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Journal of Clinical Microbiology, December 1999, p. 4167-4169, Vol. 37, No. 12
Institute of Microbiology,
Received 6 May 1999/Returned for modification 29 July 1999/Accepted 7 September 1999
Fifteen nosocomial cases of extended-spectrum
In recent years, extended-spectrum
Between 1 June and 30 November 1998, 21 ESBL-KP strains and one
ESBL-producing Serratia marcescens strain were isolated from 15 patients in the PICU of the "Géza Hetényi" County
Hospital, Szolnok, Hungary. In order to find other sources of the
ESBL-KP strains, 143 inanimate environmental samples for culture were collected in August and October, and 22 members of the staff were screened. Ceftazidime was used for empiric therapy in all clinical cases starting in 1990 and was restricted in June 1998. Additionally, the strict isolation of ESBL-KP-infected patients, emphasizing hand
washing and the use of disposable gloves, was implemented in September 1998.
Biochemical identification of all strains was done with the ATB test.
Antibiotic susceptibility testing was performed by standard disk
diffusion as recommended by the National Committee for Clinical Laboratory Standards (13). ESBL production was confirmed by the double-disk synergy test and E-test as described elsewhere (18). The isoelectric points of ESBLs were determined by the method of Matthew et al. (9).
Plasmid DNA of all strains, extracted by the modified alkaline-lysis
method, was transformed into Escherichia coli DH5- A case was defined if ESBL production was confirmed by genotyping and
by isoelectric focusing regardless of colonization or clinical
infection. Isolates were considered repeated if they were cultured from
the same patient and if they were indistinguishable from the previous
isolates by genotyping. The incidence with 95% confidence interval was
estimated by using a person-days denominator, taking colonized and
clinical cases once into the calculation. Clinical infection was
defined according to the diagnosis by a perinatologist. The cluster was
defined if cases were indistinguishable by genotyping and if they
showed the same plasmid restriction profile.
From 1 March, when the ESBL screening test was introduced, until 29 June 1998, 663 strains of the family Enterobacteriaceae were
isolated throughout the hospital without the detection of ESBL. The
retrospective search back to 1 January 1997 had shown no multiresistant
K. pneumoniae isolates in the PICU. The first ESBL-KP strain
was detected in the PICU on 30 June (Fig.
1). By the end of November, another 14 primary isolates and five repeated isolates were detected. There was no
ESBL-KP strain isolated on the other wards. In the PICU, one case
occurred in June, five cases occurred in July, two cases occurred in
August, five cases occurred in September, one case occurred in October,
and one case occurred in November. In August, ESBL-producing S. marcescens appeared in a premature neonate who had been colonized
with an ESBL-KP strain 1 week before. One ESBL-KP strain was detected in the bath soap, and there was no ESBL-KP strain isolated among the
screened staff. During this period, 132 neonates were admitted, giving
a total of 1,835 patient days. The monthly incidence rates of ESBL-KP
and the 95% confidence interval from June until November were 0.29 (0.04 to 2.06), 1.07 (0.45 to 2.57), 0.84 (0.21 to 3.36), 3.05 (1.27 to
7.33), 0.40 (0.06 to 2.84), and 0.52 (0.07 to 3.69) per 100 patient
days, respectively (Fig. 1). Six cases were male, and nine cases were
female. Five cases were colonized. One neonate among nine clinically
infected neonates died.
Fourteen clinical isolates of ESBL-KP (isolates 1 to 14), the
environmental ESBL-KP isolate, and the S. marcescens isolate had the same resistance pattern, while the 15th ESBL-KP isolate had a
different pattern, the MIC90 of cefepime, cefpirome,
trimethoprim-sulfamethoxazole, and tetracycline for this isolate being
much lower than those for the others. The plasmid analysis showed that
all primary clinical strains, the environmental ESBL-KP strain, and the
S. marcescens strain harbored a single large plasmid. The
presence of the SHV-encoding gene on the plasmid was confirmed for all
strains. The ESBL-encoding plasmid of ESBL-KP isolates 1 to 14 and the
S. marcescens isolate could be transferred to the E. coli J53-2 Rifr strain by conjugation and to E. coli DH5-
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Molecular Epidemiology of a Cluster of Cases Due to
Klebsiella pneumoniae Producing SHV-5 Extended-Spectrum
-Lactamase in the Premature Intensive Care Unit of a
Hungarian Hospital
ABSTRACT
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Abstract
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References
-lactamase-producing Klebsiella pneumoniae occurred
among 132 neonates in a premature intensive care unit in Hungary in
June through November 1998. Fourteen strains were indistinguishable by
molecular biological typing and harbored the same single conjugative
extended-spectrum -lactamase-encoding plasmid that was spontaneously
found in a Serratia marcescens strain in the same patient.
TEXT
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Abstract
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References
-lactamase-producing Klebsiella pneumoniae (ESBL-KP)
strains of the types TEM and SHV have become important pathogens in
hospital-acquired infections, showing multiresistance and causing more
and more outbreaks in hospitals since the first ESBL was isolated in
1984 (1, 8, 14, 15, 17, 19). In premature intensive care
units (PICUs), Klebsiella spp. appear as the most common
pathogens (2, 5).
cells (7). E. coli J53-2 rif was used for
conjugation (3). The MICs at which 90% of the K. pneumoniae isolates, the S. marcescens isolates, and
the E. coli DH5- transformants tested were inhibited (MIC90) were determined by E-test (AB Biodisk, Solna,
Sweden) as recommended by the manufacturer. E. coli ATCC
25922 was used as a reference strain. The purified plasmid DNA was
digested with EcoRI and HindIII (Sigma) as
recommended by the manufacturer. SHV PCR was done as prescribed by
M'Zali et al. (11). Chromosome fingerprinting was performed
by AP-PCR with the ERIC2 primer (4).
View larger version (17K):
[in a new window]
FIG. 1.
Distribution of ESBL-KP (solid bars) and non-ESBL-KP
(striped bars) strains and other species of the
Enterobacteriaceae (open bars) and incidence of ESBL-KP in
the PICU (line with asterisks) (January through November 1998).
by transformation. MIC90 for the E. coli transformants Tf1 to Tf14 were slightly lower than those for
the donor strains, but the -lactam resistance pattern with ESBL
production was the same as that for the donor strains (Table
1). The transformants also became
resistant to gentamicin, tobramycin, netilmicin, and
trimethoprim-sulfamethoxazole, which suggested that the genes encoding
the resistance to these antibiotics could be carried on the same
plasmid as the SHV gene. The clinical ESBL-KP isolates 1 to 14, the
environmental ESBL-KP isolate, and the S. marcescens isolate
had the same restriction plasmid DNA profile with
HindIII (three bands) and EcoRI (eight bands), which confirmed the similarity of their plasmids. The restriction profile of ESBL-KP isolate 15, which had a different antibiotic resistance pattern, was invisible.
TABLE 1.
MIC90 for ESBL-KP isolates 1 to 15, the
S. marcescens strain, and the E. coli
DH5- transformant
All ESBL-KP strains including the environmental strains, the S. marcescens isolate, and the transformant strains had -lactamase isoelectric bands at pI of 5.6 and 8.2. The pI of 8.2 corresponded to
the SHV-5 enzyme.
All ESBL-KP strains showed two chromosome patterns by AP-PCR. The clinical ESBL-KP isolates 1 to 14 and the environmental strain had pattern A1, containing four bands giving lines at 5.0, 3.0, between 3.0 and 2.5, 2.5, and 2.0 kb. ESBL-KP isolate 15 showed the pattern A2, also containing four bands, but it gave lines at 5.0, 2.5, 2.0, and between 2.0 and 1.5 kb (Fig. 2). During the epidemiological investigation, 14 cases, ESBL-KP cases 1 to 14, could be defined as a nosocomial cluster showing propagation. ESBL-KP case 15 was a sporadic case.
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In Hungary, the SHV-2 and SHV-5 types of ESBL-KP have been reported for sporadic cases (12, 16). Our study proved the nosocomial and epidemic nature of the SHV-5 type among premature infants with environmental occurrence. The consecutive isolation of ESBL-KP and ESBL-producing S. marcescens harboring a similar plasmid from the same patient suggested the transfer of the SHV-5-encoding plasmid between these strains. The promiscuous nature of the plasmid-encoded ESBL resistance was proved by conferring the salient antibiotic resistance on E. coli by transformation and conjugation.
It was unclear whether the first ESBL-KP strain was imported into the PICU or whether there was a conversion of non-ESBL-KP to ESBL-KP as a result of the antibiotic pressure of ceftazidime, which was suggested by other authors (6, 10, 14). In our study, the restricted use of ceftazidime could not prevent new cases. Only strict isolation was able to decrease the incidence. The absence of new cases during a 2-month follow-up period suggests the effectiveness of the control measures.
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ACKNOWLEDGMENTS |
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We thank Andy J. Hall at the London School of Hygiene and Tropical Medicine for his comments on the manuscript. The technical assistance of Orsolya Dobay, Katalin Katona, Alexandra Komáromi, and Klára Tóth is appreciated.
This work is a part of the 10th accredited Ph.D. program at the Semmelweis University of Medicine, Hungary, and was supported by the Hungarian National Scientific Research Fund, grant no. OTKA T021251.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Microbiology, Semmelweis University of Medicine, P.O. Box 370, H-1445 Budapest, Hungary. Phone and fax: 36-1210-29-59. E-mail: szabdor{at}net.sote.hu.
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Journal of Clinical Microbiology, December 1999, p. 4167-4169, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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