A New Fimbrial Antigen Harbored by CAZ-5/SHV-4-Producing Klebsiella pneumoniae Strains Involved in Nosocomial Infections

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1 INFECTION AND IMMUNITY, June 1996, p Vol. 64, No /96/$ Copyright 1996, American Society for Microbiology A New Fimbrial Antigen Harbored by CAZ-5/SHV-4-Producing Klebsiella pneumoniae Strains Involved in Nosocomial Infections PATRICK DI MARTINO, VALÉRIE LIVRELLI, DANIELLE SIROT, BERNARD JOLY, AND ARLETTE DARFEUILLE-MICHAUD* Laboratoire de Bactériologie, Facultés de Pharmacie et Médecine, Clermont-Ferrand, France Received 27 December 1995/Returned for modification 5 February 1996/Accepted 2 March 1996 We purified and characterized a new fimbria termed KPF-28 (Klebsiella pneumoniae fimbria with a fimbrin molecular mass of 28 kda) involved in K. pneumoniae adherence to the human carcinoma cell line Caco-2. Electron microscopy of bacterial surface protein preparations and immunogold labeling of bacterial cells showed that KPF-28 was a long, thin, and flexible fimbria about 4 to 5 nm in diameter and 0.5 to 2 m long. The N-terminal amino acid sequence of the KPF-28 major fimbrial subunit showed no homology with type 1 and type 3 pili of K. pneumoniae but showed 61.7% identity with residues 6 to 19 of the N-terminal amino acid sequence of PapA, the Pap major pilus subunit expressed by uropathogenic Escherichia coli strains. Total amino acid content determination showed that the KPF-28 major subunit composition was close to that of the GVVPQ fimbrial family major subunits expressed by pathogenic E. coli strains. The study of the prevalence of KPF-28 among K. pneumoniae strains involved in nosocomial infections revealed that KPF-28 was found in the great majority of the K. pneumoniae strains producing the CAZ-5/SHV-4 extended-spectrum -lactamase. As shown by curing and mating experiments, the R plasmid encoding the CAZ-5/SHV-4 enzyme was found to be involved in but not solely responsible for KPF-28 expression. Hybridization experiments using an oligonucleotide probe corresponding to the N-terminal part of the 28-kDa protein revealed that the structural gene encoding the KPF-28 major subunit was localized on this R plasmid. KPF-28 is a putative colonization factor of the human gut, since the ceftazidine-sensitive derivative strain CF914-1C no longer adhered and since the Fab fragments of antibodies raised against KPF-28 inhibited adhesion of K. pneumoniae CF914-1 to the Caco-2 cell line. The incidence of nosocomial infections caused by gramnegative bacilli has increased markedly over the past three decades (13, 20). Klebsiella pneumoniae is involved in various human infections which occur after bacterial colonization of the gastrointestinal tracts of patients (6, 28). Bacterial adherence to epithelial cell surfaces is an important step in the colonization process. The adherence of K. pneumoniae strains to eucaryotic cells may be mediated by surface factors, such as type 1 and type 3 fimbriae and the nonfimbrial adhesin CF29K (5, 9). The type 1 fimbriae of K. pneumoniae are closely related to type 1 fimbriae expressed by other member species of the family Enterobacteriaceae (15). They confer a mannose-specific adhesion phenotype to ciliated hamster tracheal cells and to rat bladder epithelial cells in vitro (10, 11). The type 3 fimbriae of K. pneumoniae mediate bacterial attachment to the basolateral surfaces of tracheal epithelial cells and to components of basement membranes (17). However, a recent study indicated that the type 3 MrkD adhesin is frequently found in strains of Klebsiella oxytoca but is rarely associated with the type 3 fimbriae of K. pneumoniae (27). Genetic determinants of type 1 fimbriae are located only on the bacterial chromosome, but genes encoding type 3 adhesin and the major fimbrial subunit can be found on the chromosome and on a plasmid, which suggests there is gene transfer between Klebsiella species (18). The transfer of adhesive factor-encoding genes between Escherichia coli and K. pneumoniae strains in the human gut is probably the cause of the emergence of CF29K in K. pneumoniae. The R-plasmid-encoded CF29K adhesin, identical to * Corresponding author. Mailing address: Laboratoire de Bactériologie, Faculté de Pharmacie, 28, Place Henri Dunant, Clermont-Ferrand, France. Phone: (33) Fax: (33) the CS31A-L protein harbored by E. coli strains, promotes the ability of E. coli and K. pneumoniae to adhere to human intestinal cell lines Caco-2 and Intestine-407 (5, 8). Many K. pneumoniae strains adhere to intestinal cell lines in vitro but do not express CF29K, which therefore indicates the expression of nonidentified adhesive factors (5). A phenotype characterized by aggregative adhesion to the embryogenic Intestine-407 cell line in which a capsule-like extracellular material seems to be involved has been described and is under study (12). It is characterized by the formation of bacterial aggregates on the eucaryotic cell surface. The aggregative adhesion phenotype is widely found among K. pneumoniae strains isolated in hospitals in Clermont-Ferrand, France. Recently, a third phenotype, termed localized adhesion, has been identified in clinical isolates (25). In an effort to identify other fimbriae or factors that may contribute to the ability of the K. pneumoniae strains involved in nosocomial infections to colonize the host, we studied the bacterial surface proteins expressed by clinical isolates which adhered to the intestinal cell line Caco-2. MATERIALS AND METHODS Bacterial strains. K. pneumoniae CF914-1 was isolated from urine samples from a patient hospitalized in an intensive care unit in a hospital of Clermont- Ferrand, France. The strain was resistant to -lactams, including ceftazidime (CAZ), by production of a CAZ-5/SHV-4 -lactamase and was also resistant to kanamycin, neomycin, tobramycin, gentamicin, netilmicin, chloramphenicol, tetracycline, trimethoprim, and sulfonamides. Seventy-eight K. pneumoniae, one E. coli, and five Enterobacter aerogenes clinical isolates, all involved in nosocomial infections and belonging to a bacterial strain collection of Jacques Sirot (teaching hospital, Clermont-Ferrand, France), were studied. Of the K. pneumoniae strains, 5 produced only the SHV-1 chromosomal -lactamase, while the other 73 produced, in addition to SHV-1, a plasmid-encoded -lactamase: 14 produced CTX-1/TEM-3, 4 produced CAZ-1/TEM-5, 10 produced CAZ-2/TEM-8, 36 produced CAZ-5/SHV-4, 6 produced CAZ-6/TEM-24, and 3 produced CAZ-7/ 2266

2 VOL. 64, 1996 IDENTIFICATION OF KPF-28, A NEW K. PNEUMONIAE FIMBRIA 2267 TEM-16. The E. coli and E. aerogenes strains produced the CAZ-5/SHV-4 enzyme. Biotypes were determined with the API 20E system (API System, Montalieu- Vercieu, France). Depending on the experiments, the strains were grown in Mueller-Hinton broth or Mueller-Hinton agar (Institut Pasteur Production, Marnes-la-Coquette, France) at 37 C for 18 to 24 h. Extraction and purification of surface proteins. Bacterial surface proteins were extracted essentially as described by Stirm et al. (30). Cultures grown overnight on 10 plates (15 by 15 cm) with Mueller-Hinton agar were harvested in 0.1 M phosphate-buffered saline (PBS; ph 7.2). The bacterial surface proteins were separated from bacterial cells by heating the suspension at 60 C for 20 min with gentle agitation. Cells and bacterial debris were sedimented at 10,000 g for 10 min. The supernatant was brought to ph 4.0 and stored overnight at 4 C. The precipitated proteins were collected by centrifugation at 20,000 g for 30 min and suspended in 0.1 M PBS (ph 7.2). This constituted the crude extract of bacterial surface proteins. The extracts of the bacterial surface proteins were fractionated through a 10 to 50% (wt/vol) sucrose gradient prepared in PBS (ph 7.2) by centrifugation for 20 h at 22,000 rpm in an SW41.1 rotor (Beckman Instruments) at 4 C. Fractions (0.5 ml) were collected from the bottom of the gradients. Each fraction was analyzed by spectrophotometry at 280 nm, dialyzed against PBS overnight at 4 C, and fractionated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Electrophoretic study. Bacterial surface proteins were analyzed by SDS-PAGE as described by Laemmli (21), with 12% (wt/vol) acrylamide 0.8% (wt/vol) bisacrylamide for the separation gel. Electrophoresis was performed in a solution of M Tris, 0.28 M glycine, and 0.1 (wt/vol) SDS (ph 8.6). Samples were denatured in 1.5% (wt/vol) SDS 1.5% (vol/vol) -mercaptoethanol in 0.50 M Tris (ph 6.8) for 5 min at 100 C just before being loaded onto the gel. The molecular weight standards (Pharmacia LKB, Uppsala, Sweden) were phosphorylase b (94,000), albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100), and -lactalbumin (14,000). Gels were stained with Coomassie blue (Sigma Chemical Co., St. Louis, Mo.). Preparation of antiserum. Rabbit antiserum against the 28-kDa surface protein was prepared as follows. Protein was purified after sucrose gradient centrifugation of bacterial surface proteins of strain CF914-1 by preparative SDS- PAGE (3-mm-thick 12% acrylamide gel). The 28-kDa band was cut from an unstained gel corresponding to a Coomassie blue-stained part of the gel. Gel slices were homogenized by being passed through a needle with complete Freund adjuvant (Sigma) and injected subcutaneously into an adult female rabbit weighing 2 kg. The animal was injected five times at 6-day intervals with 200 mg of protein. It was bled on day 8 after the last injection. The antiserum was adsorbed with E. coli K-12 C600 and K. pneumoniae CF104 producing CTX-1/TEM-3 -lactamase that did not express KPF-28. Immunoblotting. Western blotting (immunoblotting) was performed by the technique of Towbin et al. (31), with minor modifications. Bacterial surface proteins were first subjected in duplicate to electrophoresis in the presence of SDS. One part of the gel was stained with Coomassie blue, and the other was electroblotted onto nitrocellulose paper (Schleicher & Schuell, Inc., Dassel, Germany). Adsorbed antiserum raised against the 28-kDa surface protein was applied to the nitrocellulose filter at a dilution of 1:300, and the filter was incubated for 1 h at room temperature. After a thorough washing, the filter was incubated for 1 h at room temperature with peroxidase-labeled goat anti-rabbit immunoglobulin G (Nordic Immunological Laboratories, Tilburg, The Netherlands) at a dilution of 1:2,000. The blot was washed and soaked in a solution composed of 0.6 mg of 4-chloro-1-naphthol (Sigma) per ml, 0.1% H 2 O 2, and 16.5% methanol in 10 mm Tris 0.9% NaCl (ph 7.5). Colony immunoblotting was performed as follows. Bacterial strains were grown in Mueller-Hinton broth (Institut Pasteur Production) overnight at 37 C. Samples (5 l) of each broth were plated onto nitrocellulose paper placed on Mueller-Hinton agar and incubated for7hat37 C. The membrane was dried for 2hat80 C and blocked overnight at room temperature with Tris-buffered saline (10 mm Tris, 0.9% NaCl [ph 7.5]) and 2% (wt/vol) bovine serum albumin (Sigma). Immunoblotting was then performed as described above. Electron microscopy. K. pneumoniae cells were grown at 37 C on Mueller- Hinton agar, harvested in PBS (ph 7.2), and observed with a transmission electron microscope (HU 12A; Hitachi) after staining with 1% phosphotungstic acid (ph 6.8). Gold immunolabeling was performed by the method of Levine et al. (23). A washed bacterial suspension or a fimbrial preparation was placed on carboncoated grids. Excess liquid was removed, and the grid was placed face down on a suitable dilution of adsorbed antiserum raised against the 28-kDa protein for 15 min. After 10 washings, the grid was placed on a drop of gold-labeled goat anti-rabbit serum (Jansen Life Sciences Products, Olen, Belgium) for 15 min. After a further thorough washing, the grids were negatively stained with 1% ammonium molybdate. To prevent nonspecific labeling, 1% bovine serum albumin and 1% Tween 20 were added to the wash solutions. Amino acid composition analysis. The purified 28-kDa surface protein was analyzed by SDS-PAGE as described above, transferred to Immobilon P (Schleicher & Schuell), and hydrolyzed in 6 N HCl at 110 C for 20 h. The hydrolysates were analyzed on a Beckman model amino acid analyzer (Beckman Instruments, Inc., Fullerton, Calif.). Amino acids were separated by ion-exchange chromatography (sulfonated divinylbenzene-cross-linked polystyrene beads) as described by Slocum and Cummings (29). Ninhydrin was mixed continuously with the column eluent to form colored products with amines. The absorbance of the amino acid-ninhydrin complex was measured at two wavelengths, 570 nm for primary amino groups and 440 nm for secondary amino groups. A two-channel recorder monitoring the colorimeter provided a record of the amino acid profiles. Amino acids were identified and concentrations were calculated by a data system that determined the retention time and area under each sample peak. The percentage of each residue was determined as follows: P, A, V, M, I, L, Y, and F were considered hydrophobic residues, and G, S, T, C, and Y were considered polar uncharged residues. Determination of the NH 2 -terminal amino acid sequence. The primary structures of the NH 2 -terminal parts of the 22- and 28-kDa proteins were determined by automatic Edman degradation on a model 470A amino acid sequencer (Applied Biosystems, Foster City, Calif.). Phenylthiohydantoin amino acid derivatives were identified by high-performance liquid chromatography as described by Zimmerman et al. (32). Transfer of R plasmids and curing. Conjugation experiments were carried out as previously described (22). The R plasmids were transferred to mutants of E. coli K-12 C600 resistant to rifampin. Transconjugants were selected on Mueller- Hinton agar containing rifampin (300 mg/liter) and CAZ (6 mg/liter). Curing of R plasmids was performed by successive bacterial subcultures at 44 C. One hundred fifty milliliters of Mueller-Hinton broth was inoculated with approximately 1,000 cells of K. pneumoniae CF Successive subcultures were incubated overnight at 44 C without agitation and then plated for growth of well-separated colonies, which were subsequently tested for loss of plasmiddetermined characteristics. The CAZ-5/SHV-4-encoding R plasmid was conjugated from E. coli transconjugants back into the cured K. pneumoniae CF914-1C. The selection was done on Mueller-Hinton agar containing nalidixic acid (150 mg/liter) and CAZ (6 mg/ liter). Adhesion to the human Caco-2 cell line in vitro. Adherence of K. pneumoniae strains and E. coli transconjugants to the intestinal cell line Caco-2 was achieved as previously described (4). Briefly, monolayers of differentiated Caco-2 cells were prepared in 24-well Falcon tissue culture plates (Becton Dickinson Labware, Oxnard, Calif.). The cells were seeded at 10 4 /cm 2 in Dulbecco modified Eagle medium (Flow Laboratories, McLean, Va.) containing 20% (vol/vol) fetal bovine serum (Seromed, Berlin, Germany), 1% L-glutamine (Boehringer Mannheim, Meylan, France), 1% nonessential amino acids (Boehringer), 10,000 U of penicillin per ml, 10 mg of streptomycin per ml, and 25 mg of amphotericin B (American Type Culture Collection) per ml in an atmosphere of 10% CO 2 at 37 C. Monolayers of Caco-2 cells were used at postconfluence after 15 days of culture. Before the adhesion test, cells were washed once with PBS (ph 7.2). A suspension of 10 8 bacteria per ml in the cell line culture medium containing 2% (wt/vol) D-mannose was added to the tissue culture and incubated for3hat37 C. After three washes with PBS, the cells were fixed in methanol, stained with 20% Giemsa solution, and examined microscopically under oil immersion. Adhesion indices representing the mean number of bacteria per cell were calculated. Statistical analysis. The results were expressed as means standard deviations. Each adhesion index represents the results of four separate experiments. Statistical significance was determined by analysis of variance, using the F distribution of Fisher. Inhibition of adhesion to the Caco-2 cell line was performed with the anti- KPF-28 serum. Papain treatment of this antiserum was done as described by Harlow and Lane (16). Suitable dilutions of antiserum were added to 10 8 bacteria in cell line culture medium containing 2% (wt/vol) D-mannose, and the mixture was allowed to incubate at room temperature for 20 min. It was mixed with the Caco-2 cell culture, and the adhesion test was performed as described above. Adhesion inhibition was also performed with preimmune serum as a control. DNA manipulation and analysis. Small-scale preparation of plasmid DNA from E. coli transconjugants was done as previously described (5). Chromosomal DNA was extracted from K. pneumoniae CF914-1 as described by Hull et al. (19). Agarose gel electrophoresis and isolation of restriction enzyme-generated DNA fragments were performed as described by Maniatis et al. (26). Southern hybridization with an N-terminal oligonucleotide probe. The NH 2 - terminal amino acid sequence of the 28-kDa major surface protein was studied for a stretch of amino acids in which the codon degeneracy was very small. On the basis of the codon usage in the nucleotide sequence of the structural gene for type 3 pili, type 1 pili, and CF29K major subunits (8, 14, 15), the amino acid sequence TLRGTIV was chosen, and the 20-mer oligonucleotide sequence 5 - AC(CT)-CTG-CG(CT)-GG(CT)-AC(CT)-AT(CT)-GT-3 was synthesized. The synthetic oligonucleotide was 5 end labeled with [ - 32 P]ATP by using T4 polynucleotide kinase (Boehringer Mannheim). Southern blot hybridization was done at 45 C as described by Maniatis et al. (26). RESULTS Isolation and purification of major surface proteins. Most bacterial adhesive factors are assembled into polymeric surface

3 2268 DI MARTINO ET AL. INFECT. IMMUN. FIG. 1. SDS-PAGE and Western blot analyses of bacterial surface components (A) and purified fimbriae (B). Lanes 1 to 4, SDS-PAGE of surface proteins extracted from K. pneumoniae CF914-1, from the CAZ-sensitive derivative CF914-1C, and from CF914-1C after CAZ-5/SHV-4 R-plasmid reintroduction and of purified fimbriae from K. pneumoniae CF914-1, respectively. Lanes 1 to 4, corresponding Western blotting of the extracts in lanes 1 to 4, using rabbit antiserum raised against the 28-kDa purified major subunit at a dilution of 1:300. Sizes are indicated in kilodaltons. FIG. 2. Comparison of amino-terminal sequences of the 22- and 28-kDa surface proteins of K. pneumoniae CF914-1 with amino-terminal sequences of MrkA and PapA fimbrial subunits. Asterisks indicate amino acid positions in the mature subunits. structures which are predominantly composed of one major structural protein. We looked for such major surface proteins in K. pneumoniae strains involved in nosocomial infections. The bacterial surface components of the prototype CF914-1 strain were obtained by heating a bacterial suspension at 60 C for 20 min. Analysis by SDS-PAGE revealed the presence of a major protein with a subunit molecular mass of 28 kda (Fig. 1A, lane 1). The major surface proteins from the crude extract of strain CF914-1 were fractionated by sucrose gradient centrifugation. Fractions were collected and analyzed by spectrophotometry at 280 nm. The fraction corresponding to a major protein peak was analyzed by SDS-PAGE with Coomassie blue staining. This fraction showed two bands with molecular masses of 22 and 28 kda (Fig. 1B, lane 4). NH 2 -terminal amino acid sequences of the 22- and 28-kDa surface proteins. The NH 2 -terminal amino acid sequences of the surface proteins copurified by sucrose gradient centrifugation are shown in Fig. 2. Computer analysis using the FAST program and the National Biomedical Research Foundation Protein Bank revealed 100% identity between the N-terminal sequence of the 22-kDa protein and that of MrkA, the major subunit of type 3 fimbriae, among the first 19 amino acids (14). Consequently, the type 3 pili were copurified with the native form of the 28-kDa surface protein. The 28-kDa protein exhibited 61.7% identity in sequence between its residues 2 to 14 and residues 7 to 19 of PapA, a major pilus subunit expressed by E. coli strains involved in human urinary tract infections (1). Amino acid analysis of the 28-kDa protein. The amino acid composition of the 28-kDa protein determined after HCl hydrolysis revealed unusual features similar to those of the GVVPQ fimbrial subunits expressed by some pathogenic E. coli strains (3). Glycine residues were predominant, since they constituted 21% of the protein. The percentages of potentially acidic, hydrophobic, and polar uncharged residues were 22, 35, and 35%, respectively, for the 28-kDa protein, compared with 10, 40, and 42%, respectively, for the PapA major subunit expressed by uropathogenic E. coli strains (1) and 9, 42, and 48%, respectively, for the MrkA protein expressed by K. pneumoniae strains (14). Immunoblot analysis using the antiserum raised against the 28-kDa surface protein. Western immunoblot analysis showed that the serum raised against the SDS-denatured form of the 28-kDa protein extracted from K. pneumoniae CF914-1 reacted specifically with the 28-kDa protein present in the bacterial surface components of the crude extract (Fig. 1A, lane 1 ). Analysis of the sucrose gradient centrifugation-fractionated fractions indicated that among the 22- and 28-kDa copurified proteins, this antiserum recognized only the 28-kDa surface protein (Fig. 1B, lane 4 ). Thus, the two proteins were antigenically unrelated. Electron microscopic examination of bacterial cells and surface protein extract of K. pneumoniae CF Electron micrographs of a negatively stained preparation of K. pneumoniae CF914-1 showed numerous filamentous structures on the cell surface (Fig. 3A). Long, thin, and flexible fimbriae approximately 4 to 5 nm in diameter and 0.5 to 2 m long were observed. Immunogold labeling assays using the antiserum raised against the 28-kDa surface protein showed that the gold particles were located along the long and flexible fimbrial structures on the surface of K. pneumoniae CF914-1 cells (Fig. 3B) and along the long and aggregated fimbriae present in the crude bacterial surface protein extracts from the same strain (Fig. 4A and C). This fimbria composed of 28-kDa major subunits was termed KPF-28 (K. pneumoniae fimbria with a fimbrin molecular mass of 28 kda). Prevalence of KPF-28 among K. pneumoniae clinical isolates. The 78 K. pneumoniae strains involved in nosocomial infections included in this study were screened for KPF-28 expression by colony immunoblotting with the antiserum raised against the 28-kDa fimbrin. Of the 78 strains tested, 30 reacted with the antiserum in colony immunoblotting. Western blotting experiments were done with surface proteins extracted from strains positive in colony immunoblotting. All of them expressed a 28-kDa surface protein that reacted with the specific anti-28-kda protein serum in Western immunoblotting experiments after SDS-PAGE fractionation of crude bacterial surface component extracts (data not shown). Interestingly, all of the KPF-28-producing strains expressed a CAZ-5/SHV-4 extended-spectrum -lactamase. Moreover, of the 36 CAZ-5/ SHV-4-producing K. pneumoniae strains tested, 83% (30 strains) harbored KPF-28. None of the E. coli and E. aerogenes strains producing the CAZ-5/SHV-4 enzyme reacted with the antiserum raised against KPF-28 in colony immunoblotting experiments. To determine if KPF-28 expression was related to a particular biotype, we performed biotyping with the API 20E system. On the basis of urease activity and saccharose fermentation, three different biotypes were observed: 14 of the strains tested were positive for both urease activity and saccharose fermentation, 15 were negative, and 7 had weak urease activity but

4 VOL. 64, 1996 IDENTIFICATION OF KPF-28, A NEW K. PNEUMONIAE FIMBRIA 2269 FIG. 3. (A) Transmission electron micrograph of negatively stained K. pneumoniae CF (B and C) Immunogold labeling of K. pneumoniae CF914-1 (B) and of the CAZ-sensitive derivative CF914-1C (C) with the rabbit antiserum raised against the 28-kDa purified major subunit. Bars, 0.5 m.

5 2270 DI MARTINO ET AL. INFECT. IMMUN. FIG. 4. Transmission electron micrographs of negatively stained preparations of crude bacterial surface components of K. pneumoniae CF914-1 (A) and CAZ-sensitive derivative CF914-1C (B). (C) Immunogold labeling of crude bacterial surface extracts of K. pneumoniae CF914-1 with rabbit antiserum raised against the 28-kDa purified major subunit. Bars, 0.2 m. were positive for saccharose fermentation. Thus, there was no correlation between KPF-28 expression and a specific biotype. Involvement of R plasmid in KPF-28 expression. Since all of the K. pneumoniae strains expressing KPF-28 produced the plasmid-encoded CAZ-5/SHV-4 -lactamase, we looked for a genetic link between production of the CAZ-5/SHV-4 enzyme and expression of KPF-28. This was done by plasmid mating and curing experiments with the prototype strain K. pneumoniae CF914-1 and by hybridization experiments using an N-terminal oligonucleotide probe. K. pneumoniae CF914-1 was resistant to -lactams, including CAZ, by production of the extended-spectrum -lactamase CAZ-5/SHV-4 and was also resistant to kanamycin, neomycin, tobramycin, gentamicin, netilmicin, chloramphenicol, tetracycline, trimethoprim, and sulfonamides. During conjugative transfer between K. pneumoniae CF914-1 and the recipient strain E. coli K-12 C600, selection for CAZ resistance revealed the transfer of plasmid DNA conferring resistance to CAZ, kanamycin, neomycin, tobramycin, gentamicin, netilmicin, chloramphenicol, trimethoprim, and sulfonamides. This E. coli transconjugant, termed CF914-1A, harbored a very large plasmid of 200 kb (data not shown). The bacterial surface proteins were extracted from E. coli CF914-1A as described in Materials and Methods. The surface protein extract did not react with the antiserum raised against the 28-kDa protein in Western immunoblotting experiments after SDS-PAGE fractionation (data not shown). Thus, the E. coli transconjugant CF914-1A did not express KPF-28, indicating that the R plasmid harbored by K. pneumoniae CF914-1 was not sufficient to promote KPF-28 expression in E. coli K-12 C600. A CAZ-sensitive derivative strain of the prototype strain K. pneumoniae CF914-1 designated CF914-1C was obtained by successive subcultures at 44 C. It was highly sensitive to CAZ, which suggests that the transferable R plasmid encoding the CAZ-5/SHV-4 -lactamase was lost. The bacterial surface proteins were extracted from the CAZ-sensitive derivative strain K. pneumoniae CF914-1C. The 28-kDa major surface protein was not observed upon SDS-PAGE fractionation of this bacterial extract (Fig. 1A, lane 2). Electron micrographs of a negatively stained preparation revealed that the long, aggregated fimbriae observed in the bacterial surface extract of the wild-type strain K. pneumoniae CF914-1 were not present in that of K. pneumoniae CF914-1C (Fig. 4B). In addition, Western immunoblotting and immunogold labeling experiments using the antiserum raised against the 28-kDa major surface protein confirmed that the CAZ-sensitive strain K. pneumoniae CF914-1C did not express KPF-28 (Fig. 1A, lane 2, and Fig. 3C). The CAZ-5/SHV-4-encoding R plasmid was reintroduced into K. pneumoniae CF914-1C in order to restore KPF-28 expression. SDS-PAGE and Western blotting analyses of the crude bacterial extract of the resulting strain showed expression of the KPF-28 major subunit (Fig. 1A, lanes 3 and 3 ). Thus, the presence of the R plasmid encoding CAZ-5/SHV-4 -lactamase in K. pneumoniae CF914-1 was correlated with the expression of KPF-28. Hybridization experiments were performed by using as a probe a 20-mer synthetic oligonucleotide based on the NH 2 - terminal sequence of the 28-kDa surface protein. No hybridization was observed with EcoRI-digested chromosomal DNA extracted from K. pneumoniae CF914-1 (Fig. 5). In contrast, hybridization experiments revealed that the structural gene encoding the KPF-28 major subunit was located on a 20-kb EcoRI restriction fragment of the CAZ-5/SHV-4-encoding R plasmid (Fig. 5). Bacterial adhesion to Caco-2 cells. The prototype strain K. pneumoniae CF914-1 adhered to the Caco-2 cell line (Fig. 6A), and the adhesion index was bacteria per cell. The adhesion observed was not mediated by type 1 pili, since the cell binding assay was performed in the presence of 2% D- mannose. The E. coli transconjugant CF914-1A harboring the

6 VOL. 64, 1996 IDENTIFICATION OF KPF-28, A NEW K. PNEUMONIAE FIMBRIA 2271 FIG. 5. Hybridization of an oligonucleotide probe derived from the NH 2 - terminal sequence of the KPF-28 major subunit with EcoRI restriction fragments of plasmid DNA extracted from the E. coli transconjugant CF914-1A (lanes 1) and chromosomal DNA extracted from K. pneumoniae CF914-1 (lanes 2). (A) Agarose gel electrophoresis. Lane L, molecular size standards (1-kb ladder [Gibco BRL SARL, Cergy Pontoise, France]). (B) Southern blot showing hybridization of the oligonucleotide probe with a 20-kb EcoRI restriction fragment of the CAZ-5/SHV-4-encoding R plasmid. R plasmid encoding CAZ-5/SHV-4 -lactamase did not adhere to the Caco-2 cell line; its adhesion index of bacterium per cell was significantly different from that of strain CF914-1 (P 0.001). However, the derivative strain K. pneumoniae CF914-1C lacking the R plasmid encoding CAZ-5/ SHV-4 -lactamase, which did not produce KPF-28, did not adhere to Caco-2 cells (Fig. 6B); its adhesion index of bacterium per cell was significantly different from that of strain CF914-1 (P 0.001). When the CAZ-5/SHV-4-encoding R plasmid was reintroduced into K. pneumoniae CF914-1C, the resulting strain expressing KPF-28 adhered to Caco-2 cells with the same adhesion pattern as K. pneumoniae CF914-1 (data not shown), and the adhesion index of bacteria per cell was not significantly different from that of strain CF914-1 (P 0.05). Thus, the CAZ-5/SHV-4-encoding R plasmid is involved in the adhesion of the wild-type strain K. pneumoniae CF914-1 to the Caco-2 cell line. Anti-KPF-28 serum was used to inhibit the adhesion of K. pneumoniae CF914-1 to the Caco-2 cell line. K. pneumoniae CF914-1 did not adhere to Caco-2 cells when bacteria were preincubated with specific polyclonal antibodies raised against the KPF-28 major subunit, and similar inhibition results were obtained when these anti-kpf-28 antibodies were treated with papain. The adhesion indices were and bacteria per cell, respectively. There was a highly significant difference (P 0.001) between the adhesion indices of K. pneumoniae CF914-1 with or without pretreatment of the cells with anti-kpf-28 antibodies. This inhibition appeared to be specific, since when the bacteria were treated with preimmune serum, the adhesion index was bacteria per cell, which is not significantly different (P 0.05) from that of untreated controls. Thus, KPF-28 is an adhesive structure involved in adherence of K. pneumoniae CF914-1 to the Caco-2 cell line. DISCUSSION K. pneumoniae is a widespread opportunistic pathogen implicated in numerous nosocomial outbreaks (13, 20). Gastrointestinal acquisition and carriage of klebsiellae by patients is an important intermediate step in the development of infections (6, 28). This colonization process may be the result of the ability of klebsiellae to adhere to intestinal epithelial cells in the human gut. To study Klebsiella adhesiveness, we used FIG. 6. Micrographs showing adhesion to tissue-cultured Caco-2 cells of K. pneumoniae CF914-1 (A) and CAZ-sensitive derivative CF914-1C (B). Caco-2 and Intestine-407 cell lines as human intestinal models in our laboratory. In postconfluent cultures, Caco-2 cell monolayers exhibit structural and functional differentiation patterns characteristic of mature enterocytes such as brush border microvillus expression and alkaline phosphatase, sucrase isomaltase, and aminopeptidase activities, whereas Intestine-407 cells are undifferentiated cells derived from human embryonic jejunum and ileum. Several adhesive phenotypes have been observed with these two intestinal cell models. A phenotype characterized by aggregative adhesion to Intestine-407 cells, widely expressed among K. pneumoniae clinical isolates, has been described (12, 25). A diffuse adhesion phenotype has been observed with Caco-2 and Intestine-407 cell lines (5, 8). A nonfimbrial surface protein termed CF29K has been found to be involved in this adhesion. It is identical to the CS31A-L antigen expressed by E. coli strains associated with diarrhea and septicemia in bovines and humans (8). A study of the ability of K. pneumoniae clinical isolates to adhere to the Caco-2 cell line showed that numerous strains which did not express CF29K adhered to the intestinal cells in vitro (5). We reported in this study the finding of a previously unidentified fimbria, termed KPF-28, involved in K. pneumoniae CF914-1 adherence to the Caco-2 cell line. KPF-28 is a long, thin, and flexible fimbria with a diameter of about 4 to 5 nm and a length

7 2272 DI MARTINO ET AL. INFECT. IMMUN. of 0.5 to 2 m. It is formed by the polymerization of a 28-kDa major subunit. The native surface protein extract of K. pneumoniae CF914-1 contained numerous fimbrial structures that aggregated with one another. In an immunogold labeling assay using an antiserum raised against the 28-kDa protein, gold particles were fixed along fimbrial structures exposed at the bacterial surface of strain CF The total amino acid composition of the KPF-28 major subunit revealed unusual features comparable to those of major subunits of the GVVPQ fimbrial family, including relatively low percentages of hydrophobic and polar uncharged residues and large amounts of glycine and potentially acidic residues (3). However, the extreme insolubility of the GVVPQ fimbriae and the difficulty in separating them from the bacterial cells were not encountered with KPF-28. Moreover, the conserved GVVPQ N-terminal amino acid sequence characteristic of this fimbrial family was not found in the KPF-28 major subunit N-terminal amino acid sequence. The N-terminal sequence of the KPF-28 subunit was unrelated to the previously described surface proteins found in the fibrillar or nonfibrillar adhesive structures of K. pneumoniae (5, 14, 15). In contrast, it showed significant homology with protein PapA, the major subunit of Pap pili expressed by uropathogenic strains of E. coli (1). This homology was observed between residues 2 and 14 of the KPF-28 subunit and residues 7 and 19 of PapA. The KPF-28 major subunit migrated with an apparent molecular mass of 28 kda in SDS- PAGE, and PapA has a molecular mass of 16.5 kda (1). The significance of the homology between KPF-28 and Pap is unknown. Many amino acids in the N- and C-terminal sequences of the E. coli pilins are often conserved, probably because of the involvement of these sequences in subunit-subunit interactions (24). KPF-28 was extracted from K. pneumoniae CF914-1, which adhered to the Caco-2 cell line. To determine the involvement of KPF-28 in this adhesion, we performed adhesion inhibition experiments with specific antibodies raised against the KPF-28 major subunit. A specific adhesion inhibition of K. pneumoniae CF914-1 was obtained by pretreating the bacteria with anti- KPF-28 antibodies or corresponding Fab fragments. Hence, KPF-28 is an adhesive structure that may be involved in bacterial colonization of the human gut. We studied the expression of KPF-28 in 78 K. pneumoniae strains involved in nosocomial infections by colony and Western blotting experiments using the antiserum raised against the 28-kDa surface protein. Of the 78 strains tested, 30 were found to express KPF-28. All of these strains produced the CAZ-5/ SHV-4 -lactamase, and KPF-28 was found in 83% of the 36 strains tested that produced this enzyme. In the last few years, a single epidemic K. pneumoniae strain producing CAZ-5/ SHV-4 -lactamase has disseminated in many hospitals in France (2). This strain was of a special biotype, characterized by weak urease activity and no fermentation of sucrose, features which were found only in 15 Clermont-Ferrand clinical isolates expressing the KPF-28 fimbria. Hence, the expression of KPF-28 seems not to be restricted to a single strain of a special biotype but is found in most of the K. pneumoniae strains producing the plasmid-encoded CAZ-5/SHV-4 enzyme isolated in the hospitals in Clermont-Ferrand. Such a linkage between the expression of a defined adhesive factor and the production of an extended-spectrum -lactamase has already been observed (5). We previously showed that the CF29K adhesive factor was expressed only by K. pneumoniae strains producing the plasmid-encoded CAZ-1/TEM-5 -lactamase. Moreover, CF29K genetic determinants and the CAZ-1/TEM- 5-encoding gene were located on the same EcoRI restriction fragment of a 185-kb R plasmid (5). To determine the role of the R plasmid encoding CAZ-5/ SHV-4 -lactamase in KPF-28 expression, we cured it from the prototype strain K. pneumoniae CF914-1 and transferred it to E. coli K-12 C600. In curing experiments, we obtained a K. pneumoniae CAZ-sensitive variant which did not harbor the R plasmid encoding the CAZ-5/SHV-4 enzyme and failed to express KPF-28. The introduction of this R plasmid back into the CAZ-sensitive variant restored KPF-28 expression. However, E. coli transconjugants obtained by the transfer of this R plasmid into the recipient strain E. coli K-12 C600 did not express KPF-28, which indicates that this R plasmid is not solely responsible for promoting KPF-28 expression. Hybridization experiments with an oligonucleotide probe based on the NH 2 - terminal sequence of the KPF-28 major subunit showed that the structural gene encoding this major subunit is located on the CAZ-5/SHV-4-encoding R plasmid. This linkage between antibiotic and colonization factor genes may contribute to the emergence and persistence of multiresistant K. pneumoniae strains in vivo in the human gut, even in the absence of antibiotherapy selection pressure. This possibility is consistent with the high occurrence of CAZ-5/SHV-4-producing K. pneumoniae strains in hospitals in Clermont-Ferrand (7). The CAZ- 5/SHV-4 enzyme is produced primarily by K. pneumoniae isolates and rarely by E. coli and E. aerogenes strains. Moreover, the CAZ-5/SHV-4-producing E. coli and E. aerogenes strains isolated in the Clermont-Ferrand hospitals failed to express KPF-28. We showed that the CAZ-5/SHV-4-encoding R plasmid was not able to promote KPF-28 expression alone. Thus, the dissemination of this R plasmid alone does not lead to the expression of KPF-28 and to the colonization of the human gut by bacterial strains belonging to species different from K. pneumoniae. In this study, we characterized a new fimbria termed KPF-28 which may be an important virulence determinant involved in the colonization of the human gut by multiresistant K. pneumoniae strains. We determined that the structural gene encoding the KPF-28 major subunit was located on the R plasmid encoding the CAZ-5/SHV-4 -lactamase. Cloning experiments are in progress to localize and identify all of the genes involved in KPF-28 expression. Additional studies of the CAZ-5/SHV- 4-encoding R plasmids may shed further light on the acquisition of KPF-28-encoding genes and on the evolution of these plasmids. ACKNOWLEDGMENTS This work was supported by the Institut National de la Santé etde la Recherche Médicale through grant CRE We thank the Microscopy Department of Michel Bourges for technical assistance with electron microscopy analysis. REFERENCES 1. Baga, M., S. Normark, J. Hardy, P. O Hanley, D. Lark, O. Olsson, G. Schoolnik, and S. Falkow Nucleotide sequence of the papa gene encoding the Pap pilus subunit of human uropathogenic Escherichia coli. J. Bacteriol. 157: Buré, A., P. Legrand, G. Arlet, V. Jarlier, G. Paul, and A. Philippon Dissemination in five French hospitals of Klebsiella pneumoniae serotype K25 harbouring a new transferable enzymatic resistance to third generation cephalosporins and aztreonam. Eur. J. Clin. Microbiol. Infect. Dis. 7: Collinson, S. K., L. Emödy, T. J. Trust, and W. W. 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