Extraintestinal pathogenic Escherichia coli (ExPEC), apart from being a seasoned nosocomial pathogen is also an important cause of community-acquired (Johnson and Russo, 2002) and other infections such as urinary tract infection, sepsis, neonatal meningitis, and colibacillosis in humans and animals (Kaper et al., 2004; Smith et al., 2007). Genome analysis revealed that pathogenic E. coli have genome sizes in excess of about 1.0 Mb than the commensal strains mainly due to the presence of genes encoding multiple virulence factors, such as adhesins, toxins, invasins and siderophores that are absent or unlikely to be present in commensal strains (Croxen and Finlay, 2010). The virulence factors are mostly associated with phages and pathogenicity islands and undergo horizontal gene transfer which disseminates traits, thereby offering fitness advantages to recipient organisms (Ahmed et al., 2008).

Secreted toxins are the virulence factors that play an important role in long term colonization and pathogenesis of ExPEC. Some of these known secreted toxins comprise of important genotoxins, such as cytolethal distending toxins (CDT’s), cycle inhibiting factors (cif’s) and cytotoxic necrotizing factors (CNF’s) which can directly regulate the cell cycle of the host (Nougayrède et al., 2005). Colibactin is another important genotoxin produced by a 54-kb pathogenicity island known as pks island harbored by the members of Enterobacteriaceae (Nougayrède et al., 2006). This genomic island consists of a clbA-S gene cluster that encodes non-ribosomal peptides and polyketide synthases along with accessory and tailoring enzymes (Nougayrède et al., 2006). Colibactin acts as a cyclomodulin and blocks the eukaryotic cell cycle causing progressive enlargement of the nucleus as well as the cell body eventually leading to cell death (Cuevas-Ramos et al., 2010). The cytopathic effect of these genotoxins is mediated by live bacteria and requires a direct contact with the host cell (Nougayrède et al., 2006).

The pks island was first identified in sequenced genomes of ExPEC prototype strains and was detected predominantly in strains of phylogenetic group B2 (Johnson et al., 2008). The island was shown to display several signatures reminiscent of its horizontal acquisition (Putze et al., 2009; Messerer et al., 2017). The origin and prevalence of the colibactin island among enteric pathogens is largely unexplored. However, it was also found to be present in other members of Enterobacteriaceae such as Citrobacter koseri, Klebsiella pneumoniae, and Enterobacter aerogenes (Putze et al., 2009). Epidemiological studies demonstrated the prevalence of pks in ExPEC and their association with severe infections in different host populations of varied geographical locations (Johnson et al., 2008; Buc et al., 2013; Shimpoh et al., 2017).

Epidemiological studies on ExPEC in India have so far been focused on understanding antimicrobial resistance, evolution and presence of pandemic clones (Avasthi et al., 2011; Jadhav et al., 2011; Hussain et al., 2012, 2014; Shaik et al., 2017). However, a better understanding of ExPEC associated virulence factors may help in the development of therapeutic interventions, such as diagnostics and/or vaccines against ExPEC infections, and this would also facilitate risk assessment of ExPEC strains.

While colibactin is regarded as a virulence factor in ExPEC, not much is known in the context of its molecular epidemiology entailing Indian clinical isolates. Therefore, the present study was performed in order to investigate the prevalence and carriage of pks island, and to decipher the genotypic and functional characteristics of pks harboring clinical ExPEC isolates from India.

The pks island encodes enzymes that are able to synthesize colibactin, a genotoxin that could induce host DNA damage and its presence may contribute to increased virulence and severe disease outcomes. Ever since the description of colibactin by Nougayrède et al. (2006), many studies were undertaken to develop a comprehensive understanding of this bacterial genotoxin (Bossuet-Greif et al., 2018; Faïs et al., 2018). However, epidemiological data on the prevalence of the same and factors associated with pks-positive E. coli isolates, particularly from the Southern World have not been documented. The present study showed that 35 out of the 462 clinical ExPEC isolates were positive for all the four genes, indicating the presence of complete pks island(s) which might be able to synthesize functional colibactin. Thus, the overall prevalence was found to be 7.6% and this constitutes first epidemiological data on pks island harboring E. coli from India. In contrast, reports from other countries, such as those by Johnson et al. (2008) and Shimpoh et al. (2017) demonstrated high pks prevalence among clinical E. coli.

Our previous studies have suggested that majority of the pks negative clinical E. coli from India were genetically diverse and had a high prevalence of antibiotic resistance; these studies also identified and characterized the clonally evolving pandemic sequence type 131 E. coli isolates in India (Jadhav et al., 2011; Ranjan et al., 2016; Hussain et al., 2017; Shaik et al., 2017). However, the role of colibactin in the emergence of lineage specific virulence in E. coli that were comparatively less resistant to antibiotics has been shown herein for the first time from India. Such lineages could become a matter of public health concern and analysis of the underlying strains would be of great importance given the high burden of infectious diseases in this region. Thus, the findings of this study have implications for better understanding of the epidemiological context of pathogenic E. coli in human diseases.

Escherichia coli harboring pks island was reported to be strongly associated with bacteremia and human colorectal tumors (Johnson et al., 2008; Buc et al., 2013). Recent reports suggest that mice infected with colibactin positive E. coli had significantly lower survival rates compared to those infected with isogenic colibactin-negative mutant(s) (Marcq et al., 2014). It has been further demonstrated that pks-positive E. coli infection induces cellular senescence and concurrently produces growth factors which promote tumor growth (Secher et al., 2013; Cougnoux et al., 2014; Dalmasso et al., 2015). The pks-positive E. coli isolates in the present study were detected in different specimen types including urine, blood and pus. Therefore, it can be surmised that the pks-positive E. coli might contribute in many invasive and non-invasive infections at different anatomic sites.

Previous studies reported that the pks island was majorly detected in E. coli phylogenetic group B2 strains, which are mainly documented as extraintestinal pathogens (Taieb et al., 2016; Sarshar et al., 2017). Our results were in line with these observations as the pks-positive isolates in our study also belonged predominantly to phylogroup B2 (97%), except for one isolate which belonged to phylogroup D (3%) (Table 1). The pathogenic strains of E. coli mainly belong to group B2 and, to a lesser extent, group D and frequently harbor higher number of virulence-factors than group A and group B1 strains (Picard et al., 1999; Johnson et al., 2008).

Several studies have demonstrated a strong correlation between the presence of virulence genes and the pathogenic spectrum of E. coli strains (Bien et al., 2012). These include multiple ExPEC-associated virulence genes such as adhesins, invasins, secretory toxins, and iron scavenging systems (Johnson, 1991). Accordingly, the virulent nature of pks-positive E. coli isolates was supported by our findings as 100% of them were positive for fimH (D mannose specific adhesin of minor fimbrial component), sfaD/E (s-fimbrial adhesin) and usp (uropathogenic specific protein) while ≥ 30% of them were positive for iucD (enzyme for siderophore aerobactin synthesis), sat (secreted autotransporter vacuolating cytotoxin), cvaC (colicin-V precursor) and ibeA (invasion protein). The afimbrial adhesin gene, afa was found to be completely absent (Table 1). These findings could be attributed to the pathogenic potential of the pks-positive strains along with the presence of many other virulence determinants. Furthermore, in the present study, siderophore production was detected in all pks-positive isolates; this observation was consistent with previous reports on the positive correlation between the presence of pks island and iron scavenging systems among the B2 E. coli strains (Martin et al., 2017). The localization of pks island within the High-Pathogenicity Island (HPI) and its physical association with siderophore biosynthesis gene cluster has also been described in other members of Enterobacteriaceae (Putze et al., 2009). A majority of pks-positive E. coli isolates demonstrated high biofilm forming capabilities in M63 medium (Figure 1) and all the pks-positive isolates tested were found to be resistant to serum bactericidal activity (Figure 2). We speculate that these genotypic and phenotypic virulence traits expressed by B2 pks-positive E. coli could act as fitness factors in order to colonize and initiate/establish infection in intestinal and extra-intestinal sites.

Antibiotic susceptibility of the pks-positive isolates was performed and the isolates were observed to be uniquely associated with low antimicrobial resistance, this finding is consistent with the previous reports which have suggested that pks island harboring E. coli exhibit reduced antibiotic resistance (Chen et al., 2017; Sarshar et al., 2017). This observation could likely be due to opportunist E. coli infections arising from intestinal microbiota. Additionally, detailed characterization of such isolates from fecal and clinical samples is needed to certainly understand the reason behind such an observation. Multidrug resistance and ESBL production was observed in only 11.42 and 37% isolates, respectively (Table 1). Previous studies from our group have shown MDR rates of 95% for clinical ST131 strains, and 91% for clinical and stool non-ST131 strains (Hussain et al., 2014). We have also previously reported MDR rates of 100% for metallo-(β-lactamase (MBL) producing E. coli isolates (Ranjan et al., 2016) and 67% for isolates from skin and soft tissue infections (Ranjan et al., 2017). The pks positive isolates in contrast demonstrated lesser rates of antimicrobial resistance compared to the pks negative isolates characterized from the similar settings in our previous studies. All nine bla_CTXM-15 positive E. coli isolates were observed to be ESBL producers (Table 1), although further screening for other groups of CTX-M genes, and other ESBL classes are warranted. In concordance with the antimicrobial susceptibility results, molecular detection of antimicrobial resistance genes showed a low prevalence of sul1 (sulphonamide resistance), tetA (tetracycline resistance), aac(6^′)-Ib (aminoglycoside resistance), bla_CTX-M-15 (extended spectrum-β-lactamases) and bla_TEM-1 (broad spectrum-β-lactamases) genes (Table 1).

The prevalence of colibactin producing E. coli was found to be moderate among clinical E. coli isolates in our collection. These isolates harbored multiple virulence genes/traits and demonstrated relatively low antimicrobial resistance. These findings comprise essential baseline data required to understand aspects of functional molecular infection epidemiology of possibly genotoxic phenotypes of E. coli and their clinical significance. We hope to extend these studies at genomic and landscape scales to gain further insights into evolution, adaptation and dissemination of such isolates at clinical, community and ecosystem levels.

AS designed and performed all experiments with assistance from AR, SJ, AH, and SS. AS was responsible for all of the data and served as the guarantor for the manuscript. RB, MA, and LW participated in detailed discussions, advised on the interpretation of some of the results and, contributed to the writing and editing of the manuscript. NA conceived the study and provided overarching supervision, laboratory facilities and resources, interpreted and discussed results, and wrote/edited the draft and final versions of the manuscript. All authors contributed to the development of the manuscript and its display items.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.