isoniazid
(INH) and another to rifampin (RIF) only; all were susceptible to
ethambutol (EMB) and streptomycin (SM). The sequencing re-
sults corroborated resistance to INH by presenting the S315T mu-
tation in katG in seven isolates, with one of these also carrying the
C(⫺17)T SNP in inhA; one strain did not show genetic evidence
for resistance to INH. Among the nine isolates that were resistant
to RIF, six presented a duplication of 12 nucleotides (CCA GAA
CAA CCC) located before the last nucleotide of codon 516 of
rpoB, and all these also presented a substitution of GAC to GGC in
codon 516, causing the amino acid change D516V, besides the
insertion of the glutamine, asparagine, isoleucine, and proline
(QNIP). One isolate (M. tuberculosis 100285) that did not carry
the 12-nucleotide (nt) duplication presented the GAC to GTC
SNP, which was responsible for the amino acid change D513V. No
other mutations were observed in the studied part of rpoB in this
set of isolates, including the RIF-susceptible isolate (M. tuberculo-
sis 100056), and one isolate (RG621257) that was RIF resistant and
was submitted to whole-genome sequencing presented the wild-
type rpoB allele. Finally, none of the isolates presented missense
mutations in the SNPs in rpoA and rpoC described by de Vos et al.
(37).
Genotyping for lineage classification. All nine isolates pre-
sented SIT863, which is characteristic of M. pinnipedii (Fig. 1).
Upon 24-locus MIRU-VNTR typing, eight isolates presented the
same MIRU pattern, while one isolate presented five fewer copies
of MIRU21 (see Table S4 in the supplemental material). In a com-
parison of these MIRU patterns to those present in the SITVIT2
database, they were clearly different from the patterns that are
characteristic of M. pinnipedii. Using the neighbor-joining-based
phylogenetic tree building tool of MIRU-VNTRplus, the patterns
of these isolates were organized within the MIRU patterns char-
acteristic of LAM9 (Fig. 2). In addition, when constructing a
neighbor-joining tree together with 24-locus MIRU patterns from
a recent sample set from Brazil (26), the MIRU patterns were
closest to those of LAM strains (Fig. 2). Upon analysis of the pres-
ence of other M. tuberculosis markers characteristic of LAM, their
LAM nature was confirmed, and all were TbD1 negative and RD9
positive while not presenting the RD
Rio
genotype.
Whole-genome sequencing-based typing. The raw sequence
reads for RG74, RG112, and RG621257 were aligned to the H37Rv
reference sequence, and 869, 877, and 881 SNPs were identified,
respectively. Upon excluding SNPs present in proline-glutamate
(PE)/proline-proline-glutamate (PPE) genes and other repeat-
rich regions, the three isolates shared 816 SNPs, each strain bear-
ing between one and five unique SNPs (Fig. 1). In addition, 72
(RG74), 74 (RG112), and 87 (RG621257) short indels (⬍100 bp)
were present. According to the 62-SNP barcoding scheme recently
proposed by Coll et al. (15), the three strains were classified as
belonging to lineage 4.3.3, part of the Euro-American lineage (lin-
eage 4), confirming their LAM classification. Whole-genome se-
quencing confirmed the mutations observed by conventional se-
quencing of the hot spot region of rpoB, including the 12-nt
insertion at genomic position 761123 (WGS codon 435). The iso-
late without the insertion did not present an SNP in the rest of
rpoB (see Table S4 in the supplemental material). In addition, the
absence of a mutation in rpoA and rpoC was confirmed, except for
the presence of a synonymous SNP (GCC to GCG) at position
763370 (amino acid position 542).
DISCUSSION
M. pinnipedii, formally known as the “seal bacillus,” was described
in 2003 on the basis of a characterization of isolates of M. tuber-
culosis-like organisms obtained from seals (6) and being the caus-
ative agent of TB in this mammal host. Later, the involvement of
M. pinnipedii in TB transmission to humans was observed in a
European zoo, where sea lion keepers had been infected but did
not develop active TB during the time of the study (20). This M.
pinnipedii isolate sourced from the animals was susceptible to the
main antituberculosis drugs, isoniazid and rifampin, and strepto-
mycin, ethambutol, and para-aminosalicylic acid (6).
In the spolDB4 database, the spoligotype profile obtained for
the isolates under analysis in the present study corresponded to
SIT863, initially described as characteristic of the M. tuberculosis U
family (40) and later on redefined and presented by SITVITWEB
as PINI2 (17). Isolates with the genotype SIT863 were reported in
three earlier studies carried out in Porto Alegre, the first reported
by Cafrune et al. (41), who observed six human isolates between
2005 and 2007. Subsequently, Perizzolo et al. (18) identified
two isolates between 2004 and 2006, while Khuleis et al. (42)
reported five additional isolates in a study carried out between
2007 and 2008. Outside Brazil, there have been reports of a
single case in the United States and two more in Venezuela (43,
44). At SITVITWEB, four cases were reported, and three of
these were isolated in Pelotas (Rio Grande do Sul, Brazil) but
without information about the isolation data; the other case
was isolated in the United States in 2003. These four SIT863
strains were classified as PINI2 based on revised rules for PINI,
PINI1, and PINI2 (unpublished data). Accordingly, PINI2 (to
which SIT863 belongs) is characterized by the obligate pres-
ence of spacer 25 only; therefore, the SIT863 pattern (presence
of spacers 25 to 28) is compatible with the PINI2 definition and
does not match any other signature in our updated SITVIT2
database. A review of the SITVIT2 database showed the pres-
ence of a total of 159 isolates with spoligotypes characteristic of
M. pinnipedii (PINI1, n⫽33; PINI2, n⫽105; PINI-like, n⫽7)
but with the same number of isolates with the SIT863 pattern as in
the earlier version of the database (data not shown). These data
FIG 1 Venn diagram depicting the number of SNPs shared by the three M.
tuberculosis clinical isolates subjected to WGS.
Lineage Wrongly Identified as Mycobacterium pinnipedii
December 2015 Volume 53 Number 12 jcm.asm.org 3807Journal of Clinical Microbiology

suggest that human infection rates for M. pinnipedii might be
higher than has been suggested in earlier studies (20).
However, the misclassification of lineages of M. tuberculosis by
spoligotyping has been reported, and it is now widely accepted
that this genotyping method does not present the same level of
discrimination of M. tuberculosis isolates as that with MIRU-
VNTR typing (45). Convergent evolution has been described as
relatively common when using the direct repeat (DR) region as a
genetic marker, and incorrect classification of lineages by spoligo-
typing was recently evidenced in Brazil (26) and more frequently
when the deletion of large blocks of spacers occurs, such as causing
misclassification of M. tuberculosis of the Beijing type (46). There-
fore, our observation of human isolates with the SIT863 spoligo-
type characteristic of PINI2, together with the lack of notification
of M. pinnipedii as a cause of human TB, lead us to believe that we
might be dealing with cases of misclassification.
It has been shown that MIRU-VNTR typing is a robust proce-
dure for strain typing and phylogenetic classification (26,27,47),
being either in a 9-, 12-, 15-, or 24-allele format in a comparison of
large databases (MIRU-VNTRplus and SITVIT2) and 24-locus
MIRU-VNTR typing as a single procedure; it is capable of reliable
identification and discrimination of M. tuberculosis strains (25).
FIG 2 Phylogenetic tree positioning the MIRUs of the isolates in MIRU-VNTRplus. CAS, Central Asian; EAI, Eastern African and Indian; TUR, Turkey.
Dalla Costa et al.
3808 jcm.asm.org December 2015 Volume 53 Number 12Journal of Clinical Microbiology

The pattern we observed in the nine isolates was clearly different
from that obtained with the two M. pinnipedii control strains, as
can be observed in Fig. 2, and different from those characteristic of
M. pinnipedii in SITVITWEB (256324222321, 216424222322,
256424222321, and 226424253522). In addition, the construction
of a neighbor-joining tree using MIRU-VNTRplus demonstrated
that our MIRU-VNTR profile was close to that of LAM genotypes
(Fig. 2), and this was confirmed when we compared the genotype
with that obtained in a recent study in Rio de Janeiro (data not
shown).
The LAM lineage of the isolates was confirmed by the addi-
tional genotyping procedures specific for LAM strains, including
the presence of IS6110 at a particular site of the genome (48).
Another marker is present in the polyketide synthase (pks)15/1
locus, reported to be polymorphic among members of the MTBC
(49). This genetic region has an intact open reading frame in Indo-
Oceanic, East Asian, and East African-Indian lineages. However,
the Euro-American lineage contains a typical 7-bp deletion in the
pks15/1 gene, while other MTBC species, such as M. pinnipedii,
contain a 6-bp deletion (31,33,49–51). Presently, a 7-bp insertion
instead of the 6-bp insert specific for M. pinnipedii was observed.
A third marker, the fbpC SNP, which changed nucleotide G to A at
codon 103 (E103N), differentiates LAM strains from non-LAM
strains (52); this also confirmed the LAM nature of the present
isolates. Finally, we verified the absence of TbD1, which is charac-
teristic of M. tuberculosis, and the presence of RD9, which is char-
acteristic of M. africanum and MTBC species that infect mostly
animals (53). Interestingly, the phenotype of the isolates also cor-
roborated them being M. tuberculosis and not M. pinnipedii, since
all were niacin positive, and M. pinnipedii is niacin negative.
While all M. pinnipedii isolates reported so far are susceptible
to antibiotics, seven of the nine isolates were MDR; INH resistance
was due to S315T in katG in six cases, while one isolate presented
S315T and an additional T-to-G transition at position ⫺17 of
inhA. Interestingly, in seven of the nine isolates, a duplication of
12 nucleotides was observed in codon 516, which was reported
earlier in Porto Alegre by Perizzolo et al. (18), causing the inser-
tion of four amino acids. Because isolate RG621257 did not pres-
ent a mutant rpoB allele, we aimed to discover if mutations in the
rest of the genome and not present in RG74 and RG112 might
explain resistance to RIF. We observed some SNPs in genes asso-
ciated with efflux pumps and impermeability of cell membranes
(data not shown), and whether these mutations are directly asso-
ciated with resistance to RIF is under investigation.
Because of the size of this insertion and its possible putative
influence on the structure of the beta polymerase unit (unpub-
lished data), we expected to observe compensatory mutations in
either rpoA or rpoC, as described recently in M. tuberculosis strains
(37,54,55). Surprisingly, we did not observe any non-synony-
mous mutations in the rpoA and rpoC regions covered by our
sequencing approach, and this might mean that (i) the duplication
does not interfere with the biological function of the polymerase,
which seems unlikely due to the size of the duplication; (ii) un-
known and undescribed compensatory mutations are present in
other regions of the genome; or (iii) this is a recent evolutionary
event for which no compensatory mutation has yet been acquired
and is fixed in the population that contains the clinical isolate.
Interestingly, in a RIF-resistant isolate of M. smegmatis, an inser-
tion of six amino acids was observed at codons 434 and 435 of
rpoB, which led to growth attenuation (56).
To classify our strains to the lineage level, confirm their LAM
nature also based on the SNP barcode (15), and better understand
their similarity on a genetic level, three strains were submitted to
whole-genome sequencing. The observed genetic distances be-
tween the three isolates whose genomes have been sequenced also
point toward recent transmission of this strain. The three isolates
were found to be within 5 to 11 SNPs in distance, which strongly
indicates an epidemiological link between the patients, as reported
by Walker et al. (56); the strongest link was between isolates
RG112 and RG621257, with a genome difference of only 5 SNPs.
The remaining isolate (RG74), which presented differences of 8 or
11 SNPs from the other strains, falls into the indeterminate inter-
val (6 to 12 SNPs) (56) for the delineation of TB outbreaks using
WGS data. This demonstrated that independent of the lineage
nature of these isolates, they are part of an active transmission
chain of MDR-TB in Porto Alegre over a period of several years.
Earlier outbreaks of MDR strains have been reported in the city
of Porto Alegre, including a LAM5 strain reported by Perizzolo et
al. (18) and a recent MDR-TB LAM2 strain (57), both being of the
RD
Rio
genotype; the nine isolates described here, however, were
not of the RD
Rio
genotype.
This work further reinforces the extreme care needed to be
taken when using databases for comparisons of genotype identi-
fications of local M. tuberculosis isolates, and it highlights the need
to use multiple markers for correct species and/or lineage assigna-
tion of isolates with infrequent spoligotypes.
ACKNOWLEDGMENTS
Miguel Viveiros was supported by “Ciências em Fronteiras/Professor
Visitante Especial” (no. 88881.064961/2014-01) (Jose R. Lapa e Silva/
UFRJ coordinator) by CAPES/MEC/Brazil. This study was partially sup-
ported by a research grant from the European Society of Clinical Micro-
biology and Infectious Diseases, for which we would like to acknowledge
the Study Group for Mycobacterial Infections; the Fundação para a Ciên-
cia e Tecnologia, Portugal (project reference no. PTDC/SAU-EPI/122400/
2010); a postdoctoral fellowship (SFRH/BPD/95406/2013) awarded to
João Perdigão; and by CNPq (441499/2014-7 MCTI/CNPQ/Universal 14/
2014).
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