Cases and clinical data
This is a descriptive cross-sectional study investigating an outbreak of gastroenteritis in a daycare center in Vila Velha, Espírito Santo State, Brazil. The investigation began on March 22, 2019, the date of the first notification of the outbreak to the municipal health authorities of Vila Velha. Clinical and epidemiological data were obtained by health agents. Sample collection started on March 23 and finished on April 10.
Thirty-three individuals were included in the study. Stool samples were obtained from 32 of them for bacterial (n = 32) and viral (n = 30) investigation. A total of 27 cases of diarrhea and/or vomiting occurred in the daycare center, affecting fourteen children, six staff members (teachers, assistants, and general service workers), six family members (three children and three adults), and one nurse. Six asymptomatic staff members were also included in this study. The criteria for inclusion of asymptomatic staff were as follows: being a food handler or working in a class with a large number of severe cases, such as HC or HUS (classes 2 and 3A).
The daycare center was closed for 30 days starting from March 27. Diarrhea was defined as loose stools occurring at least three times a day. The primary case was defined as the one that appeared without known direct contact with other patients, and secondary cases as those that arose more than 24 h after the onset of the primary case.
Epidemiological data were obtained from the Espírito Santo State Central Laboratory, Vitória, through an authorization term for database use.
This study was approved by the Research Ethics Committee of the Health Sciences Center of the Federal University of Espírito Santo (Protocol no. 3.584.448, September 18, 2019), with a waiver of informed consent, in accordance with Brazilian Resolution on Human Research no. 466 (Certificate of Presentation for Ethical Appreciation no. 20181519.7.0000.5060). All methods were carried out in accordance with relevant guidelines and regulations.
Bacterial isolation, identification of E. coli pathotypes, EHEC serotyping, and antimicrobial susceptibility testing
Stool samples from 32 individuals were transported in Cary–Blair transport medium to the state public health laboratory for bacterial isolation on MacConkey (Basingstoke, UK) and Hektoen agar (Kasvi, Roseto degli Abruzzi, Italy). Phenotypic identification of genera/species of E. coli, Shigella, and Salmonella was performed by biochemical tests . Antimicrobial susceptibility tests were performed by the disk and strip-diffusion method, according to standards and guidelines from the Clinical and Laboratory Standards Institute (CLSI, 2019). Bacterial isolates were tested for antimicrobial resistance to the following antimicrobial agents: amikacin, gentamicin, tobramycin, ampicillin, ampicillin-sulbactam, amoxicillin-clavulanate, piperacillin-tazobactam, cefoxitin, cefotaxime, ceftazidime, cefepime, aztreonam, imipenem, meropenem, ertapenem, doxycycline, nitrofurantoin, ciprofloxacin, levofloxacin, ofloxacin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole (disks from CECON, São Paulo, Brazil; M.I.C. Evaluator Strips from Oxoid, Basingstoke, UK).
Two to four E. coli colonies from each specimen were subjected to two multiplex PCRs, as previously described . PCR 1 assay contained the primer mix for detection of the E. coli attaching and effacing gene (eae), bundle-forming pilus gene (bfp), and anti-aggregation protein transporter gene (aat, previously known as CVD432). PCR 2 assay contained specific primers for thermolabile (elt) and thermostable toxin (est) genes, invasion plasmid-encoded antigen H gene (ipaH), and Shiga toxin genes (stx1 and stx2). These assays allow identifying typical (eae+, bfp+) and atypical (eae+, bfp−, stx1−, stx2−) EPEC; typical enteroaggregative E. coli (aat+) and enterotoxigenic E. coli (elt+ and/or est+); Shigella or enteroinvasive E. coli (ipaH+); EHEC (eae+, bfp−, stx1+, and/or stx2+); and STEC eae− (eae−, bfp−, stx1+, and/or stx2+). The reference strains enteroaggregative E. coli EAEC 042 (aat+), tEPEC E2342/69 (bfp+, eae+), enterotoxigenic E. coli H10407 (st+, lt+), EHEC EDL933 (eae+, stx2+), and entero-invasive E. coli EDL1284 (ipaH+) were included in the PCR assays as positive controls.
EHEC isolates were serotyped by tube agglutination using absorbed antisera for somatic antigens O1 to O183 and flagellar antigens H1 to H56. Somatic antigens O184, O185, O186, O187, and O188 were screened by multiplex PCR [40, 42].
PFGE, MLST, and genetic characterization of virulence factors in EHEC
The clonal relationship of EHEC isolates was analyzed according to Durmaz et al. (2009) . PFGE was performed after macrorestriction with XbaI in a CHEF-DR III system (Bio-Rad, USA) and analyzed using GelJ software  by the unweighted pair-group method with arithmetic mean (UPGMA) and Dice coefficient. Isolates were considered to belong to the same pulsotype if they shared at least 80% similarity in band patterns. The ST of the EHEC strain was characterized by MLST analysis after the sequencing of seven E. coli housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA), and comparison of data against the E. coli MLST database (http://enterobase.warwick.ac.uk/species/ecoli/search_strains), according to previous recommendations .
Virulence genes (efa1, nleE, nleb, sem, pagC, terE, katP, ehxA, toxB, espP, iha, astA, sat, set1A, and chuA) were investigated by PCR, as previously described [16, 46,47,48,49]. The stx2 and eae genes were subtyped by Sanger sequencing on a Thermo Fisher Scientific ABI 3500 platform [50, 51].
Detection and molecular characterization of gastroenteric viruses
Rotavirus and norovirus GI and GII were investigated in 24 symptomatic and 6 asymptomatic cases. Viral nucleic acids were purified from 140 μL of stool suspension (10% w/v) by an automatic nucleic acid extraction procedure using a QIAamp® Viral RNA Mini kit (QIAGEN, CA, USA) in a QIAcube® automated system (QIAGEN). Viruses were detected and quantified by using TaqMan®-based qPCR protocols, as previously described [52, 53]. Primers (COG1F and R; COG2F and R) and probes (RING1C and RING2) targeting ORF1/2 were used to detect norovirus GI and GII, respectively. For rotavirus detection, primers (NSP3F and R) and probe (NSP3p) targeting the conserved NSP3 gene were used. Primers targeting the 3′-end of ORF1 and 5′-end of ORF2 (Mon 431 and G2SKR), which generated a ∼ 557 bp amplicon, were used for molecular characterization of norovirus GII . Sanger sequencing was performed using both forward and reverse primers with the BigDye™ Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA), and reactions were run at the FIOCRUZ Institutional Sequencing Platform (PDTIS) on an ABI Prism 3730xl genetic analyzer (Applied Biosystems). Consensual sequences were obtained using Geneious prime (Biomatters Ltd., Auckland, New Zealand). Norovirus genotypes were firstly assigned using two norovirus typing tools (https://www.rivm.nl/mpf/typingtool/norovirus and https://norovirus.ng.philab.cdc.gov). Phylogenetic trees were constructed by the maximum-likelihood method and Kimura two-parameter model (2000 bootstrap replications for branch support) in MEGA X  using norovirus reference sequences obtained from the National Center for Biotechnology Information (NCBI) database. Norovirus GII nucleotide sequences were submitted to GenBank and assigned the following accession numbers: MT129134 to MT129138.