RESUMEN
Glucan-dependent biofilm formation is a crucial process in the establishment of Streptococcus mutans as a cariogenic oral microbe. The process of glucan formation has been investigated in great detail, with glycosyltransferases GtfB, GtfC, and GtfD shown to be indispensable for the synthesis of glucans from sucrose. Glucan production can be visualized during biofilm formation through fluorescent labeling, and its abundance, as well as the effect of glucans on general biofilm architecture, is a common phenotype to study S. mutans virulence regulation. Here, we describe an entirely new phenotype associated with glucan production, caused by a mutation in the open reading frame SMU_848, which is located in an operon encoding ribosome-associated proteins. This mutation led to the excess production and accumulation of glucan-containing droplets on the surface of biofilms formed on agar plates after prolonged incubation. While not characterized in S. mutans, SMU_848 shows homology to the phage-related ribosomal protease Prp, essential in cleaving off the N-terminal extension of ribosomal protein L27 for functional ribosome assembly in Staphylococcus aureus. We present a further characterization of SMU_848/Prp, demonstrating that the deletion of this gene leads to significant changes in S. mutans gtfBC expression. Surprisingly, it also profoundly impacts the interkingdom interaction between S. mutans and Candida albicans, a relevant dual-species interaction implicated in severe early childhood caries. The presented data support a potential broader role for SMU_848/Prp, possibly extending its functionality beyond the ribosomal network to influence important ecological processes. IMPORTANCE: Streptococcus mutans is an important member of the oral biofilm and is implicated in the initiation of caries. One of the main virulence mechanisms is the glucan-dependent formation of biofilms. We identified a new player in the regulation of glucan production, SMU_848, which is part of an operon that also encodes for ribosomal proteins L27 and L21. A mutation in SMU_848, which encodes a phage-related ribosomal protease Prp, leads to a significant accumulation of glucan-containing droplets on S. mutans biofilms, a previously unknown phenotype. Further investigations expanded our knowledge about the role of SMU_848 beyond its role in glucan production, including significant involvement in interkingdom interactions, thus potentially playing a global role in the virulence regulation of S. mutans.
Asunto(s)
Proteínas Bacterianas , Biopelículas , Glucanos , Streptococcus mutans , Streptococcus mutans/genética , Streptococcus mutans/metabolismo , Streptococcus mutans/enzimología , Biopelículas/crecimiento & desarrollo , Glucanos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Ribosomas/metabolismo , Mutación , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/genéticaRESUMEN
Historically, the study of microbe-associated diseases has focused primarily on pathogens, guided by Koch's postulates. This pathogen-centric view has provided a mechanistic understanding of disease etiology and microbial pathogenesis. However, next-generation sequencing approaches have revealed a far more nuanced view of the roles various microbes play in disease, highlighting the importance of microbial diversity beyond individual pathogens. This broader perspective acknowledges the roles of host and microbial communities in disease development and resistance. In particular, the concept of dysbiosis, especially within the oral cavity, has gained attention for explaining the emergence of complex polymicrobial diseases. Such diseases often stem from resident microbes rather than foreign pathogens, complicating their treatment and even clouding our understanding of disease etiology. Oral health is maintained through a delicate balance between commensal microbes and the host, with diseases like caries and periodontal disease arising from pathogenic perturbations of this balance. Commensal microbes, such as certain streptococci and Corynebacterium spp., play crucial roles in maintaining oral health through mechanisms involving hydrogen peroxide production and membrane vesicle secretion, which can inhibit pathogenic species and modulate host immune responses. Recent research focused upon the mechanisms of molecular commensalism has expanded our understanding of these key functions of the commensal microbiome, demonstrating their central role in promoting oral health and preventing disease. These abilities represent a largely untapped reservoir of potential innovative strategies for disease prevention and management, emphasizing the need to bolster a symbiotic microbiome that inherently suppresses pathogenesis.
RESUMEN
During oral biofilm development, interspecies interactions drive species distribution and biofilm architecture. To understand what molecular mechanisms determine these interactions, we used information gained from recent biogeographical investigations demonstrating an association of corynebacteria with streptococci. We previously reported that Streptococcus sanguinis and Corynebacterium durum have a close relationship through the production of membrane vesicle and fatty acids leading to S. sanguinis chain elongation and overall increased fitness supporting their commensal state. Here we present the molecular mechanisms of this interspecies interaction. Coculture experiments for transcriptomic analysis identified several differentially expressed genes in S. sanguinis. Due to its connection to fatty acid synthesis, we focused on the glycerol-operon. We further explored the differentially expressed type IV pili genes due to their connection to motility and biofilm adhesion. Gene inactivation of the glycerol kinase glpK had a profound impact on the ability of S. sanguinis to metabolize C. durum secreted glycerol and impaired chain elongation important for their interaction. Investigations on the effect of type IV pili revealed a reduction of S. sanguinis twitching motility in the presence of C. durum, which was caused by a decrease in type IV pili abundance on the surface of S. sanguinis as determined by SEM. In conclusion, we identified that the ability to metabolize C. durum produced glycerol is crucial for the interaction of C. durum and S. sanguinis. Reduced twitching motility could lead to a closer interaction of both species, supporting niche development in the oral cavity and potentially shaping symbiotic health-associated biofilm communities.
Asunto(s)
Glicerol , Streptococcus , Glicerol/metabolismo , Streptococcus sanguis/genética , Biopelículas , Simbiosis , Streptococcus mutansRESUMEN
Oral microbiome sequencing efforts revealed the presence of hundreds of different microbes. Interindividual differences at strain and species resolution suggest that microbiome diversity could lead to mechanistically distinct gene regulation as well as species-related differences in phenotypes. Commonly, gene regulation and related phenotypes are studied in a few selected strains of a particular species with conclusions that are mostly generalized. The aim of this study was to isolate several species of Corynebacterium using an established protocol that led to the previous isolation of C. durum. Characterization of C. durum interspecies interactions revealed a specific mechanism for chain elongation in Streptococcus sanguinis that was the result of corynebacterial fatty acid production and secretion. While the protocol was successfully applied to isolate what we presumed to be additional Corynebacterium based on several phenotypic traits that seem to be identical to C. durum, genome sequencing of the newly isolated strains placed them closer to Actinomyces. Both Corynebacterium and Actinomyces are suborders of the Actinobacteridae and related species. Our study suggests to take several comprehensive strategies into consideration when taxonomically identifying closely related microorganisms. Furthermore, it seems to be important to test common core phenotypes in bacterial ecology to understand the behavior of specific groups of microbes, rather than simply relying upon genome sequence homology to establish relationships in the microbiome.
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Corynebacterium , Microbiota , Actinomyces/genética , Corynebacterium/genética , ADN Bacteriano/genética , Ácidos Grasos , Microbiota/genética , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Streptococcus sanguis/genéticaRESUMEN
Neutrophil accumulation is associated with lung pathology during active tuberculosis (ATB). However, the molecular mechanism or mechanisms by which neutrophils accumulate in the lung and contribute to TB immunopathology are not fully delineated. Using the well-established mouse model of TB, our new data provide evidence that the alarmin S100A8/A9 mediates neutrophil accumulation during progression to chronic TB. Depletion of neutrophils or S100A8/A9 deficiency resulted in improved Mycobacterium tuberculosis (Mtb) control during chronic but not acute TB. Mechanistically, we demonstrate that, following Mtb infection, S100A8/A9 expression is required for upregulation of the integrin molecule CD11b specifically on neutrophils, mediating their accumulation during chronic TB disease. These findings are further substantiated by increased expression of S100A8 and S100A9 mRNA in whole blood in human TB progressors when compared with nonprogressors and rapidly decreased S100A8/A9 protein levels in the serum upon TB treatment. Furthermore, we demonstrate that S100A8/A9 serum levels along with chemokines are useful in distinguishing between ATB and asymptomatic Mtb-infected latent individuals. Thus, our results support targeting S100A8/A9 pathways as host-directed therapy for TB.
Asunto(s)
Antígeno CD11b/inmunología , Calgranulina A/inmunología , Calgranulina B/inmunología , Mycobacterium tuberculosis/inmunología , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , Tuberculosis/inmunología , Animales , Antígeno CD11b/genética , Calgranulina A/genética , Calgranulina B/genética , Ratones , Ratones Noqueados , Neutrófilos/patología , Tuberculosis/genética , Tuberculosis/patología , Tuberculosis/terapiaRESUMEN
Two-component signal transduction systems (TCSTS) are abundant among prokaryotes and regulate important functions, including drug resistance and virulence. The Gram-negative bacterium Burkholderia pseudomallei, which causes the severe infectious disease melioidosis, encodes 136 putative TCSTS components. In silico analyses of these TCSTS indicated that the predicted BbeR-BbeS system (BPSL1036-BPSL1037) displayed significant amino acid sequence similarity to the Shigella flexneri virulence-associated OmpR-EnvZ osmoregulator. To assess the function of the B. pseudomallei BbeR-BbeS system, we constructed by allelic exchange a ΔbbeRS double mutant strain lacking both genes, and single ΔbbeR and ΔbbeS mutants. All three mutant strains caused disease in the BALB/c acute melioidosis model at the same rate as the wild-type strain, displayed unchanged swarming motility on semi-solid medium, and were unaffected for viability on high-osmolarity media. However, when cultured at 37 °C for at least 14 days, ΔbbeS and ΔbbeR colonies developed a distinct, hypermucoid morphology absent in similarly-cultured wild-type colonies. At both 30 °C and 37 °C, these hypermucoid strains produced wild-type levels of type I capsule but released increased quantities of extracellular DNA (eDNA). Upon static growth in liquid medium, all B. pseudomallei strains produced pellicle biofilms that contained DNA in close association with bacterial cells; however, the ΔbbeS and ΔbbeR strains produced increased biofilms with altered microscopic architecture compared to the wild-type. Unusually, while the ΔbbeS and ΔbbeR single-deletion mutants displayed clear phenotypes, the ΔbbeRS double-deletion mutant was indistinguishable from the wild-type strain. We propose that BbeR-BbeS indirectly affects eDNA secretion and biofilm formation through cross-talk with one or more other TCSTS.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/fisiología , ADN/metabolismo , Eliminación de Gen , Transducción de Señal/genética , Animales , Proteínas Bacterianas/genética , Melioidosis/microbiología , Ratones Endogámicos BALB C , Mutación , Fenotipo , VirulenciaRESUMEN
The oral microbiome engages in a diverse array of highly sophisticated ecological interactions that are crucial for maintaining symbiosis with the host. Streptococci and corynebacteria are among the most abundant oral commensals and their interactions are critical for normal biofilm development. In this study, we discovered that Streptococcus sanguinis specifically responds to the presence of Corynebacterium durum by dramatically altering its chain morphology and improving its overall fitness. By employing gas chromatography-mass spectrometry (GC-MS) analysis, specific fatty acids were identified in C. durum supernatants that are responsible for the observed effect. Membrane vesicles (MVs) containing these fatty acids were isolated from C. durum supernatants and were able to replicate the chain morphology phenotype in S. sanguinis, suggesting MV as a mediator of interspecies interactions. Furthermore, S. sanguinis responds to C. durum lipids by decreasing the expression of key FASII genes involved in fatty acid synthesis. Several of these genes are also essential for the chain elongation phenotype, which implicates a regulatory connection between lipid metabolism and chain elongation. In addition, C. durum was found to affect the growth, cell aggregation, and phagocytosis of S. sanguinis, revealing a complex association of these species that likely supports oral commensal colonization and survival.
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Corynebacterium/fisiología , Streptococcus sanguis/fisiología , Simbiosis , Biopelículas/crecimiento & desarrollo , Microbiota , Streptococcus , Streptococcus sanguis/genética , Streptococcus sanguis/metabolismoRESUMEN
Many commensal oral streptococci generate H2O2 via pyruvate oxidase (SpxB) to inhibit the growth of competing bacteria like Streptococcus mutans, a major cariogenic species. In Streptococcus sanguinis SK36 (SK36) and Streptococcus gordonii DL1 (DL1), spxB expression and H2O2 release are subject to carbon catabolite repression by the catabolite control protein A (CcpA). Surprisingly, ccpA deletion mutants of SK36 and DL1 fail to inhibit S. mutans despite their production of otherwise inhibitory levels of H2O2. Using H2O2-deficient spxB deletion mutants of SK36 and DL1, it was subsequently discovered that both strains confer protection in trans to other bacteria when H2O2 is added exogenously. This protective effect depends on the direct detoxification of H2O2 by the release of pyruvate. The pyruvate dependent protective effect is also present in other spxB-encoding streptococci, such as the pneumococcus, but is missing from spxB-negative species like S. mutans. Targeted and transposon-based mutagenesis revealed Nox (putative H2O-forming NADH dehydrogenase) as an essential component required for pyruvate release and oxidative protection, while other genes such as sodA and dps play minor roles. Furthermore, pyruvate secretion is only detectable in aerobic growth conditions at biofilm-like cell densities and is responsive to CcpA-dependent catabolite control. This ability of spxB-encoding streptococci reveals a new facet of the competitive interactions between oral commensals and pathobionts and provides a mechanistic basis for the variable levels of inhibitory potential observed among H2O2-producing strains of commensal oral streptococci.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Ácido Pirúvico/metabolismo , Streptococcus/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Piruvato Oxidasa/genética , Piruvato Oxidasa/metabolismo , Streptococcus gordonii/genética , Streptococcus gordonii/metabolismo , Streptococcus mutans , Streptococcus pneumoniae , Streptococcus sanguis/genética , Streptococcus sanguis/crecimiento & desarrollo , Streptococcus sanguis/metabolismo , SimbiosisRESUMEN
BACKGROUND: Burkholderia pseudomallei is the causative agent of melioidosis, a severe invasive disease of humans and animals. Initial screening of a B. pseudomallei signature-tagged mutagenesis library identified an attenuated mutant with a transposon insertion in a gene encoding the sensor component of an uncharacterised two-component signal transduction system (TCSTS), which we designated BprRS. RESULTS: Single gene inactivation of either the response regulator gene (bprR) or the sensor histidine kinase gene (bprS) resulted in mutants with reduced swarming motility and reduced virulence in mice. However, a bprRS double mutant was not attenuated for virulence and displayed wild-type levels of motility. The transcriptomes of the bprS, bprR and bprRS mutants were compared with the transcriptome of the parent strain K96243. Inactivation of the entire BprRS TCSTS (bprRS double mutant) resulted in altered expression of only nine genes, including both bprR and bprS, five phage-related genes and bpss0686, encoding a putative 5, 10-methylene tetrahydromethanopterin reductase involved in one carbon metabolism. In contrast, the transcriptomes of each of the bprR and bprS single gene mutants revealed more than 70 differentially expressed genes common to both mutants, including regulatory genes and those required for flagella assembly and for the biosynthesis of the cytotoxic polyketide, malleilactone. CONCLUSIONS: Inactivation of the entire BprRS TCSTS did not alter virulence or motility and very few genes were differentially expressed indicating that the definitive BprRS regulon is relatively small. However, loss of a single component, either the sensor histidine kinase BprS or its cognate response regulator BprR, resulted in significant transcriptomic and phenotypic differences from the wild-type strain. We hypothesize that the dramatically altered phenotypes of these single mutants are the result of cross-regulation with one or more other TCSTSs and concomitant dysregulation of other key regulatory genes.
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Burkholderia pseudomallei/patogenicidad , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Factores de Virulencia/genética , Proteínas Bacterianas/genética , Burkholderia pseudomallei/genética , Regulación Bacteriana de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Mutación , VirulenciaRESUMEN
Many Gram-negative pathogens use a type III secretion system (TTSS) for the injection of bacterial effector proteins into host cells. The injected effector proteins play direct roles in modulation of host cell pathways for bacterial benefit. Burkholderia pseudomallei, the causative agent of melioidosis, expresses three different TTSSs. One of these systems, the TTSS3, is essential for escape from host endosomes and therefore intracellular survival and replication. Here we have characterized three putative TTSS3 proteins; namely BapA, BapB and BapC. By employing a tetracysteine (TC)-FlAsH™ labelling technique to monitor the secretion of TC-tagged fusion proteins, BapA and BapC were shown to be secreted during in vitro growth in a TTSS3-dependant manner, suggesting a role as TTSS3 effectors. Furthermore, we constructed B. pseudomallei bapA, bapB and bapC mutants and used the well-characterized TTSS3 effector BopE as a marker of secretion to show that BapA, BapB and BapC are not essential for the secretion process. However, BopE transcription and secretion were significantly increased in the bapB mutant, suggesting that BapB levels modulate BopE expression. In a BALB/c mouse model of acute melioidosis, the bapA, bapB and bapC mutants showed a minor reduction of in vivo fitness. Thus, this study defines BapA and BapC as novel TTSS3 effectors, BapB as a regulator of BopE production, and all three as necessary for full B. pseudomallei in vivo fitness.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Animales , Transporte Biológico/genética , Femenino , Melioidosis/genética , Melioidosis/metabolismo , Melioidosis/microbiología , Ratones , Ratones Endogámicos BALB C , Mutación/genética , Virulencia/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Melioidosis is an infectious disease of high mortality for humans and other animal species; it is prevalent in tropical regions worldwide. The pathogenesis of melioidosis depends on the ability of its causative agent, the Gram-negative bacterium Burkholderia pseudomallei, to enter and survive in host cells. B. pseudomallei can escape from the phagosome into the cytosol of phagocytic cells where it replicates and acquires actin-mediated motility, avoiding killing by the autophagy-dependent process, LC3 (microtubule-associated protein light chain 3)-associated phagocytosis (LAP). The type III secretion system cluster 3 (TTSS3) facilitates bacterial escape from phagosomes, although the mechanism has not been fully elucidated. Given the recent identification of small-molecule inhibitors of the TTSS ATPase, we sought to determine the potential of the predicted TTSS3 ATPase, encoded by bsaS, as a target for chemotherapeutic treatment of infection. A B. pseudomallei bsaS deletion mutant was generated and used as a control against which to assess the effect of inhibitor treatment. Infection of RAW 264.7 cells with wild-type bacteria and subsequent treatment with the ATPase inhibitor compound 939 resulted in reduced intracellular bacterial survival, reduced escape from phagosomes, and increased colocalization with both LC3 and the lysosomal marker LAMP1 (lysosome-associated membrane protein 1). These changes were similar to those observed for infection of RAW 264.7 cells with the bsaS deletion mutant. We propose that treatment with the ATPase inhibitor compound 939 decreased intracellular bacterial survival through a reduced ability of bacteria to escape from phagosomes and increased killing via LAP. Therefore, small-molecule inhibitors of the TTSS3 ATPase have potential as therapeutic treatments against melioidosis.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Sistemas de Secreción Bacterianos/inmunología , Burkholderia pseudomallei/efectos de los fármacos , Burkholderia pseudomallei/enzimología , Melioidosis/tratamiento farmacológico , Animales , Proteínas Bacterianas/genética , Burkholderia pseudomallei/genética , Línea Celular , Femenino , Evasión Inmune , Estimación de Kaplan-Meier , Proteínas de Membrana de los Lisosomas/inmunología , Melioidosis/patología , Ratones , Ratones Endogámicos BALB C , Proteínas Asociadas a Microtúbulos/inmunología , Fagocitosis/inmunología , Factores de Virulencia/genéticaRESUMEN
Burkholderia pseudomallei, the causal agent of melioidosis, employs a number of virulence factors during its infection of mammalian cells. One such factor is the type three secretion system (TTSS), which is proposed to mediate the transport and secretion of bacterial effector molecules directly into host cells. The B. pseudomallei genome contains three TTSS gene clusters (designated TTSS1, TTSS2, and TTSS3). Previous research has indicated that neither TTSS1 nor TTSS2 is involved in B. pseudomallei virulence in a hamster infection model. We have characterized a B. pseudomallei mutant lacking expression of the predicted TTSS1 ATPase encoded by bpscN. This mutant was significantly attenuated for virulence in a respiratory melioidosis mouse model of infection. In addition, analyses in vitro showed diminished survival and replication in RAW264.7 cells and an increased level of colocalization with the autophagy marker protein LC3 but an unhindered ability to escape from phagosomes. Taken together, these data provide evidence that the TTSS1 bpscN gene product plays an important role in the intracellular survival of B. pseudomallei and the pathogenesis of murine infection.
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Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos/genética , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/patogenicidad , Melioidosis/microbiología , Animales , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Familia de Multigenes , Fagosomas , Eliminación de Secuencia , Factores de Virulencia/genéticaRESUMEN
Burkholderia pseudomallei is the causative agent of melioidosis, a fatal infectious disease endemic in tropical regions worldwide, and especially prevalent in southeast Asia and northern Australia. This intracellular pathogen can escape from phagosomes into the host cytoplasm, where it replicates and infects adjacent cells. We previously demonstrated that, in response to B. pseudomallei infection of macrophage cell line RAW 264.7, a subset of bacteria co-localized with the autophagy marker protein, microtubule-associated protein light chain 3 (LC3), implicating autophagy in host cell defence against infection. Recent reports have suggested that LC3 can be recruited to both phagosomes and autophagosomes, thereby raising questions regarding the identity of the LC3-positive compartments in which invading bacteria reside and the mechanism of the autophagic response to B. pseudomallei infection. Electron microscopy analysis of infected cells demonstrated that the invading bacteria were either free in the cytosol, or sequestered in single-membrane phagosomes rather than double-membrane autophagosomes, suggesting that LC3 is recruited to B. pseudomallei-containing phagosomes. Partial or complete loss of function of type III secretion system cluster 3 (TTSS3) in mutants lacking the BopA (effector) or BipD (translocator) proteins respectively, resulted in delayed or no escape from phagosomes. Consistent with these observations, bopA and bipD mutants both showed a higher level of co-localization with LC3 and the lysosomal marker LAMP1, and impaired survival in RAW264.7 cells, suggesting enhanced killing in phagolysosomes. We conclude that LC3 recruitment to phagosomes stimulates killing of B. pseudomallei trapped in phagosomes. Furthermore, BopA plays an important role in efficient escape of B. pseudomallei from phagosomes.
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Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/inmunología , Evasión Inmune/inmunología , Proteínas Asociadas a Microtúbulos/metabolismo , Fagocitosis/inmunología , Animales , Autofagia , Proteínas Bacterianas/genética , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/ultraestructura , Línea Celular , Citosol/metabolismo , Regulación Bacteriana de la Expresión Génica , Espacio Intracelular/microbiología , Proteínas de Membrana de los Lisosomas/metabolismo , Ratones , Mutación/genética , Fagosomas/metabolismo , Fagosomas/microbiología , Transporte de Proteínas , Vacuolas/metabolismo , Vacuolas/microbiología , Vacuolas/ultraestructuraRESUMEN
Pseudomonas aeruginosa and Burkholderia cepacia are the major pathogens that colonize the airway surface and cause progressive respiratory failure and high mortality, especially in cystic fibrosis (CF) patients. Tobramycin is the treatment of choice, but persistent usage enables the infectious organisms to activate defence mechanisms, making eradication rarely successful. Combinations of antibiotic and nonantibiotic compounds have been tested in vitro against P. aeruginosa and B. cepacia, but with mixed results. Sodium ions interfere with the bacterial tobramycin uptake system, but amiloride partially reverses this antagonism. In this pilot study, we extend previous findings of the effectiveness of tobramycin in combination with amiloride and other nonantibiotics against a P. aeruginosa type strain, and against four P. aeruginosa strains and one Burkholderia cenocepacia strain isolated from CF patients. Significantly, the four clinical P. aeruginosa strains were tobramycin resistant. We also find that Na+ and K+, but not Cl(-), are the chief antagonists of tobramycin efficacy. These results suggest that chemotherapy for CF patients might not only be compromised by antibiotic-resistant pathogens alone, but by a lack of penetration of antibiotics caused either by bacterial biofilms or the high sodium flux in the CF lung, or by antagonistic effects of some drug combinations, any of which could allow the persistence of drug-susceptible bacteria.