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. 2019 Feb 8:(144):10.3791/58978.
doi: 10.3791/58978.

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

Liang-Chun Wang  1 , Jacob Wagner  2 , Annabelle Capino  2 , Elizabeth Nesbit  2 , Wenxia Song  2 , Daniel C Stein  2
Affiliations

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

Liang-Chun Wang et al. J Vis Exp. .

Abstract

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail treatment. This Gram-negative bacterium causes gonorrhea exclusively in humans. During infection, it is able to form aggregates and/or biofilms. The minimum inhibitory concentration (MIC) test is used for to determine susceptibility to antibiotics and to define appropriate treatment. However, the mechanism of the eradication in vivo and its relationship to laboratory results are not known. A method that examines how GC aggregation affects antibiotic susceptibility and shows the relationship between aggregate size and antibiotic susceptibility was developed. When GC aggregate, they are more resistant to antibiotic killing, with bacteria in the center surviving ceftriaxone treatment better than those in the periphery. The data indicate that N. gonorrhoeae aggregation can reduce its susceptibility to ceftriaxone, which is not reflected using the standard agar plate-based MIC methods. The method used in this study will allow researchers to test bacterial susceptibility under clinically relevant conditions.

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Figures

Figure 1:
Figure 1:. Representative data of survival rate of aggregated GC under ceftriaxone treatment by ATP-utilization assay.
(A) Survival rate comparison of MS11Opa+Pil+ suspension without pre-aggregating, pre-aggregating for 6 h, or disrupting after pre-aggregating for 6 h. (B) Survival rate comparison of 6 h aggregated MS11Opa+Pil+ with MS11ΔOpaPil+, MS11Opa+Pil-, or MS11ΔLgtEPil+. Shown are the average values (±SD) obtained from three independent experiments. ***p < 0.001; **p < 0.01; *p < 0.05. This figure was previously published and is used with permission.
Figure 2:
Figure 2:. Representative data of live/dead bacteria distribution within aggregates under ceftriaxone treatment.
(A) Pr-aggregated MS11Opa+Pil+, MS11ΔOpaPil+, MS11Opa+Pil-, or MS11ΔLgtEPil+ was either incubated in the presence or absence of 1 μg/mL ceftriaxone for 2 h. Aggregates were then stained to visualize viable (green) and dead (red) GC and visualized with confocal fluorescence microscope. Scale bar: 50 μm. Images were then analyzed for (B) aggregation size and (C) ratio of live-to-dead GC and (D) a correlation graph was then created. Shown are the average values (SD) obtained from > 40 images of three independent experiments. ***p < 0.001; **p < 0.01; *p < 0.05. This figure was previously published 24and is used with permission.
See this image and copyright information in PMC

References

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