Survey of Minas frescal cheese from Southwest Minas Gerais for virulence factors and antimicrobial resistance in Escherichia coli isolates

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Escherichia coli strains isolated from the uteri horn, mouth, and rectum of bitches suffering from pyometra: virulence factors, antimicrobial susceptibilities, and clonal relationships among strains

Pyometra is recognized as one of the main causes of disease and death in the bitch, and Escherichia coli is the major pathogen associated with this disease. In this study, 70 E. coli isolates from the uteri horn, mouth, and rectum of bitches suffering from the disease and 43 E. coli isolates from the rectum of clinically healthy bitches were examined for the presence of uropathogenic virulence genes and susceptibility to antimicrobial drugs. DNA profiles of isolates from uteri horn and mouth in bitches with pyometra were compared by REP, ERIC, and BOX-PCR. Virulence gene frequencies detected in isolates from canine pyometra were as follows: 95.7% fim, 27.1% iss, 25.7% hly, 18.5% iuc, and 17.1% usp. Predominant resistance was determined for cephalothin, ampicillin, and nalidixic acid among the isolates from all sites examined. Multidrug resistance was found on ∼ 50% pyometra isolates. Using the genotypic methods some isolates from uteri, pus, and saliva of the same bitch proved to have identical DNA profiles which is a reason for concern due to the close relationship between household pets and humans.

Photodynamic inactivation of virulence factors of Candida strains isolated from patients with denture stomatitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Impact of sub-inhibitory concentrations of amoxicillin on helicobacter pylori virulence factors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Vesicles as carriers of virulence factors in parasitic protozoan diseases

Different types of shed vesicles as, for example, exosomes, plasma-membrane-derived vesicles or microparticles, are the focus of intense research in view of their potential role in cell cell communication and under the perspective that they might be good tools for immunotherapy, vaccination or diagnostic purposes. This review discusses ways employed by pathogenic trypanosomatids to interact with the host by shedding vesicles that contain molecules important for the establishment of infection, as opposed to previous beliefs considering them as a waste of cellular metabolism. Trypanosomatids are compared with Apicomplexa, which circulate parasite antigens bound to vesicles shed by host cells. The knowledge of the origin and chemical composition of these different vesicles might lead to the understanding of the mechanisms that determine their biological function. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

The presence of genes encoding for different virulence factors in clonally related Escherichia coli that produce CTX-Ms

Successful international clones have recently emerged among Escherichia coli that produce CTX-M beta-lactamases as important causes of community-onset urinary tract and bloodstream infections. One hundred and seven isolates that belong to sequence types (STs) ST38, ST131, ST405, ST648, and 38 nonrelated CTX-M producing E. coli from Canada and the Netherlands were assigned to phylogenetic groups and tested for the presence of genes encoding for virulence factors (VFs) using established multiplex polymerase chain reaction. The STs E. coli were significantly more resistant to antibiotics-ST38, ST405, and ST648 belonged to phylogenetic group D while ST131 belonged to B2. Secreted autotransporter toxin (sat), aerobactin receptor, and pathogenicity island marker were significantly more common among the STs; the heat-resistant agglutinin (hra) was present in ST38, sat, and uropathogenic-specific protein, and putative adhesin-siderophore receptor was more common in ST131, while outer membrane protease T was present in ST648. ST131 had a significantly higher VF score. In conclusion, the precise role of these VFs remains to be elucidated; however, we have identified certain putative VFs that possibly contribute to the fitness and success of certain sequence types. (C) 2012 Elsevier Inc. All rights reserved.

Exploring the biofilm of Streptococcus agalactiae to identify virulence factors

Streptococcus agalactiae, also known as Group B Streptococcus (GBS) is the primary colonizer of the anogenital mucosa of up to 40% of healthy women and an important cause of invasive neonatal infections worldwide. Among the 10 known capsular serotypes, GBS type III accounts for 30-76% of the cases of neonatal meningitis. Biofilms are dense aggregates of surface-adherent microorganisms embedded in an exopolysaccharide matrix. Centers for Disease Control and Prevention estimate that 65% of human bacterial infections involve biofilms (Post et al., 2004). In recent years, the ability of GBS to form biofilm attracted attention for its possible role in fitness and/or virulence. Here, a new in vitro biofilm formation protocol was developed to guarantee more stringent conditions, to better discriminate between strong-, low- and non- biofilm forming strains and reduce ambiguous data interpretation. This protocol was applied to screen the in vitro biofilm formation ability of more than 350 GBS clinical isolates from pregnant women and neonatal infections belonging to different serotype, in relation to media composition and pH. The results showed the enhancement of GBS biofilm formation in acidic condition and identified a subset of isolates belonging to serotypes III and V that forms strong biofilms in these conditions. Interestingly, the best biofilm formers belonged to the serotype III hypervirulent clone ST-17.It was also found that pH 5.0 induces down-regulation of the capsule but that this reduction is not enough by itself to ensure biofilm formation. Moreover, the ability of proteinase K to strongly inhibit biofilm formation and to disaggregate mature biofilms suggested that proteins play an essential role in promoting GBS biofilm formation and contribute to the biofilm structural stability. Finally, a set of proteins potentially expressed during the GBS in vitro biofilm formation were identified by mass spectrometry.

Surface antigens and potential virulence factors from parasites detected by comparative genomics of perfect amino acid repeats

ABSTRACT: BACKGROUND: Many parasitic organisms, eukaryotes as well as bacteria, possess surface antigens with amino acid repeats. Making up the interface between host and pathogen such repetitive proteins may be virulence factors involved in immune evasion or cytoadherence. They find immunological applications in serodiagnostics and vaccine development. Here we use proteins which contain perfect repeats as a basis for comparative genomics between parasitic and free-living organisms. RESULTS: We have developed Reptile http://reptile.unibe.ch, a program for proteome-wide probabilistic description of perfect repeats in proteins. Parasite proteomes exhibited a large variance regarding the proportion of repeat-containing proteins. Interestingly, there was a good correlation between the percentage of highly repetitive proteins and mean protein length in parasite proteomes, but not at all in the proteomes of free-living eukaryotes. Reptile combined with programs for the prediction of transmembrane domains and GPI-anchoring resulted in an effective tool for in silico identification of potential surface antigens and virulence factors from parasites. CONCLUSION: Systemic surveys for perfect amino acid repeats allowed basic comparisons between free-living and parasitic organisms that were directly applicable to predict proteins of serological and parasitological importance. An on-line tool is available at http://genomics.unibe.ch/dora.

Patient characteristics but not virulence factors discriminate between asymptomatic and symptomatic E. coli bacteriuria in the hospital

Background Escherichia coli is a common cause of asymptomatic and symptomatic bacteriuria in hospitalized patients. Asymptomatic bacteriuria (ASB) is frequently treated with antibiotics without a clear indication. Our goal was to determine patient and pathogen factors suggestive of ASB. Methods We conducted a 12-month prospective cohort study of adult inpatients with E. coli bacteriuria seen at a tertiary care hospital in St. Louis, Missouri, USA. Urine cultures were taken at the discretion of treating physicians. Bacterial isolates were tested for 14 putative virulence genes using high-throughput dot-blot hybridization. Results The median age of the 287 study patients was 65 (19–101) years; 78% were female. Seventy percent had community-acquired bacteriuria. One-hundred ten (38.3%) patients had ASB and 177 (61.7%) had symptomatic urinary tract infection (sUTI). Asymptomatic patients were more likely than symptomatic patients to have congestive heart failure (p = 0.03), a history of myocardial infarction (p = 0.01), chronic pulmonary disease (p = 0.045), peripheral vascular disease (p = 0.04), and dementia (p = 0.03). Patients with sUTI were more likely to be neutropenic at the time of bacteriuria (p = 0.046). Chronic pulmonary disease [OR 2.1 (95% CI 1.04, 4.1)] and dementia [OR 2.4 (95% CI 1.02, 5.8)] were independent predictors for asymptomatic bacteriuria. Absence of pyuria was not predictive of ASB. None of the individual virulence genes tested were associated with ASB nor was the total number of genes. Conclusions Asymptomatic E. coli bacteriuria in hospitalized patients was frequent and more common in patients with dementia and chronic pulmonary disease. Bacterial virulence factors could not discriminate symptomatic from asymptomatic bacteriurias. Asymptomatic E. coli bacteriuria cannot be predicted by virulence screening.

The Aeromonas salmonicida subsp. salmonicida exoproteome: determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors

BACKGROUND Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF2267) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential and stationary phases of growth. RESULTS Results confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the ΔascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected. CONCLUSIONS We demonstrated the whole in vitro secretome and T3SS repertoire of hypervirulent A. salmonicida. Several toxins, adhesins and enzymes that are not part of the T3SS secretome were secreted to a higher extent in the extremely low-virulent ΔascV mutant. All together, our results show the high importance of an intact T3SS to initiate the furunculosis and offer new information about the pathogenesis.

Vancomycin-resistant Enterococcus faecalis endocarditis: linezolid failure and strain characterization of virulence factors.

Infective endocarditis due to vancomycin-resistant (VR) Enterococcus faecalis has only rarely been reported. We report a case of VR E. faecalis endocarditis that failed to respond to linezolid therapy, outline the virulence traits of the isolate, and review previously published cases of VR E. faecalis endocarditis.

KLIKK proteases of Tannerella forsythia: putative virulence factors with a unique domain structure.

Comparative genomics of virulent Tannerella forsythia ATCC 43037 and a close health-associated relative, Tannerella BU063, revealed, in the latter, the absence of an entire array of genes encoding putative secretory proteases that possess a nearly identical C-terminal domain (CTD) that ends with a -Lys-Leu-Ile-Lys-Lys motif. This observation suggests that these proteins, referred to as KLIKK proteases, may function as virulence factors. Re-sequencing of the loci of the KLIKK proteases found only six genes grouped in two clusters. All six genes were expressed by T. forsythia in routine culture conditions, although at different levels. More importantly, a transcript of each gene was detected in gingival crevicular fluid (GCF) from periodontitis sites infected with T. forsythia indicating that the proteases are expressed in vivo. In each protein, a protease domain was flanked by a unique N-terminal profragment and a C-terminal extension ending with the CTD. Partially purified recombinant proteases showed variable levels of proteolytic activity in zymography gels and toward protein substrates, including collagen, gelatin, elastin, and casein. Taken together, these results indicate that the pathogenic strain of T. forsythia secretes active proteases capable of degrading an array of host proteins, which likely represents an important pathogenic feature of this bacterium.

Identification and characterization of virulence factors in Enterococcus faecalis

Enterococci are one of the leading causes of nosocomial infections, and Enterococcus faecalis causes the majority of enterococcal infections. However, the mechanisms of enterococcal pathogenesis are still not yet understood. In our initial screening of E. faecalis strain OG1RF genomic libraries, autolysin and a homolog of a protein of Enterococcus faecium previously designated P54 were found to be two major antigens that reacted with human patient sera, and an antigen designated MH-1 antigen that reacted with serum from a endocarditis patient was also identified. To explore a possible role for these antigens in enterococcal infections, the genes encoding these three antigens were disrupted in Enterococcus faecalis OG1RF. ^ To explore a possible role of an E. faecalis gelatinase (encoded by gelE), which belongs to a family of Zn-metalloproteases that have been shown to be virulence factors in other organisms, in enterococcal infections, an insertion mutant was constructed in OG1RF and tested in the mouse peritonitis model. The mice infected with the gelE mutant showed a significantly prolonged survival compared to the wild type strain. To study the expression of gelE, the regions flanking gelE were sequenced. Sequence analysis of the gelE flanking regions revealed three genes (fsrA, fsrB and fsrC) upstream of gelE that show homology to the genes in a locus (agr) that globally regulates the expression of virulence factors in Staphylococcus aureus and one open reading frame (sprE) with homology to bacterial serine protease downstream of gelE. ^ In conclusion, in this study of identification of possible virulence factors in E. faecalis surface and secreted proteins, of three genes encoding antigens detected by human patient sera, none could be shown to effect virulence in the mouse peritonitis model. Inactivation of one of these antigens (autolysin) was shown to slightly increase the tolerance of E. faecalis to penicillin. A serine protease and a locus (fsr) that regulates the expression of gelE and sprE were shown to be important for enterococcal infection in the mouse peritonitis model. (Abstract shortened by UMI.)^

Identification and characterization of antigens and potential virulence factors in Enterococcus

Enterococci are normal flora in the human intestinal tract, and also one of the leading causes of nosocomial infections, with most of the clinical isolates being Enterococcus faecalis and Enterococcus faecium. Despite extensive studies on the antibiotic resistance, the pathogenicity of enterococci is not well understood, especially for E. faecium. To identify potential virulence factors based on their antigenicity during infection, E. faecium genomic libraries were constructed and screened using sera from patients with E. faecium endocarditis. ^ As one of my projects, total polysaccharides were extracted from E. faecalis OG1RF and from two epa mutants constructed previously, TX5179 and TX5180, and western blots with patient sera showed that an immuno-reactive polysaccharide present in wild type OG1RF was not produced by either of the two epa mutants. The epa mutants were more sensitive to ethanol stress, neutrophil killing and neutrophil phagocytosis than the wild type OG1RF. ^ Expression of virulence factors is commonly regulated by two component systems. A BLAST search was performed to identify potential two component systems in the E. faecalis V583 genome database using PhoP/PhoS as query sequences, and 11 gene pairs were identified, seven of which were disrupted in E. faecalis OGIRF. ^ Finally, an in vitro translocation model was established for enterococci. E. faecalis strain OG1RF and E. faecium strain DO were shown to be able to translocate across a T84 monolayer, while E. coli strain DH5α and E. faecalis strain E1 could not. ^ In conclusion, several E. faecium antigens expressed in infection (whose antibodies present in sera from patients with E. faecium endocarditis) were identified, two of which, SagA and GlyA, were characterized and suggested to be involved in cell wall metabolism. E. faecalis epa gene cluster (involving in polysaccharide biosynthesis and known to be involved in virulence of E. faecalis in mice) was shown to be involved in hindering neutrophil killing. Several two-component systems were identified in E. faecalis and two of which, EtaRS and EtbRS, were involved in E. faecalis virulence in a mouse peritonitis model.^

Jasmonate-dependent modifications of the pectin matrix during potato development function as a defense mechanism targeted by Dickeya dadantii virulence factors

The plant cell wall constitutes an essential protection barrier against pathogen attack. In addition, cell-wall disruption leads to accumulation of jasmonates (JAs), which are key signaling molecules for activation of plant inducible defense responses. However, whether JAs in return modulate the cell-wall composition to reinforce this defensive barrier remains unknown. The enzyme 13-allene oxide synthase (13-AOS) catalyzes the first committed step towards biosynthesis of JAs. In potato (Solanum tuberosum), there are two putative St13-AOS genes, which we show here to be differentially induced upon wounding. We also determine that both genes complement an Arabidopsis aos null mutant, indicating that they encode functional 13-AOS enzymes. Indeed, transgenic potato plants lacking both St13-AOS genes (CoAOS1/2 lines) exhibited a significant reduction of JAs, a concomitant decrease in wound-responsive gene activation, and an increased severity of soft rot disease symptoms caused by Dickeya dadantii. Intriguingly, a hypovirulent D. dadantii pel strain lacking the five major pectate lyases, which causes limited tissue maceration on wild-type plants, regained infectivity in CoAOS1/2 plants. In line with this, we found differences in pectin methyl esterase activity and cell-wall pectin composition between wild-type and CoAOS1/2 plants. Importantly, wild-type plants had pectins with a lower degree of methyl esterification, which are the substrates of the pectate lyases mutated in the pel strain. These results suggest that, during development of potato plants, JAs mediate modification of the pectin matrix to form a defensive barrier that is counteracted by pectinolytic virulence factors from D. dadantii.