Evaluation of the efficacy of Cephalosporin antibiotics Sold in Kano Nigeria against Some Clinical Bacterial Isolates.

  1. Safiyya Atiku Ibrahim
  2. Bashir Muhammad
  3. Ismail Rabiu
  4. Abdulazeez Muhammed

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Abstract

Introduction: The circulation of substandard antibiotics is believed to aggravate the evil effect of Antibiotic resistance (AR) globally.  AR knows no boundaries and pose a serious threat to existing antibiotics. Gap statement: Sabon Gari market is chosen due to several formal and informal reports of substandard antibiotics. It's a major distribution centre for pharmaceuticals, yet research on its impact and extent of substandard drugs is lacking. Aim: This study aims to evaluate the susceptibility status of some clinical Bacterial isolates (E. coli, K. pneumoniae, S. aureus, S. pneumoniae, P. aeruginosa and Salmonella sp.) to generations of Cephalosporins purchased from drug distributors in Sabon Gari Market Kano. Methodology: Antimicrobial susceptibility pattern of the test isolates was determined by disc diffusion method. Fourier Transform Infrared Spectroscopy (FT-IR) analysis was used to confirm the functional group of the active ingredients of all the antibiotics tested. Molecular identification of resistant gene (CTX-M1) were carried out using PCR. Results: Market survey reveals that Cephalexin 61% (first generation), Cefuroxime 72% (Second generation) and Cefixime 68%, Cefpodoxime 79%, Ceftriaxone 63%, Ceftazidime 70% and Cefotaxime 45% (third generation) and Cefepime 84% (fourth generation) were the most commonly sold Cephalosporins, with different brands and company names.  In all the antibiotics tested, similar activity was observed in the branded antibiotics as compared with the standard antibiotics, with no significant difference observed. Exactly 20% of E. coli and K. pneumoniae were resistant while 80% of S. aureus, S. pneumoniae, Pseudomonas, and Salmonella sp. were susceptible. CTX-M1 resistant gene was identified E. coli and K. pneumoniae which further confirms their resistance to Cefotaxime and ceftriaxone antibiotics. Conclusion: Branded cephalosporins sold in Kano were effective as standard antibiotics with good bioavailability and compliance with the standard formulation of medicine, thus fit for human intake.

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  1. Access Microbiology

    I have issued a decision of major revisions, as whilst most changes are rewriting issues and therefore minor, there are some questions about the classification of the market sold antibiotics as "substandard" and the availability of data to back up that claim. Please respond to each reviewer comment, providing a tracked changes document highlighting the changes made. In addition, please review the titles of tables 3-8. They need changing so that the bacteria are susceptible- "Susceptibility of bacteria X to ........" I think that tables 3-8 would convey their information better as a series of multi-panel graphs. Each table would convert nicely to an 8 bar chart panel. Each bar chart would be an antibiotic, with 10 strains along the X axis (2 bars per strain- SD and PD), zone diameter on the Y axis, with a hashed line to indicate the resistance cut offs. I have attached a sketch of what I think it could look like. This is a suggestion to improve your manuscript, but is not mandatory. I would suggest a minor tweak to the title to remove the word "some".

  2. Access Microbiology

    Comments to Author

    The authors investigate whether cephalosporin antibiotics sold at the Sabon Gari market in Kano, Nigeria, are counterfeit/substandard. This is an important question because, as the authors well explain, substandard antibiotics can be very problematic, leading to treatment failures and sub-optimal concentrations of antibiotics accelerating the emergence of resistance evolution. They first used questionnaires to determine which cephalosporin antibiotics were most commonly sold for each generation (1-4) at the Sabon Gari market. They then selected a panel of clinical isolates of gram-positive and -negative bacteria to test the antibiotic susceptibility of the purchased drugs compared to quality-assured standard drugs. No significant difference was found, indicating that substandard cephalosporins are not circulating in this market. Overall, this is a relevant study, however, I think the manuscript in its current form is not easy to follow and lacks clarity on some occasions. Especially, the results and discussion section could be strengthened by rearranging some sections. Some specific points are listed below with the intention to help the authors improve the manuscript. 1) Line 21: Aim: The aim of the study is slightly confusing. As I understand it is mainly to test if the purchased cephalosporins are substandard (or not) by testing them on a panel of clinical isolates in comparison to standard drugs. And not "to evaluate the susceptibility status" of clinical isolates. Please amend and/or clarify. 2) Lines 28-30: (results abstract) Market survey reveals that Cephalexin 61% (first generation), Cefuroxime 72% (Second generation) and Cefixime 68%, Cefpodoxime 79%, Ceftriaxone 63%, Ceftazidime 70% and Cefotaxime 45% (third generation) and Cefepime 84% (fourth generation) were the most commonly sold Cephalosporins, with different brands and company names. In this context, it's not clear to me what those percentages mean? Please clarify here in the abstract. 3) The key finding that there is no significant difference between drugs purchased in the market versus standard drugs should be mentions in the results paragraph of the abstract. 4) Lines 33-35: Exactly 20% of E. coli and K. pneumoniae were resistant while 80% of S. aureus, S. pneumoniae, Pseudomonas, and Salmonella sp. were susceptible. This sentence needs some clarification. Does that mean S. aureus, S. pneumoniae, Pseudomonas, and Salmonella sp are also 20% resistant (if 80% are susceptible)? 5) Lines 38-40: Conclusion: Branded cephalosporins sold in Kano were effective as standard antibiotics with good bioavailability and compliance with the standard formulation of medicine, thus fit for human intake. Bioavailability or compliance was not assessed as part of this study I thus recommend not to state this as a conclusion. 6) lines 76-78: The primary aim of this study is to evaluate the susceptibility status of certain clinical bacterial isolates to different generations of cephalosporins purchased from drug distributors in Sabon Gari Market, Kano, Nigeria. See comment 1) I think this needs amending or clarification if this was the primary aim. 7) Lines 121-127: Please specify how many of the 100 questionnaires were returned. 8) Lines 136-142: What were the selection criteria for those 10 isolates per species? Were all the isolates from stool? 9) Line 192-200: Please explain how the samples were process before being read on the FT-IR spec. 10) 215-218: Add the temperature and times of the annealing and the elongations step and how many cycles were run. 11) There is no narrative in the results section explaining the tables or figure. The Manuscript would immensely benefit from this to guide the reader though each table and figure. Some parts are in the discussion but I strongly recommend to put this in the results section. 12) Table 3-8, add the meaning of * in the legend. 13) Table 9. Cefotaxime did not match for P. aeruginosa. Please explain and discuss this result. 14) Table 10: Correct P.seumonas to P. aerogunosa 15) In your discussion you argue that some of the difference between standard versus branded drugs could have arisen due to the disc preparation. Could this not be that some are sub-standard? Please expand on this. (Lines 333-342) 16) Line 325: Correct to 60 isolates. 17) Line 412: CTX-M was found in 2 isolates but in figure 1 it shows 4 isolates are positive - please correct. 18) Please do a thorough check of your manuscript for spelling and for consistency. E.g. Gram positive vs gram positive vs gram-positive.

    Please rate the manuscript for methodological rigour

    Satisfactory

    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

  3. Access Microbiology

    Comments to Author

    Language: The authors should thoroughly reread this manuscript to check for grammatical errors. Introduction: The authors have attempted to introduce the topic and justify the essence of this study, but there are several areas of concern that need to be addressed. 1. The authors mentioned that "Cephalosporins are the most widely prescribed class of antibiotics in the world". I think it will be better to say "Cephalosporins are one of the most prescribed classes of antibiotics in the world." 2. Is it only cephalosporins that are being sold in Sabon Gari Market? Generalizing the efficacy of all antibiotics sold in this market based on your findings on only cephalosporins is not sufficient, and therefore suggests limitation. This limitation can be stated or mentioned in your discussion. 3. Are there any previous reports or standardized tests that have been conducted on all the antibiotics sold in this market to justify that they are substandard? 4. There are a lot of repetitions in the introduction section. It is requested to start introduction section by using key words from the title to get it focused directly on topic and provides the background information on the topic of the research article. Show that the present study is important, central, interesting and problematic in some way. 5. Authors should maintain consistency in the terminology by ensuring that "Gram-positive" and "Gram-negative" are written uniformly throughout the manuscript. Materials and Methods: 1. The authors should merge the study area and study site together. There is no difference between a study area and a study site. 2. The authors mentioned that both standard antibiotics and prepared (substandard) antibiotics were used in this study. How did you make the "prepared antibiotics". Consider including the methods or procedures under materials and methods section. 3. In line 216-217, the authors mentioned the PCR steps and their corresponding temperatures. Did the authors actually carry out the annealing step at 94oC? Results: 1. The authors can remove the data for RS and RP from the tables, as these are already covered in another table. 2. While the authors have presented FT-IR analysis for the substandard antibiotics, a comparable analysis for the standard antibiotics was not conducted. This raises concerns about the basis for concluding that cephalosporins from the specified companies are substandard. What components are found in substandard antibiotics that are missing in standard antibiotics? Discussion: 1. This part needs to be rewritten for clarity. Each paragraph should address a particular or separate finding. Also, check the entire section for grammatical corrections and punctuations. Some sentences are too long. Document your findings and compare them with previous reports. 2. In lines 314-317, the authors stated that third and fourth-generation cephalosporins exhibit greater activity against Gram-negative organisms compared to first and second-generation cephalosporins. However, in lines 355-356, they reported that third-generation cephalosporins demonstrated the lowest activity. What could have caused these conflicting observations? 3. There are variants of CTX-M enzymes. Why did author only focus on CTX-M1? Conclusion: This section contains a lot of repetitions of what has been discussed before. The authors should just summarize their findings.

    Please rate the manuscript for methodological rigour

    Good

    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

  4. Version published to 10.1099/acmi.0.000837.v2 on Access Microbiology
  5. Access Microbiology

    Thank you for your submission. The investigation into the efficacy of antibiotics sold at markets is undoubtably an important area of research. Unfortunately, I am not able to send this manuscript to peer review in its current form. I encourage the authors to consider the below suggestions in a revised manuscript. 1. The Methods are lacking in detail at points. There is almost no information provided on where the strains have been isolated from, or on how the biochemical tests were performed. 2. Combining the Results and Discussion into a single section has resulted in a confusing narrative. I strongly encourage the authors to split this section into two. 3. The keys for Tables 3-8 do not match the abbreviations in the tables. These tables, whilst presenting useful content, are also not readable in their current form. I suggest moving them to a single supplementary data sheet/table and presenting efficacy/resistance for all antibiotics and strains as a figure. 4. Please conduct a thorough proofread of the manuscript, paying particular attention to the spelling of species names (for example, in Table 10). 5. The images in Fig 12 are distorted. Please provide new figures that are not stretched or squashed. 6. The arrow is drawn over the ladder which makes it difficult to read the relevant band sides. Please move all labels to the sides or at least clear of the ladder. The primer sequences are not required in the legend (or in the Conclusion section), it is more appropriate to detail those in Methods. 7. The rationale behind the study is not overly clear from the Introduction. I encourage the authors to include the literature that prompted their research, including specifically work that suggests antibiotics sold at such markets are sub-standard. 8. A questionnaire is presented but I am not clear as to the purpose of this or what was found from it. 9. The title suggests that the manuscript is investigating the resistance status of a panel of strains, whereas the manuscript focuses more on testing antibiotic efficacy. Please amend the title so that it is a better representation of the body of work. Please also include the country that the market is in.

  6. Version published to 10.1099/acmi.0.000837.v1 on Access Microbiology