Impacts of L1 Promoter Variation and L2 Clavulanate Susceptibility on Ticarcillin-Clavulanate Susceptibility of Stenotrophomonas maltophilia

Inducible expression of L1 and L2 β-lactamases is the principal mechanism responsible for β-lactam resistance in Stenotrophomonas maltophilia. Ticarcillin-clavulanate (TIM) is one of the few effective β-lactams for S. maltophilia treatment.

ABSTRACT

Inducible expression of L1 and L2 β-lactamases is the principal mechanism responsible for β-lactam resistance in Stenotrophomonas maltophilia. Ticarcillin-clavulanate (TIM) is one of the few effective β-lactams for S. maltophilia treatment. Clavulanate (CA) is a β-lactamase inhibitor that specifically targets class A, C, and D β-lactamases. In view of the presence of class B L1 β-lactamase, it is of interest to elucidate why TIM is valid for S. maltophilia treatment. The L1-L2 allelic variation and TIM susceptibilities of 22 clinical isolates were established. Based on L1 and L2 protein sequences and TIM susceptibility, three L1-based phylogenetic clusters (L1-A, L1-B, and L1-C) and three L2-based phylogenetic clusters (L2-A, L2-B1, and L2-B2) were classified. The contribution of each L1- and L2-based phylogenetic cluster to ticarcillin (TIC) and TIM susceptibility was investigated. All the L1s and L2s tested contributed to TIC resistance. The L1s tested were inert to CA; nevertheless, the sensitivities of L2s to CA were widely different. In addition, the genetic organizations upstream of the L1 gene varied greatly in these isolates. At least three different L1 promoter structures (K279a type, D457 type, and none) were found among the 22 isolates assayed. The differences in the L1 promoter structure had a great impact on TIC-induced L1 β-lactamase activities. Collectively, the L1 promoter activity in response to TIC challenge and L2 susceptibility to CA are critical factors determining TIM susceptibility in S. maltophilia.

INTRODUCTION

β-Lactam antibiotics function by inhibiting cell wall biosynthesis and causing bacterial-cell lysis. The emergence and spread of β-lactamases is the most common mechanism of β-lactam resistance in Gram-negative bacteria. Based on the primary sequences of the enzymes, β-lactamases are grouped into four molecular classes: Amber classes A, B, C, and D. To solve the problems of β-lactamase-mediated β-lactam resistance, β-lactamase inhibitors have been developed (1). The β-lactamase inhibitors inhibit β-lactamase activity and prevent degradation of β-lactam antibiotics, and thereby expand the useful spectrum of β-lactam antibiotics (2). Currently available β-lactamase inhibitors include tazobactam, clavulanate (CA), sulbactam, and avibactam, which are effective against serine active-site β-lactamases, such as class A, C, and D β-lactamases, but not class B metallo-β-lactamases (3). Commercially available coformulations include piperacillin-tazobactam, amoxicillin-clavulanate, ticarcillin-clavulanate (TIM), ampicillin-sulbactam, cefoperazone-sulbactam, and ceftazidime-avibactam (1, 4, 5).

Stenotrophomonas maltophilia, an aerobic, nonfermentative, Gram-negative bacterium, has emerged as an important nosocomial pathogen, and treatment of its infections remains a challenge owing to its resistance to multiple common antibiotics, including various β-lactams (6). Resistance to β-lactams is intrinsically mediated by two chromosomally encoded β-lactamases, L1 and L2 (7). L1, an Amber class B β-lactamase, hydrolyzes penicillins, cephalosporins, and carbapenems (8). L2 is an Amber class A cephalosporinase, and penicillins, cephalosporins, and monobactams are its substrates (9). In view of the broad variety of substrates of L1 and L2 β-lactamases, most clinical isolates of S. maltophilia show resistance to β-lactam antibiotics, thereby limiting treatment options. TIM and ceftazidime are the most effective among β-lactam drugs against S. maltophilia (6).

TIM is a commercially available coformulation of ticarcillin (TIC) and CA. CA is a β-lactamase inhibitor that specifically inhibits the serine active-site β-lactamases (3), such as L2 β-lactamase. The L1 β-lactamase, a member of the class B β-lactamases, is inert to CA action; therefore, TIM treatment for S. maltophilia infection can reasonably be expected to be ineffective. However, the rate of susceptibility of S. maltophilia to TIM still ranged from 27% to 46.1% in clinics (6). Clinical S. maltophilia isolates have manifested a high degree of genetic diversity, as much as 30% (10, 11). Some studies have revealed that the divergences of L1 and L2 proteins range from 1% to 21% and from 7% to 32%, respectively (12–14). It stands to reason that the genetic diversity of S. maltophilia should have an impact on TIM potency in S. maltophilia infections; however, little research has been conducted in this area. In this study, we tried to elucidate the relationship between genetic diversity and TIM susceptibility in S. maltophilia.

RESULTS

TIC and TIM susceptibilities of clinical isolates.

The susceptibilities of 22 S. maltophilia isolates to TIC and TIM were assessed. All the isolates tested were resistant to TIC, with MICs ranging from 128 to >1,024 μg/ml. For all the isolates, the corresponding MICs of TIC decreased 2- to 64-fold in the presence of 2 mg/liter CA; however, only eight isolates (A, H, I, J, L, M, Q, and R) manifested susceptibility to TIM (MIC ≤ 16 μg/ml) (Table 1).

TABLE 1.

L1 phylogeny, L2 phylogeny, L1 promoter strucutre, and MIC values of TIC and TIM for 22 clinical S. maltophilia isolates

Isolate	Phylogeny		L1 promoter structurea	MIC (μg/ml)
	L1	L2		TIC	TIM
A	L1-B	L2-B2	D457 type	256	16
B	L1-A	L2-A	K279a type	1,024	256
C	L1-C	L2-A	None	1,024	512
D	L1-B	L2-B2	D457 type	256	32
E	L1-A	L2-A	K279a type	256	32
F	L1-A	L2-A	K279a type	512	32
G	L1-A	L2-A	K279a type	256	32
H	L1-B	L2-B2	D457 type	128	16
I	L1-C	L2-B2	None	256	16
J	L1-B	L2-B2	D457 type	128	8
K	L1-C	L2-B1	None	1,024	256
L	L1-C	L2-B2	D457 type	256	8
M	L1-C	L2-B2	None	256	4
N	L1-A	L2-A	K279a type	1,024	256
O	L1-A	L2-A	K279a type	>1,024	512
P	L1-A	L2-A	K279a type	512	32
Q	L1-A	L2-A	K279a type	128	16
R	L1-C	L2-B2	None	128	4
S	L1-B	L2-A	D457 type	128	32
T	L1-B	L2-B1	None	512	128
U	L1-C	L2-B1	None	256	32
V	L1-C	L2-A	None	1,024	512

^aNone, neither K279a type nor D457 type.

Phylogenetic analysis of L1 and L2 proteins.

In view of the high diversity of the L1 and L2 genes (12–14), which is closely linked to the discrepancy of TIC-hydrolyzing activity, the genetic variation of L1 and L2 genes was assessed.

Sequencing of the 22 PCR amplicons revealed that the L1 genes had two lengths, 873 and 870 bp, encoding preproteins of 290 or 289 amino acid residues (aa), respectively. According to analysis on the SignalP3.0 server (http://www.cbs.dtu.dk/services/SignalP-3.0/), the 290-aa and 289-aa L1 proteins harbored predicted signal peptides of 21 and 20 aa, respectively, yielding mature L1 proteins of 269 aa. Figure 1A shows a phylogenetic tree for mature L1 proteins, which can be subdivided into three distinct phylogenetic clusters: L1-A, L1-B, and L1-C, comprising eight, six, and eight isolates, respectively. Figure 1B shows the intracluster and intercluster L1 protein identities of the isolates.

FIG 1.

Phylogenetic analysis of L1 protein sequences. (A) Dendrogram of L1 protein sequences. Phylogenetic trees of L1 protein sequences from 22 S. maltophilia clinical isolates were constructed by the neighbor-joining method. The numbers near the branch points refer to the percentages of bootstrap resampling based on 1,000 replicates. (B) Intracluster and intercluster L1 protein identities of the isolates. The pairwise L1 protein identities were analyzed using the website http://workbench.sdsc.edu.

Among the 22 PCR amplicons of the L2 gene, there were three different lengths, 915, 912, and 906 bp, resulting in predicted mature L2 proteins of 277, 276, and 274 aa, respectively. The phylogenetic tree of mature L2 proteins was subdivided into two distinct clusters, L2-A and L2-B (Fig. 2A). The lengths of L2-A cluster proteins were 276 aa, and those of L2-B cluster were 274 or 277 aa. There were 11 isolates in each cluster. Intracluster and intercluster identities of the L2 protein among the isolates are summarized in Fig. 2B.

FIG 2.

Phylogenetic analysis of L2 protein sequences. (A) Dendrogram of L2 protein sequences. Phylogenetic trees of L2 protein sequences from 22 S. maltophilia clinical isolates were constructed by the neighbor-joining method. (B) Intracluster and intercluster L2 protein identities of the isolates. The pairwise L2 protein identities were analyzed using the website http://workbench.sdsc.edu.

The divergences of L1 and L2 proteins among the 22 isolates ranged from 0% to 17% and from 0% to 33%, respectively (Fig. 1B and 2B). This tendency is generally consistent with the previously reported L1 and L2 genetic variation of clinical isolates (12–14), indicating that the isolates in this study are clinically representative.

L2 allelic variations displayed correlation with TIM susceptibility.

The L1 and L2 phylogenies and TIM susceptibilities of the 22 isolates were integrated to elucidate their possible linkages. By surveying the links between L1-based clusters and TIM susceptibility individually, we found that all the L1-A isolates, except isolate Q, were moderately or highly resistant to TIM; however, the TIM susceptibility levels of L1-B and L1-C isolates varied from high resistance to susceptibility (Fig. 3A). On the other hand, we also surveyed the relationship between L2-based clusters and TIM susceptibility. We noticed that, in contrast to the L1-based clusters, L2-based clusters showed better correlation with TIM susceptibility. (i) Except for isolate Q, isolates of the L2-A cluster were moderately or highly resistant to TIM. (ii) Isolates belonging to the L2-B cluster had two distinct TIM susceptibility levels. Isolates T, K, U, and D were resistant, and the other isolates were susceptible (Fig. 3B). By carefully examining the L2 phylogenetic tree (Fig. 2A), we found that isolates T, K, and U formed an outlying subcluster among the L2-B-type isolates. Therefore, we further classified the L2-B cluster into two subclusters, L2-B1 (isolates T, K, and U) and L2-B2 (isolates L, M, R, D, A, I, H, and J) (Fig. 3A and Table 1). L2-B2 isolates, except for isolate D, were susceptible to TIM; in contrast, L2-B1 isolates were resistant to TIM (Fig. 3B).

FIG 3.

Relationships of L1 and L2 phylogenies with TIM susceptibility. The dashed lines indicate the MIC value for a TIM-susceptible strain according to the CLSI guidelines. (A) Relationship between L1 phylogeny and TIM susceptibility. (B) Relationship between L2 phylogeny and TIM susceptibility.

Impacts of L1 and L2 phylogenies on susceptibility to TIC and CA inhibition.

To study the relatedness of L1 phylogeny to TIC susceptibility, we chose two isolates from each L1 phylogenetic cluster (Fig. 3A) as the representatives of resistant and susceptible strains for further study (isolates N and Q from the L1-A cluster, isolates S and H from the L1-B cluster, and isolates K and L from the L1-C cluster). The TIC susceptibilities of an L2 deletion mutant and an L1/L2 deletion mutant of each representative were determined for comparison. For convenience of comparison, the MIC values of TIC for each isolate and its L1 isogenic mutant were also included (Fig. 4A, gray bars). The L1 β-lactamases of six isolates tested made significant contributions to TIC resistance, elevating the MICs of TIC from 4- to 128-fold (Fig. 4A, ΔL2 mutant versus ΔL1 ΔL2 double mutant). Furthermore, the L1-mediated TIC resistances were not affected by CA (Fig. 4B), which is consistent with the notion that CA is an inhibitor of the serine active sites of β-lactamases (1).

FIG 4.

Impacts of L1 and L2 phylogenies on susceptibility to TIC and CA inhibition. The susceptibility levels were tested by the serial 2-fold dilution method in Mueller-Hinton (MH) agar according to the guidelines of the CLSI. The asterisks indicate significance based on a ratio of the MIC for the parental strain to the MIC for the deletion mutant of ≥4. (A and B) Impacts of L1 phylogeny on TIC susceptibility (A) and on susceptibility to CA inhibition (B). The dashed lines indicate the MIC value for a TIM-susceptible strain according to the CLSI guidelines. (C and D) Impacts of L2 phylogeny on TIC susceptibility (C) and on susceptibility to CA inhibition (D). The dashed lines indicate the MIC value for a TIM-susceptible strain according to the CLSI guidelines.

A similar strategy was implemented to evaluate the impact of the L2 phylogeny on TIC and CA inhibition. Isolates N, K, and L were chosen as the representatives of L2-A, L2-B1, and L2-B2, respectively. All the representative isolates tested manifested increased TIC resistance when their L2 genes were intact (Fig. 4C, ΔL1 mutant versus ΔL1 ΔL2 double mutant), indicating that all the L2 β-lactamases tested play significant roles in TIC resistance. Next, we used the individual L1 isogenic mutants to test the inhibitory effect of CA on the activities of different L2 β-lactamases. Of the three isolates tested, LΔL1 (cluster L2-B2) was the most sensitive to CA inhibition because the TIC and TIM MIC values for LΔL1 showed a 128-fold difference, which makes LΔL1 TIM susceptible (Fig. 4D). The presence of CA decreased the TIC MICs for KΔL1 and NΔL1 2- and 4-fold, respectively, but both strains still remained resistant to TIM (Fig. 4D), meaning that the mild inhibitory effect of CA on L2-B1- and L2-A-type β-lactamases was not enough to shift the TIC susceptibility from resistant to susceptible.

Impact of L1 promoter variation on TIM susceptibility.

Based on the above-mentioned results, all the L1s tested in this study clearly contributed to TIC resistance (Fig. 4A) and were inert to CA inhibition (Fig. 4B). Therefore, it can be reasoned that a TIM-susceptible isolate should have an L1 with less potency toward TIC. Considering the weak relationship between L1 phylogeny and TIM susceptibility (Fig. 3A), we wondered whether L1 promoter variation has better correlation with TIC-induced L1 expression and is thus linked to TIM resistance. To test this notion, a pair of PCR primers, K279aL1-F and K279aL1-R (see Table S1 in the supplemental material), were designed to amplify the promoter region of the L1 gene, based on the genome sequence of S. maltophilia K279a (16). Unexpectedly, of the 22 isolates, the expected PCR amplicons were successfully obtained for only 8 isolates, isolates B, E, F, G, N, O, P, and Q. This observation prompted us to consider the possibility that the DNA sequences upstream of the L1 gene are highly variable among different isolates. Sequence similarity searches of the available whole-genome sequences of S. maltophilia in the NCBI database were performed. An open reading frame (ORF) annotated as a TonB-dependent ferric siderophore receptor gene upstream of the L1 gene, was highly conserved in most of the sequenced S. maltophilia strains (such as strains K279a, JV3, and R551-3), except S. maltophilia D457 (17). Thus, the genomic organizations upstream of the L1 gene in strains K279a and D457 were further compared. Compared to strain D457, a putative three-gene operon (Smlt2664-Smlt2665-Smlt2666) was found in K279a; it was located just upstream of the L1 gene (Fig. 5A and B), which made the possible promoter sequences of the L1 genes in both strains quite different (Fig. 5C). We tentatively described the two different L1 promoter structures as the K279a and D457 types. To survey the L1 promoter structures of the 22 isolates, two pairs of PCR primers were designed: K279aL1-F/K279aL1-R and D457L1-F/D457L1-R (Fig. 5; see Table S1 in the supplemental material), specific for the K279a and D457 types, respectively. Based on the PCR patterns, of the 22 isolates tested, 8 isolates (B, E, F, G, N, O, P, and Q) and 6 isolates (A, D, H, J, L, and S) had the K279a- and D457-type L1 promoter structures, respectively (Table 1). The L1 promoters of isolates C, I, K, M, R, T, U, and V were neither K279a type nor D457 type, further supporting the high genetic diversity in the L1 promoter region of S. maltophilia.

FIG 5.

Comparison of the genomic organizations upstream of the L1 gene between S. maltophilia K279a and S. maltophilia D457. The genomic organization, gene annotation, and DNA sequence were obtained from the released whole-genome sequences of S. maltophilia K279a and S. maltophilia D457 at the website of the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov). (A) Genomic organization surrounding the L1 gene. The solid bars represent the expected PCR amplicons primed by the indicated primers. (B) Annotation of the L1 cluster genes. The Smlt code and SMD code are the assigned numbers for annotated genes in S. maltophilia K279a and S. maltophilia D457, respectively. (C) DNA sequences of the intergenic regions between the L1 gene and its upstream gene.

The influence of the L1 promoter structures on TIC-mediated L1 inducibility was thus assessed by determining TIC-induced β-lactamase activities in strains NΔL2, QΔL2, SΔL2, HΔL2, KΔL2, and LΔL2. Compared to its own noninduced counterpart, the TIC-induced L1 activities were apparently high in NΔL2 and KΔL2, moderate in SΔL2, and slight in QΔL2, HΔL2, and LΔL2 (Fig. 6), indicating that L1 promoter variation indeed impacts TIC-induced L1 activity. A more interesting finding was that the extents of TIC-induced L1 activities in the assayed strains seemed not to be related to their L1 promoter structures; in contrast, they displayed good correlation with their TIC MIC values (Fig. 6).

FIG 6.

Relationship among TIC-induced L1 β-lactamase activities, L1 promoter structure, and L1-mediated TIC resistance. The overnight cultures assayed were diluted to an optical density at 450 nm of 0.15 and subsequently grown at 37°C for 2 h. Induction was carried out using ticarcillin at a concentration of 1/4 the MIC for 1 h. The noninduced and induced β-lactamase activities were determined. The L1 promoter organization (Table 1) and TIC susceptibility (Fig. 4A) are included for comparison.

DISCUSSION

L1 and L2 β-lactamases are the known determinants contributing to β-lactam resistance in clinical S. maltophilia isolates (18). A question may arise as to whether S. maltophilia harbors another active β-lactamase(s) in addition to L1 and L2. In our previous study, we assessed the active β-lactamases from 20 clinical S. maltophilia isolates by isoelectric focusing electrophoresis and in-gel β-lactamase activity staining. Of all the isolates we tested, no active β-lactamase was observed except L1 and L2 β-lactamases (15). Furthermore, no detectable β-lactamase activities were detected in the L1/L2 double-deletion mutants constructed in this study (NΔL1ΔL2, KΔL1ΔL2, LΔL1ΔL2, QΔL1ΔL2, SΔL1ΔL2, and HΔL1ΔL2) (data not shown). This evidence supports the notion that S. maltophilia harbors two active β-lactamases.

All the L1s tested in this study made a clear contribution to TIC resistance (Fig. 4A) and were inert to CA inhibition (Fig. 4B), consistent with former ideas about L1 β-lactamase (8). However, the DNA sequences upstream of the L1 gene in clinical S. maltophilia isolates were quite distinct (Fig. 5), which resulted in the TIC-induced L1 activities greatly varying in different clinical isolates, ranging from extremely high to slight (Fig. 6). Thus, the key parameter determining the contribution of L1 β-lactamase to TIM resistance is the L1 promoter activity in response to TIC challenge.

The L2-mediated TIC resistance (with 64- to 512-fold MIC changes) (Fig. 4C) was more profound than L1-mediated TIC resistance (with 4- to 128-fold MIC changes) (Fig. 4A), suggesting that L2 generally has higher potency in hydrolyzing TIC than L1. Although all the L2 samples tested were sensitive to CA inhibition, the extents of inhibition varied from mild (e.g., the L2s of isolates N and K) to high (e.g., the L2 of isolate L) (Fig. 4D). Therefore, the critical factor determining the contribution of L2 to TIM resistance appears to be its sensitivity to CA inhibition, rather than its potency against TIC hydrolysis (Fig. 4C and D).

Based on these observations, it can be reasoned that a TIM-susceptible isolate should have an L1 promoter with low activity in response to TIC challenge and an L2 β-lactamase whose activity is highly sensitive to CA inhibition. Although the L1 and L2 phylogenies are not totally well correlated with TIM susceptibility, we found some interesting characteristics. For example, of the eight TIM-susceptible isolates, seven isolates harbored the L2-B2 type (Fig. 3B and Table 1), supporting the positive relationship between L2-B2 phylogeny and high CA susceptibility of L2 β-lactamase. In contrast, of eight isolates harboring a K279a-type L1 promoter structure, only isolate Q was TIM susceptible, positively linking the K279a-type L1 promoter structure to TIM resistance. However, no significant relatedness could be found between L1 phylogeny and TIM susceptibility. Collectively, L1 promoter activity in response to TIC challenge and L2 CA susceptibility determine the TIM susceptibility of S. maltophilia.

MATERIALS AND METHODS

Collection and identification of S. maltophilia isolates.

All the strains, plasmids, and primers used in this study are summarized in Table S1 in the supplemental material. A total of 22 nonduplicate S. maltophilia isolates were collected from the routine clinical microbiology laboratory in Taiwan. All the isolates were identified by the ID32 GN system (bioMérieux, Marcy l'Etoile, France) and confirmed by PCR amplification of the 16S rRNA region, using SM-F and SM-R as the primers (13).

Sequence analysis of L1 and L2 genes.

The PCR amplicons containing intact L1 and L2 genes were obtained by PCR from the 22 isolates, using primers L1-F and L1-R and primers L2-F and L2-R, respectively, and the PCR amplicons were directly sequenced using the same primers. To avoid PCR errors, the sequences for each isolate were confirmed by using PCR products obtained from at least three independent experiments. To avoid the effects of the PCR primer sequences on the divergence percentages, the DNA sequences of the primers were trimmed from the resultant sequences.

Phylogenetic analysis of L1 and L2 proteins.

Multiple sequence alignments of L1 and L2 proteins were performed using the ClustalW program. Phylogenetic trees were constructed by a neighbor-joining method. The optimal global sequence alignment program (http://workbench.sdsc.edu/) was used for pairwise comparison of the sequences and for calculation of the divergence percentages among the sequences.

Construction of L1 and L2 deletion mutants.

The L1 deletion mutants of isolates K, L, and N were constructed by double-crossover homologous recombination involving a two-step allelic exchange, as previously described (19). Recombinant plasmids pKΔL1, pLΔL1, and pNΔL1 were constructed for allelic exchange to make L1 deletion mutations. Two DNA fragments containing the partial 5′ terminus and partial 3′ terminus of the L1 gene were amplified by PCR using the chromosomes of isolates K, L, and N as templates. The primer pairs used were L1-F/LL1N-R and LL1C-F/L1-R for isolates K and L and L1-F/L1N-R and L1C-F/L1-R for isolate N (see Table S1 in the supplemental material). The PCR amplicons were subsequently cloned into pEX18Tc, generating pLΔK1, pLΔL1, and pNΔL1.

A similar strategy was used for the construction of L2 deletion mutants, HΔL2, KΔL2, LΔL2, NΔL2, QΔL2, and SΔL2. The primer sets used were L2IG-F/L2(B, C, E)N-R and L2(B, C, E)C-F/L2IG-R for the construction of pΗΔ2, pKΔL2, and pSΔL2; L2IG-F/LL2N-R and LL2C-F/L2IG-R for pLΔL2; and L2IG-F/VL2N-R and VL2C-F/L2IG-R for pQΔL2 (see Table S1 in the supplemental material). A partial L2 gene was amplified by PCR using primer sets L2-F/L2-R (see Table S1) and cloned into pEX18Tc, yielding pEXNL2. To construct pNΔL2, the internal 354-bp SphI-SphI DNA fragment was removed from pEXNL2. Plasmid mobilization, transconjugant selection, and L1 and L2 mutant confirmation were performed as described previously (19). The L1 and L2 double mutant was constructed from the single mutant sequentially via the same procedure.

Antibiotic susceptibility test.

The TIC and TIM susceptibilities of clinical isolates were evaluated by the 2-fold serial agar dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (20). The MIC was defined as the lowest concentration of antibiotic that could inhibit visible bacterial growth.

Determination of TIC-induced β-lactamase activity.

An overnight culture was adjusted to an optical density at 45 nm (OD₄₅₀) of 0.15. After incubation at 37°C for 2 h, induction was carried out using TIC at 1/4 the MIC for 1 h. The bacterial cells were harvested by centrifugation and resuspended in phosphate buffer, and the β-lactamase activities were determined. The β-lactamase activities of bacterial extracts can be determined by monitoring the increase in the A₄₈₆ after the addition of 10 μM nitrocefin, as described previously (21). The specific activity (in units per milligram) was defined as the amount of nitrocefin that could be hydrolyzed by β-lactamase per minute per milligram of protein. The protein concentration was measuring using the Bio-Rad protein assay reagent.