Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 6.990
Filtrar
1.
Pol J Microbiol ; 68(3): 323-329, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31880878

RESUMO

This study conducts a comparative phenotypic and genetic analysis of C. perfringens strains isolated from two patients hospitalized at the same time in 2017 in the surgical ward of the Provincial Specialist Hospital in Wloclawek (Kujawsko-Pomorskie Province) who developed necrotizing soft tissue infections (NSTI). To explain the recurring cases of this infection, a comparative analysis was performed for these strains and the ones originating from infections recorded at the same hospital in three patients with gas gangrene in 2015. The two C. perfringens isolates studied in 2017 (8554/M/17 from patient No. 1 and 8567/M/17 from patient No. 2) had identical biochemical profiles. A comparison of research results using multiplex PCR from 2017 with a genetic analysis of strains from 2015 enabled us to demonstrate that the strains currently studied have the genes encoding the same toxins (α and ß2) as the two strains analyzed in 2015: no. 7143 (patient No. 3) and no. 7149 (patient No. 2). A comparative analysis of the strain profiles obtained with pulsed-field gel electrophoresis (PFGE) in 2017 with the results from 2015 has found one identical and genetically unique restriction profile, corresponding to one clone of C. perfringens comprising of two strains: no. 8567/M/17 (patient No. 2 in 2017) and no. 7143 (patient No. 3 in 2015). The epidemiological data and detailed analysis of the course of both events suggest that this clone of C. perfringens possibly survived in adverse conditions of the external environment in the operating block of this hospital for many months.This study conducts a comparative phenotypic and genetic analysis of C. perfringens strains isolated from two patients hospitalized at the same time in 2017 in the surgical ward of the Provincial Specialist Hospital in Wloclawek (Kujawsko-Pomorskie Province) who developed necrotizing soft tissue infections (NSTI). To explain the recurring cases of this infection, a comparative analysis was performed for these strains and the ones originating from infections recorded at the same hospital in three patients with gas gangrene in 2015. The two C. perfringens isolates studied in 2017 (8554/M/17 from patient No. 1 and 8567/M/17 from patient No. 2) had identical biochemical profiles. A comparison of research results using multiplex PCR from 2017 with a genetic analysis of strains from 2015 enabled us to demonstrate that the strains currently studied have the genes encoding the same toxins (α and ß2) as the two strains analyzed in 2015: no. 7143 (patient No. 3) and no. 7149 (patient No. 2). A comparative analysis of the strain profiles obtained with pulsed-field gel electrophoresis (PFGE) in 2017 with the results from 2015 has found one identical and genetically unique restriction profile, corresponding to one clone of C. perfringens comprising of two strains: no. 8567/M/17 (patient No. 2 in 2017) and no. 7143 (patient No. 3 in 2015). The epidemiological data and detailed analysis of the course of both events suggest that this clone of C. perfringens possibly survived in adverse conditions of the external environment in the operating block of this hospital for many months.


Assuntos
Toxinas Bacterianas/metabolismo , Clostridium perfringens/isolamento & purificação , Infecção Hospitalar/microbiologia , Gangrena Gasosa/microbiologia , Toxinas Bacterianas/genética , Clostridium perfringens/classificação , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Vestuário/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia
2.
BMC Infect Dis ; 19(1): 873, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640587

RESUMO

BACKGROUND: There have been no reports regarding the molecular characteristics, virulence features, and antibiotic resistance profiles of Staphylococcus aureus (S. aureus) from Hainan, the southernmost province of China. METHODS: Two hundred twenty-seven S. aureus isolates, consisting of 76 methicillin-resistant S. aureus (MRSA) and 151 methicillin-susceptible S. aureus (MSSA), were collected in 2013-2014 and 2018-2019 in Hainan, and investigated for their molecular characteristics, virulence genes, antibiotic resistance profiles and main antibiotic resistance genes. RESULTS: Forty sequence types (STs) including three new STs (ST5489, ST5492 and ST5493), and 79 Staphylococcal protein A (spa) types were identified based on multilocus sequence typing (MLST) and spa typing, respectively. ST398 (14.1%, 32/227) was found to be the most prevalent, and the prevalence of ST398-MSSA increased significantly from 2013 to 2014 (5.5%, 5/91) to 2018-2019 (18.4%, 25/136). Seventy-six MRSA isolates were subject to staphylococcus chromosomal cassette mec (SCCmec) typing. SCCmec-IVa was the predominant SCCmec type, and specifically, ST45-SCCmec IVa, an infrequent type in mainland China, was predominant in S. aureus from Hainan. The antibiotic resistance profiles and antibiotic resistance genes of S. aureus show distinctive features in Hainan. The resistant rates of the MRSA isolates to a variety of antibiotics were significantly higher than those of the MSSA isolates. The predominant erythromycin and tetracycline resistance genes were ermC (90.1%, 100/111) and tetK (91.8%, 78/85), respectively. Eleven virulence genes, including the Panton-Valentine leukocidin (pvl) and eta, were determined, and the frequency of eta and pvl were found to be 57.3 and 47.6%. Such high prevalence has never been seen in mainland China before. CONCLUSION: S. aureus isolates in Hainan have unique molecular characteristics, virulence gene and antibiotic resistance profiles, and main antibiotic resistance genes which may be associated with the special geographical location of Hainan and local trends in antibiotic use.


Assuntos
Farmacorresistência Bacteriana/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Fatores de Virulência/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , China , Farmacorresistência Bacteriana/efeitos dos fármacos , Exotoxinas/genética , Humanos , Leucocidinas/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação , Virulência/genética
3.
BMC Infect Dis ; 19(1): 899, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31660878

RESUMO

BACKGROUND: Several reports designate the recent increase in community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) nasal carriage. Because of the scanty information regarding the nasal carriage sate of MRSA in the west of Iran, the purpose of the present study was to determine the frequency of CA-MRSA in Sanandaj city. METHODS: Swabs collected from anterior nares of 600 volunteers were analyzed for the presence of S. aureus. The isolates were further investigated for methicillin resistance by using the cefoxitin disk diffusion test, followed by PCR-amplification of the mecA gene. SCCmec types and the presence of the Panton-Valentine Leukocidin (pvl) encoding genes were determined through PCR. Finally, the antimicrobial susceptibility of the isolates was determined by the agar diffusion method. RESULTS: Nasal screening identified 181 S. aureus, of which 55 isolates were MRSA. SCCmec types IV and V were detected in MRSA at frequencies of 80 and 20%, respectively. The overall frequency of pvl genes among the MRSA isolates was 14.54%. MRSA isolates were highly susceptible (98.18%) to mupirocin, gentamicin, and fusidic acid. CONCLUSIONS: The high prevalence of CA-MRSA carriage in the population could pose a serious public health concern for the region. Additionally, advent of drug-resistant pvl-positive strains demands continuous surveillance on the colonization state of CA-MRSA in order to prevent dissemination of the bacterium in the community.


Assuntos
Resistência a Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/genética , Cavidade Nasal/microbiologia , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Adolescente , Adulto , Idoso , Toxinas Bacterianas/genética , Criança , Pré-Escolar , Estudos Transversais , Exotoxinas/genética , Feminino , Frequência do Gene , Humanos , Lactente , Irã (Geográfico)/epidemiologia , Leucocidinas/genética , Masculino , Programas de Rastreamento , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prevalência , Saúde Pública , Fatores de Risco , Adulto Jovem
4.
PLoS Genet ; 15(10): e1008232, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31622331

RESUMO

In nature, most bacteria live in biofilms where they compete with their siblings and other species for space and nutrients. Some bacteria produce antibiotics in biofilms; however, since the diffusion of antibiotics is generally hindered in biofilms by extracellular polymeric substances, i.e., the biofilm matrix, their function remains unclear. The Bacillus subtilis yitPOM operon is a paralog of the sdpABC operon, which produces the secreted peptide toxin SDP. Unlike sdpABC, yitPOM is induced in biofilms by the DegS-DegU two-component regulatory system. High yitPOM expression leads to the production of a secreted toxin called YIT. Expression of yitQ, which lies upstream of yitPOM, confers resistance to the YIT toxin, suggesting that YitQ is an anti-toxin protein for the YIT toxin. The alternative sigma factor SigW also contributes to YIT toxin resistance. In a mutant lacking yitQ and sigW, the YIT toxin specifically inhibits biofilm formation, and the extracellular neutral protease NprB is required for this inhibition. The requirement for NprB is eliminated by Δeps and ΔbslA mutations, either of which impairs production of biofilm matrix polymers. Overexpression of biofilm matrix polymers prevents the action of the SDP toxin but not the YIT toxin. These results indicate that, unlike the SDP toxin and many conventional antibiotics, the YIT toxin can pass through layers of biofilm matrix polymers to attack cells within biofilms with assistance from NprB. When the wild-type strain and the YIT-sensitive mutant were grown together on a solid medium, the wild-type strain formed biofilms that excluded the YIT-sensitive mutant. This observation suggests that the YIT toxin protects B. subtilis biofilms against competitors. Several bacteria are known to produce antibiotics in biofilms. We propose that some bacteria including B. subtilis may have evolved specialized antibiotics that can function within biofilms.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Antibacterianos/biossíntese , Bacillus subtilis/crescimento & desenvolvimento , Endopeptidases/genética , Regulação Bacteriana da Expressão Gênica/genética , Mutação , Óperon/genética
5.
Ann Agric Environ Med ; 26(3): 392-395, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31559791

RESUMO

Existing research for using the protective antigen (PA) of Bacillus anthracis as a vaccine component shows that protection against anthrax may be obtained using fragments of this protein. The aim of the research is to check whether the selected protein fragment of the protective antigen (domain 4) encoded by an appropriate nucleotide sequence of gene pag of B. anthracis, was expressed in the bacterial system of E. coli. In order to examine the selected sequence of the pag gene, a PCR reaction and a highly effective TOPO cloning strategy were used, followed by purification of the recombinant proteins and their detection by a western-blot method. In the planning of the PA4 antigen expression a higher level of effectiveness in production of small protein - domain 4 - was anticipated. As a result, the 139 amino acids protein fragment of B. anthracis PA (domain 4) was isolated. The research may have found the basis for in vivo research aimed at finding potential anthrax vaccine components.


Assuntos
Vacinas contra Antraz/imunologia , Antraz/microbiologia , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/imunologia , Animais , Antraz/imunologia , Antraz/prevenção & controle , Vacinas contra Antraz/administração & dosagem , Vacinas contra Antraz/genética , Vacinas contra Antraz/isolamento & purificação , Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Bacillus anthracis/química , Bacillus anthracis/genética , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Western Blotting , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Domínios Proteicos
6.
Lett Appl Microbiol ; 69(5): 385-390, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31529707

RESUMO

Clostridium perfringens is the main cause of sudden death in dogs and currently there is no vaccine to prevent it. In this study, a canine C. perfringens type A strain was used to prepare a vaccine. C. perfringens was inactivated by formaldehyde and adjuvants were added. The safety and immunological characteristics of the inactivated C. perfringens vaccine were evaluated in mice and dogs. The results showed that the C. perfringens vaccine was safe and had immunoprotective activity. The serum antibody titre of immunized mice reached up to 6·25 × 104 . Both single immunization of 4 ml and dual immunizations of 2 ml each provided good immune protection, with five of five immunized dogs surviving. This study also studied a detoxified crude α-toxin extract vaccine. The results showed that a single immunization with 0·5 ml of the detoxified crude α-toxin extract vaccine provided immune protection, with five of five immunized dogs surviving. The inactivated C. perfringens type A vaccine can be used to prevent canine C. perfringens infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Clostridium perfringens is the main cause of sudden death in dogs and currently there is no vaccine to prevent it. In this study, an inactivated canine C. perfringens vaccine and a detoxified crude α-toxin vaccine were prepared. The safety and protective effects of these vaccines were evaluated using mouse and dog models. The vaccines were shown to be safe and to provide immune protection effects that can be used to prevent canine C. perfringens infection.


Assuntos
Toxinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/imunologia , Animais , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/genética , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Infecções por Clostridium/microbiologia , Clostridium perfringens/genética , Cães , Avaliação Pré-Clínica de Medicamentos , Humanos , Imunização , Camundongos , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/genética , Vacinas de Produtos Inativados/imunologia
7.
Nucleic Acids Res ; 47(19): 10400-10413, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31501867

RESUMO

Chromosomally-encoded toxin-antitoxin complexes are ubiquitous in bacteria and regulate growth through the release of the toxin component typically in a stress-dependent manner. Type II ribosome-dependent toxins adopt a RelE-family RNase fold and inhibit translation by degrading mRNAs while bound to the ribosome. Here, we present biochemical and structural studies of the Escherichia coli YoeB toxin interacting with both a UAA stop and an AAU sense codon in pre- and post-mRNA cleavage states to provide insights into possible mRNA substrate selection. Both mRNAs undergo minimal changes during the cleavage event in contrast to type II ribosome-dependent RelE toxin. Further, the 16S rRNA decoding site nucleotides that monitor the mRNA in the aminoacyl(A) site adopt different orientations depending upon which toxin is present. Although YoeB is a RelE family member, it is the sole ribosome-dependent toxin that is dimeric. We show that engineered monomeric YoeB is active against mRNAs bound to both the small and large subunit. However, the stability of monomeric YoeB is reduced ∼20°C, consistent with potential YoeB activation during heat shock in E. coli as previously demonstrated. These data provide a molecular basis for the ability of YoeB to function in response to thermal stress.


Assuntos
Toxinas Bacterianas/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Estabilidade Proteica , Ribonucleases/química , Sequência de Aminoácidos/genética , Toxinas Bacterianas/genética , Códon/química , Códon/genética , Dimerização , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Resposta ao Choque Térmico/genética , Estabilidade de RNA/genética , RNA Mensageiro , RNA Ribossômico 16S/genética , Ribonucleases/genética , Ribossomos/química , Ribossomos/genética
8.
Infect Immun ; 87(10)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31383747

RESUMO

Postinfluenza methicillin-resistant Staphylococcus aureus (MRSA) infection can quickly develop into severe, necrotizing pneumonia, causing over 50% mortality despite antibiotic treatments. In this study, we investigated the efficacy of antibiotic therapies and the impact of S. aureus alpha-toxin in a model of lethal influenza virus and MRSA coinfection. We demonstrate that antibiotics primarily attenuate alpha-toxin-induced acute lethality, even though both alpha-toxin-dependent and -independent mechanisms significantly contribute to animal mortality after coinfection. Furthermore, we found that the protein synthesis-suppressing antibiotic linezolid has an advantageous therapeutic effect on alpha-toxin-induced lung damage, as measured by protein leak and lactate dehydrogenase (LDH) activity. Importantly, using a Panton-Valentine leucocidin (PVL)-negative MRSA isolate from patient sputum, we show that linezolid therapy significantly improves animal survival from postinfluenza MRSA pneumonia compared with vancomycin treatment. Rather than improved viral or bacterial control, this advantageous therapeutic effect is associated with a significantly attenuated proinflammatory cytokine response and acute lung damage in linezolid-treated mice. Together, our findings not only establish a critical role of alpha-toxin in the extreme mortality of secondary MRSA pneumonia after influenza but also provide support for the possibility that linezolid could be a more effective treatment than vancomycin to improve disease outcomes.


Assuntos
Antibacterianos/farmacologia , Toxinas Bacterianas/antagonistas & inibidores , Proteínas Hemolisinas/antagonistas & inibidores , Linezolida/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Infecções por Orthomyxoviridae/complicações , Pneumonia Estafilocócica/tratamento farmacológico , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Feminino , Expressão Gênica , Gentamicinas/farmacologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , L-Lactato Desidrogenase/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Masculino , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Plasmídeos/química , Plasmídeos/metabolismo , Pneumonia Estafilocócica/complicações , Pneumonia Estafilocócica/microbiologia , Pneumonia Estafilocócica/mortalidade , Análise de Sobrevida , Vancomicina/farmacologia
9.
Genome Biol ; 20(1): 163, 2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31405375

RESUMO

BACKGROUND: Like many bacteria, Vibrio cholerae deploys a harpoon-like type VI secretion system (T6SS) to compete against other microbes in environmental and host settings. The T6SS punctures adjacent cells and delivers toxic effector proteins that are harmless to bacteria carrying cognate immunity factors. Only four effector/immunity pairs encoded on one large and three auxiliary gene clusters have been characterized from largely clonal, patient-derived strains of V. cholerae. RESULTS: We sequence two dozen V. cholerae strain genomes from diverse sources and develop a novel and adaptable bioinformatics tool based on hidden Markov models. We identify two new T6SS auxiliary gene clusters and describe Aux 5 here. Four Aux 5 loci are present in the host strain, each with an atypical effector/immunity gene organization. Structural prediction of the putative effector indicates it is a lipase, which we name TleV1 (type VI lipase effector Vibrio). Ectopic TleV1 expression induces toxicity in Escherichia coli, which is rescued by co-expression of the TliV1a immunity factor. A clinical V. cholerae reference strain expressing the Aux 5 cluster uses TleV1 to lyse its parental strain upon contact via its T6SS but is unable to kill parental cells expressing the TliV1a immunity factor. CONCLUSION: We develop a novel bioinformatics method and identify new T6SS gene clusters in V. cholerae. We also show the TleV1 toxin is delivered in a T6SS manner by V. cholerae and can lyse other bacterial cells. Our web-based tool can be modified to identify additional novel T6SS genomic loci in diverse bacterial species.


Assuntos
Genoma Bacteriano , Sistemas de Secreção Tipo VI/genética , Vibrio cholerae/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Genes Bacterianos , Variação Genética , Software , Vibrio cholerae/isolamento & purificação
10.
Nat Commun ; 10(1): 3460, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371723

RESUMO

Bacteria often produce antimicrobial toxins to compete in microbial communities. Here we identify a family of broad-spectrum peptide toxins, named bacteroidetocins, produced by Bacteroidetes species. We study this toxin family using phenotypic, mutational, bioinformatic, and human metagenomic analyses. Bacteroidetocins are related to class IIa bacteriocins of Gram-positive bacteria and kill members of the Bacteroidetes phylum, including Bacteroides, Parabacteroides, and Prevotella gut species, as well as pathogenic Prevotella species. The bacteroidetocin biosynthesis genes are found in horizontally acquired mobile elements, which likely allow dissemination within the gut microbiota and may explain their wide distribution in human populations. Bacteroidetocins may have potential applications in microbiome engineering and as therapeutics for polymicrobial diseases such as bacterial vaginosis and periodontal disease.


Assuntos
Antibacterianos/biossíntese , Toxinas Bacterianas/biossíntese , Bacteriocinas/biossíntese , Bacteriocinas/genética , Bacteroidetes/metabolismo , Microbioma Gastrointestinal/fisiologia , Peptídeos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacologia , Bacteriocinas/farmacologia , Bacteroidetes/efeitos dos fármacos , Bacteroidetes/genética , Sequência de Bases , Feminino , Microbioma Gastrointestinal/genética , Trato Gastrointestinal/microbiologia , Transferência Genética Horizontal/genética , Humanos , Sequências Repetitivas Dispersas , Metagenômica , Testes de Sensibilidade Microbiana , Peptídeos/genética , Peptídeos/farmacologia , Prevotella/efeitos dos fármacos , Análise de Sequência de Proteína , Vaginose Bacteriana
11.
Nat Commun ; 10(1): 3684, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31417089

RESUMO

Bacterial toxins with an AB5 architecture consist of an active (A) subunit inserted into a ring-like platform comprised of five delivery (B) subunits. Salmonella Typhi, the cause of typhoid fever, produces an unusual A2B5 toxin known as typhoid toxin. Here, we report that upon infection of human cells, S. Typhi produces two forms of typhoid toxin that have distinct delivery components but share common active subunits. The two typhoid toxins exhibit different trafficking properties, elicit different effects when administered to laboratory animals, and are expressed using different regulatory mechanisms and in response to distinct metabolic cues. Collectively, these results indicate that the evolution of two typhoid toxin variants has conferred functional versatility to this virulence factor. More broadly, this study reveals a new paradigm in toxin biology and suggests that the evolutionary expansion of AB5 toxins was likely fueled by the plasticity inherent to their structural design coupled to the functional versatility afforded by the combination of homologous toxin components.


Assuntos
Toxinas Bacterianas/genética , Multimerização Proteica/genética , Salmonella typhi/genética , Fatores de Virulência/genética , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Subunidades Proteicas/genética , Homologia de Sequência do Ácido Nucleico
12.
Cell Host Microbe ; 26(3): 426-434.e6, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31447308

RESUMO

Salmonella enterica serovar Typhi causes typhoid fever only in humans. Murine infection with S. Typhimurium is used as a typhoid model, but its relevance to human typhoid is limited. Non-obese diabetic-scid IL2rγnull mice engrafted with human hematopoietic stem cells (hu-SRC-SCID) are susceptible to lethal S. Typhi infection. In this study, we use a high-density S. Typhi transposon library in hu-SRC-SCID mice to identify virulence loci using transposon-directed insertion site sequencing (TraDIS). Vi capsule, lipopolysaccharide (LPS), and aromatic amino acid biosynthesis were essential for virulence, along with the siderophore salmochelin. However, in contrast to the murine S. Typhimurium model, neither the PhoPQ two-component system nor the SPI-2 pathogenicity island was required for lethal S. Typhi infection, nor was the CdtB typhoid toxin. These observations highlight major differences in the pathogenesis of typhoid and non-typhoidal Salmonella infections and demonstrate the utility of humanized mice for understanding the pathogenesis of a human-specific pathogen.


Assuntos
Estudo de Associação Genômica Ampla/métodos , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Salmonella typhi/genética , Salmonella typhi/patogenicidade , Aminoácidos Aromáticos/biossíntese , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Ilhas Genômicas/genética , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Ferro/metabolismo , Lipopolissacarídeos/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Obesos , Camundongos SCID , Salmonella typhi/crescimento & desenvolvimento , Sideróforos/metabolismo , Células THP-1/microbiologia , Febre Tifoide , Virulência/genética
13.
J Appl Microbiol ; 127(6): 1859-1868, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31429177

RESUMO

AIMS: Given the extreme toxicity of some of the toxins of toxin-antitoxin (TA) systems, we were curious how the cell silences toxins, if the antitoxin is inactivated or independent toxins are obtained via horizontal gene transfer. METHODS AND RESULTS: Growth curves of Escherichia coli K12 BW25113 harbouring plasmid pCA24N to produce RalR, MqsR, GhoT or Hha toxins, showed toxin inactivation after 3 h. Sequencing plasmids from these cultures revealed toxin inactivation occurred primarily due to consistent deletions in the promoter. The lack of mutation in the structural genes was corroborated by a bioinformatics analysis of 1000 E. coli genomes which showed both conservation and little variability in the four toxin genes. For those strains that lacked a mutation in the plasmid, single nucleotide polymorphism analysis was performed to identify that chromosomal mutations iraM and mhpR inactivate the toxins GhoT and MqsR/GhoT respectively. CONCLUSION: We find that the RalR (type I), MqsR (type II), GhoT (type V) and Hha (type VII) toxins are inactivated primarily by a mutation that inactivates the toxin promoter or via the chromosomal mutations iraM and mhpR. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates toxins of TA systems may be inactivated by mutations that primarily affect the toxin gene promoter instead of the toxin structural gene.


Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Mutação/genética , Regiões Promotoras Genéticas/genética , Sistemas Toxina-Antitoxina/genética , Cromossomos Bacterianos/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Plasmídeos/genética
14.
PLoS Negl Trop Dis ; 13(8): e0007644, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31430284

RESUMO

Bacillus anthracis and Yersinia pestis are zoonotic bacteria capable of causing severe and sometimes fatal infections in animals and humans. Although considered as diseases of antiquity in industrialized countries due to animal and public health improvements, they remain endemic in vast regions of the world disproportionally affecting the poor. These pathogens also remain a serious threat if deployed in biological warfare. A single vaccine capable of stimulating rapid protection against both pathogens would be an extremely advantageous public health tool. We produced multiple-antigen fusion proteins (MaF1 and MaF2) containing protective regions from B. anthracis protective antigen (PA) and lethal factor (LF), and from Y. pestis V antigen (LcrV) and fraction 1 (F1) capsule. The MaF2 sequence was also expressed from a plasmid construct (pDNA-MaF2). Immunogenicity and protective efficacy were investigated in mice following homologous and heterologous prime-boost immunization. Antibody responses were determined by ELISA and anthrax toxin neutralization assay. Vaccine efficacy was determined against lethal challenge with either anthrax toxin or Y. pestis. Both constructs elicited LcrV and LF-specific serum IgG, and MaF2 elicited toxin-neutralizing antibodies. Immunizations with MaF2 conferred 100% and 88% protection against Y. pestis and anthrax toxin, respectively. In contrast, pDNA-MaF2 conferred only 63% protection against Y. pestis and no protection against anthrax toxin challenge. pDNA-MaF2-prime MaF2-boost induced 75% protection against Y. pestis and 25% protection against anthrax toxin. Protection was increased by the molecular adjuvant CARDif. In conclusion, MaF2 is a promising multi-antigen vaccine candidate against anthrax and plague that warrants further investigation.


Assuntos
Antraz/prevenção & controle , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Vacinas Bacterianas/imunologia , Peste/prevenção & controle , Proteínas Recombinantes de Fusão/imunologia , Yersinia pestis/imunologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Bacillus anthracis/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Vacinas Bacterianas/isolamento & purificação , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos BALB C , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/imunologia , Proteínas Recombinantes de Fusão/genética , Análise de Sobrevida , Resultado do Tratamento , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Yersinia pestis/genética
15.
N Z Vet J ; 67(6): 329-332, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31378159

RESUMO

Aims: To determine if presence of the Bacteroides fragilis toxin (bft) gene, a molecular marker of colonic carriage of entertoxigenic Bacteroides fragilis (ETBF) in humans, was associated with a finding of small intestinal adenocarcinomas (SIA) in sheep in New Zealand. Methods: Samples of jejunal tissue were collected from the site of tumours and from grossly normal adjacent tissue in 20 sheep, in different consignments, diagnosed with SIA based on gross examination of viscera following slaughter. Two jejunal samples were also collected from a control sheep in the same consignment that had no gross evidence of SIA. A PCR assay was used to detect the presence of the bft gene in the samples. Results: Of the sheep with SIA, the bft gene was amplified from one or both samples from 7/20 (35%) sheep, and in sheep that had no gross evidence of SIA the bft gene was amplified from at least one sample in 11/20 (55%) sheep (RR 0.61; 95% CI = 0.30-1.25; p = 0.34). Of 11 positive samples analysed, ETBF subtype bft-1 was detected in one, bft-2 was detected in 10, and none were bft-3. Conclusions and Clinical Relevance: There was a high prevalence of detection of the bft gene in both SIA-affected and non-affected sheep, but there was no apparent association between carriage of ETBF, evidenced by detection of the bft gene, and the presence of SIA. ETBF are increasingly implicated in the aetiology of human colorectal cancer, raising the possibility that sheep may provide a zoonotic reservoir of this potentially carcinogenic bacterium. Abbreviation: Bft: Bacteroides fragilis toxin; ETBF: Enterotoxigenic Bacteroides fragilis; SIA: Small intestinal adenocarcinoma.


Assuntos
Adenocarcinoma/veterinária , Toxinas Bacterianas/genética , Neoplasias Intestinais/veterinária , Metaloendopeptidases/genética , Doenças dos Ovinos/diagnóstico , Adenocarcinoma/diagnóstico , Adenocarcinoma/microbiologia , Animais , Toxinas Bacterianas/isolamento & purificação , DNA Bacteriano/análise , Genes Bacterianos , Neoplasias Intestinais/diagnóstico , Neoplasias Intestinais/microbiologia , Metaloendopeptidases/isolamento & purificação , Ovinos , Doenças dos Ovinos/microbiologia
16.
Mol Cell ; 75(5): 1031-1042.e4, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31327636

RESUMO

Every bacterial population harbors a small subpopulation of so-called persisters that are transiently antibiotic tolerant. These persisters are associated with the recalcitrance of chronic infections because they can recolonize the host after antibiotic removal. Although several effectors have been described to induce persistence, persister cell awakening is poorly understood. We previously reported that the toxin HokB induces persistence via pore formation, resulting in membrane depolarization and ATP leakage. We now delineate mechanisms responsible for the awakening of HokB-induced persisters. We show that HokB dimerization by the oxidoreductase DsbA is essential for pore formation and peptide stability. Pores are disassembled via DsbC-mediated monomerization, which targets HokB for DegQ-mediated degradation. Finally, pore disassembly allows membrane repolarization by the electron transport chain, supporting cells to resume growth. These results provide a detailed view of both the formation and awakening of HokB-induced persister cells.


Assuntos
Toxinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Potenciais da Membrana/fisiologia , Proteólise , Serina Endopeptidases/metabolismo , Toxinas Bacterianas/genética , Membrana Celular/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Serina Endopeptidases/genética
17.
Lett Appl Microbiol ; 69(4): 264-270, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31323126

RESUMO

Contaminated wastewater plays an important role in the transmission of Listeria monocytogenes in the environment. In this study, a loop-mediated isothermal amplification (LAMP) assay for sensitive detection of L.  monocytogenes in wastewater from treatment plants was developed, validated and compared to conventional PCR. The lmo0753 gene which codes for a Crp/Fnr family transcription factor, was targeted to design four specific primers to detect L.  monocytogenes in 60 min at 63°C in a water bath. Amplification products were visualized by agarose gel electrophoresis. The detection limit of the LAMP assay was 65 fg µl-1 of DNA and 38 CFU per ml. Conventional PCR was 10 times less sensitive than LAMP assay with primers targeting the HlyA gene. A total of 70 crude wastewater samples collected at different treatment stages (aeration tank, pre chlorination and post chlorination), were tested directly by LAMP and PCR. Samples from aeration and pre-chlorination stages tested positive with LAMP and culture method but not with conventional PCR. LAMP assay was tolerant to inhibitors present in wastewater and circumvented the need for isolation of pure DNA for detection. Both LAMP assay and culture method failed to detect L.  monocytogenes in post-chlorinated wastewater, confirming the efficiency of the treatment process in the removal of L.  monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Treated wastewater effluent contains Listeria monocytogenes which survives conventional wastewater treatment processes and can re-enter human food chain, thus it is imperative to detect L.  monocytogenes using a rapid and an inexpensive method. To the best of our knowledge, this is the first report of a loop-mediated isothermal amplification (LAMP) assay, targeting the lmo0753 gene for detection of L.  monocytogenes in wastewater from treatment plants. The LAMP assay detects L.  monocytogenes in 60 min at 63°C in a water bath. LAMP does not require isolation of pure genomic DNA hence it is a user friendly method for L.  monocytogenes detection.


Assuntos
Toxinas Bacterianas/genética , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/genética , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Águas Residuárias/microbiologia , Domínio AAA , Primers do DNA/genética , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Purificação da Água
18.
Biomed Environ Sci ; 32(7): 520-530, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31331436

RESUMO

OBJECTIVE: To investigate the molecular characteristics and intracellular growth ability of Legionella pneumophila (L. pneumophila) strains from 1989 to 2016 in Sichuan Province, China. METHODS: Seventy-nine isolates of L. pneumophila were collected from environmental and clinical sources, including cooling towers, hot springs, bath water, fountains, and patients, and identified with 16S rRNA gene analysis and serum agglutination assay. The isolates were then typed by Sequence-Based Typing (SBT), and Genotyping of forty-two LP1 strains were analyzed by means of multiple-locus VNTR analysis with 8 loci (MLVA-8). All strains were further analyzed for two virulence genes: Legionella vir homologue (lvh) and repeats in structural toxin (rtxA). The intracellular growth ability of 33 selected isolates was determined by examining their interaction with J774 cells. RESULTS: All isolates were identified to L. pneumophila including 11 serogroups, among which the main serogroup were LP1, accounting for 54.43%. Thirty-three different sequence types (STs) from five main clonal groups and five singletons were identified, along with 8 different MLVA patterns. Both the lvh and rtxA loci were found in all 79 strains. Thirty isolates showed high intracellular growth ability in J774 cells. CONCLUSION: L. pneumophila is a potential threat to public health, and effective control and prevention strategies are urgently needed.


Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Legionella pneumophila/genética , RNA Ribossômico 16S/genética , China , Técnicas de Genotipagem , Humanos , Legionella pneumophila/crescimento & desenvolvimento , Legionella pneumophila/isolamento & purificação , Microbiologia da Água
19.
J Food Sci ; 84(8): 2250-2255, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31313323

RESUMO

The ability to produce various extracellular enzymes is considered as an important virulence feature in Aeromonas spp., in addition to producing specific virulence factors such as aerolysin and hemolysin. In this study, the effect of salinity and pH on the production of extracellular virulence factors by Aeromonas was investigated. Aeromonas was obtained from different food sources. A comparative study of the activities of extracellular enzymes secreted by these bacteria at different environmental conditions can widen our understanding on their pathogenicity. The activities of various extracellular enzymes such as amylase, gelatinase, and caseinase, which are implicated as virulence factors, were measured in vitro by calculating the enzymatic activity index (EAI) of each enzyme using standard laboratory protocols. For all enzymes, a significant change (P < 0.05) in the EAI was observed when the concentration of NaCl in the media increased from 0.5% to 3%. Among three enzymes tested, caseinase was found to be affected the most by salinity, with a significant difference in EAI when NaCl concentration in the media increased from 0.5% to 2%. Similarly, amylase was found to be affected the most by acidity. The pH values ranging from 6 to 9 did not exert any significant change in EAI of amylase; however, a pH value of 5 had a significant effect. Overall, compared to salinity, the change in pH was found to be less effective in controlling the extracellular virulence factor production in Aeromonas. PRACTICAL APPLICATIONS: The practical application is to minimize the extracellular virulence factor production by Aeromonas in food commodities by altering the salt content and pH. The results demonstrate that an increase in salinity and a decrease in pH can minimize the extracellular virulence factor production by Aeromonas spp.


Assuntos
Aeromonas/metabolismo , Proteínas de Bactérias/metabolismo , Cloreto de Sódio/metabolismo , Fatores de Virulência/metabolismo , Aeromonas/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Microbiologia de Alimentos , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Salinidade , Cloreto de Sódio/análise , Fatores de Virulência/genética
20.
Chem Commun (Camb) ; 55(63): 9311-9314, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31310244

RESUMO

Discrimination between cysteine and homocysteine at the single-molecule level is achieved within a K238Q mutant aerolysin nanopore, which provides a confined space for high spatial resolution to identify the amino acid difference with a 5'-benzaldehyde poly(dA)4 probe. Our strategy allows potential detection and characterization of various amino acids and their modifications, and provides a crucial step towards developing nanopore protein sequencing devices.


Assuntos
Toxinas Bacterianas/química , Cisteína/análise , Homocisteína/análise , Nanoporos , Proteínas Citotóxicas Formadoras de Poros/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Cromatografia Líquida de Alta Pressão , Mutagênese Sítio-Dirigida , Poli A/química , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Espectrometria de Massas por Ionização por Electrospray
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA