Bristol Centre for Antimicrobial Research and Evaluation (BCARE), Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK
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Abstract |
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Introduction |
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The mechanisms of antibiotic resistance in S. maltophilia have not been studied in detail, though isolates resistant to all known aminoglycosides, quinolones and ß-lactams, and to chloramphenicol, rifampicin, tetracycline and trimethoprim, have been reported.3,4 A multi-drug efflux system has recently been described in S. maltophilia,5 but it is believed to play only a minor role in resistance to ß-lactams, which is mediated mainly by the inducible expression of two ß-lactamases, L1 and L2.68 L1 is a broad-spectrum metallo-ß-lactamase that hydrolyses carbapenems,6 while L2, a serine enzyme, is active principally against cephalosporins.7 The genes encoding these enzymes have been cloned and sequenced,9,10 but little is known about the regulation of their transcription.
Strains of S. maltophilia that express additional ß-lactamases have been described.1113 In this study, we were particularly interested in investigating the presence of TEM-type ß-lactamases in clinical isolates of S. maltophilia, because the spread of such enzymes into this species has not been reported.
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Materials and methods |
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Clinical isolates of S. maltophilia were obtained over a period of several years from blood cultures of bacteraemic oncology patients being treated at the Bristol Royal Infirmary. Isolates were plated on to nutrient agar (Oxoid plc, Basingstoke, UK) to check culture purity and the identity of each was confirmed using API 20NE test strips (bioMérieux, La Balme les Grottes, France). Bacteria were grown at 37°C in air unless otherwise stated. Bacterial strains and plasmids used in this study are listed in Table I.
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Unless otherwise stated, media used were nutrient broth and nutrient agar (Oxoid plc, Basingstoke, UK). ß-Lactams used were nitrocefin, clavulanic acid and BRL 42715 (SmithKline Beecham, Worthing, UK); ampicillin, carbenicillin, oxacillin, cephalothin, cephaloridine and ceftazidime (Sigma Chemical Co., St Louis, MO, USA); piperacillin (Lederle, Carolina, Puerto Rico) and meropenem (Zeneca Pharmaceuticals, Macclesfield, UK). PCR primers were purchased from SigmaGenosys Ltd (Pampisford, UK). All other general reagents were from Sigma Chemical Co. (Poole, UK) or BDH (Poole, UK).
Susceptibility tests
Antibiotic susceptibility was determined by Etest (AB Biodisks, Solna, Sweden) on Isosensitest agar (Oxoid) with an inoculum of 0.5 McFarland. The MIC of the test ß-lactam was defined as the lowest concentration of the antibiotic that prevented growth after incubation at 37°C for 24 h.
Preparation of ß-lactamases
Bacterial strains were cultured overnight in nutrient broth with shaking at 37°C. Bacteria were harvested by centrifugation (10 min, 4°C, 3500g) and the pellet was washed twice in 10 mL of ice-cold extraction buffer {50 mM MOPS [3-(N-morpholino) propane-sulphonic acid], pH 7.0}. After resuspension in 1 mL of extraction buffer, the cells were disrupted using a Hybaid Ribolyser (Hybaid, Teddington, UK) in tubes containing silica beads (Hybaid Blue matrix), with a single 30 s burst (amplitude 6). Cell debris and silica beads were pelleted by centrifugation (10 min, 4°C, 15000g) and the supernatant was transferred to a clean tube and used directly as a source of ß-lactamase.
ß-Lactamase assays
Hydrolysis of ß-lactam antibiotics was examined by spectrophotometric assay (LKB Ultraspec III; Pharmacia, St Albans, UK) in 1 cm light-path cuvettes with readings recorded at 2 s intervals for 3 min at the wavelength of optimal absorbance of the ß-lactam ring of each drug.10 Antibiotic solutions (100 µM) were prepared in 50 mM MOPS, pH 7.0. The protein concentration of each bacterial extract was determined using the Bio-Rad protein assay reagent (Bio-Rad, München, Germany) according to the manufacturer's instructions. One unit of ß-lactamase activity is defined as that required to hydrolyse 1 µmol substrate per minute at 25°C. Specific activity is therefore defined as the number of units/mg of protein in the assay.
Isoelectric focusing
Ten micrograms of total protein from each bacterial extract (above) were resolved by isoelectric focusing gel electrophoresis and ß-lactamases were visualized as described previously.14
Preparation of DNA template and polymerase chain reaction
For colony (genomic) polymerase chain reaction (PCR), a single bacterial colony was suspended in 50 µL of water, the suspension was boiled for 5 min and cell debris was pelleted by centrifugation (15000g, 10 min). Twenty microlitres of the supernatant was used directly as a source of DNA. PCR was performed using 5 U of Super-Taq DNA polymerase (HT Biotechnology Ltd, Cambridge, UK) in a final volume of 50 µL of 10 mM TrisHCl pH 9.0 containing 50 mM KCl, 1.5 mM MgCl2, 0.1% (v/v) Triton X-100, 0.2 mM each of dATP, dCTP, dGTP and dTTP and 2 µM of the appropriate reverse and forward primers. The TEM PCR primers were (forward) 5'-CTGGATCTCAACAGCGGT-3' and (reverse) 5'-CAGCCGGAAGGGCCGAGC-3' and the TN-TEM primers were (forward) 5'-CGACATGATCCAACTGAT-3' and (reverse) 5'-CTGACAGTTACCAATGCT-3'. After a 96°C denaturation for 5 min, PCR was performed for 30 cycles of 1 min incubations at 96°C, 55°C then 72°C.
TA cloning of the TN-TEM PCR product
The 1.4 kb TN-TEM PCR amplicon, obtained by PCR as described above, was purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The amplicon was ligated into the pCR 2.1 TA cloning vector (Invitrogen, Leek, The Netherlands) using the manufacturer's protocol and Escherichia coli DH5 cells were transformed with the ligation mixture by electroporation.15 Transformants were selected on nutrient agar containing 30 mg/L kanamycin, 1 mM isopropyl-ß-d-thiogalactopyranoside and 40 mg/L of 5-bromo-4-chloro-3-indolyl phosphate. Cells containing the vector with an insert grew as white colonies, while those containing just the empty vector grew as blue colonies.15 Several white colonies were chosen and cultured overnight in nutrient broth; plasmid DNA was then isolated and purified (Plasmid Recovery Kit; Hybaid). The cloned insert of each recombinant was cut from the vector by digestion with EcoRI and DNA fragments of the expected size were excised from the gel, cleaned using a Qiagen QIAquick gel extraction kit and then cloned into EcoRI-linearized cloning vector pK18.16 One such construct was denoted pUB6051. Constructs based on pK18 are replicated to a higher copy number than those based on pCR 2.1 (M. B. Avison, unpublished data), so this procedure enabled the production of large amounts of plasmid DNA for sequencing of the pUB6051 insert.
DNA sequencing and sequence analysis
Sequencing of the insert from pUB6051 was initiated by using primers targeted to the multiple cloning site of pK18.16 A primer-walking strategy15 was then employed to complete sequencing of the insert on both strands. The entire cloning and sequencing procedure was repeated three times using separate PCR products to limit the possibility of PCR error altering the sequence obtained. All three sequences were identical.
Bacterial mating experiments
An overnight culture of each bacterium to be mated was prepared using nutrient broth. One hundred microlitres of each culture was mixed in the centre of a nutrient agar plate containing no antibiotics and the mixture was incubated overnight at 37°C. A loopful of the mixed growth was then streaked on a nutrient agar plate containing the appropriate selective agents and incubated overnight at 37°C.
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Results |
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TEM ß-lactamases are sometimes encoded within Tn1- or Tn3-type transposable elements.19,20 To determine if this was the case with the blaTEM gene from isolate J675Ia, PCR was performed using J576Ia genomic DNA as a template and primers (TN-TEM primers) based on sequences found in the resolvase gene and blaTEM 5'-flanking region that are identical in Tn1 and Tn3 (EMBL accession numbers L10085 and J01832).19,20 An appropriately sized PCR product (1.4 kb) was observed, indicating that blaTEM is present in a Tn1/Tn3 context (data not shown). To discover more about the blaTEM gene and the transposon of which it is a part, the TN-TEM amplicon was cloned in the pCR 2.1 TA vector and sequenced as set out in Materials and methods (EMBL accession number AJ251946). The differences between the J676Ia TN-TEM and the equivalent regions of the published Tn1 and Tn3 sequences19,20 are shown in Table IV. When examining the 1.4 kb region of J675Ia Tn that had been sequenced, three nucleotide differences from Tn1 and 11 nucleotide differences from Tn3 were detected (Table IV
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Discussion |
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Mobile ß-lactamase genes have been an increasing problem in clinical practice since the first use of ß-lactams.24 Several such genes have become embedded within transposable elements and/or transferred on to plasmids, and so have been disseminated from one organism to another, often crossing from one genus to another. In most cases, in fact, it is not certain from which organism the ß-lactamase gene originated.24 The most common of these enzymes are the TEM and SHV penicillinases and the various extended-spectrum and inhibitor-resistant variants derived from them.25 Additionally, there are the OXA and PSE enzymes with broad-spectrum penicillinase activities,25 and the more recently emerging metallo-carbapenemases IMP-1 and VIM-1.2628 These enzymes, particularly the extended-spectrum ß-lactamases, have undoubtedly been responsible for many therapeutic failures worldwide.25
Mobile ß-lactamase genes may be lost, together with the genetic element carrying them, if they do not confer a survival advantage to the organism that replicates them.24 These genes are often found in otherwise ß-lactam-sensitive organisms, for example E. coli, Salmonella typhimurium and Klebsiella pneumoniae, where they confer an obvious advantage.24,25 Expression of the TEM enzyme significantly increases the MIC of ampicillin and piperacillin for J675Ia when compared with those S. maltophilia isolates that lack the blaTEM gene. The TEM-negative strains are, however, already resistant to these ß-lactams (Table II),17 so the survival advantage conferred by blaTEM in J675Ia is not obvious when simple breakpoints are examined. However, it is likely that a combination of a higher intrinsic ß-lactamase activity, together with constitutive production, offers a selective advantage in vivo.
Given that J675Ia, like the other S. maltophilia isolates examined, does not appear to have acquired a plasmid (data not shown), the blaTEM-containing transposon is presumably present on the chromosome. As expected, therefore, the blaTEM gene cannot be transferred directly to E. coli via conjugation, even if the conjugative machinery is provided by R388. After a mating of J675Ia:R388 with E. coli UB1832, transconjugants with ampicillin (500 mg/L) and rifampicin (50 mg/L) resistance were obtained; all those examined were trimethoprim resistant and contained a single 35 kb plasmid, the predicted size of R388 plus the transposon (data not shown). Thus the transposon in J675Ia can be mobilized on to R388 and then conjugated into other bacteria.
This report is the first confirmed example of a transposon-mediated TEM ß-lactamase in the genus Stenotrophomonas and, as such, widens the known repertoire of TEM. Clearly, even multi-drug-resistant organisms such as S. maltophilia harbour, and so act as a reservoir for, mobile resistance determinants, and can exchange genetic material with other bacteria, e.g. E. coli, which are often found in the same clinical environment.
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Acknowledgments |
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Notes |
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References |
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Received 27 March 2000; returned 16 June 2000; revised 22 June 2000; accepted 16 August 2000