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1.
Curr Genet ; 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31925496

RESUMO

In all living organisms, genome replication and cell division must be coordinated to produce viable offspring. In the event of DNA damage, bacterial cells employ the SOS response to simultaneously express damage repair systems and halt cell division. Extensive characterization of SOS-controlled cell division inhibition in Escherichia coli has laid the ground for a long-standing paradigm where the cytosolic SulA protein inhibits polymerization of the central division protein, FtsZ, and thereby prevents recruitment of the division machinery at the future division site. Within the last decade, it has become clear that another, likely more general, paradigm exists, at least within the broad group of Gram-positive bacterial species, namely membrane-localized, SOS-induced cell division inhibition. We recently identified such an inhibitor in Staphylococci, SosA, and established a model for SosA-mediated cell division inhibition in Staphylococcus aureus in response to DNA damage. SosA arrests cell division subsequent to the septal localization of FtsZ and later membrane-bound division proteins, while preventing progression to septum closure, leading to synchronization of cells at this particular stage. A membrane-associated protease, CtpA negatively regulates SosA activity and likely allows growth to resume once conditions are favorable. Here, we provide a brief summary of our findings in the context of what already is known for other membrane cell division inhibitors and we emphasize how poorly characterized these intriguing processes are mechanistically. Furthermore, we put some perspective on the relevance of our findings and future developments within the field.

2.
Transbound Emerg Dis ; 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31838770

RESUMO

Staphylococcus aureus is a commensal bacterium and an important opportunistic pathogen in humans and animals. The increase in multi-drug resistant (MDR) strains of S. aureus is a growing concern due to their impact on animal health and potential for zoonotic transmission. Increasing evidence has shown that MRSA could be transmitted by faeces. The present study determined the prevalence, antibiotic resistance profile and genotypic characteristics of S. aureus isolated from monkey faecal samples in China. Thirty-eight out of 145 (26.21%) macaque faecal samples were S. aureus positive, which eight (5.5%) isolates were identified as MRSA. Antimicrobial susceptibility tests showed that most of the S. aureus isolates were resistant to tetracycline (TE, 44.74%), followed by penicillin (P, 21.05%), cefoxitin (FOX, 21.05%) and ciprofloxacin (CIP, 18.42%). The predominant spa types were t13638 (44.74%) and t189 (13.16%), which are reported to be closely associated with human infections in China. All MRSA isolates belonged to the SCCmecV type, which six of MRSA isolates were ST3268, while the other two isolates belonged to ST4981. This study for the first time describes the prevalence of S. aureus and MRSA in monkey faeces in China, indicating that faeces could be a potential factor of transmitting S. aureus between humans and monkeys.

3.
Front Microbiol ; 10: 2212, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31611856

RESUMO

Staphylococcus aureus is a commensal colonizer of both humans and animals, but also an opportunistic pathogen responsible for a multitude of diseases. In recent years, colonization of pigs by methicillin resistant S. aureus has become a problem with increasing numbers of humans being infected by livestock strains. In S. aureus colonization and virulence factor expression is controlled by the agr quorum sensing system, which responds to and is activated by self-generated, autoinducing peptides (AIPs). AIPs are also produced by coagulase negative staphylococci (CoNS) commonly found as commensals in both humans and animals, and interestingly, some of these inhibit S. aureus agr activity. Here, we have addressed if cross-communication occurs between S. aureus and CoNS strains isolated from pig nares, and if so, how properties such as host factor binding and biofilm formation are affected. From 25 pig nasal swabs we obtained 54 staphylococcal CoNS isolates belonging to 8 different species. Of these, none were able to induce S. aureus agr as monitored by reporter gene fusions to agr regulated genes but a number of agr-inhibiting species were identified including Staphylococcus hyicus, Staphylococcus simulans, Staphylococcus arlettae, Staphylococcus lentus, and Staphylococcus chromogenes. After establishing that the inhibitory activity was mediated via AgrC, the receptor of AIPs, we synthesized selective AIPs to explore their effect on adhesion of S. aureus to fibronectin, a host factor involved in S. aureus colonization. Here, we found that the CoNS AIPs did not affect adhesion of S. aureus except for strain 8325-4. When individual CoNS strains were co-cultured together with S. aureus we observed variable degrees of biofilm formation which did not correlate with agr interactions. Our results show that multiple CoNS species can be isolated from pig nares and that the majority of these produce AIPs that inhibit S. aureus agr. Further they show that the consequences of the interactions between CoNS and S. aureus are complex and highly strain dependent.

4.
Antibiotics (Basel) ; 8(4)2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569631

RESUMO

Biocides are chemical compounds widely used for sterilization and disinfection. The aim of this study was to examine whether exposure to subinhibitory biocide concentrations influenced transcriptional expression of genes that could improve a pathogen's drug resistance or fitness. We used DNA microarrays to investigate the transcriptome of the uropathogenic Escherichia coli strain CFT073 in response to prolonged exposure to subinhibitory concentrations of four biocides: benzalkonium chloride, chlorhexidine, hydrogen peroxide and triclosan. Transcription of a gene involved in polymyxin resistance, arnT, was increased after treatment with benzalkonium chloride. However, pretreatment of the bacteria with this biocide did not result in cross-resistance to polymyxin in vitro. Genes encoding products related to transport formed the functional group that was most affected by biocides, as 110 out of 884 genes in this category displayed altered transcription. Transcripts of genes involved in cysteine uptake, sulfate assimilation, dipeptide transport, as well as cryptic phage genes were also more abundant in response to several biocides. Additionally, we identified groups of genes with transcription changes unique to single biocides that might include potential targets for the biocides. The biocides did not increase the resistance potential of the pathogen to other antimicrobials.

5.
Microbiol Spectr ; 7(5)2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31562736

RESUMO

Most Staphylococcus aureus isolates carry multiple bacteriophages in their genome, which provide the pathogen with traits important for niche adaptation. Such temperate S. aureus phages often encode a variety of accessory factors that influence virulence, immune evasion and host preference of the bacterial lysogen. Moreover, transducing phages are primary vehicles for horizontal gene transfer. Wall teichoic acid (WTA) acts as a common phage receptor for staphylococcal phages and structural variations of WTA govern phage-host specificity thereby shaping gene transfer across clonal lineages and even species. Thus, bacteriophages are central for the success of S. aureus as a human pathogen.

6.
Sci Rep ; 9(1): 11460, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391518

RESUMO

The clinical use of the antibiotic erythromycin (ery) is hampered owing to the spread of resistance genes that are mostly mutating rRNA around the ery binding site at the entrance to the protein exit tunnel. Additional effective resistance mechanisms include deletion or insertion mutations in ribosomal protein uL22, which lead to alterations of the exit tunnel shape, located 16 Å away from the drug's binding site. We determined the cryo-EM structures of the Staphylococcus aureus 70S ribosome, and its ery bound complex with a two amino acid deletion mutation in its ß hairpin loop, which grants the bacteria resistance to ery. The structures reveal that, although the binding of ery is stable, the movement of the flexible shorter uL22 loop towards the tunnel wall creates a wider path for nascent proteins, thus enabling bypass of the barrier formed by the drug. Moreover, upon drug binding, the tunnel widens further.

7.
Mol Microbiol ; 112(4): 1116-1130, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31290194

RESUMO

Inhibition of cell division is critical for viability under DNA-damaging conditions. DNA damage induces the SOS response that in bacteria inhibits cell division while repairs are being made. In coccoids, such as the human pathogen, Staphylococcus aureus, this process remains poorly studied. Here, we identify SosA as the staphylococcal SOS-induced cell division inhibitor. Overproduction of SosA inhibits cell division, while sosA inactivation sensitizes cells to genotoxic stress. SosA is a small, predicted membrane protein with an extracellular C-terminal domain in which point mutation of residues that are conserved in staphylococci and major truncations abolished the inhibitory activity. In contrast, a minor truncation led to SosA accumulation and a strong cell division inhibitory activity, phenotypically similar to expression of wild-type SosA in a CtpA membrane protease mutant. This suggests that the extracellular C-terminus of SosA is required both for cell division inhibition and for turnover of the protein. Microscopy analysis revealed that SosA halts cell division and synchronizes the cell population at a point where division proteins such as FtsZ and EzrA are localized at midcell, and the septum formation is initiated but unable to progress to closure. Thus, our findings show that SosA is central in cell division regulation in staphylococci.

8.
PLoS Pathog ; 15(7): e1007888, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31276485

RESUMO

Temperate phages are bacterial viruses that as part of their life cycle reside in the bacterial genome as prophages. They are found in many species including most clinical strains of the human pathogens, Staphylococcus aureus and Salmonella enterica serovar Typhimurium. Previously, temperate phages were considered as only bacterial predators, but mounting evidence point to both antagonistic and mutualistic interactions with for example some temperate phages contributing to virulence by encoding virulence factors. Here we show that generalized transduction, one type of bacterial DNA transfer by phages, can create conditions where not only the recipient host but also the transducing phage benefit. With antibiotic resistance as a model trait we used individual-based models and experimental approaches to show that antibiotic susceptible cells become resistant to both antibiotics and phage by i) integrating the generalized transducing temperate phages and ii) acquiring transducing phage particles carrying antibiotic resistance genes obtained from resistant cells in the environment. This is not observed for non-generalized transducing temperate phages, which are unable to package bacterial DNA, nor for generalized transducing virulent phages that do not form lysogens. Once established, the lysogenic host and the prophage benefit from the existence of transducing particles that can shuffle bacterial genes between lysogens and for example disseminate resistance to antibiotics, a trait not encoded by the phage. This facilitates bacterial survival and leads to phage population growth. We propose that generalized transduction can function as a mutualistic trait where temperate phages cooperate with their hosts to survive in rapidly-changing environments. This implies that generalized transduction is not just an error in DNA packaging but is selected for by phages to ensure their survival.


Assuntos
Bacteriófagos/genética , Bacteriófagos/patogenicidade , Transdução Genética , Bacteriófagos/fisiologia , Simulação por Computador , Empacotamento do DNA/genética , Farmacorresistência Bacteriana/genética , Evolução Molecular , Humanos , Lisogenia/genética , Modelos Biológicos , Prófagos/genética , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/virologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/virologia , Virulência/genética
9.
Int J Biol Macromol ; 135: 931-939, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31170488

RESUMO

The sarcotesta of Punica granatum fruit contains an antimicrobial lectin called PgTeL. In this work, we evaluated the antibacterial activity of PgTeL against five drug-resistant Escherichia coli isolates able to produce ß-lactamases. Minimum inhibitory (MIC) and bactericidal (MBC) concentrations were determined by broth dilution. Morphometric and viability analyses were performed by flow cytometry, and ultrastructural changes were evaluated by scanning electron microscopy. Potential synergistic effects of PgTeL with antibiotics and anti-biofilm effect were also evaluated. PgTeL showed antibacterial activity against all isolates with MIC and MBC values ranging from 12.5 to 50.0 µg/mL and from 25.0 to 100.0 µg/mL, respectively. For most isolates, PgTeL postponed the growth start by at least ten hours. At the MIC, the lectin caused alterations in size, shape and structure of bacterial cells. The combination PgTeL-ceftazidime showed a synergistic effect for all isolates. Synergy was also detected with ampicillin (one isolate), carbenicillin (one isolate), cefotaxime (one isolate), cephalexin (four isolates) and cefuroxime (three isolates). PgTeL exhibited anti-biofilm activity against all isolates, causing ≥50% inhibition of biofilms at or above 6.25 µg/mL. The antibacterial effect of PgTeL and its synergy with antibiotics indicate that this fruit-derived molecule may have potential for future treatment of multidrug-resistant infections.


Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Lythraceae/química , Lectinas de Plantas/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Biofilmes/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Frutas/química , Testes de Sensibilidade Microbiana , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Resistência beta-Lactâmica , beta-Lactamases/biossíntese
10.
Nat Chem ; 11(5): 463-469, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31011175

RESUMO

Staphylococci secrete autoinducing peptides (AIPs) as signalling molecules to regulate population-wide behaviour. AIPs from non-Staphylococcus aureus staphylococci have received attention as potential antivirulence agents to inhibit quorum sensing and virulence gene expression in the human pathogen Staphylococcus aureus. However, only a limited number of AIP structures from non-S. aureus staphylococci have been identified to date, as the minute amounts secreted in complex media render it difficult. Here, we report a method for the identification of AIPs by exploiting their thiolactone functionality for chemoselective trapping and enrichment of the compounds from the bacterial supernatant. Standard liquid chromatography mass spectrometry analysis, guided by genome sequencing data, then readily provides the AIP identities. Using this approach, we confirm the identity of five known AIPs and identify the AIPs of eleven non-S. aureus species, and we expect that the method should be extendable to AIP-expressing Gram-positive bacteria beyond the Staphylococcus genus.


Assuntos
Proteínas de Bactérias/análise , Depsipeptídeos/análise , Staphylococcus/química , Sequência de Aminoácidos , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Cisteína/química , Depsipeptídeos/síntese química , Depsipeptídeos/isolamento & purificação , Depsipeptídeos/farmacologia , Limite de Detecção , Listeria monocytogenes/química , Estrutura Molecular , Percepção de Quorum/efeitos dos fármacos , Staphylococcus/metabolismo
11.
Int J Food Microbiol ; 300: 14-21, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-30991234

RESUMO

It is well established, that certain bacteria within the Brochothrix, Carnobacterium, Lactobacillus, Lactococcus, and Leuconostoc genera have an important role in the spoilage of chill stored poultry meat packaged in modified atmosphere. However, little is known about the role of microorganisms that are difficult to culture and the microbiota during poultry spoilage. We combined traditional cultivation and culture-independent 16S rRNA amplicon sequencing to investigate the microbiota encompassing putative bacteria of whole broiler meat, packaged in modified atmosphere, during and exceeding shelf-life. Samples were taken from 6 flocks during independent slaughter days. Additional samples were analysed from the production line. There was a significant difference in the microbial community structure of 80%O2/20%CO2 retail packaged broiler meat during different times of shelf-life, mainly due to an increase of species within the Brochothrix, Carnobacterium, Vagococcus, and Janthinobacterium genera. These genera were already detected four to eight days after slaughter. However, no significant difference between flocks with respect to the microbiota encompassing putative spoilage bacteria was observed when examined in retail packaged broilers, slaughtered at the same abattoir on different days. Our study also showed that lactic acid bacteria within the Vagococcus genus can constitute a dominating part of the later shelf-life microbiota in fresh whole broiler meat packaged in 80%O2/20%CO2 modified atmosphere. A single operational taxonomic unit (OTU) assigned as Janthinobacterium lividum, an occasional spoiler of meat products, was identified as a major part of the microbiota in late shelf life broiler meat and swab samples in the cooling facility at the slaughter house production line. The combination of traditional cultivation and culture-independent methods provided a great insight into the microbiota of broiler meat during shelf-life and identified a potential point of contamination in the production line for cold tolerant Janthinobacterium.


Assuntos
Fenômenos Fisiológicos Bacterianos , Galinhas/microbiologia , Microbiologia de Alimentos , Carne/microbiologia , Microbiota/fisiologia , Matadouros , Animais , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Carnobacterium/genética , Carnobacterium/fisiologia , Galinhas/genética , Embalagem de Alimentos , Microbiota/genética , RNA Ribossômico 16S/genética
12.
Microbiol Spectr ; 7(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30900543

RESUMO

Staphylococcus aureus is capable of becoming resistant to all classes of antibiotics clinically available and resistance can develop through de novo mutations in chromosomal genes or through acquisition of horizontally transferred resistance determinants. This review covers the most important antibiotics available for treatment of S. aureus infections and a special emphasis is dedicated to the current knowledge of the wide variety of resistance mechanisms that S. aureus employ to withstand antibiotics. Since resistance development has been inevitable for all currently available antibiotics, new therapies are continuously under development. Besides development of new small molecules affecting cell viability, alternative approaches including anti-virulence and bacteriophage therapeutics are being investigated and may become important tools to combat staphylococcal infections in the future.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/uso terapêutico , Replicação do DNA/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Drogas em Investigação/farmacologia , Drogas em Investigação/uso terapêutico , Genes Bacterianos , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Viabilidade Microbiana/efeitos dos fármacos , Mutação , Inibidores da Síntese de Proteínas , Infecções Estafilocócicas , Staphylococcus aureus/genética , Virulência
13.
Int J Antimicrob Agents ; 53(6): 716-723, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30825504

RESUMO

Resveratrol is a naturally occurring polyphenolic antioxidant that has received massive attention for its potential health benefits, including anticarcinogenesis, anti-aging and antimicrobial properties. The compound is well tolerated by humans and in recent years has been widely used as a nutraceutical. Its common use makes it interesting to investigate with respect to antimicrobial properties both as a single agent and in combination with conventional antibiotics. Resveratrol displays antimicrobial activity against a surprisingly wide range of bacterial, viral and fungal species. At subinhibitory concentrations, resveratrol can alter bacterial expression of virulence traits leading to reduced toxin production, inhibition of biofilm formation, reduced motility and interference with quorum sensing. In combination with conventional antibiotics, resveratrol enhances the activity of aminoglycosides against Staphylococcus aureus, whereas it antagonises the lethal activity of fluoroquinolones against S. aureus and Escherichia coli. Whilst the antimicrobial properties of the compound have been extensively studied in vitro, little is known about its efficacy in vivo. Nonetheless, following topical application resveratrol has alleviated acne lesions caused by the bacterium Propionibacterium acnes. There are currently no in vivo studies addressing its effect in combination with antibiotics, but recent research suggests that there may be a potential for enhancing the antimicrobial efficacy of certain existing antibiotic classes in combination with resveratrol. Given the difficulties associated with introducing new antimicrobial agents to the market, nutraceuticals such as resveratrol may prove to be interesting candidates when searching for solutions for the growing problem of antimicrobial resistance.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Antioxidantes/farmacologia , Bactérias/efeitos dos fármacos , Reposicionamento de Medicamentos , Fungos/efeitos dos fármacos , Resveratrol/farmacologia , Animais , Infecções Bacterianas/tratamento farmacológico , Humanos , Micoses/tratamento farmacológico
14.
Int J Biol Macromol ; 123: 600-608, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30414418

RESUMO

In this work, we evaluated the ability of Punica granatum sarcotesta lectin (PgTeL) to impair the growth and viability of the Staphylococcus aureus clinical isolates 8325-4 (non-resistant) and LAC USA300 (MRSA strain). The effects of this lectin on aggregating, hemolytic activity, biofilm-forming ability, and expression of virulence genes (hla, rnaIII, and spa) were also investigated. PgTeL showed antibacterial activity against 8325-4 and LAC USA300 strains by interfering with both the growth (MIC50 of 6.25 and 12.5 µg/mL, respectively) and survival (MBC values of 25.0 and 50.0 µg/mL, respectively). Culture growth started only at the ninth (8325-4) and tenth (LAC USA300) hour in the presence of PgTeL at MIC50, while growth was detected since the first hour in the control. The lectin caused markedly altered cell morphology in both the strains. Although, at the MIC50, PgTeL caused structural alterations, most cells were still viable, while at the MBC it promoted cell injury and death. PgTeL showed anti-aggregation effect and exhibited antibiofilm activity against both the isolates. However, the lectin did not interfere with the hemolytic activity of LAC USA300 and with the expression of hla, rnaIII, and spa genes. In conclusion, PgTeL is a lectin with multiple inhibitory effects on S. aureus clinical isolates.


Assuntos
Biofilmes/efeitos dos fármacos , Lectinas/química , Lythraceae/química , Staphylococcus aureus/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Agregação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Lectinas/farmacologia , Staphylococcus aureus/patogenicidade
15.
Vet Clin Pathol ; 47(4): 560-574, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30586190

RESUMO

BACKGROUND: Staphylococcus aureus is an opportunistic pathogen with the ability to form mobile planktonic aggregates during growth, in vitro. The in vivo pathophysiologic effects of S aureus aggregates on host responses are unknown. Knowledge of these could aid in combating infections. OBJECTIVE: This study aimed to investigate the effect of increasing concentrations of two different aggregating S aureus strains on the hemostatic and inflammatory host responses in canine whole blood. The hypothesis was that aggregating bacteria would induce pronounced hemostatic and inflammatory responses. METHODS: Citrate-stabilized whole blood from 10 healthy dogs was incubated with two strains of aggregating S aureus at three different concentrations. Each sample was analyzed using tissue factor-thromboelastography (TF-TEG) and the formed clot was investigated with electron microscopy. The plasma activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen, and D-dimer tests were measured. Bacteria-leukocyte binding was evaluated with flow cytometry, and neutrophil phagocytosis was assessed using light and transmission electron microscopy. RESULTS: The highest concentration of bacteria resulted in a significantly shortened TF-TEG initiation time, decreased alpha, maximum amplitude, global strength, and increased lysis. In addition, significantly shortened PT, decreased fibrinogen, and increased D-dimers were demonstrated at the highest concentration of bacteria. Lower concentrations of bacteria showed no differences in TF-TEG when compared with controls. The findings were similar for both S aureus strains. Increased concentration-dependent binding of bacteria and leukocytes and neutrophil bacterial phagocytosis was observed. CONCLUSIONS: Two strains of S aureus induced alterations of clot formation in concentrations where bacterial aggregates were formed. A concentration-dependent cellular inflammatory response was observed.


Assuntos
Coagulação Sanguínea , Doenças do Cão/microbiologia , Fibrinólise , Linfócitos/microbiologia , Monócitos/microbiologia , Neutrófilos/microbiologia , Fagocitose , Infecções Estafilocócicas/veterinária , Animais , Aderência Bacteriana , Doenças do Cão/sangue , Doenças do Cão/fisiopatologia , Cães , Feminino , Linfócitos/patologia , Masculino , Monócitos/patologia , Neutrófilos/patologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/fisiopatologia , Staphylococcus aureus/fisiologia
16.
BMC Res Notes ; 11(1): 503, 2018 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-30041686

RESUMO

OBJECTIVE: In the human pathogen, Staphylococcus aureus, the agr quorum sensing system controls expression of a multitude of virulence factors and yet, agr negative cells frequently arise both in the laboratory and in some infections. The aim of this study was to examine the possible reasons behind this phenomenon. RESULTS: We examined viability of wild type and agr mutant cell cultures using a live-dead stain and observed that in stationary phase, 3% of the wild type population became non-viable whereas for agr mutant cells non-viable cells were barely detectable. The effect appears to be mediated by RNAIII, the effector molecule of agr, as ectopic overexpression of RNAIII resulted in 60% of the population becoming non-viable. This effect was not due to toxicity from delta toxin that is encoded by the hld gene located within RNAIII as hld overexpression did not cause cell death. Importantly, lysed S. aureus cells promoted bacterial growth. Our data suggest that RNAIII mediated cell death of agr positive but not agr negative cells provides a selective advantage to the agr negative cell population and may contribute to the common appearance of agr negative cells in S. aureus populations.


Assuntos
Morte Celular , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum , Staphylococcus aureus/patogenicidade , Proteínas de Bactérias , Humanos , RNA Bacteriano/metabolismo , Transativadores , Virulência
17.
Sci Rep ; 8(1): 10849, 2018 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-30022089

RESUMO

Persister cells constitute a small subpopulation of bacteria that display remarkably high antibiotic tolerance and for pathogens such as Staphylococcus aureus are suspected as culprits of chronic and recurrent infections. Persisters formed during exponential growth are characterized by low ATP levels but less is known of cells in stationary phase. By enrichment from a transposon mutant library in S. aureus we identified mutants that in this growth phase displayed enhanced persister cell formation. We found that inactivation of either sucA or sucB, encoding the subunits of the α-ketoglutarate dehydrogenase of the tricarboxylic acid cycle (TCA cycle), increased survival to lethal concentrations of ciprofloxacin by 10-100 fold as did inactivation of other TCA cycle genes or atpA encoding a subunit of the F1F0 ATPase. In S. aureus, TCA cycle activity and gene expression are de-repressed in stationary phase but single cells with low expression may be prone to form persisters. While ATP levels were not consistently affected in high persister mutants they commonly displayed reduced membrane potential, and persistence was enhanced by a protein motive force inhibitor. Our results show that persister cell formation in stationary phase does not correlate with ATP levels but is associated with low membrane potential.


Assuntos
Trifosfato de Adenosina/metabolismo , Antibacterianos/farmacologia , Ciclo do Ácido Cítrico , Complexo Cetoglutarato Desidrogenase/antagonistas & inibidores , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Farmacorresistência Bacteriana Múltipla , Regulação Bacteriana da Expressão Gênica , Potenciais da Membrana , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo
18.
Int J Antimicrob Agents ; 52(3): 390-396, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29906565

RESUMO

Development of new antibiotics is costly and time-consuming, and therefore increasing the efficacy of conventional antibiotics is extremely attractive. For the human pathogen, Staphylococcus aureus, inactivation of the ATP synthase increases its susceptibility to gentamicin (an aminoglycoside) 16-fold. Aminoglycosides are rarely used as monotherapy against S. aureus due to the risk of development of resistance and toxic effects. This study explored the possibility of enhancing the efficacy of aminoglycosides against S. aureus and other Gram-positive pathogens by inhibiting the ATP synthase with resveratrol, a polyphenolic ATP synthase inhibitor that is commonly used as a dietary supplement. Co-administration of subinhibitory concentrations of resveratrol increased the activity of aminoglycosides, including gentamicin, kanamycin, neomycin, streptomycin and tobramycin, up to 32-fold against S. aureus, while the effect was lower (2-4-fold reduction in minimum inhibitory concentration) for other Gram-positive pathogens (i.e. Staphylococcus epidermidis, Enterococcus faecium and Enterococcus faecalis). The mechanism by which resveratrol increases the efficacy of aminoglycosides appears to be unrelated to membrane hyperpolarization and disruption of membrane integrity, which have been associated previously with increased aminoglycoside susceptibility. These results demonstrate that inhibition of the ATP synthase increases the efficacy of aminoglycosides against important Gram-positive pathogens, and the ATP synthase should be explored further as a target that may extend the clinical applicability of aminoglycosides.


Assuntos
Complexos de ATP Sintetase/antagonistas & inibidores , Aminoglicosídeos/farmacologia , Resveratrol/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/farmacologia , Quimioterapia Combinada , Humanos , Testes de Sensibilidade Microbiana
19.
J Dairy Sci ; 101(8): 7322-7333, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29778469

RESUMO

The role of non-aureus staphylococci (NAS) in the risk of acquisition of intramammary infections with Staphylococcus aureus is vague and still under debate. The objectives of this study were to (1) investigate the distribution patterns of NAS species from milk and teat skin in dairy herds with automatic milking systems, and (2) examine if the isolated NAS influences the expression of S. aureus virulence factors controlled by the accessory gene regulator (agr) quorum sensing system. In 8 herds, 14 to 20 cows with elevated somatic cell count were randomly selected for teat skin swabbing and aseptic quarter foremilk samples from right hind and left front quarters. Teat skin swabs were collected using the modified wet-dry method and milk samples were taken aseptically for bacterial culture. Colonies from quarters with suspicion of having NAS in milk or teat skin samples (or both) were subjected to MALDI-TOF assay for species identification. To investigate the interaction between S. aureus and NAS, 81 isolates NAS were subjected to a qualitative ß-galactosidase reporter plate assay. In total, 373 NAS isolates were identified representing 105 from milk and 268 from teat skin of 284 quarters (= 142 cows). Sixteen different NAS species were identified, 15 species from teat skin and 10 species from milk. The most prevalent NAS species identified from milk were Staphylococcus epidermidis (50%), Staphylococcus haemolyticus (15%), and Staphylococcus chromogenes (11%), accounting for 76%. Meanwhile, the most prevalent NAS species from teat skin were Staphylococcus equorum (43%), S. haemolyticus (16%), and Staphylococcus cohnii (14%), accounting for 73%. Using reporter gene fusions monitoring transcriptional activity of key virulence factors and regulators, we found that out of 81 supernatants of NAS isolates, 77% reduced expression of hla, encoding a-hemolysin, 70% reduced expression of RNAIII, the key effector molecule of agr, and 61% reduced expression of spa encoding protein A of S. aureus, respectively. Our NAS isolates showed 3 main patterns: (1) downregulation effect such as S. chromogenes (milk) and Staphylococcus xylosus (milk and teat), (2) no effect such as Staphylococcus sciuri (teat) and S. vitulinus (teat), and the third pattern (c) variable effect such as S. epidermidis (milk and teat) and S. equorum (milk and teat). The pattern of cross-talk between NAS species and S. aureus virulence genes varied according to the involved NAS species, habitat type, and herd factors. The knowledge of how NAS influences S. aureus virulence factor expression could explain the varying protective effect of NAS on S. aureus intramammary infections.


Assuntos
Glândulas Mamárias Animais/microbiologia , Mastite Bovina/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus/isolamento & purificação , Animais , Bovinos , Feminino , Leite , Infecções Estafilocócicas/microbiologia , Staphylococcus/patogenicidade , Staphylococcus aureus
20.
Eur J Med Chem ; 152: 370-376, 2018 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-29738955

RESUMO

Emergence of antibiotic-resistant bacteria constitutes an increasing threat to human health. For example, treatment options for Staphylococcus aureus infections is declining with the worldwide spreading of highly virulent community-associated methicillin-resistant S. aureus (CA-MRSA) strains. Anti-virulence therapy has been proposed as an alternative treatment strategy, as it typically involves inhibition of expression of virulence factors rather than direct bacterial killing, thereby attenuating the risk of resistance development. An intriguing target is the agr quorum-sensing system, which is a major inducer of virulence in CA-MRSA upon activation by agr-encoded staphylococcal autoinducing peptides (AIPs). In the present work a previously identified lactam hybrid analogue based on the marine depsipeptide solonamide B and the general structure of AIPs was investigated with respect to structure-function relationships. An array of 27 analogues exploring expansion of ring size, type of side chain, amino acid substitutions, and stereochemistry was designed and tested for AgrC-inhibitory activity. Interestingly, it was found that an analogue derived from the mirror image of the original hit proved to be the hitherto most efficient AgrC inhibitor resembling solonamide B in amino acid sequence. This and closely related compounds were 20- to 40-fold more potent in AgrC inhibition than the starting hit compound.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/farmacologia , Depsipeptídeos/farmacologia , Lactamas/farmacologia , Peptídeos Cíclicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Depsipeptídeos/síntese química , Depsipeptídeos/química , Relação Dose-Resposta a Droga , Lactamas/química , Conformação Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Quinases/metabolismo , Relação Estrutura-Atividade , beta-Lactamases/metabolismo
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