Assessment of the In Vitro and In Vivo Antifungal Activity of NSC319726 against Candida auris

ABSTRACT Candida auris is an emerging yeast pathogen of candidemia with the ability to develop resistance to all current antifungal drug classes. Novel antifungal therapies against C. auris are warranted. NSC319726 is a thiosemicarbazone with an inhibitory effect on fungal ribosome biogenesis that has demonstrated some antifungal activity. In this study, we assessed the in vitro activity and in vivo efficacy of NSC319726 against C. auris. NSC319726 was active in vitro against 22 C. auris isolates from different clades, with MICs ranging from 0.125 to 0.25 mg/liter. Despite complete visual growth inhibition, the effect was described as fungistatic in time-kill curves. Interactions with fluconazole, amphotericin B, and micafungin, as tested by the checkerboard dilution method, were described as indifferent. NSC319726 demonstrated significant effects in rescuing G. mellonella larvae infected with two distinct C. auris isolates, compared to the untreated group. In conclusion, NSC319726 demonstrated in vitro activity against C. auris and in vivo efficacy in an invertebrate model of infection. Its potential role as a novel antifungal therapy in humans should be further investigated. IMPORTANCE Candida auris is emerging as a major public health threat because of its ability to cause nosocomial outbreaks of severe invasive candidiasis. Management of C. auris infection is difficult because of its frequent multidrug-resistant profile for currently licensed antifungals. Here, we show that the thiosemicarbazone NSC319726 was active in vitro against a large collection of C. auris isolates from different clades. Moreover, the drug was well tolerated and effective for the treatment of C. auris infection in an invertebrate model of Galleria mellonella. We conclude that NSC319726 might represent an interesting drug candidate for the treatment of C. auris infection.

E-mail: spectrum@asmusa.org Reviewer comments: Reviewer #1 (Comments for the Author): This manuscript describes the antifungal activity of a investigational chemotherapeutic NSC319726 against Candida auris clinical isolates both in vitro and in vivo. Overall, while the antifungal activity of this agent has been described previously, as mentioned by the authors of this manuscript, these studies expand what is known to an emerging healthcare associated and multidrug resistant pathogen of great clinical concern. The manuscript is well written and the procedures are easy to follow and understand.
-In the methods the authors describe that MIC testing was performed per CLSI methods, however the description of the endpoint used for micafungin was complete growth inhibition rather than thẽ 50% inhibition as detailed by CLSI M27 S4. IF the authors did indeed use an alternative and more stringent endpoint for micafungin than is standard, this should be clearly mentioned and justification for the decision to do so detailed.
-The authors should include a description of the media, incubation time, and incubation temperatures used for MIC testing particularly if the differ from the CLSI M27 S4 as with the previous point. These parameters all can significantly impact MIC determination, particularly with Candida auris, and as the comparative MIC values are a key aspect of the manuscript they should be as clearly defined/ described as possible.
-The supplementary figure should be moved to the main text. The finding that this investigational agent is fungistatic against Candida auris but was previously shown to be fungicidal against Candida albicans is important and of interest.
-The arrows denoting the significance of comparisons between treatments in Figure 1 are a bit confusing as they are placed between the endpoint of the graph and the legend. These should be moved or changed to more clearly show which of the two they are corresponding to.
-In line 35-36, the authors describe the clades of C. auris to be geographic clades. While references like this are common, this is somewhat misleading. While the clades where each associated with a geographic region where they were first found, assignment of isolates to clades is done based up on genetic relatedness and the global dissemination of C. auris has resulted in isolates from various clades being identified in many countries (as seen in Table 1). It would seem simply changing the word "geographic" to "genetic" would be more appropriate.
-lines 82 through 86 are very redundant and should be revised/ reworded to address this.
Reviewer #2 (Comments for the Author): The manuscript is well written and presents novel preliminary data on NSC319726 activity against Candida auris. Please see my detailed comments below.
1. It seems the tested drug concentration ranges were rather unusual -judging by fluconazole and micafungin MICs {greater than or equal to} 1024. Please clarify (in the Materials and Methods section) what drug concentration ranges were applied for each drug.
2. The time-kill curves should be performed in biological triplicates, and error bars presented on the graph. Moreover, it is not clear why the graph presenting time-kill curves was made a Supplementary Material and not a Figure in the main text.
3. Please provide some explanation why stains I.3 and IV.1 were selected for in vivo studies.
4. Please provide a comment (in the discussion) on the in vivo results in light of the discovery of fungistatic activity of NSC319726.

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Spectrum 01395-21 : responses to reviewers comments
Line numbering refers to the "clean" revised manuscript.

Reviewer #1 (Comments for the Author):
-In the methods the authors describe that MIC testing was performed per CLSI methods, however the description of the endpoint used for micafungin was complete growth inhibition rather than the ~50% inhibition as detailed by CLSI M27 S4. IF the authors did indeed use an alternative and more stringent endpoint for micafungin than is standard, this should be clearly mentioned and justification for the decision to do so detailed.
Response: We agree that the CLSI M27 S4 document defines the cut-off at 50% inhibition for micafungin. Actually, we did not observe any trailing effect regarding micafungin (clear cut inhibition) for all isolates, which means that the MIC 50% inhibition and MIC 100% inhibition are the same. We agree that strict adherence to CLSI method is warranted and we have modified the MIC definitions for micafungin (50% instead of complete) in methods (lines 126-129), which actually does not affect the results presented in text and in Table 1.
-The authors should include a description of the media, incubation time, and incubation temperatures used for MIC testing particularly if the differ from the CLSI M27 S4 as with the previous point. These parameters all can significantly impact MIC determination, particularly with Candida auris, and as the comparative MIC values are a key aspect of the manuscript they should be as clearly defined/ described as possible.
Response: We have adhered to CLSI procedure. We have now added details about media, inocula, incubation time and temperature in methods (lines 118-129).
-The supplementary figure should be moved to the main text. The finding that this investigational agent is fungistatic against Candida auris but was previously shown to be fungicidal against Candida albicans is important and of interest.
Response: We agree. The paper was initially submitted as an "observation", which according to journal guidelines allow only 2 figures/tables. This is why we have moved the time-kill curve as Supplemental material. We agree that it makes perfect sense to move it as a figure in the main manuscript (now inserted as new Figure 1).
-The arrows denoting the significance of comparisons between treatments in Figure 1 are a bit confusing as they are placed between the endpoint of the graph and the legend. These should be moved or changed to more clearly show which of the two they are corresponding to.
Response: We have moved the arrows in the graph to clearly show the curves that have been compared. We have also added more precisions in the legend.
-In line 35-36, the authors describe the clades of C. auris to be geographic clades. While references like this are common, this is somewhat misleading. While the clades where each associated with a geographic region where they were first found, assignment of isolates to clades is done based up on genetic relatedness and the global dissemination of C. auris has resulted in isolates from various clades being identified in many countries (as seen in Table 1). It would seem simply changing the word "geographic" to "genetic" would be more appropriate.
Response: We agree and we have changed the sentence as follows: five genetic clades have been identified from distinct geographical origin: South Asian (1), etc… (lines 36-38).
-lines 82 through 86 are very redundant and should be revised/ reworded to address this. Response: We agree. We have suppressed the first sentence.
Reviewer #2 (Comments for the Author): 1. It seems the tested drug concentration ranges were rather unusual -judging by fluconazole and micafungin MICs {greater than or equal to}1024. Please clarify (in the Materials and Methods section) what drug concentration ranges were applied for each drug.
Response: We have indeed tested some higher concentrations than those recommended in the range of the CLSI procedure. For a strict adherence to CLSI recommendations (as also suggested by Reviewer 1), we have now provided only the results within the CLSI ranges (see modifications in methods, lines 123-125, results lines 53-56, and Table 1). Thank you for resubmitting your revised manuscript. All of the reviewer's previous comments were addressed.

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