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Previous Article | Next Article 
Journal of Clinical Microbiology, November 2001, p. 3976-3981, Vol. 39, No. 11
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.3976-3981.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Heterogeneity of Pseudomonas
aeruginosa in Brazilian Cystic Fibrosis Patients
Suzane
Silbert,1,2,*
Afonso
Luis
Barth,2 and
Hélio S.
Sader1
Laboratório Especial de Microbiologia
Clínica, Disciplina de Doenças Infecciosas e
Parasitárias, Universidade Federal de São Paulo,
São Paulo,1 and Hospital de
Clínicas de Porto Alegre, Unidade de Pesquisa
Biomédica, Serviço de Patologia Clínica,
Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande
do Sul,2 Brazil
Received 16 May 2001/Returned for modification 23 June
2001/Accepted 4 August 2001
 |
ABSTRACT |
The aim of this study was to assess the diversity and genomic
variability of Pseudomonas aeruginosa isolates from
cystic fibrosis (CF) patients being treated at a university hospital in
Brazil. Ninety-seven isolates of P. aeruginosa from 43 CF patients were characterized by macrorestriction analysis of
chromosomal DNA by pulsed-field gel electrophoresis (PFGE) and tested
for susceptibility to 20 antimicrobial agents by broth microdilution.
It was possible to evaluate single isolates from 20 patients and
multiple isolates (two to seven) from 23 patients collected during a
22-month period. Among all of the unrelated patients, we detected only
one pair of patients sharing a common strain. Among the 77 isolates
from 23 patients who had multiple isolates analyzed, we identified 37 major types by PFGE, and five different colonization patterns were
recognized. The isolates were susceptible to several antimicrobial agents, although consecutive isolates from the same patient may display
differences in their susceptibilities. Mucoid isolates were more
resistant (P < 0.001) than nonmucoid isolates to
five antibiotics. Our results indicate that CF patients remain
colonized by more than one strain of P. aeruginosa for
long periods of time. In addition, the finding of several different
genotypes in the same patient suggests that the colonizing strain may
occasionally be replaced.
| |
INTRODUCTION |
When cystic fibrosis (CF) was first
described in 1938, 80% of affected babies died within the first year
of life (2). The better understanding of the disease and
the improvements in treatment conditions in recent years have resulted
in a dramatic increase in the median survival time for patients with CF
(7). Nowadays, most patients reach adulthood, but
Pseudomonas aeruginosa continues to be the most prevalent
cause of infections in these patients. This pathogen is one of the main
causes of morbidity and mortality in CF patients (3, 8, 9, 11,
13).
It is generally accepted that once colonized with P. aeruginosa, most patients tend to harbor a conserved type (or
clone) during the course of the disease (5, 21, 26, 28, 29, 30). Patients may also harbor variants of the original clone, which are characterized by minor differences in genome fingerprints. Conversely, Boukadida et al. (4) found distinct strains
within the same patient, indicating that there may be a relatively high heterogeneity of strains of P. aeruginosa in the pulmonary
microbiota of chronically infected CF patients. Those authors also
found that the emergence of distinct strains in the same CF patient was
often associated with previous courses of antibiotic therapy. It
therefore appears that the epidemiology of P. aeruginosa in CF patients may vary in different medical centers.
Hospital de Clínicas de Porto Alegre (HCPA) is a 700-bed
university hospital located in southern Brazil. HCPA includes a center
for treatment of individuals with CF where more than 150 patients have
been monitored. The present study aimed to characterize isolates of
P. aeruginosa from CF patients being treated at HCPA according to their susceptibility to antimicrobial agents and their DNA
macrorestriction profiles.
| |
MATERIALS AND METHODS |
Bacterial isolates.
We collected P. aeruginosa isolates from 43 CF patients attending the CF
center of HCPA from March 1996 to December 1997. The clinical
specimens were processed for qualitative aerobic culture according
to conventional diagnostic methods (18). The identification of isolates was performed with the Vitek systems (BioMerieux, Hazelwood, Mo.) using the GNI card. All isolates were subcultured on nutrient agar slants and stored at room temperature before complementary tests (antibiogram and molecular typing).
Antibiogram.
The antimicrobial susceptibilities of isolates
to 20 compounds were evaluated by broth microdilution using MicroScan
Dried Gram Negative MIC/Combo panels according to NCCLS instructions (19). A standard suspension of the organism was used to
inoculate the panels, which were incubated at 35°C for a minimum of
16 h. The MIC was determined as the lowest antimicrobial
concentration showing inhibition of growth. The following antimicrobial
agents were tested: cefoxitin, ceftizoxime, ceftazidime, cefotaxime, ceftriaxone, cefoperazone, cefpodoxime, cefepime, imipenem, meropenem, ampicillin-sulbactam, ciprofloxacin, aztreonam,
amoxicillin-clavulanate, ticarcillin-clavulanate, mezlocillin,
sparfloxacin, piperacillin-tazobactam, netilmicin, and
azlocillin. P. aeruginosa ATCC 27853 was used as
the quality control strain. The results were interpreted according to
the NCCLS criteria (20).
Seven of the 20 antimicrobial agents tested were chosen to establish
the antibiotype of each isolate: ceftazidime, cefepime, imipenem,
ciprofloxacin, netilmicin, aztreonam, and piperacilin-tazobactam. These
compounds were selected because they represent the different classes of
antimicrobial agents and because they are commonly used for the
treatment of P. aeruginosa infections. Differences between
the results from susceptible to resistant and from resistant to
susceptible with at least one of these seven antimicrobial agents
characterized a different antibiotype, which was represented by a
capital letter. Differences in the results from susceptible to
intermediate or from intermediate to susceptible characterized a
variation of that antibiotype (subtype), and an arabic number was added
to the capital letter. The MIC at which 50% of the isolates tested are
inhibited (MIC50), MIC90,
and percent susceptibility for these compounds were also calculated.
The isolates were classified according to their colonial morphology on
blood agar as mucoid or nonmucoid, and the rates of susceptibility to each antimicrobial agent of these two groups were
compared using the Fisher exact test (10).
Molecular typing.
All isolates were characterized by
macrorestriction analysis of chromosomal DNA by pulsed-field gel
electrophoresis (PFGE) (24). Genomic DNA inserts were
digested at 37°C overnight (12 to 16 h) with 10 U of
SpeI enzyme (New England Biolabs, Inc., Beverly, Mass.).
Electrophoresis was performed in a CHEF-DRII apparatus (Bio-Rad,
Richmond, Calif.) with the following conditions: 0.5 Tris-borate-EDTA, 1% agarose, 13°C, and 200 V. The electrophoresis was run for 24 h, and the switch interval was ramped from 5 to 90 s (24). Photographs of ethidium bromide-stained
gels were examined visually. Isolates were considered distinct strains
if there were more than six fragment (band) differences between
PFGE profiles. Isolates were considered related (subtypes) if there were only two to six band differences between PFGE profiles. Isolates with the same PFGE profile were considered indistinguishable
(31).
| |
RESULTS |
A total of 97 clinical isolates of P. aeruginosa from
43 CF sputum samples were obtained between March 1996 and December
1997. The carbapenens meropenem (96.9% susceptibility;
MIC90, 2 µg/ml) and imipenem (93.8%
susceptibility; MIC90, 4 µg/ml) were the most active compounds against the P. aeruginosa isolates
evaluated in this study. Piperacillin-tazobactam (85.6%
susceptibility), ticarcillin-clavulanate (83.5% susceptibility), and
ceftazidime (81.4% susceptibility) also displayed reasonable in vitro
antimicrobial activity. The results of MIC50,
MIC90, and susceptibility rate determinations
were classified according to colonial morphology (mucoid and nonmucoid)
of the isolates (Table 1). The nonmucoid isolates were significantly more susceptible (P < 0.001) to cefoperazone, cefepime, ciprofloxacin, aztreonam, and
sparfloxacin (Table 1). The nonmucoid isolates also displayed higher
rates of susceptibility than the mucoid isolates to ceftazidime,
imipenem, ticarcillin-clavulanate, piperacillin-tazobactam, and
meropenem, but with no statistical significance.
The isolates were separated into two different groups: group 1, which
was composed of 20 isolates from 20 patients, and group 2, which
included 77 isolates from 23 patients (two to seven isolates from each
patient). A total of 19 distinct strains (major PFGE profiles)
were observed in group 1 isolates. Only two unrelated patients were
colonized with indistinguishable strains of P. aeruginosa in
this group (Fig. 1). On the other hand,
it was possible to identify 37 major PFGE profiles among the
isolates of group 2, indicating a considerable genomic diversity of
P. aeruginosa from different patients (Fig.
2). Two patients (siblings) of group 2 shared indistinguishable strains of P. aeruginosa. In
total, 13 isolates of these two patients were typed, and 12 of them
displayed the same PFGE pattern.
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FIG. 1.
PFGE of 20 clinical isolates of P.
aeruginosa from 20 different patients. Lane  , lambda ladder
(48.5 kb). Lanes 1 to 20, clinical isolates of P.
aeruginosa from 20 different patients.
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FIG. 2.
Genotypes of P. aeruginosa isolates from
six CF patients. Lanes 1, 2, and 3, patient 9; lanes 4, 5, and 6, patient 10; lanes 7, 8, and 9, patient 17; lanes 10, 11, 12, and 13, patient 6; lanes 14, 15, and 16, patient 5; lanes 17, 18, 19, and 20, patient 14; lane , lambda ladder (48.5 kb).
|
|
It was possible to obtain multiple isolates from the 23 patients of
group 2 over a period of time of up to 17 months. Thirteen patients
(56%) harbored at least two distinct strains over their follow-up
period, although most of these patients tended to harbor a single
strain (genotype) over many months (persistence of genotype) (Table 2). P. aeruginosa of
antibiotype A (susceptible to ceftazidime, ticarcillin-clavulanate, piperacillin-tazobactam, imipenem, and meropenem) or its subtypes was seen in 14 patients, but no
association between genotype and antibiotype was observed, as
isolates of the same genotype displayed different antibiotypes and vice
versa (Table 2).
View this table:
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|
TABLE 2.
Distribution of P. aeruginosa isolates from
the 23 patients of group 2 according to genome macrorestriction type
(genotype) and antimicrobial susceptibility profile (antibiotype)
|
|
We identified 18 patients harboring the same strain over different
periods of time, and 12 of them were colonized with P. aeruginosa displaying the same resistance profile. In the
remaining six patients the subsequent isolates displayed higher rates
of antimicrobial resistance than the initial isolate.
Twelve patients were colonized with both mucoid and nonmucoid isolates,
which were compared by molecular typing. Five patients presented
mucoid and nonmucoid isolates with distinct PFGE profiles, while
seven patients harbored indistinguishable isolates regardless of
colonial morphology. Among these seven patients, a change of the
colonies from nonmucoid to mucoid was observed in five patients, and a
change from mucoid to nonmucoid was observed in two patients.
| |
DISCUSSION |
Analysis of the antimicrobial susceptibilities of isolates
showed that high rates of resistance to antimicrobial agents indicated for the treatment of CF P. aeruginosa occurred, especially
among mucoid isolates. However, the carbapenens (imipenem and
meropenem) remained very active against both mucoid and nonmucoid
isolates, with susceptibility rates of >90%. The P. aeruginosa strains evaluated in the present study showed higher
rates of susceptibility than P. aeruginosa strains from CF
patients evaluated in other studies (6, 27). We also
observed that mucoid isolates showed a tendency for higher rates of
resistance (P < 0.001) than nonmucoid isolates to
several antimicrobial agents, including cefperazone, cefepime, aztreonam, sparfloxacin, and ciprofloxacin (23).
Despite the great number of studies on the epidemiology of P. aeruginosa infection in CF (1, 4, 5, 12, 14, 15, 16, 22, 26,
30), no data have been published on the colonization or
infection of Brazilian CF patients with P. aeruginosa. We
evaluated 97 isolates of P. aeruginosa from 43 CF patients,
and we found 39 distinct PFGE patterns among isolates from 41 patients
that were not epidemiologically related. Only two nonrelated
patients shared indistinguishable isolates. On the other hand, we
also found that 12 out of 13 isolates obtained from two CF siblings (closely related patients) displayed the same PFGE pattern. Similarly to other reports, these results suggest that cross-infection of P. aeruginosa among CF patients would occur more frequently
when contact was more intense (12).
Our study also documented the permanence of a single genotype in a
patient over time. We observed the presence of the original strain
isolated two to six times over 22 months in 18 patients. Ten of these
18 patients harbored a single genotype with a conserved macrorestriction pattern over time (3 to 17 months). These results suggest that even during periods of antibiotic therapy, the original strain is not eradicated from these patients. Another interesting finding was the presence of variations of the same genotype (subtypes) of P. aeruginosa in two single patients, as published by
other authors (30).
The remaining six patients carried two totally different genotypes, and
usually each one of these strains was isolated from different specimens
and on different days (Table 2). A mixed colonization of different
clones of P. aeruginosa can explain this finding. The clone
that predominates would vary over time, with one clone predominating in
one period and another clone predominating in another period. This
event suggests that P. aeruginosa infection and colonization
of the CF patient's lung is a very dynamic process, as proposed by
Renders et al. (25).
The most relevant result of our study was the finding of several
different genotypes in five patients of group 2 (21.7%). These
patients had two to four distinct strains (distinct major PFGE
patterns) isolated during the follow-up period (2 to 13 months). All
PFGE patterns were detected only once in these subgroups of patients. In contrast to other studies (1, 5, 12, 14, 15, 16, 26,
30), these results suggest that the colonizing strain may
occasionally be replaced. Boukadida et al. (4) also found different strains in the same patient and observed that the
emergence of distinct strains in the same CF patient was often associated with periods of antibiotic therapy.
Among the 97 clinical isolates of P. aeruginosa included in
our study, 40 presented the mucoid appearance, confirming that this
phenotypic alteration is also very common among P. aeruginosa strains from Brazilian CF patients (13).
Moreover, it was possible to determine that mucoid and nonmucoid
isolates were indistinguishable by PFGE in 7 out of 12 patients
colonized with these colonial variants. This finding confirmed the
clonal relationship between mucoid and nonmucoid isolates of P. aeruginosa from the same CF patient, as previously demonstrated
(17, 26, 32).
In conclusion, the results of this study indicate that in spite of the
fact that the CF patients had received courses of antibiotic therapy
periodically, nonmucoid P. aeruginosa strains isolated from
their lung showed relatively low rates of antimicrobial resistance. Among unrelated patients, we detected only one pair of patients sharing
isolates with identical PFGE patterns, and we concluded that
cross-infection is not common in our population. Our results also
indicate that CF patients remain colonized by more than one strain of
P. aeruginosa for long periods of time. In addition, the
finding of several isolates with distinct genotypes in the same patient
suggests that the colonizing strain may occasionally be replaced.
| |
ACKNOWLEDGMENTS |
We thank the Unidade de Microbiologia and the Cystic Fibrosis
Division, HCPA, for assistance in collecting and processing the
specimens for our study. We also thank the Molecular Epidemiology and
Fungus Testing Lab (Pathology Department, University of Iowa) for
assistance with the molecular techniques.
This work was supported by Fundação de Amparo a Pesquisa do
Estado de São Paulo (FAPESP) (grant 97/02795-6) and Fundo de Incentivo a Pesquisa e Ensino from HCPA.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Laboratório Especial de Microbiologia Clínica (LEMC),
Disciplina de Doenças Infecciosas e Parasitárias, Escola
Paulista de Medicina-Universidade Federal de São Paulo, Rua
Leandro Dupret, 188, São Paulo, SP-CEP 04025-010, Brazil. Phone:
55 11 5081-2819. Fax: 55 11 5571-5180. E-mail: lemc{at}ajato.com.br.
| |
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Journal of Clinical Microbiology, November 2001, p. 3976-3981, Vol. 39, No. 11
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.3976-3981.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
