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1.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299005

RESUMO

Nanoparticles can interact with the complement system and modulate the inflammatory response. The effect of these interactions on the complement activity strongly depends on physicochemical properties of nanoparticles. The interactions of silver nanoparticles with serum proteins (particularly with the complement system components) have the potential to significantly affect the antibacterial activity of serum, with serious implications for human health. The aim of the study was to assess the influence of graphite oxide (GO) nanocomposites (GO, GO-PcZr(Lys)2-Ag, GO-Ag, GO-PcZr(Lys)2) on the antibacterial activity of normal human serum (NHS), serum activity against bacteria isolated from alveoli treated with nanocomposites, and nanocomposite sensitivity of bacteria exposed to serum in vitro (using normal human serum). Additionally, the in vivo cytotoxic effect of the GO compounds was determined with application of a Galleria mellonella larvae model. GO-PcZr(Lys)2, without IR irradiation enhance the antimicrobial efficacy of the human serum. IR irradiation enhances bactericidal activity of serum in the case of the GO-PcZr(Lys)2-Ag sample. Bacteria exposed to nanocomposites become more sensitive to the action of serum. Bacteria exposed to serum become more sensitive to the GO-Ag sample. None of the tested GO nanocomposites displayed a cytotoxicity towards larvae.


Assuntos
Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Grafite/química , Nanopartículas Metálicas/química , Nanocompostos/química , Óxidos/química , Soro/efeitos dos fármacos , Animais , Antibacterianos/química , Anti-Infecciosos/química , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/efeitos da radiação , Humanos , Raios Infravermelhos , Larva/efeitos dos fármacos , Larva/efeitos da radiação , Lepidópteros/efeitos dos fármacos , Lepidópteros/efeitos da radiação , Nanopartículas Metálicas/administração & dosagem , Nanocompostos/administração & dosagem , Soro/microbiologia , Prata/química
2.
Int J Med Microbiol ; 311(6): 151518, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34237624

RESUMO

Many models assessing the risk of sepsis utilize the knowledge of the constituents of the plasminogen system, as it is proven that some species of bacteria can activate plasminogen, as a result of interactions with bacterial outer membrane proteins. However, much is yet to be discovered about this interaction since there is little information regarding some bacterial species. This study is aimed to check if Klebsiella pneumoniae, one of the major factors of nosocomial pneumonia and a factor for severe sepsis, has the ability to bind to human plasminogen. The strain used in this study, PCM 2713, acted as a typical representative of the species. With use of various methods, including: electron microscopy, 2-dimensional electrophoresis, immunoblotting and peptide fragmentation fingerprinting, it is shown that Klebsiella pneumoniae binds to human plasminogen, among others, due to plasminogen-bacterial enolase-like protein interaction, occurring on the outer membrane of the bacterium. Moreover, the study reveals, that other proteins, such as: phosphoglucomutase, and phosphoenolpyruvate carboxykinase act as putative plasminogen-binding factors. These information may virtually act as a foundation for future studies investigating: the: pathogenicity of Klebsiella pneumoniae and means for prevention from the outcomes of Klebsiella-derived sepsis.


Assuntos
Klebsiella pneumoniae , Plasminogênio , Proteínas da Membrana Bacteriana Externa , Humanos , Immunoblotting , Fosfopiruvato Hidratase
3.
Pathogens ; 10(7)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209937

RESUMO

OBJECTIVE: the main purpose of this work was to compare the genetic and phenotypic changes of E. coli treated with silver nanoformulations (E. coli BW25113 wt, E. coli BW25113 AgR, E. coli J53, E. coli ATCC 11229 wt, E. coli ATCC 11229 var. S2 and E. coli ATCC 11229 var. S7). Silver, as the metal with promising antibacterial properties, is currently widely used in medicine and the biomedical industry, in both ionic and nanoparticles forms. Silver nanoformulations are usually considered as one type of antibacterial agent, but their physical and chemical properties determine the way of interactions with the bacterial cell, the mode of action, and the bacterial cell response to silver. METHODS: the changes in the bacterial genome, resulting from the treatment of bacteria with various silver nanoformulations, were verified by analyzing of genes (selected with mutfunc) and their conservative and non-conservative mutations selected with BLOSUM62. The phenotype was verified using an outer membrane proteome analysis (OMP isolation, 2-DE electrophoresis, and MS protein identification). RESULTS: the variety of genetic and phenotypic changes in E. coli strains depends on the type of silver used for bacteria treatment. The most changes were identified in E. coli ATCC 11229 treated with silver nanoformulation signed as S2 (E. coli ATCC 11229 var. S2). We pinpointed 39 genes encoding proteins located in the outer membrane, 40 genes of their regulators, and 22 genes related to other outer membrane structures, such as flagellum, fimbria, lipopolysaccharide (LPS), or exopolysaccharide in this strain. Optical density of OmpC protein in E. coli electropherograms decreased after exposure to silver nanoformulation S7 (noticed in E. coli ATCC 11229 var. S7), and increased after treatment with the other silver nanoformulations (SNF) marked as S2 (noticed in E. coli ATCC 11229 var. S2). Increase of FliC protein optical density was identified in turn after Ag+ treatment (noticed in E.coli AgR). CONCLUSION: the results show that silver nanoformulations (SNF) exerts a selective pressure on bacteria causing both conservative and non-conservative mutations. The proteomic approach revealed that the levels of some proteins have changed after treatment with appropriate SNF.

4.
Int J Mol Sci ; 22(3)2021 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-33530493

RESUMO

The impact of the Gram-negative bacterium Escherichia coli (E. coli) on the microbiomic and pathogenic phenomena occurring in humans and other warm-blooded animals is relatively well-recognized. At the same time, there are scant data concerning the role of E. coli strains in the health and disease of cold-blooded animals. It is presently known that reptiles are common asymptomatic carriers of another human pathogen, Salmonella, which, when transferred to humans, may cause a disease referred to as reptile-associated salmonellosis (RAS). We therefore hypothesized that reptiles may also be carriers of specific E. coli strains (reptilian Escherichia coli, RepEC) which may differ in their genetic composition from the human uropathogenic strain (UPEC) and avian pathogenic E. coli (APEC). Therefore, we isolated RepECs (n = 24) from reptile feces and compared isolated strains' pathogenic potentials and phylogenic relations with the aforementioned UPEC (n = 24) and APEC (n = 24) strains. To this end, we conducted an array of molecular analyses, including determination of the phylogenetic groups of E. coli, virulence genotyping, Pulsed-Field Gel Electrophoresis-Restriction Analysis (RA-PFGE) and genetic population structure analysis using Multi-Locus Sequence Typing (MLST). The majority of the tested RepEC strains belonged to nonpathogenic phylogroups, with an important exception of one strain, which belonged to the pathogenic group B2, typical of extraintestinal pathogenic E. coli. This strain was part of the globally disseminated ST131 lineage. Unlike RepEC strains and in line with previous studies, a high percentage of UPEC strains belonged to the phylogroup B2, and the percentage distribution of phylogroups among the tested APEC strains was relatively homogenous, with most coming from the following nonpathogenic groups: C, A and B1. The RA-PFGE displayed a high genetic diversity among all the tested E. coli groups. In the case of RepEC strains, the frequency of occurrence of virulence genes (VGs) was lower than in the UPEC and APEC strains. The presented study is one of the first attempting to compare the phylogenetic structures of E. coli populations isolated from three groups of vertebrates: reptiles, birds and mammals (humans).


Assuntos
Doenças dos Animais/microbiologia , Infecções por Escherichia coli/veterinária , Filogenia , Répteis/microbiologia , Escherichia coli Uropatogênica/classificação , Escherichia coli Uropatogênica/genética , Animais , Proteínas de Escherichia coli/genética , Especificidade de Hospedeiro , Humanos , Tipagem de Sequências Multilocus , Doenças das Aves Domésticas/microbiologia , Virulência/genética , Fatores de Virulência/genética
5.
Pathogens ; 10(1)2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33450948

RESUMO

Yersinia enterocolitica, widespread within domestic and wild-living animals, is a foodborne pathogen causing yersiniosis. The goal of this study was to assess a genetic similarity of Y. enterocolitica and Y. enterocolitica-like strains isolated from different hosts using Multiple Locus Variable-Number Tandem Repeat Analysis (MLVA) and Pulsed-Field Gel Electrophoresis (PFGE) methods, and analyze the prevalence of virulence genes using multiplex-Polymerase Chain Reaction (PCR) assays. Among 51 Yersinia sp. strains 20 virulotypes were determined. The most common virulence genes were ymoA, ureC, inv, myfA, and yst. Yersinia sp. strains had genes which may contribute to the bacterial invasion and colonization of the intestines as well as survival in serum. One wild boar Y. enterocolitica 1A strain possessed ail gene implying the possible pathogenicity of 1A biotype. Wild boar strains, represented mainly by 1A biotype, were not classified into the predominant Variable-Number Tandem Repeats (VNTR)/PFGE profile and virulotype. There was a clustering tendency among VNTR/PFGE profiles of pig origin, 4/O:3, and virulence profile. Pig and human strains formed the most related group, characterized by ~80% of genetic similarity what suggest the role of pigs as a potential source of infection for the pork consumers.

6.
Adv Clin Exp Med ; 29(12): 1433-1441, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33245853

RESUMO

BACKGROUND: Enolase is generally known as the glycolytic pathway enzyme present in the cytoplasm of eukaryotic cells and in some microorganisms. In human cells, it is also a component of cell surface membranes, where it functions as a human plasminogen receptor. OBJECTIVES: The study aimed to purify Salmonella enterica serovar Typhimurium cytosolic enolase and obtain the antibodies against this protein; to identify enolase on the surface of bacteria; and to find cross-reactivity and plasminogen binding properties. MATERIAL AND METHODS: Cytosolic enolase from S. Typhimurium was purified using a five-step preparation method. Anti-cytosolic enolase antibodies combined with scanning electron microscopy (SEM) allowed us to detect enolase on the surface of intact S. Typhimurium cells. The binding of plasminogen to surface enolase and the cross-reactivity of this protein with antibodies against human enolases were tested with western blot. RESULTS: Antibodies against human α- and ß-enolases cross-reacted with S. Typhimurium membrane protein, the identity of which was further confirmed using a mass spectrometry analysis of enolase tryptic peptides. The enolase form bacterial membrane also bound plasminogen. CONCLUSIONS: The cross-reactivity of membrane enolase with antibodies against human enolases suggests that this bacterium shares epitopes with human proteins. Surface exposition of enolase and the demonstrated affinity for human plasminogen indicates that Salmonella membrane enolase could play a role in the interaction of S. Typhimurium with host cells.


Assuntos
Plasminogênio , Salmonella typhimurium , Proteínas de Transporte , Humanos , Proteínas de Membrana , Fosfopiruvato Hidratase/metabolismo , Plasminogênio/metabolismo , Salmonella typhimurium/enzimologia
7.
BMC Vet Res ; 15(1): 312, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477105

RESUMO

BACKGROUND: Salmonella is generally considered as a human pathogen causing typhoid fever and gastrointestinal infections called salmonellosis, with S. Enteritidis and S. Typhimurium strains as the main causative agents. Salmonella enterica strains have a wide host array including humans, birds, pigs, horses, dogs, cats, reptiles, amphibians and insects. Up to 90% of reptiles are the carriers of one or more serovars of Salmonella. Extraintestinal bacterial infections associated with reptiles pose serious health threat to humans. The import of exotic species of reptiles as pet animals to Europe correlates with the emergence of Salmonella serotypes, which not found previously in European countries. The presented study is a new report about Salmonella serotypes associated with exotic reptiles in Poland. The goal of this research was to examine the zoonotic potential of Salmonella strains isolated from reptiles by comparative analysis with S. Enteritidis strains occurring in human population and causing salmonellosis. RESULTS: The main findings of our work show that exotic reptiles are asymptomatic carriers of Salmonella serovars other than correlated with salmonellosis in humans (S. Enteritidis, S. Typhimurium). Among the isolated Salmonella strains we identified serovars that have not been reported earlier in Poland, for example belonging to subspecies diarizonae and salamae. Restriction analysis with Pulsed-field Gel Electrophoresis (PFGE), showed a great diversity among Salmonella strains isolated from reptiles. Almost all tested strains had distinct restriction patterns. While S. Enteritidis strains were quite homogeneous in term of phylogenetic relations. Most of the tested VGs were common for the two tested groups of Salmonella strains. CONCLUSIONS: The obtained results show that Salmonella strains isolated from reptiles share most of virulence genes with the S. Enteritidis strains and exhibit a greater phylogenetic diversity than the tested S. Enteritidis population.


Assuntos
Eletroforese em Gel de Campo Pulsado , Répteis/microbiologia , Infecções por Salmonella/microbiologia , Salmonella enterica/genética , Animais , Portador Sadio , Cromatografia Gasosa , DNA Bacteriano , Genótipo , Humanos , Salmonella enterica/patogenicidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Virulência , Zoonoses
8.
Curr Med Chem ; 26(11): 1960-1978, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30378478

RESUMO

Salmonellosis continues to be a significant worldwide health problem. Despite rapid progress in identifying mechanisms of Salmonella virulence and resistance to chemicals, our knowledge of these mechanisms remains limited. Furthermore, it appears that the resistance to antibiotics can be amplified by ubiquitous usage of the disinfectants (biocides), both by industry and by ordinary households. Salmonella, as other Gram-negative bacteria possess outer membrane proteins (OMPs), which participate in maintaining cell integrity, adapting to environment, and interacting with infected host. Moreover, the OMPs may also contribute to resistance to antibacterials. This review summarizes the role of OMPs in Salmonella serum resistance, antibiotics resistance and cross-resistance to biocides. Although collected data do not allow to assign OMPs as markers of the Salmonella susceptibility to the above-mentioned factors, some of these proteins retain a dominant presence in certain types of resistance.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Farmacorresistência Bacteriana/fisiologia , Salmonella/metabolismo , Antibacterianos/efeitos adversos , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Biomarcadores , Desinfetantes/efeitos adversos , Desinfetantes/farmacologia , Farmacorresistência Bacteriana/genética , Humanos , Salmonella/genética , Salmonella/imunologia
9.
PLoS One ; 13(10): e0205205, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30356274

RESUMO

Herein we present an innovative method of coating the surface of Titanium-Aluminium-Niobium bone scaffold implants with bacterial cellulose (BC) polymer saturated with antibiotic. Customized Ti6Al7Nb scaffolds manufactured using Selective Laser Melting were immersed in a suspension of Komagataeibacter xylinus bacteria which displays an ability to produce a 3-dimensional structure of bio-cellulose polymer. The process of complete implant coating with BC took on average 7 days. Subsequently, the BC matrix was cleansed by means of alkaline lysis and saturated with gentamycin. Scanning electron microscopy revealed that BC adheres and penetrates into the implant scaffold structure. The viability and development of the cellular layer on BC micro-structure were visualized by means of confocal microscopy. The BC-coated implants displayed a significantly lower cytotoxicity against osteoblast and fibroblast cell cultures in vitro in comparison to non-coated implants. It was also noted that gentamycin released from BC-coated implants inhibited the growth of Staphylococcus aureus cultures in vitro, confirming the suitability of such implant modification for preventing hostile microbial colonization. As demonstrated using digital microscopy, the procedure used for implant coating and BC chemical cleansing did not flaw the biomaterial structure. The results presented herein are of high translational value with regard to future use of customized, BC-coated and antibiotic-saturated implants designed for use in orthopedic applications to speed up recovery and to reduce the risk of musculoskeletal infections.


Assuntos
Celulose/química , Materiais Revestidos Biocompatíveis/química , Gentamicinas/química , Titânio/química , Antibacterianos/química , Antibacterianos/uso terapêutico , Desenvolvimento Ósseo/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/microbiologia , Celulose/uso terapêutico , Materiais Revestidos Biocompatíveis/uso terapêutico , Humanos , Osteoblastos/química , Próteses e Implantes/microbiologia , Staphylococcus aureus , Tecidos Suporte , Titânio/uso terapêutico
10.
Int J Mol Sci ; 18(10)2017 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-28934165

RESUMO

Complement is one of the most important parts of the innate immune system. Some bacteria can gain resistance against the bactericidal action of complement by decorating their outer cell surface with lipopolysaccharides (LPSs) containing a very long O-antigen or with specific outer membrane proteins. Additionally, the presence of sialic acid in the LPS molecules can provide a level of protection for bacteria, likening them to human cells, a phenomenon known as molecular mimicry. Salmonella O48, which contains sialic acid in the O-antigen, is the major cause of reptile-associated salmonellosis, a worldwide public health problem. In this study, we tested the effect of prolonged exposure to human serum on strains from Salmonella serogroup O48, specifically on the O-antigen length. After multiple passages in serum, three out of four tested strains became resistant to serum action. The gas-liquid chromatography/tandem mass spectrometry analysis showed that, for most of the strains, the average length of the LPS O-antigen increased. Thus, we have discovered a link between the resistance of bacterial cells to serum and the elongation of the LPS O-antigen.


Assuntos
Complemento C3/farmacologia , Farmacorresistência Bacteriana/imunologia , Interações Hospedeiro-Patógeno/imunologia , Ácido N-Acetilneuramínico/química , Antígenos O/química , Salmonella/efeitos dos fármacos , Ativação do Complemento , Complemento C3/química , Meios de Cultura/química , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Viabilidade Microbiana , Mimetismo Molecular , Ácido N-Acetilneuramínico/imunologia , Antígenos O/imunologia , Salmonella/imunologia , Soro/química , Soro/imunologia
11.
Int J Mol Sci ; 18(7)2017 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-28696348

RESUMO

A new emerging phenomenon is the association between the incorrect use of biocides in the process of disinfection in farms and the emergence of cross-resistance in Salmonella populations. Adaptation of the microorganisms to the sub-inhibitory concentrations of the disinfectants is not clear, but may result in an increase of sensitivity or resistance to antibiotics, depending on the biocide used and the challenged Salmonella serovar. Exposure of five Salmonella enterica subsp. enterica serovar Senftenberg (S. Senftenberg) strains to triamine-containing disinfectant did not result in variants with resistance to antibiotics, but has changed their susceptibility to normal human serum (NHS). Three biocide variants developed reduced sensitivity to NHS in comparison to the sensitive parental strains, while two isolates lost their resistance to serum. For S. Senftenberg, which exhibited the highest triamine tolerance (6 × MIC) and intrinsic sensitivity to 22.5% and 45% NHS, a downregulation of flagellin and enolase has been demonstrated, which might suggest a lower adhesion and virulence of the bacteria. This is the first report demonstrating the influence of biocide tolerance on NHS resistance. In conclusion, there was a potential in S. Senftenberg to adjust to the conditions, where the biocide containing triamine was present. However, the adaptation did not result in the increase of antibiotic resistance, but manifested in changes within outer membrane proteins' patterns. The strategy of bacterial membrane proteins' analysis provides an opportunity to adjust the ways of infection treatments, especially when it is connected to the life-threating bacteremia caused by Salmonella species.


Assuntos
Anti-Infecciosos/farmacologia , Proteínas de Bactérias/metabolismo , Desinfetantes/farmacologia , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/metabolismo , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Humanos , Testes de Sensibilidade Microbiana
12.
PLoS One ; 11(10): e0164069, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27695090

RESUMO

Differential analysis of outer membrane composition of S. Enteritidis strains, resistant to 50% normal human serum (NHS) was performed in order to find factors influencing the resistance to higher concentrations of NHS. Ten S. Enteritidis clinical strains, resistant to 50% NHS, all producing very long lipopolysaccharide, were subjected to the challenge of 75% NHS. Five extreme strains: two resistant and three sensitive to 75% NHS, were chosen for the further analysis of outer membrane proteins composition. Substantial differences were found in the levels of particular outer membrane proteins between resistant and sensitive strains, i.e. outer membrane protease E (PgtE) was present mainly in resistant strains, while sensitive strains possessed a high level of flagellar hook-associated protein 2 (FliD) and significantly higher levels of outer membrane protein A (OmpA).


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Soros Imunes/imunologia , Proteoma , Proteômica , Salmonella enteritidis/imunologia , Salmonella enteritidis/metabolismo , Complemento C3/imunologia , Feminino , Humanos , Lipopolissacarídeos/imunologia , Masculino , Viabilidade Microbiana/imunologia , Testes de Neutralização , Estabilidade Proteica , Proteômica/métodos , Temperatura
13.
Postepy Hig Med Dosw (Online) ; 70(0): 610-7, 2016 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-27333931

RESUMO

The presented study is to analyze the participation of outer membrane proteins of Gram- negative bacteria in sensitivity to silver nanomaterials. The mechanism of interaction of silver with the bacterial cell is best described in this group of microorganisms. There are several theories regarding the effectiveness of antimicrobial ions and nanosilver, and at the indicated differences in the way they work. Outer membrane proteins of Gram-negative bacteria are involved in the procurement of silver from the environment and contribute to the development mechanisms of resistance to nanometals. They are measurable parameter in the field of cell phenotypic response to the presence of Gram-negative bacteria in the environment silver nanoforms: its properties, chemical composition, content or times of action. Proteomic methods (including two dimensional electrophoresis and MALDI­TOF MS) are therefore relevant techniques for determining the susceptibility of bacteria to silver and the changes taking place in the outer membrane under the influence: uptime/exposure and physical and chemical parameters of silver nanomaterials. Many products containing nanosilver is still in the research phase in terms of physico­chemical characteristics and biological activity, others have been already implemented in many industries. During the very fast nanotechnology developing and introduction to the market products based on the nanosilver the bacterial answer to nanosilver is needed.


Assuntos
Anti-Infecciosos/uso terapêutico , Infecções Bacterianas/tratamento farmacológico , Bactérias Gram-Negativas/efeitos dos fármacos , Proteínas de Membrana/efeitos dos fármacos , Nanoestruturas/uso terapêutico , Prata/uso terapêutico
14.
Curr Microbiol ; 72(5): 570-82, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26804795

RESUMO

In diagnostic microbiology as well as in microbiological research, the identification of a microorganism is a crucial and decisive stage. A broad choice of methods is available, based on both phenotypic and molecular properties of microbes. The aim of this study was to compare the application of phenotypic and molecular tools in bacterial identification on the example of Gram-negative intestine rod with an ambiguous phenotype. Different methods of identification procedure, which based on various properties of bacteria, were applied, e.g., microscopic observation of single-bacterial cells, macroscopic observation of bacterial colonies morphology, the automated system of microorganism identification (biochemical tests), the mass spectrometry method (analysis of bacterial proteome), and genetic analysis with PCR reactions. The obtained results revealed discrepancies in the identification of the tested bacterial strain with an atypical phenotype: mucous morphology of colonies, not characteristic for either E. coli and Citrobacter spp., mass spectrometry analysis of proteome initially assigned the tested strain to Citrobacter genus (C. freundii) and biochemical profiles pointed to Escherichia coli. A decisive method in the current study was genetic analysis with PCR reactions which identified conserved genetic sequences highly specific to E. coli species in the genome of the tested strain.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/microbiologia , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Técnicas de Tipagem Bacteriana/instrumentação , Fezes/microbiologia , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/diagnóstico , Humanos , Fenótipo
15.
PLoS One ; 8(11): e79525, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24260242

RESUMO

The EnvZ/OmpR two-component system constitutes a regulatory pathway involved in bacterial adaptive responses to environmental cues. Our previous findings indicated that the OmpR regulator in Yersinia enterocolitica O:9 positively regulates the expression of FlhDC, the master flagellar activator, which influences adhesion/invasion properties and biofilm formation. Here we show that a strain lacking OmpR grown at 37°C exhibits extremely high resistance to the bactericidal activity of normal human serum (NHS) compared with the wild-type strain. Analysis of OMP expression in the ompR mutant revealed that OmpR reciprocally regulates Ail and OmpX, two homologous OMPs of Y. enterocolitica, without causing significant changes in the level of YadA, the major serum resistance factor. Analysis of mutants in individual genes belonging to the OmpR regulon (ail, ompX, ompC and flhDC) and strains lacking plasmid pYV, expressing YadA, demonstrated the contribution of the respective proteins to serum resistance. We show that Ail and OmpC act in an opposite way to the OmpX protein to confer serum resistance to the wild-type strain, but are not responsible for the high resistance of the ompR mutant. The serum resistance phenotype of ompR seems to be multifactorial and mainly attributable to alterations that potentiate the function of YadA. Our results indicate that a decreased level of FlhDC in the ompR mutant cells is partly responsible for the serum resistance and this effect can be suppressed by overexpression of flhDC in trans. The observation that the loss of FlhDC enhances the survival of wild-type cells in NHS supports the involvement of FlhDC regulator in this phenotype. In addition, the ompR mutant exhibited a lower level of LPS, but this was not correlated with changes in the level of FlhDC. We propose that OmpR might alter the susceptibility of Y. enterocolitica O:9 to complement-mediated killing through remodeling of the outer membrane.


Assuntos
Proteínas de Bactérias/metabolismo , Yersinia enterocolitica/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Yersinia enterocolitica/genética
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