Genotypic Determination of Extended Spectrum β-Lactamases and Carbapenemase Production in Clinical Isolates of Klebsiella pneumoniae in Southwest Nigeria

Round 1

Reviewer 1 Report

The article introduces a topic of great relevance: the study of resistance and its epidemiology. Understanding the global distribution of resistant pathogens is crucial, and the presented work provides valuable data and results for further analysis. However, to fully leverage this information, some aspects of the methodology and results require clarification.

Specifically, it would be beneficial to examine the relationship between the presented genes and their respective resistance profiles. Additionally, the article raises some questions and comments that merit further discussion. Overall, this work contributes to the ongoing efforts to combat resistance, but additional analysis and clarification would enhance its impact.

line 111.- reference method, primer?

line 123: Susceptibility to polymixyn should not be studied by the diffusion method. Refer to laboratory standard. Eg CLSI or EUCAST

Line 113: Antibiotic Susceptibility testing:

 

A basic susceptibility study was carried out. What was the criteria for the selection of antimicrobials? It is suggested to incorporate new compounds or those that are used as an alternative to cbp-resistant klebsiella infections, such as eg. hunting/avi Expand study to caz-avi, tigecycline, fosfomycin. Refer to the official methodologies according to CLSI or EUCAST.

Line 128: what is the difference between the detection of blaCTX-M VERSUS CTX-M-2; M9 and M-15? This analysis is confusing.

line 114: corrects the methology name

Line 163: klebsiella is intrinsically resistant to ampicillin given the chromosomal carriage of SHV.

 

 

Line 170: Why if the other CTX-M could you identify the group, which one is this one?

Line 268: presents resistance gene frequency data which is very interesting. It does not perform an analysis between the presence of certain genes versus the resistance profile versus ST. that analysis is more relevant since the data is present

 

 

 

 

 

 

 

 

it is possible to improve the wording and eliminate some spelling problems.

Author Response

Reviewer 1……Response in Red.

The article introduces a topic of great relevance: the study of resistance and its epidemiology. Understanding the global distribution of resistant pathogens is crucial, and the presented work provides valuable data and results for further analysis. However, to fully leverage this information, some aspects of the methodology and results require clarification.

Specifically, it would be beneficial to examine the relationship between the presented genes and their respective resistance profiles. Additionally, the article raises some questions and comments that merit further discussion. Overall, this work contributes to the ongoing efforts to combat resistance, but additional analysis and clarification would enhance its impact.

line 111.- reference method, primer?

            We used 16s RNA gene

line 123: Susceptibility to polymixyn should not be studied by the diffusion method. Refer to laboratory standard. Eg CLSI or EUCAST

The antibiotic susceptibility testing was performed by the Kirby- Bauer disc diffusion method and broth microdilution method as modified by the Clinical and               Laboratory Standards Institute .

Line 113: Antibiotic Susceptibility testing:

A basic susceptibility study was carried out. What was the criteria for the selection of antimicrobials? It is suggested to incorporate new compounds or those that are used as an alternative to cbp-resistant klebsiella infections, such as eg. hunting/avi Expand study to caz-avi, tigecycline, fosfomycin. Refer to the official methodologies according to CLSI or EUCAST.

Line 128: what is the difference between the detection of blaCTX-M VERSUS CTX-M-2; M9 and M-15? This analysis is confusing.

            Response; CTX-M-2, CTX-M-9 and CTX-M-15  were variants of CTX-M

line 114: corrects the methology name
            Kirby- Bauer Disc Diffusion method and broth microdilution method

Line 163: klebsiella is intrinsically resistant to ampicillin given the chromosomal carriage of SHV.

Response;   Correct. Thank you.

Line 170: Why if the other CTX-M could you identify the group, which one is this one?

            CTX-M-2, CTX-M-9 and CTX-M-15 were variants of CTX-M

(As explained previously in line 128 response. Thank you). CTX-M-type enzymes are a group of class A extended-spectrum β-lactamases (ESBLs) that are rapidly spreading among Enterobacteriaceae worldwide. More that 50 allotypes are known, clustered into six sub-lineages. The CTX-M-encoding genes have been captured from the chromosome of Kluyvera spp.

Line 268: presents resistance gene frequency data which is very interesting. It does not perform an analysis between the presence of certain genes versus the resistance profile versus ST. that analysis is more relevant since the data is present

 Table 7: Analysis of Carbapenem Phenotypes and Carbapenem genes

Antibiotics	Number/% Resistant (Phenotype)	KPC gene (17)		IMP gene (29)		VIM gene (55)
		Number(%)	P value	Number/%	P value	Number/%	P value
Levofloxacin	66(51.6)	2(11.8)	0.505	16(55.2)	0.062	29(52.7)	0.480
Cefoxitin	69(53.9)	4(23.5)	0.447	11(37.9)	0.510	32(58.2)	0.254
Ceftazidime	68(53.1)	1(5.9)	0.274	14(48.3)	0.107	29(52.7)	0.540
Tetracycline	86(67.2)	6(35.2)	0.381	17(58.6)	0.403	37(67.3)	0.570
Aztreonam	70(54.7)	5(29.4)	0.325	12(41.4)	0.406	33(60.0)	0.193
Gentamicin	69(53.9)	4(23.5)	0.447	14(48.3)	0.130	31(56.4)	0.380
Cefepime	57(44.5)	2(11.8)	0.315	8(27.6)	0.395	27(49.1)	0.235
Imipenem	62(48.4)	5(29.4)	0.041*	16(55.2)	0.000*	30(54.5)	0.036*
Amikacin	70(54.7)	4(23.5)	0.486	8(27.6)	0.181	32(58.2)	0.305
Meropenem	55(43.0)	8(47.1)	0.001*	16(55.2)	0.000*	35(63.6)	0.000*
Ofloxacin	69(53.9)	7(41.2)	0.081	12(41.4)	0.361	33(60.0)	0.154
Cephalexin	69(53.9)	3(17.6)	0.553	13(44.8)	0.230	32(58.2)	0.254
Amoxycillin/ Clavulanic acid	68(53.1)	1(5.9)	0.274	6(20.7)	0.076	28(50.9)	0.398
Ciprofloxacin	75(58.6)	7(41.2)	0.209	14(48.3)	0.342	36(65.4)	0.118
Cefuroxime	74(57.8)	3(17.6)	0.359	12(41.4)	0.556	31(56.4)	0.457
Ampicillin	77(60.2)	7(41.2)	0.270	11(37.9)	0.271	34(61.8)	0.441
Oxacillin	79(61.7)	10(58.8)	0.041	15(51.7)	0.377	41(74.5)	0.080
Cefotaxime	66(51.6)	1(5.9)	0.343	12(41.4)	0.273	31(56.4)	0.222
Chloramphenicol	72(56.3)	3(17.6)	0.436	9(31.0)	0.221	28(50.9)	0.190
Ceftriaxone	69(53.9)	4(23.5)	0.447	9(31.0)	0.342	31(56.4)	0.380

*Carbapenem phenotype showed significant relationship with KPC, IMP and VIM

Comments on the Quality of English Language

it is possible to improve the wording and eliminate some spelling problems.: Done

Submission Date

20 April 2023

Date of this review

Author Response File: Author Response.doc

Reviewer 2 Report

The study entitled as “Genotypic Determination of Extended spectrum β-lactamases and Carbapenamase Production in Clinical Isolates of Klebsiella pneumoniae in South-west Nigeria” is interesting. However, it needs improvement and authors need to address the following points:

1.    The manuscript has a lot of syntax error.

2.    The image quality of gels is not acceptable.

3.    The labeling of images are missing/incomplete.

4.    I recommend to make a table indicating the age of patient, sample type, gene found positive and the name of identified bacteria in the sample to summaries the data.

5.    Did authors perform the 16s RNA sequence to identified the bacteria?

6.    What is the rational for selecting only 10 samples for multilocus sequence typing (MLST).

7.    Did authors submit the identified sequence of the bacteria in the public database (such as NCBI)? If not, please submit it and provide the accession number.

 

 

 

Author Response

Reviewer 2…….Response in Red

 

 

Comments and Suggestions for Authors….

The study entitled as “Genotypic Determination of Extended spectrum β-lactamases and Carbapenamase Production in Clinical Isolates of Klebsiella pneumoniae in South-west Nigeria” is interesting. However, it needs improvement and authors need to address the following points:

 

1. The manuscript has a lot of syntax error.

Errors corrected in Green Highlights in body of the work. Thanks for the observation.

 

2. The image quality of gels is not acceptable.:

More graphical input has been added, however the editor may decide to add them as supplemental data. Thanks also for this observation. Grateful.

 

3. The labeling of images are missing/incomplete. DONE. Proper labeling effected.

 

4. I recommend to make a table indicating the age of patient, sample type, gene found positive and the name of identified bacteria in the sample to summaries the data.

 

Unfortunately this is not done since it’s not part of the scope and objective set out to achieve. However, it’s a good observation, which we will look into in our next project.

Thanks for this observation.

5. Did authors perform the 16s RNA sequence to identified the bacteria?

           

            YES

 

6. What is the rational for selecting only 10 samples for multilocus sequence typing (MLST).

 

            10 samples were selected due to financial constraints, this research did not receive   fund from any agency.

And we are from Low Medium Income Resource nation. Thank you.

7. Did authors submit the identified sequence of the bacteria in the public database (such as NCBI)? If not, please submit it and provide the accession number.

 We didn’t but we will look into. Thank you

 

Comments on the Quality of English Language

 

 

Submission Date

20 April 2023

Date of this review

11 May 2023 23:29:02

 

 

 

 

Reviewer 3 Report

The paper by Odewale et al. aims to survey the extent of ESBL and carbapenem resistant Klebsiella pneumoniae in regional hospitals in Nigeria. They use standard established methods of culture based determination, PCR, and multi-locus sequence typing. Papers such as this can be useful in establishing regional rates of antibiotic resistance. This paper would be greatly improved by the following additions and expanding the analysis to provide more context to the rates of resistance given. My suggestions are as follows:

1.       More information is needed as to why and how these particular samples were chosen for analysis. A summary table with an overall breakdown of site of collection (urine, BAL, blood etc.) and other demographic info would be very helpful to start the results section (This is currently Table 3?). This study initially begins with 420 samples- are all of these already confirmed as K. pneumoniae? I ask because in the conclusion, it mentions only 128 samples. This is unclear and the 128 needs to be more fully explained in the results section.

2.       The primers used to confirm these samples as K. pneumoniae (lines 110-112) need to be included in Table 1.

3.       Why were only 10 samples used for MLST analysis? Explanation is needed as to how these 10 samples were chosen and why more were not included. This analysis is of limited utility given this small sample size without more explanation.

4.       All of the figures showing the PCR gels would be better off as Supplemental figures, rathe than part of the paper itself. They are more appropriate as supplemental figures because they serve primarily to show the accuracy of the PCR reaction itself, rather than trends within the data set.

5.       The tables need to be properly set to fit each within 1 page.

6.       The main point of the analysis for this paper is of the relative rates of antibiotic resistance in these samples. Much more analysis and discussion is needed as to how these rates compare to other areas either within Nigeria or in greater Africa. Terms such as High rates of resistance are entirely relative- high compared to what? More discussion should also be included as to the relative availability or carbapenems in this region of Nigeria. If these drugs are readily and easily available, this indicates a different type of issue that one in which this class of drugs is rare. Are these drugs used extensively in the clinics that were the source of these samples?

7.       A discussion and table of how many samples exhibit resistance to multiple of these drugs would greatly enhance the analysis. Especially when paired with genotype information as to the type of beta-lactamase.

While the English is readable, there are numerous typos, examples of poor phrasing, and incomplete sentences in this article. In particular look at the sentence sat lines: 23-25, 63, 65-67, 290-293, 315-317, and 367-369 to improve clarity, fix typos, and add in extra needed information.

Author Response

Reviewer 3……………….Responses in Red

 

Comments and Suggestions for Authors

The paper by Odewale et al. aims to survey the extent of ESBL and carbapenem resistant Klebsiella pneumoniae in regional hospitals in Nigeria. They use standard established methods of culture based determination, PCR, and multi-locus sequence typing. Papers such as this can be useful in establishing regional rates of antibiotic resistance. This paper would be greatly improved by the following additions and expanding the analysis to provide more context to the rates of resistance given. My suggestions are as follows:

1. More information is needed as to why and how these particular samples were chosen for analysis. A summary table with an overall breakdown of site of collection (urine, BAL, blood etc.) and other demographic info would be very helpful to start the results section (This is currently Table 3?). This study initially begins with 420 samples- are all of these already confirmed as K. pneumoniae? I ask because in the conclusion, it mentions only 128 samples. This is unclear and the 128 needs to be more fully explained in the results section.

Two hundred and fifty three bacteria were isolated from 420 clinical samples and these           isolates were identified using Microbact GNB 12E. The bacterial isolates included : Klebsiella pneumoniae 128 (30.5%), Klebsiella oxytoca 41(9.8%),  Citrobacter  diversus 3(0.7%),  Citrobacter freundii 4(0.9%), Escherichia coli 26(6.2%),  Enterobacter agglomerans 14(3.3%), Enterobacter gergoviae 11(2.6%), Acinetobacter baumannii 19(4.5%), Proteus mirabilis 4(0.9%), and Providencia rettgeri 3(0.7%).

 

2. The primers used to confirm these samples as K. pneumoniae (lines 110-112) need to be included in Table 1.

                  It was included. Thank you

3. Why were only 10 samples used for MLST analysis? Explanation is needed as to how these 10 samples were chosen and why more were not included. This analysis is of limited utility given this small sample size without more explanation.

10 samples were selected due to financial constraints, this research did not receive fund from any agency.  Selected samples were multiple drug resistant and carried carbapenem resistant genes.   Three (3) samples were selected from Lagos state because it had the highest prevalence of  K. pneumoniae, followed by Oyo state where two (2) samples were  selected. Two samples were also selected from Osun because samples               were collected from two tertiary hospitals. One sample was selected from each other state

                                                                                                                        

1. All of the figures showing the PCR gels would be better off as Supplemental figures, rather than part of the paper itself. They are more appropriate as supplemental figures because they serve primarily to show the accuracy of the PCR reaction itself, rather than trends within the data set.

Noted. The Editor may which to do that. We have no objection to that. Thanks for the suggestion.

2. The tables need to be properly set to fit each within 1 page.

Done as requested.

6. The main point of the analysis for this paper is of the relative rates of antibiotic resistance in these samples. Much more analysis and discussion is needed as to how these rates compare to other areas either within Nigeria or in greater Africa. Terms such as High rates of resistance are entirely relative- high compared to what? More discussion should also be included as to the relative availability or carbapenems in this region of Nigeria. If these drugs are readily and easily available, this indicates a different type of issue that one in which this class of drugs is rare. Are these drugs used extensively in the clinics that were the source of these samples?

There is increased usage of carbapenems in Nigeria recently and that calls for concern. 

In Africa, the problem of carbapenem-resistant Enterobacteriaceae (CRE) is aggravated by factors such as the high rate of infections, poor diagnostic tools, sub-optimal disease surveillance, and abuse of antibiotics. Besides, the problem of CRE in Africa is actually underreported.

7. A discussion and table of how many samples exhibit resistance to multiple of these drugs would greatly enhance the analysis. Especially when paired with genotype information as to the type of beta-lactamase.

Done: Table 7 included.

 

Comments on the Quality of English Language

While the English is readable, there are numerous typos, examples of poor phrasing, and incomplete sentences in this article. In particular look at the sentence sat lines: 23-25, 63, 65-67, 290-293, 315-317, and 367-369 to improve clarity, fix typos, and add in extra needed information.               DONE

23-25

            Clinical samples were cultured on blood and MacConkey agar and the isolated           bacteria were  identified by Microbact GNB          12E. All K. pneumoniae isolates       were confirmed by Polymerase Chain Reaction (PCR) using 16s rRNA gene.       Antibiotic       susceptibility testing (AST) was done on these isolates and the           PCR    was used for   evaluation of the common ESBL-encoding genes and       carbapenem             resistance genes.

63

            The emergence of Extended Spectrum Beta lactamases (ESBL) producing organism was considered.....

65-67

            The development and selection of multiple drug resistant bacteria such as ESBL         producers have also been attributed to the rise in the use of second and third      generation cephalosporins to K. pneumoniae treat infections

290-293

                The major proportion of  the sample used in this study were urine samples. Hence, the highest number of K. pneumoniae were observed in urine samples and this is in agreement to the finding of         [38]

 

 

315-317

                The 43.8% prevalence of the blaCTX-M gene among our clinical K. pneumoniae isolates is slightly higher                 than 41.67% reported in Port Harcourt two years earlier [46] and also higher than 35.71% in          Sokoto State [35] and the 32%  in Southwestern Nigeria four years ago [45].

367-369

                A  total number of 128 non-duplicate K. pneumoniae were isolated and characterized from       hospitalized patients in southwestern Nigeria.

 

Submission Date

20 April 2023

Date of this review

17 May 2023 23:29:20

 

 

 

 

 

Round 2

Reviewer 1 Report

The description of local ESBL is always an interesting topic. The article aims to describe the epidemiological and molecular characteristics of ESBL and carbapenemases carriage in samples of infections of clinical origin. Although, the article can be improved, the answers provided are satisfactory and allow to advance in the publication of the article. On the other hand, it would be interesting to be able to select some of the strains carrying carbapenemases, or to identify the co-carriage of these genes, and try to perform genomic studies by means of WGS. We invite you to continue in this line of work. 

 

 

Reviewer 3 Report

Comments appropriately addressed. This paper will need the help of a layout editor to fit tables on 1 page etc.