Antifungal susceptibility profile and local epidemiological cut-off values of Yarrowia (Candida) lipolytica: an emergent and rare opportunistic yeast

ABSTRACT The antifungal susceptibility profile and epidemiological cut-off values (ECOFFs) of Yarrowia lipolytica, a rare opportunistic yeast, remain unclear. We conducted a comprehensive multi-method study on clinical isolates from various central hospitals, based on the China Hospital Invasive Fungal Surveillance Network (2009–2022). Our objective was to evaluate the antifungal susceptibility of Y. lipolytica, establish its local ECOFFs (L-ECOFFs), and compare the performance of the ATB FUNGUS 3 (ATB), Sensititre YeastOne (SYO), and minimum inhibitory concentration (MIC) test strip (MTS) with that of the broth microdilution (BMD) method. L-ECOFFs were established using ECOFFinder, and we examined ERG11 mutations to assess the reliability of the L-ECOFFs. The L-ECOFF for fluconazole was 8 µg/mL. Non-wild-type isolates of antifungal drugs, such as flucytosine and azoles, were exclusively isolated from patients. Additionally, we detected that four strains with the ERG11 A395T mutation (azole MIC >L-ECOFF) may be associated with the exposure to azole drugs. For azoles, ATB showed the highest essential agreement with the BMD (98.18%–100%), followed by SYO (85.45%–100%). However, ATB could not detect susceptibility to echinocandins, while SYO exhibited the highest agreement (98.18%–100%) in detecting echinocandin susceptibility. Our findings indicate that acquired azole cross-resistance has emerged despite Y. lipolytica infections being rare. This research provides crucial antifungal susceptibility data and establishes the initial L-ECOFFs for Y. lipolytica. The SYO is recommended as the optimal laboratory antifungal susceptibility testing method for Y. lipolytica, followed by ATB, whereas the use of MTS requires caution. We hope that this study will facilitate improved clinical management of Y. lipolytica infections. IMPORTANCE Yarrowia lipolytica, also known as Candida lipolytica, is an emerging opportunistic “rare pathogenic yeast”. Due to the limited data on its antifungal susceptibility, the clinical treatments become challenging. Based on the China Hospital Invasive Fungal Surveillance Network (2009–2022), we conducted a comprehensive multi-method study on clinical isolates from various central hospitals. This study is currently the largest study carried out to assess the antifungal susceptibility of Y. lipolytica. It is also the first to establish local epidemiological cut-off values (L-ECOFFs), identify its ERG11 mutations, and assess the consistency between the three prevalent commercial antifungal susceptibility testing methods and the broth microdilution method. We recommend the Sensititre YeastOne as the best option for antifungal susceptibility testing for Y. lipolytica, followed by the ATB FUNGUS 3. Nevertheless, practitioners should use the MIC test strip with discretion.

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Reviewer #1 (Comments for the Author): The present manuscript describes the establishment of local ECOFFs for Y. lipolytic and compares three different commercial kits with the standard method according to CLSI.74 isolates ( both clinical and non clinical) were included in the study.In addition, the authors looked for mutation of ERG11.
The paper is interesting and data for Y. lipolytica is scare.Therefore, more data is definitely important.The paper is well written, however, there are some inconsistencies which should be revised.1. the number of isolates is very small.It is doubtful, if this number is sufficient for establishing sound ECOFFS.For the establishment of ECOFFS it is crucial to ensure that methodological variation (e.g. the variation created by using material from different manufacturers, incubation times varying between 16 and 20 hours, several staff members, thermostats), interlaboratory variation, and whatever biological variation there may be between isolates are incorporated into the MIC distribution and the ECOFF.It seems that these criteria are not completely fulfilled.The authors should therefore explain in a more detailed way how the results were obtained.2. the comparison and evaluation of commercial kits is very important and valuable for diagnostic work up in routine laboratories.However, the rate of concordance and discordance should be stated explicitly.It is unclear how accuracy was calculated, more information should be given.3. Specific comments: Material and Methods: Page 6, lines 123 +130, page 7, line 136: it would be easier to read if the exact name of the test would be stated and not the abbreviation page 6, line 131: please insert City and Country of the company's name Results: page 8, lines 166-167: please explain why these strains did not yield any result.Was there no growth?What about the positive controls when testing these isolates?What could be the reason that you could not achieve any result?Page 10, line 194: only caspofungin is mentioned, why are micafungin and anidulafungin missing?Table 2 and page 9, lines 178 -183: it is unclear if the results are from your work or if data from other isolates are included ( 135 isolates are quoted, but you tested 74 isolates).The information given is for readers without sound knowledge of ERG11 mutations not easy to understand.More explanations would therefore be needed.
Reviewer #2 (Comments for the Author): Authors present an important dataset of antifungal susceptibility profiles for Y. lipolityca clinical and environmental isolates.This species is a rare opportunistic pathogen and antifungal profiles associated with some molecular data is helpful for understand potential resistance of those rare species.I don't understand why authors indicate that number of isolates is not sufficient.In my opinion, some sentences need clarification.Additional technical details are also essential in the materials and methods section.Other remarks: Line 26: lipolytica and not "lipolytic" Authors indicate that the antifungal susceptibility profile of the Y. lipolytica is unknown but there are some studies that already give MIC distribution for isolates of these species and they mentioned some like ref 25 and 26.They could also add one reference with 34 isolates of Y. lipolytica for which antifungal MIC were determined (doi: 10.1111/myc.13095)Line 40 "ATB could not detect echinocandins" do you mean that ATB is not good to determine susceptibility to echinocandins or that ATB is not able to distinguish between wild-type and non wild-type isolates?Line 43: authors say that their study give information about epidemiology of Y. lipolytica but there is no clinical data presented in this study so I am not sure we could say that it's increase the knowledge concerning the epidemiology of this species.Line 83 could you explain what do you mean by azoles are important for the treatment ?azoles are recommended, frequently used?Line 87: not known is maybe more appropriate that unclear Line 93: maybe also important to remind that commercialized methods are expensive Line 102: could you give more details about the species identification?Were the isolates identified by MALDI Tof or sequencing or other techniques?Line 111: Many isolates of Y. lipolytica don't grow well at 35{degree sign}C, did you test to determine antifungal susceptibility profile at 30{degree sign}C?Line 114 for the strains with differing results did you redo the test or did you check the purity of the strains, is it possible that some samples contained mixture of isolates explaining the discordance?Line 149 could you give more details concerning the method of extraction and sequencing because the paragraph is not clear for me, Did you sequence the region corresponding to the YALI0_B05126g gene for all your isolates?Line 167: did you perform the tets at 30{degree sign}C for the isolates which did not grow at 35{degree sign}C?Line 176 echinomycin is echinocandin?Line 179 replace codon by nucleotide maybe Line 181: "the two patients with isolated Y08...." Isolated corresponds to isolates?Sometimes you indicate strains and in some other sentences isolates, could you harmonize the term Line 192: could you clarify, do you mean ATB method could not detect echinocandin resistant ?Line 206: some references could be interesting about the treatment of infection due to Y. lipolytica In the references 25 the 5flucytosine is not tested and in the reference 26 the value of 5FC MIC are high so I don't understand why do you indicate that isolates have high MIC except for 5flucytosine.
Line 218: could you conclude that AMPHOB and voriconazole are the most effective treatment?Lines 220-222 could you clarify the sentence Line did you sequence the entire ERG11 gene or only the coding region?Maybe sequenced is more appropriate than analysed.Line : Y132F is a mutation in the protein Erg11 Line could you develop the last sentence Line the use of 5flucytpsine with other antifungal agents is frequent for many fungal species and not only Y. lipolytica Line 249: Authors indicate that they determine antifungal profiles for 74 isolates which is a huge quantity for a rare opporutinistic pathogen, why do they say that they have a restricted number of isolates?

Peking Union Medical College Hospital
No.1 Shuaifuyuan Wangfujing Dongcheng District, Beijing, China 100730 November 10, 2023 Dear Reviewer, Thank you for your thorough review, insightful comments, and constructive suggestions, all of which have considerably enhanced the presentation of our manuscript.We have meticulously revised the manuscript in line with your remarks.
Below, we provide a summary of our responses to each point raised.We trust that our revisions have adequately addressed all the issues raised, and we are hopeful that our manuscript is now suitable for publication.

Response to Reviewer 1 Comment 1:
the number of isolates is very small.It is doubtful, if this number is sufficient for establishing sound ECOFFS.For the establishment of ECOFFS it is crucial to ensure that methodological variation (e.g. the variation created by using material from different manufacturers, incubation times varying between 16 and 20 hours, several staff members, thermostats), interlaboratory variation, and whatever biological variation there may be between isolates are incorporated into the MIC distribution and the ECOFF.It seems that these criteria are not completely fulfilled.The authors should therefore explain in a more detailed way how the results were obtained.
Response：Thank you for bringing attention to the concerns about sample size and methodological variation in our study.We acknowledge these concerns and, after a comprehensive review of the standards for testing the susceptibility of rare fungal pathogens and establishing Epidemiological Cutoff Values (ECOFFs), we maintain that our findings are clinically significant.Despite the smaller sample size, which is a common challenge when dealing with rare fungi such as those with an isolation rate below 1%, the established ECOFFs remain pertinent, as detailed in the corresponding section of our article.
Moreover, it is important to highlight that our study encompasses all strains from China's most extensive fungal disease surveillance network, CHIF-NET, which spans the entire country.This wide coverage lends considerable reliability to our results concerning the drug susceptibility of Yarrowia lipolytica in China.

Peking Union Medical College Hospital
No.1 Shuaifuyuan Wangfujing Dongcheng District, Beijing, China 100730 Addressing the issue of methodological variation, the rarity of Yarrowia lipolytica means not every laboratory has the necessary strains for testing, which presents a challenge for multi-laboratory studies.
Nevertheless, we mitigated this by conducting three separate replicate experiments, carried out by different personnel, using freshly prepared media for each, and with results assessed blind to the experiment.While conducted in a single central laboratory, we used the term "Local ECOFF (L-ECOFF)" to align our terminology with similar studies, ensuring consistency and clarity in our findings.

Comment 2:
the comparison and evaluation of commercial kits is very important and valuable for diagnostic work up in routine laboratories.However, the rate of concordance and discordance should be stated explicitly.It is unclear how accuracy was calculated; more information should be given.
Response： Thank you for your astute suggestion, which we had initially overlooked.We have replaced any inappropriate terminology in the article, such as "accuracy," with more precise terms such as "essential agreement" or "concordance" to better reflect the intended meaning.The antifungal susceptibility tests for the strains sourced from diverse locations-flies (CGMCC 2.3222), East China garbage stations (CGMCC 2.1556), and CGMCC 2.1502 from Kyoto University's Faculty of Agriculture in Japan-did not produce results due to their poor growth at 35°C.This outcome, which also affected the growth of positive controls, is consistent with the environmental nature of Yarrowia lipolytica, known for its limited tolerance to elevated temperatures.Following your advice, which was echoed by another reviewer, we have now carried out additional drug sensitivity assays at the more suitable temperature of 30°C for these three strains.The strain CGMCC 2.1502 is non-wild type for Voriconazole and Fluconazole, and CGMCC 2.3222 is also non-wild type for Fluconazole.This may also suggest that Yarrowia lipolytica naturally has insensitivity to some azole drugs.

The results can be seen in
In line 194 on Page 10, which we had previously overlooked, we have now added descriptions for the other two drugs, micafungin and anidulafungin.
Comment 5: isolates are included (135 isolates are quoted, but you tested 74 isolates).The information given is for readers without sound knowledge of ERG11 mutations not easy to understand.More explanations would therefore be needed.
Response：Thank you for your valuable feedback.
The fungal cytochrome P450 lanosterol 14α-demethylase, encoded by the ERG11 gene, plays a crucial role in synthesizing ergosterol, unique to fungi, and serves as the target for azole drugs.Notably, the ERG11 mutation (A395T) has been identified exclusively in the fluconazole-resistant Y. lipolytica strain CBS 18115.Its presence in other strains remains to be determined.
The presumed ERG11 gene in Y. lipolytica, YALI0_B05126g (Gene ID:2906856), was pinpointed using the protein BLAST algorithm.This gene encodes a 523 amino acid protein sharing 59.21% similarity with the Candida albicans homolog (GenBank accession number X13296.1).
For gene amplification and sequencing, we crafted primers for YALI0_B05126g with the NCBI Blast Primer tool.The ERG11 gene was amplified from 77 strains using the forward primer "TGATCATTCTCACGACGCTCA," and the reverse primer "TCTAGTTACGCTCTCGCTTGC." To thoroughly investigate the ERG11 mutation spectrum in Y. lipolytica, we accessed next-generation sequencing genome data for 58 strains, including all clinical isolates up to December 2021, from the NCBI Sequence Read Archive Database.The strains analyzed for YALI0_B05126g are listed in Supplementary Table S1.We aligned the clean read sequences with the reference genome (ASM252v1) using bowtie 2 software (v2.3.5.1), sorted the alignments with samtools, and annotated variation sites with SnpEff software to identify gene variants, categorize mutations as synonymous or non-synonymous, and assess their impact on the amino acids.

Response to Reviewer 2
Comment 1: I don't understand why authors indicate that number of isolates is not sufficient.
Line 249: Authors indicate that they determine antifungal profiles for 74 isolates which is a huge

Peking Union Medical College Hospital
No.1 Shuaifuyuan Wangfujing Dongcheng District, Beijing, China 100730 quantity for a rare opporutinistic pathogen, why do they say that they have a restricted number of isolates?
Response：We apologize for any lack of clarity in our previous description.
When referring to a small sample size, we meant it in relation to other studies that establish Epidemiological Cutoff Values (ECOFFs) for more commonly encountered clinical pathogens.
Nevertheless, we do not view this as a limitation in the context of our research.Given that Y. lipolytica is a rare and emerging opportunistic yeast, a low prevalence is inherent.Our study is comprehensive in that it includes clinical isolates of Y. lipolytica gathered from the National China Hospital Invasive Fungal Surveillance Network (CHIF-NET) over the last decade, which is the largest network of its kind in China.
Moreover, in cases involving rare fungi like Y. lipolytica, even a seemingly small dataset can significantly inform clinical treatments, as supported by literature.In essence, for rare fungi, the sample size we have is considered adequate.

Comment 2:
Additional technical details are also essential in the materials and methods section.
Line 26: lipolytica and not "lipolytic" Line 87: not known is maybe more appropriate that unclear Line 93: maybe also important to remind that commercialized methods are expensive Line 176 echinomycin is echinocandin?
Line 179 replace codon by nucleotide maybe Line 227 Y132F is a mutation in the protein Erg11 Sometimes you indicate strains and in some other sentences isolates, could you harmonize the term Response: Thank you for your insightful writing suggestions; they have been incredibly beneficial.
We have thoroughly reviewed and revised the manuscript, clarifying any ambiguous descriptions and correcting the inaccuracies you pointed out.Additionally, we have enriched the methodology section with a comprehensive account of the antifungal susceptibility tests and refined the approach for analyzing the ERG11 mutations.
Our distinction between "strains" and "isolates" is well-founded and aligns with the terminology used within the scientific literature.An isolate is indeed something that has been obtained from a specific source, such as an animal or a patient.Subsequent investigation allows us to classify these isolates, assigning a name and identifying them as a particular strain.Hence, the use of both terms in our manuscript is appropriate and accurately reflects the process of identification and classification within your research.

Comment 3:
Authors indicate that the antifungal susceptibility profile of the Y. lipolytica is unknown but there are some studies that already give MIC distribution for isolates of these species and they mentioned some like ref 25 and 26.They could also add one reference with 34 isolates of Y. lipolytica for which antifungal MIC were determined (doi: 10.1111/myc.13095) Response: Thank you for your insightful suggestions.In our manuscript, the article 'Mycoses.
2020;63(7):737-745.doi:10.1111/myc.13095' is indeed acknowledged as the ninth citation.We recognize that the literature contains reports on the antifungal susceptibility of Yarrowia lipolytica; these reports tend to be case studies involving a limited number of strains.Critically, there appears to be an absence of studies employing the CLSI standard broth microdilution method.This absence casts doubts on the reliability of the reported drug sensitivity findings.Taking your advice into account, we agree that the term 'unclear' is more fitting than 'unknown' in our manuscript.This alteration more accurately conveys the present knowledge and underscores the necessity for standardized methodological practices in forthcoming studies.

Comment 4:
Line 246 the use of 5flucytpsine with other antifungal agents is frequent for many fungal species and not only Y. lipolytica.
Line 43: authors say that their study give information about epidemiology of Y. lipolytica but there is no clinical data presented in this study so I am not sure we could say that it's increase the knowledge concerning the epidemiology of this species.
Response: Thank you for your valuable writing advice; it has been greatly beneficial.

Peking Union Medical College Hospital
No.1 Shuaifuyuan Wangfujing Dongcheng District, Beijing, China 100730 We have revised reads: Due to the propensity for rapid development of resistance, it is generally not recommended to use 5-flucytosine as a monotherapy in clinical practice.
In our manuscript, the use of 'epidemiology' was intended to denote the variability and distribution of drug sensitivity among Yarrowia lipolytica strains.We accept that this term may not be ideally suited, and we will adjust the text in our manuscript to better reflect our intended meaning.

Comment 5:
Line 40 "ATB could not detect echinocandins" do you mean that ATB is not good to determine susceptibility to echinocandins or that ATB is not able to distinguish between wild-type and non wild-type isolates?
Line 192: could you clarify, do you mean ATB method could not detect echinocandin resistant?
Response： We apologize for any lack of clarity in our initial description.ATB is suitable for Candida and Cryptococcus species and can detect susceptibility to five antifungal drugs: 5-flucytosine, amphotericin B, fluconazole, itraconazole, and voriconazole.However, it cannot detect susceptibility to echinocandins.Additionally, it cannot perform drug susceptibility testing for Aspergillus species.We will provide a detailed description in the manuscript to avoid any ambiguity.Reports indicate that azoles are effective in treating Yarrowia lipolytica infections, with clinical Page 6, lines 123 +130, page 7, line 136: it would be easier to read if the exact name of the test would be stated and not the abbreviation page 6, line 131: please insert City and Country of the company's name Response：Thank you for your recommendation.I have amended the manuscript by spelling out the previously unclear abbreviations on Page 6, lines 123 and 130, and on Page 7, line 136, to their full terms for clarity.Additionally, the omitted city and country names at line 131 have been duly inserted.Comment 4: page 8, lines 166-167: please explain why these strains did not yield any result.Was there no growth?What about the positive controls when testing these isolates?What could be the reason that you could not Peking Union Medical College Hospital No.1 Shuaifuyuan Wangfujing Dongcheng District, Beijing, China 100730 achieve any result?Page 10, line 194: only caspofungin is mentioned, why are micafungin and anidulafungin missing?Response：Thank you for your suggestions.
Line 83 could you explain what do you mean by azoles are important for the treatment?azoles are recommended, frequently used?Response：Thank you for your recommendations.Azole antifungals, including fluconazole, itraconazole, voriconazole, and posaconazole, function by disrupting ergosterol synthesis within fungal cell membranes, leading to cellular death.Their efficacy spans a wide spectrum of fungal species, establishing them as a frequent choice for treating various fungal infections.

Table 2
and page 9, lines 178 -183: it is unclear if the results are from your work or if data from other Peking Union Medical College Hospital No.1 Shuaifuyuan Wangfujing Dongcheng District, Beijing, China 100730