Sputum containing zinc enhances carbapenem resistance,biofilm formation and virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa chronic lung infections are the leading cause of mortality in cystic fibrosis patients, a serious problem which is notably due to the numerous P. aeruginosa virulence factors, to its ability to form biofilms and to resist the effects of most antibiotics. Production of virulence factors and biofilm formation by P. aeruginosa is highly coordinated through complex regulatory systems. We recently found that CzcRS, the zinc and cadmium-specific two-component system is not only involved in metal resistance, but also in virulence and carbapenem antibiotic resistance in P. aeruginosa. Interestingly, zinc has been shown to be enriched in the lung secretions of cystic fibrosis patients. In this study, we investigated whether zinc might favor P. aeruginosa pathogenicity using an artificial sputum medium to mimic the cystic fibrosis lung environment. Our results show that zinc supplementation triggers a dual P. aeruginosa response: (i) it exacerbates pathogenicity by a CzcRS two-component system-dependent mechanism and (ii) it stimulates biofilm formation by a CzcRS-independent mechanism. Furthermore, P. aeruginosa cells embedded in these biofilms exhibited increased resistance to carbapenems. We identified a novel Zn-sensitive regulatory circuit controlling the expression of the OprD porin and modifying the carbapenem resistance profile. Altogether our data demonstrated that zinc levels in the sputum of cystic fibrosis patients might aggravate P. aeruginosa infection. Targeting zinc levels in sputum would be a valuable strategy to curb the increasing burden of P. aeruginosa infections in cystic fibrosis patients.     

Azithromycin retards Pseudomonas aeruginosa biofilm formation

Using a flow cell biofilm model, we showed that a sub-MIC of azithromycin (AZM) can delay but not inhibit Pseudomonas aeruginosa biofilm formation and results in the development of a stable AZM resistance phenotype. Furthermore, mature biofilms were not affected by AZM.     

Determinants for persistence of Pseudomonas aeruginosa in hospitals: interplay between resistance,virulence and biofilm formation

Pseudomonas aeruginosa (Pa) is one of the major bacterial pathogens causing nosocomial infections. During the past few decades, multidrug-resistant (MDR) and extensively drug-resistant (XDR) lineages of Pa have emerged in hospital settings with increasing numbers. However, it remains unclear which determinants of Pa facilitated this spread. A total of 211 clinical XDR and 38 susceptible clinical Pa isolates (nonXDR), as well as 47 environmental isolates (EI), were collected at the Heidelberg University Hospital. We used RAPD PCR to identify genetic clusters. Carriage of carbapenamases (CPM) and virulence genes were analyzed by PCR, biofilm formation capacity was assessed, in vitro fitness was evaluated using competitive growth assays, and interaction with the host’s immune system was analyzed using serum killing and neutrophil killing assays. XDR isolates showed significantly elevated biofilm formation (p?<?0.05) and higher competitive fitness compared to nonXDR and EI isolates. Thirty percent (62/205) of the XDR isolates carried a CPM. Similarities in distribution of virulence factors, as well as biofilm formation properties, between CPM+ Pa isolates and EI and between CPM- and nonXDR isolates were detected. Molecular typing revealed two distinct genetic clusters within the XDR population, which were characterized by even higher biofilm formation. In contrast, XDR isolates were more susceptible to the immune response than nonXDR isolates. Our study provides evidence that the ability to form biofilms is an outstanding determinant for persistence and endemic spread of Pa in the hospital setting.     

阿奇霉素对铜绿假单胞菌生物被膜和毒力因子的干预作用

目的 研究阿奇霉素对铜绿假单胞菌生物被膜形成及毒力因子分泌的干预作用.方法 2倍稀释法测定最低抑菌浓度(MIC);结晶紫染色法测定早期黏附;建立PAO1体外生物被膜模型;扫描电镜观察生物被膜形态;连续稀释法进行活菌计数;弹性蛋白-刚果红法测定弹性蛋白酶活性;偶氮酪蛋白法测定蛋白水解酶活性;氯仿萃取法测定绿脓菌素;苔黑酚法测定鼠李糖脂.结果 PAO1阿奇霉素组对载体的黏附低于PAO1空白对照组(P＜0.05);第3、7天PAO1阿奇霉素组生物被膜少于PAO1空白对照组,且载体活菌计数少于PAO1空白对照组(P＜0.05).PAO1阿奇霉素组弹性蛋白酶活性、蛋白水解酶活性、绿脓菌素浓度、鼠李糖脂浓度均明显低于PAO1空白对照组(P＜0.01),弹性蛋白酶活性及绿脓菌素浓度与PA-JP3株组相当(P＞0.05),而蛋白水解酶活性及鼠李糖脂浓度高于PA-JP3株组(P＜0.05).结论 阿奇霉素可以抑制铜绿假单胞菌的黏附及其生物被膜的形成,并可以抑制其毒力因子的释放.     

Cross-sectional analysis of clinical and environmental isolates of Pseudomonas aeruginosa: biofilm formation, virulence, and genome diversity

Chronic lung infections with Pseudomonas aeruginosa biofilms are associated with refractory and fatal pneumonia in cystic fibrosis (CF). In this study, a group of genomically diverse P. aeruginosa isolates were compared with the reference strain PAO1 to assess the roles of motility, twitching, growth rate, and overproduction of a capsular polysaccharide (alginate) in biofilm formation. In an in vitro biofilm assay system, P. aeruginosa displayed strain-specific biofilm formation that was not solely dependent on these parameters. Compared with non-CF isolates, CF isolates expressed two opposing growth modes: reduced planktonic growth versus efficient biofilm formation. Planktonic cells of CF isolates showed elevated sensitivity to hydrogen peroxide, a reactive oxygen intermediate, and decreased lung colonization in an aerosol infection mouse model. Despite having identical genomic profiles, CF sequential isolates produced different amounts of biofilm. While P. aeruginosa isolates exhibited genomic diversity, the genome size of these isolates was estimated to be 0.4 to 19% (27 to 1,184 kb) larger than that of PAO1. To identify these extra genetic materials, random amplification of polymorphic DNA was coupled with PAO1-subtractive hybridization. Three loci were found within the genomes of two CF isolates encoding one novel homolog involved in retaining a Shigella virulence plasmid (mvpTA) and two divergent genes that function in removing negative supercoiling (topA) and biosynthesis of pyoverdine (PA2402). Together, P. aeruginosa biodiversity could provide one cause for the variation of morbidity and mortality in CF. P. aeruginosa may possess undefined biofilm adhesins that are important to the development of an antibiofilm therapeutic target.     

Motility of Pseudomonas aeruginosa contributes to SOS-inducible biofilm formation

DNA-damaging antibiotics such as ciprofloxacin induce biofilm formation and the SOS response through autocleavage of SOS-repressor LexA in Pseudomonas aeruginosa. However, the biofilm-SOS connection remains poorly understood. It was investigated with 96-well and lipid biofilm assays. The effects of ciprofloxacin were examined on biofilm stimulation of the SOS mutant and wild-type strains. The stimulation observed in the wild-type in which SOS was induced was reduced in the mutant in which LexA was made non-cleavable (LexAN) and thus SOS non-inducible. Therefore, the stimulation appeared to involve SOS. The possible mechanisms of inducible biofilm formation were explored by subproteomic analysis of outer membrane fractions extracted from biofilms. The data predicted an inhibitory role of LexA in flagellum function. This premise was tested first by functional and morphological analyses of flagellum-based motility. The flagellum swimming motility decreased in the LexAN strain treated with ciprofloxacin. Second, the motility-biofilm assay was performed, which tested cell migration and biofilm formation. The results showed that wild-type biofilm increased significantly over the LexAN. These results suggest that LexA repression of motility, which is the initial event in biofilm development, contributes to repression of SOS-inducible biofilm formation.     

Roles for flagellar stators in biofilm formation by Pseudomonas aeruginosa

While Pseudomonas aeruginosa has only a single flagellum, its genome encodes two flagellar stators, called MotAB and MotCD. Here we report that despite no apparent alterations in swimming motility, mutations in either the MotAB or the MotCD stator render the strains defective for biofilm formation in both static and flow cell systems. Our data suggest distinct roles for the stators in early biofilm formation, with both the MotAB and MotCD stators playing a role in initial polar attachment of the bacterial cell to the surface (reversible attachment) and the MotAB stator also participating in the downstream adherence event of irreversible attachment. We also show that the initial polar attachment of P. aeruginosa to two different abiotic surfaces occurs largely at the flagellated end of the cell, a finding that should help develop models for early attachment events. Interestingly, in flowing conditions, a mutation in either stator alone revealed a more severe biofilm defect than mutating both stators or mutating the flagellum. Our data suggest that defects in biofilm formation observed for the stator mutants may be in part due to impacting flagellar reversal rates.     

Aspirin is an efficient inhibitor of quorum sensing,virulence and toxins in Pseudomonas aeruginosa

Quorum sensing (QS) plays a vital role in regulation of virulence factors and toxins in Pseudomonas aeruginosa, which can cause serious human infections. Therefore, the QS system in P. aeruginosa may be an important target for pharmacological intervention. Activity of aspirin on the QS system was assessed using a reporter strain assay and confirmed using RT-PCR to test expression of virulence factors and toxins. In addition, molecular modeling techniques including docking, flexible alignment and surface mapping were also applied to further understand aspirin's potential QS inhibition activity. Aspirin (6 mg/ml) showed significant reduction (p < 0.01) of quorum sensing signals in P. aeruginosa, including expression of elastase, total proteases, and pyocyanin (p < 0.01) without affecting bacterial viability. Aspirin also significantly reduced organism motility and biofilm production (p < 0.01) and decreased expression of lasI, lasR, rhlI, rhlR, pqsA and pqsR genes by 38, 72, 69, 72, 74 and 43% respectively. Moreover, the expression of Pseudomonas toxins exoS and exoY was reduced by 47 and 55% respectively. The molecular modeling analysis suggests the QS inhibitory action of aspirin occurs through interaction of aspirin's aryl group and Tyr-88 of the LasR receptor, by strong π–π stacking interactions, which associated with a conformational change of the receptor–aspirin complex. The inhibitory effect of aspirin on virulence factors was specific to P. aeruginosa as aspirin at sub-MIC did not affect the biofilm or motility of Escherichia coli.To summarize, the collective data demonstrate that low concentrations of aspirin inhibit quorum sensing of P. aeruginosa.     

Effect of plant phenolic compounds on biofilm formation by Pseudomonas aeruginosa

In the natural environment, bacteria predominantly exist in matrix‐enclosed multicellular communities associated with various surfaces, referred to as biofilms. Bacteria in biofilms are extremely resistant to antibacterial agents thus causing serious problems for antimicrobial therapy. In this study, we showed that different plant phenolic compounds, at concentrations that did not or weakly suppressed bacterial growth, increased the capacity of Pseudomonas aeruginosa PAO1 to form biofilms. Biofilm formation of P. aeruginosa PAO1 was enhanced 3‐ to 7‐fold under the action of vanillin and epicatechin, and 2‐ to 2.5‐fold in the presence of 4‐hydroxybenzoic, gallic, cinnamic, sinapic, ferulic, and chlorogenic acids. At higher concentrations, these compounds displayed an inhibiting effect. Similar experiments carried out for comparison with Agrobacterium tumefaciens C58 showed the same pattern. Vanillin, 4‐hydroxybenzoic, and gallic acids at concentrations within the range of 40 to 400 μg/mL increased the production of N–3‐oxo‐dodecanoyl‐homoserine lactone in P. aeruginosa PAO1 which suggests a possible relationship between stimulation of biofilm formation and Las Quorum Sensing system of this bacterium. Using biosensors to detect N‐acyl‐homoserine lactones (AHL), we demonstrated that the plant phenolics studied did not mimic AHLs.     

Phenazines affect biofilm formation by Pseudomonas aeruginosa in similar ways at various scales

Some pseudomonads produce phenazines, a group of small, redox-active compounds with diverse physiological functions. In this study, we compared the phenotypes of Pseudomonas aeruginosa strain PA14 and a mutant unable to synthesize phenazines in flow cell and colony biofilms quantitatively. Although phenazine production does not impact the ability of PA14 to attach to surfaces, as has been shown for Pseudomonas chlororaphis Maddula et al., 2006, Maddula et al., 2008, it influences swarming motility and the surface-to-volume ratio of mature biofilms. These results indicate that phenazines affect biofilm development across a large range of scales, but in unique ways for different Pseudomonas species.     

铜绿假单胞菌感染豚鼠后生物被膜形成的研究

目的 建立体内铜绿假单胞菌生物被膜模型，研究体内细菌生物被膜的组织学及细菌学特征。方法 通过吸入法使铜绿假单胞菌以气雾剂的形式吸入豚鼠肺内并生长定植，分别观察不同时期肺组织内细菌生物被膜的特征。结果 定植在肺内的铜绿假单胞菌以肉芽肿结节的形式存在，外周包绕类上皮细胞和成纤维细胞。结节内细菌被被膜基质所包绕并彼此连结，中间镶嵌宿主炎性细胞。接种后3周，肺内仍可见结节并培养出铜绿假单胞菌。结论 用吸入法可建立较稳定的铜绿假单胞菌肺感染生物被膜，其以肉芽肿结节的形式存在，宿主的反应细胞参与了生物被膜的形成。     

Role of lipopolysaccharide in virulence of Pseudomonas aeruginosa

The role of lipopolysaccharide (LPS) in the virulence of Pseudomonas aeruginosa was studied. The virulence of several P. aeruginosa strains for burned mice was found to be directly related to the dispersion of LPS into either the phenol or the water phase after extraction. Virulence decreased as the proportion of LPS recovered from the phenol phase increased. No similar correlation was observed when several other strain characteristics were investigated. This phenomenon was studied in greater detail by using the "smooth"-specific phage E79 to select mutants altered in LPS structure. One such mutant, PA220-R2, was extensively characterized. LPS isolated from PA220-R2 was found to be completely deficient in high-molecular-weight polysaccharide material. This alteration rendered the strain serum sensitive and dramatically changed the reaction with O-specific typing sera and sensitivity to typing phages. However, motility, toxin A and elastase production, and 22 metabolic functions remained unchanged. PA220-R2 was found to be comparatively nonvirulent, with a 50% lethal dose more than 1,000-fold higher than that of its parent for burned mice. This was due to the inability of PA220-R2 to establish an infection in burned skin.     

Subinhibitory bismuth-thiols reduce virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a common pathogen in mechanically ventilated patients and produces a wide array of virulence factors. Bismuth-thiols (BTs) are active in vitro against all bacterial lung pathogens, including P. aeruginosa. The objective of these studies was to examine the biochemical and morphologic effects of sublethal BT concentrations on P. aeruginosa and to evaluate virulence in cell culture. Bismuth-dimercaprol, at a fraction of the minimal inhibitory concentration, reduced alginate expression by 67% in P. aeruginosa, whereas subinhibitory bismuth-ethanedithiol (BisEDT) reduced alginate by 92% in P. syringae. BisEDT effects on lipopolysaccharide content and type III secreted cytoxins were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Subinhibitory BisEDT reduced cell-associated lipopolysaccharide, and inhibited processing of the secreted cytotoxic protein ExoU. BisEDT-induced outer membrane blebbing and aggregation of cytoplasmic material was noted in electron microscopy. Virulence of P. aeruginosa was assessed by adherence to epithelial cells and sensitivity to serum killing. BisEDT inhibited adherence of P. aeruginosa to 16HBE14o- cells by 28% and to a collagen matrix by 53%. BisEDT-treated bacteria were also 100-fold more sensitive to serum bactericidal activity. In summary, low BT concentrations affect P. aeruginosa in a variety of ways, the combination of which may help prevent or resolve respiratory tract infection.     

Full virulence of Pseudomonas aeruginosa requires OprF

OprF is a general outer membrane porin of Pseudomonas aeruginosa, a well-known human opportunistic pathogen associated with severe hospital-acquired sepsis and chronic lung infections of cystic fibrosis patients. A multiphenotypic approach, based on the comparative study of a wild-type strain of P. aeruginosa, its isogenic oprF mutant, and an oprF-complemented strain, showed that OprF is required for P. aeruginosa virulence. The absence of OprF results in impaired adhesion to animal cells, secretion of ExoT and ExoS toxins through the type III secretion system (T3SS), and production of the quorum-sensing-dependent virulence factors pyocyanin, elastase, lectin PA-1L, and exotoxin A. Accordingly, in the oprF mutant, production of the signal molecules N-(3-oxododecanoyl)-l-homoserine lactone and N-butanoyl-l-homoserine lactone was found to be reduced and delayed, respectively. Pseudomonas quinolone signal (PQS) production was decreased, while its precursor, 4-hydroxy-2-heptylquinoline (HHQ), accumulated in the cells. Taken together, these results show the involvement of OprF in P. aeruginosa virulence, at least partly through modulation of the quorum-sensing network. This is the first study showing a link between OprF, PQS synthesis, T3SS, and virulence factor production, providing novel insights into virulence expression.     

Staphylococcus quorum sensing in biofilm formation and infection

Cell population density-dependent regulation of gene expression is an important determinant of bacterial pathogenesis. Staphylococci have two quorum-sensing (QS) systems. The accessory gene regulator (agr) is genus specific and uses a post-translationally modified peptide as an autoinducing signal. In the pathogens Staphylococcus aureus and Staphylococcus epidermidis, agr controls the expression of a series of toxins and virulence factors and the interaction with the innate immune system. However, the role of agr during infection is controversial. A possible second QS system of staphylococci, luxS, is found in a variety of Gram-positive and Gram-negative bacteria. Importantly, unlike many QS systems described in Gram-negative bacteria, agr and luxS of staphylococci reduce rather than induce biofilm formation and virulence during biofilm-associated infection. agr enhances biofilm detachment by up-regulation of the expression of detergent-like peptides, whereas luxS reduces cell-to-cell adhesion by down-regulating expression of biofilm exopolysaccharide. Significant QS activity in staphylococci is observed for actively growing cells at a high cell density, such as during the initial stages of an infection and under optimal environmental conditions. In contrast, the metabolically quiescent biofilm mode of growth appears to be characterized by an overall low activity of the staphylococcal QS systems. It remains to be shown whether QS control in staphylococci represents a promising target for the development of novel antibacterial agents.     

Pseudomonas aeruginosa relA contributes to virulence in Drosophila melanogaster

The stringent response is a mechanism by which bacteria adapt to nutritional deficiencies through the production of the guanine nucleotides ppGpp and pppGpp, produced by the RelA enzyme. We investigated the role of the relA gene in the ability of an extracellular pathogen, Pseudomonas aeruginosa, to cause infection. Strains lacking the relA gene were created from the prototypical laboratory strain PAO1 as well as the mucoid cystic fibrosis isolate 6106, which lacks functional quorum-sensing systems. The absence of relA abolished the production of ppGpp and pppGpp under conditions of amino acid starvation. We found that strains lacking relA exhibited reduced virulence in a D. melanogaster feeding assay. In conditions of low magnesium, the relA gene enhanced production of the cell-cell signal N-[3-oxododecanoyl]-l-homoserine lactone, whereas relA reduced the production of the 2-heptyl-3-hydroxy-4-quinolone signal during serine hydroxamate induction of the stringent response. In the relA mutant, alterations in the Pseudomonas quinolone system pathways seemed to increase the production of pyocyanin and decrease the production of elastase. Deletion of relA also resulted in reduced levels of the RpoS sigma factor. These results suggest that adjustment of cellular ppGpp and pppGpp levels could be an important regulatory mechanism in P. aeruginosa adaptation in pathogenic relationships.     

Influence of biofilm formation in the susceptibility of Pseudomonas aeruginosa from Brazilian patients with cystic fibrosis

Ferreira AG, Leão RS, Carvalho‐Assef APD, Folescu TW, Barth AL, Marques EA. Influence of biofilm formation in the susceptibility of Pseudomonas aeruginosa from Brazilian patients with cystic fibrosis. APMIS 2010; 118: 606–12. Biofilms play a key role in the occurrence of lung infections by Pseudomonas aeruginosa in patients with cystic fibrosis (CF). In this study, we examined 40 isolates of P. aeruginosa from CF patients according to their capacity to form biofilm. We also compared their in vitro response to antimicrobials according to different modes of growth (planktonic vs biofilm) and performed molecular typing. All isolates proved capable of forming biofilm. However, there was no difference in biofilm development according to the mucoid and nonmucoid phenotypes and among isolates obtained at different periods of the chronic infection. All isolates tested for antimicrobial susceptibility in the biofilm state (BIC) were consistently more resistant to antibiotics than the same isolate tested in the planktonic state. The molecular typing indicates a considerable clonal diversity among isolates. We identified five patients harboring the same strain over different periods. These strains, however, displayed different levels of biofilm formation and BIC values for antibiotics tested. The results of the present study demonstrate that there is a marked difference in the susceptibility profile according to the mode of growth of CF P. aeruginosa, as cells tested in the biofilm state proved consistently more resistant to antibiotics.     

Influence of gentamicin and tobramycin on binary biofilm formation by co-cultures of Burkholderia cepacia and Pseudomonas aeruginosa

The aim of this study was to investigate the stability and dynamics of binary culture biofilm formation following antibiotic treatment. Pseudo steady-state biofilm cultures of clinical strains of P. aeruginosa and B. cepacia concurrently isolated from a single CF patient were established in separate Constant Depth Film Fermenters (CDFFs). Pans, containing established biofilms, were swapped between CDFFs. Biofilms were treated either for 5 days with tobramycin (0.3 mg/ml) prior to pan-swapping, or with gentamicin (1 mg/ml) immediately following pan-swapping. In both instances stable binary biofilms were formed. In addition, fresh un-colonised surfaces added at the time of pan-swapping and exposed simultaneously to biofilm derived P. aeruginosa and B. cepacia and subjected to antibiotic challenge also established stable binary communities. Treatment of P. aeruginosa and B. cepacia with tobramycin or gentamicin, either prior to or immediately after surface colonisation had little effect on the establishment of stable binary biofilms. Such treatment also had little effect on the immigration of one organism into an established biofilm of a second organism.