Pooled Antibiotic Susceptibility Testing Performs Within CLSI Standards for Validation When Measured Against Broth Microdilution and Disk Diffusion Antibiotic Susceptibility Testing of Cultured Isolates

<i>Study Design and Identifying Candidate Specimens</i>This study is an analysis of consecutive fresh clinical urine specimens collected in the US and submitted with sufficient volume (minimum 2 mL) in boric acid stabilizer for diagnostic testing along with ICD-10-CM codes consistent with a diagnosis of UTI. Specimens with the same monomicrobial non-fastidious bacteria identified by both M-PCR and SUC were selected for analysis on matched cases.Samples for the study were collected via a biobank. The Western Institutional Review Board deemed the use of the data to be exempt under 45 CFR § 46.104(d)(4) as the information was used in a manner that the identity of the subject could not be readily ascertained directly or through identifiers linked to the subjects, the subject was not contacted, and the investigator did not re-identify subjects.<i>Bacterial Identification with Multiplex- Polymerase Chain Reaction (M-PCR)</i>The M-PCR assay was performed as previously<b>[10]</b>. Briefly, extracted DNA from urine samples was mixed with a universal PCR master mix, amplified using TaqMan technology, and spotted in duplicate on OpenArray chips. Probes and primers were used to detect 23 bacteria, four yeast, three bacterial groups, and 32 antibiotic resistance genes. However, only specimens with a single non-fastidious bacterial species or group were included in the current study.<i>Bacterial Identification with Standard Urine Culture (SUC)</i>Bacterial identification by SUC was performed as previously described ​<b>[33]</b>.<i>Pooled Antibiotic Susceptibility Testing (P-AST)</i>The fluorescence-based P-AST test component determines susceptibility to 19 antibiotics commonly used for UTI treatment. The assay was performed as described previously<b>[34]</b>.​​ Briefly, 1 mL of urine specimen was aliquoted into a 1.7 mL microcentrifuge tube. After centrifugation, the supernatant was aspirated and discarded, and the pellet was suspended with 1 mL of Mueller Hinton Growth (MHG) Media for incubation at 35ºC in a non-CO₂ incubator for 6 hours. Samples reaching a predetermined density threshold at the end of the incubation were diluted by aliquoting an appropriate volume of the sample into a 50 mL conical tube containing 29 mL to achieve a final concentration of around 500 thousand cells/mL in MHG Media. Then, the diluted sample was inoculated into a 96-well plate pre-loaded with antibiotics and incubated along with the control plates for 12-16 hours at 35ºC. Resazurin was used as a fluorescent probe to measure cell growth. The fluorescent density of the samples was measured on an Infinite M Nano+ Microplate Reader (TECAN, Switzerland).<i>Broth Microdilution (BMD) Antibiotic Susceptibility Testing</i>BMD AST was performed on isolates from the SUC plates following standard procedures outlined in the CLSI M07 12^th edition<b>[35]</b>.<i>Disk Diffusion (DD) Antibiotic Susceptibility Testing</i>Initial DD AST was performed on organisms isolated by SUC following the standard procedures outlined in the CLSI M100 34^th edition<b>[11]</b>. DD AST performed during heteroresistance analysis was performed on diluted subculture from a P-AST well displaying resistance. Briefly, the content of each P-AST-resistant antibiotic well was diluted (100 µL of well culture + 900 µL of fresh MH) and precultured individually overnight at 35°C.In every case, the suspended isolate or pre-cultured well dilution was grown as a lawn on an MH plate with a single antibiotic disk. After incubation at 35°C for 16 hours, clearance zones were measured and interpreted according to the CLSI M100 34^th edition<b>[11]</b>.<i>Four Times Antibiotic Concentration Culture for Resistance Verification</i>Briefly, the content of each P-AST-resistant antibiotic well was diluted (100 µL of well culture + 900 µL of fresh MH) and precultured individually overnight at 35°C. Using a 10 µL loop, each diluted preculture was plated on MH impregnated with antibiotic at a concentration 4X the highest MIC tested in the P-AST well exhibiting resistance. After incubating 12 – 18 hours at 35°C, any growth was interpreted as confirming the resistant phenotype.<i>Analysis Workflow</i>Upon receipt, urine samples were subjected, in parallel, to standard urine culture and M-PCR for microbial identification and quantification. Specimens with a single organism detected by M-PCR were chosen for inclusion in the study. Specimens were excluded if the SUC results were: negative for microbial detection, “mixed flora” or “contaminated”, or positive detection of multiple species (polymicrobial). Antibiotic susceptibility testing was conducted on these samples. Isolated colonies from SUC were tested by BMD, while P-AST was conducted as described in Methods. Susceptibility results were then compared between the two techniques. Cases in which susceptibility results were discrepant between the two techniques underwent DD AST using the isolates from SUC to resolve the discrepancy (Figure 1).If BMD and DD results were in agreement, the result was defined as “Isolate AST Consensus.” If BMD and DD were conflicting, no susceptibility “Isolate AST Consensus” was determined and the sample was excluded from the study. Natural technical variability of these two standardized isolate AST techniques sometimes results in such discrepancies<b>[36–38]</b>. For specimens with P-AST-sensitive and BMD/DD consensus-resistant discrepant results, no further testing was performed. If P-AST was resistant and BMD/DD consensus was sensitive, the specimen was worked up for heteroresistance as described below.<i>Analysis Workflow for Detecting Heteroresistant Phenotypes</i>For specimens with P-AST-resistant and BMD/DD consensus-susceptible results, 1µL from the resistant P-AST culture well was plated onto BAP. Simultaneously, a specimen from the resistant P-AST well was plated with 4X antibiotic concentration on MH and subjected to DD AST. If no growth was observed, the “Isolate AST Consensus” was affirmed as sensitive, and the P-AST results were deemed falsely resistant. If growth was observed in DD, BMD or both; the resistant P-AST result was affirmed and the BMD/DD initial “Isolate AST Consensus” result determination was deemed falsely sensitive due to heteroresistance. These are cases where heteroresistance was detected by the P-AST technique and missed by the standard isolate AST techniques BAP and DD. The “Heteroresistance-Corrected Consensus” in the Results reflect the heteroresistant corrected analysis. Both analyses with and without accounting for heteroresistance are included in the results.<i>Statistical analyses</i>Metrics of P-AST validation were calculated according to CLSI standards<b>[30]</b>. Essential agreement (EA%) = Number of tests with minimum inhibitory concentration (MIC) within ± one two-fold dilution/total tests x 100. Categorical agreement (CA%) = Number of tests with same category results/total tests x 100. Very major errors (VME%) = Number of tests where the P-AST result is “S” and the “Consensus” result is “R”/total tests x 100. Major errors (ME%) = Number of tests where the P-AST result is “R” and the “Consensus” result is “S”/total tests x 100. Minor errors (mE%) = % of tests where 1) the P-AST result is “I” and the “Consensus” result is either “S” or “R” OR 2) the P-AST result is either “S” or “R” and the “Consensus” result is “I”. In the heteroresistance-corrected analysis, cases that showed heteroresistance were categorized as VMEs for BMD and DD AST instead of MEs by P-AST, as the resistance subpopulation was missed by the standard techniques and detected by P-AST. For all measures, 95% confidence intervals were calculated using the Agresti-Coull method.