Characterization of the extended-spectrum beta-lactamase reference strain,Klebsiella pneumoniae K6 (ATCC 700603), which produces the novel enzyme SHV-18

Klebsiella pneumoniae K6 (ATCC 700603), a clinical isolate, is resistant to ceftazidime and other oxyimino-beta-lactams. A consistent reduction in the MICs of oxyimino-beta-lactams by at least 3 twofold dilutions in the prese nce of clavulanic acid confirmed the utility of Ii. pneumoniae K6 as a qual ity control strain for extended-spectrum beta-lactamase (ESBL) detection. I soelectric-focusing analysis of crude lysates of K6 demonstrated a single b eta-lactamase with a pi of 7.8 and a substrate profile showing preferential hydrolysis of cefotaxime compared to ceftazidime. PCR analysis of total ba cterial DNA from K6 identified the presence of a bla(SHV) gene. K6 containe d two large plasmids with molecular sizes of approximately 160 and 80 kb, H ybridization of plasmid DNA with a bla(SHV)-specific probe indicated that a bla(SHV) gene was encoded on the 80-kb plasmid, which was shown to transfe r resistance to ceftazidime in conjugal mating experiments With Escherichia coli HB101. DNA sequencing of this bLa(SHV)-related gene revealed that it differs from bla(SHV-1) at nine nucleotides, five of which resulted in amin o acid substitutions: Ile to Phe at position 8, Arg to Ser at position 43, Gly to Na at position 238, and Glu to Lys at position 240. In addition to t he production of this novel ESBL, designated SHV-18, analysis of the outer membrane proteins of K6 revealed the loss of the OmpK35 and OmpK37 porins.