Extended-spectrum-β-lactamase (ESBL)-producing strains are increasing worldwide, limiting therapeutic options. Taniborbactam (VNRX-5133) is a newly developed β-lactamase inhibitor with a wide spectrum of activity covering both serine and metallo enzymes.
KEYWORDS: ESBL, Klebsiella pneumoniae, Pseudomonas aeruginosa, β-lactamase inhibitor, cefepime, checkerboard, combination, taniborbactam
ABSTRACT
Extended-spectrum-β-lactamase (ESBL)-producing strains are increasing worldwide, limiting therapeutic options. Taniborbactam (VNRX-5133) is a newly developed β-lactamase inhibitor with a wide spectrum of activity covering both serine and metallo enzymes. We therefore evaluated cefepime-taniborbactam activity against ESBL-producing isolates and determined the concentrations to be used in MIC determinations in the clinical laboratory. The in vitro activity of cefepime (0.06 to 256 mg liter−1) combined with taniborbactam (0.03 to 32 mg liter−1) against 129 clinically and molecularly well-documented ESBL-producing isolates (42 Escherichia coli, 39 Klebsiella pneumoniae, 28 Pseudomonas aeruginosa, 16 Enterobacter cloacae, 2 Citrobacter freundii, and 2 Enterobacter aerogenes) was tested with a broth microdilution checkerboard method based on the ISO standard. The MICs of cefepime alone and in combination, together with percentage resistance at different concentrations of taniborbactam, were calculated for each species and resistance mechanism. The median (range)/MIC90 of cefepime was 32 (0.125 to 256)/256 mg liter−1 for all Enterobacterales isolates (n = 101), with 72% being resistant, and 32 (8 to 256)/128 mg liter−1 for the 28 P. aeruginosa isolates, with 86% being resistant. The median (range)/90th percentile concentration of taniborbactam required to restore Enterobacterales susceptibility to cefepime (MIC ≤1 mg liter−1) was 0.06 (≤0.03 to 32)/4 mg liter−1 and P. aeruginosa susceptibility to increased exposure to cefepime (MIC ≤8 mg liter−1) 1 (≤0.032 to 32)/32 mg liter−1. At a fixed concentration of 4 mg liter−1 of taniborbactam, cefepime median (range)/MIC90 were reduced to 0.125 (0.06 to 4)/1 mg liter−1 for Enterobacterales with no resistant isolates found, and to 8 (2 to 64)/16 mg liter−1 for P. aeruginosa isolates, where 36% remained resistant. The combination cefepime-taniborbactam demonstrated a potent activity against ESBL isolates, restoring susceptibility of all Enterobacterales and two-thirds of P. aeruginosa isolates.
INTRODUCTION
The global spread of extended-spectrum β-lactamases (ESBLs) has been an ongoing problem since the first report appeared in 1983 (1). β-Lactamases are enzymes produced by some bacteria that confer resistance to β-lactam antibiotics. They are most commonly found in members of the Enterobacterales and in nonfermenting Gram negative bacteria. ESBLs are enzymes that can hydrolyze what were supposed to be β-lactamase-resistant oxyimino-β-lactams. Currently, many third and fourth generation cephalosporins have little activity against these ESBL-producing strains (2).
The development of new drugs has always played a major role in providing the solution to these resistance mechanisms. Venatorx Pharmaceuticals has developed a new β-lactamase inhibitor, called taniborbactam (formerly VNRX-5133), to overcome resistance (3). The compound has a wide spectrum of activity, including inhibition of Ambler class A extended spectrum β-lactamases (ESBLs), KPC class A enzymes, class B enzymes such as NDM and VIM, class C (AmpC) enzymes, and class D enzymes such as OXA-48 (4). Cefepime is a broad-spectrum cephalosporin often used clinically but susceptible to degradation by many of the existing β-lactamases, as are most cephalosporins. Studies in vitro have shown that the MICs of the cephalosporins ceftazidime and ceftolozane against resistant strains were drastically reduced in the presence of the β-lactamase inhibitors avibactam and tazobactam, respectively, restoring susceptibility (5).
We therefore determined the in vitro activity of the combination of cefepime with taniborbactam against ESBL-producing isolates that in most cases also expressed serine (KPC) or metallo (NDM, VIM) carbapenemases with a broth microdilution checkerboard technique. The potential clinical feasibility of the combination was assessed, and the taniborbactam concentration for MIC testing of the combination was determined. Subsequently, these data will be used for in vivo pharmacokinetic and pharmacodynamic studies of cefepime-taniborbactam combinations.
RESULTS
Taniborbactam alone at a concentration up to and including 32 mg liter−1 did not have an inhibitory effect on Enterobacterales or P. aeruginosa isolates (not shown). Overall, the median (range)/MIC90 of cefepime alone was 32 (0.125 to 256)/256 mg liter−1 for Enterobacterales and 32 (8 to 256)/128 mg liter−1 for P. aeruginosa, with 72% and 86% of isolates being resistant, respectively. The addition of increasing concentrations of taniborbactam to either Enterobacterales or P. aeruginosa isolates resulted in decreasing MICs and % resistance for cefepime (Table 1).
TABLE 1.
Antimicrobial activity of cefepime at various concentrations of taniborbactam
| Taniborbactam concn (mg liter−1) |
E. coli (n = 42) |
K. pneumoniae (n = 39) |
P. aeruginosa (n = 28) |
Othersa (n = 20) |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Median (range) MIC (mg liter−1) | MIC90 | %Rb | Median (range) MIC (mg liter−1) | MIC90 | %Rb | Median (range) MIC (mg liter−1) | MIC90 | %Rb | Median MIC (mg liter−1) | MIC90 | %Rb | |
| 0 | 32 (0.125–256) | 256 | 83 | 32 (0.25–256) | 256 | 72 | 32 (8–256) | 128 | 86 | 8 | 32 | 50 |
| 0.03 | 4 (0.06–>256) | 128 | 40 | 8 (0.06–>256) | 128 | 51 | 32 (4–>256) | 128 | 82 | 2 | 16 | 25 |
| 0.06 | 1 (0.06–>256) | 32 | 29 | 2 (0.06–>256) | 64 | 44 | 32 (8–>256) | 128 | 82 | 1 | 8 | 10 |
| 0.125 | 0.5 (0.06–128) | 32 | 14 | 2 (0.06–>256) | 64 | 33 | 32 (8–>256) | 128 | 86 | 0.5 | 4 | 10 |
| 0.25 | 0.25 (0.06–128) | 8 | 14 | 1 (0.06–128) | 32 | 28 | 16 (4–>256) | 128 | 79 | 0.5 | 2 | 10 |
| 0.5 | 0.25 (0.06–16) | 2 | 7 | 0.5 (0.06–32) | 32 | 15 | 16 (4–>256) | 64 | 71 | 0.5 | 2 | 5 |
| 1 | 0.125 (0.06–8) | 1 | 2 | 0.5 (0.06–16) | 4 | 8 | 16 (2–256) | 64 | 54 | 0.25 | 1 | 0 |
| 2 | 0.125 (0.06–8) | 1 | 2 | 0.25(0.06–8) | 2 | 5 | 8 (2–64) | 32 | 36 | 0.25 | 0.5 | 0 |
| 4 | 0.125 (0.06–2) | 0.5 | 0 | 0.25 (0.06–4) | 1 | 0 | 8 (2–64) | 16 | 36 | 0.25 | 0.5 | 0 |
| 8 | 0.06 (0.06–2) | 0.25 | 0 | 0.125 (0.06–2) | 1 | 0 | 8 (1–64) | 16 | 25 | 0.25 | 0.5 | 0 |
| 16 | 0.06 (0.06–2) | 0.25 | 0 | 0.125 (0.06–1) | 1 | 0 | 8 (1–32) | 16 | 21 | 0.125 | 0.5 | 0 |
| 32 | 0.06 (0.06–0.5) | 0.125 | 0 | 0.125 (0.06–1) | 0.5 | 0 | 8 (1–32) | 16 | 25 | 0.125 | 0.5 | 0 |
Other isolates: 2 Citrobacter freundii, 2 Enterobacter aerogenes, and 16 Enterobacter cloacae.
%R, percentage of resistant isolates (cefepime MIC above 4 and 8 mg liter−1 for Enterobacterales and P. aeruginosa, respectively).
The decrease in MICs was, however, more pronounced in Enterobacterales than in P. aeruginosa isolates. To obtain susceptibility to cefepime (according to EUCAST criteria) (6) in ≥90% of the Enterobacterales isolates (MIC ≤1 mg liter−1 cefepime), 4 mg liter−1 taniborbactam was required, whereas even at the highest taniborbactam concentration (32 mg liter−1), only 75% of P. aeruginosa isolates were susceptible to increased exposure (MIC ≤8 mg liter−1 cefepime). At 4 mg liter−1 taniborbactam, none of the Enterobacterales isolates were resistant to cefepime, while 36% of P. aeruginosa isolates were still resistant to cefepime (Table 1).
Table 2 includes taniborbactam concentrations required to reduce cefepime MICs to different levels, including the EUCAST (7) and CLSI (8) breakpoints of cefepime for Enterobacterales (susceptible at ≤1 mg liter−1 cefepime) and P. aeruginosa (susceptible to increased exposure at ≤8 mg liter−1 cefepime). The median (range)/90th percentile concentrations of taniborbactam required to reduce the cefepime MIC to 1 mg liter−1 were 0.06 (≤0.03 to 32)/4 mg liter−1 for E. coli isolates and 0.25 (≤0.03 to 32)/16 mg liter−1 for K. pneumoniae isolates. For P. aeruginosa isolates, a median (range)/90th percentile taniborbactam concentration of 1 (≤0.03 to 32)/32 mg liter−1 was required to reduce the cefepime MIC to 8 mg liter−1.
TABLE 2.
Concentration of taniborbactam required to obtain a given MIC of cefepime for the median (range) and the 90th percentile of isolates
| Cefepime target MIC (mg liter−1) | Median (range)/90th percentile of taniborbactam concentrations in mg liter−1 |
|||
|---|---|---|---|---|
| E. coli (n = 42) | K. pneumoniae (n = 39) | P. aeruginosa (n = 28) | Othersa (n = 20) | |
| 1 | 0.06 (≤0.03–32)/4 | 0.25 (≤0.03–32)/16 | 32 (8–32)/32 | 0.06 (≤0.03–2)/0.5 |
| 2 | 0.03 (≤0.03–32)/0.5 | 0.06 (≤0.03–32)/4 | 32 (1–32)/32 | ≤0.03 (≤0.03–1)/0.25 |
| 4 | 0.03 (≤0.03–32)/0.25 | 0.06 (≤0.03–32)/2 | 32 (0.03–32)/32 | ≤0.03 (≤0.03–1)/0.125 |
| 8 | ≤0.03 (≤0.03–32)/0.25 | 0.03 (≤0.03–32)/1 | 1 (≤0.03–32)/32 | ≤0.03 (≤0.03–0.5)/0.06 |
| 16 | ≤0.03 (≤0.03–0.5)/0.125 | ≤0.03 (≤0.03–32)/0.5 | ≤0.03 (≤0.03–32)/4 | ≤0.03 (≤0.03–0.25)/≤0.03 |
Other isolates: 2 Citrobacter freundii, 2 Enterobacter aerogenes, and 16 Enterobacter cloacae.
In Fig. 1, the maximum decrease in cefepime MIC in number of 2-fold dilutions is shown. The addition of taniborbactam to cefepime reduced the MIC of cefepime by 8 to 12 2-fold dilutions for the majority of the Enterobacterales isolates. The maximum reduction effect for 50% of the Enterobacterales was reached at 4 mg liter−1 taniborbactam. The maximum effect for P. aeruginosa isolates was less pronounced; the majority only reached 2 to 6 2-fold reductions. Although higher concentrations of taniborbactam were required to significantly potentiate cefepime activity against P. aeruginosa, 50% of the isolates already reached their maximum possible reduction effect at 2 mg liter−1 taniborbactam. Of the 28 P. aeruginosa isolates, 3 did not show any change in MIC at all.
FIG 1.
Distribution of the maximum effect of cefepime-taniborbactam combinations over the tested concentration range expressed as a 2-fold dilution in the MIC decrease of cefepime. The maximum MIC-lowering effect of the addition of taniborbactam to cefepime, compared to the MIC of cefepime alone, for percentage of isolates of both Enterobacterales and P. aeruginosa is presented on a 2-log transformation x axis. Bars are segmented by the most common groups of resistance genes.
Fig. 1 also shows the groups of resistance genes harbored by the isolates in each bar of the graph. Enterobacterales and P. aeruginosa isolates harbor different sets of resistance mechanisms, but the maximum MIC-lowering effect did not appear to be explained by the presence of certain mechanisms. The influence of specific resistance mechanisms on the MIC of cefepime is detailed in Table 3. Enterobacterales isolates harboring a TEM-1, VIM, KPC, KPC-2, or CTX-M-15 gene showed the highest cefepime median MICs (>32 mg liter−1) and also showed the greatest median 2-fold MIC reduction (≥7) compared to other resistance mechanisms. For P. aeruginosa isolates, the median 2-fold reduction of cefepime MIC was 1 and 2 for both AmpC- and VIM-producing isolates, respectively.
TABLE 3.
Effect of taniborbactam on the antimicrobial activity of cefepime for the most frequently found resistance mechanisms (≥5 isolates per mechanism)
| Resistance mechanism |
P. aeruginosa (n = 28) |
Enterobacterales (n = 101)a |
||||||
|---|---|---|---|---|---|---|---|---|
| No. isolates | Cefepime median (range)/MIC90 |
Median (range) 2-fold MIC reductiona | No. isolates | Cefepime median (range)/MIC90 |
Median (range) 2-fold MIC reductiona | |||
| Cefepime alone | With 4 mg liter−1 TAN | Cefepime alone | With 4 mg liter−1 TAN | |||||
| TEM-1 | 0 | - | - | - | 26 | 32 (0.5–256)/256 | 0.125 (0.06–1)/1 | 8 (1–12) |
| TEM-84 | 0 | - | - | - | 7 | 1 (0.5–64)/32 | 0.06 (0.06–0.25)/0.25 | 5 (2–10) |
| SHV-1 | 0 | - | - | - | 5 | 256 (4–256)/256 | 0.5 (0.06–0.5)/0.5 | 5 (2–10) |
| SHV-12 | 0 | - | - | - | 9 | 4 (1–256)/256 | 0.125 (0.06–0.5)/0.5 | 5 (1–12) |
| OXA-1 | 0 | - | - | - | 23 | 32 (1–256)/256 | 0.25 (0.06–1)/1 | 5 (1–12) |
| VIM | 9 | 32 (8–128)/128 | 8 (1–32)/32 | 2 (0–6) | 11 | 256 (8–256)/256 | 0.25 (0.125–2)/1 | 8 (0–12) |
| KPC | 0 | - | - | - | 8 | 128 (16–256)/256 | 0.25 (0.06–1)/1 | 7 (0–11) |
| KPC-2 | 0 | - | - | - | 13 | 256 (8–256)/256 | 0.125 (0.06–1)/1 | 10 (0–12) |
| CTX-M-1 | 0 | - | - | - | 5 | 32 (8–128)/128 | 0.06 (0.06–0.125)/0.125 | 9 (2–11) |
| CTX-M-9 | 0 | - | - | - | 11 | 4 (1–32)/16 | 0.06 (0.06–1)/0.25 | 5 (1–8) |
| CTX-M-15 | 0 | - | - | - | 23 | 128 (8–256)/256 | 0.125 (0.06–1)/0.5 | 9 (1–12) |
| AmpC | 14 | 32 (8–128)/64 | 8 (2–64)/16 | 1 (0–4) | - | - | - | - |
The median (range) 2-fold MIC reduction over the entire taniborbactam range (0.03 to 32 mg liter−1) tested. TAN, taniborbactam; -, not applicable.
DISCUSSION
The current study shows that addition of taniborbactam renders cefepime-resistant, ESBL-producing Enterobacterales and P. aeruginosa isolates susceptible to cefepime. The concentration of taniborbactam required to achieve this, however, is species and isolate dependent. It should be taken into account that the isolates included in the present study were selected for their cefepime resistance so as to study the concentration-effect profile of taniborbactam when combined with cefepime against β-lactamase-producing isolates, and are therefore not in any way representative of any current population of clinical isolates. The results obtained will be essential for ensuing pharmacokinetic/pharmacodynamic (PK/PD) studies.
The maximum effective taniborbactam concentration was determined for the different species. From this comparison, it can be concluded that the cefepime-taniborbactam combination has a more pronounced effect on Enterobacterales than on P. aeruginosa isolates. Seven P. aeruginosa isolates (25%) did not reach susceptibility according to EUCAST criteria (6), even at 32 mg liter−1 taniborbactam. Of these 7 isolates, 3 harbored a VIM gene, whereas the remaining 4 harbored a combination of OprD and/or AmpC constitutive/inducible/transcript overexpressed, blapoxB, or nitrocefinase activity. Of the Enterobacterales, all except 3 isolates reached susceptibility at 4 mg liter−1 taniborbactam. Of these 3 isolates, 1 harbored a VIM gene and the other two harbored VIM-1, CMY-13, and qnrA1 genes. It seems that VIM and AmpC may reduce cefepime/taniborbactam activity for both Enterobacterales and P. aeruginosa, or else other underlying resistance mechanisms are responsible given the potent inhibition of those enzymes in engineered isolates (4).
The optimum inhibitory concentrations of the in vitro cefepime-taniborbactam combination were also explored, in which the ideal concentration of taniborbactam is one at which the majority of the isolates are inhibited by the combination. Of the 28 P. aeruginosa isolates, 86% were resistant (>8 mg liter−1, EUCAST criteria) (6) for cefepime alone. Adding 4 mg liter−1 taniborbactam brought the percentage of cefepime-resistant isolates down to 36%.
For Enterobacterales (n = 101 isolates), the presence of a KPC (n = 23 isolates), VIM (n = 24), CTX-M-1 (n = 5), CTX-M-2 (n = 3), CTX-M-3 (n = 2), or CTX-M-15 (n = 23) enzyme always rendered the isolate cefepime resistant (>4 mg liter−1, EUCAST criteria). This was also the case for all except one of the isolates harboring SHV-1 (n = 5) or CTX-M-9 (n = 11). Addition of 1 mg liter−1 taniborbactam reduced the percentage of cefepime-resistant isolates carrying any of these β-lactamases to 0% to 3% of all Enterobacterales isolates, rendering them susceptible (≤1 mg liter−1, EUCAST criteria) or susceptible to increased exposure (2 to 4 mg liter−1, EUCAST criteria). The greatest cefepime MIC reduction among Enterobacterales isolates was found for the most resistant isolates harboring TEM-1, VIM, KPC, KPC-2, or CTX-M-15 genes. Not all resistance could be attributed to the presence of these specific β-lactamases, however, as 4 mg liter−1 taniborbactam only reduced the cefepime MIC of 90% of the P. aeruginosa isolates to 16 mg liter−1, and to 2 mg liter−1 for 90% of the Enterobacterales when 1 mg liter−1 taniborbactam was added (Table 2).
Taniborbactam has shown significant activity in Enterobacterales against β-lactamases of all classes. Compared to other β-lactamase inhibitors, such as avibactam (9), taniborbactam was able to successfully inhibit isolates harboring the metallo-β-lactamase VIM, as well as KPC. This makes taniborbactam a promising inhibitor. Compared to avibactam concentrations required to restore ceftazidime’s activity in 90% of the same collection of isolates, higher taniborbactam concentrations were required to restore cefepime activity against P. aeruginosa (8 versus 32 mg liter−1), E. coli (2 versus 4 mg liter−1), and K. pneumoniae (8 versus 16 mg liter−1).
In conclusion, taniborbactam in combination with cefepime enhanced the overall in vitro activity against most Enterobacterales and P. aeruginosa isolates in this study. Four mg liter−1 taniborbactam was required to reduce the cefepime MIC90 of the Enterobacterales from 256 to 1 mg liter−1, thus reversing resistance in all of them, whereas for P. aeruginosa isolates, the cefepime MIC90 was reduced from 128 to 16 mg liter−1, reducing resistance levels from 86% to 36%. Whether these concentrations of taniborbactam will prove safe and achievable in vivo will have to be explored in further experiments.
MATERIALS AND METHODS
Antibacterials.
Cefepime (Maxipime, lot no. 143F005) and taniborbactam (VNRX5133-[HCl]2; lot no. A/2191/31/1) were provided by Venatorx Pharmaceuticals, Inc. The drugs were reconstituted in sterile water for Maxipime or DMSO for VNRX-5133 to a stock solution of 10,240 mg liter−1 and further dilutions were prepared in Mueller-Hinton broth (MHB; Becton, Dickinson and Company).
Bacterial isolates.
We used 129 well-characterized ESBL-producing isolates, including 101 Enterobacterales (42 Escherichia coli, 39 Klebsiella pneumoniae, 16 Enterobacter cloacae, 2 Enterobacter aerogenes, and 2 Citrobacter freundii) and 28 Pseudomonas aeruginosa isolates (Table 4). Presumptive ESBLs were previously confirmed (10). Of the Enterobacterales isolates, 14.9% harbored a VIM gene and 20.8% harbored a KPC gene.
TABLE 4.
Resistance specifications of clinical isolates included in the checkerboard assay
| Species | No. of isolates | Resistance specificationsa,b |
|---|---|---|
| Pseudomonas aeruginosa | 28 | KPC-2+, VIM, carbapenem resistant, AmpCcon, AmpCind |
| Escherichia coli | 42 | TEM-1, 12, 52, 84, SHV-12, OXA-1, CTX-M-1, 2, 3, 9, 14, 15, KPC-2, VIM, VIM-1, LEN, GES-1, qnrB2 |
| Klebsiella pneumoniae | 39 | TEM-1, 84, SHV-1, 2, 2A, 5,11, 12, 33, OXA-1, 48, CTX-M-1, 2, gr. 9, 15, 39, KPC, KPC-2, 3, VIM, VIM-1, LEN, GES-1, OmpK35red, OmpK36red, CMY-13, qnrA1 |
| Enterobacter cloacae | 16 | TEM-1, 84, SHV-12, OXA-1, CTX-M-9, 15, 39, KPC, VIM |
| Enterobacter aerogenes | 2 | TEM-1, KPC-2, VIM-1 |
| Citrobacter freundii | 2 | TEM-1, CTX-M-39, KPC-2 |
Isolates harbored one or multiple of the shown resistance mechanisms, but never all of them.
AmpCcon, AmpC derepressed; AmpCind, AmpC inducible.
Susceptibility testing.
MICs were determined by broth microdilution in accordance with ISO (11), CLSI (12), and EUCAST (7) methods. Working stock solutions of cefepime and taniborbactam were made in freshly prepared MHB (Becton, Dickinson and Company). For the checkerboard assay of cefepime with taniborbactam, both agents were tested in 2-fold dilutions over a range of 0.06 to 256 mg liter−1 cefepime and 0.03 to 32 mg liter−1 for taniborbactam. Two disposable plastic microdilution trays were required to cover all the different combinations and single compounds. Each well contained 50 μl of 2× the final concentrations of cefepime and taniborbactam alone and in combination. Every tray contained a negative broth and a positive growth control. Microdilution trays were stored at −80°C until use for a maximum of one month. On the day of the experiment, trays were thawed, inoculated with 50 μl of 0.5 × 106 CFU/ml bacterial suspension prepared from a 24-h culture grown on a blood agar plate, and incubated at 37°C in ambient air. Each set of MIC determinations included three ATCC control strains, E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), and K. pneumoniae (ATCC 700603), and all showed cefepime-taniborbactam MICs within the current CLSI QC ranges (13). After 18 to 20 h, MICs were manually read, using an angled mirror with a support stand, as the lowest concentration of the agent that competently inhibited visible growth.
Analysis.
The median (range) MIC and MIC90 of cefepime were calculated together with the percentage of resistant isolates (>4 mg liter−1 for Enterobacterales and >8 mg liter−1 for P. aeruginosa) (6) at different concentrations of taniborbactam. The median (range) and 90th percentile concentration of taniborbactam required to obtain a given MIC of cefepime were calculated for each cefepime MIC. The effect of taniborbactam on the MIC of cefepime was expressed as the number of 2-fold dilution decreases compared to the MIC of cefepime alone. The effect of taniborbactam on the activity of cefepime was graphed as cumulative percentage isolates inhibited. The susceptibility to cefepime was interpreted according to EUCAST criteria (6).
ACKNOWLEDGMENT
This study was supported by an unrestricted grant from Venatorx Pharmaceuticals, Inc.
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