P-1349. Multi-hospital Dissemination of Ampicillin Resistance Associated with a Frame-shift Deletion in pbp4 of Vancomycin-Resistant Enterococcus faecalis (VREfs)
Authors: Diaz Lorena , Singh Kavindra , Tran Truc Cecilia , Quiroz Valeria E , Soto Katherine , Peters Anne S , de Paula Baptista Rodrigo , Martínez Jose R W , Ugalde Juan , Hoppe Guillermo , Rafael Araos , Acuna Maria Paz , Martinez Ignacio , Garcia Patricia , Miller William R , Munita José M , Arias Cesar A , Panesso Diana 
Published: January 2025

Abstract

Background
Penicillin (PEN) and ampicillin (AMP) resistance is rare in E. faecalis, and may arise via increased expression of penicillin-binding protein 4 (PBP4), mutations affecting PBP4’s affinity to β-lactams, and rarely β-lactamase production. We recently identified 23 vanA vancomycin-resistant E. faecalis (VREfs), recovered from 19 patients in 4 hospitals in Chile (2020-2023) that exhibited PEN resistance and unusually elevated AMP MICs (4-16 µg/mL). Here, we characterize the molecular basis of the reduced β-lactam susceptibility in these VREfs isolates.Table 1.AMP susceptibility after inducible expression of pbp4SCL10298 in the pbp4-defective and AMP hypersusceptible strain JH2-2Δpbp4

Methods
We analyzed 23 VREfs including sequence type (ST), resistome, PBP sequences and pbp4 promoter. PBP4 production was assessed by ELISA and Western blot using goat anti-rPBP4 sera, in the Chilean VREfs isolate SCL10298 and using pbp4-defective strains (JH2-2Δpbp4 and OG117Δpbp4). We recreated the pbp4 changes of SCL10298 by allelic replacement of E. faecalis OG117 (OG117
Δpbp4SCL10298) using CRISPR-Cas9. Expression of pbp4SCL10298 was also assessed in-trans in a JH2-2 background (JH2-2Δpbp4) using a nisin-inducing vector (pMSP3535).Figure 1.Schematic representation of predicted open reading frames (ORFs) on the JH2-2 and SCL10298
Black line represents the nucleotide sequence for the PBP4 promoter region, followed by the predicted ORFs indicated with grey arrows. Text in red represents the amino acid sequence sharing 100% identity between JH2-2 ORF1 and SCL10298 ORF1-p1 and ORF1-p2.

Results
All 23 isolates belonged to ST1518 (an ST6 single-locus variant). No β-lactamase-encoding genes or changes in pbppromoter regions were detected. A frameshift deletion of 100 nt was observed in pbp4 in all isolates, resulting in 2 open reading frames, including a potential transcript of a shorter pbp4 (ORF1-p1, fig1). All the other PBP sequences were identical to those of E. faecalis JH2-2. PBP4 was detected in SCL10298 by whole-cell ELISA. Further, western blots revealed no differences in PBP4 amounts between SCL10298 and control strains. Allelic replacement of OG117Δ did not result in decreased AMP susceptibility as compared to the parental OG117 strain (AMP MIC 0.5 µg/mL) nor did the inducible expression of the truncated version of pbp4 in JH2-2Δpbp4 (0.19 µg/ml, table1).

Conclusion
We document the emergence of a new lineage of VREfs in Chile with alteration in PBP4 that exhibits decreased susceptibility to B-lactams (akin E. faecium). Our molecular experiments suggest that an alteration of PBP4 is necessary, but not sufficient to increase the MICs of AMP. Therefore, we propose the strain background might be key for the full expression of the B-lactam-resistant phenotype.

Disclosures
William R. Miller, M.D., Merck: Grant/Research Support|UptoDate: Royalties José M Munita, MD, MSD: Grant/Research Support|Pfizer: Grant/Research Support