==== Front PLoS Negl Trop Dis PLoS Negl Trop Dis plos plosntds PLoS Neglected Tropical Diseases 1935-2727 1935-2735 Public Library of Science San Francisco, CA USA 32479497-50-3807/journal.pntd.0008373 PNTD-D-19-01830 Research Article Biology and Life Sciences Evolutionary Biology Evolutionary Systematics Phylogenetics Biology and Life Sciences Taxonomy Evolutionary Systematics Phylogenetics Computer and Information Sciences Data Management Taxonomy Evolutionary Systematics Phylogenetics People and places Geographical locations South America Brazil Biology and Life Sciences Computational Biology Comparative Genomics Biology and Life Sciences Genetics Genomics Comparative Genomics Medicine and Health Sciences Pathology and Laboratory Medicine Pathogens Virulence Factors Biology and Life Sciences Microbiology Bacteriology Bacterial Genetics Bacterial Genomics Biology and Life Sciences Genetics Microbial Genetics Bacterial Genetics Bacterial Genomics Biology and Life Sciences Genetics Genomics Microbial Genomics Bacterial Genomics Biology and Life Sciences Microbiology Microbial Genomics Bacterial Genomics Biology and Life Sciences Computational Biology Genome Analysis Genomic Libraries Biology and Life Sciences Genetics Genomics Genome Analysis Genomic Libraries Biology and Life Sciences Biogeography Phylogeography Ecology and Environmental Sciences Biogeography Phylogeography Earth Sciences Geography Biogeography Phylogeography Biology and Life Sciences Evolutionary Biology Population Genetics Phylogeography Biology and Life Sciences Genetics Population Genetics Phylogeography Biology and Life Sciences Population Biology Population Genetics Phylogeography Biology and Life Sciences Evolutionary Biology Evolutionary Systematics Phylogenetics Phylogenetic Analysis Biology and Life Sciences Taxonomy Evolutionary Systematics Phylogenetics Phylogenetic Analysis Computer and Information Sciences Data Management Taxonomy Evolutionary Systematics Phylogenetics Phylogenetic Analysis Comparative genomic analysis provides insight into the phylogeny and virulence of atypical enteropathogenic Escherichia coli strains from Brazil Genomic analysis from Brazilian atypical EPEChttp://orcid.org/0000-0001-6695-6003Hernandes Rodrigo T. ConceptualizationFormal analysisFunding acquisitionInvestigationMethodologyProject administrationValidationWriting – original draft1* Hazen Tracy H. Formal analysisInvestigationMethodologySupervisionValidationWriting – review & editing2 dos Santos Luís F. InvestigationMethodologyWriting – review & editing3 http://orcid.org/0000-0002-6547-793XRichter Taylor K. S. InvestigationMethodologyWriting – review & editing2 Michalski Jane M. InvestigationMethodologyWriting – review & editing2 http://orcid.org/0000-0002-7337-7154Rasko David A. ConceptualizationFormal analysisFunding acquisitionProject administrationSupervisionValidationWriting – review & editing2 1 Departamento de Microbiologia e Imunologia, Instituto de Biociências, Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP), Botucatu, SP, Brasil 2 Institute for Genome Sciences, Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America 3 Instituto Adolfo Lutz, São Paulo, SP, Brasil Yang Ruifu Editor Beijing Institute of Microbiology and Epidemiology, CHINA The authors have declared that no competing interests exist. * E-mail: martinezjared@example.net 1 6 2020 6 2020 14 6 e00083497-50-3807 7 5 2020 © 2020 Hernandes et al2020Hernandes et alThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background Atypical enteropathogenic Escherichia coli (aEPEC) are one of the most frequent intestinal E. coli pathotypes isolated from diarrheal patients in Brazil. Isolates of aEPEC contain the locus of enterocyte effacement, but lack the genes of the bundle-forming pilus of typical EPEC, and the Shiga toxin of enterohemorrhagic E. coli (EHEC). The objective of this study was to evaluate the phylogeny and the gene content of Brazilian aEPEC genomes compared to a global aEPEC collection. Methodology Single nucleotide polymorphism (SNP)-based phylogenomic analysis was used to compare 106 sequenced Brazilian aEPEC with 221 aEPEC obtained from other geographic origins. Additionally, Large-Scale BLAST Score Ratio was used to determine the shared versus unique gene content of the aEPEC studied. Principal Findings Phylogenomic analysis demonstrated the 106 Brazilian aEPEC were present in phylogroups B1 (47.2%, 50/106), B2 (23.6%, 25/106), A (22.6%, 24/106), and E (6.6%, 7/106). Identification of EPEC and EHEC phylogenomic lineages demonstrated that 42.5% (45/106) of the Brazilian aEPEC were in four of the previously defined lineages: EPEC10 (17.9%, 19/106), EPEC9 (10.4%, 11/106), EHEC2 (7.5%, 8/106) and EPEC7 (6.6%, 7/106). Interestingly, an additional 28.3% (30/106) of the Brazilian aEPEC were identified in five novel lineages: EPEC11 (14.2%, 15/106), EPEC12 (4.7%, 5/106), EPEC13 (1.9%, 2/106), EPEC14 (5.7%, 6/106) and EPEC15 (1.9%, 2/106). We identified 246 genes that were more frequent among the aEPEC isolates from Brazil compared to the global aEPEC collection, including espG2, espT and espC (P<0.001). Moreover, the nleF gene was more frequently identified among Brazilian aEPEC isolates obtained from diarrheagenic patients when compared to healthy subjects (69.7% vs 41.2%, P<0.05). Conclusion The current study demonstrates significant genomic diversity among aEPEC from Brazil, with the identification of Brazilian aEPEC isolates to five novel EPEC lineages. The greater prevalence of some virulence genes among Brazilian aEPEC genomes could be important to the specific virulence strategies used by aEPEC in Brazil to cause diarrheal disease. Author summary Atypical EPEC (aEPEC) is one of the most frequent diarrheagenic Escherichia coli pathotypes isolated from patients in Brazil and is associated with diarrheal outbreaks. This study is the first to sequence the genomes of a collection of aEPEC isolates from a South American country, Brazil, and compare their phylogenetic relationships and gene content with a global collection of aEPEC. This approach identified Brazilian aEPEC genomes in previously characterized EPEC/EHEC phylogenomic lineages and resulted in the identification of five novel EPEC phylogenomic lineages, designated EPEC11 to EPEC15. We also observed that virulence genes, such as espG2, espT and espC were more frequently identified among the Brazilian aEPEC genomes, demonstrating potential differences in the virulence repertoire of this pathogen in Brazil. National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human ServicesU19AI110820http://orcid.org/0000-0002-7337-7154Rasko David A. http://dx.doi.org/10.13039/501100001807Fundação de Amparo à Pesquisa do Estado de São Paulo2015/26207-6 and 2016/17584-3http://orcid.org/0000-0001-6695-6003Hernandes Rodrigo T. This study was funded by federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services (grant number U19AI110820 to DAR) and São Paulo Research Foundation - FAPESP (grant numbers: +1-778-630-9783 and +1-778-630-9783 to RTH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PLOS Publication Stagevor-update-to-uncorrected-proofPublication Update2020-06-11Data AvailabilityAll relevant data are within the manuscript and in its Supporting Information files.Data Availability All relevant data are within the manuscript and in its Supporting Information files. ==== Body Introduction The enteropathogenic Escherichia coli (EPEC) pathotype is divided into two subgroups: typical EPEC (tEPEC) and atypical EPEC (aEPEC) [1]. The main difference between these two groups is the presence of the EPEC adherence factor plasmid (pEAF) encoding the bundle forming pilus (BFP) in tEPEC, and its absence in aEPEC [1, 2]. A previous epidemiological study demonstrated that tEPEC was a significant cause of moderate to severe diarrhea and increased mortality in infants [3]. In recent decades aEPEC has emerged as a frequent cause of diarrheal disease in children and adults, and has been linked to several diarrheal outbreaks [2, 4–12]. In particular, aEPEC isolates of serotypes O2:H16, O33:H34, O39:H9, O108:H- and ONT:H19 were implicated in five distinct diarrheal outbreaks in São Paulo State, Brazil, in 2013 [8]. The main virulence feature of EPEC is its ability to produce the histopathological attaching and effacing (A/E) lesions on the surface of infected host cells [13]. The A/E lesion is characterized by the destruction of the microvilli brush border, intimate bacteria-cell adherence and formation of pedestal-like structures rich in F-actin and other cytoskeleton elements, which causes intestinal epithelial cells to have a reduced absorptive capacity [13, 14]. The 41 genes associated with this phenotype are located in a pathogenicity island (PAI) termed the locus of enterocyte effacement (LEE) [15]. The LEE region encodes all the structural components of the type 3 secretion system (T3SS), as well as seven effector proteins, the intimin outer membrane protein, the intimin translocated receptor protein (Tir), chaperones, and transcriptional regulators [15–18]. Although enterohemorrhagic E. coli (EHEC) similarly contain the LEE and produce the A/E lesion on infected epithelial cells, EHEC also contain the stx genes, encoding the Shiga toxin, which is not present in EPEC [13]. Besides the T3SS effectors located in the LEE region, effectors encoded outside of LEE, known as non-LEE encoded effectors, have emerged as important accessory virulence factors in A/E pathogens [19, 20]. These effectors are responsible for inducing several modifications in the host cells during infection, including inhibition of apoptosis, interference with inflammatory signaling pathways, invasion, tight junction disruption and phagocytosis [19, 20]. Previous comparative genomic studies have demonstrated that EPEC exhibit considerable genomic diversity, with representatives in at least 10 different phylogenomic lineages [21, 22]. This is most likely due to the acquisition of the LEE region and its stable retention by genomically diverse lineages of E. coli [23]. These prior studies have focused on investigating the genomic diversity of EPEC isolates from locations in Europe, Africa, and Asia [22, 23]. While aEPEC poses a significant diarrheal burden in South America [2, 9, 24], to our knowledge there have been no previous comparative genomics studies that have investigated the genetic diversity of aEPEC from Brazil or other countries in South America. Thus, the goal of this study was to analyze the genetic relatedness and virulence gene content of Brazilian aEPEC isolates compared to a globally distributed collection of aEPEC. Methods DNA extraction, genome sequencing, and assembly Genomic DNA of the 106 aEPEC isolates from Brazil (S1 Table) was extracted from overnight cultures using the Sigma GenElute bacterial genomic DNA kit (Sigma-Aldrich; St. Louis, MO). The genomes were sequenced using paired-end libraries on the Illumina HiSeq 4000. The 150 bp Illumina reads were assembled using SPAdes v.3.7.1 [25], and the final assemblies were filtered to contain contigs that were ≥500 bp in length and had ≥5X kmer coverage. Phylogenomic analysis A single nucleotide polymorphism (SNP)-based phylogenomic analysis was performed on the 106 Brazilian aEPEC (S1 Table), 221 global aEPEC publicly available from previous studies [22, 23] (S2 Table, S1 Fig), and a collection of 48 diverse E. coli and Shigella genomes (S3 Table). A total of 179,202 SNPs were detected relative to the completed genome sequence of the phylogroup F laboratory isolate E. coli IAI39 (accession number: NC_011750) using the In Silico Genotyper (ISG) [26]. A maximum-likelihood phylogeny with 100 bootstrap replicates was generated using RAxML v.7.2.8 [27] with the GTR model of nucleotide substitution, the GAMMA model of rate heterogeneity, and visualized using FigTree v.1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). In silico Multilocus Sequence Typing (MLST) and serotyping The seven loci of the multilocus sequence typing (MLST) scheme (adk, gyrB, fumC, icd, mdh, purA, and recA) developed by Wirth et al. [28] were identified in each of the genomes listed in S1 and S2 Tables using BLASTN, and MLST sequence type (ST) were determined by querying the PubMLST database (https://pubmlst.org). The molecular serotype of each genome analyzed was determined using Serotype Finder 1.1 (https://cge.cbs.dtu.dk/services/SerotypeFinder/) with the default settings of an 85% identity threshold and 60% minimum alignment length [29]. Large-Scale BLAST Score Ratio (LS-BSR) analysis The 106 Brazilian aEPEC and additional A/E E. coli genomes (S1 to S3 Tables) analyzed in this study were compared by BLASTN LS-BSR analysis as previously described [30]. Briefly, the predicted protein-encoding genes of each genome that had ≥90% nucleotide sequence identity and ≥90% alignment length were assigned to gene clusters using CD-HIT v.4.6.4 [31] (S1 Data Set). The predicted protein function of each gene cluster was determined with an ergatis-based [32] in-house annotation pipeline [33]. The presence of gene clusters with significant similarity in each of the five novel phylogenomic lineages identified in this study (EPEC11, EPEC12, EPEC13, EPEC14 and EPEC15) were defined by using an LS-BSR value ≥0.9; while the absence in the remaining A/E genomes were defined by using an LS-BSR value ≤0.4. Additionally, we examined the occurrence of genes that were more common among the Brazilian aEPEC isolates when compared to the isolates from outside of Brazil, as well as among Brazilian and global aEPEC identified in the same phylogenomic lineage. The presence of each gene cluster was defined as having a BLASTN LS-BSR value ≥0.8, and differences in genes frequency among the two group of aEPEC studied (Brazilian versus Global) were tested by using the Chi-square test with Yates correlation and two-tailed Fisher's exact test. In silico detection of virulence factor-encoding genes and intimin subtyping TBLASTN LS-BSR was used to detect the presence of known EPEC virulence factor-encoding genes in the 106 Brazilian aEPEC genomes, as previously described [22, 34]. The virulence factor-encoding genes investigated included the repertoire of Non-LEE effectors present in the typical EPEC prototypes E2348/69 [35] and B171 [36]; since many of them has been associated with the EPEC pathogenesis [20], as well as espT [37] and espV [38], first indentified and functionally characterized in the outbreak-associated aEPEC E110019. In addition, we searched for genes reponsible for encoding adhesins (efa1/lifA, iha, paa, toxB, cah, ehaA, ehaB, ehaC, ehaD, ehaG, and ehaJ) and toxins (astA, pic, pet ehxA, espP, sepA, sigA, and espC) that haven been frequentelly deteceded among aEPEC isolates, demontrated to contribute to aEPEC pathogenesis and/or previously associated with diarrheal disease [8, 39–42]. The intimin subtypes [43, 44] were identified in the 106 aEPEC genomes analyzed using BLASTN LS-BSR as described above. Results Characteristics of the Brazilian aEPEC genomes A total of 106 aEPEC isolates obtained from previous studies performed in Brazil [5, 8, 9, 45] were sequenced. This collection of Brazilian aEPEC were isolated over a span of seven years from 2009 to 2016, and were associated with multiple clinical presentations, including 17 isolates from subjects with asymptomatic infection, 76 from patients with diarrhea, and 13 from diarrheal patients investigated in five distinct diarrheal outbreaks (S1 Table). The average genome size of the Brazilian aEPEC genome assemblies was 4,927,030 bp (range 4,551,111 to 5,442,868), and the average GC content was 50.53% (range: 50.13% to 50.78%), which is consistent with previous studies of sequenced E. coli genomes [22, 46] (S1 Table). The majority of the aEPEC Brazilian genomes were identified in phylogroup B1 The 106 Brazilian aEPEC genomes were identified in phylogroup B1 (47.2%, 50/106), followed by phylogroups B2 (23.6%, 25/106), A (22.6%, 24/106), and E (6.6%, 7/106) (Fig 1, Table 1). The 221 global aEPEC genomes analyzed had a similar phylogroup distribution with phylogroup B1 (55.7%, 123/221) being the most common, followed by phylogroups A (22.6%, 50/221) and B2 (11.3%, 25/221), while the aEPEC genomes of phylogroups E (9.5%, 21/221) and D (0.9%, 2/221) were the least common (Table 1). While the phylogroup distributions of Brazilian aEPEC were similar to those of the global aEPEC genomes, there were significantly more phylogroup B2 genomes identified among the Brazilian aEPEC (23.6%, 25/106) compared with the global collection (11.3%, 25/221) (Table 1) (P<0.05). The reasons for a greater prevalence of phylogroup B2 aEPEC in Brazil compared with the global collections are not clear at this time and may be representative of an isolation or sequencing bias. 10.1371/journal.pntd.0008373.g001Fig 1 Phylogenomic analysis of aEPEC from Brazil compared to aEPEC from other geographic origins. Phylogenomic analysis of atypical EPEC genomes (labeled in blue), compared to typical EPEC (labeled in green), EHEC (labeled in red) and reference E. coli and Shigella genomes (labeled in black). Red circles indicate the 106 Brazilian aEPEC genomes. The clades in the phylogeny are colored by phylogenomic lineage, and the E. coli phylogroups are indicated by the letters: A, B1, B2, D, E, and F. An asterisk (*) indicates the five novel EPEC phylogenomic lineages identified in this study. The nodes with bootstrap values >80% are labeled with a black circle. 10.1371/journal.pntd.0008373.t001Table 1 Classification of the aEPEC genomes in the distinct phylogroups and phylogenomic lineages. Phylogroup/ Phylogenomic lineage No. (%) of atypical EPEC (aEPEC) P value Brazil (n = 106) Global (n = 221) Total (n = 327) Phylogroup A 24 (22.6) 50 (22.6) 74 (22.6) 0.9972b EPEC5 3 (2.8) 3 (1.4) 6 (1.8) 0.3937c EPEC10 19 (17.9) 30 (13.6) 49 (15.0) 0.3865b EPEC15 2 (1.9) 13 (5.9) 15 (4.6) 0.157c uEPECa 0 4 (1.8) 4 (1.2) 0.3086c Phylogroup B1 50 (47.2) 123 (55.7) 173 (52.9) 0.1866b EHEC2 8 (7.5) 37 (16.7) 45 (13.8) 0.0368b EPEC2 2 (1.9) 13 (5.9) 15 (4.6) 0.1570c EPEC7 7 (6.6) 17 (7.7) 24 (7.3) 0.8991b EPEC11 15 (14.2) 30 (13.6) 45 (13.8) 0.8874b EPEC12 5 (4.7) 7 (3.2) 12 (3.7) 0.5348c EPEC13 2 (1.9) 10 (4.5) 12 (3.7) 0.3499c EPEC14 6 (5.7) 2 (0.9) 8 (2.4) 0.0159c uEPECa 5 (4.7) 7 (3.2) 12 (3.7) 0.5348c Phylogroup B2 25 (23.6) 25 (11.3) 50 (15.3) 0.0065b EPEC4 2 (1.9) 8 (3.6) 10 (3.1) 0.5090c EPEC6 6 (5.7) 3 (1.4) 9 (2.8) 0.0629c EPEC9 11 (10.4) 2 (0.9) 13 (4.0) 0.0001c uEPECa 6 (5.7) 12 (5.4) 18 (5.5) 0.9318b Phylogroup D 0 2 (0.9) 2 (0.6) 1.0000c uEPECa 0 2 (0.9) 2 (0.6) 1.0000c Phylogroup E 7 (6.6) 21 (9.5) 28 (8.6) 0.5056b EHEC1 5 (4.7) 19 (8.6) 24 (7.3) 0.3017b uEPECa 2 (1.9) 2 (0.9) 4 (1.2) 0.5978c aUnclassified EPEC (uEPEC) refer to atypical EPEC genomes not classified in any of the pre-existing EPEC/EHEC phylogenomic lineages or in the five novel EPEC lineages described in this study. b,cDifferences observed between aEPEC genomes from Brazil and from a global distribution were determined using Chi-square test with Yates correlation (b) or two-tailed Fisher's exact test (c). Brazilian aEPEC genomes were identified in previously described EPEC/EHEC phylogenomic lineages Previous genetic studies focusing on understanding the evolution of EPEC have demonstrated that isolates from this pathotype can belong to at least ten distinct EPEC phylogenomic lineages, which have been designated EPEC1 to EPEC10 [21, 47, 48], in addition to unclassified EPEC isolates, that exist outside of these defined EPEC lineages. We observed that 59.4% (63/106) of the aEPEC Brazilian genomes could be classified into previously defined phylogenomic lineages: EPEC2 (1.9%, 2/106), EPEC4 (1.9%, 2/106), EPEC5 (2.8%, 3/106), EPEC6 (5.7%, 6/106), EPEC7 (6.6%, 7/106), EPEC9 (10.4%, 11/106), EPEC10 (17.9%, 19/106), as well as a small proportion in EHEC lineages, EHEC1 (4.7%, 5/106) and EHEC2 (7.5%, 8/106) (Fig 1, Table 1). The greatest number of Brazilian aEPEC genomes were identified in the EPEC10 lineage (17.9%, 19/106). The majority of the Brazilian aEPEC in this lineage were MLST ST10 (78.9%, 15/19), including seven aEPEC isolates with serotype O2:H16 (36.8%, 7/19). We also observed a significantly greater number of Brazilian aEPEC genomes than global aEPEC (10.4% vs 0.9%, P<0.001) in the EPEC9 lineage (Table 1). In this lineage we observed eight Brazilian aEPEC of serotype O33:H34 (72.7%, 8/11). Of note, the Brazilian aEPEC genomes identified with serotypes (sequence types) O2:H16 (ST10) and O33:H34 (ST1951), were obtained from diarrheal outbreaks that occurred in Brazil in 2013 (4 isolates O2:H16 and 4 isolates O33:H34, each serotype from a distinct outbreak), as well as from sporadic cases of diarrhea that occurred in Brazil over several years (3 isolates O2:H16 and 4 isolates O33:H34) (S1 Table), while in the global aEPEC collection, only one isolate, from Mali (isolate 200135), was identified with serotype O33:H34 (ST2346); and aEPEC of serotype O2:H16 were not detected (S2 Table). The differences observed in the frequency of aEPEC of serotypes O2:H16 (6.6% vs 0.0%, P<0.001) and O33:H34 (7.5% vs 0.5%, P<0.001), among the Brazilian vs Global aEPEC collections, may be due to these serotypes being associated with Brazilian diarrheal outbreaks. Brazilian aEPEC identified in the lineages EHEC1 and EHEC2 had the serotypes O55:H7 (100.0%, 5/5) and O26:H11 (75.0%, 6/8), respectively. The five O55:H7 were ST335 (Table 2) and were most closely related to the O157:H7 of the EHEC1 lineage (Fig 1). The six aEPEC with serotype O26:H11 in the EHEC2 lineage were one of two MLST sequence types: ST21 (50.0%, 3/6) or ST29 (50.0%, 3/6) (Table 2). The number of aEPEC genomes identified in the EHEC2 lineage was significantly higher among the global collection of aEPEC than the Brazilian aEPEC (7.5% vs 16.7%, P<0.05) (Table 1). 10.1371/journal.pntd.0008373.t002Table 2 Characteristics of the 106 Brazilian aEPEC genomes analyzed in this study. Phylogroup Phylogenomic Lineage MLST ST Intimin Subtype In Silico Serotype (No. of aEPEC isolates) A (22.6%) EPEC5 (2.8%) 206 kappa O88:H5 (2), O118/O151:H5 (1) EPEC10 (17.9%) 10 beta ONT:H16 (7)a epsilon O157:H16 (3) omicron O13/O135:H11 (1) theta O51:H40 (1), O117:H40 (3) 34 lambda O101:H33 (2) ND theta O127:H40 (2) EPEC15 (1.9%) 301 omicron O76:H2 (1) xi O80:H2 (1) B1 (47.2%) EHEC2 (7.5%) 21 beta O26:H11 (3) 29 beta O26:H11 (3) theta ONT:H9 (1) 4550 beta O103:H8 (1) EPEC2 (1.9%) 20 beta O119:H2 (1), O128ab/ac:H2 (1) EPEC7 (6.6%) 642 omicron O85:H4 (3), O103:H4 (2), O184:H4 (1), ONT:H4 (1) EPEC11 (14.2%) 517 epsilon2 O35:H19 (1), O71:H19 (1), O88:H25 (2), O123/O186:H19 (3), O126:H19 (3), ONT:H19 (1) eta O160:H19 (2) 5241 eta O108:HNT (2) EPEC12 (4.7%) 40 theta O66:H21 (1) beta O109:H21 (1) 101 eta O21:H21 (1) 224 epsilon O182:H23 (1) ND eta O91:H23 (1) EPEC13 (1.9%) 337 theta O108:H21 (1) 442 theta O146:H21 (1) EPEC14 (5.7%) 381 iota O39:H9 (2) zeta O177:H9 (1), ONT:H12 (1) 590 iota O98:H8 (1), ONT:H8 (1) uEPEC (4.7%) 300 zeta O182:H25 (1) 327 theta O111:H8 (1), O115:H8 (1) 2178 theta O170:H49 (1) 2338 theta O131:H46 (1) B2 (23.6%) EPEC4 (1.9%) 4601 beta2 ONT:H6 (1) 6323 beta2 O56:H6 (1) EPEC6 (5.7%) 122 alpha2 O63:H6 (1) 526 iota O145:H34 (1) 582 alpha2 O132:H34 (1) 583 alpha2 O63:H6 (2) 713 iota O145:H34 (1) EPEC9 (10.4%) 1951 lambda O33:H34 (8) 2346 alpha O142:H34 (3) uEPEC (5.7%) 941 zeta O156:H1 (1) 1040 alpha O177:H45 (1) 2201 mu O63:H34 (1) 5428 kappa O71:H49 (1) 5575 alpha2 O125ac:H49 (1) ND alpha O37:H45 (1) E (6.6%) EHEC1 (4.7%) 335 gamma O55:H7 (5) uEPEC (1.9%) 32 gamma O145:HNT (1) 2569 theta ONT:H31 (1) aPreviously defined as serotype O2:H16 by standard agglutination tests with absorbed antisera (9, 45). Designation of five novel EPEC phylogenomic lineages Phylogenomic analysis of Brazilian aEPEC genomes along with aEPEC genomes from a global distribution, led to the identification of five novel phylogenomic lineages with five or more genomes in each, which we have designated EPEC11 to EPEC15 (Fig 1). The EPEC11 to EPEC14 lineages were identified in phylogroup B1, whereas EPEC15 was identified in phylogroup A (Fig 1). Among the aEPEC Brazilian genomes analyzed, we observed that 28.3% (30/106) were identified in one of the five novel EPEC lineages. The distribution of these isolates is as follow: EPEC11 (14.2%, 15/106), EPEC12 (4.7%, 5/106), EPEC13 (1.9%, 2/106), EPEC14 (5.7%, 6/106) and EPEC15 (1.9%, 2/106) (Table 1). It is important to highlight the number of Brazilian aEPEC genomes identified in the EPEC11 lineage (14.2%, 15/106) (Table 1), which are second in abundance after the Brazilian aEPEC isolates identified in the EPEC10 lineage (17.9%, 19/106) (Table 1). The Brazilian aEPEC identified in EPEC11 had eight distinct serotypes (O35:H19, O71:H19, O88:H25, O108:HNT, O123/O186:H19, O126:H19, O160:H19 and ONT:H19), harbored intimin subtype epsilon2 (73.3%, 11/15) or eta (26.7%, 4/15), and the majority (86.7%, 13/15) were ST517 (Tables 2 and 3). Moreover, we observed that the aEPEC archetype isolate E110019, from an outbreak that occurred in Finland [49], was present in the newly designated EPEC14 lineage (Fig 1), along with two Brazilian aEPEC isolates (IAL5152 and IAL 5153) which were serotype O39:H9 and obtained from an outbreak (S1 Table). It is important to emphasize that the number of Brazilian aEPEC genomes identified in EPEC14, a novel EPEC lineage, is statistically different when comparing with aEPEC from outside of Brazil (5.7% vs 0.9%, P<0.05) (Table 1). Additionally, 12.3% (13/106) of the Brazilian aEPEC genomes identified in phylogroups B1 (5 isolates), B2 (6 isolates) and E (2 isolates) were not classified into any of the described EPEC or EHEC phylogenomic lineages (Tables 1 and 2). 10.1371/journal.pntd.0008373.t003Table 3 Serotypes and Multilocus sequence typing (MLST) of the aEPEC isolates classified in the five novel phylogenomic lineages of EPEC described in this study. Phylogroup Phylogenomic Lineage MLST ST aEPEC in silico Serotype: From Brazil From other origins B1 EPEC11 (n = 45) 517 O35:H19 (1) O71:H19 (1) O88:H25 (2) O123/O186:H19 (3) O126:H19 (3) O160:H19 (2) ONT:H19 (1) O71:H19 (6) O88:H25 (1) O111:H19 (4) O116:H9 (3) O123/O186:H19 (3) O136:H51 (1) O160:H19 (2) O165:H9 (2) O171:H19 (1) ONT:H19 (1) 5241 O108:HNT (2) O8:H19 (5) 5485 - O171:H19 (1) EPEC12 (n = 12) 40 O66:H21 (1) O109:H21 (1) O109:H21 (6) ONT:H21 (1) 101 O21:H21 (1) - 224 O182:H23 (1) - ND O91:H23 (1) - EPEC13 (n = 12) 337 O108:H21 (1) O108:H21 (2) 442 O146:H21 (1) O146:H21 (2) 443 - O64:H21 (4) O161:H21 (1) 5508 - O104:H7 (1) EPEC14 (n = 8) 381 O39:H9 (2) O177:H9 (1) ONT:H12 (1) O177:H9 (1) ONT:H9 (1) 590 O98:H8 (1) ONT:H8 (1) - A EPEC15 (n = 15) 301 O76:H2 (1) O80:H2 (1) O45:H19 (6) O61:HNT (1) O80:H2 (2) 382 - ONT:H5 (3) 4590 - ONT:H2 (1) Identification of genes that are unique among the novel EPEC phylogenomic lineages We used LS-BSR to identify gene content that is present in one group of isolates and lacking in another. First, we examined the existence of genes that were present in each of the five novel EPEC lineages (EPEC11, EPEC12, EPEC13, EPEC14 and EPEC15) and absent in all other A/E E. coli genomes included in this study. A total of 936 gene clusters were found exclusively, at a variable frequency, in the five novel EPEC phylogenomic lineages as follows: 196 (EPEC11), 181 (EPEC12), 241 (EPEC13), 140 (EPEC14), and 178 gene clusters (EPEC15) (Table 4). However, only one of these gene clusters was identified in all of the genomes within its associated lineage and absent from all other genomes analyzed. The EPEC11 lineage harbors a single gene cluster (termed CYCE01000006.1_37), that encodes a hypothetical protein, present in all 45 (100.0%) aEPEC genomes from this phylogenomic lineage and absent in all other A/E genomes analyzed (Table 4). A summary of the LS-BSR analyses can be found in Table 4, and all genes exclusively present in each of the five novel phylogenomic lineages are listed in S4 Table. 10.1371/journal.pntd.0008373.t004Table 4 Detection of gene clusters that are unique to each of the novel EPEC phylogenomic lineages. Phylogenomic Lineages (number of genomes)c No. of gene clusters exclusively present in each phylogenomic lineagea,b: 100% of the Genomes ≥90% of the Genomes ≥75% of the Genomes ≥50% of the Genomes ≥1 of the Genomes EPEC11 (n = 45) 1 2 2 2 196 EPEC12 (n = 12) 0 0 2 11 181 EPEC13 (n = 12) 0 0 0 0 241 EPEC14 (n = 8) 0 0 3 13 140 EPEC15 (n = 15) 0 0 0 12 178 aTotal gene clusters: 24,383 bThe presence of gene clusters in each of the five novel phylogenomic lineages was defined using a LS-BSR value ≥0.9, and absence in the remaining A/E genomes analyzed were defined by a LS-BSR value ≤0.4. The gene clusters detected exclusively in each of the five novel phylogenomic lineage are listed in S4 Table. cThe number of attaching and effacing (typical EPEC, atypical EPEC and EHEC) genomes analyzed was 349, as described in S1, S2 and S3 Tables. Differential prevalence of gene content between Brazilian aEPEC compared to a global collection of aEPEC Since so few genes were exclusively present or absent in the novel phylogenomic lineages we wanted to examine if there were any genes that were more statistically prevalent among the Brazilian aEPEC isolates when compared to the isolates obtained from locations outside of Brazil. This analysis of the gene content led to the identification of 246 genes that were statistically more frequent (P<0.001) among the Brazilian aEPEC genomes than aEPEC from other geographic origins (Brazilian aEPEC vs Global aEPEC). Included among these genes were two T3SS-effectors: espG2, gene cluster 128_11_7_153 (20.8% vs 6.8%), and espT, gene clusters 148_12_48_29 (21.7% vs 3.6%) and CYEQ01000057.1_8 (17.9% vs 3.2%). The espC gene, encodes an autotransporter protein [50], represented by gene clusters 1024_15_1_31 (27.4% vs 7.2%) and 276_12_73_9 (26.4% vs 8.1%) was also significantly more prevalent among the Brazilian aEPEC than the global aEPEC (Table 5). 10.1371/journal.pntd.0008373.t005Table 5 Virulence and antimicrobial resistance genes differentially detected between Brazilian versus Global distributed aEPEC genomes. Gene Gene Cluster No. (%) of aEPEC genomes P valuea Brazil (n = 106) Global (n = 221) Gene clusters more frequent among Brazilian aEPEC (P<0.001) espC 1024_15_1_31 29 (27.4) 16 (7.2) <0.0001 276_12_73_9 28 (26.4) 18 (8.1) <0.0001 espT 148_12_48_29 23 (21.7) 8 (3.6) <0.0001 CYEQ01000057.1_8 19 (17.9) 7 (3.2) <0.0001 espG2 128_11_7_153 22 (20.8) 15 (6.8) 0.0004 Gene clusters more frequent among aEPEC from other origins (P<0.001) clpV 401091_31_7 52 (49.1) 154 (69.7) 0.0005 nleA 148_12_80_8 39 (36.8) 132 (59.7) 0.0002 espW 103338_65_6 8 (7.5) 56 (25.3) 0.0003 iha 100329_168_1 7 (6.6) 50 (22.6) 0.0006 ospB C341_10_332_45 26 (24.5) 98 (44.3) 0.0009 sul2 CYGQ01000108.1_5 16 (15.1) 90 (40.7) <0.0001 blaTEM-1 100329_76_20 18 (17.0) 86 (38.9) 0.0001 aph(6)-Id 100329_76_18 12 (11.3) 86 (38.9) <0.0001 tetA DEC11A_c104_2 8 (7.5) 53 (24.0) 0.0006 aDifferences in the prevalence of gene clusters among the two groups of aEPEC studied (Brazilian aEPEC versus Global aEPEC) were tested using the Chi-square test with Yates correlation. Conversely, 446 genes were statistically less frequent (P<0.001) among the Brazilian aEPEC when compared to the global aEPEC from outside Brazil, including genes encoding a type VI secretion ATPase (49.1% vs 69.7%), the T3SS-effectors nleA (36.8% vs 59.7%), ospB (24.5% vs 44.3%) and espW (7.5% vs 25.3%), and the IrgA homolog adhesin iha (6.6% vs 22.6%) (Table 5). Curiously, genes encoding proteins that mediate antimicrobial resistance were also statistically less frequent (P<0.001) among the Brazilian aEPEC genomes compared to the aEPEC genomes from outside Brazil, as follows: blaTEM-1 (17.0% vs 38.9%) encoding a TEM-1 beta-lactamase, sul2 (15.1% vs 40.7%) encoding a sulfonamide resistant dihydropteroate synthase, aph(6)-Id (11.3% vs 38.9%) encoding an aminoglycoside phosphotransferase, and tetA (7.5% vs 24.0%) encoding an antibiotic efflux pump (Table 5). Other genes that were differentially detected among Brazilian and global aEPEC have predicted functions associated with central metabolism and transcriptional regulation, as well as numerous hypothetical proteins (S5 Table). Virulence genes identified among the Brazilian aEPEC that exhibit lineage specific distributions We also investigated the differences in gene content between Brazilian and global aEPEC isolates identified in the same phylogenomic lineage. Statistically different prevalences (P<0.001) were observed in five phylogenomic lineages (EHEC1, EHEC2, EPEC7, EPEC10 and EPEC11) between gene content of the groups of aEPEC genomes analyzed (S6 Table). These included the toxB gene, which was first characterized in the pO157 plasmid of EHEC O157:H7 [51]. Interestingly, toxB was detected in four Brazilian aEPEC (50.0%, 4/8) that were serotype O26:H11 and identified in the EHEC2 lineage, while the EHEC2 isolates analyzed from outside Brazil lack this gene (S6 Table). In addition, the autotransporter-encoding gene sepA (85.7% vs 5.9%) and the T3SS-effector gene espK (71.4% vs 0.0%) were statistically more prevalent (P<0.001) among the EPEC7 Brazilian aEPEC genomes than the EPEC7 aEPEC from outside Brazil (S6 Table). We also observed that genes from the PAI OI-122 were statistically (P<0.001) different among Brazilian aEPEC compared to global aEPEC in two phylogenomic lineages: EPEC7 and EPEC10 (S6 Table). The OI-122 is a 23-kb chromosomal PAI that consists of three distinct modules, and has been associated with virulence in EHEC and EPEC [52, 53]. Virulence factor-encoding genes can be found in each one of these three modules, as follows: module 1: pagC; module 2: espL2, nleB and nleE; and module 3: efa1 and efa2 (in O157:H7 EHEC EDL933), or efa1/lifA (in tEPEC E2348/69) [35, 52, 54]. All seventeen global aEPEC identified in the EPEC7 lineage contained genes of module 2 and module 3, while the Brazilian aEPEC isolates lack these genes (S6 Table). In contrast, the pagC (module 1) and efa1/lifA (module 3) genes were detected in seven (36.8%, 7/19) Brazilian aEPEC identified in the EPEC10 lineage (serotype O2:H16), and in none of the global aEPEC (S6 Table). NleF-encoding gene is associated with aEPEC diarrheal disease in Brazil Since the Brazilian aEPEC isolates studied here were obtained from patients with diarrhea (89 isolates) as well from healthy subjects (17 isolates), we decided to compare if any of the 43 genes encoding virulence factors investigated was associated with their ability to cause illness in the human host. The number of virulence factor-encoding genes detected in aEPEC isolates obtained from patients with diarrhea ranged from 3 to 23, whereas in those from healthy subjects ranged from 5 to 18 (Fig 2). Of importance, the nleF gene was significantly more prevalent among aEPEC isolates obtained from diarrheagenic patients than from healthy subjects (69.7% vs 41.2%, P<0.05) (S7 Table). 10.1371/journal.pntd.0008373.g002Fig 2 In silico detection of known EPEC virulence factor-encoding genes in the 106 Brazilian aEPEC genomes studied. The virulence factor-encoding genes were detected in each of the 106 Brazilian aEPEC genomes analyzed in this study using large-scale BLAST score ratio (LS-BSR) analysis. Each row is a different aEPEC genome, grouped by phylogenomic lineage, and each column represents a single gene (listed in the S7 Table). The presence or absence of all predicted protein-encoding genes in the 106 Brazilian aEPEC genomes is indicated by the BSR values represented in the heatmap generated by using the heatmap2 function of gplots v.3.0.1 in R v.3.3.2. Colors of the heat map indicate virulence genes that were detected with significant similarity (blue), with divergent similarity (gray) or were absent (white) in each of the Brazilian aEPEC genomes analyzed. On the far right red circles indicate aEPEC isolates obtained from patients with diarrhea, while green circles indicate aEPEC isolates obtained from healthy subjects. Discussion As prior studies have focused on investigating the genomic diversity of EPEC isolates from locations in Europe, Africa, and Asia [22, 23], the genomic diversity of aEPEC from Brazil or other countries in South America remains largely unexplored. Thus, in this study we describe the genomic diversity of a collection of 106 human aEPEC isolates from Brazil. We also considered the genomic diversity of the Brazilian aEPEC compared to a global collection of aEPEC isolates to gain insight into how Brazilian aEPEC may differ in their evolutionary relatedness and virulence gene content compared with aEPEC from outside of Brazil. By examining the prevalence of known EPEC virulence factors, we determined that the T3SS non-LEE encoded effectors espG2 [55] and espT [37, 56], and the autotransporter gene espC [50] were statistically more prevalent among aEPEC from Brazil compared to the global aEPEC from outside Brazil. Of particular interest is EspT, which was functionally characterized in aEPEC archetype isolate E110019 from one of the more several diarrheal outbreaks associated with aEPEC as there were 650 individuals in a school and 137 associated household members in Finland that developed diarrhea [49]. EspT is responsible for triggering the formation of lamellipodia and membrane ruffles, through the activation of Rac-1 and Cdc42 [37], thus facilitating the bacterial invasion into non-phagocytic cells [56]. Although the occurrence of invasive aEPEC in the Brazilian settings have already been reported [57], no correlation between this phenotype and the presence of the espT gene has been observed to date. EspG2 is also of importance as it is involved in microtubule disruption [55]. EspC, a member of the autotransporter family, is involved in EPEC-mediated cell death and induces both apoptosis and necrosis in epithelial cells [58]. In tEPEC archetype isolate E2348/69 the espC and espG2 (orf3) are located in the same PAI, termed integrative element (IE) 5 or EspC PAI [35, 59], which may explain the simultaneous dissemination of these two genes among the Brazilian aEPEC isolates. In addition, we observed that nleF was the only virulence factor-encoding gene that demonstrated a statistical association with the isolates from diarrheal disease cases when compared to Brazilian aEPEC isolates from asymptomatic individuals. NleF is a Non-LEE effector, secreted to the host cells by the T3SS, that binds to caspase-4, -8, and -9 inhibiting the catalytic activity of these caspases in vitro, and therefore, prevents apoptosis in HeLa and Caco-2 cells [60]. We also observed that the Brazilian aEPEC differ in their gene content at the phylogenomic lineage level, when compared with aEPEC from other geographic origins. In particular, the toxB gene of pO157 from O157:H7 EHEC strains, and efa1/lifA of the PAI OI-122 [54, 61], were more prevalent among Brazilian aEPEC identified in the EHEC2 and EPEC10 phylogenomic lineages, respectively. The proteins encoded by the toxB and efa1/lifA genes are associated with the ability of EHEC strains to adhere to epithelial cells. Of note, the EHEC adherence factors efa1 (Z4332) and efa2 (Z4333) genes, located in the third module of the PAI OI-122 from EHEC O157:H7 strains [52], can encode proteins with amino acid sequences that are identical to the amino acids 1 to 433 (99.0% of identity) and 437 to 711 (100.0% identity) of the Efa1 adhesin identified in the EHEC O111:H- [62]. A previous study, using random transposon mutagenesis, demonstrated that the EHEC O157:H7 Sakai strain, harboring a transposon insertion in the efa1 gene, was significantly less adherent than the wild type strain in assays performed with Caco-2 cells [63]. The nucleotide sequence of the efa1 gene, from the EHEC O111:H- strain, is 99.0% identical to the lifA gene (also termed efa1/lifA) from the tEPEC E2348/69, that has been demonstrated to inhibit lymphocyte proliferation and lymphokine production [64]. A clinical study, that evaluated stool samples from three distinct children with diarrhea associated with an EHEC isolate with the serotype O157:HNM, demonstrated that EHEC may lose the stx2 gene during trafficking in the host [65]. This line of evidence suggests that some of the aEPEC isolates in our study, which were genomically related to EHEC lineages and/or harbored virulence genes associated with this pathotype may in fact be EHEC that have lost the Shiga toxin genes during trafficking in the host or environment, or during culture in the clinical laboratory. Overall, this study demonstrated the aEPEC from Brazil are genomically diverse, with representatives in nearly all of the E. coli phylogroups and previously described EHEC and EPEC phylogenomic lineages, which is similar to what has been previously described for aEPEC from outside of Brazil. The Brazilian aEPEC genomes described in this study not only provide insight into the genomic diversity of aEPEC in Brazil, but also allowed the description of five novel EPEC phylogenomic lineages. We also identified important differences in gene content, including known virulence associated genes, between Brazilian aEPEC compared to a global collection of aEPEC from outside Brazil. Further investigation of the genomic diversity and prevalence of select virulence genes among additional aEPEC from Brazil as well as other locations in South America may provide insight into the greater prevalence of some of these lineages of aEPEC in association with diarrheal illness in Brazil. Supporting information S1 Data Nucleotide sequence of the gene clusters in fasta format. (FASTA) Click here for additional data file. S1 Fig World map indicating the countries from which the globally distributed aEPEC collection was obtained. Atypical EPEC from the global collection was obtained from all five continents, mainly from Africa, Asia, and Europe. In parentheses is the number of aEPEC isolates obtained from each country. This world map was constructed using the free-software Q-GIS. (TIFF) Click here for additional data file. S1 Table Brazilian atypical EPEC (aEPEC) strains sequenced in the present study. (XLSX) Click here for additional data file. S2 Table Global collection of atypical enteropathogenic Escherichia coli (aEPEC) strains from outside Brazil. (XLSX) Click here for additional data file. S3 Table Escherichia coli and Shigella reference genomes included in the phylogenomic analysis. (XLSX) Click here for additional data file. S4 Table LS-BSR identification of gene clusters that are unique in the five novel phylogenomic lineage identified in this study. (XLSX) Click here for additional data file. S5 Table Gene clusters differentially detected in the Brazilian aEPEC genomes in comparison with aEPEC from outside Brazil. (XLSX) Click here for additional data file. S6 Table Gene clusters differentially detected among aEPEC from Brazil and from other origins by phylogenomic lineage. (XLSX) Click here for additional data file. S7 Table Occurrence of virulence factor-encoding genes in aEPEC obtained from diarrheagenic patients and healthy subjects in Brazil. (XLSX) Click here for additional data file. We thank Waldir Pereira Elias, from Instituto Butantan, for providing the atypical EPEC isolate BA92. ==== Refs References 1 Trabulsi LR , Keller R , Tardelli Gomes TA . Typical and atypical enteropathogenic Escherichia coli . Emerg Infect Dis . 2002 ; 8 (5 ):508 –+1-778-630-9783/eid0805.010385 .11996687 2 Hernandes RT , Elias WP , Vieira MA , Gomes TA . An overview of atypical enteropathogenic Escherichia coli . 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