Department of Microbiology and Immunology, Queens University, Kingston, Ontario, Canada K7L 3N6
Received 17 October 2002; returned 6 December 2002; revised 8 January 2003; accepted 11 January 2003
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Abstract |
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Keywords: aminoglycoside, resistance, Stenotrophomonas maltophilia
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Introduction |
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S. maltophilia shows unusually high levels of resistance to aminoglycoside antibiotics, despite the fact that most Gram-negative bacilli are generally quite susceptible to this class of antibiotics.7,1315 Mechanisms of bacterial resistance to aminoglycosides are many, and include reduced uptake, mutational modification of the 16S rRNA, mutational modification of ribosomal proteins, enzymic modification of the 16S rRNA and enzymic modification of antibiotics.16,17 More recently, active efflux of aminoglycosides has emerged as an additional mechanism of aminoglycoside resistance in Gram-negative bacteria,18,19 including S. maltophilia.10,20 Still, aminoglycoside resistance among Gram-negative bacilli is predominantly due to enzymic modification of aminoglycosides by a family of enzymes, including O-nucleotidyltransferases, O-phosphotransferases and N-acetyltransferases (AACs).16,17,21 The latter, AACs, are acetylCoA-dependent acetyltransferases that primarily modify amino groups and often lead to resistance to the aminoglycosides that possess amino groups.21,22 The presence of an AAC aminoglycoside-modifying enzyme in S. maltophilia was first suggested by King et al.,23 whereas Vanhoof et al.24 postulated an O-nucleotidyltransferase in this organism. Still, the genetic identity of the modifying enzymes in S. maltophilia remained unknown. A gene encoding the AAC(6')-Iz acetyltransferase of S. maltophilia was previously cloned and sequenced.25 This enzyme was proposed to be the determinant for intrinsic aminoglycoside resistance in S. maltophilia,25 although no direct evidence was provided. Here we report on the influence of chromosomal deletions of the aac(6')-Iz gene on aminoglycoside resistance of S. maltophilia, and the distribution of the aac(6')-Iz gene in clinical isolates of S. maltophilia.
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Materials and methods |
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The bacterial strains and plasmids used in this study are listed in Table 1. S. maltophilia strain K1668 derived from strain K1449 was a multidrug-resistant mutant selected on ciprofloxacin and cefsulodin, and this mutant strain displayed increased resistance to aminoglycosides, ß-lactams and fluoroquinolones via an efflux-mediated mechanism.20 Sixty-five clinical isolates of S. maltophilia included strains K1013 to K1029 from Kingston General Hospital (Kingston, Ontario, Canada) and strains K1319 to K1366 from Mount Sinai Hospital (Toronto, Ontario, Canada). LuriaBertani (LB) broth [1% (w/v) Difco tryptone, 0.5% (w/v) Difco yeast extract and 0.5% (w/v) NaCl] and agar [LB broth containing 1.5% (w/v) agar] were used as the growth media throughout, and bacterial cells were cultivated at 37°C. In some instances, aminoglycoside susceptibilities were carried out using nutrient broth (NB) [5% (w/v) Difco beef extract, 3% (w/v) Difco peptone]. Plasmids were maintained in E. coli with appropriate antibiotic selection [pBluescript II SK(+), 100 mg/L ampicillin; pRK41520 and pEX18Tc,26 10 mg/L tetracycline; and pRK2013,20 50 mg/L kanamycin].
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Drug susceptibility testing was carried out by two-fold serial dilution using LB or NB media (1 mL) with an inoculum of 5 x 105 cells/mL. Data were reported as MICs, which reflected the lowest concentration of antibiotic inhibiting visible cell growth after an overnight incubation at 37°C.
DNA methodology
Basic DNA procedures, including restriction endonuclease digestions, ligations, transformations and agarose gel electrophoresis were carried out as described by Sambrook et al.27 The alkaline lysis method or a plasmid midi kit (Qiagen Inc., Mississauga, Ontario, Canada) was used to isolate plasmids from E. coli DH5 and S. maltophilia.27 The genomic DNA of S. maltophilia was extracted as described previously.28 DNA fragments used in cloning were extracted from agarose gels using Prep-A-Gene (Bio-Rad Labs, Richmond, CA, USA) as per the manufacturers instructions. Nucleotide sequencing of plasmid-borne DNA or PCR products was carried out by Cortec DNA Services Inc. (Kingston, Ontario, Canada) using universal or custom primers. Compilation of DNA sequence data was carried out using DNAMAN (Version 4.11; Lynnon Biosoftware, Vaudreuil, Quebec, Canada).
PCR amplification and cloning of aac(6')-Iz
The aac(6')-Iz gene (GenBank accession no. AF140221) was amplified from genomic DNA of S. maltophilia ULA-511 on a 1 kb fragment using primers smaac1xz and smaac4xz (Table 2) and conditions described previously,20 although with a modified annealing temperature of 56°C for 1 min. The aac(6')-Iz-containing PCR product was purified using a Qiaquick PCR purification kit (Qiagen Inc.), and following digestion of the PCR products with HindIII and SstI it was cloned into HindIIISstI-restricted pBluescript II SK(+). The nucleotide sequence of aac(6')-Iz in the resultant plasmid, pLZ651, was confirmed by nucleotide sequencing. The aac(6')-Iz gene of pLZ651 was then released by digestion with HindIII and SstI, and cloned into HindIIISstI-restricted pRK415, yielding plasmid pLZ655. This plasmid was subsequently mobilized from E. coli DH5
into S. maltophilia via a triparental mating procedure employing the helper strain MM294 carrying vector pRK2013.20 Transconjugants were selected on LB agar supplemented with 5 mg/L norfloxacin (to counterselect the E. coli) and 50 mg/L tetracycline (to select plasmid-bearing transconjugants). PCR as above was also used to assess the presence of the aac(6')-Iz gene in 65 clinical isolates of S. maltophilia.
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To construct aac(6')-Iz mutants, two PCR assays were carried out to amplify two
0.5 kb DNA fragments [upstream and downstream of the aac(6')-Iz gene sequence to be deleted]. Sequences 5' to the deletion were amplified from genomic DNA of S. maltophilia ULA-511 using primers smaac1xz and smaac2xz (Table 2), whereas sequences 3' to the deletion were amplified using primers smaac3xz and smaac4xz (Table 2). The two PCR products were digested with HindIIIXbaI and XbaISstI, respectively, and cloned into HindIIIaSstI-restricted pBluescript II SK(+) via three-piece ligation, yielding pLZ650. The intragenic deletion of 107 bp generated in the aac(6')-Iz gene of pLZ650 was confirmed by nucleotide sequencing. Following digestion of pLZ650 with HindIII and SstI, the
aac(6')-Iz gene-containing fragment was cloned into gene replacement vector pEX18Tc previously digested with HindIII and SstI. The resultant plasmid, pLZ653, was used to transform E. coli S17-1, from which it was mobilized into strains K1449 and K1668 and several clinical isolates via conjugation as described.10,20 Transconjugants carrying pLZ653 in the chromosome were selected on LB agar containing tetracycline (40 mg/L for strain K1449; 60 mg/L for strain K1668; 50 mg/L for strains K1319 and K1324) and norfloxacin (2.55 mg/L; for counter-selection). Transconjugants were then streaked on to LB agar containing 10% (w/v) sucrose and sucrose-resistant colonies arising after overnight incubation at 37°C were screened for the presence of the aac(6')-Iz deletion using PCR and/or Southern hybridization (see below).
Southern hybridization
Genomic DNA of S. maltophilia was digested with PstI and the resultant DNA fragments were separated on a 0.8% (w/v) agarose gel before being blotted onto positively charged Nylon membranes (Roche, Laval, Quebec, Canada). Membranes were subjected to overnight hybridization with an aac(6')-Iz-specific probe and detected using the DIG High Prime DNA labelling and detection starter kit II (Roche) as per the manufacturers instructions. The aac(6')-Iz probe was obtained from pLZ651 by PCR amplification of a 370 bp intragenic fragment using primers smaac5xz and smaac6xz as above. The aac(6')-Iz PCR product was purified as above and labelled using the DIG High Prime DNA labelling kit (Roche) according to the manufacturers instructions.
RTPCR
RTPCR was carried out as published previously28 using the aac(6')-Iz-specific primers smaac5xz and smaac6xz and 40 cycles of amplification.
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Results and discussion |
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S. maltophilia generally displays high level resistance to a wide variety of antibiotics, including aminoglycosides,6,10,28,29 a result confirmed here (Tables 3 and 4). To assess the role of the previously described aac(6')-Iz gene in the aminoglycoside resistance of S. maltophilia, chromosomal deletions of the aac(6')-Iz gene were carried out as described in Materials and methods. Elimination of aac(6')-Iz in the wild-type S. maltophilia strain K1449 (yielding strain K1669) resulted in an increase in susceptibility to the 2-deoxystreptamine aminoglycoside antibiotics including netilmicin, sisomicin, tobramycin and neomycin. This is not unexpected as these aminoglycosides are known substrates for the AAC(6')-I-modifying enzymes since their 6'-NH2 group of the 6-aminohexose is subject to acetylation.21,22,25,30 Interestingly, gentamicin susceptibility is also increased four-fold upon loss of the aac(6')-Iz gene (Table 3). Gentamicin is a complex of four closely related subspecies C1, C1a, C2 and a minor component C2a, where all differ structurally at the site of acetylation, the 6'-NH2 group.22 Gentamicins C1a, C2 and C2a, however, do present a 6'-NH2 and are likely to be modified at this site.22 These gentamicin components are similarly modified by other AAC(6')-I enzymes.3133 The observation that susceptibility to kanamycin was not affected by loss of AAC(6')-Iz is, perhaps, surprising given the presence of the 6'-NH2 on this aminoglycoside. Still, it has previously been demonstrated that the in vitro substrate profiles of aminoglycoside-modifying enzymes do not necessarily completely mirror the aminoglycoside resistance profile of organisms expressing these enzymes.31,33,34 Also, aminoglycoside resistance is often multifactorial in nature, being affected by, for example, decreased permeability, active efflux and multiple aminoglycoside-modifying enzymes. Thus, the lack of an effect of the aac(6')-Iz deletion on kanamycin resistance in strains K1669 and K1670 might be explained by the presence of an aminoglycoside phosphotransferase [APH(3')] in these strains. Although AAC(6')-Iz is the only aminoglycoside-modifying enzyme to be described to date in S. maltophilia, Vanhoof et al.24 have proposed the presence of other aminoglycoside-modifying enzymes in this organism.
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Conservation of the aac(6')-Iz gene in S. maltophilia
To determine whether the aac(6')-Iz gene was ubiquitous in S. maltophilia, amplification of the gene was carried out from a number of clinical strains using primers that annealed upstream and downstream of the aac(6')-Iz gene present in strain ULA-511. Of the two reference strains (i.e. ATCC13637 and ULA-511) and 65 clinical isolates tested, the resultant PCR products generally displayed three patterns. First, most S. maltophilia strains (37/65) yielded a 1 kb fragment, the size expected from the sequence data (GenBank accession no. AF140221),25 and DNA sequencing of several of these (e.g. ULA-511) confirmed the presence of the intact aac(6')-Iz gene.25 Less frequently (21/65), a PCR product of 0.6 kb was obtained. Nucleotide sequencing of this smaller fragment, obtained from six representative clinical isolates, K1018 and K1025 from Kingston General Hospital, and K1337, K1352, K1354 and K1355 from Mount Sinai Hospital in Toronto, revealed the absence of the aac(6')-Iz gene. Interestingly, however, the nucleotide sequences immediately upstream and downstream of the aac(6')-Iz gene25 (GenBank accession no. AF140221, derived from ATCC13637) were retained in these apparently aac(6')-Iz strains, indicating that there was a specific loss of the aac(6')-Iz gene only. Several base pair differences were, however, noted in these upstream/downstream sequences in the various aac(6')-Iz mutants examined, indicating that they were probably not derived from a single clone. Some strains (7/65) produced inconsistent PCR products of variable size, which might indicate the absence of the aac(6')-Iz gene or, possibly, alterations in sequences immediately upstream or downstream of the gene. A second PCR was undertaken using aac(6')-Iz-specific intragenic primers (i.e. smaac5xz and smaac6xz, or smaac7xz and smaac8xz; Table 2). The expected PCR product of
0.3 kb was amplified from genomic DNA of those strains containing the aac(6')-Iz gene (e.g. strains ATCC13637, ULA-511, K1019, K1327, K1341 and K1366) (data not shown). In contrast, those strains that previously failed to amplify a 1 kb fragment using primers annealing immediately upstream and downstream of the aac(6')-Iz gene also failed to yield a PCR product with the intragenic primers (data not shown). These data are consistent with the absence of the acetyltransferase gene in these strains.
Using a combination of dot blots, Southern hybridization with an intragenic aac(6')-Iz-specific probe and PCR, Lambert et al.25 previously demonstrated that this gene was conserved in all 80 strains examined and at the same location in the genome. To reconcile these data with our own, Southern hybridization was employed to re-examine the distribution of the aac(6')-Iz gene in the aforementioned S. maltophilia clinical isolates (Figure 1). Using a 370 bp intragenic aac(6')-Iz-specific probe, strains confirmed as having the aac(6')-Iz gene hybridized with the expected 1 kb PstI fragment (e.g. Figure 1). Intriguingly, strains confirmed as having a specific deletion of the aac(6')-Iz gene also yielded a hybridization signal, which was consistently
0.6 kb in size (e.g. Figure 1). This result was consistent with the probe hybridizing to the same PstI fragment that was, however, smaller due to loss of the aac(6')-Iz sequences. Possibly, the probe is able to cross-hybridize with sequences upstream or downstream of the aac(6')-Iz gene, although no obvious aac(6')-Iz-like sequences were identified. Alternatively, a related gene conserved elsewhere in the chromosome may be cross-hybridizing in these instances.21 Interestingly, those strains that failed to yield specific PCR products with primers internal or external to the aac(6')-Iz gene also yielded modified hybridization signals, although these varied from strain to strain (e.g. Figure 1). Whether this means that an aac(6')-Iz gene is present but in a variety of locations in the genomes of these strains, in contrast to earlier reports where its position appeared to be conserved,25 or that this gene is absent and Southern hybridization is identifying related sequences is unclear. The fact that strains with precise deletions of the aac(6')-Iz gene nonetheless elicit a hybridization signal certainly suggests that some cross-reactivity is possible with this probe. Moreover, the Lambert et al.25 dot blot and PCR data were in complete agreement as regards the presence of the aac(6')-Iz gene in all strains of S. maltophilia examined, again suggesting that the less stringent Southern hybridization might here be detecting related as opposed to aac(6')-Iz-specific sequences. In any case, the aac(6')-Iz gene does not appear to be completely conserved in S. maltophilia, and so is unlikely to be a housekeeping resistance gene. The differences between the results presented here and those reported by Lambert et al.25 may reflect geographical issues (isolates from Canada versus isolates from France).
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Acknowledgements |
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Footnotes |
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References |
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