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Development of an In-House Rapid Antimicrobial Susceptibility Testing Protocol for Positive Blood Culture and Its Implementation in Routine Microbiology Laboratories.

ABSTRACT: Bloodstream infections (BSI) are associated with high morbidity and mortality and remain a leading cause of death. Blood culture (BC) including the identification and the antimicrobial susceptibility testing of the causative microorganisms should be performed as soon as possible. In this study, we developed an in-house rapid antimicrobial susceptibility testing (rAST) protocol for positive BC. First, the rAST was performed in the simulated positive BC of standard strains (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Pseudomonas aeruginosa ATCC 27853) at three different times to assess the reproducibility and operability by dispensing four drops of BC broth onto a Mueller-Hinton agar plate after a positive signal. Furthermore, the rAST was performed in clinical positive BCs. The results of rAST at 4, 6, 8, and 18 h of incubation were compared with results of the standard 16- to 20-h disk diffusion method, and the preliminary breakpoints of the rAST method were established according to the inhibition diameter of sensitive strains and resistant strains. Finally, the rAST was performed in the simulated positive BC of clinical strains to evaluate the availability of the preliminary breakpoints. The rAST results of standard strains were distributed evenly at three different times. Among the 202 clinical strains used to establish the preliminary breakpoints, the number of zone diameters that could be read and interpreted (60, 87, 98, and 100%) increased with incubation time (4, 6, 8, and 18 h), and the categorical agreement was acceptable, with total error rates of 3.0, 2.3, 2.1, and 1.3% at 4, 6, 8, and 18 h of incubation, respectively. In conclusion, the in-house rAST protocol for positive BC can be implemented in routine laboratories. It provides reliable antimicrobial susceptibility testing results for BSI pathogens after 4-6 h of incubation.

SUBMITTER: Cao M

PROVIDER: S-EPMC8669140 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionMany clinical microbiology laboratories report on cumulative antimicrobial susceptibility testing (cAST) data on a regular basis. Criteria for generation of cAST reports, however, are often obscure and inconsistent. Whereas the CLSI has published a guideline for analysis and presentation of cAST data, national guidelines directed at clinical microbiology laboratories are not available in Europe. Thus, we sought to describe the influence of different parameters in the process of cAST data analysis in the setting of a German routine clinical microbiology laboratory during 2 consecutive years.Material and methodsWe developed various program scripts to assess the consequences ensuing from different algorithms for calculation of cumulative antibiograms from the data collected in our clinical microbiology laboratory in 2013 and 2014.ResultsOne of the most pronounced effects was caused by exclusion of screening cultures for multi-drug resistant organisms which decreased the MRSA rate in some cases to one third. Dependent on the handling of duplicate isolates, i.e. isolates of the same species recovered from successive cultures on the same patient during the time period analyzed, we recorded differences in resistance rates of up to 5 percentage points for S. aureus, E. coli and K. pneumoniae and up to 10 percentage points for P. aeruginosa. Stratification by site of care and specimen type, testing of antimicrobials selectively on resistant isolates, change of interpretation rules and analysis at genus level instead of species level resulted in further changes of calculated antimicrobial resistance rates.ConclusionThe choice of parameters for cAST data analysis may have a substantial influence on calculated antimicrobial resistance rates. Consequently, comparability of cAST reports from different clinical microbiology laboratories may be limited. We suggest that laboratories communicate the strategy used for cAST data analysis as long as national guidelines for standardized cAST data analysis and reporting do not exist in Europe.

Project description:Highlights • SEM-based method can be applied for rapid phenotypic AST on Gram-positive cocci towards 2 vancomycin based on morphological changes 3.• The ratio of septa as a marker of bacterial division and size of grape-like clusters enabled the 4 profiling of E. faecalis, E. faecium and S. aureus after brief incubation with vancomycin 5.• SEM-AST strategy showed the feasibility of detecting antibiotic susceptibility or 6 resistance on Gram-positive cocci within one hour of exposition to vancomycin 7. The rapid detection of resistant bacteria has become a challenge for microbiologists worldwide. Numerous pathogens that cause nosocomial infections are still being treated empirically and have developed resistance mechanisms against key antibiotics. Thus, one of the challenges for researchers has been to develop rapid antimicrobial susceptibility testing (AST) to detect resistant isolates, ensuring better antibiotic stewardship. In this study, we established a proof-of-concept for a new strategy of phenotypic AST on Gram-positive cocci towards vancomycin using scanning electron microscopy (SEM). Our study evaluated the profiling of Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus after brief incubation with vancomycin. Sixteen isolates were analysed aiming to detect ultrastructural modifications at set timepoints, comparing bacteria with and without vancomycin. After optimising slides preparation and micrographs acquisition, two analytical strategies were used. The high magnification micrographs served to analyse the division of cocci based on the ratio of septa, along with the bacterial size. Susceptible strains with vancomycin showed a reduced septa percentage and the average surface area was consequently double that of the controls. The resistant bacteria revealed multiple septa occurring at advanced timepoints. Low magnification micrographs made it possible to quantify the pixels at different timepoints, confirming the profiling of cocci towards vancomycin. This new phenotypic AST strategy proved to be a promising tool to discriminate between resistant and susceptible cocci within an hour of contact with vancomycin. The analysis strategies applied here would potentially allow the creation of artificial intelligence algorithms for septa detection and bacterial quantification, subsequently creating a rapid automated SEM-AST assay. Graphical abstract Image, graphical abstract

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Development of an In-House Rapid Antimicrobial Susceptibility Testing Protocol for Positive Blood Culture and Its Implementation in Routine Microbiology Laboratories.

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