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Modified nitrocefin-edta method to differentially quantify the induced l1 and l2 β-lactamases in stenotrophomonas maltophilia

Letters in Applied Microbiology ISSN 0266-8254 Modified nitrocefin-EDTA method to differentially quantifythe induced L1 and L2 b-lactamases in StenotrophomonasmaltophiliaR.-M. Hu1, K.-H. Chiang2, C.-W. Lin2 and T.-C. Yang2 1 Department of Biotechnology and Bioinformatics, Asia University, Taichung, Taiwan2 Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan b-lactamase, ethylene diamine tetraaceticacid, L1, nitrocefin, Stenotrophomonas Aim: To estimate the ethylene diamine tetraacetic acid (EDTA) concentration at which the L1 enzyme activity in the cell extracts of Stenotrophomonas malto-philia can be mostly inhibited.
Methods and Results: The effective inhibition concentration of EDTA against the L1 enzyme in the cell extracts was firstly evaluated by using the L2 isogenic Laboratory Science and Biotechnology, China mutant of S. maltophilia KJ, KJDL2, as the assayed strain. Approximately 92% Medical University, Taichung 404, Taiwan.
E-mail: tcyang@mail.cmu.edu.tw L1 activity was inhibited by 10 mmol l)1 EDTA, which is 100-fold higher thanthat from previously reported protocols (0Æ1 mmol l)1). Three phylogenetic 2008 ⁄ 0724: received 28 April 2008, revised clusters of L1 proteins were revealed from 11 clinical S. maltophilia isolates, with a L1 protein divergence of 0–11%. The EDTA concentration required toinhibit the L1 enzymes of different phylogenetic clusters was estimated to be 10 mmol l)1.
Conclusion: The previous nitrocefin-EDTA protocol for differentially quantify-ing the L1 and L2 activity in the cell extracts has been modified by raising theadded EDTA concentration to 10 mmol l)1.
Significance and Impact of the Study: A rapid and accurate method for deter-mination of L1 and L2 activity will provide a convenient tool for enzyme char-acterization and induction mechanism study of S. maltophilia.
the enzyme activity (Saino et al. 1982). L2 is a serine active-site b-lactamase, and its activity is inert to the inhi- Stenotrophomonas maltophilia has gained increasing atten- bition of EDTA (Saino et al. 1984). Based on the charac- tion owing to its multiple antimicrobial resistances (Vart- teristics of EDTA inhibition against L1, a strategy for the ivarian et al. 1994; Alonso and Martinez 1997). Two differential quantification of L1 and L2 in induced soni- chromosomal-encoded b-lactamases, L1 and L2, have cated extract preparations has been proposed (Gould been reported as the candidates of b-lactam resistance. It et al. 2004; Okazaki and Avison 2008). Briefly, the total is well known that both b-lactamases are co-inducibly induced b-lactamases activities, i.e. L1 and L2, are mea- expressed, but the real induction mechanism has not yet sured using nitrocefin as the substrate. Meanwhile, L2 been uncovered (Mett et al. 1988; Avison et al. 2002; activity is determined with nitrocefin plus 0Æ1 mmol l)1 Okazaki and Avison 2008). Developing a convenient EDTA. The difference between the two measurements is method for differentially quantifying the induced L1 and then used to represent the activity of the L1 enzyme. In L2 of S. maltophilia is therefore a necessity for under- our previous study, a constructed L2 mutant, KJDL2 (Hu standing the expression of L1 and L2 b-lactamases. L1 is et al. 2008), was used as the material for characterization a Zn2+-dependent metalloenzyme, and 0Æ1 mmol l)1 eth- ylene diamine tetraacetic acid (EDTA) can inhibit 93% of 0Æ1 mmol l)1 EDTA only partially inhibited b-lactamase ª 2008 The AuthorsJournal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 457–461 Differential quantification of L1 and L2 b-lactamases activity expressed by the induced KJDL2 (to be mentioned focusing electrophoresis with in-gel nitrocefin staining to later). This indicates that the aforementioned method ensure that the sole active b-lactamase expressed in may overestimate L2 activity, while the calculated L1 KHDL2 (and YWDL2) was the L1 enzyme.
activity is underestimated. As S. maltophilia is a species ofhigh genetic diversity, we further evaluated the inhibition property of EDTA towards the L1 enzymes from differentisolates. Accordingly, a modified method for differentially An overnight culture of each bacterium to be mated was quantifying L1 and L2 b-lactamase activity in cell extracts prepared using Luria-Bertani (LB) broth. The cultures of donor strain and recipient strain were mixed in a 10 : 1ratio. Mixed cultures were spread on the nitrocellulosemembrane in the centre of a nutrient agar plate (no antibiotics added) and then incubated overnight at 37°C.
The recombinant strains were selected on LB agar containing tetracycline (40 lg ml)1) and norfloxacin Stenotrophomonas maltophilia KJ and its L1, L2 isogenic mutants, KJDL1 and KJDL2, have been characterized inour previous study (Hu et al. 2008). Ten S. maltophilia Preparation of sonicated b-lactamase extracts isolates were isolated from clinical patients and confirmedby species-specific PCR (Whitby et al. 2000).
Sonicated extract preparations for the b-lactamase activityassay were done according to the procedure reported inour previous paper (Hu et al. 2008). In brief, an over- Cloning and sequence analysis of L1 genes night culture was inoculated into 20-ml fresh LB at a final To amplify the L1 genes of the 10 isolates, a primer pair turbidity of 0Æ15 OD450 and cultured at 37°C for 0Æ5 h.
L1–F2 (5¢-AAGGAGGCCCATGCTAGTTT-3¢) and L1–R2 Cefoxitin of 50 lg ml)1 was then added and incubated at (5¢-TTCTGACCGGCACCCTTC-3¢) was designed (http:// 37°C for 2 h. Bacterial cells were harvested, washed, and www.sanger.ac.uk/Projects/Microbes/). The 50-ll PCR resuspended in 2 ml of 50 mmol l)1 phosphate buffer reaction mixture contained 10 ng genomic DNA, 50 pmol (pH 7Æ0). The cell suspension was sonicated in an ultra- forward and reverse primers, 2Æ5 mmol l)1 each of deoxy- sonic disrupter (S-3000; Misonix, NY, USA) for 5 min in nucleoside triphosphate, 1 · PCR buffer (Yeastern Bio- an ice bath. The supernatant was used as a source for tech Co., Taiwan) and 2Æ5 U of Taq DNA polymerase.
b-lactamase activity assay after centrifuging at 10 000 g The PCR reaction consisted of one cycle of 10 min at 94°C, 30 cycles of 1 min at 94°C, 1 min at 52°C, and1 min at 72°C, followed by a final extension step at 72°C for 10 min. The approximate 1-kb PCR product wasligated into T-vector (Yeastern Biotech Co., Taiwan) and The b-lactamase activity of crude sonicated extracts was measured by UV spectrophotometer as described previ-ously (Hu et al. 2008) with nitrocefin (Oxoid, UK)(100 lmol l–1) as the substrate. The wavelength and absorption coefficient for nitrocefin were 486 nm and Multiple sequence alignments were performed using the 20 500 mol)1 cm)1, respectively. One unit of b-lactamase ClustalX program. Phlyogenetic trees were constructed activity was defined as the amount of b-lactamase that by a neighbor-joining method. The phylogenetic ‘cluster’ hydrolyzed 1 nmol of nitrocefin in 1 min at 30°C. Pro- was defined as having a cutoff value of 96% identity.
tein concentration was determined using a Bio-Rad pro-tein assay reagent. The specific activity of the enzymewas defined in terms of units per mg protein in the cell Construction of the L2 isogenic mutants of Stenotropho- extracts. For the inhibition assay, various concentrations of EDTA were preincubated with the assayed extracts for The L2 genes of strains KH and YW were obtained by 10 min at 30°C prior to the b-lactamase activity PCR strategy, and two recombinant plasmids for the con- determination. The total reaction volume was 0Æ5 ml in struction of the L2 isogenic mutants, pKHDL2 and pYWDL2, were constructed as described previously (Hu 50 mmol l)1 phosphate buffer (pH 7Æ0). At least three et al. 2008). Mutants KHDL2 and YWDL2 were obtained independent experiments were conducted to determine by conjugation and confirmed by PCR and isoelectric Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 457–461 Differential quantification of L1 and L2 b-lactamases mature L1 protein divergence among these 11 isolates ranged from 0% to 11%, which is consistent with the The sequences determined for the L1 genes of the 10 S.
previous findings (Hauben et al. 1999; Avison et al.
maltophilia isolates and for the L2 genes of strains KH 2001). Figure 1 shows a phylogenetic tree for mature L1 and YW have been deposited in GenBank under accession proteins, which can be divided into three distinct phylo- numbers EF126061 (KJa, L1), EF126053 (KJb, L1), genetic clusters (L1-1 to L1-3). The inter-cluster L1 EF126054 (KJc, L1), EU441218 (KH, L1), EF126056 (YW, enzymes displayed a protein identity ranging from 89% to 92%. The members with an 873-nt L1 gene, including EF126058 (YWc, L1), EF126063 (YWd, L1), EF126065 our previous isolate KJ, belonged to cluster L1-1, whereas (YWe, L1), EU032534 (KH, L2), and EF126080 (YW, L2), the members of L1-2 and L1-3 all contained 870 nt. Iso- lates KJ, KH and YW were selected as the representativesfor clusters L1-1, L1-2 and L1-3, respectively, for furtherstudy.
EDTA-inhibition property of Stenotrophomonas EDTA-inhibition property on the b-lactamase activity of different Stenotrophomonas maltophilia isolates andmutants The sole active b-lactamases inducibly expressed in KJDL1and KJDL2 are L2 and L1 enzymes, respectively (Hu et al.
Table 1 shows the cell-extract b-lactamase activity of 2008). Table 1 shows the inhibitory effect of EDTA on S. maltophilia KHDL2 and YWDL2, both with and with- the b-lactamase activity expressed in the cell extracts of out EDTA as an inhibitor. The percentages of b-lactamase KJDL1 and KJDL2. Apparently, EDTA effectively inhibited activity inhibited by 0Æ1 mmol l)1 and 10 mmol l)1 EDTA L1 activity, but hardly affected L2 activity. An EDTA con- were 51% and 98% for mutant KHDL2, and 87% and centration to inhibit the most L1 b-lactamase activity 93% for mutant YWDL2. To gain a better understanding (>90%) was as high as 10 mmol l)1.
of the inhibition effect of 0Æ1 mmol l)1 and 10 mmol l)1EDTA on the cell-extract b-lactamase activity of theS. maltophilia isolates; the b-lactamase activity of the 11 isolates were determined in the presence of 0, 0Æ1 and Sequencing of these 10 PCR amplicons revealed that the 10 mmol l)1 EDTA, respectively (Table 2). For each L1 genes were of two different lengths, 870 and 873 bp, isolate, EDTA displayed a significant inhibition of b-lac- encoding a preprotein of 289 and 290 amino acid resi- tamase activity. Activity in the presence of 10 mmol l)1 dues. After the predicted signal peptide of 20 or 21 amino EDTA was notably lower than that of 0Æ1 mmol l)1.
acids had been deducted, all the tested isolates had amature 269-amino acid L1 protein. As the reported L1 sequence of isolate KJ (Hu et al. 2008) was included, the The active b-lactamase solely presented in mutantsKJDL2, KHDL2 and YWDL2 is the L1 enzyme, as con- Table 1 The inhibitory effect of ethylene diamine tetraacetic acid firmed by isoelectric focusing electrophoresis with in-gel (EDTA) on the b-lactamase activity expressed in the cell extracts of nitrocefin staining (data not shown). Furthermore, the L1 enzymes of different phylogenetic clusters show different susceptibility towards the action of EDTA (Table 1). The L1 enzyme of strain YW is the one most susceptible to EDTA; 0Æ1 mmol l)1 EDTA is enough to inhibit most L1activity (87%). Conversely, L1 enzymes of strains KJ and KH display less susceptibility to EDTA; therefore, a higher EDTA concentration, i.e. 10 mmol l)1, is essential to impose significant inhibition. Saino et al. (1982) purified L1 enzyme from S. maltophilia GN12873 and demon- strated that 0Æ1 mmol l)1 EDTA is enough to inhibit thepurified L1 enzyme. However, only 36–87% of L1 enzyme *One unit of b-lactamase is defined as 1 nm of nitrocefin hydrolysed activity in sonicated extracts in the present study is inhib- per minute per mg protein. Results are geometric means of threeindependent determinations. Standard deviations were within 10% of ited by 0Æ1 mmol l)1 EDTA. The EDTA inhibition effect on L1 enzyme in the sonicated extract preparations is ª 2008 The AuthorsJournal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 457–461 Differential quantification of L1 and L2 b-lactamases protein sequences of Stenotrophomonasmaltophilia isolates. L1 genes from the 11 clinical strains of S. maltophilia isolated in Phylogenetic distances were determined byneighbor-joining analysis. Numbers near a node represent the percentage of bootstrap apparently less significant than that on the purified L1 different strains. As can be seen in Table 2, the EDTA enzyme, implying that there are factors within the extracts susceptibility for L1 enzymes of the three phylogenetic that counteract the inhibition effect of EDTA.
clusters is generally, in order, L1-3, L1-2 and L1-1. Gould Because S. maltophilia is a genus of high genetic diver- et al. proposed a strategy for differential quantification of sity, as reported in the L1 allele with a diversity of 20% L1 and L2 in an induced sonicated extracts preparation (Hauben et al. 1999; Avison et al. 2001), it is likely that based on the characteristics of EDTA inhibition against the EDTA-inhibited level of the L1 enzymes differs in the L1 (Gould et al. 2004). In Gould’s study, 0Æ1 mmol l)1EDTA was employed, which apparently overestimated theL2 activity, while underestimating the L1 activity. This is Table 2 The inhibitory effect of ethylene diamine tetraacetic acid because 0Æ1 mmol l)1 EDTA is insufficient for inhibiting (EDTA) on the b-lactamase activity expressed in the cell extracts ofStenotrophomonas maltophilia strains the activity of L1 enzyme. Instead, 10 mmol l)1 EDTA isthe optimal concentration for the inhibition of L1 10 mmol l)1 EDTA can inhibit 92–98% L1 activities of different S. maltophilia L2 isogenic mutants. The inhibi-tion of L1 activity is enhanced insignificantly even though the EDTA concentration is as high as 50 mmol l)1.
Table 2 also shows that the L1 b-lactamase activity of strain KJ can be obtained in two different ways: one by the mutant KJDL2 in the assayed condition of 0 mmol l)1 EDTA (432 U mg)1) and the other by subtracting the b-lactamase activity of strain KJ in 0 mmol l)1 EDTA from that in 10 mmol l)1 EDTA (402 U mg). Both show good consistency, indicating that 10 mmol l)1 EDTA indeed effectively inhibited L1 activity. Pair-wise compari- sons among strains KH ⁄ KHDL2 and YW ⁄ YWDL2 also gave similar results. Therefore, we propose a modified nitrocefin–EDTA method to differentially quantify theinduced L1 and L2 b-lactamases in the cell extracts of S.
*One unit of b-lactamase is defined as 1 nm of nitrocefin hydrolysed maltophilia. The b-lactamase activities of the assayed per minute per mg protein. Results are geometric means of threeindependent determinations. Standard deviations were within 10% of extracts were determined with and without 10 mmol l)1 EDTA. In the absence of EDTA, the detected b-lactamase Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 457–461 Differential quantification of L1 and L2 b-lactamases activity (Unitrocefin) represents the sum of L1 and L2 activ- Hauben, L., Vauterin, L., Moore, E.R.B., Hoste, B. and Swings, ity. Because most L1 activity is inhibited by 10 mmol l)1 J. (1999) Genomic diversity of the genus Stenotrophomon- as. Int J Syst Bacteriol 49, 1749–1760.
Hu, R.-M., Huang, K.-J., Wu, L.-T., Hsiao, Y.-J. and Yang, T.-C.
from the L2 enzyme. Accordingly, the L1 enzyme can be (2008) Induction of L1 and L2 b-lactamases of Stenotroph- calculated by subtracting the activity of U omonas maltophilia. Antimicrob Agents Chemother 52, Mett, H., Rosta, S., Schacher, B. and Frei, R. (1988) Outer membrane permeability and b-lactamase content in Pseu- domonas maltophilia clinical isolates and laboratorymutants. Rev Infect Dis 10, 765–769.
This research was supported by grant nos. CMC93-MT- Okazaki, A. and Avison, M.B. (2008) Induction of L1 and L2 03 and CMU95-102 to T.-C. Yang from the China Medi- b-lactamase production in Stenotrophomonas maltophilia is dependent on an AmpR-type regulator. Antimicrob AgentsChemother 38, 2143–2149.
Saino, Y., Kobayashi, F., Inoue, M. and Mitsuhashi, S. (1982) Purification and properties of inducible penicillin b-lac- Alonso, A. and Martinez, J.I. (1997) Multiple antimicrobial tamase isolated from Pseudomonas maltophilia. Antimicrob resistance in Stenotrophomonas maltophilia. Antimicrob Saino, Y., Inoue, M. and Mitsuhashi, S. (1984) Purification Avison, M.B., Higgins, C.S., von Heldreich, C.J., Bennett, P.M.
and properties of an inducible cephalosporinase from and Walsh, T.R. (2001) Plasmid location and molecular Pseudomonas maltophilia GN12873. Antimicrob Agents heterogeneity of the L1 and L2 b-lactamase genes of Steno- trophomonas maltophilia. Antimicrob Agents Chemother 45, Vartivarian, S., Anaissie, E., Bodey, G., Sprigg, H. and Rol- ston, K. (1994) A changing pattern of susceptibility of Avison, M.B., Higgins, C.S., Ford, P.J., von Heldreich, C.J., Xanthomonas maltophilia to antimicrobial agents: impli- Walsh, T.R. and Bennett, P.M. (2002) Differential regula- cations for therapy. Antimicrob Agents Chemother 38, tion of L1 and L2 b-lactamase expression in Stenotropho- monas maltophilia. J Antimicrob Chemother 49, 387–389.
Whitby, P.W., Carter, K.B., Burns, J.L., Royall, J.A., Lipuma, Gould, V.C., Okazaki, A., Howe, R.A. and Avison, M.B. (2004) J.J. and Stull, T.L. (2000) Identification and detection of Analysis of sequence variation among smeDEF multi drug Stenotrophomonas maltophilis by rRNA-directed PCR.
efflux pump genes and flanking DNA from defined 16S rRNA subgroups of clinical Stenotrophomonas maltophiliaisolates. J Antimicrob Chemother 54, 348–353.
ª 2008 The AuthorsJournal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 457–461

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