The collection of stool samples from children and animals will be performed in the following way and with the material as described below:
Children: a) A stool sampling kit that includes: 1) Sterile wide mouth plastic container along with a spoon for transferring the stool to the container; 2) A sterile polyethylene sheet measuring 15x15cm for collecting stool. The container and polythene sheet to be enclosed in a zip locked polyethylene bag. b) The stool sampling kit will be distributed in the households of the selected children one day prior to the day of sample collection, c) The caregivers will be trained by the research assistants how to use the stool sampling kit to collect early morning stool on the day of sample collection, d) The parents/caregivers will be re-briefed regarding stool collection procedure at every time point of sample collection.
Animals: Fresh stool samples from animals will be collected in a sterile container using spatula with a precaution to avoid contamination by soil.
Laboratory methods
1)
Microbiological and molecular methods
a)
Isolation and confirmation of E. coli
E.coli will be isolated from the following sources: stools from cohort of children, animal stool samples, household- and source-drinking water from village and village waste- water.
The stool samples (human and animal) will be directly plated on selective (for all coliforms, inhibits other bacteria) and differential (differentiate E. coli from other coliforms) media HiCrome® Agar (HiMedia laboratories, Mumbai, India).
The household- and source-drinking water will be directly subjected to Membrane Filtration Technique (MFT) while waste-water will undergo sufficient serial dilution with normal saline (0.9 %) depending on the visual turbidity [
16]. The filtration will be done by using through 47 mm diameter and 0.45 μm pore size nylon membrane filters for a minimum of two and half hours [
16,
17]. If less than six
E.coli colonies after 24 h of incubation, the original sample will further be filtered once with lower dilution. Following filtration the membrane will be placed on HiCrome® Coliform Agar plates incubated at 37 °C for 24 h [
17]. The number of colonies of coliforms and
E.coli (identified as blue-violet colonies on HiCrome® Coliform Agar) will be estimated after considering the dilution factor of total number of colony forming units (CFU) of coliforms and
E. coli per 100 ml of filtered sample. The number of CFUs per 100 ml of filtered sample will be estimated for the number of total coliforms. The number of
E. coli (identified as blue-violet colonies on HiCrome® Coliform Agar) will be counted. The comparison of CFUs will provide a snapshot of coliform load and the relative abundance of other coliforms over
E. coli or vice versa in the samples tested [
17].
All the cultured plates will be photographed. A total of six isolated colonies of
E.coli (to get at least five confirmed
E.coli per sample to be able to detect variation) and two colonies of different coliforms (to store for future study) will be placed in 3 ml of Luria-Bertani (LB) media [
18]. The LB broth will be incubated for 24 h in shaking incubator to get sufficient growth. Thereafter 2 ml of the broth will be processed for DNA extraction while 1 ml of broth will be cryo-preserved at -80 °C in 1:1 solution with sterile autoclaved glycerol [
18]. Confirmation of all
E.coli isolates will be done by PCR (see below).
b)
Antibiotic susceptibility testing
Antibiotic susceptibility testing (AST) will be performed by the Kirby-Bauer disc diffusion method. Disc strengths used will be as recommended by the current Clinical and Laboratory Standards Institute (CLSI) guidelines [
19]. Antibiotics tested will be: ampicillin, ceftriaxone, cefepime, ciprofloxacin, tetracycline, tigecycline, meropenem, imipenem, gentamicin, amikacin, sulphamethoxazole, co-trimoxazole, nalidixic acid and nitrofurantoin. The choice of antibiotics is based on the CLSI guidelines and use of antibiotics in the study area for the treatment of infections caused by gram negative coliforms. The diameter of inhibition zones will be measured to the nearest millimeter independently by two technical experts. If there will be variation of more than three millimeters between two readings, the procedure will be repeated. If any further discrepancy will be there in zone diameter the susceptibility pattern will be confirmed by minimum inhibitory concentration method according to CLSI guidelines. For calculations, all isolates with intermediate susceptibility readings will be considered resistant. Results will be recorded in the form of mono-resistance (resistance to a single antibiotic), co-resistance (resistance to two antibiotics of same or different groups) and multi-drug resistance (MDR) (resistance to at least three different antibiotic classes) [
20]. Extended Spectrum Beta-Lactamase (ESBL) will be detected phenotypically by the combined disc diffusion method using Muller Hinton agar with cefotaxime (30 μg) and cefotaxime/clavulanic acid (30/10 μg) and ceftazidime (30 μg) and ceftazidime/clavulanic acid (30/10 μg) according to CLSI guidelines [
19].
Various quality control measures will be taken at each step of the procedure to ensure laboratory quality check. The quality controls will be: a) checking of samples from each batch of all types of prepared media b) confirmation of sterility of inoculation loops, swabs and micro tips; c) check of reagents - normal saline and distilled water; d) checking of laminar flow at the beginning and at the end of procedures; Reference strains will be;
E. coli ATCC 25922 strain for AST; an ESBL-producing
K. pneumoniae ATCC 700603 strain as per CLSI guideline [
19].
The confirmation of all
E.coli isolates will be done through PCR based on genus-specific primers and whole genome re-sequencing will be used for pan-genomic identification of molecular determinants of observed antibiotic resistance in
E. coli.
The genomic DNA will be extracted using standard procedures from pure
E.coli isolates and confirmed by PCR based on genus-specific primers. The confirmed
E.coli isolates per time point will be classified into six phenotypically different categories based on the susceptibility patterns to the tested antibiotics – purely susceptible (where the isolate is susceptible to all the fourteen antibiotics tested), purely resistant (where the isolate is resistant to all the fourteen antibiotics tested), purely intermediate (where the isolate is intermediately susceptible/resistant to all the fourteen antibiotics tested), any resistance (where the isolate is resistant to one or more but not all the fourteen antibiotics tested), any intermediate (where the isolate is intermediately susceptible/resistant to one or more but not all the fourteen antibiotics tested) and mixed (where the isolate shows a mixed combination susceptible, resistant and intermediately susceptible/resistant to the fourteen antibiotics tested). The genomic and plasmid DNA will be extracted and purified using standard procedures from confirmed and pure
E. coli isolates which exhibit antibiotic resistance and susceptibility, respectively, to the selected antibiotics. DNA samples from all resistant isolates and from 20 % of susceptible isolates (as controls) will be subjected to whole genome sequencing using Ion-proton Technology [
21]. The sequences will be assembled on the reference genome of
E. coli available and the abundance of known antibiotic resistance genes will be estimated using the depth of coverage of the sequences assembled on the known loci of the resistance genes identified in the reference genome.
PCR-positive and PCR-negative controls will be processed in all PCR assays as: a) E. coli ATCC 25922 strain DNA, b) positive PCR product from previous PCR assay, c) an extraction control (with all the steps but without any E. coli), (b) a reaction tube with substitution of distilled water for the test template, and (c) a sample that previously yielded a negative result on PCR. In a run, the PCR-negative controls will be processed first, followed by test samples and then PCR-positive control. The run will be accepted when all the PCR controls will be consistent with the results.