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.
Highlights
To date, most studies of environmental antimicrobial resistant (AMR) bacteria have relied on two approaches
The results demonstrated that a large proportion of the culturable bacteria in River Thames were antibiotic resistant, of which 23%, 67% and 3% of the total culturable bacteria respectively resisted to carbenicillin, kanamycin and both two antibiotics at the concentrations of 10 × minimum inhibitory concentration (MIC)
Eight isolates were randomly picked from LB agar plates containing antibiotics, and their identities based on 16S-rRNA were shown in Table 1, which were 5 different Gram-negative strains, all belonged to the class of γ-proteobacteria
Summary
The first approach is conventional cultivation method for obtaining AMR isolates from the community and subsequently studying their response to changing environment factors[4,5,6,7,8], and the second approach is metagenomic method coupled with antimicrobial resistance gene (ARG) identification[9,10,11,12,13]. The Raman Activated Cell Ejection (RACE)[27] was employed to link AMR phenotype and genotype (16S-rRNA sequencing) It demonstrates that Raman-DIP should be a powerful culture-independent approach for rapid and quantitative assessment of AMR bacteria in situ
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