Guiding Empiric Treatment for Serious Bacterial Infections via Point of Care [Formula: see text]-Lactamase Characterization

Abstract

Fever is one of the most common symptoms of illness in infants and represents a clinical challenge due to the potential for serious bacterial infection. As delayed treatment for these infections has been correlated with increased morbidity and mortality, broad-spectrum [Formula: see text]-lactam antibiotics are often prescribed while waiting for microbiological lab results (1-3 days). However, the spread of antibiotic resistance via the [Formula: see text]-lactamase enzyme, which can destroy [Formula: see text]-lactam antibiotics, has confounded this paradigm; empiric antibiotic regimens are increasingly unable to cover all potential bacterial pathogens, leaving some infants effectively untreated until the pathogen is characterized. This can lead to lifelong sequela or death. Here, we introduce a fluorescent, microfluidic assay that can characterize [Formula: see text]-lactamase derived antibiotic susceptibility in 20 min with a sensitivity suitable for direct human specimens. The protocol is extensible, and the antibiotic spectrum investigated can be feasibly adapted for the pathogens of regional relevance. This new assay fills an important need by providing the clinician with hitherto unavailable point of care information for treatment guidance in an inexpensive and simple diagnostic format.

Keywords: Antibiotic resistance; beta-lactamase; microfluidic; point of care.

FIGURE 1.

-lactam antibiotic structure.

FIGURE 2.

-LEAF assay principle. a) The -LEAF probe comprises a -lactam core structure (green) including the cleavable lactam ring, flanked by two fluorophores (encircled), which undergo static quenching when the probe is intact. Following cleavage by -lactamase, the fluorophores move apart and show fluorescence. b) Assay profile for -lactamase producing bacteria. c) Assay profile for lactamase non-producing bacteria.

FIGURE 3.

-LEAF Assay on 96 well plate. Increasing fluorescence indicates a -lactamase positive organism (green). The addition of cefazolin to the lactamase-positive assay conditions leads to negligible fluorescence change (blue) due to competitive inhibition and indicates the destruction of cefazolin by -lactamase. 1e8 CFU per well was used.

FIGURE 4.

Microfluidic Assembly. a) Schematic of single chamber. Polycarbonate filter (pink) is compressed between two sheets of clear polymethyl methacrylate (grey). A 50 micron deep channel is defined in a layer of double sided adhesive (yellow). Another layer of double sided adhesive (not shown) is placed above the filter to seal it against the outlet. b) Array of chambers is designed into a standard well-plate format. c) Photograph of microfluidic plate. d) Vacuum fitting used to drive fluids through the microfluidic.

FIGURE 5.

Microfluidic principle of operation. a) sample insertion. b) -LEAF probe insertion, c) fluorescent detection.

FIGURE 6.

Representative fluorescence time course on microfluidic device. l of 5e8 CFU/ml S. aureus was inserted into the device followed by the -LEAF probe. Fluorescence was measured with a commercial plate reader.

FIGURE 7.

Comparison of 96 well plate assay and new microfluidic design. Average and std. dev. from 2 separate experiments. Two different concentrations of -lactamase positive S. aureus were studied, and the change in fluorescent emission was determined after a 20 minute incubation. Two different bacterial volume conditions were investigated with the microfluidic assay to probe its enhanced sensitivity as a function of volume. Results were normalized for instrument drift using a -LEAF probe only condition.

FIGURE 8.

Microfluidic -LEAF antibiotic susceptibility assay. 1e8 CFU S. aureus in PBS was injected into the device. Cleavage rate was measured over a 45 minute incubation. Average and std. error from 5 independent experiments on -lactamase positive and negative strains of S. aureus. * indicates (ANOVA with 2-tailed t-test).

FIGURE 9.

Validation of microfluidic -LEAF assay with pathogen-spiked CSF. 1e8 CFU S. aureus in either CSF or PBS were injected into the microfluidic device. PBS data was taken with 5 independent experiments; CSF data was taken with 3 experiments. Cleavage rate was measured over 20 minutes. Error bars are standard error. * indicates (Mann-Whitney).

FIGURE 10.

Clinical interpretation flowchart. (a) A simple assay setup is depicted. Multiple reaction can be setup simultaneously allowing several antibiotics to be tested concurrently. A representative small footprint fluorimeter from Promega France (GloMax Discover) is shown as an example, though smaller handheld fluorimeters could also be used in principle. (b) Assay provides simple yes/no outputs as to whether -lactamase is produced and whether an antibiotic would be stable against cleavage. These outputs can be programmed into commercially available portable fluorimeters.

FIGURE 11.

Application of microfluidic -LEAF assay. One scenario for use is presented, comparing the current practice with potential future application.