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Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes diverse human infections including chronic airway infection in patients with cystic fibrosis (CF). Comparing the genomes of CF and non-CF PA isolates has great potential to identify the genetic basis of pathogenicity. To gain a deeper understanding of PA adaptation in CF airways, we performed a genome-wide association study (GWAS) on 1,001 PA genomes. Genetic variations identified among CF isolates were categorized into (i) alterations in protein-coding regions, either large- or small-scale, and (ii) polymorphic variation in intergenic regions. We introduced each CF-associated genetic alteration into the genome of PAO1, a prototype PA strain, and validated the outcomes experimentally. Loci readily mutated among CF isolates included genes encoding a probable sulfatase, a probable TonB-dependent receptor (PA2332~PA2336), L-cystine transporter (YecS, PA0313), and a probable transcriptional regulator (PA5438). A promoter region of a heme/hemoglobin uptake outer membrane receptor (PhuR, PA4710) was also different between the CF and non-CF isolate groups. Our analysis highlights ways in which the PA genome evolves to survive and persist within the context of chronic CF infection., Competing Interests: The authors have declared that no competing interests exist. more...

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen whose virulence is dependent on quorum sensing (QS). DksA1, an RNA polymerase-binding transcriptional regulator, plays a role in determining a number of phenotypes, including QS-mediated virulence. We therefore envisioned that DksA1 inhibitors may help to control P. aeruginosa infection. Here, we screened a library of 6970 chemical compounds and identified two compounds (henceforth termed Dkstatins) that specifically suppressed DksA1 activity. Treatment with these two compounds also substantially decreased the production of elastase and pyocyanin, dominant virulence determinants of P. aeruginosa, and protected murine hosts from lethal infection from a prototype strain of P. aeruginosa, PAO1. The Dkstatins also suppressed production of homoserine lactone (HSL)-based autoinducers that activate P. aeruginosa QS. The level of 3-oxo-C12-HSL produced by Dkstatin-treated wildtype PAO1 closely resembled that of the ΔdksA1 mutant. RNA-Seq analysis showed that transcription levels of QS- and virulence-associated genes were markedly reduced in Dkstatin-treated PAO1 cells, indicating that Dkstatin-mediated suppression occurs at the transcriptional level. Importantly, Dkstatins increased the antibiotic susceptibilities of PAO1, particularly to protein synthesis inhibitors, such as tobramycin and tetracycline. Co-immunoprecipitation assays demonstrated that these Dkstatins interfered with DksA1 binding to the β subunit of RNA polymerase, pointing to a potential mechanism of action. Collectively, our results illustrate that inhibition of P. aeruginosa QS may be achieved via DksA1 inhibitors and that Dkstatins may serve as potential lead compounds to control infection., Competing Interests: Conflict of interest We declare that there are no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.) more...

The stringent response (SR) is a highly conserved stress response in bacteria. It is composed of two factors, (i) a nucleotide alarmone, guanosine tetra- and pentaphosphate ((p)ppGpp), and (ii) an RNA polymerase-binding protein, DksA, that regulates various phenotypes, including bacterial virulence. The clinically significant opportunistic bacterial pathogen Pseudomonas aeruginosa possesses two genes, dksA1 and dksA2 , that encode DksA proteins. It remains elusive, however, which of these two genes plays a more important role in SR regulation. In this work, we compared genome-wide, RNA-Seq-based transcriptome profiles of Δ dksA1 , Δ dksA2 , and Δ dksA1 Δ dksA2 mutants to globally assess the effects of these gene deletions on transcript levels coupled with phenotypic analyses. The Δ dksA1 mutant exhibited substantial defects in a wide range of phenotypes, including quorum sensing (QS), anaerobiosis, and motility, whereas the Δ dksA2 mutant exhibited no significant phenotypic changes, suggesting that the dksA2 gene may not have an essential function in P. aeruginosa under the conditions used here. Of note, the Δ dksA1 mutants displayed substantially increased transcription of genes involved in polyamine biosynthesis, and we also detected increased polyamine levels in these mutants. Because SAM is a shared precursor for the production of both QS autoinducers and polyamines, these findings suggest that DksA1 deficiency skews the flow of SAM toward polyamine production rather than to QS signaling. Together, our results indicate that DksA1, but not DksA2, controls many important phenotypes in P. aeruginosa We conclude that DksA1 may represent a potential target whose inhibition may help manage recalcitrant P. aeruginosa infections., (© 2020 Min and Yoon.) more...

IL-17 family cytokines are directly involved in host immune responses and the critical mediators for host defense against infection or inflammation. IL-17C is highly expressed in respiratory epithelium and is induced after acute bacterial lung infection. However, the definite function of IL-17C induced by Pseudomonas aeruginosa (PAO1 strain) is not fully understood, and our study was designed to demonstrate IL-17C-induced immune response against PAO1 infection in nasal epithelium. Passage-2 normal human nasal epithelial (NHNE) cells were infected with PAO1 and the relationship between IL-17C-related immune responses and the iron absorption of PAO1, depending on inoculation of recombinant human IL-17C (rhIL-17C), was assessed by measuring the siderophore activity of PAO1. Microarray data showed that IL-17C expression increased 34.7 times at 8 hours postinfection (hpi) in NHNE cells, and IL-17C mRNA levels increased until 48 hpi. The PAO1 colonies significantly increased from 8 hpi in NHNE cells, and siderophore activity of PAO1 was enhanced in the supernatants of PAO1-infected NHNE cells. Interestingly, PAO1 colonies were reduced in PAO1-infected NHNE cells treated with rhIL-17C, and supernatants from NHNE cells treated with rhIL-17C also exhibited decreased PAO1 colonies. We found that the siderophore activity of PAO1 was significantly reduced in the supernatants of NHNE cells treated with rhIL-17C where LCN2 expression was highly elevated. Our findings indicate that IL-17C mediates an antibacterial effect against PAO1 by inhibiting siderophore activity in nasal epithelium. We propose that IL-17C might be an efficient mediator to suppress PAO1 infection through disturbing iron absorption of PAO1 in nasal epithelium. more...

In the inner ear, endolymph fluid surrounds the organ of Corti, which is important for auditory function; notably, even slight environmental changes mediated by trauma or infection can have significant consequences. However, it is unclear how the immune response is modulated in these tissues. Here, we report the local immune surveillance role of cleaved cochlin LCCL (Limulus factor C, Cochlin, and Lgl1) during Pseudomonas aeruginosa infection in the cochlea. Upon infection, the LCCL domain is cleaved from cochlin and secreted into the perilymph. This cleaved fragment sequesters infiltrating bacteria in the scala tympani and subsequently recruits resident immune cells to eliminate the bacteria. Importantly, hearing loss in a cochlin knockout mouse model is remedied by treatment with a cochlin LCCL peptide. These findings suggest cleaved cochlin LCCL constitutes a critical factor in innate immunity and auditory function and may be a potential therapeutic target to treat chronic otitis media-induced hearing loss., (Copyright © 2019 Elsevier Inc. All rights reserved.) more...

Pseudomonas aeruginosa displays intrinsic resistance to many antibiotics and known to acquire actively genetic mutations for further resistance. In this study, we attempted to understand genomic and transcriptomic landscapes of P. aeruginosa clinical isolates that are highly resistant to multiple antibiotics. We also aimed to reveal a mode of antibiotic resistance by elucidating transcriptional response of genes conferring antibiotic resistance. To this end, we sequenced the whole genomes and profiled genome-wide RNA transcripts of three different multi-drug resistant (MDR) clinical isolates that are phylogenetically distant from one another. Multi-layered genome comparisons with genomes of antibiotic-susceptible P. aeruginosa strains and 70 other antibiotic-resistance strains revealed both well-characterized conserved gene mutations and distinct distribution of antibiotic-resistant genes (ARGs) among strains. Transcriptions of genes involved in quorum sensing and type VI secretion systems were invariably downregulated in the MDR strains. Virulence-associated phenotypes were further examined and results indicate that our MDR strains are clearly avirulent. Transcriptions of 64 genes, logically selected to be related with antibiotic resistance in MDR strains, were active under normal growth conditions and remained unchanged during antibiotic treatment. These results propose that antibiotic resistance is achieved by a "constitutive" response scheme, where ARGs are actively expressed even in the absence of antibiotic stress, rather than a "reactive" response. Bacterial responses explored at the transcriptomic level in conjunction with their genome repertoires provided novel insights into (i) the virulence-associated phenotypes and (ii) a mode of antibiotic resistance in MDR P. aeruginosa strains. more...

Objectives: Pseudomonas aeruginosa frequently colonizes the lungs of chronic obstructive pulmonary disease (COPD) patients. Mucoid conversion is a hallmark of chronic P. aeruginosa infection, which is mediated by mucA gene mutations. The aim of this study is to identify predictive factors for mortality and the influence of mucA gene mutation in COPD patients with P. aeruginosa pneumonia., Methods: This study assessed 75 COPD patients with P. aeruginosa pneumonia at two university hospitals. The clinical and laboratory data were collected, and the P. aeruginosa isolates analyzed for the presence of mucA gene mutations., Results: MucA gene mutation of P. aeruginosa was an independent predictor of mortality (odds ratio [OR] 10.43, 95% confidence interval [CI]: 1.53-70.90, p = 0.017). In addition, the APACHE II score and C-reactive protein/Albumin (CA) ratio were independent predictive factors for mortality (OR 1.25, 95% CI: 1.07-1.46, p = 0.004; and OR 1.06, 95% CI: 1.02-1.10, p = 0.003, respectively). The optimal cutoff value of CA ratio for the greatest sensitivity and specificity was calculated as 31.27 (sensitivity, 85.7%; specificity, 80.3%)., Conclusions: CA ratio and mucA gene mutation of P. aeruginosa could be used as predictors to identify poor prognosis in COPD patients with P. aeruginosa pneumonia., (Copyright © 2018 Elsevier Ltd. All rights reserved.) more...

To address the drug-resistance of bacterial pathogens without imposing a selective survival pressure, virulence and biofilms are highly attractive targets. Here, we show that terrein, which was isolated from Aspergillus terreus, reduced virulence factors (elastase, pyocyanin, and rhamnolipid) and biofilm formation via antagonizing quorum sensing (QS) receptors without affecting Pseudomonas aeruginosa cell growth. Additionally, the effects of terrein on the production of QS signaling molecules and expression of QS-related genes were verified. Interestingly, terrein also reduced intracellular 3,5-cyclic diguanylic acid (c-di-GMP) levels by decreasing the activity of a diguanylate cyclase (DGC). Importantly, the inhibition of c-di-GMP levels by terrein was reversed by exogenous QS ligands, suggesting a regulation of c-di-GMP levels by QS; this regulation was confirmed using P. aeruginosa QS mutants. This is the first report to demonstrate a connection between QS signaling and c-di-GMP metabolism in P. aeruginosa, and terrein was identified as the first dual inhibitor of QS and c-di-GMP signaling. more...

Pseudomonas aeruginosa, a Gram-negative bacterium, is characterized by its versatility that enables persistent survival under adverse conditions. It can grow on diverse energy sources and readily acquire resistance to antimicrobial agents. As an opportunistic human pathogen, it also causes chronic infections inside the anaerobic mucus airways of cystic fibrosis patients. As a strict respirer, P. aeruginosa can grow by anaerobic nitrate ( [Formula: see text] ) respiration. Nitric oxide (NO) produced as an intermediate during anaerobic respiration exerts many important effects on the biological characteristics of P. aeruginosa. This review provides information regarding (i) how P. aeruginosa grows by anaerobic respiration, (ii) mechanisms by which NO is produced under such growth, and (iii) bacterial adaptation to NO. We also review the clinical relevance of NO in the fitness of P. aeruginosa and the use of NO as a potential therapeutic for treating P. aeruginosa infection., (© 2018 Elsevier Ltd. All rights reserved.) more...

Biofilms are microbial communities that inhabit various surfaces and are surrounded by extracellular matrices (ECMs). Clinical microbiologists have shown that the majority of chronic infections are caused by biofilms, following the introduction of the first biofilm infection model by J. W. Costerton and colleagues (J. Lam, R. Chan, K. Lam, and J. W. Costerton, Infect Immun 28:546-556, 1980). However, treatments for chronic biofilm infections are still limited to surgical removal of the infected sites. Pseudomonas aeruginosa and Enterococcus faecalis are two frequently identified bacterial species in biofilm infections; nevertheless, the interactions between these two species, especially during biofilm growth, are not clearly understood. In this study, we observed phenotypic changes in a dual-species biofilm of P. aeruginosa and E. faecalis , including a dramatic increase in biofilm matrix thickness. For clear elucidation of the spatial distribution of the dual-species biofilm, P. aeruginosa and E. faecalis were labeled with red and green fluorescence, respectively. E. faecalis was located at the lower part of the dual-species biofilm, while P. aeruginosa developed a structured biofilm on the upper part. Mutants with altered exopolysaccharide (EPS) productions were constructed in order to determine the molecular basis for the synergistic effect of the dual-species biofilm. Increased biofilm matrix thickness was associated with EPSs, not extracellular DNA. In particular, Pel and Psl contributed to interspecies and intraspecies interactions, respectively, in the dual-species P. aeruginosa and E. faecalis biofilm. Accordingly, targeting Pel and Psl might be an effective part of eradicating P. aeruginosa polymicrobial biofilms. IMPORTANCE Chronic infection is a serious problem in the medical field. Scientists have observed that chronic infections are closely associated with biofilms, and the vast majority of infection-causing biofilms are polymicrobial. Many studies have reported that microbes in polymicrobial biofilms interact with each other and that the bacterial interactions result in elevated virulence, in terms of factors, such as infectivity and antibiotic resistance. Pseudomonas aeruginosa and Enterococcus faecalis are frequently isolated pathogens in chronic biofilm infections. Nevertheless, while both bacteria are known to be agents of numerous nosocomial infections and can cause serious diseases, interactions between the bacteria in biofilms have rarely been examined. In this investigation, we aimed to characterize P. aeruginosa and E. faecalis dual-species biofilms and to determine the molecular factors that cause synergistic effects, especially on the matrix thickening of the biofilm. We suspect that our findings will contribute to the development of more efficient methods for eradicating polymicrobial biofilm infections., (Copyright © 2017 American Society for Microbiology.) more...

A biofilm is a community of microbes that typically inhabit on surfaces and are encased in an extracellular matrix. Biofilms display very dissimilar characteristics to their planktonic counterparts. Biofilms are ubiquitous in the environment and influence our lives tremendously in both positive and negative ways. Pseudomonas aeruginosa is a bacterium known to produce robust biofilms. P. aeruginosa biofilms cause severe problems in immunocompromised patients, including those with cystic fibrosis or wound infection. Moreover, the unique biofilm properties further complicate the eradication of the biofilm infection, leading to the development of chronic infections. In this review, we discuss the history of biofilm research and general characteristics of bacterial biofilms. Then, distinct features pertaining to each stage of P. aeruginosa biofilm development are highlighted. Furthermore, infections caused by biofilms on their own or in association with other bacterial species ( i.e. , multispecies biofilms) are discussed in detail. more...

Pseudomonas aeruginosa is capable of establishing airway infections. Human airway mucus contains a large amount of lysozyme, which hydrolyzes bacterial cell walls. P. aeruginosa , however, is known to be resistant to lysozyme. Here, we performed a genetic screen using a mutant library of PAO1, a prototype P. aeruginosa strain, and identified two mutants (Δ bamB and Δ fabY ) that exhibited decrease in survival after lysozyme treatment. The bamB and fabY genes encode an outer membrane assembly protein and a fatty acid synthesis enzyme, respectively. These two mutants displayed retarded growth in the airway mucus secretion (AMS). In addition, these mutants exhibited reduced virulence and compromised survival fitness in two different in vivo infection models. The mutants also showed susceptibility to several antibiotics. Especially, Δ bamB mutant was very sensitive to vancomycin, ampicillin, and ceftazidime that target cell wall synthesis. The Δ fabY displayed compromised membrane integrity. In conclusion, this study uncovered a common aspect of two different P. aeruginosa mutants with pleiotropic phenotypes, and suggests that BamB and FabY could be novel potential drug targets for the treatment of P. aeruginosa infection. more...

Pseudomonas aeruginosa has been identified as an important causative agent of airway infection, mainly in cystic fibrosis. This disease is characterized by defective mucociliary clearance induced in part by mucus hyper-production. Mucin is a major component of airway mucus and is heavily O-glycosylated, with a protein backbone. Airway infection is known to be established with bacterial adhesion to mucin. However, the genes involved in mucin degradation or utilization remain elusive. In this study, we sought to provide a genetic basis of P. aeruginosa airway growth by identifying those genes. First, using RNASeq analyses, we compared genome-wide expression profiles of PAO1, a prototype P. aeruginosa laboratory strain, grown in M9-mucin (M9M) and M9-glucose (M9G) media. Additionally, a PAO1 transposon (Tn) insertion mutants library was screened for mutants defective in growth in M9M medium. One mutant with a Tn insertion in the xcpU gene (PA3100) was determined to exhibit faulty growth in M9M medium. This gene contributes to the type II secretion system, suggesting that P. aeruginosa uses this secretion system to produce a number of proteins to break down and assimilate the mucin molecule. Furthermore, we screened the PAO1 genome for genes with protease activity. Of 13 mutants, one with mutation in PA3247 gene exhibited defective growth in M9M, suggesting that the PA3247-encoded protease plays a role in mucin utilization. Further mechanistic dissection of this particular process will reveal new drug targets, the inhibition of which could control recalcitrant P. aeruginosa infections. more...

Pseudomonas aeruginosa is an opportunistic pathogen, known to develop robust biofilms. Its biofilm development increases when antibiotics are presented at subminimal inhibitory concentrations (MICs) for reasons that remain unclear. In order to identify genes that affect biofilm development under such a sublethal antibiotic stress condition, we screened a transposon (Tn) mutant library of PAO1, a prototype P. aeruginosa strain. Among ∼5000 mutants, a fiuA gene mutant was verified to form very defective biofilms in the presence of sub-MIC carbenicillin. The fiuA gene encodes ferrichrome receptor A, involved in the iron acquisition process. Of note, biofilm formation was not decreased in the ΔpchΔpvd mutant defective in the production of pyochelin and pyoverdine, two well-characterized P. aeruginosa siderophore molecules. Moreover, ΔfiuA, a non-polar fiuA deletion mutant, produced a significantly decreased level of elastase, a major virulence determinant. Mouse airway infection experiments revealed that the mutant expressed significantly less pathogenicity. Our results suggest that the fiuA gene has pleiotropic functions that affect P. aeruginosa biofilm development and virulence. The targeting of FiuA could enable the attenuation of P. aeruginosa virulence and may be suitable for the development of a drug that specifically controls the virulence of this important pathogen., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.) more...

Pseudomonas aeruginosa is a Gram-negative bacterium of clinical significance. Although the genome of PAO1, a prototype strain of P. aeruginosa, has been extensively studied, approximately one-third of the functional genome remains unknown. With the emergence of antibiotic-resistant strains of P. aeruginosa, there is an urgent need to develop novel antibiotic and anti-virulence strategies, which may be facilitated by an approach that explores P. aeruginosa gene function in systems-level models. Here, we present a genome-wide functional network of P. aeruginosa genes, PseudomonasNet, which covers 98% of the coding genome, and a companion web server to generate functional hypotheses using various network-search algorithms. We demonstrate that PseudomonasNet-assisted predictions can effectively identify novel genes involved in virulence and antibiotic resistance. Moreover, an antibiotic-resistance network based on PseudomonasNet reveals that P. aeruginosa has common modular genetic organisations that confer increased or decreased resistance to diverse antibiotics, which accounts for the pervasiveness of cross-resistance across multiple drugs. The same network also suggests that P. aeruginosa has developed mechanism of trade-off in resistance across drugs by altering genetic interactions. Taken together, these results clearly demonstrate the usefulness of a genome-scale functional network to investigate pathogenic systems in P. aeruginosa. more...

Background: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infections. A rapid and sensitive test to detect CRPA and CRAB is required for appropriate antibiotic treatment. We optimized a loop-mediated isothermal amplification (LAMP) assay to detect the presence of bla(VIM-2), bla(IMP-1), and bla(OXA-23), which are critical components for carbapenem resistance., Methods: Two sets of primers, inner and outer primers, were manually designed as previously described. The LAMP buffer was optimized (at 2mM MgSO₄) by testing different concentrations of MgSO₄. The optimal reaction temperature and incubation time were determined by using a gradient thermocycler. Then, the optimized bla(VIM-2), bla(IMP-1), and bla(OXA-23) LAMP reactions were evaluated by using 120 P. aeruginosa and 99 A. baumannii clinical isolates., Results: Only one strain of the 100 CRPA isolates harbored bla(IMP-1), whereas none of them harbored bla(VIM-2). These results indicate that the acquisition of bla(VIM-2) or bla(IMP-1) may not play a major role in carbapenem resistance in Korea. Fifty two strains of the 75 CRAB isolates contained bla(OXA-23), but none contained bla(VIM-2) and bla(IMP-1) alleles., Conclusions: Our results demonstrate the usefulness of LAMP for the diagnosis of CRPA and CRAB. more...

Pseudomonas aeruginosa establishes airway infections in Cystic Fibrosis patients. Here, we investigate the molecular interactions between P. aeruginosa and airway mucus secretions (AMS) derived from the primary cultures of normal human tracheal epithelial (NHTE) cells. PAO1, a prototype strain of P. aeruginosa, was capable of proliferating during incubation with AMS, while all other tested bacterial species perished. A PAO1 mutant lacking PA4834 gene became susceptible to AMS treatment. The ΔPA4834 mutant was grown in AMS supplemented with 100 μM ferric iron, suggesting that the PA4834 gene product is involved in iron metabolism. Consistently, intracellular iron content was decreased in the mutant, but not in PAO1 after the AMS treatment. Importantly, a PAO1 mutant unable to produce both pyoverdine and pyochelin remained viable, suggesting that these two major siderophore molecules are dispensable for maintaining viability during incubation with AMS. The ΔPA4834 mutant was regrown in AMS amended with 100 μM nicotianamine, a phytosiderophore whose production is predicted to be mediated by the PA4836 gene. Infectivity of the ΔPA4834 mutant was also significantly compromised in vivo. Together, our results identify a genetic element encoding a novel iron acquisition system that plays a previously undiscovered role in P. aeruginosa airway infection. more...

Pseudomonas aeruginosa, a Gram-negative bacterium of clinical significance, produces elastase as a predominant exoprotease. Here, we screened a library of chemical compounds currently used for human medication and identified diethylene triamine penta-acetic acid (DTPA, pentetic acid) as an agent that suppresses the production of elastase. Elastase activity found in the prototype P. aeruginosa strain PAO1 was significantly decreased when grown with a concentration as low as 20 μM DTPA. Supplementation with Zn(2+) or Mn(2+) ions restored the suppressive effect of DTPA, suggesting that the DTPA-mediated decrease in elastase activity is associated with ion-chelating activity. In DTPA-treated PAO1 cells, transcription of the elastase-encoding lasB gene and levels of the Pseudomonas quinolone signal (PQS), a molecule that mediates P. aeruginosa quorum sensing (QS), were significantly downregulated, reflecting the potential involvement of the PQS QS system in DTPA-mediated elastase suppression. Biofilm formation was also decreased by DTPA treatment. When A549 alveolar type II-like adenocarcinoma cells were infected with PAO1 cells in the presence of DTPA, A549 cell viability was substantially increased. Furthermore, the intranasal delivery of DTPA to PAO1-infected mice alleviated the pathogenic effects of PAO1 cells in the animals. Together, our results revealed a novel function for a known molecule that may help treat P. aeruginosa airway infection., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.) more...

Alginate-overproducing mucoid Pseudomonas aeruginosa, responsible for chronic airway infections in cystic fibrosis (CF) patients, is resistant to antibiotic treatments and host immune clearance. In this study, we performed a phenotype microarray screen and identified sulfate ion as a molecule that can suppress alginate production. When a mucoid P. aeruginosa strain CM21 and additional mucoid isolates were grown with 5% sodium sulfate, significantly decreased levels of alginate were produced. Suppression of alginate production was also induced by other sulfate salts. Expression of a reporter gene fused to the algD promoter was considerably decreased when grown with sulfate. Furthermore, bacterial cell shape was abnormally altered in CM21, but not in PAO1, a prototype nonmucoid strain, suggesting that sulfate-stimulated cell shape change is associated with transcriptional suppression of the alginate operon. Finally, a CM21 lpxC mutant defective in lipid A biosynthesis continued to produce alginate and maintained the correct cell shape when grown with sulfate. These results suggest a potential involvement of lipoploysaccharide biosynthesis in the sulfate-induced reversion to nonmucoid phenotype. This study proposes a novel strategy that can be potentially applied to treat persistent infection by recalcitrant mucoid P. aeruginosa., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.) more...

The incidence of carbapenem-resistant Pseudomonas aeruginosa (CRPA) bacteremia has increased in recent years, and infections caused by CRPA result in higher mortality than those caused by susceptible strains. This study was performed to evaluate the risk factors for mortality and to study the impact of virulence factors and bacterial strains on clinical outcomes in patients with CRPA bacteremia. Data on 63 episodes of CRPA bacteremia that have occurred between January 1, 2007, and December 31, 2009, in a teaching hospital (2000 beds) in Seoul, Korea, were analyzed. The Acute Physiology and Chronic Health Evaluation II (APACHE II) score at the time of CRPA bacteremia and the capacity of CRPA to form biofilm were independent predictive factors for mortality in patients with CRPA bacteremia. In addition, the biofilm-forming ability and elastase activity of strains were correlated with APACHE II scores to measure the severity of disease and estimate predicted mortality in the patients., (Copyright © 2014 Elsevier Inc. All rights reserved.) more...

Objectives: To investigate the epidemiological traits of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa (MPPA) clinical isolates collected by the Asian Network for Surveillance of Resistant Pathogens (ANSORP)., Methods: A total of 16 MPPA clinical isolates were collected from six Asian countries in 2000 to 2009 by ANSORP. The MBL gene was detected by PCR amplification. The genetic organization of the class 1 integron carrying the MBL gene cassette was investigated by PCR mapping and sequencing. Southern blotting, repetitive sequence-based PCR and multilocus sequence typing (MLST) experiments were performed to characterize the isolates., Results: PCR and sequencing experiments detected the blaVIM-2 (n = 12), blaVIM-3 (n = 1), blaIMP-6 (n = 2) and blaIMP-26 (n = 1) genes. The MBL genes were located on the chromosome in all isolates except one. Furthermore, all the MBL genes were located in a class 1 integron. All the MPPA isolates from Malaysia, Thailand, Sri Lanka and Korea were identified as sequence type (ST) 235 by MLST. Three VIM-2-producing isolates from India were identified as ST773, and one isolate harbouring VIM-3 from Taiwan was identified as ST298., Conclusions: P. aeruginosa ST235 might play a role in dissemination of MBL genes in Asian countries. more...

In this study, biosurfactant-producing strain N2 and non-biosurfactant producing stain KB18 were used to investigate the effects of microbial treatment on the prevention and removal of paraffin deposits on stainless steel surfaces. Strain N2, with a biosurfactant production capacity, reduced the contact angle of stainless steel to 40.04°, and the corresponding adhesion work of aqueous phase was decreased by 26.5 mJ/m(2). By contrast, KB18 could only reduce the contact angle to 50.83°, with a corresponding 7.6 mJ/m(2) decrease in the aqueous phase work adhesion. The paraffin removal test showed that the paraffin removal efficiencies of strain N2 and KB18 were 79.0% and 61.2%, respectively. Interestingly, the N2 cells could attach on the surface of the oil droplets to inhibit droplets coalescence. These results indicate that biosurfactant-producing strains can alter the wettability of stainless steel and thus eliminate paraffin deposition., (Copyright © 2012 Elsevier Ltd. All rights reserved.) more...

We isolated a new lytic Pseudomonas aeruginosa phage that requires type IV pili for infection. PA1Ø has a broad bactericidal spectrum, covering Gram-positive and Gram-negative bacteria, and can eradicate biofilm cells. PA1Ø may be developed as a therapeutic agent for biofilm-related mixed infections with P. aeruginosa and Staphylococcus aureus. more...

Pseudomonas aeruginosa undergoes cell elongation and forms robust biofilms during anaerobic respiratory growth using nitrate (NO(3)(-)) as an alternative electron acceptor. Understanding the mechanism of cell shape change induced upon anaerobiosis is crucial to the development of effective treatments against P. aeruginosa biofilm infection. Here, we uncovered the molecular basis of anaerobiosis-triggered cell elongation and identified vitamin B(12) to be a molecule that can reinstate defective anaerobic growth of P. aeruginosa. The ratio of total cellular DNA content to protein content was significantly decreased in the PAO1 strain grown under anaerobic conditions, indicating that DNA replication is impaired during anaerobic growth. Anaerobic growth of PAO1 reached a higher cell density in the presence of vitamin B(12), an essential coenzyme of class II ribonucleotide reductase. In addition, cell morphology returned to a normal rod shape and transcription of stress-response genes was downregulated under the same anaerobic growth conditions. These results suggest that vitamin B(12), the production of which was suppressed during anaerobic growth, can restore cellular machineries for DNA replication and therefore facilitate better anaerobic growth of P. aeruginosa with normal cell division. Importantly, biofilm formation was substantially decreased when grown with vitamin B(12), further demonstrating that anaerobiosis-induced cell elongation is responsible for robust biofilm formation. Taken together, our data reveal mechanistic details of a morphological change that naturally occurs during anaerobic growth of P. aeruginosa and illustrates the ability of vitamin B(12) to modulate the biofilm-forming capacity of P. aeruginosa under such condition. more...

Pseudomonas aeruginosa, an opportunistic pathogen of clinical importance, causes chronic airway infections in patients with cystic fibrosis (CF). Current literature suggests that pockets with reduced oxygen tension exist in the CF airway mucus. However, virulence features of this opportunistic pathogen under such conditions are largely unknown. Cell-free supernatant of the standard laboratory P. aeruginosa strain PAO1 obtained from anaerobic culture, but not aerobic culture, failed to kill A549 human airway epithelial cells. Further investigation revealed that this reduced cytotoxicity upon anaerobiosis was due to the suppressed secretion of elastase, a virulence factor controlled by P. aeruginosa quorum sensing (QS). Both a lacZ-reporter fusion assay and quantitative real-time PCR (RT-PCR) analysis demonstrated that transcription of the elastase-encoding lasB gene was substantially decreased during anaerobic growth compared with aerobic growth. Moreover, transcription of other genes controlled by the LasI/R QS system, such as rhlR, vqsR, mvfR, and rsaL, was also repressed under the same anaerobic growth conditions. Importantly, synthesis of 3-oxo-C(12)-HSL (PAI-1), an autoinducer molecule that mediates induction of the LasI/R QS system, was >22-fold decreased during anaerobic growth while C(4)-HSL (PAI-2), which mediates RhlI/R QS, was nondetectable under the same growth conditions. Transcription of the lasB gene was restored by exogenous supplementation with autoinducers, with PAI-2 more effective than PAI-1 or Pseudomonas quinolone signal (PQS) at restoring transcription of the lasB gene. Together, these results suggest that anaerobiosis deprives P. aeruginosa of the ability to regulate its virulence via QS and this misregulation attenuates the pathogenic potential of this important pathogen. more...

Pseudomonas aeruginosa, a gram-negative bacterium of clinical importance, forms more robust biofilm during anaerobic respiration, a mode of growth presumed to occur in abnormally thickened mucus layer lining the cystic fibrosis (CF) patient airway. However, molecular basis behind this anaerobiosis-triggered robust biofilm formation is not clearly defined yet. Here, we identified a morphological change naturally accompanied by anaerobic respiration in P. aeruginosa and investigated its effect on the biofilm formation in vitro. A standard laboratory strain, PAO1 was highly elongated during anaerobic respiration compared with bacteria grown aerobically. Microscopic analysis demonstrated that cell elongation likely occurred as a consequence of defective cell division. Cell elongation was dependent on the presence of nitrite reductase (NIR) that reduces nitrite (NO(2) (-)) to nitric oxide (NO) and was repressed in PAO1 in the presence of carboxy-PTIO, a NO antagonist, demonstrating that cell elongation involves a process to respond to NO, a spontaneous byproduct of the anaerobic respiration. Importantly, the non-elongated NIR-deficient mutant failed to form biofilm, while a mutant of nitrate reductase (NAR) and wild type PAO1, both of which were highly elongated, formed robust biofilm. Taken together, our data reveal a role of previously undescribed cell biological event in P. aeruginosa biofilm formation and suggest NIR as a key player involved in such process. more...

Pseudomonas aeruginosa is an opportunistic human pathogen of clinical importance that causes airway infections in immunocompromised patients. Here, we report the virulence-associated characteristics of strains of P. aeruginosa, isolated from the sputa of 25 Korean pneumonia patients. A high degree of genomic plasticity was observed by random amplified polymorphic DNA genotype analysis, suggesting that the infections were caused by strains with diverse genomic backgrounds. Biofilm formation of each isolate was heterogeneous in terms of their relative motilities. In addition, 48% of isolates were defective in the production of 3-oxo-C(12)-HSL (PAI-1), a quorum sensing signal molecule. In these strains, PAI-1-dependent elastase production was correspondingly decreased, suggesting that a large number of strains were presumed to be quorum sensing deficient. Multidrug resistance (MDR) was seen in 56% of the isolates tested, and 44% of the MDR strains were resistant to five or more antibiotics. Taken together, our results provide additional insights into the virulence traits of P. aeruginosa clinical isolates, which will aid in treating P. aeruginosa infections in pneumonia patients. more...

Protection from NO gas, a toxic byproduct of anaerobic respiration in Pseudomonas aeruginosa, is mediated by nitric oxide (NO) reductase (NOR), the norCB gene product. Nevertheless, a norCB mutant that accumulated approximately 13.6 microM NO paradoxically survived anaerobic growth. Transcription of genes encoding nitrate and nitrite reductases, the enzymes responsible for NO production, was reduced >50- and 2.5-fold in the norCB mutant. This was due, in part, to a predicted compromise of the [4Fe-4S](2+) cluster in the anaerobic regulator ANR by physiological NO levels, resulting in an inability to bind to its cognate promoter DNA sequences. Remarkably, two O(2)-dependent dioxygenases, homogentisate-1,2-dioxygenase (HmgA) and 4-hydroxyphenylpyruvate dioxygenase (Hpd), were derepressed in the norCB mutant. Electron paramagnetic resonance studies showed that HmgA and Hpd bound NO avidly, and helped protect the norCB mutant in anaerobic biofilms. These data suggest that protection of a P. aeruginosa norCB mutant against anaerobic NO toxicity occurs by both control of NO supply and reassignment of metabolic enzymes to the task of NO sequestration. more...

Mucoid, mucA mutant Pseudomonas aeruginosa cause chronic lung infections in cystic fibrosis (CF) patients and are refractory to phagocytosis and antibiotics. Here we show that mucoid bacteria perish during anaerobic exposure to 15 mM nitrite (NO2) at pH 6.5, which mimics CF airway mucus. Killing required a pH lower than 7, implicating formation of nitrous acid (HNO2) and NO, that adds NO equivalents to cellular molecules. Eighty-seven percent of CF isolates possessed mucA mutations and were killed by HNO2 (3-log reduction in 4 days). Furthermore, antibiotic-resistant strains determined were also equally sensitive to HNO2. More importantly, HNO2 killed mucoid bacteria (a) in anaerobic biofilms; (b) in vitro in ultrasupernatants of airway secretions derived from explanted CF patient lungs; and (c) in mouse lungs in vivo in a pH-dependent fashion, with no organisms remaining after daily exposure to HNO2 for 16 days. HNO2 at these levels of acidity and NO2 also had no adverse effects on cultured human airway epithelia in vitro. In summary, selective killing by HNO2 may provide novel insights into the important clinical goal of eradicating mucoid P. aeruginosa from the CF airways. more...

Recent evidence indicates that Pseudomonas aeruginosa residing as biofilms in airway mucus of cystic fibrosis (CF) patients is undergoing anaerobic metabolism, a form of growth requiring gene products that are not utilized during aerobic growth. The outer membrane protein, OprF, and the rhl quorum sensing circuit are two previously unrecognized cellular factors that are required for optimal anaerobic biofilm viability. Without OprF, bacteria grow extremely poorly because they lack nitrite reductase activity while lacking rhlR or rhlI forces bacteria to undergo metabolic suicide by overproduction of nitric oxide. Furthermore, anaerobic growth favors maintenance of the mucoid, alginate-overproducing phenotype. Thus, with increasing age of CF patients, mucoid populations predominate, indicating that anaerobic bacteria reside in the inspissated airway mucus. Because many frontline antibiotics used in the treatment of CF airway disease are either ineffective or show reduced efficacy during anaerobic conditions, we propose development of new drugs to combat anaerobic metabolism by P. aeruginosa for more effective treatment of chronic CF lung infections., (Copyright 2002 Elsevier Science B.V.) more...

Recent data indicate that cystic fibrosis (CF) airway mucus is anaerobic. This suggests that Pseudomonas aeruginosa infection in CF reflects biofilm formation and persistence in an anaerobic environment. P. aeruginosa formed robust anaerobic biofilms, the viability of which requires rhl quorum sensing and nitric oxide (NO) reductase to modulate or prevent accumulation of toxic NO, a byproduct of anaerobic respiration. Proteomic analyses identified an outer membrane protein, OprF, that was upregulated approximately 40-fold under anaerobic versus aerobic conditions. Further, OprF exists in CF mucus, and CF patients raise antisera to OprF. An oprF mutant formed poor anaerobic biofilms, due, in part, to defects in anaerobic respiration. Thus, future investigations of CF pathogenesis and therapy should include a better understanding of anaerobic metabolism and biofilm development by P. aeruginosa. more...