Characterization of the alpha-haemolysin determinant from the human enteropathogenic Escherichia coli O26 plasmid pEO5

The 157-kb conjugative plasmid pEO5 encoding alpha-haemolysin in strains of human enteropathogenic Escherichia coli (EPEC) O26 was investigated for its relationship with EHEC-haemolysin-encoding plasmids of enterohaemorrhagic E. coli (EHEC) O26 and O157 strains. Plasmid pEO5 was found to be compatible with EHEC-virulence plasmids and did not hybridize in Southern blots with plasmid pO157 from the EHEC O157:H7 strain EDL933, indicating that both plasmids were unrelated. A 9227-bp stretch of pEO5 DNA encompassing the entire alpha-hlyCABD operon was sequenced and compared for similarity to plasmid and chromosomally inherited alpha-hly determinants. The alpha-hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of the murine E. coli alpha-hly plasmid pHly152, in particular, the structural alpha-hlyCABD genes (99.2% identity) and the regulatory hlyR regions (98.8% identity). pEO5 and alpha-hly plasmids of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural alpha-hlyCABD genes. The major difference found between the hly regions of pHly152 and pEO5 is caused by the insertion of an IS2 element upstream of the hlyC gene in pHly152. The presence of transposon-like structures at both ends of the alpha-hly sequence indicates that this pEO5 virulence factor was probably acquired by horizontal gene transfer.

Influenza virus vaccination in kidney transplant recipients: serum antibody response to different immunosuppressive drugs

Introduction: This study prospectively accessed the immune response to the inactivated influenza vaccine in renal transplant recipients receiving either azathioprine or mycophenolate mofetil (MMF). Side effects were investigated. Methods: Sixty-nine patients received one dose of inactivated trivalent influenza vaccine. Antihemagglutinin (HI) antibody response against each strain was measured before and one to six months after vaccination. Results: Geometric mean HI antibody titers for H1N1 and H3N2 strains increased from 2.57 and 2.44 to 13.45 (p = 0.001) and 7.20 (p < 0.001), respectively. Pre- and post-vaccination protection rates for H1N1 and H3N2 increased from 8.7% to 49.3% (p < 0.001); and 36.3% (p < 0.001) and seroconversion rates were 36% and 25.3%, respectively. There was no response to influenza B. The use of MMF reduced the H1N1 and H3N2 protection rates and the seroconversion rate for the H1N1 strain when compared with the use of azathioprine, and subjects transplanted less than 87 months also had inferior antibody response. Adverse events were mild and there were no change on renal function post-vaccination. Conclusion: Renal transplant patients vaccinated against influenza responded with antibody production for in. uenza A virus strains, but not for in. uenza B. Use of MMF and shorter time from transplantation decreased the immune response to the vaccine.

Network genealogy of 195-bp satellite DNA supports the superimposed hybridization hypothesis of Trypanosoma cruzi evolutionary pattern

Trypanosoma cruzi is highly diverse genetically and has been partitioned into six discrete typing units (DTUs), recently re-named T. cruzi I-VI. Although T. cruzi reproduces predominantly by binary division, accumulating evidence indicates that particular DTUs are the result of hybridization events. Two major scenarios for the origin of the hybrid lineages have been proposed. It is accepted widely that the most heterozygous TcV and TcVI DTUs are the result of genetic exchange between TcII and TcIII strains. On the other hand, the participation of a TcI parental in the current genome structure of these hybrid strains is a matter of debate. Here, sequences of the T. cruzi-specific 195-bp satellite DNA of TcI, TcII, Tat, TcV, and TcVI strains have been used for inferring network genealogies. The resulting genealogy showed a high degree of reticulation, which is consistent with more than one event of hybridization between the Tc DTUs. The data also strongly suggest that Tat is a hybrid with two distinct sets of satellite sequences, and that genetic exchange between TcI and TcII parentals occurred within the pedigree of the TcV and TcVI DTUs. Although satellite DNAs belong to the fast-evolving portion of eukaryotic genomes, in >100 satellite units of nine T. cruzi strains we found regions that display 100% identity. No DTU-specific consensus motifs were identified, inferring species-wide conservation. (C) 2010 Elsevier B.V. All rights reserved.

Clonal Relationship among Atypical Enteropathogenic Escherichia coli Strains Isolated from Different Animal Species and Humans

Forty-nine typical and atypical enteropathogenic Escherichia coli (EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.

Molecular detection of enterotoxins in environmental strains of Aeromonas hydrophila and Aeromonas jandaei

Aeromonas species are widely distributed in aquatic environments and recent studies include the genus in the emergent pathogens group because of its frequent association with local and systemic infections in immunocompetent humans. Aiming to search for virulence genes in environmental strains of Aeromonas hydrophila and Aeromonas jandaei, we designed specific primers to detect act/hly A/aer complex and alt genes. Primers described elsewhere were used to detect ast. Eighty-seven strains previously identified using phenotypic and genotypic tests as A. hydrophila (41) and A. jandaei (46) were analysed for the presence of the virulence genes using PCR. DNA fragments of expected size were purified and directly sequenced. Among the 41 strains of A. hydrophila 70.7% (29), 97.6% (40) and 26.8% (11) possessed act/hly A/aer complex, ast and alt genes, respectively. Among the 46 strains of A. jandaei, 4.4% (2), 0% (0) and 32.6% (15) were positive for act/hly A/aer complex, ast and alt genes, respectively. Sequencing allowed for the confirmation of amplified products using BLAST. The present work proposes a specific and rapid diagnostic method to detect the main virulence determinants of Aeromonas, a genus potentially pathogenic to humans.

Effects of kinetin and nitrogen on growth rates, pigment and protein contents in wild and phycoerythrin-deficient strains of Hypnea musciformis (Rhodophyta)

Hypnea musciformis (Wulfen in Jacqu.) J.V. Lamour. is the main source for carrageenan production in Brazil and strains with selected characteristics could improve the production of raw material. The effects of kinetin on growth rates, morphology, protein content, and concentrations of pigments (chlorophyll a, phycoerythrin, phycocyanin, and allophycocyanin) were assessed in the wild strain (brown phenotype) and in the phycoerythrin-deficient strain (green phenotype) of H. musciformis. Concentrations of kinetin ranging from 0 to 50 mu M were tested in ASP 12-NTA synthetic medium with 10 mu M nitrate (N-limited) and 100 mu M nitrate (N-saturated). In N-limited condition, kinetin stimulated growth rates of the phycoerythrin-deficient strain and formation of lateral branches in both colour strains. Kinetin stimulated protein biosynthesis in both strains. However, differences between both nitrogen conditions were significant only in the phycoerythrin-deficient strain. In the wild strain, effects of kinetin on concentrations of phycobiliproteins were not significant in both nitrogen conditions, except for chlorophyll content. However, the phycoerythrin-deficient strain showed an opposite response, and kinetin stimulated the phycobiliprotein biosynthesis, with the highest concentrations of phycoerythrin in N-saturated medium, while the highest concentrations of allophycocyanin and phycocyanin were observed in N-limited medium. These results indicate that the effects of kinetin on growth, morphology, protein and phycobiliprotein contents are influenced by nitrogen availability, and the main nitrogen storage pools in phycoerythrin-deficient strain of H. musciformis submitted to N-limited conditions were phycocyanin and allophycocianin, the biosynthesis of which was enhanced by kinetin.

Clinical correlations of human cytomegalovirus strains and viral load in kidney transplant recipients

Little is known about clinical differences associated with cytomegalovirus (CMV) infection by distinct strains in renal transplant patients. Different clinical pictures may be associated with specific viral genotypes. viral load, as well as host factors. The objective of this study was to identify CMV strains to determine viral load (antigenemia), and their correlation with clinical data in renal transplant recipients. Seventy-one patients were enrolled, comprising 91 samples. After selection, polymorphonuclear cells were used to amplify and sequence the gB region of CMV DNA. The sequences were analyzed to ascertain the frequency of different genotypes. Additionally, the results of this Study showed that the gB coding gene presents a great variability, revealing a variety of patterns: classical gB (1.4%), gB1V (46.4%), classical gB2 (35.2%), gB2V (2.8%), gB3 (1.4%), classical gB4 (4.9%) and gB4V (4.9%). The mean viral load in kidney transplant patient was 75.1 positive cells (1-1000). A higher viral load was observed in patients with genotype 4 infection. Statistically significant differences were detected between gB1 and gB4 (p=0.010), and between gB2 and gB4 (p=0.021). The average numbers of positive cells in relation to clinical presentation were: 34.5 in asymptomatic, 49.5 in CMV associated syndrome and 120.7 in patients with invasive disease (p=0.048). As a group, gB1 was the most frequent strain and revealed a potential risk for developing invasive disease. Viral load also seemed to be important as a marker associated with clinical presentation of the disease. (C) 2008 Elsevier B.V. All rights reserved.

Strain variation in ppGpp concentration and RpoS levels in laboratory strains of Escherichia coli K-12

Laboratory strains and natural isolates of Escherichia coli differ in their level of stress resistance due to strain variation in the level of the sigma factor sigma(S) (or RpoS), the transcriptional master controller of the general stress response. We found that the high level of RpoS in one laboratory strain (MC4100) was partially dependent on an elevated basal level of ppGpp, an alarmone responding to stress and starvation. The elevated ppGpp was caused by two mutations in spoT, a gene associated with ppGpp synthesis and degradation. The nature of the spoT allele influenced the level of ppGpp in both MC4100 and another commonly used K-12 strain, MG1655. Introduction of the spoT mutation into MG1655 also resulted in an increased level of RpoS, but the amount of RpoS was lower in MG1655 than in MC4100 with either the wild-type or mutant spoT allele. In both MC4100 and MG1655, high ppGpp concentration increased RpoS levels, which in turn reduced growth with poor carbon sources like acetate. The growth inhibition resulting from elevated ppGpp was relieved by rpoS mutations. The extent of the growth inhibition by ppGpp, as well as the magnitude of the relief by rpoS mutations, differed between MG1655 and MC4100. These results together suggest that spoT mutations represent one of several polymorphisms influencing the strain variation of RpoS levels. Stress resistance was higher in strains with the spoT mutation, which is consistent with the conclusion that microevolution affecting either or both ppGpp and RpoS can reset the balance between self-protection and nutritional capability, the SPANC balance, in individual strains of E coli.

Boosting systemic and secreted antibody responses in mice orally immunized with recombinant Bacillus subtilis strains following parenteral priming with a DNA vaccine encoding the enterotoxigenic Escherichia coli (ETEC) CFA/I fimbriae B subunit

Recombinant Bacillus subtilis strains, either spores or vegetative cells, may be employed as safe and low cost orally delivered live vaccine vehicles. In this study, we report the use of an orally delivered B. subtilis vaccine strain to boost systemic and secreted antibody responses in mice i.m. primed with a DNA vaccine encoding the structural subunit (CfaB) of the CFA/I fimbriae encoded by enterotoxigenic Escherichia coli (ETEC), an important etiological agent of diarrhea among travelers and children living in endemic regions. DBA/2 female mice submitted to the prime-boost immunization regimen developed synergic serum (IgG) and mucosal (IgA) antibody responses to the target CfaB antigen. Moreover, in contrast to mice immunized only with one vaccine formulation, sera harvested from prime-boosted vaccinated individuals inhibited adhesion of ETEC cells to human red blood cells. Additionally, vaccinated dams conferred full passive protection to suckling newborn mice challenged with a virulent ETEC strain. Taken together the present results further demonstrate the potential use of recombinant B. subtilis strains as an alternative live vaccine vehicle. (C) 2008 Elsevier Ltd. All rights reserved.

Alkaline phosphatase as a reporter of sigma(S) levels and rpoS polymorphisms in different E-coli strains

sigma(S) is responsible for the transcriptional regulation of genes related to protection against stresses and bacterial survival and it accumulates in the cell under conditions of stress, such as nutrient limitation. An increase in the levels of sigma(S) causes a reduction in the expression of genes that are transcribed by RNA polymerase associated with the principal sigma factor, sigma(70). phoA, that encodes alkaline phosphatase (AP) is expressed under phosphate shortage conditions, and is also repressed by sigma(S). Here we show that in a Pi-limited chemostat, accumulation of rpoS mutations is proportional to the intrinsic level of sigma(S) in the cells. Acquisition of mutations in rpoS relieves repression of the PHO genes. We also devised a non-destructive method based on the rpoS effect on AP that differentiates between rpo(S+) and rpoS mutants, as well as between high and low-sigma(S) producers. Using this method, we provide evidence that sigma(S) contributes to the repression of AP under conditions of Pi excess and that AP variation among different strains is at least partly due to intrinsic variation in sigma(S) levels. Consequently, a simple and non-destructive AP assay can be employed to differentiate between strains expressing different levels of sigma(S) on agar plates.

Toxicokinetics and toxicological effects of single oral dose of fumonisin B1 containing Fusarium verticillioides culture material in weaned piglets

Toxicokinetics and the toxicological effects of culture material containing fumonisin B(1) (FB(1)) were studied in male weaned piglets by clinical, pathological, biochemical and sphingolipid analyses. The animals received a single oral dose of 5 mg FB(1)/kg of body weight. obtained from Fusarium verticillioides culture material. FB(1) was detected by H PLC in plasma collected at 1-h intervals up to 6 h and at 12-h intervals up to 96 h. FB(1) eliminated in feces and urine was quantified over a 96-h period and in liver samples collected 96 h post-intoxication. Blood samples were obtained at the beginning and end of the experiment to determine serum enzyme activity, total bilirubin, cholesterol, sphinganine (Sa), sphingosine (So) and the Sa/So ratio. FB(1) was detected in plasma between 30 min and 36 h after administration. The highest concentration of FB(1) was observed after 2 h, with a mean concentration of 282 mu g/ml. Only 0.93% of the total FB(1) was detected in urine between 75 min and 41 h after administration, the highest mean concentration (561 mu g/ml) was observed during the interval after 8 at 24 h. Approximately 76.5% of FB(1) was detected in feces eliminated between 8 and 84 h after administration, with the highest levels observed between 8 and 24 h. Considering the biochemical parameters, a significant increase only occurred in cholesterol, alkaline phosphatase and aspartate aminotransferase activities. In plasma and urine, the highest Sa and Sa/So ratios were obtained at 12 and 48 h, respectively. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin

Aims: To evaluate the sensitivity and specificity of polyclonal and monoclonal antibodies (Mabs) against intimin in the detection of enteropathogenic and enterohaemorrhagic Escherichia coli isolates using immunoblotting. Methods and Results: Polyclonal and Mabs against the intimin-conserved region were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti-intimin IgG-enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of 1 center dot 3 x 10-8 mol l-1, failed in the detection of some of these isolates. Conclusion: All employed antibodies showed 100% specificity, not reacting with any of the eae-negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti-intimin IgG-enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. Significance and Impact of the Study: The rabbit anti-intimin IgG-enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.

PHA(MCL) biosynthesis systems in Pseudomonas aeruginosa and Pseudomonas putida strains show differences on monomer specificities

The production of PHA from plant oils by Pseudomonas species soil isolated from a sugarcane crop was evaluated. Out of 22 bacterial strains three were able to use efficiently plant oils to grow and to accumulate PHA. Pseudomonas putida and Pseudomonas aeruginosa strains produced PHA presenting differences on monomer composition compatible with variability on monomer specificity of their PHA biosynthesis system. The molar fraction of 3-hydroxydodecanoate detected in the PHA was linearly correlated to the oleic acid supplied. A non-linear relationship between the molar fractions of 3-hydroxy-6-dodecenoate (3HDd Delta(6)) detected in PHA and the linoleic acid supplied was observed, compatible with saturation in the biosynthesis system capability to channel intermediate of P-oxidation to PHA synthesis. Although P. putida showed a higher 3HDd Delta(6) yield from linoleic acid when compared to P. aeruginosa, in both species it was less than 10% of the maximum theoretical value. These results contribute to the knowledge about the biosynthesis of PHA with a controlled composition from plant oils allowing in the future establishing the production of these polyesters as tailor-made polymers. (C) 2009 Elsevier B.V. All rights reserved.

Shiga toxin-producing Escherichia coli and atypical enteropathogenic Escherichia coli strains isolated from healthy sheep of different populations in Sao Paulo, Brazil

Aims: Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent. Methods and Results: About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx(1), stx(2), eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)-PCR was performed to investigate the variants of stx(1) and stx(2), and the flagellar antigen (fliC) genes in nonmotile isolates. Five isolates were eae(+) and stx(-), and belonged to serotypes O128:H2/beta-intimin (2), O145:H2/gamma, O153:H7/beta and O178:H7/epsilon. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx(1c)stx(2d-O118) (46.9%), stx(1c) (27.2%), stx(2d-O118) (23.4%), and stx(1c)stx(2dOX3a) (2.5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates. Conclusions: This study demonstrated that healthy sheep in Sao Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC. Significance and Impact of the Study: As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.

Epidemiology and Genetic Variability of Human Metapneumovirus During a 4-Year-Long Study in Southeastern Brazil

Epidemiological and molecular characteristics of human metapneumovirus (hMPV) were compared with human respiratory syncytial virus (hRSV) in infants and young children admitted for acute lower respiratory tract infections in a prospective study during four consecutive years in subtropical Brazil. GeneScan polymerase chain assays (GeneScan RT-PCR) were used to detect hMPV and hRSV in nasopharyngeal aspirates of 1,670 children during January 2003 to December 2006. hMPV and hRSV were detected, respectively, in 191 (11.4%) and in 702 (42%) of the children admitted with acute lower respiratory tract infections at the Sao Paulo University Hospital. Sequencing data of the hMPV F gene revealed that two groups of the virus, each divided into two subgroups, co-circulated during three consecutive years. It was also shown that a clear dominance of genotype B1 occurred during the years 2004 and 2005, followed by genotype A2 during 2006. J. Med. Virol. 81:915-921,2009. (C) 2009 Wiley-Liss, Inc.