Genome analyses of bla_NDM-4 carrying ST 315 Escherichia coli isolate from sewage water of one of the Indian hospitals

Escherichia coli share a commensal relationship with humans and animals. The extensive acquisition of virulence genes has potentiated E. coli to become pathogenic [1]. The E. coli pathotypes are identified on the basis of virulence determinants present in the genome. A pathotype, ExPEC (extra intestinal pathogenic E. coli) has been reported in extra intestinal, neonatal meningitis and septicaemia infections. Over the years ExPECs are being increasingly recognised for plasmid-mediated carriage of extended spectrum β-lactamases and carbapenemases (metallo β-lactamases, MBLs) [2]. The emergence of a carbapenemase, New Delhi-Metallo β-lactamase (NDM-1) has conferred resistance to last resort β-lactams which has further made the management of ExPECs difficult [3]. A single amino acid variation (Met154Leu) in NDM-1 has resulted in the emergence of a novel NDM-4 which has extended and increased hydrolytic activity towards β-lactams, especially towards carbapenem [4].

The bacterial isolate AK-1 found in hospital sewage water was subjected to antibiotic susceptibility testing which revealed an exorbitantly elevated MIC values against β-lactams [5]. This unusual resistance in AK-1 strain intrigued us to further explore other genetically predisposed features through whole genome sequencing.

One of the highlight of this study was an NDM-4 carrying multidrug plasmid. It harboured 15 resistance markers (Additional file 2: Table S1) and some of them were in association with mobile genetic elements (MGEs). Genetic context of the resistance markers on the plasmid was determined to assess their association with mobile genetic elements and their potential for horizontal gene transfer. Genetic environment of bla_NDM-1 have revealed the downstream presence of ble_MBL and upstream presence of remnants of, or entire ISAba125 [18]. The plasmid pAK1 shared similar arrangement for bla_NDM-4 with partial ISAba125 (Fig. 1a).

The genetic environment of bla_TEM1b was identified to be flanked by one truncated copy of IS26. The bla_TEM1b gene was associated with partial Tn2, it was present distally to tnpR-encoding gene and proximally to an IR (Fig. 1b) [19].

A truncated Tn2 transposon unit was found to be associated with bla_CTX-M-15. It was revealed that both DRL and DRR were bracketing this unit where ISEcp1 was distally located to bla_CTX-M-15 gene (Fig. 1c). This arrangement is very common and ISEcp1 is reported to mediate the mobilization of bla_CTX-M-15 [20, 21].

Other previously reported resistance genes and associated mobile genetic elements were found on AK-1 plasmid. (i) Class I integron carrying qacE∆ 1, sul 2, dfrA12 and aadA2 (Fig. 1d) [22], (ii) rmt B was found to be associated with partial sequence of IS 26 (Fig. 1e) and upstream to bla_TEM1b (not shown) [23], (iii) sul1 gene upstream to strA and strB genes which are bracketed by Tn 5903 [24], (iv) tet (A) efflux protein and its regulator tet R (A) associated with Tn1721 mobile element [25].

AK-1 chromosome carries wide range of resistance markers towards major classes of antibiotics like flouroquinoles, macrolides, aminoglycosides, tetracycline, trimethoprim isoniazid, triclosan, elfamycin and β-lactams (Additional file 3: Table S2). Extensive numbers of genes conferring resistance towards β-lactams were found; four types of penicillin binding proteins and class C β-lactamases, bla_{CFE 1} and bla_{PEDO 2}. Accumulative effect of these genes explains the high level of phenotypic resistance towards β-lactams in AK-1 [5].

ST 315 E. coli has been reported earlier in urosepsis, intra-abdominal infections and primary sepsis in medical cases [16]. Therefore, comparative analysis [with NC_017646 (NMEC), NC_008253 (UPEC), NC_017631 (UPEC), NC_007946 (UPEC)] and exploration of virulence genes in AK-1 was performed which resulted in identification of assorted virulence factors. These are commonly associated with ExPEC isolates [26] as shown in Table 2. E. coli type III secretion system 2 (ETT2) identified in AK-1 has been previously reported in E. coli strains in partial or complete form [27]. ETT2 is associated with virulence regulation in some ExPEC strains and pathogenicity in septicemic E. coli [28]. Pathogenicity island (PAI), type 6 secretion system was identified in AK-1 [29]. Multiple PAI, invested in fimbriae and adhesions expression, were observed in AK-1 strain which are described as (i) Type 1 fimbriae is common in UPEC, causes infection in mucous surfaces by inducing adhesion and virulence [30], (ii) Chaperone usher (CU) fimbriae clusters yad and sfm provide additional adhesion to the host [31], (iii) Mat (meningitis associated and temperature regulated) fimbria or E. coli common pilus (ECP) responsible for colonisation and adherence in host [32], (iv) Curli fibres binds to hosts matrix and plasma protein, and is reported to cause haemagglutination, fibronectin binding and formation of proteolytically active plasmin which aids in bacterial diffusion through tissue disintegration [30], (v) ExPEC specific FdeC (factor adherence E. coli) responsible for bacterial fitness and colonization in UTI [33].

Haemolytic toxins were also identified in AK-1 (i) Hemolysin α is associated with ExPEC virulence and attacks immune cell [34], (ii) membrane pore-forming toxin HlyE lyses mammalian cells and erythrocytes [35].

ExPECs cope up with low iron availability by secreting siderophores which retrieves sequestered iron from host proteins [36]. AK-1 was identified to harbour aerobactin, enterobactin and chuA siderophores. Proectins/invasins like ibeB, ompA and K1 capsule have been reported in invasion of brain microvasular endothelial cells [37].

Presence of emerging NDM variant along with multidrug resistances on a mobile Inc F plasmid prompted us to compare the pattern of their dissemination and relatedness. Phylogenetic analysis was performed using plasmid sequences which produced significant alignment with AK-1 plasmid, and the query coverage was found between 44 and 53% of all the plasmid sequences with ~ 99% identity. Furthermore, plasmid sequences having alleles, repFIB and/or repFII and acquired genes, bla_TEM1 and/or bla_CTX-M-15, and/or bla_NDM variants (NDM-4/NDM-1/NDM-5), were specifically selected for comparisons with AK-1 plasmid (Fig. 2). The result showed overall dissimilarity in the backbone sequences except for plasmid pGUE NDM (France) which differs by nine single nucleotide variations with AK-1 plasmid. Distribution and backbone sequences of Inc F plasmids harbouring NDM variants and/or ESBLs (CTX-M-15/TEM1b) are inconsistent. Active mobilome could account for such high variation in plasmid sequences. This implies ExPECs carrying such plasmids could have fluctuating resistance profile leading to a concern for clinicians.

The genetic context of NDM-4 in AK-1 plasmid was similar to plasmids (pM109 FII, pMC-NDM, pGUE NDM, pCRKP-2297, pCRKP-1215, pM214 FII, and pNDM5-IBAC) carrying NDM variants. The genetic environment for bla_NDM remained conserved in these plasmids. This suggests that alteration in bla gene originated new NDM variants. Furthermore, genetic context of bla_TEM1b and bla_CTX-M-15 were almost similar in these plasmids.

Presence of plasmid harbouring bla_NDM-4 and other β-lactamase genes near the hospital setting environment is a serious concern in context to its circulation and spread in hospital settings. It is the first time NDM-4 producing ST 315 E. coli was detected in India. Moreover, no genome based information on ST 315 strain is yet available. Lack of information on mechanism of virulence, transmission sources and other genetic characteristics have become an impasse for alleviation of ExPECs infections. AK-1 genome based virulence profile provides cause of serious infections by ST 315 ExPEC, a common microflora of healthy individuals. Genome informed virulence and resistance mechanisms will definitely help in identification of this ExPECs in healthcare settings and controlling the clonal spread of carbapenamase carrying E. coli pathovars.