The osmoregulated metabolism of trehalose contributes to production of type 1 fimbriae and bladder colonization by extraintestinal Escherichia coli strain BEN2908.

In Escherichia coli, the disaccharide trehalose can be metabolized as a carbon source or be accumulated as an osmoprotectant under osmotic stress. In hypertonic environments, E. coli accumulates trehalose in the cell by synthesis from glucose mediated by the cytosolic enzymes OtsA and OtsB. Trehalose in the periplasm can be hydrolyzed into glucose by the periplasmic trehalase TreA. We have previously shown that a treA mutant of extraintestinal E. coli strain BEN2908 displayed increased resistance to osmotic stress by 0.6 M urea, and reduced production of type 1 fimbriae, reduced invasion of avian fibroblasts, and decreased bladder colonization in a murine model of urinary tract infection. Since loss of TreA likely results in higher periplasmic trehalose concentrations, we wondered if deletion of otsA and otsB genes, which would lead to decreased internal trehalose concentrations, would reduce resistance to stress by 0.6 M urea and promote type 1 fimbriae production. The BEN2908ΔotsBA mutant was sensitive to osmotic stress by urea, but displayed an even more pronounced reduction in production of type 1 fimbriae, with the consequent reduction in adhesion/invasion of avian fibroblasts and reduced bladder colonization in the murine urinary tract. The BEN2908ΔtreAotsBA mutant also showed a reduction in production of type 1 fimbriae, but in contrast to the ΔotsBA mutant, resisted better than the wild type in the presence of urea. We hypothesize that, in BEN2908, resistance to stress by urea would depend on the levels of periplasmic trehalose, but type 1 fimbriae production would be influenced by the levels of cytosolic trehalose.

Enteroaggregative Escherichia coli (EAEC) isolates obtained from non-diarrheic children carry virulence factor-encoding genes from Extraintestinal Pathogenic E. Coli (ExPEC).

Enteroaggregative E. coli (EAEC) is one of the most frequent pathogens isolated from diarrheal patients as well as from healthy individuals in Brazil and has recently also been implicated as an extraintestinal pathogenic E. coli (ExPEC) associated with bloodstream and urinary tract infections. In this study, 37 EAEC isolates, obtained from fecal samples of non-diarrheic children, were molecularly and phenotypically characterized to access the pathogenic features of these isolates. The EAEC isolates were assigned into the phylogroups A (54.1%), D (29.7%), B1 (13.5%) and B2 (2.7%); and harbored genes responsible for encoding the major pilin subunit of the aggregative adherence fimbriae (AAFs) or aggregate-forming pili (AFP) adhesins as follows: aggA (24.3%), agg3A (5.4%), agg4A (27.0%), agg5A (32.4%) and afpA (10.8%). The most frequent O:H serotypes were O15:H2 (8.1%), O38:H25 (5.4%) and O86:H2 (5.4%). Twenty-one isolates (56.8%) produce the aggregative adherence (AA) pattern on HeLa cells, and biofilm formation was more efficient among EAEC isolates harboring the aggA and agg5A genes. PFGE analysis showed that 31 (83.8%) of the isolates were classified into 10 distinct clusters, which reinforces the high diversity found among the isolates studied. Of note, 40.5% (15/37) of the EAEC isolates have a genetic profile compatible with E. coli isolates with intrinsic potential to cause extraintestinal infections in healthy individuals, and therefore, classified as EAEC/ExPEC hybrids. In conclusion, we showed the presence of EAEC/ExPEC hybrids in the intestinal microbiota of non-diarrheic children, possibly representing the source of some endogenous extraintestinal infections.

Can Extraintestinal Pathogenic Escherichia coli with Heat Resistance Profile Overcome Nonthermal Technologies?

Ultraviolet-C light-emitting diode (UVC-LED) and ultrasound (US) are two nonthermal technologies with the potential to destroy pathogens. However, little is known about their effectiveness in strains with a history of heat resistance. Thus, this study aimed to evaluate the phenotype and genotype of heat-resistant extraintestinal pathogenic Escherichia coli (ExPEC) with heat resistance genes after the application of US, UVC-LED, and UVC-LED+US. For this, two central composite rotatable designs were used to optimize the UVC-LED and US conditions in four ExPEC isolated from beef. From the genome of these isolates obtained in a previous study, possible genes for UVC resistance were analyzed. Results showed that US was ineffective in reducing >0.30 log colony-forming unit/mL, and that when used after UVC-LED, it showed a nonsynergic or antagonistic effect. Also, UVC-LED had the greatest effect at the maximum dose (4950 mJ/cm2 from 1.65 mW/cm2 for 50 min). However, the strains showed some recovery after that, which could be implicated in the expression of genes included in SOS system genes, some others present in the transmissible Locus of Stress Tolerance (trxBC and degP), and others (terC). Thus, ExPEC can overcome the conditions used in this study for US, UVC-LED, and UVC-LED+US, probably due to the history of resistance to other cellular damage. The result of this study will contribute to future studies that aim to find better treatment conditions for each food product.

Detecção da secreção da toxina Sat pela Escherichia coli extraintestinal (EC071) na infecção de células endoteliais in vitro

Escherichia coli é uma bactéria gram-negativa da família das Enterobacteriaceae, inicialmente descrita como uma bactéria comensal, mas que pela aquisição de fatores de virulência tornar-se patogênica. Dentre os fatores de virulência importantes na patogenicidade destacam-se membros da família das Serino-proteases Autotransportadoras de Enterobacteriaceae. (SPATEs). Sat (toxina autotransportadora secretada) tem se destacado como fator de virulência presente nas E. coli patogênicas, sendo a SPATE mais prevalente em E. coli isolada de sepse. No entanto, até o momento, não está claro se Sat poderia conferir alguma vantagem no escape da E. coli do intestino e/ou na sua colonização e manutenção da bactéria em nichos extraintestinais, tais como circulação sanguínea, acarretando a bacteremia que, dependendo das condições do hospedeiro, pode vir a evoluir para um quadro grave de sepse. Desta forma, o objetivo deste trabalho foi investigar se uma cepa de E. coli extraintestinal, portadora do gene sat, isolada de bacteremia (EC071) secreta a toxina durante a infecção de células endoteliais, in vitro. Inicialmente as condições ideais para o cultivo bacteriano e estabelecimento do MOI foram determinadas. Em seguida, células endoteliais da linhagem HUVEC foram infectadas com a cepa EC071. Na análise da cinética de infecção, por Western blotting detectou-se a presença de Sat no sobrenadante das células a partir de 4h de infecção. Contudo, nos ensaios preliminares para avaliação da citotoxicidade que avaliou morfologia e destacamento celular a partir da comparação com células infectadas com a cepa HB101, utilizada como controle, os danos celulares observados não foram significativos. Desta forma, o esclarecimento sobre a ação de Sat sobre o endotélio e manutenção da bactéria dentro do contexto da infecção na corrente sanguínea, dependerá de novos ensaios.

Identification of Escherichia coli isolated from flies (Insecta: Diptera) that inhabit the environment of dairy farms harboring extraintestinal virulence markers.

AIMS: We investigate extraintestinal pathogenic genes (ExPEC) related to virulence of Escherichia coli in flies from the dairy environment. METHODS AND RESULTS: We collected 217 flies from nine dairy farms, which were submitted to microbiological culture. Fifty-one E. coli were identified using mass spectrometry. Eleven dipteran families were identified, with a predominance of Muscidae, and a minor frequency of Tachinidae, Drosophilidae, Sphaeroceridae, Ulidiidae, Syrphidae, Chloropidae, Calliphoridae, Sarcophagidae, and Piophilidae. A panel of 16 virulence-encoding genes related to ExPEC infections were investigated, which revealed predominance of serum resistance (traT, 31/51 = 60.8%; ompT, 29/51 = 56.9%), iron uptake (irp2, 17/51 = 33.3%, iucD 11/51 = 21.6%), and adhesins (papC, 6/51 = 11.8%; papA, 5/51 = 9.8%). CONCLUSIONS: Our findings reveal Dipterans from milking environment carrying ExPEC virulence-encoding genes also identified in clinical bovine E. coli-induced infections.

Strain belonging to an emerging, virulent sublineage of ST131 Escherichia coli isolated in fresh spinach, suggesting that ST131 may be transmissible through agricultural products.

Food contamination with pathogenic Escherichia coli can cause severe disease. Here, we report the isolation of a multidrug resistant strain (A23EC) from fresh spinach. A23EC belongs to subclade C2 of ST131, a virulent clone of Extraintestinal Pathogenic E. coli (ExPEC). Most A23EC virulence factors are concentrated in three pathogenicity islands. These include PapGII, a fimbrial tip adhesin linked to increased virulence, and CsgA and CsgB, two adhesins known to facilitate spinach leaf colonization. A23EC also bears TnMB1860, a chromosomally-integrated transposon with the demonstrated potential to facilitate the evolution of carbapenem resistance among non-carbapenemase-producing enterobacterales. This transposon consists of two IS26-bound modular translocatable units (TUs). The first TU carries aac(6')-lb-cr, bla OXA-1, ΔcatB3, aac(3)-lle, and tmrB, and the second one harbors bla CXT-M-15. A23EC also bears a self-transmissible plasmid that can mediate conjugation at 20°C and that has a mosaic IncF [F(31,36):A(4,20):B1] and Col156 origin of replication. Comparing A23EC to 86 additional complete ST131 sequences, A23EC forms a monophyletic cluster with 17 other strains that share the following four genomic traits: (1) virotype E (papGII+); (2) presence of a PAI II536-like pathogenicity island with an additional cnf1 gene; (3) presence of chromosomal TnMB1860; and (4) frequent presence of an F(31,36):A(4,20):B1 plasmid. Sequences belonging to this cluster (which we named "C2b sublineage") are highly enriched in septicemia samples and their associated genetic markers align with recent reports of an emerging, virulent sublineage of the C2 subclade, suggesting significant pathogenic potential. This is the first report of a ST131 strain belonging to subclade C2 contaminating green leafy vegetables. The detection of this uropathogenic clone in fresh food is alarming. This work suggests that ST131 continues to evolve, gaining selective advantages and new routes of transmission. This highlights the pressing need for rigorous epidemiological surveillance of ExPEC in vegetables with One Health perspective.

E. coli Common pili promote the fitness and virulence of a hybrid aEPEC/ExPEC strain within diverse host environments.

Pathogenic subsets of Escherichia coli include diarrheagenic (DEC) strains that cause disease within the gut and extraintestinal pathogenic E. coli (ExPEC) strains that are linked with urinary tract infections, bacteremia, and other infections outside of intestinal tract. Among DEC strains is an emergent pathotype known as atypical enteropathogenic E. coli (aEPEC), which can cause severe diarrhea. Recent sequencing efforts revealed that some E. coli strains possess genetic features that are characteristic of both DEC and ExPEC isolates. BA1250 is a newly reclassified hybrid strain with characteristics of aEPEC and ExPEC. This strain was isolated from a child with diarrhea, but its genetic features indicate that it might have the capacity to cause disease at extraintestinal sites. The spectrum of adhesins encoded by hybrid strains like BA1250 are expected to be especially important in facilitating colonization of diverse niches. E. coli common pilus (ECP) is an adhesin expressed by many E. coli pathogens, but how it impacts hybrid strains has not been ascertained. Here, using zebrafish larvae as surrogate hosts to model both gut colonization and extraintestinal infections, we found that ECP can act as a multi-niche colonization and virulence factor for BA1250. Furthermore, our results indicate that ECP-related changes in activation of envelope stress response pathways may alter the fitness of BA1250. Using an in silico approach, we also delineated the broader repertoire of adhesins that are encoded by BA1250, and provide evidence that the expression of at least a few of these varies in the absence of functional ECP.

Genomic features and heat resistance profiles of Escherichia coli isolated from Brazilian beef.

AIMS: Characterize Escherichia coli and E. coli -producing (STEC) isolates from Brazilian beef to determine heat resistance and the presence of the transmissible locus of stress tolerance (tLST). METHODS AND RESULTS: Twenty-two STEC previously isolated from beef and characterized as STEC by PCR were subjected to different heat survival challenges (60°C and 71°C). Furthermore, the three tLST-positive isolates and one tLST-negative isolate by PCR were selected for WGS analysis. Phenotypic results indicated that 3/22 (13.64%) were heat resistant, 12/22 (54.54%) were moderately resistant, and 7/22 (31.82%) were sensitive to heat treatments. WGS analyses showed that three isolates with heat resistance showed tLST with up to 80% and 42% of similarity by BLAST analysis, with the major tLST genes being responsible for the homeostasis module. However, WGS showed the absence of stx genes associated with tLST-positive isolates, albeit with virulence and resistance genes found in extraintestinal pathogenic E. coli (ExPEC). CONCLUSION: Our findings demonstrate the presence of heat-resistant E. coli as well as confirm some tLST genes in E. coli isolated from Brazilian beef.

Characterization of unconventional pathogenic Escherichia coli isolated from bloodstream infection: virulence beyond the opportunism.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of urinary tract infection worldwide and a critical bloodstream infection agent. There are more than 50 virulence factors (VFs) related to ExPEC pathogenesis; however, many strains isolated from extraintestinal infections are devoid of these factors. Since opportunistic infections may occur in immunocompromised patients, E. coli strains that lack recognized VFs are considered opportunist, and their virulence potential is neglected. We assessed eleven E. coli strains isolated from bloodstream infections and devoid of the most common ExPEC VFs to understand their pathogenic potential. The strains were evaluated according to their capacity to interact in vitro with human eukaryotic cell lineages (Caco-2, T24, HEK293T, and A549 cells), produce type 1 fimbriae and biofilm in diverse media, resist to human sera, and be lethal to Galleria mellonella. One strain displaying all phenotypic traits was sequenced and evaluated. Ten strains adhered to Caco-2 (colon), eight to T24 (bladder), five to HEK-293 T (kidney), and four to A549 (lung) cells. Eight strains produced type 1 fimbriae, ten adhered to abiotic surfaces, nine were serum resistant, and seven were virulent in the G. mellonella model. Six of the eleven E. coli strains displayed traits compatible with pathogens, five of which were isolated from an immune-competent host. The genome of the EC175 strain, isolated from a patient with urosepsis, reveals that the strain belonged to ST504-A, and serotype O11:H11; harbors thirteen VFs genes, including genes encoding UpaG and yersiniabactin as the only ExPEC VFs identified. Together, our results suggest that the ExPEC pathotype includes pathogens from phylogroups A and B1, which harbor VFs that remain to be uncovered.

E. coli Common pili promote the fitness and virulence of a hybrid aEPEC/ExPEC strain within diverse host environments

Pathogenic subsets of Escherichia coli include diarrheagenic (DEC) strains that cause disease within the gut and extraintestinal pathogenic E. coli (ExPEC) strains that are linked with urinary tract infections, bacteremia, and other infections outside of intestinal tract. Among DEC strains is an emergent pathotype known as atypical enteropathogenic E. coli (aEPEC), which can cause severe diarrhea. Recent sequencing efforts revealed that some E. coli strains possess genetic features that are characteristic of both DEC and ExPEC isolates. BA1250 is a newly reclassified hybrid strain with characteristics of aEPEC and ExPEC. This strain was isolated from a child with diarrhea, but its genetic features indicate that it might have the capacity to cause disease at extraintestinal sites. The spectrum of adhesins encoded by hybrid strains like BA1250 are expected to be especially important in facilitating colonization of diverse niches. E. coli common pilus (ECP) is an adhesin expressed by many E. coli pathogens, but how it impacts hybrid strains has not been ascertained. Here, using zebrafish larvae as surrogate hosts to model both gut colonization and extraintestinal infections, we found that ECP can act as a multi-niche colonization and virulence factor for BA1250. Furthermore, our results indicate that ECP-related changes in activation of envelope stress response pathways may alter the fitness of BA1250. Using an in silico approach, we also delineated the broader repertoire of adhesins that are encoded by BA1250, and provide evidence that the expression of at least a few of these varies in the absence of functional ECP.

Transmission of Escherichia coli Causing Pyometra between Two Female Dogs.

Despite its clinical relevance, the pathogenesis of canine pyometra remains poorly understood. To date, it is recognized as a non-transmissible infectious disease. In this study, the simultaneous occurrence of pyometra and Escherichia coli in two cohabitant female dogs underwent in-depth investigation due to the hypothesis of transmission between these animals. Two 5-year-old Chow Chow dogs (namely, dogs 23 and 24-D23 and D24) were referred to a veterinary hospital with suspected pyometra. Both animals showed prostration, anorexia, and purulent vulvar discharge over a 1-week period. After ovariohysterectomy, uterine tissue, uterine contents, and rectal swabs were collected for histopathological and microbiological analysis. Uterine histology demonstrated purulent material and multifocal necrosis with endometrial ulceration, and a morphological diagnosis of pyometra was confirmed. Furthermore, E. coli from the same phylogroup (B2) and positive for the same virulence factors with the same antimicrobial susceptibility profile was isolated from the uterine contents of both dogs and the rectum of D23. Conversely, the E. coli strains recovered from D24 differed in phylogroup (one isolate), virulence factors (all three isolates), and antimicrobial susceptibility (all three isolates). Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) suggested that all isolates from the uterine content of both dogs and the rectal swab of D23 were 100% the same, but different from all isolates in the rectal swab of D24. One isolate from the uterine content of each animal as well as rectal swabs were subjected to whole-genome sequencing (WGS). Both whole-genome multilocus sequence typing(wgMLST) and single-nucleotide polymorphism (SNP) analysis supported the hypothesis that the isolates from the uterine content of both animals and the rectal swab of D23 were clonal. Taken together, these clinical features, pathology, microbiology, and molecular findings suggest, to the best of our knowledge, the first transmission of E. coli associated with pyometra between two animals. These results could impact the management of sites where several females cohabit in the same local area such as kennels.

Virulence Profile, Antibiotic Resistance, and Phylogenetic Relationships among Escherichia coli Strains Isolated from the Feces and Urine of Hospitalized Patients.

Extra-intestinal pathogenic Escherichia coli (ExPEC) may inhabit the human gut microbiota without causing disease. However, if they reach extra-intestinal sites, common cystitis to bloodstream infections may occur, putting patients at risk. To examine the human gut as a source of endogenous infections, we evaluated the E. coli clonal diversity of 18 inpatients' guts and their relationship with strains isolated from urinary tract infection (UTI) in the same hospital. Random amplified polymorphic DNA evaluated the clonal diversity, and the antimicrobial susceptibility was determined by disk diffusion. One isolate of each clone detected was sequenced, and their virulome and resistome were determined. Overall, 177 isolates were screened, among which 32 clones were identified (mean of two clones per patient), with ExPEC strains found in over 75% of the inpatients' guts. Endogenous infection was confirmed in 75% of the cases. ST10, ST59, ST69, ST131, and ST1193 clones and critical mobile drug-resistance encoding genes (blaCTX-M-15, blaOXA-1, blaDHA-1, aac(6')-lb-cr, mcr-1.26, qnrB4, and qnrB19) were identified in the gut of inpatients. The genomic analysis highlighted the diversity of the fecal strains, colonization by lactose-negative E. coli, the high frequency of ExPEC in the gut of inpatients without infections, and the presence of ß-lactamase producing E. coli in the gut of inpatients regardless of the previous antibiotics' usage. Considering that we found more than one ExPEC clone in the gut of several inpatients, surveillance of inpatients' fecal pathogens may prevent UTI caused by E. coli in the hospital and dissemination of risk clones.

Evaluation of the Pathogenic Potential of Escherichia coli Strains Isolated from Eye Infections.

While primarily Gram-positive bacteria cause bacterial eye infections, several Gram-negative species also pose eye health risks. Currently, few studies have tried to understand the pathogenic mechanisms involved in E. coli eye infections. Therefore, this study aimed to establish the pathogenic potential of E. coli strains isolated from eye infections. Twenty-two strains isolated between 2005 and 2019 from patients with keratitis or conjunctivitis were included and submitted to traditional polymerase chain reactions (PCR) to define their virulence profile, phylogeny, clonal relationship, and sequence type (ST). Phenotypic assays were employed to determine hemolytic activity, antimicrobial susceptibility, and adhesion to human primary corneal epithelial cells (PCS-700-010). The phylogenetic results indicated that groups B2 and ST131 were the most frequent. Twenty-five virulence genes were found among our strains, with ecp, sitA, fimA, and fyuA being the most prevalent. Two strains presented a hemolytic phenotype, and resistance to ciprofloxacin and ertapenem was found in six strains and one strain, respectively. Regarding adherence, all but one strains adhered in vitro to corneal cells. Our results indicate significant genetic and virulence variation among ocular strains and point to an ocular pathogenic potential related to multiple virulence mechanisms.

Carbapenemase-Producing Extraintestinal Pathogenic Escherichia coli From Argentina: Clonal Diversity and Predominance of Hyperepidemic Clones CC10 and CC131.

Extraintestinal pathogenic Escherichia coli (ExPEC) causes infections outside the intestine. Particular ExPEC clones, such as clonal complex (CC)/sequence type (ST)131, have been known to sequentially accumulate antimicrobial resistance that starts with chromosomal mutations against fluoroquinolones, followed with the acquisition of bla CTX-M-15 and, more recently, carbapenemases. Here we aimed to investigate the distribution of global epidemic clones of carbapenemase-producing ExPEC from Argentina in representative clinical isolates recovered between July 2008 and March 2017. Carbapenemase-producing ExPEC (n = 160) were referred to the Argentinean reference laboratory. Of these, 71 were selected for genome sequencing. Phenotypic and microbiological studies confirmed the presence of carbapenemases confirmed as KPC-2 (n = 52), NDM-1 (n = 16), IMP-8 (n = 2), and VIM-1 (n = 1) producers. The isolates had been recovered mainly from urine, blood, and abdominal fluids among others, and some were from screening samples. After analyzing the virulence gene content, 76% of the isolates were considered ExPEC, although non-ExPEC isolates were also obtained from extraintestinal sites. Pan-genome phylogeny and clonal analysis showed great clonal diversity, although the first phylogroup in abundance was phylogroup A, harboring CC10 isolates, followed by phylogroup B2 with CC/ST131, mostly H30Rx, the subclone co-producing CTX-M-15. Phylogroups D, B1, C, F, and E were also detected with fewer strains. CC10 and CC/ST131 were found throughout the country. In addition, CC10 nucleated most metalloenzymes, such as NDM-1. Other relevant international clones were identified, such as CC/ST38, CC155, CC14/ST1193, and CC23. Two isolates co-produced KPC-2 and OXA-163 or OXA-439, a point mutation variant of OXA-163, and three isolates co-produced MCR-1 among other resistance genes. To conclude, in this work, we described the molecular epidemiology of carbapenemase-producing ExPEC in Argentina. Further studies are necessary to determine the plasmid families disseminating carbapenemases in ExPEC in this region.

Molecular Epidemiology and Presence of Hybrid Pathogenic Escherichia coli among Isolates from Community-Acquired Urinary Tract Infection.

Urinary tract infections (UTI) affect community and healthcare patients worldwide and may have different clinical outcomes. We assessed the phylogenetic origin, the presence of 43 virulence factors (VFs) of diarrheagenic and extraintestinal pathogenic Escherichia coli, and the occurrence of hybrid strains among E. coli isolates from 172 outpatients with different types of UTI. Isolates from phylogroup B2 (46%) prevailed, followed by phylogroups A (15.7%) and B1 (12.2%), with similar phylogenetic distribution in symptomatic and asymptomatic patients. The most frequent VFs according to their functional category were fimA (94.8%), ompA (83.1%), ompT (63.3%), chuA (57.6%), and vat (22%). Using published molecular criteria, 34.3% and 18.0% of the isolates showed intrinsic virulence and uropathogenic potential, respectively. Two strains carried the eae and escV genes and one the aggR gene, which classified them as hybrid strains. These hybrid strains interacted with renal and bladder cells, reinforcing their uropathogenic potential. The frequency of UPEC strains bearing a more pathogenic potential in the outpatients studied was smaller than reported in other regions. Our data contribute to deepening current knowledge about the mechanisms involved in UTI pathogenesis, especially among hybrid UPEC strains, as these could colonize the host's intestine, leading to intestinal infections followed by UTI.

Unveiling the Virulent Genotype and Unusual Biochemical Behavior of Escherichia coli ST59.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a leading cause of human and animal infections worldwide. The utilization of selective and differential media to facilitate the isolation and identification of E. coli from complex samples, such as water, food, sediment, and gut tissue, is common in epidemiological studies. During a surveillance study, we identified an E. coli strain isolated from human blood culture that displayed atypical light cream-colored colonies in chromogenic agar and was unable to produce ß-glucuronidase and ß-galactosidase in biochemical tests. Genomic analysis showed that the strain belongs to sequence type 59 (ST59) and phylogroup F. The evaluation in silico of 104 available sequenced lineages of ST59 complex showed that most of them belong to serotype O1:K1:H7, are ß-glucuronidase negative, and harbor a virulent genotype associated with the presence of important virulence markers such as pap, kpsE, chuA, fyuA, and yfcV. Most of them were isolated from extraintestinal human infections in diverse countries worldwide and could be clustered/subgrouped based on papAF allele analysis. Considering that all analyzed strains harbor a virulent genotype and most do not exhibit biochemical behavior typical of E. coli, we report that they could be misclassified or underestimated, especially in epidemiological studies where the screening criteria rely only on typical biochemical phenotypes, as happens when chromogenic media are used. IMPORTANCE The use of selective and differential media guides presumptive bacterial identification based on specific metabolic traits that are specific to each bacterial species. When a bacterial specimen displays an unusual phenotype in these media, this characteristic may lead to bacterial misidentification or a significant delay in its identification, putting a patient at risk depending on the infection type. In the present work, we describe a virulent E. coli sequence type (ST59) that does not produce beta-glucuronidase (GUS negative), production of which is the metabolic trait widely used for E. coli presumptive identification in diverse differential media. The recognition of this unusual metabolic trait may help in the proper identification of ST59 isolates, the identification of their reservoir, and the evaluation of the frequency of these pathogens in places where automatic identification methods are not available.

International high-risk clone of fluoroquinolone-resistant Escherichia coli O15:H1-D-ST393 in remote communities of Brazilian Amazon.

The global dissemination of multidrug-resistant Escherichia coli lineages belonging to high- risk clones poses a significant public health threat. Herein we report the identification and genomic profiling of two multidrug-resistant E. coli strains [BL-II-03(2) and BL-II-11(3)] belonging to the O15:H1-D-ST393 (clonal complex 31) worldwide spread clone, isolated from fecal samples of indigenous peoples belonging to two different ethnic groups of remote communities of Brazilian Amazon. Genomic analysis revealed genes and mutations conferring resistance to ß-lactams [blaTEM-1], aminoglycosides [aadA5, aph(3″)-Ib, aph(6)-Id], tetracyclines [tetB], sulfamethoxazole/trimethoprim [sul1, sul2, dfrA17], and fluoroquinolones [gyrA (D87N, S83L), parC (S80I, S57T), parE (L416F)]; and presence of IncQ1, IncFIA, and IncFIB(pB171) plasmids. On the other hand, phylogenomics of globally reported E. coli ST393 assigned E. coli strains BL-II-03(2) and BL-II-11(3) to a cluster comprising human isolates from Australia, Canada, China, Sweden, and United States of America. These results might provide valuable information for understanding dissemination of intercontinental multidrug-resistant clones in remote communities with low levels of antibiotic exposure.

Genetic and Virulence Characteristics of a Hybrid Atypical Enteropathogenic and Uropathogenic Escherichia coli (aEPEC/UPEC) Strain.

Hybrid strains of Escherichia coli combine virulence traits of diarrheagenic (DEC) and extraintestinal pathogenic E. coli (ExPEC), but it is poorly understood whether these combined features improve the virulence potential of such strains. We have previously identified a uropathogenic E. coli (UPEC) strain (UPEC 252) harboring the eae gene that encodes the adhesin intimin and is located in the locus of enterocyte effacement (LEE) pathogenicity island. The LEE-encoded proteins allow enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) to form attaching and effacing (A/E) lesions in enterocytes. We sought to characterize UPEC 252 through whole-genome sequencing and phenotypic virulence assays. Genome analysis unveiled that this strain harbors a complete LEE region, with more than 97% of identity comparing to E2348/69 (EPEC) and O157:H7 Sakai (EHEC) prototype strains, which was functional, since UPEC 252 expressed the LEE-encoded proteins EspB and intimin and induced actin accumulation foci in HeLa cells. Phylogenetic analysis performed comparing 1,000 single-copy shared genes clustered UPEC 252 with atypical EPEC strains that belong to the sequence type 10, phylogroup A. Additionally, UPEC 252 was resistant to the bactericidal power of human serum and colonized cells of the urinary (T24 and HEK293-T) and intestinal (Caco-2 and LS174T) tracts. Our findings suggest that UPEC 252 is an atypical EPEC strain that emerges as a hybrid strain (aEPEC/UPEC), which could colonize new niches and potentially cause intestinal and extraintestinal infections.

Diversity of Hybrid- and Hetero-Pathogenic Escherichia coli and Their Potential Implication in More Severe Diseases.

Although extraintestinal pathogenic Escherichia coli (ExPEC) are designated by their isolation site and grouped based on the type of host and the disease they cause, most diarrheagenic E. coli (DEC) are subdivided into several pathotypes based on the presence of specific virulence traits directly related to disease development. This scenario of a well-categorized E. coli collapsed after the German outbreak of 2011, caused by one strain bearing the virulence factors of two different DEC pathotypes (enteroaggregative E. coli and Shiga toxin-producing E. coli). Since the outbreak, many studies have shown that this phenomenon is more frequent than previously realized. Therefore, the terms hybrid- and hetero-pathogenic E. coli have been coined to describe new combinations of virulence factors among the classic E. coli pathotypes. In this review, we provide an overview of these classifications and highlight the E. coli genomic plasticity that results in some mixed E. coli pathotypes displaying novel pathogenic strategies, which lead to a new symptomatology related to E. coli diseases. In addition, as the capacity for genome interrogation has grown in the last few years, it is clear that genes encoding some virulence factors, such as Shiga toxin, are found among different E. coli pathotypes to which they have not traditionally been associated, perhaps foreshowing their emergence in new and severe outbreaks caused by such hybrid strains. Therefore, further studies regarding hetero-pathogenic and hybrid-pathogenic E. coli isolates are necessary to better understand and control the spread of these pathogens.