Genotypic characterization of bacterial isolates causing urinary tract infections among adults at Kiambu Level 5 Hospital, Kenya: selected extended-spectrum β-lactamase genes and biofilm formation

  1. Fredrick K. Wanja
  2. Eric O. Omwenga
  3. Caroline W. Ngugi
  4. John N. Maina
  5. John N. Kiiru

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Abstract

The menace of antimicrobial resistance affecting public health is rising globally. Many pathogenic bacteria use mechanisms such as mutations and biofilm formation, significantly reducing the efficacy of antimicrobial agents. In this cross-sectional study, we aimed to determine the prevalence of selected extended-spectrum β-lactamase (ESβL) genes and analyse the biofilm formation abilities of the isolated bacteria causing urinary tract infection among adult patients seeking Medicare at Kiambu Level 5 Hospital, Kenya. The double-disc synergy test was used for phenotypic identification of ESβL-producing isolates, while microtitre plate assays with some modifications were used for the biofilm formation test. Ten isolates were bioassayed for ESβL genes out of 57 bacterial isolates obtained from urine samples. This study found the bla TEM genes to be the most prevalent ESβL type [10/10 (100 %)], followed by bla OXA and bla SHV genes at 4/10 (40 %) and 3/10 (30 %), respectively. In addition, co-carriage of bla TEM and bla SHV was 50 % lower than that of bla TEM +bla OXA genes at 66.7 % among Escherichia coli isolates studied. Biofilm formation was positive in 36/57 (63.2 %) of the isolates tested, with most being Gram-negative [25/36 (69.4 %)]. Escherichia coli [15/36 (41.7 %)], Klebsiella species [7/36 (19.4 %)] and Staphylococcus aureus [7/36 (19.4 %)] were the dominant biofilm formers. However, there was no significant difference in biofilm formation among all tested isolates, with all isolates recording P -values >0.05. In light of these findings, biofilm formation potential coupled with antimicrobial resistance genes in urinary tract infection isolates may lead to difficult-to-treat infections.

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  1. Version published to 10.1099/acmi.0.000632.v4 on Access Microbiology
  3. Version published to 10.1099/acmi.0.000632.v3 on Access Microbiology
  5. Access Microbiology

    Comments to Author

    Most comments from previous review round were corrected but below are minor suggestions: 1. The revised manuscript still has not defined "biofilm former" versus "biofilm non-former". These isolates might have been tested in previous studies, but here still need to provide clear definition. Otherwise, it is difficult to understand Table 3. 2. Figure 1. Gel images are better but still can be improved. More bands in DNA markers need to be labeled, especially around target bands. In the figure or figure legend, each lane needs to be labeled to indicate which lane represents which sample. In addition, it is unclear what is the meaning of "500bp" in the figure legend or the text? There is no band around 500bp in these three gel images. 3. Typo: Lane 41, it would be "beta-lactam" or "β-lactam", not βeta-lactam.

    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?

    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

  6. Version published to 10.1099/acmi.0.000632.v2 on Access Microbiology
  8. Access Microbiology

    Comments to Author

    In the manuscript, the authors isolated bacteria from patient urine samples, tested antibiotic resistance, conducted genotyping of selected antibiotics, as well as evaluated biofilm-forming capabilities of certain bacterial strains and anti-biofilm capabilities of selected antibiotics. Studies with clinical isolates are crucial to understand true status of antibiotic resistance and this study showed good merits and efforts doing so. However, this manuscript has quite a few areas to improve, in particular seemingly two isolated parts of antibiotic gene-genotyping and later anti-biofilm testing. Specific comments are listed below: 1. Biofilm is a lifestyle of bacteria, and typically is not associated with genetic changes. Therefore, Biofilm may exhibit more tolerance against biocides, but it is not resistance. Resistance typically refers to genetic changes that allow bacteria to resist antibiotic actions. 2. As the manuscript focuses on beta-lactam antibiotics, it would be helpful for readers if the authors explained more about those antibiotics, especially current clinical applications and differences of antibiotics selected in the study. 3. Is more demography information of those patients available? Is there any association between antibiotic resistance to patient disease, symptoms, received antibiotic treatments? 4. Electrophoresis images in Figure 1 need to be reworked to meet publication quality and layout. 5. Please clarify in the text of the definition and criteria determining whether an isolate is biofilm-former or not? 6. In the antibiotic anti-biofilm tests, what are those selected strains? Are those strains with four-digit numbers isolated selected from disk tests, or from a previous study? If these strains were from a previous study, clinical isolates studied in this manuscript need to be tested instead. If these strains were from the same study, please unify naming strategy of bacterial strains, show specific antibiotic resistance results in disk assays and discuss potential difference in disk assays and biofilm assays. 7. Describe in more detail how and when the antibiotics were added in the antibiofilm tests. 8. It is still puzzling that decreased antibiofilm efficiency with increased antibiotic concentrations, in Figure 4 B, E, F, G, H. Although the authors discussed this in Discussion, did authors see bacterial aggregation during experiments? Are these tests repeatable? Discuss clinical doses of these antibiotics relative to this biofilm assay. 9. The writing is generally in good quality, but there occasionally appear typos, such as singular vs plural versions of nouns, and full names of a term need to be described when it first appears. For instance, bacterial species names in Abstract and Introduction. Also recommend authors to go through the manuscript again, perfect writing and correct other typos.

    Please rate the manuscript for methodological rigour

    Satisfactory

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

    Good

    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

  9. Access Microbiology

    Comments to Author

    I would like to commend the authors on the work presented in this manuscript. However, some remarks need to be addressed: General comments: - Only present information about the data investigated in this manuscript, especially in the results. - The introduction is unreasonably long, please remove repetitive and unnecessary information. - Always include the number besides the percentage, e.g. n(%) or % (n), especially in the discussion section. - The discussion needs to be more concise, focused, and direct to the point. I would suggest using subheadings. Specific comments: - L39: change "becoming fewer" to less effective. - L41: "extra-drug…" please change it to either extensively drug resistant or extended drug resistance. - L41-42: "soon reaching an antibiotic-free world on the horizon" that is not scientifically appropriate, please remove or re-phrase. - L44: add reference. - L46-48: Please re-phrase, the meaning is lost. - L55-62: The is an abundance in reports from Arican countries, could you include examples from Africa? or nearby countries. - L73: "even though" I would suggest changing it to 'in fact'. - L74: biofilm-producing bacteria. - L79: "genetic diversity" maybe change it to gene expression alterations. - L80-82: Please re-phrase. - L116: Please mention the manufacturer. - Tables: Change species to sp. - Figure 1: remove the unnecessary zeros.

    Please rate the manuscript for methodological rigour

    Very good

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

    Satisfactory

    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

  10. Access Microbiology

    Comments to Author

    1. Methodology: Method is integrated , many bacterial isolates and they are not getting enough information for each in molecular study and isolation method DNA template for the PCR test using the boiling method (should be more clarified) 2. Presentation of result s: integrated results for molecular study of bacteria, needs to be simple and more clear. 3. Literature analysis or discussion: very Long literature review 4.conclusion need to be more matching with title and goal of the study

    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?

    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

  11. Version published to 10.1099/acmi.0.000632.v1 on Access Microbiology