JOURNAL OF CLINICAL MICROBIOLOGY,
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Mar. 1999, p. 788–791Vol. 37, No. 3
The IS6110 Restriction Fragment Length Polymorphism in
Particular Multidrug-Resistant Mycobacterium tuberculosis
Strains May Evolve Too Fast for Reliable Use in
ALICIA ALITO,1NORA MORCILLO,2SILVIA SCIPIONI,2ALBERTO DOLMANN,2
MARI´A I. ROMANO,3ANGEL CATALDI,3AND DICK VAN SOOLINGEN4*
Pathobiology Institute CICV/INTA1and Biotechnology Institute CICV/INTA,3Moro ´n, and Dr. Cetra ´ngolo Hospital,
Vte. Lo ´pez,2Argentina, and National Institute of Public Health and the Environment, 3720 BA Bilthoven,
Received 22 June 1998/Returned for modification 18 August 1998/Accepted 3 November 1998
To study possible nosocomial transmission of multidrug-resistant (MDR) Mycobacterium tuberculosis, strain
types and other information on 24, mostly human immunodeficiency virus-positive patients, were collected.
Isolates from 11 patients had identical IS6110 restriction fragment length polymorphism (RFLP) patterns as
well as spoligotype patterns and resistance profiles. Noticeably, nine other isolates from related cases also
exhibited identical spoligotypes but slightly different RFLP patterns. These results indicate that for some MDR
strains, the evolutionary clock of IS6110 RFLP may run too fast for reliable interpretation of strain typing
results over a period of a few years.
The high tuberculosis (TB) morbidity and mortality due to
multidrug-resistant (MDR) TB have caused major concern
regarding the clinical management and prevention of dissem-
ination of the disease (10, 21). In several countries, hospital
outbreaks due to MDR TB were reported in association with
human immunodeficiency virus (HIV)-positive patients (6, 15,
16). The short intervals of only 4 to 16 weeks for HIV-positive
patients from diagnosis to death and the high transmission
rates from patients to health care providers (4) have posed an
important public health problem.
From 1989 on, the number of HIV-positive patients admit-
ted for medical treatment at the Dr. Cetra ´ngolo Hospital
(CH), located in the northern suburbs of Buenos Aires, Ar-
gentina, began to increase (13). About 29% of the HIV-posi-
tive patients between 1992 and 1997 had one or more TB
episodes. In this period, 19% of the TB cases were caused by
MDR Mycobacterium tuberculosis strains. The number of HIV-
negative or noninvestigated patients with MDR TB remained
steady and amounted to about 2% of the total number of TB
cases recorded in the hospital. Both HIV-positive and HIV-
negative patients, with or without TB, received medical treat-
ment as in- or outpatients. However, all of them shared the
same hospital facilities, and most of the time, no effective
containment was implemented to prevent transmission of TB
Restriction fragment length polymorphism (RFLP) typing of
M. tuberculosis isolates has proven to be a useful tool to inves-
tigate nosocomial outbreaks (4, 12, 15, 16). Spoligotyping is a
new method for typing M. tuberculosis complex isolates. This
method is based on the polymorphic nature of short sequences
which are interspersed among the conserved direct repeats in
the genomic direct repeat region (8, 9, 11). In this study, the
spoligotyping method, with a lower level of discrimination than
that of IS6110 RFLP typing (5), was used as an additional tool
to validate the IS6110 RFLP typing results. In order to exam-
ine the transmission of TB in our hospital, both methodologies
A total of 74 M. tuberculosis isolates were obtained from 58
patients. Thirty-six of these patients were coinfected with HIV.
The isolates were identified by AccuProbe tests for M. tuber-
culosis complex (GenProbe, San Diego, Calif.) and by standard
biochemical procedures (3). The susceptibility to antitubercu-
lostatics was determined by the proportion method (2) on
Lo ¨wenstein-Jensen medium.
The 18 MDR TB patients among the HIV-negative patients
had undergone several episodes of anti-TB treatment while
receiving medical treatment in the hospital during an average
period of 3 years (range, 2 to 5 years). In contrast, all HIV-
positive patients infected with MDR TB had a single episode
of the disease, and they had an average survival period of 5.8
months (range, 4 to 18 months) from the onset of the disease.
Seventy-four M. tuberculosis strains, isolated from 58 pa-
tients, were analyzed by RFLP typing and spoligotyping (11,
17, 18, 20). A group of 24 MDR TB patients, formed by 20
clustered patients and 4 nonclustered patients, was selected for
further investigation. During alternate periods from 1992 to
1997, all of these patients were hospitalized or received med-
ical treatment as outpatients, and they sometimes shared the
same rooms. Figure 1 shows the RFLP and the spoligotyping
patterns as well as the drug resistance profiles of the MDR
isolates retrieved from the 20 patients, epidemiologically
linked on the basis of the strain typing results. In this figure,
the data on hospitalization period and HIV status of the pa-
tients also are indicated.
The MDR strains from HIV-positive patients 1 to 11 (group
1) exhibited identical RFLP patterns (type II) and spoligotype
patterns (type B), as well as the same resistance profiles. Since
the patients concerned had overlapping dates of admission to
and/or release from the hospital and they did not receive an-
ti-TB treatment previously, it was highly likely that a person-
* Corresponding author. Mailing address: National Institute of Pub-
lic Health and the Environment, P.O. Box 1, 3720 BA Bilthoven, The
Netherlands. Phone: 31-30-2742363. Fax: 31-30-2744414. E-mail: d.van
to-person transmission of MDR TB occurred among these
The second group comprised nine MDR-TB strains isolated
from patients 12 to 20. Although five highly similar RFLP
patterns (type VIII) were found in this group of nine strains, all
had identical spoligotype patterns (type C). The spoligotype
patterns of the two described outbreak strains were not found
in the database comprising 285 spoligotype patterns of M.
tuberculosis isolates from Argentina. The five RFLP patterns
distinguished among the nine isolates related to the second
outbreak were pattern VIII (five instances) and the patterns
VIIIa to VIIId (once each). RFLP pattern VIII was found in
FIG. 1. Patient information, drug resistance profiles, and DNA fingerprints of M. tuberculosis isolates related to the two described TB outbreaks. (A) First outbreak;
(B) second outbreak. The column “Drug susc.” indicates resistance to the drugs abbreviated as follows: H, isoniazid; S, streptomycin; R, rifampin; E, ethambutol; Z,
pyrazinamide. ND, not determined. Dates are given as month/year. ?, period of patient’s attendance at hospital H1; ??, period of patient’s attendance at hospital H2.
VOL. 37, 1999NOTES 789
five MDR TB isolates recovered from five HIV-positive pa-
tients (patients 14, 15, and 18 to 20). Patient 20 was a health
care worker at the CH. The diagnosis of patients 14 and 15
occurred during 1993 to 1994 in a different hospital in Buenos
Aires (H1). The RFLP pattern VIIIa was found in an isolate
from an HIV-negative person (patient 12) who had a disease
progression of 6 years, 1991 to 1997, and received medical
assistance in several hospitals including CH and H1 (1992 to
present). RFLP pattern type VIIIb (patient 13) was exhibited
by two isolates from one HIV-negative patient. These isolates
were obtained at an interval of 1 year. All nine of these strains
were resistant to isoniazid, streptomycin, and rifampin, and
some of the strains were resistant also to pyrazinamide (pa-
tients 12 and 16 to 20) and ethambutol (patients 12 and 17 to
The third group of isolates comprised nonclustered patients
21 to 24. The RFLP patterns V, XIII, and XIV and the spo-
ligotypes D, G, and E were obtained from different MDR TB
isolates from three HIV-negative patients (patients 21, 22, and
24, respectively [data not shown]). The patients had a disease
progression of 3 years; they remained positive by smear exam-
ination during this period although they were receiving appro-
priate therapy. From the remaining HIV-negative patient of
the third group, patient 23, we obtained two isolates, at an
interval of 1 year (Fig. 2). The RFLP patterns of these isolates
differed in a single band of 1.5 kb, present in the first isolate
and absent in the second. Both strains had the same spoligo-
All isolates from the first group of patients (patients 1 to 11)
had identical RFLP and spoligotype patterns as well as resis-
tance profiles. In contrast, the isolates from the second group
of patients (patients 12 to 20) had identical spoligotype pat-
terns and highly similar, but not identical, RFLP patterns. The
second outbreak strain provoked an outbreak in hospital H1
which also extended to other hospitals (15). Since we are quite
sure that all of the isolates from the second group of patients
are derived from a common ancestor within a period of 4 years,
it is tempting to speculate on the instability of IS6110 RFLP in
these strains with regard to bacterial factors and host-depen-
dent influences. Also, the serial isolates recovered from MDR
TB patient 23 showed a change in the RFLP pattern. Many
reports have proved the stability of IS6110 RFLP during in
vivo and in vitro (7, 19) incubation of M. tuberculosis strains. In
contrast, this study indicates that particular strains, such as
those causing the second outbreak, may have a higher muta-
tion frequency. In a recent study by Yeh et al. (22), it was
found that serial isolates from 14 (29%) of 49 patients showed
changes in their DNA genotypes between isolates (12 in
IS6110 RFLPs and 2 in polymorphic GC-rich sequence
RFLPs). However, the changed IS6110 RFLPs were not re-
lated to the bacterial drug susceptibility or to the patient’s HIV
serum status or adherence to therapy. In contrast, in this study
the instability of IS6110 RFLP found in the second outbreak
strain may be somehow related to the selective pressure of a
combination of drugs, as these drug-resistant patients were
difficult to treat. On the other hand, the patient population in
the first outbreak did not differ significantly from the patient
group in the second microepidemic; however, we did not ob-
serve any alteration in the IS6110 RFLP of this strain. There-
fore, the factor influencing the transposition frequency may be
strain dependent. For instance, IS6110 elements in the second
outbreak strain may be inserted in particular genomic pro-
moter regions, resulting in a higher transposition pressure.
Spoligotyping, in addition to IS6110 RFLP, can be useful in
determining more distant relationships among isolates. This
has been proven for the first time in the disclosure of the
Beijing genotype family of M. tuberculosis strains (20). One
representative of this type of M. tuberculosis strain, the W
variant, has been transmitted in New York hospitals since the
early 1990s (14). In the study by Moss et al. (14), 8 of 128
(6.5%) W-type MDR TB strains isolated in New York City
hospitals in 1992 also exhibited IS6110 RFLP patterns slightly
different from those of the predominant type. Within a rela-
tively short period (a few years), this W-type strain developed
several lineages with slightly different, but recognizable, IS6110
RFLPs (1). In our current study, the relative instability of
IS6110 RFLP was found in one of two MDR outbreak strains;
however, not fewer than four of nine of the IS6110 RFLP
patterns showed a minor and different alteration. Therefore,
the transposition rate may be strongly related to the M. tuber-
culosis genotype represented.
The work in Argentina was supported by the Centro Argentino-
Brasilen ˜o de Biotecnologı ´a (CABBIO), AC and MIR, and fellowships
of CONICET, Argentina.
We acknowledge the help of Simone van de Pas and Kristin Kremer
in preparing the manuscript.
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