The Fourier-transform infrared spectroscopy-based method as a new typing tool for Candida parapsilosis clinical isolates

ABSTRACT The Fourier-transform infrared spectroscopy-based IR Biotyper is a straightforward typing tool for bacterial species, but its use with Candida species is limited. We applied IR Biotyper to Candida parapsilosis, a common cause of nosocomial bloodstream infection (BSI), which is aggravated by the intra-hospital spread of fluconazole-resistant isolates. Of 59 C. parapsilosis isolates studied, n = 56 (48 fluconazole-resistant and 8 fluconazole-susceptible) and n = 3 (2 fluconazole-resistant and 1 fluconazole-susceptible) isolates, respectively, had been recovered from BSI episodes in 2 spatially distant Italian hospitals. The latter isolates served as an outgroup. Of fluconazole-resistant isolates, n = 40 (including one outgroup) harbored the Y132F mutation alone and n = 10 (including one outgroup) harbored both Y132F and R398I mutations in the ERG11-encoded azole-target enzyme. Using a microsatellite typing method, which relies on the amplification of genomic short tandem repeats (STR), two major clusters were obtained based on the mutation(s) (Y132F or Y132F/R398I) present in the isolates. Regarding IR Biotyper, each isolate was analyzed in quintuplicate using an automatic (i.e., proposed by the manufacturer’s software) or tentative (i.e., proposed by us) cutoff value. In the first case, four clusters were identified, with clusters I and II formed by Y132F or Y132F/R398I isolates, respectively. In the second case, six subclusters (derived by the split of clusters I and II) were identified. This allowed to separate the outgroup isolates from other isolates and to increase the IR Biotyper typeability. The agreement of IR Biotyper with STR ranged from 47% to 74%, depending on type of cutoff value used in the analysis. IMPORTANCE Establishing relatedness between clinical isolates of Candida parapsilosis is important for implementing rapid measures to control and prevent nosocomial transmission of this Candida species. We evaluated the FTIR-based IR Biotyper, a new typing method in the Candida field, using a collection of fluconazole-resistant C. parapsilosis isolates supposed to be genetically related due to the presence of the Y132F mutation. We showed that IR Biotyper was discriminatory but not as much as the STR method, which is still considered the method of choice. Further studies on larger series of C. parapsilosis isolates or closely related Candida species will be necessary to confirm and/or extend the results from this study.


Methods:
How were the replicates treated?Were the spectra averaged, or do the results show individual results?Were there any differences among the replicates?Was the ability to form biofilms on solid artificial surfaces tested?It was shown that biofilm formation changes the FTIR spectra, and it is well-known that some C. parapsilosis strains have this ability.It is not clear how the tentative value was determined.I believe it is crucial since it improved the results so significantly.
Reviewer #2 (Comments for the Author): The article "The Fourier-Transform Infrared Spectroscopy Based Method as a New Typing Tool for Candida parapsilosis Clinical Isolates" is relevant, considering the importance of developing techniques for typing microorganisms that are faster and more appropriate in the hospital context.Methods based on spectroscopy are particularly relevant, considering the advantages and numerous possibilities of these techniques.The authors showed that the IR Biotyper is capable of detecting differences in isolates considered clonal by the STR, increasing its discriminatory power in relation to the standard method.The work is written concisely and clearly and is interesting and relevant.However, I have some aspects to point out that needs to be better explored in the writing, mainly in the Materials and Methods section: 1) Line 139: "To our knowledge, only one study to date has investigated IR Biotyper as a typing tool for Candida species (25)" Please include the following article in the Discussion: Contreras DA, Morgan MA.Surveillance diagnostic algorithm using realtime PCR assay and strain typing method development to assist with the control of C. auris amid COVID-19 pandemic. Front Cell Infect Microbiol. 2022;12:887754. doi: 10.3389/fcimb.2022.887754.In this article, the authors used the IR Biotyper to genotype 28 hospital isolates of Candida auris, using whole genome sequencing as a reference method.In addition, they were also able to use the IR Biotyper to successfully differentiate several yeast species, such as C. albicans, C. glabrata, C. auris, and Cryptococcus neoformans.
2) Line 238: "Spectra were acquired (in the 1300-800 cm−1 carbohydrate region) using the IR Biotyper system..." I know that this is the default of the instrument and also the "fingerprint" region.However, considering that the maximum Simpson's index of diversity value obtained was 0.839, was it considered that analyzing other regions of the spectrum could improve this index?
3) Was any kind of pre-processing used in the spectra (normalization, derivatives...)?Could these pre-processes improve the discriminatory power of the IR Biotyper?I suggest better detailing the chemometric analyzes in the methodology.4) Line 116: "Using a tentative cutoff value (0.995), which was based on our experimental input..." The tentative cutoff was suggested by IR Biotyper software?Explain better how the tentative cutoff value was defined.It would be interesting to add this information in the methdology.

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"We showed that IR Biotyper was comparable to the STR method, which is s ll considered the method of choice" -seems to be an inadequate and very strong statement since the results showed 47 to 74% agreement.In my opinion, a comparison with WGS would be jus fied here.Otherwise, the results are not very convincing.
Results: The authors should provide characteris c spectra for C. parapsilosis and discuss the spectral changes between fluconazole-suscep ble and fluconazole-resistant strains.

Methods:
How were the replicates treated?Were the spectra averaged, or do the results show individual results?
Were there any differences among the replicates?
Was the ability to form biofilms on solid ar ficial surfaces tested?It was shown that biofilm forma on changes the FTIR spectra, and it is well-known that some C. parapsilosis strains have this ability.
It is not clear how the tenta ve value was determined.I believe it is crucial since it improved the results so significantly.
The article "The Fourier-Transform Infrared Spectroscopy Based Method as a New Typing Tool for Candida parapsilosis Clinical Isolates" is relevant, considering the importance of developing techniques for typing microorganisms that are faster and more appropriate in the hospital context.Methods based on spectroscopy are particularly relevant, considering the advantages and numerous possibilities of these techniques.The authors showed that the IR Biotyper is capable of detecting differences in isolates considered clonal by the STR, increasing its discriminatory power in relation to the standard method.
The work is written concisely and clearly and is interesting and relevant.However, I have some aspects to point out that needs to be better explored in the writing, mainly in the Materials and Methods section: In this article, the authors used the IR Biotyper to genotype 28 hospital isolates of Candida auris, using whole genome sequencing as a reference method.In addition, they were also able to use the IR Biotyper to successfully differentiate several yeast species, such as C. albicans, C. glabrata, C. auris, and Cryptococcus neoformans.
2) Line 238: "Spectra were acquired (in the 1300-800 cm −1 carbohydrate region) using the IR Biotyper system..." I know that this is the default of the instrument and also the "fingerprint" region.However, considering that the maximum Simpson's index of diversity value obtained was 0.839, was it considered that analyzing other regions of the spectrum could improve this index?
3) Was any kind of pre-processing used in the spectra (normalization, derivatives...)?Could these pre-processes improve the discriminatory power of the IR Biotyper?I suggest better detailing the chemometric analyzes in the methodology.4) Line 116: "Using a tentative cutoff value (0.995), which was based on our experimental input..." The tentative cutoff was suggested by IR Biotyper software?Explain better how the tentative cutoff value was defined.It would be interesting to add this information in the methdology.
Spectrum02388-23 (The Fourier-Transform Infrared Spectroscopy Based Method as a New Typing Tool for Candida parapsilosis Clinical Isolates)

Reviewer_1
"We showed that IR Biotyper was comparable to the STR method, which is still considered the method of choice" -seems to be an inadequate and very strong statement since the results showed 47 to 74% agreement.In my opinion, a comparison with WGS would be justified here.Otherwise, the results are not very convincing.Answer: We agree with the reviewer that the above statement may be too strong considering that the agreement of IR Biotyper with the STR method is not greater than 74%.Accordingly, we have edited the statement to mitigate its strength.See page 2 of the revised manuscript.We also agree that using WGS to validate the comparison of IR Biotyper with the STR method in this study could have strengthened our findings.Therefore, we have added a sentence that emphasizes the limitation of our study.See page 9 of the revised manuscript.Methods: How were the replicates treated?Were the spectra averaged, or do the results show individual results?Were there any differences among the replicates?Answer: As requested, we have provided all information on how the replicates were treated or how an average spectrum was obtained.See page 11 of the revised manuscript.

Results
Was the ability to form biofilms on solid artificial surfaces tested?It was shown that biofilm formation changes the FTIR spectra, and it is well-known that some C. parapsilosis strains have this ability.Answer: While agreeing that biofilm formation may change FTIR spectra, isolates in our study were not tested while growing in biofilms.We have previously tested some C. parapsilosis isolates from another collection, but we did not observe any difference in FTIR spectra between high or low biofilm forming isolates.Thus, it is plausible that biofilm-related cell surface components do not influence the FTIR analysis results in our study.
It is not clear how the tentative value was determined.I believe it is crucial since it improved the results so significantly.Answer: As requested we have added details on how the tentative cutoff value was determined.See page 11 of the revised manuscript.

Reviewer_2
The article "The Fourier-Transform Infrared Spectroscopy Based Method as a New Typing Tool for Candida parapsilosis Clinical Isolates" is relevant, considering the importance of developing techniques for typing microorganisms that are faster and more appropriate in the hospital context.Methods based on spectroscopy are particularly relevant, considering the advantages and numerous possibilities of these techniques.The authors showed that the IR Biotyper is capable of detecting differences in isolates considered clonal by the STR, increasing its discriminatory power in relation to the standard method.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred 1) Line 139: "To our knowledge, only one study to date has investigated IR Biotyper as a typing tool for Candida species (25)" Please include the following article in the Discussion: Contreras DA, Morgan MA.Surveillance diagnostic algorithm using real-time PCR assay and strain typing method development to assist with the control of C. auris amid COVID-19 pandemic.Front Cell Infect Microbiol.2022;12:887754.doi: 10.3389/fcimb.2022.887754.
: The authors should provide characteristic spectra for C. parapsilosis and discuss the spectral changes between fluconazole-susceptible and fluconazole-resistant strains.Answer: As requested, we have added a new Figure (Fig. 4) showing the spectral profiles of representative fluconazole-resistant and fluconazole-susceptible C. parapsilosis isolates.As shown the profiles overlap and only subtle differences in absorbance are visible.See Fig. 4, and pages 6 and 18 of the revised manuscript.