A TEM-2 ß-lactamase encoded on an active Tn1-like transposon in the genome of a clinical isolate of Stenotrophomonas maltophilia

Matthew B. Avison*, Charlotte J. von Heldreich, Catherine S. Higgins, Peter M. Bennett and Timothy R. Walsh

Bristol Centre for Antimicrobial Research and Evaluation (BCARE), Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A constitutively expressed ß-lactamase gene from a clinical isolate of Stenotrophomonas maltophilia, J675Ia, has been cloned. Its DNA sequence is almost identical to that of blaTEM2 (one nucleotide change) and the expressed enzyme is a Bush type 2a penicillinase with an amino acid sequence identical to that of TEM-2. The blaTEM gene was present within a novel Tn1/Tn3-type transposon in the genome of isolate J675Ia and the transposon was able to mobilize blaTEM on to the broad host-range conjugative plasmid, R388. When transferred to an Escherichia coli recipient, R388::Tn conferred high-level ampicillin resistance. This represents the first identification of a TEM ß-lactamase in S. maltophilia and the first evidence that this important clinical pathogen is able to act as a reservoir for mobile ß-lactamase genes in the hospital environment.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Stenotrophomonas maltophilia is a classic example of an emerging pathogen.1,2 Its tolerance to silver-lined catheters and its inherent resistance to many antibacterial drugs, including most ß-lactam antibiotics,14 has given it a survival advantage over other potential pathogens in the hospital environment. As a cause of nosocomial nonfermenting Gram-negative bacteraemia, it is second only to Pseudomonas aeruginosa, and the frequency of its isolation is increasing rapidly.1,2

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

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 IGo.


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Table I. The bacterial strains and plasmids used in this study
 
Materials

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 Sigma–Genosys 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 Tris–HCl 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{alpha} 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clinical isolates of S. maltophilia were collected from patients suffering from bacteraemias that had not responded to ß-lactam therapy. MICs of six ß-lactam antibiotics for the isolates were determined (Table IIGo). All isolates were found to be resistant (according to BSAC breakpoints for the Enterobacteriaceae)17 to imipenem (MIC > 32 mg/L) and cefotaxime (MIC 1–4 mg/L) and susceptible to meropenem (MIC 0.75 mg/L) and ceftazidime (MIC 0.25–0.38 mg/L). One isolate, J675Ia, showed greater resistance to piperacillin (>256 mg/L) and ampicillin (>32 mg/L) than the other four (MIC 16–32 and 3–4 mg/L, respectively). Measurement of basal ß-lactamase levels (performed using extracts of cells grown in the absence of a ß-lactam) revealed that isolate J675Ia expressed an enzyme that rapidly hydrolysed ampicillin (Table IIIGo). The enzyme must have a serine active site, because its activity was completely inhibited by the serine ß-lactamase inhibitors clavulanic acid and BRL42715 (10 µM) but not affected by EDTA (100 mM) (Table IIIGo), which is known to inhibit metallo-ß-lactamases.8 The rest of the S. maltophilia isolates tested showed the more usual pattern of low-level basal ß-lactamase production (Table IIIGo) that has been reported previously in S. maltophilia.610


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Table II. MICs of various ß-lactams for the S. maltophilia isolates
 

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Table III. The substrate profiles of ß-lactamase activities found in extracts from uninduced clinical S. maltophilia isolates
 
To help assess the nature of the constitutive penicillinase, isoelectric focusing gel electrophoresis was performed to separate the ß-lactamases of isolate J675Ia. The ß-lactamases were visualized with the chromogenic ß- lactam nitrocefin.14 Besides the usual L1 (pI = 6.5) and L2 (pI = 8.4) ß-lactamases,610 J675Ia also expressed a third enzyme with a pI of 5.6 (data not shown). This pI is similar to those of the widely distributed TEM ß-lactamases.18 To determine if J675Ia carried a blaTEM gene, PCR was performed using primers based on two intragenic regions conserved within all known blaTEM genes.18 When J675Ia genomic DNA was the template, an amplicon of the expected size (750 bp) was obtained. No such product was obtained when genomic DNA from the other isolates was used (data not shown).

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 IVGo. 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 IVGo).


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Table IV. Differences in the nucleotide sequence of Tn1-type transposons
 
Isolate J675Ia (susceptible to rifampicin and trimethoprim; resistant to ampicillin) was not able to transfer ampicillin resistance to E. coli strain UB1832 (resistant to rifampicin; susceptible to trimethoprim and ampicillin)21 after a standard bacterial mating (see Materials and methods). The 30 kb conjugative plasmid, R388 (which confers trimethoprim resistance),22 was successfully transferred to J675Ia by conjugation from donor E. coli strain C600 (ampicillin-susceptible),23 assessed by the recovery of colonies that showed high-level resistance to trimethoprim (200 mg/L) and ampicillin (500 mg/L). The J675Ia:R388 construct was then mated with E. coli UB1832 and colonies that were resistant to ampicillin (500 mg/L), trimethoprim (50 mg/L) and rifampicin (50 mg/L) were recovered. These transconjugants contained a single 35 kb plasmid, which is likely to represent R388::Tn (data not shown).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
S. maltophilia J675Ia carries a transposon with very high homology to Tn1 and Tn3 (Table IVGo). The transposon contains a blaTEM gene encoding a TEM-2 ß-lactamase, i.e. the expressed enzyme has the same amino acid sequence as TEM-2 (Table IVGo). This represents the first confirmed example of a TEM ß-lactamase in S. maltophilia.

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 IIGo),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.


    Acknowledgments
 
We thank Dr Emma Williamson, Bristol Royal Infirmary, Bristol, UK, for donating the clinical isolates used and Dr Jenny Jury, Department of Biochemistry, University of Bristol, for DNA sequencing. This work was funded by a grant from the Wellcome Trust. C.S.H. is in receipt of a Biotechnology and Biological Sciences Research Council CASE studentship in collaboration with SmithKline Beecham Pharmaceuticals.


    Notes
 
* Corresponding author. Tel: +44-117-9287541; Fax: +44-117-9287896; Email: Matthewb.Avison{at}bris.ac.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 27 March 2000; returned 16 June 2000; revised 22 June 2000; accepted 16 August 2000