Mauricio J. Farfan

MOLECULAR MECHANISMS THAT MEDIATE COLONIZATION OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI STRAINS

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) is a group of pathogens which cause gastrointestinal disease in humans and have been associated with numerous food-borne outbreaks worldwide. The intimin adhesin has been considered for many years to be the only colonization factor in these strains. However, the rapid progress in whole-genome sequencing of different STEC serotypes has accelerated the discovery of other adhesins (fimbrial and afimbrial), which have emerged as important contributors to the intestinal colonization occurring during STEC infection. This review summarizes recent progress to identify and characterize, at the molecular level, novel adhesion and colonization factors in STEC strains, with an emphasis on their contribution to virulence traits, their host-pathogen interactions, the regulatory mechanisms controlling their expression, and their role as targets eliciting immune responses in the host.

The Major Pilin Subunit of the AAF/II Fimbriae from Enteroaggregative Escherichia coli Mediates Binding to Extracellular Matrix Proteins

ABSTRACT Enteroaggregative Escherichia coli (EAEC) adherence to human intestinal tissue is mediated by aggregative adherence fimbriae (AAF); however, the receptors involved in EAEC adherence remain uncharacterized. Adhesion to extracellular matrix proteins is commonly observed among enteric pathogens, so we addressed the hypothesis that EAEC may bind to extracellular matrix proteins commonly found in the intestine. We found that EAEC prototype strain 042 adhered more abundantly to surfaces that were precoated with the extracellular matrix proteins fibronectin, laminin, and type IV collagen. Differences in fibronectin binding of almost 2 orders of magnitude were observed between EAEC 042 and a mutant in the AAF/II major pilin gene, aafA. Purified AafA, refolded as a donor strand complementation construct, bound fibronectin in a dose-dependent manner. Addition of fibronectin to the apical surfaces of polarized T84 cell monolayers augmented EAEC 042 adherence, and this effect required expression of aafA. Finally, increased bacterial adherence was observed when apical secretion of fibronectin was induced by adenosine in polarized T84 cells. Binding to fibronectin may contribute to colonization of the gastrointestinal tract by EAEC.

Long polar fimbriae of enterohemorrhagic Escherichia coli O157:H7 bind to extracellular matrix proteins.

ABSTRACT Adherence to intestinal cells is a key process in infection caused by enterohemorrhagic Escherichia coli (EHEC). Several adhesion factors that mediate the binding of EHEC to intestinal cells have been described, but the receptors involved in their recognition are not fully characterized. Extracellular matrix (ECM) proteins might act as receptors involved in the recognition of enteric pathogens, including EHEC. In this study, we sought to characterize the binding of EHEC O157:H7 to ECM proteins commonly present in the intestine. We found that EHEC prototype strains as well as other clinical isolates adhered more abundantly to surfaces coated with fibronectin, laminin, and collagen IV. Further characterization of this phenotype, by using antiserum raised against the LpfA1 putative major fimbrial subunit and by addition of mannose, showed that a reduced binding of EHEC to ECM proteins was observed in a long polar fimbria (lpf) mutant. We also found that the two regulators, H-NS and Ler, had an effect in EHEC Lpf-mediated binding to ECM, supporting the roles of these tightly regulated fimbriae as adherence factors. Purified Lpf major subunit bound to all of the ECM proteins tested. Finally, increased bacterial adherence was observed when T84 cells, preincubated with ECM proteins, were infected with EHEC. Taken together, these findings suggest that the interaction of Lpf and ECM proteins contributes to the EHEC colonization of the gastrointestinal tract.

Frequency and clinical outcome of respiratory viral infections and mixed viral-bacterial infections in children with cancer, fever and neutropenia.

Background: The role of respiratory viral infections (RVIs) as a cause of overall fever and neutropenia (FN) episodes in children with cancer has been less characterized than bacterial infections. We conducted a study aimed to determine the frequency of RVI in children with low compared with high risk for invasive bacterial infection (IBI) FN episodes and compare the clinical outcome of RVI and mixed RV-bacterial infections. Methods: Prospective, multicenter study in children with cancer and FN admitted to pediatric hospitals in Chile between May 2009 and January 2011. Children were evaluated by clinical examination and laboratory tests, including bacterial cultures and their risk for IBI. Nasopharyngeal sample was obtained for the detection of 17 respiratory viruses using polymerase chain reaction−DNA microarray platform. Results: A total of 331 episodes of FN in 193 children were enrolled of whom 55% were male, with the median age of 7 years and 61% had a hematological malignancy. A viral and/or bacterial pathogen was detected in 67% (224/331) episodes. Overall, RVIs were associated with 57% of FN of which one-third were mixed RV-bacterial infections. Bacterial infection was detected in 29% (97/331). Children classified at admission as high risk for IBI had a similar overall proportion of RVI compared with low-risk group. Respiratory syncytial virus (31%) and rhinovirus (23%) were the most frequently detected respiratory viruses, followed by parainfluenza (12%) and influenza A (11%). Children detected with any respiratory virus had fewer days of hospitalization and a significantly lower probability of hypotension and admission to pediatric intensive care unit irrespective of their risk classification status at admission when compared with children with mixed RV-bacterial or bacterial infections (P < 0.05). All children with a sole RVI had favorable outcome. Conclusions: RVIs were the most frequently detected agents irrespective of their initial risk assessment for IBI. The clinical outcome of mixed RVI was similar to sole RVI episodes as well as for bacterial infections compared with mixed viral-bacterial infections. Systematic and early detection of RVI in children with cancer and FN might help to optimize their management by reducing hospitalization and antimicrobial use.

Prevalence of TPMT and ITPA gene polymorphisms and effect on mercaptopurine dosage in Chilean children with acute lymphoblastic leukemia

BackgroundMercaptopurine (6-MP) plays a pivotal role in treatment of childhood acute lymphoblastic leukemia (ALL); however, interindividual variability in toxicity of this drug due to genetic polymorphism in 6-MP metabolizing enzymes has been described. We determined the prevalence of the major genetic polymorphisms in 6-MP metabolizing enzymes in Chilean children with ALL.Methods103 Chilean pediatric patients with a confirmed diagnosis of ALL were enrolled. DNA was isolated from whole blood and genetic polymorphism in thiopurine S-methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) coding genes were detected by polymorphism chain reaction-restriction fragment length (PCR-RFLP) assay.ResultsThe total frequency of variant TPMT alleles was 8%. TPMT*2, TPMT*3A and TPMT*3B alleles were found in 0%, 7%, and 1% of patients, respectively. For ITPA, the frequency of P32T allele was 3%. We did not observe any homozygous variant for TPMT and ITPA alleles. We also analyzed a subgroup of 40 patients who completed the maintenance phase of ALL treatment, and we found that patients carrying a TPMT gene variant allele required a significantly lower median cumulative dosage and median daily dosage of 6-MP than patients carrying wild type alleles.ConclusionTMPT genotyping appears an important tool to further optimize 6-MP treatment design in Chilean patients with ALL.

Diagnosis of bacteremia in febrile neutropenic episodes in children with cancer: microbiologic and molecular approach.

Background: Bacterial isolation using conventional microbiologic techniques rarely surpasses 25% in children with clinical and laboratory findings indicative of an invasive bacterial infection. The aim of this study was to determine the role of real-time polymerase chain reaction (RT-PCR) from whole blood samples compared with automated blood cultures (BC) in detection of relevant microorganisms causing bacteremia in episodes of high-risk febrile neutropenia (HRFN) in children with cancer. Methods: Children presenting with HRFN at 6 hospitals in Santiago, Chile, were invited to participate. Blood samples were obtained at admission for BC, and at admission and 24 hours for RT-PCR targeting DNA of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa causing bacteremia in children with HRFN. Results: A total of 177 HRFN episodes were evaluated from May 2009 to August 2010, of which 29 (16.3%) had positive BC, 9 (5%) positive for 1 of the 3 selected bacterial species: 5 for E. coli, 3 for S. aureus, and 1 for P. aeruginosa. RT-PCR detected 39 bacteria in 36 episodes (20%): 14 E. coli, 20 S. aureus, and 5 P. aeruginosa. The sensitivity, specificity, and positive and negative predictive values of RT-PCR compared with BC were 56%, 80%, 13%, and 97%. The final clinical diagnosis was compatible with an invasive bacterial infection in 30/36 (83%) RT-PCR-positive episodes. Conclusions: In our series, RT-PCR significantly improved detection of the most relevant bacteria associated with HRFN episodes. Large number of patients and close clinical monitoring, in addition to improved RT-PCR techniques will be required to fully recommend RT-PCR-based diagnosis for the routine workup of children with cancer, fever, and neutropenia.

Characterization of the most prevalent colonization factor antigens present in Chilean clinical enterotoxigenic Escherichia coli strains using a new multiplex polymerase chain reaction.

Current methods to detect the colonization factor antigens (CFAs) associated with enterotoxigenic Escherichia coli (ETEC) strains are cumbersome, with some methods requiring antibodies that are not readily available. To achieve a gene-based method, we designed 2 multiplex polymerase chain reaction reactions to detect genes encoding the most common ETEC fimbrial colonization factors, including CFA/I and coli surface (CS) antigens CS1, CS2, CS3, CS4, CS5, and CS6. Analysis of 183 clinical ETEC strains shows that the most prevalent colonization factors were CFA/I only, CS1 and CS3, CS2 and CS3, and CS6 only. Interestingly, we identified 3 clinical isolates expressing CS1 only without its regulator rns. The method described here proved to be rapid and robust and correlates well with phenotypic expression of the CFAs, becoming a novel molecular diagnostic and research tool for future epidemiologic studies.

Enterovirus D68 Infection, Chile, Spring 2014

To the Editor: Enterovirus D68 (EV-D68) is an emergent viral pathogen associated with severe respiratory illness, especially in children with asthma (1). The ongoing epidemic in the United States has expanded to 47 states; as of November 25, 2014, a total of 1,121 persons were affected (http://www.cdc.gov/non-polio-enterovirus/outbreaks/EV-D68-outbreaks.html).

The long polar fimbriae of STEC O157:H7 induce expression of pro-inflammatory markers by intestinal epithelial cells

Infection with Shiga toxin-producing Escherichia coli (STEC) O157:H7 is characterized by acute inflammation of the colonic mucosa. STEC O157:H7 contains two non-identical loci encoding long polar fimbriae (Lpf), which play a role in the STEC colonization of the intestinal epithelial cells. However, no information is available regarding the involvement of Lpf in the STEC-induced host inflammatory response. Hence, in this study we assess the role of Lpf as an inducer of inflammation on intestinal epithelial cells. Secretion of pro-inflammatory cytokines in response to STEC wild type and lpf isogenic mutants was evaluated on intestinal T84 cells. Of the 27 cytokines assayed, IL-6, IL-8, IL-15, FGF, GM-CSF and IP-10 were significantly reduced, when compared to the wild-type strain, in the lpfA1 lpfA2 double mutant. Further, the host intracellular signaling pathways activated in response to Lpf were determined by using an array containing genes representative of 18 different signal transduction pathways. The analysis indicated that the NF-κB pathway is activated in response to Lpf-expressing STEC. Therefore, our study supports the role of Lpf as a STEC factor mediating intestinal inflammation.

Locus of Adhesion and Autoaggregation (LAA), a pathogenicity island present in emerging Shiga Toxin–producing Escherichia coli strains

Shiga Toxin-producing Escherichia coli (STEC) are a group of foodborne pathogens associated with diarrhea, dysentery, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Shiga toxins are the major virulence factor of these pathogens, however adhesion and colonization to the human intestine is required for STEC pathogenesis. A subset of STEC strains carry the Locus of Enterocyte Effacement (LEE) pathogenicity island (PAI), which encodes genes that mediate the colonization of the human intestine. While LEE-positive STEC strains have traditionally been associated with human disease, the burden of disease caused by STEC strains that lacks LEE (LEE-negative) has increased recently in several countries; however, in the absence of LEE, the molecular pathogenic mechanisms by STEC strains are unknown. Here we report a 86-kb mosaic PAI composed of four modules that encode 80 genes, including novel and known virulence factors associated with adherence and autoaggregation. Therefore, we named this PAI as Locus of Adhesion and Autoaggregation (LAA). Phylogenomic analysis using whole-genome sequences of STEC strains available in the NCBI database indicates that LAA PAI is exclusively present in a subset of emerging LEE-negative STEC strains, including strains isolated from HC and HUS cases. We suggest that the acquisition of this PAI is a recent evolutionary event, which may contribute to the emergence of these STEC.