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1.
Microbiology (Reading) ; 169(11)2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37909284

RESUMO

Within the vaginal ecosystem, lactobacilli and Gardnerella spp. likely interact and influence each other's growth, yet the details of this interaction are not clearly defined. Using medium simulating vaginal fluid and a two-chamber co-culturing system to prevent cell-to-cell contact between the bacteria, we examined the possibility that Lactobacillus jensenii 62B (Lj 62B) and/or G. piotii (Gp) JCP8151B produce extracellular factors through which they influence each other's viability. By 24 h post-inoculation (hpi) in the co-culture system and under conditions similar to the vaginal environment - pH 5.0, 37 °C, and 5% CO2, Lj 62B viability was not affected but Gp JCP8151B had been eliminated. Cell-free supernatant harvested from Lj 62B cultures (Lj-CFS) at 20 hpi, but not 16 hpi, also eliminated Gp JCP8151B growth. Neither lactic acid nor H2O2 production by Lj 62B was responsible for this effect. The Lj-CFS did not affect viability of three species of lactobacilli or eight species of Gram-positive and Gram-negative uropathogens but eliminated viability of eight different strains of Gardnerella spp. Activity of the inhibitory factor within Lj-CFS was abolished by protease treatment and reduced by heat treatment suggesting it is most likely a bacteriocin-like protein; fractionation revealed that the factor has a molecular weight within the 10-30 kDa range. These results suggest that, in medium mimicking vaginal fluid and growth conditions similar to the vaginal environment, Lj 62B produces a potential bacteriocin-like inhibitory substance (Lj-BLIS) that clearly targets Gardnerella spp. strains. Once fully characterized, Lj-BLIS may be a potential treatment for Gardnerella-related BV that does not alter the vaginal microflora.


Assuntos
Bacteriocinas , Feminino , Humanos , Bacteriocinas/farmacologia , Bacteriocinas/metabolismo , Gardnerella/metabolismo , Peróxido de Hidrogênio/metabolismo , Ecossistema , Vagina/metabolismo , Vagina/microbiologia , Gardnerella vaginalis
2.
BMC Microbiol ; 23(1): 186, 2023 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-37442975

RESUMO

BACKGROUND: Glycogen metabolism by Lactobacillus spp. that dominate the healthy vaginal microbiome contributes to a low vaginal pH (3.5-4.5). During bacterial vaginosis (BV), strict and facultative anaerobes including Gardnerella vaginalis become predominant, leading to an increase in the vaginal pH (> 4.5). BV enhances the risk of obstetrical complications, acquisition of sexually transmitted infections, and cervical cancer. Factors critical for the maintenance of the healthy vaginal microbiome or the transition to the BV microbiome are not well defined. Vaginal pH may affect glycogen metabolism by the vaginal microflora, thus influencing the shift in the vaginal microbiome. RESULTS: The medium simulating vaginal fluid (MSVF) supported growth of L. jensenii 62G, L. gasseri 63 AM, and L. crispatus JV-V01, and G. vaginalis JCP8151A at specific initial pH conditions for 30 d. L. jensenii at all three starting pH levels (pH 4.0, 4.5, and 5.0), G. vaginalis at pH 4.5 and 5.0, and L. gasseri at pH 5.0 exhibited the long-term stationary phase when grown in MSVF. L. gasseri at pH 4.5 and L. crispatus at pH 5.0 displayed an extended lag phase over 30 d suggesting inefficient glycogen metabolism. Glycogen was essential for the growth of L. jensenii, L. crispatus, and G. vaginalis; only L. gasseri was able to survive in MSVF without glycogen, and only at pH 5.0, where it used glucose. All four species were able to survive for 15 d in MSVF with half the glycogen content but only at specific starting pH levels - pH 4.5 and 5.0 for L. jensenii, L. gasseri, and G. vaginalis and pH 5.0 for L. crispatus. CONCLUSIONS: These results suggest that variations in the vaginal pH critically influence the colonization of the vaginal tract by lactobacilli and G. vaginalis JCP8151A by affecting their ability to metabolize glycogen. Further, we found that L. jensenii 62G is capable of glycogen metabolism over a broader pH range (4.0-5.0) while L. crispatus JV-V01 glycogen utilization is pH sensitive (only functional at pH 5.0). Finally, our results showed that G. vaginalis JCP8151A can colonize the vaginal tract for an extended period as long as the pH remains at 4.5 or above.


Assuntos
Gardnerella vaginalis , Vaginose Bacteriana , Feminino , Humanos , Lactobacillus , Glicogênio/metabolismo , Vagina/microbiologia , Vaginose Bacteriana/microbiologia , Concentração de Íons de Hidrogênio
3.
Microorganisms ; 11(4)2023 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-37110338

RESUMO

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes high morbidity and mortality in cystic fibrosis (CF) and immunocompromised patients, including patients with ventilator-associated pneumonia (VAP), severely burned patients, and patients with surgical wounds. Due to the intrinsic and extrinsic antibiotic resistance mechanisms, the ability to produce several cell-associated and extracellular virulence factors, and the capacity to adapt to several environmental conditions, eradicating P. aeruginosa within infected patients is difficult. Pseudomonas aeruginosa is one of the six multi-drug-resistant pathogens (ESKAPE) considered by the World Health Organization (WHO) as an entire group for which the development of novel antibiotics is urgently needed. In the United States (US) and within the last several years, P. aeruginosa caused 27% of deaths and approximately USD 767 million annually in health-care costs. Several P. aeruginosa therapies, including new antimicrobial agents, derivatives of existing antibiotics, novel antimicrobial agents such as bacteriophages and their chelators, potential vaccines targeting specific virulence factors, and immunotherapies have been developed. Within the last 2-3 decades, the efficacy of these different treatments was tested in clinical and preclinical trials. Despite these trials, no P. aeruginosa treatment is currently approved or available. In this review, we examined several of these clinicals, specifically those designed to combat P. aeruginosa infections in CF patients, patients with P. aeruginosa VAP, and P. aeruginosa-infected burn patients.

4.
Antibiotics (Basel) ; 11(11)2022 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-36358169

RESUMO

Despite the implementation of stringent guidelines for the prevention of catheter-associated (CA) urinary tract infection (UTI), CAUTI remains one of the most common health care-related infections. We previously showed that an antimicrobial/antibiofilm agent inhibited biofilm development by Gram-positive and Gram-negative bacterial pathogens isolated from human infections. In this study, we examined the ability of a novel biofilm preventative agent (BPA) coating on silicone urinary catheters to inhibit biofilm formation on the catheters by six different bacterial pathogens isolated from UTIs: three Escherichia coli strains, representative of the most common bacterium isolated from UTI; one Enterobacter cloacae, a multidrug-resistant isolate; one Pseudomonas aeruginosa, common among patients with long-term catheterization; and one isolate of methicillin-resistant Staphylococcus aureus, as both a Gram-positive and a resistant organism. First, we tested the ability of these strains to form biofilms on urinary catheters made of red rubber, polyvinyl chloride (PVC), and silicone using the microtiter plate biofilm assay. When grown in artificial urine medium, which closely mimics human urine, all tested isolates formed considerable biofilms on all three catheter materials. As the biofilm biomass formed on silicone catheters was 0.5 to 1.6 logs less than that formed on rubber or PVC, respectively, we then coated the silicone catheters with BPA (benzalkonium chloride, polyacrylic acid, and glutaraldehyde), and tested the ability of the coated catheters to further inhibit biofilm development by these uropathogens. Compared with the uncoated silicone catheters, BPA-coated catheters completely prevented biofilm development by all the uropathogens, except P. aeruginosa, which showed no reduction in biofilm biomass. To explore the reason for P. aeruginosa resistance to the BPA coating, we utilized two specific lipopolysaccharide (LPS) mutants. In contrast to their parent strain, the two mutants failed to form biofilms on the BPA-coated catheters, which suggests that the composition of P. aeruginosa LPS plays a role in the resistance of wild-type P. aeruginosa to the BPA coating. Together, our results suggest that, except for P. aeruginosa, BPA-coated silicone catheters may prevent biofilm formation by both Gram-negative and Gram-positive uropathogens.

5.
Int Wound J ; 19(3): 553-572, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34263993

RESUMO

Topical antimicrobials that reduce the bacterial bioburden within a chronically-infected wound may have helpful or harmful effects on the healing process. We used murine models of full-thickness skin wounds to determine the effects of the novel biofilm-dispersing wound gel (BDWG) and its gel base on the healing of uninfected wounds. The rate of wound closure over 19 days was comparable among the BDWG-treated (BT) wounds and the controls. Compared with the controls, histology of the BT wounds showed formation of a stable blood clot at day 1, more neovascularisation and reepithelialisation at day 3, and more organised healing at day 7. Fluorescence-activated cell sorting analysis showed a lower percentage of neutrophils in wounded tissues of the BT group at days 1 and 3, and significantly more M2 macrophages at day 3. Levels of proinflammatory cytokines and chemokines were increased over the uninjured baseline within the wounds of all treatment groups but the levels were significantly lower in the BT group at day 1, modulating the inflammatory response. Our results suggest that BDWG does not interfere with the wound healing process and may enhance it by lowering inflammation and allowing transition to the proliferative stage of wound healing by day 3.


Assuntos
Anti-Infecciosos Locais , Infecção dos Ferimentos , Animais , Anti-Infecciosos Locais/farmacologia , Biofilmes , Géis , Camundongos , Pele/lesões , Cicatrização , Infecção dos Ferimentos/tratamento farmacológico
6.
Can J Microbiol ; 67(12): 919-932, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34437812

RESUMO

Pseudomonas aeruginosa, a gram-negative opportunistic pathogen, is one of the major species isolated from infected chronic wounds. The multidrug resistance exhibited by P. aeruginosa and its ability to form biofilms that are difficult to eradicate, along with the rising cost of producing new antibiotics, has necessitated the search for alternatives to standard antibiotics. Pyocins are antimicrobial compounds produced by P. aeruginosa that protect themselves from their competitors. We synthesized and purified recombinant P. aeruginosa R2 pyocin and used it in an aqueous solution (rR2P) or formulated in polyethylene glycol (rR2PC) to treat P. aeruginosa-infected wounds. Clinical strains of P. aeruginosa were found to be sensitive (completely), partially sensitive, or resistant to rR2P. In the in vitro biofilm model, rR2P inhibited biofilm development by rR2P-sensitive isolates, while rR2PC eliminated partial biofilms formed by these strains in an in vitro wound biofilm model. In the murine model of excision wounds, and at 24 h post-infection, rR2PC application significantly reduced the bioburden of the clinical isolate BPI86. Application of rR2PC containing two glycoside hydrolase antibiofilm agents eliminated BPI86 from infected wounds. These results suggest that the topical application of rR2PC is an effective therapy for treating wounds infected with R2P-senstive P. aeruginosa strains.


Assuntos
Infecções por Pseudomonas , Infecção dos Ferimentos , Animais , Biofilmes , Camundongos , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa , Piocinas , Infecção dos Ferimentos/tratamento farmacológico
7.
Mol Microbiol ; 116(2): 516-537, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33892520

RESUMO

Pseudomonas aeruginosa is an opportunistic pathogen that uses malonate among its many carbon sources. We recently reported that, when grown in blood from trauma patients, P. aeruginosa expression of malonate utilization genes was upregulated. In this study, we explored the role of malonate utilization and its contribution to P. aeruginosa virulence. We grew P. aeruginosa strain PA14 in M9 minimal medium containing malonate (MM9) or glycerol (GM9) as a sole carbon source and assessed the effect of the growth on quorum sensing, virulence factors, and antibiotic resistance. Growth of PA14 in MM9, compared to GM9, reduced the production of elastases, rhamnolipids, and pyoverdine; enhanced the production of pyocyanin and catalase; and increased its sensitivity to norfloxacin. Growth in MM9 decreased extracellular levels of N-acylhomoserine lactone autoinducers, an effect likely associated with increased pH of the culture medium; but had little effect on extracellular levels of PQS. At 18 hr of growth in MM9, PA14 formed biofilm-like structures or aggregates that were associated with biomineralization, which was related to increased pH of the culture medium. These results suggest that malonate significantly impacts P. aeruginosa pathogenesis by influencing the quorum sensing systems, the production of virulence factors, biofilm formation, and antibiotic resistance.


Assuntos
Biofilmes/crescimento & desenvolvimento , Farmacorresistência Bacteriana/fisiologia , Malonatos/metabolismo , Pseudomonas aeruginosa/patogenicidade , Percepção de Quorum/fisiologia , Antibacterianos/farmacologia , Biomineralização/fisiologia , Catalase/biossíntese , Decanoatos , Dissacarídeos/biossíntese , Glicerol/metabolismo , Norfloxacino/farmacologia , Oligopeptídeos/biossíntese , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Piocianina/biossíntese , Serina Endopeptidases/biossíntese , Virulência , Fatores de Virulência/metabolismo
8.
PLoS One ; 15(10): e0240351, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33057423

RESUMO

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that produces numerous virulence factors and causes serious infections in trauma patients and patients with severe burns. We previously showed that the growth of P. aeruginosa in blood from severely burned or trauma patients altered the expression of numerous genes. However, the specific influence of whole blood from healthy volunteers on P. aeruginosa gene expression is not known. Transcriptome analysis of P. aeruginosa grown for 4 h in blood from healthy volunteers compared to that when grown in laboratory medium revealed that the expression of 1085 genes was significantly altered. Quorum sensing (QS), QS-related, and pyochelin synthesis genes were downregulated, while genes of the type III secretion system and those for pyoverdine synthesis were upregulated. The observed effect on the QS and QS-related genes was shown to reside within serum fraction: growth of PAO1 in the presence of 10% human serum from healthy volunteers significantly reduced the expression of QS and QS-regulated genes at 2 and 4 h of growth but significantly enhanced their expression at 8 h. Additionally, the production of QS-regulated virulence factors, including LasA and pyocyanin, was also influenced by the presence of human serum. Serum fractionation experiments revealed that part of the observed effect resides within the serum fraction containing <10-kDa proteins. Growth in serum reduced the production of many PAO1 outer membrane proteins but enhanced the production of others including OprF, a protein previously shown to play a role in the regulation of QS gene expression. These results suggest that factor(s) within human serum: 1) impact P. aeruginosa pathogenesis by influencing the expression of different genes; 2) differentially regulate the expression of QS and QS-related genes in a growth phase- or time-dependent mechanism; and 3) manipulate the production of P. aeruginosa outer membrane proteins.


Assuntos
Pseudomonas aeruginosa/metabolismo , Percepção de Quorum/genética , Virulência/genética , Bacteriemia/microbiologia , Bacteriemia/patologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Meios de Cultura/química , Regulação para Baixo , Humanos , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/patogenicidade , Soro/química , Sideróforos/genética , Transcriptoma , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Regulação para Cima
9.
Metabolomics ; 16(3): 40, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170472

RESUMO

INTRODUCTION: Sepsis is a leading cause of mortality in burn patients. One of the major causes of sepsis in burn patients is Pseudomonas aeruginosa. We hypothesized that during dissemination from infected burn wounds and subsequent sepsis, P. aeruginosa affects the metabolome of the blood resulting in changes to specific metabolites that would serve as biomarkers for early diagnosis of sepsis caused by P. aeruginosa. OBJECTIVES: To identify specific biomarkers in the blood after sepsis caused by P. aeruginosa infection of burns. METHODS: Gas chromatography with time-of-flight mass spectrometry was used to compare the serum metabolome of mice that were thermally injured and infected with P. aeruginosa (B-I) to that of mice that were neither injured nor infected, mice that were injured but not infected, and mice that were infected but not injured. RESULTS: Serum levels of 19 metabolites were significantly increased in the B-I group compared to controls while levels of eight metabolites were significantly decreased. Thymidine, thymine, uridine, and uracil (related to pyrimidine metabolism), malate and succinate (a possible sign of imbalance in the tricarboxylic acid cycle), 5-oxoproline (related to glutamine and glutathione metabolism), and trans-4-hydroxyproline (a major component of the protein collagen) were increased. Products of amino acid metabolism were significantly decreased in the B-I group, including methionine, tyrosine, indole-3-acetate, and indole-3-propionate. CONCLUSION: In all, 26 metabolites were identified, including a unique combination of five metabolites (trans-4-hydroxyproline, 5-oxoproline, glycerol-3-galactoside, indole-3-acetate, and indole-3-propionate) that could serve as a set of biomarkers for early diagnosis of sepsis caused by P. aeruginosa in burn patients.


Assuntos
Queimaduras/metabolismo , Pseudomonas aeruginosa/metabolismo , Sepse/metabolismo , Infecção dos Ferimentos/metabolismo , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Queimaduras/sangue , Queimaduras/microbiologia , Cromatografia Gasosa , Modelos Animais de Doenças , Feminino , Espectrometria de Massas , Metabolômica , Camundongos , Sepse/sangue , Sepse/microbiologia , Infecção dos Ferimentos/sangue , Infecção dos Ferimentos/microbiologia
10.
J Med Microbiol ; 68(10): 1560-1572, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31460863

RESUMO

Introduction. Severely burned patients are susceptible to bacterial infection within their burn wounds, which frequently leads to sepsis, multiple organ failure and death. The opportunistic pathogen Pseudomonas aeruginosa, an organism inherently resistant to multiple antibiotics, is a common cause of sepsis in these patients.Aim. Development of a topical treatment unrelated to conventional antibiotics is essential for prevention of P. aeruginosa infection and sepsis, leading to a role for the direct application of probiotics or their by-products.Methodology. We examined the effectiveness of 20× concentrated supernatant from Lactobacillus gasseri strain 63 AM (LgCS) grown in de Man, Rogosa and Sharpe broth in inhibiting P. aeruginosa biofilms in vitro, as well as in reducing wound bioburden and P. aeruginosa sepsis in vivo.Results. LgCS inhibited the growth of P. aeruginosa strain PAO1, prevented its biofilm development and eliminated partially developed PAO1 biofilms. In the murine model of thermal injury, a single injection of LgCS following injury and PAO1 infection reduced mortality to 0 % and prevented systemic spread (sepsis). Furthermore, a second injection of LgCS 24 h after the first eliminated PAO1 from the wound. In the murine dorsal excision infection model, either LgCS or ceftazidime treatment of the PAO1-infected wound significantly reduced the mortality rate among infected mice, while combining LgCS with ceftazidime eliminated mortality.Conclusion. These results suggest the potential of LgCS in preventing sepsis from P. aeruginosa infection in severely burned and other immunocompromised patients.


Assuntos
Queimaduras/complicações , Lactobacillus gasseri/fisiologia , Infecções por Pseudomonas/terapia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Sepse/terapia , Músculos Superficiais do Dorso/lesões , Animais , Antibiose , Biofilmes , Terapia Biológica , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Pseudomonas/etiologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/mortalidade , Pseudomonas aeruginosa/fisiologia , Sepse/etiologia , Sepse/microbiologia , Sepse/mortalidade , Músculos Superficiais do Dorso/microbiologia , Músculos Superficiais do Dorso/cirurgia , Infecção dos Ferimentos
11.
mSystems ; 4(4)2019.
Artigo em Inglês | MEDLINE | ID: mdl-31086830

RESUMO

Trauma patients (TPs) are highly susceptible to infections, which often lead to sepsis. Among the numerous causative agents, Pseudomonas aeruginosa is especially important, as P. aeruginosa sepsis is often fatal. Understanding the mechanism of its pathogenesis in bloodstream infections is imperative; however, this mechanism has not been previously described. To examine the effect of trauma-induced changes in blood on the expression of P. aeruginosa genes, we grew strain UCBPP-PA14 (PA14) in blood samples from eight TPs and seven healthy volunteers (HVs). Compared with its growth in blood from HVs, the growth of PA14 in blood from TPs significantly altered the expression of 285 genes. Genes whose expression was significantly increased were related to carbon metabolism, especially malonate utilization and mannitol uptake, and efflux of heavy metals. Genes whose expression was significantly reduced included genes of the type VI secretion system, genes related to uptake and metabolism of amino acids, and genes related to biosynthesis and transport of the siderophores pyoverdine and pyochelin. These results suggest that during systemic infection in trauma patients, and to adapt to the trauma-induced changes in blood, P. aeruginosa adjusts positively and negatively the expression of numerous genes related to carbon metabolism and virulence, respectively. IMPORTANCE While a considerable body of knowledge regarding sepsis in trauma patients is available, the potential influence of trauma-induced changes in the blood of these patients on the pathogenesis of Pseudomonas aeruginosa is basically an unexplored area. Rather than using standard laboratory media, we grew P. aeruginosa in whole blood from either healthy volunteers or trauma patients. The specific changes in the P. aeruginosa transcriptome in response to growth in blood from trauma patients reflect the adaptation of this organism to the bloodstream environment. This knowledge is vital for understanding the strategies this pathogen uses to adapt and survive within the host during systemic infection. Such information will help researchers and clinicians to develop new approaches for treatment of sepsis caused by P. aeruginosa in trauma patients, especially in terms of recognizing the effects of specific therapies (e.g., iron, zinc, or mannitol) on the organism. Further, this information can most likely be extrapolated to all patients with P. aeruginosa septicemia.

12.
Microb Pathog ; 127: 288-295, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30528249

RESUMO

Paerucumarin synthesized by pvc operon pvcABCD is an iron binding molecule which modulates biofilm formation in Pseudomonas aeruginosa but its direct function in bacterial pathogenesis needs further investigation. pvcA synthesizes isonitrile functionalized tyrosine (IFT) which is converted to mature paerucumarin by the proteins encoded by pvcB, pvcC and pvcD genes. Interruption of pvcB in MPAO1 resulted in accumulation of IFT as it cannot be converted to mature molecule. The MPAO1 pvcB mutant (PW4832) showed enhanced swarming motility, while complementation with plasmid pLL2 carrying pvcB reduced swarming motility. Enhanced levels of rhlA expression and rhamnolipid production were observed in PW4832 compared to the parent strain. Overexpression of ptxR, the positive regulator of pvcABCD, in PW4832 caused accumulation of more IFT and further elevated the level of rhlA expression. Expression of the quorum sensing system transcriptional activators lasR and rhlR, as well as the synthase genes lasI and rhlI, was enhanced in PW4832 compared to MPAO1, as was PQS accumulation. Exogenously added IFT, but not paerucumarin, enhanced the production of rhamnolipids in P. aeruginosa. These results suggest that IFT enhances swarming motility in P. aeruginosa either directly by enhancing rhamnolipid production or indirectly through modulation of the quorum sensing systems. This is the first report assigning an independent function to IFT in P. aeruginosa.


Assuntos
Locomoção/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Nitrilas/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum/efeitos dos fármacos , Tirosina/metabolismo , Proteínas de Bactérias/genética , Expressão Gênica , Técnicas de Inativação de Genes , Teste de Complementação Genética , Óperon , Plasmídeos
13.
Infect Immun ; 86(1)2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29061710

RESUMO

The opportunistic pathogen Pseudomonas aeruginosa is a major cause of sepsis in severely burned patients. If it is not eradicated from the wound, it translocates to the bloodstream, causing sepsis, multiorgan failure, and death. We recently described the P. aeruginosa heparinase-encoding gene, hepP, whose expression was significantly enhanced when P. aeruginosa strain UCBPP_PA14 (PA14) was grown in whole blood from severely burned patients. Further analysis demonstrated that hepP contributed to the in vivo virulence of PA14 in the Caenorhabditis elegans model. In this study, we utilized the murine model of thermal injury to examine the contribution of hepP to the pathogenesis of P. aeruginosa during burn wound infection. Mutation of hepP reduced the rate of mortality from 100% for mice infected with PA14 to 7% for mice infected with PA14::hepP While comparable numbers of PA14 and PA14::hepP bacteria were recovered from infected skin, only PA14 was recovered from the livers and spleens of infected mice. Despite its inability to spread systemically, PA14::hepP formed perivascular cuffs around the blood vessels within the skin of the thermally injured/infected mice. Intraperitoneal inoculation of the thermally injured mice, bypassing the need for translocation, produced similar results. The rate of mortality for mice infected with PA14::hepP was 0%, whereas it was 66% for mice infected with PA14. As before, only PA14 was recovered from the livers and spleens of infected mice. These results suggest that hepP plays a crucial role in the pathogenesis of PA14 during burn wound infection, most likely by contributing to PA14 survival in the bloodstream of the thermally injured mouse during sepsis.


Assuntos
Proteínas de Bactérias/genética , Queimaduras/microbiologia , Heparina Liase/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Virulência/genética , Infecção dos Ferimentos/microbiologia , Animais , Feminino , Camundongos , Mutação/genética , Sepse/microbiologia , Pele/microbiologia
14.
BMC Microbiol ; 17(1): 233, 2017 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-29246112

RESUMO

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that causes serious infections in immunocompromised hosts including severely burned patients. In burn patients, P. aeruginosa infection often leads to septic shock and death. Despite numerous studies, the influence of severe thermal injuries on the pathogenesis of P. aeruginosa during systemic infection is not known. Through RNA-seq analysis, we recently showed that the growth of P. aeruginosa strain UCBPP-PA14 (PA14) in whole blood obtained from severely burned patients significantly altered the expression of the PA14 transcriptome when compared with its growth in blood from healthy volunteers. The expression of PA14_23430 and the adjacent gene, PA14_23420, was enhanced by seven- to eightfold under these conditions. RESULTS: Quantitative real-time PCR analysis confirmed the enhancement of expression of both PA14_23420 and PA14_23430 by growth of PA14 in blood from severely burned patients. Computer analysis revealed that PA14_23430 (hepP) encodes a potential heparinase while PA14_23420 (zbdP) codes for a putative zinc-binding dehydrogenase. This analysis further suggested that the two genes form an operon with zbdP first. Presence of the operon was confirmed by RT-PCR experiments. We characterized hepP and its protein product HepP. hepP was cloned from PA14 by PCR and overexpressed in E. coli. The recombinant protein (rHepP) was purified using nickel column chromatography. Heparinase assays using commercially available heparinase as a positive control, revealed that rHepP exhibits heparinase activity. Mutation of hepP resulted in delay of pellicle formation at the air-liquid interface by PA14 under static growth conditions. Biofilm formation by PA14ΔhepP was also significantly reduced. In the Caenorhabditis elegans model of slow killing, mutation of hepP resulted in a significantly lower rate of killing than that of the parent strain PA14. CONCLUSIONS: Changes within the blood of severely burned patients significantly induced expression of hepP in PA14. The heparinase encoded by hepP is a potential virulence factor for PA14 as HepP influences pellicle formation as well as biofilm development by PA14 and the protein is required for full virulence in the C. elegans model of slow killing.


Assuntos
Proteínas de Bactérias/genética , Regulação Enzimológica da Expressão Gênica , Heparina Liase/genética , Heparina Liase/metabolismo , Infecções por Pseudomonas/enzimologia , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidade , Animais , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Queimaduras/sangue , Queimaduras/imunologia , Queimaduras/microbiologia , Caenorhabditis elegans/microbiologia , Escherichia coli/genética , Perfilação da Expressão Gênica , Heparina Liase/isolamento & purificação , Humanos , Hospedeiro Imunocomprometido , Mutação/genética , Óperon/genética , Infecções por Pseudomonas/sangue , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
15.
Int J Otolaryngol ; 2017: 3037191, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28798773

RESUMO

PURPOSE: The goal of this study was to understand the potential interaction between Pseudomonas aeruginosa and Fusobacterium nucleatum within the middle ear. METHODS: We examined the microbiota of ear fluid and tympanostomy tubes (TTs) obtained from patients with posttympanostomy tube otorrhea. We also examined biofilms formed by P. aeruginosa and F. nucleatum, singly or together, under aerobic or anaerobic conditions. RESULTS: While the facultative anaerobe P. aeruginosa dominated the bacterial population within the ear fluid, strict anaerobes, including F. nucleatum, dominated bacterial populations within the TTs. F. nucleatum was able to grow under aerobic conditions only in the presence of P. aeruginosa, whose growth reduced the level of dissolved oxygen within the broth to nearly anoxic condition within 4 h after inoculation. The presence of P. aeruginosa allowed F. nucleatum to maintain its growth for 72 h within the dual-species biofilm but not within the planktonic growth. Visualization of the biofilms revealed coaggregation of P. aeruginosa and F. nucleatum. CONCLUSION: Extrapolation of these results suggests that, within the middle ear fluid, the growth of P. aeruginosa produces the anaerobic conditions required for the growth of F. nucleatum, both within effusion and within biofilms.

16.
J Microbiol ; 54(8): 573-81, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27480638

RESUMO

Proteins encoded by the Pseudomonas aeruginosa pvcA-D operon synthesize a novel isonitrile functionalized cumarin termed paerucumarin. The pvcA-D operon enhances the expression of the P. aeruginosa fimbrial chaperone/usher pathway (cup) genes and this effect is mediated through paerucumarin. Whether pvcA-D and/or paerucumarin affect the expression of other P. aeruginosa genes is not known. In this study, we examined the effect of a mutation in pvcA-D operon the global transcriptome of the P. aeruginosa strain PAO1-UW. The mutation reduced the expression of several ironcontrolled genes including pvdS, which is essential for the expression of the pyoverdine genes. Additional transcriptional studies showed that the pvcA-D operon is not regulated by iron. Exogenously added paerucumarin enhanced pyoverdine production and pvdS expression in PAO1-UW. Iron-chelation experiments revealed that purified paerucumarin chelates iron. However, exogenously added paerucumarin significantly reduced the growth of a P. aeruginosa mutant defective in pyoverdine and pyochelin production. In contrast to other secondary metabolite, Pseudomonas quinolone signal (PQS), paerucumarin is not localized to the P. aeruginosa membrane vesicles. These results suggest that paerucumarin enhances the expression of iron-controlled genes by chelating iron within the P. aeruginosa extracellular environment. Although paerucumarin chelates iron, it does not function as a siderophore. Unlike PQS, paerucumarin is not associated with the P. aeruginosa cell envelope.


Assuntos
Vesículas Extracelulares/metabolismo , Ferro/metabolismo , Pseudomonas aeruginosa/metabolismo , Metabolismo Secundário , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vesículas Extracelulares/genética , Regulação Bacteriana da Expressão Gênica , Mutação , Oligopeptídeos/metabolismo , Óperon , Pseudomonas aeruginosa/genética
17.
PLoS One ; 11(3): e0149229, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26933952

RESUMO

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes serious infections in immunocompromised hosts including severely burned patients. After multiplying within the burn wound, P. aeruginosa translocate into the bloodstream causing bacterial sepsis frequently leading to organ dysfunction and septic shock. Although the pathogenesis of P. aeruginosa infection of thermally-injured wounds has been extensively analyzed, little is known regarding the ability of P. aeruginosa to adapt and survive within the blood of severely burned patients during systemic infection. To identify such adaptations, transcriptome analyses (RNA-seq) were conducted on P. aeruginosa strain PA14 that was grown in whole blood from a healthy volunteer or three severely burned patients. Compared with growth in blood from healthy volunteers, growth of PA14 in the blood from severely burned patients significantly altered the expression of 2596 genes, with expression of 1060 genes enhanced, while that of 1536 genes was reduced. Genes whose expression was significantly reduced included genes related to quorum sensing, quorum sensing-controlled virulence factors and transport of heme, phosphate, and phosphonate. Genes whose expression was significantly enhanced were related to the type III secretion system, the pyochelin iron-acquisition system, flagellum synthesis, and pyocyanin production. We confirmed changes in expression of many of these genes using qRT-PCR. Although severe burns altered the levels of different blood components in each patient, the growth of PA14 in their blood produced similar changes in the expression of each gene. These results suggest that, in response to changes in the blood of severely burned patients and as part of its survival strategy, P. aeruginosa enhances the expression of certain virulence genes and reduces the expression of others.


Assuntos
Queimaduras/complicações , Queimaduras/microbiologia , Regulação Bacteriana da Expressão Gênica , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/genética , Adulto , Queimaduras/sangue , Humanos , Ferro/metabolismo , Masculino , Pessoa de Meia-Idade , Infecções por Pseudomonas/sangue , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum , Transcriptoma , Sistemas de Secreção Tipo III/genética
18.
Int J Pediatr Otorhinolaryngol ; 79(11): 1909-14, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26388185

RESUMO

OBJECTIVE: The purpose of this study was to determine if the recently developed novel antimicrobial/antibiofilm agent Next-Science (NS) inhibits biofilm development by Staphylococcus aureus or Pseudomonas aeruginosa on tympanostomy tubes (TT) and to define the concentration of NS at which this inhibition occurs. METHODS: Preliminary titration experiments determined the effective concentrations of NS that completely inhibit the planktonic growth of S. aureus and P. aeruginosa. Since NS has the potential to inhibit both planktonic growth and biofilm development, we examined the antibiofilm effect using the established concentrations that inhibited planktonic growth. Biofilms developed on TT using the microtiter plate assay were assessed quantitatively by determining the number of microorganisms per tube (CFU/tube) and qualitatively by visualization with confocal laser scanning microscopy (CLSM). RESULTS: Planktonic growth of S. aureus and P. aeruginosa was inhibited by 20.3 µg/mL and 325 µg/mL of NS, respectively. While S. aureus and P. aeruginosa formed well-developed biofilms on TT at 24 h without treatment, addition of the indicated concentrations of NS at the time of inoculation of the TT inhibited the formation of biofilms by both organisms. CLSM confirmed the absence of biofilms on either the inner or outer surface of the treated TTs. At 8 h post-inoculation, P. aeruginosa formed a partial biofilm on the TT when untreated. In comparison, the NS-treated biofilms failed to develop further and the CFU/TT were significantly reduced. CONCLUSION: The novel antimicrobial agent NS inhibited the development of S. aureus and P. aeruginosa biofilms on TTs. The same concentrations of NS inhibited both planktonic growth and biofilm development.


Assuntos
Anti-Infecciosos/farmacologia , Biofilmes/efeitos dos fármacos , Géis , Ventilação da Orelha Média/instrumentação , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Pseudomonas aeruginosa/fisiologia , Staphylococcus aureus/fisiologia
19.
Can J Microbiol ; 60(3): 155-66, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24588389

RESUMO

In Pseudomonas aeruginosa, type IV pili (TFP)-dependent twitching motility is required for development of surface-attached biofilm (SABF), yet excessive twitching motility is detrimental once SABF is established. In this study, we show that mucin significantly enhanced twitching motility and decreased SABF formation in strain PAO1 and other P. aeruginosa strains in a concentration-dependent manner. Mucin also disrupted partially established SABF. Our analyses revealed that mucin increased the amount of surface pilin and enhanced transcription of the pilin structural gene pilA. Mucin failed to enhance twitching motility in P. aeruginosa mutants defective in genes within the pilin biogenesis operons pilGHI/pilJK-chpA-E. Furthermore, mucin did not enhance twitching motility nor reduce biofilm development by chelating iron. We also examined the role of the virulence factor regulator Vfr in the effect of mucin. In the presence or absence of mucin, PAOΔvfr produced a significantly reduced SABF. However, mucin partially complemented the twitching motility defect of PAOΔvfr. These results suggest that mucin interferes with SABF formation at specific concentrations by enhancing TFP synthesis and twitching motility, that this effect, which is iron-independent, requires functional Vfr, and only part of the Vfr-dependent effect of mucin on SABF development occurs through twitching motility.


Assuntos
Biofilmes/crescimento & desenvolvimento , Fímbrias Bacterianas/fisiologia , Mucinas/fisiologia , Pseudomonas aeruginosa/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Movimento , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade
20.
Antimicrob Agents Chemother ; 58(6): 3060-72, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24637684

RESUMO

Loss of the skin barrier facilitates the colonization of underlying tissues with various bacteria, where they form biofilms that protect them from antibiotics and host responses. Such wounds then become chronically infected. Topical antimicrobials are a major component of chronic wound therapy, yet currently available topical antimicrobials vary in their effectiveness on biofilm-forming pathogens. In this study, we evaluated the efficacy of Next Science wound gel technology (NxtSc), a novel topical agent designed to kill planktonic bacteria, penetrate biofilms, and kill the bacteria within. In vitro quantitative analysis, using strains isolated from wounds, showed that NxtSc inhibited biofilm development by Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae by inhibiting bacterial growth. The gel formulation NxtSc-G5, when applied to biofilms preformed by these pathogens, reduced the numbers of bacteria present by 7 to 8 log10 CFU/disc or CFU/g. In vivo, NxtSc-G5 prevented biofilm formation for 72 h when applied at the time of wounding and infection and eliminated biofilm infection when applied 24 h after wounding and infection. Storage of NxtSc-G5 at room temperature for 9 months did not diminish its efficacy. These results establish that NxtSc is efficacious in vitro and in vivo in preventing infection and biofilm development by different wound pathogens when applied immediately and in eliminating biofilm infection already established by these pathogens. This novel antimicrobial agent, which is nontoxic and has a usefully long shelf life, shows promise as an effective agent for the prevention and treatment of biofilm-related infections.


Assuntos
Infecções por Acinetobacter/prevenção & controle , Anti-Infecciosos/farmacologia , Biofilmes/efeitos dos fármacos , Infecções por Klebsiella/prevenção & controle , Infecções por Pseudomonas/prevenção & controle , Infecções Estafilocócicas/prevenção & controle , Infecção dos Ferimentos/prevenção & controle , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Administração Tópica , Animais , Biofilmes/crescimento & desenvolvimento , Feminino , Géis/farmacologia , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Camundongos , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacos , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/microbiologia
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