Correlation between genotype and ß-lactamases of clinical and environmental strains of Stenotrophomonas maltophilia

Miles Dentona,*, Valerie Keera and Peter M. Hawkeyb

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Heterogeneity of ß-lactamase production by 17 clinical and nine environmental isolates of Stenotrophomonas maltophilia was investigated using MICs of six different ß-lactam antibiotics, isoelectric focusing (IEF) and pulsed field gel electrophoresis. There was no clear correlation between the results of IEF, genotype and MIC determination. Environmental isolates were more susceptible than clinical isolates; eight clinical and none of the environmental isolates expressed high-level resistance to meropenem. Only two isolates expressed high-level resistance to ceftazidime. These results indicate that further studies are required to elucidate the extent of genetic heterogeneity within the L1 and L2 ß-lactamase genes.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Stenotrophomonas maltophilia is an increasingly recognized cause of nosocomial infection, particularly in immunocompromised patients. One contributory factor is resistance to ß-lactams, mediated by a combination of ß-lactamase production and reduced outer membrane permeability.1

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).


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

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 Luria–Betani 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 3–10 or pH 5–6) (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 4.7–10.6 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 Tris–HCl 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 5–35 s linear ramping at six V/cm. PFGE profiles were interpreted using the criteria of Tenover et al, 11 and assigned to `types'.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The MICs of various ß-lactam antibiotics, IEF results and genotypes determined for each S. maltophilia isolates are shown in Table I. Despite repeated attempts we were unable to extract enough ß-lactamase from strain C1d for analysis, due to its poor growth in LBB.


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Table I. MICs of different ß-lactam antibiotics, ß-lactamase IEF results and PFGE type for clinical and environmental strains of S. maltophilia
 
There was a disparity between meropenem and imipenem MICs (i.e. >4-fold difference) for nine of the 17 clinical strains and all of the environmental strains. However, there was also a significant correlation between increasing MICs of meropenem and increases in MICs observed for ampicillin, aztreonam and ceftazidime. These and other correlations noted are shown in Table II. All isolates possessed both metallo- and serine ß-lactamases. PFGE profiles for isolates C1b, C1c and C1d were indistinguishable (data not shown) and differed from C1a by only one band. Environmental strains E1–E6 (PFGE types IIc–IIg) were epidemiologically related, having been isolated from various sites on the same hospital ward. E6 was indistinguishable from C7 (both type IIc). C2 and C3 also possessed PFGE pro-files that differed by fewer than three bands from C7 and environmental isolates E1–E6. All other strains were clearly distinguishable.


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Table II. Correlation coefficients (r) of ß-lactam MICs for all tested strains of S. maltophilia
 
There was no clear correlation between the results of MIC determination, IEF and genotyping results. C1a differed from C1b–1d by only one band but had a serine ß-lactamase pI of 8.8, compared with 9.4 for strains 1b and 1c. There was little difference for the metallo-ß-lactamase. These changes correlated with a >=4-fold difference in MICs of meropenem, ampicillin, ceftazidime and aztreonam (Table I). C2 and C3, differentiated by only one band on PFGE, had >=4-fold differences in MICs of meropenem, ampicillin, ceftazidime and aztreonam, but little difference in pI for their metallo- and serine ß-lactamases. Conversely, E3 and C2, differing by only one band on PFGE, had virtually identical ß-lactam MICs but differences in pI for both their serine and metallo-ß-lactamase. Similar results were observed for the pairs E1 and E4, and E6 and C7.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although there was heterogeneity of ß-lactamase production by S. maltophilia, the variation in pI of enzymes from isolates with indistinguishable genotypes and ß-lactam MICs may reflect difficulties in objective marking of band positions on IEF gels and inter-gel variation. However, recent sequencing of L1 ß-lactamase genes 7,8 has confirmed genetic heterogeneity of around 12–14%, although each of these studies only used a single strain. The L2 gene sequence has also been published 12 but as yet there is no reported comparison with that of another strain. Further studies are needed to ascertain the extent of molecular heterogeneity of the L1 and L2 ß-lactamase genes and what effect this has on the properties of the enzymes produced.

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.


    Acknowledgments
 
We would like to thank the Biomedical Scientists at St James' University Hospital Microbiology Department for their assistance in saving clinical strains of S. maltophilia from cystic fibrosis patients. This work was supported by the British Society for Antimicrobial Chemotherapy.


    Notes
 
* Tel: +44-113-392-2922; Fax: +44-113-233-5649; E-mail: milesd{at}pathology.leeds.ac.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Akova, M., Bonfiglio, G. & Livermore, D. M. (1991). Susceptibility to ß-lactam antibiotics of mutant strains of Xanthomonas maltophilia with high- and low-level constitutive expression of L1 and L2 ß -lactamases. Journal of Medical Microbiology 35, 208–13.[Abstract]

2 . Saino, Y., Kobayashi, F., Inoue, M. & Mitsuhashi, S. (1982). Purification and properties of inducible penicillin ß-lactamase isolated from Pseudomonas maltophilia. Antimicrobial Agents and Chemotherapy 22, 564 –70.[ISI][Medline]

3 . Saino, Y., Inoue, M. & Mitsuhashi, S. (1984). Purification and properties of an inducible cephalosporinase from Pseudomonas maltophilia GN12873. Antimicrobial Agents and Chemotherapy 25, 362–5.[ISI][Medline]

4 . Cullmann, W. & Dick, W. (1990). Heterogeneity of ß-lactamase production in Pseudomonas maltophilia: a nosocomial pathogen. Chemotherapy 36, 117–26.[ISI][Medline]

5 . Payne, D. J., Cramp, R., Bateson, J. H., Neale, J. & Knowles, D. (1994). Rapid identification of metallo- and serine ß -lactamases.Antimicrobial Agents and Chemotherapy 38, 991–6.[Abstract]

6 . Paton, R., Miles, R. S. & Amyes, S. G. B. (1994). Biochemical properties of inducible ß-lactamases produced from Xanthomonas maltophilia. Antimicrobial Agents and Chemotherapy 38, 2143–9.[Abstract]

7 . Sanschagrin, F., Dusfresne, J. & Levesque, R. C. (1998). Molecular heterogeneity of the L1 metallo-ß-lactamase family from Stenotrophomonas maltophilia. Antimicrobial Agents and Chemotherapy 42, 1245–8.[Abstract/Free Full Text]

8 . Walsh, T. R., Hall, L., Assinder, S. J., Nichols, W. W., Cartwright, S. J., MacGowan, A. P. et al. (1994). Sequence analysis of the L1 metallo- ß-lactamase from Xanthomonas maltophilia. Biochimica et Biophysica Acta 1218, 199–201.[ISI][Medline]

9 . Mathew, A., Harris, A. M., Marshall, M. J. & Ross, G. W. (1975). The use of analytical isoelectric focusing for detection and identification of ß-lactamases. Journal of General Microbiology 88, 169–78.[ISI][Medline]

10 . Denton, M., Todd, N. J., Kerr, K. G., Hawkey, P. M. & Littlewood, J. M. (1998). Molecular epidemiology of Stenotrophomonas maltophilia isolated from clinical specimens from patients with cystic fibrosis and associated environmental samples. Journal of Clinical Microbiology 36, 1953–8.[Abstract/Free Full Text]

11 . Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing D. H. et al. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 2233–9.[Free Full Text]

12 . Walsh, T. R., MacGowan, A. P. & Bennett, P. M. (1997). Sequence analysis and enzyme kinetics of the L2 serine ß-lactamase from Stenotrophomonas maltophilia. Antimicrobial Agents and Chemotherapy 41,1460 –4.

Received 3 August 1998; returned 9 October 1998; revised 7 December 1998; accepted 10 December 1998