First Report of Enteroaggregative E. Coli Harbouring NDM-4 and NDM-7 From Asymptomatic Children in India

For the rst time in the world, we report Enteroaggregative E.coli harbouring bla NDM−4 and bla NDM−7 that were isolated from healthy children. NDM-4 and NDM-7 are associated with increased carbapenemase activity. Clonal spread, inter-strain and interspecies horizontal transfer of such resistant determinants in a healthy population will be a cause of concern.


Abstract
For the rst time in the world, we report Enteroaggregative E.coli harbouring bla NDM−4 and bla NDM−7 that were isolated from healthy children. NDM-4 and NDM-7 are associated with increased carbapenemase activity. Clonal spread, inter-strain and interspecies horizontal transfer of such resistant determinants in a healthy population will be a cause of concern.
Main Text New Delhi metallo-beta-lactamase (NDM) is a carbapenemase having a broad range of hydrolysing activity against all beta lactam antibiotics (except aztreonam) including carbapenems which are last resort antibiotics to treat a multitude of health-care associated infections. Since its rst discovery, 20 variants of NDM have been discovered so far (1). These variants differ from NDM-1 by one to two residues at different positions of the active site of the NDM protein. NDM-4 has a single amino acid substitution (Met154Leu) while NDM-7 has double mutations [(Asp130 Asn) and (Met154Leu)], all of these mutations have been con rmed to increase the carbapenemase activity (2). Till now, only sporadic isolations of carbapenem resistant Enterobacteriaceae (CRE) carrying NDM-4 and NDM-7 variants have been reported from patients admitted to ICUs, and hospital sewage mainly from Asian countries (3)(4)(5).
Commensal carriage of NDM-1 which is the most prevalent NDM type has been reported from healthy adults and children from many countries across the world (6) (). Enteroaggregative Escherichia coli (EAEC) is an evolving enteric pathogen that is signi cantly associated with acute and persistent diarrhoea, malnutrition in children, HIV infection, and traveller's diarrhoea (7). Asymptomatic EAEC carriage are common early in life specially in developing countries which can lead to intestinal in ammation and clinical growth shortfalls (8).
Here for the rst time, we report EAEC harbouring NDM-4 and NDM-7 variants isolated from healthy children. We collected stool samples from 550 healthy children <5 years of age from 25 anganwadi centers of Chandigarh which offer primary healthcare to children as a part of the government public healthcare system (9). The EAEC were identi ed by using pCVD432 gene coding for CVD432 on pAA plasmid of EAEC (9). Whole genomes of EAEC strains (n= 33) were sequenced on Illumina Hiseq platform to generate 150 x 2 bp paired end reads and the sequences were analysed for in silico MLST, serotyping, identi cation of antimicrobial resistance (AMR) genes, plasmid replicons by SRST2 v0.2.0 (10). The genomes were assembled with SPADES v3.14.0 (11), annotated with RAST v2.0 and viewed on SEED genome browser v2.0 (12). The raw reads from this study are available under the BioProject ID: PRJNA760671; accession numbers: SAMN21220438 and SAMN21220439.
We believe this to be the rst report in world literature where EAEC with bla NDM−4 and bla NDM−7 were isolated from healthy children. NDM-4 and NDM-7are both associated with increased carbapenemase activity and clonal spread of such strains, inter-strain and interspecies horizontal transfer of such resistant determinants in healthy population will be a cause of concern. The gut is a very highly Ethics Committee (INT/IEC/2017/173). Written informed consent was obtained from the parent/guardian to participate in the study. All methods were performed in accordance to the relevant guidelines and regulations of the Institute Ethics Committee.

Consent to publish
A written consent from the parent/guardian of the children were taken to publish the results. All the patient identi ers were removed and sample IDs have been coded to safeguard the patient identity.

Data availability
The raw reads generated in this study have been deposited to NCBI under BioProject ID: PRJNA760671; accession numbers: SAMN21220438 and SAMN21220439.

Acknowledgement
We acknowledge kind support of local health authorities for facilitating the sample access in their areas