Modification of the penicillin-binding-protein 3 as a source of resistance to broad-spectrum cephalosporins in Escherichia coli

Multidrug-resistant Gram-negative bacteria are one of the major threats to human health, largely due to the dissemination of some high-risk successful multidrug-resistant clones. 1 Of particular concern is the resistance to ß-lactams, because those antibiotics are the most extensively used antimicrobial ones, accounting for half of all prescriptions in Europe.

Multidrug-resistant Gram-negative bacteria are one of the major threats to human health, largely due to the dissemination of some high-risk successful multidrug-resistant clones. 1 Of particular concern is the resistance to ß-lactams, because those antibiotics are the most extensively used antimicrobial ones, accounting for half of all prescriptions in Europe. 2 Resistance to broad-spectrum cephalosporins is of particular concern, as related infections require usage of carbapenems considered as last-resort antibiotics supposed to be preserved.Although the production of β-lactamases is the most common mechanism of resistance to broad-spectrum β-lactams in Gram-negative bacteria, and particularly in Enterobacterales, other mechanisms have also been reported including efflux, permeability changes and modification of PBP targets that contribute to this resistance. 3ur study was initiated by the isolation in March 2023 of an Escherichia coli strain recovered from a urine sample of an 86-year-old female patient hospitalized in Zürich, Switzerland, suffering from recurrent urinary tract infections.Corresponding antibiotic regimen were either cotrimoxazole or fosfomycin, but no ß-lactam has been used.Susceptibility testing by disc diffusion and broth microdilution showed that this isolate was resistant to broad-spectrum cephalosporins (ceftazidime, cefotaxime, cefepime) and to aztreonam, but remained susceptible to ceftazidime-avibactam and carbapenems, according to the EUCAST 2023 breakpoints. 4In addition, it was resistant to fluoroquinolones, nitrofurantoin, cotrimoxazole and fosfomycin.
Multilocus sequence typing performed as described in ref. 5 identified that strain as belonging to ST1193, that has been reported as an emerging high-risk multidrug-resistant global clone. 1 Resistance to broad-spectrum cephalosporins (ceftazidime, cefotaxime, cefepime) could not be explained by common mechanisms of resistance such as broad-spectrum ß-lactamases.Indeed, the Rapid ESBL NP test remained negative and PCR assays for all ESBL-encoding genes also remained negative. 6Likewise, PCR for all known AmpC β-lactamase encoding genes remained negative.
Whole-genome sequencing was therefore performed, using a short-read technology (MiSeq platform, Illumina, San Diego, CA, USA), 7 and allowed the identification of mutations in gyrA/parC (gyrA S83L, D87N; parC S80I), responsible for fluroquinolone resistance, 1 and an 8-bp insertion and subsequent disruption of nfsA, conferring resistance to nitrofurantoin. 8Regarding β-lactam resistance, the gene encoding the narrow-spectrum TEM-1 was the sole ß-lactamase gene identified, that explained the resistance to penicillins but not to the broad-spectrum cephalosporins. 9However, a four amino-acid insertion (YRVP) just after the amino-acid residue 333 (close to the β-lactam binding pocket) of the PBP-3 sequence was identified.
To investigate the role of this PBP-3 insertion in the resistance to cephalosporins, a wild-type PBP-3 was amplified from E. coli ATCC25922 reference strain using primers PBP3_F (5′-CCACGGA AAAGCTGCAAATG-3′) and PBP3_R (5′-CATCGGTCGCCTCATCTT TC-3′) and cloned into plasmid pCR-Blunt II-TOPO, 7 then the corresponding recombinant plasmid transformed into the E. coli clinical isolate.Hence, recovery of susceptibility to broad-spectrum cephalosporins was observed in the complemented strain (Table 1).
A similar PBP-3 modification has been recently evidenced among NDM-1 and NDM-5 E. coli isolates already been reported, 1,10 with either YRIN or YRIK 4-amino-acid insertions also identified after position 333.It was shown that these PBP-3 modifications were responsible for resistance to aztreonam and consequently also to the aztreonam-avibactam combination, which is a significant concern as those isolates were co-producing NDM-like carbapenemases along with AmpC-type CMY ß-lactamases, resulting in high-level resistance to all ß-lactams including carbapenems.
Overall, we showed that the YRVP insertion in the PBP-3 was involved in the decreased susceptibility to cephalosporins, such as ceftazidime, cefotaxime, cefepime and aztreonam, a phenotype observed without production of any acquired broad-spectrum ß-lactamase.This implies that recovery of an increased susceptibility cannot be expected when considering combinations made of ß-lactam/ß-lactamase inhibitor such as ceftazidime-avibactam, as highlighted here with an MIC at 8 mg/L either for ceftazidime and for ceftazidime-avibactam (a value being at the exact EUCAST breakpoint for the latter, whereas that of ceftazidime is actually at 1 mg/L).Zhang et al. previously highlighted that PBP-3 modifications so far described did not confer resistance to CAZ-AVI but recommended accurate monitoring. 11Our observations are fully in accordance with this warning.In addition, the isolate investigated here belonged to the ST1193, recently considered to be an emerging high-risk multidrug-resistant global clone responsible for urinary tract and bloodstream infections.