p53 gene delivery via a recombinant Salmonella enterica Typhimurium leads to human bladder carcinoma cell death in vitro.

Numerous studies have attempted to restore the function of the tumour suppressor p53 as an anti-cancer strategy through gene delivery. However, most studies employed non-bacterial vectors to deliver p53. Various facultative and obligate anaerobic bacteria have been proposed as vectors because of their intrinsic tumour targeting ability and anti-tumour activity. Salmonella enterica Typhimurium is the most studied bacterial vector in anti-cancer therapy. We used the previously designed χ11218 strain of S. enterica Typhimurium, displaying regulated delayed lysis, as a vector for delivering p53 to human bladder carcinoma cells, restoring wild-type p53 protein function. We cloned p53 into pYA4545 (containing a eukaryotic expression system) to generate the χ11218 pYA4545p53 strain. Cloning of p53 did not affect the growth or interfere with the invasive and replicative capacity of χ11218 bacteria in tumour cells. Human bladder carcinoma cells (expressing mutated p53) transfected with pYA4545p53 showed a significant increase in the expression of p53 protein. We demonstrated that p53 supplied by χ11218 significantly decreased the viability of human bladder cancer cells in a dose-dependent manner. This study demonstrates the applicability of the attenuated χ11218 strain as a vector for DNA plasmids expressing tumour suppressor genes.

Salmonella enterica Typhimurium fljBA operon stability: implications regarding the origin of Salmonella enterica I 4,[5],12:i:.

Salmonella enterica subsp enterica serovar 4,5,12:i:- has been responsible for many recent Salmonella outbreaks worldwide. Several studies indicate that this serovar originated from S. enterica subsp enterica serovar Typhimurium, by the loss of the flagellar phase II gene (fljB) and adjacent sequences. However, at least two different clones of S. enterica 4,5,12:i:- exist that differs in the molecular events responsible for fljB deletion. The aim of this study was to test the stability of the fljBA operon responsible for the flagellar phase variation under different growth conditions in order to verify if its deletion is a frequent event that could explain the origin and dissemination of this serovar. In fact, coding sequences for transposons are present near this operon and in some strains, such as S. enterica Typhimurium LT2, the Fels-2 prophage gene is inserted near this operon. The presence of mobile DNA could confer instability to this region. In order to examine this, the cat (chloramphenicol acetyltransferase) gene was inserted adjacent to the fljBA operon so that deletions involving this genomic region could be identified. After growing S. enterica chloramphenicol-resistant strains under different conditions, more than 104 colonies were tested for the loss of chloramphenicol resistance. However, none of the colonies were sensitive to chloramphenicol. These data suggest that the origin of S. enterica serovar 4,5,12:i:- from Typhimurium by fljBA deletion is not a frequent event. The origin and dissemination of 4,5,12:i:- raise several questions about the role of flagellar phase variation in virulence.

Development of nanostructured silver vanadates decorated with silver nanoparticles as a novel antibacterial agent.

In this work we report the synthesis, characterization and application of silver vanadate nanowires decorated with silver nanoparticles as a novel antibacterial agent. These hybrid materials were synthesized by a precipitation reaction of ammonium vanadate and silver nitrate followed by hydrothermal treatment. The silver vanadate nanowires have lengths of the order of microns and diameters around 60 nm. The silver nanoparticles decorating the nanowires present a diameter distribution varying from 1 to 20 nm. The influence of the pH of the reaction medium on the chemical structure and morphology of silver vanadates was studied and we found that synthesis performed at pH 5.5-6.0 led to silver vanadate nanowires with a higher morphological yield. The antimicrobial activity of these materials was evaluated against three strains of Staphylococcus aureus and very promising results were found. The minimum growth inhibiting concentration value against a MRSA strain was found to be ten folds lower than for the antibiotic oxacillin.

Genomic diversity of vibrios associated with the Brazilian coral Mussismilia hispida and its sympatric zoanthids (Palythoa caribaeorum, Palythoa variabilis and Zoanthus solanderi).

AIMS: A taxonomic survey of the vibrios associated with the Brazilian endemic coral Mussismilia hispida and the sympatric zoanthids (i.e. Palythoa caribaeorum, Palythoa variabilis and Zoanthus solanderi). METHODS AND RESULTS: Mucus of 54 cnidarian specimens collected in three different places at São Sebastião in two consecutive years (i.e. 2005 and 2006) was used for taxonomic characterization of the cnidarian microbiota. Ninety-eight of the 151 vibrio isolates fell within the vibrio core group according to partial 16S rDNA sequences. We performed the sequencing of recA and pyrH genes of all vibrio isolates. The most abundant taxa belonged to the vibrio core group (Vibrio harveyi, Vibrio rotiferianus, Vibrio campbellii and Vibrio alginolyticus), Vibrio mediterranei (=Vibrio shillonii) and Vibrio chagasii. With the exception of V. chagasii which was found only in the mucus of M. hispida, the other species appeared in different hosts with no evidence for the presence of host-specific clones or species. Using rep-PCR analysis, we observed a high genomic heterogeneity within the vibrios. Each vibrio isolate generated a different rep-PCR fingerprint pattern. There was a complete agreement between the grouping based on rep-PCR and concatenated sequences of pyrH, recA and 16S rDNA, but the pyrH gene has the highest discriminatory power for vibrio species identification. CONCLUSION: The vibrio core group is dominant in the mucus of these cnidarians. There is a tremendous diversity of vibrio lineages within the coral mucus. pyrH gene sequences permit a clear-cut identification of vibrios. SIGNIFICANCE AND IMPACT OF THE STUDY: The taxonomic resolution provided by pyrH (but not recA) appears to be enough for identifying species of vibrios and for disclosing putative new taxa. The vibrio core group appears to be dominant in the mucus of the Brazilian cnidarians. The overrepresentation of these vibrios may reflect as yet unknown ecological functions in the coral holobiont.

Involvement of the Helicobacter pylori plasticity region and cag pathogenicity island genes in the development of gastroduodenal diseases.

Infection by Helicobacter pylori is associated with the development of several gastroduodenal diseases, including gastritis, peptic ulcer disease (gastric ulcers and duodenal ulcers), and gastric adenocarcinoma. Although a number of putative virulence factors have been reported for H. pylori, there are conflicting results regarding their association with specific H. pylori-related diseases. In this work, we investigated the presence of virB11 and cagT, located in the left half of the cag pathogenicity island (cagPAI), and the jhp917-jhp918 sequences, components of the dupA gene located in the plasticity zone of H. pylori, in Brazilian isolates of H. pylori. We also examined the association between these genes and H. pylori-related gastritis, peptic ulcer disease, and gastric and duodenal ulcers in an attempt to identify a gene marker for clinical outcomes related to infection by H. pylori. The cagT gene was associated with peptic ulcer disease and gastric ulcers, whereas the virB11 gene was detected in nearly all of the samples. The dupA gene was not associated with duodenal ulcers or any gastroduodenal disease here analyzed. These results suggest that cagT could be a useful prognostic marker for the development of peptic ulcer disease in the state of São Paulo, Brazil. They also indicate that cagT is associated with greater virulence and peptic ulceration, and that this gene is an essential component of the type IV secretion system of H. pylori.

Epidemiological characterization of resistance and PCR typing of Shigella flexneri and Shigella sonnei strains isolated from bacillary dysentery cases in Southeast Brazil.

Shigella spp are Gram-negative, anaerobic facultative, non-motile, and non-sporulated bacilli of the Enterobacteriaceae family responsible for "Shigellosis" or bacillary dysentery, an important cause of worldwide morbidity and mortality. However, despite this, there are very few epidemiological studies about this bacterium in Brazil. We studied the antibiotic resistance profiles and the clonal structure of 60 Shigella strains (30 S. flexneri and 30 S. sonnei) isolated from shigellosis cases in different cities within the metropolitan area of Campinas, State of São Paulo, Brazil. We used the following well-characterized molecular techniques: enterobacterial repetitive intergenic consensus, repetitive extragenic palindromic, and double-repetitive element-polymerase chain reaction to characterize the bacteria. Also, the antibiotic resistance of the strains was determined by the diffusion disk method. Many strains of S. flexneri and S. sonnei were found to be multi-resistant. S. flexneri strains were resistant to ampicillin in 83.3% of cases, chloramphenicol in 70.0%, streptomycin in 86.7%, sulfamethoxazole in 80.0%, and tetracycline in 80.0%, while a smaller number of strains were resistant to cephalothin (3.3%) and sulfazotrim (10.0%). S. sonnei strains were mainly resistant to sulfamethoxazole (100.0%) and tetracycline (96.7%) and, to a lesser extent, to ampicillin (6.7%) and streptomycin (26.7%). Polymerase chain reaction-based typing supported the existence of specific clones responsible for the shigellosis cases in the different cities and there was evidence of transmission between cities. This clonal structure would probably be the result of selection for virulence and resistance phenotypes. These data indicate that the human sanitary conditions of the cities investigated should be improved.

DNA sequencing of a pathogenicity-related plasmid of an avian septicemic Escherichia coli strain.

A 43-MDa conjugative plasmid isolated from an avian septicemic Escherichia coli (APEC) strain possessing genes related to the adhesion and invasion capacities of in vitro-cultured cells was sequenced. The results demonstrated that the 43-MDa plasmid harbors bacterial pathogenicity-related sequences which probably allow the wild-type pathogenic strain to adhere to and invade tissues and to cause septicemia in poultry. The existence of homology sequences to sequences belonging to other human pathogenic Enterobacteriaceae like Escherichia coli O157:H7, Shigella and Salmonella was also observed. The presence of these sequences in this plasmid could indicate that there is horizontal genetic transfer between bacterial strains isolated from different host species. In conclusion, the present study suggests that APEC strains harbor high-molecular weight plasmids that present pathogenicity-related sequences and that these are probably responsible for the pathogenicity exhibited by these strains. The presence of human pathogenicity-associated sequences in APEC conjugative plasmids suggests that these strains could represent a zoonotic risk.

Pathogenic characteristics of Escherichia coli strains isolated from newborn piglets with diarrhea in Brazil.

Ninety-one Escherichia coli isolates obtained from diarrheic and normal feces of newborn piglets (0-11 days of age) from three states of Brazil were assessed for phenotypic and genotypic characteristics associated with pathogenic processes. These isolates expressed fimbriae F18ac and type 1, but not fimbriae K88, K99, 987P or F41. Genes for toxins (LT-I, STa, SLT-I, SLT-II, SLT-IIv) either individually or combined were found to be present in most of the diarrheic strains (65.7%) and in 42.8% of the non-diarrheic ones. The eaeA gene was present in 25.7% of the diarrheic isolates and in 9.5% of the non-diarrheic ones. Colicin, hemolysin and aerobactin were also found to be produced by some strains from both sources. Because of the great variety of biological characteristics associated with different illness processes, we suggest that, in Brazil, pigs may act as a reservoir for transmission of Escherichia coli strains to other animals.

Biological and genetic characteristics of uropathogenic Escherichia coli strains.

The aim of the present study was to determine biological characteristics such as expression of fimbriae, Congo red binding, production of hemolysin and aerobactin, adhesion to HeLa and uroepithelial cells and invasion of HeLa cells by Escherichia coli isolates obtained from patients showing clinical signs of urinary tract infection (UTI). Also, the presence of genes (apa, afa, spa) for fimbria expression and cytotoxic necrotizing factors (CNF1, CNF2) was assayed using specific primers in PCR. The data obtained were compared with the clonal relationships obtained by analysis of multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphism (RFLP) of the rDNA (ribotyping) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). All isolates but one presented a combination of at least two of the characteristics studied, a fact suggesting the presence of pathogenicity islands (PAIs). Diffuse adherence type to HeLa cells was observed to occur in most of the strains, but adhesion to uroepithelial cells seems to be a more reliable test to verify pathogenicity. Although four strains seemed to be able to invade HeLa cells when assayed by light microscopy, electron microscopy studies demonstrated that these strains were not invasive. MLEE, RFLP and ERIC-PCR were able to group the isolates differently into main clusters that were not correlated with the presence of pathogenic traits.

Molecular genotyping and epidemiology of Mycobacterium tuberculosis isolates obtained from inmates of correctional institutions of Campinas, Southeast Brazil.

The objective of this study was to investigate the possible transmission of tuberculosis among 39 inmates with positive Mycobacterium tuberculosis smears in four correctional institutions located in Campinas City, SP, Brazil over a 19-month period. Fifty-one M. tuberculosis isolates from these inmates were characterized according to the number of IS6110 insertion elements present in their genomic DNA. The number of insertion elements in M. tuberculosis isolates varied from two to twelve. The dendrogram of similarity resulted in the grouping the isolates in six main clusters. These results, associated to epidemiological data, suggested the transmission of tuberculosis among inmates of the same and different institutions inmates. Univariate analysis of epidemiological data (total delay for beginning of treatment, previous treatment, and HIV status) and clustering occurrence showed that only 'previous treatment' (OR = 7.65, p = 0.032) was associated with the possible transmission of tuberculosis in the studied prisons.

Integration of minitransposons for expression of the Escherichia coli elt genes at a preferred site in Salmonella typhimurium identifies a novel putative fimbrial locus.

An asd-complementing mini-Tn5 transposon was constructed for random insertion of the Escherichia coli LT enterotoxin genes (elt) into the genome of Deltaasd attenuated strains of Salmonella typhimurium. Transfer of the minitransposon to different S. typhimurium strains resulted in random integration only in strain chi4072, while in strain chi3987, which harbours the virulence plasmid, over 20% of the insertions occurred at the same site. Expression of elt was found to be highest in Salmonella isolates carrying the mini-Tn5 integrated at the preferred site, which was mapped to an uncharacterised region of the virulence plasmid. Sequence analysis of the integration site showed that it lies within an open reading frame with sequence similarity to E. coli leuO and contiguous to a novel fimbrial locus.

Levels of expression and immunogenicity of attenuated Salmonella enterica serovar typhimurium strains expressing Escherichia coli mutant heat-labile enterotoxin.

The effects of heterologous gene dosage as well as Salmonella typhimurium strain variability on immune response toward both the heterologous antigen, the nontoxic mutant of the Escherichia coli heat-labile enterotoxin LTK63, and the carrier Salmonella strain have been analyzed. Effects of a single integration into the host DNA and different-copy-number episomal vectors were compared in S. typhimurium delta cya delta crp delta asd strains of two different serotypes, UK-1 and SR-11. Expression of the enterotoxin in the different Salmonella isolates in vitro was found to vary considerably and, for the episomal vectors, to correlate with the plasmid copy number. LTK63-specific serum immunoglobulin G (IgG) and mucosal immunoglobulin A (IgA) antibodies were highest in mice immunized with the high-level-expression strain. High anti-LTK63 IgG and IgA titers were found to correspond to higher anti-Salmonella immunity, suggesting that LTK63 exerts an adjuvant effect on response to the carrier. Statistically significant differences in anti-LTK63 immune response were observed between groups of mice immunized with the attenuated delta cya delta crp UK-1 and SR-11 derivatives producing the antigen at the same rate. These data indicate that the same attenuation in S. typhimurium strains of different genetic backgrounds can influence significantly the immune response toward the heterologous antigen. Moreover, delivery of the LTK63 enterotoxin to the immune system by attenuated S. typhimurium strains is effective only when synthesis of the antigen is very high during the initial phase of invasion, while persistence of the S. typhimurium strain in deep tissues has only marginal influence.

Virulence characteristics and epidemiology of Yersinia enterocolitica and Yersiniae other than Y. pseudotuberculosis and Y. pestis isolated from water and sewage.

AIMS: To determine the species, bio-sero-phagetypes, antimicrobial drug resistance and also the pathogenic potential of 144 strains of Yersinia spp. isolated from water sources and sewage in Brazil. METHODS AND RESULTS: The 144 Yersinia strains were characterized biochemically, serologically and had their antibiotic resistance and phenotypic virulence markers determined by microbiological and serological standard techniques. The Y. enterocolitica strains related to human diseases were also tested for the presence of virulence genes, by the PCR technique. The isolates were classified as Y. enterocolitica, Y. intermedia, Y. frederiksenii, Y. kristensenii and Yersinia biochemically atypical. The 144 isolates belonged to various bio-serogroups. Half of the strains showed resistance to three or more drugs. The Y. enterocolitica strains related to human diseases exhibited phenotypic virulence characteristics and virulence genes. CONCLUSIONS: Water from various sources and sewage are contaminated with Yersinia spp. in Brasil. Among these bacteria, virulent strains of Y. enterocolitica were found, with biotypes and serogroups related to human diseases. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first documented description of the occurrence of pathogenic Y. enterocolitica in water sources and sewage in Brazil. The occurrence of virulence strains of Y. enterocolitica shows that the environment is a potential source of human infection by this species in this country.

Determination of the clonal structure of avian Escherichia coli strains by isoenzyme and ribotyping analysis.

Forty-nine avian Escherichia coli strains isolated from different outbreak cases of septicemia (24), swollen head syndrome (14) and omphalitis (11), and 20 strains isolated from poultry with no signs of the mentioned illnesses, for a total of 69 strains, were typed by isoenzyme profile and ribotyping analysis by restriction fragment length polymorphism (RFLP). Isoenzyme analysis discriminated better among strains (0-0.07 degree of genetic dissimilarity) than ribotyping analysis (0- 0.02 degree of genetic dissimilarity). The enzyme profiles of the E. coli isolates allowed the identification of 33 clones that were organized into six main clusters (A-F). Cluster A comprised 87% of the pathogenic strains and had no commensal strains, while commensal strains were assigned to clusters B-F. The ribotyping analysis resulted in a more heterogenous distribution of strains but most of those that cause the same type of infection were kept close together. Taken as a whole, these results demonstrate that pathogenic clones are more similar to one another when compared with commensal strains and suggest a correlation between the genetic background and the pathogenic characteristics of avian pathogenic E. coli strains.

Molecular genotyping and epidemiology of Mycobacterium tuberculosis isolates obtained from inmates of correctional institutions of Campinas, Southeast Brazil

The objective of this study was to investigate the possible transmission of tuberculosis among 39 inmates with positive Mycobacterium tuberculosis smears in four correctional institutions located in Campinas City, SP, Brazil over a 19-month period. Fifty-one M. tuberculosis isolates from these inmates were characterized according to the number of IS6110 insertion elements present in their genomic DNA. The number of insertion elements in M. tuberculosis isolates varied from two to twelve. The dendrogram of similarity resulted in the grouping the isolates in six main clusters. These results, associated to epidemiological data, suggested the transmission of tuberculosis among inmates of the same and different institutions inmates. Univariate analysis of epidemiological data (total delay for beginning of treatment, previous treatment, and HIV status) and clustering occurrence showed that only "previous treatment" (OR = 7.65, p = 0.032) was associated with the possible transmission of tuberculosis in the studied prisons.

DNA sequencing of a pathogenicity-related plasmid of an avian septicemic Escherichia coli strain

A 43-MDa conjugative plasmid isolated from an avian septicemic Escherichia coli (APEC) strain possessing genes related to the adhesion and invasion capacities of in vitro-cultured cells was sequenced. The results demonstrated that the 43-MDa plasmid harbors bacterial pathogenicity-related sequences which probably allow the wild-type pathogenic strain to adhere to and invade tissues and to cause septicemia in poultry. The existence of homology sequences to sequences belonging to other human pathogenic Enterobacteriaceae like Escherichia coli O157:H7, Shigella and Salmonella was also observed. The presence of these sequences in this plasmid could indicate that there is horizontal genetic transfer between bacterial strains isolated from different host species. In conclusion, the present study suggests that APEC strains harbor high-molecular weight plasmids that present pathogenicity-related sequences and that these are probably responsible for the pathogenicity exhibited by these strains. The presence of human pathogenicity-associated sequences in APEC conjugative plasmids suggests that these strains could represent a zoonotic risk.

Epidemiological characterization of resistance and PCR typing of Shigella flexneri and Shigella sonnei strains isolated from bacillary dysentery cases in Southeast Brazil

Shigella spp are Gram-negative, anaerobic facultative, non-motile, and non-sporulated bacilli of the Enterobacteriaceae family responsible for "Shigellosis" or bacillary dysentery, an important cause of worldwide morbidity and mortality. However, despite this, there are very few epidemiological studies about this bacterium in Brazil. We studied the antibiotic resistance profiles and the clonal structure of 60 Shigella strains (30 S. flexneri and 30 S. sonnei) isolated from shigellosis cases in different cities within the metropolitan area of Campinas, State of São Paulo, Brazil. We used the following well-characterized molecular techniques: enterobacterial repetitive intergenic consensus, repetitive extragenic palindromic, and double-repetitive element-polymerase chain reaction to characterize the bacteria. Also, the antibiotic resistance of the strains was determined by the diffusion disk method. Many strains of S. flexneri and S. sonnei were found to be multi-resistant. S. flexneri strains were resistant to ampicillin in 83.3 percent of cases, chloramphenicol in 70.0 percent, streptomycin in 86.7 percent, sulfamethoxazole in 80.0 percent, and tetracycline in 80.0 percent, while a smaller number of strains were resistant to cephalothin (3.3 percent) and sulfazotrim (10.0 percent). S. sonnei strains were mainly resistant to sulfamethoxazole (100.0 percent) and tetracycline (96.7 percent) and, to a lesser extent, to ampicillin (6.7 percent) and streptomycin (26.7 percent). Polymerase chain reaction-based typing supported the existence of specific clones responsible for the shigellosis cases in the different cities and there was evidence of transmission between cities. This clonal structure would probably be the result of selection for virulence and resistance phenotypes. These data indicate that the human sanitary conditions of the cities investigated should be improved.

Bacteriophages and insertion sequences of Chromobacterium violaceum ATCC 12472

A fluid genome is a great advantage to prokaryotes, enabling quick adaptation to various types of ecological niches and to diverse environmental selective pressures. A substantial portion of these sudden changes is mediated by lateral gene transfer (LGT), through genetic recombination mechanisms, such as transformation, conjugation and transduction. The recent sequencing of several organisms has offered a new approach to the study of LGT, using comparison and analysis of nucleotide sequences dispersed throughout the genome of these species. This analysis in Choromobacterium violaceum has revealed four prophage and 12 insertion sequences, suggesting genetic exchange with several other bacterial species, including Salmonella enterica, Ralstonia and Xanthomonas. An Rhs (recombination hot spot) element (containing a vgr-like gene) was also observed, the function of which remains unknown, but it has a sequence related to species of Acinetobacter and Sphingomonas. These results support the role of LGT in the acquisition of new traits by C. violaceum

Drug resistance in Chromobacterium violaceum

Chromobacterium violaceum is a free-living bacterium commonly found in aquatic habitats of tropical and subtropical regions of the world. This bacterium is able to produce a large variety of products of biotechnological and pharmacological use. Although C. violaceum is considered to be non-pathogenic, some cases of severe infections in humans and other animals have been reported. Genomic data on the type strain ATCC 12472(T) has provided a comprehensive basis for detailed studies of pathogenicity, virulence and drug resistance genes. A large number of open reading frames associated with various mechanisms of drug resistance were found, comprising a remarkable feature of this organism. Amongst these, beta-lactam (penicillin and cephalosporin) and multidrug resistance genes (drug efflux pumps) were the most numerous. In addition, genes associated with bacitracin, bicyclomycin, chloramphenicol, kasugamycin, and methylenomycin were also found. It is postulated that these genes contribute to the ability of C. violaceum to compete with other bacteria in the environment, and also may help to explain the common drug resistance phenotypes observed in infections caused by this bacterium