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Antimicrob Agents Chemother. Oct 2006; 50(10): 3396–3406.
PMCID: PMC1610099

High-Level Carbapenem Resistance in a Klebsiella pneumoniae Clinical Isolate Is Due to the Combination of blaACT-1 β-Lactamase Production, Porin OmpK35/36 Insertional Inactivation, and Down-Regulation of the Phosphate Transport Porin PhoE

Abstract

Clinical isolates of Klebsiella pneumoniae resistant to carbapenems and essentially all other antibiotics (multidrug resistant) are being isolated from some hospitals in New York City with increasing frequency. A highly related pair of K. pneumoniae strains isolated on the same day from one patient in a hospital in New York City were studied for antibiotic resistance. One (KP-2) was resistant to imipenem, meropenem, and sulopenem (MICs of 16 to 32 μg/ml) while the other (KP-1) was susceptible (MIC of 0.5 μg/ml); both contained the blaACT-1, blaSHV-1, and blaTEM-1 β-lactamases. blaACT-1 in both strains was encoded on a large ~150-kb plasmid. Both isolates contained an identical class 1 integron encoding resistance to aminoglycosides and chloramphenicol. They each had identical insertions in ompK35 and ompK36, resulting in disruption of these key porin genes. The carbapenem-resistant and -susceptible isolates were extensively studied for differences in the structural and regulatory genes for the operons acrRAB, marORAB, romA-ramA, soxRS, micF, micC, phoE, phoBR, rpoS, and hfq. No changes were detected between the isolates except for a significant down-regulation of ompK37, phoB, and phoE in KP-2 as deduced from reverse transcription-PCR analysis of mRNA and polyacrylamide gel electrophoresis separation of outer membrane proteins. Backcross analysis was conducted using the wild-type phoE gene cloned into the vector pGEM under regulation of its native promoter as well as the lacZ promoter following transformation into the resistant KP-2 isolate. The wild-type gene reversed carbapenem resistance only when under control of the heterologous lacZ promoter. In the background of ompK35-ompK36 gene disruption, the up-regulation of phoE in KP-1 apparently compensated for porin loss and conferred carbapenem susceptibility. Down-regulation of phoE in KP-2 may represent the normal state of this gene, or it may have been selected from KP-1 in vivo under antibiotic pressure, generating the carbapenem-resistant clone. This is the first study in the Enterobacteriaceae where expression of the phosphate-regulated PhoE porin has been associated with resistance to antimicrobials. Our results with this pair of Klebsiella clinical isolates highlight the complex and evolving nature of multiple drug resistance in this species.

While Klebsiella pneumoniae has long been a serious respiratory tract pathogen in hospitalized patients, it has recently surfaced as one of the most antibiotic-resistant organisms in localized outbreaks (7, 9, 10, 53). Isolates of this species have been reported to be resistant to almost all classes of antibiotics through progressive mutations in chromosomally encoded genes and acquisition of genes from mobile plasmids and integrons. Recent outbreaks of K. pneumoniae containing KPC carbapenemases have occurred in hospitals in New York City. A single isolate of Klebsiella oxytoca and 18 K. pneumoniae isolates from seven hospitals in New York City contained the KPC-2 carbapenemase as well as the inhibitor-resistant TEM-30 enzyme (7). Such strains may also possess porin deletions and are often resistant to virtually all antibiotics.

K. pneumoniae contains three known porins in the outer membrane: OmpK35, -36, and -37 (4, 21, 29). OmpK37 is a small porin related to OmpN of Escherichia coli and is not normally expressed, while porins OmpK35 and OmpK36 play an important role in the penetration of antibiotics into the cell. Their loss can confer resistance to cephalosporins and carbapenems, particularly in strains containing Ambler group A, B, C, or D β-lactamases (5, 8, 19, 30, 38, 40, 44). Loss of OmpK35 plays a major role in antibiotic resistance, as illustrated in the K. pneumoniae CSUB10R deletion mutant (ΔompK35 and ΔompK36) under complementation following transformation with a plasmid-encoded ompK35 gene (22). The transformant had ≥128-fold lower MICs to cephalosporins and meropenem and ≥8-fold lower MICs to imipenem, ciprofloxacin, and chloramphenicol than did the deletion mutant, illustrating the importance of a functional OmpK35 in antibiotic permeation (22). Since K. pneumoniae normally lacks a chromosomally encoded, class C β-lactamase, acquisition of a plasmid-encoded blaAmpC or another broad-spectrum β-lactamase can confer high-level carbapenem resistance in porin-deficient strains (8, 19, 30, 43, 49). In an example of this “synergistic” resistance, a K. pneumoniae clinical isolate encoding the blaSHV-2 along with reduced expression of OmpK36 was resistant to cefoxitin, ceftazidime-clavulanate, and piperacillin-tazobactam with MICs of >256 μg/ml (19). MICs for meropenem and imipenem were also elevated (8 to 16 μg/ml). Since carbapenems can be the drugs of “last choice” in multidrug-resistant strains of Klebsiella, it is extremely important to characterize mechanisms that can lead to clinically relevant levels of carbapenem resistance in this species.

As a result of surveillance efforts to identify carbapenem-resistant K. pneumoniae from hospitals in New York City, we obtained two strains (KP-1 and KP-2) that are highly related as measured by pulsed-field gel electrophoresis (PFGE), yet KP-1 was susceptible to carbapenems and KP-2 was resistant to imipenem, meropenem, and sulopenem (MICs of 16 to 32 μg/ml). While both strains contained the Ambler class C β-lactamase blaACT-1, the difference in resistance to carbapenems between these strains turned out to be due to loss of porins OmpK35 and OmpK36 as well as a complicated regulatory mechanism that controls expression of the phosphate-regulated porin PhoE. This is the first study that implicates regulation of PhoE in conferring antibiotic resistance, and our results illustrate the propensity for K. pneumoniae to become resistant to antibiotics.

(This work was presented in part at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., 2005.)

MATERIALS AND METHODS

Strains and antimicrobial susceptibility testing.

K. pneumoniae 13094-1 (KP-1) and 13094-2 (KP-2) were isolated from an abdominal wound culture on 17 February 1998 from the same patient in the intensive care unit of the Westchester Medical Center. The antibiotic history of this patient was not available. The carbapenem-susceptible K. pneumoniae ATCC 13883 (wild-type ompK35 and ompK36) was used as a control strain in several experiments (44). K. pneumoniae UAB1 (qnr+), Enterobacter cloacae SSW1 (NMC-A), and K. pneumoniae UMM3 (KPC-2) were gifts from George Jacoby, Thomas Fritsche, and Nancy Hanson, respectively. Acinetobacter baumannii isolates NCTC 13301 (OXA-23), NCTC 13302 (OXA-25), NCTC 13303 (OXA-26), NCTC 13304 (OXA-27), and NCTC 13305 (OXA-58) control strains were obtained from the Health Protection Agency, London, United Kingdom. Pseudomonas aeruginosa 296 (VIM-2), A. baumannii MAR (OXA-23), A. baumannii MAD (OXA-58), A. baumannii CLA (OXA-40), K. pneumoniae 11978 (OXA-48), Salmonella enterica serovar Typhimurium (CTX-M-2), and Enterobacter cloacae CTX-M-9 were kind gifts from Patrice Nordmann. All other control strains used in the PCR studies were genetically characterized strains in the Pfizer culture collection.

Antibiotic susceptibility testing was conducted in cation-supplemented Mueller-Hinton broth according to methods of the Clinical Laboratory Standards Institute (18). Imipenem, meropenem, and ciprofloxacin powders were obtained from their respective manufacturers. Sulopenem sodium was prepared at Pfizer, Groton, Conn. Kanamycin, amikacin, chloramphenicol, and piperacillin-tazobactam were purchased from Sigma (St. Louis, MO). Imipenem and sulopenem concentrations were determined from 18-h broth cultures using a bioassay with Bacillus subtilis ATCC 6633 (57).

Mutation frequency studies.

Mutation frequencies to carbapenem resistance were determined for strain KP-1 by plating at least 1 × 10 9 cells from an overnight culture onto the surface of agar plates containing imipenem, meropenem, sulopenem, or cefepime at a concentration 4× MIC for each drug. After overnight incubation at 37°C, the resulting colonies were counted and the mutation frequency was calculated by comparing the number of colonies counted on drug-free medium.

Pulse-field gel electrophoresis.

PFGE was performed according to the procedure described by Saito et al. (47).

Isolation of large plasmids.

Plasmid DNA was isolated from KP-1 and KP-2 using a QIAGEN large construct kit according to the manufacturer's instructions. Plasmids were separated on a 0.85% agarose gel prepared with 0.5% Tris-borate-EDTA and electrophoresed at 90 V for 15 h at 4°C.

Gene amplification experiments.

The primers used in amplification experiments to identify the ompK and operon genes present in both Klebsiella strains are listed in Table Table1.1. PCR products obtained from two independent reactions were analyzed for each gene. The primers used in the amplification reactions to identify the class 1 integron were identical to the ones described by Rosser and Young (45). In order to screen for bla genes, primers for the major bla families were designed and tested, including Ambler class A (TEM, SHV, CTX-M, KPC, Sme-1, NMC-A, and IMI), class B (IMP, VIM, and SPM), class C (AmpC, MIR-1, ACT-1, CMY-2, LAT-1, FOX, and MOX), and class D (OXA) β-lactamases. PCR amplification was carried out with cell lysates. Template DNA was obtained by placing several small colonies of each strain in 100 μl of TE (10 mM Tris, 1 mM EDTA, pH 8.0) and boiling the sample for 10 min. After being cooled on ice, 1 to 2 μl of each lysate was used in the PCR. Standard PCR conditions were used with either high-fidelity Taq polymerase or elongase (Invitrogen, Carlsbad, CA). PCR products from a minimum of two independent reactions were sequenced in order to unambiguously identify specific bla genes.

TABLE 1.
Primers used in this study

Analysis of gene expression.

Analysis of mRNA was performed using reverse transcription-PCR (RT-PCR) as follows. RNA was extracted from early-exponential-phase cultures using an RNeasy kit (QIAGEN, Valencia, CA). RT-PCR was performed using a OneStep RT-PCR kit (QIAGEN) following the manufacturer's recommendations. 16S rRNA was used to normalize the levels of mRNA expression in different strains. Each RT-PCR was run in duplicate such that one reaction was subjected to the entire cycle to determine the level of expression and the other was first heat-inactivated to inactivate the reverse transcriptase and to activate the HotStar Taq polymerase. The second reaction was used to detect DNA contamination in the purified RNA.

Construction of plasmids bearing ompK37 and phoE.

Plasmid-containing transformants used in this study are listed in Table Table2.2. For overexpression of ompK37 under control of its native promoter, a 1,500-bp fragment was amplified from KP-1 and KP-2 by the use of the primers given in Table Table1,1, subcloned into pGEM-T Easy vector, and then cloned into pBR322 as an EcoRI fragment. For overexpression of phoE under control of its native promoter, a 3,000-bp fragment was amplified from KP-1 and KP-2 by the use of the primers given in Table Table1.1. This phoE fragment was then fused to the tetracycline gene of pBR322 by sequential PCR amplifications, and the fused tet-phoE product was cloned into pGEM-T Easy vector. Both ompK37 and phoE were also overexpressed under control of the lac promoter of pGEM-T Easy. In both cases, the tetracycline gene from pBR322 was fused to either ompK37 or phoE by sequential PCR amplifications before cloning into pGEM-T Easy. Plasmid DNA was isolated using a QIAGEN plasmid mini kit according to the manufacturer's instructions. Plasmid DNA was transferred to KP-1 and KP-2 by electroporation.

TABLE 2.
Susceptibility results for study strains

DNA sequencing and analysis of sequence data.

Terminator kits were obtained from Applied Biosystems (Foster City, CA). Primers were synthesized by Operon/QIAGEN (Valencia, CA.). Cycle sequencing was performed according to the Applied Biosystems BigDye Terminator TaqFS v. 3.0 cycle sequencing protocol with the following specific modifications: quarter reaction in a 10-μl volume with a final 1× concentration of dilution buffer (200 mM Tris-HCl [pH 9.0], 5 mM MgCl2), 10 pmol of primer, and 5% dimethyl sulfoxide. A modified thermal cycle program was performed on a DNA Engine Tetrad cycler (M. J. Research, Watertown, MA) as follows: 95°C for 1 min (1 cycle); 98°C for 45 s, 50°C for 10 s, and 60°C for 4 min (1 cycle); 98°C for 15 s, 50°C for 10 s, and 60°C for 4 min (29 cycles); 4°C for 5 min (1 cycle); and 10°C until needed. Unincorporated terminators were removed as per the Applied Biosystems isopropanol precipitation protocol. Samples were analyzed on an ABI PRISM 3700 DNA analyzer (Applied Biosystems) with Data Collection 2.0 with POP6 polymer. Chromatograms were analyzed by using the Sequencher program, enhanced for Sequence Collector v. 4.1.4b5 (Gene Codes Corp., Ann Arbor, MI).

Analysis of outer membrane proteins.

Outer membrane proteins were isolated according to the Rapid Outer Membrane Protein Procedure of Carlone et al. (12). Samples were normalized by resuspending the outer membrane pellet to a standard optical density at 660 nm of 0.75 in 10 mM HEPES buffer, pH 7.4. Samples were boiled and analyzed by polyacrylamide gel electrophoresis (PAGE) utilizing Novex 10% Bis-Tris gels (Invitrogen, Carlsbad, CA) and protein bands from different strains, and constructs were compared following staining with Coomassie blue.

Protein sequencing.

Protein bands of interest were cut from gels stained with Coomassie blue. Automated Edman sequencing was performed on an Applied Biosystems Procise sequencer, model 494. Data were collected and analyzed using the Applied Biosystems model 610A software.

Purification of blaACT-1 β-lactamase and determination of specific activity.

The blaACT-1 β-lactamase from KP-1 and KP-2 was purified to near homogeneity from E. coli DH10B transformants using boronic acid affinity and HiLoad 26/60 Superdex G-75 column chromatography (13). Each strain was incubated overnight in Mueller-Hinton broth, after which the cells were centrifuged at 5,000 × g for 30 min, resuspended in 0.05 M phosphate buffer, pH 7.0, and centrifuged again. Cells were resuspended in cold buffer and broken by sonication. Cleared sonicate was obtained from the supernatant after centrifugation of the broken cells. From 7 g of cell paste, approximately 0.8 mg of ACT-1 β-lactamase was purified, representing a 79% recovery by total activity. Purified β-lactamases were diluted in phosphate buffer, and hydrolysis of the β-lactam ring of each antibiotic was followed spectrophotometrically at 25°C from 10 to 60 min at the wavelength for maximum absorption of each compound.

Efflux measurements.

KP-1 and KP-2 were used in an assay to quantitatively measure accumulation of ethidium bromide. Cells grown overnight were diluted 1:10 into fresh Luria-Bertani broth and cultured for an additional 4 h at 37°C with shaking at 180 rpm. Cells were centrifuged at 1,500 × g for 10 min, and the cell pellets were washed once in 100 ml of 0.05 M Na2PO4 buffer, pH 7.0, containing 100 mM NaCl. Cells were again centrifuged at 1,500 × g for 10 min, and the pellets were resuspended to an optical density at 600 nm of 0.5 in 0.05 M Na2PO4, buffer pH 7.0, containing 100 mM NaCl and 50 mM sodium formate. Ethidium bromide accumulation was measured by adding 180 μl of cells to each well of a 96-well plate containing 20 μl of 100 μM ethidium bromide (final concentration, 10 μM). Whole-cell fluorescence was measured in a SpectraMax Gemini EM plate reader (Molecular Devices, Sunnyvale, CA) for 5 min using an excitation wavelength of 530 nm and an emission wavelength of 600 nm. Intact efflux limits the amount of ethidium bromide accumulation in the assay. The inhibition of this efflux was quantitatively measured after addition of 50 μM CCCP (carbonyl cyanide m-chlorophenylhydrazone; Sigma Chemical) to each cell sample.

RESULTS

Antimicrobial susceptibility and PFGE.

Susceptibility data for KP-1 and KP-2 are found in Table Table2.2. Both of these strains are multiply antibiotic resistant, with MICs above the breakpoints for expanded-spectrum cephalosporins, cefoxitin, and piperacillin-tazobactam. In addition, KP-1 and KP-2 MICs for cefepime were 0.5 and 4 μg/ml, respectively (data not shown). Most notably, while KP-1 was susceptible to imipenem, meropenem, and sulopenem, KP-2 was resistant to these agents with MICs of 16 to 32 μg/ml. Both strains were also resistant to kanamycin and chloramphenicol. MICs to ciprofloxacin were 0.5 μg/ml with both strains, and both strains remained susceptible to tetracycline.

KP-1 and KP-2 were shown to be highly related by PFGE, having only a one-band difference after partial digestion with SmaI (data not shown), and both strains gave identical identification numbers using the Phoenix system (Becton-Dickinson, Sparks, MD).

Amplification and sequencing of resistance genes.

Gene amplification and DNA sequencing studies revealed that KP-1 and KP-2 contained the class 1 integron encoding intact gene cassettes for the IntI integrase, catB3 for chloramphenicol acetyltransferase, the aadB aminoglycoside-modifying enzyme, and the multidrug exporter qacD1 (data not shown). No β-lactamase gene was found as part of this integron. Both strains also contained the Thr83Phe substitution in gyrA; neither strain contained the qnr gene when tested by PCR.

The TEM-1 and SHV-1 β-lactamase genes were amplified and sequenced from both strains (Table (Table3).3). blaACT-1 was also identified in both strains, having an identical sequence as that deposited in GenBank (accession no. U58495). In order to compare the blaACT-1 genes in both KP-1 and KP-2, sequencing was also conducted on the region 500 bp upstream of the structural gene including the −35 and −10 promoter and ampR regulatory regions; again, the sequences were identical in both strains. The level of expression of blaACT-1 in both strains was found to be identical by measuring mRNA levels from RT-PCR determinations (Fig. (Fig.3A).3A). Despite using primers to identify the most common carbapenemase genes, we did not observe PCR products for either the Ambler class A, class B metallo, or the OXA carbapenemase. We also did not detect the presence of a metallo-β-lactamase by using a commercial E-test system with EDTA.

FIG. 3.
(A) RT-PCR results for expression of mRNA for the ompK37 porin gene and blaACT-1 from KP-1 (lanes 4 and 6) and KP-2 (lanes 5 and 7). Lanes 2 and 3 contain control 16S mRNA for each preparation. (B) Graph reflecting the relative difference in ompK37 mRNA ...
TABLE 3.
β-Lactamases contained in KP-1 and KP-2

Plasmid localization of β-lactamase genes.

Plasmid analysis of both KP-1 and KP-2 indicated that each strain contained several large and small plasmids. Both strains contained an approximately 150-kb plasmid that was transformed into E. coli DH10B, selecting for resistance to cefoxitin (Fig. (Fig.1).1). The blaACT-1 gene was amplified in the respective E. coli transformants containing this plasmid, but as expected, resistance to carbapenems was not transferred to E. coli with this plasmid. PCR methods did not detect the presence of blaTEM-1 or blaSHV-1 in the transformants containing the 150-kb plasmid.

FIG. 1.
Plasmid profile of clinical K. pneumoniae strains and transformants containing a 150-kb plasmid. Lanes: 1, 2- to 16-kb supercoiled DNA standards; 2, plasmid BAC, 181 kb; 3, plasmid pR1, 97 kb; 4, plasmid RP4, 57 kb; 5, KP-1; 6, E. coli DH10B transformant, ...

Selection of carbapenem-resistant variants from KP-1.

Mutation rates for strain KP-1 were relatively high, ranging from 2.0 × 10−6 for cefepime to 5.3 × 10−6 for meropenem (data not shown). As in the case of KP-2, MICs for the KP-1 variants selected with the carbapenems were in the 16- to 32-μg/ml range for these agents (Table (Table2).2). The MICs of variants selected with cefepime were 16 μg/ml for this compound, but MICs ranged from 0.5 to 8 μg/ml for sulopenem and the carbapenems. MICs of these variants remained elevated after multiple passages on drug-free plates. Selected variants were evaluated further to compare their mechanism of carbapenem resistance to that of clinical strain KP-2.

Isolation and characterization of blaACT-1 from Klebsiella isolates.

While a genetic analysis failed to indicate differences in the sequence of the structural or regulatory genes of blaACT-1, this enzyme was purified to near homogeneity from both KP-1 and KP-2 by a two-step chromatography method. As would be expected for an AmpC-type β-lactamase, the purified blaACT-1 hydrolzyed expanded-spectrum cephalosporins as well as cefoxitin (Fig. (Fig.2).2). No hydrolytic activity of imipenem or sulopenem was detected, consistent with our sequence data indicating that the blaACT-1 in both isolates is typical of this type of AmpC β-lactamase. When flasks containing Mueller-Hinton broth containing either imipenem or sulopenem at 14 μg/ml were seeded with KP-2 and incubated at 37°C for 18 h, 50% and 100% of drug, respectively, was inactivated compared to uninoculated control flasks with drug. A positive control culture of Serratia marcescens S-6 encoding blaIMP-1 metallo-β-lactamase completely inactivated both drugs (data not shown). This suggests that despite a failure to detect substantial hydrolysis of these agents by the purified enzyme in vitro, significant inactivation occurs with intact cells during prolonged incubation.

FIG. 2.
Comparison of hydrolytic activity of purified ACT-1 in transformants. Shown is a specific activity plot of the purified blaACT-1 beta-lactamase from KP-1 and KP-2. On the right, the y axis indicates the specific activity against cephalexin; on the left, ...

Assessment of whole-cell efflux.

Both KP-1 and KP-2 possessed similar efflux activities that limited the accumulation of ethidium bromide into the cell. Accumulation of ethidium bromide was markedly increased in both strains to an equal degree when 50 μM CCCP was added to whole cells to inhibit the efflux pump(s) (data not shown).

Amplification and sequencing of ompK genes.

Since neither KP-1 nor KP-2 contained a class A, B, or D carbapenemase, the ompK35, ompK36, and ompK37 porin genes from these strains were examined in detail to determine if their absence acted in concert with blaACT-1 to confer carbapenem resistance in KP-2. Using the primers given in Table Table1,1, sequencing of DNA amplified from all three genes indicated that both KP-1 and KP-2 contained identical gene sequences for ompK35 and ompK36. In both strains, ompK35 contained a 5-bp insert (CGCAC) after nucleotide 612 (amino acid 204) that presumably alters translation resulting in early termination at amino acid 254. In ompK36, disruption of the coding sequence occurred in both genes as well via the presence of IS903, inserted after nucleotide 126. Wild-type sequence was found in the ompK37 gene including the upstream regulatory region. Interestingly, analysis of ompK37 expression by RT-PCR revealed that mRNA levels for this gene were at least fivefold lower in KP-2 than in KP-1 (Fig. (Fig.3A,3A, lanes 4 and 5, B). This is in contrast to levels of mRNA for blaACT-1 that were equal for both strains (Fig. (Fig.3A,3A, lanes 6 and 7).

Sequencing of antibiotic resistance operons.

Since the carbapenem resistance observed in KP-2 could not be explained by differences in beta-lactamase content or OmpK porin differences from susceptible KP-1, a number of additional antibiotic resistance genes and operons were amplified and sequenced, comparing results for both Klebsiella strains. A schematic comparison of the DNA regions sequenced and the relative expression of the mRNA for each gene/operon determined by RT-PCR are given in Table Table4.4. For the majority of genes and operons (acrRAB, marORAB, romA-ramA, soxRS, micF, and micC), including upstream regulatory and downstream sequences, no differences were detected in the gene sequences or mRNA levels between strains KP-2 and KP-1. For four genes (ompK37, phoE, and phoBR) there were also no differences detected at the gene sequence level; however, using the 16S mRNA isolated from each strain as a control in RT-PCR, a reduction in the expression of mRNA was found in the carbapenem-resistant KP-2 compared with susceptible KP-1 (Table (Table44).

TABLE 4.
Comparison of DNA sequences and mRNA expression levels for strain KP-2 and KP-1 genes/operons (KP-2 relative to KP-1)

Although the phosphate-regulatable porin PhoE has not been previously implicated in antibiotic resistance, more detailed studies were performed in order to characterize the expression of PhoE in strains KP-1 and KP-2.

Analysis of cloned phoE under regulation by native and heterologous promoters.

Our results suggest that in the genetic background of blaACT-1 and ΔompK35 and ΔompK36, susceptibility to carbapenems in strain KP-2 is dramatically influenced by the expression of phoE. In order to validate this hypothesis, we performed a backcross experiment in which a wild-type phoE was cloned into the vector pGEM, first under regulation by the native phoE promoter and then under regulation by the heterologous promoter lacZ. As shown in Fig. Fig.4A,4A, complementation of chromosomal phoE in KP-2 by plasmid-encoded pGEM-phoE with the native promoter greatly increased phoE mRNA compared with that obtained from KP-2 alone (compare lanes 6 and 5). However, KP-2(pGEM-phoE) remained resistant to imipenem, meropenem, and sulopenem with no change in the MICs to these compounds (Table (Table2).2). In contrast, in Fig. Fig.4C4C (compare lanes 6 and 5) a parallel increase in phoE mRNA levels was observed under the regulation of the heterologous promoter construct KP-2(pGEM-lacZ-phoE), and it conferred an imipenem-susceptible phenotype with MICs similar to those of KP-1 (Table (Table2).2). PhoE protein in membrane extracts prepared from KP-1 and KP-2(pGEM-lacZ-phoE) was identified by N-terminal amino acid sequencing of protein blotted from PAGE gels. As shown in Fig. Fig.5A,5A, outer membranes prepared from KP-1 and the construct KP-2(pGEM-lacZ-phoE) analyzed by PAGE contained visible amounts of PhoE, while membranes prepared from the original KP-2 do not (compare lanes 2 and 7 with lane 3). Likewise, membranes from two imipenem-resistant variants selected from KP-1 also do not contain PhoE under the same gel conditions (Fig. (Fig.5A,5A, lanes 4 and 5). These results establish that carbapenem resistance in KP-2 correlates with the absence of PhoE porin in the cytoplasmic membrane.

FIG. 4.
RT-PCR results evaluating expression of phoE mRNA in KP-1, KP-2, and trans complementation constructs under regulation of (A and B) the native promoter and (C and D) the heterologous lacZ promoter. Lanes 1 to 3 in each instance contain control 16S mRNA ...
FIG. 5.
Protein-stained PAGE gel of outer membranes from strains and constructs for presence of porin PhoE (A) and OmpK37 (B).

Role of OmpK37 in conferring carbapenem resistance in KP-2.

Given the results obtained implicating the expression of PhoE in carbapenem susceptibility, a similar set of experiments were conducted involving parallel constructs with the ompK37 gene. As shown in Fig. Fig.5B,5B, lane 4, under the control of its native promoter strain, KP-2(pBR322-ompK37) does not make sufficient levels of OmpK37 to detect in membranes examined by PAGE, while under the heterologous promoter strain, KP-2(pGEM-lacZ-ompK37) makes levels of OmpK37 that are detected in the gel (lane 5). Identification of OmpK37 obtained from the gel was confirmed by N-terminal protein sequencing. The susceptibility data in Table Table22 indicate that successful translation of OmpK37 in KP-2(pGEM-lacZ-ompK37) lowers the MICs to carbapenems only two- to fourfold compared with KP-2. Thus, down-regulation of ompK37 in KP-2 plays a minimal role in conferring carbapenem resistance compared to the down-regulation of phoE. This is the first time in the Enterobacteriaceae that the phosphate-regulatable porin PhoE has been shown to play a major role in conferring resistance to antimicrobials.

DISCUSSION

Multidrug-resistant K. pneumoniae strains are becoming a problem worldwide. In many parts of the world, K. pneumoniae is the most common species of the Enterobacteriaceae to contain one or more extended-spectrum β-lactamases that can confer resistance to expanded-spectrum cephalosporins (24, 38, 53). Several Ambler class C β-lactamases, many of them encoded on mobile genetic elements, have also been found in this species, further expanding β-lactam resistance to include cefoxitin and piperacillin-tazobactam (41).

While the ESBLs and class C enzymes present a problem in terms of conferring resistance to expanded-spectrum cephalosporins, representatives of the Ambler class A (NMC-A, SME-1 to -3, IMI-1, KPC-1 to -3, GES-2, and SHV-38), class B (IMP-1 to -13, VIM-1 to -7, and SPM-1), and class D (OXA-23 to -27 and -40, -48, -54, and -58) β-lactamases broaden this resistance profile to include carbapenems (24, 27, 40, 41, 42). There has been endemic spread of imipenem-resistant Klebsiella spp. containing the class A, KPC-1 to KPC-3 β-lactamases in New York City hospitals for some time (7-10, 54, 56). There is also a growing concern that enteric organisms such as K. pneumoniae will acquire class B or D carbapenemase genes, leading to increased carbapenem resistance in this group. While several publications describe such strains from outside the United States (26, 32, 37), rapid acquisition of carbapenemase genes from P. aeruginosa and other nonfermenters may not be easily accomplished due to low rates of transfer, maintenance, and expression of these genes in the Enterobacteriaceae (14).

While much attention is being directed toward detecting K. pneumoniae strains that have acquired carbapenemases of the Ambler classes A, B, and D, conferring high-level resistance to carbapenems, this species has already evolved ways to become resistant to almost all β-lactams without having acquired carbapenemase genes. These mechanisms have involved utilizing multiple resistance mechanisms such as acquisition of an Ambler class A or C β-lactamase in combination with the loss of the OmpK35 and OmpK36 porins (8, 19, 30, 49).

Given the complexity of mechanisms leading to carbapenem resistance, our strains KP-1 and KP-2 represent an unusual opportunity to study two highly related clinical K. pneumoniae isolates from a wound culture from one patient on the same day. Although KP-1 is multidrug resistant, it is susceptible to carbapenems, while KP-2 is resistant with MICs for imipenem and meropenem of 16 to 32 μg/ml. An in-depth analysis of the antibiotic resistance genotype of these strains indicates that they both contain a class I integron, encoding resistance to chloramphenicol and aminoglycosides. Both strains have a single mutation in gyrA, although the ciprofloxacin MIC in both was 0.5 μg/ml. In addition, both strains encode the class C β-lactamase blaACT-1 that inactivates expanded-spectrum cephalosporins and cefoxitin (8). Our analysis of the regulatory and structural genes of blaACT-1 in both KP-1 and KP-2, as well as an initial substrate utilization study with purified enzyme, indicated that the ACT-1 in both strains was identical. While the purified ACT-1 β-lactamases from these isolates did not hydrolyze carbapenems in vitro, KP-2 cells incubated overnight with imipenem or sulopenem produced significant degradation of both drugs. Such slow inactivation of drug in whole cells may be an important contributing factor for carbapenem resistance, particularly in strains such as KP-2 which undoubtedly has compromised drug permeation through porin loss.

Since there did not appear to be a variant of blaACT-1 in KP-2 accounting for carbapenem resistance, we looked for other resistance determinants in this strain. While a number of different genes and operons may influence the susceptibility of K. pneumoniae to β-lactams, we compared the DNA sequences and mRNA expression of several such genes for strains KP-1 and KP-2. As shown in Table Table4,4, no differences between the two strains were detected with the efflux operon acrRAB (35, 46, 48), the multidrug-resistant operons sox-RS or marORAB (1-3, 16), the mic genes that in E. coli act as transcriptional regulators of porin genes (15, 20, 23), or in the romA-ramA operon that can regulate multidrug resistance (17, 25, 46). Furthermore, there was no measurable difference between the strains in terms of the rate of efflux of ethidium bromide or the sensitivity of ethidium efflux to the energy uncoupler CCCP.

While both ompK35 and -36 genes contained insertion sequence interruptions, it was obvious that the proposed functional loss of these porins in concert with blaACT-1 could not account for the differences in carbapenem MICs between KP-1 and KP-2, since both strains were identical with respect to these properties. Insertional inactivation of porin genes in K. pneumoniae has been observed previously (28). At this point in the study, there appeared to be some other determinant or regulatory element responsible for carbapenem resistance in KP-2.

Our initial analysis of the mRNA expression of the third porin gene, ompK37, suggested that little or no expression occurred in KP-2 while low levels of expression existed in KP-1. This observation was not immediately informative, since OmpK37 is known as a quiescent porin, not visible in stained gels of outer membrane proteins from K. pneumoniae and unlike OmpK35 and -36 (6, 22, 29, 30), its absence in isolated membranes has never been associated with antibiotic resistance (21). Our backcross experiments using a plasmid-encoded ompK37 gene under the control of the lacZ promoter illustrated that overexpression of ompK37 in KP-2 (Fig. (Fig.44 to to55 and Table Table2)2) lowered carbapenem MICs only two- to fourfold (MICs of 8 to 16 μg/ml). These results are consistent with previous studies indicating that OmpK37 is not a major determinant of antibiotic resistance.

Further analysis of the outer membrane proteins of both strains indicated the presence of an outer membrane protein in KP-1 that was absent in KP-2, and this protein migrated in PAGE gels slightly ahead of OmpA (Fig. (Fig.5A).5A). N-terminal amino acid sequencing of this protein isolated from gels identified it as the phosphate-regulated porin PhoE. Further characterization revealed that the carbapenem-susceptible strain KP-1 had a constitutively expressed PhoE porin, while this porin was absent in the membranes isolated from the carbapenem-resistant variants selected from KP-1 with β-lactams. While there is considerable amino acid conservation among the porins of E. coli (39), the phosphate-regulated porin appears to have evolved to promote cell entry of negatively charged substrates. The PhoE porin contains a number of positively charged amino acids pointing to the interior of the porin. It is therefore a porin that favors permeation of negatively charged molecules such as inorganic phosphate (50, 51). Our data suggest that in a background of insertional inactivation of ompK35 and ompK36 as in our strains, PhoE could also serve as an important entry path into the cell for zwitterionic carbapenems as well. While PhoE expression has not previously been associated with antibiotic resistance in E. coli, PhoP-PhoQ levels have been shown to cause resistance to aminoglycosides in P. aeruginosa (33).

Markedly decreased expression of the phoE/phoBR genes in the resistant strain KP-2 was confirmed by comparison of mRNA products using RT-PCR. A backcross experiment conducted with a plasmid-encoded phoE construct under control of a heterologous lac promoter in the resistant isolate KP-2 completely reversed the carbapenem resistance phenotype and was associated with the presence of visible PhoE in cytoplasmic membrane preparations.

In E. coli, the Pho regulon is composed of a two-component signal transduction system where PhoR is a histidine kinase sensor in the cytoplasmic membrane and PhoB is a response regulator (31, 50, 52, 55). Under low extracellular phosphate levels, PhoR phosphorylates PhoB, which then recognizes and binds to DNA sequences upstream of phoE, referred to as the Pho box. PhoB binding upstream of the promoter activates transcription of phoE, leading to increases in the anion-selective porin PhoE in the cytoplasmic membrane (31, 50). Studies with E. coli indicate that the phoBR operon is transcribed as a bicistronic mRNA from multiple sigma-dependent promoters. This complicated regulation of phoE expression offers several hypotheses linking PhoE porin expression with the carbapenem resistance we have observed in our K. pneumoniae strains. Our data indicate that both the phoBR regulatory and phoE genes are down-regulated in strain KP-2 compared with KP-1. One explanation for the involvement of PhoE in carbapenem resistance is that in strain KP-1, mutation leading to PhoE overexpression is selected by the cell in order to compensate for the loss of the major porins OmpK35 and OmpK36. We have not identified the metabolic selective pressure that would promote such overexpression of phoE. Such a compensatory mutation may be overridden under the selective pressure of carbapenems or other β-lactams, leading to repression of phoE. This hypothesis is supported by our preliminary observation that PhoE expression in KP-1 or KP-2 is not responsive to the phosphate content of the minimal growth medium used (data not shown). Alternatively, the repressed expression of phoE in strain KP-2 may be the normal state of the gene when under conditions of high extracellular phosphate such as when the cells are grown in rich medium.

In our trans complementation experiments involving a plasmid-encoded phoE gene under regulation of a heterologous lacZ promoter, expression of phoE and translation of the mRNA product was achieved in the transformant KP-2(pGEM-lacZ-phoE). Since successful translation was not observed with the phoE regulated by the native promoter, it is suggested that mutation has occurred in another unidentified protein involved in promoter recognition required for correct transcription/translation of phoE. This hypothesis is reinforced by our DNA sequencing data that show identity in the upstream regulatory and structural gene regions of phoE in both KP-1 and KP-2, indicating that the mutation affecting phoE expression is not in one of these genes. Potential candidates for this mutant protein would be the sigma factors necessary for correct transcription of phoE. Studies in E. coli show that multiple sigma-dependent promoters are necessary for successful transcription of the phoBR/phoE operon (50). Our analysis of KP-2(pGEM-phoE) by RT-PCR indicates that phoE mRNA is being made, yet it may not be translated since no PhoE protein can be detected in PAGE gel analysis of outer membrane proteins. In the analysis with KP-2(pGEM-lacZ-phoE) where expression of phoE is through the heterologous promoter, phoE mRNA is made, PhoE protein is detected in the outer membrane, and carbapenem resistance is lost. Our limited analysis of two genes, rpoS and hfq, failed to detect any differences between KP-1 and KP-2 with respect to DNA sequence or expression of mRNA (Table (Table4).4). The general stress sigma factor RpoS is widely used in promoting gene expression (34), and hfq encodes an RNA binding protein that helps pair noncoding RNAs with complementary regions of target mRNAs (36). While the exact gene involved in controlling expression of phoE in our strain pair has not been identified, the high mutation rate of KP-1 to carbapenem resistance under selection with β-lactams indicates that the mutation(s) can occur with a frequency that could lead to carbapenem resistance in clinical strains of K. pneumoniae strains that have β-lactamases and insertional inactivation of porins similar to those of strain KP-1.

Our study results indicate that in the background of blaACT-1 and ompK35/36 insertional inactivation, regulation of phoE can affect resistance to carbapenems, contributing to a very complicated multidrug-resistant genotype. Importantly, strain KP-2 represents yet another example of the generation of carbapenem resistance in K. pneumoniae that can occur in clinical strains in the absence of acquisition of genes encoding a carbapenemase. It would be expected that such strains will continue to occur in the clinical environment in the absence of further dissemination of genes encoding Ambler class A, B, and D carbapenemases.

Acknowledgments

We thank Andrea Marra for helpful comments on the manuscript.

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