Structural and kinetic studies on metallo-ß-lactamase IMP-1.

In an effort to probe for metal binding to metallo-ß-lactamase (MßL) IMP-1, the enzyme was overexpressed, purified, and characterized. The resulting enzyme was shown to bind 2 equiv of Zn(II), exhibit significant catalytic activity, and yield EXAFS results similar to crystallographic data previously reported. Rapid kinetic studies showed that IMP-1 does not stabilize a nitrocefin-derived reaction intermediate; rather, the enzyme follows a simple Michaelis mechanism to hydrolyze nitrocefin. Metal-substituted and metal-reconstituted analogues of IMP-1 were prepared by directly adding metal ion stocks to metal-free enzyme, which was generated by dialysis versus EDTA. UV-vis studies on IMP-1 containing 1 equiv of Co(II) showed a strong ligand-to-metal charge transition at 340 nm, and the intensity of this feature increased when the second equivalent of Co(II) was added to the enzyme. EXAFS fits on IMP-1 containing 1 equiv of Co(II) strongly suggest the presence of a metal-metal interaction, and EPR spectra of the IMP-1 containing 1 and 2 equiv of Co(II) are very similar. Taken together, steady-state kinetic and spectroscopic studies suggest that metal binding to metal-free IMP-1 follows a positive-cooperative mode.

Virulence factors of Escherichia coli isolated from cows with acute mastitis.

A total of 184 Escherichia coli isolates recovered from cows with acute mastitis were examined for recognized pathogenic mechanisms and virulence factors commonly found in pathogenic groups of E coli. A modification of the Eng procedure (for detecting complement deficiencies in serum) was used to test for resistance to different animal sera. The Sereny test (for invasiveness), infant mouse test (for heat-stable enterotoxin), and Y-1 adrenal tumor cell assay (for heat-labile enterotoxin) were used. Hemagglutination tests, using rabbit, sheep, and guinea pig RBC, were done with and without added mannose. All of the 184 isolates were serum resistant in all tested sera. None of the isolates was invasive. Only 1 isolate was positive for heat-stable enterotoxin and 2 cultures were positive for heat-labile enterotoxin. Multiple patterns of hemagglutination were observed. The majority of the isolates exhibited both mannose-sensitive and mannose-resistant hemagglutinins with guinea pig and rabbit RBC. A few strains were positive only in mannose-sensitive or mannose-resistant hemagglutination tests. A few strains were negative in all hemagglutination tests. Based on our results, E. coli from cows with acute mastitis lack the virulence factors commonly observed in other E coli groups associated with disease. Serum resistance was the only characteristic that could be related to virulence.

Biochemical and serologic properties of Escherichia coli isolated from cows with acute mastitis.

Biochemical and serologic characteristics of 184 Escherichia coli isolates from cows with acute mastitis were determined. Trends or patterns unique to bovine isolates were not observed, except for the frequency of adonitol fermentation, which was positive in 84 (45.6%) of the cultures. Growth on Tergitol-7 agar had 3 types of colony morphologic characteristics; of these, the rough and intermediate textures predominated over those with smooth colony surfaces. A large variety of serovars was detected; the majority occurring as single isolates within a herd. Few K antigens were typeable. Only 2 isolates contained the K99 antigen. Seemingly, E coli organisms isolated from cows with acute mastitis cannot be distinguished as a specific pathogenic group on the basis of biochemical and serotest reactions.