RESUMEN
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
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
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
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
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
The lungs are a major site of Pseudomonas aeruginosa infection in patients with compromised immune systems. We have shown that a large number of cells of the P. aeruginosa wild-type PAO1 strain invade cultured human bronchial epithelial cells (BEAS-2B). In the present study, we investigated whether P. aeruginosa pilus protein PilA might modulate cellular functions by binding to unknown factor(s) in human host cells. Using a yeast two-hybrid screen, we showed that the calcium-modulating cyclophilin ligand (CAMLG), which is involved in Ca(2+) signaling, was the major host cell PilA binding protein. Overexpression of the pilA gene in BEAS-2B cells resulted in a significant increase in cytoplasmic Ca(2+) and consequent upregulation of the activity of the nuclear factor of activated T cells, followed by induction of cyclooxygenase 2 gene expression. Infection of BEAS-2B cells with the P. aeruginosa wild-type strain, but not with the pilA gene knockout strain (ΔpilA), caused a significant increase in intracellular Ca(2+) concentration in infected cells. Therefore, we propose a novel bacterial strategy for PilA modulation of intracellular Ca(2+) signaling through intracellular PilA-CAMLG interaction.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Señalización del Calcio/fisiología , Proteínas Fimbrias/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Células CACO-2 , Calcio/metabolismo , Línea Celular , Citoplasma/metabolismo , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Interacciones Huésped-Patógeno , Humanos , Microscopía Confocal , Unión Proteica , Infecciones por PseudomonasRESUMEN
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.
Asunto(s)
Movimiento Celular/fisiología , Flagelos/fisiología , Mucinas/metabolismo , Pseudomonas aeruginosa/fisiología , Proteínas Bacterianas/genética , Células CACO-2 , Eliminación de Gen , Interacciones Huésped-Patógeno , Humanos , Mutación , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidad , Serina Endopeptidasas/genéticaRESUMEN
BACKGROUND: Bacteria release a wide variety of small molecules including cell-to-cell signaling compounds. Gram-negative bacteria use a variety of self-produced autoinducers such as acylated homoserine lactones (acyl-HSLs) as signal compounds for quorum sensing (QS) within and between bacterial species. QS plays a significant role in the pathogenesis of infectious diseases and in beneficial symbiosis by responding to acyl-HSLs in Pseudomonas aeruginosa. It is considered that the selection of bacterial languages is necessary to regulate gene expression and thus it leads to the regulation of virulence and provides a growth advantage in several environments. In this study, we hypothesized that RND-type efflux pump system MexAB-OprM of P. aeruginosa might function in the selection of acyl-HSLs, and we provide evidence to support this hypothesis. RESULTS: Loss of MexAB-OprM due to deletion of mexB caused increases in QS responses, as shown by the expression of gfp located downstream of the lasB promoter and LasB elastase activity, which is regulated by a LasR-3-oxo-C12-HSL complex. Either complementation with a plasmid containing wild-type mexB or the addition of a LasR-specific inhibitor, patulin, repressed these high responses to 3-oxo-acyl-HSLs. Furthermore, it was shown that the acyl-HSLs-dependent response of P. aeruginosa was affected by the inhibition of MexB transport activity and the mexB mutant. The P. aeruginosa MexAB-OprM deletion mutant showed a strong QS response to 3-oxo-C10-HSL produced by Vibrio anguillarum in a bacterial cross-talk experiment. CONCLUSION: This work demonstrated that MexAB-OprM does not control the binding of LasR to 3-oxo-Cn-HSLs but rather accessibility of non-cognate acyl-HSLs to LasR in P. aeruginosa. MexAB-OprM not only influences multidrug resistance, but also selects acyl-HSLs and regulates QS in P. aeruginosa. The results demonstrate a new QS regulation mechanism via the efflux system MexAB-OprM in P. aeruginosa.
Asunto(s)
Acil-Butirolactonas/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Homoserina/análogos & derivados , Lactonas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Pseudomonas aeruginosa/fisiología , Percepción de Quorum , Fusión Artificial Génica , Proteínas de la Membrana Bacteriana Externa/genética , Eliminación de Gen , Genes Reporteros , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Homoserina/metabolismo , Proteínas de Transporte de Membrana/genética , Modelos Biológicos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Transducción de Señal , Vibrio/metabolismo , Vibrio/fisiologíaRESUMEN
Translocation of endogenous Pseudomonas aeruginosa from the colonized intestinal tract is an important pathogenic phenomenon. Comparative genome hybridization analysis of high virulent and low virulent strains allowed us to identify bacterial genes that are associated with bacterial translocation from gut in infected hosts. Here we focused on the pvdE pyoverdine synthesis gene among the identified bacterial genes, showing that the pvdE gene is required for bacterial penetration through epithelial cell monolayers and for bacterial translocation from gut to hemolymph in infected silkworms. We next revealed that mRNA expression level of the exoS gene in a pvdE-deficient mutant (ΔpvdE) after incubation with Caco-2 cells was greatly reduced as compared with that in the wild-type strain. The pvdE- and exoS-complemented ΔpvdE strains (ΔpvdE/pvdE and ΔpvdE/exoS) showed recovery of the ability of bacterial penetration through Caco-2 cell monolayers and of the ability of bacterial translocation from gut to hemolymph in infected silkworms. However, there were differences between the ability of ΔpvdE/pvdE and ΔpvdE/exoS to kill silkworms after intestinal infection and to replicate in hemolymph following direct injection into the hemolymph: ΔpvdE/pvdE could kill silkworms after intestinal infection and could replicate in hemolymph to levels similar to those of the wild-type strain, but ΔpvdE/exoS could not. Taken together, our results suggest that the virulence of the wild-strain mediated by the pvdE gene is the result of the ability to both penetrate through the intestinal epithelial cell barrier depending on ExoS and to replicate in hemolymph independently of ExoS.
Asunto(s)
ADP Ribosa Transferasas/genética , Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Bombyx/microbiología , Mucosa Intestinal/microbiología , Pseudomonas aeruginosa/genética , ADP Ribosa Transferasas/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos , Toxinas Bacterianas/metabolismo , Células CACO-2 , Hibridación Genómica Comparativa , Perros , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Prueba de Complementación Genética , Hemolinfa/microbiología , Interacciones Huésped-Patógeno , Humanos , Mucosa Intestinal/citología , Células de Riñón Canino Madin Darby , Mutación , Oligopéptidos/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Supervivencia , Virulencia/genéticaRESUMEN
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
OBJECTIVES: In vitro and in vivo antibacterial activities of modithromycin against Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae were examined. METHODS: MICs were determined by the broth microdilution method. Experimental infection of epithelial cell line A549 was performed to compare the intracellular activity and lasting effects of the antimicrobial agents. To evaluate in vivo efficacy, the rat pulmonary infection model was used. RESULTS: Modithromycin had MICs of ≤ 1 mg/L against all the clinical strains of both streptococci, including erythromycin-resistant strains. In particular, the MICs of modithromycin for erm(B)- or mef(A)-carrying S. pyogenes were 16-32 times or 2-4 times lower than those of telithromycin, respectively. The MIC(90) of modithromycin for H. influenzae was 8 mg/L, which was 4 times higher than that of telithromycin. Modithromycin, as well as azithromycin, showed a lasting inhibitory effect on bacterial growth of cell-associated H. influenzae compared with telithromycin and levofloxacin after removal of the agents from the apical medium. In the pulmonary infection model, modithromycin showed greater or comparable efficacy against erm(B)-carrying S. pneumoniae and H. influenzae, respectively, than telithromycin, regardless of having an MIC that was 2 or 4 times higher for these strains. CONCLUSIONS: Modithromycin has the most potent anti-S. pyogenes activity of the antimicrobial agents tested. Modithromycin also has the better in vivo efficacy against S. pneumoniae and H. influenzae, which might be due to its lasting intracellular activity.
Asunto(s)
Infecciones por Haemophilus/tratamiento farmacológico , Haemophilus influenzae/efectos de los fármacos , Macrólidos/farmacología , Macrólidos/uso terapéutico , Infecciones Estreptocócicas/tratamiento farmacológico , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pyogenes/efectos de los fármacos , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Modelos Animales de Enfermedad , Células Epiteliales/microbiología , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/aislamiento & purificación , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Neumonía Bacteriana/tratamiento farmacológico , Neumonía Bacteriana/microbiología , Ratas , Enfermedades de los Roedores/tratamiento farmacológico , Enfermedades de los Roedores/microbiología , Infecciones Estreptocócicas/microbiología , Streptococcus pneumoniae/aislamiento & purificación , Streptococcus pyogenes/aislamiento & purificación , Resultado del TratamientoRESUMEN
We investigated the influence of the type III effector, ExoS, on the host epithelial cell response to Pseudomonas aeruginosa infection, and we found that disruption of the exoS gene caused a significant increase in the amount of interleukin-8 (IL-8) in the culture medium of Caco-2 cells. We show that IL-8 was degraded in the culture medium following infection of the cells with the wild-type (PAO1), but not the exoS knock-out (the ΔexoS) strain. Purified ExoS protein itself did not degrade IL-8. We next show that IL-8 degradation by PAO1 was inhibited by the addition of serine protease inhibitors. These results strongly suggest that a bacterial serine protease that degrades IL-8 is expressed and secreted into the culture medium of Caco-2 cells infected with PAO1, and that the expression of this protein is repressed in cells infected with the ΔexoS strain. The PAO1 genome encodes 28 different protease genes, including two serine proteases: PA3535 and mucD. PA3535 and mucD gene knock-outs were constructed (ΔmucD and ΔPA3535), and ΔmucD but not ΔPA3535 showed reduced IL-8 degradation. To understand the significance of IL-8 degradation, we next evaluated neutrophil infiltration in lungs excised from mice intranasally infected with the P. aeruginosa strains. Increased neutrophil infiltration was observed in PAO1-infected mice, but not in ΔexoS- or ΔmucD-infected mice. Taken together, our results suggest that P. aeruginosa escapes from phagocytic killing due to IL-8 degradation following the secretion of the MucD serine protease, whose expression appears to be influenced by ExoS.
Asunto(s)
Proteínas Bacterianas/metabolismo , Interleucina-8/metabolismo , Pseudomonas aeruginosa/metabolismo , Serina Endopeptidasas/metabolismo , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/metabolismo , Animales , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Células CACO-2 , Femenino , Técnicas de Inactivación de Genes , Histocitoquímica , Interacciones Huésped-Patógeno , Humanos , Pulmón/química , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila , Neumonía Bacteriana/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Serina Endopeptidasas/genéticaRESUMEN
The intestinal tract is considered the most important reservoir of Pseudomonas aeruginosa in intensive care units (ICUs). Gut colonization by P. aeruginosa underlies the development of invasive infections such as gut-derived sepsis. Intestinal colonization by P. aeruginosa is associated with higher ICU mortality rates. The translocation of endogenous P. aeruginosa from the colonized intestinal tract is an important pathogenic phenomenon. Here we identify bacterial and host proteins associated with bacterial penetration through the intestinal epithelial barrier. We first show by comparative genomic hybridization analysis that the exoS gene, encoding the type III effector protein, ExoS, was specifically detected in a clinical isolate that showed higher virulence in silkworms following midgut injection. We further show using a silkworm oral infection model that exoS is required both for virulence and for bacterial translocation from the midgut to the hemolymph. Using a bacterial two-hybrid screen, we show that the mammalian factor FXYD3, which colocalizes with and regulates the function of Na,K-ATPase, directly binds ExoS. A pulldown assay revealed that ExoS binds to the transmembrane domain of FXYD3, which also interacts with Na,K-ATPase. Na,K-ATPase controls the structure and barrier function of tight junctions in epithelial cells. Collectively, our results suggest that ExoS facilitates P. aeruginosa penetration through the intestinal epithelial barrier by binding to FXYD3 and thereby impairing the defense function of tight junctions against bacterial penetration.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Toxinas Bacterianas/metabolismo , Bombyx/microbiología , Células Epiteliales/microbiología , Hemolinfa/microbiología , Intestinos/microbiología , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Pseudomonas aeruginosa/patogenicidad , Animales , Transporte Biológico , Células CACO-2 , Línea Celular , Hibridación Genómica Comparativa , Células Epiteliales/metabolismo , Humanos , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Uniones Estrechas/metabolismo , VirulenciaRESUMEN
Pseudomonas aeruginosa is an important opportunistic human pathogen. Certain strains can transmigrate across epithelial cells, and their invasive phenotype is correlated with capacity to cause invasive human disease and fatal septicemia in mice. Four multidrug efflux systems have been described in P. aeruginosa, however, their contribution to virulence is unclear. To clarify the role of efflux systems in invasiveness, P. aeruginosa PAO1 wild-type (WT) and its efflux mutants were evaluated in a Madin-Darby canine kidney (MDCK) epithelial cell monolayer system and in a murine model of endogenous septicemia. All efflux mutants except a deltamexCD-oprJ deletion demonstrated significantly reduced invasiveness compared with WT. In particular, a deltamexAB-oprM deletion strain was compromised in its capacity to invade or transmigrate across MDCK cells, and could not kill mice, in contrast to WT which was highly invasive (P < 0.0006) and caused fatal infection (P < 0.0001). The other mutants, including deltamexB and deltamexXY mutants, were intermediate between WT and the deltamexAB-oprM mutant in invasiveness and murine virulence. Invasiveness was restored to the deltamexAB-oprM mutant by complementation with mexAB-oprM or by addition of culture supernatant from MDCK cells infected with WT. We conclude that the P. aeruginosa MexAB-OprM efflux system exports virulence determinants that contribute to bacterial virulence.
Asunto(s)
Proteínas Bacterianas/genética , Traslocación Bacteriana/genética , Farmacorresistencia Bacteriana/genética , Proteínas de Transporte de Membrana , Pseudomonas aeruginosa/patogenicidad , Virulencia/genética , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Portadoras/genética , Línea Celular , Medios de Cultivo Condicionados/farmacología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Perros , Células Epiteliales/citología , Células Epiteliales/microbiología , Células Epiteliales/ultraestructura , Gentamicinas/farmacología , Riñón/citología , Ratones , Pruebas de Sensibilidad Microbiana , Mutación , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/fisiología , Sepsis/microbiología , Sepsis/patología , Tasa de Supervivencia , Factores de Tiempo , Virulencia/efectos de los fármacosRESUMEN
A mini-Tn5 insertion into a ciprofloxacin (CIP)-resistant mutant of Vibrio cholerae O1 revealed that overexpression of the vca0421 gene, which encodes a hypothetical protein, in the CIP-resistant mutant carrying a mutation in the quinolone resistance-determining region (QRDR) of the gyrA gene causes sensitization to CIP. We propose a new intrinsic mechanism of resistance to fluoroquinolones due to the inherently reduced expression of the vca0421 gene in V. cholerae O1.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Ciprofloxacina/farmacología , Vibrio cholerae O1/efectos de los fármacos , Fluoroquinolonas/farmacología , Quinolonas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vibrio cholerae O1/genéticaRESUMEN
OBJECTIVES: The aim of this study was to evaluate the dissemination of metallo-beta-lactamase (MBL)-encoding genes among multidrug-resistant (MDR) Pseudomonas aeruginosa isolates recovered from major hospitals in the Hiroshima region. METHODS: During July to December from 2004 to 2006, a surveillance of eight major hospitals in the Hiroshima region identified 387 non-duplicate isolates resistant to imipenem (MIC >or= 16 mg/L). They were screened for resistance to amikacin (MIC >or= 64 mg/L) and ciprofloxacin (MIC >or= 4 mg/L) and MBL-encoding genes. The structure of the variable regions of the integrons was determined using PCR mapping. Clonality was assessed using PFGE and multilocus sequence typing (MLST). RESULTS: The frequency of MBL-positive isolates in MDR P. aeruginosa isolates significantly increased from 42.3% in 2004 to 81.4% in 2006. Most of the MBL-positive isolates produced IMP-1 followed by VIM-2. The bla(IMP-1) and bla(VIM-2) genes were present in class 1 integrons. Characterization of the variable regions of the integron showed the presence of six different gene cassette arrays in bla(IMP-1) cassettes and a single array in bla(VIM-2) cassettes. The IMP-1 producers belonged to two clonal lineages using PFGE and MLST analyses and the integron variations correlated well with the clonal complexes. Among them, strains positive for a newly identified In113-derived bla(IMP-1) gene cassette array were most widely distributed in Hiroshima. CONCLUSIONS: This study shows a dramatic increase in MBL genes, primarily bla(IMP-1), in MDR P. aeruginosa isolates in Hiroshima during these 3 years. In addition, MDR P. aeruginosa with the newly discovered In113-derived bla(IMP-1) gene cassette array appears to be clonally expanding.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple , Infecciones por Pseudomonas/epidemiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/clasificación , Pseudomonas aeruginosa/enzimología , beta-Lactamasas/genética , Antibacterianos/farmacología , Técnicas de Tipificación Bacteriana , Análisis por Conglomerados , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , Dermatoglifia del ADN/métodos , ADN Bacteriano/genética , Genotipo , Hospitales , Humanos , Integrones , Japón/epidemiología , Pruebas de Sensibilidad Microbiana , Epidemiología Molecular , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , beta-Lactamasas/biosíntesisRESUMEN
BACKGROUND: Serratia marcescens is a gram-negative bacterium and often causes nosocomial infections. There have been few studies of the virulence factors of this bacterium. The only S. marcescens hemolytic and cytotoxic factor reported, thus far, is the hemolysin ShlA. RESULTS: An S. marcescens shlAB deletion mutant was constructed and shown to have no contact hemolytic activity. However, the deletion mutant retained hemolytic activity on human blood agar plates, indicating the presence of another S. marcescens hemolytic factor. Functional cloning of S. marcescens identified a phospholipase A (PhlA) with hemolytic activity on human blood agar plates. A phlAB deletion mutant lost hemolytic activity on human blood agar plates. Purified recombinant PhlA hydrolyzed several types of phospholipids and exhibited phospholipase A1 (PLA1), but not phospholipase A2 (PLA2), activity. The cytotoxic and hemolytic activities of PhlA both required phospholipids as substrates. CONCLUSION: We have shown that the S. marcescens phlA gene produces hemolysis on human blood agar plates. PhlA induces destabilization of target cell membranes in the presence of phospholipids. Our results indicated that the lysophospholipids produced by PhlA affected cell membranes resulting in hemolysis and cell death.