A biosensor platform for rapid antimicrobial susceptibility testing directly from clinical samples

Abstract

Purpose: A significant barrier to efficient antibiotic management of infection is that the standard diagnostic methodologies do not provide results at the point of care. The delays between sample collection and bacterial culture and antibiotic susceptibility reporting have led to empirical use of antibiotics, contributing to the emergence of drug resistant pathogens. As a key step toward the development of a point of care device for determining the antibiotic susceptibility of urinary tract pathogens, we report on a biosensor based antimicrobial susceptibility test.

Materials and methods: For assay development bacteria were cultured with or without antibiotics, and growth was quantitated by determining viable counts and electrochemical biosensor measurement of bacterial 16S rRNA. To determine antibiotic susceptibility directly from patient samples, urine was cultured on antibiotic plates for 2.5 hours and growth was determined by electrochemical measurement of bacterial 16S rRNA. For assay validation 252 urine samples were collected from patients at the Spinal Cord Injury Service at Veterans Affairs Palo Alto Health Care System. The biosensor based antimicrobial susceptibility test was completed for samples containing gram-negative organisms. Pathogen identification and antibiotic susceptibility results were compared between our assay and standard microbiological analysis.

Results: A direct biosensor quantitation of bacterial 16S rRNA can be used to monitor bacterial growth for a biosensor based antimicrobial susceptibility test. Clinical validation of a biosensor based antimicrobial susceptibility test with patient urine samples demonstrated that this test was 94% accurate in 368 pathogen-antibiotic tests compared to standard microbiological analysis.

Conclusions: This biosensor based antimicrobial susceptibility test, in concert with our previously described pathogen identification assay, can provide culture and susceptibility information directly from a urine sample within 3.5 hours.

Figure 1

Comparison of conventional and biosensor based culture susceptibility analysis of urine. Urine sample is first tested for presence and identity of pathogens. For detection of pathogens sensors are functionalized with capture oligonucleotides targeting 16S rRNA of E. coli, P. mirabilis, P. aeruginosa, Enterococcus spp., Klebsiella-Enterobacter group, Enterobacteriaceae group, universal eubacterial and negative control. Electrochemical detection of pathogen 16S rRNA hybridization with capture and detector probes provides culture data within 1 hour of urine sample collection. If pathogens are identified, level of 16S rRNA from sample incubated without and with antibiotic is quantified on biosensor providing susceptibility data within 3.5 hours of urine sample collection. In contrast to b-AST, conventional culture and susceptibility can take up to 72 hours.

Figure 2

Biosensor detection 16S rRNA as bacterial growth marker. Growth of uropathogenic E. coli strain CFT073 measured over time by biosensor detection (nA) and quantitative plating data (cfu/ml). Result indicates that biosensor can measure bacterial growth over 4-log unit range, from 5 × 10⁵ to 1 × 10⁹ cfu/ml.

Figure 3

Antibiotic susceptibility determination of E. coli clinical isolates. E. coli isolated from patient urine samples with differing antibiotic resistance profiles was grown under 4 conditions of no antibiotic (purple line), AMP (red line), CIP (green line) and SXT (blue line). Time zero represents inoculation at early log phase growth. Biosensor measurements of 16S rRNA using E. coli probes were taken at 15-minute intervals. Antibiotic susceptibility profiles for clinical isolates as determined by bio-sensor assay were same as determined by conventional methods (VITEK® 2) at clinical microbiology laboratory.

Figure 4

Biosensor pathogen identification and antibiotic susceptibility directly from urine sample. A, biosensor signals more than 3 standard deviations over negative control (NC) for E. coli (EC). Enterobacteriaceae (EB) and universal (UNI) probes indicate urine sample contains E. coli. EF, E. faecalis. KP, K. pneumonia. PA, P. aeruginosa. PM, P. mirabilis. B, difference in growth between no antibiotic (POS), and AXO and FEP indicate that E. coli from sample is sensitive to AXO and FEP but resistant to other antibiotic tested. Interpretation of biosensors assay was confirmed by clinical microbiology culture and susceptibility analysis.