Regulation and Anaerobic Function of the Clostridioides difficile β-Lactamase

Clostridioides difficile causes severe antibiotic-associated diarrhea and colitis. C. difficile is an anaerobic, Gram-positive sporeformer that is highly resistant to β-lactams, the most commonly prescribed antibiotics. The resistance of C. difficile to β-lactam antibiotics allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of β-lactam resistance in C. difficile are not fully understood.

ABSTRACT

Clostridioides difficile causes severe antibiotic-associated diarrhea and colitis. C. difficile is an anaerobic, Gram-positive sporeformer that is highly resistant to β-lactams, the most commonly prescribed antibiotics. The resistance of C. difficile to β-lactam antibiotics allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of β-lactam resistance in C. difficile are not fully understood. Our data reinforce prior evidence that C. difficile produces a β-lactamase, which is a common β-lactam resistance mechanism found in other bacterial species. Here, we characterize the C. difficile bla operon that encodes a lipoprotein of unknown function and a β-lactamase that was greatly induced in response to several classes of β-lactam antibiotics. An in-frame deletion of the operon abolished β-lactamase activity in C. difficile strain 630Δerm and resulted in decreased resistance to the β-lactam ampicillin. We found that the activity of this β-lactamase, BlaCDD, is dependent upon the redox state of the enzyme. In addition, we observed that transport of BlaCDD out of the cytosol and to the cell surface is facilitated by an N-terminal signal sequence. Our data demonstrate that a cotranscribed lipoprotein, BlaX, aids in BlaCDD activity. Further, we identified a conserved BlaRI regulatory system and demonstrated via insertional disruption that BlaRI controls transcription of the blaXCDD genes in response to β-lactams. These results provide support for the function of a β-lactamase in C. difficile antibiotic resistance and reveal the unique roles of a coregulated lipoprotein and reducing environment in C. difficile β-lactamase activity.

INTRODUCTION

Clostridioides difficile is an anaerobic, Gram-positive, spore-forming bacterial pathogen that causes antibiotic-associated diarrhea (1–3). C. difficile infection (CDI) can be severe, resulting in pseudomembranous colitis, intestinal rupture, and death. The Centers for Disease Control and Prevention (CDC) estimates that almost half a million people in the United States suffer from CDI per year, resulting in approximately 29,000 deaths per year (4). As a result, CDI cases add approximately $4.8 billion per year to U.S. health care costs (5). C. difficile was first linked to antibiotic-associated diarrhea in 1978, and antibiotic treatment is still one of the highest risk factors for CDI (2, 3). Antibiotic treatment results in gastrointestinal dysbiosis, eliminating important indigenous anaerobes, thereby allowing for C. difficile population expansion (6, 7). Antibiotic treatment of CDI is limited to the use of vancomycin, fidaxomicin, or metronidazole due to the high resistance C. difficile exhibits for a wide array of antibiotics (8–10).

The most commonly prescribed class of antibiotics are the β-lactams, which comprise 62% of all prescribed antibiotics in the United States and are strongly associated with C. difficile infections (11–13). β-Lactams are inhibitors of bacterial cell wall synthesis and are characterized by a four-membered core lactam ring (14). β-Lactams are further classified into four groups based on adjoining structures: the penicillins, cephalosporins, monobactams, and carbapenems (15). All β-lactam antibiotics bind to, and thus disable, cell wall synthesizers called penicillin-binding proteins (PBPs) of bacteria (16, 17). Since the introduction of β-lactams into modern medicine, multiple mechanisms of resistance to these antibiotics have been discovered in a variety of bacterial species. β-Lactam resistance mechanisms include the production of β-lactamases, which hydrolyze the β-lactam ring and render the antibiotic ineffective; mutations acquired in PBPs that prevent binding of the β-lactams; reduced outer membrane permeability due to reduced porin expression; and efflux pumps, which prevent the antibiotic from reaching the cell wall (18–23).

The most common mechanism of β-lactam resistance occurs through the production of β-lactamase enzymes. Most of the characterized β-lactamases have been identified in Gram-negative species; in these bacteria, the β-lactamase is generally secreted into the periplasm, where the enzyme is concentrated, allowing for high levels of β-lactam resistance (24). Less common are the outer membrane-anchored β-lactamases, which may be further packaged into outer membrane vesicles, enabling the inactivation of nearby β-lactams (25–27). β-Lactam resistance in Gram-positive bacteria, however, is more commonly conferred by the modification of the intended targets of the β-lactam, the PBPs (28). Still, β-lactamases do exist in Gram-positive bacteria (29–33). Although Gram-positive bacteria lack a periplasmic space, some species do produce membrane-bound β-lactamases (29, 34–37). A few of these enzymes are proteolytically cleaved, producing an exoenzyme that can be released from the membrane (31, 36, 38).

β-Lactamase enzymes are classified into four classes: A, B, C, and D. Classes A, C, and D are serine hydrolases, while class B β-lactamases are metallohydrolases (18). Whereas β-lactamases of all classes have been discovered in Gram-negative bacteria, most Gram-positive β-lactamases belong to class A or class B (32). Class D β-lactamases were recently identified in Gram-positive bacteria, including one that is highly conserved among C. difficile isolates (33, 39). A recent study demonstrated that a β-lactamase in C. difficile confers resistance to the penicillin, cephalosporin, and monobactam class of β-lactams (39). According to the substrate profile of this enzyme, this β-lactamase belongs to the 2de functional group of β-lactamases (39, 40). The purpose of our study was to characterize the genetic organization, activity, and regulation of the C. difficile β-lactamase. To accomplish this, we deleted the genes encoding the β-lactamase and the upstream predicted membrane protein in C. difficile and examined the resulting resistance profiles, biochemical activity, and regulation of this operon. Notably, we observed that the C. difficile β-lactamase is inactivated by oxygen, which has not been described for other class D β-lactamases. We also examined how this β-lactamase enzyme is transported, and detail its mechanism of regulation. We demonstrate that unlike other described β-lactamases, the C. difficile β-lactamase is cotranscribed with a membrane protein that facilitates β-lactamase processing and function. These results further our understanding of β-lactam resistance in C. difficile, which may expose approaches to prevent or treat β-lactam-associated CDI.

RESULTS

C. difficile produces an inducible, anaerobic β-lactamase.

C. difficile was recently reported to produce a β-lactamase that can cleave β-lactam antibiotics (39). We further investigated the regulation and potential inducibility of C. difficile β-lactamase activity and examined the environmental conditions required for its function. Four diverse strains of C. difficile, 630Δerm (ribotype 012), R20291 (ribotype 027), M120 (ribotype 078), and VPI 10463 (ribotype 003), were grown in the presence or absence of ampicillin (a penicillin), pelleted, resuspended in ∼30 μl of spent media, and incubated either in an anaerobic chamber at 37°C or in an aerobic incubator at 37°C for 15 min. After incubation, the cells were applied to a membrane disk impregnated with nitrocefin, a chromogenic cephalosporin, and incubated for another 20 min either anaerobically or aerobically at 37°C. As shown in Fig. 1A, all four strains of C. difficile grown in the presence of ampicillin under anaerobic conditions caused a color change from yellow to red, indicating cleavage of nitrocefin. In the absence of ampicillin, none of the strains demonstrated observable nitrocefin cleavage. These results suggested that C. difficile produces a β-lactamase that is inducible by β-lactams and is present in diverse strains. During optimization of these assays, we observed markedly higher β-lactamase activity under anaerobic conditions, suggesting that this activity was impaired by oxygen. Indeed, as indicated by Fig. 1A, when the nitrocefin assay was performed in the presence of oxygen, the disks did not change color, even under induction by ampicillin, indicating a loss of β-lactamase activity. Quantification of the β-lactamase activities is shown in Fig. 1B. All four C. difficile strains exhibited significantly increased β-lactamase activity in the absence of oxygen. These results demonstrate that C. difficile strains produce an inducible β-lactamase, and that the activity of this enzyme is quenched by oxygen.

FIG 1.

C. difficile strains exhibit inducible, anaerobic β-lactamase activity. Hydrolysis of the chromogenic cephalosporin nitrocefin was assessed for strains 630Δerm, R20291, M120, and VPI 10463. Strains were grown anaerobically to mid-log phase in BHIS medium with or without 2 μg/ml ampicillin and then pelleted. (A) Cell pellets were resuspended in nonreduced (+O₂) or reduced (–O₂) BHIS and incubated anaerobically or aerobically for 15 min. Cell pellets in ∼30 μl of the remaining media were spotted onto nitrocefin disks for 20 min. A color change from yellow to red indicates cleavage of nitrocefin. (B) Frozen cell pellets were resuspended in reduced or nonreduced BHIS and assayed for nitrocefin cleavage either anaerobically or aerobically, respectively. Columns represent the means ± the standard errors of the mean (SEM) from three independent replicates. Data were analyzed by a two-tailed unpaired Student t test, compared to aerobic conditions for each strain. Adjusted P values indicated by asterisks (*, P ≤ 0.05; ***, P ≤ 0.0005; ****, P < 0.0001).

blaCDD (CD0458) is the only β-lactam-induced β-lactamase gene in C. difficile.

Based on the observed induction of β-lactamase activity, we hypothesized that the expression of one or more putative β-lactamases would be induced upon exposure to β-lactams. To test this, C. difficile strain 630Δerm was grown in the presence of three classes of β-lactams: cefoperazone (a cephalosporin), ampicillin (a penicillin), and imipenem (a carbapenem). Using quantitative reverse transcription-PCR analysis (qRT-PCR), we measured the gene expression for 17 putative β-lactamases identified in the C. difficile genome (8, 41, 42). Figure S1 in the supplemental material demonstrates that the expression of one of these genes, CD0458, was robustly induced upon exposure to each of the three types of β-lactams. None of the other putative β-lactamase genes were induced by any of the β-lactam classes tested (Fig. S1). Expression of the homologous gene in C. difficile strain R20291 was also greatly induced by these three β-lactams (CDR20291_0399, 99% identity; Fig. S1).

CD0458 is analogous to the loci described recently by Toth et al. as CDD-2 (630 genome) and CDD-1 (ATCC 43255 genome) (39). However, other genes are already annotated as cdd, cdd2, cdd3, and cdd4 in C. difficile (43, 44). In addition, these β-lactamases share high sequence similarity (≥94% identity), with the greatest variability in a putative N-terminal signal sequence, suggesting that they are gene alleles rather than distinct genes. To avoid confusion with the previously established cdd loci and to adhere to the guidelines on genotypic designation of operons, the locus was renamed bla and the β-lactamase gene was named blaCDD by the National Center for Biotechnology Information, in accordance with its function as a class D β-lactamase (45).

CD0457 encodes a putative membrane protein, BlaX, which is cotranscribed with blaCDD.

Analysis of the region surrounding blaCDD revealed the presence of another gene, CD0457, which appeared to be part of an operon with blaCDD. Figure 2A illustrates the putative bla operon, in which CD0457 is located 27 nucleotides upstream of the start codon of CD0458. To determine whether expression of CD0457 is similarly induced upon β-lactam exposure, we measured the transcription of CD0457 in C. difficile strain 630Δerm upon exposure to cefoperazone, ampicillin, and imipenem. Figure 2B demonstrates that expression of CD0457 is comparably induced upon exposure to all three β-lactams. This coregulation by β-lactams strongly suggested that CD0457 is cotranscribed with CD0458 and that the CD0457 predicted membrane protein product could play a role in the β-lactam resistance. The expression of the homologous gene in C. difficile strain R20291 was also comparably induced upon exposure to these β-lactams, indicating a similar organization in divergent strains (Fig. S2).

FIG 2.

The putative β-lactamase gene, CD0458, and the upstream gene CD0457 are induced by β-lactams. (A) The putative β-lactamase gene CD0458 is located 27 bp downstream of the predicted membrane protein, CD0457. (B) The relative expression of each gene was measured via qRT-PCR. C. difficile strain 630Δerm was grown to mid-log phase in BHIS medium supplemented with subinhibitory concentrations of β-lactams (Cfp, cefoperazone at 50 μg/ml; Amp, ampicillin at 2 μg/ml; Ipm, imipenem at 1.5 μg/ml). The mRNA levels are normalized to the expression levels in BHIS alone. Columns represent the means ± the SEM from three independent replicates. Data were analyzed by one-way ANOVA with Dunnett’s multiple-comparison test, compared to no antibiotic. Adjusted P values indicated by an asterisk (*, P ≤ 0.05).

To determine whether the CD0457 and blaCDD genes are part of a single cistronic unit, we assessed the linkage of these transcripts by amplifying the region between CD0457 and blaCDD from cDNA generated after exposure of C. difficile strains 630Δerm and R20291 to ampicillin (Fig. S3A). Figure S3B illustrates the results of the PCR from cDNA that generated a product of 1 kb, which matches the genomic DNA product from the same strain. These data demonstrate that the transcription of CD0457 and blaCDD are linked, indicating that they comprise a monocistronic unit. Since CD0457 and blaCDD form an operon whose function of CD0457 is unknown, we named the CD0457 gene blaX.

To further define the transcriptional organization of the bla operon, we examined promoter activity within the bla locus. Potential promoter activity was measured for putative promoter regions within the locus using phoZ reporter fusions, which produce alkaline phosphatase (46). As illustrated in Fig. 3, regions of 300 nucleotides directly upstream of the start codons of blaX or blaCDD were fused to phoZ and expressed in C. difficile. The results of these reporter assays indicate that the region 300 nucleotides upstream of blaX, but not the region 300 nucleotides upstream of blaCDD, is able to promote transcription, resulting in measurable activity. To confirm the absence of a cryptic blaCDD promoter located within the blaX coding region, the entire region from the translational start of blaX to the start codon of blaCDD was also examined for possible promoter activity. However, no transcriptional activity was observed from this region (Fig. 3). The only segment that produced significant and inducible activity contained the region upstream of the blaX coding sequence, strongly suggesting that solely this region drives blaX and blaCDD expression.

FIG 3.

Alkaline phosphatase activity from P_blaXCDD::phoZ is induced in the presence of ampicillin. C. difficile 630Δerm cultures were grown to an OD₆₀₀ of ∼0.5 in BHIS with 2 μg/ml thiamphenicol for plasmid maintenance in the presence or absence of 2 μg/ml ampicillin. Strains MC448 (::phoZ-empty vector), MC1317 (P_blaXCDD::phoZ), MC1318 (5′ blaCDD::phoZ), and MC1369 (blaX::phoZ) were tested. The means and SEM of three biological replicates are shown. Data were analyzed by one-way ANOVA with Dunnett’s multiple-comparison test. The adjusted P value is indicated by asterisks (****, P < 0.0001).

BlaX and BlaCDD contribute to ampicillin resistance in C. difficile.

Notably, 36% of complete C. difficile genomes contain a homolog of blaX. Other sequenced genomes simply contain the same promoter and blaCDD region without the membrane protein. The membrane protein only shares approximately 23 to 40% amino acid identity to uncharacterized proteins found in a few other bacterial species. Thus, the function of this membrane protein cannot be inferred from other systems. To define the roles of BlaX and BlaCDD in β-lactam resistance and in β-lactamase activity, we created mutants of the 630Δerm strain with an insertional mutation in the blaX gene (MC905) or complete deletion of the blaX-blaCDD locus (MC1327). Compared to the parent strain, blaX::erm displayed decreased, but still inducible blaCDD expression (Fig. S4). Although blaX transcription is measurable in the blaX::erm mutant, the product is presumably nonfunctional because of the insertional mutation. We confirmed that neither the blaX nor the blaCDD transcript was expressed in the ΔblaXCDD mutant (Fig. S4).

Based on the induction of β-lactamase activity and the induction of the bla operon by β-lactams, we hypothesized that both genes contribute to C. difficile resistance to β-lactams. In order to measure the contribution of the bla operon to β-lactam resistance in C. difficile, we performed time-kill curves in 8× MIC of the β-lactams cefoperazone, ampicillin, or imipenem, as well as growth curves in 60 μg/ml cefoperazone, 4 μg/ml ampicillin, or 2 μg/ml imipenem. The time-kill curves shown in Fig. 4A to C demonstrate that the ΔblaXCDD demonstrated no significant difference in cell death in the different β-lactams. However, Fig. 4D to F show a larger impairment of growth of the ΔblaXCDD and blaX::erm strains in ampicillin compared to the parent strain. These data suggest that the bla operon contributes modestly to ampicillin resistance in C. difficile. Interestingly, the deletion of blaX and blaCDD improved growth in imipenem. This could be a result of BlaCDD sequestering imipenem to the cell wall. In the wild-type cell, imipenem would be sequestered to the cell wall by this mechanism. This hypothesis is supported by the finding by Toth et al. that BlaCDD binds to but does not hydrolyze imipenem (39).

FIG 4.

blaX and blaCDD contribute to ampicillin resistance in C. difficile. C. difficile strains 630Δerm (gray), blaX::erm (MC905; pink), and ΔblaXCDD (MC1327; blue) were grown to mid-log phase. The strains were then backdiluted to 1E7 CFU/ml (A, B, and C) or to an OD₆₀₀ of 0.02 (D, E, and F) and grown in BHIS (no marker) or BHIS supplemented (filled marker) with cefoperazone (Cfp) at 8× MIC (A), ampicillin (Amp) at 8× MIC (B), imipenem (Ipm) at 8× MIC (C), Cfp at 60 μg/ml (D), Amp at 4 μg/ml (E), or Ipm at 2 μg/ml (F). The lines represent the means ± the SEM from four independent replicates (except for panel B, h 4, and panel C, which represent the means ± the SEM from three independent replicates). Data were analyzed by one-tailed paired Student t test, compared to strain 630Δerm. No statistically significant differences found.

To further define the contribution of blaX and blaCDD to β-lactam resistance in C. difficile, we measured the MIC of β-lactams in the 630Δerm, ΔblaXCDD, and blaX::erm strains. Although the parent strain grew better in ampicillin, the MICs for both cefoperazone and ampicillin were similar in all three strains (Table S2) and higher for the 630Δerm strain in imipenem, indicating a modest difference in imipenem resistance.

The bla operon exhibits high-level, dose-dependent expression in β-lactams.

The induction of both blaX and blaCDD by β-lactams suggested that these genes are important for β-lactam resistance in C. difficile. To determine whether these genes could be induced by other cell wall targeting antimicrobials or if the induction is specific to β-lactam exposure, we measured the levels of gene expression for C. difficile strain 630Δerm in various cell wall targeting antibiotics (vancomycin, polymyxin B, and lysozyme) and cationic antimicrobial peptides (nisin and LL-37), as well as a ribosome-targeting antibiotic (kanamycin). The concentrations of each of these antimicrobials was at sub-MIC value and sufficient for robust induction of resistance gene expression based on previous work (47–50). Figure 5 shows that expression of blaX and blaCDD was induced in the presence of vancomycin and polymyxin B. However, these levels of expression are not statistically significant and were <3% of the levels seen for expression after β-lactam exposure, suggesting that the high levels of induction of blaX and blaCDD are specific to β-lactams.

FIG 5.

blaXCDD transcription is modestly induced by vancomycin and polymyxin B. The relative expression of each gene was measured via qRT-PCR. C. difficile strain 630Δerm was grown to mid-log phase in BHIS medium supplemented with subinhibitory concentrations of cell wall targeting antimicrobials (Van, vancomycin at 0.75 μg/ml; PmB, polymyxin B at 75 μg/ml; Lys, lysozyme at 1 mg/ml; Nis, nisin at 7.5 μg/ml; LL-37, LL-37 at 2 μg/ml; Kan, kanamycin at 250 μg/ml). mRNA levels are normalized to expression levels in BHIS alone. Columns represent the means ± the SEM from four independent replicates. Data were analyzed by one-way ANOVA with Dunnett’s multiple-comparison test, compared to expression in strain 630Δerm without antibiotic. No statistically significant values found.

Although the levels of blaX and blaCDD induction were high in all three β-lactams, expression varied greatly between each β-lactam. These results suggested that the level of induction of the bla operon is dependent upon the type of β-lactam C. difficile is exposed to and could be dose dependent. To determine whether the bla operon exhibits dose-dependent expression in β-lactams, we measured the relative expression of blaX and blaCDD in the 630Δerm strain in various concentrations of cefoperazone, ampicillin, and imipenem. Figure S5 shows that the bla operon did indeed exhibit dose-dependent induction by β-lactams and that the response was different for the various classes of β-lactams. In increased concentrations of cefoperazone, induction of the bla operon trended downward, whereas expression trended upward in increased concentrations of ampicillin. Expression of the bla operon was high in all concentrations of imipenem, exhibiting only a modest increase in expression as the concentration of imipenem was increased. Furthermore, the level of induction of the bla operon was high even at concentrations of β-lactams far below the MIC (0.03125× MIC of cefoperazone, 0.125× MIC of ampicillin, and 0.0625× MIC of imipenem). These results suggest that bla expression is controlled in a dose-dependent manner specific to the class of β-lactam administered.

BlaX is not required for β-lactamase activity.

Of the 1747 amino acid sequence variants retrieved from a 630Δerm BlaCDD BLASTp search of C. difficile (>90% coverage and >80% identity), 736 isolates (42%) also encode the upstream putative membrane protein (>97% coverage and >86% identity), suggesting that the membrane protein BlaX may be important for β-lactamase activity in some strains but not in others. The BlaCDD enzyme from strains M120 and VPI 10463, which lack BlaX, and strains 630Δerm and R20291 are highly similar, but the 4% variability clearly lies within the N termini of these proteins (Fig. S6). As shown in Fig. 1A, all four of these strains exhibit β-lactamase activity. The variability in the amino acid sequence of these enzymes may be due to differences in signal sequence recognition, but a potential interaction with another protein cannot be ruled out.

Since the function of BlaX was not immediately apparent, we examined whether BlaX is necessary to observe the β-lactamase activity of BlaCDD in strain 630Δerm. To test this, we complemented the ΔblaXCDD strain with blaX and/or blaCDD in trans. As expected, no apparent β-lactamase activity was observed for the ΔblaXCDD strain (Fig. 6A). In comparison, the blaX::erm strain exhibits a slight change in color to a light pink, indicating that this mutant does not fully abolish production and activity of the β-lactamase, a finding in agreement with the decrease in blaCDD gene expression observed for this strain (Fig. S4). Nitrocefin disk assays (Fig. 6B) demonstrated that expression of blaCDD alone can restore β-lactamase activity in the ΔblaXCDD mutant, indicating that BlaCDD can act independently of BlaX, despite the cotranscription of these two genes. This result is further supported by the observation that the blaX::erm strain exhibits some β-lactamase activity (Fig. 6A).

FIG 6.

The N terminus of BlaCDD is necessary for β-lactamase secretion, independent of BlaX. Hydrolysis of the chromogenic cephalosporin nitrocefin was assessed for strains 630Δerm, blaX::erm (MC905), and ΔblaXCDD (MC1327) (A); strain ΔblaXCDD complemented with blaX and/or blaCDD, expressed from their native promoter (B); and strains 630Δerm, blaX::erm 1.0, blaX::erm 2.0 (C). Strains were grown anaerobically to mid-log phase in BHIS medium (with 2 μg/ml thiamphenicol for plasmid maintenance in panel B with or without 2 μg/ml ampicillin) and pelleted. Cell pellets in ∼30 μl of remaining media were incubated anaerobically on nitrocefin disks for 2 h. A color change from yellow to red indicates nitrocefin cleavage. The results shown are representative of three independent assays.

BlaCDD contains a predicted signal sequence and is associated with the cell membrane.

A common characteristic of β-lactamases is an N-terminal signal sequence that directs the protein out of the cytoplasm. We hypothesized that the N terminus of BlaCDD encodes a signal sequence based on the signal sequence prediction within the first 18 amino acid residues (51, 52). We generated a truncated version of BlaCDD missing these first 18 residues (BlaCDDΔ18; pblaCDDΔ18). As shown in Fig. 6B, the expression of BlaCDDΔ18 is unable to complement the absence of β-lactamase activity in the ΔblaXCDD mutant in a whole-cell assay. The qRT-PCR results shown in Fig. S7 confirm that blaX and/or blaCDD are expressed in the complemented strains, indicating that the absence of gene expression is not the cause of the lack of observable β-lactamase activity. This suggested that BlaCDDΔ18 either is not translated, is an unstable or inactive protein, or is active but trapped in the cytosol and unable to hydrolyze nitrocefin.

All of the characterized β-lactamases in Gram-positive bacteria are membrane-bound enzymes, although many of these proteins are cleaved, resulting in a smaller, soluble form that can be found in culture supernatants (29, 31, 34, 36). These findings are consistent with the lack of a periplasmic space for β-lactamases accumulation in Gram-positive bacteria. To determine whether a soluble form of BlaCDD is secreted into the culture medium, we performed a nitrocefin hydrolysis assay using culture supernatants. As shown in Fig. 7A and C, neither the supernatants of ΔblaXCDD cells harboring pblaCDD or pblaX-blaCDD nor the wild-type strains 630Δerm or M120 reacted with nitrocefin, indicating that BlaCDD is not secreted into the medium. To confirm that BlaCDD is a membrane-associated enzyme, we lysed the cells and performed a nitrocefin hydrolysis assay using lysates containing cell debris (denoted as “lysates”) or the cleared cell lysates (denoted as “lysate filtrate”). Figure 7B and D show that when we compared the level of activity in the lysate to that in the lysate filtrate in strains containing a full-length blaCDD, 74 to 80% of the total β-lactamase activity is found in the cell debris, indicating that BlaCDD is associated with the cell surface. Furthermore, BlaCDDΔ18 activity is not associated with the cell surface, as demonstrated by the similar levels of activity in the lysate and the lysate filtrate (Fig. 7B). This result indicates that BlaCDDΔ18 is an active, soluble form of BlaCDD that is trapped in the cytosol and strongly suggests that the first 18 residues at the N terminus of BlaCDD encode a signal sequence. Together, these results support the presence of a signal sequence that helps bring the protein to the cell surface.

FIG 7.

BlaCDD utilizes a signal sequence to act at the cell membrane. C. difficile strain ΔblaXCDD (A and B) or C. difficile strains 630Δerm, ΔblaXCDD, and M120 (C and D) were grown to mid-log phase in 2 μg/ml thiamphenicol (A and B) and 2 μg/ml ampicillin and assayed for β-lactamase activity via a nitrocefin (A), supernatant or cell suspension (C), and cell lysate or cell lysate filtrate (B and D) assays. Strains ΔblaXCDD pMC123 (MC 1400), ΔblaXCDD pblaXCDD (MC1399), ΔblaXCDD pblaCDD (MC1466), ΔblaXCDD pblaCDDΔ18 (MC1338), and ΔblaXCDD pM120blaCDD (MC1494) were evaluated. Columns represent the means ± the SEM from at least three independent replicates. Data were analyzed by one-way ANOVA with Dunnett’s multiple-comparison test, compared to pblaCDD (A and B) or 630Δerm (C and D), or using a two-tailed unpaired Student t test, where indicated by bars. No absence of asterisk indicates there was no statistically significant difference found. Adjusted P values are indicated by asterisks (*, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005; ****, P < 0.0001).

BlaX aids in BlaCDD activity.

Although BlaX is not necessary for BlaCDD activity (Fig. 6A and B), blaX is conserved in many C. difficile strains. Thus, we examined whether BlaX enhances BlaCDD activity. The results shown in Fig. 7A and B demonstrate that the presence of BlaX increases β-lactamase activity of the 630Δerm BlaCDD 2- to 3-fold, suggesting that BlaX plays a role in the function of BlaCDD. To investigate the activity of a BlaCDD from a C. difficile genome that lacks BlaX, we also complemented the ΔblaXCDD strain with blaCDD cloned from the M120 genome, under the M120 native promoter. Figure 7A shows that in cell suspensions of ΔblaXCDD complemented strains, the M120 BlaCDD (pM120blaCDD) exhibits 2-fold-higher activity than the 630Δerm BlaCDD (pblaCDD). This result suggests that the M120 BlaCDD is superior to the 630Δerm BlaCDD at translocating to the cell surface when BlaX is not present. However, M120 BlaCDD is only two-thirds as active as the 630Δerm BlaXD complement (pblaXCDD). In lysed cells, the M120 BlaCDD β-lactamase activity levels are slightly higher than the 630Δerm BlaCDD (Fig. 7B). Interestingly, the wild-type strains 630Δerm and M120 exhibit similar β-lactamase activity levels in both cell suspension and lysate samples, indicating that their overall efficacy is comparable (Fig. 7C and D). Together, these results demonstrate that in 630Δerm strain BlaX enhances BlaCDD activity, whereas in M120 the β-lactamase activity is not dependent on BlaX. Finally, because the M120 BlaCDD does not fully complement the ΔblaXCDD strain, the N-terminal sequence variability of the BlaCDD proteins likely plays a role in strain-dependent translocation of BlaCDD to the cell surface.

The bla operon is regulated by BlaRI.

Transcription of most β-lactamase genes in Gram-positive bacteria is regulated by the two-component BlaRI system (53–55). The C. difficile genome encodes several orthologs of the two genes that make up this system, blaI and blaR. In other bacteria, BlaR is a sensor that is activated upon β-lactam binding (56). Activated BlaR cleaves the BlaI repressor, which is bound as a dimer to the bla operon promoter in the absence of β-lactams (57–59). Once cleaved, BlaI can no longer bind to the bla promoter, thus allowing for active transcription. Two candidate orthologs CD0471 (blaI) and CD0470 (blaR) are located 11 kb downstream of the blaXCDD operon. To determine whether these blaRI orthologs regulate the blaXCDD operon in C. difficile, we created an insertional disruption in blaI. Figure S8 shows that transcription of blaR is decreased in the blaI::erm mutant, confirming that blaI and blaR are organized in an operon, as is consistent with other bacteria. As seen in Fig. S9B, transcription of blaI is high, even in the absence of β-lactams, which demonstrates that BlaI regulates itself, as the primers used were upstream of the disruptional insertion. As shown in Fig. 8, in the absence of β-lactams, blaX and blaCDD are transcribed at high levels in the blaI::erm mutant compared to the wild-type 630Δerm strain. These results confirm that BlaI acts as a repressor of the bla operon. Further, the induction of blaXCDD in β-lactams in the wild-type strain, but not in the mutant, strongly suggests that BlaI repression is relieved by the presence of β-lactams in wild-type strain. To verify that relief of BlaI repression results in β-lactamase production, we performed a nitrocefin hydrolysis assay on the blaI::erm mutant. Figure 6C confirms that the absence of BlaI results in active β-lactamase, independent of β-lactam presence. Together, these results show that C. difficile encodes a BlaRI system that represses bla transcription in the absence of β-lactams. Efforts to complement blaRI resulted in poor growth of Escherichia coli mating strains, as well as C. difficile, and were not successful; however, two independent strains of blaI::erm were successfully created, as shown in Fig. 6C.

FIG 8.

blaXCDD is derepressed in the blaI::erm strain. qRT-PCR was performed to measure expression of blaX (A) and blaCDD (B) in C. difficile 630Δerm and blaI::erm strains grown to mid-log phase in BHIS media with or without β-lactam (Cfp, cefoperazone at 60 μg/ml; Amp, ampicillin at 2 μg/ml; Ipm, imipenem at 1.5 μg/ml). The mRNA levels are normalized to the expression levels in strain 630Δerm in BHIS alone. Columns represent the means ± the SEM from three independent replicates. Data were analyzed by one-way ANOVA with Dunnett’s multiple-comparison test, compared to expression in 630Δerm without antibiotic. Adjusted P values are indicated by asterisks (*, P ≤ 0.05; **, P ≤ 0.005).

BlaRI contributes to ampicillin resistance in C. difficile.

To further confirm that the BlaRI system regulates the bla operon and to define its contribution to ampicillin resistance, we examined the death, as well as the growth, of the blaI::erm mutant in multiple β-lactams via time-kill and growth curves. Figure 9A to C demonstrate that the blaI::erm strain exhibited slightly less cell death in cefoperazone and significantly less death in ampicillin. Figure 9D illustrates that growth of the blaI mutant is not significantly different than the wild-type 630Δerm strain in the presence of cefoperazone. However, growth of the blaI mutant is significantly improved in the presence of ampicillin compared to the 630Δerm strain (Fig. 9E). Finally, the blaI::erm mutant shows slightly impaired growth in imipenem compared to 630Δerm (Fig. 9F). These results are reversed, but consistent, with the results observed with the blaCDD mutant in imipenem. These data show that BlaRI contributes to ampicillin resistance in C. difficile at least partially through regulation of the bla operon.

FIG 9.

blaI regulates ampicillin resistance in C. difficile. The C. difficile strains 630Δerm (gray) and blaI::erm (MC985; red) were grown to mid-log phase, backdiluted to 1E7 CFU/ml (A, B, and C) or to an OD₆₀₀ of 0.02 (D, E, and F), and then grown in BHIS (no marker) or BHIS supplemented (filled marker) with cefoperazone (Cfp) at 8× MIC (A), ampicillin (Amp) at 8× MIC (B), imipenem (Imp) at 8× MIC (C), Cfp at 60 μg/ml (D), Amp at 4 μg/ml (E), or Ipm at 2 μg/ml (F). Lines represent the means ± the SEM from four independent replicates (except for panel B, h 4, and panel C, which represent the means ± the SEM from three independent replicates). Data were analyzed by one-tailed paired Student t test, compared to strain 630Δerm. Adjusted P values are indicated by an asterisk (*, P ≤ 0.05).

DISCUSSION

This study provides evidence for robust β-lactam-dependent expression of the β-lactamase, BlaCDD. The blaCDD gene is located in an operon with blaX, which encodes a putative membrane protein (Fig. S3). Our data indicate that the promoter for the blaXCDD operon is located within a 300-nucleotide region located directly upstream of the blaX start codon (Fig. 3). The high level of blaCDD and blaX expression in response to β-lactams far below the MICs (Fig. S5) indicates that the promoter of the bla operon is quite strong, in contrast to a previous report in which part of the blaCDD locus was expressed in a heterologous host (39).

Our work has demonstrated that BlaCDD is a β-lactamase that is only active under anaerobic (reducing) conditions (Fig. 1). Analysis of the protein via DiANNA (Fig. S6) revealed that all of the cysteines encoded in the four BlaCDD proteins analyzed have a predicted oxidation state probability of 1 (60). The high probability of these cysteines oxidizing under aerobic conditions renders this enzyme sensitive to changes in the redox state of the environment. To our knowledge, no other anaerobic-restricted β-lactamases have been reported, which is not surprising given that β-lactamase assays are generally performed in the presence of oxygen (61, 62). This, however, may be one reason that so few β-lactamases have been identified in anaerobic, Gram-positive bacteria (63–66). Indeed, the addition of 0.2 mM dithiothreitol to the nitrocefin hydrolysis assays or steady-state enzyme kinetics assays (39) allows for observation of BlaCDD activity (Fig. 7) by maintaining reducing conditions. Assaying β-lactamases from other anaerobic, Gram-positive bacteria under reducing conditions may lead to the identification of additional anaerobic β-lactamases in other species.

Our data indicate that BlaCDD acts at the cell surface, which is facilitated by the signal sequence at the N terminus, which allows for translocation of BlaCDD to the membrane. BlaCDD is not secreted into the environment but remains associated with the cell surface (Fig. 7). While the exact function of BlaX is unknown, the data demonstrate that BlaCDD activity is enhanced by the presence of BlaX (Fig. 7B). BlaX has five predicted transmembrane domains, with an ∼125-residue-long extracellular loop (67). Because the activity of BlaCDD is membrane associated across all samples except BlaCDDΔ18, and because BlaCDD activity in cell lysates lacking BlaX is 60% less than when BlaX is present, it is possible that BlaX interacts with BlaCDD in a way that makes BlaCDD more accessible to substrates on the cell surface. Alternatively, BlaX may interact with β-lactams to facilitate their interaction with BlaCDD. Nitrocefin hydrolysis assays showed that in cell lysates the activity of full-length BlaCDD (pblaCDD) is 45% less than that of BlaCDDΔ18 (Fig. 7B). This could result from BlaCDD cleavage at the N terminus after translocation to the cell membrane or from BlaX helping to relieve a steric hindrance caused by insertion into the cell membrane. The absence of β-lactamase activity in cell supernatants does not support cleavage of BlaCDD, unless BlaCDD remains anchored to the cell membrane after cleavage. Although BlaCDD does not contain a canonical lipobox immediately downstream of the signal peptide, BlaCDD has a putative transmembrane domain at the N terminus, which may allow for membrane anchoring via a noncanonical mechanism (67, 68). Further experiments are needed to determine how BlaCDD is processed by C. difficile.

To date, only one other published β-lactamase is reported to be cotranscribed with a membrane protein (69). This membrane-bound β-lactamase, PenA, found in the Gram-negative Burkholderia pseudomallei, is encoded in an operon with nlpD1, a gene annotated as an outer membrane lipoprotein and thought to be involved in cell wall hydrolytic amidase activation (70). However, C. difficile does not contain an outer membrane, and nlpD1 does not exhibit homology with blaX. Analysis of the blaCDD locus in the C. difficile strain M120, which does not contain a full blaX coding sequence, revealed regions of partial homology to the 5′ and 3′ ends of blaX, located between the promoter and the blaCDD start codon. This suggests that over the course of evolution of C. difficile, the majority of this gene was deleted. A search of the rest of the M120 genome revealed no other proteins similar to BlaX, further supporting the model that in many C. difficile strains, BlaX is not necessary for sufficient BlaCDD activity. However, the superior activity levels of M120 BlaCDD (Fig. 7A and B) and the 74% of cell surface-associated activity of M120 BlaCDD (Fig. 7B), as well as the equal levels of β-lactamase activity of the 630Δerm strain compared to M120 (Fig. 7D), suggest that M120 may have a different mechanism of translocation.

We have shown that the bla operon confers partial resistance to ampicillin and is regulated by the BlaRI system in C. difficile (Fig. 4, 8, and 9). The discrepancy between the time-kill curves and the growth curves of the ΔblaXCDD strain compared to the wild-type strain is likely due to the difference in concentrations of antibiotic used. It is probable that the bla operon is able to contribute some, but not all resistance to ampicillin in C. difficile and that ampicillin levels greater than 2× the MIC eliminate any opportunity for BlaCDD to inactivate all antibiotic molecules. Disruption of blaI resulted in the constitutive expression of blaX and blaCDD (Fig. 8), which resulted in slowed death and improved growth in ampicillin (Fig. 9B and E), supporting the model that BlaI is a direct repressor of the bla operon. We identified a 52-nucleotide region of dyad symmetry in the promoter of the bla operon, which contains a canonical BlaI binding site, supporting the model of BlaI-PblaX binding, but does not rule out other binding partners. Our results align with previously reported data that BlaCDD confers resistance to penicillins (39). It is possible that the BlaRI system regulates other penicillin resistance mechanism(s) in C. difficile based on the data that disruption of blaI contributes to increased ampicillin resistance, but it is unlikely to be another β-lactamase. The discrepancy of the MIC values versus the growth curves can be attributed to the greater sensitivity of growth curves in assessing the impact of antimicrobials on cell growth. Further investigation is needed to fully define the mechanisms of β-lactam resistance in C. difficile. Identification and characterization of any additional β-lactam resistance mechanisms may aid in preventing C. difficile infections and recurrence in the future.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

Bacterial strains and plasmids used in this study are listed in Table 1 . Escherichia coli was grown at 37°C in Luria-Bertani medium with 100 μg/ml ampicillin (Sigma-Aldrich) and 20 μg/ml chloramphenicol (Sigma-Aldrich) when necessary (71). C. difficile was grown anaerobically at 37°C as previously described (72) in brain heart infusion medium supplemented with 2% yeast extract (BHIS; Βecton Dickinson Company) or Mueller-Hinton broth (MHB; Difco) with 2 μg/ml thiamphenicol (Sigma-Aldrich), 3.125 to 60 μg/ml cefoperazone (Sigma-Aldrich), 0.25 to 2 μg/ml ampicillin or 0.125 to 1.5 μg/ml imipenem (US Pharmacopeia), 0.75 μg/ml vancomycin (Sigma-Aldrich), 75 μg/ml polymyxin B (Sigma-Aldrich), 1 mg/ml lysozyme (Fisher Scientific), 7.5 μg/ml nisin (MP Biomedicals), 2 μg/ml LL-37 (Anaspec), or 250 μg/ml kanamycin (Sigma-Aldrich) when specified.

TABLE 1.

Bacterial strains and plasmids

Plasmid or strain	Relevant genotype or features	Source, construction, or reference
Strains
E. coli
HB101	F– mcrB mrr hsdS20(rB– mB–) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20	B. Dupuy
DH5α	F–Φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK− mK+) phoA supE44 λ- thi-1 gyrA96 relA1	Invitrogen
C. difficile
630	Clinical isolate	85
630Δerm	Erms derivative of strain 630	86
M120	Clinical isolate	87
MC324	630Δerm pMC123	78
MC448	630Δerm pMC358	46
MC905	blaX::erm	This study
MC985	blaI::erm 1.0	This study
MC1316	630Δerm pMC822	This study
MC1317	630Δerm pMC826	This study
MC1318	630Δerm pMC827	This study
MC1327	630Δerm ΔblaXCDD	This study
MC1369	630Δerm pMC842	This study
MC1338	ΔblaXCDD pMC811	This study
MC1399	ΔblaXCDD pMC867	This study
MC1400	ΔblaXCDD pMC123	This study
MC1438	blaI::erm pMC123	This study
MC1466	ΔblaXCDD pMC897	This study
MC1494	ΔblaXCDD pMC896	This study
MC1538	blaI::erm 2.0	This study
Plasmids
pRK24	Tra+ Mob+; bla tet	88
pCR2.1	bla kan	Invitrogen
pCE240	C. difficile TargeTron construct based on pJIR750ai (group II intron, ermB::RAM, ltrA); catP	73
pMTL-SC7215	Pseudo-suicide plasmid used for allelic exchange in C. difficile	74
pMC123	E. coli-C. difficile shuttle vector; bla catP	79
pMC358	pMC123::phoZ	46
pMC585	pCR2.1 + group II intron targeted to blaX	This study
pMC586	pCE240 + group II intron targeted to blaX	This study
pMC593	pCR2.1 + group II intron targeted to blaI	This study
	pCE240 + group II intron targeted to blaI	This study
pMC622	pMC123 + group II intron targeted to blaX, ermB::RAM, ltrA, catP	This study
pMC664	pMC123 + group II intron targeted to blaI, ermB::RAM, ltrA, catP	This study
pMC810	pMC123 + PblaXCDD + blaX	This study
pMC811	pMC123 + PblaXCDD + blaCDDΔ18	This study
pMC822	pMTL-SC7215 + 500 bp 5′ + 500 bp 3′ of blaXCDD	This study
pMC826	pMC358 + PblaXCDD (300 bp 5′ UTR of blaX)	This study
pMC827	pMC358 + 300-bp 5′ UTR of blaCDD	This study
pMC842	pMC358 + blaX	This study
pMC867	pMC123 + PblaXCDD + blaXCDD	This study
pMC896	pMC123 + PM120blaCDD + M120blaCDD	This study
pMC897	pMC123 + PblaXCDD + blaCDD (cdd-2 in reference 39)	This study

Strain and plasmid construction.

The oligonucleotide primers used in this study are listed in Table S1. Primer design and the template for PCRs were based on C. difficile strain 630 (GenBank accession NC_009089.1), except for pMC896, which was based on strain M120 (GenBank accession FN665653.1).

The blaX::erm and blaI::erm mutant strains were created by retargeting the group II intron from pCE240 with the primers listed in Table S1, as previously described (73). To generate insertional disruptions, transconjugants were selected on 5 μg/ml erythromycin (Sigma-Aldrich) and 50 μg/ml kanamycin (Sigma-Aldrich) to select against E. coli.

The ΔblaXCDD mutant strain was created using a pseudo-suicide plasmid technique, as described previously, with slight variation (74). Details of the strain construction can be found in Fig. S2.

Detailed construction of plasmids can be found in Fig. S1. Plasmids were transferred to C. difficile as previously described, with slight variation (75, 76). Briefly, plasmids were chemically transformed into E. coli strain HB101 pRK24 and mated with C. difficile on agar plates for 48 h. Transconjugants were selected on BHIS agar containing 10 μg/ml thiamphenicol for plasmid selection and 100 μg/ml kanamycin to counterselect against E. coli.

Nitrocefin hydrolysis disk assays.

β-lactamase activity was assessed by hydrolysis of nitrocefin, a chromogenic cephalosporin (Sigma-Aldrich). Briefly, C. difficile was grown overnight in BHIS to log phase, then diluted to an optical density at 600 nm (OD₆₀₀) of 0.05 in BHIS medium with or without 2 μg/ml ampicillin. Cultures were grown to an OD₆₀₀ of 0.45 to 0.55, and 1 ml of culture was collected and centrifuged for 5 min at 21,130 relative centrifugal force (rcf). For wild-type anaerobic versus aerobic assays, pellets were resuspended in 1 ml of nonreduced or reduced BHIS and incubated at 37°C for 15 min, either aerobically or anaerobically. The cells were then centrifuged for 5 min at 21,130 rcf. For all disk assays, all but approximately 30 μl of the supernatant was decanted, the pellets were resuspended, and the cells were spotted onto a nitrocefin disk. The disks were incubated aerobically or anaerobically for 20 min to 2 h at 37°C, as noted.

Nitrocefin liquid hydrolysis assays.

The β-lactamase activity was determined for wild-type or complemented strains via anaerobic (or aerobic, where noted) liquid nitrocefin assays, as previously reported, with some modifications (77). Details of the assays can be found in Fig. S3. The results reported are the means of at least three independent experiments.

qRT-PCR.

Actively growing C. difficile were diluted to an OD₆₀₀ of 0.02 in 10 to 25 ml BHIS with appropriate antibiotic and grown to log phase. RNA was isolated as described previously (75, 78). Briefly, 3-ml samples were taken at an OD₆₀₀ of 0.45 to 0.55, mixed with 3 ml of ice-cold 1:1 acetone-ethanol, and stored immediately at –80°C. RNA was isolated (using a Qiagen RNeasy kit) and treated for contaminating DNA (using an Invitrogen Turbo DNA-free kit), and RNA was reverse transcribed into cDNA (using a Bioline Tetro cDNA synthesis kit). cDNA samples were used for qPCR (Bioline SensiFAST SYBR and fluorescein kit) in technical triplicates on a Roche LightCycler 96 as described previously (79). The comparative cycle threshold method was used to determine mRNA levels of the genes of interest via normalization to an internal control transcript (rpoC) (80). The results are presented as means and standard errors of the means for three biological replicates. The statistical significance was determined using a one-way analysis of variance (ANOVA), followed by Dunnett’s multiple-comparison test (GraphPad Prism v6.0).

Alkaline phosphatase activity assays.

Alkaline phosphatase activity assays in C. difficile were performed as described previously, with minor modifications to the original published assay (46, 81). Briefly, C. difficile cultures were grown anaerobically at 37°C overnight in BHIS with thiamphenicol (2 μg/ml) to log phase and then diluted to an OD₆₀₀ of 0.05 in 10 ml of BHIS with thiamphenicol. Next, 1-ml portions of cells were collected in duplicate when the OD₆₀₀ reached 0.5. The cells were centrifuged at 21,130 rcf for 3 min, and the pellets were stored at –20°C at least overnight. For the assay, cell pellets were thawed, resuspended in 500 μl of cold wash buffer (10 mM Tris [pH 8.0], 10 mM MgSO₄), and pelleted for 3 min at 21,130 rcf. Alkaline phosphatase assays were performed as previously described (46) without the addition of chloroform (82). The OD₅₅₀ (cell debris) and the OD₄₂₀ (pNP cleavage) were measured in a BioTek microplate reader. Values were averaged between the triplicate wells and then between duplicate technical samples. AP units were calculated as {[OD₄₂₀ − (1.75 × OD₅₅₀)] × 1,000}/(OD₆₀₀ × time), where OD₆₀₀ is the value at the time of collection. The results reported are the averages of three independent experiments. Statistical significance was determined using a one-way ANOVA, followed by Dunnett’s multiple-comparison test (GraphPad Prism v6.0).

Time-kill curves.

Cell death in β-lactams was measured as described previously with minor modifications (83, 84). Briefly, active C. difficile cultures were diluted in MHB to an OD₆₀₀ of 0.1 and then diluted to ∼10⁷ CFU per ml (CFU/ml) in 10 ml of MHB plus cefoperazone, ampicillin, or imipenem at 8× MIC (800, 16, or 16 μg/ml, respectively). Cultures were serially diluted and plated onto BHIS agar plates without antibiotic at the time points indicated, and colonies were counted after a 24-h incubation in the anaerobic chamber at 37°C. The results are presented as means and standard errors of the means for at least three biological replicates. Statistical significance was determined using a one-tailed unpaired Student t test.

MIC determination.

The β-lactam susceptibility of C. difficile was determined as described previously (83). Briefly, active C. difficile cultures were diluted in MHB to an OD₆₀₀ of 0.1, which were then grown to an OD₆₀₀ of 0.45 and further diluted 1:10 in MHB. Then, 15 μl of this diluted culture (∼5 × 10⁵ CFU/ml) was plated in a prereduced 96-well round-bottom polystyrene plate that contained 135 μl of MHB with appropriate β-lactams in each well. The MIC was determined as the concentration at which there was no visible growth after 24 h of anaerobic incubation at 37°C.

Data availability.

The accession numbers for the BlaCDD β-lactamases in different C. difficile strains are YP_001086931 (strain 630), CBE02158 (strain R20291), WP_003417462 (strain M120), and WP_009901927 (strain VPI 10463/ATCC 43255).