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.

Characterisation and development of aspirin inducible biosensors inE. coliNissle 1917 and SimCells

“…It has been demonstrated that Raman micro-spectroscopy - deuterium isotope probing (Raman-DIP) can identify metabolically active cells by adding a certain percentage of heavy water (D 2 O) in a medium (36, 37). If cells are metabolically active in the presence of D 2 O, a C-D Raman band (2070 – 2300 cm -1 ) forms via NADPD+ ((H/D free exchange) electon transport chain.…”

“…If cells are metabolically active in the presence of D 2 O, a C-D Raman band (2070 – 2300 cm -1 ) forms via NADPD+ ((H/D free exchange) electon transport chain. As this approach has been demonstrated to be more sensitive compared to tranditional growth curve comparisons, we applied Raman-DIP to explore the activity of the biosensor in response to different carbon sources and thereby the activation pathway (36, 37). Fig.…”

Imaging the invisible—Bioorthogonal Raman probes for imaging of cells and tissues

“…E, Raman mean spectra of ampicillin resistant strain E coli WH1274, characterized by the emergence of the C D signal in presence of different ampicillin concentrations. Reproduced from Song et al [58] with permission of Springer Nature antibiotic resistance assays. Comprehensive investigations of the "nongrowing but metabolically active" status will promote recognition of treatment deficiencies as well as the development of more effective approaches to overcome the risks of antimicrobial resistant bacteria.…”

“…The presence of antimicrobial resistant bacteria in natural environmental fields poses a great risk to human health. Raman-deuterium labeling has been advertised for investigating the bacterial response to antibiotics in river water [58]. The study revealed that environmental isolates of Pseudomonas veronii and Citrobacter freundii are metabolically active in presence of carbenicillin and kanamycin.…”

“…The majority of bacteria (76%) in river water are metabolically active, that means able to incorporate deuterated substrates. The study enabled to differentiate between ampicillin resistant and sensitive bacteria ( Figure 2D,E) [58]. Despite the presence of antibiotics, resistant bacteria maintained the same level of metabolic activity, resulting in higher deuterium uptake, while susceptible bacteria were characterized by a lower C-D ratio.…”