Identification of the long polar fimbriae gene variants in the locus of enterocyte effacement-negative Shiga toxin-producing Escherichia coli strains isolated from humans and cattle in Argentina

The long polar fimbriae (Lpf) is one of few adhesive factors of Shiga toxin-producing Escherichia coli (STEC) and it is associated with colonization of the intestine. Studies have demonstrated the presence of lpf genes in several pathogenic E. coli strains, and classification of variants based on polymorphisms in the lpfA1 and lpfA2 genes has been adopted. Using a collection of Argentinean locus of enterocyte effacement (LEE)-negative STEC strains, we determined that the different lpfA types were present in a wide variety of serotypes with no apparent association between the types of lpfA1 or lpfA2 genes and the severity of human disease. The lpfA2-1 was the most prevalent variant identified, which was present in 95.8% of the isolates, and lpfA1-3 and lpfA2-2, proposed as specific biomarkers of E. coli O157:H7, were not found in any of the serotypes studied. The prevalence of lpf genes in a large number of strains is useful to understand the genetic diversity of LEE-negative STEC and to define the association of some of these isolates carrying specific lpf-variants with disease.Fil: Galli, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; ArgentinaFil: Torres, Alfredo G.. University of Texas Medical Branch; Estados UnidosFil: Rivas, Marta. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentin

From ingestion to colonization: the influence of the host environment on regulation of the LEE encoded type III secretion system in enterohaemorrhagic Escherichia coli

Enterohaemorrhagic Escherichia coli (EHEC) binds to host tissue and intimately attaches to intestinal cells using a dedicated type III secretion system (T3SS). This complex multi-protein organelle is encoded within a large pathogenicity island called the locus of enterocyte effacement (LEE), which is subject to extensive regulatory control. Over the past 15 years we have gained a wealth of knowledge concerning how the LEE is regulated transcriptionally by specific, global and phage encoded regulators. More recently, significant advances have been made in our understanding of how specific signals, including host or microbiota derived metabolic products and various nutrient sources, can affect how the LEE-encoded T3SS is regulated. In this review we discuss regulation of the LEE, focusing on how these physiologically relevant signals are sensed and how they affect the expression of this major virulence factor. The implications for understanding the disease process by specific regulatory mechanisms are also discussed

Metabolic characteristics and genomic epidemiology of Escherichia coli serogroup O145 : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand

Shiga toxin-producing Escherichia coli (STEC) are a global public health concern, and can cause severe human disease. Ruminants are asymptomatic reservoirs of STEC, shedding this pathogen via their faeces. There is ‘zero tolerance’ for the Top 7 STEC serogroups (O26, O45, O103, O111, O121, O145 and O157) in ground beef products exported to the USA. STEC may contaminate carcasses during processing and therefore are a major regulatory concern for New Zealand’s meat industry. A previous study investigating the prevalence of STEC in young calves (n=1508) throughout New Zealand identified STEC O145 as the most prevalent serogroup (43%) at the dairy farm level compared to the other Top 7 serogroups. This high prevalence underlines STEC O145 as a public health concern and an issue for the meat industry. Current culture-based methods for STEC detection are not fully discriminatory due to the lack of consistent differential characteristics between STEC and non-pathogenic E. coli. This study aims to (i) investigate metabolic characteristics of E. coli O145 to facilitate the differential culture of this serogroup and (ii) understand the genomic epidemiology of E. coli O145 using whole genome sequencing (WGS). E. coli O145 strains examined in this study were genetically and metabolically diverse, according to carbon utilisation. The metabolic and genomic analyses were unable to differentiate between stx-positive and stx-negative O145 strains and there was no association with isolation source. However, clustering of O145 strains was observed according to multi-locus sequence type and at the level of eae subtype, a gene encoding the protein intimin which is involved in bacterial attachment to intestinal epithelial cells. Carbon substrates such as D-serine and D-malic acid were identified as candidate metabolites to differentiate defined O145 sequence types and may assist with identification in conjunction with currently available molecular methods. This research has demonstrated the genetic heterogeneity of serogroup O145 and has made significant progress in the identification of metabolites that may prove beneficial in the development of a differential media for certain subsets of serogroup O145. Such a medium would prove a valuable tool for maintaining and monitoring public health and providing food quality and safety assurances that New Zealand meat for export is free of this pathogen

Identification of intimin alleles in pathogenic Escherichia coli by PCR-restriction fragment length polymorphism analysis

A rapid two-step identification method based on PCR-RFLP analysis of the intimin gene was developed to differentiate specific alleles in pathogenic Escherichia coli. This technique, tested on isolates eae-positive, accurately detects eae and resolves alleles encoding the α1, α2, β, γ1, γ2/θ, κ, ɛ, ζ, and ι intimin variants

Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells.

Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these molecular syringes for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells

Virulence profile comparison between LEE-negative Shiga toxin-producing Escherichia coli (STEC) strains isolated from cattle and humans

For comparison purposes, the prevalence of 8 virulence markers was investigated, by PCR, in 153 cattle and 47 human Locus for Enterocyte Effacement (LEE)-negative Shiga toxin-producing Escherichia coli (STEC) strains isolated in Argentina. Also, their correlation with severe disease was established. The virulence markers studied comprises 5 fimbrial and nonfimbrial adhesin-encoding genes (fimA, iha, efa1, lpfAO113, and saa) and 3 toxin genes (cdt-V, subAB and astA) in addition to the Shiga toxins. The most prevalent virulence marker found was that encoded by the lpfAO113 gene (199/200, 99%). Comparatively, the lpfAO113, fimA, iha, saa, subAB, cdt-V and astA genes were detected in 100%, 92.8%, 85%, 52.9%, 36%, 11.8% and 9.8% of the cattle strains and in 97.9%, 95.7%, 89.4%, 40.4%, 32%, 17% and 10.6% of the human strains, respectively. All STEC strains were efa1 negative. The most prevalent profile observed among cattle and human STEC strains was lpfAO113 iha fimA. These results show that bovine LEE-negative STEC strains possessed genes encoding virulence factors present in human LEE-negative STEC strains that are associated with disease. Despite a great diversity of virulence profiles observed, further studies comparing wild type strains and their allelic mutants are needed to evaluate the role of each factor in the pathogenesis of LEE-negative STEC strains during human infections.Fil: Galli, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; ArgentinaFil: Miliwebsky, Elizabeth. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; ArgentinaFil: Irino, Kinue. Instituto Adolfo Lutz; BrasilFil: Leotta, Gerardo Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; ArgentinaFil: Rivas, Marta. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentin

The host metabolite D-serine contributes to bacterial niche specificity through gene selection

Escherichia coli comprise a diverse array of both commensals and niche-specific pathotypes. The ability to cause disease results from both carriage of specific virulence factors and regulatory control of these via environmental stimuli. Moreover, host metabolites further refine the response of bacteria to their environment and can dramatically affect the outcome of the host–pathogen interaction. Here, we demonstrate that the host metabolite, D-serine, selectively affects gene expression in E. coli O157:H7. Transcriptomic profiling showed exposure to D-serine results in activation of the SOS response and suppresses expression of the Type 3 Secretion System (T3SS) used to attach to host cells. We also show that concurrent carriage of both the D-serine tolerance locus (dsdCXA) and the locus of enterocyte effacement pathogenicity island encoding a T3SS is extremely rare, a genotype that we attribute to an ‘evolutionary incompatibility’ between the two loci. This study demonstrates the importance of co-operation between both core and pathogenic genetic elements in defining niche specificity

Identification and detection of iha subtypes in LEE-negative Shiga toxin-producing Escherichia coli (STEC) strains isolated from humans, cattle and food

LEE-negative Shiga toxin-producing Escherichia coli (STEC) strains are important cause of infection in humans and they should be included in the public health surveillance systems. Some isolates have been associated with haemolytic uremic syndrome (HUS) but the mechanisms of pathogenicity are is a field continuos broadening of knowledge. The IrgA homologue adhesin (Iha), encoded by iha, is an adherence-conferring protein and also a siderophore receptor distributed among LEE-negative STEC strains. This study reports the presence of different subtypes of iha in LEE-negative STEC strains. We used genomic analyses to design PCR assays for detecting each of the different iha subtypes and also, all the subtypes simultaneously. LEE-negative STEC strains were designed and different localizations of this gene in STEC subgroups were examinated.Genomic analysis detected iha in a high percentage of LEE-negative STEC strains. These strains generally carried iha sequences similar to those harbored by the Locus of Adhesion and Autoaggregation (LAA) or by the plasmid pO113. Besides, almost half of the strains carried both subtypes. Similar results were observed by PCR, detecting iha LAA in 87% of the strains (117/135) and iha pO113 in 32% of strains (43/135). Thus, we designed PCR assays that allow rapid detection of iha subtypes harbored by LEE-negative strains. These results highlight the need to investigate the individual and orchestrated role of virulence genes that determine the STEC capacity of causing serious disease, which would allow for identification of target candidates to develop therapies against HUS.Fil: Colello, Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Krüger, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Velez, María Victoria. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; ArgentinaFil: Del Canto, Felipe. Universidad de Chile. Facultad de Medicina. Instituto de Ciencias Biomédicas; ChileFil: Etcheverría, Analía Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Vidal, Roberto. Universidad de Chile. Facultad de Medicina. Instituto de Ciencias Biomédicas; Chile. Universidad de Chile. Facultad de Medicina. Instituto Milenio de Inmunología e Inmunoterapia; ChileFil: Padola, Nora Lía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentin

Pathogenetic sequences in coeliac disease : closing the jigsaw puzzle

Coeliac disease is one of the best understood models of adaptive immunity in which a known dietary component triggers over-presentation of a known autoantigen in genetically predisposed individuals. The dynamics of this gene-nutrient interaction and the mechanism responsible for accelerated programmed death of the enterocytes lining the upper small intestine are explored in order to generate insight into the large number of candidate pharmacological agents that may well aid or replace cumbersome dietary treatment in years to come.peer-reviewe

Shiga Toxin–producing Escherichia coli Strains Negative for Locus of Enterocyte Effacement

The ehx plasmids of these strains are highly related, which suggests acquisition of the large plasmid was central to the strains’ emergence