Snehal Anuj

Cough-generated aerosols of Pseudomonas aeruginosa and other Gram-negative bacteria from patients with cystic fibrosis

Background: Pseudomonas aeruginosa is the most common bacterial pathogen in patients with cystic fibrosis (CF). Current infection control guidelines aim to prevent transmission via contact and respiratory droplet routes and do not consider the possibility of airborne transmission. It was hypothesised that subjects with CF produce viable respirable bacterial aerosols with coughing. Methods: A cross-sectional study was undertaken of 15 children and 13 adults with CF, 26 chronically infected with P aeruginosa. A cough aerosol sampling system enabled fractioning of respiratory particles of different sizes and culture of viable Gram-negative non-fermentative bacteria. Cough aerosols were collected during 5 min of voluntary coughing and during a sputum induction procedure when tolerated. Standardised quantitative culture and genotyping techniques were used. Results: P aeruginosa was isolated in cough aerosols of 25 subjects (89%), 22 of whom produced sputum samples. P aeruginosa from sputum and paired cough aerosols were indistinguishable by molecular typing. In four cases the same genotype was isolated from ambient room air. Approximately 70% of viable aerosols collected during voluntary coughing were of particles ⩽3.3 μm aerodynamic diameter. P aeruginosa, Burkholderia cenocepacia, Stenotrophomonas maltophilia and Achromobacter xylosoxidans were cultivated from respiratory particles in this size range. Positive room air samples were associated with high total counts in cough aerosols (p = 0.003). The magnitude of cough aerosols was associated with higher forced expiratory volume in 1 s (r = 0.45, p = 0.02) and higher quantitative sputum culture results (r = 0.58, p = 0.008). Conclusion: During coughing, patients with CF produce viable aerosols of P aeruginosa and other Gram-negative bacteria of respirable size range, suggesting the potential for airborne transmission.

Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reaction assay targeting the ecfX and the gyrB genes.

Phenotypic identification of Gram-negative bacteria from respiratory specimens of patients with cystic fibrosis carries a high risk of misidentification. Molecular identification techniques that use single-gene targets are also susceptible to error, including cross-reaction issues with other Gram-negative organisms. In this study, we have designed a Pseudomonas aeruginosa duplex real-time polymerase chain reaction (PCR) (PAduplex) assay targeting the ecfX and the gyrB genes. The PAduplex was evaluated against a panel of 91 clinical and environmental isolates that were presumptively identified as P. aeruginosa. The results were compared with those obtained using a commercial biochemical identification kit and several other P. aeruginosa PCR assays. The results showed that the PAduplex assay is highly suitable for routine identification of P. aeruginosa isolates from clinical or environmental samples. The 2-target format provides simultaneous confirmation of P. aeruginosa identity where both the ecfX and gyrB PCR reactions are positive and may also reduce the potential for false negatives caused by sequence variation in primer or probe targets.

Virulence factor expression patterns in Pseudomonas aeruginosa strains from infants with cystic fibrosis

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.

Type 3 secretion system effector genotype and secretion phenotype of longitudinally collected Pseudomonas aeruginosa isolates from young children diagnosed with cystic fibrosis following newborn screening

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.

Rapid single-nucleotide polymorphism-based identification of clonal Pseudomonas aeruginosa isolates from patients with cystic fibrosis by the use of real-time PCR and high-resolution melting curve analysis

Pseudomonas aeruginosa genotyping relies mainly upon DNA fingerprinting methods, which can be subjective, expensive and time-consuming. The detection of at least three different clonal P. aeruginosa strains in patients attending two cystic fibrosis (CF) centres in a single Australian city prompted the design of a non-gel-based PCR method to enable clinical microbiology laboratories to readily identify these clonal strains. We designed a detection method utilizing heat-denatured P. aeruginosa isolates and a ten-single-nucleotide polymorphism (SNP) profile. Strain differences were detected by SYBR Green-based real-time PCR and high-resolution melting curve analysis (HRM10SNP assay). Overall, 106 P. aeruginosa sputum isolates collected from 74 patients with CF, as well as five reference strains, were analysed with the HRM10SNP assay, and the results were compared with those obtained by pulsed-field gel electrophoresis (PFGE). The HRM10SNP assay accurately identified all 45 isolates as members of one of the three major clonal strains characterized by PFGE in two Brisbane CF centres (Australian epidemic strain-1, Australian epidemic strain-2 and P42) from 61 other P. aeruginosa strains from Australian CF patients and two representative overseas epidemic strain isolates. The HRM10SNP method is simple, is relatively inexpensive and can be completed in <3 h. In our setting, it could be made easily available for clinical microbiology laboratories to screen for local P. aeruginosa strains and to guide infection control policies. Further studies are needed to determine whether the HRM10SNP assay can also be modified to detect additional clonal strains that are prevalent in other CF centres.

A comparison of two informative SNP-based strategies for typing Pseudomonas aeruginosa isolates from patients with cystic fibrosis

BackgroundMolecular typing is integral for identifying Pseudomonas aeruginosa strains that may be shared between patients with cystic fibrosis (CF). We conducted a side-by-side comparison of two P. aeruginosa genotyping methods utilising informative-single nucleotide polymorphism (SNP) methods; one targeting 10 P. aeruginosa SNPs and using real-time polymerase chain reaction technology (HRM10SNP) and the other targeting 20 SNPs and based on the Sequenom MassARRAY platform (iPLEX20SNP).MethodsAn in-silico analysis of the 20 SNPs used for the iPLEX20SNP method was initially conducted using sequence type (ST) data on the P. aeruginosa PubMLST website. A total of 506 clinical isolates collected from patients attending 11 CF centres throughout Australia were then tested by both the HRM10SNP and iPLEX20SNP assays. Type-ability and discriminatory power of the methods, as well as their ability to identify commonly shared P. aeruginosa strains, were compared.ResultsThe in-silico analyses showed that the 1401 STs available on the PubMLST website could be divided into 927 different 20-SNP profiles (D-value = 0.999), and that most STs of national or international importance in CF could be distinguished either individually or as belonging to closely related single- or double-locus variant groups. When applied to the 506 clinical isolates, the iPLEX20SNP provided better discrimination over the HRM10SNP method with 147 different 20-SNP and 92 different 10-SNP profiles observed, respectively. For detecting the three most commonly shared Australian P. aeruginosa strains AUST-01, AUST-02 and AUST-06, the two methods were in agreement for 80/81 (98.8%), 48/49 (97.8%) and 11/12 (91.7%) isolates, respectively.ConclusionsThe iPLEX20SNP is a superior new method for broader SNP-based MLST-style investigations of P. aeruginosa. However, because of convenience and availability, the HRM10SNP method remains better suited for clinical microbiology laboratories that only utilise real-time PCR technology and where the main interest is detection of the most highly-prevalent P. aeruginosa CF strains within Australian clinics.

Genotypic and phenotypic identification of Aeromonas species and CphA-mediated carbapenem resistance in Queensland, Australia.

Infection caused by Aeromonas spp. ranges from superficial wound infection to life-threatening septicemia. Carbapenem resistance due to metallo-beta-lactamase, CphA encoded by the cphA gene, is a significant problem. This study defines Aeromonas spp. causing clinical disease in Queensland, Australia. Phenotypic tests for carbapenemase detection were assessed. One hundred Aeromonas isolates from blood (22), wound (46), sterile sites (11), stool (18), eye (2), and sputum (1) were characterized by rpoB and gyrB sequencing. Meropenem susceptibility by VITEK2, disk diffusion, and E-test MIC were determined. Carbapenemase production was assessed by Carba NP test and cphA by PCR. Gene sequencing identified isolates as Aeromonas dhakensis (39), Aeromonas veronii (21), Aeromonas hydrophila (20), Aeromonas caviae (14), Aeromonas jandaei (4), Aeromonas bestiarum (1), and Aeromonas sanarellii (1). Disk diffusion and E-test failed to detect resistance in isolates with presence of cphA. Carba NP was performed with 97.4% sensitivity and 95.7% specificity. Carbapenem resistance gene cphA was detected in A. veronii (21; 100%), A. hydrophila (18; 90%), A. dhakensis (34; 87.2%), A. jandaei (3; 75%), and A. bestiarum (1; 100%) but not A. caviae. We found that A. dhakensis was the predominant species, a previously unrecognized pathogen in this region.