a Department of Microbiology, The General Infirmary at Leeds, Great George Street, Leeds LS1 3EX b Department of Microbiology, University of Leeds, Leeds LS2 9JT, UK
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Abstract |
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Introduction |
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ß-Lactamase production by S. maltophilia is not as simple as first thought. Studies in the early 1980s revealed the presence of two different ß-lactamases designated L1 2 and L2.3 However, heterogeneity of S. maltophilia ß-lactamase production was reported from Germany in 1990 4 and two further studies have confirmed this. In one study 17 clinical isolates from the UK were found to possess seven metallo-ß-lactamases and eight serine ß-lactamases5 and another analysing seven isolates of S. maltophilia isolated from blood cultures revealed four ß-lactamases, 6 one metallo-ß-lactamase and three serine ß-lactamases. Finally, recent studies have shown molecular heterogeneity within the L1 ß-lactamase gene. The nucleotide sequence of the L1 blaS gene of S. maltophilia GN128737 shows 88.6% homology with the published sequence of the L1 blaS gene of S. maltophilia IID1275.8
All studies demonstrating heterogeneity of ß-lactamase production have only analysed clinical isolates and none have used typing to assess clonal relationships of isolates that appear to possess the same ß-lactamases. In this study we investigated the heterogeneity of ß-lactamase production using a collection of clinical and environmental isolates of S. maltophilia and analysed their clonal relationships using pulsed field gel electrophoresis (PFGE).
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Materials and methods |
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Twenty-seven isolates of S. maltophilia were analysed; 17 of clinical (C) and nine of environmental (E) origin. One clinical isolate was collected from the sputum of each of 13 different cystic fibrosis patients over a 26 month period and a further four from another cystic fibrosis patient at different times over a 20 month period. The nine environmental isolates were collected from sites on an in-patient ward used by patients with cystic fibrosis (six), their out-patient clinic (one) and from the homes of two of the colonized cystic fibrosis patients (two). One reference strain, S. maltophilia NCTC 10258, was also used. All isolates were confirmed as S. maltophilia using API 20NE (bio-Mérieux, Marcy l'Etoile, France) and stored at -70°C in 15% glycerol broth until required.
Antibiotic susceptibilities
The MICs of six different ß-lactam antibiotics were determined using an agar dilution method. Antibiotics were obtained from the following sources: ampicillin, cefta- zidime and cephradine from Sigma Chemical Co., Poole, UK; aztreonam from Bristol Myers Squibb, Hounslow, UK; imipenem from MSD, Hoddesdon, UK; meropenem from Zeneca, Wilmslow, UK. Each strain was grown overnight in 10 mL nutrient broth and diluted 1:100 using sterile saline to give 104 cfu/spot when inoculated using a multipoint inoculator (Mast Laboratories Ltd, Liverpool, UK). Plates were incubated overnight at 37°C in air. Pseudomonas aeruginosa NCTC 10662 and Escherichia coli NCTC 10418 were included as controls. Plates were read by eye and single colonies or a barely visible haze of growth were disregarded. The results were analysed using Pearson's correlation coefficient.
Isoelectric focusing
ß-Lactamase was extracted from each strain of S. maltophilia using a method described previously.5 LuriaBetani broth was used in place of Nutrient Broth No. 2 and strains were incubated at 30°C. Isoelectric focusing (IEF) was performed by adapting the method of Mathew et al. 9 Each gel was composed of 0.3 g of IEF agarose (Pharmacia Biotech, St Albans, UK), 3.6 g of sorbitol and 27 g of ultrapure distilled water; 1.9 mL Pharmalyte (pH 310 or pH 56) (Pharmacia Biotech, St Albans, UK) was added before pouring. Electrophoresis was performed using an FBE-3000 electrophoresis unit (Pharmacia, Uppsala, Sweden) with settings at 1500 V, 15 W, for 1.5 h at 10°C. The isoelectric point of each ß-lactamase (pI) was determined by comparison with a pH 4.710.6 pI calibration kit (BDH Chemicals Ltd., Poole, UK). Enzyme inhibition characteristics were determined using overlay techniques described by Payne et al. 5 Clavulanic acid (100 µM) (SmithKline Beecham, Welwyn Garden City, UK) was used instead of BRL 42715 because the latter is no longer available.
Pulsed field gel electrophoresis
The PFGE method used has been described elsewhere, 10 except that strains were inoculated into Tryptone-Soya broth (Oxoid, Basingstoke, UK) to obtain exponential growth and a different lysis buffer (6 mM TrisHCl pH 7.6, 100 mM EDTA pH 8.0, 1 M NaCl, 0.2% deoxycholic acid, 0.5% polyoxyethelene cetyl ether (Brij 58), 0.5% lauroylsarcosine; 2 mg/mL lysozyme, 30 µg/mL RNase) was used. DNA was digested with 10 U of XbaI restriction endonuclease and electrophoresis performed in 1.2% agarose gels using a Gene-Navigator system (Pharmacia, Uppsala, Sweden) over 20 h at 14°C with 535 s linear ramping at six V/cm. PFGE profiles were interpreted using the criteria of Tenover et al, 11 and assigned to `types'.
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Results |
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Discussion |
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The variation in ß-lactam MICs for isolates with indistinguishable genotypes may relate
to differences in ß-lactamase expression. Eight clinical isolates had meropenem MICs
64 mg/L without induction, suggesting that these may have been mutants overexpressing
L1,1 but unlike those described by Akova et al,
1 all but two of these remained susceptible to ceftazidime.
Interestingly, there was still a significant correlation between meropenem and ceftazidime MICs,
despite evidence that carbapenems and penicillins are predominantly hydrolysed by L1 and
cephalosporins and aztreonam are predominantly hydrolysed by L2.3,4 The significant correlation between changes
in MICs of most of the other tested ß-lactams (Table II) also
supports the hypothesis that
genetic control of L1 and L2 is linked as previously suggested.1
Some studies found that in-vitro selection of S. maltophilia mutants overexpressing
ß-lactamase was difficult to achieve by exposure to ß-lactam antibiotics alone.
1 The clinical isolates used in this study were taken from
cystic fibrosis patients who had all received frequent courses of antibiotics, including
ß-lactams, over long periods of time. The absence of high-level meropenem resistance
amongst the environmental isolates suggests that exposure to antibiotics or other factors in
vivo selects for overexpressing variants. This was supported by isolates C1a and C1b of the
same strain, taken 9 months apart from the same patient, showing a 32-fold increase in MIC
of meropenem (4 to 128 mg/L), and ampicillin (4 mg/L to >128 mg/L). Over the same period
these isolates exhibited only 4-fold and 8-fold increases to ceftazidime (1 to 4 mg/L) and
aztreonam (4 to 32 mg/L) respectively. However, a further
16-fold increase in ceftazidime
MIC for this strain was noted over the next 8 months (strains C1c and C1d). Precise mechanisms
behind this stepwise process remain to be elucidated.
In conclusion, there appears to be no clear correlation between genotype, MIC of ß-lactams, and the pI of extractedß-lactamases for this collection of clinical and environmental isolates of S. maltophilia. The resistance of S. maltophilia to ß-lactam antibiotics is extremely complex and further work is required to clarify the mechanisms behind the wide variations observed in phenotypic characteristics.
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Acknowledgments |
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Notes |
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References |
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Received 3 August 1998; returned 9 October 1998; revised 7 December 1998; accepted 10 December 1998