| AGORA

RESEARCH LETTER

Mycobacterium abscessus in patients with

cystic fibrosis: low impact of inter-human
transmission in Italy
To the Editor:
Mycobacterium abscessus is a rapidly growing nontuberculous mycobacterial species increasingly isolated
worldwide [1]. It may cause chronic pulmonary infections, mainly in elderly patients with underlying
bronchiectasis or chronic obstructive pulmonary disease, and it may also cause soft tissue, bone and joint
infections [2]. M. abscessus has been isolated frequently from patients with cystic fibrosis (CF) [3],
although its role in the decline of lung function remains unclear as it can be found both in patients with
severe decrease in forced expiratory volume in 1 second (FEV1) and progressive worsening on computed
tomography (CT) scan [4], and in asymptomatic patients. We investigated at the whole genome level
M. abscessus isolated from all patients attending four Italian CF centres in the past decade with the aim of
assessing the role of inter-human transmission.
The four centres are located in geographically distinct regions of Italy. All of the centres routinely perform
sputum screening for mycobacteria using conventional protocols [5], together with extended incubation
culture on Burkholderia cepacia selective agar (BCSA). The selective activity of BCSA for rapidly growing
mycobacteria [6] allows the risk of missing M. abscessus through contamination or overgrowth of other
bacterial species to be minimised. No special segregation policy was in force in any of the centres during
the years covered by this study.
M. abscessus consists of three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. bolletii and
M. abscessus subsp. massiliense [7]. We identified 306 isolates at the subspecies level by rpoB gene
sequencing [8]. In our study, M. abscessus subsp. abscessus accounted for 62% of isolates, in agreement
with reported data [9]. Unexpectedly, M. abscessus subsp. bolletii was the second most prevalent subspecies
(22%), suggesting that this subspecies is not as rare as previously thought.
In total, 162 isolates were used for whole genome sequencing (WGS); one isolate was taken from each
patient and at least two (the first and the last) if multiple isolates were found. In patients shown to be
infected by more than one strain belonging either to the same or different subspecies, a representative
proportion of isolates were selected for WGS, with the objective of differentiating persistent infections
from occasional re-infections.
Cluster analysis of the isolates was performed by single nucleotide polymorphism (SNP) detection using
an arbitrary cut-off based on the average difference of isolates collected in a short time interval from the
same patient [10]. Reads were mapped to the M. abscessus ATCC19977 genome (NC_010397.1) using
BWA (http://bio-bwa.sourceforge.net), and mappings refined with the GATK and Samtools toolkits. SNPs
were called by Samtools and perl scripts with a minimum coverage of four reads in both forward and
reverse orientation, with the four reads calling the allele with a phred score of at least 20 and allele
frequency of 75%. SNP positions were then combined, and positions with a reliable base call in at least
95% of the isolates were concatenated to a sequence alignment, excluding SNPs within a window of 12 bp
from each other.
In 42 out of 48 patients with multiple isolates of M. abscessus over time, the average difference in SNPs
was seven (range 0–22), a low number suggesting the persistence of the same strain and not a re-infection.
In four patients, we isolated more than one subspecies of M. abscessus at different time points. In another

@ERSpublications
No outbreak of M. abscessus among Italian CF patients despite the presence of a widespread
circulating clone http://ow.ly/oM9l30cs6M0

Cite this article as: Tortoli E, Kohl TA, Trovato A, et al. Mycobacterium abscessus in patients with cystic
fibrosis: low impact of inter-human transmission in Italy. Eur Respir J 2017; 50: 1602525 [https://doi.org/
10.1183/13993003.02525-2016].

https://doi.org/10.1183/13993003.02525-2016 Eur Respir J 2017; 50: 1602525

 RESEARCH LETTER | E. TORTOLI ET AL.

M1
5
BN2
8 1
BN2
5 BN1
4128
8
BN1
1
BN3 M2
5034 16
BN3
16
1615
N01
N01 5
M. abscessus s. massiliense
4427 1 PA4
RO1
1
2 4
3241 CG1
CG2
PA1
1
2
BS01 3 PA2
1758 1527 BS05
3136
2777 2
1
BS10, BS11 PA3
BS08, BS09, BS12 12 VM1
2934 2
BS03 1
1 9
1538 295
CP01
BS02
VM3
5
1 PA4
3855 2 4
PA1
3 PA2
11449
M 3221
1
12
PA3
VM1
9
VM3
1
1 NL1
1
4191 3
2
NL3
NL4
5 A1
1
NL5
10 DL2
NL2
5248
713
TA1 DL1
847
AS1 2
31 1
3
BM2 7 GA4
BM3
2 A2
BM1 DA1
10
5
DL2 1
24 DL1 CP03
7
2
2
GA4 7 CP07
DA1
1
7
CP03
CP07
8 CP09
8 CP09 2
3
2
2
CP06 2 CP06
7
4 1 CP05 7
CP11
4 1 CP05
31 CP02, CP10
3
2
2
CP08 CP11
CP04
4
4
2
CR1 CP02, CP10
3 CR3
2 CP08
2
CR2 3
33 DF2
2
4
1
DF1 CP04
M. abscessus s. abscessus
4
13
3
5
GL2
4 CR1
19
GL1
2
13
1
BO1
BE2
3 CR3
4
13
2
BE4
BE5
CR2
11
1989 BE6
BE3 A3
3
7 GM2 9
3 19 416
GM5 LG2
GM1, GM3, GM4 1
14
3
FJ2 GT1
2
FJ1 3 3
4 9
LS2 GT3
1
GT1
1
19 3 3
GT3 GT2
GT2
LG1 LG1
24
PM1
30
1 LC1
2
25 TR1
TR2
43
DE1
1
6472 SG1
6
3 SG3
1
SG2
1365 153 GA5
1 GA7
4678 1
11 GA6
3674
GA1
271 1
GA3
1
GA2
2
4361 CE1
3
996 CE2
6
4293 PE2
6
PE1
635 3129 BS04
6523
1246 VM2
579 3
1755 BS07
1062 1
BS06
4
1212 SR1
4
903 SR2
404 14
18 PL1
813 8
766 225 PL2
265
EP1, EP2
441 4367
VM4
474 1245
BC1
3055
1113 ML1
1 CA2
4875

A 6331
1
CA1 A4
4123 CI1
3
924 CI2
896 30
3614
139
MO1
1
572 4663
BA1
26 FI1
SP1
1 1
1260
26
1
FI1
FI2
FI2
5 5
6
4
MC1, MC2
MC3 6 MC1, MC2
3989
3 1 4
5
IR1
IR3
MC3
98 1
IR2 3 1
15 1
GI1 IR1
5
GI2 5 IR3
TE2
3
98 1
2
TE1, TE3
IR2
4575
2
MA2 15 1
3995
4811
MA1
QE1
GI1
6483
1
1
OA1 GI2
OA2
4077 5
PI1
1
2
CS2 3 TE2
2646 4
1
CS1
CS3
TE1, TE3
6
IF3, IF4
1
5 IF6
1 IF5
1 2 1
RM4
494
11
1
RM1, RM2, RM3 B1
IF1
3394 6
3122
IF2
6
MN3
IF3, IF4
17 1
MN2 1
777 MN1 5 IF6
532 1
GG1 1 IF5
1
2
GG4
GG6
1 2 1
3227 4
1
GG3 1 RM4
GG5, GG7
728 5
GG2 11 RM1, RM2, RM3
B 13583
662
3076
3678
MM1

4
1
FR3
BE1
IF1
1089 1
1185
4035
8
FR2
FR1
IF2
2 6
3780
3
DI1
DI2
MN3
4667 17 1
4460
6
MR1
M. abscessus s. bolletii
MN2
13
MR2
MN1

FIGURE 1 Maximum parsimony phylogenetic tree constructed on 110 551 SNP positions of 162 clinical
isolates and three reference strains of Mycobacterium abscessus on a logarithmic scale. Numbers on the
branches indicate the number of distinct SNP positions between isolates. All isolates were grouped together
(as indicated by the same colour) with a maximum distance of 25 SNPs to the nearest group member. M, A
and B indicate the branches corresponding to the three subspecies: M. a. subsp. massiliense, M. a. subsp.
abscessus and M. a. subsp. bolletii respectively. The grey shaded box highlights the dominating clone of M.
abscessus subsp. abscessus. Clustered isolates are shown in circles M1, M2, A1, A2, A3, A4 and B1; each
isolate is indicated by a two-letter identifier, unique to each patient, followed by a number representing the
order of isolation. SNP: single nucleotide polymorphism.

https://doi.org/10.1183/13993003.02525-2016 2
 RESEARCH LETTER | E. TORTOLI ET AL.

two patients, two unrelated strains of the same subspecies were detected. In almost all cases presenting
more than one subspecies (5/6), we observed persistence of the same strain over time.
WGS confirmed the species identification performed by rpoB Sanger sequencing in all cases except two
strains that, despite the presence of a rpoB sequence specific of M. abscessus subsp. abscessus, were
assigned to M. abscessus subsp. massiliense by WGS.
Figure 1 shows the minimum parsimony tree of the 162 isolates analysed by WGS. Three main clusters
corresponding to the subspecies are clearly identifiable (rectangles A, B and M). Within M. abscessus
subsp. abscessus (figure 1A), we observed one group of clustered isolates (grey shading) originating from
15 (25%) different patients, whereas the remaining isolates were dispersed. The median branch length of
the clustered isolates was 21 SNPs (range 1–144).
A recent paper by BRYANT et al. analysing WGS of a vast number of M. abscessus isolated from patients
with CF worldwide [11] reported the existence of at least three dominant clones responsible for at least
three outbreaks postulated to be consequent to direct or fomite-mediated transmission. The analysis of our
genomes, together with 30 of those reported by BRYANT et al. [11] (10 for each for the three major clusters:
Abscessus 1 and 2 and Massiliense) revealed that Abscessus 1 overlapped the largest cluster identified in
our collection (grey shading in figure 1). To avoid the risk of overlooking transmission episodes, we
intentionally kept the threshold for flagging possible patient-to-patient transmission wide (⩽30 SNPs, with
our same-patient difference being ⩽21 SNPs in 99% of the cases). By applying this threshold, we detected
in the aforementioned cluster three possible patient-to-patient transmission episodes involving seven
patients in total (3 in A1 and 2 each in A2 and A3). An additional cluster was identified within M.
abscessus subsp. abscessus dispersed isolates, which involved five patients (A4). The number of SNPs
confirmed possible occurrence of transmission for only two patients (IR and GI). Two clusters were
detected within M. abscessus. subsp. massiliense (M1 and M2), each involving two patients, while one
cluster (B1) involving three patients was present for M. abscessus subsp. bolletii.
We were able to collect information on attendance of each patient at the different centres included in this
study. Of the seven “possible” transmission episodes identified by WGS analysis using a cut-off of SNPs
difference ⩽30, only three (M1, M2 and B1 [ patients IF and RM only]) involved patients who had
attended the same CF centre at the same time. For the remaining four patients, transmission appears very
unlikely based on the geographical location. In fact, these cases involved either patients attending centres
located in different cities (A1), or couples of patients (one adult and one paediatric) referred to the same
centre but cared for in different and distant locations of the clinic (A2, A3 and A4). One of our small
clusters (A4), which included strains that were not very closely related, coincided with the Abscessus 2 of
BRYANT et al. [11], while Massiliense was far from our M. abscessus subsp. massiliense genomes with the
exception of one strain (CP01), isolated only once from one patient. Importantly, our investigation could
exclude the occurrence in the past 12 years of major outbreaks of M. abscessus strains in any of the four
Italian centres participating in this study. The discrepancy with the paper by BRYANT et al. [11] that
reported large outbreaks in multiple countries may be explained by their much larger sample size;
however, in an older study by BRYANT et al. [10], which was very similar to ours in terms of number of
patients recruited, two large outbreaks of M. abscessus subsp. massiliense were reported in a single CF
centre. Another explanation may be the very low incidence of M. abscessus subsp. massiliense in our study.
We conclude that the cluster Abscessus 1 described by BRYANT et al. [11] is present in Italy in the
population of patients with CF. In addition, we identified very few highly clustered cases, each involving a
maximum of three patients, for which the possibility of healthcare-related transmission could not be
excluded. No major outbreaks were identified in our study in any of the participating centres. Our
findings, far from minimising the importance of preventive infection control measures in CF centres, will
hopefully reduce the concern in the CF community provoked by the emphasis given to the risk of
inter-human transmission of M. abscessus.

Enrico Tortoli1, Thomas A. Kohl2, Alberto Trovato1, Rossella Baldan1, Silvia Campana3, Lisa Cariani4,
Carla Colombo5, Danila Costa6, Simona Cristadoro7, M. Clelia Di Serio8, Antonio Manca9,
Giovanna Pizzamiglio 5, Paola M.V. Rancoita8, Gian Maria Rossolini10, Giovanni Taccetti3, Antonio Teri4,
Stefan Niemann2 and Daniela M. Cirillo1
1
Emerging Bacterial Pathogens Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy. 2Molecular and Experimental
Mycobacteriology, Forschungszentrum Borstel, Leibniz-Zentrum für Medizin und Biowissenschaften, Borstel, Germany.
3
Regional Reference Center for Cystic Fibrosis, Meyer University Hospital, Florence, Italy. 4Cystic Fibrosis Microbiology
Laboratory, IRCCS Ca’ Granda, Milan, Italy. 5Cystic Fibrosis Center, IRCSS Ca’ Granda, Milan, Italy. 6Microbiology
Unit, Policlinico University Hospital, Bari, Italy. 7Cystic Fibrosis Center, Messina Hospital, Messina, Italy. 8Centre for
Statistics in the Biomedical Sciences, Vita-Salute San Raffaele University, Milano, Italy. 9Reference Center for Cystic
Fibrosis, Policlinico University Hospital, Bari, Italy. 10Microbiology and Virology Unit, Careggi University Hospital,
Florence, Italy.

https://doi.org/10.1183/13993003.02525-2016 3
 RESEARCH LETTER | E. TORTOLI ET AL.

Correspondence: E. Tortoli, Emerging Bacterial Pathogens Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy.
E-mail: e.tortoli.enrico@hsr.it

Received: Dec 22 2016 | Accepted after revision: April 07 2017

Support statement: This study was supported by the research grant FFC #27/2014 from Fondazione Ricerca Fibrosi
Cistica. Funding information for this article has been deposited with the Crossref Funder Registry.

Conflict of interest: None declared.

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