Whole genome-based genetic insights of bla NDM producing clinical E. coli isolates in hospital settings of Pakistant

ABSTRACT Carbapenem resistance among Enterobacterales has become a global health concern. Clinical Escherichia coli isolates producing the metallo β-lactamase NDM have been isolated from two hospitals in Faisalabad, Pakistan. These E. coli strains were characterized by MALDI-TOF, PCR, antimicrobial susceptibility testing, XbaI and S1 nuclease pulsed-field gel electrophoresis (PFGE), conjugation assay, DNA hybridization, whole genome sequencing, bioinformatic analysis, and Galleria mellonella experiments. Thirty-four bla NDM producing E. coli strains were identified among 52 nonduplicate carbapenem-resistant strains. More than 90% of the isolates were found to be multidrug resistant by antimicrobial susceptibility testing. S1 PFGE confirmed the presence of bla NDM gene on plasmids ranging from 40 kbps to 250 kbps, and conjugation assays demonstrated transfer frequencies of bla NDM harboring plasmids ranging from 1.59 × 10−1 to 6.46 × 10−8 per donor. Whole genome sequencing analysis revealed bla NDM-5 as the prominent NDM subtype with the highest prevalence of bla OXA-1, bla CTX-M-15, aadA2, aac(6')-Ib-cr, and tet(A) associated resistant determinants. E. coli sequence types: ST405, ST361, and ST167 were prominent, and plasmid Inc types: FII, FIA, FIB, FIC, X3, R, and Y, were observed among all isolates. The genetic environment of bla NDM region on IncF plasmids included partial ISAba125, the bleomycin ble gene, and a class I integron. The virulence genes terC, traT, gad, fyuA, irp2, capU, and sitA were frequently observed, and G. mellonella experiments showed that virulence correlated with the number of virulence determinants. A strong infection control management in the hospital is necessary to check the emergence of carbapenem resistance in Gram-negative bacteria. IMPORTANCE We describe a detailed analysis of highly resistant clinical E. coli isolates from two tertiary care centers in Pakistan including carbapenem resistance as well as common co-resistance mechanisms. South Asia has a huge problem with highly resistant E. coli. However, we find that though these isolates are very difficult to treat they are of low virulence. Thus the Western world has an increasing problem with virulent E. coli that are mostly of low antibiotic resistance, whereas, South Asia has an increasing problem with highly resistant E. coli that are of low virulence potential. These observations allow us to start to devise methodologies to limit both virulence and resistance and combat problems in developing nations as well as the Western world.

Line 177-178: Accession numbers of sequenced data generated in this study should be updated if possible.
Line 214: It is recommended to correct the spelling of 'b-lactamases' to 'β-lactamases' in the manuscript.
Line 245: To provide a more comprehensive understanding of the phylogenetic relationships among the strains, additional information regarding the tree-building method is necessary.Specifically, the manuscript should include details on the sequencing data used for the analysis, such as whether SNP or core genome-based methods were employed, the length of the core genome used, the software and methodology utilized for the tree construction (such as ML or NJ), and if appropriate outgroups were used.
Line 248: While the current phylogenetic tree suggests that ST167 may not belong to a single cluster and that SNP distances between clusters are significant, it is possible that alternative tree-building methods could result in different conclusions.It may be necessary for the authors to explore additional tree-building methods beyond what is presented in the manuscript.Additionally, it is recommended to use the same color for ST1702 in Figure 1.
Line 273: Figures 2A, 2B, and 2D appear to have been stretched and distorted, and the sizes of the figures are not consistent with one another.The authors should adjust the figures to ensure that they are accurately and clearly presented.
Line 294: The bleMBL gene should be labeled on Figure 3. Line 295: Although there are some differences in the gene structures of Groups C and D, Figure 3 shows that most of the regions of the two groups share a high degree of identity.The authors may need to provide a detailed explanation to address this apparent discrepancy.
Line 298: The genetic environment of blaNDM gene located in the IncHI2 plasmid pPK-5160-blaNDM-1 was not fully described and discussed in this study.
Reviewer #2 (Comments for the Author): In the present manuscript entitled 'Whole genome-based genetic insights of blaNDM producing clinical E. coli isolates from 2 hospitals in Pakistan, the authors raised an important topic: the importance to characterise the genetic environment of antimicrobial resistance genes especially those involved in the resistance of last-resort antibiotics such as carbapenems.The authors concentrated their work on the main prevalent carbapenemase gene in India, blaNDM.They showed that distinct plasmid replicons are involved in the spread of these genes.

Methods
The methods used are well described.However, the results of AST would have provided important information.

Interpretation
The two hospitals being located in the same area of Pakistan, Faisalabad, can the blaNDM distribution described be representative of the one of Pakistan?Are some of these strains part of outbreaks?In the abstract (lane 36), the authors wrote that 'virulence correlated with the number of virulence determinants' while in lanes 307-308 and lanes 355-356 they conclude that 'most isolates... showed negligible pathogenicity.What is the real impact of such virulence factors on the pathogenicity of the strains studied?Results Table 1 -Could it be added as supplemental figure?Table 2 -Did the authors described the characteristics of the blaNDM-positive plasmids from 10 representative E. coli ST?If so, would it be pertaining to include the genes actually detected on the described plasmid i.e.PK-5160 seem to carry blaNDM-1 on the only IncHI2 plasmid.A table including antimicrobial susceptibility data should be added.Table 3 -The words 'E. coli strains' are missing in the title.The authors should better explain the similarity of the 4 groups of IncF plasmids described figure 3.Such a similarity look like a signature of genetic exchange leading to the mosaic plasmids described.

Major modifications
The authors performed AST.It could have been relevant to correlate the presence of the predicted AMR genes described in the paper to the antimicrobial susceptibility testing results.As stated in the conclusion, 'Early detection of the blaNDM ...with decreased sensitivity to carbapenems is crucial'... excepted that the 12 antimicrobials tested are not mentioned (lines 98:100), neither their MIC result provided.

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Summary
In the present manuscript entitled 'Whole genome-based genetic insights of blaNDM producing clinical E. coli isolates from 2 hospitals in Pakistan, the authors raised an important topic: the importance to characterise the genetic environment of antimicrobial resistance genes especially those involved in the resistance of last-resort antibiotics such as carbapenems.The authors concentrated their work on the main prevalent carbapenemase gene in India, blaNDM.
They showed that distinct plasmid replicons are involved in the spread of these genes.

Methods
The methods used are well described.However, the results of AST would have provided important information.

Interpretation
The two hospitals being located in the same area of Pakistan, Faisalabad, can the blaNDM distribution described be representative of the one of Pakistan?Are some of these strains part of outbreaks?
In the abstract (lane 36), the authors wrote that 'virulence correlated with the number of virulence determinants' while in lanes 307-308 and lanes 355-356 they conclude that 'most isolates… showed negligible pathogenicity.What is the real impact of such virulence factors on the pathogenicity of the strains studied?

Results
Table 1 -Could it be added as supplemental figure?Table 2 -Did the authors described the characteristics of the blaNDM-positive plasmids from 10 representative E. coli ST?If so, would it be pertaining to include the genes actually detected on the described plasmid i.e.PK-5160 seem to carry blaNDM-1 on the only IncHI2 plasmid.
A table including antimicrobial susceptibility data should be added.The authors should better explain the similarity of the 4 groups of IncF plasmids described figure 3.Such a similarity look like a signature of genetic exchange leading to the mosaic plasmids described.

Major modifications
The authors performed AST.It could have been relevant to correlate the presence of the predicted AMR genes described in the paper to the antimicrobial susceptibility testing results.As stated in the conclusion, 'Early detection of the blaNDM …with decreased sensitivity to carbapenems is crucial'… excepted that the 12 antimicrobials tested are not mentioned (lines 98:100), neither their MIC result provided.

Response to Reviewers
Reviewer #1 (Comments for the Author): The study presented herein is intriguing as it provides insights into the genomic environment of clinical E. coli carrying blaNDM in Pakistan.The article reported on the isolation and antimicrobial susceptibility testing of 34 blaNDM-carrying E. coli strains, as well as genomewide analysis.Although the authors have made valuable contributions to the understanding of the genetic environment of NDM-carrying strains, a few comments are necessary to improve the manuscript's quality: Line 43: The manuscript contains several spelling errors, including the misspelling of 'a-' as 'a' in some instances.To ensure the manuscript's accuracy and precision, these errors should be corrected.Response: Spellings of β-lactamases have been corrected throughout the manuscript.
Correction has been made.Line 245: To provide a more comprehensive understanding of the phylogenetic relationships among the strains, additional information regarding the tree-building method is necessary.Specifically, the manuscript should include details on the sequencing data used for the analysis, such as whether SNP or core genome-based methods were employed, the length of the core genome used, the software and methodology utilized for the tree construction (such as ML or NJ), and if appropriate outgroups were used.(https://cge.cbs.dtu.dk/services/CSIPhylogeny/).The pipeline comprises of various freely available programs.The paired-end reads from each isolate were aligned against the reference genome, using Burrows-Wheeler Aligner (BWA), SAMtools, "mpileup" command and bedtools.
The single nucleotide polymorphism (SNP) based phylogenetic tree was generated by calling and filtering SNPs, site validation and phylogeny based on a concatenated alignment of the highquality SNPs.For inferring phylogeny, the analysis was run with the standard parameters and the NCTC11129 strain genome (GenBank accession number NZ_LR134222.1) was used as a reference sequence.The analysis was run with the default parameters; a minimal depth at SNP positions was ten reads along with a relative depth at SNP positions of 10%, a minimal distance between SNPs was of 10 bp, a minimal SNPs quality was 30 and a minimal Z-score was of 1.96, a minimal SNP quality was 30 and a minimal read mapping quality was of 25.The Z-score expresses the confidence with which a base was called at a given position and the Z-score was 1.96.Number of SNPs exhibited among closely related isolates were calculated using distance matrix file generated as a result of phylogeny (35).The output core alignment file was used to construct the Maximum-likelihood tree with 1000 bootstrap replications, by using MEGA-X v 10.0.5 (https://www.megasoftware.net/home)(36).The phylogenetic tree of the alignments was visualized and edited by iTOL v 4.4.2software (https://itol.embl.de)(37).
Correction has been made.
Line 248: While the current phylogenetic tree suggests that ST167 may not belong to a single cluster and that SNP distances between clusters are significant, it is possible that alternative treebuilding methods could result in different conclusions.It may be necessary for the authors to explore additional tree-building methods beyond what is presented in the manuscript.
Additionally, it is recommended to use the same color for ST1702 in Figure 1.
Response: Maximum likelihood phylogenetic tree was constructed using core genome SNPS.
Lines 262-263.Notably, the reference genome grouped with three isolates belonging to ST156 and unknown ST, has been added in manuscript.
Color correction for ST1702 has been made.Response: The bleMBL gene has been labeled in Figure 3.
Line 295: Although there are some differences in the gene structures of Groups C and D, Figure 3 shows that most of the regions of the two groups share a high degree of identity.The authors may need to provide a detailed explanation to address this apparent discrepancy.
A detailed explanation has been added in the manuscript.Figure 3 has also been updated.
Line 298: The genetic environment of blaNDM gene located in the IncHI2 plasmid pPK-5160-blaNDM-1 was not fully described and discussed in this study.

Added in manuscript.
Reviewer #2 (Comments for the Author): In the present manuscript entitled 'Whole genome-based genetic insights of blaNDM producing clinical E. coli isolates from 2 hospitals in Pakistan, the authors raised an important topic: the importance to characterise the genetic environment of antimicrobial resistance genes especially those involved in the resistance of last-resort antibiotics such as carbapenems.The authors concentrated their work on the main prevalent carbapenemase gene in India, blaNDM.
They showed that distinct plasmid replicons are involved in the spread of these genes.

Methods
The methods used are well described.However, the results of AST would have provided important information.

Interpretation
The two hospitals being located in the same area of Pakistan, Faisalabad, can the blaNDM distribution described be representative of the one of Pakistan?
Response: Faisalabad is the third largest city of Pakistan, and second largest city of wider Punjab region.Allied and DHQ, two tertiary care hospitals in this city, offer a variety of healthcare facilities to a large number of populations of the city and other distant rural areas.This city could serve as a representative of large population of Pakistan.
Are some of these strains part of outbreaks?
Response: This study collected E. coli strains from laboratories of two hospitals, isolated from urine and pus cultures of in and outpatients.These strains were not part of outbreaks.
In the abstract (lane 36), the authors wrote that 'virulence correlated with the number of virulence determinants' while in lanes 307-308 and lanes 355-356 they conclude that 'most isolates... showed negligible pathogenicity.What is the real impact of such virulence factors on the pathogenicity of the strains studied?
Response: The investigation of virulence factors on the pathogenicity of studied strains may facilitate the novel therapeutics, the improvement of design of effective vaccines, and the prevention of further spreading of the multidrug resistant isolates.

Results
Table 1 -Could it be added as supplemental figure?
Response: This table has been added as a supplementary table 2.
Table 2 -Did the authors described the characteristics of the blaNDM-positive plasmids from 10 representative E. coli ST?If so, would it be pertaining to include the genes actually detected on the described plasmid i.e.PK-5160 seem to carry blaNDM-1 on the only IncHI2 plasmid.
Response: Authors selected the isolates for comparative analysis of plasmids on the basis of Inc types of plasmids as well as blaNDM genes harbored by these isolates.Isolate PK-5160 was selected for analysis as it harbored blaNDM-1 gene and IncHI2 type of different Inc types of plasmids.
A table including antimicrobial susceptibility data should be added.
Response: A supplemental table 1 showing the results of antimicrobial susceptibility data has been added.A detailed explanation has been added in the manuscript.Figure 3 has also been updated.

Major modifications
The authors performed AST.It could have been relevant to correlate the presence of the predicted AMR genes described in the paper to the antimicrobial susceptibility testing results.As stated in the conclusion, 'Early detection of the blaNDM ...with decreased sensitivity to carbapenems is crucial'... excepted that the 12 antimicrobials tested are not mentioned (lines 98:100), neither their MIC result provided. Response: A table showing the results of antibiotic susceptibility testing has been added as a supplemental table 1.
Antimicrobial susceptibility testing revealed that all 34 E. coli isolates were MDR strains, and they were resistant to multiple categories of antibiotics (n>3) (Table S1 in supplemental material).Therefore, each isolate carried at least three categories of resistance genes associated with resistance phenotype (Table 1).Almost all these isolates were non-susceptible to fluoroquinolones (95-100%), cephalosporins (87-95%) and penicillins (82-97%).Moreover, over half of the isolates were resistant to aminoglycosides (57%).The MICs results of CR-EC isolates revealed 100% susceptibility to tigecycline and colistin.
Has also been added in manuscript.
Response: Lines 42 to 44.Morbidity and mortality caused by multidrug-resistant (MDR) bacteria are increasing globally, with a recent study estimating the global burden of AMR at 4.95 million deaths in 2019 (2).Corrections have been made throughout the manuscript.Line 177-178: Accession numbers of sequenced data generated in this study should be updated if possible.Response: Lines 173 to 174.The genomes of E. coli isolates have been submitted to NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/)under the bioproject PRJNA932156.Line 214: It is recommended to correct the spelling of 'b-lactamases' to 'β-lactamases' in the manuscript.

Line 273 :
Figures 2A, 2B, and 2D appear to have been stretched and distorted, and the sizes of the figures are not consistent with one another.The authors should adjust the figures to ensure that they are accurately and clearly presented.Response: Figure: Sizes of figures have been adjusted properly for clear presentation.(Figure 2) Line 294: The bleMBL gene should be labeled on Figure 3.

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Table 3 -
The words 'E. coli strains' are missing in the title.
Response: Table

2. Characteristics of E. coli strains harboring blaNDM gene
The words 'E.Coli strains' has been added in title of table 2.