TITLE: Isolation and phenotypic characterization of antibiotic resistant E.coli and Salmonella from food animal feces
AUTHORS: Rosine Manishimwe, Paola M. Moncada, Marie Bugarel, H. Morgan Scott, Guy H. Loneragan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol was designed to help researchers with limited laboratory resources to generate valuable information on the status of antibiotic resistance among indicator  </span><span style = "font-style:italic;">E.coli</span><span> and pathogenic </span><span style = "font-style:italic;">Salmonella</span><span> from food producing animals.   </span></div><div class = "text-block"><span>The protocol uses less technology intensive methods such the disk diffusion and the combination disk test to estimate isolate- and sample-level prevalence of </span><span style = "font-style:italic;">E.coli </span><span>and </span><span style = "font-style:italic;">Salmonella</span><span>  resistance to most critical important antibiotics.</span></div><div class = "text-block"><span>In this protocol, agar-based media  without antibiotic-supplements are used to isolate </span><span style = "font-style:italic;">E. coli</span><span> and </span><span style = "font-style:italic;">Salmonella</span><span>  from fecal samples. Besides, agar-based media supplemented with antibiotics  are used to screen for </span><span style = "font-style:italic;">E. coli</span><span> and </span><span style = "font-style:italic;">Salmonella</span><span> resistant to third-generation cephalosporins  and to screen for</span><span style = "font-style:italic;"> E. coli </span><span>and </span><span style = "font-style:italic;">Salmonella</span><span>  with low susceptibility to quinolones. Furthermore, all bacterial isolates are tested for their susceptibility to a panel of twelve antibiotic disks using the disk diffusion.  Finally, the combination disk test is used to test for phenotypic production of extended spectrum beta-lactamases (ESBLs) or AmpC among all bacterial isolates resistant to third-generation cephalosporins using a second panel of 12 antibiotic disks.</span></div></div>

[STEPS]
?. [DAY 1 : Preparation of fecal samples]
For each fecal sample Use a wooden scoopula to weight   of feces
10 g
?. [DAY 1 : Preparation of fecal samples]
Transfer the feces in a 710mL Whirl Pak® bag (Whirl-Pak, Madison, Wisconsin)  with a filter
?. [DAY 1 : Preparation of fecal samples]
Add   of  to the bag
90 mL
?. [DAY 1 : Preparation of fecal samples]
Stomach at 230 rpm for
?. [DAY 1 : Preparation of fecal samples]
Put bags in a container and incubate at
42 °C
Overnight non-selective enrichments
?. [DAY 2 : Isolation of E. coli]
Remove the bags from the incubator
?. [DAY 2 : Isolation of E. coli]
Use a   loop to streak the non-selective enrichments onto    plates  with no antibiotic supplements to isolate non-type-specific E. coli.
10 µl
?. [DAY 2 : Isolation of E. coli]
Use another     loop to streak the non-selective enrichments onto  plates supplemented with  1.0 mg/L of   to screen for  E. coli resistant to third-generation cephalosporins.
10 µl
?. [DAY 2 : Isolation of E. coli]
Use another    loop to streak the non-selective enrichments onto  plates  supplemented with 0.5 mg/L of  to screen for  E. coli not susceptible to quinolones.
10 µl
?. [DAY 2 : Isolation of E. coli]
Incubate all agar-plates at
37 °C
Bacterial colonies with E.coli typical morphology
?. [DAY 2 : Isolation of Salmonella]
For each of the overnight non-selective enrichmentPipet
1 mL
?. [DAY 2 : Isolation of Salmonella]
Transfer it  into   of  in a culture tube and vortex mix
9 mL
?. [DAY 2 : Isolation of Salmonella]
For each of the overnight non-selective enrichment Pipet another
1 mL
?. [DAY 2 : Isolation of Salmonella]
Transfer it into   of  in a culture tube and vortex mix
9 mL
?. [DAY 2 : Isolation of Salmonella]
Incubate all  culture tubes at
42 °C
Selective Enrichment for Salmonella
?. [DAY 3 : Isolation of E.coli]
Remove all MacConkey agar-plates from the incubator
?. [DAY 3 : Isolation of E.coli]
On each MacConkey agar-plate : Inspected agar-plates to identify growth of bacterial colonies with typical morphology of E. coli  :  pink, convex, circular and dry colonies with a surrounding pink zone.
?. [DAY 3 : Isolation of E.coli]
From each type of MacConkey agar-plate (with and without antibiotics), select one isolated E.coli typical colony
?. [DAY 3 : Isolation of E.coli]
Use  a  loop to re-streak the colony  onto a similar MacConkey agar-plate type
1 µl
?. [DAY 3 : Isolation of E.coli]
Incubate all the agar-plates at
37 °C
Isolated and pure colonies of E.coli
?. [DAY 3 : Isolation of Salmonella]
Remove Salmonella selective enrichments from the incubator
?. [DAY 3 : Isolation of Salmonella]
For each of the enriched selective broths:- Use a   loop to streak  the enriched selective broth onto (BGS) to isolate Salmonella- Use another    loop to streak  the enriched selective broth onto    (XLD) to isolate Salmonella
10 µl
10 µl
?. [DAY 3 : Isolation of Salmonella]
For each of the enriched selective broths:- Use another loop to streak  of the selective broth onto  supplemented with 0.5 mg/L of  to screen for Salmonella not susceptible to quinolones- Use another loop to streak  of the selective broth onto     supplemented with 0.5 mg/mL of  to screen for Salmonella not susceptible to quinolones
10 µl
10 µl
?. [DAY 3 : Isolation of Salmonella]
Incubate all the inoculated plates at
37 °C
Bacterial colonies with  Salmonella typical morphology
?. [DAY 4 : Isolation of E.coli]
Remove all MacConkey plates from the incubator
?. [DAY 4 : Isolation of E.coli]
From each of the agar-plates, pick one E.coli typical  colony Transfer the colony on a filter paper
?. [DAY 4 : Isolation of E.coli]
- Drop 1 or 2 droplet of on the bacterial colony- If the colony turns blue : the colony is E. coli ( indole positive )
E.coli colonies confirmed
?. [DAY 4 : Isolation of E.coli]
For each of the MacConkey agar-plates with confirmed E.coli colonies- Use a  loop to transfer one well isolated colony into  of   in a culture tube- Vortex mix
1 µl
9 mL
?. [DAY 4 : Isolation of E.coli]
Incubate all the inoculated  culture tubes at
37 °C
?. [DAY 4 : Isolation of Salmonella]
Remove all Salmonella selective agar-plates from the incubator
?. [DAY 4 : Isolation of Salmonella]
Inspected agar-plates to identify growth of bacterial colonies with typical morphology of Salmonella : - On Brilliant Green Sulfa: pink, circular, dry, convex colonies - On Xylose Lysine Deoxycholate : black, circular convex colonies
?. [DAY 4 : Isolation of Salmonella]
From each type of Salmonella selective agar-plates, select one isolated Salmonella typical colony Use  a  loop to re-streak the colony  onto a similar agar-plate type
1 µl
?. [DAY 4 : Isolation of Salmonella]
Incubate all the inoculated plates at
37 °C
Isolated and pure colonies of Salmonella
?. [DAY 5 : Isolation of E.coli]
Remove inoculated culture tubes (with E.coli in tryptic soy broth) from the incubator
?. [DAY 5 : Isolation of E.coli]
-For each of the E.coli  culture - Use a pipettor to transfer   of E. coli culture into a microcentrifuge tube containing   of glycerol - Vortex mix
850 µl
150 µl
?. [DAY 5 : Isolation of E.coli]
Keep the isolates at    or  until further processing
-20 °C
-80 °C
?. [DAY 5 : Isolation of Salmonella]
Remove all Salmonella selective agar-plates from the incubator
?. [DAY 5 : Isolation of Salmonella]
For each of the plates:- Use a  loop to pick one isolated colony - Stab streak the colony onto   slants in culture tubes for biochemical testing
1 µl
Don't discard the agar-plates, keep them at 4 °C
?. [DAY 5 : Isolation of Salmonella]
Incubate all inoculated Lysine Iron Agar slants  at
37 °C
?. [DAY 3 : Isolation of Salmonella]
For each of the enriched selective broths:- Use another loop to streak of the selective broth onto  supplemented with 1.0 mg/L of   to screen for Salmonella resistant to third-generation cephalosporins- Use another loop to streak  of the selective broth onto      supplemented with 1.0 mg/L of  to screen for Salmonella resistant to third-generation cephalosporins
10 µl
10 µl
?. [DAY 6 : Isolation of Salmonella]
Remove culture tubes with Lysine Iron Agar slants from the incubator
?. [DAY 6 : Isolation of Salmonella]
For each of the Salmonella selective agar-plates with confirmed Salmonella colonies (Positive Lysine Iron Agar):- Use a loop to transfer one well isolated colony into   of   in a culture tube- Vortex mix
1 µl
9 mL
?. [DAY 6 : Isolation of Salmonella]
Inspect the slants for typical Salmonella reactions:      - Notice a bacterial growth on the slant     - The slant and butt are purple      - The butt blackening with H2S positive
?. [DAY 6 : Isolation of Salmonella]
Incubate all the inoculated  culture tubes at
37 °C
?. [DAY 7 : Isolation of Salmonella]
Remove inoculated culture tubes (with Salmonella in tryptic soy broth) from the incubator
?. [DAY 7 : Isolation of Salmonella]
For each of the Salmonella  culture :- use a pipettor to transfer  of the culture into a microcentrifuge tube containing  of glycerol - Vortex mix
850 µl
150 µl
?. [DAY 7 : Isolation of Salmonella]
Keep the isolates at   or   until further processing
-20 °C
-80 °C
?. [DAY 3 : Antibiotic susceptibility testing]
- After the antimicrobial susceptibility testing- Record  diameters of inhibition zones in mm
?. [DAY 3 : Antibiotic susceptibility testing]
Compare inhibition zone diameters to the CLSI clinical breakpoints to classify bacterial isolates  as resistant, intermediate or susceptible.
For Colistin : Use breakpoint by Galani et al., 2008
?. [DAY 3 : Antibiotic susceptibility testing]
For E.coli For Salmonella
1. Antibiotic resistance results for non-type-specific E.coli  Isolated from MacConkey agar without antibiotics: E.coli isolate-level prevalence of resistance to 12 antibiotics.2. Antibiotic resistance results for presumptive E.coli  resistant to third-generation cephalosporins isolated from MacConkey agar  with cefotaxime: Confirmed E.coli resistant to third-generation cephalosporins                   - Proportion of samples with E.coli resistant to third-generation cephalosporins                   - Calculate the sample-level prevalence for third-generation cephalosporin resistance :              Number of samples with  Confirmed E.coli resistant to third-generation cephalosporins                ------------------------------------------------------------------------------------------------------------------------------------------                                            Total number of samples collected
                  - Establish antibiotic resistance profiles of third-generation cephalosporin resistant E.coli3. Antibiotic resistance results for presumptive E.coli  resistant to quinolones isolated from MacConkey agar with ciprofloxacin:  Confirmed E.coli not susceptible (resistant and intermediate) to quinolones                  - Proportion of samples with E.coli not susceptible to quinolones                  - Calculate the sample-level prevalence for low susceptibility to quinolone :              Number of samples with  Confirmed E.coli not susceptible to quinolones                ------------------------------------------------------------------------------------------------------------------------------------------                                            Total number of samples collected
                  - Establish antibiotic resistance profiles of E.coli with low susceptibility to quinolones
1. Antibiotic resistance results for Non-type-specific Salmonella isolated from agar media without antibiotics: Salmonella isolate-level prevalence of resistance to 12 antibiotics.2. Antibiotic resistance results for presumptive Salmonella resistant to third-generation cephalosporins isolated from agar media with cefotaxime: Confirmed Salmonella resistant to third-generation cephalosporins                   - Proportion of samples with Salmonella resistant to third-generation cephalosporins                   - Calculate the sample-level prevalence for third-generation cephalosporin resistance :              Number of samples with  Confirmed Salmonella resistant to third-generation cephalosporins                ------------------------------------------------------------------------------------------------------------------------------------------                                            Total number of samples collected
                  - Establish antibiotic resistance profiles of third-generation cephalosporin resistant Salmonella3. Antibiotic resistance results for presumptive Salmonella not susceptible to quinolones(resistant and intermediate)  isolated from agar media with ciprofloxacin:  Confirmed Salmonella not susceptible to quinolones                  - Proportion of samples with Salmonella  not susceptible to quinolones                  - Calculate the sample-level prevalence for low susceptibility to quinolone :              Number of samples with  Confirmed Salmonella not susceptible  to quinolones                ------------------------------------------------------------------------------------------------------------------------------------------                                            Total number of samples collected
                  - Establish antibiotic resistance profiles of  Salmonella not susceptible to quinolones
?. [DAY 3 : Antibiotic susceptibility testing]
Incubate all inoculated plates at
37 °C
?. [DAY 3 : Antibiotic susceptibility testing]
Identify all E.coli and Salmonella isolates resistant to third-generation cephalosporins despites the media of isolation
?. [DAY 3 : Antibiotic susceptibility testing]
These isolates will be tested for beta-lactamases production  using the combination disk test  according to CLSI guidelines
?. [DAY 3 : Antibiotic susceptibility testing]
Streak identified third-generation cephalosporin resistant E.coli and Salmonella isolates on 5% sheep blood agar plates
?. [DAY 4 : Antibiotic susceptibility testing]
Follow the CLSI  protocol for the combination disk test to detect the production of extended spectrum beta-lactamases (ESBLs)
Apply antibiotic disks on Muller Hinton agar according to the plate 2 map
?. [DAY 5 : Antibiotic susceptibility testing]
Identify ESBL or AmpC producing bacterial isolates
ESBL producers: If the presence of clavulanic acid increases the inhibition zone diameters by at least 5 mm for either ceftazidime or cefotaxime, then the test is considered positive for the production of an ESBL. 
AmpC producers: If the presence of clavulanic acid doesn’t increase the inhibition zone diameters by at least 5 mm for either ceftazidime or cefotaxime, then the test is considered positive for the production of an AmpC.
Precaution while deciding if a bacterial isolate has an ESBLs or AmpC phenotype:     The combination disk test  can  produce several false positive E. coli producing extended spectrum beta-lactamases, especially when the results (difference in inhibition zone diameter caused by the clavulanic acid ) are close to 5 mm decision point.
?. [DAY 5 : Antibiotic susceptibility testing]
- After the combination disk test- Record  diameters of inhibition zones in mm
?. [DAY 5 : Antibiotic susceptibility testing]
Compare inhibition zone diameters to the CLSI clinical breakpoints to classify bacterial isolates  as resistant, intermediate or susceptible.
?. [DAY 1: Antibiotic susceptibility testing ]
Remove bacterial isolates to be tested from the freezer
?. [DAY 1: Antibiotic susceptibility testing ]
Use a   loop to streak the bacteria isolate on 5% sheep blood agar plates
10 µl
?. [DAY 1: Antibiotic susceptibility testing ]
Incubate the all inoculated blood agar-plates at
37 °C
?. [DAY 2: Antibiotic susceptibility testing ]
Remove all  blood agar-plates from the freezer
?. [DAY 2: Antibiotic susceptibility testing ]
Follow the Clinical and Laboratory Standards Institute (CLSI)'s  guidelines for antibiotic susceptibility testing using the disk diffusion method
Apply antibiotic disks on Muller Hinton agar according to the plate1 map  bellow