High-throughput single-nucleotide polymorphism-based typing of shared Pseudomonas aeruginosa strains in cystic fibrosis patients using the Sequenom iPLEX platform

Shared strains of Pseudomonas aeruginosa are now well recognized in people with cystic fibrosis (CF), and suitable P. aeruginosa laboratory typing tools are pivotal to understanding their clinical significance and guiding infection control policies in CF clinics. We therefore compared a single-nucleotide polymorphism (SNP)-based typing method using Sequenom iPLEX matrix-assisted laser desorption ionization with time-of-flight mass spectrometry (MALDI-TOF MS) with typing methods used routinely by our laboratory. We analysed 617 P. aeruginosa isolates that included 561 isolates from CF patients collected between 2001 and 2009 in two Brisbane CF clinics and typed previously by enterobacterial repetitive intergenic consensus (ERIC)-PCR, as well as 56 isolates from non-CF patients analysed previously by multilocus sequence typing (MLST). The isolates were tested using a P. aeruginosa Sequenom iPLEX MALDI-TOF (PA iPLEX) method comprising two multiplex reactions, a 13-plex and an 8-plex, to characterize 20 SNPs from the P. aeruginosa housekeeping genes acsA, aroE, guaA, mutL, nuoD, ppsA and trpE. These 20 SNPs were employed previously in a real-time format involving 20 separate assays in our laboratory. The SNP analysis revealed 121 different SNP profiles for the 561 CF isolates. Overall, there was at least 96% agreement between the ERIC-PCR and SNP analyses for all predominant shared strains among patients attending our CF clinics: AUST-01, AUST-02 and AUST-06. For the less frequently encountered shared strain AUST-07, 6/25 (24%) ERIC-PCR profiles were misidentified initially as AUST-02 or as unique, illustrating the difficulty of gel-based analyses. SNP results for the 56 non-CF isolates were consistent with previous MLST data. Thus, the PA iPLEX format provides an attractive high-throughput alternative to ERIC-PCR for large-scale investigations of shared P. aeruginosa strains.

Shared Pseudomonas aeruginosa genotypes are common in Australian cystic fibrosis centres

Recent molecular-typing studies suggest cross-infection as one of the potential acquisition pathways for Pseudomonas aeruginosa in patients with cystic fibrosis (CF). In Australia, there is only limited evidence of unrelated patients sharing indistinguishable P. aeruginosa strains. We therefore examined the point-prevalence, distribution, diversity and clinical impact of P. aeruginosa strains in Australian CF patients nationally. 983 patients attending 18 Australian CF centres provided 2887 sputum P. aeruginosa isolates for genotyping by enterobacterial repetitive intergenic consensus-PCR assays with confirmation by multilocus sequence typing. Demographic and clinical details were recorded for each participant. Overall, 610 (62%) patients harboured at least one of 38 shared genotypes. Most shared strains were in small patient clusters from a limited number of centres. However, the two predominant genotypes, AUST-01 and AUST-02, were widely dispersed, being detected in 220 (22%) and 173 (18%) patients attending 17 and 16 centres, respectively. AUST-01 was associated with significantly greater treatment requirements than unique P. aeruginosa strains. Multiple clusters of shared P. aeruginosa strains are common in Australian CF centres. At least one of the predominant and widespread genotypes is associated with increased healthcare utilisation. Longitudinal studies are now needed to determine the infection control implications of these findings.

Clonal complex Pseudomonas aeruginosa in horses

Pseudomonas aeruginosa is associated with infectious endometritis in horses. Although infectious endometritis is often considered a venereal infection, there is relatively limited genotypic-based evidence to support this mode of transmission. The study sought to determine the relatedness between genital P. aeruginosa isolates collected from a limited geographical region using molecular strain typing. Enterobacterial repetitive intergenic consensus PCR typing was performed on 93 isolates collected between 2005 and 2009 from 2058 thoroughbred horses (including 18 stallions) at 66 studs. While P. aeruginosa was not detected in the stallions, 53/93 (57%) mares harbouring P. aeruginosa had clonally related strains, which included a single dominant genotype detected in 42 (45%) mares from 13 different studs. These novel findings suggest that most equine genital P. aeruginosa infections in this region may have been acquired from mechanisms other than direct horse to horse transmission. Instead, other potential acquisition pathways, as well as strain specific adaptation to the equine genital tract, should be investigated.

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.

Comparison of three molecular techniques for typing Pseudomonas aeruginosa isolates in sputum samples from patients with cystic fibrosis

Monitoring the emergence and transmission of Pseudomonas aeruginosa strains among cystic fibrosis (CF) patients is important for infection control in CF centers internationally. A recently developed multilocus sequence typing (MLST) scheme is used for epidemiologic analyses of P. aeruginosa outbreaks; however, little is known about its suitability for isolates from CF patients compared with that of pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). As part of a prevalence study of P. aeruginosa strains in Australian CF clinics, we compared the discriminatory power and concordance of ERIC-PCR, PFGE, and MLST among 93 CF sputum and 11 control P. aeruginosa isolates. PFGE and MLST analyses were also performed on 30 paired isolates collected 85 to 354 days apart from 30 patients attending two CF centers separated by 3,600 kilometers in order to detect within-host evolution. Each of the three methods displayed high levels of concordance and discrimination; however, overall lower discrimination was seen with ERIC-PCR than with MLST and PFGE. Analysis of the 50 ERIC-PCR types yielded 54 PFGE types, which were related by ≤ 6 band differences, and 59 sequence types, which were classified into 7 BURST groups and 42 singletons. MLST also proved useful for detecting novel and known strains and for inferring relatedness among unique PFGE types. However, 47% of the paired isolates produced PFGE patterns that within 1 year differed by one to five bands, whereas with MLST all paired isolates remained identical. MLST thus represents a categorical analysis tool with resolving power similar to that of PFGE for typing P. aeruginosa. Its focus on highly conserved housekeeping genes is particularly suited for long-term clinical monitoring and detecting novel strains.

Low rates of Pseudomonas aeruginosa misidentification in isolates from cystic fibrosis patients

Pseudomonas aeruginosa is an important cause of pulmonary infection in cystic fibrosis (CF). Its correct identification ensures effective patient management and infection control strategies. However, little is known about how often CF sputum isolates are falsely identified as P. aeruginosa. We used P. aeruginosa-specific duplex real-time PCR assays to determine if 2,267 P. aeruginosa sputum isolates from 561 CF patients were correctly identified by 17 Australian clinical microbiology laboratories. Misidentified isolates underwent further phenotypic tests, amplified rRNA gene restriction analysis, and partial 16S rRNA gene sequence analysis. Participating laboratories were surveyed on how they identified P. aeruginosa from CF sputum. Overall, 2,214 (97.7%) isolates from 531 (94.7%) CF patients were correctly identified as P. aeruginosa. Further testing with the API 20NE kit correctly identified only 34 (59%) of the misidentified isolates. Twelve (40%) patients had previously grown the misidentified species in their sputum. Achromobacter xylosoxidans (n = 21), Stenotrophomonas maltophilia (n = 15), and Inquilinus limosus (n = 4) were the species most commonly misidentified as P. aeruginosa. Overall, there were very low rates of P. aeruginosa misidentification among isolates from a broad cross section of Australian CF patients. Additional improvements are possible by undertaking a culture history review, noting colonial morphology, and performing stringent oxidase, DNase, and colistin susceptibility testing for all presumptive P. aeruginosa isolates. Isolates exhibiting atypical phenotypic features should be evaluated further by additional phenotypic or genotypic identification techniques.

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.

Burkholderia pseudomallei:another emerging pathogen in cystic fibrosis

BACKGROUND: Burkholderia pseudomallei is an important cause of acute fulminant pneumonia and septicaemia in tropical regions of northern Australia and south east Asia. Subacute and chronic forms of the disease also occur. There have been three recent reports of adults with cystic fibrosis (CF) who presumably acquired B pseudomallei infection during extended vacations or residence in either Thailand or northern Australia.

METHODS: The clinical course, molecular characteristics, serology and response to treatment are described in four adult CF patients infected with B pseudomallei. Polymerase chain reaction (PCR) based methods were used to confirm B pseudomallei and exclude B cepacia complex. Genotyping was performed using randomly amplified polymorphic DNA (RAPD) PCR and pulsed field gel electrophoresis (PFGE).

RESULTS: Four patients are described with a mean duration of infection of 32 months. All but one patient lived in tropical Queensland. Two patients (with the longest duration of infection) deteriorated clinically and one subsequently died of respiratory failure. Both responded to intravenous treatment specifically targeting B pseudomallei. Another patient suffered two severe episodes of acute bronchopneumonia following acquisition of B pseudomallei. Eradication of the organism was not possible in any of the cases. PFGE of a sample isolate from each patient revealed the strains to be unique and RAPD analysis showed retention of the same strain within an individual over time.

CONCLUSIONS: These findings support a potential pathogenic role for B pseudomallei in CF lung disease, producing both chronic infection and possibly acute bronchopneumonia. Identical isolates are retained over time and are unique, consistent with likely environmental acquisition and not person to person spread. B pseudomallei is emerging as a significant pathogen for patients with CF residing and holidaying in the tropics.

Multiplex Touchdown PCR for Rapid Typing of the Opportunistic Pathogen Propionibacterium acnes.

The opportunistic human pathogen Propionibacterium acnes is comprised of a number of distinct phylogroups, designated types IA1, IA2, IB, IC, II and III, that vary in their production of putative virulence factors, inflammatory potential, as well as biochemical, aggregative and morphological characteristics. Although Multilocus Sequence Typing (MLST) currently represents the gold standard for unambiguous phylogroup classification, and individual strain identification, it is a labour and time-consuming technique. As a consequence, we have developed a multiplex touchdown PCR assay that will, in a single reaction, confirm species identity and phylogeny of an isolate based on its pattern of reaction with six primer sets that target the 16S rRNA (all isolates), ATPase (type IA1, IA2, IC), sodA (type IA2, IB), atpD (type II) and recA (type III) housekeeping genes, as well as a Fic family toxin gene (type IC). When applied to 312 P. acnes isolates previously characterised by MLST, and representing type IA1 (n=145), IA2 (n=20), IB (n=65), IC (n=7), II (n=45) and III (n=30), the multiplex displayed 100% sensitivity and 100% specificity for the detection of isolates within each targeted phylogroup. No cross-reactivity with isolates from other bacterial species was observed. The multiplex assay will provide researchers with a rapid, high-throughput and technically undemanding typing method for epidemiological and phylogenetic investigations. It will facilitate studies investigating the association of lineages with various infections and clinical conditions, as well as a pre-screening tool to maximise the number of genetically diverse isolates selected for downstream, higher resolution sequence-based analyses.

Complete nucleotide sequence of φCHU: A Luz24likevirus infecting Pseudomonas aeruginosa and displaying a unique host range

A complete nucleotide sequence of the new Pseudomonas aeruginosa Luz24likevirus phiCHU was obtained. This virus was shown to have a unique host range whereby it grew poorly on the standard laboratory strain PAO1, but infected 26 of 46 clinical isolates screened, and strains harboring IncP2 plasmid pMG53. It was demonstrated that phiCHU has single strand interruptions in its genome. Analysis of the phiCHU genome also suggested that recombination event(s) participated in the evolution of the leftmost portion of the genome, presumably encoding early genes.