RESUMEN
Serratia marcescens is an important nosocomial pathogen causing various opportunistic infections, such as urinary tract infections, bacteremia and sometimes even hospital outbreaks. The recent emergence and spread of multidrug-resistant (MDR) strains further pose serious threats to global public health. This bacterium is also ubiquitously found in natural environments, but the genomic differences between clinical and environmental isolates are not clear, including those between S. marcescens and its close relatives. In this study, we performed a large-scale genome analysis of S. marcescens and closely related species (referred to as the 'S. marcescens complex'), including more than 200 clinical and environmental strains newly sequenced here. Our analysis revealed their phylogenetic relationships and complex global population structure, comprising 14 clades, which were defined based on whole-genome average nucleotide identity. Clades 10, 11, 12 and 13 corresponded to S. nematodiphila, S. marcescens sensu stricto, S. ureilytica and S. surfactantfaciens, respectively. Several clades exhibited distinct genome sizes and GC contents and a negative correlation of these genomic parameters was observed in each clade, which was associated with the acquisition of mobile genetic elements (MGEs), but different types of MGEs, plasmids or prophages (and other integrative elements), were found to contribute to the generation of these genomic variations. Importantly, clades 1 and 2 mostly comprised clinical or hospital environment isolates and accumulated a wide range of antimicrobial resistance genes, including various extended-spectrum ß-lactamase and carbapenemase genes, and fluoroquinolone target site mutations, leading to a high proportion of MDR strains. This finding suggests that clades 1 and 2 represent hospital-adapted lineages in the S. marcescens complex although their potential virulence is currently unknown. These data provide an important genomic basis for reconsidering the classification of this group of bacteria and reveal novel insights into their evolution, biology and differential importance in clinical settings.
Asunto(s)
Bacteriemia , Serratia marcescens , Hospitales , Humanos , Filogenia , Plásmidos , Serratia marcescens/genéticaRESUMEN
We investigated the role of the resistance-nodulation-cell division superfamily (RND) efflux system on intrinsic multidrug resistance in Serratia marcescens. We identified eight putative RND efflux system genes in the S. marcescens Db10 genome that included the previously characterized systems, sdeXY, sdeAB, and sdeCDE. Six out of the eight genes conferred multidrug resistance on KAM32, a drug hypersensitive strain of Escherichia coli. Five out of the eight genes conferred resistance to benzalkonium, suggesting the importance of RND efflux systems in biocide resistance in S. marcescens. The energy-dependent efflux activities of five of the pumps were examined using a rhodamine 6 G efflux assay. When expressed in the tolC-deficient strain of E. coli, KAM43, none of the genes conferred resistance on E. coli. When hasF, encoding the S. marcescens TolC ortholog, was expressed in KAM43, all of the genes conferred resistance on E. coli, suggesting that HasF is a major outer membrane protein that is used by all RND efflux systems in this organism. We constructed a sdeXY deletion mutant from a derivative strain of the clinically isolated multidrug-resistant S. marcescens strain and found that the sdeXY deletion mutant was sensitive to a broad spectrum of antimicrobial agents.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/genética , Infecciones por Serratia/genética , Serratia marcescens/efectos de los fármacos , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/genética , Compuestos de Benzalconio/farmacología , División Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Genoma Bacteriano/efectos de los fármacos , Genoma Bacteriano/genética , Humanos , Pruebas de Sensibilidad Microbiana , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Infecciones por Serratia/tratamiento farmacológico , Infecciones por Serratia/microbiología , Serratia marcescens/genética , Serratia marcescens/patogenicidadRESUMEN
PURPOSE: Pseudomonas aeruginosa produces pyoverdine, encoded by the pvdE gene, for high-affinity iron uptake from transferrin and lactoferrin. This study investigated the contribution of pyoverdine to P. aeruginosa keratitis pathogenesis using in vitro and in vivo models. METHODS: The P. aeruginosa strains examined were parental strain PAO1 and isogenic mutant strain pvdE (ΔpvdE) defective in pyoverdine. Bacterial growth in vitro was determined by PAO1 and ΔpvdE optical densities in Luria-Bertani (LB) broth. PAO1 or ΔpvdE (10 colony-forming units/mL) was inoculated onto cultured human corneal epithelial cells (HCECs) for 1 hour. The monolayers were examined for bacterial adhesion and invasion. In addition, the corneas of C57BL/6 mice were infected with PAO1 or ΔpvdE. Corneal virulence was evaluated by determining clinical scores and bacterial counts during infection. RESULTS: The growth of PAO1 and ΔpvdE in LB broth was similar. Although adhesion of ΔpvdE onto HCECs was significantly increased compared with PAO1, the invasive capacity of ΔpvdE was significantly decreased. Clinical scores and bacterial numbers were significantly lower in ΔpvdE-infected eyes compared with PAO1-infected eyes at 6, 24, and 48 hours (P < 0.001). ΔpvdE was not detected in mouse corneas and did not induce corneal opacity at 6, 24, or 48 hours. CONCLUSIONS: ΔpvdE lost invasive ability toward HCECs. Moreover, ΔpvdE did not cause keratitis in vivo. Thus, pvdE pyoverdine synthesis has critical roles in proliferation and invasion on ocular surfaces and could be a target for prevention of P. aeruginosa keratitis.
Asunto(s)
Queratitis/microbiología , Oligopéptidos/fisiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Animales , Adhesión Bacteriana/fisiología , Proliferación Celular/fisiología , Modelos Animales de Enfermedad , Epitelio Corneal/microbiología , Ratones Endogámicos C57BL , Pseudomonas aeruginosa/crecimiento & desarrolloRESUMEN
Pseudomonas aeruginosa can penetrate the layer of mucus formed by host intestinal epithelial cells, often resulting in sepsis in immunocompromised patients. We have previously demonstrated that P. aeruginosa can penetrate the mucin layer by flagellar motility and the degradation of the mucin layer. However, it remains unclear how P. aeruginosa initially recognizes epithelial cells. Using the human epithelial colorectal adenocarcinoma (Caco-2) cell line, we investigated extracellular signaling that could facilitate the penetration of P. aeruginosa through the mucin layer. The supernatant from Caco-2 cell cultures increased penetration of P. aeruginosa through an artificial mucin layer. The Caco-2 cell supernatant increased bacterial flagella-dependent swarming motility, but it did not influence P. aeruginosa growth or protease activity. Filtering of the Caco-2 cell supernatant indicated that proteins weighing <10 kDa increased mucin penetration, swarming motility, and, based on a tethered cell assay, induced acceleration of the flagellar filament rotational rate. Furthermore, a capillary assay showed that <10 kDa proteins in the Caco-2 cell supernatant attracted P. aeruginosa cells. Finally, we identified that growth-regulated oncogene-α (GRO-α) secreted by Caco-2 cells was a factor facilitating flagellar filament rotation and swarming motility, although it did not attract the bacteria. We conclude that penetration of the mucin layer by P. aeruginosa is facilitated by small proteins (<10 kDa) secreted by Caco-2 cells, both by inducing acceleration of flagellar motility and increasing chemotaxis.
Asunto(s)
Traslocación Bacteriana , Quimiotaxis/fisiología , Mucosa Intestinal/microbiología , Mucinas/metabolismo , Pseudomonas aeruginosa/patogenicidad , Células CACO-2 , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Citocinas/análisis , Flagelos/fisiología , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Humanos , Plásmidos/química , Plásmidos/genéticaRESUMEN
Ozenoxacin, a novel non-fluorinated topical quinolone, is used for the treatment of acne vulgaris in Japan. We investigated bactericidal activity and post-antibiotic effect (PAE) of ozenoxacin against Propionibacterium acnes, a major causative bacterium of acne vulgaris. The minimum inhibitory concentrations (MICs) of ozenoxacin against 3 levofloxacin-susceptible strains (MIC of levofloxacin; ≤4 µg/mL) and 3 levofloxacin-resistant strains (MIC of levofloxacin; ≥8 µg/mL) ranged from 0.03 to 0.06 µg/mL and from 0.25 to 0.5 µg/mL, respectively. These MICs of ozenoxacin were almost the same or lower than nadifloxacin and clindamycin. The minimum bactericidal concentrations (MBCs) of ozenoxacin against the levofloxacin-susceptible and -resistant strains were from 0.06 to 8 µg/mL and from 0.5 to 4 µg/mL, respectively. These MBCs were lower than those of nadifloxacin and clindamycin. In time-kill assay, ozenoxacin at 1/4, 1 and 4 times the respective MIC against both levofloxacin-susceptible and -resistant strains showed a concentration-dependent bactericidal activity. Ozenoxacin at 4 times the MICs against the levofloxacin-susceptible strains showed more potent and more rapid onset of bactericidal activity compared to nadifloxacin and clindamycin at 4 times the respective MICs. The PAEs of ozenoxacin at 4 times the MICs against the levofloxacin-susceptible strains were from 3.3 to 17.1 h, which were almost the same or longer than nadifloxacin and clindamycin. In contrast, the PAEs were hardly induced by any antimicrobial agents against the levofloxacin-resistant strains. The present findings suggest that ozenoxacin has a potent bactericidal activity against both levofloxacin-susceptible and -resistant P. acnes, and a long-lasting PAE against levofloxacin-susceptible P. acnes.
Asunto(s)
Aminopiridinas/farmacología , Antibacterianos/farmacología , Propionibacterium acnes/efectos de los fármacos , Quinolonas/farmacología , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Levofloxacino/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
A type IV pilus filament, mainly composed of PilA, is retracted by the driving power generated by PilT and PilU ATPases. pilA is required for injection of type III ExoS effectors into epithelial cells thereby facilitating Pseudomonas aeruginosa penetration through the epithelial barrier by impairing the defense function of tight junctions. Here, we examined whether the pilT and pilU of the P. aeruginosa PAO1 strain are required for ExoS injection into epithelial cells. We measured the quantity of ExoS injected into epithelial cells, and found that within such cells its quantity decreased by 80% (ΔpilA strain), 75% (ΔpilT strain), and 30% (ΔpilU strain) compared with the wild-type strain. pilT deficiency decreased the disruption of human epithelial colorectal adenocarcinoma (Caco-2) cell monolayers to the same extent as that of pilA and exoS deficiency, whereas pilU deficiency decreased disruption of the monolayers less than deficiency of the other genes. pilT and pilU deficiency decreased bacterial penetration of the Caco-2 cell monolayers to the same level as pilA and exoS deficiency. Our data showed that the pilU gene expression level was reduced in the PAO1 strain after adhesion to Caco-2 cell surfaces, but the expression levels of the pilA and pilT genes did not change. We conclude that P. aeruginosa injects ExoS into cells through the function of type IV pilus retraction, and that pilT makes a greater contribution to this process than pilU.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Células Epiteliales/microbiología , Fimbrias Bacterianas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Células CACO-2 , Proteínas Fimbrias/genética , Fimbrias Bacterianas/fisiología , Humanos , Uniones Intercelulares/microbiología , Mutación , Ocludina/metabolismoRESUMEN
Benzoyl peroxide (BPO), a therapeutic agent for acne vulgaris, was assessed for in vitro antimicrobial activity against Propionibacterium acnes using a novel broth microdilution testing that improved BPO solubility. We searched for a suitable culture medium to measure the minimum inhibitory concentration (MIC) of BPO against P. acnes and finally found the Gifu anaerobic medium (GAM) broth supplemented with 0.1(v/v)% glycerol and 2(v/v)% Tween 80, in which BPO dissolved up to 1250 µg/mL and P. acnes grew well. The MICs and minimum bactericidal concentrations (MBCs) of BPO against 44 clinical isolates of P. acnes collected from Japanese patients with acne vulgaris were determined by our testing method using the supplemented GAM broth. The MICs of BPO were 128 or 256 µg/mL against all isolates of P. acnes regardless of susceptibility to nadifloxacin or clindamycin. The MBCs of BPO were also 128 or 256 µg/mL against the same isolates. Moreover, BPO at the MIC showed a rapid bactericidal activity against P. acnes ATCC11827 in time-kill assay. In conclusion, we could develop a novel assay for the MIC and MBC determinations of BPO against P. acnes, which is reliable and reproducible as a broth microdilution testing and the present results suggest that BPO has a potent bactericidal activity against P. acnes.
Asunto(s)
Antibacterianos/farmacología , Peróxido de Benzoílo/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Propionibacterium acnes/efectos de los fármacos , Acné Vulgar/microbiología , Medios de Cultivo , Humanos , Propionibacterium acnes/aislamiento & purificación , Reproducibilidad de los ResultadosRESUMEN
A Pseudomonas aeruginosa quorum-sensing system, which produces N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12 -HSL) and N-butanoyl-l-homoserine lactone (C4 -HSL), regulates the virulence factors. In our previous study, 3-oxo-C12 -HSL, encoded by lasI gene, was shown to promote wound healing. However, the effect of C4 -HSL, encoded by rhlI gene, remains to be elucidated. We addressed the effect of C4 -HSL on wounds in P. aeruginosa infection. Wounds were created on the backs of Sprague-Dawley SD rats, and P. aeruginosa PAO1 (PAO1) or its rhlI deletion mutant (ΔrhlI) or lasI deletion mutant (ΔlasI) was inoculated onto the wound. Rats were injected intraperitoneally with anti-C4 -HSL antiserum or treated with C4 -HSL at the wound surface. PAO1 inoculation led to significant acceleration of wound healing, which was associated with neutrophil infiltration and TNF-α synthesis. These responses were reversed, except for TNF-α production, when ΔrhlI was inoculated instead of PAO1 or when rats were co-treated with PAO1 and anti-C4 -HSL antiserum. In contrast, the healing process and neutrophil infiltration, but not TNF-α synthesis, were accelerated when C4 -HSL was administered in the absence of PAO1. This acceleration was not affected by anti-TNF-α antibody. These results suggest that C4 -HSL may be involved in the acceleration of acute wound healing in P. aeruginosa infection by modifying the neutrophilic inflammation.
Asunto(s)
4-Butirolactona/análogos & derivados , Pseudomonas aeruginosa/enzimología , Cicatrización de Heridas/fisiología , Heridas y Lesiones/patología , Heridas y Lesiones/terapia , 4-Butirolactona/farmacología , Enfermedad Aguda , Análisis de Varianza , Animales , Biopsia con Aguja , Modelos Animales de Enfermedad , Femenino , Homoserina/análogos & derivados , Homoserina/farmacología , Inmunohistoquímica , Neutrófilos/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Estadísticas no Paramétricas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
PURPOSE: To examine bacterial virulence factors in Pseudomonas aeruginosa isolates from contact lens (CL) wearers and non-CL wearers with P. aeruginosa keratitis, and to investigate relationships between virulence factors and clinical features of keratitis. METHODS: The study involved 25 subjects including 18 CL and 7 non-CL-related P. aeruginosa keratitis patients. Slit-lamp photographs of all subjects were captured, and the focus occupancy ratio (FOR) was defined as the total focus area/entire cornea area, using image processing software. Twenty-five clinical P. aeruginosa isolates from keratitis were assessed for protease production, elastase production, biofilm formation, bacterial swimming and swarming motility, cell surface hydrophobicity, and genes encoding the type III secretion system (TTSS) effectors (ExoU and ExoS). RESULTS: Ring abscess was found in 9 of 18 CL-related P. aeruginosa keratitis cases (CL[+] ring[+] group) but not in another 9 cases (CL[+] ring[-] group). Expression or prevalence of virulence factors in P. aeruginosa isolates from the CL(+) ring(+) group, CL(+) ring(-) group, and CL(-) group were compared. The FOR for CL(+) ring(+) or CL(-) was higher than for CL(+) ring(-) (P < 0.05 and P < 0.01, respectively). The rate of positive swimming motility for CL(+) ring(+) or CL(-) was higher than for CL(+) ring(-) (P < 0.05), whereas the rate of positive swarming motility for CL(+) ring(+) was higher than for CL(+) ring(-) or CL(-) (P < 0.05). Prevalence of an exoS+/exoU-genotype for CL(+) ring(+) or CL(-) was higher than for CL(+) ring(-) (P < 0.05). In the CL-related group, expression of elastase and swarming motility significantly correlated with FOR. CONCLUSIONS: Swimming motility, swarming motility, and TTSS ExoS could play a major role in the determination of clinical features of P. aeruginosa keratitis.
Asunto(s)
Queratitis/microbiología , Infecciones por Pseudomonas/microbiología , Factores de Virulencia/fisiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biopelículas , Lentes de Contacto/efectos adversos , Lentes de Contacto/microbiología , Córnea/microbiología , Córnea/patología , Femenino , Humanos , Queratitis/patología , Masculino , Persona de Mediana Edad , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/patogenicidad , Lámpara de Hendidura , Adulto JovenRESUMEN
Translocation of Pseudomonas aeruginosa through epithelial tissues can cause sepsis. Here, we examined whether P. aeruginosa penetrates epithelial cell layers using type IV pili (TFP). Deletion of TFP (pilA) did not affect association with Caco-2 cells, although it decreased penetration through, and disruption of, Caco-2 cell monolayers. We found that TFP are necessary for injection of the type III effector ExoS, which impairs defense against P. aeruginosa penetration, into host cells. Deletion of pilA attenuated oral infection in silkworms. We conclude that P. aeruginosa injects ExoS into cells through the function of TFP, enabling penetration of epithelial barriers.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Toxinas Bacterianas/metabolismo , Células Epiteliales/metabolismo , Fimbrias Bacterianas/metabolismo , Pseudomonas aeruginosa/citología , Pseudomonas aeruginosa/metabolismo , ADP Ribosa Transferasas/genética , Animales , Toxinas Bacterianas/genética , Bombyx/microbiología , Células CACO-2 , Regulación Bacteriana de la Expresión Génica , Humanos , Uniones Intercelulares/metabolismo , Transporte de Proteínas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologíaRESUMEN
Serratia marcescens is an important nosocomial pathogen that can cause an array of infections, most notably of the urinary tract and bloodstream. Naturally, it is found in many environmental niches, and is capable of infecting plants and animals. The emergence and spread of multidrug-resistant strains producing extended-spectrum or metallo beta-lactamases now pose a threat to public health worldwide. Here we report the complete genome sequences of two carefully selected S. marcescens strains, a multidrug-resistant clinical isolate (strain SM39) and an insect isolate (strain Db11). Our comparative analyses reveal the core genome of S. marcescens and define the potential metabolic capacity, virulence, and multidrug resistance of this species. We show a remarkable intraspecies genetic diversity, both at the sequence level and with regards genome flexibility, which may reflect the diversity of niches inhabited by members of this species. A broader analysis with other Serratia species identifies a set of approximately 3,000 genes that characterize the genus. Within this apparent genetic diversity, we identified many genes implicated in the high virulence potential and antibiotic resistance of SM39, including the metallo beta-lactamase and multiple other drug resistance determinants carried on plasmid pSMC1. We further show that pSMC1 is most closely related to plasmids circulating in Pseudomonas species. Our data will provide a valuable basis for future studies on S. marcescens and new insights into the genetic mechanisms that underlie the emergence of pathogens highly resistant to multiple antimicrobial agents.
Asunto(s)
Genoma Bacteriano , Infecciones por Serratia/microbiología , Serratia marcescens/genética , Animales , Antibacterianos/farmacología , Resistencia a Múltiples Medicamentos , Evolución Molecular , Variación Genética , Humanos , Insectos/microbiología , Infecciones por Serratia/tratamiento farmacológico , Serratia marcescens/efectos de los fármacos , Serratia marcescens/aislamiento & purificación , Serratia marcescens/patogenicidadRESUMEN
DS-8587 is a novel broad-spectrum fluoroquinolone with extended antimicrobial activity against both Gram-positive and Gram-negative pathogens. In this study, we evaluated the antibacterial activity and mechanism of DS-8587 in 31 quinolone-resistant Acinetobacter baumannii clinical isolates. Efflux pump and qnr genes, mutations in quinolone resistance-determining regions of target enzymes, and sequence types determined by multilocus sequence typing were analyzed. Forty-two quinolone-susceptible clinical isolates were analyzed for comparison. For susceptibility testing, DS-8587 exhibited more effective antibacterial activity when compared with ciprofloxacin and levofloxacin. When combined with the efflux pump inhibitor 1-(1-napthylmethyl)-piperazine, the MIC of DS-8587 was less affected when compared with the MIC exhibited by combined ciprofloxacin and 1-(1-napthylmethyl)-piperazine. The efflux pump genes adeA/adeB/adeC and regulatory elements adeR/adeS were detected in 23 of 31 quinolone-resistant isolates. The qnrA/qnrB/qnrS genes were not detected in any A. baumannii isolates analyzed. Mutations in quinolone resistance-determining regions were observed in all 31 quinolone-resistant isolates. Multilocus sequence typing analyses revealed that 22 of 31 quinolone-resistant isolates belonged to ST-2, corresponding to international clonal lineage II. In conclusion, DS-8587 exhibits potent antibacterial activity against quinolone-resistant A. baumannii isolates that harbor mutations in quinolone resistance-determining regions. In the presence of the efflux pump inhibitor 1-(1-napthylmethyl)-piperazine, no significant changes were observed in the MIC for DS-8587. DS-8587 should be considered as a treatment option for A. baumannii including ST-2 strains that are predominant among the quinolone-resistant A. baumannii isolates found in Japan.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Fluoroquinolonas/farmacología , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/aislamiento & purificación , ADN Bacteriano/análisis , ADN Bacteriano/genética , Humanos , Japón , Pruebas de Sensibilidad MicrobianaRESUMEN
The lungs are a major site of Pseudomonas aeruginosa infection in patients with compromised immune systems. P. aeruginosa secretes a number of toxins by a type III secretion system, and these are important in virulence. One of these toxins, ExoS can induce a cytotoxic effect and is associated with the ability to produce lung damage. ExoS is a bifunctional toxin, with N-terminal GTPase-activating protein (GAP) activity and a C-terminal ADP ribosyl transferase (ADPRT) domain. Although these two domains have numerous potential cellular targets, the overall mechanism of ExoS-induced cytotoxicity remains unclear. We carried out a yeast two-hybrid screen using the ExoS truncation mutant ExoSΔ (residue 1-388), which lacks the 14-3-3 binding site in the ADPRT domain, to identify unknown cellular targets associated with ExoS-induced cytotoxicity. We identified the mammalian factor, kinesin family member 7 (KIF7), which is involved in Hedgehog signaling, as a binding partner for ExoSΔC2. A pull-down assay revealed that ExoS bound to the truncated KIF7 gene encoding the N-terminal domain (residues 1-109) of KIF7. Yeast two-hybrid analysis showed that the ADPRT domain (residues 234-354) of ExoS bound to the truncated KIF7. Furthermore, exoS gene expression and silencing the expression of KIF7 both caused significant cytotoxicity in cultured human bronchial epithelial cells (BEAS-2B). Taken together, our results suggest that ExoS could induce cytotoxicity in BEAS-2B cells by interacting with KIF7.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Toxinas Bacterianas/metabolismo , Cinesinas/metabolismo , Muerte Celular/fisiología , Línea Celular , Humanos , Mucosa Respiratoria/citología , Transducción de Señal/fisiología , Técnicas del Sistema de Dos HíbridosRESUMEN
PURPOSE: Pseudomonas aeruginosa is a leading pathogen of blinding keratitis worldwide. In this study, the role of the serine protease in the pathogenesis of P. aeruginosa keratitis in the mouse cornea was investigated by comparing the parent and rescue strains. METHODS: Cornea of C57BL/6 mice were infected with P. aeruginosa strain PAO1, serine protease (MucD protease or PA3535) mutants (ΔmucD or ΔPA3535), or a complemented strain. Corneal virulence was evaluated by determining clinical scores and bacterial enumeration. A myeloperoxidase assay was performed to determine the number of polymorphonuclear (PMN) cells infiltrating the cornea. An ELISA was used to quantify inflammatory cytokines and chemokines in the cornea. RESULTS: The clinical score and bacterial numbers in eyes infected with ΔmucD were significantly lower than in those infected with PAO1, ΔPA3535, or the MucD rescue strain after 48 hours (P < 0.001). A larger number of infiltrating PMN cells was observed in eyes infected with ΔmucD at 12 and 24 hours, compared with eyes infected with PAO1. IL-1ß, KC, and MIP2 levels were higher in eyes infected with ΔmucD than in those infected with PAO1 after 12 hours. CONCLUSIONS: The MucD protease suppressed IL-1ß, KC, and MIP2 during the early stages of the infection and inhibited neutrophil recruitment in the cornea. Therefore, the MucD protease contributes significantly to the pathogenesis of keratitis. MucD protease plays a critical role in the establishment of Pseudomonas aeruginosa keratitis by facilitating evasion of the immune response.
Asunto(s)
Proteínas Bacterianas/metabolismo , Córnea/microbiología , Infecciones Bacterianas del Ojo/microbiología , Queratitis/microbiología , Neutrófilos/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/enzimología , Serina Endopeptidasas/metabolismo , Animales , Córnea/metabolismo , Córnea/patología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Infecciones Bacterianas del Ojo/metabolismo , Infecciones Bacterianas del Ojo/patología , Femenino , Inmunohistoquímica , Queratitis/metabolismo , Queratitis/patología , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/aislamiento & purificaciónRESUMEN
Bacteria secrete effector proteins required for successful infection and expression of toxicity into host cells. The type III secretion apparatus is involved in these processes. Previously, we showed that the viscous polymer polyethylene glycol (PEG) 8000 suppressed effector secretion by Pseudomonas aeruginosa. We thus considered that other viscous polymers might also suppress secretion. We initially showed that PEG200 (formed from the same monomer (ethylene glycol) as PEG8000, but which forms solutions of lower viscosity than the latter compound) did not decrease effector secretion. By contrast, alginate, a high-viscous polymer formed from mannuronic and guluronic acid, unlike PEG8000, effectively inhibited secretion. The effectiveness of PEG8000 and alginate in this regard was closely associated with polymer viscosity, but the nature of viscosity dependence differed between the two polymers. Moreover, not only the natural polymer alginate, but also mucin, which protects against infection, suppressed secretion. We thus confirmed that polymer viscosity contributes to the suppression of effector secretion, but other factors (e.g. electrostatic interaction) may also be involved. Moreover, the results suggest that regulation of bacterial secretion by polymers may occur naturally via the action of components of biofilm or mucin layer.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Sistemas de Secreción Bacterianos/efectos de los fármacos , Proteínas Activadoras de GTPasa/metabolismo , Polímeros/farmacología , Pseudomonas aeruginosa/metabolismo , ADP Ribosa Transferasas/genética , Alginatos/farmacología , Toxinas Bacterianas/metabolismo , Biopelículas , Proteínas Activadoras de GTPasa/genética , Viabilidad Microbiana , Mucinas/farmacología , Polietilenglicoles/farmacología , Pseudomonas aeruginosa/genética , ViscosidadRESUMEN
In a continuing study from Dec 2006 to Apr 2008, we characterized nine multi-drug resistant Pseudomonas aeruginosa strains isolated from four patients in a ward at the Hiroshima University Hospital, Japan. Pulsed-field gel electrophoresis of SpeI-digested genomic DNAs from the isolates suggested the clonal expansion of a single strain; however, only one strain, NK0009, was found to produce metallo-ß-lactamase. PCR and subsequent sequencing analysis indicated NK0009 possessed a novel class 1 integron, designated as In124, that carries an array of four gene cassettes: a novel aminoglycoside (AG) resistance gene, aac(6')-Iag, blaIMP-1, a truncated form of blaIMP-1, and a truncated form of aac(6')-Iag. The aac(6')-Iag encoded a 167-amino-acid protein that shows 40% identity with AAC(6')-Iz. Recombinant AAC(6')-Iag protein showed aminoglycoside 6'-N-acetyltransferase activity using thin-layer chromatography (TLC) and MS spectrometric analysis. Escherichia coli carrying aac(6')-Iag showed resistance to amikacin, arbekacin, dibekacin, isepamicin, kanamycin, sisomicin, and tobramycin; but not to gentamicin. A conjugation experiment and subsequent Southern hybridization with the gene probes for blaIMP-1 and aac(6')-Ig strongly suggested In124 is on a conjugal plasmid. Transconjugants acquired resistance to gentamicin and were resistant to virtually all AGs, suggesting that the In124 conjugal plasmid also possesses a gene conferring resistance to gentamicin.
Asunto(s)
Acetiltransferasas/genética , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Integrones , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , beta-Lactamasas/genética , Acetilación , Acetiltransferasas/metabolismo , Aminoglicósidos/química , Aminoglicósidos/metabolismo , Secuencia de Bases , Infección Hospitalaria/epidemiología , Brotes de Enfermedades , Orden Génico , Humanos , Cinética , Datos de Secuencia Molecular , Filogenia , Infecciones por Pseudomonas/epidemiología , Pseudomonas aeruginosa/clasificación , Alineación de Secuencia , beta-Lactamasas/metabolismoRESUMEN
The type III secretion apparatus (T3SA) participates in the secretion of bacterial proteins called effectors, although the detailed mechanism of effector secretion remains unclear. T3SA and flagellum were shown to branch from a common ancestor and also show structural similarity. In addition, both T3SA-dependent effector secretion and flagellar rotation were reported to require proton-motive force (PMF) for activity. From these reports, we hypothesized that T3SA, like the flagellum, would rotate via PMF and that this rotation is responsible for effector secretion. To observe T3SA rotation, we constructed a novel observation system by modifying the tip of T3SA on bacterial cell membranes with an observation probe, which allowed documentation of T3SA rotation for the first time. T3SA rotation was stopped by the addition of a protonophore that decreases PMF. Moreover, increased viscosity of the observation medium inhibited both rotation of T3SA associated with beads and effector secretion. These results suggested that effector secretion would follow the PMF-dependent rotation of T3SA and could be inhibited by preventing T3SA rotation. Moreover, the motion-track analysis of bead rotation suggested that the T3SA needle might be flexible. Consequently, we propose a "rotational secretion model" as a novel effector secretion mechanism of T3SA.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/fisiología , Fuerza Protón-Motriz/fisiología , Pseudomonas aeruginosa/fisiología , ADP Ribosa Transferasas/metabolismo , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Western Blotting , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Flagelos/metabolismo , Flagelos/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Microscopía de Fuerza Atómica , Modelos Biológicos , Modelos Moleculares , Mutación , Oligopéptidos/genética , Oligopéptidos/metabolismo , Polietilenglicoles/farmacología , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Ionóforos de Protónes/farmacología , Fuerza Protón-Motriz/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , RotaciónRESUMEN
We investigated the in vitro activity of DS-8587, a novel fluoroquinolone, against Acinetobacter baumannii. The MICs of DS-8587 against clinical isolates and its inhibitory activity against target enzymes were superior to those of ciprofloxacin and levofloxacin. Furthermore, the antibacterial activity of DS-8587 was less affected by adeA/adeB/adeC or abeM efflux pumps than was that of ciprofloxacin and the frequency of single-step mutations with DS-8587 was lower than that with ciprofloxacin. DS-8587 might be an effective agent against A. baumannii infection.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/metabolismo , Antibacterianos/farmacología , Fluoroquinolonas/farmacología , Quinolonas/farmacología , Acinetobacter baumannii/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ciprofloxacina/farmacología , Pruebas de Sensibilidad Microbiana , Mutación/genéticaRESUMEN
BACKGROUND: Pseudomonas aeruginosa is frequently isolated from chronic wounds and causes serious infection in immunocompromised hosts. N-(3-Oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) is synthesized by an autoinducer synthase encoded by the bacterial lasI gene in P. aeruginosa, which regulates the production of virulence factors and biofilm formation in this bacterium. Recent studies have suggested that 3-oxo-C12-HSL contributes to the modulation of immune responses. However, the effect of this molecule on wound healing in P. aeruginosa infection remains to be elucidated. OBJECTIVE: We used an animal model to study the effect of 3-oxo-C12-HSL on wound healing in skin infected with P. aeruginosa. METHODS: Wounds were created on the backs of Sprague-Dawley (SD) rats and the P. aeruginosa strain PAO1 (PAO1) or its lasI deletion mutant (ΔlasI) was inoculated onto the wound surface. To examine the biological activity of 3-oxo-C12-HSL, rats were injected intraperitoneally with anti-3-oxo-C12-HSL antiserum or administered 3-oxo-C12-HSL at the wound surface. The wound tissues were harvested for analysis of the healing process and inflammatory response. RESULTS: PAO1 inoculation significantly accelerated the wound healing and inflammatory response on day 3 post-wounding. These responses were reversed by inoculation with ΔlasI instead of PAO1 or treatment with anti-3-oxo-C12-HSL antiserum. In contrast, administration of 3-oxo-C12-HSL in the absence of PAO1 significantly promoted these responses, which were suppressed by the anti-TNF-α mAb. CONCLUSION: These results strongly suggest that 3-oxo-C12-HSL may be involved in healing wounds infected with P. aeruginosa through induction of inflammatory responses.
Asunto(s)
4-Butirolactona/análogos & derivados , Homoserina/análogos & derivados , Neutrófilos/metabolismo , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Cicatrización de Heridas/fisiología , 4-Butirolactona/metabolismo , Animales , Anticuerpos Antiidiotipos/farmacología , Proliferación Celular , Modelos Animales de Enfermedad , Epidermis/patología , Homoserina/metabolismo , Masculino , Neovascularización Fisiológica/fisiología , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Pseudomonas aeruginosa can penetrate the extracellular mucin barrier formed by the intestinal epithelial cell layer and establish gut-derived sepsis in immunocompromised patients. We found that two efficient mechanisms, flagellar motility and mucin degradation, are needed for penetration of P. aeruginosa through the mucin barrier. Deletion of the flagellar motility-related gene, the filament protein gene fliC, the cap protein gene fliD, and the motor complex protein genes motABCD from P. aeruginosa PAO1 decreased association of P. aeruginosa with the apical surface of human epithelial colorectal adenocarcinoma (Caco-2) cells. A penetration experiment using an artificial mucin layer suggested that the decreased penetration is caused by attenuation of mucin penetration ability. Additionally, the presence of P. aeruginosa decreased the total mucin, including the secreted mucin protein MUC2, on the surface of the Caco-2 cell monolayer, regardless of flagellar motility. Construction of the PAO1 mutant series knocked out 12 putative serine protease genes and identified the mucD gene, which participated in degradation of total mucin, including MUC2. Furthermore, decreased association with the surface of the Caco-2 cell monolayer was observed in the mucD mutant, and the decrease was synergistically amplified by double knockout with fliC. We conclude that P. aeruginosa can penetrate the mucin layer using flagellar motility and mucin degradation, which is dependent on the MucD protease or the mucD gene-related protease.