Escherichia coli is a major pathogen involved in the etiology of environmentally derived bovine mastitis and is characterized by a variety of virulence factors (VF). Mammary infections with E. coli have shown a wide range of clinical signs, causing changes in milk (score 1, or mild), abnormal appearance of milk and udder inflammation (score 2, or moderate), and abnormalities in milk, udder inflammation, and systemic signs of illness (score 3, or severe). Nevertheless, to date, the profile of the genes related to the virulence of the pathogen in mammary infections and the severity scores of cases have not been thoroughly elucidated. Therefore, a panel of 18 virulence-encoding genes associated with extra-enteric pathogenicity of E. coli (ExPEC) were investigated in addition to in vitro swimming and swarming motility profiles and antimicrobial susceptibility/resistance patterns among 114 E. coli strains isolated from cows with clinical mastitis and different severity scores. Of 114 clinical cases, 39.5, 54.4, and 6.1% were mild, moderate, and severe, respectively. The main genes related to VF harbored by isolates were adhesins (fimH 100%; ecpA 64.0%, fimA 31.6%), serum resistance (traT 81.6%; ompT 35.1%), siderophores (irp2 9.6%), and hemolysin (hlyA 7%). Among the isolates studied, 99.1% showed in vitro resistance to bacitracin and cloxacillin, and 98.2% to lincosamin. Of the total isolates, 98.2% were considered multidrug resistant based on the multiple antimicrobial resistance index. No significant difference was observed between mean swimming (13.8 mm) and swarming (13.5 mm) motility, as well as severity scores of clinical mastitis and the ExPEC genes studied. The isolation of strains resistant to various antimicrobials, even though tested only in vitro, highlights the importance of rational use of antimicrobials for mastitis treatment. The high prevalence of the genes related to serum resistance (traT and ompT) and adhesion (ecpA) of the pathogen, in addition to main associations between the genes fimH, ecpA, and traT among cows with severity scores of 1 (15%) and 2 (22.6%), indicates that the genes traT, ecpA, and ompT could be further studied as biomarkers of ExPEC for clinical intramammary infections. In addition, the ExPEC genes ompT (protectin), ibe10 (invasin), and ecpA (adhesin) were investigated for the first time among cows with mastitis, where scores of clinical severity were assessed. Results of this study contribute to the characterization of virulence mechanisms and antimicrobial resistance profile of ExPEC variants that affect dairy cows with different scores of clinical mastitis.

(1) Background: Pathogenic Escherichia coli are divided into two groups: diarrheagenic (DEC) and extraintestinal pathogenic (ExPEC) E. coli. ExPEC causing urinary tract infections (UTIs) are termed uropathogenic E. coli (UPEC) and are the most common cause of UTIs worldwide. (2) Methods: Here, we characterized 112 UPEC in terms of phylogroup, serotype, the presence of virulence factor-encoding genes, and antimicrobial resistance. (3) Results: The majority of the isolates were assigned into the phylogroup B2 (41.07%), and the serogroups O6 (12.5%) and O25 (8.9%) were the most frequent. Five hybrid UPEC (4.5%), with markers from two DEC pathotypes, i.e., atypical enteropathogenic (aEPEC) and enteroaggregative (EAEC) E. coli, were identified, and designated UPEC/aEPEC (one isolate) and UPEC/EAEC (four isolates), respectively. Three UPEC/EAEC harbored genes from the pap operon, and the UPEC/aEPEC carried ibeA. The highest resistance rates were observed for ampicillin (46.4%) and trimethoprim/sulfamethoxazole (34.8%), while 99.1% of the isolates were susceptible to nitrofurantoin and/or fosfomycin. Moreover, 9.8% of the isolates were identified as Extended Spectrum β-Lactamase producers, including one hybrid UPEC/EAEC. (4) Conclusion: Our data reinforce that hybrid UPEC/DEC are circulating in the city of Botucatu, Brazil, as uropathogens. However, how and whether these combinations of genes influence their pathogenicity is a question that remains to be elucidated.

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. 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. 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). 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. 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.

Enteroaggregative Escherichia coli (EAEC) is an important agent of acute and persistent diarrhea in children and adults worldwide. Here we report a characterization of 220 EAEC isolates, 88.2% (194/220) of which were typical and 11.8% (26/220) were atypical, obtained from diarrheal patients during seven years (2010-2016) of epidemiological surveillance in Brazil. The majority of the isolates were assigned to phylogroups A (44.1%, 97/220) or B1 (21.4%, 47/220). The aggregative adherence (AA) pattern was detected in 92.7% (204/220) of the isolates, with six of them exhibiting AA concomitantly with a chain-like adherence pattern; and agg5A and agg4A were the most common adhesin-encoding genes, which were equally detected in 14.5% (32/220) of the isolates. Each of 12 virulence factor-encoding genes (agg4A, agg5A, pic, aap, aaiA, aaiC, aaiG, orf3, aar, air, capU, and shf) were statistically associated with typical EAEC (P < 0.05). The genes encoding the newly described aggregate-forming pili (AFP) searched (afpB, afpD, afpP, and afpA2), and/or its regulator (afpR), were exclusively detected in atypical EAEC (57.7%, 15/26), and showed a significant association with this subgroup of EAEC (P < 0.001). In conclusion, we presented an extensive characterization of the EAEC circulating in the Brazilian settings and identified the afp genes as putative markers for increasing the efficiency of atypical EAEC diagnosis.

Mammary pathogenic Escherichia coli (MPEC) is one of the most common pathogens associated with clinical mastitis. We analyzed isolates obtained from milk samples of cows with clinical mastitis, collected from 10 farms in Brazil, to verify molecular and phenotypic characteristics. A total of 192 (4.5%) mammary pathogenic E. coli isolates were obtained from 4,275 milk samples analyzed, but we tested 161. We assigned most of these isolates to E. coli phylogroups B1 (52.8%) and A (36.6%), although phylogroups B2, C, D, E, and unknown also occurred. All isolates were assessed for the presence of several genes encoding virulence factors, such as adhesins (sfaDE, papC, afaBC III, ecpA, fimH, papA, and iha), toxins (hlyA, cnf1, sat, vat, and cdt), siderophores (iroN, irp2, iucD, ireA, and sitA), an invasion protein (ibeA), and serum resistance proteins (traT, KpsMTII, and ompT), and isolates from phylogroups B1, B2, and E showed up to 8 genes. Two isolates harbored the locus of enterocyte effacement (escN⁺) and lack the bundle-forming pilus (bfpB^-) operon, which corresponds to a molecular profile of a subgroup of diarrheagenic E. coli (aEPEC), thus being classified as hybrid MPEC/aEPEC isolates. These isolates displayed a localized adherence-like pattern of adherence in HeLa cells and were able to promote F-actin polymerization underneath adherent bacteria. Based on the pulsed-field gel electrophoresis analyses, considerable genetic variability was observed. A low index of antimicrobial resistance was observed and 2 extended-spectrum β-lactamase-producing E. coli were identified, both harboring bla_CTX-M15 gene, and were classified as ST10 and ST993 using multilocus sequence typing. A total of 148 (91.2%) isolates were weak biofilm producers or formed no biofilm. Because raw milk is still frequently consumed in Brazil, the occurrence of virulence factor-encoding genes from extraintestinal or diarrheagenic E. coli added to the presence of extended-spectrum β-lactamase-producing isolates can turn this veterinary medicine problem into a public health concern.