Selection of Klebsiella pneumoniae Mutants with High-Level Cefotaxime Resistance during Growth in Serum Containing Therapeutic Concentrations of Cefotaxime

Abstract

Background: In a previous investigation of the genetic characterization of extended-spectrum β-lactamases (ESBLs) in Klebsiella pneumoniae from Zagreb, Croatia, 20 strains were found to produce SHV-2 β-lactamase. Those strains displayed varying degrees of β-lactam resistance and a wide range of β-lactamase activity. We concluded that more resistant isolates were hyperproducers of SHV-2 β-lactamase. Methods: In this investigation, we tried to develop hyperproducing variants from 8 low-level SHV-2 β-lactamase-producing Klebsiella strains by subculturing them in serum containing therapeutic concentrations of cefotaxime (CTX). Results: In most cases, there was a moderate increase in CTX resistance (twofold to threefold), except in one strain which displayed a 16-fold increase in the minimum inhibitory concentration (MIC) of CTX after incubation in the serum. That strain showed a marked increase in enzyme activity as well. The strains with a moderate increase in CTX MIC did not produce more enzyme after exposure to the serum, except for one strain which had a threefold rise in β-lactamase activity after exposure to serum. Conclusions: In this investigation, it was established that the mutants with high-level CTX resistance developed very quickly in the biological fluids containing therapeutic concentrations of CTX. It is reasonable to expect that a similar process occurs in patients infected with an ESBL-producing K. pneumoniae strain during antibiotic treatment. Since most of the high-level CTX-resistant mutants did not have a marked rise in β-lactamase activity after exposure to serum, it is possible that the elevated resistance was due to some other mechanism, such as reduced penicillin-binding protein affinity, changes in outer membrane proteins or efflux by multidrug efflux pumps.