Of a total of 200 Marmots sampled between July and August 2013, 51 were from Zhongdaxiang (with an altitude of 3599.6 m above sea level (a.s.l)), 120 from Dezhuotan (3025 m a.s.l), and 29 from Dedacun (3625.6 m a.s.l), respectively. The Marmots were captured by cages in the field and sampled in the laboratory of local Centre for Disease Control (CDC). The intestinal contents were collected in 2 ml sterile tubes containing Luria-Bertani (LB) medium in 30% glycerol, which were stored at −20 °C immediately and transported to the laboratory in the National Institute for Communicable Disease Control and Prevention in Beijing. Strains were isolated and confirmed to be STEC by the methods we previously described11 (link),13 (link). Briefly, enriched samples in E. coli broth (Land Bridge, Beijing, China) were examined by PCR for the presence of stx genes with primers stx1F/Stx1R and Stx2F/Stx2R respectively13 (link). PCR-positive enrichments were then streaked onto CHROMagarTM ECC agar (CHROMagar, Paris, France), and MacConkey agar (Oxoid, Hampshire, UK). Colonies resembling E. coli were picked and tested for stx genes by single colony duplex PCR assay. Serotyping, detection of main STEC-related virulence factors (stx, eae, ehxA, efa1, saa, paa, toxB, and astA), and multilocus sequence typing (MLST) were conducted as we previously described11 (link),13 (link).
Ecc agar
Manufactured by CHROMagar
Sourced in France
CHROMagar™ ECC agar is a selective and differential chromogenic culture medium used for the isolation and identification of Escherichia coli (E. coli) and Coliform bacteria from various sample types. The medium contains specific chromogenic substrates that allow the differentiation of E. coli and Coliform colonies based on their color development.
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Lab products found in correlation
Api 20e biochemical test strips, by bioMérieux (4 mentions)
Macconkey agar, by Thermo Fisher Scientific (2 mentions)
Stec agar, by CHROMagar (2 mentions)
Sensititre ast, by Thermo Fisher Scientific (1 mentions)
Luria bertani agar, by Thermo Fisher Scientific (1 mentions)
Rainbow agar o157, by Biolog (1 mentions)
Buffered peptone water (bpw), by BD (1 mentions)
Colilert 18, by IDEXX (1 mentions)
Luria bertani broth, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Merck Group (1 mentions)
13 protocols using ecc agar
1
Shiga Toxin-Producing E. coli in Marmots
2
Isolation of STEC Strains from China
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Fecal samples from diarrheal patients, healthy carriers, animals, and meats were collected from April 2009 to March 2019 in different regions of China. STEC strains were isolated and confirmed by the methods as previously described [49 (link)]. Briefly, the samples were enriched in EC broth and then tested for the presence of stx1/stx2 genes by PCR. stx-positive samples were inoculated into two selective media, i.e., CHROMagarTM ECC agar and CHROMagarTM STEC agar (CHROMagar, Paris, France), for isolation of STEC strains, as described previously [42 (link)]. After overnight incubation at 37 ℃, presumptive colonies were picked and tested for stx genes by single a colony duplex PCR assay. API 20E biochemical test strips (bioMérieux, Lyon, France) were used for a confirmatory test. The stx1/stx2 subtyping was initially conducted by amplifying, sequencing the complete stx1/stx2 genes, and comparing against known stx subtypes, as described previously [5 (link)]. STEC strains carrying the stx2e subtype were selected for subsequent study.
3
Quantification of E. coli in Sewage Samples
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Samples were shaken vigorously before approximately 25 mL was poured into 50 mL Falcon tubes containing sterile 4 mm glass beads, which were added to improve homogenization. The tubes were vortexed for 30 s and immediately used to prepare serial dilutions of the samples using 0.85% NaCl as diluent. Dilutions were plated on ECC agar (100 µL per plate; CHROMagar, Paris, France) with 3, 15 and 3 plates of the 10−1, 10−2 and 10−3 dilutions, respectively. An increased replication of plates inoculated with the 10−2 dilution was included to allow collection of colonies from many separate agar plates. The plates were incubated at 37 °C for 18 h before blue colonies were counted to estimate E. coli concentration. Previous studies have shown that E. coli can be accurately (>99.5%) distinguished using this sewage cultivation procedure [1 (link),2 (link)]. Culture plates were divided into three different pie segments and the two first well-separated blue colonies encountered in each segment, starting from the upper left corner, were picked. To assure non-biased picking of colonies, segments and direction of picking were determined a priori.
4
Isolation and Culture of E. coli
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Eluates from pooled fecal samples were plated on ECC agar (CHROMagar, France) and incubated at 37°C for 20h. A total of 30 to 50 E. coli (blue) colonies from each agar sample were picked, pooled, and incubated in cation-adjusted MHB to log phase. The resulting bacterial suspensions were investigated as described above.
5
Antimicrobial Resistance Profiling of E. coli
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From each sample solution, a volume of 10 μL was cultured on ECC agar (CHROMagar, France), a chromogenic medium allowing the detection of E. coli. One colony per sample showing typical E. coli morphology was selected. E. coli isolates were tested for their susceptibility to 11 antimicrobials belonging to eight classes by Sensititre AST (Thermo Fisher Scientific, UK) comprising colistin (polymyxins), cefpodoxime and ceftiofur (third-generation cephalosporins), azithromycin (macrolides), neomycin and streptomycin (aminoglycosides), amoxicillin (penicillins), enrofloxacin (quinolones), florfenicol (phenicols), doxycycline and oxytetracycline (tetracyclines). Potential production of ESBLs, as indicated by resistance to ceftiofur and/or cefpodoxime, was confirmed by double disc diffusion test according to the CLSI guidelines.
6
Growth-based Antimicrobial Resistance Screening
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Eluates from pooled faecal samples were plated onto ECC agar (CHROMagar, France) and incubated at 37 o C for 20h. A total of 30-50 E. coli (blue) colonies from each agar sample were picked, pooled and incubated in CAMHB to log-phase. The resulting bacterial suspensions were investigated as described above.
The change in AUCadj over age of chicken was modeled using a random effects linear regression.
In order to allow for a nonlinear trend, we used a natural spline for the fixed effect term (knots at 0, 8, 12 and 20 weeks). We allowed for a random intercept and linear trend by age.
The overall costs (per sample) of the method described above were calculated based on expenses on medium, reagents and consumables (excluding staff time, which was estimated separately).
The estimated costs were compared with those incurred in testing one sample by broth microdilution and Etest in Vietnam as of January 2020. Our calculations were based on the investigation of 40 E. coli isolates per sample using the growth-based method, compared with 10 isolates each by broth microdilution and by Etest. We declare that we have no competing interests. TABLE 1 Description of AMU and estimated prevalence of colistin resistance in 36 small-scale chicken flocks stratified by whether farmers administered colistin or not.
The change in AUCadj over age of chicken was modeled using a random effects linear regression.
In order to allow for a nonlinear trend, we used a natural spline for the fixed effect term (knots at 0, 8, 12 and 20 weeks). We allowed for a random intercept and linear trend by age.
The overall costs (per sample) of the method described above were calculated based on expenses on medium, reagents and consumables (excluding staff time, which was estimated separately).
The estimated costs were compared with those incurred in testing one sample by broth microdilution and Etest in Vietnam as of January 2020. Our calculations were based on the investigation of 40 E. coli isolates per sample using the growth-based method, compared with 10 isolates each by broth microdilution and by Etest. We declare that we have no competing interests. TABLE 1 Description of AMU and estimated prevalence of colistin resistance in 36 small-scale chicken flocks stratified by whether farmers administered colistin or not.
7
Isolation and identification of STEC from raw meat
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A total of 131 samples of raw meat, including 64 raw mutton and 67 raw beef were purchased in Jinan city, Shandong, China, between 2018 and 2019. Only one sample per retail meat market stall was collected. STEC strains were isolated using the methods described previously with minor modification (Bai et al., 2015 (link)). Briefly, meat samples were enriched in EC broth (Beijing Landbridge Technology Co., Ltd., China), and incubated overnight at 37 °C. Given the small sample size, all enriched samples were inoculated into two selective media CHROMagar™ ECC agar and CHROMagar™ STEC agar (CHROMagar, France) for isolation of STEC strains as described previously (Bai et al., 2015 (link)). After overnight incubation at 37 °C, presumptive colonies were picked and tested for stx genes by single colony duplex PCR assay. API 20E biochemical test strips (bioMérieux, France) were used for confirmatory test. To capture O157 STEC, immunomagnetic separation (IMS) with magnetic beads coated with antibody to O157 (Tianjin Biochip Co., Ltd., China) was performed with the enrichment of stx-positive samples according to the manufacturer's protocol, the concentrated samples were inoculated onto the two selective media and following steps were repeated as described above. Only one isolate per sample was kept for further analysis.
8
Enumeration and Isolation of E. coli
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All samples were analyzed using Colilert vessel (Westbrook, ME, USA) according to the manufacturers’ protocol, and E. coli population was expressed in Most Probable Number (MPN/100 mL). For isolation of E. coli, 100 μL liquid sample was removed from positive wells, then spread plated onto Chromagar ECC agar (CHROMagar Microbiology, Paris, France), and incubated at 37 °C for 24 h. All colonies were preserved at −80 °C for further characterization. Manure samples (10 g) were diluted with 90 mL of phosphate buffered saline (PBS) water (0.0425 g/L KH2PO4 and 0.4055 g/L MgCl2) and shaken for 15 min and individual colonies were processed as above according to method 9223 [9 ].
9
Isolation and Identification of Escherichia albertii
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One loopful of each eae-positive enrichment culture was directly streaked on MacConkey agar (Oxoid, UK) and CHROMagar ECC agar (CHROMagar, France). After overnight incubation at 36 °C, colourless, round and moist, presumptive E. albertii colonies on both agars were chosen to test for the presence of eae. The eae-positive colonies were plated on Luria-Bertani (LB) agar (Oxoid) and incubated overnight for further identification. If more than one eae-positive lactose nonfermenting isolate was identified, only one from each sample was retained for further investigation.
10
Isolation of STEC from Goat Feces
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In total, 2,896 fecal samples from healthy-looking goats were collected from different family-scale farms in Lanling County, Shandong Province, China, from November 2017 to October 2021. Fecal samples were collected in 2-mL sterile tubes containing Luria-Bertani (LB) medium in 30% glycerol and transported in bags of ice to the laboratory at the National Institute for Communicable Disease Control and Prevention, China CDC, for the isolation of STEC. Strains were isolated and confirmed by methods described previously (40 (link)). Briefly, after enrichment with E. coli broth (EC broth, Land Bridge, Beijing, China), the samples were examined by PCR for the presence of stx (41 (link)). Samples positive for stx were inoculated onto CHROMagar ECC agar (CHROMagar, Paris, France). After overnight incubation at 37°C, green-blue or colorless colonies on agar were picked and tested for stx by a single-colony duplex PCR assay (40 (link)). API 20E biochemical test strips (bioMérieux, Lyon, France) were used to confirm that all stx-containing isolates were E. coli .
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