Synthesis and antibacterial activity of catecholate–ciprofloxacin conjugates

The development of an efficient route to obtain artificial siderophore–antibiotic conjugates active against Gram-negative bacteria is crucial. Herein, a practical access to triscatecholate enterobactin analogues linked to the ciprofloxacin along with their antibacterial evaluation are described. Two series of conjugates were obtained with and without a piperazine linker which is known to improve the pharmacokinetics profile of a drug. A monocatecholate–ciprofloxacin conjugate was also synthesized and evaluated. The antibacterial activities against Pseudomonas aeruginosa for some conjugates are related to the iron concentration in the culture medium and seem to depend on the bacterial iron uptake systems.     

Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies

Pseudomonas aeruginosa is an opportunistic pathogen that is a leading cause of morbidity and mortality in cystic fibrosis patients and immunocompromised individuals. Eradication of P. aeruginosa has become increasingly difficult due to its remarkable capacity to resist antibiotics. Strains of Pseudomonas aeruginosa are known to utilize their high levels of intrinsic and acquired resistance mechanisms to counter most antibiotics. In addition, adaptive antibiotic resistance of P. aeruginosa is a recently characterized mechanism, which includes biofilm-mediated resistance and formation of multidrug-tolerant persister cells, and is responsible for recalcitrance and relapse of infections. The discovery and development of alternative therapeutic strategies that present novel avenues against P. aeruginosa infections are increasingly demanded and gaining more and more attention. Although mostly at the preclinical stages, many recent studies have reported several innovative therapeutic technologies that have demonstrated pronounced effectiveness in fighting against drug-resistant P. aeruginosa strains. This review highlights the mechanisms of antibiotic resistance in P. aeruginosa and discusses the current state of some novel therapeutic approaches for treatment of P. aeruginosa infections that can be further explored in clinical practice.     

Bacterial cis-2-unsaturated fatty acids found in the cystic fibrosis airway modulate virulence and persistence of Pseudomonas aeruginosa

There is an increasing appreciation of the polymicrobial nature of many bacterial infections such as those associated with cystic fibrosis (CF) and of the potentially important role for interspecies interactions in influencing both bacterial virulence and response to therapy. Patients with CF are often co-infected with Pseudomonas aeruginosa and other pathogens including Burkholderia cenocepacia and Stenotrophomonas maltophilia. These latter bacteria produce signal molecules of the diffusible signal factor (DSF) family, which are cis-2-unsaturated fatty acids. We have previously shown by in vitro studies that DSF from S. maltophilia leads to altered biofilm formation and increased resistance to antibiotics by P. aeruginosa; these responses of P. aeruginosa require the sensor kinase PA1396. Here we show that DSF signals are present in sputum taken from patients with CF. Presence of these DSF signals was correlated with patient colonization by S. maltophilia and/or B. cenocepacia. Analysis of 50 clinical isolates of P. aeruginosa showed that each responded to the presence of synthetic DSF by increased antibiotic resistance and these strains demonstrated little sequence variation in the PA1396 gene. In animal experiments using CF transmembrane conductance regulator knockout mice, the presence of DSF promoted P. aeruginosa persistence. Furthermore, antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells was enhanced in the presence of DSF. Taken together, these data provide substantial evidence that interspecies DSF-mediated bacterial interactions occur in the CF lung and may influence the efficacy of antibiotic treatment, particularly for chronic infections involving persistence of bacteria.     

Comparison of seven structurally related coumarins on the inhibition of Quorum sensing of Pseudomonas aeruginosa and Chromobacterium violaceum

Quorum sensing (QS) is a cell-to-cell signaling communication system that controls the virulence behavior of a broad spectrum of bacterial pathogens, participating also in the development of biofilms, responsible of the antibiotic ineffectiveness in many infections. Therefore, QS system is an attractive target for antimicrobial therapy. In this study, we compare the effect of seven structurally related coumarins against bacterial growth, biofilm formation and elastase activity of Pseudomonas aeruginosa. In addition, the anti-pathogenic capacity of the seven coumarins was evaluated on the wild type and the biosensor strain of Chromobacterium violaceum.The comparative study of coumarins showed that molecules with hydroxyl groups on the aromatic ring displayed higher activity on the inhibition of biofilm formation of P. aeruginosa over coumarins with substituents in positions 3 and 4 or without the double 3,4-bond. These 3 or 4-hydroxylated positions caused a decrease in the anti-biofilm activity obtained for coumarin. However, the hydroxyl group in position 3 of the pyrone ring was important for the inhibition of C. violaceum QS and elastolytic activity of P. aeruginosa. The effects observed were active independently of any effect on growth. According to our results, coumarin and its hydroxylated derivatives represent an interesting group of compounds to use as anti-virulence agents against the human pathogen P. aeruginosa.     

Is the Improvement of CF Patients,Hospitalized for Pulmonary Exacerbation,Correlated to a Decrease in Bacterial Load?

Background

Cystic Fibrosis (CF) patients are vulnerable to airway colonization with Pseudomonas aeruginosa. In case eradication fails after antibiotic treatment, patients become chronically colonized with P. aeruginosa, with recurrent pulmonary exacerbation, for which patients typically are hospitalized for 2 weeks and receive intravenous antibiotic treatment. Normally, improvement of the patients'' health is established.

Aim

Determination of the correspondence between patient improvement and changes of the P. aeruginosa and total bacterial load in the sputum.

Methods

Eighteen CF patients with exacerbation were included for a total of 27 hospitalization episodes. At day 1, 8 and 15, inflammation and lung function parameters were determined, together with the P. aeruginosa load in the sputum using culture, quantitative PCR (qPCR) and propidium monoazide qPCR.

Results

Patients improved during hospitalization (decrease in levels of C-reactive protein, white blood cell counts and erythrocyte sedimentation rate, increase of FEV₁), reaching normal values already after one week. Also the P. aeruginosa load and the total bacterial load decreased during the first week of antibiotic treatment (p<0.05), except for patients with a low lung function (FEV₁≤39.4%), for whom no significant decrease of P. aeruginosa was established. Comparison of culture-based and propidium monoazide qPCR-based quantification of P. aeruginosa showed that at the end of the treatment on average 62% of the P. aeruginosa cells are not cultivable, indicating that many cells are alive but dormant, or dead but still structurally intact.

Conclusion

Improvement of the clinical status is accompanied with a decrease of the P. aeruginosa load, whereby both occur mainly during the first week of antibiotic treatment. However, for patients with a low lung function, no decrease of the P. aeruginosa load is observed. Comparison of detection techniques shows that a large amount of noncultivable or dead bacteria are present in the samples.     

Autophagy Enhances Bacterial Clearance during P. aeruginosa Lung Infection

Pseudomonas aeruginosa is an opportunistic bacterial pathogen which is the leading cause of morbidity and mortality among cystic fibrosis patients. Although P. aeruginosa is primarily considered an extacellular pathogen, recent reports have demonstrated that throughout the course of infection the bacterium acquires the ability to enter and reside within host cells. Normally intracellular pathogens are cleared through a process called autophagy which sequesters and degrades portions of the cytosol, including invading bacteria. However the role of autophagy in host defense against P. aeruginosa in vivo remains unknown. Understanding the role of autophagy during P. aeruginosa infection is of particular importance as mutations leading to cystic fibrosis have recently been shown to cause a blockade in the autophagy pathway, which could increase susceptibility to infection. Here we demonstrate that P. aeruginosa induces autophagy in mast cells, which have been recognized as sentinels in the host defense against bacterial infection. We further demonstrate that inhibition of autophagy through pharmacological means or protein knockdown inhibits clearance of intracellular P. aeruginosa in vitro, while pharmacologic induction of autophagy significantly increased bacterial clearance. Finally we find that pharmacological manipulation of autophagy in vivo effectively regulates bacterial clearance of P. aeruginosa from the lung. Together our results demonstrate that autophagy is required for an effective immune response against P. aeruginosa infection in vivo, and suggest that pharmacological interventions targeting the autophagy pathway could have considerable therapeutic potential in the treatment of P. aeruginosa lung infection.     

Mannitol Does Not Enhance Tobramycin Killing of Pseudomonas aeruginosa in a Cystic Fibrosis Model System of Biofilm Formation

Cystic Fibrosis (CF) is a human genetic disease that results in the accumulation of thick, sticky mucus in the airways, which results in chronic, life-long bacterial biofilm infections that are difficult to clear with antibiotics. Pseudomonas aeruginosa lung infection is correlated with worsening lung disease and P. aeruginosa transitions to an antibiotic tolerant state during chronic infections. Tobramycin is an aminoglycoside currently used to combat lung infections in individuals with CF. While tobramycin is effective at eradicating P. aeruginosa in the airways of young patients, it is unable to completely clear the chronic P. aeruginosa infections in older patients. A recent report showed that co-addition of tobramycin and mannitol enhanced killing of P. aeruginosa grown in vitro as a biofilm on an abiotic surface. Here we employed a model system of bacterial biofilms formed on the surface of CF-derived airway cells to determine if mannitol would enhance the antibacterial activity of tobramycin against P. aeruginosa grown on a more clinically relevant surface. Using this model system, which allows the growth of robust biofilms with high-level antibiotic tolerance analogous to in vivo biofilms, we were unable to find evidence for enhanced antibacterial activity of tobramycin with the addition of mannitol, supporting the observation that this type of co-treatment failed to reduce the P. aeruginosa bacterial load in a clinical setting.     

The microbiota protects against Pseudomonas aeruginosa pneumonia via γδ T cell-neutrophil axis in mice

Exposure to antimicrobials leading to microbiota dysbiosis has been found to be an independent risk factor for extensively drug-resistant Pseudomonas aeruginosa acquisition. Microbiota dysbiosis may induce imbalanced immune responses and can affect disease susceptibility. However, the potential role of commensal microbiota in bacterial pneumonia is poorly defined. The aim of this study was to investigate the mechanistic basis for the defective host defenses against P. aeruginosa pneumonia induced by antibiotic pretreatment perturbing microbiota. We found that antibiotic pretreatment significantly perturbed the composition of intestinal microbiota. The microbiota dysbiosis impaired host defenses against P. aeruginosa pneumonia, as reflected by the increased bacterial burden and dissemination, compromised local inflammatory responses and shortened survival time in microbiota-depleted mice compared with controls. This impairment correlated with a defective γδ T17 cell and downstream neutrophil responses. Anti-TCRγδ-treated mice had changes similar to those in the microbiota-depleted mice. Overall, our results suggest the importance of microbiota in supporting the host defense against pneumonia, define a crucial role for the γδ T cell-neutrophil axis in the potential mechanism, and delineate the deleterious effects of antibiotic treatment on antibacterial defenses.     

Crosstalk between Pseudomonas aeruginosa antibiotic resistance and virulence mediated by phenylethylamine

Multidrug efflux pumps are among the main Pseudomonas aeruginosa antibiotic-resistance determinants. Besides, efflux pumps are also involved in other relevant activities of bacterial physiology, including the quorum sensing-mediated regulation of bacterial virulence. Nevertheless, despite the relevance of efflux pumps in bacterial physiology, their interconnection with bacterial metabolism remains obscure. The effect of several metabolites on the expression of P. aeruginosa efflux pumps, and on the virulence and antibiotic resistance of this bacterium, was studied. Phenylethylamine was found to be both inducer and substrate of MexCD-OprJ, an efflux pump involved in P. aeruginosa antibiotic resistance and in extrusion of precursors of quorum-sensing signals. Phenylethylamine did not increase antibiotic resistance; however, the production of the toxin pyocyanin, the tissue-damaging protease LasB and swarming motility were reduced in the presence of this metabolite. This decrease in virulence potential was mediated by a reduction of lasI and pqsABCDE expression, which encode the proteins that synthesise the signalling molecules of two quorum-sensing regulatory pathways. This work sheds light on the interconnection between virulence and antibiotic-resistance determinants, mediated by bacterial metabolism, and points to phenylethylamine as an anti-virulence metabolite to be considered in the study of therapies against P. aeruginosa infections.     

Pseudomonas aeruginosa zinc homeostasis: Key issues for an opportunistic pathogen

Zinc is an essential trace element for almost all living organisms. In the opportunistic bacterial pathogen Pseudomonas aeruginosa, zinc has been shown to play an important role in virulence, in colonization of the host organism and has also been shown to be involved in antibiotic resistance. P. aeruginosa possesses numerous systems enabling it to thrive in zinc-depleted conditions as well as high-zinc situations, two environments that are encountered during human infection. These capabilities account for its pathogenic strength. The main aim of this review is to focus on zinc homeostasis in P. aeruginosa and the genetic regulation of the systems involved. The interconnection with virulence, as well as the mechanism of co-regulation between metal and antibiotic resistance, are of prime interest for understanding the molecular mechanisms allowing P. aeruginosa to switch from its existence as a common environmental bacterium to a severe opportunistic pathogen. This article is part of a Special Issue entitled: Dynamic gene expression, edited by Prof. Patrick Viollier.     

PA2800 Plays an Important Role in Both Antibiotic Susceptibility and Virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important human pathogen which causes a variety of infections. P. aeruginosa infections are often difficult to treat due to the pathogen’s resistance to many antibiotics. Previously, it has been reported that a transposon insertion mutant in gene PA2800 of P. aeruginosa PAO1 was more sensitive to tetracycline and ciprofloxacin. Further characterization of this gene, a vacJ homolog, in this study indicated that this gene plays an important role in both antibiotic susceptibility and virulence in P. aeruginosa. The role of PA2800 in antibiotic susceptibility probably signifies its involvement in maintaining outer membrane stability, similar to the role of vacJ in E. coli and Shigella flexneri. However, in contrast to vacJ in other bacteria, PA2800 also affects antibiotic susceptibility by affecting the expression of oprH in P. aeruginosa. As shown by in vivo studies using a Drosophila melanogaster infection model, significantly increased virulence was observed in the PA2800 mutant when compared to the wild type, and such a difference is likely a result of disrupted outer membrane stability and altered expression of znuA in the mutant. The role of PA2800 or vacJ in antibiotic susceptibility and pathogenicity seems to be unique in P. aeruginosa in which it affects both outer membrane stability as well as gene expression.     

Nitrite modulates bacterial antibiotic susceptibility and biofilm formation in association with airway epithelial cells

Pseudomonas aeruginosa is the major pathogenic bacteria in cystic fibrosis and other forms of bronchiectasis. Growth in antibiotic-resistant biofilms contributes to the virulence of this organism. Sodium nitrite has antimicrobial properties and has been tolerated as a nebulized compound at high concentrations in human subjects with pulmonary hypertension; however, its effects have not been evaluated on biotic biofilms or in combination with other clinically useful antibiotics. We grew P. aeruginosa on the apical surface of primary human airway epithelial cells to test the efficacy of sodium nitrite against biotic biofilms. Nitrite alone prevented 99% of biofilm growth. We then identified significant cooperative interactions between nitrite and polymyxins. For P. aeruginosa growing on primary CF airway cells, combining nitrite and colistimethate resulted in an additional log of bacterial inhibition compared to treating with either agent alone. Nitrite and colistimethate additively inhibited oxygen consumption by P. aeruginosa. Surprisingly, whereas the antimicrobial effects of nitrite in planktonic, aerated cultures are nitric oxide (NO) dependent, antimicrobial effects under other growth conditions are not. The inhibitory effect of nitrite on bacterial oxygen consumption and biofilm growth did not require NO as an intermediate as chemically scavenging NO did not block growth inhibition. These data suggest an NO-radical independent nitrosative or oxidative inhibition of respiration. The combination of nebulized sodium nitrite and colistimethate may provide a novel therapy for chronic P. aeruginosa airway infections, because sodium nitrite, unlike other antibiotic respiratory chain “poisons,” can be safely nebulized at high concentration in humans.     

Bacterial membrane disrupting dodecapeptide SC4 improves survival of mice challenged with Pseudomonas aeruginosa

Background

Dodecapeptide SC4 is a broad-spectrum bactericidal agent that functions by disintegrating bacterial membranes and neutralizing endotoxins. For insight into which SC4 amino acids are functionally important, we assessed Gram-negative bactericidal effects in structure–activity relationship experiments. Subsequently, SC4 was tested in a murine bacteremia model to combine and compare the efficacy with Zosyn, a first-line antibiotic against Pseudomonas aeruginosa (P. aeruginosa).

Methods

SC4 alanine-scanning analogs and their activities on were tested on P. aeruginosa. Survival studies in P. aeruginosa challenged mice were executed to monitor overall efficacy of SC4 and Zosyn, as a single modality and also as combination treatment. ELISAs were used to measure blood serum levels of selected inflammatory cytokines during treatment.

Results

Cationic residues were found to play a crucial role in terms of bactericidal activity against P. aeruginosa. In vivo, while only 9% (3/34) of control animals survived to day two and beyond, 44% (12/27) to 41% (14/34) of animals treated with SC4 or Zosyn, respectively, survived beyond one week. Combination treatment of SC4 and Zosyn demonstrated improved survival, i.e. 60% (12/20). The TNFα, IL-1, and IL-6 serum levels were attenuated in each treatment group compared to the control group.

Conclusions

These data show that combination treatment of SC4 and Zosyn is most effective at killing P. aeruginosa and attenuating inflammatory cytokine levels in vivo.

General significance

Combination treatment of SC4 and Zosyn may be useful in the clinic as a more effective antibiotic therapy against Gram-negative infectious diseases.     

Phage Therapy: a Step Forward in the Treatment of Pseudomonas aeruginosa Infections

Antimicrobial resistance constitutes one of the major worldwide public health concerns. Bacteria are becoming resistant to the vast majority of antibiotics, and nowadays, a common infection can be fatal. To address this situation, the use of phages for the treatment of bacterial infections has been extensively studied as an alternative therapeutic strategy. Since Pseudomonas aeruginosa is one of the most common causes of health care-associated infections, many studies have reported the in vitro and in vivo antibacterial efficacy of phage therapy against this bacterium. This review collects data of all the P. aeruginosa phages sequenced to date, providing a better understanding about their biodiversity. This review further addresses the in vitro and in vivo results obtained by using phages to treat or prevent P. aeruginosa infections as well as the major hurdles associated with this therapy.     

Extracellular DNA Chelates Cations and Induces Antibiotic Resistance in Pseudomonas aeruginosa Biofilms

Biofilms are surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, bacterial polysaccharides and proteins, which are up to 1000-fold more antibiotic resistant than planktonic cultures. To date, extracellular DNA has been shown to function as a structural support to maintain Pseudomonas aeruginosa biofilm architecture. Here we show that DNA is a multifaceted component of P. aeruginosa biofilms. At physiologically relevant concentrations, extracellular DNA has antimicrobial activity, causing cell lysis by chelating cations that stabilize lipopolysaccharide (LPS) and the outer membrane (OM). DNA-mediated killing occurred within minutes, as a result of perturbation of both the outer and inner membrane (IM) and the release of cytoplasmic contents, including genomic DNA. Sub-inhibitory concentrations of DNA created a cation-limited environment that resulted in induction of the PhoPQ- and PmrAB-regulated cationic antimicrobial peptide resistance operon PA3552–PA3559 in P. aeruginosa. Furthermore, DNA-induced expression of this operon resulted in up to 2560-fold increased resistance to cationic antimicrobial peptides and 640-fold increased resistance to aminoglycosides, but had no effect on β-lactam and fluoroquinolone resistance. Thus, the presence of extracellular DNA in the biofilm matrix contributes to cation gradients, genomic DNA release and inducible antibiotic resistance. DNA-rich environments, including biofilms and other infection sites like the CF lung, are likely the in vivo environments where extracellular pathogens such as P. aeruginosa encounter cation limitation.     

siRNA-mediated gene silencing of MexB from the MexA-MexB-OprM efflux pump in Pseudomonas aeruginosa

To gain insights into the effect of MexB gene under the short interfering RNA (siRNA), we synthesized 21 bp siRNA duplexes against the MexB gene. RT-PCR was performed to determine whether the siRNA inhibited the expression of MexB mRNA. Changes in antibiotic susceptibility in response to siRNA were measured by the E-test method. The efficacy of siRNAs was determined in a murine model of chronic P. aeruginosa lung infection. MexB-siRNAs inhibited both mRNA expression and the activity of P. aeruginosa in vitro. In vivo, siRNA was effective in reducing the bacterial load in the model of chronic lung infection and the P. aeruginosa-induced pathological changes. MexB-siRNA treatment enhanced the production of inflammatory cytokines in the early infection stage (P ＜ 0.05). Our results suggest that targeting of MexB with siRNA appears to be a novel strategy for treating P. aeruginosa infections. [BMB Reports 2014; 47(4): 203-208]