The lack of a rapid, point-of-care antibiotic susceptibility test(AST) has led to misuse of antibiotics in urinary tract infections (UTI). To address this issue, we present a rapid point-of-care phenotypic AST device, which can report antibiotic susceptibility/resistance of a uropathogen against a panel of antibiotics in as fast as 2hrs by utilizing fluorescent labelling chemistry and a highly sensitive particle counting instrument.

BiesseBioscreen is an instrument developed by ASI that allows measuring the concentration of fluorescent particles into a liquid. Measurements were performed diluting at first 30μl of urine samples in 1ml of isotonic solution (i.s.) and incubating for 7mins at 80°C. After dilution,30μl 0.05mM of nucleic fluorescent probe (SYTOX®Orange Nucleic Acid Stain, INVITROGEN) were added to the samples. The probe entered the bacterial wall and concentrated into it allowing its recognition by the instrument. After 60sec the operator could read on the instrument screen the CFU/ml measurement. A CFU/ml≥5x104CFU/ml was considered a cut-off for positivity. A panel of 5 antibiotics(amoxicillin clavulanate, ciprofloxacin, ceftazidime, fosfomycin and nitrofurantoin) was selected. For each unique patient sample that tested positive for UTI screening, an aliquot of the raw urine sample was spiked into each of the 6 culture tubes for a final concentration of 5x104CFU/ml. At time points of 0,1,2, and 3hrs, 30μl from each tube was collected and added to a cuvette containing 3ml of i.s. plus 30μl of Sytox orange(0.05 mM), heated for 7mins at 80°C and scanned for 30sec on the particle counter unit. Tubes were incubated at 37°C between subsequent time points. Presence of at least 5x104CFU/ml was considered resistance to the selected antibiotic. Results were compared to standard urine cultures (VITEK® 2ASTinst,BioMérieux).

We considered 87 UTI-positive patient urine samples; using a panel of 5 common UTI antibiotics plus a growth control, a total of 1740 measurements were performed. Comparing our results to the urine cultures, it was obtained an overall sensitivity=81%, specificity=83%, a sensitive predictive value=95% and a resistant predictive value=54%.

We demonstrated the ability of BiesseBioscreen device [1] to differentiate between sensitive and resistant bacteria with specificity up to 100% to over a 2 hour growth period. By reducing the timeframe to accurately assess antibiotic resistance from 2-3 days to 2 hours, our point-of- care phenotypic AST has potential to provide critical information to clinicians prior the administration of antibiotic therapy. For all the samples measured at least one antibiotic among those chosen for the measure was in accordance for sensitive with the gold standard of the microbiology laboratory.

According to our preliminary data, the sensitive predictive value for a single antibiotic agent is 95%, thus allowing (in most cases) an early (within 2hrs) recognition of an effective agent in the patient. Further studies are needed to confirm these results.

Nicolai et al., “Evaluation of BiesseBioscreen as a new methodology for bacteriuria screening”, New Microbiologica, 37, 495-501, 2014.
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Guidelines on urological infections, EAU 2015;