Rapid susceptibility profiling of carbapenem-resistant Klebsiella pneumoniae

Mulroney, Kieran T.; Hall, Jarrad M.; Huang, Xiaoshui; Turnbull, E.; Bzdyl, Nicole M.; Chakera, Aron; Naseer, Umaer; Corea, Enoka; Ellington, Matthew J.; Hopkins, Katie L.; Wester, Astrid Lousie; Ekelund, Oskar; Woodford, Neil; Inglis, Timothy J. J.

doi:10.1038/s41598-017-02009-3

Cited by 44 publications

(43 citation statements)

References 34 publications

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“…Novel approaches, including high-powered optical microscopy, microfluidic assays, flow cytometry, and dielectrophoresis systems, allow for cellular-level observation of bacterial morphologies [20, 21, 23, 51]. These analyses have been used to develop rapid antimicrobial susceptibility tests for several Gram-negative organisms.…”

Section: Discussionmentioning

confidence: 99%

Optical microscopy reveals the dynamic nature ofB. pseudomalleimorphology during β-lactam antimicrobial susceptibility testing

McLaughlin

Bugrysheva

Sue

2020

Preprint

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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. Length of B. pseudomallei cells was 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.

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Section: Discussionmentioning

confidence: 99%

Optical microscopy reveals the dynamic nature ofB. pseudomalleimorphology during β-lactam antimicrobial susceptibility testing

McLaughlin

Bugrysheva

Sue

2020

Preprint

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“…Each flow cytometer procedure was staffed by one cytometer operator and another scientist preparing bacterial suspensions for FAST and other cytometer assays, plus at least one of the above authors engaged in the air sampling procedure. The majority of flow cytometer experiments analyzed the K. pneumoniae isolates as previously reported 13 . The other two species analyzed were S. pneumoniae and B. thailandensis .…”

Section: Methodsmentioning

confidence: 99%

“…Our use of flow cytometry was not for cell sorting, but for a less aerosol-prone cellular analysis. We commenced use of our analytic flow cytometer in a physical containment level two laboratory while developing a flow cytometry-assisted antimicrobial susceptibility test (FAST) assay method with Klebsiella pneumoniae 13 . Though the cytometer we used had no cell sorting function and therefore would not generally produce aerosols, 14 , 15 we decided to conduct an assessment to confirm that viable bacteria are not aerosolized during use before progressing with any analysis of potentially more hazardous aerosol-transmitted species such as Neisseria meningitidis, Mycobacterium tuberculosis and Burkholderia pseudomallei .…”

Section: Introductionmentioning

confidence: 99%

Air sampling to assess potential generation of aerosolized viable bacteria during flow cytometric analysis of unfixed bacterial suspensions

Carson

Inglis

2018

Gates Open Res

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This study investigated aerosolized viable bacteria in a university research laboratory during operation of an acoustic-assisted flow cytometer for antimicrobial susceptibility testing by sampling room air before, during and after flow cytometer use. The aim was to assess the risk associated with use of an acoustic-assisted flow cytometer analyzing unfixed bacterial suspensions. Air sampling in a nearby clinical laboratory was conducted during the same period to provide context for the existing background of microorganisms that would be detected in the air. The three species of bacteria undergoing analysis by flow cytometer in the research laboratory were Klebsiella pneumoniae, Burkholderia thailandensis and Streptococcus pneumoniae. None of these was detected from multiple 1000 L air samples acquired in the research laboratory environment. The main cultured bacteria in both locations were skin commensal and environmental bacteria, presumed to have been disturbed or dispersed in laboratory air by personnel movements during routine laboratory activities. The concentrations of bacteria detected in research laboratory air samples were reduced after interventional cleaning measures were introduced and were lower than those in the diagnostic clinical microbiology laboratory. We conclude that our flow cytometric analyses of unfixed suspensions of K. pneumoniae, B. thailandensis and S. pneumoniae do not pose a risk to cytometer operators or other personnel in the laboratory but caution against extrapolation of our results to other bacteria and/or different flow cytometric experimental procedures.

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“…We recently reported a flow cytometry assisted susceptibility testing (FAST) assay method to determine a carbapenem minimal inhibitory concentration in pure cultures of carbapenem-resistant Klebsiella pneumoniae using the nucleic acid intercalating dye, SYTO ® 9 [25]. This assay proved applicable to accurate detection of bacteria with the non-specific dye SYTO ® 9 in both unexposed and antibiotic-exposed bacteria in pure cultures.…”

Section: Introductionmentioning

confidence: 99%

Accelerated detection of Gram negative bacteria in blood culture by enhanced acoustic flow cytometry (AFC) following peptide nucleic acid fluorescence in situ hybridization (PNA-FISH)

Huang

Urosevic

2018

Preprint

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25 2 26 Bacteraemia is a risk factor for subsequent clinical deterioration and death. Current 27 reliance on culture-based methods for detection of bacteraemia delays identification and 28 assessment of this risk until after the optimal period for positively impacting treatment 29 decisions has passed. Therefore, a method for rapid detection and identification of bacterial 30 infection in the peripheral bloodstream in acutely ill patients is crucial for improved patient 31 survival through earlier targeted antibiotic treatment. The turnaround time for current 32 clinical laboratory methods ranges from 12 to 48 hours, emphasizing the need for a faster 33 diagnostic test. Here we describe a novel assay for accelerated detection of bacterial 34 infection in blood culture (BC) using peptide nucleic acid fluorescence in situ hybridization 35 enhanced acoustic flow cytometry (PNA-FISH-AFC). For assay development, we used 36 simulated blood cultures (BCs) spiked with one of three bacterial species: Escherichia coli, 37 Klebsiella pneumoniae or Pseudomonas aeruginosa at a low concentration of 10 CFU/mL.38 Under current clinical settings, it takes a minimum of 12 hours incubation to reach positivity 39 on the BacTEC system, corresponding to a bacterial concentration of 10 7 -10 9 CFU/mL 40 optimal for further analyses. In contrast, our PNA-FISH-AFC assay detected 10 3 -10 4 41 CFU/mL bacteria in BC following a much shorter incubation of 5 to 10 hours in culture. Using 42 either PCR-based FilmArray® assay or MALDI-TOF for bacterial detection, it took 7-10 and 43 12-24 hours of incubation, respectively, to reach the positive result. These findings indicate 44 a potential time advantage of PNA-FISH-AFC assay over currently used laboratory 45 techniques for rapid bacterial detection in BC with significantly improved turnaround time. 46 47 48 Introduction 49 Sepsis is one of the leading causes of death from infection, usually accompanied by 50 bacteraemia. In its most fulminant form sepsis has a mortality rate of up to 40-60% [1-3]. 51 Sepsis-associated bacteraemia may be mono-or polymicrobial and requires lengthy 52 laboratory processes to identify and characterize the causative agent(s). The time required 53 for initial bacterial detection in the diagnostic laboratory ranges from 12 to 48 hours and 54 depends in part on bacterial growth rate and the number of culture-dependent laboratory 55 procedures required after initial isolation. 56 The standard method for detection of bacteremia boosts bacterial cell numbers by 57 culture of peripheral blood (blood culture, BC) to aid detection and subsequent analysis. To 58 do this, an 8-10mL sample of patient's blood is aseptically inoculated into each of a pair of 59 aerobic and anaerobic BC bottles that contain bacterial growth media, lytic agents to 60 release phagocytosed bacteria from leukocytes and ion exchange resin beads to neutralize 61 the effect of residual antibiotics and improve bacterial recovery [4]. Once inoculated BCs 62 reach the clinical laboratory, they are i...

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Rapid susceptibility profiling of carbapenem-resistant Klebsiella pneumoniae

Cited by 44 publications

References 34 publications

Optical microscopy reveals the dynamic nature ofB. pseudomalleimorphology during β-lactam antimicrobial susceptibility testing

Optical microscopy reveals the dynamic nature ofB. pseudomalleimorphology during β-lactam antimicrobial susceptibility testing

Air sampling to assess potential generation of aerosolized viable bacteria during flow cytometric analysis of unfixed bacterial suspensions

Accelerated detection of Gram negative bacteria in blood culture by enhanced acoustic flow cytometry (AFC) following peptide nucleic acid fluorescence in situ hybridization (PNA-FISH)

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