Jacinta Sanchez Pelayo

Detection of diarrheagenic Escherichia coli strains isolated from dogs and cats in Brazil

Escherichia coli are gut microbiota bacteria that can cause disease in some humans and other animals, including dogs and cats that humans often keep as pets. Diarrheagenic E. coli (DEC) strains are classified into six categories: enteropathogenic (EPEC), enterotoxigenic (ETEC), Shiga toxin-producing (STEC), enteroinvasive (EIEC), enteroaggregative (EAEC), and diffuse-adhering E. coli (DAEC). In this study 144 and 163 E. coli colonies were isolated from the fecal samples of 50 dogs and 50 cats, respectively, with and without diarrhea from a Veterinary Hospital (clinical isolates). The virulence factors were determined using multiplex Polymerase Chain Reaction. Adherence assays, antibacterial susceptibility and serotyping (somatic or flagellar antigens) were performed on DEC isolates. We found 25 (17.4%) and 4 (2.5%) DEC strains isolated from dogs and cats, respectively. Only the EPEC and EAEC pathotypes were found in both animals. Meanwhile, genes from other pathotypes (STEC, EIEC, and ETEC) were not found in these clinical isolates. All of the DEC strains showed mannose-resistant adherence to HEp-2 and HeLa cells, and aggregative adherence was predominant in these isolates. Multiresistant strains to antimicrobials were found in most DEC strains including usual and unusual antimicrobials in veterinary practices. The serotypes of these DEC isolates were variable. The ONT serotype was predominant in these isolates. Some serotypes found in our study were described to human DEC. Here, we demonstrate that pets carry virulent DEC genes, which are mainly strains of EPECs and EAECs. The presence of these virulence factors in isolates from animals without diarrhea suggests that pets can act as a reservoir for human infection.

Detection of OXA-231, a new variant of blaOXA-143, in Acinetobacter baumannii from Brazil: a case report

Department of Microbiology, Universidade Estadual de Londrina, Londrina, Paraná, Brazil; Department of Science Pathological, Universidade Estadual de Londrina, Londrina, Paraná, Brazil; Laboratório ALERTA, Division of Infectious Diseases, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil; Department of Pathology, Clinical and Toxicological Analysis, Hospital Universitário de Londrina, Universidade Estadual de Londrina, Londrina, Paraná, Brazil

Metallo-β-lactamase-production in meropenem-susceptible Pseudomonas aeruginosa isolates: risk for silent spread

The aim of this study was to characterize two metallo-β-lactamases (MBLs)-producing Pseudomonas aeruginosa clinical isolates showing meropenem susceptibility. Antimicrobial susceptibility was assessed by automated testing and Clinical and Laboratory Standards Institute agar dilution method. MBL production was investigated by phenotypic tests. Molecular typing was determined by pulsed field gel electrophoresis (PFGE). MBL-encoding genes, as well as their genetic context, were identified by polymerase chain reaction (PCR) and sequencing. The location of blaIMP-16 was determined by plasmid electrophoresis, Southern blot and hybridization. Transcriptional levels of blaIMP-16, mexB, mexD, mexF, mexY, ampC and oprD were determined by semi-quantitative real time PCR. The P. aeruginosa isolates studied, Pa30 and Pa43, showed imipenem and meropenem susceptibility by automated testing. Agar dilution assays confirmed meropenem susceptibility whereas both isolates showed low level of imipenem resistance. Pa30 and Pa43 were phenotypically detected as MBL producers. PFGE revealed their clonal relatedness. blaIMP-16 was identified in both isolates, carried as a single cassette in a class 1 integron that was embedded in a plasmid of about 60-Kb. Pa30 and Pa43 overexpressed MexAB-OprM, MexCD-OprJ and MexXY-OprM efflux systems and showed basal transcriptional levels of ampC and oprD. MBL-producing P. aeruginosa that are not resistant to meropenem may represent a risk for therapeutic failure and act as silent reservoirs of MBL-encoding genes.

Diversity of Shiga toxin-producing Escherichia coli in sheep flocks of Paraná State, southern Brazil

Sheep constitute an important source of zoonotic pathogens as Shiga toxin-producing Escherichia coli (STEC). In this study, the prevalence, serotypes and virulence profiles of STEC were investigated among 130 healthy sheep from small and medium farms in southern Brazil. STEC was isolated from 65 (50%) of the tested animals and detected in all flocks. A total of 70 STEC isolates were characterized, and belonged to 23 different O:H serotypes, many of which associated with human disease, including hemolytic-uremic syndrome (HUS). Among the serotypes identified, O76:H19 and O65:H- were the most common, and O75:H14 and O169:H7 have not been previously reported in STEC strains. Most of the STEC isolates harbored only stx1, whereas the Stx2b subtype was the most common among those carrying stx2. Enterohemolysin (ehxA) and intimin (eae) genes were detected in 61 (87.1%) and four (5.7%) isolates, respectively. Genes encoding putative adhesins (saa, iha, lpfO113) and toxins (subAB and cdtV) were also observed. The majority of the isolates displayed virulence features related to pathogenesis of STEC, such as adherence to epithelial cells, high cytotoxicity and enterohemolytic activity. Ovine STEC isolates belonged mostly to phylogenetic group B1. PFGE revealed particular clones distributed in some farms, as well as variations in the degree of genetic similarity within serotypes examined. In conclusion, STEC are widely distributed in southern Brazilian sheep, and belonged mainly to serotypes that are not commonly reported in other regions, such as O76:H19 and O65:H-. A geographical variation in the distribution of STEC serotypes seems to occur in sheep.

Resistance profile to antimicrobials of Salmonella spp. isolated from human infections

O objetivo deste estudo foi analisar o perfil de resistencia a antimicrobianos, de Salmonella isoladas de 21 amostras provenientes de pacientes com sintomatologia de gastroenterite. Verificou-se que S. enteritidis foi o sorotipo com maior prevalencia. Estas cepas mostraram-se sensiveis a maioria dos antimicrobianos testados, no entanto, as cepas S. typhimurium e S. enterica subsp. enterica sorotipo 4,5,12:i:-, mostraram-se resistentes a varios antimicrobianos. Vigilância e um monitoramento eficiente, para diminuir a resistencia antimicrobiana em microrganismos que causam infeccoes veiculadas por alimentos, devem ser prioridade para a saude publica.

The nitroxyl donor Angeli's salt ameliorates Staphylococcus aureus-induced septic arthritis in mice

Abstract Septic arthritis is a severe and rapidly debilitating disease associated with severe joint pain, inflammation and oxidative stress. Nitroxyl (HNO) has become a nitrogen oxide of significant interest due to its pharmacological endpoints that are potentially favorable for treating varied diseases. However, whether HNO also serves as a treatment to septic arthritis is currently unknown. The aim of this study was to investigate the effect of the HNO donor, Angelis salt (AS), in the outcome of chronic Staphylococcus aureus (S. aureus)‐induced septic arthritis in mice. Daily treatment with AS inhibited mechanical hyperalgesia and inflammation (edema, leukocyte migration, cytokines release and NF‐&kgr;B activation, and oxidative stress) resulting in reduced disease severity (clinical course, histopathological changes, proteoglycan levels in the joints, and osteoclastogenesis). In addition, AS decreased the number of S. aureus colony forming unities in synovial tissue, enhanced the bactericidal effect of macrophages and inhibited the worsening of systemic inflammatory response (leukocyte counts in the lung and systemic proinflammatory cytokine concentration). Our results suggest for the first time the therapeutic potential of AS in a model of septic arthritis by mechanisms involving microbicidal effects, anti‐inflammatory actions and reduction of disease severity. Graphical abstract Figure. No Caption available. HighlightsAngelis salt (AS) reduces S. aureus‐induced articular pain and inflammation.AS reduced S. aureus‐induced cartilage damage and osteoclastogenesis.AS reduced S. aureus‐induced cytokines production and NF‐&kgr;B activation.AS reduced S. aureus‐induced oxidative stress.AS is microbicide and prevents systemic inflammatory response.

Lambs are an important source of atypical enteropathogenic Escherichia coli in southern Brazil

Food-producing animals can harbor Escherichia coli strains with potential to cause diseases in humans. In this study, the presence of enteropathogenic E. coli (EPEC) was investigated in fecal samples from 130 healthy sheep (92 lambs and 38 adults) raised for meat in southern Brazil. EPEC was detected in 19.2% of the sheep examined, but only lambs were found to be positive. A total of 25 isolates was characterized and designated atypical EPEC (aEPEC) as tested negative for bfpA gene and BFP production. The presence of virulence markers linked to human disease as ehxA, paa, and lpfAO113 was observed in 60%, 24%, and 88% of the isolates, respectively. Of the 11 serotypes identified, eight were described among human pathogenic strains, while three (O1:H8, O11:H21 and O125:H19) were not previously detected in aEPEC. Associations between intimin subtypes and phylogroups were observed, including eae-θ2/A, eae-β1/B1, eae-α2/B2 and eae-γ1/D. Although PFGE typing of 16 aEPEC isolates resulted in 14 unique pulsetypes suggesting a genetic diversity, specific clones were found to be distributed in some flocks. In conclusion, potentially pathogenic aEPEC strains are present in sheep raised for meat, particularly in lambs, which can better contribute to dissemination of these bacteria than adult animals.

First description of a Shiga toxin-producing Escherichia coli O103:H2 strain isolated from sheep in Brazil

1 Departamento de Microbiologia, Universidade Estadual de Londrina, Londrina, Brazil 2 Departamento de Microbiologia, Parasitologia e Imunologia, Universidade Federal de São Paulo, São Paulo, Brazil 3 Laboratório de Bacteriologia, Instituto Butantan, São Paulo, Brazil 4 Laboratorio de Referencia de E. coli, Departamento de Microbiología y Parasitología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain

New approach for detection of Escherichia coli invasion to HeLa cells

To establish a successful infection, microorganisms have developed strategies to invade host cells. One of the most important human pathogens and the greatest cause of urinary tract infections, Escherichia coli, still do not have its invasion mechanisms fully understood. This work aims to present a new approach for detecting bacterial invasion of lineage cells, based on an enzymatic-fluorogenic method. The focus of this technique is the detection of E. coli invasion of HeLa cells, exploring β-glucuronidase, a specific constitutive enzyme of this bacterium. This enzyme hydrolyses the key substrate of this work, 4-methylumbelliferyl-β-d-glucuronide (MUG), resulting in a fluorogenic molecule, 4-methylumbelliferone. The fluorescence curve created by this method, analyzed by Tukey statistical test, demonstrated that this detection can be efficiently performed after 5 h incubation with MUG. When testing uropathogenic E. coli and E. coli isolated from human gastrointestinal microbiota, the proposed method presented similar results to those exhibited by plate counting invasion detection. Data examination by Duncan statistical test allowed the creation of an intensity range of bacterial invasion, which is part of the process of results interpretation. Detection by this enzymatic-fluorogenic method, compared to other existing bacterial invasion detection techniques, is less burdensome, more sensitive and allows fast achievement of reliable results.

Interleukin-33 Receptor (ST2) Deficiency Improves the Outcome of Staphylococcus aureus-Induced Septic Arthritis

The ST2 receptor is a member of the Toll/IL-1R superfamily and interleukin-33 (IL-33) is its agonist. Recently, it has been demonstrated that IL-33/ST2 axis plays key roles in inflammation and immune mediated diseases. Here, we investigated the effect of ST2 deficiency in Staphylococcus aureus-induced septic arthritis physiopathology. Synovial fluid samples from septic arthritis and osteoarthritis individuals were assessed regarding IL-33 and soluble (s) ST2 levels. The IL-33 levels in samples from synovial fluid were significantly increased, whereas no sST2 levels were detected in patients with septic arthritis when compared with osteoarthritis individuals. The intra-articular injection of 1 × 107 colony-forming unity/10 μl of S. aureus American Type Culture Collection 6538 in wild-type (WT) mice induced IL-33 and sST2 production with a profile resembling the observation in the synovial fluid of septic arthritis patients. Data using WT, and ST2 deficient (−/−) and interferon-γ (IFN-γ)−/− mice showed that ST2 deficiency shifts the immune balance toward a type 1 immune response that contributes to eliminating the infection due to enhanced microbicide effect via NO production by neutrophils and macrophages. In fact, the treatment of ST2−/− bone marrow-derived macrophage cells with anti-IFN-γ abrogates the beneficial phenotype in the absence of ST2, which confirms that ST2 deficiency leads to IFN-γ expression and boosts the bacterial killing activity of macrophages against S. aureus. In agreement, WT cells achieved similar immune response to ST2 deficiency by IFN-γ treatment. The present results unveil a previously unrecognized beneficial effect of ST2 deficiency in S. aureus-induced septic arthritis.