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1.
J Med Microbiol ; 69(9): 1151-1168, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32840477

RESUMO

Introduction. Enterococcus faecalis is a facultative, anaerobic, opportunistic pathogen associated with medical and dental diseases. Bacterial phenotypic traits and pathogenesis are often influenced by lysogeny.Aim. The aim of this study was to characterize both the morphology and complete genome sequences of induced prophages purified from E. faecalis clinical isolates.Methodology. E. faecalis isolates were recovered from the roots of teeth of patients attending an endodontic clinic. The morphological features of isolated phage were characterized using transmission electron microscopy (TEM). DNA sequencing was performed using the Illumina MiSeq platform.Results. TEM indicated that the isolated φEf-vB1 prophage belongs to the family Siphoviridae. The φEf-vB1 prophage was stable over a wide range of temperatures and pH. Sequencing of φEf-vB1 DNA revealed that the phage genome is 37 561 bp in length with a G+C content of 37.6mol% and contained 53 ORFs. Comparison with previously predicted prophage genomes using blast revealed that φEf-vB1 has a high sequence similarity to previously characterized phage genomes. The lysogenic E. faecalis strain exhibited a higher biofilm formation capacity relative to the non-lysogenic strain.Conclusion. The current findings highlight the role of lysogeny in modification of E. faecalis properties and reveal the potential importance of prophages in E. faecalis biology and pathogenesis.


Assuntos
Bacteriófagos/fisiologia , Enterococcus faecalis/fisiologia , Enterococcus faecalis/virologia , Prófagos/fisiologia , Siphoviridae/isolamento & purificação , Composição de Bases , Cavidade Pulpar/microbiologia , Enterococcus faecalis/genética , Enterococcus faecalis/isolamento & purificação , Genoma Viral , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Lisogenia , Fases de Leitura Aberta , Periodontite , Prófagos/classificação , Prófagos/genética , Prófagos/isolamento & purificação , Siphoviridae/classificação , Siphoviridae/genética , Siphoviridae/fisiologia
2.
PLoS Biol ; 18(8): e3000814, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32797039

RESUMO

Plasmid-mediated horizontal gene transfer of antibiotic resistance and virulence in pathogenic bacteria underlies a major public health issue. Understanding how, in the absence of antibiotic-mediated selection, plasmid-bearing cells avoid being outnumbered by plasmid-free cells is key to developing counterstrategies. Here, we quantified the induction of the plasmidial sex pheromone pathway of Enterococcus faecalis to show that the integration of the stimulatory (mate-sensing) and inhibitory (self-sensing) signaling modules from the pCF10 conjugative plasmid provides a precise measure of the recipient-to-donor ratio, agnostic to variations in population size. Such ratiometric control of conjugation favors vertical plasmid transfer under low mating likelihood and allows activation of conjugation functions only under high mating likelihood. We further show that this strategy constitutes a cost-effective investment into mating effort because overstimulation produces unproductive self-aggregation and growth rate reduction. A mathematical model suggests that ratiometric control of conjugation increases plasmid fitness and predicts a robust long-term, stable coexistence of donors and recipients. Our results demonstrate how population-level parameters can control transfer of antibiotic resistance in bacteria, opening the door for biotic control strategies.


Assuntos
Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Enterococcus faecalis/genética , Transferência Genética Horizontal , Feromônios/genética , Percepção de Quorum/efeitos dos fármacos , Antibacterianos/farmacologia , Carga Bacteriana , Proteínas de Bactérias/metabolismo , Conjugação Genética , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/metabolismo , Expressão Gênica , Aptidão Genética , Modelos Estatísticos , Feromônios/biossíntese , Plasmídeos/química , Plasmídeos/metabolismo , Percepção de Quorum/genética , Virulência
3.
BMC Infect Dis ; 20(1): 467, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32615925

RESUMO

BACKGROUND: Urinary tract infection (UTI) caused by various pathogenic microorganisms is ubiquitous in the parts of the urinary system such as kidney, ureter, bladder, and urethra. Currently, clinical detection of UTI is mainly focused on urine culture; however, the diagnostic value of urine culture remains limited due to the time-consuming procedure and low detection rate, especially in patients who have used antibiotics. Generally, treatment for UTI relies on empirical medication rather than pathogen diagnosis, which leads to the inappropriate use of antimicrobial agents and a significant increase in resistant strains. Comparatively, metagenomic next-generation sequencing (mNGS) is capable of overcoming the disadvantages of clinical culture, and identifying pathogens for further treatment. CASE PRESENTATION: A 33-year-old male patient was admitted to hospital with a high fever and chills. None of his autoimmune disease or thyroid function related indicators were positive, and he had no risk of endocarditis. His white blood cell count, C-reactive protein, procalcitonin, interleukin 6, and neutrophil proportion were markedly elevated. He was initially diagnosed as having an infection of unknown etiology. Since empirical treatment of Sulperazon and Metronidazole did not relieve his symptoms, both the blood and urine specimens were examined using traditional culture, serological testing, and mNGS assay. Traditional culture and serological testing produced negative results, while the mNGS assay revealed the presence of a potential pathogen, Enterococcus faecalis, in the urine specimen, which was further confirmed by both Sanger sequencing and qPCR analysis. A CT scan of the patient's whole abdomen showed stones in the right kidney. Once targeted antibiotic therapy was administered, the patient recovered quickly. CONCLUSIONS: Our case illustrated that mNGS, as a novel culture-independent approach, demonstrated the capability of rapid, sensitive, and accurate pathogen identification. Furthermore, this technology provides strong support for guiding clinicians to determine appropriate treatment.


Assuntos
Enterococcus faecalis/genética , Infecções por Bactérias Gram-Positivas/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/métodos , Infecções Urinárias/diagnóstico , Adulto , Antibacterianos/uso terapêutico , DNA Bacteriano/genética , Seguimentos , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/urina , Humanos , Linezolida/uso terapêutico , Masculino , Resultado do Tratamento , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/urina , Infecções Urinárias/virologia
4.
BMC Infect Dis ; 20(1): 356, 2020 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-32517758

RESUMO

BACKGROUND: Vancomycin-resistant Enterococcus spp. (VRE) have spread all over the world. The present study aims to investigate the species distribution, specimen type and susceptibilities of Enterococcal species collected from Nanjing Drum Tower Hospital from 2013 to 2018. Additionally, distribution of VRE and prevalence of van gene among VRE isolates were also analyzed. METHODS: The susceptibilities of 3913 Enterococcus isolates were retrospectively investigated. Among these strains, 60 VRE strains were further anazlyed in this study. The minimum inhibitory concentrations (MICs) of the VRE strains towards vancomycin, teicoplanin and linezolid were determined by E-test. Polymerase chain reaction (PCR) and DNA sequencing were used to investigate the prevalence of van genes among VRE. Furthermore, the sequence types (STs) of VRE strains were explored by multi-locus sequence typing (MLST). RESULTS: Among the 3913 enterococci isolates, Enterococcus faecalis (n = 1870, 47.8%) and Enterococcus faecium (1738, 44.4%) were the main isolates. These Enterococcus strains were mainly isolated from urine (n = 1673, 42.8%), followed by secretions (n = 583, 14.9%) and ascites (n = 554, 14.2%). VRE displayed a decreasing trend year by year. Molecular analysis revealed that 49 out of 60 VRE isolates carried vanA gene, 10 carried vanM, and 1 carried both vanA and vanM genes. Sixteen distinct STs were identified among the 58 VREM, with ST78 (n = 16), ST192 (n = 8) and ST570 (n = 7) being the most dominant ones. CONCLUSIONS: E. faecalis and E. faecium were the major enterococci strains which are the main pathogens of urinary traction infections; vanA and vanM were the main determinants conferring resistance to vancomycin; ST78, ST192 and ST570 were the leading STs of VREM which displayed a decreasing trend of prevalence year by year.


Assuntos
Enterococcus/isolamento & purificação , Infecções por Bactérias Gram-Positivas/microbiologia , Enterococos Resistentes à Vancomicina/isolamento & purificação , Antibacterianos/farmacologia , China , DNA Bacteriano/metabolismo , Enterococcus/efeitos dos fármacos , Enterococcus/genética , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/genética , Enterococcus faecalis/isolamento & purificação , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/genética , Enterococcus faecium/isolamento & purificação , Infecções por Bactérias Gram-Positivas/diagnóstico , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Estudos Retrospectivos , Centros de Atenção Terciária , Vancomicina/farmacologia , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Enterococos Resistentes à Vancomicina/genética
5.
Nat Struct Mol Biol ; 27(5): 489-499, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32367067

RESUMO

Cas1 integrase associates with Cas2 to insert short DNA fragments into a CRISPR array, establishing nucleic acid memory in prokaryotes. Here we applied single-molecule FRET methods to the Enterococcus faecalis (Efa) Cas1-Cas2 system to establish a kinetic framework describing target-searching, integration, and post-synapsis events. EfaCas1-Cas2 on its own is not able to find the CRISPR repeat in the CRISPR array; it only does so after prespacer loading. The leader sequence adjacent to the repeat further stabilizes EfaCas1-Cas2 contacts, enabling leader-side integration and subsequent spacer-side integration. The resulting post-synaptic complex (PSC) has a surprisingly short mean lifetime. Remarkably, transcription effectively resolves the PSC, and we predict that this is a conserved mechanism that ensures efficient and directional spacer integration in many CRISPR systems. Overall, our study provides a complete model of spacer acquisition, which can be harnessed for DNA-based information storage and cell lineage tracing technologies.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Enterococcus faecalis/enzimologia , Integrases/metabolismo , Eletroporação , Enterococcus faecalis/genética , Escherichia coli/genética , Transferência Ressonante de Energia de Fluorescência , Integrases/genética , Cinética , Microrganismos Geneticamente Modificados , Mutação , Transcrição Genética
6.
Poult Sci ; 99(5): 2675-2683, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32359604

RESUMO

Enterococcus faecalis (E. faecalis) has rapidly acquired resistance to multiple antimicrobials, and the antimicrobial resistance of E. faecalis from broiler breeders has been implicated in its vertical transmission to their offspring. The objective of this study was to investigate the antimicrobial resistance and genetic diversity of commensal E. faecalis isolated from the broiler breeder farms. Among a total of 229 E. faecalis isolates from 9 broiler breeder farms, the highest resistance rate was observed in tetracycline (78.2%), followed by doxycycline (58.1%) and erythromycin (43.7%), and the prevalence of antimicrobial resistance showed significant differences among the 9 broiler breeder farms (P < 0.05). The tetM gene (77.1%) and ermB gene (85.0%) were detected at the highest levels in 179 TE-and 100 E-resistant isolates, respectively. Twenty-four high-level gentamicin-resistant isolates carried aac(6″)Ie-aph(2″)-la gene, and 9 high-level ciprofloxacin-resistant isolates showed point mutations in both gyrA and parC genes. All high-level gentamicin-resistant or high-level ciprofloxacin-resistant isolates showed one of the two different virulence gene patterns, ace-asa1-efaA-gelE complex or ace-efaA-gelE complex. These results indicate that constant epidemiological monitoring at the breeder level is required to prevent the pyramidal transmission of antimicrobial-resistant E. faecalis.


Assuntos
Antibacterianos/farmacologia , Galinhas , Farmacorresistência Bacteriana/genética , Enterococcus faecalis/efeitos dos fármacos , Variação Genética , Infecções por Bactérias Gram-Positivas/veterinária , Doenças das Aves Domésticas/epidemiologia , Animais , Farmacorresistência Bacteriana Múltipla/genética , Enterococcus faecalis/genética , Enterococcus faecalis/fisiologia , Infecções por Bactérias Gram-Positivas/epidemiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Doenças das Aves Domésticas/microbiologia , Prevalência , República da Coreia/epidemiologia
7.
Lett Appl Microbiol ; 71(1): 39-45, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32390273

RESUMO

Filter feeding is a biotic process that brings waterborne bacteria in close contact with each other and may thus support the horizontal transfer of their antimicrobial resistance genes. This laboratory study investigated whether the freshwater sponge Ephydatia fluviatilis supported the transfer of vancomycin resistance between two Enterococcus faecalis strains that we previously demonstrated to exhibit pheromone responsive plasmid conjugation. Microcosm experiments exposed live and dead colonies of laboratory-grown sponges to a vancomycin-resistant donor strain and a rifampicin-resistant recipient strain of Ent. faecalis. Enterococci with both resistance phenotypes were detected on double selection plates. In comparison to controls, abundance of these presumed transconjugants increased significantly in water from sponge microcosms. Homogenized suspensions of sponge cells also yielded presumed transconjugants; however, there was no significant difference between samples from live or dead sponges. Fluorescent in situ hybridization analysis of the sponge cell matrix using species-specific probes revealed the presence of enterococci clusters with cells adjacent to each other. The results demonstrated that sponge colonies can support the horizontal transfer of antimicrobial resistance although the mechanism underlying this process, such as binding of the bacteria to the sponge collagen matrix, has yet to be fully elucidated.


Assuntos
Antibacterianos/farmacologia , Conjugação Genética/genética , Farmacorresistência Bacteriana/genética , Enterococcus faecalis/genética , Poríferos/microbiologia , Resistência a Vancomicina/genética , Animais , Enterococcus faecalis/efeitos dos fármacos , Água Doce , Hibridização in Situ Fluorescente , Feromônios/farmacologia , Plasmídeos/genética , Vancomicina/farmacologia
8.
PLoS One ; 15(4): e0232165, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32343730

RESUMO

We have recently demonstrated that collagenolytic Enterococcus faecalis plays a key and causative role in the pathogenesis of anastomotic leak, an uncommon but potentially lethal complication characterized by disruption of the intestinal wound following segmental removal of the colon (resection) and its reconnection (anastomosis). Here we hypothesized that comparative genetic analysis of E. faecalis isolates present at the anastomotic wound site before and after surgery would shed insight into the mechanisms by which collagenolytic strains are selected for and predominate at sites of anastomotic disruption. Whole genome optical mapping of four pairs of isolates from rat colonic tissue obtained following surgical resection (herein named "pre-op" isolates) and then 6 days later from the anastomotic site (herein named "post-op" isolates) demonstrated that the isolates with higher collagenolytic activity formed a distinct cluster. In order to perform analysis at a deeper level, a single pair of E. faecalis isolates (16A pre-op and 16A post-op) was selected for whole genome sequencing and assembled using a hybrid assembly algorithm. Comparative genomics demonstrated absence of multiple gene clusters, notably a pathogenicity island in the post-op isolate. No differences were found in the fsr-gelE-sprE genes (EF1817-1822) responsible for regulation and production of collagenolytic activity. Analysis of unique genes among the 16A pre-op and post-op isolates revealed the predominance of transporter systems-related genes in the pre-op isolate and phage-related and hydrolytic enzyme-encoding genes in the post-op isolate. Despite genetic differences observed between pre-op and post-op isolates, the precise genetic determinants responsible for their differential expression of collagenolytic activity remains unknown.


Assuntos
Anastomose Cirúrgica , Colo/cirurgia , Enterococcus faecalis/genética , Anastomose Cirúrgica/efeitos adversos , Fístula Anastomótica/etiologia , Fístula Anastomótica/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mapeamento Cromossômico , Colagenases/genética , Colagenases/metabolismo , Enterococcus faecalis/enzimologia , Enterococcus faecalis/isolamento & purificação , Microbioma Gastrointestinal/genética , Genoma Bacteriano , Intestinos/microbiologia , Ratos , Virulência/genética
9.
Gene ; 748: 144704, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32339624

RESUMO

Resistance to antibiotics have created havoc around the globe due to the emergence of multi-drug resistant (MDR) pathogenic bacterial strains. To decipher this problem, a detailed understanding of the antimicrobial resistance (AMR) genes and their resistant mechanisms are obligatory. The present study is mainly focused on an opportunistic, nosocomial bacterial strain Enterococcus faecalis V583, which possess acquired exogenous AMR genes portraying resistance against Chloramphenicol, Tetracycline, Vancomycin, Linezolid, Ampicillin and other antibiotics. An interaction network of eight AMR genes along with 40 functional partners have been constructed and analysed. Functional enrichment analysis highlighted 20, 21 and 22 genes having significant roles in Cellular Component (CC), Molecular Functions (MF) and Biological Process (BP) respectively. Clustering analysis resulted in four densely interconnected clusters (C1-C4) which were associated with three AMR mechanisms that include the alteration in drug target (pbps, mur and van genes), complete replacement/bypass of target sites (van genes) and ATP Binding Cassette (ABC) transporter efflux pump mechanisms (msrA, EF_1680, EF_1682 and pbps). Our results showed that the genes responsible for ß-lactams resistance (pbp1A, 1C, 2A, 2B); glycopeptide resistance (ddl, vanBHBRBSBWXYB); Erythromycin, Macrolides, Lincosamide and Streptogramin-B (MLSB) resistance (msrA, EF_1680, EF_1682) along with mur genes (murABBCDEFG) played an important role in MDR mechanisms. Network analysis has shown the genes mraY, pbpC, murE, murG and murD possessed 26, 24, 23, 22 and 22 interactions respectively. With more number of direct interactions, these genes can be considered as hub genes that could be exploited as potential drug targets for new drug discovery.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Enterococcus faecalis/efeitos dos fármacos , Redes Reguladoras de Genes , Enterococcus faecalis/genética , Genes Bacterianos
10.
PLoS Pathog ; 16(4): e1008310, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32240270

RESUMO

Enterococci are robust gram-positive bacteria that are found in a variety of surroundings and that cause a significant number of healthcare-associated infections. The genus possesses a high-efficiency pheromone-responsive plasmid (PRP) transfer system for genetic exchange that allows antimicrobial-resistance determinants to spread within bacterial populations. The pCF10 plasmid system is the best characterised, and although other PRP systems are structurally similar, they lack exact functional homologues of pCF10-encoded genes. In this review, we provide an overview of the enterococcal PRP systems, incorporating functional details for the less-well-defined systems. We catalogue the virulence-associated elements of the PRPs that have been identified to date, and we argue that this reinforces the requirement for elucidation of the less studied systems.


Assuntos
Proteínas de Bactérias/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Enterococcus faecalis/genética , Infecções por Bactérias Gram-Positivas/microbiologia , Feromônios/fisiologia , Plasmídeos/genética , Animais , Conjugação Genética , Humanos , Virulência
11.
PLoS Pathog ; 16(3): e1008278, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32119717

RESUMO

Antibiotic combinations are increasingly used to combat bacterial infections. Multidrug therapies are a particularly important treatment option for E. faecalis, an opportunistic pathogen that contributes to high-inoculum infections such as infective endocarditis. While numerous synergistic drug combinations for E. faecalis have been identified, much less is known about how different combinations impact the rate of resistance evolution. In this work, we use high-throughput laboratory evolution experiments to quantify adaptation in growth rate and drug resistance of E. faecalis exposed to drug combinations exhibiting different classes of interactions, ranging from synergistic to suppressive. We identify a wide range of evolutionary behavior, including both increased and decreased rates of growth adaptation, depending on the specific interplay between drug interaction and drug resistance profiles. For example, selection in a dual ß-lactam combination leads to accelerated growth adaptation compared to selection with the individual drugs, even though the resulting resistance profiles are nearly identical. On the other hand, populations evolved in an aminoglycoside and ß-lactam combination exhibit decreased growth adaptation and resistant profiles that depend on the specific drug concentrations. We show that the main qualitative features of these evolutionary trajectories can be explained by simple rescaling arguments that correspond to geometric transformations of the two-drug growth response surfaces measured in ancestral cells. The analysis also reveals multiple examples where resistance profiles selected by drug combinations are nearly growth-optimized along a contour connecting profiles selected by the component drugs. Our results highlight trade-offs between drug interactions and resistance profiles during the evolution of multi-drug resistance and emphasize evolutionary benefits and disadvantages of particular drug pairs targeting enterococci.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Enterococcus faecalis/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/microbiologia , Adaptação Fisiológica , Evolução Biológica , Interações Medicamentosas , Enterococcus faecalis/genética , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/fisiologia , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Humanos , Testes de Sensibilidade Microbiana
12.
Sci Rep ; 10(1): 3937, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32127598

RESUMO

For a One-Health investigation of antimicrobial resistance (AMR) in Enterococcus spp., isolates from humans and beef cattle along with abattoirs, manured fields, natural streams, and wastewater from both urban and cattle feedlot sources were collected over two years. Species identification of Enterococcus revealed distinct associations across the continuum. Of the 8430 isolates collected, Enterococcus faecium and Enterococcus faecalis were the main species in urban wastewater (90%) and clinical human isolates (99%); Enterococcus hirae predominated in cattle (92%) and feedlot catch-basins (60%), whereas natural streams harbored environmental Enterococcus spp. Whole-genome sequencing of E. faecalis (n = 366 isolates) and E. faecium (n = 342 isolates), revealed source clustering of isolates, indicative of distinct adaptation to their respective environments. Phenotypic resistance to tetracyclines and macrolides encoded by tet(M) and erm(B) respectively, was prevalent among Enterococcus spp. regardless of source. For E. faecium from cattle, resistance to ß-lactams and quinolones was observed among 3% and 8% of isolates respectively, compared to 76% and 70% of human clinical isolates. Clinical vancomycin-resistant E. faecium exhibited high rates of multi-drug resistance, with resistance to all ß-lactam, macrolides, and quinolones tested. Differences in the AMR profiles among isolates reflected antimicrobial use practices in each sector of the One-Health continuum.


Assuntos
Antibacterianos/farmacologia , Enterococcus/patogenicidade , Farmacorresistência Bacteriana/genética , Enterococcus/efeitos dos fármacos , Enterococcus/genética , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/genética , Enterococcus faecalis/patogenicidade , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/genética , Enterococcus faecium/patogenicidade , Humanos , Macrolídeos/farmacologia , Filogenia , Quinolonas/farmacologia , Tetraciclinas/farmacologia , Virulência , Sequenciamento Completo do Genoma , Resistência beta-Lactâmica/genética
13.
Mikrobiyol Bul ; 54(1): 26-39, 2020 Jan.
Artigo em Turco | MEDLINE | ID: mdl-32050876

RESUMO

Enterococci, which are commonly found in the environment, cause serious infections despite the absence of well-defined virulence factors and toxins. Knowing the virulence properties of enterococci is important to understand the complex pathogenic structures. In this study, we aimed to investigate the virulence factors (asa1, hyl, cylA, efa, ebp, ace, esp, gelE, sprE, fsrA, fsrB, fsrC genes, gelatinase activity, hemolysin, hydrogen peroxide and biofilm production) and antibiotic resistance of Enterococcus faecium and Enterococcus faecalis strains isolated from clinical specimens. A total of 110 enterococcus isolates which were accepted as infectious agents were included in the study. The polymerase chain reaction method was used to identify the isolates and to detect virulence genes. Characteristics of hemolysis, biofilm formation, hydrogen peroxide production and gelatinase activity were investigated by phenotypic methods. The antibiotic susceptibility test was performed with VITEK 2 automated system. E.faecalis ATCC 29212 standard strain was used as a quality control in all tests. Of the 110 enterococci isolates included in the study, 61 were identified as E.faecium and 49 as E.faecalis. The efa gene was the most frequently detected virulence gene (92.7%), followed by ace (83.6%), esp (66.4%), ebp (60.0%), cylA (50.9%), hyl (46.4%), asa1 (45.5%), gelE, sprE, fsrC (33.6%), fsrA (12.7%) and fsrB (11.8%). All genes except hyl were higher in E.faecalis isolates and the difference was statistically significant (p<0.05). Twenty-five (51%) E.faecalis and 1 (1.6%) E.faecium isolates had beta-hemolysis and the difference was statistically significant (p= 0.000). Seven (11.5%) E.faecium and 4 (8.2%) E.faecalis isolates formed biofilm, but the difference was not statistically significant (p> 0.05). Two (3.3%) E.faecium and 14 (28.6%) E.faecalis isolates exhibited gelatinase activity and the difference between the two species was statistically significant (p= 0.000). Hydrogen peroxide production was not detected in any of the isolates. The highest resistance rate was determined against ciprofloxacin (70.9%). The resistance to ampicillin was 69.1%, high level streptomycin 65.1%, high level gentamicin 39.4%, vancomycin and teicoplanin 4.5%, and linezolid 1.8%. In conclusion, our data indicated that virulence factors except hyl gene and biofilm production were higher in E.faecalis isolates but E.faecium isolates were more resistant to antibiotics. In order to prevent infection of such virulent or resistant isolates in the hospital setting, infection control measures must be followed. In vivo studies are needed for the better understanding of the virulence of enterococci.


Assuntos
Enterococcus faecalis , Enterococcus faecium , Infecções por Bactérias Gram-Positivas , Fatores de Virulência , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/genética , Enterococcus faecalis/patogenicidade , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/genética , Enterococcus faecium/patogenicidade , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Fatores de Virulência/genética
14.
Nat Microbiol ; 5(4): 554-561, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32094585

RESUMO

Polyamines are essential metabolites that play an important role in cell growth, stress adaptation and microbial virulence1-3. To survive and multiply within a human host, pathogenic bacteria adjust the expression and activity of polyamine biosynthetic enzymes in response to different environmental stresses and metabolic cues2. Here, we show that ornithine capture by the ribosome and the nascent peptide SpeFL controls polyamine synthesis in γ-proteobacteria by inducing the expression of the ornithine decarboxylase SpeF4, via a mechanism involving ribosome stalling and transcription antitermination. In addition, we present the cryogenic electron microscopy structure of an Escherichia coli ribosome stalled during translation of speFL in the presence of ornithine. The structure shows how the ribosome and the SpeFL sensor domain form a highly selective binding pocket that accommodates a single ornithine molecule but excludes near-cognate ligands. Ornithine pre-associates with the ribosome and is then held in place by the sensor domain, leading to the compaction of the SpeFL effector domain and blocking the action of release factor 1. Thus, our study not only reveals basic strategies by which nascent peptides assist the ribosome in detecting a specific metabolite, but also provides a framework for assessing how ornithine promotes virulence in several human pathogens.


Assuntos
Proteínas de Bactérias/química , Escherichia coli/genética , Ornitina Descarboxilase/química , Ornitina/química , Ribossomos/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Modelos Moleculares , Ornitina/metabolismo , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Filogenia , Poliaminas/química , Poliaminas/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Domínios e Motivos de Interação entre Proteínas , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Thermus thermophilus/genética , Thermus thermophilus/metabolismo , Virulência
15.
Folia Microbiol (Praha) ; 65(1): 79-85, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31041600

RESUMO

In Slovakia, dairy products made from ewes' milk have a long tradition. These products include the lactic acid product called "zincica" which is a by-product occurring during the preparation of ewes' lump cheese. There is no information in the literature regarding the special properties of the microbiota, especially lactic acid Firmicutes, which can survive in "zincica." From the safety aspect, enterococci are a controversial group of bacteria, and those from "zincica" have never been tested for their properties. The "zincica" used in our study was supplied by several different agrofarms producing ewes' lump cheese in central Slovakia. The species Enterococcus faecium (strains EF30E1, EF32E1, EF34E1, EF34E5) and Enterococcus faecalis (strains EE30E4, EE35E1, E31E2, altogether 7) were detected in samples from "zincica" identified using MALDI-TOF spectrometry with secure genus identification/probable species identification and then confirmed by means of PCR. Enterococci were hemolysis-negative and the genes of the typical enterococcal virulence factors were mostly absent; the gelE gene was found in two E. faecium strains (EF30E1 and EF32E1), the agg gene was detected in E. faecalis EE35E1, and the esp gene was found in two E. faecalis strains (EE30E4 and EE31E2). No strains harbored the cytolysin A gene. Biofilm formation was detected in four strains (EF30E1, EF32E1, EF34E1, and EF34E5), indicating highly positive and low-grade positive biofilm formation. Enterococci were mostly susceptible to antibiotics tested for their phenotype. This is the first study to analyze enterococci in "zincica."


Assuntos
Queijo/microbiologia , Enterococcus/classificação , Microbiologia de Alimentos , Inocuidade dos Alimentos , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Enterococcus/efeitos dos fármacos , Enterococcus/patogenicidade , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/genética , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/genética , Feminino , Testes de Sensibilidade Microbiana , Microbiota , Ovinos , Eslováquia , Fatores de Virulência/genética
16.
Gene ; 726: 144197, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31669636

RESUMO

Enterococcus faecalis is one of the main components of symbiotic bacteria in the intestine of silkworm (Bombyx mori L.). The abundance of E. faecalis in the intestine of silkworm is affected by fluoride exposure. However, the response mechanism of E. faecalis toward fluoride remains largely unknown. In this study, a strain of E. faecalis (named TV4), which is a symbiotic bacteria of silkworm, was isolated and characterized. Inhibition assay showed that fluoride can significantly inhibit the growth of the TV4 strain (P < 0.05) after culture for 4 h. Finally, Illumina X-Ten platform was used to investigate the response mechanism of E. faecalis TV4 under fluoride exposure. We found that the TV4 strain demonstrated significant changes in its carbohydrate transport and metabolism and energy metabolism. The transcriptome sequencing results revealed that 237 genes were differentially expressed for TV4 grown after fluoride exposure, i.e., 92 genes were differentially up-regulated and 145 genes were differentially down-regulated. Many of the down-regulated genes were involved in cell carbohydrate transport and metabolism and energy production, whereas the up-regulated genes were mostly related to ethanolamine utilization and amino acid synthesis and metabolism. Our results revealed that strain TV4 reduced its carbohydrate metabolism and energy metabolism and increased ethanolamine utilization and amino acid metabolism to adapt and survive under fluoride exposure. This study enhances our understanding about the response mechanism of E. faecalis after fluoride exposure and has important implications for investigations on the three-way interaction among fluoride, symbiotic bacteria and silkworm.


Assuntos
Bombyx/microbiologia , Enterococcus faecalis/genética , Fluoretos/efeitos adversos , Animais , Regulação para Baixo/genética , Perfilação da Expressão Gênica/métodos , Intestinos/microbiologia , RNA-Seq , Transcriptoma/genética , Regulação para Cima/genética , Sequenciamento Completo do Exoma/métodos
17.
Mol Microbiol ; 113(2): 464-477, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31755602

RESUMO

Enterococci are gram-positive pathogens and lead to cause hospital-acquired infections worldwide. Central carbon metabolism was shown as highly induced in Enterococcus faecalis during infection context. Metabolism of α-polysaccharides was previously described as an important factor for host colonisation and biofilm formation. A better characterisation of the adaptation of this bacterium to carbohydrate availabilities may lead to a better understanding of the link between carbohydrate metabolism and the infection process of E. faecalis. Here we show that MalR, a LacI/GalR transcriptional regulator, is the main factor in the regulation of the two divergent operons involved in maltose metabolism in this bacterium. The malR gene is transcribed from the malP promoter, but also from an internal promoter inside the gene located upstream of malR. In the absence of maltose, MalR acts as a repressor and in the presence of glucose, it exerts efficient CcpA-independent carbon catabolite repression. The central PTS protein P-Ser-HPr interacts directly with MalR and enhances its DNA binding capacity, which allows E. faecalis to adapt its metabolism to environmental conditions.


Assuntos
Proteínas de Bactérias/metabolismo , Enterococcus faecalis/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Proteínas Repressoras/metabolismo , Metabolismo dos Carboidratos/fisiologia , Enterococcus faecalis/genética , Regulação Bacteriana da Expressão Gênica , Maltose/metabolismo , Óperon , Regiões Promotoras Genéticas
18.
J Biol Chem ; 295(7): 2136-2147, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31796629

RESUMO

PlsX plays a central role in the coordination of fatty acid and phospholipid biosynthesis in Gram-positive bacteria. PlsX is a peripheral membrane acyltransferase that catalyzes the conversion of acyl-ACP to acyl-phosphate, which is in turn utilized by the polytopic membrane acyltransferase PlsY on the pathway of bacterial phospholipid biosynthesis. We have recently studied the interaction between PlsX and membrane phospholipids in vivo and in vitro, and observed that membrane association is necessary for the efficient transfer of acyl-phosphate to PlsY. However, understanding the molecular basis of such a channeling mechanism remains a major challenge. Here, we disentangle the binding and insertion events of the enzyme to the membrane, and the subsequent catalysis. We show that PlsX membrane binding is a process mostly mediated by phospholipid charge, whereas fatty acid saturation and membrane fluidity remarkably influence the membrane insertion step. Strikingly, the PlsXL254E mutant, whose biological functionality was severely compromised in vivo but remains catalytically active in vitro, was able to superficially bind to phospholipid vesicles, nevertheless, it loses the insertion capacity, strongly supporting the importance of membrane insertion in acyl-phosphate delivery. We propose a mechanism in which membrane fluidity governs the insertion of PlsX and thus regulates the biosynthesis of phospholipids in Gram-positive bacteria. This model may be operational in other peripheral membrane proteins with an unprecedented impact in drug discovery/development strategies.


Assuntos
Proteínas de Bactérias/genética , Bactérias Gram-Positivas/genética , Fluidez de Membrana/genética , Fosfolipídeos/biossíntese , Bacillus subtilis/genética , Enterococcus faecalis/genética , Escherichia coli/genética , Fosfatos/metabolismo , Fosfolipídeos/genética
19.
Curr Microbiol ; 77(3): 369-387, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31832841

RESUMO

Bacteria isolated from different segments of the gastro-intestinal tract (GIT) of healthy free-range broilers were screened for probiotic properties. Six strains were selected and identified as Lactobacillus gallinarum, Lactobacillus johnsonii, Lactobacillus salivarius, Lactobacillus crispatus, Enterococcus faecalis and Bacillus amyloliquefaciens based on 16S rRNA, gyrB and recA gene sequence analyses. All six strains produced exopolysaccharides (EPS) and formed biofilms under conditions simulating the broiler GIT. Lactobacillus johnsonii DPN184 and L. salivarius DPN181 produced hydrogen peroxide, and L. crispatus DPN167 and E. faecalis DPN94 produced bile salt hydrolase (BSH) and phytase. Bacillus amyloliquefaciens DPN123 produced phytase, amylase, surfactin and iturin A1. No abnormalities were observed when broilers were fed the multi-strain combination, suggesting that it could be used as a probiotic.


Assuntos
Bacillus amyloliquefaciens/genética , Galinhas/microbiologia , Enterococcus faecalis/genética , Lactobacillus/genética , Probióticos/classificação , Animais , Bacillus amyloliquefaciens/enzimologia , Biofilmes/crescimento & desenvolvimento , Enterococcus faecalis/fisiologia , Lactobacillus/classificação , Lactobacillus/fisiologia , RNA Ribossômico 16S/genética
20.
Microb Pathog ; 139: 103907, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31811888

RESUMO

Enterococcus faecalis is one of the important causes of nosocomial infections. Nowadays, increasing prevalence of antibiotic-resistant bacteria and slow progress in recognizing new antimicrobial agents has limited the efficiency of conventional antibiotics, which cause to find novel strategies to overcome bacteria. Therefore, in this study, we aimed to assess the role of efaA gene in the biofilm formation and the role of ftsZ gene in the controlling of bacterial growth by the anti-sense PNAs(Peptide Nucleic Acid).E. faecalis ATCC® 29212™was used for the study of PNAs designed to targeting the start codon section of the ftsZ andefaA genes. PNA attachment to RNA was confirmed by blotting. Electroporation technique was used for the intracellular transfer of anti-ftsZ PNAs. The spot-plating method was used to the assessment of alteration in bacterial growth. Biofilm formation assay and real-time PCR were used for detection of biofilm inhibitory effect of cell penetrating peptide (CPP) conjugated to anti-efaA PNAs.ByftsZ PNAs treatment, no growth was seen from the strain in agar by a spot plating method and the inhibition zone of anti-ftsZ PNAs was not seen. PNAs against the efaA gene decreased by 95% the expression of the efaA gene and biofilm formation. In addition, the(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) MTT assay showed no toxicity on MCF7 cells for both of anti-ftsZand anti-efaA PNAs.This study used new genetic and molecular tools to inhibit pathogenicity and infection by E. faecalis. In this study, we suggested that efaA gene plays a critical role in the biofilm formation and anti-efaA PNAs could decrease the formation of biofilm, as well as, anti-ftsZ PNAs could eliminate bacterial growth.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Biofilmes , Proteínas do Citoesqueleto/genética , Enterococcus faecalis/genética , Ácidos Nucleicos Peptídicos/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/fisiologia , Regulação Bacteriana da Expressão Gênica
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