Carbapenem-resistant Enterobacterales is an urgent antibiotic resistance threat.1 In the U.S., carbapenem resistance in Enterobacterales is primarily due to the production of serine-based carbapenemase (e.g., KPC). Clinical isolates expressing KPC could be resistant to a broad range of antimicrobial agents, limiting effective treatment options. Notably, the hydrolytic activity of these isolates varies as a function of bacterial density at the site of infection (e.g., empyema vs. uncomplicated urinary tract infection), enzyme variants (e.g., KPC-2 vs. KPC-3), presence of other beta-lactamases and their transcriptional levels, as well as substrate stability. Novel beta-lactamase inhibitors (e.g., avibactam, vaborbactam, relebactam) have been used to restore the activity of beta-lactam agents, when they are used in combination.2 These beta-lactamase inhibitors are expected to reduce enzymatic degradation of the partnering beta-lactam agents and preserve their therapeutic effect. Theoretically, the effective concentration needed for a beta-lactamase inhibitor should match (or exceed) the hydrolytic activity of the target isolates. Where available, molecular testing in microbiology laboratories aims to detect the presence of target beta-lactamase genes, but not the functional capacity quantitatively. Accordingly, routine testing may not be very informative to guide dosing of beta-lactamase inhibitors. We examined the hydrolytic activity of several KPC-positive isolates to provide insights on the utility of this approach.
Six clinical isolates of Klebsiella pneumoniae resistant to imipenem were examined. The presence of gene(s) encoding KPC and other beta-lactamase(s) were confirmed previously by PCR and DNA sequencing.3,4 The relatedness of the isolates was assessed in triplicate using Fourier-transform infrared spectroscopy (FTIR) (Bruker, Billerica, MA). Enzymatic activity of crude cell lysate (normalized to 40 μg of total protein content) was assessed using a spectrophotometric assay. Ceftazidime (50 μM) degradation in phosphate buffered saline (pH = 7.4) was tracked by absorbance at 260 nm over 20 minutes at 35 °C. K. pneumoniae ATCC 13883 (American Type Culture Collection, Manassas, VA) was used as a negative control. For comparison, ceftazidime susceptibility (MIC) was determined by a modified broth dilution method, in the presence of escalating avibactam concentration (up to 64 mg/L). The concentration-response (i.e., MIC profiling) observed at 24h was characterized by the sigmoid inhibitory maximum effect (Emax) model.5
Pertinent isolate characteristics are shown in Table 1. Three isolates (KP1438, KP1491 and KP1587) from the same institution were deemed to be related (data not shown). In contrast to common belief, the presence of multiple beta-lactamase genes did not result in a higher hydrolytic activity. It appeared that not all genes were expressed to the maximal extent and thus they did not contribute additively to the overall hydrolytic activity. Lysates from all isolates susceptible to ceftazidime/avibactam (MIC ≤ 4/4 mg/L) were found to have a similar hydrolytic activity against ceftazidime. However, a higher degradation rate was noted for an isolate resistant to ceftazidime/avibactam (KP 1491, Appendix 1). In this isolate, a higher avibactam concentration was needed to restore ceftazidime MIC to the susceptible range (data not shown). These insights are important and will be used to develop a quantitative method guiding optimal beta-lactamase inhibitor dosing.
Table 1.
Known beta-lactamase(s) and susceptibility of the isolates
| Isolate | KPC variant | Concurrent beta-lactamase(s) | IMI MIC (mg/L) |
CAZ MIC (mg/L) |
CAZ /AVI MIC (mg/L)* |
Initial hydrolytic rate (μmol/min/μg of protein) |
|---|---|---|---|---|---|---|
| KP 1438 | KPC-3 | TEM-1, SHV-11, OXA-9 | 64 | >512 | 2 | 39.7 |
| KP 1491 | KPC-8 | TEM-1, SHV-11, OXA-9 | 16 | >512 | 16 | 122.4 |
| KP 1510 | KPC-2 | CTX-M15, SHV-28, OXA-1 | 64 | 512 | 1 | 42.4 |
| KP 1575 | KPC-19 | ND | 32 | >512 | 2 | 41.3 |
| KP 1587 | KPC-3 | TEM-1B, SHV-11, OXA-9 | 32 | 512 | 1 | 44.4 |
| KP 1804 | KPC-2 | ND | 128 | 512 | 1 | 39.9 |
Note – IMI: imipenem; CAZ: ceftazidime; AVI: avibactam; MIC: minimum inhibitory concentration; ND: not detected
MIC determined with 4 mg/L of avibactam
Despite harboring diverse genes encoding for different beta-lactamases, the hydrolytic activity of various KPC-positive isolates appeared to be reasonably predicted by ceftazidime/avibactam susceptibility. Additional isolates harboring other beta-lactamases (e.g., VIM, NDM) should be examined in future studies.
Acknowledgments
This study was supported by the National Institutes of Health (R01AI140287-03) and the Summer Undergraduate Research Fellowship program at the University of Houston.
Appendix 1.
Hydrolytic profiles of ceftazidime.
Footnotes
Disclosure of interest
The authors report no conflicts of interest.
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