Genomic context as well as sequence of both and penicillin-binding protein 5 contributes to β-lactam resistance in .

UNLABELLED

Penicillin-binding protein 5 (PBP5) of () is vital for ampicillin resistance (AMP-R). We previously designated three forms of PBP5, namely, PBP5-S in clade B strains [ampicillin susceptible (AMP-S)], PBP5-S/R (AMP-S or R), and PBP5-R (AMP-R) in clade A strains. Here, deletion resulted in a marked reduction in AMP minimum inhibitory concentrations (MICs) to 0.01-0.09 µg/mL for clade B and 0.12-0.19 µg/mL for clade A strains; complementation restored parental AMP MICs. Using D344SRF (lacking ), constructs with (from a clade A1 strain) cloned upstream of and alleles resulted in modest increases in MICs to 3-8 µg/mL, while high MICs (64 µg/mL) were seen using from A1 strains. Next, using from clade B and clade A/B and B/A hybrid constructs, the presence of , even alone or in , resulted in much lower AMP MICs (3-8 µg/mL) than when was present (MICs 64 µg/mL). qRT PCR showed relatively greater expression ( = 0.007) with cloned downstream of clade A1 (MIC 128 µg/mL) vs when cloned downstream of clade B (MIC 4-16 µg/mL), consistent with results in western blots. In conclusion, we report the effect of clade A vs B on AMP MICs as well as the impact of alleles from different clades. While previously, Psr was not thought to contribute to AMP MICs in our results showed that the presence of resulted in a major decrease in AMP MICs.

IMPORTANCE

The findings of this study shed light on ampicillin resistance in clade A strains. They underscore the significance of alterations in the amino acid sequence of penicillin-binding protein 5 (PBP5) and the pivotal role of the psr region in PBP5 expression and ampicillin resistance. Notably, the presence of a full-length psrB leads to reduced PBP5 expression and lower minimum inhibitory concentrations (MICs) of ampicillin compared to the presence of a shorter psrA, regardless of the pbp5 allele involved. Additionally, clade B strains exhibit lower AMP MICs when both alleles from clades A and B are present, although it is important to consider other distinctions between clade A and B strains that may contribute to this effect. It is intriguing to note that the divergence between clade A and clade B and the subsequent evolution of heightened AMP MICs in hospital-associated strains appear to coincide with changes in and . These changes in may have resulted in an inactive Psr, facilitating increased PBP5 expression and greater ampicillin resistance. These results raise the possibility that a mimicker of PsrB, if one could be designed, might be able to lower MICs of ampicillin-resistant , thus potentially resorting ampicillin to our therapeutic armamentarium for this species.