A Decade-Long Evaluation of Neonatal Septicaemic Escherichia coli: Clonal Lineages, Genomes, and New Delhi Metallo-Beta-Lactamase Variants

ABSTRACT Longitudinal studies of extraintestinal pathogenic Escherichia coli (ExPEC) and epidemic clones of E. coli in association with New Delhi metallo-β-lactamase (blaNDM) in septicaemic neonates are rare. This study captured the diversity of 80 E. coli isolates collected from septicaemic neonates in terms of antibiotic susceptibility, resistome, phylogroups, sequence types (ST), virulome, plasmids, and integron types over a decade (2009 to 2019). Most of the isolates were multidrug-resistant and, 44% of them were carbapenem-resistant, primarily due to blaNDM. NDM-1 was the sole NDM-variant present in conjugative IncFIA/FIB/FII replicons until 2013, and it was subsequently replaced by other variants, such as NDM-5/-7 found in IncX3/FII. A core genome analysis for blaNDM+ve isolates showed the heterogeneity of the isolates. Fifty percent of the infections were caused by isolates of phylogroups B2 (34%), D (11.25%), and F (4%), whereas the other half were caused by phylogroups A (25%), B1 (11.25%), and C (14%). The isolates were further distributed in approximately 20 clonal complexes (STC), including five epidemic clones (ST131, ST167, ST410, ST648, and ST405). ST167 and ST131 (subclade H30Rx) were dominant, with most of the ST167 being blaNDM+ve and blaCTX-M-15+ve. In contrast, the majority of ST131 isolates were blaNDM-ve but blaCTX-M-15+ve, and they possessed more virulence determinants than did ST167. A single nucleotide polymorphism (SNP)-based comparative genome analysis of epidemic clones ST167 and ST131 in a global context revealed that the study isolates were present in close proximity but were distant from global isolates. The presence of antibiotic-resistant epidemic clones causing sepsis calls for a modification of the recommended antibiotics with which to treat neonatal sepsis. IMPORTANCE Multidrug-resistant and virulent ExPEC causing sepsis in neonates is a challenge to neonatal health. The presence of enzymes, such as carbapenemases (blaNDM) that hydrolyze most β-lactam antibiotic compounds, result in difficulties when treating neonates. The characterization of ExPECs collected over 10 years showed that 44% of ExPECs were carbapenem-resistant, possessing transmissible blaNDM genes. The isolates belonged to different phylogroups that are considered to be either commensals or virulent. The isolates were distributed in around 20 clonal complexes (STC), including two predominant epidemic clones (ST131 and ST167). ST167 possessed few virulence determinants but was blaNDM+ve. In contrast, ST131 harbored several virulence determinants but was blaNDM-ve. A comparison of the genomes of these epidemic clones in a global context revealed that the study isolates were present in close proximity but were distant from global isolates. The presence of epidemic clones in a vulnerable population with contrasting characteristics and the presence of resistance genes call for strict vigilance.

Executive Summary. There is no information on the source of the isolates considered in this study (e.g., information about the population, geographic region, etc.).
L93 "This versatility is observed in its genome and also in its function or potential to cause disease" A reference to this statement is needed.
L106 What is "[WHO]"? L171 "Whole genome sequencing (WGS)" WGS is spelled out in the sentence. The description of the method is redundant. It is too long compared to other items and should be shortened or moved to a supplement file.
Result. L272 "Seventy E. coli were identified from the blood of septicaemic neonates (2009 to 2019)." The number of cases, patient background, and outcomes (treatment outcomes) are unknown. Also, whether the results are similar to other parts of India or unique to this facility needs to be added to the discussion.
L231 "Isolates were resistant to second (52/80, 65%) and third-generation cephalosporins (67/80, 84%)" In the Methods section, the study on cephalosporins I am aware that the study regarding cephalosporins is not explicitly mentioned in the Methods section.
L267 "Transmissibility of metallo-β-lactamases and plasmid profile:" Is a colon necessary? Discussion L434 "under-5 mortality" The phrase "under-five" is also mentioned. L480-489 "Several studies have shown that commensal E. coli are " The sentence is redundant due to the extensive use of literature citations. I think that you should state shortly and precisely what you want to assert from the results of this review. Overall, we believe that the authors over-explain the contents of the cited references. The description of the discussion items is polite and clear, but is it possible to separate the items to make them more readable?
The reviewer has studied several reports of genetic testing of bacterial isolates, and it is not particularly rare to find such a heterogeneous population of isolates in a single institution's study?
Reviewer #2 (Comments for the Author): Would be interesting to know if clinical outcomes changed after 2013 when transition from NDM-1 to NDM -5/ -7.

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Reviewer #1
The fact that a long-term study of ExPECs was conducted on newborns admitted to the NICU is valuable information. Although the study was conducted in a single region, the content of the study was substantial, and I believe it will contribute to future research activities. On the other hand, I cannot find any evidence that the understanding of the transition of resistant strains itself will change the current practice of antimicrobial chemotherapy. In order to understand that the emergence of resistant strains is a critical situation, it is necessary to analyze the relationship between detected bacteria or isolates and outcomes (e.g., death, prolonged hospital stay, etc.). I have attached a comment on how. Please refer to the reference.

Executive Summary
There is no information on the source of the isolates considered in this study (e.g., information about the population, geographic region, etc.). Answer: Isolates were collected primarily from the blood of septicaemic neonates admitted to a tertiary care hospital (IPGME&R and SSKM hospital of Kolkata, India). This tertiary care hospital in Kolkata, West Bengal caters to a population in Kolkata and also patients from different districts (at a distance of 100 km radius from Kolkata) of West Bengal.
2. L93 "This versatility is observed in its genome and also in its function or potential to cause disease" A reference to this statement is needed. Answer: Authors have included the reference in reference number 9. 4. L171 "Whole genome sequencing (WGS)" WGS is spelled out in the sentence. The description of the method is redundant. It is too long compared to other items and should be shortened or moved to a supplement file.

Answer:
This is the sub-heading for the following section, hence it is not spelled out.  Table S2A, S2B) highlighting studies on E. coli causing neonatal sepsis from different parts of India and across the globe have been incorporated. Authors have compared data from these studies wherever applicable.
6. L231 "Isolates were resistant to second (52/80, 65%) and third-generation cephalosporins (67/80, 84%)" In the Methods section, the study on cephalosporins I am aware that the study regarding cephalosporins is not explicitly mentioned in the Methods section. Answer: Antibiotic susceptibility pattern was determined for cephalosporins (cefoxitin, cefuroxime as 2 nd gen and cefotaxime, ceftriaxone as 3 rd gen cephalosporins) as a part of the antibiotic susceptibility test [disk diffusion data of cefoxitin, cefotaxime (2009-2017) and cefuroxime, ceftriaxone in VITEK®2 AST 280 (2018)(2019). This has been clarified in the foot note of 12. The description of the discussion items is polite and clear, but is it possible to separate the items to make them more readable? Answer: Authors have now modified the discussion.
13. The reviewer has studied several reports of genetic testing of bacterial isolates, and it is not particularly rare to find such a heterogeneous population of isolates in a single institution's study? Answer: Yes, the authors agree to this comment. However, since this unit had a heterogeneous population of isolates, hence it is mentioned in the study.
14. Reviewer #2: Would be interesting to know if clinical outcomes changed after 2013 when transition from NDM-1 to NDM -5/ -7. Answer: A descriptive table with patient data and outcomes has been incorporated ( Table 4) in the main manuscript. The possible relationship between mortality and sepsis due to a bla NDM +ve isolate was tested (Supplementary Table S3B) but not found to be statistically significant. It was noted that mortality increased from 2013 onwards (before 2013-39%, after 2013-57%). This has been incorporated on Page No. 19 & Line No 432-433.

Note:
New sequence types have been obtained for two study isolates which have been included in Table 1