Dataset Information

Rapid Antimicrobial Susceptibility Testing of Patient Urine Samples Using Large Volume Free-Solution Light Scattering Microscopy.

ABSTRACT: The emergence of antibiotic resistance has prompted the development of rapid antimicrobial susceptibility testing (AST) technologies that will enable evidence-based treatment and promote antimicrobial stewardship. To date, many rapid AST methods have been developed, but few are able to be performed on clinical samples directly. Here we developed a large volume light scattering microscopy technique that tracks phenotypic features of single bacterial cells directly in clinical urine samples without sample enrichment or culturing. The technique demonstrated rapid (90 min) detection of Escherichia coli in 24 clinical urine samples with 100% sensitivity and 83% specificity and rapid (90 min) AST in 12 urine samples with 87.5% categorical agreement with two antibiotics, ampicillin and ciprofloxacin.

SUBMITTER: Mo M

PROVIDER: S-EPMC7003966 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Rapid Antimicrobial Susceptibility Testing of Patient Urine Samples Using Large Volume Free-Solution Light Scattering Microscopy.

Mo Manni M Yang Yunze Y Zhang Fenni F Jing Wenwen W Iriya Rafael R Popovich John J Wang Shaopeng S Grys Thomas T Haydel Shelley E SE Tao Nongjian N

Analytical chemistry 20190710 15

The emergence of antibiotic resistance has prompted the development of rapid antimicrobial susceptibility testing (AST) technologies that will enable evidence-based treatment and promote antimicrobial stewardship. To date, many rapid AST methods have been developed, but few are able to be performed on clinical samples directly. Here we developed a large volume light scattering microscopy technique that tracks phenotypic features of single bacterial cells directly in clinical urine samples withou ...[more]

PMID: 31251566

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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

Project description:BackgroundIn Gram-negative species, β-lactam antibiotics target penicillin binding proteins (PBPs) resulting in morphological alterations of bacterial cells. Observations of antibiotic-induced cell morphology changes can rapidly and accurately differentiate drug susceptible from resistant bacterial strains; however, resistant cells do not always remain unchanged. Burkholderia pseudomallei is a Gram-negative, biothreat pathogen and the causative agent of melioidosis, an often fatal infectious disease for humans.ResultsHere, we identified β-lactam targets in B. pseudomallei by in silico analysis. Ten genes encoding putative PBPs, including PBP-1, PBP-2, PBP-3 and PBP-6, were detected in the genomes of susceptible and resistant strains. Real-time, live-cell imaging of B. pseudomallei strains demonstrated dynamic morphological changes in broth containing clinically relevant β-lactam antibiotics. At sub-inhibitory concentrations of ceftazidime (CAZ), amoxicillin-clavulanic acid (AMC), and imipenem (IPM), filamentation, varying in length and proportion, was an initial response of the multidrug-resistant strain Bp1651 in exponential phase. However, a dominant morphotype reemerged during stationary phase that resembled cells unexposed to antibiotics. Similar morphology dynamics were observed for AMC-resistant strains, MSHR1655 and 724644, when exposed to sub-inhibitory concentrations of AMC. For all B. pseudomallei strains evaluated, increased exposure time and exposure to increased concentrations of AMC at and above minimal inhibitory concentrations (MICs) in broth resulted in cell morphology shifts from filaments to spheroplasts and/or cell lysis. B. pseudomallei morphology changes were more consistent in IPM. Spheroplast formation followed by cell lysis was observed for all strains in broth containing IPM at concentrations greater than or equal to MICs, however, the time to cell lysis was variable. B. pseudomallei cell lengths were strain-, drug- and drug concentration-dependent.ConclusionsBoth resistant and susceptible B. pseudomallei strains exhibited filamentation during early exposure to AMC and CAZ at concentrations used to interpret susceptibility (based on CLSI guidelines). While developing a rapid β-lactam antimicrobial susceptibility test based on cell-shape alone requires more extensive analyses, optical microscopy detected B. pseudomallei growth attributes that lend insight into antibiotic response and antibacterial mechanisms of action.

Dataset Information

Rapid Antimicrobial Susceptibility Testing of Patient Urine Samples Using Large Volume Free-Solution Light Scattering Microscopy.

Publications

Rapid Antimicrobial Susceptibility Testing of Patient Urine Samples Using Large Volume Free-Solution Light Scattering Microscopy.

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