Rapid Detection and Antibiotic Susceptibility of Uropathogenic Escherichia coli by Flow Cytometry

Abstract

Background: Early preliminary data on antibiotic resistance patterns available before starting the empiric therapy of urinary tract infections (UTIs) in patients with risk factors for acquiring antibiotic resistance could improve both clinical and epidemiological outcomes. The aim of the present study was two-fold: (i) to assess the antibiotic susceptibility of uropathogenic Escherichia coli isolates, exhibiting different antibiotic resistance phenotypes, directly in artificially contaminated urine samples using a flow cytometry (FC) based protocol; (ii) to optimize the protocol on urine samples deliberately contaminated with bacterial suspensions prepared from uropathogenic E. coli strains. Results: The results of the FC based antimicrobial susceptibility testing (AST) protocol were compared with the reference AST methods results (disk diffusion and broth microdilution) for establishing the sensitivity and specificity. The proposed FC protocol allowed the detection and quantification of uropathogenic E. coli strains susceptibility to nitrofurantoin, trimethoprim–sulfamethoxazole, ciprofloxacin, and ceftriaxone within 4 h after the inoculation of urine specimens. The early availability of preliminary antibiotic susceptibility results provided by direct analysis of clinical specimens could essentially contribute to a more targeted emergency therapy of UTIs in the anticipation of AST results obtained by reference methodology. Conclusions: This method will increase the therapeutic success rate and help to prevent the emergence and dissemination of drug resistant pathogens.

Current and Future Flow Cytometry Applications Contributing to Antimicrobial Resistance Control

“…However, they take several days to provide a result because they require the isolation of bacteria isolated from patients who were then exposed to the antibiotics. To overcome this delay in conventional AST assays, new phenotypic strategies have been proposed for the rapid measurement of various phenotypic characteristics of bacteria, such as morphology, metabolism, biochemical composition, and growth after exposure to antibiotics, including isothermal microcalorimetry [ 28 , 29 ], electrochemical impedance spectroscopy [ 30 , 31 ], microscopy [ 32 , 33 ], electrochemical ASTs [ 34 , 35 ], spectroscopy [ 36 ], flow cytometry [ 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 ], and spectrometry [ 46 ]. Rapid phenotypic AST methods have detected microbial growth and/or metabolism as well as morphological changes in biological samples with very low microbial loads.…”

“…Table 1 provides examples of FCM-based AST approaches developed for the detection of AMR in pathogenic bacteria. FCM assays were demonstrated to accurately and rapidly detect AMR profiles of pathogens directly in clinical samples [ 37 , 38 , 42 , 47 , 48 , 49 , 50 , 51 , 52 , 53 ]. Most of the developed AST methods based on FCM used fluorescent dyes to assess the viability of microbial cells after exposure to antibiotics.…”

“…ASTs based on FCM methods offer the great advantage of a short indication time for the microbial mortality level in clinical samples exposed to antibiotics, in contrast to classical culture-based methods that take a minimum of 24 h ( Table 2 ). FCM was shown to be able to detect bacterial pathogens in clinical urine samples and to differentiate among their susceptibility or resistance to ciprofloxacin, trimethoprim–sulfamethoxazole nitrofurantoin, and ceftriaxone after 4 h of incubation versus the minimum of 24 h required by the conventional ASTs test based on cultivation [ 37 ]. Molecular ASTs are faster than plating and can predict phenotypic drug resistance but with variable sensitivity.…”

Colibactin possessing E. coli isolates in association with colorectal cancer and their genetic diversity among Pakistani population

“…The used antibiotic disks were Doxycycline (DO) (30 μg), Nalidixic acid (NA) (30 μg), Ceftazidime (CAZ) (30 μg), Erythromycin (E) (15 μg), Amoxicillin (AML) (20 μg), Gentamicin (CN) (10 μg), Trimethoprim-sulfamethoxazole (SXT) (25 μg), Rifampin (RD) (5 μg), Ampicillin (Amp) (10 μg), Amikacin (AK) (30 μg), Levofloxacin (LEV) (5 μg), Ofloxacin (OFX) (5 μg), Colistin (CT) (10 μg), Amoxicillin-clavulanic Acid (AMC) (20 μg) and Ciprofloxin (CIP) (5 μg). Antibiotic susceptibility was ascertained after 18-24 hours of PLOS ONE incubation at 37˚C as described by (Velican et al, 2020) [22]. E. coli strains were classified as resistant, susceptible and intermediate by comparison of diameter of inhibition to the CLSI, 2017 guidelines.…”

Rapid, label‐free antibiotic susceptibility determined directly from positive blood culture

“…Flow cytometry has demonstrated promise in the rapid assessment of bacterial antibiotic susceptibilities [31][32][33][34][35][36][37][38][39][40][41][42][43]. One such approach, FASTinov (Porto, Portugal), classifies bacteria as sensitive, intermediate, or resistant by monitoring antibiotic-induced changes in flow cytometric fluorescence signals following 1-h incubation of antibiotics and dye with bacteria [32,44,45].…”