Rapid Determination of Antibiotic Resistance in Klebsiella pneumoniae by a Novel Antibiotic Susceptibility Testing Method Using SYBR Green I and Propidium Iodide Double Staining

Due to the broad-spectrum antibiotic usage and empirical treatments, the pathogenic bacterium, Klebsiella pneumoniae, has shown extremely high detection rates at hospitals with an increasing antibiotic resistance. Therefore, rapid detection of the antibiotic resistance is urgently required and essential for effective treatments. In this study, we evaluated the performance of a newly developed method for ultra-rapid detection of antibiotic resistance in 30–60 min in K. pneumoniae by using the SYBR Green I and propidium iodide (PI) staining. A total of 100 clinical isolates were tested for antibiotic resistance using four different antibiotics (ceftriaxone, cefepime, meropenem, and ciprofloxacin). The results showed that the SYBR Green I/PI rapid antibiotic susceptibility test (AST) could reliably detect antibiotic resistance to the four drugs in 60 min, and the results were highly concordant with the conventional AST (i.e., Kirby-Bauer method and broth microdilution method) for detection of ceftriaxone, cefepime, meropenem, and ciprofloxacin resistance with a high accuracy of 99, 96, 96, and 93%, respectively. Therefore, the rapid AST established in our study helps to enable targeted therapy to save lives and reduce the empirical use of antibiotics and ultimately the health and economic burdens of antibiotic resistance.

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Ultra-Rapid Drug Susceptibility Testing for Klebsiella pneumoniae Clinical Isolates in 60 Min by SYBR Green I/Propidium Iodide Viability Assay

Frontiers in Microbiology ◽

10.3389/fmicb.2021.694522 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jiazhen Chen ◽

Xuyang Wang ◽

Shiyong Wang ◽

Chen Chen ◽

Wenhong Zhang ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Stationary Phase ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Sybr Green ◽

Sybr Green I ◽

Clinical Isolates ◽

Pi Method ◽

Microdilution Method ◽

Broth Microdilution Method

BackgroundWe aimed to optimize and validate the drug susceptibility test (DST) assay by SYBR Green I/PI (SG-PI) method using a panel of 89 Klebsiella pneumoniae clinical isolates in comparison with the conventional DST method to three most important antibiotics used for treatment of this bacterial infection, including imipenem, cefmetazole, and gentamicin.MethodsBy staining with SYBR Green I and PI dyes, green fluorescence and red fluorescence, which linearly correlated with the percentages of live and dead or membrane damaged cells, respectively, were used to produce two standard curves to calculate the relative cell membrane impermeable rates for each log and stationary phase cultures. Stationary phase K. pneumoniae cells were used in imipenem and cefmetazole SG-PI DST assay whereas log phase cells were used in the gentamicin assay. The conventional broth microdilution method was used as a gold standard for DST for comparison.ResultsData showed that after antibiotic treatment for 30–60 min, the antibiotic-resistant K. pneumoniae strains had significantly higher numbers of surviving cells than the susceptible strains at different concentrations of imipenem, cefmetazole, and gentamicin, where the average relative membrane impermeable rates were 88.5, 92.5, and 103.8% for resistant clinical strains, respectively, and 9.1, 49.3, and 71.5% for susceptible strains, respectively. Overall, the total concordances between the ultra-rapid SG-PI method and conventional minimal inhibitory concentration assay in diagnosing imipenem, cefmetazole and gentamicin resistance were high and were 96.6% (86/89), 95.4% (83/87), and 95.5% (85/89), respectively.ConclusionWe demonstrate that our novel SG-PI assay can accurately and stably detect resistance to different antibiotics in clinical isolates of K. pneumoniae in an ultra-fast manner in 60–90 min.

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Association of Efflux Pump and OMPs with Antibiotic Susceptibility Among ESBL-Producing Klebsiella Pneumoniae Clinical Strains in Malaysia

10.21203/rs.3.rs-375267/v1 ◽

2021 ◽

Author(s):

Golnaz Mobasseri ◽

Thong Kwai Lin ◽

Cindy Shuan Ju Teh

Keyword(s):

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Antibiotic Susceptibility ◽

Efflux Pump ◽

Outer Membrane Proteins ◽

Multidrug Resistant ◽

Efflux Pump Inhibitor ◽

Pump System ◽

Extended Spectrum Beta Lactamases ◽

Amoxicillin Clavulanate

Abstract Multidrug-resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) poses a serious public health threat. K. pneumoniae strains that produce extended-spectrum beta-lactamases (ESBL) are becoming increasingly reported in nosocomial and community-acquired infections. Besides resistance genes, integrons, and plasmids, altered membrane permeability caused by porin loss and energy-dependent efflux have also contributed to antibiotic resistance in K. pneumoniae. The objective of this study was to determine the correlation between the reduction of antibiotic susceptibility and overexpression of efflux pump as well as the lack of outer membrane proteins (OMPs) among clinical ESBLs resistant K. pneumoniae. The expression levels of ramA, acrA, ompK35 and ompK36 in 12 MDR K. pneumoniae strains with varying MICs levels were analyzed using quantitative real time-Polymerase Chain Reaction (qRT-PCR). The role of efflux pump on antibiotic resistance was also studied by using minimum inhibitory concentration (MICs) method with//without efflux pump inhibitor. The result indicated that the strains with highest resistance to cefotaxime showed the lowest level of ompK35 and ompK36 genes expression while the strains with lowest MIC level of resistance to cefotaxime showed the highest level of expression of acrA and ramA. Our finding also revealed the effect of efflux pump inhibitor phenyl-arginine-b-naphthylamide (PAβN) on the MIC levels of ceftazidime, amoxicillin-clavulanate and cefotaxime which were significantly reduced around 1–7 folds MIC levels. These results suggest that Efflux pump system and deficiently of OMPs contributing role in antibiotic susceptibility which should be taken seriously to prevent the treatment failure due to antimicrobial resistance.

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A Duplex SYBR Green I-based Real-time Polymerase Chain Reaction Assay for Rapid Detection of Canine Kobuvirus and Canine Circovirus

10.21203/rs.3.rs-960132/v1 ◽

2021 ◽

Author(s):

Yang Pan ◽

Jing Chen ◽

Junhuang Wu ◽

Yongxia Wang ◽

Junwei Zou ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Rapid Detection ◽

Simultaneous Detection ◽

Sybr Green ◽

Sybr Green I ◽

Chain Reaction ◽

Pcr Assay ◽

Polymerase Chain ◽

Canine Kobuvirus

Abstract Background: Canine Kobuvirus (CaKoV) and Canine Circovirus (CaCV) are viruses that infect dogs causing diarrheal symptoms that are very similar. However, there is no clinical method to detect a co-infection of these two viruses.Results: In this study, a duplex SYBR Green I-based quantitative real-time polymerase chain reaction (PCR) assay for the rapid and simultaneous detection of CaKoV and CaCV was established. CaKoV and CaCV were distinguished by their different melting temperature which was 86℃ for CaKoV and 78℃ for CaCV. The assay was highly specific, with no cross-reactivity with other common canine viruses and demonstrated high sensitivity. The detection limits of CaKoV and CaCV were 8.924 × 101 copies/μL and 3.841 × 101 copies/μL, respectively. The highest intra- and inter-assay Ct value variation coefficients (CV) of CaKoV were 0.40% and 0.96%, respectively. For CaCV, the highest intra- and inter-assay Ct value variation coefficients were 0.26% and 0.70%, respectively. In 57 clinical samples, positive detection rates of CaKoV and CaCV were 8.77% (7/57) and 15.79% (9/57), respectively. The co-infection rate was 7.02% (4/57). Conclusions: The duplex SYBR Green I-based real-time PCR assay established in this study is a fast, efficient, and sensitive method for the simultaneous detection of the two viruses and provides a powerful tool for the rapid detection of CaKoV and CaCV in clinical practice.

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Establishment of a duplex SYBR green I-based real-time polymerase chain reaction assay for the rapid detection of canine circovirus and canine astrovirus

Molecular and Cellular Probes ◽

10.1016/j.mcp.2020.101666 ◽

2020 ◽

Vol 54 ◽

pp. 101666

Author(s):

Yong Wang ◽

Yeqiu Li ◽

Yongqiu Cui ◽

Shudong Jiang ◽

Guangqing Liu ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Polymerase Chain Reaction Assay ◽

Rapid Detection ◽

Sybr Green ◽

Sybr Green I ◽

Chain Reaction ◽

Polymerase Chain

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Simple and Rapid Detection of Human Norovirus from Produce Using SYBR Green I-based Real-time RT-PCR

Food and Environmental Virology ◽

10.1007/s12560-011-9066-5 ◽

2011 ◽

Vol 3 (3-4) ◽

pp. 121-129 ◽

Cited By ~ 6

Author(s):

Dragoslava Radin ◽

Doris H. D’Souza

Keyword(s):

Real Time ◽

Rapid Detection ◽

Sybr Green ◽

Sybr Green I ◽

Rt Pcr ◽

Human Norovirus

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Effects of CLSI and EUCAST clinical breakpoints on antibiotic susceptibility test reporting of CTX-M-15 ESBL and KPC-3 carbapenemase Klebsiella pneumoniae isolates

10.26226/morressier.56d5ba32d462b80296c969eb ◽

2016 ◽

Author(s):

Cátia Caneiras

Keyword(s):

Klebsiella Pneumoniae ◽

Antibiotic Susceptibility ◽

Susceptibility Test ◽

Antibiotic Susceptibility Test ◽

Clinical Breakpoints

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Assessing and analysing contamination of a dairy products processing plant byStaphylococcus aureususing antibiotic resistance and PFGE

Canadian Journal of Microbiology ◽

10.1139/w00-111 ◽

2000 ◽

Vol 46 (12) ◽

pp. 1108-1114 ◽

Cited By ~ 26

Author(s):

E C Tondo ◽

MC M Guimarães ◽

J AP Henriques ◽

M AZ Ayub

Keyword(s):

Staphylococcus Aureus ◽

Antibiotic Resistance ◽

Antibiotic Susceptibility ◽

Dairy Products ◽

Dairy Product ◽

Raw Milk ◽

Food Handler ◽

Processing Plant ◽

Pfge Analysis ◽

Antibiotic Susceptibility Test

A dairy product processing plant was studied for 2.5 years to examine contamination with Staphylococcus aureus and try to correlate the source of contamination. Cultures were submitted to an antibiotic susceptibility test (AST) and characterised by Pulsed-field Gel Electrophoresis (PFGE) analysis. Results showed that 35.2% (19/51) of food handlers were asymptomatic carriers of S. aureus, and that 90.4% (19/21) of raw milk sampled was contaminated. Staphylococcus aureus was isolated from only 10 samples among more than 3200 investigated dairy products. No S. aureus contamination was found on machinery. The AST analysis demonstrated sensitivity of tested S. aureus to oxacillin, cephalothin, vancomycin, gentamicin, and sulfamethoxazole/trimethoprim. AST analysis generated eight different phenotypic profiles, but did not allow us to identify the source of contamination in seven of ten final products. PFGE analysis proved to be a sensitive method as it generated 42 different DNA banding profiles among the 48 S. aureus investigated, demonstrating a lack of predominance of endemic strains in the plant, contrary to suggestions raised by antibiotic resistance typing. Based on PFGE genotyping, S. aureus strains isolated from four contaminated final products were similar to four S. aureus isolated from raw milk. Five final products contained S. aureus different from all other strains collected, and one showed similarity to a strain isolated from a food handler. These results suggest contamination by raw milk as the main source of contamination of the final dairy products.Key words: Staphylococcus aureus, dairy products, antibiotic susceptibility, PFGE.

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