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 first 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 confirmed 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 (3–5). 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 significantly 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 inflammation and clinical growth shortfalls (8).
Here for the first 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 identified 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, identification 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.
In two of the EAEC isolates (S56 and S76) originating respectively from a four year and a one year old female children, variants of blaNDM−1 gene i.e. blaNDM−4 and blaNDM−7 were detected. Both these females had no history of antibiotic exposure or underlying conditions at the time of sample collection and were located at two different anganwadis situated in the Chandigarh city. Phenotypically both strains were multi-drug resistant (MDR) showing resistance to ampicillin, ciprofloxacin, co-trimoxazole, levofloxacin, cefoxitin, cefepime, ceftriaxone, ertapenem and imipenem. In addition to NDM-4, sample S56 also carried genes encoding AmpC cephalosporinases (ampH, ampC1, ampC2), various ESBLs (blaCTX−M−15, blaTEM−206, blaOXA−1), aminoglycosides (aac3-IIa, aac6Ib-cr, aadA12, sat-2A, strA, strB), chloramphenicol (catB4), trimethoprim (dfrA1, dfrA12), mrdA (encodes PBP2), rmtB (16srRNA methylase), sulphonamides (sulII, sulI), and tetracycline (tetA) resistances. This strain belonged to ST6303 from clonal complex 31 with O15:H34 antigenic serotype. Additionally IncFII (pRSB107) plasmid was also identified in this genome. Plasmids of IncFII group are widely known to be involved in the worldwide dissemination of AMR genes as in the case blaCTXM−15 gene (13). The virulence profile of this isolate composed of signature EAEC genes such as aggACDR, air, aap, aar, aatA. The second isolate (S76) carrying NDM-7, also harboured genes encoding AmpC cephalosporinases (ampH, ampC1, ampC2), ESBLs (blaCMY−42, blaCTX−M−15), and mrdA (encodes PBP2) resistances. This isolate belonged to ST448 (clonal complex CC448) with O188:H19 antigenic serotype and possessed a self-transmissible plasmid IncX3. ST448 as well as IncX3 plasmid associated with NDM variants have also been reported from clinical cases in India (5, 14, 15). The major virulence gene present in this isolate was aar gene encoding AggR-activated regulator. In both strains, immediately upstream of blaNDM genes contained bleomycin resistance gene (ble) along with dsbD encoding oxidoreductase and trp1 encoding putative phosphoribosylanthranilate isomerase (for synthesis of amino acid tryptophan) were observed as reported earlier from India (Figure) (5).
We believe this to be the first report in world literature where EAEC with blaNDM−4 and blaNDM−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 populated ecosystem and at a later stage such strains may be a source of extra intestinal infections, may spread to other hosts or transfer genetic resistance element to other members of micro-biota including pathogens.