Detection of ERG11 gene mutation in coding and non-coding region of clinical Candida glabrata isolates from Pakistan, a short communication
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Azoles inhibit cytochrome P450 dependent enzyme lanosterol-14α-demethylase (CYP51) that is encoded by ERG11gene. Mutations in the ERG11gene confer azole resistance in Candida species through different mechanisms, like ERG11 overexpression, CDR1,2 overexpression, tolerance to methylation, biofilm formation and import of host cholesterol. We sequenced ERG11 gene to determine mutations in coding and non-coding region of ERG11 in clinical isolates of Candida glabrata from Pakistan. Eight C. glabrata strains with fluconazole minimum inhibitory concentrations ranging from 2-256 mg/L were included in this study. The ERG11 gene was amplified by PCR, sequenced using Sanger methodology, and analyzed using bioinformatic tools. We identified mutation at position I166S in non-coding region of ERG11gene in C. glabrata. Two novel synonymous mutations at position I100 and D278, and previously reported synonymous mutations D256, L341, P519 and R527 were also observed. This is the first study evaluating ERG11 mutations in C. glabrata species from Pakistan. Such ERG11 gene mutations which are unique to geographical regions should be investigated through resistance and genotypic surveillance.
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Thank you very much for submitting you manuscript to Access Microbiology. It has now been reviewed by two experts in the field whose comments are attached at the bottom of this email. In general, substantial amendments need to be applied to the manuscript before being considered for publication, and the reviewers provide a list of constructive comments and suggestions that you may find useful for producing a revised version. Additionally, they both have concerns about the use of English, which may impact the understanding of the work. I would suggest that you ask a native English speaking colleague to proof it for you, or alternatively you may want to contact our Editorial Office for assistance with article English correction services. Please provide a revised version of the manuscript (including a tracked changes version), along with …
Thank you very much for submitting you manuscript to Access Microbiology. It has now been reviewed by two experts in the field whose comments are attached at the bottom of this email. In general, substantial amendments need to be applied to the manuscript before being considered for publication, and the reviewers provide a list of constructive comments and suggestions that you may find useful for producing a revised version. Additionally, they both have concerns about the use of English, which may impact the understanding of the work. I would suggest that you ask a native English speaking colleague to proof it for you, or alternatively you may want to contact our Editorial Office for assistance with article English correction services. Please provide a revised version of the manuscript (including a tracked changes version), along with a point-by-point response to the reviewer comments within 2 months.
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Comments to Author
In this study, the author analyzes the presence of mutations in coding and non-coding regions of clinical isolates of Candida glabrata, aiming to correlate them with azole resistance. To achieve this, they amplified the ERG11 gene, including both coding and non-coding regions, sequenced it, and compared it with the reference sequence to identify mutations. The study is interesting; however, several issues need to be addressed. First, the authors should clearly specify the exact region they sequenced and explicitly indicate which parts correspond to the coding and non-coding regions. The supplementary figure should be corrected to accurately indicate the mutations found. If a mutation occurs in a non-coding region, it should not be described using amino acid notation (e.g., I166S), as this is not …
Comments to Author
In this study, the author analyzes the presence of mutations in coding and non-coding regions of clinical isolates of Candida glabrata, aiming to correlate them with azole resistance. To achieve this, they amplified the ERG11 gene, including both coding and non-coding regions, sequenced it, and compared it with the reference sequence to identify mutations. The study is interesting; however, several issues need to be addressed. First, the authors should clearly specify the exact region they sequenced and explicitly indicate which parts correspond to the coding and non-coding regions. The supplementary figure should be corrected to accurately indicate the mutations found. If a mutation occurs in a non-coding region, it should not be described using amino acid notation (e.g., I166S), as this is not applicable. A major concern is that a single sequencing reaction can introduce errors. I strongly recommend repeating the sequencing using at least two independent PCR reactions for each strain to confirm the presence of the mutations. I believe the use of English and grammar still require a thorough revision. Specific comments: Lines 27-28: Please correct the following sentence: "We identified mutation at 28 position I166S in non-coding region of ERG11 gene in C. glabrata." If the mutation is in a non-coding region, it should not be described using an amino acid substitution format. Line 44: In the introduction, please clarify the sentence: "Several enzymes involved in sterol biosynthesis are encoded by ERG11 gene." How does a single gene encode multiple enzymes? Are they isoforms? You should explain it. Lines 59-60: Please rephrase the following sentence for clarity, as "revived" may not be the most appropriate verb: "Eight retrospectively archived invasive C. glabrata isolates were revived and analysed for ERG1160 gene mutation." Line 68: The manuscript refers to CHROMagar as both "CROM agar" and "CHROMagar". Please ensure consistency throughout the text. Line 69: If Biggy is an abbreviation for Bismuth Glucose Glycine Yeast Agar, it should be explicitly stated the first time it appears in the text. Lines 83-84: The following sentence needs rewording for clarity: "The susceptibility of C. glabrata isolates was interpreted as MIC≤ 32 mg/L was SDD and MIC ≥ 64 mg/L was resistant." Lines 85-89: The description of the DNA extraction process is unclear and should be rephrased to improve readability. Line 119: The authors mention amplifying a 950 bp region of ERG11, which includes both coding and non-coding sequences. Please specify the exact boundaries of this amplification (e.g., from the promoter to the stop codon). This is a key point in the study and should be clarified. Lines 124-125: The description of the mutation I166S needs correction or clarification. If it is in a non-coding region, what do the letters represent? It cannot refer to amino acids. Lines 126-129: The description of the mutations does not match the information in Supplementary Figure 1. In this figure, the DNA and protein sequences from the different strains (10 strains) appear almost identical, except for a highlighted T to G substitution at position 495 in the DNA sequence of one of the strains and an I to S substitution at position 166 in the amino acid sequence of another strain. This discrepancy should be addressed. Supplementary Figure 1b: The figure is labeled as "Non-coding fragment of the ERG11 gene in C. glabrata," but it displays an amino acid sequence. This inconsistency needs to be corrected.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
Manuscript ACMI-D-24-00236 describes the sequencing of ERG11 gene variants and the identification of synonymous and nonsynonymous mutations in a few Nakaseomyces glabratus (Candida glabrata) isolates from Pakistan. The authors attempted to establish a relationship between the presence of specific mutations with resistance to fluconazole. The manuscript requires revision of the content and the English language before final publication. Specific comments: Title: _ The current name of the fungus, Nakaseomyces glabratus, should be provided here, in the keywords, or elsewhere in the manuscript. Abstract: _ L21-23: This sentence has no clear meaning. Perhaps: "C. glabrata resistance to azoles is conferred by different mechanisms, like ...chloesterol." _ L26: Were these MIC values determined in this …
Comments to Author
Manuscript ACMI-D-24-00236 describes the sequencing of ERG11 gene variants and the identification of synonymous and nonsynonymous mutations in a few Nakaseomyces glabratus (Candida glabrata) isolates from Pakistan. The authors attempted to establish a relationship between the presence of specific mutations with resistance to fluconazole. The manuscript requires revision of the content and the English language before final publication. Specific comments: Title: _ The current name of the fungus, Nakaseomyces glabratus, should be provided here, in the keywords, or elsewhere in the manuscript. Abstract: _ L21-23: This sentence has no clear meaning. Perhaps: "C. glabrata resistance to azoles is conferred by different mechanisms, like ...chloesterol." _ L26: Were these MIC values determined in this study? (see comment below). _ L30: Delete "species". _ L31-32: No argument or data is presented in the manuscript indicating that these mutations are unique to the geographical region investigated. Keywords: _ The current name of the fungus should appear here. Introduction: _ L37: The current name of the fungus should be added in parentheses after Candida glabrata. _ L44-45: This sentence seems redundant with the previous one. _ L45: Replace "change… sequence" with "result in amino acid changes in the enzyme sequence". _ L51: The frequency of mutations was not estimated in this study, only their presence. Evaluation of a larger fungal population is required to accurately assess such a frequency. Also, in L178. Materials and methods: _ L55: What does cross-sectional mean? _ L58-71: These two sections could be merged. _ L59: "retrospectively archived" is unnecessary. "were revived" should be replaced with "were retrieved". Where and under which conditions are these isolates deposited/stored? _ L60: Were these MIC results previously published? If so, a reference needs to be provided. If not, the methods used for MIC determination and their results should be placed in the appropriate sections of the manuscript. Also, in L120. _ L61-63: If the resistance to fluconazole was determined in this study, the resistance of the isolates should be placed in the results. Otherwise, a reference must be provided. _ L66: delete "fungal". _ L67: Add "Biggy" after "Yeast". _ L65-71: In this section, only L65-68 are methods, the additional lines are results. _ L63: The meaning of the abbreviation SDD should be provided before using it. _ L79: Replace "performed" with "determined". _ L86: Delete "For extraction". _ L96: Indicate whether a partial or complete sequence was amplified. Delete "cyclic". Also, in L119. _ L98-99: The final concentrations of the reagents in the reaction mix must be provided. _ L99-100: The description implies that 30 cycles of initial denaturation followed by a single temperature cycling step was utilized for amplification. Normally, a single denaturation step followed by 30 cycles of temperature cycling is used. Revise this description. _ L103: The characteristics of the sequencer must be provided. _ L109 and elsewhere: The supplementary figures and the main figures are the same. _ L109: Only one accession number for a reference sequence is mentioned here, but two reference sequences are mentioned in the legend of the phylogenetic tree. Also, the geographic origin, year of collection and other relevant aspects of those isolates should be provided. Why were those two references chosen? _ L110: Why only two sequences are mentioned here since a larger number of sequences were analyzed and are shown in the phylogenetic tree? Instead, the accession numbers and geographic origin of all isolates included in the phylogenetic tree should be added in a column in table 1. Alternatively, add a new table with such an information. Results: _ L123-124: This sentence is redundant with the last one in the previous paragraph. _ L125: Add AKU-2019-408 after "isolate". _ L132: Which strain from Switzerland? Provide its identification code. _ L132-133: This sentence is not clear; better contextualization is required _ L137: Add the full identification code for strain AKU-2029-408. Also, in L176. Strain AKU-2019-408 does not show the same MIC as the others being referred to in this sentence. _ L144: Why were reference ERG11 sequences from C. albicans not included as outgroups in the phylogenetic analysis? _ L145: The current name for C. auris, Candidozyma auris, must be provided. _ L151-153: This sentence needs to be improved for better understanding. _ L159: Explain or better describe the meaning of "whisper… exchange". _ L179: The meaning of WGS must be described before using the abbreviation. _ L182: Add "present" before "study". Tables: _ Table 1: Nothing is mentioned in the text with respect to the information in columns VOR, POS, ITR, AMP and CAS. It indicates that this information is irrelevant to understand the message of the manuscript. If that is the case, the columns should be deleted from the table. Otherwise, add information in the main text to show the relevance. _ Table 1 footnote: L297-300: This description needs to be considerably improved for clarity. _ Figure 1: The origin of the isolates must be added after their identification codes. What does the number on the branches mean? If they are bootstrap values, they are very low, indicating low confidence, which needs to be explained. A bar indicating the meaning of branch lengths is missing. It is advisable to add at least one outgroup sequence (from a different Candida species) to the tree. _ Figure 1 legend: The origin of the reference sequences should be better explained in this legend, or else, in the main text. The meaning of a bar referred to in the previous comment (missing) should also be described.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Thank you for submitting your manuscript to Access Microbiology. The study is interesting for the understanding of regional fuconazol resistance in C. glabrata. However, a major revise is needed before progressing to peer-review. The methods need a more comprehensive description and a division in different subsections for each experiment. The results could be further described (for example, the phylogenetic analysis). The DNA sequences obtained throughout the work need to be made publicly available in a related repository and the respective reference should be cited in the Data availability section. Figure 2 is included in supplementary material needs to be cited in the main text. Figures included in the supplementary material need to be named accordingly (Supplementary Figure S...). Can a mutation occurring in in a non-coding region …
Thank you for submitting your manuscript to Access Microbiology. The study is interesting for the understanding of regional fuconazol resistance in C. glabrata. However, a major revise is needed before progressing to peer-review. The methods need a more comprehensive description and a division in different subsections for each experiment. The results could be further described (for example, the phylogenetic analysis). The DNA sequences obtained throughout the work need to be made publicly available in a related repository and the respective reference should be cited in the Data availability section. Figure 2 is included in supplementary material needs to be cited in the main text. Figures included in the supplementary material need to be named accordingly (Supplementary Figure S...). Can a mutation occurring in in a non-coding region of a gene be synonymous or non-synonymous if that region would not be translated to a protein? The use of English and the grammar need an exhaustive revision (please contact the Editorial Office if you would like to explore English proofing options). Please provide a revised version of the manuscript (including a tracked-changes document) addressing these comments, along with a point-by-point response to them within 2 months.
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