Piperacillin-tazobactam resistance in Klebsiella pneumoniae is often associated with IS26-mediated blaSHV-1 amplification in a widespread Klebsiella-adapted plasmid.

Piperacillin-tazobactam (TZP) resistance in Klebsiella pneumoniae involves diverse mechanisms with unclear prevalence and phenotypic impact. To elucidate these mechanisms, we analyzed K. pneumoniae clinical isolates resistant to TZP but susceptible to cefotaxime and cefepime. Among 53 isolates, 14 were further studied by MIC testing for TZP, amoxicillin-clavulanic acid (AMC), and ceftazidime (CAZ). Short-read sequencing was performed for all 14 isolates and long-read sequencing for two. Core-genome MLST showed that all were unrelated. Two had a blaOXA-1 gene, one also carrying an ompK35 porin gene mutation; two others had the same mutation in the promoter of the chromosomal copy of blaSHV usually associated with overexpression. In the remaining 10, resistance correlated with plasmid-borne blaSHV-1 copies. Nine isolates carried blaSHV-1v1 in the same IS26 pseudocompound transposon (PTn), corresponding to PTnSHV-L and located on a conserved IncFIB(K)_1_Kpn3 plasmid in eight. The tenth isolate carried PTnSHV-L with a distinct blaSHV-1 variant on both an IncHI1B_1_pNDM-MAR plasmid and a high-copy-number Col-type plasmid. Read depth analysis confirmed that blaSHV copy number correlated with TZP, AMC, and CAZ MICs. Large-scale database screening identified related IncFIB(K)_1_Kpn3 plasmids, strongly associated with K. pneumoniae and frequently carrying a PTnSHV-L marker. Analysis of a K. pneumoniae genome data set confirmed the frequent co-occurrence of this plasmid and the PTnSHV-L marker in strains with multiple blaSHV copies. These findings suggest the emergence of an epidemic plasmid adapted to K. pneumoniae and driving TZP resistance through blaSHV-1 amplification, underscoring the need for genomic surveillance to detect amplification-based resistance overlooked by standard phenotypic or PCR assays.