Detection of SHV beta-lactamases in Gram-negative bacilli using fluorescein-labeled antibodies.
BACKGROUND
beta-lactam resistance in Gram-negative bacteria is a significant clinical problem in the community, long-term care facilities, and hospitals. In these organisms, beta-lactam resistance most commonly results from the production of beta-lactamases. In Gram-negative bacilli, TEM-, SHV-, and CTX-M-type beta-lactamases predominate. Therefore, new and accurate detection methods for these beta-lactamase producing isolates are needed.


RESULTS
E. coli DH10B cells producing SHV-1 beta-lactamase and a clinical isolate of K. pneumoniae producing SHV-5 beta-lactamase were rendered membrane permeable, fixed and adhered to poly-L-lysine coated slides, and stained with purified polyclonal anti-SHV antibodies that were fluorescein labeled. E. coli DH10B cells without a blaSHV gene were used as a negative control. The procedure generated a fluorescence signal from those slides containing cells expressing SHV beta-lactamase that was sufficient for direct imaging.


CONCLUSIONS
We developed a rapid and accurate method of visualizing the SHV family of enzymes in clinical samples containing Gram-negative bacilli using a fluorescein-labeled polyclonal antibody.
Hujer
Andrea M
AM
Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Research Service, Cleveland, OH, USA. - ammh20@yahoo.com
Keslar
Karen S
KS
Dietenberger
Nicole J
NJ
Bethel
Christopher R
CR
Endimiani
Andrea
A
Bonomo
Robert A
RA
D000907:Antibodies, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D019793:Fluorescein / Q000737:chemistry; D005455:Fluorescent Antibody Technique / Q000379:methods; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D008856:Microscopy, Fluorescence; D001618:beta-Lactamases / Q000302:isolation & purification*
D000907:Antibodies, Bacterial; C117193:beta-lactamase SHV-5; D001618:beta-Lactamases; D019793:Fluorescein