No abstract

Clinical isolates of Klebsiella pneumoniae resistant to carbapenems and essentially all other antibiotics (multidrug resistant) are being isolated from some hospitals in New York City with increasing frequency. A highly related pair of K. pneumoniae strains isolated on the same day from one patient in a hospital in New York City were studied for antibiotic resistance. One (KP-2) was resistant to imipenem, meropenem, and sulopenem (MICs of 16 to 32 g/ml) while the other (KP-1) was susceptible (MIC of 0.5 g/ml); both contained the bla ACT-1 , bla SHV-1 , and bla TEM-1 ␤-lactamases. bla ACT-1 in both strains was encoded on a large ϳ150-kb plasmid. Both isolates contained an identical class 1 integron encoding resistance to aminoglycosides and chloramphenicol. They each had identical insertions in ompK35 and ompK36, resulting in disruption of these key porin genes. The carbapenem-resistant and -susceptible isolates were extensively studied for differences in the structural and regulatory genes for the operons acrRAB, marORAB, romA-ramA, soxRS, micF, micC, phoE, phoBR, rpoS, and hfq. No changes were detected between the isolates except for a significant down-regulation of ompK37, phoB, and phoE in KP-2 as deduced from reverse transcription-PCR analysis of mRNA and polyacrylamide gel electrophoresis separation of outer membrane proteins. Backcross analysis was conducted using the wild-type phoE gene cloned into the vector pGEM under regulation of its native promoter as well as the lacZ promoter following transformation into the resistant KP-2 isolate. The wild-type gene reversed carbapenem resistance only when under control of the heterologous lacZ promoter. In the background of ompK35-ompK36 gene disruption, the up-regulation of phoE in KP-1 apparently compensated for porin loss and conferred carbapenem susceptibility. Down-regulation of phoE in KP-2 may represent the normal state of this gene, or it may have been selected from KP-1 in vivo under antibiotic pressure, generating the carbapenemresistant clone. This is the first study in the Enterobacteriaceae where expression of the phosphate-regulated PhoE porin has been associated with resistance to antimicrobials. Our results with this pair of Klebsiella clinical isolates highlight the complex and evolving nature of multiple drug resistance in this species.

Ceftobiprole exhibited tight binding to PBP2a in methicillin-resistant Staphylococcus aureus, PBP2x in penicillin-resistant Streptococcus pneumoniae, and PBP3 and other essential penicillin-binding proteins in methicillin-susceptible S. aureus, Escherichia coli, and Pseudomonas aeruginosa. Ceftobiprole also bound well to PBP2 in the latter organisms, contributing to the broad-spectrum antibacterial activity against gram-negative and gram-positive bacteria.

Doripenem, a parenteral carbapenem, exhibited high affinity for penicillin-binding protein 2 (PBP2) and PBP3 in Pseudomonas aeruginosa and PBP2 in Escherichia coli, the primary PBPs whose inhibition leads to cell death. This PBP affinity profile correlates with the broad-spectrum gram-negative activity observed with doripenem.

The stability of doripenem to hydrolysis by ␤-lactamases from molecular classes A to D was compared to the stability for imipenem and meropenem. Doripenem was stable to hydrolysis by extended-spectrum ␤-lactamases and AmpC type ␤-lactamases and demonstrated high affinity for the AmpC enzymes. For the serine carbapenemases SME-3 and KPC-2 and metallo-␤-lactamases IMP-1 and VIM-2, doripenem hydrolysis was generally 2-to 150-fold slower than imipenem hydrolysis. SPM-1 hydrolyzed meropenem and doripenem fourfold faster than imipenem.Doripenem is a parenteral carbapenem with broad-spectrum activity against many aerobic and anaerobic pathogens. Doripenem MICs against gram-negative clinical isolates are frequently Յ0.5 g/ml, even in Enterobacteriaceae expressing extended-spectrum ␤-lactamases (ESBLs) or overproduced AmpC (7,12,14). Resistance to doripenem and other carbapenems is observed in isolates producing metallo-␤-lactamases (MBLs) or class A or class D carbapenemases (12,13). In this study, we evaluated the hydrolysis of doripenem by a spectrum of ␤-lactamases from almost every functional group (3) and