**4. Discussion**

We studied antibiotic resistance and selected antibiotic resistance genes in human stool together with their shared and neighboring environment in a rural community from Central India with a 'one-health' approach. We found that the antibiotic resistance pattern and its genetic make-up are essentially the same in commensal bacteria from humans and their environment. The percentage of resistant isolates, including MDR (Figure 1A,B), is higher in humans than in the environment (animal stool and water samples), but the load (number of resistant isolates/sample) is higher in the environment than in humans. The appearance of antibiotic-resistant bacteria in healthy individuals and their environment should be evaluated together to accomplish effective antibiotic resistance control.

The antibiotic resistance profile including certain patterns of co-resistance and MDR (i.e., cephalosporin-quinolone-penicillin, sulphonamide + tetracycline + cephalosporin, quinolones + carbapenem + sulfonamide or + tetracycline) in *E. coli* obtained from humans, animals, sourceand household-drinking water are high (57%–69%) in our study area. The presence of co-resistance and MDR signifies that there might be high use of antibiotics inhuman and non-human use in the community. The non-human use of highly-important antibiotics contributes to the resistance against a range of antibiotics [1,2,15]. Van den Bogaard et al. and others have shown that the selective pressure on the commensal microflora due to antibiotic misuse determine the frequency and pattern of resistance in a population [16]. The relatively cheap and commonly prescribed drugs commonly favour high co-resistance [17,18].

We found similar patterns of co-resistance, MDR, and gene carriage in various sources. Nearly 90% of MDR *E. coli* isolates are carrying plasmid-encoded (*blaCTX-M1*, *blaCTX-M9*, *qnrS*, and *qnrS*) genes, which may indicate the possible spread of the resistance genes between diverse sources. This is similar to another study from India [19]. *CTX-M*–producing *E. coli* is the dominant MDR *E. coli* in all parts of Asia and of major clinical significance [20]. The patterns of antibiotic use in the community favor the persistence of plasmids carrying antibiotic resistance genes. The intestine is considered as a 'hot spot' for the transfer of resistance genes between bacteria as the exposure of frequently-used antibiotics to

a high density of bacteria favours evolution and dissemination of antibiotic resistance by cell-to-cell contact [21,22]. Additionally, the existing various species of MDR bacteria, as we noticed in MDR non-*E. coli* coliform species, (Table 6, Figure 1C), might also be contributing to the spread of antibiotic resistance genes in the intestine with *E. coli*.

The resistant isolates are distributed in a higher percentage of drinking-water samples compared to human samples. In rural communities, the high level of bacterial contamination is reported in source-water to the extent that it lacks the criteria of safe-water supply for domestic purposes [23]. Studies illustrate that surface water contamination occurs mainly from livestock operations and human sewage and that decreasing livestock access to surface water reduced the fecal coliforms levels by an average of 94% [24]. Treatment processes of water, however, might further result in a selective increase of antibiotic-resistant bacteria and might, therefore, increase the occurrence of multidrug-resistant organisms [11,25]. It has also been observed that the microbiological quality of water in vessels in households is lower than that at the source, suggesting that bacterial contamination is widespread during collection, transport, storage, and drawing of water [26].

In our study, phylogenetic group D (extra-intestinal virulent) *E. coli* isolates with resistant genes are more often found from human stool than from environmental samples (30%–52% vs. 0%–24%). It has been reported that co-location of genes in plasmids not only results in resistance to multiple antibiotics, but also in the increased presence of virulence determinants, which facilitates infections [27]. Indeed, the exposure of commensal bacteria to antibiotics increases the carriage level of resistant organisms that might result in the transmission of resistance to a virulent organism [28]. Johnson et al. [29] reported the horizontal transfer of antibiotic resistance not only between isolates from one source to another, but also from resistant to susceptible isolates in the same source. The number of virulent strains carrying resistant genes in human commensal samples is a matter of public health concern, as it may give rise to infection with an increased risk of treatment failure.

We have not identified any *E. coli* or non-*E. coli* isolates (including all forms of MDR strains) with colistin resistance or *mcr-1* gene carriage. With the emergence of MDR and extensive drug resistant (XDR) strains of Gram-negative bacteria, colistin is considered as one of the few last resort antibacterial agents. Recently, sporadic clinical cases infected with colistin-resistant *E. coli* carrying the *mcr-1* gene has been described in India [30,31]. The plasmid-mediated *mcr-1* gene to colistin resistance is a matter of global alarm as its spread within the human commensal flora could lead to epidemics of virtually untreatable infections. Measures with the 'one-health' approach, such as colistin susceptibility testing of MDR isolates from patients, testing of food, animal, environmental isolates, and the reduction of colistin use in food-producing animals would be crucial for effective minimization of *mcr*-*1*-positive commensal dissemination in the community and healthcare facilities.

Our study has some methodological limitations. The study, being from a village, cannot be generalized. There is no reason, however, to believe that the situation in this village is very different from many other villages with similar low socio-economic levels in India. Additionally, in our study, none of the carbapenem-resistant isolates (six imipenem resistant and 29 meropenem resistant isolates) from all sources are carrying any of the tested (*NDM-*1, *VIM*, and *IMP*) carbapenemases encoding genes. Studies showed the presence of *OXA-*48 and *NDM-*1 genes in clinical isolates from India [32,33]. However, in another study from our setting, we did not find any of these genes in either clinical or in hospital waste water [34]. We, however, cannot rule out some different resistance mechanisms in these isolates, which we have not tested. Although our study involves a limited number of animals and sewage water samples, the comparison of multiple types of environmental samples with apparently healthy human samples from community provides us better understanding about the current scenario of antibiotic resistance at the community level. This is required in scientific research for establishing effective measures to mitigate resistance in clinically relevant bacteria.
