JCM Accepts, published online ahead of print on 11 June 2014 J. Clin. Microbiol. doi:10.1128/JCM.00549-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Multidrug resistant non-tuberculous mycobacteria
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isolated from cystic fibrosis patients
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Pedro Henrique Campanini Cândido1, Luciana de Souza Nunes2, Elizabeth
4
Andrade Marques3, Tânia Wrobel Folescu4, Fábrice Santana Coelho5, Vinicius
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Calado Nogueira de Moura6, Marlei Gomes da Silva6, Karen Machado Gomes6,
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Maria Cristina da Silva Lourenço7, Fábio Silva Aguiar1, Fernanda Chitolina8,
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Derek T. Armstrong9, Sylvia Cardoso Leão10, Felipe Piedade Gonçalves
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Neves11, Fernanda Carvalho de Queiroz Mello8, Rafael Silva Duarte6 *.
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Programa de Pós Graduação em Clínica Médica, Hospital Universitário
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Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, RJ, Brazil1;
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Programa de Pós Graduação em Ciências Médicas, Universidade Federal do Rio
12
Grande do Sul, RS, Brazil2; Departamento de Microbiologia, Imunologia e
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Parasitologia, Faculdade de Ciências Médicas, Universidade do Estado do Rio de
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Janeiro, RJ, Brazil3; Instituto Fernandes Figueira, RJ, Brazil4; Hospital Universitário
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Pedro Ernesto, Universidade do Estado do Rio de Janeiro, Brazil5; Instituto de
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Microbiologia, Universidade Federal do Rio de Janeiro, RJ, Brazil6; Instituto de
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Pesquisa Evandro Chagas, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil7;
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Instituto de Doenças do Tórax, Rio de Janeiro, Brazil8; Johns Hopkins University,
19
Baltimore, MD, USA9, Departamento de Microbiologia, Imunologia e Parasitologia,
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Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo,
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Brazil10; Instituto Biomédico, Universidade Federal Fluminense, Niterói, RJ,
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Brazil11. 1
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Running title: Multidrug resistant non-tuberculous mycobacteria strains in cystic
25
fibrosis patients
26
Keywords: cystic fibrosis, colonization, infection, multidrug resistance, non-
27
tuberculous mycobacteria, PFGE, Rio de Janeiro, Brazil.
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*Corresponding author
29
Mailing address:
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Instituto de Microbiologia, Universidade Federal do Rio de Janeiro,
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CCS, Bloco I, Cidade Universitária
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Rio de Janeiro, RJ 21941-902, Brazil
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Phone: 55 21 2560-8344 r. 134
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Fax: 55 21 2560-8344 r. 108
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E-mail: [email protected]
36 37 38 39
2
Summary
40 41
Worldwide,
nontuberculous
mycobacteria
(NTM)
have
become
emergent
42
pathogens of pulmonary infections in cystic fibrosis (CF) patients, with an
43
estimated prevalence varying from 5 to 20%. This work investigated the presence
44
of NTM in sputum samples of 129 CF patients (2 to 18 years old) submitted to
45
longitudinal clinical supervision at a regional reference center in Rio de Janeiro,
46
Brazil. From June 2009 to March 2012, 36 NTM isolates recovered from 10
47
(7.75%) out of 129 children were obtained. Molecular identification of NTM was
48
performed by PRA-hsp65 and sequencing of rpoB gene, and susceptibility tests
49
followed Clinical and Laboratory Standards Institute recommendations. For
50
evaluating the genotypic diversity, PFGE and/or ERIC-PCR were performed. The
51
species identified were Mycobacterium abscessus subsp. bolletii (n=24), M.
52
abscessus subsp. abscessus (n=6), M. fortuitum (n=3), M. marseillense (n=2) and
53
M. timonense (n=1). Most of the isolates presented resistance to five or more
54
antimicrobials tested. Typing profiles were mainly patient-specific. The PFGE
55
profiles indicated the presence of two clonal groups for M. abscessus subsp.
56
abscessus and five clonal groups for M. abscesssus subsp. bolletii, with just one
57
clone detected in two patients. Given the observed multidrug resistance patterns
58
and the possibility of transmission among different patients, we suggest the
59
implementation of continuous and routine investigation of NTM infections or
60
colonization in CF patients, including countries with high burden of tuberculosis
61
disease.
62 63 3
64
INTRODUCTION
65
The survival rate of cystic fibrosis (CF) patients has increased along recent
66
years, mainly due to early diagnosis, nutritional therapies and the discovery of new
67
antimicrobial agents. Acute or chronic pulmonary infections caused by bacteria are
68
the major causes of morbidity and mortality in CF patients [1]. For the last 20 - 25
69
years, non-tuberculous mycobacteria (NTM) have emerged as newly detected
70
pathogens in CF patients. Several centers around the world have reported the
71
isolation of NTM in the respiratory tract of CF patients. Nowadays this prevalence
72
varies between 5 and 20% [2,3,4,5,6,7] with most of those cases (>90%) caused
73
by species of the Mycobacterium avium complex (MAC) and the Mycobacterium
74
abscessus group [5,8,9].
75
NTM have been described in diverse environments and clinical scenarios
76
due to their adaptive biological properties and opportunistic potentials. It is
77
considered that selective pressure in hospital environments predominantly caused
78
by excessive antibiotics use, has contributed to NTM resistance to a large number
79
of antibiotics and disinfection processes, such as anti-tuberculosis agents (i.e.
80
isoniazid and pyrazinamide), glutaraldehyde and organomercury compounds [10].
81
However, NTM usually present as susceptible to aminoglycosides and macrolides
82
[11,12,13]. Cefoxitin, quinolone and tetracyclines are other options for drug
83
treatment, specifically for rapidly growing mycobacteria (RGM) infections.
84
However, similarly to other bacteria commonly associated with CF infections, NTM
85
resistant strains have been reported. Broda et al. [14] reports clarithromycin
86
susceptibility rates of 57% in Mycobacterium abscessus isolates. Binder et al. [13]
4
87
suggest that all CF patients undertake evaluation for NTM disease, due to
88
continual exposure to macrolides for other bacterial infections.
89
In Brazil, microbiological studies involving CF patients are rare, with most
90
focused on infections caused by Burkholderia cenocepacia and/or Pseudomonas
91
aeruginosa [2]. The Instituto Fernandes Figueira (IFF) is a large unit that belongs to
92
the Oswaldo Cruz Foundation (FIOCRUZ) and is the major clinical research center
93
for CF children in Rio de Janeiro state and in the country of Brazil. Microbiological
94
surveillance of these patients is common, however investigation of NTM disease
95
has only been performed since June 2009. Given the lack of epidemiological
96
information on NTM in Brazilian CF children and the emergence of NTM as
97
pathogens in this population worldwide, the significance of developing research on
98
NTM and its association to CF is definitely warranted. Epidemiological studies in
99
European cohorts have demonstrated a high prevalence of NTM infections in this
100
population, but these countries have lower disease-burdens of tuberculosis (TB).
101
Brazil is one of the main high TB-burden countries and has seen large epidemics
102
of NTM infections described up in recent literature [10]. Therefore, it is necessary
103
to undergo studies looking at prevalence of NTM in CF patients, to better
104
understand the magnitude of this emergence in different countries globally.
105
This is the first complete study providing genotypic identification,
106
antimicrobial susceptibility profiles and strain diversity of non-tuberculous
107
mycobacterial isolates recovered from CF patients in Brazil.
108 109
5
110
MATERIAL AND METHODS
111
Mycobacterial isolates. From June 2009 to March 2012, spontaneous
112
sputa from CF patients monitored at a local reference center (IFF-FIOCRUZ, Rio
113
de Janeiro, Brazil) were sent monthly to the Bacteriology and Mycobacteriology
114
laboratories at Hospital Universitário Pedro Ernesto, Rio de Janeiro, Brazil for
115
routine microbiological investigation. Sputa were processed by NALC-NaOH and
116
5% oxalic acid decontamination as recommended by the National Guideline for TB
117
and NTM Laboratory Surveillance [15]. A total of 36 NTM isolates were recovered
118
from sputa obtained from 10 (7.75%, 2 to 18 years old) out of 129 children.
119
Species identification. A loopful of bacterial growth from Löwenstein–
120
Jensen media was mixed in 100 µL of TE (Tris-HCl 10 mM, EDTA 1 mM, pH 8,0).
121
The suspensions were boiled at 100°C for 10 min and immediately incubated at
122
20°C for 12 hours. Genotypic identification of the 36 isolates was performed by the
123
PRA-hsp65 technique using the primers TB11 5´-ACCAACGATGGTGTGTCCAT-
124
3’ and TB12 5´-CTTGTCGAACCGCATACCCT-3’ [16]. A denaturation step of 95ºC
125
for 5 min was followed by 45 cycles of 94ºC for 1 min, 60ºC for 1 min and 72ºC for
126
1 min and by a final extension of 72ºC for 7 min. Amplicons were submitted to
127
individual digestions using the restriction enzymes BstEII and HaeIII (Promega
128
Corporation, Madison, WI) for 3 hours at 60 and 37°C, respectively. The digestion
129
products were analyzed after a 4% agarose gel electrophoresis at 3V/cm using 25
130
and 50 bp DNA ladders (Invitrogen). The PRA-hsp65 profiles were analyzed using
131
the algorithm available at the PRAsite (http://app.chuv.ch/prasite/index).
132
All the results were confirmed by partial sequencing of the rpoB gene using
133
the primers Myco F (5’-GCAAGGTCACCCCGAAGGG-3’) and MycoR (5’6
134
AGCGGCTGCTGGGTGATCATC-3’), according to the methodology described by
135
Adekambi, Colson & Drancourt [17]. Region V of rpoB gene sequences were
136
submitted
137
http://www.ncbi.nlm.nih.gov/BLAST) for similarity analysis and comparison to the
138
sequences from M. abscessusT ATCC 19977 (JF346872), “M. massilienseT” CIP
139
108297 (EU254721.1) and “M. bolletiiT” CCUG 50184 (DQ987717.1), the M.
140
abscessus subsp. bolletii epidemic strain INCQS 594 (EU117207.1), M. fortuitum
141
ATCC 6841 (JF346874.1), M. marseillense strain 62863 (EF584444.1) and M.
142
timonense strain 3256799 (EF584435.1).
143
to
BLAST
–
Basic
Local
Alignment
Tool
(URL:
Antimicrobial susceptibility testing. The RGM isolates were tested
144
against
amikacin
(128-1µg/mL),
145
0,125µg/mL),
146
moxifloxacin (16-0,125 µg/mL), sulfamethoxazole-trimethoprim (16/304-0,5/9,5
147
µg/mL). Slowly growing mycobacteria (SGM) were tested against amikacin (128-
148
1µg/mL), clarithromycin (64-0,5µg/mL), streptomycin (16–0,125µg/mL), ethambutol
149
(16-0,125µg/mL), moxifloxacin (16-0,125µg/mL) and rifampicin (16-0,125µg/mL).
150
Minimum inhibitory concentrations (MIC) were measured using microdilution broth
151
as proposed by the Clinical Laboratory and Standards Institute (CLSI) [18]. Only
152
non-inducible clarithromycin patterns (3-5 days reading) were evaluated in this
153
study for M. abscessus.
clarithromycin
cefoxitin
(64-0,5
(256-2µg/mL),
µg/mL),
ciprofloxacin
doxycycline
(16-
(32-0,25µg/mL),
154
The breakpoints proposed by Siddiqii and collaborators [19] were used to
155
determine the resistance profiles for amikacin, streptomycin, ethambutol and
156
rifampicin for SGM.
7
157
Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR).
158
ERIC-PCR was performed for Pulsed-field gel electrophoresis (PFGE) non-
159
typeable isolates in order to verify the genotypic diversity among them. The primers
160
ERIC1R
161
AAGTAAGTGACTGGGGTGAGCG-3’) were applied [20]. The mixture was heated
162
at 95ºC for 4 min, followed by 35 cycles of 95ºC for 45 s, 52ºC for 1 min, 70ºC for
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10 min and a last step of 70ºC for 20 min. PCR products were analyzed after a 2%
164
gel electrophoresis (5V/cm) and stained with ethidium bromide using a 100 bp
165
DNA ladder (Invitrogen). The profiles obtained by electrophoresis were analyzed
166
and compared using the Bionumerics version 6.6 (Applied Maths, Belgium). Dice’s
167
coefficient was applied and the dendrogram based on percentages of similarity
168
was generated by the Unweighted-Pair Group with Arithmetic Mean (UPGMA)
169
method. The genotypic grouping was done by automated and visual analysis of the
170
gels using a cut-off value of 85% of similarity and a tolerance index of 1.5%.
(5’-ATGTAAGCTCCTGGGGATTCAC-3’)
and
ERIC2
(5’-
171
PFGE. PFGE was performed when multiple RGM isolates of the same
172
species were obtained in order to verify the genotypic diversity. The M. abscessus
173
subsp. bolletii strain CRM 0019, which belongs to the BRA100 clone [10], and
174
CCUG 48898 were used as control strains. The cells were grown in Mueller-Hinton
175
Agar (Becton Dickinson and Company, Brazil) at 35-37°C for seven days. Then, 3
176
isolated colonies were transferred to a flask containing 40ml of Mueller-Hinton
177
Broth (Becton Dickinson and Company, Brazil) with 0.1% Tween 80 (Sigma-
178
Aldrich, Brazil) and incubated at 37°C. Flasks were shaken at 300 g, for at least 4
179
days until the optical density (OD) at 650 nm reached 0.7. The cells from the liquid
180
culture were harvested by centrifugation at 18.000 g at 4°C for 25 min (Centrifuge 8
181
5810 R Eppendorf). The pellet was frozen at -20°C for 2 hours and then re-
182
suspended in STE – Tween 80 buffer (100mM NaCl, 10mM Tris pH 8.0, 50mM
183
EDTA pH 8.0 and 0.1% Tween 80) and mixed v/v with 2% low melting point
184
agarose (Sigma-Aldrich, Brazil). The plugs were incubated at 4°C for 20 min. Each
185
plug was individually treated with the solution containing STE plus 10 mg/mL
186
lysozyme and incubated at 35-37°C for 16 hours, followed by a new incubation at
187
4°C for 1 hour. The lysis solution was replaced by a solution containing 0.5M EDTA
188
and 1% Sarkosyl and incubated at 4°C for 1 hour. It was then replaced by 0.5M
189
EDTA, 1% Sarkosyl and 50mg/ml Proteinase K (Invitrogen Life Technologies) for
190
each sample. The plugs were incubated at 55°C for 24 hours and at 4°C for 1 hour.
191
Each plug was washed with 1x TE and incubated for 30 min at 4°C. After this step
192
a new incubation at 55°C for 1 hour with 1x TE and 0.12mg/mL of
193
phenylmethylsulfonyl fluoride (PMSF) was done. Each plug was washed three
194
times with 1x TE, incubating for 30 min at room temperature. The plugs were
195
stored in 0.5M EDTA (Sigma-Aldrich) at 4°C. For the restriction enzyme digestion,
196
a 3mm piece was cut and washed twice with 1x TE at 4°C for 30 min, washed in
197
0.1% Triton at 4°C for two hours and then digested with 30 U DraI (Promega).
198
Plugs were incubated for 16 hours at 37°C and washed with 0.05M at 4°C for 15
199
min. The plugs were melted at 70°C and the samples were loaded on a 1%
200
agarose gel (Sigma-Aldrich) in 5x TE for the PFGE. The Lambda Ladder PFG
201
Marker (Uniscience) was used as a molecular weight marker. The PFGE was
202
performed on a CHEF DR III, using the following settings: initial time of 1.6
203
seconds, final time of 21.3 seconds, running time of 24 hours, 6V/cm and 120°
204
included angle. Gels were stained and photographed, and PFGE patterns obtained 9
205
were analyzed by Bionumerics version 6.6 (Applied Maths, Belgium) software.
206
Dice’s coefficient was applied and the dendrogram based on similarity percentages
207
was generated by the UPGMA method. The genotypic grouping was done by an
208
automated and visual analysis of the gels using a tolerance index of 1.5%.
209
Statistical analysis
210
Evaluation of discriminatory power was performed for M. abscessus subsp.
211
bolletii isolates PFGE patterns by using the numerical index of discrimination (D)
212
based on Simpson’s index of diversity, as described by Hunter and Gaston [21]
213
Ethics: This study was approved by the Brazilian Ethics Committee and
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Regional committee with the following number: 213/08 - CEP HUCFF UFRJ, Title:
215
Caracterização Epidemiológica e Laboratorial das Micobacterioses do Trato
216
Respiratório Inferior no Estado do Rio de Janeiro. Consent terms were obtained for
217
all cased included in this study.
218 219
10
220
RESULTS
221
M. tuberculosis was not detected among any CF sputa samples.
222
Considering NTM, 36 isolates were recovered from 10 patients, and multiple
223
isolation of the same RGM species were observed. Although considering RGM
224
isolated as associated to respiratory infections, as recommended by the American
225
Thoracic Society (ATS) [22], these organisms were not related to clinical
226
syndromes. A letter identified each patient that had a positive isolate for NTM. The
227
numbers in parenthesis correspond to how many positive NTM isolations were
228
found in each patient: A (3), B (3), C (2), D (3), E (2), F (2), G (1), H (15), I (4) and
229
J (1).
230
The analysis of the PRA-hsp65 results showed 22 M. abscessus type II
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(BstE II 235/210 – Hae III 200/70/60) restriction profiles, 08 M. abscessus type I
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(BstE II 235/210 - Hae III 145/70/60), 02 M. chimerae / M. intracellulare (BstE II
233
235/120/100 – Hae III 145/130/60) and 04 M. fortuitum type II (BstE II 235/120/85 -
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Hae III 140/120/60). The partial sequencing of the rpoB gene identified all isolates,
235
obtaining 98 - 100% of similarity among 24 M. abscessus subsp. bolletii isolates
236
when compared to reference strains at BLAST database, 98% for 06 M. abscessus
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subsp. abscessus, 99% for 03 M. fortuitum, 100% for 02 M. marseillense and
238
100% for 01 M. timonense. However it was different from the PRA-hsp65 results
239
for 02 isolates (CRM 694, CRM 696) from patients G and C, identified as M.
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chimerae / M. intracellulare. These presented 100% of similarity to M. marseillense
241
and for CRM 747 (from patient C), showed 98% of similarity to M. abscessus
242
subsp. bolletii. Table 1 shows the comparison between the techniques.
11
243
Tables 2 show the MIC against the antimicrobials tested for RGM.
244
Considering the three MAC isolates, all of them presented full resistance to
245
streptomycin (MIC = 5 µg/ml) and they also showed intermediate resistance to
246
ethambutol (MIC = 2.5 to 5 µg/ml), moxifloxacin (MIC = 2 µg/ml) and rifampicin
247
(MIC = 1 to 4 µg/ml). The M. marseillense isolates (CRM 694 and 696) were highly
248
resistant to amikacin (MIC = 8 µg/ml) and the isolate 695 showed intermediate
249
levels of resistance to this drug (MIC = 4 µg/ml). All isolates were susceptible to
250
clarithromycin (MIC = 1 to 2 µg/ml).
251
The M. abscessus subsp. bolletii isolate CRM 803 from patient J presented
252
susceptibility to amikacin, ciprofloxacin and doxycycline. Isolates from patient A
253
initially presented susceptibility to one of the 07 drugs tested and evolved to
254
complete non-susceptibility over a period of four months. Isolates from patient B
255
presented similar characteristics in a period of 15 months. Considering patient H,
256
only the first two isolates were susceptible to one of the tested drugs, followed by
257
13 complete non-susceptible isolates during the period of study.
258
A predominant genotype was found among the isolates of M. fortuitum, with
259
2 isolates sharing an identical profile (CRM 725 and CRM 726) and another isolate
260
(CRM 724) with similarity sharing 95% similarity with the others. All isolates were
261
obtained from the same patient, found non-typeable by PFGE and analyzed by
262
ERIC-PCR (Figure 1).
263
Thirty M. abscessus spp. isolates were analyzed by PFGE, only CRM 473,
264
512, 915 e 949 were classified as nontypeable due to DNA degradation using
265
PFGE or ERIC-PCR.
12
266
Based on PFGE analysis, two distinct genotypes were obtained among the
267
six isolates of M. abscessus subsp. abscessus (Figure 2). The isolates CRM 641
268
and 697 presented an identical profile and were considered as clones. Even
269
though the isolate CRM 720 was isolated from the same patient (B), it does not
270
belong to the same clone and in this case was classified within the same genotype
271
- MAA 1. The isolates CRM 511, 718 e 740, obtained from the same patient (A),
272
were identical and classified as genotype MAA 2.
273
Among 20 isolates of M. abscessus subsp. bolletii that were studied, five
274
different genotypes were obtained (Figure 3) among seven related different
275
profiles. The isolates CRM 569, 639, 642, 719, 723, 732, 747, 835, 916, 917, 927,
276
928, 950, 1009 and 1010 were classified as genotype MAB 1. With the exception
277
of the isolate CRM 639, the genotype MAB 1 presented as identical profiles for all
278
isolates. CRM 639 and CRM 747 were the only ones not isolated from the same
279
patient. Genotype MAB 2 presented with two distinctly related profiles. The isolates
280
CRM 836 and 901 were identical and were obtained from the same patient. Isolate
281
CRM 803 and the reference strain CCUG 48898 presented unique genotypes,
282
respectively, MAB 3 and MAB 4, respectively. We observed that the genotype MAB
283
5 comprised CRM 640 and 920, respecting the patient-specific profile. None of the
284
CF clinical isolates presented a profile that was similar to the control strains. The D
285
values for PFGE typing of MAA and MAB were 1.00 [Non-Approximated
286
Confidence Interval (CNIA) 95% 0.456-1.000] and 0.952 (CINA 95% 0.761-1.000),
287
respectively.
288 289 13
290
DISCUSSION
291
Several studies worldwide have reported the prevalence of NTM in CF
292
patients. In France, a prevalence of 6.6% of NTM among 1582 CF patients was
293
reported [6]. In Toronto, Canada, the prevalence of NTM among 98 CF patients
294
between 6 and 18 years old was 6.1% [5]. In Spain, the prevalence of NTM among
295
220 CF patients was 7.72%; however, the patient’s age group varied between 6
296
and 74 years old [3]. A study developed in Israel that showed a prevalence of
297
22.6% of NTM among 186 CF patients, differs considerably from several other
298
studies around the world [4]. In our country, we observed only one study that
299
showed a prevalence of 11% of NTM among 54 CF adult patients in Brazil [2]. Our
300
study showed a prevalence of 7.75% of CF patients that were colonized/infected
301
with NTM.
302
The PRA-hsp65 had a concordance of 86,11% with the results obtained
303
from the partial sequencing of the rpoB. The isolates CRM 720, 747 and 803 were
304
identified by PRA-hsp65 as M. abscessus type I, whereas by rpoB sequencing they
305
were identified as M. abscessus subsp. bolletii. The isolates identified as M.
306
chimerae / M. intracellulare by PRA-hsp65 were identified as M. marseillense by
307
rpoB sequencing. The isolate CRM 695 identified as M. fortuitum by PRA-hsp65
308
was the one that presented the largest discordance with the rpoB sequencing,
309
even after several repetitions. However, the PRA-hsp65 database does not contain
310
the profile for M. timonense, which may have caused this difference in identification
311
between the methods used.
312
The M. abscessus group was the predominant NTM in our study (eight
313
patients), followed by MAC (three patients) and M. fortuitum (one patient). With the 14
314
exception of patients C (M. marseillense and M. abscessus subsp. boletii) and
315
patient I (M. fortuitum and M. timonense) who presented isolation of two different
316
species, the remaining patients presented isolation of only one species, allowing
317
better investigation of clonality. These results were similar to previous studies in
318
which the main species belonged to the M. abscessus group [7,23,24]. However,
319
our results differ from other results in which most of the isolated species were MAC
320
[3,5]. Additional, characterization of the microbiological profile of CF patients
321
developed by Paschoal et al [2] did not identify NTM to the species level. Currently,
322
CF NTM colonization/infection may be related to distinct NTM species prevalence
323
among distinct countries, reinforcing the importance of species identification in
324
therapeutic decision-making.
325
Brown-Elliott et al [25] reported that most of the strains of the M. abscessus
326
group show resistance to doxycycline, ciprofloxacin and moxifloxacin. Only one
327
isolate of M. abscessus subsp. boletii, obtained from patient J, was susceptible to
328
doxycycline (MIC ≤ 0,25 µg/ml) and ciprofloxacin (MIC 1µg/ml). It may be expected
329
that the isolates belonging to the M. abscessus obtained from CF patients may
330
show resistance to amikacin and other aminoglycosides, drugs that are commonly
331
used for treatment of bacterial infections, although amikacin resistance is rare
332
among M. abscessus recovered from different sources worldwide.
333
Although multidrug resistant NTM have been reported in studies involving
334
CF patients [6,7,26], the present work has shown the highest rates compared to
335
other studies in the scientific literature. Nevertheless, even when considering
336
infections by ATS criteria, only patient H presented with development of pulmonary
337
disease without resolution to date. The majority of the isolates from our study 15
338
(97.2%) were resistant to five or more antimicrobials, with the exception of the
339
isolate CRM 803, obtained from patient J. Fourteen M. abscessus subsp. bolletii
340
isolates (13 from patient H and 1 from patient B) presented amikacin MIC values
341
higher than 128µg/ml, one M. abscessus subsp. abscessus isolate with a MIC of
342
64 µg/ml (patient C) and two isolates with MIC of 32 µg/ml (patient A). The first
343
isolate from patient A showed susceptibility to amikacin, however the two following
344
isolates, obtained afterwards, showed intermediate resistance. The same event
345
was observed for patients C and H. The mechanisms for the acquisition of NTM
346
resistance to amikacin are unknown. Nevertheless, mutations in specific regions of
347
rrs and rpsL genes may confer aminoglycoside resistance in mycobacteria [26,27,
348
28].
349
We recovered only three MAC, two M. marseillense (CRM 694 and 696) one
350
M. timonense (CRM 695) isolates and all of them were susceptible to
351
clarithromycin. According to the CLSI 2011 guidelines, clarithromycin is the drug
352
chosen for the treatment of MAC infections, followed by moxifloxacin. The drug
353
resistance of MAC in CF patients is rare, which facilitates longer antimicrobial
354
treatment [25]. Other drugs such as ethambutol and rifampicin are used in
355
association with clarithromycin therapy. In some cases, amikacin is used together
356
with clarithromycin. However, due to the long duration of the treatment, which may
357
last longer than 06 months, and the side nephrotoxic and ototoxic effects caused
358
by amikacin this therapy has been abandoned [25, 28, 29]. Furthermore, the
359
present results, based on the breakpoints described by Siddiqii et al [19], indicate
360
that the therapy with amikacin, ethambutol, moxifloxacin, rifampicin or streptomycin
16
361
may be ineffective due to the high amikacin MIC values for the isolates CRM 694
362
and 696 (8µg/ml) and intermediate resistance found for other drugs.
363
According to the review published by Pitombo et al [30], most of M. fortuitum
364
strains are susceptible to amikacin, cefoxitin, ciprofloxacin, imipenem, linezolid and
365
sulfamethoxazole / trimethoprim, however M. fortuitum isolates from CF patients in
366
the present study were resistant to cefoxitin, ciprofloxacin, doxycycline, and
367
sulfamethoxazole / trimethoprim. The isolates CRM 724 and 725 also showed
368
resistance to clarithromycin. According to Brown-Elliott, et al [25], the M. fortuitum
369
group presents sulfonamide susceptibility of 100%, however, all M. fortuitum
370
isolates from CF patients were resistant to sulfamethoxazole/trimethoprim with MIC
371
values higher than 8 / 152 µg/mL.
372
The ERIC PCR is a simple molecular tool and can be used for genotyping
373
mycobacteria. A study developed by Sampaio et al [31] compared different
374
genotyping techniques in 17 isolates of M. fortuitum from an outbreak of infection
375
post mammoplasty and in this comparison the ERIC PCR showed clonal profiles
376
that were identical to the gold standard technique (PFGE). It is considered a fast
377
and economic alternative to PFGE. In a study developed with 40 Ugandan children
378
and teenagers with pulmonary infection caused by M. fortuitum, ERIC PCR
379
revealed the presence of a clonal group of three isolates, another group comprised
380
by two isolates and also 35 distinct profiles [32].
381
A clonal group of M. fortuitum was recovered from patient I with over 95% of
382
similarity. It is interesting to note that even though they presented with the same
383
genotypic restriction pattern, CRM 725 and 726 showed different susceptibility
384
profiles. The isolate CRM 724 showed the same susceptibility profile as CRM 725, 17
385
being both isolated from the same clinical sample. In conclusion, a single sputum
386
sample presented two different types of colonies with slightly different genotypic
387
profiles; the small colony named CRM 724 and the larger one named CRM 725.
388
The isolation of CRM 726 occurred 30 days after the isolation of the cited isolates
389
(CRM 724 and 725) and showed a profile identical to CRM 725. The susceptibility
390
profile and the genotypic profile results from isolates obtained from this clinical
391
case may suggest multiclonal NTM colonization belonging to the same species,
392
which may represent recent genomic mutations in mycobacterial populations.
393
The PFGE of the M. abscessus subsp. abscessus isolates revealed two
394
distinct genotypes: MAA1 and MAA2. The MAA 1 PFGE pattern comprised three
395
isolates from patient B, and showed two identical strains (CRM 641 and 697) and
396
another (CRM-720) with similarity of 88%. The MIC data of these isolates suggest
397
that the most recent isolated strain (CRM-720) presented resistance to amikacin
398
and different PFGE patterns from the others. A recent study showed a patient
399
infected by M. abscessus subsp. bolletii acquired resistance to amikacin during
400
therapy with the same drug [26]. The genotype MAA 2 comprised 3 clonal isolates
401
from patient A. However, the resistance profile was different among the three
402
isolates. These aspects may indicate that the molecular basis involved in the
403
alteration of susceptibility to drugs may not be related to the M. abscessus subsp.
404
abscessus genotypic profile by PFGE. Further studies are required for better
405
comprehension.
406
The PFGE results for isolates identified as M. abscessus subsp. bolletii were
407
classified as five different genotypes (MAB 1 to 5). Among those five, MAB 1 was
408
the one composed by most of the M. abscessus subsp. bolletii isolates [15]. All the 18
409
isolates from patient H were included in this genotype, as well as the isolate CRM
410
747 from patient C and CRM 639, obtained from patient E. CRM 639 showed a
411
profile that differed from the other isolates by only one band. After review of
412
medical records, we verified that no other isolation of mycobacteria occurred from
413
patients C and E up to the date we finished this study, suggesting these bacteria
414
may have composed the most transient microbiota of these patients. It is also very
415
startling that the MAB 1 genotype was circulating among three patients, and that
416
thissame clone remained causing severe disease in patient H up to the present
417
day (data not shown).
418
The genotype MAB 2 was comprised of the isolates CRM 836 and 901
419
isolated from patient D, presenting identical molecular and susceptibility profiles,
420
which confirm a patient-specific pattern. Similarly, the genotype MAB 5 had two
421
clones isolated from the same patient (D), with a difference of 18 months after the
422
date of the first isolation.
423
PFGE for MAA and MAB 2 were evaluated by a discriminatory index which
424
presented values higher than 0.90. These results confirm the elevated
425
discriminatory power of this method for diversity studies of these species, as
426
already showed in previous studies [33, 34].
427
The data obtained in this work will contribute to a better comprehension of
428
NTM colonization/infection of CF patients. The observed NTM characteristics are
429
alarming, since most of the isolates circulating among the patients present
430
resistance to multiple drugs. Isolation of high resistant strains from CF patients
431
must be considered a significant threat to public health mainly due to the paucity of
19
432
alternative therapy or potentially untreatable disease. Based on these findings, it is
433
highly recommended to monitor NTM infections in CF patients.
434
These findings, unprecedented in a country with a high incidence of TB
435
reinforce the importance of systematic and routine research for the identification of
436
mycobacteria to species level and susceptibility evaluation to antimicrobials.
437
Studies on monitoring of NTM transmission through respiratory equipment among
438
CF patients may also be useful to better understand rates of colonization and
439
prevalence of these organisms in these settings. ACKNOWLEDGMENTS
440 441 442
The Plataform-DNA Sequencing (PDTIS / FIOCRUZ), Rio de Janeiro, RJ,
443
Brazil is acknowledged for performing the rpoB sequencing procedures. We
444
appreciated the collaboration of Alcon Laboratórios do Brasil Ltda. by moxifloxacin
445
donation.
446
This study was supported in part by the Fundação de Amparo à Pesquisa
447
do Estado do Rio de Janeiro (FAPERJ, projects no. 103.225/2011, 103.287/2011,
448
110.272/2010, 110.761/2010 and 111.497/2008), the Conselho Nacional de
449
Desenvolvimento Científico e Tecnológico (MCT/CNPq, projects no. 476536/2012-
450
0, 473444/2010-0 and 567037/2008-8) and PDTIS – FIOCRUZ.
451
20
452 453
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589
FIGURE TITLES
590 591
Figure 1 – Dendrogram obtained from the automated analysis of the ERIC
592
PCR profiles of M. fortuitum isolates, showing the similarity coefficients (in %).
593
Dice’s coefficient was used as the algorithym for the construction of the
594
dendrogram.
595 596
Figure 2 – Dendrogram obtained from the automated analysis of the PFGE
597
profiles of the M. abscessus subsp. abscessus isolates, showing the similarity
598
coefficients (in %). Dice’s coefficient was used as the algorithym for the
599
construction of the dendrogram.
600 601
Figure 3 – Figure 1 – Dendrogram obtained from the automated analysis of
602
the PFGE profiles of the M. abscessus subsp. bolletii isolates, showing the
603
similarity coefficients (in %). Dice’s coefficient was used as the algorithym for the
604
construction of the dendrogram.
605
27
TABLES
606 607 608
Table 1 – Comparison of PRA-hsp65 and rpoB sequencing results for identification
609
of NTM strains isolated from sputa of 10 CF patients in Rio de Janeiro, Brazil.
Patient
Isolate
A
CRM 511 CRM 718 CRM 740 CRM 641 CRM 697 CRM 720 CRM 696 CRM 747 CRM 512 CRM 640 CRM 920 CRM 473 CRM 639 CRM 836 CRM 901 CRM 694 CRM 569 CRM 642 CRM 719 CRM 723 CRM 732 CRM 835 CRM 915 CRM 916 CRM 917 CRM 927 CRM 928 CRM 949 CRM 950 CRM 1009 CRM 1010 CRM 695 CRM 724a CRM 725 CRM 726 CRM 803
B C D E F G
H
I
610
J
a
Isolation date (day/month/year) 17/06/2009 24/02/2010 23/06/2010 21/10/2009 11/01/2010 16/04/2011 03/12/2009 12/07/2010 22/06/2009 19/12/2009 20/06/2011 03/06/2009 02/09/2009 19/01/2011 21/03/2011 02/09/2009 06/07/2009 21/10/2009 31/03/2010 24/04/2010 02/06/2010 13/01/2011 14/04/2011 14/04/2011 14/04/2011 14/07/2011 04/08/2011 02/06/2010 08/11/2011 12/03/2012 12/03/2012 03/12/2009 17/06/2009 17/06/2009 15/07/2009 30/08/2010
Molecular Identification PRA-hsp65 rpoB M. abscessus type 1 M. abscessus ssp abscessus M. abscessus type 1 M. abscessus ssp abscessus M. abscessus type 1 M. abscessus ssp abscessus M. abscessus type 1 M. abscessus ssp abscessus M. abscessus type 1 M. abscessus ssp abscessus M. abscessus type 1 M. abscessus ssp abscessus M. chimerae /M. intracellulare M. marseillense M. abscessus type 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/bolletii 1 M. abscessus ssp bolletii M. abscessus 2/bolletii 1 M. abscessus ssp bolletii M. chimerae /M. intracellulare M. marseillense M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. fortuitum type 2 M. timonense M. fortuitum type 2 M. fortuitum M. fortuitum type 2 M. fortuitum M. fortuitum type 2 M. fortuitum M. abscessus type 1 M. abscessus ssp bolletii
CRM-724 and CRM725 were distinct colonies isolated from the same sputum.
611 612 613 614 615 616
28
617
Table 2 – Antimicrobial susceptibility of the NTM isolates recovered from sputa of
618
CF patients in Rio de Janeiro, Brazil MIC (µg/ml)a
Patient
Isolate
Species AMI
CEF
CIP
CLA
DOX
MOX
T/S
A
CRM 511
M. abscessus subsp. abscessus
8(S)
64(I)
8(R)
8(R)
>32(R)
8(R)
> 04/76(R)
A
CRM 718
M. abscessus subsp.abscessus
32(I)
32(I)
>16(R)
1(S)
>32(R)
16(R)
>08/152(R)
A
CRM 740
M. abscessus subsp.abscessus
32(I)
128
>16(R)
32(R)
>32(R)
16(R)
> 04/76(R)
B
CRM 641
M. abscessus subsp.abscessus
8(S)
256(R)
4(R)
4(I)
>32(R)
16(R)
>16/304(R)
B
CRM 697
M. abscessus subsp.abscessus
16(S)
32(I)
16(R)
16(R)
>32(R)
16(R)
>16/304(R)
B
CRM 720
M. abscessus subsp.abscessus
64(R)
64(I)
>16(R)
16(R)
>32(R)
64(R)
>16/304(R)
C
CRM 747
M. abscessus subsp.bolletii
>128(R)
256(R)
>16(R)
32(R)
>32(R)
16(R)
> 04/76(R)
D
CRM 512
M. abscessus subsp.bolletii
8(S)
64(I)
8(R)
2(S)
>32(R)
16(R)
>16/304(R)
D
CRM 640
M. abscessus subsp.bolletii
8(S)
64(I)
2(I)
32(R)
32(R)
>16/304(R)
D
CRM 920
M. abscessus subsp.bolletii
8(S)
256(R)
>16(R) 32(R)
4(R)
>16/304(R)
E
CRM 473
M. abscessus subsp.bolletii
8(S)
64(I)
8(R)
32(R)
16(R)
>16/304(R)
E
CRM 639
M. abscessus subsp.bolletii
8(S)
256(R)
16(R)
32(R)
32(R)
>16/304(R)
F
CRM 836
M. abscessus subsp.bolletii
8(S)
32(I)
>16(R) 32(R)
32(R)
> 04/76(R)
F
CRM 901
M. abscessus subsp.bolletii
8(S)
64(I)
>16(R) 32(R)
32(R)
> 04/76(R)
H
CRM 569
M. abscessus subsp.bolletii
16(S)
128(R)
16(R)
64(R)
>32(R)
32(R)
> 04/76(R)
H
CRM 642
M. abscessus subsp.bolletii
8(S)
128(R)
16(R)
>64(R)
>32(R)
32(R)
> 04/76(R)
H
CRM 719
M. abscessus subsp.bolletii
>128(R)
64(I)
>16(R)
32(R)
>32(R)
128(R)
>16/304(R)
H
CRM 723
M. abscessus subsp.bolletii
>128(R)
128(R)
>16(R)
32(R)
>32(R)
32(R)
>16/304(R)
H
CRM 732
M. abscessus subsp.bolletii
>128(R)
256(R)
>16(R)
32(R)
>32(R)
128(R)
>16/304(R)
H
CRM 835
M. abscessus subsp.bolletii
>128(R)
64(I)
>16(R) >64(R)
>32(R)
32(R)
>16/304(R)
H
CRM 915
M. abscessus subsp.bolletii
>128(R)
256(R)
>16(R) >64(R)
>32(R)
16(R)
>16/304(R)
H
CRM 916
M. abscessus subsp.bolletii
>128(R)
64(I)
>16(R) >64(R)
>32(R)
32(R)
>16/304(R)
H
CRM 917
M. abscessus subsp.bolletii
>128(R)
64(I)
>16(R) >64(R)
>32(R)
32(R)
>16/304(R)
H
CRM 927
M. abscessus subsp.bolletii
>128(R)
64(I)
>16(R) >64(R)
>32(R)
16(R)
>16/304(R)
H
CRM 928
M. abscessus subsp.bolletii
>128(R)
64(I)
>16(R) >64(R)
>32(R)
256(R)
>16/304(R)
H
CRM 949
M. abscessus subsp.bolletii
>128(R)
64(I)
>8(R)
>16(R)
>32(R)
>2(I)
>16/304(R)
H
CRM 950
M. abscessus subsp.bolletii
>128(R)
64(I)
>16(R) >16(R)
>32(R)
>2(I)
>16/304(R)
H
CRM 1009
M. abscessus subsp.bolletii
>128(R)
64(I)
>8(R)
>16(R)
>32(R)
>2(I)
>16/304(R)
H
CRM 1010
M. abscessus subsp. Bolletii
>128(R)
64(I)
>8(R)
>16(R)
>32(R)
>2(I)
>16/304(R)
I
CRM 724
M. fortuitum
8(S)
>256(R)
8(R)
16(R)
>32(R)
04/76(R)
I
CRM 725
M. fortuitum
8(S)
>256(R)
8(R)
4(I)
>32(R)
04/76(R)
I
CRM 726
M. fortuitum
256(R) 0,5(S)
16(R)
32(R)
04/76(R)
J
CRM 803
M. abscessus subsp.bolletii
1
Multidrug resistant non-tuberculous mycobacteria
2
isolated from cystic fibrosis patients
3
Pedro Henrique Campanini Cândido1, Luciana de Souza Nunes2, Elizabeth
4
Andrade Marques3, Tânia Wrobel Folescu4, Fábrice Santana Coelho5, Vinicius
5
Calado Nogueira de Moura6, Marlei Gomes da Silva6, Karen Machado Gomes6,
6
Maria Cristina da Silva Lourenço7, Fábio Silva Aguiar1, Fernanda Chitolina8,
7
Derek T. Armstrong9, Sylvia Cardoso Leão10, Felipe Piedade Gonçalves
8
Neves11, Fernanda Carvalho de Queiroz Mello8, Rafael Silva Duarte6 *.
9
Programa de Pós Graduação em Clínica Médica, Hospital Universitário
10
Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, RJ, Brazil1;
11
Programa de Pós Graduação em Ciências Médicas, Universidade Federal do Rio
12
Grande do Sul, RS, Brazil2; Departamento de Microbiologia, Imunologia e
13
Parasitologia, Faculdade de Ciências Médicas, Universidade do Estado do Rio de
14
Janeiro, RJ, Brazil3; Instituto Fernandes Figueira, RJ, Brazil4; Hospital Universitário
15
Pedro Ernesto, Universidade do Estado do Rio de Janeiro, Brazil5; Instituto de
16
Microbiologia, Universidade Federal do Rio de Janeiro, RJ, Brazil6; Instituto de
17
Pesquisa Evandro Chagas, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil7;
18
Instituto de Doenças do Tórax, Rio de Janeiro, Brazil8; Johns Hopkins University,
19
Baltimore, MD, USA9, Departamento de Microbiologia, Imunologia e Parasitologia,
20
Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo,
21
Brazil10; Instituto Biomédico, Universidade Federal Fluminense, Niterói, RJ,
22
Brazil11. 1
23 24
Running title: Multidrug resistant non-tuberculous mycobacteria strains in cystic
25
fibrosis patients
26
Keywords: cystic fibrosis, colonization, infection, multidrug resistance, non-
27
tuberculous mycobacteria, PFGE, Rio de Janeiro, Brazil.
28
*Corresponding author
29
Mailing address:
30
Instituto de Microbiologia, Universidade Federal do Rio de Janeiro,
31
CCS, Bloco I, Cidade Universitária
32
Rio de Janeiro, RJ 21941-902, Brazil
33
Phone: 55 21 2560-8344 r. 134
34
Fax: 55 21 2560-8344 r. 108
35
E-mail: [email protected]
36 37 38 39
2
Summary
40 41
Worldwide,
nontuberculous
mycobacteria
(NTM)
have
become
emergent
42
pathogens of pulmonary infections in cystic fibrosis (CF) patients, with an
43
estimated prevalence varying from 5 to 20%. This work investigated the presence
44
of NTM in sputum samples of 129 CF patients (2 to 18 years old) submitted to
45
longitudinal clinical supervision at a regional reference center in Rio de Janeiro,
46
Brazil. From June 2009 to March 2012, 36 NTM isolates recovered from 10
47
(7.75%) out of 129 children were obtained. Molecular identification of NTM was
48
performed by PRA-hsp65 and sequencing of rpoB gene, and susceptibility tests
49
followed Clinical and Laboratory Standards Institute recommendations. For
50
evaluating the genotypic diversity, PFGE and/or ERIC-PCR were performed. The
51
species identified were Mycobacterium abscessus subsp. bolletii (n=24), M.
52
abscessus subsp. abscessus (n=6), M. fortuitum (n=3), M. marseillense (n=2) and
53
M. timonense (n=1). Most of the isolates presented resistance to five or more
54
antimicrobials tested. Typing profiles were mainly patient-specific. The PFGE
55
profiles indicated the presence of two clonal groups for M. abscessus subsp.
56
abscessus and five clonal groups for M. abscesssus subsp. bolletii, with just one
57
clone detected in two patients. Given the observed multidrug resistance patterns
58
and the possibility of transmission among different patients, we suggest the
59
implementation of continuous and routine investigation of NTM infections or
60
colonization in CF patients, including countries with high burden of tuberculosis
61
disease.
62 63 3
64
INTRODUCTION
65
The survival rate of cystic fibrosis (CF) patients has increased along recent
66
years, mainly due to early diagnosis, nutritional therapies and the discovery of new
67
antimicrobial agents. Acute or chronic pulmonary infections caused by bacteria are
68
the major causes of morbidity and mortality in CF patients [1]. For the last 20 - 25
69
years, non-tuberculous mycobacteria (NTM) have emerged as newly detected
70
pathogens in CF patients. Several centers around the world have reported the
71
isolation of NTM in the respiratory tract of CF patients. Nowadays this prevalence
72
varies between 5 and 20% [2,3,4,5,6,7] with most of those cases (>90%) caused
73
by species of the Mycobacterium avium complex (MAC) and the Mycobacterium
74
abscessus group [5,8,9].
75
NTM have been described in diverse environments and clinical scenarios
76
due to their adaptive biological properties and opportunistic potentials. It is
77
considered that selective pressure in hospital environments predominantly caused
78
by excessive antibiotics use, has contributed to NTM resistance to a large number
79
of antibiotics and disinfection processes, such as anti-tuberculosis agents (i.e.
80
isoniazid and pyrazinamide), glutaraldehyde and organomercury compounds [10].
81
However, NTM usually present as susceptible to aminoglycosides and macrolides
82
[11,12,13]. Cefoxitin, quinolone and tetracyclines are other options for drug
83
treatment, specifically for rapidly growing mycobacteria (RGM) infections.
84
However, similarly to other bacteria commonly associated with CF infections, NTM
85
resistant strains have been reported. Broda et al. [14] reports clarithromycin
86
susceptibility rates of 57% in Mycobacterium abscessus isolates. Binder et al. [13]
4
87
suggest that all CF patients undertake evaluation for NTM disease, due to
88
continual exposure to macrolides for other bacterial infections.
89
In Brazil, microbiological studies involving CF patients are rare, with most
90
focused on infections caused by Burkholderia cenocepacia and/or Pseudomonas
91
aeruginosa [2]. The Instituto Fernandes Figueira (IFF) is a large unit that belongs to
92
the Oswaldo Cruz Foundation (FIOCRUZ) and is the major clinical research center
93
for CF children in Rio de Janeiro state and in the country of Brazil. Microbiological
94
surveillance of these patients is common, however investigation of NTM disease
95
has only been performed since June 2009. Given the lack of epidemiological
96
information on NTM in Brazilian CF children and the emergence of NTM as
97
pathogens in this population worldwide, the significance of developing research on
98
NTM and its association to CF is definitely warranted. Epidemiological studies in
99
European cohorts have demonstrated a high prevalence of NTM infections in this
100
population, but these countries have lower disease-burdens of tuberculosis (TB).
101
Brazil is one of the main high TB-burden countries and has seen large epidemics
102
of NTM infections described up in recent literature [10]. Therefore, it is necessary
103
to undergo studies looking at prevalence of NTM in CF patients, to better
104
understand the magnitude of this emergence in different countries globally.
105
This is the first complete study providing genotypic identification,
106
antimicrobial susceptibility profiles and strain diversity of non-tuberculous
107
mycobacterial isolates recovered from CF patients in Brazil.
108 109
5
110
MATERIAL AND METHODS
111
Mycobacterial isolates. From June 2009 to March 2012, spontaneous
112
sputa from CF patients monitored at a local reference center (IFF-FIOCRUZ, Rio
113
de Janeiro, Brazil) were sent monthly to the Bacteriology and Mycobacteriology
114
laboratories at Hospital Universitário Pedro Ernesto, Rio de Janeiro, Brazil for
115
routine microbiological investigation. Sputa were processed by NALC-NaOH and
116
5% oxalic acid decontamination as recommended by the National Guideline for TB
117
and NTM Laboratory Surveillance [15]. A total of 36 NTM isolates were recovered
118
from sputa obtained from 10 (7.75%, 2 to 18 years old) out of 129 children.
119
Species identification. A loopful of bacterial growth from Löwenstein–
120
Jensen media was mixed in 100 µL of TE (Tris-HCl 10 mM, EDTA 1 mM, pH 8,0).
121
The suspensions were boiled at 100°C for 10 min and immediately incubated at
122
20°C for 12 hours. Genotypic identification of the 36 isolates was performed by the
123
PRA-hsp65 technique using the primers TB11 5´-ACCAACGATGGTGTGTCCAT-
124
3’ and TB12 5´-CTTGTCGAACCGCATACCCT-3’ [16]. A denaturation step of 95ºC
125
for 5 min was followed by 45 cycles of 94ºC for 1 min, 60ºC for 1 min and 72ºC for
126
1 min and by a final extension of 72ºC for 7 min. Amplicons were submitted to
127
individual digestions using the restriction enzymes BstEII and HaeIII (Promega
128
Corporation, Madison, WI) for 3 hours at 60 and 37°C, respectively. The digestion
129
products were analyzed after a 4% agarose gel electrophoresis at 3V/cm using 25
130
and 50 bp DNA ladders (Invitrogen). The PRA-hsp65 profiles were analyzed using
131
the algorithm available at the PRAsite (http://app.chuv.ch/prasite/index).
132
All the results were confirmed by partial sequencing of the rpoB gene using
133
the primers Myco F (5’-GCAAGGTCACCCCGAAGGG-3’) and MycoR (5’6
134
AGCGGCTGCTGGGTGATCATC-3’), according to the methodology described by
135
Adekambi, Colson & Drancourt [17]. Region V of rpoB gene sequences were
136
submitted
137
http://www.ncbi.nlm.nih.gov/BLAST) for similarity analysis and comparison to the
138
sequences from M. abscessusT ATCC 19977 (JF346872), “M. massilienseT” CIP
139
108297 (EU254721.1) and “M. bolletiiT” CCUG 50184 (DQ987717.1), the M.
140
abscessus subsp. bolletii epidemic strain INCQS 594 (EU117207.1), M. fortuitum
141
ATCC 6841 (JF346874.1), M. marseillense strain 62863 (EF584444.1) and M.
142
timonense strain 3256799 (EF584435.1).
143
to
BLAST
–
Basic
Local
Alignment
Tool
(URL:
Antimicrobial susceptibility testing. The RGM isolates were tested
144
against
amikacin
(128-1µg/mL),
145
0,125µg/mL),
146
moxifloxacin (16-0,125 µg/mL), sulfamethoxazole-trimethoprim (16/304-0,5/9,5
147
µg/mL). Slowly growing mycobacteria (SGM) were tested against amikacin (128-
148
1µg/mL), clarithromycin (64-0,5µg/mL), streptomycin (16–0,125µg/mL), ethambutol
149
(16-0,125µg/mL), moxifloxacin (16-0,125µg/mL) and rifampicin (16-0,125µg/mL).
150
Minimum inhibitory concentrations (MIC) were measured using microdilution broth
151
as proposed by the Clinical Laboratory and Standards Institute (CLSI) [18]. Only
152
non-inducible clarithromycin patterns (3-5 days reading) were evaluated in this
153
study for M. abscessus.
clarithromycin
cefoxitin
(64-0,5
(256-2µg/mL),
µg/mL),
ciprofloxacin
doxycycline
(16-
(32-0,25µg/mL),
154
The breakpoints proposed by Siddiqii and collaborators [19] were used to
155
determine the resistance profiles for amikacin, streptomycin, ethambutol and
156
rifampicin for SGM.
7
157
Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR).
158
ERIC-PCR was performed for Pulsed-field gel electrophoresis (PFGE) non-
159
typeable isolates in order to verify the genotypic diversity among them. The primers
160
ERIC1R
161
AAGTAAGTGACTGGGGTGAGCG-3’) were applied [20]. The mixture was heated
162
at 95ºC for 4 min, followed by 35 cycles of 95ºC for 45 s, 52ºC for 1 min, 70ºC for
163
10 min and a last step of 70ºC for 20 min. PCR products were analyzed after a 2%
164
gel electrophoresis (5V/cm) and stained with ethidium bromide using a 100 bp
165
DNA ladder (Invitrogen). The profiles obtained by electrophoresis were analyzed
166
and compared using the Bionumerics version 6.6 (Applied Maths, Belgium). Dice’s
167
coefficient was applied and the dendrogram based on percentages of similarity
168
was generated by the Unweighted-Pair Group with Arithmetic Mean (UPGMA)
169
method. The genotypic grouping was done by automated and visual analysis of the
170
gels using a cut-off value of 85% of similarity and a tolerance index of 1.5%.
(5’-ATGTAAGCTCCTGGGGATTCAC-3’)
and
ERIC2
(5’-
171
PFGE. PFGE was performed when multiple RGM isolates of the same
172
species were obtained in order to verify the genotypic diversity. The M. abscessus
173
subsp. bolletii strain CRM 0019, which belongs to the BRA100 clone [10], and
174
CCUG 48898 were used as control strains. The cells were grown in Mueller-Hinton
175
Agar (Becton Dickinson and Company, Brazil) at 35-37°C for seven days. Then, 3
176
isolated colonies were transferred to a flask containing 40ml of Mueller-Hinton
177
Broth (Becton Dickinson and Company, Brazil) with 0.1% Tween 80 (Sigma-
178
Aldrich, Brazil) and incubated at 37°C. Flasks were shaken at 300 g, for at least 4
179
days until the optical density (OD) at 650 nm reached 0.7. The cells from the liquid
180
culture were harvested by centrifugation at 18.000 g at 4°C for 25 min (Centrifuge 8
181
5810 R Eppendorf). The pellet was frozen at -20°C for 2 hours and then re-
182
suspended in STE – Tween 80 buffer (100mM NaCl, 10mM Tris pH 8.0, 50mM
183
EDTA pH 8.0 and 0.1% Tween 80) and mixed v/v with 2% low melting point
184
agarose (Sigma-Aldrich, Brazil). The plugs were incubated at 4°C for 20 min. Each
185
plug was individually treated with the solution containing STE plus 10 mg/mL
186
lysozyme and incubated at 35-37°C for 16 hours, followed by a new incubation at
187
4°C for 1 hour. The lysis solution was replaced by a solution containing 0.5M EDTA
188
and 1% Sarkosyl and incubated at 4°C for 1 hour. It was then replaced by 0.5M
189
EDTA, 1% Sarkosyl and 50mg/ml Proteinase K (Invitrogen Life Technologies) for
190
each sample. The plugs were incubated at 55°C for 24 hours and at 4°C for 1 hour.
191
Each plug was washed with 1x TE and incubated for 30 min at 4°C. After this step
192
a new incubation at 55°C for 1 hour with 1x TE and 0.12mg/mL of
193
phenylmethylsulfonyl fluoride (PMSF) was done. Each plug was washed three
194
times with 1x TE, incubating for 30 min at room temperature. The plugs were
195
stored in 0.5M EDTA (Sigma-Aldrich) at 4°C. For the restriction enzyme digestion,
196
a 3mm piece was cut and washed twice with 1x TE at 4°C for 30 min, washed in
197
0.1% Triton at 4°C for two hours and then digested with 30 U DraI (Promega).
198
Plugs were incubated for 16 hours at 37°C and washed with 0.05M at 4°C for 15
199
min. The plugs were melted at 70°C and the samples were loaded on a 1%
200
agarose gel (Sigma-Aldrich) in 5x TE for the PFGE. The Lambda Ladder PFG
201
Marker (Uniscience) was used as a molecular weight marker. The PFGE was
202
performed on a CHEF DR III, using the following settings: initial time of 1.6
203
seconds, final time of 21.3 seconds, running time of 24 hours, 6V/cm and 120°
204
included angle. Gels were stained and photographed, and PFGE patterns obtained 9
205
were analyzed by Bionumerics version 6.6 (Applied Maths, Belgium) software.
206
Dice’s coefficient was applied and the dendrogram based on similarity percentages
207
was generated by the UPGMA method. The genotypic grouping was done by an
208
automated and visual analysis of the gels using a tolerance index of 1.5%.
209
Statistical analysis
210
Evaluation of discriminatory power was performed for M. abscessus subsp.
211
bolletii isolates PFGE patterns by using the numerical index of discrimination (D)
212
based on Simpson’s index of diversity, as described by Hunter and Gaston [21]
213
Ethics: This study was approved by the Brazilian Ethics Committee and
214
Regional committee with the following number: 213/08 - CEP HUCFF UFRJ, Title:
215
Caracterização Epidemiológica e Laboratorial das Micobacterioses do Trato
216
Respiratório Inferior no Estado do Rio de Janeiro. Consent terms were obtained for
217
all cased included in this study.
218 219
10
220
RESULTS
221
M. tuberculosis was not detected among any CF sputa samples.
222
Considering NTM, 36 isolates were recovered from 10 patients, and multiple
223
isolation of the same RGM species were observed. Although considering RGM
224
isolated as associated to respiratory infections, as recommended by the American
225
Thoracic Society (ATS) [22], these organisms were not related to clinical
226
syndromes. A letter identified each patient that had a positive isolate for NTM. The
227
numbers in parenthesis correspond to how many positive NTM isolations were
228
found in each patient: A (3), B (3), C (2), D (3), E (2), F (2), G (1), H (15), I (4) and
229
J (1).
230
The analysis of the PRA-hsp65 results showed 22 M. abscessus type II
231
(BstE II 235/210 – Hae III 200/70/60) restriction profiles, 08 M. abscessus type I
232
(BstE II 235/210 - Hae III 145/70/60), 02 M. chimerae / M. intracellulare (BstE II
233
235/120/100 – Hae III 145/130/60) and 04 M. fortuitum type II (BstE II 235/120/85 -
234
Hae III 140/120/60). The partial sequencing of the rpoB gene identified all isolates,
235
obtaining 98 - 100% of similarity among 24 M. abscessus subsp. bolletii isolates
236
when compared to reference strains at BLAST database, 98% for 06 M. abscessus
237
subsp. abscessus, 99% for 03 M. fortuitum, 100% for 02 M. marseillense and
238
100% for 01 M. timonense. However it was different from the PRA-hsp65 results
239
for 02 isolates (CRM 694, CRM 696) from patients G and C, identified as M.
240
chimerae / M. intracellulare. These presented 100% of similarity to M. marseillense
241
and for CRM 747 (from patient C), showed 98% of similarity to M. abscessus
242
subsp. bolletii. Table 1 shows the comparison between the techniques.
11
243
Tables 2 show the MIC against the antimicrobials tested for RGM.
244
Considering the three MAC isolates, all of them presented full resistance to
245
streptomycin (MIC = 5 µg/ml) and they also showed intermediate resistance to
246
ethambutol (MIC = 2.5 to 5 µg/ml), moxifloxacin (MIC = 2 µg/ml) and rifampicin
247
(MIC = 1 to 4 µg/ml). The M. marseillense isolates (CRM 694 and 696) were highly
248
resistant to amikacin (MIC = 8 µg/ml) and the isolate 695 showed intermediate
249
levels of resistance to this drug (MIC = 4 µg/ml). All isolates were susceptible to
250
clarithromycin (MIC = 1 to 2 µg/ml).
251
The M. abscessus subsp. bolletii isolate CRM 803 from patient J presented
252
susceptibility to amikacin, ciprofloxacin and doxycycline. Isolates from patient A
253
initially presented susceptibility to one of the 07 drugs tested and evolved to
254
complete non-susceptibility over a period of four months. Isolates from patient B
255
presented similar characteristics in a period of 15 months. Considering patient H,
256
only the first two isolates were susceptible to one of the tested drugs, followed by
257
13 complete non-susceptible isolates during the period of study.
258
A predominant genotype was found among the isolates of M. fortuitum, with
259
2 isolates sharing an identical profile (CRM 725 and CRM 726) and another isolate
260
(CRM 724) with similarity sharing 95% similarity with the others. All isolates were
261
obtained from the same patient, found non-typeable by PFGE and analyzed by
262
ERIC-PCR (Figure 1).
263
Thirty M. abscessus spp. isolates were analyzed by PFGE, only CRM 473,
264
512, 915 e 949 were classified as nontypeable due to DNA degradation using
265
PFGE or ERIC-PCR.
12
266
Based on PFGE analysis, two distinct genotypes were obtained among the
267
six isolates of M. abscessus subsp. abscessus (Figure 2). The isolates CRM 641
268
and 697 presented an identical profile and were considered as clones. Even
269
though the isolate CRM 720 was isolated from the same patient (B), it does not
270
belong to the same clone and in this case was classified within the same genotype
271
- MAA 1. The isolates CRM 511, 718 e 740, obtained from the same patient (A),
272
were identical and classified as genotype MAA 2.
273
Among 20 isolates of M. abscessus subsp. bolletii that were studied, five
274
different genotypes were obtained (Figure 3) among seven related different
275
profiles. The isolates CRM 569, 639, 642, 719, 723, 732, 747, 835, 916, 917, 927,
276
928, 950, 1009 and 1010 were classified as genotype MAB 1. With the exception
277
of the isolate CRM 639, the genotype MAB 1 presented as identical profiles for all
278
isolates. CRM 639 and CRM 747 were the only ones not isolated from the same
279
patient. Genotype MAB 2 presented with two distinctly related profiles. The isolates
280
CRM 836 and 901 were identical and were obtained from the same patient. Isolate
281
CRM 803 and the reference strain CCUG 48898 presented unique genotypes,
282
respectively, MAB 3 and MAB 4, respectively. We observed that the genotype MAB
283
5 comprised CRM 640 and 920, respecting the patient-specific profile. None of the
284
CF clinical isolates presented a profile that was similar to the control strains. The D
285
values for PFGE typing of MAA and MAB were 1.00 [Non-Approximated
286
Confidence Interval (CNIA) 95% 0.456-1.000] and 0.952 (CINA 95% 0.761-1.000),
287
respectively.
288 289 13
290
DISCUSSION
291
Several studies worldwide have reported the prevalence of NTM in CF
292
patients. In France, a prevalence of 6.6% of NTM among 1582 CF patients was
293
reported [6]. In Toronto, Canada, the prevalence of NTM among 98 CF patients
294
between 6 and 18 years old was 6.1% [5]. In Spain, the prevalence of NTM among
295
220 CF patients was 7.72%; however, the patient’s age group varied between 6
296
and 74 years old [3]. A study developed in Israel that showed a prevalence of
297
22.6% of NTM among 186 CF patients, differs considerably from several other
298
studies around the world [4]. In our country, we observed only one study that
299
showed a prevalence of 11% of NTM among 54 CF adult patients in Brazil [2]. Our
300
study showed a prevalence of 7.75% of CF patients that were colonized/infected
301
with NTM.
302
The PRA-hsp65 had a concordance of 86,11% with the results obtained
303
from the partial sequencing of the rpoB. The isolates CRM 720, 747 and 803 were
304
identified by PRA-hsp65 as M. abscessus type I, whereas by rpoB sequencing they
305
were identified as M. abscessus subsp. bolletii. The isolates identified as M.
306
chimerae / M. intracellulare by PRA-hsp65 were identified as M. marseillense by
307
rpoB sequencing. The isolate CRM 695 identified as M. fortuitum by PRA-hsp65
308
was the one that presented the largest discordance with the rpoB sequencing,
309
even after several repetitions. However, the PRA-hsp65 database does not contain
310
the profile for M. timonense, which may have caused this difference in identification
311
between the methods used.
312
The M. abscessus group was the predominant NTM in our study (eight
313
patients), followed by MAC (three patients) and M. fortuitum (one patient). With the 14
314
exception of patients C (M. marseillense and M. abscessus subsp. boletii) and
315
patient I (M. fortuitum and M. timonense) who presented isolation of two different
316
species, the remaining patients presented isolation of only one species, allowing
317
better investigation of clonality. These results were similar to previous studies in
318
which the main species belonged to the M. abscessus group [7,23,24]. However,
319
our results differ from other results in which most of the isolated species were MAC
320
[3,5]. Additional, characterization of the microbiological profile of CF patients
321
developed by Paschoal et al [2] did not identify NTM to the species level. Currently,
322
CF NTM colonization/infection may be related to distinct NTM species prevalence
323
among distinct countries, reinforcing the importance of species identification in
324
therapeutic decision-making.
325
Brown-Elliott et al [25] reported that most of the strains of the M. abscessus
326
group show resistance to doxycycline, ciprofloxacin and moxifloxacin. Only one
327
isolate of M. abscessus subsp. boletii, obtained from patient J, was susceptible to
328
doxycycline (MIC ≤ 0,25 µg/ml) and ciprofloxacin (MIC 1µg/ml). It may be expected
329
that the isolates belonging to the M. abscessus obtained from CF patients may
330
show resistance to amikacin and other aminoglycosides, drugs that are commonly
331
used for treatment of bacterial infections, although amikacin resistance is rare
332
among M. abscessus recovered from different sources worldwide.
333
Although multidrug resistant NTM have been reported in studies involving
334
CF patients [6,7,26], the present work has shown the highest rates compared to
335
other studies in the scientific literature. Nevertheless, even when considering
336
infections by ATS criteria, only patient H presented with development of pulmonary
337
disease without resolution to date. The majority of the isolates from our study 15
338
(97.2%) were resistant to five or more antimicrobials, with the exception of the
339
isolate CRM 803, obtained from patient J. Fourteen M. abscessus subsp. bolletii
340
isolates (13 from patient H and 1 from patient B) presented amikacin MIC values
341
higher than 128µg/ml, one M. abscessus subsp. abscessus isolate with a MIC of
342
64 µg/ml (patient C) and two isolates with MIC of 32 µg/ml (patient A). The first
343
isolate from patient A showed susceptibility to amikacin, however the two following
344
isolates, obtained afterwards, showed intermediate resistance. The same event
345
was observed for patients C and H. The mechanisms for the acquisition of NTM
346
resistance to amikacin are unknown. Nevertheless, mutations in specific regions of
347
rrs and rpsL genes may confer aminoglycoside resistance in mycobacteria [26,27,
348
28].
349
We recovered only three MAC, two M. marseillense (CRM 694 and 696) one
350
M. timonense (CRM 695) isolates and all of them were susceptible to
351
clarithromycin. According to the CLSI 2011 guidelines, clarithromycin is the drug
352
chosen for the treatment of MAC infections, followed by moxifloxacin. The drug
353
resistance of MAC in CF patients is rare, which facilitates longer antimicrobial
354
treatment [25]. Other drugs such as ethambutol and rifampicin are used in
355
association with clarithromycin therapy. In some cases, amikacin is used together
356
with clarithromycin. However, due to the long duration of the treatment, which may
357
last longer than 06 months, and the side nephrotoxic and ototoxic effects caused
358
by amikacin this therapy has been abandoned [25, 28, 29]. Furthermore, the
359
present results, based on the breakpoints described by Siddiqii et al [19], indicate
360
that the therapy with amikacin, ethambutol, moxifloxacin, rifampicin or streptomycin
16
361
may be ineffective due to the high amikacin MIC values for the isolates CRM 694
362
and 696 (8µg/ml) and intermediate resistance found for other drugs.
363
According to the review published by Pitombo et al [30], most of M. fortuitum
364
strains are susceptible to amikacin, cefoxitin, ciprofloxacin, imipenem, linezolid and
365
sulfamethoxazole / trimethoprim, however M. fortuitum isolates from CF patients in
366
the present study were resistant to cefoxitin, ciprofloxacin, doxycycline, and
367
sulfamethoxazole / trimethoprim. The isolates CRM 724 and 725 also showed
368
resistance to clarithromycin. According to Brown-Elliott, et al [25], the M. fortuitum
369
group presents sulfonamide susceptibility of 100%, however, all M. fortuitum
370
isolates from CF patients were resistant to sulfamethoxazole/trimethoprim with MIC
371
values higher than 8 / 152 µg/mL.
372
The ERIC PCR is a simple molecular tool and can be used for genotyping
373
mycobacteria. A study developed by Sampaio et al [31] compared different
374
genotyping techniques in 17 isolates of M. fortuitum from an outbreak of infection
375
post mammoplasty and in this comparison the ERIC PCR showed clonal profiles
376
that were identical to the gold standard technique (PFGE). It is considered a fast
377
and economic alternative to PFGE. In a study developed with 40 Ugandan children
378
and teenagers with pulmonary infection caused by M. fortuitum, ERIC PCR
379
revealed the presence of a clonal group of three isolates, another group comprised
380
by two isolates and also 35 distinct profiles [32].
381
A clonal group of M. fortuitum was recovered from patient I with over 95% of
382
similarity. It is interesting to note that even though they presented with the same
383
genotypic restriction pattern, CRM 725 and 726 showed different susceptibility
384
profiles. The isolate CRM 724 showed the same susceptibility profile as CRM 725, 17
385
being both isolated from the same clinical sample. In conclusion, a single sputum
386
sample presented two different types of colonies with slightly different genotypic
387
profiles; the small colony named CRM 724 and the larger one named CRM 725.
388
The isolation of CRM 726 occurred 30 days after the isolation of the cited isolates
389
(CRM 724 and 725) and showed a profile identical to CRM 725. The susceptibility
390
profile and the genotypic profile results from isolates obtained from this clinical
391
case may suggest multiclonal NTM colonization belonging to the same species,
392
which may represent recent genomic mutations in mycobacterial populations.
393
The PFGE of the M. abscessus subsp. abscessus isolates revealed two
394
distinct genotypes: MAA1 and MAA2. The MAA 1 PFGE pattern comprised three
395
isolates from patient B, and showed two identical strains (CRM 641 and 697) and
396
another (CRM-720) with similarity of 88%. The MIC data of these isolates suggest
397
that the most recent isolated strain (CRM-720) presented resistance to amikacin
398
and different PFGE patterns from the others. A recent study showed a patient
399
infected by M. abscessus subsp. bolletii acquired resistance to amikacin during
400
therapy with the same drug [26]. The genotype MAA 2 comprised 3 clonal isolates
401
from patient A. However, the resistance profile was different among the three
402
isolates. These aspects may indicate that the molecular basis involved in the
403
alteration of susceptibility to drugs may not be related to the M. abscessus subsp.
404
abscessus genotypic profile by PFGE. Further studies are required for better
405
comprehension.
406
The PFGE results for isolates identified as M. abscessus subsp. bolletii were
407
classified as five different genotypes (MAB 1 to 5). Among those five, MAB 1 was
408
the one composed by most of the M. abscessus subsp. bolletii isolates [15]. All the 18
409
isolates from patient H were included in this genotype, as well as the isolate CRM
410
747 from patient C and CRM 639, obtained from patient E. CRM 639 showed a
411
profile that differed from the other isolates by only one band. After review of
412
medical records, we verified that no other isolation of mycobacteria occurred from
413
patients C and E up to the date we finished this study, suggesting these bacteria
414
may have composed the most transient microbiota of these patients. It is also very
415
startling that the MAB 1 genotype was circulating among three patients, and that
416
thissame clone remained causing severe disease in patient H up to the present
417
day (data not shown).
418
The genotype MAB 2 was comprised of the isolates CRM 836 and 901
419
isolated from patient D, presenting identical molecular and susceptibility profiles,
420
which confirm a patient-specific pattern. Similarly, the genotype MAB 5 had two
421
clones isolated from the same patient (D), with a difference of 18 months after the
422
date of the first isolation.
423
PFGE for MAA and MAB 2 were evaluated by a discriminatory index which
424
presented values higher than 0.90. These results confirm the elevated
425
discriminatory power of this method for diversity studies of these species, as
426
already showed in previous studies [33, 34].
427
The data obtained in this work will contribute to a better comprehension of
428
NTM colonization/infection of CF patients. The observed NTM characteristics are
429
alarming, since most of the isolates circulating among the patients present
430
resistance to multiple drugs. Isolation of high resistant strains from CF patients
431
must be considered a significant threat to public health mainly due to the paucity of
19
432
alternative therapy or potentially untreatable disease. Based on these findings, it is
433
highly recommended to monitor NTM infections in CF patients.
434
These findings, unprecedented in a country with a high incidence of TB
435
reinforce the importance of systematic and routine research for the identification of
436
mycobacteria to species level and susceptibility evaluation to antimicrobials.
437
Studies on monitoring of NTM transmission through respiratory equipment among
438
CF patients may also be useful to better understand rates of colonization and
439
prevalence of these organisms in these settings. ACKNOWLEDGMENTS
440 441 442
The Plataform-DNA Sequencing (PDTIS / FIOCRUZ), Rio de Janeiro, RJ,
443
Brazil is acknowledged for performing the rpoB sequencing procedures. We
444
appreciated the collaboration of Alcon Laboratórios do Brasil Ltda. by moxifloxacin
445
donation.
446
This study was supported in part by the Fundação de Amparo à Pesquisa
447
do Estado do Rio de Janeiro (FAPERJ, projects no. 103.225/2011, 103.287/2011,
448
110.272/2010, 110.761/2010 and 111.497/2008), the Conselho Nacional de
449
Desenvolvimento Científico e Tecnológico (MCT/CNPq, projects no. 476536/2012-
450
0, 473444/2010-0 and 567037/2008-8) and PDTIS – FIOCRUZ.
451
20
452 453
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589
FIGURE TITLES
590 591
Figure 1 – Dendrogram obtained from the automated analysis of the ERIC
592
PCR profiles of M. fortuitum isolates, showing the similarity coefficients (in %).
593
Dice’s coefficient was used as the algorithym for the construction of the
594
dendrogram.
595 596
Figure 2 – Dendrogram obtained from the automated analysis of the PFGE
597
profiles of the M. abscessus subsp. abscessus isolates, showing the similarity
598
coefficients (in %). Dice’s coefficient was used as the algorithym for the
599
construction of the dendrogram.
600 601
Figure 3 – Figure 1 – Dendrogram obtained from the automated analysis of
602
the PFGE profiles of the M. abscessus subsp. bolletii isolates, showing the
603
similarity coefficients (in %). Dice’s coefficient was used as the algorithym for the
604
construction of the dendrogram.
605
27
TABLES
606 607 608
Table 1 – Comparison of PRA-hsp65 and rpoB sequencing results for identification
609
of NTM strains isolated from sputa of 10 CF patients in Rio de Janeiro, Brazil.
Patient
Isolate
A
CRM 511 CRM 718 CRM 740 CRM 641 CRM 697 CRM 720 CRM 696 CRM 747 CRM 512 CRM 640 CRM 920 CRM 473 CRM 639 CRM 836 CRM 901 CRM 694 CRM 569 CRM 642 CRM 719 CRM 723 CRM 732 CRM 835 CRM 915 CRM 916 CRM 917 CRM 927 CRM 928 CRM 949 CRM 950 CRM 1009 CRM 1010 CRM 695 CRM 724a CRM 725 CRM 726 CRM 803
B C D E F G
H
I
610
J
a
Isolation date (day/month/year) 17/06/2009 24/02/2010 23/06/2010 21/10/2009 11/01/2010 16/04/2011 03/12/2009 12/07/2010 22/06/2009 19/12/2009 20/06/2011 03/06/2009 02/09/2009 19/01/2011 21/03/2011 02/09/2009 06/07/2009 21/10/2009 31/03/2010 24/04/2010 02/06/2010 13/01/2011 14/04/2011 14/04/2011 14/04/2011 14/07/2011 04/08/2011 02/06/2010 08/11/2011 12/03/2012 12/03/2012 03/12/2009 17/06/2009 17/06/2009 15/07/2009 30/08/2010
Molecular Identification PRA-hsp65 rpoB M. abscessus type 1 M. abscessus ssp abscessus M. abscessus type 1 M. abscessus ssp abscessus M. abscessus type 1 M. abscessus ssp abscessus M. abscessus type 1 M. abscessus ssp abscessus M. abscessus type 1 M. abscessus ssp abscessus M. abscessus type 1 M. abscessus ssp abscessus M. chimerae /M. intracellulare M. marseillense M. abscessus type 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/bolletii 1 M. abscessus ssp bolletii M. abscessus 2/bolletii 1 M. abscessus ssp bolletii M. chimerae /M. intracellulare M. marseillense M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. abscessus 2/ bolletii 1 M. abscessus ssp bolletii M. fortuitum type 2 M. timonense M. fortuitum type 2 M. fortuitum M. fortuitum type 2 M. fortuitum M. fortuitum type 2 M. fortuitum M. abscessus type 1 M. abscessus ssp bolletii
CRM-724 and CRM725 were distinct colonies isolated from the same sputum.
611 612 613 614 615 616
28
617
Table 2 – Antimicrobial susceptibility of the NTM isolates recovered from sputa of
618
CF patients in Rio de Janeiro, Brazil MIC (µg/ml)a
Patient
Isolate
Species AMI
CEF
CIP
CLA
DOX
MOX
T/S
A
CRM 511
M. abscessus subsp. abscessus
8(S)
64(I)
8(R)
8(R)
>32(R)
8(R)
> 04/76(R)
A
CRM 718
M. abscessus subsp.abscessus
32(I)
32(I)
>16(R)
1(S)
>32(R)
16(R)
>08/152(R)
A
CRM 740
M. abscessus subsp.abscessus
32(I)
128
>16(R)
32(R)
>32(R)
16(R)
> 04/76(R)
B
CRM 641
M. abscessus subsp.abscessus
8(S)
256(R)
4(R)
4(I)
>32(R)
16(R)
>16/304(R)
B
CRM 697
M. abscessus subsp.abscessus
16(S)
32(I)
16(R)
16(R)
>32(R)
16(R)
>16/304(R)
B
CRM 720
M. abscessus subsp.abscessus
64(R)
64(I)
>16(R)
16(R)
>32(R)
64(R)
>16/304(R)
C
CRM 747
M. abscessus subsp.bolletii
>128(R)
256(R)
>16(R)
32(R)
>32(R)
16(R)
> 04/76(R)
D
CRM 512
M. abscessus subsp.bolletii
8(S)
64(I)
8(R)
2(S)
>32(R)
16(R)
>16/304(R)
D
CRM 640
M. abscessus subsp.bolletii
8(S)
64(I)
2(I)
32(R)
32(R)
>16/304(R)
D
CRM 920
M. abscessus subsp.bolletii
8(S)
256(R)
>16(R) 32(R)
4(R)
>16/304(R)
E
CRM 473
M. abscessus subsp.bolletii
8(S)
64(I)
8(R)
32(R)
16(R)
>16/304(R)
E
CRM 639
M. abscessus subsp.bolletii
8(S)
256(R)
16(R)
32(R)
32(R)
>16/304(R)
F
CRM 836
M. abscessus subsp.bolletii
8(S)
32(I)
>16(R) 32(R)
32(R)
> 04/76(R)
F
CRM 901
M. abscessus subsp.bolletii
8(S)
64(I)
>16(R) 32(R)
32(R)
> 04/76(R)
H
CRM 569
M. abscessus subsp.bolletii
16(S)
128(R)
16(R)
64(R)
>32(R)
32(R)
> 04/76(R)
H
CRM 642
M. abscessus subsp.bolletii
8(S)
128(R)
16(R)
>64(R)
>32(R)
32(R)
> 04/76(R)
H
CRM 719
M. abscessus subsp.bolletii
>128(R)
64(I)
>16(R)
32(R)
>32(R)
128(R)
>16/304(R)
H
CRM 723
M. abscessus subsp.bolletii
>128(R)
128(R)
>16(R)
32(R)
>32(R)
32(R)
>16/304(R)
H
CRM 732
M. abscessus subsp.bolletii
>128(R)
256(R)
>16(R)
32(R)
>32(R)
128(R)
>16/304(R)
H
CRM 835
M. abscessus subsp.bolletii
>128(R)
64(I)
>16(R) >64(R)
>32(R)
32(R)
>16/304(R)
H
CRM 915
M. abscessus subsp.bolletii
>128(R)
256(R)
>16(R) >64(R)
>32(R)
16(R)
>16/304(R)
H
CRM 916
M. abscessus subsp.bolletii
>128(R)
64(I)
>16(R) >64(R)
>32(R)
32(R)
>16/304(R)
H
CRM 917
M. abscessus subsp.bolletii
>128(R)
64(I)
>16(R) >64(R)
>32(R)
32(R)
>16/304(R)
H
CRM 927
M. abscessus subsp.bolletii
>128(R)
64(I)
>16(R) >64(R)
>32(R)
16(R)
>16/304(R)
H
CRM 928
M. abscessus subsp.bolletii
>128(R)
64(I)
>16(R) >64(R)
>32(R)
256(R)
>16/304(R)
H
CRM 949
M. abscessus subsp.bolletii
>128(R)
64(I)
>8(R)
>16(R)
>32(R)
>2(I)
>16/304(R)
H
CRM 950
M. abscessus subsp.bolletii
>128(R)
64(I)
>16(R) >16(R)
>32(R)
>2(I)
>16/304(R)
H
CRM 1009
M. abscessus subsp.bolletii
>128(R)
64(I)
>8(R)
>16(R)
>32(R)
>2(I)
>16/304(R)
H
CRM 1010
M. abscessus subsp. Bolletii
>128(R)
64(I)
>8(R)
>16(R)
>32(R)
>2(I)
>16/304(R)
I
CRM 724
M. fortuitum
8(S)
>256(R)
8(R)
16(R)
>32(R)
04/76(R)
I
CRM 725
M. fortuitum
8(S)
>256(R)
8(R)
4(I)
>32(R)
04/76(R)
I
CRM 726
M. fortuitum
256(R) 0,5(S)
16(R)
32(R)
04/76(R)
J
CRM 803
M. abscessus subsp.bolletii
