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Antibiotic resistance in Pseudomonas aeruginosa biofilms necessitates the need for novel antimicrobial therapy with anti-biofilm properties. Bacteriophages (phages) are recognized as an ideal biopharmaceutical for combating antibiotic-resistant bacteria especially when used in combination with antibiotics. However, previous studies primarily focused on using phages against of P. aeruginosa biofilms of laboratory strains. In the present study, biofilms of six P. aeruginosa isolated from cystic fibrosis and wound patients, and one laboratory strain was treated singly and with combinations of anti-Pseudomonas phage PEV20 and ciprofloxacin. Of these strains, three were highly susceptible to the phage, while one was partially resistant and one was completely resistant. Combination treatment with PEV20 and ciprofloxacin enhanced biofilm eradication compared with single treatment. Phage and ciprofloxacin synergy was found to depend on phage-resistance profile of the target bacteria. Furthermore, phage and ciprofloxacin combination formulation protected the lung epithelial and fibroblast cells from P. aeruginosa and promoted cell growth. The results demonstrated that thorough screening of phage-resistance is crucial for designing phage-antibiotic formulation. The addition of highly effective phage could reduce the ciprofloxacin concentration required to combat P. aeruginosa infections associated with biofilm in cystic fibrosis and wound patients.

Pulsed-field gel electrophoresis (PFGE) is a technique used for the separation of high molecular weight restriction fragments from digested bacterial genomic DNA on a gel matrix by applying an electric field that periodically changes direction. PFGE has been shown to be the most discriminatory and reproducible bacterial strain typing technique available. It is considered the 'gold standard' in epidemiological studies of pathogenic and nonpathogenic microorganisms. This chapter provides a detailed method for using PFGE in the genotyping of the opportunistic Gram-negative bacterial pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis (CF). This study examines the role of organism-specific factors in the pathogenesis of very early P. aeruginosa infection in the CF airway. A total of 168 longitudinally collected P. aeruginosa isolates from children diagnosed with CF following newborn screening were genotyped by pulsed-field gel electrophoresis (PFGE) and phenotyped for 13 virulence factors. Ninety-two strains were identified. Associations between virulence factors and gender, exacerbation, persistence, timing of infection and infection site were assessed using multivariate regression analysis. Persistent strains showed significantly lower pyoverdine, rhamnolipid, haemolysin, total protease, and swimming and twitching motility than strains eradicated by aggressive antibiotic treatments. Initial strains had higher levels of virulence factors, and significantly higher phospholipase C, than subsequent genotypically different strains at initial isolation. Strains from males had significantly lower pyoverdine and swimming motility than females. Colony size was significantly smaller in strains isolated during exacerbation than those isolated during non-exacerbation periods. All virulence factors were higher and swimming motility significantly higher in strains from bronchoalveolar lavage (BAL) and oropharyngeal sites than BAL alone. Using unadjusted regression modelling, age at initial infection and age at isolation of a strain showed U-shaped profiles for most virulence factors. Among subsequent strains, longer time since initial infection meant lower levels of most virulence factors. This study provides new insight into virulence factors underpinning impaired airway clearance seen in CF infants, despite aggressive antibiotic therapy. This information will be important in the development of new strategies to reduce the impact of P. aeruginosa in CF.

Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis (CF) patients and causes a wide range of infections among other susceptible populations. Its inherent resistance to many antimicrobials also makes it difficult to treat infections with this pathogen. Recent evidence has highlighted the diversity of this species, yet despite this, the majority of studies on virulence and pathogenesis focus on a small number of strains. There is a pressing need for a P. aeruginosa reference panel to harmonize and coordinate the collective efforts of the P. aeruginosa research community. We have collated a panel of 43 P. aeruginosa strains that reflects the organism's diversity. In addition to the commonly studied clones, this panel includes transmissible strains, sequential CF isolates, strains with specific virulence characteristics, and strains that represent serotype, genotype or geographic diversity. This focussed panel of P. aeruginosa isolates will help accelerate and consolidate the discovery of virulence determinants, improve our understanding of the pathogenesis of infections caused by this pathogen, and provide the community with a valuable resource for the testing of novel therapeutic agents., (© 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Chronic Pseudomonas aeruginosa infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa isolates undergo significant transcriptomic and proteomic modulation as they adapt to the niche environment of the CF lung and the host defences. This study characterized the in vitro virulence of isogenic strain pairs of P. aeruginosa epidemic or frequent clonal complexes (FCCs) and non-epidemic or infrequent clonal complexes (IFCCs) that were collected 5-8 years apart from five chronically infected adult CF patients. Strains showed a significant decrease in virulence over the course of chronic infection using a Caenorhabditis elegans slow-killing assay and in phenotypic tests for important virulence factors. This decrease in virulence correlated with numerous differentially expressed genes such as oprG, lasB, rsaL and lecB. Microarray analysis identified a large genomic island deletion in the IFCC strain pair that included type three secretion system effector and fimbrial subunit genes. This study presents novel in vitro data to examine the transcriptomic profiles of sequentially collected P. aeruginosa from CF adults. The genes with virulence-related functions identified here present potential targets for new therapies and vaccines against FCCs and IFCCs.

Studies of the type 3 secretion system (T3SS) in Pseudomonas aeruginosa isolates from chronically infected older children and adults with cystic fibrosis (CF) show a predominantly exoS+/exoU- (exoS+) genotype and loss of T3SS effector secretion over time. Relatively little is known about the role of the T3SS in the pathogenesis of early P. aeruginosa infection in the CF airway. In this longitudinal study, 168 P. aeruginosa isolates from 58 children diagnosed with CF following newborn screening and 47 isolates from homes of families with or without children with CF were genotyped by pulsed-field gel electrophoresis (PFGE) and T3SS genotype and phenotype determined using multiplex PCR and western blotting. Associations were sought between T3SS data and clinical variables and comparisons made between T3SS data of clinical and environmental PFGE genotypes. Seventy-seven of the 92 clinical strains were exoS+ (71% secretors (ExoS+)) and 15 were exoU+ (93% secretors (ExoU+)). Initial exoS+ strains were five times more likely to secrete ExoS than subsequent exoS+ strains at first isolation. The proportion of ExoS+ strains declined with increasing age at acquisition. No associations were found between T3SS characteristics and gender, site of isolation, exacerbation, a persistent strain or pulmonary outcomes. Fourteen of the 23 environmental strains were exoS+ (79% ExoS+) and nine were exoU+ (33% ExoU+). The exoU+ environmental strains were significantly less likely to secrete ExoU than clinical strains. This study provides new insight into the T3SS characteristics of P. aeruginosa isolated from the CF airway early in life., (© 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.)

The invasive properties of Pseudomonas aeruginosa pose a serious threat to the wellbeing of cystic fibrosis (CF) patients; however the specific factors affecting invasiveness are not well understood, especially in chronic infection. This study characterises the invasive profiles of sequential isolates of the same P. aeruginosa strain collected five to eight years apart from five chronically infected adult CF patients. Strains from three patients were characterised as unique isolates and from two patients as the Australian Epidemic strain (AES-1) by pulsed field gel electrophoresis. The capacity of these strains to invade the human alveolar A549 cell line was examined. Later isolates were significantly more invasive than earlier counterparts from the same patient. Quantitative real-time PCR and Western blotting showed that the increase in invasiveness over time was independent of ExoS expression and secretion. A link between clonality and invasiveness was also identified, with AES-1 isolates more invasive than unique isolates. These results suggest that despite a reduction in some virulence factors such as the Type-3 Secretion System (T3SS) during chronic infection, a particular strain can become more invasive over time. Defining mechanisms behind the increased invasiveness during chronic infection may help identify new therapeutic targets for CF patients., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire P. aeruginosa from the environment, person-person transmissible strains have been identified in CF clinics worldwide. The molecular basis for transmissibility and colonization of the CF lung remains poorly understood., Results: A dual proteomics approach consisting of gel-based and gel-free comparisons were undertaken to analyse protein profiles in a transmissible, early (acute) isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1. Over 1700 P. aeruginosa proteins were confidently identified. AES-1R protein profiles revealed elevated abundance of proteins associated with virulence and siderophore biosynthesis and acquisition, antibiotic resistance and lipopolysaccharide and fatty acid biosynthesis. The most abundant protein in AES-1R was confirmed as a previously hypothetical protein with sequence similarity to carbohydrate-binding proteins and database search revealed this gene is only found in the CF-associated strain PA2192. The link with CF infection may suggest that transmissible strains have acquired an ability to rapidly interact with host mucosal glycoproteins., Conclusions: Our data suggest that AES-1R expresses higher levels of proteins, such as those involved in antibiotic resistance, iron acquisition and virulence that may provide a competitive advantage during early infection in the CF lung. Identification of novel proteins associated with transmissibility and acute infection may aid in deciphering new strategies for intervention to limit P. aeruginosa infections in CF patients.

Background and Objective: Earlier reports suggested that Pseudomonas aeruginosa frequent epidemic clones circulating in cystic fibrosis (CF) centres had increased virulence. However, recent data show no consistent associations with virulence, and suggest attenuation of virulence in chronic infection. Changes to infection control programmes in relation to frequent epidemic clones should be based on their frequency, virulence across all age groups and mode of acquisition. The Australian epidemic strain-1 (AES-1) (or the Melbourne epidemic strain) and AES-2 are common in CF clinics in mainland eastern Australia, but not in the environment. Both have shown increased virulence, but there are no data specifically in adults. This study examines the frequency and virulence of P. aeruginosa frequent epidemic clones in the adult CF clinic at Royal Prince Alfred Hospital, Sydney, Australia., Methods: Two hundred and fifty-eight P. aeruginosa isolates from 112 participants were genotyped by pulsed field gel electrophoresis. Ninety-eight patients were followed up for 1 year and associations sought between infection with a frequent epidemic clone, clinical outcome and antibiotic resistance., Results: Four frequent P. aeruginosa epidemic clones (AES-1, AES-2, S-1, S-2) affected almost 50% of participants. AES-1 predominated (38%). AES-1, AES-2 and S-1 were associated with increased exacerbations and hospital-admission days. AES-1 showed increased resistance to aminoglycosides and ticarcillin-clavulanate., Conclusions: This study supports the potential threat of frequent P. aeruginosa epidemic clones in adult CF populations.

The emergence of virulent Pseudomonas aeruginosa clones is a threat to cystic fibrosis (CF) patients globally. Characterization of clonal P. aeruginosa strains is critical for an understanding of its clinical impact and developing strategies to meet this problem. Two clonal strains (AES-1 and AES-2) are circulating within CF centers in eastern Australia. In this study, phenotypic characteristics of 43 (14 AES-1, 5 AES-2, and 24 nonclonal) P. aeruginosa isolates were compared to gain insight into the properties of clonal strains. All 43 isolates produced bands of the predicted size in PCRs for vfr, rhlI, rhlR, lasA, lasB, aprA, rhlAB, and exoS genes; 42 were positive for lasI and lasR, and none had exoU. Thirty-seven (86%) isolates were positive in total protease assays; on zymography, 24 (56%) produced elastase/staphylolysin and 22 (51%) produced alkaline protease. Clonal isolates were more likely than nonclonal isolates to be positive for total proteases (P = 0.02), to show elastase and alkaline protease activity by zymography (P = 0.04 and P = 0.01, respectively), and to show elastase activity by the elastin-Congo red assay (P = 0.04). There were no other associations with genotype. Overall, increasing patient age was associated with decreasing elastase activity (P = 0.03). Thirty-two (74%) isolates had at least one N-acylhomoserine lactone (AHL) by thin-layer chromatography. rhl-associated AHL detection was associated with the production and level of total protease and elastase activity (all P < 0.01). Thirty-three (77%) isolates were positive for ExoS by Western blot analysis, 35 (81%) produced rhamnolipids, and 34 (79%) showed chitinase activity. Findings suggest that protease activity during chronic infection may contribute to the transmissibility or virulence of these clonal strains.

Protease IV is important in the pathogenesis of Pseudomonas aeruginosa-induced microbial keratitis, but little is known of its role in cystic fibrosis (CF) lung infection. In this study protease IV production was examined in 43 P. aeruginosa isolates (24 non-clonal and 19 clonal) from the lungs of chronically infected adult patients attending the Royal Prince Alfred Hospital CF Clinic, Sydney, Australia. Overall, 32/43 (74 %) isolates were positive for protease IV protein by Western blotting and 22/43 (51 %) had evidence of active protease IV on gelatin zymography. Clonal strains were 1.6 times more likely than non-clonal strains to produce protease IV [18/19 (95 %) versus 14/24 (58 %), RR=1.6, CI 1.1-2.3, P=0.007] and 3 times more likely to secrete the protein [16/19 (84 %) versus 6/24 (25 %), RR=3.4, CI 1.6-6.9, P<0.001]. Nine of the ten strains negative by both Western blotting and zymography were non-clonal, and all but one of these was positive for the protease IV gene. There was a marked strain-to-strain variation in the amount of protease IV produced. Secretion of protease IV by clonal strains may enhance their infectivity and ability to adapt to the changing CF lung environment. Overall the findings suggest that protease IV plays an important role in the pathogenesis of P. aeruginosa infection in the CF lung.