RESUMO
Bacterial viruses known as bacteriophages have been demonstrated to be effective in killing foodborne pathogens such as Shigella flexneri. Adsorption is the first step in the phage-host interaction. In the present work, 10 Shigella phages were used to characterize the adsorption process on Shigella flexneri ATCC12022 in several physicochemical conditions related to food and in a food matrix. One-step growth curves were drawn for all the Shigella-phages evaluated. Furthermore, the adsorption rate for each of the 10 phages was determined. In addition, the influence of temperature, Na+, Mg2+, pH, sucrose and glycerol on phage adsorption was investigated. Two phages (Shi22 and Shi30) showed higher burst sizes values (67 and 64 PFU cell-1, respectively) and burst times of 25 min to 30 min, while the other eight phages exhibited burst sizes ranging from 14 to 17 PFU cell-1 with slower burst times. Furthermore, most phages achieved a high adsorption rate, and the adsorption constants (k) ranged from ~10-9 to 10-10 mL min-1. Regarding the influence of temperature, cations and pH, a high or moderate percentage of adsorption was observed for most of the phages evaluated. The adsorption decreased at increasing concentrations of Na+, sucrose and glycerol, although at different levels, since adsorption was more affected by sucrose than by glycerol and Na+ for most phages. The adsorption obtained in Triptein soy broth (TSB) for most of the phages/strain systems evaluated was moderate or high, as well as those observed in a food matrix. Thus, our phages could potentially be used to improve food safety under a wide range of environmental conditions against foodborne pathogens.
Assuntos
Bacteriófagos , Shigella , Shigella flexneri , Adsorção , Glicerol , SacaroseRESUMO
BACKGROUND: Shigella specie is a globally important intestinal pathogen disseminated all over the world. In this study we analyzed the genome and the proteomic component of two Shigella flexneri 2a clinical isolates, collected from pediatric patients with gastroenteritis of the Northwest region of Argentina (NWA) in two periods of time, with four years of difference. Our goal was to determine putative changes at molecular levels occurred during these four years, that could explain the presence of this Shigella`s serovar as the prevalent pathogen in the population under study. RESULTS: As previously reported, our findings support the idea of Shigella has a conserved "core" genome, since comparative studies of CI133 and CI172 genomes performed against 80 genomes obtained from the NCBI database, showed that there is a large number of genes shared among all of them. However, we observed that CI133 and CI172 harbors a small number of strain-specific genes, several of them present in mobile genetic elements, supporting the hypothesis that these isolates were established in the population by horizontal acquisition of genes. These differences were also observed at proteomic level, where it was possible to detect the presence of certain secreted proteins in a culture medium that simulates the host environment. CONCLUSION: Great similarities were observed between the CI133 and CI172 strains, confirming the high percentage of genes constituting the "core" genome of S. flexneri 2. However, numerous strain specific genes were also determined. The presence of the here identified molecular elements into other strain of our culture collation, is currently used to develop characteristic markers of local pathogens. In addition, the most outstanding result of this study was the first description of a S. flexneri 2 producing Colicin E, as one of the characteristics that allows S. flexneri 2 to persist in the microbial community. These findings could also contribute to clarify the mechanism and the evolution strategy used by this pathogen to specifically colonize, survive, and cause infection within the NWA population.
Assuntos
Disenteria Bacilar , Shigella , Argentina/epidemiologia , Criança , Genômica , Humanos , Lactente , Proteômica , Shigella flexneri/genéticaRESUMO
Bacteria may enter into a viable but nonculturable (VBNC) state as a response to stresses, such as those found in food processing. Cells in the VBNC state lose the ability to grow in a conventional culture medium but man recover culturability. The viability, culturability and intracellular reactive oxygen species (ROS) of Salmonella Enteritidis and Shigella flexneri were evaluated under stress conditions to induce a VBNC state. Cells were maintained under nutritional, osmotic and cold stresses (long-term induction) in Butterfield's phosphate solution plus 1.2 M of NaCl at 4°C and under nutritional and oxidative stresses (short-term induction) in 10 mM of H2O2. Culture media, recovery agents, sterilization methods of media and incubation temperature, were combined and applied to recover the culturability of the VBNC cells. Salmonella entered in the VBNC state after 135 days under long-term induction, while Shigella maintained culturability after 240 days. Under short-term induction, Salmonella and Shigella lose culturability after 135 and 240 min, respectively. Flow cytometric analysis revealed viable cells and intracellular ROS in both species in VBNC. It was not possible to recover the culturability of VBNC cells using the 42 combinations of different factors.
Assuntos
Salmonella enteritidis/fisiologia , Shigella flexneri/fisiologia , Meios de Cultura/química , Microbiologia de Alimentos , Viabilidade Microbiana , Espécies Reativas de Oxigênio/metabolismo , Estresse FisiológicoRESUMO
Shigella flexneri has been a major public health problem in developing countries. This work analyzed the frequency of 16 virulence genes, the genotypic diversity, and the antimicrobial resistance profiles of 130 S. flexneri strains isolated in Brazil. The ipaH gene was found in all the 130 strains. The frequencies of the other genes were variable ial (88.5%), sigA (82.3%), iuc (74.6%), virA (73%), pic (72.3%), virF (57.7%), sat (48.5%), ipaBCD (37%), sen (36%), set1A (35.4%), sepA (30%), set1B (30%), virB (14%), icsA (10%), and ipgD (5.4%). A total of 57 (43.8%) strains were multidrug-resistant. ERIC-PCR grouped 96 of the strains into a single cluster with ≥ 70.4% of similarity, 75 of these strains presented a similarity ≥ 80.9%. PFGE grouped 120 of the strains into a single cluster with 57.4% of similarity and 82 of these strains presented a similarity ≥ 70.6%. In conclusion, the high frequency of some virulence genes reinforces the pathogenic potential of the strains studied. The high rates of MDR strains are alarming once it may lead to failure when antimicrobial treatment is necessary. Genotype techniques reveled a major cluster with high genetic similarity including S. flexneri strains from the different Brazilian states and distinct years of isolation, showing that they probably emerged from a common ancestor.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Disenteria Bacilar/microbiologia , Shigella flexneri , Fatores de Virulência/genética , Brasil/epidemiologia , Variação Genética , Humanos , Shigella flexneri/classificação , Shigella flexneri/isolamento & purificação , Shigella flexneri/patogenicidadeRESUMO
Shigellosis is a diarrheal disease that causes high mortality every year, especially in children, elderly and immunocompromised patients. Recently, resistance strains to antibiotic therapy are in the rise and the World Health Organization prioritizes the development of a safe vaccine against the most common causal agent of shigellosis, Shigella flexneri. This pathogen uses autotransporter proteins such as SigA, Pic and Sap to increase virulence and some of them have been described as highly immunogenic proteins. In this study, we used immune-informatics analysis to identify the most antigenic epitope as a vaccine candidate on three passenger domains of auto-transporter proteins encoded on the pathogenic island SHI-1, to induce immunity against S. flexneri. Epitope identification was done using various servers such as Bepipred, Bcepred, nHLAPRED, NetMHCII, Rankpep and IEDB and the final selection was done based on its antigenicity using the VaxiJen server. Moreover, to enhance immunity, the GroEL adjuvant was added to the final construct as a Toll-like receptor 2 (TLR2) agonist. On the other hand, to predict the tertiary structure, the I-TASSER server was used, and the best model was structurally validated using the ProSA-web software and the Ramachandran plot. Subsequently, the model was refined and used for docking and molecular dynamics analyses with TLR2, which demonstrated an appropriate and stable interaction. In summary, a potential subunit vaccine candidate, that contains B and T cell epitopes with proper physicochemical properties was designed. This multiepitope vaccine is expected to elicit robust humoral and cellular immune responses and vest protective immunity against S. flexneri.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Disenteria Bacilar/terapia , Serina Proteases/imunologia , Shigella flexneri/imunologia , Sistemas de Secreção Tipo V/imunologia , Adjuvantes Imunológicos/farmacologia , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/uso terapêutico , Chaperonina 60/imunologia , Chaperonina 60/farmacologia , Biologia Computacional , Simulação por Computador , Disenteria Bacilar/microbiologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Imunogenicidade da Vacina , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Domínios Proteicos/imunologia , Receptor 2 Toll-Like/agonistas , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/uso terapêuticoRESUMO
In recent years, multidrug resistance of Shigella strains associated with genetic elements like pathogenicity islands, have become a public health problem. The Shigella resistance locus pathogenicity island (SRL PAI) of S. flexneri 2a harbors a 16Kbp region that contributes to the multidrug resistance phenotype. However, there is not much information about other functions such as metabolic, physiologic or ecological ones. For that, wild type S. flexneri YSH6000 strain, and its spontaneous SRL PAI mutant, 1363, were used to study the contribution of the island in different growth conditions. Interestingly, when both strains were compared by the Phenotype Microarrays, the ability to metabolize D-aspartic acid as a carbon source was detected in the wild type strain but not in the mutant. When D-aspartate was added to minimal medium with other carbon sources such as mannose or mannitol, the SRL PAI-positive strain was able to metabolize it, while the SRL PAI-negative strain did not. In order to identify the genetic elements responsible for this phenotype, a bioinformatic analysis was performed and two genes belonging to SRL PAI were found: orf8, coding for a putative aspartate racemase, and orf9, coding for a transporter. Thus, it was possible to measure, by an indirect analysis of racemization activity in minimal medium supplemented only with D-aspartate, that YSH6000 strain was able to transform the D-form into L-, while the mutant was impaired to do it. When the orf8-orf9 region from SRL island was transformed into S. flexneri and S. sonnei SRL PAI-negative strains, the phenotype was restored. Although, when single genes were cloned into plasmids, no complementation was observed. Our results strongly suggest that the aspartate racemase and the transporter encoded in the SRL pathogenicity island are important for bacterial survival in environments rich in D-aspartate.
Assuntos
Isomerases de Aminoácido/metabolismo , Ácido D-Aspártico/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Ilhas Genômicas , Shigella flexneri/genética , Isomerases de Aminoácido/genética , Proteínas de Bactérias/metabolismo , Ácido D-Aspártico/análise , Genes Bacterianos , Manose/metabolismo , Fases de Leitura Aberta/genética , Fenótipo , Shigella flexneri/enzimologia , Shigella flexneri/crescimento & desenvolvimento , Shigella sonnei/genéticaRESUMO
Fish is the most indispensable source of proteins for individuals and have high nutritional value. On the other hand, the fish culturing raised issues of fish health due to close contact between the aquatic environment and the fish pathogens. So, the aim of the current study was to identify the bacterial pathogens and screen the injured Rainbow trout rearing in different trout hatcheries run under fisheries department of the government of Azad Jammu and Kashmir, Pakistan. Seven bacterial pathogens such as Shigella flexneri, Enterobacter amnigenus, Salmonella Typhimurium, Serratia odorifera, Pseudomonas aeruginosa, Streptococcus pyogenes, and Bacillus cereus were isolated and identified. Results revealed that the injury of fish specimens was due to overcrowding. Instead of rainbow coloration, specimens have darker black in color. The water of ponds was not clean and clear and such conditions was because of the greater quantity of feed thrown in the water. It was concluded that poor hygienic water condition and overloading allowed the opportunistic bacterial contaminations to succeed which cause a serious threat to hatcheries.(AU)
O peixe é a fonte mais indispensável de proteínas para os indivíduos e tem alto valor nutricional. Por outro lado, a cultura dos peixes levantou questões sobre a saúde dos peixes devido ao próximo contato entre o ambiente aquático e os agentes patogênicos desses peixes. Assim, o objetivo do presente estudo foi identificar os patógenos bacterianos e rastrear a criação da truta arco-íris que apresentou lesões em diferentes incubadoras de trutas, com supervisão do departamento de pesca do governo de Azad Jammu e Caxemira, Paquistão. Sete patógenos bacterianos foram isolados e identificados, tais como: Shigella flexneri, Enterobacter amnigenus, Salmonella typhimurium, Serratia odorifera, Pseudomonas aeruginosa, Streptococcus pyogenes e Bacillus cereus. Os resultados revelaram que a lesão de espécimes de peixes foi devido à superlotação. Em vez da coloração do arco-íris, os espécimes tiveram uma coloração preta mais escura. A água das lagoas não era limpa e nem clara, e tais condições ocorreram devido a maior quantidade de alimento lançada na água. Concluiu-se que a precária condição higiênica da água e também a sobrecarga permitiram que as contaminações bacterianas oportunistas fossem bem-sucedidas, causando séria ameaça às incubadoras.(AU)
Assuntos
Animais , Peixes/microbiologia , Incubadoras , Shigella flexneri/patogenicidade , Enterobacter/patogenicidade , Salmonella typhimurium/patogenicidade , Serratia/patogenicidade , Pseudomonas aeruginosa/patogenicidade , Noxas/análiseRESUMO
Shigella spp. are a leading cause of human diarrheal disease worldwide, with Shigella flexneri being the most frequently isolated species in developing countries. This serogroup is presently classified into 19 serotypes worldwide. We report here a multicenter validation of a multiplex-PCR-based strategy previously developed by Q. Sun, R. Lan, Y. Wang, A. Zhao, et al. (J Clin Microbiol 49:3766-3770, 2011) for molecular serotyping of S. flexneri This study was performed by seven international laboratories, with a panel of 71 strains (researchers were blind to their identity) as well as 279 strains collected from each laboratory's own local culture collections. This collaborative work found a high extent of agreement among laboratories, calculated through interrater reliability (IRR) measures for the PCR test that proved its robustness. Agreement with the traditional method (serology) was also observed in all laboratories for 14 serotypes studied, while specific genetic events could be responsible for the discrepancies among methodologies in the other 5 serotypes, as determined by PCR product sequencing in most of the cases. This work provided an empirical framework that allowed the use of this molecular method to serotype S. flexneri and showed several advantages over the traditional method of serological typing. These advantages included overcoming the problem of availability of suitable antisera in testing laboratories as well as facilitating the analysis of multiple samples at the same time. The method is also less time-consuming for completion and easier to implement in routine laboratories. We recommend that this PCR be adopted, as it is a reliable diagnostic and characterization methodology that can be used globally for laboratory-based shigella surveillance.
Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Sorotipagem/métodos , Shigella flexneri/classificação , Técnicas de Tipagem Bacteriana/métodos , Técnicas de Tipagem Bacteriana/normas , DNA Bacteriano/genética , Humanos , Internacionalidade , Reação em Cadeia da Polimerase Multiplex/normas , Sorogrupo , Shigella flexneri/imunologiaRESUMO
Shigella spp. is the most frequent micro-biological isolation in bacterial diarrhea in Argentina. It causes a watery diarrhea or dysenteric disease. It rarely causes extraintestinal problems. It has an incidence of bacteremia of 0,4-7,3%, and its appearance compels us to look for associated risk factors, as children under one year of age and immunodeficiency, among others. We describe two children with Shigella flexneri bacteremia. They presented with fever and diarrhea. One of them had primary immune deficiency.
Assuntos
Bacteriemia/microbiologia , Disenteria Bacilar/microbiologia , Shigella flexneri/isolamento & purificação , Bacteriemia/diagnóstico , Disenteria Bacilar/diagnóstico , Humanos , Lactente , MasculinoRESUMO
Shigella infections account for a considerable burden of acute diarrheal diseases worldwide and remain a major cause of childhood mortality in developing countries. Although, all four species of Shigella (S. dysenteriae, S. flexneri, S. boydii, and S. sonnei) cause bacillary dysentery, historically only S. dysenteriae type 1 has been recognized as carrying the genes for Shiga toxin (stx). Recent epidemiological data, however, have suggested that the emergence of stx carrying S. flexneri strains may have originated from bacteriophage-mediated inter-species horizontal gene transfer in one specific geographical area, Hispaniola. To test this hypothesis, we analyzed whole genome sequences of stx-encoding phages carried by S. flexneri strains isolated in Haiti and S. flexneri S. boydii and S. dysenteriae strains isolated from international travelers who likely acquired the infection in Haiti or the Dominican Republic. Phylogenetic analysis showed that phage sequences encoded in the Shigella strains from Hispaniola were bacteriophage φPOC-J13 and they were all closely related to a phage isolated from a USA isolate, E. coli 2009C-3133 serotype O119:H4. In addition, despite the low genetic heterogeneity of phages from different Shigella spp. circulating in the Caribbean island between 2001 and 2014, two distinct clusters emerged in Haiti and the Dominican Republic. Each cluster possibly originated from phages isolated from S. flexneri 2a, and within each cluster several instances of horizontal phage transfer from S. flexneri 2a to other species were detected. The implications of the emergence of stx-producing non-S. dysenteriae type 1 Shigella species, such as S. flexneri, spans not only the basic science behind horizontal phage spread, but also extends to medical treatment of patients infected with this pathogen.