Raman-Deuterium Isotope Probing for in-situ identification of antimicrobial resistant bacteria in Thames River

Abstract

The emergence and widespread distribution of antimicrobial resistant (AMR) bacteria has led to an increasing concern with respect to potential environmental and public health risks. Culture-independent and rapid identification of AMR bacteria in-situ in complex environments is important in understanding the role of viable but non-culturable and antibiotic persistent bacteria and in revealing potential pathogens without waiting for colony formation. In this study, a culture-independent and non-destructive phenotyping approach, so called Raman Deuterium Stable Isotope Probing (Raman-DIP), was developed to identify AMR bacteria in the River Thames. It is demonstrated that Raman-DIP was able to accurately identify resistant and susceptible bacteria within 24 hours. The work shows that, in the River Thames, the majority of the bacteria (76 ± 2%) were metabolically active, whilst AMR bacteria to carbenicillin, kanamycin and both two antibiotics were 35 ± 5%, 28 ± 3%, 25 ± 1% of the total bacterial population respectively. Raman activated cell ejection (RACE) was applied to isolate single AMR bacteria for the first time, linking AMR phenotype (reistance to antibiotics) and genotype (DNA sequence). The sequences of the RACE sorted cells indicate that they were potential human pathogens Aeromonas sp., Stenotrophomonas sp. and an unculturable bacterium. This work demonstrates Raman-DIP and RACE are effective culture-independent approach for rapid identification of AMR bacteria at the single cell level in their natural conditions.

Figure 1

SCRS of E. coli after 24-hour incubation in heavy water amended with different concentrations of ampicillin. Each SCRS is averaged with standard deviation. (a) Ampicillin susceptible strain E. coli DH5α. (b) Ampicillin resistant strain E. coli WH1274. The lines represent average of SCRS from individual cells and the grey shadow represents standard deviation of SCRS (n = 10 to 46). The bands of C-D (2040 to 2300 cm⁻¹) and C-H (2800–3100 cm⁻¹) are marked. Spectra were stacked for illustration purpose. (c) The C-D ratio of each SCRS obtained for E. coli cells after 24-hour incubation in heavy water amended with or without ampicillin. The data for ‘DH5α + Amp’ and ‘WH1274 + Amp’ are combinative data collected form samples with ampicillin concentration of 5, 10, 15, 30 and 60 μg/ml.

Figure 2

(a) SCRS of Pseudomonas veronii Carb03 after 24-hour incubation. Each SCRS is averaged with standard deviation. P. veronii Carb03 is a carbenicillin resistant isolate from the River Thames in this study. (b) SCRS of Citrobacter freundii Km03 after 24-hour incubation. C. freundii Km03 is a double drug resistant strain isolated from the Thames in this study which can resist kanamycin and carbenicillin. The lines represent average of SCRS from individual cells grey shadow represents standard deviation of SCRS (n = 50). Spectra were stacked for illustration purpose.

Figure 3

(a) Some example of the Raman spectra obtained on the single cell from the River Thames after incubation in amended river water for 24 hours. Carb: carbenicillin; kan: kanamycin. Spectra were stacked for illustration purpose. (b) The C-D/C-D + C-H ratio of SCRS for bacterial cells from the Thames sample in Feb 2017 after 24-hour incubation. The replicates in each conditions are 50, 62, 86, 96, 88, 83, 50, 52. Legends: C: carbenicillin; K: kanamycin; M: minimum inhibitory concentration (8 μg/ml for carbenicillin and 2 μg/ml for kanamycin; H: 10 X MIC concentration. The significant difference between the treatment were calculated using student-t test and showed in the figure with double asterisk (p < 0.01). The dashed line represents the selection benchmark of 4.5% (D %) for active bacteria.

Figure 4

The percentage of active bacteria in the samples from the River Thames revealed by Raman-SIP and the CFU counts obtained by LB agar plate. (a) Percentage of deuterium labelled cells revealed by SCRS. Legends: C: carbenicillin; K: kanamycin; H: 10 × MIC concentration. (b) The correlation between Raman active cell and CFU counts for cells exposed to Carbenicillin (carb) and Kanamycin (kan). Error bar represents the error for 3 replicates. The R values of linear regression are marked.