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Antimicrobial Agents and Chemotherapy, April 1999, p. 937-939, Vol. 43, No. 4
Received 10 August 1998/Returned for modification 23 November
1998/Accepted 22 January 1999
We have demonstrated by using an in vitro approach that
interruption of the OmpK36 porin gene by insertion sequences
(ISs) is a common type of mutation that causes loss of porin expression and increased resistance to cefoxitin in Klebsiella
pneumoniae. This mechanism also operates in vivo: of 13 porin-deficient cefoxitin-resistant clinical isolates of
K. pneumoniae, 4 presented ISs in their ompK36 gene.
Klebsiella pneumoniae is
a major nosocomial pathogen, and successful chemotherapy is critical
for the treatment of infections caused by this microorganism.
Antibiotic-resistant strains emerge with variable frequency,
particularly in the hospital environment. K. pneumoniae
resistance to expanded-spectrum cephalosporins caused by plasmid-borne
extended-spectrum We have described porins OmpK36 and OmpK35 (2, 7), the
K. pneumoniae homologues of Escherichia coli
porins OmpC and OmpF, respectively. Clinical isolates of this species
express either OmpK36 or OmpK35 porins or both (6).
Recently, we reported that loss of porins OmpK36 and OmpK35 in
extended-spectrum These analyses revealed that different events occurred in the
ompK36 gene contained in pSHA2: point mutations or small
deletions, deletions of the entire gene, and insertions. These mutant
plasmids derived from pSHA2 were designated pSHA followed by an
arbitrary number (Table 1). As shown in
Table 1, the most frequent types of changes detected in the mutant
plasmids were insertions. Among 14 randomly selected mutants, 9 (64.3%) presented an insertion. Detailed characterization of the
nature and position of the inserted DNA was carried out by sequencing
the mutant plasmids. DNA sequencing was performed with primers U681
(5'-CGGTTACGGCCAGTGGGAATA-3') and L1316
(5'-GACGCAGACCGAAATCGAACT-3'), which represent DNA sequences 230 and 345 bp downstream of the ompK36 start codon, and
primers U228 and L1730. We detected four different insertion sequences (ISs; IS26, IS5, IS903, and
IS1) located in different sites of the ompK36
gene. Furthermore, with probes specific for the identified ISs
(obtained by elution of an IS internal restriction fragment from the
corresponding mutant plasmid), we verified by Southern blot analysis
that the ISs were present in many copies in the chromosome of the
original strain LB4 (data not shown).
Since resistance to cefoxitin in laboratory-derived mutants was
dependent on mutations of the OmpK36 porin gene, predominantly insertion of ISs, we investigated whether this type of mechanism also
occurred in vivo. For this purpose, we amplified the entire ompK36 gene from two pairs of strains isolated from two
patients before and after antimicrobial therapy: strains LB1 and LB4
(9) and strains CMD1 and CMD2 (19). LB4 and CMD2
are porin-deficient cefoxitin-resistant clinical isolates derived from
the porin-sufficient, cefoxitin-sensitive strains LB1 and CMD1,
respectively. Additionally, we characterized the OmpK36 porin gene in
11 additional porin-deficient K. pneumoniae clinical
isolates: strains HUSR2/94, C1, LB66, LB68, LB73, CSUB2, CSUB8R,
CSUB9R, CSUB10R, CSUB11, and CSUB12.
Analysis of the amplicons with the ompK36 gene probe
detected insertions, seen as increases in the size of
ompK36, in some of these clinical isolates (Fig.
1A). In addition, we observed in strain
HUSR2/94 a deletion on the OmpK36 porin gene (Fig. 1A, lane 2). Further
characterization of the above-described insertions was obtained by
sequencing the PCR amplicons. We identified the presence of
IS102 in strain CMD2, and we identified insertions with a
high degree of sequence identity to IS5 in the amplicons from strains LB73, CSUB2, and CSUB11. These ISs were located in different positions within the ompK36 coding region (Fig.
1B). These results demonstrate that porin loss due to insertions of ISs
within the porin genes is a mechanism that operates in vivo in patients
undergoing antibiotic treatment.
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Copyright © 1999, American Society for Microbiology. All rights reserved.
Development of Resistance during Antimicrobial Therapy Caused
by Insertion Sequence Interruption of Porin Genes
rea de
Microbiología,
ABSTRACT
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-lactamases is a well-documented example and has
increased since the 1980s (11, 14, 17). Additionally,
effects in porin expression contribute to increase the resistance
levels provided by the mechanism described above (12).
-lactamase-producing clinical isolates of
K. pneumoniae caused increased resistance to
cefoxitin and expanded-spectrum cephalosporins (3, 9). The
relationship between porin loss and antimicrobial resistance has
also been demonstrated in other bacterial species (1, 10). However, the mechanisms leading to porin deficiency and subsequent antimicrobial resistance are largely unknown. The purpose of the present study was to identify the mechanisms causing porin deficiency in clinical isolates of K. pneumoniae. For this
purpose, we first used an in vitro approach. We selected
cefoxitin-resistant mutants derived from strain LB4(pSHA2)
(9). Strain LB4 is a porin-deficient, cefoxitin-resistant
(MIC, 128 µg/ml) clinical isolate that reverted to cefoxitin
sensitivity (MIC, 4 µg/ml) after the plasmid pSHA2 carrying the
OmpK36 porin gene was cloned. Cefoxitin-resistant mutants were obtained
by plating LB4(pSHA2) on increasing concentrations of cefoxitin.
Mutants with increased resistance to cefoxitin (MIC, >48
µg/ml), determined according to the National Committee
for Clinical Laboratory Standards recommendations, were
selected and further characterized. The outer membrane proteins
(OMPs) from these mutants were isolated from bacterial envelopes as
sodium lauryl sarcosinate-insoluble material (5) and
analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
We found that expression of porin OmpK36 was completely abolished in
those mutants. In order to study possible mutations affecting the
ompK36 gene, we isolated the pSHA2 plasmid from the
cefoxitin-resistant mutants. Plasmids were analyzed by Southern
blotting (4) with an ompK36 probe obtained by PCR
amplification of plasmid pSHA2 with primers U228
(5'-GGTAAAAAAAACCGGATGCG-3') and L1730
(5'-CGTGCTTAGAACTGGTAAACC-3'), which anneal to sequences 427 bp upstream and 1,097 bp downstream of the ompK36 start
codon (accession no. Z33506), respectively.
TABLE 1.
Analysis of randomly selected mutant plasmids conferring
increased resistance to cefoxitin (MIC, >48 µg/ml) derived from
strain LB4(pSHA2)
View larger version (42K):
[in a new window]
FIG. 1.
(A) PCR and Southern blot analysis of the OmpK36 porin
gene from porin-deficient clinical isolates. The entire
ompK36 gene was amplified by using PCR and the U228-L1730
primer pair. Amplification products were analyzed with the
ompK36 gene probe. Lanes: 1, strain CMD1; 2, strain
HUSR2/94; 3, strain CMD2; 4, strain LB73; 5, strain CSUB2; 6, strain
CSUB11. (B) Scheme of the ompK36 gene, its upstream
micF gene, and the positions of the ISs. ISs are represented
by inverted triangles and the numbers above them correspond to the
lanes in panel A. The positions (in base pairs) of genes and ISs with
respect to the ompK36 start codon are indicated on the
bottom line.
It has been suggested that a low level of random transposition might help cells to adapt to environmental changes and to increase the survival rate (18), and it is also well known that ISs can interrupt or alter gene expression. There are many examples in the literature where ISs have been involved in resistance to certain antibiotics by creating novel promoters for antimicrobial resistance genes (8, 15). However, to our knowledge, this is the first report where antimicrobial resistance is caused by interruption of porin genes by ISs. Mechanisms of porin loss and increased resistance to imipenem were also studied in Pseudomonas aeruginosa by Yoneyama and Nakae by using an in vitro approach (20): only deletions in the OprD2 porin gene were observed. However, we have shown that, both in vitro and in vivo, ISs cause porin deficiency in K. pneumoniae. ISs are widespread in all bacterial species studied, and, although their presence has not been correlated with antimicrobial resistance, they have also been found in E. coli porins (13, 16). Thus, interruption of porin genes by ISs is therefore not an exclusive phenomenon of K. pneumoniae. Further studies will be required to determine the importance of this mechanism in other bacterial species.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (CICYT). S.H.A. and S.A. were supported by a predoctoral fellowship and a postdoctoral contract from CICYT, respectively.
We thank J. A. M. van de Klundert (University Hospital Leiden), G. Jacoby (Lahey Hitchcock Clinic), and J. Liñares and C. Ardanuy (Hospital de Bellvitge) for clinical isolates, the Servicio de Secuenciación del Centro de Investigaciones Biológicas for DNA sequencing, and J. Casadesús (Universidad de Sevilla) for helpful discussions throughout the work.
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FOOTNOTES |
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* Corresponding author. Mailing address: UIB-Microbiología, Carretera de Valldemosa Km. 7.5, 07071-Palma de Mallorca, Spain. Phone: 34-971-173335. Fax: 34-971-173184. E-mail: dbasas3{at}clust.uib.es.
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Antimicrobial Agents and Chemotherapy, April 1999, p. 937-939, Vol. 43, No. 4
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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