ABSTRACT
The impact of penicillin-binding protein 3 (PBP3) modifications that may be identified in Escherichia coli was evaluated with respect to susceptibility to β-lactam/β-lactamase inhibitor combinations including ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, cefepime-taniborbactam, and to cefiderocol. A large series of E. coli recombinant strains producing broad-spectrum β-lactamases was evaluated. While imipenem-relebactam showed a similar activity regardless of the PBP3 background, susceptibility to other molecules tested was affected at various levels. This was particularly the case for ceftazidime-avibactam, aztreonam-avibactam, and cefepime-taniborbactam.
KEYWORDS: PBP3, YRIN, YRIK, cefiderocol, taniborbactam, relebactam, vaborbactam, avibactam, susceptibility testing, β-lactamase
INTRODUCTION
Resistance to β-lactam antibiotics is driven by several different enzymatic and non-enzymatic mechanisms, such as β-lactamases, alterations in penicillin-binding proteins (PBPs), efflux, and decreased outer membrane permeability (1). Those mechanisms are most often combined, eventually leading to multidrug resistance. The recent development of new β-lactamase inhibitors such as avibactam (AVI), relebactam (REL), vaborbactam (VAB), and taniborbactam (TAN) has promoted the development of new β-lactam/β-lactamase inhibitor (BL/BLI) combinations including ceftazidime-avibactam (CZA), aztreonam-avibactam (AZA), imipenem-relebactam (I/R), meropenem-vaborbactam (MVB), and cefepime-taniborbactam (FEP-TAN). Hence, those novel combinations (except the latter) are already commercially available or considered for clinical development and have already demonstrated activities against multidrug-resistant Gram-negative bacteria, including producers of metallo-β-lactamases (MBLs) for the AZA and FEP-TAN combinations (2–4).
Although β-lactamase production is the most common source of resistance to broad-spectrum cephalosporins in Enterobacterales (5, 6), the role of PBP modifications seems to play now a significant role at least regarding the novel BL/BLI, such as AZA (7). The new combination, AZA, represents a valuable therapy against infections associated with MBL producers. However, recent studies showed that alterations of the PBP3 sequence, and particularly insertions of four amino acids YRIN or YRIK, contributed to significantly reduce AZA activity against Escherichia coli (7–14). This was particularly evidenced with co-expression of acquired broad-spectrum β-lactamases such as the class C β-lactamase CMY-42 and the class B β-lactamase NDM-5 (10–14), and more recently the latter with the co-expression of CTX-M-15, which has also a high affinity for aztreonam as a substrate (15).
Similarly, the efficacy of cefepime (therefore involving the FEP-TAN combination) and cefiderocol (FDC) may also be affected by PBP3 modifications (8, 16–20).
The aim of our study was, therefore, to precisely evaluate the impact of PBP3 modifications on susceptibility to CZA, AZA, I/R, MVB, FEP-TAN, and FDC using isogenic E. coli recombinant strains producing a wide range of β-lactamases and using several isogenic backgrounds [wild type (WT) or modified PBP3 proteins].
In order to assess the impact of a wide range of β-lactamases on the susceptibility to the above-mentioned antibiotics, the corresponding genes were amplified by PCR with specific primers for β-lactamase genes, and corresponding amplicons were cloned into plasmid pUCp24, as described previously (21). For the present study, all recombinant plasmids harboring the β-lactamase genes generated in the previous work were extracted and transformed into three different genetic backgrounds of E. coli MG1655, respectively, producing a WT PBP3, a modified PBP3 with a four amino-acid insertion (YRIN), and a modified PBP3 with another four amino-acid insertion (YRIK) (8). A wide range of β-lactamase genes was selected, encoding either narrow-spectrum or broad-spectrum β-lactamases, the latter being either ESBLs or carbapenemases of different classes. Hence, the activity of the following β-lactamases could be evaluated, namely class A penicillinases (TEM-1), class A ESBLs (CTX-M-2, CTX-M-3, CTX-M-14, CTX-M-15, GES-1, BEL-1, SHV-1, SHV-2a, SHV-12, PER-1, PER-2, PER-6, PER-7, VEB-1), class A ESBLs with weak carbapenemase activity (CTX-M-33, GES-2, GES-5), class A carbapenemases (FRI-1, IMI-1, KPC-2, KPC-3, KPC-41, KPC-46, KPC-50, GES-6), class B carbapenemases (VIM-1, VIM-2, VIM-53, VIM-83, NDM-1, NDM-5, NDM-7, NDM-9, IMP-1), class C cephalosporinases (CMY-2, CMY-42), narrow-spectrum class D β-lactamases (OXA-1), and carbapenem-hydrolyzing class D β-lactamases (OXA-23, OXA-48, OXA-181, OXA-427). Noteworthy, those enzymes were chosen as representatives of clinically relevant β-lactamases being sources of resistance to broad-spectrum β-lactams in Gram-negative bacteria, particularly in E. coli for most of them.
MICs were determined by broth microdilution (BMD) for ceftazidime (CAZ), imipenem (IPM), meropenem (MER), aztreonam (ATM), FEP, FDC, and their combinations with their respective β-lactamase inhibitor. CAZ, FEP, and ATM were purchased from Sigma-Aldrich (Saint-Louis, USA), IPM, MER from HuiChem (Shanghai, China), and FDC from Shionogi (Osaka, Japan). All the inhibitors (AVI HY-14879, REL HY-16752, VAB HY-19930, TAN HY-109124) were purchased from MedChem Express (Luzern, Switzerland). The concentration of β-lactamase inhibitors such as AVI, REL, and TAN was fixed at 4 mg/L, whereas VAB was fixed at 8 mg/L (21). BMD assays were performed in cation-adjusted Mueller-Hinton (MH) broth (Bio-Rad, Marnes-la-Coquette, France) for all antibiotics or antibiotic combinations listed above, except for FDC and FDC-based combinations for which iron-depleted MH was used, according to the EUCAST guidelines (22). The results were interpreted according to the increase in the MIC value for the same antibiotic or drug combination of the corresponding E. coli MG1655 WT PBP3 lacking any β-lactamase production. The reference E. coli ATCC 25922 and E. coli NCTC 13353 strains were used as quality control for all testings (23).
Results obtained for recombinant E. coli strains highlighted a series of important features (Table 1). Firstly, the MIC values for CAZ, ATM, FEP, and their respective combinations with their β-lactamase inhibitors (CZA, AZA, and FEP-TAN) were consistently higher in PBP3-modified E. coli compared to PBP3-WT E. coli. This was observed even in strains that did not produce any β-lactamase, which is consistent with a previous study evaluating susceptibility to ATM, CAZ, and FEP in isogenic E. coli mutants exhibiting modified PBP3s (8).
TABLE 1.
Susceptibility testing of recombinant E. coli MG1655 strains according to the PBP3 modification statusa
| Strain (β-lactamase produced) | β-Lactamase spectrum | Ambler class | Minimal inhibitory concentrations (µg/mL)b,c | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Ceftazidime | Ceftazidime-avibactam | Aztreonam | Aztreonam-avibactam | Cefepime | Cefepime-taniborbactam | |||||||||||||||
| E. coli ATCC25922 | – | – | 0.125 | 0.125 | ≤0.06 | ≤0.06 | ≤0.06 | ≤0.06 | ||||||||||||
| E. coli NCTC13353 | ESBL | A | >64 | 0.25 | >64 | ≤0.06 | >64 | 0.5 | ||||||||||||
| WT | YRIN | YRIK | WT | YRIN | YRIK | WT | YRIN | YRIK | WT | YRIN | YRIK | WT | YRIN | YRIK | WT | YRIN | YRIK | |||
| E. coli MG1655 | – | – | 0.125 | 0.5 | 0.5 | 0.125 | 0.5 | 0.5 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | ≤0.06 | 0.5 | 0.5 | ≤0.06 | 0.25 | 0.25 |
| E. coli TEM-1 | Narrow | A | 0.125 | 0.5 | 0.5 | 0.125 | 0.5 | 0.5 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | ≤0.06 | 0.5 | 0.5 | ≤0.06 | 0.25 | 0.25 |
| E. coli CTX-M-2 | ESBL | A | 0.5 | 2 | 2 | 0.125 | 0.5 | 0.5 | 0.25 | 4 | 4 | ≤0.06 | 1 | 1 | 0.5 | 1 | 1 | ≤0.06 | 0.25 | 0.25 |
| E. coli CTX-M-3 | ESBL | A | 4 | 32 | 32 | 0.125 | 1 | 1 | 4 | 64 | 64 | ≤0.06 | 1 | 1 | 4 | 64 | 64 | ≤0.06 | 0.25 | 0.25 |
| E. coli CTX-M-14 | ESBL | A | 1 | 4 | 4 | 0.125 | 0.5 | 0.5 | 4 | 64 | 64 | ≤0.06 | 1 | 1 | 4 | 32 | 32 | ≤0.06 | 0.25 | 0.25 |
| E. coli CTX-M-15 | ESBL | A | 256 | 2,048 | 2,048 | 0.5 | 8 | 8 | 1024 | >2,048 | >2,048 | ≤0.06 | 1 | 1 | 512 | >1,024 | >1,024 | 0.125 | 2 | 2 |
| E. coli CTX-M-33 | Carba | A | 16 | 64 | 64 | 0.25 | 2 | 2 | 64 | 1,024 | 1,024 | ≤0.06 | 1 | 1 | 128 | >1,024 | >1,024 | 0.125 | 1 | 1 |
| E. coli GES-1 | ESBL | A | 16 | 32 | 32 | 0.25 | 2 | 2 | 0.25 | 2 | 2 | ≤0.06 | 1 | 1 | 0.125 | 2 | 2 | ≤0.06 | 0.25 | 0.25 |
| E. coli GES-2 | Carba | A | 8 | 8 | 8 | 0.125 | 0.5 | 1 | 0.125 | 4 | 4 | ≤0.06 | 1 | 1 | 0.125 | 2 | 2 | ≤0.06 | 0.25 | 0.25 |
| E. coli GES-5 | Carba | A | 1 | 2 | 2 | 0.125 | 0.5 | 1 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | ≤0.06 | 0.5 | 0.5 | ≤0.06 | 0.25 | 0.25 |
| E. coli GES-6 | Carba | A | 16 | 64 | 64 | 1 | 16 | 16 | 0.25 | 8 | 8 | ≤0.06 | 1 | 1 | ≤0.06 | 0.5 | 0.5 | ≤0.06 | 0.25 | 0.25 |
| E. coli BEL-1 | ESBL | A | 8 | 16 | 16 | 0.5 | 1 | 1 | 8 | 128 | 128 | 0.25 | 1 | 1 | 0.5 | 4 | 4 | ≤0.06 | 0.25 | 0.25 |
| E. coli SHV-1 | Narrow | A | 32 | 1,024 | 1,024 | 0.25 | 2 | 2 | 256 | >2,048 | >2,048 | ≤0.06 | 2 | 2 | 1 | 64 | 64 | ≤0.06 | 0.25 | 0.25 |
| E. coli SHV-2a | ESBL | A | 4 | 16 | 16 | 0.25 | 1 | 2 | 4 | 32 | 32 | ≤0.06 | 2 | 2 | 2 | 64 | 64 | ≤0.06 | 0.25 | 0.25 |
| E. coli SHV-12 | ESBL | A | 128 | 2,048 | 2,048 | 0.125 | 1 | 1 | 128 | >2,048 | >2,048 | 0.25 | 4 | 4 | 8 | 128 | 128 | ≤0.06 | 0.25 | 0.25 |
| E. coli VEB-1 | ESBL | A | 128 | 512 | 512 | 0.5 | 2 | 2 | 8 | 512 | 512 | ≤0.06 | 2 | 2 | 2 | 32 | 64 | ≤0.06 | 0.25 | 0.25 |
| E. coli FRI-1 | Carba | A | 1 | 4 | 4 | 0.25 | 1 | 1 | 512 | 2,048 | 2,048 | 0.5 | 8 | 8 | 0.125 | 1 | 1 | ≤0.06 | 0.25 | 0.25 |
| E. coli PER-1 | ESBL | A | 128 | 512 | 256 | 1 | 8 | 8 | 32 | 512 | 512 | 0.25 | 4 | 4 | 8 | 64 | 64 | ≤0.06 | 0.25 | 0.25 |
| E. coli PER-2 | ESBL | A | 256 | 1,024 | 1,024 | 2 | 16 | 16 | 128 | 2,048 | 2,048 | 2 | 16 | 16 | 8 | 64 | 64 | ≤0.06 | 0.25 | 0.25 |
| E. coli PER-6 | ESBL | A | >2,048 | >2,048 | >2,048 | 16 | 256 | 256 | 2,048 | >2,048 | >2,048 | 16 | 256 | 256 | 64 | 256 | 256 | 0.125 | 2 | 2 |
| E. coli PER-7 | ESBL | A | >2,048 | >2,048 | >2,048 | 16 | 256 | 256 | 1,024 | >2,048 | >2,048 | 16 | 256 | 256 | 64 | 256 | 256 | 0.125 | 2 | 2 |
| E. coli KPC-2 | Carba | A | 16 | 64 | 64 | 0.125 | 1 | 1 | 32 | 1024 | 1024 | ≤0.06 | 1 | 1 | 4 | 64 | 64 | ≤0.06 | 0.5 | 0.5 |
| E. coli KPC-3 | Carba | A | 32 | 256 | 256 | 0.125 | 2 | 2 | 64 | 2,048 | 2,048 | ≤0.06 | 1 | 1 | 4 | 256 | 256 | ≤0.06 | 0.5 | 0.5 |
| E. coli KPC-41 | Carba | A | 128 | 256 | 256 | 16 | 64 | 64 | 16 | 1,024 | 1,024 | ≤0.06 | 1 | 1 | 4 | 64 | 64 | 0.125 | 1 | 1 |
| E. coli KPC-46 | Carba | A | 64 | 256 | 256 | 0.25 | 4 | 4 | 128 | 2,048 | 2,048 | ≤0.06 | 1 | 1 | 8 | 256 | 256 | ≤0.06 | 0.5 | 0.5 |
| E. coli KPC-50 | Carba | A | 64 | 512 | 512 | 16 | 128 | 128 | 16 | 256 | 256 | ≤0.06 | 1 | 1 | 4 | 64 | 64 | 0.125 | 1 | 1 |
| E. coli IMI-1 | Carba | A | 1 | 4 | 4 | 0.25 | 1 | 1 | 128 | 2,048 | 2,048 | 0.5 | 4 | 4 | 0.125 | 2 | 2 | ≤0.06 | 0.25 | 0.25 |
| E. coli VIM-1 | Carba | B | 128 | 2,048 | 2,048 | 128 | >1,024 | >1,024 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | 8 | 64 | 64 | 0.25 | 4 | 4 |
| E. coli VIM-2 | Carba | B | 32 | 256 | 256 | 32 | 256 | 256 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | 0.125 | 1 | 1 | ≤0.06 | 0.25 | 0.25 |
| E. coli VIM-53 | Carba | B | 4 | 32 | 32 | 4 | 32 | 32 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | 0.125 | 1 | 1 | ≤0.06 | 0.25 | 0.25 |
| E. coli VIM-83 | Carba | B | 512 | >2,048 | >2,048 | 512 | >1,024 | >1,024 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | 16 | 512 | 512 | 16 | 512 | 512 |
| E. coli NDM-1 | Carba | B | 1,024 | >2,048 | >2,048 | 1,024 | >1,024 | >1,024 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | 4 | 64 | 64 | 0.125 | 4 | 4 |
| E. coli NDM-5 | Carba | B | 1,024 | >2,048 | >2,048 | 1,024 | >1,024 | >1,024 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | 2 | 64 | 64 | 0.125 | 4 | 4 |
| E. coli NDM-9 | Carba | B | 1,024 | >2,048 | >2,048 | 1,024 | >1,024 | >1,024 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | 8 | 64 | 64 | 8 | 64 | 64 |
| E. coli IMP-1 | Carba | B | 512 | >2,048 | >2,048 | 512 | >1,024 | >1,024 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | 8 | 128 | 128 | 8 | 128 | 128 |
| E. coli CMY-2 | Case | C | 64 | 512 | 512 | 0.5 | 2 | 2 | 32 | 256 | 256 | 0.5 | 4 | 4 | 0.5 | 16 | 8 | ≤0.06 | 0.25 | 0.25 |
| E. coli CMY-42 | Case | C | 128 | 512 | 512 | 1 | 4 | 4 | 64 | 256 | 256 | 1 | 8 | 8 | 2 | 8 | 8 | ≤0.06 | 0.25 | 0.25 |
| E. coli OXA-1 | Narrow | D | 0.125 | 0.5 | 1 | 0.125 | 0.5 | 1 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | 0.5 | 16 | 16 | 0.125 | 2 | 2 |
| E. coli OXA-23 | Carba | D | 0.125 | 0.5 | 1 | 0.125 | 0.5 | 1 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | ≤0.06 | 2 | 2 | ≤0.06 | 2 | 2 |
| E. coli OXA-48 | Carba | D | 0.125 | 0.5 | 0.5 | 0.125 | 0.5 | 1 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | ≤0.06 | 0.25 | 0.25 |
| E. coli OXA-181 | Carba | D | 0.125 | 0.5 | 1 | 0.125 | 0.5 | 1 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | ≤0.06 | 1 | 1 | ≤0.06 | 0.25 | 0.25 |
| E. coli OXA-427 | Carba | D | 32 | 128 | 128 | 1 | 8 | 8 | 1 | 32 | 32 | 0.25 | 2 | 2 | ≤0.06 | 2 | 2 | ≤0.06 | 0.25 | 0.25 |
WT: E. coli MG1655 wild type; YRIN: E. coli MG1655 with an amino-acid insertion (Tyr-Arg-Ile-Asn) in the PBP3 sequence; and YRIK: E. coli MG1655 with an amino-acid insertion (Tyr-Arg-Ile-Lys) in the PBP3 sequence. Narrow, narrow-spectrum; Case, cephalosporinase; ESBL, extended-spectrum β-lactamase; and Carba, carbapenemase.
MIC values shown in bold are those corresponding to at least a 64-fold change from the wild-type E. coli MG1655 MIC value of the corresponding antibiotic tested.
Shaded MIC values are those corresponding to a significantly elevated MIC value compared to wild-type E. coli MG1655, defined as an MIC value higher than eightfold compared to wild-type E. coli MG1655.
Overall, most E. coli recombinant strains producing an acquired broad-spectrum β-lactamase and exhibiting modification on their PBP3 sequence showed higher MIC values ranging from 16-fold and 64-fold change for the CZA, AZA, and FEP-TAN combinations (Table 1). Of note, much higher MIC values (≥64-fold change in comparison to WT E. coli MG1655 producing no β-lactamase) were observed for PBP3-modified E. coli recombinants strains producing CTX-M-15, GES-6, PER-1, PER-2, PER-6, PER-7, KPC-41, KPC-50, OXA-427, and all tested class B β-lactamases when testing CZA. On the other hand, PBP3-modified E. coli producers also exhibited much higher MIC values of AZA when producing SHV-12, FRI-1, PER-1, PER-2, PER-6, PER-7, IMI-1, CMY-2, and CMY-42. Concerning FEP-TAN, only VIM-1, VIM-83, NDM-1, NDM-5, NDM-9, and IMP-1 PBP3-modified E. coli producers displayed higher MIC values (≥64-fold change) in comparison with E. coli WT MG1655 producing no β-lactamase. Noteworthy, no difference in terms of MIC values was observed between the two types of modified PBP3 variants.
On the other hand, alterations of the PBP3 sequence did not affect MIC values of I/R for the corresponding recombinant E. coli strains. Moreover, MIC values of MVB were found to be slightly increased for PBP3-modified recombinant E. coli strains producing meropenem-hydrolyzing β-lactamases. Also, when testing FDC on PBP3-modified E. coli, moderately increased MIC values were observed for almost all recombinant strains. However, the addition of a β-lactamase inhibitor such as AVI or TAN allowed for the recovery of the basic MICs, in line with a low hydrolysis of FDC by several β-lactamases, except for PER-like enzymes (24). Susceptibility testing of the commercially available combinations of carbapenems with inhibitors, namely I/R and MVB, as well as data with FDC and in combination with AVI or TAN are provided in Table S1.
All the results we obtained here were basically consistent with previous works evaluating the respective PBP affinities of E. coli to IPM and MER (25, 26), and showing that carbapenems bound most strongly to PBP2, even if MER has a 10-fold higher affinity for PBP3 than IPM. In addition, Davies et al. showed that CAZ has the highest affinity for PBP3 and that ATM tightly binds only to PBP3 in E. coli, supporting our data (26).
A detailed analysis of the respective MIC values for all antibiotics and combinations tested against all β-lactamases producers is provided in Table 1 and Table S1. A wider range of β-lactamases with high affinity for CAZ or ATM (i.e., different AmpC variants) has been recently investigated with different genetic backgrounds of PBP3 protein sequence for their susceptibility to commercially available BL/BLI combinations (9).
To conclude, this work highlights the potential impact of the PBP3 modification in acquired-broad spectrum β-lactamases producing E. coli on susceptibility to last-line treatments, not only for AZA as previously reported in several studies, but also for CZA, FEP-TAN and to some extent, MVB and FDC. However, a notable exception was observed with I/R that was not affected by the PBP3 mutations tested in this study.
ACKNOWLEDGMENTS
This work was financed by the University of Fribourg, Switzerland and the NARA. We thank Shionogi & Co., Ltd (Doshomachi 3-chome, Chuo-ku, Osaka 541-0045, Japan) for providing us with the isolates Escherichia coli MG1655 PBP3::YRIK and Escherichia coli MG1655 PBP3::YRIN, and Pierre Bogaerts for sharing an OXA-427-positive isolate.
Contributor Information
Laurent Poirel, Email: laurent.poirel@unifr.ch.
Alessandra Carattoli, Universita degli studi di roma La Sapienza, Rome, Italy.
SUPPLEMENTAL MATERIAL
The following material is available online at https://doi.org/10.1128/aac.01548-23.
MIC data.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
MIC data.