Distribution of Antibiotic-Resistant Enterobacteriaceae Pathogens in Potable Spring Water of Eastern Indian Himalayas: Emphasis on Virulence Gene and Antibiotic Resistance Genes in Escherichia coli

Abstract

Every year millions of people die due to fatal waterborne diseases around the world especially in developing countries like India. Sikkim, a northeastern state of India, greatly depends on natural water sources. About 80% of the population of Sikkim depends on natural spring water for domestic as well as agricultural use. Recent waterborne disease outbreaks in the state raises a concerning question on water quality. In this study, we analyzed water quality especially for the detection of Enterobacteriaceae members from four districts of the state. Isolation with selective culture media techniques and taxonomic characterization of Enterobacteriaceae bacteria with 16S rRNA gene showed the prevalence of Escherichia coli (37.50%), Escherichia fergusonii (29.41%), Klebsiella oxytoca (36.93%), Citrobacter freundii (37.92%), Citrobacter amalonaticus (43.82%), Enterobacter sp. (43.82%), Morganella morganii (43.82%), Hafnia alvei (32.42%), Hafnia paralvei (38.74%), and Shigella flexneri (30.47%) in the spring water of Sikkim. Antibiotic susceptibility test (AST) showed resistance of the isolates to common antibiotics like ampicillin, amoxicillin as well as to third generation antibiotics like ceftazidime and carbapenem. None of the isolates showed resistance to chloramphenicol. E. coli isolated from spring water of Sikkim showed presence of different virulence genes such as stx1 (81.81%), elt (86.66%), and eae (66.66%) along with resistance gene for ampicillin (CITM) (80%), quinolones (qnrB) (44.44%), tetracycline (tetO) (66.66%), and streptomycin (aadA1) (66.66%). The data indicates a high incidence rate of multiple antibiotic resistant enteric bacteria in the spring water of Sikkim. Additionally, the presence of enteric bacteria in the water samples indicates widespread fecal contamination of the spring water.

Keywords: Enterobacteriaceae; antibiotic resistance gene; multidrug resistant (MDR); spring; virulence gene; water quality; waterborne pathogens.

FIGURE 1

The photograph showing (A) Spring Water and (B) Map of the study site (blue dots specify the villages from where samples were collected) as previously reported (Singh et al., 2019). The map was collected from the Spring Detail (http://www.Sikkimsprings.org/dv/view.php) [Map chart was prepared in Microsoft Excel (office 365)] (Supplementary File: Table S1).

FIGURE 2

Dominant bacteria present in the spring water of Sikkim. ANOVA didn’t show any significant difference between the data.

FIGURE 3

16S rRNA PCR gel electrophoresis (L, Ladder; GD, Genomic DNA; PC, positive control of E. coli amplified product; NC, Negative control; 1–13: isolates code).

FIGURE 4

Antibiotic resistance pattern in (A) Escherichia coli, (B) Escherichia fergusonii, (C) Enterobacter sp., (D) Klebsiella oxytoca, (E) Citrobacter freundii, (F) Citrobacter amalonaticus, (G) Shigella flexneri, (H) Morganella morganii, (I) Hafnia alvei, (J) Hafnia paralvei (showed statistical significance at p-value ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^****p < 0.0001).

FIGURE 5

(A) The graph showing the MAR index of bacterial species from different districts of Sikkim. (B) Statistical significance between the data of different districts (^∗showed statistical significance at p-value ^****p < 0.0001).

FIGURE 6

Prevalence of virulence gene in Escherichia coli isolated from spring water of different districts of Sikkim (+ve, positive control; –ve, negative control).

FIGURE 7

Prevalence of antibiotic resistance gene in Escherichia coli isolates from spring water of different districts of Sikkim. ^∗Showed statistical significance at p value ^∗∗p < 0.01.

FIGURE 8

Correlation between antibiotic resistance gene and virulence gene of Escherichia coli isolated from spring water of Sikkim (+ve, positive control; –ve, negative control; (^∗ = statistically significant).