Direct antimicrobial susceptibility testing (AST) from positive blood cultures using Microscan system for early detection of bacterial resistance phenotypes

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Highlights

Abstract

Antimicrobial Susceptibility Testing is mandatory for Bloodstream Infections management in order to establish appropriate antimicrobial therapy. Herein we evaluated new approach based on AST results directly from positive blood cultures, using Microscan WA to carry out rapid phenotypical profile of antibiotic resistance. Our investigations allow to reduce time versus traditional results

Introduction

The increase of multidrug-resistant Gram-negative bacteria (MDR-GNB) in healthcare settings is recognized as a global public health problem (Nelson et al., 2017). In order to reduce exposure time to broad-spectrum antibiotics and to monitor local trends in antibiotic resistance, antimicrobial susceptibility testing (AST) is a crucial step for an appropriate therapy of infectious diseases. However, typical AST requires an additional 16 to 24 hours of isolated pathogens culture. Therefore, the aim of our study was to evaluate a new approach based on early readings of antimicrobial susceptibility testing directly from positive blood cultures, using a broth microdilution method, to carry out a rapid profile of antibiotic resistance. Our investigations can be useful to report timely resistance phenotypes.

Despite the availability of innovative molecular technologies, a phenotypical antibiogram is considered the gold standard for an appropriate antimicrobial stewardship (Clinical and Laboratory Standards Institute, CLSI,)

Several genotypic assays are able to detect specific microorganism species but even more relevant resistance genes within hours of blood culture positivity (Timbrook et al., 2017). However, these approaches have some limitations: the risk of overcalling resistance due to the presence of targeted resistance genes that are not expressed as well as the relatively narrow spectrum of detectable resistance mechanisms (Ginn et al., 2017; Tziolos and Giamarellos-Bourboulis, 2016).

Conventional antimicrobial susceptibility testing requires 48 to 72 hours to produce final results and broad-spectrum antimicrobials are often used as empiric therapy, which has been shown to negatively impact patient outcomes, particularly in the setting of multidrug-resistant organisms (Opota et al., 2015; Rhodes et al., 2017 ).

Rapid antimicrobial susceptibility testing (AST) is hopefull to provide early definitive therapeutic guidance to optimize patient outcome. Recently, EUCAST guidelines (EUCAST, 2019) have published recommendations for rapid AST directly from positive blood culture bottles with a direct inoculation of disk diffusion Mueller-Hinton and Mueller-Hinton Fastidious agar plates (guidelines for management of sepsis and septic shock 2016).

The purpose of the present study was to evaluate a new approach with same-day antimicrobial susceptibility testing using a broth microdilution method, directly from positive blood cultures at 4, 6, 8 and 16 hours. This alternative method could lead to a positive impact on the management and outcome of bloodstream infections as well as, a future applicability in sepsis diagnostic flowchart.

Forty positive blood cultures were obtained from among hospitalized patients. The blood cultures system used was BACT/ALERT VIRTUO (BioMérieux, Italy). Following a routine Gram-stain, mono-microbial bacteremia by gram-negative strains were selected on the basis of a larger prevalence of such gram-negative isolates found in both bacteremia samples and other nosocomial infection specimens. All the isolates were identified by MALDI-TOF (Vitek MS, BioMérieux, Italy) or molecular approaches (FilmArray, BioMérieux, Italy or ePlex System, GenMark Diagnostics, Carlsbad, CA). The following genera and species made up the list of isolated bacteria: Escherichia coli (17 isolates), Acinetobacter baumannii (3 isolates), Klebsiella pneumoniae (12 isolates), Pseudomonas aeruginosa (3 isolates), Enterobacter cloacae complex (3 isolates) and one isolate each of Aeromonas hydrophila complex and Serratia marcescens.

AST MDR panels (EUCAST criteria) for gram-negative bacteria were directly inoculated with a volume of 250 µl of positive blood cultures using MicroScan WA System (Beckman Coulter). AST readings were assayed after 4, 6 and 8 hours of incubation and validated after 16 hours (direct-AST). AST with MicroScan WA system was also carried out from overnight blood subcultures, in accordance with the manufacturer's instructions (standard-AST). The results, obtained by the direct and standard methods, were compared and expressed as: agreement, very major error (false susceptibility), major error (false resistance) or minor error (susceptibility/resistant vs intermediate susceptibility).

A total of 40 isolates were evaluated. Results of susceptibility testing profile by the direct method (direct-AST) were available for all samples processed. The most of these isolates were clinically susceptible strains. Our data showed an agreement of 60% for the readings of AST at 4 hours versus standard-AST (N = 24/40). This concordance increases with incubation times increase; infact at 16 hours the agreement percentage was 100% (Table 1).

The overall rate of discrepant susceptibilities observed was referred to a multi-drug resistance profile of antibiotic susceptibility testing. Although early readings were not available, our experimental approach allowed us to put forward the susceptibility for the Ceftazidime/Avibactam already at 16 hours, compared to the standard method.

At 8 hours we observed an agreement of 67.5% between two methods used. At this time we found some false susceptibility (Very Major Errors) concerning 2/3 Acinetobacter baumannii isolates, 2/3 Pseudomonas aeruginosa isolates and 4 out of 17 E. coli isolates. We did not find any false resistance (Major Error) in studied isolates. Moreover, in all tested Klebsiella pneumoniae isolates we did not observe any error, neither very major nor major/minor errors (Table 2).

Our study underlines the temporal gain in the phenotypic AST, using the method proposed here, that can allow to save 22 hours vs time spent following traditional method (about 48 hours).

Procedure starts directly from positive blood culture bottle instead of from subcultured colonies.

Most of the isolates were Multi Drug Resistant bacteria and the Microscan WA System included the whole panel of the antibiotics often used against these pathogens.

Early and fast antibiotic susceptibility testing of bacteria causing bloodstream infections is considered to be a very high priority in microbiological laboratories. This rapid approach can enable clinicians to decide appropriate and targeted antimicrobial therapy at 16 hours after the blood culture positivity, however as soon as 6/8 hours we can have an estimate although with the 35% disagreement. One limit of the present study is the small number of studied isolates. However our group is continuing on the present design including a larger number of isolates both MDR Gram-negative and Gram-positive, the last ones lacked in the dataset included in the present investigation. This simple method can be a useful alternative versus the conventional procedures, in order to report timely results, such as to allow a future applicability in sepsis diagnostic flowchart.

Section snippets

Author's contributions

AQ conceived the study, drafted the manuscript and participated in its design. MN contributed in the draft and editing of the manuscript. CP conceived the study, drafted the manuscript and contributed to perform and validate assays. LG and GSB contributed to editing of the manuscript. AG and AGL contributed to the evaluation and interpretation of data analysis. MC and PM contributed to the discussed AST data. ET and CT contributed to the draft and editing of the manuscript. MCL conceived the

Declarations of competing interest

There are no conflicts of interest.

Funding

“This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors”.

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