<xhtml:span xmlns:xhtml="http://www.w3.org/1999/xhtml" xml:lang="en">Detection of <em>aac3IIc,</em> <em>aac(6)Ib</em> and <em>armA</em> gene encoding aminoglycoside resistance in <em>Klebsiella pneu (2025)

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Abstract

BackgroundKlebsiella pneumoniae is the second most frequently isolated bacterium in medical bacteriology laboratory after Escherichia coli. It can be responsible for many infections including urinary tract infections and pneumonia. The treatment of these infections is often prolonged because of the resistance of this bacterium to antibiotics. The production of aminoglycoside-modifying enzymes and the production of RNA methylases confer resistance to aminoglycosides. In Burkina Faso, studies on bacterial resistance to aminoglycosides, an antibiotic regularly prescribed and consumed by patients, are limited, hence the purpose of this study was to determine the prevalence of aminoglycoside resistance genes in K. pneumoniae isolated from urine and pus samples.Methods and FindingsA total of 150 Klebsiella strains from pus and urine cultures were collected from October 2018 to June 2019 in two hospitals in Ouagadougou and were included in this study. After plating on Muller Hinton (MH) medium to obtain pure colonies, antibiotic susceptibility testing was performed with aminoglycosides, β-lactams, fluoroquinolones and sulfonamides. PCR testing for the phoE gene allowed genotypic identification of Klebsiella pneumoniae. PCRs were then performed on strains with at least one aminoglycoside resistance for identification of the aac(6')-Ib, aac(3)-IIc, and armA resistance genes.Antibiotic susceptibility testing showed that 38.67% (58/150) of the strains were resistant to Tobramycin, 28.67% (43/150) to Gentamicin, 10.00% (15/150) to Netilmicin, 8.67% (13/150) to Kanamycin, 4.67% (7/150) to Amikacin and 0.67% (1/150) to Neomycin. Of the 63 strains (42.66%) with at least one aminoglycoside resistance, the resistance genes, aac(3)-IIc and aac(6')-Ib were detected in 49 strains (77.77%) and 39 strains (61.90%), respectively; and 34 strains (53.97%) had both genes. Among the 16S rRNA genes, the armA gene was present in 19.04% of the strains (12/63).ConclusionAminoglycoside resistance in K. pneumoniae strains in this study is primarily due to aac(3)-IIc and aac(6')-Ib acetyltransferases. Although armA RNA methylases genes are less frequent, increased surveillance is necessary due to the high level of aminoglycoside resistance conferred by this gene.

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  1. <xhtml:span xmlns:xhtml="http://www.w3.org/1999/xhtml" xml:lang="en">Detection of <em>aac3IIc,</em> <em>aac(6)Ib</em> and <em>armA</em> gene encoding aminoglycoside resistance in <em>Klebsiella pneumoniae</em> in Burkina Faso.</xhtml:span> (2)

    Access Microbiology

    Thank you for responding to the reviewer comments, however after carefully reading through the manuscript after this round of revisions I do not think it is in a publishable state as it is. I have several comments which I have listed below;1) A lack of key controls within the PCRs. This was flagged by reviewer 1 and hasn't been rectified within the revisions. For the phoE PCR used to identify K. pneumoniae there are no highly related strains included to demonstrate the specificity of these primers for K. pneumoniae. For the pehX PCR to identify K. oxytoca the manuscript states that no PCR positive results were obtained, but no details of a positive control are included to demonstrate that the PCR primers and conditions worked. Therefore it is not possible to know if none of the strains were K. oxytoca or if the PCR didn't work. For …

    Thank you for responding to the reviewer comments, however after carefully reading through the manuscript after this round of revisions I do not think it is in a publishable state as it is. I have several comments which I have listed below;1) A lack of key controls within the PCRs. This was flagged by reviewer 1 and hasn't been rectified within the revisions. For the phoE PCR used to identify K. pneumoniae there are no highly related strains included to demonstrate the specificity of these primers for K. pneumoniae. For the pehX PCR to identify K. oxytoca the manuscript states that no PCR positive results were obtained, but no details of a positive control are included to demonstrate that the PCR primers and conditions worked. Therefore it is not possible to know if none of the strains were K. oxytoca or if the PCR didn't work. For the AMR gene PCRs you should include known +ve control (which you have for the armA PCR) and also a known sensitive strain which doesn't contain the target genes, as well as a no DNA H2O negative, which you have included in figure 1 but not 2.2) The manuscript states that 150 K. pneumoniae strains were isolated, but PCR positive results are only provided for 12. Which strains are they? The gel image doesn't add anything to the manuscript without the controls demonstrating the PCR is specific for K. pneumoniae. I would therefore simply state in the manuscript that all 150 strains isolated tested PCR positive for the phoE gene and remove this gel image. The gel image that should be included is one testing the primers on a bank of Klebsiella species to show the primer specificity for K. pneumoniae.3) AMR gene PCR results follow, but no gel image is provided for the multiplexed aac3(IIC) and aac(6')-Ib PCR. This needs to be included.4) The gel image for the armA PCR has been redone for this revision. It now includes more samples than the first version (12 samples are now 18), with only 7 positive for the armA gene. However, 12 armA positive samples are described in the table- which strains are represented in the armA PCR gel?There is a lack of continuity between the 150 strains isolated and those tested in each gel image. For clarity it would be useful for to include an extended version of table 5 showing all the AMR data for all 150 strains, and then the gel images need details of which strains are in which lanes of the gels.5) table 4 is missing data that was in the original version. What was the breakdown of pus and urine samples? What was the breakdown of samples from each age group? The ESBL data has been deleted with no care for how that has left the table. Is this table useful? What do the odds ratios refer to?Overall, I think the manuscript is quite confused and would benefit from some clarity about what its focus is. The title and introduction talks about aminoglycoside resistance, but data presented in table 5 shows B-lactam, fluoroquinolone, and sulfonamide resistance. I think including this data is good but the focus of the manuscript should be characterisation of AMR profiles of Klebsiella strains isolated from your study sites, with further scrutiny of the aminoglycoside resistance genes (with justification as to why you want to investigate aminoglycoside resistance specifically). If it were my manuscript I would expand table 5 to include all 150 strains and their AMR susceptibility data, and make this the leading piece of data in the main body of the text. Then go on to do the aminoglycoside resistance gene PCRs (including the AMR gene gel images for all 63 K. pneumoniae strains resistant to at least one aminoglycoside antibiotic) and state why I am interested in these specific genes.For these reasons I have chosen to remove this manuscript from the peer review process as it requires the PCRs to be carried out in a more rigorous manner (which was asked for in the first round of peer review and wasn't addressed) and the manuscript would benefit from a rewrite to start more broad to include all of your AMR data before focusing down on the aminoglycoside data, resulting in a different manuscript. I would advise you to make these changes and resubmit as a new manuscript.

  2. <xhtml:span xmlns:xhtml="http://www.w3.org/1999/xhtml" xml:lang="en">Detection of <em>aac3IIc,</em> <em>aac(6)Ib</em> and <em>armA</em> gene encoding aminoglycoside resistance in <em>Klebsiella pneumoniae</em> in Burkina Faso.</xhtml:span> (4)

    Access Microbiology

    Please carefully consider the comments made by both reviewers, especially comments about the lack of appropriate negative controls in the PCR identification of K. pneumoniae, and the presence of bands in the "negative samples" in the armA PCR.Please also check the Fig.2 Y axis title for a typo, and change the maximum value to 100% not 120%.Please disregard the following comment from reviewer 2- "Genomic DNA extraction Page 6 Line 109-118 had better be deleted. Reference 17 gave the detailed description." Please do not delete this detail. Methodology details given in a manuscript, rather than referencing other publications, is more useful for our readers.

  3. <xhtml:span xmlns:xhtml="http://www.w3.org/1999/xhtml" xml:lang="en">Detection of <em>aac3IIc,</em> <em>aac(6)Ib</em> and <em>armA</em> gene encoding aminoglycoside resistance in <em>Klebsiella pneumoniae</em> in Burkina Faso.</xhtml:span> (5)

    Access Microbiology

    Comments to Author

    1. Methodological rigour, reproducibility and availability of underlying data1)Page 2 Line28 antibiotic susceptibility testing inclide fluoroquinolones, but no fluoroquinolones found in the part of Methods and Findings.2)The purpose of this study was to determine the prevalence of aminoglycoside resistance genes. Please give the reseaon why another common RNA methylase resistance gene, rmtB, was not included in the study? 3)Why DNA sequencing was not given to some positive PCR product to identify the target genes?4) ESBL-producing strains should be defined definitly. 2. Presentation of results1)Page 3 Line 37, '16S rRNA genes' should be changed.2)Page 10 Line 161-167, β-lactamase production was not defined in the methods. 3)Page 11, Table V, Resistance genes, what BLSE indicated. 3. How the …

    Comments to Author

    1. Methodological rigour, reproducibility and availability of underlying data1)Page 2 Line28 antibiotic susceptibility testing inclide fluoroquinolones, but no fluoroquinolones found in the part of Methods and Findings.2)The purpose of this study was to determine the prevalence of aminoglycoside resistance genes. Please give the reseaon why another common RNA methylase resistance gene, rmtB, was not included in the study? 3)Why DNA sequencing was not given to some positive PCR product to identify the target genes?4) ESBL-producing strains should be defined definitly. 2. Presentation of results1)Page 3 Line 37, '16S rRNA genes' should be changed.2)Page 10 Line 161-167, β-lactamase production was not defined in the methods. 3)Page 11, Table V, Resistance genes, what BLSE indicated. 3. How the style and organization of the paper communicates and represents key findings1)Genomic DNA extraction Page 6 Line 109-118 had better be deleted. Reference 17 gave the detailed description. 2)Page 5 Line 92-97, the sort order of the antibiotic discs tested should be in accordance with that in the abstract. 4. Literature analysis or discussion1)Page 4 Line 61-70, there are several mechanisms of aminoglycoside resistance, …Page 4 Line 73-74, Little is known about the mechanisims of Klebsiella resistance to aminoglycosides. It seems to be self-contradiction. Why the authors selected only three target genes associated with aminoglycoside resistance.2)It was suggested that the first paragraph in the discussion should give a summarizing statements. 3)Page 16, Line 269, This prevalence is lower to that obtained in our study.4)Page 16, Line 274, 'The percentage of the prevalence of ' was duplicated 5. Any other relevant comments1) The language should be modified further. 2) The references should be updated

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    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

  4. <xhtml:span xmlns:xhtml="http://www.w3.org/1999/xhtml" xml:lang="en">Detection of <em>aac3IIc,</em> <em>aac(6)Ib</em> and <em>armA</em> gene encoding aminoglycoside resistance in <em>Klebsiella pneumoniae</em> in Burkina Faso.</xhtml:span> (6)

    Access Microbiology

    Comments to Author

    Summary: Klebsiella pneumoniae (Kp) is a notorious multi-drug resistant (MDR) pathogen, which makes treatments challenging. This study aims to understand the potential resistance mechanism, especially the aminoglycoside resistance in Kp isolated from hospitals in Burkina Faso. There needs to be significant changes to the details provided in the manuscript, including proper controls for the various PCR presented and better rationale provided. Below I outline some of my major concerns regarding clarity and organization of this review:(1)In the introduction (and title of the manuscript), it is not clear why the authors focused on these 3 aminoglycoside resistance genes and not others (e.g. ant(3")-I, aph(3′)-II, and ant(2")-I). The introduction needs to provide more background on what is currently …

    Comments to Author

    Summary: Klebsiella pneumoniae (Kp) is a notorious multi-drug resistant (MDR) pathogen, which makes treatments challenging. This study aims to understand the potential resistance mechanism, especially the aminoglycoside resistance in Kp isolated from hospitals in Burkina Faso. There needs to be significant changes to the details provided in the manuscript, including proper controls for the various PCR presented and better rationale provided. Below I outline some of my major concerns regarding clarity and organization of this review:(1)In the introduction (and title of the manuscript), it is not clear why the authors focused on these 3 aminoglycoside resistance genes and not others (e.g. ant(3")-I, aph(3′)-II, and ant(2")-I). The introduction needs to provide more background on what is currently known about the different resistance genes for different aminoglycosides, so that it makes it clearer why we are looking at these three different resistance genes. I would recommend providing additional rationale.(2)Along the same line, why were ESBL testing done if the authors are specifically interested in aminoglycoside resistance? Are there co-occurrence of ESBL and aminoglycoside resistance? If so, need to provide additional statistics to demonstrate this(3)The Methods section is severely lacking in details. Further information is needed:a.Top of page 6, age group of the patient cohort is listed as 0-90 years which is a huge range. Can you provide mean/median age, as well as the interquartile range so we understand the cohort a bit betterb.How are Klebsiella strains isolated? Is this with McConkey and Simmons? Need this as the assumption that the characterization is all Kp isolatesc.The agar media disc diffusion - what is the agar media used here? Briefly describe the method and not just reference EUCASTd.In the methods section on "champagne cork" synergy test - need to provide reference citation.e.What is the rationale of testing lots of different classes of antibiotics?(4)One major methodological issue I have with this work is how were Klebsiella pneumoniae isolated and their species identify confirmed? Specifically, no method was provided for how Kp were isolated in the clinic (beyond alluding to standard clinical microbiology method). Then the genomic DNA extraction is done using 8-10 bacterial colonies of similar morphology, which implied it could've been a mixture of strains/species (with different resistance profile!). Then Kp species identities were determined by phoE PCR, without proper controls provided. The negative control (Fig 1) only included a no-DNA PCR control, but did not include other Enterobacterciae or Klebsiella species (e.g. oxytoca) to show specificity of the phoE PCR, so it's hard to know whether the PCR conditions and primers truly only identify Kp. Lastly in Fig 1, the arrows blocks the negative control, so can't even be certain that there is not a faint band there.(5)In Figure 3, which looks at the identification of armA gene. The authors considered some of the lanes to contain negative strains, but there is clearly a faint band in lanes 4, 7, 10, 13, and even possibly 9. Which makes one concerned about the resistance gene presence/ absence based on PCR. Similar to my point above, no true negative controls were provi

    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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&lt;xhtml:span xmlns:xhtml="http://www.w3.org/1999/xhtml" xml:lang="en"&gt;Detection of &lt;em&gt;aac3IIc,&lt;/em&gt; &lt;em&gt;aac(6)Ib&lt;/em&gt; and &lt;em&gt;armA&lt;/em&gt; gene&amp;#160;encoding aminoglycoside resistance in &lt;em&gt;Klebsiella pneu (2025)
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