JAC Advance Access originally published online on April 5, 2006
Journal of Antimicrobial Chemotherapy 2006 57(6):1070-1076; doi:10.1093/jac/dkl106
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SmeDEF-mediated antimicrobial drug resistance in Stenotrophomonas maltophilia clinical isolates having defined phylogenetic relationships
Bristol Centre for Antimicrobial Research and Evaluation, Department of Cellular and Molecular Medicine, University of Bristol, School of Medical Sciences, University Walk Bristol BS8 1TD, UK
*Corresponding author. Tel: +44-117-9287528; Fax: +44-117-9287896; E-mail: Matthewb.Avison{at}bris.ac.uk
Received 7 February 2006; returned 6 March 2006; revised 7 March 2006; accepted 7 March 2006
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Abstract |
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Objectives: To test whether smeDEF overexpression leads to a predictable multi-drug resistance phenotype in Stenotrophomonas maltophilia and to measure the frequency with which smeDEF overexpression occurs in clinical isolates and in spontaneous drug-resistant mutants.
Methods: Overexpression of smeDEF was induced in clinical isolates by the introduction of chromosomal mutations in smeT using a gene-replacement approach. Spontaneous drug-resistant mutants were selected using greater than MIC concentrations of various antimicrobial agents. Levels of smeE and smeF mRNAs were quantified using RT–PCR; MICs were determined using Etest.
Results: Of 20 spontaneous S. maltophilia drug-resistant mutants tested, four overexpressed smeDEF, but only two carried mutations within smeT. Of 30 clinical isolates tested, 6 significantly overexpressed smeDEF. One of these had an IS1246-like element embedded within the putative SmeT binding site in the smeDEF promoter. All smeDEF overexpressing derivatives of an isolate had the same resistance profile; derivatives that did not overexpress smeDEF did not share this resistance profile. However, no consistent phenotype could be associated with smeDEF overexpression in S. maltophilia isolates per se.
Conclusions: SmeT is not the only gene product that affects smeDEF expression. IS element insertion is a viable mechanism by which smeDEF expression can be derepressed. There is evidence for a background-specific, predictable effect on resistance profile when smeDEF is overexpressed, but the variability of backgrounds encountered means no general SmeDEF-mediated phenotype can be defined. There is strong evidence for the existence of as yet unidentified multi-drug efflux pumps in this species.
Keywords: efflux pumps , SmeT , nosocomial infections
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Introduction |
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Stenotrophomonas maltophilia is a Gram-negative non-fermenting bacterium found extensively in the environment, where it is capable of surviving in a wide variety of niches. In recent years, S. maltophilia has become an increasing nosocomial threat. Within hospitals, the bacterium is most often found in water sources, and can be a contaminant of hospital equipment such as nebulizers and intravenous catheters. From these sources, the organism can infect patients, resulting in a wide spectrum of symptoms dependent upon the site of infection, though, most commonly, S. maltophilia causes bacteraemias or respiratory tract infections.1,2 Prior exposure to antimicrobial agents, particularly ß-lactams and aminoglycosides, significantly increases the risk of S. maltophilia infection, as the organism is commonly resistant to these compounds.1,2 ß-Lactam resistance is due to the expression of the ß-lactamases L1 and L2, which together hydrolyse the full gamut of ß-lactam drugs, with the exception of monobactams.3 L1 and L2 expression is often induced following ß-lactam challenge through different, overlapping induction mechanisms.4 Aminoglycoside resistance, at least to a subset of these drugs, has been unequivocally linked to the expression of an aac(6')-Iz determinant,5,6 and we have recently characterized an aph(3')-IIa, which encodes resistance to the rest of the aminoglycoside class, with the exception of gentamicin.7 Other resistance genes so far found in S. maltophilia include those encoding resistance to macrolides8 and sulphonamides.9
As well as specific resistance genes, two putative multi-drug resistance (MDR) loci have been cloned and sequenced. Both encode resistance nodulation division (RND)-based tripartite efflux pumps named SmeABC10 and SmeDEF.11 SmeA and SmeB are unlikely to be involved in clinical resistance, though the outer membrane protein (OMP) SmeC might contribute to resistance through its interaction with an as yet unknown efflux pump.10 SmeDEF, on the other hand, has been shown to confer multiple antimicrobial drug resistance when overproduced in an Escherichia coli heterologous host.11 Furthermore, in the S. maltophilia MDR mutant, D457R, smeDEF is overexpressed due to a mutation in the transcriptional repressor, SmeT, which is encoded upstream of smeDEF in the opposite orientation.12
Variability amongst clinical isolates of S. maltophilia is a recurring observation. Several recent reports have highlighted genotypic and phenotypic differences amongst collections of isolates, even from the same hospital.13,14 Phenotypic variation between members of a species causes problems both in the clinic, where isolates may not behave predictably, and in the research laboratory, where it is difficult to come to generalized conclusions. We have developed methods for phylogenetically grouping clinical S. maltophilia isolates in an attempt to see whether there is more phenotypic homogeneity amongst groups of closely phylogenetically related isolates. Our first approach to phylogenetic analysis was through comparisons of L1 and L2 ß-lactamase and 16S rRNA gene sequences carried by a group of isolates from the Bristol Royal Infirmary. In this way, we were able to define three phylogenetic groups: ‘A’, typified by isolate K279a (and being most similar to the S. maltophilia type strain); ‘B’, typified by isolate N531 and ‘C’, typified by isolate J675a.3 More recently, we have shown that a hypervariable sequence located between smeD and smeT is suitable for sub-grouping isolates with identical 16S rRNA sequences,15 and have now defined four phylogenetic groups using a worldwide collection of isolates (named A–D) with the most populous groups being A, which encompasses highly genetically homogeneous isolates, and B, which includes highly heterogeneous isolates that are genotypically very different from those in group A.16
There has been some debate concerning the role of SmeDEF in multiple antimicrobial drug resistance in S. maltophilia. A clear role for this efflux pump in reduced susceptibility to a cross section of antimicrobials (macrolides, tetracyclines, chloramphenicol and quinolones, but not aminoglycosides or ß-lactams) has been defined in MDR mutants derived from isolate D457.11 However, using the parent isolate ULA-511 to select hyper-resistant mutants, smeDEF overexpression was only linked to a modest increase in MICs of a small group of antimicrobials (some quinolones and chloramphenicol).17 One explanation for this seeming disparity is that ULA-511 and D457 come from different phylogenetic groups, and that these groups have predictably different SmeDEF-mediated resistance phenotypes. Indeed, we recently demonstrated that D457 is phylogenetically a group B isolate and is very different from ULA-511, which is a group A isolate.15 If it turns out to be the case that phylogenetic group-specific antimicrobial drug resistance properties exist, then this information may be of use clinically. Accordingly, the main hypothesis being tested in the study reported here was that SmeDEF-mediated antimicrobial drug resistance phenotypes are phylogenetic group-specific. This hypothesis was tested by selecting mutants displaying reduced susceptibility to antimicrobial drugs from parent isolates of phylogenetic groups A and B (K279a and N531, respectively) and then attempting to relate smeDEF expression level and resistance profile in these mutants. In addition, we examined a collection of 30 phylogenetically grouped clinical S. maltophilia isolates from Europe and North, South and Central America and compared their resistance profiles and smeDEF expression levels.
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Materials and methods |
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Bacterial strains and materials
S. maltophilia isolates K279a and N531 have been described previously.3 Nitrocefin was obtained from Becton–Dickinson (Cockeysville, USA). PCR primers were purchased from Qiagen (Crawley, UK) and PCR enzymes were from ABGene (Epsom, UK). Restriction enzymes were obtained from NEB (Hitchin, UK); general reagents for DNA manipulation were obtained from Roche (Lewes, UK). All other general reagents were from Sigma Chemical Co, or BDH, both of Poole, Dorset, UK.
Construction of smeT frameshift mutations on the K279a and N531 chromosomes
The smeT gene from isolate K279a was amplified by PCR using the protocol described previously18 in two sections, one using the primers ‘KsmeT+ve’ (5'-AGGGTCGGATTCGGCTCA-3') and ‘KsmeTHindIII–ve’ (5'-AAGCTTGCACCCGTTCGACGATC-3'), and another using the primers ‘KsmeTHindIII+ve’ (5'-AAGCTTCATGCAGGAACTGGAGCG-3') and ‘KsmeT–ve’ (5'-CCACTCACGCTTCGGGCAG-3'). The result was two non-overlapping amplicons, each having a HindIII restriction site artificially introduced at one end. The two amplicons were treated with HindIII and ligated together using T4 DNA ligase. The result was the conversion of the sequence GCACCTGCCCTT into GCAAGCTT, and thus, the generation of an smeT frameshift mutant allele (named smeTKFS) carrying a 4 bp deletion (marked in bold in the wild-type sequence) flanked by a single nucleotide substitution (underlined) centred at position 202 in the gene.
The smeT gene from isolate N531 was amplified by PCR using the primers ‘NsmeT+ve’ (5'-ATGGCCCGCAAGACCAA-3') and ‘NsmeT–ve’ (5'-GCCGGGAACGAACA-3'). The amplicon was digested with HaeII to give two equally sized fragments, which were made blunt-ended using T4 DNA polymerase (in the absence of dNTPs, so the 5' overhang was removed in each case), and the two fragments were blunt-end ligated using T4 DNA ligase. The result was the generation of an smeT frameshift mutant allele (named smeTNFS) carrying a 4 bp deletion centred at position 315 in the gene.
The frameshift mutant alleles smeTKFS and smeTNFS were amplified by PCR using primers ‘KsmeT+ve’ and ‘KsmeT–ve’ or ‘NsmeT+ve’ and ‘NsmeT–ve’, respectively, using the ligation reactions (after filter dialysis) as a source of DNA template. Both the resulting amplicons were inserted separately into the Invitrogen TA cloning vector, pCR2.1 TOPO (KanR); recombinants were used to chemically transform E. coli strain TOP10 (Invitrogen) to kanamycin resistance (30 mg/L) according to the manufacturer's instructions. Transformants were screened by PCR, HaeII/HindIII digestion as appropriate, and finally by DNA sequencing of the inserts. Those that carried pCR2.1 including a confirmed smeTKFS or smeTNFS insert were used to harvest large quantities of plasmid, and the smeT mutant alleles were removed using EcoRI digestion, and ligated separately into the EcoRI linearized gene-replacement vector, pEX18Tc,19 to produce plasmids pEX::smeTKFS and pEX::smeTNFS, which were used to replace the wild-type S. maltophilia chromosomal smeT genes, of isolate K279a or N531, respectively, using the method described previously.17,19 The S. maltophilia mutant derivatives produced (mutations were checked by PCR, restriction digestion and DNA sequencing) were named K279a smeTFS and N531 smeTFS.
Susceptibility tests, selection of hyper-resistant mutants and ß-lactamase assays
All susceptibility data were determined using Mueller–Hinton agar (Oxoid). MIC values were quantified using Etest strips (AB Biodisk, Solna, Sweden) following incubation at 37°C for 24 h with a starting inoculum having a turbidity equivalent to that of a 0.5 McFarland standard. Single-step hyper-resistant mutants of isolates K279a and N531 were selected on Mueller–Hinton agar plates containing one of a number of antimicrobials (see Results) at doubling concentrations above their MICs for the parent isolate. Plates were inoculated using a spiral plater, and the outermost colonies (if any) from each plate (i.e. those where the inoculum was most dilute) were selected.
Assays to measure the combined activities of the L1 and L2 ß-lactamases in crude cell extracts were carried out according to the methods described previously,4 using nitrocefin as substrate. No ß-lactam inducer was added to the cells before extraction and assay of ß-lactamase.
RT–PCR
Single bacterial colonies were used to inoculate 10 mL of nutrient broth in universal bottles and cultures were incubated overnight at 37°C with shaking. Each overnight culture was used to inoculate a fresh 10 mL of nutrient broth to an initial turbidity (OD600) of 0.05–0.1. Cultures were incubated as above until the OD600 reached 0.5, at which time 1 mL of the culture was removed, the cells were pelleted and total RNA was purified using a Qiagen RNeasy extraction kit according to the manufacturer's instructions; the optional Ribolyser (Hybaid) step was used in addition to the standard enzymatic digestion, as this significantly improved RNA yield from S. maltophilia. Once total RNA had been purified, it was treated with RNAase-free DNAase to remove any DNA contamination, and the pure RNA was quantified and diluted to 100 ng/µL. All batches of RNA were confirmed DNA free before use by performing smeT–smeD intergenic PCR, as described previously.15 RT–PCR determinations of mRNA levels for smeE, smeF or the housekeeping gyrA control in each total RNA sample were performed using a single tube reaction (Qiagen One-Step RT–PCR Kit) according to the manufacturer's instructions with 100 ng of RNA as template; the optional ‘Solution Q’ was used since it enhanced amplification. Primers for smeE (‘smeE+’: 5'-AGCTCGACGCCACGGTA-3' and ‘smeE–ve’: 5'-TGGCCTGGATCGAGAGCA-3') and smeF (‘smeF+’: 5'-GCCACGCTGAAGACCTA-3' and ‘smeF–ve’: 5'-CACCTTGTACAGGGTGA-3') were designed from regions that are invariant in the four known examples of this gene;15 primers for gyrA (‘gyrA+’: 5'-AACTCAACGCGCACA-3' and ‘gyrA–ve’: 5'-CCGATTCCTTTTCGTCGTAGTTGGG-3') were from a section predicted to encode the quinolone resistance-determining region of S. maltophilia GyrA, as reported previously.20 The sequences of this section of the gyrA genes from S. maltophilia isolates K279a and N531 are identical (A. Okazaki and M. B. Avison, unpublished results). Following RT–PCRs, amplicons were separated by gel electrophoresis and images of the ethidium bromide-stained gels were acquired using a Kodak Image Station 440CF (Kodak Ltd, Hemel Hempstead, UK) and analysed using the ImageQuant suite of programs (Molecular Dynamics Inc., CA, USA) to determine band intensities. In all cases, the intensities of smeE and smeF RT–PCR product bands were normalized for RNA loading using the intensity of the corresponding gyrA RT–PCR band from each RNA preparation.
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Results and discussion |
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Expression of smeDEF in spontaneous S. maltophilia mutants with reduced susceptibility to antimicrobial drugs
Ten spontaneous, single-step hyper-resistant mutants were selected from each of S. maltophilia parent isolates K279a (phylogenetic group A) and N531 (phylogenetic group B). Selection was for generation of reduced susceptibility to one of a variety of single antimicrobials. MICs of a cross selection of antimicrobials for each mutant were then determined. To complement this analysis, RT–PCR was used to measure the levels of smeE and smeF mRNAs in the mutants and parent isolates relative to a gyrA control. To produce smeDEF overexpression controls, smeTFS loss-of-function mutants were made from each parent isolate. These mutants were also used as indicators of the resistance phenotypes expected when smeDEF expression is maximally derepressed from control by SmeT. For spontaneous mutants with reduced ß-lactam susceptibility, ß-lactamase activities in cell extracts were measured in the absence of ß-lactam challenge in order to detect whether constitutive overproduction of ß-lactamase is involved in resistance.
Analysis of the K279a-derived mutants (Table 1) reveals three distinct resistance phenotypes. ‘Resistance profile 1’ is decreased susceptibility to ciprofloxacin, with a very modest decrease in susceptibility to chloramphenicol. Three spontaneous mutants (K279a M1, M6 and M7) expressed this phenotype. All of them were initially selected for resistance to fluoroquinolones, and all were shown to overexpress smeE and smeF relative to K279a. The control mutant, K279a smeTFS, overexpresses smeE and smeF to the same extent as the spontaneous mutants, and has the same resistance phenotype, confirming that this is the phenotype caused by smeDEF overexpression in a K279a background. Interestingly, all ‘resistance profile 1’ mutants except K279a M1 have a subtly increased susceptibility to amikacin. In contrast, K279a M1 has a slightly decreased amikacin susceptibility level. Therefore, it may be that K279a M1 has a change in addition to that which results in smeE/smeF overexpression. Resistance profile 1 is identical to that displayed by the smeDEF overexpressing drug-resistant mutant K1385 derived from another phylogenetic group A isolate, ULA-511.17 Therefore, there is evidence for a consistent, phylogenetically group A-specific phenotype associated with smeDEF overexpression. We sequenced smeT from the K279a-derived mutants. In K-M1, there is a frameshift in smeT due to the insertion of a cytosine base at position 480 within the gene. In K-M7, a single point mutation was found in smeT, predicted to cause a Thr to Pro change at residue 82 in SmeT. However, in the smeDEF overexpressing mutant N-M6, no mutation is present within smeT or the smeT–smeD intergenic region. Thus, it appears that when smeDEF overexpression is seen in phylogenetically group A isolates, mutations in smeT can be found that are likely to have significant effects on the activity of SmeT. However, mutants that overexpress smeDEF but do not have a mutation in smeT or in the smeT–smeD intergenic region can also be obtained. The obvious explanation for this is that another regulatory mechanism exists to control smeDEF expression. A similar conclusion was drawn in an earlier study of clinical isolates that were shown to overexpress smeDEF, but appeared to have smeT and smeT–smeD intergenic region sequences identical to other isolates that did not overexpress smeDEF.21 It may be that the mutated regulatory locus in K-M6 encodes a protein whose function is to regulate the concentration of a ligand that signals for SmeT to derepress smeDEF expression, as has been shown to be the case with NalC mutants of Pseudomonas aeruginosa, where MexR ceases to be a transcriptional repressor of mexAB oprM expression due to the overproduction of an enzyme that affects MexR ligand concentration.22 Work is in process to address the identity of the mutant locus in K279a M6.
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‘Resistance profile 2’ is reduced susceptibility to amikacin and tetracycline, and is displayed by K279a M2 and M3. ‘Resistance profile 3’ is reduced susceptibility to amikacin and ciprofloxacin, with a slight reduction in tetracycline MIC, seen in K279a M4 and M5. These two phenotypes are not associated with smeE or smeF overexpression; so if an efflux pump is involved, it must be one that has not previously been discovered.
In addition to the MDR profiles observed, three K279a mutants have reduced susceptibility to ß-lactams only (i.e. K279a M8 to M10). However, ß-lactamases were not found to be constitutively overproduced in these mutants. This is further evidence that some non-exclusively-ß-lactamase-mediated ß-lactam resistance mechanism must exist in S. maltophilia, as was suggested in our earlier report.4
The N531 spontaneous mutants (Table 2) also displayed three multi-resistance profiles. ‘Resistance profile 1’, which is expressed by the N531 smeTFS control mutant (i.e. due to smeDEF overexpression), is seen in one spontaneous mutant, N531 M5. This mutant is the only one that overexpresses smeE and smeF, and the same phenotype is expressed by the phylogenetically group B mutant, D457R, which has a mutation in smeT, and overexpresses smeDEF, again further evidence for phylogenetic group-specificity in SmeDEF-mediated resistance in S. maltophilia. We sequenced smeT and the smeT–smeD intergenic region in the N531 M5, but no mutations were found. As with K279a M6, therefore, it is possible that the derepressed phenotype observed is due to the mutation of a locus whose product regulates the concentration of the SmeT ligand.
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‘Resistance profile 2’ is very similar to resistance profile 1, but includes dramatically increased susceptibility to amikacin and reduced susceptibility to ß-lactams. This phenotype is expressed by N531 M3, which was selected using meropenem. Intriguingly, this mutant constitutively overproduces ß-lactamases and also overexpresses smeF (though not smeE). Since SmeF is very unlikely to be able to cause reduced antimicrobial drug susceptibility on its own, it will be very interesting to find which other (novel) efflux pump it can partner in N531, and whether there is a more than coincidental link between efflux pump and ß-lactamase expression in N531 M3. Evidence has already been published that suggests the OMP SmeC might interact with other efflux pumps in S. maltophilia in addition to the non-antimicrobial drug efflux pump SmeB.10 Furthermore, it is well established that OprM, an OMP in P. aeruginosa, can interact with at least two efflux pumps: MexB and MexY.23 Therefore, the possibility that SmeF might interact with another pump is an interesting, but not entirely surprising, finding.
‘Resistance profile 3’ is also similar to profile 1, but MICs are slightly lower and there is a dramatic elevation in the MIC of amikacin. This phenotype is expressed by three mutants, N531 M7, M9 and M10, and is not associated with overexpression of smeE or smeF. This phenotype is reminiscent of K279a resistance profile 3, and it will be interesting to see whether an aminoglycoside MDR efflux pump exists in S. maltophilia.
In addition to the five MDR mutants selected, five other mutants with reduced susceptibility to only one of the tested antimicrobials were observed. These were chloramphenicol (M1), ß-lactams (M2—though ß-lactamase overproduction is not involved), tetracycline (M4 and M6) and ciprofloxacin (M8). None of these mutants overexpresses smeE or smeF.
Analysis of smeDEF expression and resistance phenotypes in a collection of clinical S. maltophilia isolates from around the world
The data reported above suggest that when smeDEF is overexpressed, a predictable, phylogenetic group-specific drug resistance phenotype is generated in S. maltophilia. We therefore tested whether there is any phylogenetic group-specificity in antimicrobial drug resistance and a correlation with smeE and smeF expression in a wider collection of clinical isolates. We examined 30 isolates from Europe and North, South and Central America that have already been phylogenetically grouped according to smeT–smeD intergenic and 16S rRNA sequence. Of 30 isolates, 15 fall into group A; 10 into group B; 2 into group C; and 3 into group D.16 RT–PCR was used to measure mRNA levels for smeE and smeF relative to the gyrA control in these isolates. Only 6/30 isolates have obvious (i.e. more than 5-fold relative to either K279a or N531) overexpression of smeE and smeF: 3/15 from group A, 2/10 from group B and 1/2 from group C. MICs of a variety of known SmeDEF substrate antimicrobials for the 30 isolates were determined (Table 3). There is no correlation between MIC and smeE and smeF expression data. Isolates that overexpress smeDEF exist, but have higher levels of susceptibility to antimicrobials than non-smeDEF overexpressing isolates. Applying a cut-off threshold lower than 5 to identify smeE and smeF overexpressing isolates did mean more were identified. However, we were reticent to use these data in our analysis, since the smeE and smeF sequences are not known for the clinical isolates tested, and might be variable,15 making the RT–PCR data potentially more prone to error (i.e. primer binding sequences might not be exact matches in some cases). Even using a lower cut-off threshold, however, no significant correlations between the MICs of antimicrobials for these isolates and their levels of smeE/smeF expression could be identified. Thus, it appears that whilst smeDEF overexpression in a given background consistently gives a very similar phenotypic result, it is not possible to generalize about the effect of smeDEF overexpression in all S. maltophilia backgrounds. Undoubtedly, this is because of the complex interplay of a wide variety of resistance mechanisms in individual isolates coupled with the fact that S. maltophilia represents a genotypically and phenotypically diverse group of organisms.
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It appears that, as seen previously when looking at ß-lactamase expression and ß-lactam resistance phenotypes, there is really no difference in antimicrobial drug resistance phenotypes of phylogenetically group A and B clinical S. maltophilia isolates—the former are not generally more resistant than the latter. This further begs the question as to why group A isolates are more common, and phylogenetically highly homogeneous, whereas group B isolates are less abundant and highly heterogeneous.16 Group A isolates must have some phenotype that makes them more successful clinically. As far as we can see, however, this phenotype is not related to antimicrobial drug resistance.
Insertion sequences can cause overexpression of smeDEF
The transcriptional repressor SmeT controls expression of smeDEF. However, clinical isolates that overexpress smeDEF, but which have ‘wild-type’ smeT, have been found, implicating the involvement of at least one additional gene product in control of smeDEF expression21—as we have seen using the spontaneous MDR mutants in this report. The problem with interpreting studies of clinical isolates, however, and not spontaneous mutants, is that there is no real wild-type comparator for each clinical isolate; so it is not certain what ‘wild-type’ actually means. Given that the species S. maltophilia is known to be diverse, it is never really clear whether differences between the smeT genes of smeDEF overexpressing isolates compared with unrelated isolates that do not overexpress smeDEF are simply due to phylogenetic drift. Because of this, we did not attempt to compare smeT sequences of those six clinical isolates from our study that were found to overexpress smeDEF with sequences carried by those isolates with lower-level smeDEF expression. We did notice one interesting point, however, when analysing smeT–smeD intergenic sequences of the isolates during phylogenetic analysis. Isolate number 4, which overexpresses smeDEF, consistently gave an 
1270 bp larger than expected smeT–smeD intergenic PCR amplicon. We sequenced around 400 bp of each end of this amplicon, which revealed the presence of an insertion element with high levels of identity (86% over the region sequenced) to IS1246; an IS5-type insertion sequence that can exist as a composite transposon in order to mobilize metabolic genes in Pseudomonas spp.24 Terminal 12 bp inverted repeats of 5'-GGGCACCTCGAA-3' have been previously identified at the extremities of IS1246 and are likely to be transposase recognition sites.24 The same sequence was identified at the 5' terminus of the insert from S. maltophilia clinical isolate 4, and a slight variant, 5'-GGGCGCCTCGAA-3', at the 3' terminus. Figure 1 shows the positioning of the obtained sequence from the insertion in isolate 4 in relation to the sequence of IS1246. In S. maltophilia clinical isolate 4, the new IS element has been inserted into the smeT–smeD intergenic region 65 bp upstream of smeT and 160 bp upstream of smeD. The insertion site is in-between the proposed –35 and –10 elements for smeD (Figure 1). It is highly likely that this insertion derepresses smeDEF transcription from control by SmeT by providing a promoter for smeD without an SmeT binding site. It is not uncommon to find the insertion of IS elements derepressing resistance gene expression, and this has been found before for efflux pumps,25 but it is interesting to note that in 30 randomly selected clinical isolates, and of six that overexpress smeDEF, one has this method of derepression.
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The data presented in this report coupled with that already published show that (i) there is a consistent isolate-specific resistance phenotype associated with smeDEF overexpression in S. maltophilia; this background specificity is due to the complex interplay of multiple mechanisms of resistance in a phylogenetically diverse group of organisms. (ii) Loss-of-function mutations in smeT and mutations or insertions that disrupt SmeT binding to the smeD operator region, lead to overexpression of smeDEF but smeT is not the only regulatory locus contributing to smeDEF expression. (iii) At least two efflux pumps are present in S. maltophilia in addition to smeE: one effluxes multiple antimicrobial drugs plus amikacin and the other effluxes multiple antibiotics and its overexpression is linked to increased susceptibility to amikacin. (iv) There is some evidence for the co-ordinated regulation of ß-lactamase expression and smeF expression, and for smeF having its own promoter independent of the smeDEF operon promoter.
In some respects, this study highlights our lack of knowledge concerning MDR mechanisms in this rapidly emerging pathogen, but the results generated will fuel a series of targeted experiments aimed at understanding these mechanisms in more detail.
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None to declare (both authors).
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Acknowledgements |
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Efflux pump research at the Bristol Centre for Antimicrobial Research and Evaluation is funded by the British Society for Antimicrobial Chemotherapy. We are grateful to Sharon Tomaselli, Department of Microbiology, Southmead Hospital, for selecting the S. maltophilia drug-resistant mutants used in this study and to Professor Keith Poole, Queens University, Kingston, Canada, for providing the suicide vector and E. coli strains required to generate the loss-of-function mutations in S. maltophilia.
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References |
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Sanchez P, Alonso A, Martinez JL. (2002) Cloning and characterization of SmeT, a repressor of the Stenotrophomonas maltophilia multi-drug efflux pump SmeDEF. Antimicrob Agents Chemotherapy 46:3386–93.
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Gould VC, Okazaki A, Howe RA, et al. (2004) Analysis of sequence variation amongst smeDEF multi-drug efflux pump genes and flanking DNA from defined 16S rRNA sub-groups of clinical Stenotrophomonas maltophilia isolates. J Antimicrob Chemother 54:348–53.
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