Genotypic Detection of Antibiotic-Resistant Genes in Emerging Bacterial Pathogens From Neonatal Sepsis Patients in Lagos

  1. Helen Opeoluwa Adegboyega
  2. Taiwo Abayomi Banjo
  3. Anotu Mopelola Deji-Agboola
  4. Bamidele Abiodun Iwalokun
  5. Wasiu Bamidele Mutiu
  6. Olubunmi Adetokunbo Osinupebi
  7. Tessie Owolabi Shorunmu
  8. Farouk Adedeji Oladoja
  9. Oluwabunmi Motunrayo Fatungase
  10. Muinat Moronke Adeyanju
  11. Raheem Akinwunmi Akindele
  12. Ponle Bamidele Fakunle
  13. Ayobami Babatunde
  14. Olusola Ajayi
  15. Olufemi Onadeko
  16. Abimbola Adeola Akintunde Oyelekan
  17. Solomon Olubodunrin Ariyibi
  18. Kolawole Sunday Oritogun
  19. Emmanuel Sunday Omirin

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Abstract

High-risk neonatal sepsis resulting in up to four million infants’ mortality and organ damages during the first 28 days of life predominates in low-income countries. We investigated genotypic antibiotic-resistant genes in emerging bacterial pathogens from newborn sepsis cases in Lagos.

A cross-sectional study of 294 at-risk hospitalised newborns in prominent paediatrics hospitals throughout three senatorial districts of Lagos utilised the socio-demographic data of their mothers and informed consents. We aseptically collected approximately 3 mL of venous blood from neonates, subsequently isolated and identified the associated bacteria using agar culture on Mueller Hinton agar. To evaluate the antibiotic resistance genes, we used Kirby-Bauer disc diffusion technique; MICROBACT TM Identification System and molecular techniques including PCR, RAPD-PCR, sequencing and docking.

From 294 patient samples, 110 (37.4%) were cultured positive, with men (47%), females (53%), ages ≤ 72 hours (71.8%), and ages > 72 hours (28.1%). Klebsiella pneumoniae, K. oxytoca, Enterobacter gergoviae, Serratia rubidiae, Escherichia coli, and Cronobacter sakazakii were the involved Gram- negative bacteria, whereas Coagulase-negative Staphylococci , Staphylococcus aureus , and Enterococcus faecalis were the involved Gram-positive bacteria. Fourteen (12.7%) positive bacteria were resistant to only three antibiotic classes, whereas 73 (66.4%) were resistant to over three antibiotics. Antibiotic- resistant genes bla-CTX, bla-SHV, and bla-TEM were found in Serratia rubidiae and Enterobacter gergoviae , unusual aetiology of newborn sepsis. Molecular docking proved Meropenem’s antibacterial effectiveness.

The identified antimicrobial-resistant genes are crucial for improved patient management, as high- resistance strains of ampicillin, gentamicin, and cefotaxime remain ineffective against bacterial sepsis, leaving Meropenem as the sole feasible therapeutic option.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/15873809.

    Brief summary of the study

    The cross-sectional study isolated bacteria causing neo-natal sepsis among newborns hospitalized in several hospitals in Lagos, Nigeria, over a period of 3 years. It then determined antimicrobial resistance patterns among the isolates, used molecular detection to identify antibiotic-resistant genes and used molecular docking to evaluate antibiotic effectiveness as one of the approaches to combat resistance. Identification of the bacterial isolates is supposedly done also but it is not clear from the methods and results sections.

    The authors identified the prevalent pathogens, their antimicrobial resistance patterns as well as emerging resistant strains. The study also identifies effective antimicrobials using molecular docking.

    Major comments 

    • The presentation of the paper from methods, results and discussion section need a serious revision to improve clarity and provide a logical flow. In addition, important details are omitted in the methods section, presentation of the results has several issues, and the discussion needs to provide a stronger literature comparison. We provide detailed comments on each section below

    • In the methodology, the combination of conventional methods (agar culture, Kirby-Bauer disc diffusion, MICROBACTTM Identification System) and molecular techniques (PCR, RAPD-PCR, sequencing, docking) provided a comprehensive approach to bacterial identification and characterization of antibiotic resistance, however, the study could be strengthened by discussing the limitations of the cross-sectional study design used in this study in assessing trends over time and the potential for selection bias in the choice of hospitals and patients.

    • For the statistical analyses, providing more details on the specific parameters, how the stated statistical tools and tests were used would enhance understanding and reproducibility.

    Minor comments

    • The article needs keen proofreading and editing

    • Several areas need clarification such as:

      • This statements from the abstract: "From 294 patient samples, 110 (37.4%) were cultured positive, with men (47%), females (53%), ages ≤ 72 hours (71.8%), and ages > 72 hours (28.1%)", "Molecular docking proved Meropenem's antibacterial effectiveness."

      • Writing numbers as numerals, for example "One hundred and ten" in the results section should be written as 110

    Comments on reporting - information on the statistical analyses or availability of data.

    •  Several statistical tools are mentioned but no details are given on how they were used

    • Data, especially sequencing data and other supplementary information such as the questionnaires used are missing

    • A statement at the end of the manuscript simply states "All data generated or analyzed during this study are included in this manuscript" but that is not the case, for example, genomic data is supposed to be deposited in public repositories like DDBJ, ENA or NCBI

    Conflicts of interest of reviewers

    None declared

    Ethical issues

    • Provide a reference number for the ethical clearance in addition to stating "Ethical approval and permission were obtained"

    Comments by section

    Abstract

    Abstract statements should be results-driven and paired with the techniques used, rather than the opposite. For example, instead of "we evaluated antibiotic resistance genes using the Kirby-Bauer disk diffusion method", the statement could be written as the Kirby-Bauer disk diffusion method showed that 78% of the bacterial isolates were highly resistant to drugs x and y OR High resistance levels to drug x and drug y were observed in 78% of the tested bacterial isolates using the Kirby-Bauer disk diffusion method.

    Methods

    • The methods section needs to be rewritten for clarity ang logical flow. For example, the statement "Gram staining techniques were carried out on all isolated bacterial agents then identified by Agar culturing, and Mueller Hinton agar" is confusing because agar culture is not an identification method, at least without much more detail. Similar statements that are unclear are present throughout the methods section

    • Please identify the abbreviation at first use, for example Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR)

    • Clarify whether they were detecting "the presence of the ESBL enzyme in the bacteria" or detecting genes that code for ESBL enzymes, not the enzymes themselves. We think it is the latter is based on the method written

    • Include information on how data generation proceeded from the PCR products to library preparation and finally to sequencing for reproducibility purposes.

    • Genomic data information for the resistance genes and bacterial identification and analysis details are missing

    • The authors mention using 4 different software programs but do not specify the role of each program or how it was used for which data type. For example, Epi Info is a suite of public domain computer programs for public health professionals used for rapid questionnaire design, data entry and validation, data analysis, including mapping and graphing, and creation of reports WHILE Prism is useful for analysis of quantitative and categorical data and can create many graphs that Excel simply cannot, including scatter graphs, box-and-whiskers plots, and survival curves. It is not clear how these tools were used in the study/paper

    Results

    • Tables and data presentation in tables

      • It is not clear what the benefit of the mean value is in Table 2. Also, a legend to the table should be added to illustrate the meaning of the signs (stars). In addition, it is not clear what the labels mean

      • The data presented in Table 3 is not clear (resolution needs enhancement), and the headings of the 10 columns starting from the 3rd column need to be identified

      • Tables 4-6 are not cited in the article, and the data presented in them need clarification

      • The information presented in Table 5 is already presented in the methods section (duplication) and it should not be in the results section at all

      • The abbreviations in Table 6 need to be defined, and the significance of the data presented in this table needs to be explained

      • Table 7 shows BLAST results but the genes used for BLAST identification were not appropriate, as using the AMR gene would lead to false results because most of these genes are found in more than one species of bacteria. The proper way to use these sequenced genes may be to identify mutations in the identified genes (SNPs, MNPs). For isolates identification, they could have used the 16s gene or genes used for multi-locus sequence typing.

      • Tables 8-9 are not cited in the article, and the significance of the illustrated data needs to be identified

    • Figures

      • The figures 2-3 are not cited in the article

      • It is not clear what abbreviations 6YD9 and 1SHV in the captions of figure 2 and 3, respectively, mean. They were previously mentioned and not presented in the abbreviation section

    • Some parts of the methods section could be moved to the discussion section, such as this part: "Sequenced Enterobacter gergoviae revealed Desemzia incerta, Klebsiella variicola, Enterobacter asburiae, Escherichia coli, and Pluralibacter gergovaie, and necessitated reconsideration of new antibiotics regime–Piperacillin-Tazobactam, Ciprofloxacin, Meropenem and/or Imipenem (11)); Amoxicillin/Clavulanate (12–14). Meanwhile, the new sequence identities of Serratia rubidiae were susceptible to the following antibiotic regimes: Ciprofloxacin and Sulfamethoxazole (10), Cefepime (15) Piperacillin- Piperacillin-tazobactam, Cefepime (16–15)"

    Discussion

    • In the methods and abstract, it was mentioned that the participants are at-risk neonates, as the term "at-risk newborn" describes a neonate with an increased risk of morbidity and/or mortality but who is currently maintaining homeostasis and does not require a special or intensive care level of monitoring or medical intervention. This includes a variety of cases, not just sepsis. Please revise doi: 10.9745/GHSP-D-22-00099. As presented in the results, only 110 participants were proven to have sepsis. Please be specific about this term. It is recommended to add clear inclusion and exclusion criteria for the participants.

    • How was this association in the statement "Multiple risk factors associated with NS in this cohort (Table 3)" confirmed? No comparisons were performed in Table 3; all the data in the paragraph were illustrated in Table 1, and the comparisons in Table 1 were insignificant (p˃0.05).

    • The statement "CTX-M1, being one of the most widely disseminated ESBL genes globally, has been documented as the predominant variant in sub-Saharan Africa" need references

    • In the statement "The co-expression of multiple β-lactamase genes within the same isolate further underscores the complexity of resistance mechanisms and the growing trend observed in contemporary studies", the appropriate term could be detection; however, the manner in which the study was conducted cannot ascertain whether the detected genes were expressed. Gene detection does not always indicate that a gene is expressed.

    • A large part of the discussion section is dedicated to description of the results of molecular docking; however, interpretation of the results and comparison with other studies is not presented

    References

    There are several issues with the references, from wrong links to wrong years, as outlined below.

    • The link of the reference "Clinical and Laboratory Standards Institute (CLSI) (2013) Guidelines—Performance Standards for Antimicrobial Susceptibility Testing. CLSI Document M100" shows that it is published 2020 not 2013

    • The link refers to another article "Adegboyega, H. O., Banjo, T. A., Deji-Agboola, A. M., Iwalokun, B., Mutiu, W. B., Osinupebi, O. A., ... & Omirin, E. S. (2025). GENOTYPIC DETECTION OF ANTIBIOTIC-RESISTANT GENES IN EMERGING BACTERIAL PATHOGENS FROM NEONATAL SEPSIS PATIENTS IN LAGOS. bioRxiv, 2025-06" and NOT "Sharma Babita. (2023). Beta-lactamase test: principle, types, procedure, results. https://microbenotes.com/beta-β-lactamase-test-objectives-principle-procedure-and-results"

    • The year in reference "Clinical and Laboratory Standards Institute (2012). Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Second Informational Supplement. CLSI Document M 100–S22. Clinical and Laboratory Standards Institute, Wayne" is 2011 not 2012

    • The link of article "Kramer, M. F; & Coen, D.M. (2001). Enzymatic amplification of DNA by PCR: standard procedures and optimization. Curr Protoc Toxicol May; Appendix 3:A.3C.1-14. doi: 10.1002/0471140856.txa03cs03pmid:. PMID: 20972963" was not found. The correct citation is Kramer, M. F., & Coen, D. M. (2006). Enzymatic amplification of DNA by PCR: standard procedures and optimization. Current protocols in cytometry, Appendix 3.  https://doi.org/10.1002/0471142956.cya03ks37. PMID: 18770830

    Competing interests

    The authors declare that they have no competing interests.

  2. Version published to 10.1101/2025.06.02.657359 on bioRxiv