Antimicrobial susceptibility testing for Gram positive cocci towards vancomycin using scanning electron microscopy

Abstract

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.

Keywords: Enterococcus; Gram-positive cocci; Microbiology; Rapid AST; Scanning electron microscopy; Staphylococcus; Vancomycin.

Graphical abstract

Fig. 1

Detailed strategy applied to a selection of Gram-positive cocci incubated with and without vancomycin, then imaged at selected timepoints (0, 15, 30, 60, and 120 min) using TM4000 Plus tabletop SEM, followed by two analyses strategies. Yellow lines represent the septa.

Fig. 2

Micrographs of E. faecalis incubated with vancomycin at the starting time and at the one- and two-hour timepoints. a, b, c: high magnification micrographs of the vancomycin-susceptible E. faecalis isolate (Q3202). d, e, f: high magnification micrographs of the vancomycin-resistant E. faecalis (Q3205). Micrographs were recorded using TM4000 Plus SEM at a 7,000x magnification. Imaging settings are visible on the micrographs. Scale bars: 5 µm.

Fig. 3

Graphs representing the mean ±SD of total septa percentage for each phenotypic group (Susceptible and Resistant) conducted in triplicate.

Fig. 4

Graphs representing the mean ±SD of the surface area (µm²) for each phenotypic group (Susceptible and Resistant) conducted in triplicate.

Fig. 5

Low magnification micrographs of E. faecalis at the studied timepoints, after threshold adjustment on Image-J. a, b: Vancomycin-susceptible E. faecalis isolate (Q3202) incubated without (a) and with vancomycin (b). c, d: vancomycin-resistant E. faecalis isolate (Q3205) incubated without (c) and with vancomycin (d). Micrographs were recorded at a 500x magnification. Scale bars: 100 µm.

Fig. 6

Graphs representing the mean ±SD of total cluster area measurement for each phenotypic group (Susceptible and Resistant) conducted in triplicate.

Fig. 7

Micrographs of E. faecium incubated with vancomycin at the starting time and at the one- and two-hour timepoints. a, b, c: High magnification micrographs of the vancomycin-susceptible isolate (Q3188). d, e, f: High magnification micrographs of the vancomycin-resistant E. faecium (Q3193). Micrographs were recorded using TM4000 Plus SEM at a 7,000x magnification. Imaging settings are visible on the micrographs. Scale bars: 5 µm.

Fig. 8

Low magnification micrographs of E. faecium at the studied timepoints, after threshold adjustment on Image-J. a, b: vancomycin-susceptible E. faecium isolate (Q3188) incubated without (a) and with vancomycin (b). c, d: vancomycin-resistant E. faecium isolate (Q3193) incubated without (c) and with vancomycin (d). Micrographs were recorded at a 500x magnification. Scale bars: 100 µm.

Fig. 9

Micrographs of S. aureus incubated with vancomycin at the starting timepoint and at the one- and two-hour timepoints. a, b, c: High magnification micrographs of the vancomycin-susceptible S. aureus isolate (Q5878). Micrographs were recorded using TM4000 Plus SEM at a 7,000x magnification. Imaging settings are visible on the micrographs. Scale bars: 5 µm.

Fig. 10

Low magnification micrographs of S. aureus at the studied timepoints, after threshold adjustment on Image-J. a, b: Vancomycin-susceptible S. aureus isolate (Q5878) incubated without (a) and with vancomycin (b). Micrographs were recorded at a 500x magnification. Scale bars: 100 µm.