Identification of E. coli pathotypes in association with blood stream infections is limited in many developing countries because routine diagnostic screens only the micro-organism, the conventional microbiological testing is unable to distinguish between normal flora and pathogenic strains of E. coli [27]. Entero pathogenic E.coli (EPEC) is a major etiological agent of infant diarrhea predominantly in developing countries [28,29,30]. ETEC defined by their production of the plasmid-encoded heat-labile (LT) and heat-stable (STIa/STIb) toxins is the leading cause of traveler’s diarrhea [31]. ETEC & EAEC are the most commonly identified pathogens in our study. It was found that the proportion of ETEC was significantly higher among the blood isolates as compared to fecal E.coli isolates (p = 0.029). However, we did not find any significant difference in the proportion of EAEC between blood and fecal E.coli isolates. Our data showed a large proportion of the isolates with localized adherence, which is a known characteristic of EPEC however none of the isolates were found positive for EPEC by PCR.

All the blood isolates analysed by ERIC-PCR were found to be clustered into two groups while fecal E.coli isolates were clustered into four groups. The principal component analysis (PCA) of the blood E.coli isolates were more similar among themselves with respect to the ERIC band profiles while the fecal E.coli isolates were more diverse. We can correlate the observation of PCA and cluster analysis with the hypothesis that a single strain from the gut may be the source of endogenous infection which may prompt an “overspill” bacteraemia.

Antibiotic susceptibility results showed that more than 70% of the fecal E.coli isolates and more than 90% of the blood isolates were resistant to all of the cephalosporins tested. Among the fecal isolates, we observed a slight decrease in susceptibility to cephalosporin in combination with an inhibitor clavulanic acid. Fecal E.coli isolates were resistant to Cefpodoxime/clavulanic acid (89%), Ceftazidime/clavulanic acid (55%) and Cefotaxime/clavulanic acid (81%). While blood E.coli isolates were resistant to Cefpodoxime/clavulanic acid (98.5%), Cefotaxime/clavulanic acid (90%), Ceftazidime (91%) and Ceftazidime/clavulanic acid (69%). A significant decrease in susceptibility to Ceftazidime in combination with clavulanic acid was observed as compared to Ceftazidime alone.

However, among the other classes of antibiotics aminoglycosides, fluoroquinolone, and monobactams studied, 85% of the fecal isolates were resistant to Ciprofloxacin, 75% were resistant to Piperacillin, 72% for Aztreonam, 34% for Amikacin and 51% for Gentamicin. Whereas, 91% of the blood isolates were resistant to Ciprofloxacin, 76% were resistant to Piperacillin, 88% for Aztreonam, 63% for Amikacin, and 70% for Gentamicin. In comparison to blood isolates, fecal isolates were more susceptible to amikacin (34% verses 63%) and gentamicin (51% verses 70%). Overall, 68% of the blood isolates were found to be ESBL producers whereas 44% of the fecal isolates were confirmed as ESBL producers by observing the susceptibility patterns in disc synergy tests with clavulanic acid.

Antibiotic resistance in E.coli can be conferred by both chromosomal and plasmid-encoded genes. Resistance to ciprofloxacin was observed in concurrence with cephalosporin resistance. ESBLs degrade the β-lactam moiety of penicillin derivatives, cephalosporins, monobactams, and Carbapenems. The ESBL genes are frequently encoded on transferable plasmids that encode resistance genes. Acquisition of such resistant genes by commensal or fecal isolates leads to MDR pathogens. The three major groups of ESBL enzymes are TEM, SHV and CTX-M. Among the CTX-M-type ESBLs, CTX-M-15 is widely distributed worldwide [32], and are the most prevalent in India [33]. In our study, we found that 83% of the blood E.coli isolates whereas 90% of the fecal isolates showed CTX-M15; CTXM-15 producing isolates were reported to have reduced susceptibility to Cefepime [34]. Our results are in line with the observation since 91 and 79% of the blood and fecal isolates respectively are resistant to Cefepime. Therefore, Cefepime which is a fourth generation cephalosporin is administered intravenously and used primarily for treatment of pneumonia, UTIs, and intra-abdominal infections, can no longer be a choice of drug.

The gut has been thought to be a repository of pathogens and an incredible source for the development of antibiotic resistance [35]. Overall the carriage of ESBL genes is more in fecal isolates than that of the blood isolates CTX-M9 (63% verses 21%), CTX-M15 (90% verses 83%), TEM (88% verses 74%), and OXA-1 (96% verses 74%). Previously, it was demonstrated as an intestinal colonization by gram-negative organisms before the onset of the disease [36]. We propose a similar scenario since we have found CTX-M15 as a predominant ESBL gene among the fecal isolates in our study. There is a significant increase in the prevalence of CTX-M enzyme producing E.coli worldwide. We report the detection of CTX-M group 9 genes and the CTX-M15 as a predominant ESBL gene among fecal isolates. Our results also highlight the importance of studying gut flora in assessing the changing repertoire of organisms to investigate the pattern of antibiotic susceptibilities. We found prevalence of ESBL-producers more among the blood isolates whereas the isolates showing the ESBL genes were found predominantly among fecal isolates.

The high resistance patterns against all of the 15 antibiotics we studied compelled us to further analyse the isolates for the ST131 clone. E.coli ST131 clone is well known affiliated with the worldwide spread of CTX-M15 enzyme. ST131 isolates were associated with extra-intestinal infections, frequently in UTI and bacteraemia. Initially detected in a community, later ST131 isolates were also obtained from health care settings [13]. ST131 strains are MDR and patients with such infections are at high risk of having constrained treatment choices with a prolonged duration of disease. We observed that 80% of the fecal isolates and 92% of the blood isolates belonged to ST131 clone, a sub-clonal lineage of E.coli ST131 that contains the type 1 fimbriae fimH30 (H30) allele and is termed as FimH 30 sub group. Isolates of this sub group were reported resistant to fluoro-quinolones (FQ) with only < 1% of FQ-susceptible isolates. The abrupt expansion and genetic similarity among the H30 strains clue that the emergence of FQ-resistant ST131 strains was driven by clonal expansion and dissemination. Isolates of H30 ST131 sub-clone were found to be resistant for more than 3 antibiotic classes and with CTX-M1. Within the H30 lineage, Price et al., identified a close distinct sub lineage with more extensive antimicrobial resistance profile called H30-Rx. This sub lineage was formed from H30 strains that carried CTX-M15 distinguished from ESBL-negative H30 strains by 3 core genome SNPs [26]. Interestingly, in association with the high resistance to Ciprofloxacin (85% fecal and 91% blood isolates), we found 83% of the fecal and 91.5% of the blood E.coli isolates belonged to FimH 30 and FimH 30-Rx sub groups.

A few studies reported the prevalence of ST131 clone from India. Among them is a study from neonatal isolates, which reported 9% prevalence of ST131 [37]. Another study reported 70% prevalence of ST131 among the ESBL producing strains [38]. MLST is the most accurate and competent method for detection of ST131 clones. But it is tedious and costly. Especially, its application for investigating the MDR clone for clinical diagnosis is not possible. ST131 clone rapid detection assays were previously reported [23, 39]. Even though those methods may validate ST131, the results usually vary and need a confirmation by MLST. It is alarming to find a higher prevalence of ST131 clone isolates in our study evidenced by extreme antibiotic resistant and carriage of ESBL genes.