Genomic insights of high-risk clones of ESBL-producing Escherichia coli remoted from neighborhood infections and business meat in southern Brazil

E. coli remoted from human urine samples

Throughout June 2016 to Might 2019, 195,080 urine cultures had been carried out in a public well being providers, in a metropolis in south of Brazil. A complete of 34,293 (17.6%) had been optimistic for Gram-positive or Gram-negative microorganisms; of those 22,698 (66.2%) had been E. coli strains and a complete of 2033 (6.2%) ESBL producing micro organism, being 1389 (51.2%) ESBL-producing-E. coli.

As screening standards for performing the ERIC-PCR, we chosen 1,389 E. coli isolates from human urine that introduced ESBL manufacturing and/or had been proof against quinolones, beforehand recognized by the automated VITEK 2 system.

Via the evaluation of the dendrogram constructed by the ERIC-PCR method, we chosen 59 E. coli isolates from human urine that introduced an attention-grabbing genetic similarity profile to carry out the whole genome sequencing.


All contributors supplied the written knowledgeable consent for this examine and the examine was authorized by the Ethics and Analysis Committee of the State College of Londrina by a doc numbered as CAAE 56869816.0.0000.5231. In addition to, we affirm that every one experiments had been carried out in accordance with moral rules.

E. coli remoted from hen meat and pork samples

A surveillance examine from January to Might 2019 was carried out, to analysis ESBL-producing E. coli, in hen and pork meat, purchased at markets and butcher store close to public well being providers. Quinolone-resistant and ESBL-producing E. coli had been investigated in hen meat (n = 50), and pork (n = 50) samples. A complete of 102 E. coli was remoted from hen meat marketed, with 52 ESBL optimistic. And 67 resistant E. coli had been remoted in pork meat, 31 ESBL optimistic.

We carried out ERIC-PCR on all 169 E. coli isolates derived from hen meat and pork that confirmed ESBL manufacturing and/or quinolone resistance. Via dendrogram evaluation, we chosen 32 E. coli isolates from hen meat (n = 24) and pork (n = 8) that introduced an attention-grabbing genetic similarity profile to carry out the whole genome sequencing.


Research inhabitants

The examine depends on the whole sequencing of 91 E. coli isolates derived from human urine (n = 59), hen meat (n = 24) and pork (n = 8).

Microbiological strategies

All of the 195,080 urines collected from girls sufferers throughout June 2016 to Might 2019 was inoculated on CHROMagar (Becton Dickinson, Heidelberg, Germany) and MacConkey (Merck, Darmstadt, Germany) plates utilizing a calibrated inoculating loop with a capability of 10 μl and incubated at 37 °C for twenty-four h.


The samples of hen meat (n = 50) and pork (n = 50) had been dipped in Mind Coronary heart Infusion (BHI) broth (Oxoid) with cefotaxime (4 µg/mL), ciprofloxacin (4 µg/mL), and each (Sigma-Aldrich, Munich, Germany) to chose resistant E. coli strains. After incubation, the answer was inoculated in the identical approach used for urine samples. We evaluated the expansion of E. coli from the three BHI options and we preferentially chosen the isolates that grew in broth with each antimicrobials (cefotaxime and ciprofloxacin). In case there was no development of any isolate on that broth, we chosen from the broth containing separate cefotaxime and ciprofloxacin. All of the isolates chosen had been saved in Tryptic Soy Broth (TSB) with 15% glycerol (− 20◦C).

The identification and bacterial susceptibility had been carried out by the automated VITEK 2 system, utilizing the VITEK 2 AST 239 card and the VITEK 2 GN ID card (BioMérieux, USA). The bacterial susceptibility was examined for 14 antibiotics: ampicillin, amoxicillin/clavulanate, ceftriaxone, cefepime, ertapenem, meropenem, nalidixic acid, ciprofloxacin, norfloxacin, gentamicin, amikacin, nitrofurantoin, trimethoprim-sulfamethoxazole, and piperacillin-tazobactam. The CLSI 2020 (Medical and Laboratory Requirements Institute) standards had been used for interpretation. E. coli ATCC25922 pressure was used as high quality management.


On a regular basis of examine, samples had been phenotypically confirmed for ESBL manufacturing utilizing the chromogenic agar medium (ChromID ESBL bioMèrieux, Marcy L’Etoile, France).


1389 ESBL-producing isolates had been subjected to Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR), by Versalovicet al. (1991)37. Evaluation of genomic fingerprinting was carried out utilizing GelJ v.2.0 software program by the Cube similarity methodology (HERAS et al., 2015)38. Strains had been thought-about genetically associated if the similarity index was ≥ 85%.

DNA isolation and whole-genome sequencing

For DNA extraction, strains had been grown on Mueller–Hinton Agar in a single day at 37 °C. Subsequently, a single colony was inoculated in 2 mL of Luria–Bertani broth for 12 h at 37 °C. The suspension was used to proceed extraction and purification by the DNA extraction equipment (Invitrogen, Carlsbad, CA). The extracted DNA was quantified by Qubit dsDNA (double-stranded DNA) BR assay equipment (Invitrogen, Carlsbad, CA). After quantification, the DNA was used to assemble a paired-end library (150 bp), sequenced utilizing the NextSeq platform (Illumina). The directions of every producer had been adopted in all steps.

Bioinformatic evaluation

Genome high quality filter and assemblies had been carried out by the CLC Genomics Workbench model 7.0 (Aarhus, Denmark). Multilocus sequence kind (MLST), resistome, and virulome had been recognized utilizing MLST v2.0 (Larsen et al., 2012), ResFinder v3.1(Bortolaia et al., 2020), VirulenceFinder v2.0, (Joensen et al., 2014), PlasmidFinder v2.1 (Carattoli et al., 2014), FimTyper v1.0 (Roer et al., 2017) and SerotypeFinder v.2.0 (Joensen et al., 2015), respectively. The BacMet database (Pal et al., 2013) was used to establish biocides and heavy steel (HM)32,39,40,41,42,43,44,45.

In an effort to evaluate our strains belonging to ST38, ST117, ST131, ST354, ST410 and ST648 with different genetically associated strains from totally different sources and nations, we carried out a seek for every ST on the Enterobase Escherichia/Shigella database46 (, then we downloaded all genome assemblies with knowledge for nation, supply and assortment yr. Number of assemblies to phylogenetic evaluation was based mostly on fimH kind, obtained from Enterobase, and on common nucleotide id (ANI), assessed with FastANI v1.3247 (

CSI Phylogeny v1.448 ( was used with default settings to generate phylogenetic timber for every ST, utilizing as reference genome the chromosome sequences of E. coli ST38 pressure 144 (RefSeq accession numberNZ_CP023364.1), ST117 pressure 14EC020 (NZ_CP024138.1), ST131 pressure p4A (NZ_CP049085.2), ST354 pressure SMS-3-5 (NC_010498.1), ST410 pressure Ecol_517 (NZ_CP018965.1) and ST648 pressure Ecol_881 (NZ_CP019029.1).

Phylogenetic timber had been rooted at midpoint and annotated with knowledge from Enterobase utilizing iTOL v6 ( As a result of nice variety of ST131, the tree was break up into two subtrees, one for fimH22 and the opposite for fimH30 and fimH485.

Moral approval

The examine was authorized by the Ethics and Analysis Committee of the State College of Londrina CAAE 56869816.0.000.5231.

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