A multi-phase mathematical model of quorum sensing in a maturing Pseudomonas aeruginosa biofilm

It is well known that sessile bacteria have a strong tendency to exist in a biofilm phenotype, whereby bacterial cells aggregate and produce a gel-like extracellular matrix, which, in an infection scenario, offers a significant barrier to attack by conventional antibiotics and the immune system. In this paper we develop a multi-phase model of a maturing Pseudomonas aeruginosa biofilm, allowing for the production and secretion of exopolysaccharide (EPS). The primary quorum-sensing system of P. aeruginosa (namely the lasR system) is believed to be required for full biofilm development, and we thus take the synthesis of EPS to be regulated by the cognate signal molecule, 3-oxo-C12-HSL. We also take EPS and signal production, along with bacterial growth, to be limited by oxygen availability, thus factoring in the nutrient poor conditions deep inside the biofilm. We use simulations to examine the role played by quorum sensing in the biofilm maturation process, and to investigate the effect of anti-quorum sensing and antibiotic treatments on EPS concentration, signal level, bacterial numbers and biofilm growth rate. In addition, we undertake analysis of the associated travelling-wave behaviour.     

Metabolism Dependent Chemotaxis of Pseudomonas aeruginosa N1 Towards Anionic Detergent Sodium Dodecyl Sulfate

Sodium dodecyl sulfate (SDS) is one of the most commonly used detergent, which exhibits excellent biocidal activity against various bacteria and fungi. It is commonly employed in many detergent formulations and is employed for disinfection purposes. It is shown to be toxic to fishes, aquatic animals and is also inhibitory to microbes and cyanobacteria. We had isolated a strain belonging to Pseudomonas aeruginosa N1, from a detergent contaminated pond situated in Varanasi city India, which was able to degrade and metabolize SDS as a source of carbon. In the present investigation, we have studied chemotactic response of this strain towards SDS. The results clearly indicate that this strain showed chemotactic response towards SDS. The nature of chemotaxis was found to be metabolism dependent as glucose grown cells showed a delayed chemotactic response towards SDS. This is first study that reported chemotaxis response for P. aeruginosa towards anionic detergent SDS.     

A trade-off between oxidative stress resistance and DNA repair plays a role in the evolution of elevated mutation rates in bacteria

The dominant paradigm for the evolution of mutator alleles in bacterial populations is that they spread by indirect selection for linked beneficial mutations when bacteria are poorly adapted. In this paper, we challenge the ubiquity of this paradigm by demonstrating that a clinically important stressor, hydrogen peroxide, generates direct selection for an elevated mutation rate in the pathogenic bacterium Pseudomonas aeruginosa as a consequence of a trade-off between the fidelity of DNA repair and hydrogen peroxide resistance. We demonstrate that the biochemical mechanism underlying this trade-off in the case of mutS is the elevated secretion of catalase by the mutator strain. Our results provide, to our knowledge, the first experimental evidence that direct selection can favour mutator alleles in bacterial populations, and pave the way for future studies to understand how mutation and DNA repair are linked to stress responses and how this affects the evolution of bacterial mutation rates.     

Protist predation can favour cooperation within bacterial species

Here, we studied how protist predation affects cooperation in the opportunistic pathogen bacterium Pseudomonas aeruginosa, which uses quorum sensing (QS) cell-to-cell signalling to regulate the production of public goods. By competing wild-type bacteria with QS mutants (cheats), we show that a functioning QS system confers an elevated resistance to predation. Surprisingly, cheats were unable to exploit this resistance in the presence of cooperators, which suggests that resistance does not appear to result from activation of QS-regulated public goods. Instead, elevated resistance of wild-type bacteria was related to the ability to form more predation-resistant biofilms. This could be explained by the expression of QS-regulated resistance traits in densely populated biofilms and floating cell aggregations, or alternatively, by a pleiotropic cost of cheating where less resistant cheats are selectively removed from biofilms. These results show that trophic interactions among species can maintain cooperation within species, and have further implications for P. aeruginosa virulence in environmental reservoirs by potentially enriching the cooperative and highly infective strains with functional QS system.     

PslG,a self-produced glycosyl hydrolase,triggers biofilm disassembly by disrupting exopolysaccharide matrix

Biofilms are surface-associated communities of microorganism embedded in extracellular matrix. Exopolysaccharide is a critical component in the extracellular matrix that maintains biofilm architecture and protects resident biofilm bacteria from antimicrobials and host immune attack. However, self-produced factors that target the matrix exopolysaccharides, are still poorly understood. Here, we show that PslG, a protein involved in the synthesis of a key biofilm matrix exopolysaccharide Psl in Pseudomonas aeruginosa, prevents biofilm formation and disassembles existing biofilms within minutes at nanomolar concentrations when supplied exogenously. The crystal structure of PslG indicates the typical features of an endoglycosidase. PslG mainly disrupts the Psl matrix to disperse bacteria from biofilms. PslG treatment markedly enhances biofilm sensitivity to antibiotics and macrophage cells, resulting in improved biofilm clearance in a mouse implant infection model. Furthermore, PslG shows biofilm inhibition and disassembly activity against a wide range of Pseudomonas species, indicating its great potential in combating biofilm-related complications.     

The antimicrobial susceptibility,biofilm formation and genotypic profiles of Staphylococcus haemolyticus from bloodstream infections

We analysed the antimicrobial susceptibility, biofilm formation and genotypic profiles of 27 isolates of Staphylococcus haemolyticus obtained from the blood of 19 patients admitted to a hospital in Rio de Janeiro, Brazil. Our analysis revealed a clinical significance of 36.8% and a multi-resistance rate of 92.6% among these isolates. All but one isolate carried the mecA gene. The staphylococcal cassette chromosome mec type I was the most prevalent mec element detected (67%). Nevertheless, the isolates showed clonal diversity based on pulsed-field gel electrophoresis analysis. The ability to form biofilms was detected in 66% of the isolates studied. Surprisingly, no icaAD genes were found among the biofilm-producing isolates.     

抗微生物肽CRAMP联合抗生素分散铜绿假单胞菌生物被膜的增效作用研究

【目的】研究修饰后的鼠源抗微生物肽CRAMP联用抗生素对铜绿假单胞菌PAO1成熟生物被膜的分散作用,为临床联合应用抗生物被膜药物提供理论依据。【方法】采用微量肉汤稀释法测定CRAMP修饰肽和抗生素对PAO1的最低抑菌浓度(MIC)、最低杀菌浓度(MBC)和生物被膜最小根除浓度(MBEC);采用时间杀菌曲线(time-kill curve,TKC)法测定CRAMP修饰肽及抗生素单用和联用对PAO1成熟生物被膜的杀菌活性;采用菌落计数法和激光共聚焦扫描显微镜(CLSM)评估CRAMP修饰肽联用抗生素对PAO1成熟生物被膜的分散作用。【结果】与单用抗生素组相比,除2种碳青霉烯类药物和4种β-内酰胺类药物,其他抗生素联合CRAMP修饰肽后的MBEC值均有不同程度的下降,万古霉素、罗红霉素和阿奇霉素下降倍数最明显(4倍)。TKC试验结果表明,CRAMP修饰肽分别联用万古霉素、罗红霉素和阿奇霉素均具有比单用药更快且更强的杀菌作用,尤其是与万古霉素联用仅在3h时杀灭了全部(100%)的生物被膜细菌。随后,通过CLSM观察发现生物被膜数量、体积、面积和单位面积荧光强度均有明显的变化。【结论】CRAMP...     

Preparation,Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more “temperate swarmers” that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. “Wettability”, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.     

The draft genome sequence of multidrug-resistant Pseudomonas aeruginosa strain CCBH4851, a nosocomial isolate belonging to clone SP (ST277) that is prevalent in Brazil

The high occurrence of nosocomial multidrug-resistant (MDR) microorganisms isconsidered a global health problem. Here, we report the draft genome sequence of aMDR Pseudomonas aeruginosa strain isolated in Brazil that belongsto the endemic clone ST277. The genome encodes important resistance determinantgenes and consists of 6.7 Mb with a G+C content of 66.86% and 6,347 predictedcoding regions including 60 RNAs.     

Co-inoculation of Urea and DAP Tolerant <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> as Integrated Approach for Growth Enhancement of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Two plant growth promoting rhizobacteria––Sinorhizobium meliloti RMP1 and Pseudomonas aeruginosa GRC₂ were studied for integrated nutrient management to obtain improved yield of Brassica juncea. Low concentrations of urea and diammonium phosphate (DAP) stimulated the growth of both S. meliloti RMP1 and P. aeruginosa GRC₂. 1 M of urea and 0.35 M of DAP was found lethal for RMP1, while 1.3 M and 0.37 M concentrations of urea and DAP proved to be toxic for GRC₂. Lc₅₀ was observed as 0.49 M of urea and 0.15 M of DAP for RMP1, and 0.66 M urea and 0.18 M of DAP for GRC₂. Urea and DAP adaptive variants of RMP1 and GRC₂ was isolated. Adaptive bacterial variants had better growth rates at sub-lethal (Lc₅₀) concentrations of urea and DAP as compared to non-adaptive variants. They also retained plant growth promoting attributes similar to non adaptive variants. GRC₂ and RMP1 did not affect the growth of each other and were chemotactically active for DAP, urea as well as root exudates of B. juncea. Both the isolates colonized well in the rhizosphere of B. juncea, as their populations were recorded ≈5 log₁₀ cfu g⁻¹ after 120 days. Interestingly, the colonization ability was found even better when both strains were co-inoculated, as their population was recorded in the range of ≈6 log₁₀ cfu g⁻¹ after 120 days. In field trials, application of RMP1 and GRC₂ resulted in significant increase in biomass and yield of B. juncea as compared to control. However, yield was better with application of half dose and full dose of recommended fertilizers. Interestingly, the biomass as well as yield improved further when both isolates were applied together along with half dose of recommended fertilizers.     

Binding of protegrin-1 to Pseudomonas aeruginosa and Burkholderia cepacia

Background  

Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy. Protegrins are antimicrobial peptides with potent activity against many bacteria, including P. aeruginosa. The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P. aeruginosa and B. cepacia.     

PH826噬菌体与抗生素联用有效抑制多重耐药铜绿假单胞菌生物被膜的形成

【目的】铜绿假单胞菌(Pseudomonas aeruginosa)是一种重要的革兰氏阴性病原体,可以加重囊性纤维化患者的肺部感染,最终会导致患者的死亡。然而由于多重耐药(multi-drug resistant,MDR)和泛耐药(pan-drug resistant, PDR)铜绿假单胞菌菌株的出现,使其防控变得更为严峻。【方法】从养殖场污水中分离能有效裂解多重耐药铜绿假单胞菌的噬菌体,研究其形态特征、生物学特性、宿主谱范围、基因组特征和体外抑菌能力等,并采用噬菌体和抗生素联用的方法进行生物被膜的抑制试验。【结果】透射电子显微镜的形态分析和基因组分析结合表明,该噬菌体属于Nankokuvirus病毒属。生物学特性试验表明,PH826具有广泛的温度稳定性(4-60℃)和pH稳定性(3.0-11.0)。宿主谱测试显示,PH826可以裂解13株铜绿假单胞菌(包括人源和动物源),体外抑菌试验显示,PH826在感染复数(multiplicity of infection, MOI)分别为10、1、0.1时对铜绿假单胞菌均有强烈的裂解作用。根据基因组分析,PH826噬菌体的基因组大小为87 95...     

Study of the initial phase of biofilm formation using a biofomic approach

Aims

The purpose of this work was to study the initial steps of formation of a biofilm using the BioFilm Ring Test® and the Crystal violet staining technique.

Methods and results

Eight strains of Pseudomonas aeruginosa were studied. The two methods revealed that four strains formed a rapid biofilm. The biofilm formed by these strains was detected after only 45 min with the BioFilm Ring Test® and after 6 h with the Crystal violet method. The enumeration of bacteria of the PA01 strain confirmed that, after 30 min, a significant amount of bacteria had attached on the bottom of the culture wells. After 48 h the Crystal violet method detected a biofilm with all strains. The four strains which rapidly formed a biofilm did not differ from the slow-forming strains by their mucoid character or their swarming motility or their synthesis of rhamnose. They showed higher swimming mobility.

Conclusions

Our results show that the BioFilm Ring Test® is a method specially suited for the study of the initial phase of the formation of a biofilm.

Significance and impact of study

The BioFilm Ring Test® is an easy and rapid alternative to the Crystal violet staining and the enumeration methods.     

Quorum quenching quandary: resistance to antivirulence compounds

Quorum sensing (QS) is the regulation of gene expression in response to the concentration of small signal molecules, and its inactivation has been suggested to have great potential to attenuate microbial virulence. It is assumed that unlike antimicrobials, inhibition of QS should cause less Darwinian selection pressure for bacterial resistance. Using the opportunistic pathogen Pseudomonas aeruginosa, we demonstrate here that bacterial resistance arises rapidly to the best-characterized compound that inhibits QS (brominated furanone C-30) due to mutations that increase the efflux of C-30. Critically, the C-30-resistant mutant mexR was more pathogenic to Caenorhabditis elegans in the presence of C-30, and the same mutation arises in bacteria responsible for chronic cystic fibrosis infections. Therefore, bacteria may evolve resistance to many new pharmaceuticals thought impervious to resistance.     

One shot-two pathogens blocked: Exposure of Arabidopsis to hexadecane,a long chain volatile organic compound,confers induced resistance against both Pectobacterium carotovorum and Pseudomonas syringae

Bacteria and plant derived volatile organic compounds have been reported as the chemical triggers that elicit induced resistance in plants. Previously, volatile organic compounds (VOCs), including acetoin and 2,3-butanediol, were found to be emitted from plant growth-promoting rhizobacteria (PGPR) Bacillus subtilis GB03, which had been shown to elicit ISR and plant growth promotion. More recently, we reported data that stronger induced resistance could be elicited against Pseudomonas syringae pv maculicola ES4326 in plants exposed to C13 VOC from another PGPR Paenibacillus polymyxa E681 compared with that of strain GB03. Here, we assessed whether another long hydrocarbon C16 hexadecane (HD) conferred protection to Arabidopsis from infection of a biotrophic pathogen, P. syringae pv maculicola and a necrotrophic pathogen, Pectobacterium carotovorum subsp carotovorum. Collectively, long-chain VOCs can be linked to a plant resistance activator for protecting plants against both biotrophic and necrotrophic pathogens at the same time.     

An experimental study of strong reciprocity in bacteria

Strong reciprocity, whereby cooperators punish non-cooperators, may help to explain the evolutionary success of cooperative behaviours. However, theory suggests that selection for strong reciprocity can depend upon tight genetic linkage between cooperation and punishment, to avoid the strategy being outcompeted by non-punishing cooperators. We tested this hypothesis using experimental populations of the bacterium Pseudomonas aeruginosa, which cooperate by producing iron-scavenging siderophores and, in this context, punish non-cooperators with toxins. Consistent with theory, we show that cooperative punishers can indeed invade cheats, but only when the traits are tightly linked. These results emphasize that punishment is only likely to be favoured when the punishment itself leads to a direct or indirect fitness benefit to the actor.