Differential regulation of L1 and L2 ß-lactamase expression in Stenotrophomonas maltophilia
Matthew B. Avison,*,
Catherine S. Higgins,
Peter J. Ford,
Charlotte J. von Heldreich,
Timothy R. Walsh and
Peter M. Bennett
Bristol Centre for Antimicrobial Research and Evaluation, 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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It has been reported that Stenotrophomonas maltophilia L1 and L2 ß-lactamase expression is coordinated. We have isolated S. maltophilia mutants where (i) L1 is constitutively hyper-expressed and L2 is inducible; (ii) L2 is hyper-expressed and L1 is inducible; and (iii) L1 and L2 are constitutively hyper-expressed. The frequency of isolating type 1 and 2 mutants is c. 10-7, indicating that promoter mutations are probably not involved and providing strong evidence that L1 and L2 expression is not, after all, coordinated. The frequency of isolating type 3 mutants is c. 10-9, however, implying that there is a significant overlap between the regulatory mechanisms.
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Introduction
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In a recent survey of British medical microbiologists, Stenotrophomonas maltophilia was voted one of the most important emergent multidrug-resistant pathogens.1 As well as causing bacteraemias, S. maltophilia has been reported to be responsible for many different organ-specific infections and is an important cause of respiratory tract colonization in young adults with cystic fibrosis.2
S. maltophilia isolates are resistant to many clinically useful antibiotics, with ß-lactam resistance being mediated through two ß-lactamases, L1 and L2, whose expression is induced when cells are exposed to ß-lactam antibiotics.2,3 Little is known about the mechanism(s) used to regulate L1 and L2 expression, so the experiments presented in this report were designed to investigate the regulation of L1 and L2 expression in S. maltophilia by isolating mutants in which L1 and L2 expression were altered.
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Materials and methods
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Bacterial strains and materials
The 10 clinical S. maltophilia isolates used in this study have been described previously.3 They are divided into groups A, B and C according to 16S rRNA gene sequence. Group A strains are K279a, H33b, J530b and J675Ib; group B strains are N531 and P567II; and group C strains are J675a, J675b, O132II and O133.3 All the materials used in this study have been described elsewhere.35
Induction, preparation of crude cell extracts and ß-lactamase assay
Induction of ß-lactamase activity was attempted where appropriate, crude cell extracts were made, ß-lactamase assays were carried out and specific ß-lactamase activity was calculated using the methods described previously.4 One unit of ß-lactamase activity was defined as that required to hydrolyse 1 micromole of substrate per minute at 25°C.
Isolation of total RNA and RTPCR analysis
Isolation of RNA and RTPCR were carried out as described previously.5 The RTPCR primers used were L1-MID (forward) 5'-CTGGCCGTCGCTCTTCCG-3' and (reverse) 5'-TCCAGGCGGTGCTGCCC-3' and L2-MID (forward) 5'-CGATGATCACCAGCGACA-3' and (reverse) 5'-CGGTTACCTCATCCGATC-3'. Although these primers were designed from the L1c and L2b gene sequences located in S. maltophilia group A isolates, they are known to amplify equivalent regions from the L1 and L2 isoforms carried by group B and C S. maltophilia isolates.3
Selection of ß-lactamase hyper-producing mutants
Etest MIC determinations were carried out on IsoSensitest agar with an inoculum of 0.5 McFarland. Following incubation at 37°C for 24 h, colonies that grew within the zone of clearing were selected.
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Results
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Using crude cell extracts from uninduced or cefoxitin-induced group A, B and C S. maltophilia isolates (see Materials and methods), the activities of L1 and L2 ß-lactamases were determined with meropenem as a specific L1 substrate and ceftazidime as an L2 substrate (Figure 1a
). In the latter case, EDTA (100 mM) was added to inhibit L1, which also hydrolyses ceftazidime.6 RTPCR analysis was used to determine L1 and L2 transcript levels in similarly incubated cells (Figure 1b
). The data reveal that L2 is expressed at higher levels following induction of group A strains, but very little L2 expression was detected in group B or C strains, even following induction. The same level of L1 induction was seen in group A and B strains, but there was no detectable L1 expression in group C strains (Figure 1
).

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Figure 1. Induction of S. maltophilia L1 and L2 transcription. S. maltophilia isolates from group A, B and C strains are represented in the figure by isolates K279a, N531 and J675a, respectively. (a) Crude extracts from cefoxitin-induced (labelled A In, B In or C In) or uninduced (labelled A, B or C) cultures were prepared as described in Materials and methods. ß-Lactamase activities in the extracts were measured against meropenem (white bar) or ceftazidime in the presence of 100 mM EDTA (black bar). Error bars represent s.e.m. values; n = 3. (b) From the same cultures used in (a), total RNA was prepared and assayed for the abundance of L1 and L2 transcripts by RTPCR (see Materials and methods). The figure represents an ethidium bromide-stained 1.2% w/v agarose gel under UV illumination following resolution of the L1 mid and L2 mid PCR amplicons.
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S. maltophilia K279a (group A) has inducible L1 and L2 ß-lactamases (Figure 1
). K279a mutants that display high-level resistance to meropenem, ceftazidime or ampicillin were isolated (see Materials and methods). Of 50 hyper-resistant mutants examined, eight showed increased levels of ß-lactamase expression. Three classes of ß-lactamase hyper-producing mutants were isolated, denoted K279a M1, K279a M2 and K279a M3. Using crude cell extracts of uninduced K279a M1, K279a M2 and K279a M3, the activities of L1 and L2 ß-lactamases (Figure 2a
) and the levels of L1 and L2 transcripts (Figure 2b
) were determined. Compared with K279a, K279a M1 has increased expression of L1 but not L2; K279a M2 has increased expression of L2 but not L1; and K279a M3 has increased expression of both L1 and L2 (Figure 2
). The ß-lactamases that are not over-expressed in uninduced K279a M1 or K279a M2 are inducible upon cefoxitin challenge (data not shown).

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Figure 2. L1 and L2 transcription in S. maltophilia K279a mutants. S. maltophilia isolate K279a was used to produce the mutants denoted M1, M2 and M3 as described in the text. (a) Crude extracts from uninduced cultures of the parent strain or from the three mutants were prepared as described in Materials and methods. ß-Lactamase activities in the extracts were measured using meropenem (white bar) or ceftazidime in the presence of 100 mM EDTA (black bar). Error bars represent s.e.m. values; n = 3. (b) The same cultures were used to prepare total RNA, which was assayed for the abundance of L1 and L2 transcripts by RTPCR (see Materials and methods). The figure represents an ethidium bromide-stained 1.2% w/v agarose gel under UV illumination following resolution of the L1 mid and L2 mid PCR amplicons.
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Discussion
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Both sets of data indicate that expression of the L1 and L2 genes in S. maltophilia is differential, a conclusion that contrasts with those of previous studies.2 It is clear from several reports that challenging S. maltophilia isolates with ß-lactams induces L1 and L2 expression simultaneously. From this, it has been concluded that the same mechanism is used to control the expression of both enzymes.2 In contrast, Akova et al.7 isolated an S. maltophilia mutant where L1 was constitutively repressed and L2 remained inducible; Bonfiglio et al.8 found a clinical isolate with identical properties; and we have found clinical isolates where L2 is repressed and L1 is inducible (Figure 1
). Promoter mutations could explain all of these findings, however, uncoupling one gene in each case from the induction machinery.
We also report the isolation of S. maltophilia K279a mutants where either L1 or L2 is constitutively hyper-expressed and the partner enzyme remains inducible (Figure 2
). Again, promoter mutations could explain this, but the frequency at which differentially hyper-expressing mutations arise argues against this theory. Promoter mutations that lead to over-expression of ß-lactamases are rare events. For example, the promoter mutation frequency resulting in ampC over-expression in Escherichia coli is c. 10-9.9 Although the Etest method used here does not allow determination of accurate mutation frequencies, the frequency to L1 over-expression is c. 10-7 and that to L2 over-expression is similar. Hence, mutation to L1 or L2 over-expression is about two orders of magnitude greater than that for ampC over-expression in E. coli due to a promoter mutation.9 A similar high mutation frequency (c. 10-7) leading to ß-lactamase over-expression of ampC in Citrobacter freundii and Enterobacter cloacae is primarily a reflection of non-specific loss-of-function mutations in ampD.10 Hence, we think it likely that non-specific mutations are the cause of ß-lactamase hyper-production in the S. maltophilia mutants isolated, and not promoter mutations. Whereas the predicted frequency of isolating L1 and L2 double over-expressing mutants would be 10-14 (10-7 for L2 multiplied by 10-7 for L1), if both systems were entirely independent, the observed frequency is c. 10-9. This frequency indicates that some single-site mutations result in over-expression of both genes, implying that there is a common component(s) to control of L1 and L2 gene expression.
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Acknowledgements
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This work was funded by grants from the British Society for Antimicrobial Chemotherapy (to M.B.A. and P.M.B.) and the Wellcome Trust (to P.M.B. and T.R.W.). C.S.H. was in receipt of a Biotechnology and Biological Sciences Research Council CASE studentship in collaboration with GlaxoSmithKline pharmaceuticals.
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Notes
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* Correspondence address. Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK. Tel: +44-117-9287439; Fax: +44-117-9287896; E-mail: matthewb.avison{at}bris.ac.uk 
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Received 15 May 2001;
returned 26 October 2001; revised 5 November 2001;
accepted 8 November 2001