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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, February 2002, p. 333-343, Vol. 46, No. 2
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.46.2.333-343.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Department of Microbiology and Immunology, Queen’s University, Kingston, Ontario, Canada K7L 3N6
Received 25 April 2001/ Returned for modification 29 August 2001/ Accepted 26 October 2001
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INTRODUCTION |
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Multiple antibiotic resistance in S. maltophilia, as in other gram-negative bacteria, is also attributable, in part, to limited outer membrane permeability (27) and active antibiotic extrusion (3, 4, 53), although these mechanisms are poorly characterized to date. Multidrug efflux pumps responsible for multiple antibiotic resistance of Pseudomonas aeruginosa, Escherichia coli, and Neisseria gonorrhoeae have, however, been well characterized (for a review, see reference 38). These gram-negative multidrug efflux systems are composed of three components—a cytoplasmic membrane transporter, a periplasmic but cytoplasmic membrane-associated fusion protein, and an outer membrane efflux channel protein (outer membrane factor [OMF])—which work together to extrude antimicrobials across both membranes simultaneously (35). The cytoplasmic membrane transporter belongs to the resistance-nodulation-cell division (RND) family of the secondary transporters, drug-proton antiporters that use proton motive force across the cytoplasmic membrane to actively extrude drugs from the cell (23, 34, 38, 45). Intriguingly, the various tripartite multidrug efflux systems in gram-negative bacteria share high identity and similarity at the levels of nucleotide and amino acid sequences, allowing ready identification of homologous multidrug efflux systems in the other bacterial species (see reference 45 for a review).
Multidrug efflux systems have also been described in S. maltophilia. Multidrug-resistant (MDR) strains have, for example, been reported that hyperexpress SmeM (53), a homologue of the outer membrane components of multidrug efflux systems in several gram-negative bacteria (38). Recently, MDR strains expressing the SmeDEF multidrug efflux pump have also been described in this organism (3, 4) and shown to play an important role in the resistance of clinical strains (5). Further study of the SmeDEF pump has demonstrated that this pump contributes to intrinsic multidrug resistance in S. maltophilia (53a). Here we report the identification and characterization of a novel multidrug efflux system of S. maltophilia, SmeABC. The SmeABC multidrug system is unique among the known multidrug efflux systems in gram-negative bacteria, being regulated by a two-component regulatory system encoded by the smeSR genes.
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MATERIALS AND METHODS |
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and S. maltophilia. The genomic DNA of S. maltophilia was extracted by the method of Barcak et al. (8). DNA fragments used in cloning were extracted from agarose gels using Prep-A-Gene (Bio-Rad Labs, Richmond, Calif.) as per the manufacturer’s instructions. Selections of multiple-antibiotic-resistant strains. Selection of multiple-antibiotic-resistant mutants was carried out as described previously (53). Briefly, 50 µl of an overnight culture of K1449 (an L1 and L2 ß-lactamase-deficient strain derived from ULA-511) was plated onto antibiotic-containing LB agar (cefsulodin [16 µg/ml] and ciprofloxacin [4 µg/ml]) and incubated for 24 to 48 h at 37°C. Resistant colonies were subsequently tested for cross-resistance to additional antibiotics, and those exhibiting resistance to at least two structurally unrelated antibiotics were saved for further study.
Antimicrobial susceptibility assay. Susceptibility testing was carried out by twofold serial dilution using LB broth, with an inoculum of 5 x 105 cells/ml. In some instances, this was carried out on LB agar plates containing twofold serial dilution of antibiotics with inoculum of 104 cells/spot (5 µl). Data were reported as MICs, which reflect the lowest concentrations of antibiotics inhibiting visible cell growth after an overnight incubation at 37°C.
Construction of a genomic library of S. maltophilia.
The genomic DNA of S. maltophilia ULA-511 was subjected to partial Sau3A digestion (46) and fractionated on a 10 to 40% (wt/vol) continuous sucrose gradient. Fractionations were examined by agarose gels, and those fractions enriched for DNA of ca. 20 kb in length were recovered, extensively dialyzed against 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA to remove the sucrose, and ligated to the broad-host-range cosmid cloning vector pLAFR3 (49), previously treated with BamHI and calf intestinal alkaline phosphatase (46). Following in vitro packaging of the ligated DNA with a Gigapack III Gold Package Extract Kit (Stratagene Cloning Systems, La Jolla, Calif.) and infection of E. coli DH5 cells according to instructions provided by the manufacturer, cosmid-containing DH5 clones were recovered on LB agar containing 10 µg of tetracycline per ml. Overall, more than 100,000 transductants were obtained, and these were pooled in groups and stored as 15% (vol/vol) glycerol stocks at -80°C for future use. Random screening of cosmids isolated from several transductants confirmed the presence of inserts of ca. 15 to 25 kb.
Amplification of a mexB homologue from S. maltophilia by PCR. To identify multidrug efflux system-encoding genes in S. maltophilia, two PCR primers, mexb1xz (5'-GTGTTCCTGGTGATGTACCTGT-3') and mexb3xz (5'-CGATGAGAATGGCGTTCTTCG-3'), derived from the internal sequences of the mexB gene of the P. aeruginosa mexAB-oprM operon (GenBank accession number L11616) (40, 41), were used to amplify a fragment of ca. 1.8 kb in a PCR using the genomic DNA of S. maltophilia (ATCC 13637 or ULA-511) as the template. The reaction mixture contained 0.1 µg of genomic DNA as the template, 40 pmol of each primer, a 200 µM concentration of each deoxynucleoside triphosphate (dNTP), 2 mM MgSO4, 10% (vol/vol) dimethyl sulfoxide, and 2 U of Vent DNA polymerase (New England Biolabs, Mississauga, Ontario, Canada) in 1x Thermo reaction buffer and was heated for 2 min at 94°C followed by 30 cycles of 1 min at 94°C, 1 min at 52°C, and 1.5 min at 72°C. PCR products were purified using a QIAquick-spin PCR purification kit (Qiagen) and cloned into a SmaI-digested pBluescript II SK(+). The cloned insert in the resulting plasmid, dubbed pLZ338, was then sequenced using the universal T3 and T7 promoter primers (see below). Sequencing of pLZ338 identified the 1.8-kb insert as a mexB homologue. Thus, this gene was designated smeB (for Stenotrophomonas multidrug efflux).
Southern and colony hybridizations. To obtain the intact smeB gene (and flanking DNA) directly from the genomic DNA of S. maltophilia or from a genomic library of S. maltophilia, Southern and colony hybridizations were carried out, respectively, with a 759-bp smeB-containing DNA fragment as the probe. This DNA fragment was obtained by PCR amplification of plasmid pLZ338 using primers smexb5 (5'-CCAGTTCTCGGTGACGAT-3'; anneals within smeB [bp 5171 to 5188 of GenBank accession no. AF173226]) and smexb6xz (5'-CGTAGCGCACGTTGACCA-3'; anneals within smeB [bp 5911 to 5928 of GenBank accession no. AF173226]) as described above. Following purification, the PCR product was labeled using a digoxygenin (DIG) High Prime DNA labeling kit (Boehringer Mannheim, Montreal, Quebec, Canada) as per the manufacturer’s instructions.
Southern hybridization was carried out in order to identify DNA fragments that were complementary to the 759-bp DIG-labeled smeB probe. Following digestion of S. maltophilia genomic DNA with a variety of restriction enzymes, the DNA fragments were separated on 0.8% (wt/vol) agarose gel and blotted onto a positively charged nylon membrane (Boehringer Mannheim), and smeB-complementary DNA was detected according to a protocol provided with the DIG High Prime DNA labeling and detection kit II (Boehringer Mannheim). A strong hybridization signal was obtained with a ca. 5-kb SalI fragment, and the corresponding region of an equivalent, unblotted gel of SalI-digested genomic DNA was excised, purified using Prep-A-Gene (Bio-Rad), and shotgun cloned into SalI-digested pBluescript II SK(+). One resultant plasmid, pLZ369, was shown by PCR and Southern hybridization to carry the 5-kb fragment. Nucleotide sequencing confirmed that the SalI-digested insert of pLZ369 carried the smeB gene in association with an upstream mexA-like gene (dubbed smeA) and a downstream oprM-like gene (smeC).
For colony hybridization, E. coli DH5 cells carrying cosmids with S. maltophilia genomic DNA were grown overnight on LB agar supplemented with tetracycline. Following the printing of the colonies onto a positively charged nylon membrane, the membrane was processed and screened with the aforementioned 759-bp DIG-labeled smeB probe according to instructions provided with DIG High Prime DNA labeling and detection kit II (Boehringer Mannheim). From the colony hybridization, a clone that positively reacted with the smeB probe was identified, and its cosmid, dubbed pLZ370, was further studied. PCR was used to amplify a product of 759 bp from pLZ370 using primers smexb5xz and smexb6xz (see above). Nucleotide sequencing indicated that this PCR fragment was identical to the aforementioned smeB gene (see above). The digestion of pLZ370 by SstI resulted in two DNA fragments (ca. 15 kb and ca. 10 kb), which were subcloned into an SstI-digested pAK1900, yielding plasmids pLZ380 and pLZ381, respectively. These plasmids were then subjected to nucleotide sequencing.
Cloning of sme genes. Plasmid pLZ380 carries the intact smeABC genes as well as substantial unsequenced DNA beyond smeC. In order to better assess the contribution of smeABC alone to antimicrobial resistance, it was necessary to remove this downstream DNA. Suitable restriction sites for subcloning were not readily identified downstream of smeC, necessitating the use of a multistep cloning procedure. Initially, the intact smeABC genes and additional unknown sequence downstream of smeC were recovered on a ca. 10-kb MluI-XbaI fragment, which was cloned into SmaI-restricted pAK1900 following the polishing of its ends with T4 DNA polymerase. The resultant vector, pLZ465, was digested with SstI-EcoRV to release a ca.-7-kb fragment carrying the intact smeAB genes and the 5' end of smeC. Next, the 3' end of smeC (ca. 600 bp) with ca. 120 bp downstream sequence (i.e., ca. 720 bp total) was amplified from plasmid pLZ380 using primers soprm11xz (5'-AATACCCGGGATGGCAGCGTCGCATTGGC-3'; anneals within smeC; SmaI site underlined) and soprm12xz (5'-TATCAAGCTTCTGGTGGGTGCCGAC-3'; anneals ca. 120 bp downstream from smeC stop codon; HindIII site underlined). Following digestion with SmaI and HindIII, the PCR product was cloned into SmaI-HindIII-restricted pBluescript II SK(+) to produce pLZ509. Digestion of this plasmid with SstI-EcoRV generated an intact pBluescript II SK(+) fragment carrying the 3' end of smeC, which was then ligated to the aforementioned SstI-EcoRV fragment of pLZ465 (see above) to produce pLZ519. The smeABC genes were then recovered on a ca.-8-kb SstI-HindIII fragment and cloned into pVLT31, yielding pLZ526.
The smeC gene was liberated from plasmid pLZ370 on a ca. 3-kb fragment following digestion with KpnI, as confirmed by Southern hybridization using a DIG-labeled smeC probe. The smeC probe was obtained by labeling of a PCR product, which was amplified from plasmid pLZ380 using primers soprm3xz (5'-ATGCTCTAGAACAACCGCGATCTGCGCGT-3'; anneals within smeC; XbaI site underlined) and soprm4xz (5'-TGAGAAGCTTGCCATCAACGTCGACAAC-3'; anneals within smeC; HindIII underlined). The PCR was formulated and processed as described above for smeB. The smeC-containing KpnI fragment was cloned into KpnI-digested pBluescript II SK(+), producing plasmid pLZ410. Subsequently, the smeC-containing fragment of pLZ410 was subcloned into KpnI-digested pRK415, yielding plasmids pLZ436 and pLZ437, which carried the smeC gene in the same and opposite orientation to the pRK415 lac promoter, respectively.
A blunt-ended ca.-1.1-kb fragment containing intact smeR gene was generated following digestion of pLZ415A with MluI and treatment with T4 DNA polymerase. This fragment was cloned into the SmaI-EcoRV-restricted pBluescript II SK(+) to produce pLZ515. The smeR gene was then released from pLZ515 by digestion with HindIII and XbaI, and the 1.1-kb fragment was subcloned into pRK415 to yield pLZ518, which permits transcription of the smeR gene under the control of plac.
Construction of sme deletion mutants.
To construct 
smeBC mutants, PCRs were performed to amplify sequences upstream (1.2 kb) and downstream (0.8 kb) of the intended deletion. The upstream fragment was amplified from pLZ369 using primers smexb23xz (5'-AGATGAATTCGCGCAGGTGGTGCTCAAG-3'; anneals upstream of smeB; EcoRI site underlined) and smexb24xz (5'-AGTATCTAGACTGGTCCAATGCGTGCTG-3'; anneals downstream of the smeB start codon; XbaI site underlined), while the downstream fragment was amplified with primers, soprm3xz (see above) and soprm4xz (see above). PCRs were formulated and processed as described above for smeB. The two PCR products were digested with EcoRI-XbaI or XbaI-HindIII, as appropriate, and cloned into the EcoRI-HindIII-digested gene replacement vector pEX18Tc via a three-piece ligation. The resultant plasmid, pLZ403, was introduced into E. coli S17-1 by transformation and mobilized into S. maltophilia strains ULA-511, K1385, K1449, and K1668 via conjugation (48). Transconjugants carrying pLZ403 in the chromosome were selected on LB agar containing tetracycline (40 µg/ml for ULA-511 and K1449; 80 µg/ml for K1385 and K1668) and norfloxacin (10 µg/ml; for counterselection). Transconjugants were then streaked onto LB agar containing 10% (wt/vol) sucrose, and sucrose-resistant colonies arising after overnight incubation at 37°C were screened for the presence of the smeBC deletion using PCR with three pairs of primers (soprm5xz-smexbxz8xz, soprm5xz-smexb27xz, and soprm5xz-smexb5xz [see above]).
To construct in-frame smeB deletion mutants, plasmid pLZ605 carrying the intact smeABC operon was digested by KpnI to remove two KpnI fragments that together contained smeA and the 5' end of smeB. KpnI-restricted pLZ605 was then self-ligated to create pLZ613, which contained the 3' end of smeB and the entirety of smeC. Subsequent digestion of pLZ519, which also carried the intact smeABC operon, with KpnI-ScaI released several fragments, including a 2.8-kb KpnI fragment carrying the entirety of smeA and the 5' end of smeB. This fragment was then cloned into KpnI-restricted pLZ613. One of the resulting plasmids, pLZ622, in which the orientation of the smeA-smeB insert was the same as the smeB-smeC genes of pLZ613 (confirmed by PCR), carried a ca. 2.7-kb deletion in smeB. The smeB deletion was then recovered on a 4.5-kb fragment from pLZ622 and cloned into the HindIII-SstI restricted pEX18Tc to yield pLZ623, which was introduced to E. coli S17-1 by transformation. Plasmid pLZ623 was then mobilized into S. maltophilia strains K1449 and K1668 via conjugation (see above). Transconjugants carrying pLZ623 in the chromosome were selected on LB agar containing tetracycline (80 µg/ml) and norfloxacin (10 µg/ml; for counterselection). Transconjugants were then streaked onto LB agar containing 10% (wt/vol) sucrose, and sucrose-resistant colonies arising after overnight incubation at 37°C were screened for the presence of the smeB deletion using PCR with primers smebaxz and smebbxz.
To construct a smeC strain of S. maltophilia, an intact smeC gene was initially released from plasmid pLZ471 (see below) by digestion with EcoRI and HindIII. The 1.4-kb smeC-containing fragment was then cloned into EcoRI-HindIII-treated pTZ19U to yield pLZ633. Digestion of pLZ633 with BamHI-EcoRV removed a 130-bp DNA fragment from the middle of smeC. Following blunt ending of BamHI-EcoRV-digested pLZ633 with T4 DNA polymerase, the plasmid was recircularized via ligation to produce the smeC-carrying plasmid pLZ634. The smeC gene was then released from pLZ634 via EcoRI-HindIII digestion and cloned into pEX18Tc previously digested with EcoRI-HindIII, to produce pLZ635. This vector was introduced to E. coli S17-1 by transformation and mobilized into S. maltophilia strains K1449 and K1668 via conjugation (see above). Transconjugants carrying pLZ623 in the chromosome were selected on LB agar containing tetracycline (50 µg/ml) and norfloxacin (5 µg/ml; for counterselection). Transconjugants were then streaked onto LB agar containing 10% (wt/vol) sucrose, and sucrose-resistant colonies arising after overnight incubation at 37°C were screened for the presence of the smeC deletion using PCR with primers smexb17xz and soprm5xz.
To construct smeR mutants, two PCRs were performed to amplify DNA fragments 5' (ca. 1 kb) and 3' (ca. 0.6 kb) to the sequences to be deleted within smeR. Using plasmid pLZ370 as a template, sequences 5' to the deletion were amplified with primers smer5xz (5'-TGACTCTAGACCGGTACATCGCTGCT-3'; anneals within smeS; XbaI site underlined) and smer6xz (5'-CTACAAGCTTCAACCGGATGGATGGCGCA-3'; anneals downstream of smeR start codon; HindIII underlined), while sequences 3' to deletion were amplified with primers smer7xz (5'-CTCTGAATTCGAGCGCGCCATGTTG-3'; anneals downstream of smeR; EcoRI site underlined) and smer4xz (5'-TGAATCTAGACAGCCATGTGCGCAAC-3'; anneals upstream of smeR stop codon; XbaI site underlined). The PCR was formulated and processed as described above for smeB. The two PCR products were digested with EcoRI-XbaI or XbaI-HindIII, as appropriate, and cloned into EcoRI-HindIII-digested pEX18Tc via a three-piece ligation. The resultant plasmid, pLZ517, which contained an internal deletion of 340 bp in the smeR gene, was introduced into E. coli S17-1 and then mobilized into S. maltophilia strains K1449 and K1668 via conjugation. Transconjugants carrying pLZ517 in the chromosome were selected on LB agar containing tetracycline (80 µg/ml) and norfloxacin (10 µg/ml; for couterselection). Transconjugants were then streaked onto LB agar containing 10% (wt/vol) sucrose, and sucrose-resistant colonies arising after overnight incubation at 37°C were screened for the presence of the smeR deletion using PCR with primers smer6xz (see above) and smerbxz (see below).
Construction of an L2 bla insertion mutation. Disruption of the L2 bla gene in K1668 was originally carried out by insertion of 4 bp near the middle of the gene, although this could still yield a truncated (and perhaps partially functional) L2 enzyme of 187 amino acids (c.f. 303 amino acids in the native enzyme). To create a null mutation, then, an internal fragment of the L2 bla gene was amplified by PCR and cloned into the gene replacement vector pEX18Tc. Following homologous recombination, the chromosomal L2 bla gene would be disrupted by insertion of pEX18Tc sequences. A ca.-400-bp fragment of the L2 bla gene (beginning immediately after the ATG start codon) was amplified with primers sml8xz (5'-ACTTGTCGACCGTCGCCGATTCCTGCAGTT-3'; SalI site underlined) and sml9xz (5'-AGATGGATCCTGATCGTGGCACGGCACAGA-3'; BamHI site underlined) using plasmid pLZ416 as a template. Reaction mixtures were formulated as described above and subjected to 30 cycles of 50 s at 94°C, 40 s at 56°C, and 30 s at 73°C before finishing with a 5-min incubation at 72°C. The resultant PCR product was restricted with SalI and BamHI and cloned into SalI-BamHI-restricted pEX18Tc, yielding pLZ736. Following introduction of this vector into E. coli S17-1, it was mobilized into S. maltophilia K1668 as described above, and transconjugants (e.g., K1975) were selected on tetracycline (25 µg/ml) and norfloxacin (2.5 µg/ml; for counterselection). K1795 would be effectively be L2-, containing as it would two incomplete versions of the L2 bla gene, one truncated at the 5' end (immediately after the start codon) and one truncated at the 3' end (400 bp into the gene).
RT-PCR. Total bacterial RNA was isolated from log-phase cultures (1 to 2 ml) of S. maltophilia strains using the Qiagen RNeasy Mini Kit (Qiagen), treated with RNase-free DNase (Promega, Madison, Wis.; 2 U of enzyme/µg of RNA for 60 min at 37°C), and repurified using the same kit. A 0.2-µg sample of DNase-treated RNA was used as a template for reverse transcription (RT)-PCR with the Qiagen OneStep RT-PCR kit according to a protocol supplied by the manufacturer. Primer pairs specific for and internal to smeA (smeaaxz, 5'-TACAGAATTCATGTCTCTCCTGCGCCCG-3' [forward]; smexa2xz, 5'-ACCTTAACCTGTGCCTTG-3' [reverse]; and smexa3xz, 5'-GTCGACCTGGTACAGCA-3' [reverse]) were used to amplify and quantitate the corresponding mRNA, as a measure of smeABC expression. Thirty picomoles of each primer was used per reaction mixture (final volume of 50 µl), which involved a 30-min incubation at 50°C, followed by 15 min at 95°C and 40 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C before finishing with 10 min at 72°C. A 15-µl sample of each reaction product was analyzed by agarose (1.4% [wt/vol]) gel electrophoresis for the expected RT-PCR products (742 and 296 bp).
Expression of SmeC and SmeR. To identify the products of the smeC and smeR genes, it was necessary to clone these genes into a suitable expression system. PCR was initially used to amplifying smeC and smeR from pLZ380 with two pairs of primers, smecaxz (5'-TCTGGAATTCATGAAGCCGATGCTGCTGC-3'; EcoRI site underlined) and smecbxz (5'-TCATAAGCTTCCGCGCGTCCGCGCCACCAC-3'; HindIII site underlined) for smeC and smeraxz (5'-TACAGAATTCATGAGCACGTCGCCCGCCAC-3'; EcoRI site underlined) and smerbxz (5'-TCATAAGCTTGGGCTCGTAGCTGTAGCCCAT-3'; HindIII site underlined) for smeR. The smeC-containing (1.4-kb) and smeR-containing (0.7-kb) PCR products were directly cloned into pCR-Blunt-II-TOPO vector as instructed by the manufacturer (Invitrogen, Carlsbad, Calif.), yielding plasmids pLZ471 and pLZ473, respectively. Cloned inserts were confirmed by DNA sequencing. Following digestion of pLZ471 and pLZ473 with EcoRI and HindIII, the smeC- and smeR-containing fragments were cloned into EcoRI-HindIII-restricted pET21-d(+) to yield pLZ477 and pLZ479, respectively. These plasmids were then transformed into E. coli BL21(DE3) carrying the pLysS plasmid, respectively, and expression of smeC and smeR was examined in whole-cell lysates of BL21(DE3). Briefly, cells grown overnight in LB broth were subcultured in 5 ml of LB broth to early exponential phase of growth (optical density at 600 nm, 0.2). The cells were then incubated with (to induce expression of the cloned genes) or without 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) for 3 h. One milliliter of cells was subsequently harvested, washed once with 50 mM sodium phosphate buffer (pH 7.5), and resuspended in the same buffer before being subjected to sodium dodecyl sulfate-polyacrylmide (11% [wt/vol]) gel electrophoresis (SDS–11% PAGE) as described previously (46).
Cloning of smeABC and smeSR promoter regions. To evaluate expression of the smeABC and smeSR operons, the intergenic region encompassing sequences upstream of both was recovered on a ca.-0.7-kb PCR product produced using pLZ380 as the template and primers smexa6xz (5'-ACGGGTCGACCTGGTACAGC-3'; anneals within smeA) and smes1xz (5'-ACCGTCTAGATGCTCCCAGCCACCGT-3'; anneals within smeS). The PCR product was directly cloned into pCR-Blunt II-TOPO vector (Invitrogen) as per the manufacturer’s instructions, producing plasmid pLZ474. The insert DNA was then sequenced to ensure no error had been introduced during PCR. Digestion of pLZ474 with KpnI and XbaI released a 0.7-kb fragment whose cloning into KpnI-XbaI-restricted pMP190 (to yield pLZ497) placed the smeABC promoter region upstream of and in the proper orientation relative to the promoterless lacZ gene of this vector. Similarly, digestion of pLZ474 with SalI and XbaI released a 0.7-kb fragment whose cloning into SalI-XbaI-restricted pMP190 (to yield pLZ499) placed the putative smeSR promoter region upstream of and in the proper orientation relative to the promoterless lacZ gene of this vector.
Plasmid mobilization.
Introduction of plasmids pRK415 and pVLT31 and their derivatives from E. coli DH5 into S. maltophilia required a triparental mating procedure employing the helper vector pRK2013 (39), while introduction of plasmid pMP190 and its derivatives from E. coli S17-1 into S. maltophilia employed a biparental mating procedure. Briefly, in triparental mating, 100-µl aliquots of overnight cultures of plasmid-containing E. coli DH5, pRK2013-containing E. coli MM294, and ULA-511 derivatives of S. maltophilia were mixed in a microcentrifuge tube, and in biparental mating, 100 µl of plasmid-containing E. coli S17-1 and S. maltophilia K1449 were mixed. The cell mixtures were subsequently pelleted by centrifugation, resuspended in 40 µl of LB broth, and spotted onto the center of an LB agar plate. Following incubation overnight at 37°C, bacterial cells were resuspended in 1 ml of LB broth and appropriate dilutions (10-2 to 10-4) were plated on LB agar containing norfloxacin (5 to 10 µg/ml; to counterselect the E. coli strains) and tetracycline (25 to 50 µg/ml; for pRK415 and pVLT31 and their derivatives) or chloramphenicol (20 µg/ml; for pMP190 and its derivatives). Plasmid DNA was prepared from S. maltophilia recipients using the miniprep procedure to confirm successful plasmid transfer.
ß-Galactosidase assays. Bacteria harboring plasmids pMP190 and pRK415 and their derivatives were cultured overnight at 37°C in LB broth supplemented with chloramphenicol (30 µg/ml) and tetracycline (25 µg/ml) and subsequently diluted 50-fold into fresh LB broth. Following growth to late log phase (optical density at 600 nm, 0.8 to 1.0), cultures were assayed for ß-galactosidase activity as described previously (29)
ß-Lactamase preparation and activity assays. The extraction and assay of the S. maltophilia chromosomal ß-lactamases were carried out as described previously (without imipenem induction) using nitrocefin as the chromogenic substrate (24). Large-scale extraction of ß-lactamase from strain K1668 was carried out using a cold-osmotic shock method (33), followed by purification of the ß-lactamase on a borate-affinity column (9). Six hundred milliliters of cells at the late-exponential phase of growth in LB broth was harvested and washed once with 100 ml of 30 mM Tris-HCl (pH 7.5). The cells were then resuspended in 30 ml of 30 mM Tris-HCl (pH 7.5), followed by the addition of 30 ml of 40% (wt/vol) sucrose (prepared in 30 mM Tris-HCl [pH 7.5]-2 mM EDTA). The cell suspension was centrifuged at 8,000 x g for 20 min, and the cell pellet resuspended in 25 ml of chilled H2O containing 1 mM MgCl2 before being centrifuged at 12,000 x g for 20 min. The ß-lactamase-containing supernant recovered after centrifugation was mixed with an equal volume of 20 mM triethanolamine (pH 7.0) and then loaded onto an m-aminophenylboronic acid agarose (Sigma) column equilibrated with 20 mM triethanolamine (pH 7.0). The column was then washed with 50 ml of 20 mM triethanolamine (pH 7.0) containing 0.5 M NaCl, and the ß-lactamase was eluted with 20 ml of 0.5 M sodium borate (pH 7.0), also containing 0.5 M NaCl. One-milliliter fractions were collected and those enriched for ß-lactamase activity (assayed using the nitrocefin assay described above) and demonstrating high purity (assayed by SDS-PAGE with silver staining [46]) were pooled and dialyzed against 10 mM Tris-HCl (pH 7.5). The 22-kDa ß-lactamase was then concentrated using a Centricon concentrator (Amicon, Inc., Beverly, Mass.) as previously described (53). Hydrolysis of ß-lactams by the enzyme was determined spectrophotometrically at 230 nm (for penicillins) or at 260 nm (cephalosporins) using a 0.5 mM (penicillins) or 0.1 mM (cephalosporins) final substrate concentration. The enzyme activity was expressed in nanomoles of substrate hydrolyzed per minute per milligram of protein at room temperature. The rates of hydrolysis for each substrate were calculated relative to that of nitrocefin.
DNA sequencing. Nucleotide sequencing of plasmid-borne DNA or PCR products was carried out by Cortec DNA Services Inc. (Queen’s University) using universal or custom primers. Compilation of DNA sequence data was performed using the PC Gene software package (version 2.1; Intelligenetics Inc., Mountain View, Calif.) and DNAMAN (version 4.11; Lynnon Biosoftware, Vaudreuil, Quebec, Canada). Promoter prediction was carried out using neural network promoter prediction software (http://www.fruitfly.org/seq_tools/promoter.html).
Nucleotide sequence accession number. The nucleotide sequences of the smeABC and smeSR operons have been deposited with the GenBank sequence database under accession number AF173226.
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L1 L2 parent strain K1449 were measured. Unexpectedly, the MDR strain demonstrated a marked increase in ß-lactamase activity relative to K1449, though still well below that seen for the L1-L2-producing strain (Table 6 and reference 53). Moreover, elimination of smeBC (K1781) or smeC (K1785) reduced the ß-lactamase activity to levels seen for the K1449 parent strain, while a smeB deletion had no effect (Table 6). Deletion of smeR from K1668 (K1787), on the other hand, had an intermediate effect on ß-lactamase activity, reducing it to a level below that of the MDR strain but above that seen for its parent (Table 6). Indeed, the relative ß-lactamase activities exactly paralleled the MICs seen for ß-lactams, indicating that the ß-lactam resistance of K1668 was likely explained not by any efflux activity provided by a pump that includes SmeC but by this ß-lactamase.
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The observation that the smeC gene cloned in the opposite orientation relative to the resident lac promoter of the pRK415 cloning vector was able to complement, to some extent, the antibiotic susceptibility of an OprM-deficient mutant of P. aeruginosa is consistent with the smeC gene possessing its own, albeit weak, promoter. Similarly, the oprM gene of the mexAB-oprM multidrug efflux operon of P. aeruginosa was also shown to possess a weak promoter, which permitted its expression independent of mexAB (54). This was likely necessary since this protein functions as the OMF of several multidrug efflux systems in this organism (1, 30, 52), and thus, its expression would likely be needed even under circumstances when MexAB-OprM expression is not. The fact that smeC but not smeAB contributes to antimicrobial resistance and can be expressed independently of these genes suggests that SmeC also functions as part of an additional, as-yet-unidentified efflux system(s) which does accommodate antimicrobials. Similarly, a three-component RND-type efflux system, AmeABC, was recently described in Agrobacterium tumefaciens, where loss of the inner-membrane-associated components (AmeAB, homologues of SmeAB) did not impact antimicrobial susceptibility while loss of the OMF component, AmeC, did (37a). The putative AmeABC pump was implicated in export of intermediates of purine biosynthesis, while AmeC was proposed to also function in drug efflux as part of an as-yet-unidentified efflux system (37a). That smeC expression is driven by a promoter upstream of smeA as well as its own promoter is also supported by the demonstration that inactivation of smeR, which encodes a positive regulator of smeABC expression, has only an intermediate effect on resistance levels. While loss of smeR will negatively impact the expression of smeC from the smeA upstream promoter (which is the target for SmeR [see Table 5]) smeC expression is still possible from its own promoter in these mutants. Given the correlation between SmeC production and antimicrobial resistance in K1668, then, smeR knockouts are expected to provide at least some resistance while smeC knockouts are not, and this is, in fact, what is seen.
The discovery that the ß-lactam resistance of a SmeABC (i.e., SmeC)-overexpressing strain is due to increased ß-lactamase activity and not efflux is interesting. While it might reflect coregulation of the smeC and ß-lactamase genes, it appears more likely that the status of SmeC impacts, e.g., cell physiology (perhaps via accumulation or release of certain substrates) in such a way as to induce (or activate) the enzyme. This is supported by the observation that deletion of smeC itself produces a decline in ß-lactamase activity which is more striking than the loss provided by the smeR knockout (i.e., it is likely that the smeR effect on ß-lactamase activity results from its impact on SmeC levels and not loss of, e.g., SmeR-dependent activation of the ß-lactamase gene). Certainly the presence or absence of SmeC has a major impact on the cell, inasmuch as the SmeABC-hyperexpressing strain grows markedly more slowly than its parent or smeC deletion derivatives, forms smaller cells, and is stained much less intensely with Gram stain (data not shown). Nonetheless, given the homology between SmeR and other regulators of ß-lactamase gene expression (e.g., BlrAB [2] and CreB [6]) (Table 2), the possibility of SmeR regulating ß-lactamase gene expression is intriguing. A connection between expression of multidrug efflux pumps and ß-lactamase was made previously in P. aeruginosa, where it was noted that increased expression of the MexCD-OprJ multidrug efflux system in an nfxB mutant was accompanied by a decrease in expression of the organism’s AmpC ß-lactamase (26). Thus, efflux systems can both positively and negatively influence ß-lactamase production.
The nature of the connection between the L2 ß-lactamase and SmeABC is unknown at this point. While it would be worthwhile to study this in a wild-type background, SmeABC-expressing derivatives of, e.g., ULA-511 are not currently available, and the nature of the mutation(s) leading to SmeABC expression in MDR strain K1668 is unknown (precluding construction of a SmeABC-expressing derivative of ULA-511). Still, L2 activity is readily measurable in wild-type S. maltophilia (which does not express SmeABC), indicating that expression of these two resistance determinants is not intimately linked, though SmeABC (or SmeC) expression may necessitate production of some L2 enzyme.
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