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Department of Medical Microbiology and Virology, University of Kiel, Kiel, Germany,1 Central Public Health Laboratory, London, United Kingdom,2 Area de Microbiologia, Departmento de Biologia, Universidad de las Islas Baleares, Palma de Mallorca,3 Departmento de Microbiologia, Facultad de Biologia, Universidad de Barcelona, Barcelona, Spain,7 Laboratoire de Bactériologie, Facultés de Médecine-Pharmacie, Clermont-Ferrand, France,4 Department of Gastrointestinal Infections, Statens Serum Institut, Copenhagen, Denmark,5 Department of Human Microbiology, University of Tel Aviv, Tel Aviv, Israel6
Received 27 October 2003/ Returned for modification 28 January 2004/ Accepted 30 April 2004
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ABSTRACT |
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INTRODUCTION |
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Extensive use of antibiotics has contributed greatly to the emergence of multidrug-resistant strains that cause hospital infections. In Klebsiella species, various types of plasmid-encoded, extended-spectrum ß-lactamases (ESBLs), especially TEM and SHV enzymes, have been described worldwide (13, 14). Recent surveys (17, 34) have shown that approximately 20 years after the first outbreaks in Germany and France, the prevalence of ESBL-producing K. pneumoniae isolates from intensive-care units (ICUs) varies from 9% in southern Germany to 40% in France, 49% in Portugal, and 59% in Turkey. Of particular interest are reports on the association between ESBL production and the expression of pathogenicity factors in Klebsiella. Several studies have shown that ESBL-producing Klebsiella strains have an increased ability to adhere to human epithelial cells, probably due to plasmid-coded production of fimbrial or nonfimbrial adhesins (6, 8, 9, 18). It is anticipated that Klebsiella strains will become more virulent and resistant to antibiotics in the future, an eventuality that could contribute to the spread of certain Klebsiella clones by virtue of their greater resistance to antibiotics and better adherence to host tissues.
After the onset of inflammation, invading Klebsiella strains meet the cellular and humoral bactericidal components of the innate immune system. The host's first line of defense against invading microorganisms includes the bactericidal effect of serum, which is mediated primarily by complement proteins. Lipopolysaccharides (LPS) have been implicated as a major factor in the ability of bacteria to resist serum bactericidal activity by the host (4, 19, 24, 30). Nine different LPS serotypes (O antigens) in K. pneumoniae have been described, the O1 serotype being the most common O antigen found in clinical isolates (11). In the present study, we examined whether an association exists between ESBL production and the ability of K. pneumoniae to resist the bactericidal potency of human serum. We also evaluated the distribution of the O serotypes among the different strains.
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MATERIALS AND METHODS |
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The origin of each strain was noted, and only one strain per patient was included in this collection. The strains were identified biochemically with the API 20E system (API bioMérieux, Nürtingen, Germany) or by conventional methods as described previously (29).
ESBLs were detected according to NCCLS recommendations (20). All strains were phenotypically screened for ESBL production by the broth microdilution method. ESBL production in isolates for which cefpodoxime, cefotaxime, ceftriaxone and ceftazidime MICs were 
2 mg/liter (by initial screen test) was confirmed by testing for synergy between ceftazidime or cefotaxime and clavulanic acid by the broth microdilution method, by following NCCLS criteria (20) and by the double-disk synergy test with disks (BD Biosciences) containing ceftazidime or cefotaxime (concentration of each, 30 µg) and either ceftazidime and clavulanic acid or cefotaxime and clavulanic acid (concentration of ceftazidime or cefotaxime, 30 µg; concentration of clavulanic acid, 10 µg), as described by Jarlier et al. (15). Either a 3-fold concentration decrease in the MIC of ceftazidime or cefotaxime tested in combination with clavulanic acid, compared to the MIC recorded when ceftazidime or cefotaxime was tested alone, or a difference in zone diameter of 5 mm indicated phenotypic confirmation of ESBL production.
To determine the ESBL types, DNA from all strains was prepared for PCR as previously described (25). The blaSHV gene was amplified with primers designed to cover the more-conserved terminal point of the gene. The primers shv-F (CGC CTG TGT ATT ATC TCC CTG TTA GCC) and shv-B (TTG CCA GTG CTC GAT CAG CG) were derived from GenBank accession no. AF178850, which resulted in a PCR product of 842 bp. Likewise, for the blaTEM gene, primers were made to amplify most of the single-point mutation variations of blaTEM. Primers used were tem-F (GTA TCC GCT CAT GAG ACA ATA ACC CTG) and tem-B (CCA ATG CTT AAT CAG TGA GGC ACC), obtained from GenBank accession no. AF309824. This strain yielded an amplicon of 918 bp, which was separated on 1.5% agarose gels.
PCR conditions were as follows: initial denaturation at 95°C for 120 s, followed by denaturation at 95°C for 45 s, annealing at 62°C for 45 s, and elongation at 72°C for 60 s; except for the initial denaturation, all steps were repeated for a total of 30 cycles. Control strains for positive amplifications, K. pneumoniae KP15 (blaSHV2) and K. pneumoniae KP6T (blaTEM-4), were kindly obtained from Teresa Coque, Madrid, Spain (5). The PCR products were purified with a spin column (QIAGEN PCR purification kit), and sequencing on both strands was carried out as described previously (25) with 10 pmol of one of the primers in combination with the Big Dye terminator cycle sequencing kit (PE Biosystems, Foster City, Calif.) according to the manufacturer's instructions. Sequencing reaction mixtures were analyzed with an ABI 377 acrylamid gel DNA genetic analyzer (Applied Biosystems, Foster City, Calif.). Raw sequences were compared to the reversed counterpart of the DNA, and unresolved nucleotides in one sequencing direction were ignored if the complementary position was unambiguously determined. If the position was ambiguously determined in both sequencing directions, the sequencing reactions were repeated. The sequences were processed with a BioNumerics GeneBuilder (Applied Maths BVBA, Sint-Martens-Latem, Belgium), and the consensus sequence was compared to the Entrez Nucleotides database by the use of the National Center for Biotechnology Information BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/).
Expression of the different O serotypes by the strains was tested by enzyme-linked immunosorbent assay (ELISA) with O-type-specific rabbit antibodies as described by Hansen et al. (11). LPS from all strains whose O serotypes were not detectable by ELISA were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described previously (11). Strains with smooth LPS (S-LPS; non-O-typeable but with the O antigen expressed [NTO+]) were differentiated from rough strains (R-LPS; non-O-typeable but with the O antigen unexpressed [NTO–]) by the presence of high-molecular-weight LPS. K-serotype isolates were determined by the capsular swelling method with K-specific antisera (22) or by countercurrent immunoelectrophoresis (29).
All strains whose O serotypes were not detectable by ELISA were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gels were stained with silver. According to the resulting LPS ladder, strains were divided into groups of nontypeable strains with S-LPS (NTO+) or nontypeable strains with R-LPS (NTO–).
The clonality of the individual strains was analyzed with regard to their ribotype, K serotype, and O serotype by means of automated ribotyping (RiboPrinting) according to the manufacturer's instructions with the RiboPrinter and the restriction enzyme EcoRI as previously described by Bruce (3). Strains were regarded as clonal if they expressed an identical ribotype, K serotype, and O serotype. In order to ensure that prevalent outbreak clones could not skew the results and to ensure clonal diversity, only one isolate of each clone (strains with identical K and O serotypes and ribotypes) was included in sequential analysis. NTO+ antigens and untypeable K antigens were considered different from strains expressing typeable O and K antigens (12). Strains which expressed no O antigen (NTO–) were assigned to the clones of those strains with otherwise identical typing characteristics.
Serum bactericidal assay. The ability of the isolates to resist killing by serum was tested as described previously (22). Bacteria were grown in nutrient broth, harvested during the early logarithmic phase of growth, and adjusted to a concentration of 2 x 106 bacteria/ml of physiological saline. Twenty-five microliters of the bacterial suspension was mixed with 75 µl of pooled normal human serum in microtiter plates. Viable counts (VCs) of bacteria were determined for a period of 3 h. Responses were graded as follows. For grade 1, the VC after 1 and 2 h was <10% of the inoculum; after 3 h, it was <0.1%. For grade 2, the VC after 1 h was 10 to 100%; after 3 h, it was <10%. For grade 3, the VC after 1 h was >100% of the inoculum; after 2 and 3 h, it was <100%. For grade 4, the VC after 1 and 2 h was >100%; after 3 h, the VC was <100%. For grade 5, the VC after 1, 2, and 3 h was >100%; but the VC fell sometime during the 3-h period. For grade 6, the VC after 1, 2, and 3 h was >100% of the inoculum and rose throughout the 3-h period. Each strain was tested at least three times. A strain was considered serum resistant or serum sensitive if the grading was the same in all experiments.
Preparation of plasmid DNA and plasmid curing. To analyze the relation between harboring ESBL-coding plasmids and serum resistance in Klebsiella, six strains producing ESBL type TEM3, TEM 8, TEM16, SHV4, SHV5, or SHV12, all of which were shown to be resistant to serum (grade 5 or 6), were subjected to plasmid curing as described previously (9). The preparation of the plasmids was performed with a QIAGEN preparation kit for high-molecular-weight plasmids, according to the manufacturer's instructions. The loss of the plasmids was visualized by agarose gel electrophoresis as described previously (9). All plasmid-cured derivatives were screened for ESBL production and for their ability to resist killing by serum, as described above.
Statistical analysis. The significance of differences between groups of isolates was evaluated by Yates' correction of the chi-square test. For tables showing results where less than five positive isolates were found within a cell, Fisher's exact test was used. The odds ratio (OR) and 95% confidence intervals (CI) were determined, and values were taken as gauges of association.
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RESULTS |
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4% (Table 2). For seven strains, ESBLs were not determined, as the sequences of the PCR products of the blaTEM or blaSHV genes were not homologous to published sequences.
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The percentage of serum-resistant isolates (grades 5 to 6) was significantly higher among the ESBL-producing strains than among the non-ESBL-producing strains (P = 0.037; OR = 1.96; 95% CI = 1.08 to 3.53). All told, 30% (27/90 strains) of the ESBL-producing strains but only 17.9% (32/178 strains) of the non-ESBL-producing strains showed serum resistance (Table 3).
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Distribution of the O serotypes related to ESBL production and serum resistance. The O1 serotype had the highest incidence among both ESBL-producing (58.8%; 53/90 strains) and non-ESBL-producing (35.9%; 64/178 strains) Klebsiella isolates (P = 0.0006; OR = 2.5; 95% CI = 1.52 to 4.29) (Table 4). The O serotypes O2a (19.1%; 34/178 strains), O3 (18%; 32/178 strains), and O5 (10.7%; 19/178 strains) had significantly higher frequencies among the non-ESBL-producing isolates than among ESBL producers (for O2a, 6.7%; 6/90 strains; for O3, 6.7%; 6/90 strains; and for O5, 1.1%; 1/90 strains) (P < 0.02) (Table 4). Details of the frequencies of all other serotypes are listed in Table 4.
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As with the entire bacterial collection, the clonally revised collection was found to have a significantly higher frequency of serum-resistant strains among ESBL-producing strains (36%; 17/47 strains) than among non-ESBL-producing strains (16%; 27/166 strains) (P = 0.0056) (OR = 2.9; 95% CI = 1.41 to 6.01) (Table 3). Among serum-resistant strains, the O1 frequency in both ESBL-producing strains (65%; 11/17 strains) and non-ESBL-producing strains (74%; 20/27 strains) was significantly higher among serum-resistant strains than among ESBL-producing (16.7%; 5/30 strains) and non-ESBL-producing (25.4%; 35/138 strains) serum-sensitive strains (P < 0.0025) (Table 4 and 5). The data on the frequencies of all other serotypes are detailed in Tables 4 and 5.
Relationship among ESBL-coding plasmids and serum resistance. To better assess the contribution of the ESBL-coding plasmid in conferring serum resistance to bacteria, six serum-resistant strains that harbor the ESBL-coding plasmid were subjected to a plasmid-curing procedure. The plasmid-cured derivatives were retested for ESBL production and for their ability to resist the bactericidal activity of serum, as described above. The loss of the ESBL-coding plasmids in the strains was demonstrated by agarose gel electrophoresis (data not shown). To confirm the consecutive lack of ESBL production, MICs of cefpodoxime, ceftazidime, and cefotaxime for all plasmid-cured derivatives were determined, by following NCCLS guidelines. In all strains, the MICs of the tested cephalosporins were <2 mg/liter. The absence of the blaTEM and blaSHV ESBL genes was confirmed by PCR. All six plasmid-cured derivatives showed the same serum resistance grade (5 or 6), indicating that plasmid curing did not influence the ability of the strains to resist the bactericidal effect of serum (data not shown).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we investigated whether there is a relationship between ESBL production and the serum resistance of K. pneumoniae isolates. ESBL-producing Klebsiella strains proved to be significantly more resistant to the bactericidal effect of human serum than their non-ESBL-producing counterparts. Moreover, our results demonstrate that the O1 serotype, the most frequent O serotype among clinical K. pneumoniae isolates (11, 32), predominates among both ESBL-producing and non-ESBL-producing Klebsiella strains. Its frequency among ESBL-producing strains, however, was significantly higher (59%) than among non-ESBL producers (36%).
Because both serum resistance and expression of the O1 serotype are thought to be factors in the pathogenicity of Klebsiella (22), it can be hypothesized that the higher frequency of the O1 serotype among ESBL-producing strains and their superior resistance to the bactericidal activity of serum are indicators of the greater pathogenic potential of ESBL-producing strains than that of non-ESBL-producing strains.
The molecular basis of serum resistance is not entirely clear. O side chains of the LPS of pathogenic bacteria are thought to play a major role in conferring serum resistance. Gram-negative bacteria which express intact O side chains (S-LPS) are generally more serum resistant than those which do not express O side chains (R-LPS) (28). For K. pneumoniae, a number of investigations have shown that serum sensitivity is determined by its LPS molecule (1, 2, 4, 16, 19, 33). Cell-free LPS from serum-resistant K. pneumoniae O1 strains inhibit serum's bactericidal effect on serum-sensitive strains but does not render serum-resistant strains susceptible to serum (4).
Interestingly, our analysis of the distribution of O serotypes in serum-resistant isolates of both ESBL-producing and non-ESBL-producing strains showed they had equally high incidences of the O1 serotype. If it is assumed that S-LPS confer serum resistance on bacteria, further studies are needed to clarify why all O serotypes do not protect the bacteria against the bacteriolytic activity of complement. Further studies are also needed to show how the concept of the O1 serotype as a factor that mediates serum resistance harmonizes with the fact that the O1 serotype is expressed by 52% of ESBL-producing and by 27% of non-ESBL-producing serum-sensitive isolates.
One aspect of this study was to examine whether the high incidence of particular serotypes and serum-resistant isolates among ESBL-producing strains can be assigned to clonal groups. Among all isolates tested, we were able to define 18 clones, most from among ESBL-producing strains.
The higher incidence of the O1 serotype in ESBL-producing strains of the entire bacterial collection was apparently due to the three major clones 1, 18, and 26. A sequential analysis revealed that serum-resistant strains were significantly more frequent among ESBL-producing isolates than among non-ESBL-producing isolates, even after the exclusion of clonal strains. Among ESBL-producing isolates, the TEM producers showed a significantly higher frequency of serum resistance than the SHV producers, although no correlation between the expression of certain ESBL types and serum resistance could be detected.
The linkage between the O serotypes, especially O1, serum resistance, and the presence of certain ESBL-encoding plasmids is not clear at this stage.
Because the incidence of the O1 serotype among ESBL producers after the exclusion of clonal strains was not higher than that among non-ESBL producers, it is conceivable that the higher frequency of serum-resistant strains among ESBL producers was due to factors other than the O1 antigen. Moreover, it appears to be unlikely that the ESBL plasmids also code for specific factors that confer serum resistance, because curing the R plasmid did not impair the ability of the bacteria to resist killing by serum and because no association could be shown in the tested strains between serum resistance and the harboring of particular ESBL types.
It could be speculated that virulent "successful" Klebsiella strains possessing certain serotype genes have higher competence for acquiring R plasmids. Such an association between particular genetic background and plasmid compatibility has been shown. For instance, although ß-lactam antibiotics have been used extensively for the last 4 decades to treat infections caused by Streptococcus pyogenes, the bacteria fail to acquire the ß-lactamase-encoding plasmid, and all S. pyogenes isolates identified to date are de facto sensitive to ß-lactam antibiotics (7, 31). Alternatively, both biological selection by the innate immune system (e.g., complement) and the increased use of ß-lactam antibiotics are responsible for the emergence of strains harboring ESBL plasmids and exhibiting serum resistance.
The fact that different ESBL types are harbored in strains belonging to one clone supports the notion that the general plasmid compatibility phenomenon also applies for Klebsiella. However, in order to clarify whether or not certain Klebsiella serotypes are better fit for acquiring R plasmids, additional conjugation experiments which consider all Klebsiella serotypes are needed.
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FOOTNOTES |
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This study was submitted in memory of Vicente Javier Benedí, who died prior to submission of the manuscript.
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REFERENCES |
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