Though ABT-737 supplier cephalosporins are used as standard treatment, they can be hydrolyzed by β-lactamases at high inocula (‘inoculum effect’), resulting in clinical failures [33–40]. Conventional ASTs typically utilize 5*105 CFU/ml as standard test inoculums [41, 42]. Koing et al. studied the efficacy of several antibiotics against Escherichia coli and S. aureus, and cited much higher bacterial numbers in infections compared to numbers used in standard susceptibility tests as a major reason for predicted antibiotic susceptibility

not matching with observed efficacy [68]. Pus and infected peritoneal samples, for example, contain an average of 2*108 CFU/ml, a concentration 400 times higher Wortmannin than the inocula used for standard conventional ASTs [68]. The β-LEAF assay is compatible with usage of high bacterial numbers

(i.e. ~108 CFU and higher), by virtue of which it may facilitate assessments at clinically relevant numbers based on infection sites. Some conventional AST methods, such as those relying on turbidometric detection of bacterial growth, may not BV-6 research buy be able to utilize higher bacterial numbers as the starting inoculum. Although PCR-based diagnostics have been employed to detect antibiotic resistance factors relatively rapidly [69–72], the presence of a gene does not necessarily reflect expression of the protein (e.g. enzyme), actually responsible for conferring Celecoxib resistance. For instance, Bacillus anthracis contains genes for lactamases bla1 and bla2, but usually resistance is not observed [73]. In the current study also, despite the different diagnostic methodologies for β-lactamase

enzyme production being consistent (nitrocefin disk test, zone edge test and the β-LEAF assay), the blaZ genotype did not match for some of the isolates (Table 2). In these isolates (e.g. #9, #15) no β-lactamase production was observed, although they contained the gene for β-lactamase (blaZ). Thus, investigating the protein resistance factor phenotypically can be of value. Rapid determination of functional β-lactamase and its correlation to antibiotic activity/usability by assaying for enzyme activity is a distinctive feature of the β-LEAF assay. Conclusions This study reports a fluorescence quenching-dequenching guided method for rapid β-lactamase detection and prediction of antibiotic activity in the context of β-lactamase. The initial results with standard ATCC bacterial strains and clinical isolates are encouraging, though further validation in a large number of isolates is required. The technology merits further rigorous and broader investigations with bacterial strains, antibiotics and direct biological samples to be a viable routine methodology. This requires the development of more sensitive probes and perhaps some novel engineering, which are currently being evaluated.