Are non-lactose-fermenting Escherichia coli important diarrhoeal pathogens in children and adults?
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Introduction. Diarrhoeagenic Escherichia coli (DEC) remains one of the major causes of acute diarrhoea episodes in developing countries. The percentage of acute diarrhoea cases caused by DEC is 30–40 % in these countries. Approximately 10% of E. coli isolates obtained from stool specimens have been reported to be non-lactose-fermenting (NLF). The available literature is sparse regarding the pathogenicity of NLF E. coli causing infectious diarrhoea.
Aim. We aimed to elucidate the importance of NLF E. coli in causing diarrhoea in both adults and children by detecting various DEC pathotypes among NLF E. coli in stool samples taken from gastroenteritis cases.
Material and Methods. A total of 376 NLF E. coli isolates from 3110 stool samples from diarrhoea/gastroenteritis patients were included in the study. Up to three NLF colonies that were not confirmed as Vibrio cholerae , Aeromonas spp., Salmonella spp. or Shigella spp., but were identified as E. coli using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), were carefully picked up from each MacConkey agar plate and then meticulously streaked onto freshly prepared, sterilized nutrient agar plates, and biochemical reactions were conducted. Multiplex PCR was conducted for the EAEC, EPEC, ETEC and EHEC pathotypes and PCR for the ipaH gene was conducted for EIEC. The disc diffusion method was used for antibiotic sensitivity testing.
Results. Using multiplex PCR and ipaH PCR, a total of 63 pathotypes of DEC were obtained, with EAEC being the most predominant ( n =31) followed by EIEC ( n =22), EPEC ( n =8) and ETEC ( n =2). To further differentiate EIEC from Shigella , additional biochemical tests were performed, including acetate utilization, mucate and salicin fermentation, and aesculin hydrolysis. Antimicrobial susceptibility testing (AST) showed that maximum resistance was seen against ciprofloxacin (82.5 %) followed by ampicillin (77.8 %) and cotrimoxazole (68.2 %), and minimum resistance was seen against ertapenem (4.8 %).
Conclusion. In our study two pathotypes (EAEC, EIEC) were predominant among NLF E. coli and these were not only important aetiological agents in children, but also in adults. Our study also sheds light on the epidemiology of EIEC, which is one of the most neglected DEC pathotypes, as hardly any microbiological laboratories process NLF E. coli for EIEC.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. All comments by the reviewers were satisfactorily addressed.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
Overall I think that the manuscript submitted here is of good quality and suitable for publication at this journal, with a few minor modifications. My general comments are below. General Comments 1) Based on the amount of tests that were performed in the manuscript (Biochemical testing, Serotyping, Antibiotic Sensitivity Testing) I was expecting to see a more diverse array of figures, showcasing the results of those experiments. Instead it appears to be just the PCR results and a flow chart of the study. I know these results are represented more in tables than in figures, but I think it would be useful if the authors added more figures representative of those experiments, to aide the reader in the interpretation of the data. 2) There are a few grammatical errors and phrases that I dont fully …
Comments to Author
Overall I think that the manuscript submitted here is of good quality and suitable for publication at this journal, with a few minor modifications. My general comments are below. General Comments 1) Based on the amount of tests that were performed in the manuscript (Biochemical testing, Serotyping, Antibiotic Sensitivity Testing) I was expecting to see a more diverse array of figures, showcasing the results of those experiments. Instead it appears to be just the PCR results and a flow chart of the study. I know these results are represented more in tables than in figures, but I think it would be useful if the authors added more figures representative of those experiments, to aide the reader in the interpretation of the data. 2) There are a few grammatical errors and phrases that I dont fully understand that are used throughout - for example, "biochemical reactions were PUT UP". I assume that means that "biochemical reactions were CONDUCTED" or a similar phrase. I think this manuscript would benefit from a thorough re-read to remove grammar and spelling issues, as I spotted a not-insignificant amount.
Please rate the manuscript for methodological rigour
Very 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
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Comments to Author
While up to 1-% of E. coli can be non fermenting, the clinical significance remains largely unknown as they are not routinely screened for role in Diarrheagenic disease. This study has been developed to elucidate the role non fermenting E. coli plays in disease in adult and children populations from 3110 stool samples analysed. 1. Methodological rigour The authors present a number of culture, MALDI-TOF-MS, antimicrobial resistance testing and PCR methods to determine isolates present in the stool samples and for appropriate characterization of microorganisms and resistances present. Appropriate consideration has been employed to differentiate Shigella from non fermenting E. coli. Antimicrobial resistance testing has been performed in line with CLSI guidelines. Molecular assays are well considered for …
Comments to Author
While up to 1-% of E. coli can be non fermenting, the clinical significance remains largely unknown as they are not routinely screened for role in Diarrheagenic disease. This study has been developed to elucidate the role non fermenting E. coli plays in disease in adult and children populations from 3110 stool samples analysed. 1. Methodological rigour The authors present a number of culture, MALDI-TOF-MS, antimicrobial resistance testing and PCR methods to determine isolates present in the stool samples and for appropriate characterization of microorganisms and resistances present. Appropriate consideration has been employed to differentiate Shigella from non fermenting E. coli. Antimicrobial resistance testing has been performed in line with CLSI guidelines. Molecular assays are well considered for inclusion. 2. Presentation of results Overall the results sections are clear with the percentage of isolates identified in EAEC, EIEC, EPEC and ETEC. Table 2 and 3 are clear. Figures 2 and 3 need modification. An appropriate legend to describe each multiplex would help the reader understand more clearly what is being presented. Also samples on gel images should not be labelled as "negative samples". They should be given a name and stated not detected in the legend. Negative sample may just indicate the assay was not sensitive enough to detect the target of interest. 3. How the style and organization of the paper communicates and represents key findings Style and organization of manuscript are easy to follow and logical. The key findings are clear and address the aims of the study- to elucidate the role that non fermenting E. coli play in diarrheagenic disease. 4. Literature analysis or discussion This study is aimed at a topic that lacks literature. Where appropriate literature exists, it has been referenced. It compares results to a similar study in children in Bangladesh and highlights the differences in study population which may account for some discrepancies in findings which are plausible. 5. Any other relevant comments There are a number of edits which should be considered by the authors: L133- Reference how these methods are performed Section 149- PCR conditions for DEC genes There appears to be an initial PCR mix used followed by a final PCR reaction which contains difference concentrations of MGcl2 and primers. Please clarify which should be used in each instance L152 - "1.5 mM of MgCl2, at a final concentration of 1.5mM"- this is confusing, it is sufficient to state a final concentration. L154- 10μMconcentration should be 10 μM concentration L154- were as follows. Should be were as follows; L157- Amplified samples were tested for 1.5% agarose gel electrophoresis in Tris-borate-EDTA and EtBr staining. Perhaps consider PCR products were subsequently analysed on a 1.5% agarose gel electrophoresis in Tris-borate-EDTA and EtBr staining L160- "were future subjected to" please consider were subsequently tested for L161- remove reference 14 ad it is provided in Table 1 Section 162 PCR for ipaH gene- same comment as for section 149: There appears to be an initial PCR mix used followed by a final PCR reaction which contains difference concentrations of MGcl2 and primers. Please clarify which should be used in each instance L169- Would suggest formatting PCR conditions in same format as lines 155-156 L182-183- states "All EAEC and EIEC isolates were sent to National Salmonella and Escherichia Centre, Central Research Institute, Kasauli (HP), India for Escherichia coli O serotyping". Please reference the method used for serotyping Line 206 - "most common serotype was O22, O88, O149 (4 in number each)" suggest reword to most common serotype was O22, O88, O149 (n=4) Line 207- (3) suggest change to (n=3) Line 217- Please provide a reference to Fischers test L225- "Though NLF E.coli is commonly isolated, they are usually not processed further" - please provide supporting references for this statement. Also place a space between E. and coli L229-231 please provide references for % figures used L237- In the present study,376 please insert space between comma and 376 L255- et al change to et al. L270 "but none of them was immunocompromised" change to but none of them were immunocompromised Throughout text of manuscript Tables and tables are used. Please change table to Table throughout e.g L200, 218 etc. Table 1: State which is forward, and which is reverse primer Table 1: Please state if primers are in 5' to 3' orientation
Please rate the manuscript for methodological rigour
Very 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
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