Identical Burkholderia cepacia complex strain types isolated from multiple patients attending a hospital in Brazil.
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Adult↗
Biomedical subjects
Craig Winstanley
Publications and source records attributed to Craig Winstanley.
Use of suppression subtractive hybridization to examine the accessory genome of the Liverpool cystic fibrosis epidemic strain of Pseudomonas aeruginosa.
The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa has been highly successful at colonizing cystic fibrosis (CF) patients throughout the UK, has replaced previously established strains in CF patients, has caused infections of non-CF parents of CF patients, and can cause greater morbidity in CF than other strains of P. aeruginosa. Using suppression subtractive hybridization (SSH) to identify strain-specific sequences, a diagnostic test for the LES based on PCR amplification of SSH sequence PS21 had previously been developed. In this study, the SSH sequence database of LES was substantially increased, using both extension of previous sequences and new rounds of subtraction. Of 92 SSH sequences identified as present in the LES but absent from strain PAO1, 25 were assessed for prevalence amongst a strain panel consisting mainly of LES and non-LES CF isolates. Preliminary analysis of genome sequence data indicated that all SSH sequences that were LES specific or found only rarely in other strains of P. aeruginosa were present on one of three contigs. All of the SSH sequences screened were either unstable amongst LES isolates or were not completely LES specific. Rare false positives were found with the PS21 test. The authors suggest that a second PCR assay designed to detect SSH sequence LESF9 can be used to confirm the identity of the most prevalent CF epidemic lineage in the UK.
Base Sequence↗
Development of a diagnostic test for the Midlands 1 cystic fibrosis epidemic strain of Pseudomonas aeruginosa.
In a previous study of isolates from cystic fibrosis (CF) patients in England and Wales, the Midlands 1 strain of Pseudomonas aeruginosa was identified as the second most common clone, representing 10% of isolates and found in nearly one-third of all CF centres [Scott, F. W. & Pitt, T. L. (2004). J Med Microbiol 53, 609-615]. Using suppression subtractive hybridization, 54 sequences were identified as present in a Midlands 1 strain but were absent from strain PAO1. The distribution of 14 of these sequences amongst representatives of Midlands 1, other CF epidemic strains and unrelated P. aeruginosa CF isolates was determined using PCR assays. Using these data, a PCR-based test was developed that was specific for the Midlands 1 clone, which was confirmed using dot-blot hybridization. By applying the test to CF isolates from a CF centre in Liverpool, a Midlands 1 clone was identified. The identity was confirmed using typing by PFGE. The PCR test should facilitate a greater understanding of the distribution of the Midlands 1 strain in the UK and elsewhere.
Cystic Fibrosis↗
Caenorhabditis elegans killing assay as an infection model to study the role of type III secretion in Burkholderia cenocepacia.
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Animals↗
Identification, typing, and insecticidal activity of Xenorhabdus isolates from entomopathogenic nematodes in United Kingdom soil and characterization of the xpt toxin loci.
Xenorhabdus strains from entomopathogenic nematodes isolated from United Kingdom soils by using the insect bait entrapment method were characterized by partial sequencing of the 16S rRNA gene, four housekeeping genes (asd, ompR, recA, and serC) and the flagellin gene (fliC). Most strains (191/197) were found to have genes with greatest similarity to those of Xenorhabdus bovienii, and the remaining six strains had genes most similar to those of Xenorhabdus nematophila. Generally, 16S rRNA sequences and the sequence types based on housekeeping genes were in agreement, with a few notable exceptions. Statistical analysis implied that recombination had occurred at the serC locus and that moderate amounts of interallele recombination had also taken place. Surprisingly, the fliC locus contained a highly variable central region, even though insects lack an adaptive immune response, which is thought to drive flagellar variation in pathogens of higher organisms. All the X. nematophila strains exhibited a consistent pattern of insecticidal activity, and all contained the insecticidal toxin genes xptA1A2B1C1, which were present on a pathogenicity island (PAI). The PAIs were similar among the X. nematophila strains, except for partial deletions of a peptide synthetase gene and the presence of insertion sequences. Comparison of the PAI locus with that of X. bovienii suggested that the PAI integrated into the genome first and then acquired the xpt genes. The independent mobility of xpt genes was further supported by the presence of xpt genes in X. bovienii strain I73 on a type 2 transposon structure and by the variable patterns of insecticidal activity in X. bovienii isolates, even among closely related strains.
Alleles↗
Use of a variable amplicon typing scheme reveals considerable variation in the accessory genomes of isolates of Burkholderia pseudomallei.
Melioidosis, a disease caused by the bacterium Burkholderia pseudomallei, is endemic in southeast Asia and northern Australia. We used suppression subtractive hybridization (SSH) to identify sequences that varied between two B. pseudomallei isolates from Australia and determined the distribution of 45 SSH-derived sequences among a panel of B. pseudomallei and B. thailandensis isolates. Sequences exhibiting variable prevalence were included in a variable amplicon typing (VAT) scheme designed to score the presence or absence of 14 PCR amplicons. VAT analysis was carried out with 48 isolates from Thailand, which were typed by multilocus sequence typing (MLST), and 44 isolates from Australia of known MLST type. The VAT scheme could be used to divide the 48 isolates from Thailand into 23 VAT types and the 44 isolates from Australia into 36 VAT types. Some of the sequences included in the VAT scheme were more commonly PCR positive among isolates from Australia than among isolates from Thailand, and vice versa. No isolate from Australia was PCR positive for genomic island 11 or a putative transposase sequence, whereas four SSH-derived sequences were far more prevalent among the Australian isolates. Analysis based on the VAT scheme indicated that the isolates clustered into groups, some of which were mainly or exclusively from one geographical origin. One cluster included Australian isolates that were mostly associated with severe disease, including rare neurological melioidosis, suggesting that the content of the accessory genome may play an important role in determining the clinical manifestation of the disease.
Bacterial Typing Techniques↗
Genotypic and phenotypic characteristics of Pseudomonas aeruginosa isolates associated with ulcerative keratitis.
A collection of 63 isolates of Pseudomonas aeruginosa associated with ulcerative keratitis, collected from six centres in England, were typed using serotyping and random amplified polymorphic DNA-PCR, and screened for several variable virulence-related genotypes and phenotypes. Sixty-one percent of the isolates were of either serotype O1 or serotype O11, but there was no evidence for a common clone. The majority of isolates (59%) were PCR-positive for exoU rather than for exoS (38%), and carried a-type fliC genes (76%) rather than b-type (24%). Isolates were PCR-positive for pyoverdine-receptor types at a prevalence of 38% for type I, 46 % for type II and 8 % for type III. All but one of the isolates exhibited twitching activity. There was a correlation between the presence of exoS and twitching activity (P = 0.04), suggesting that a combination of exoS genotype and good twitching activity may have a role to play in ExoU-independent corneal virulence.
ADP Ribose Transferases↗
A cystic fibrosis epidemic strain of Pseudomonas aeruginosa displays enhanced virulence and antimicrobial resistance.
The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa is a transmissible aggressive pathogen of cystic fibrosis (CF) patients. We compared transcriptome profiles of two LES isolates with each other and with a laboratory and genetic reference strain (PAO1) after growth to late exponential phase and following exposure to oxidative stress. Both LES isolates exhibited enhanced antimicrobial resistances linked to specific mutations in efflux pump genes. Although transcription of AmpC beta-lactamase was up-regulated in both, one LES isolate contained a specific mutation rendering the ampC gene untranslatable. The virulence-related quorum-sensing (QS) regulon of LES431, an isolate that caused pneumonia in the non-CF parent of a CF patient, was considerably up-regulated in comparison to either isolate LES400, associated with a chronic CF infection, or strain PAO1. Premature activation of QS genes was detected in isolates from both non-CF parents and the CF patient in a previously reported infection episode. LES isolates lacking the up-regulated QS phenotype contained different frameshift mutations in lasR. When fed to Drosophila melanogaster, isolate LES431 killed the fruit flies more readily than either isolate LES400 or strain PAO1, indicating that virulence varies intraclonally. The LES may represent a clone with enhanced virulence and antimicrobial resistance characteristics that can vary or are lost due to mutations during long-term colonization but have contributed to the successful spread of the lineage throughout the CF population of the United Kingdom.
Anti-Bacterial Agents↗
Isolation of Waddlia malaysiensis, a novel intracellular bacterium, from fruit bat (Eonycteris spelaea).
An obligate intracellular bacterium was isolated from urine samples from 7 (3.5%) of 202 fruit bats (Eonycteris spelaea) in peninsular Malaysia. The bacterium produced large membrane-bound inclusions in human, simian, and rodent cell lines, including epithelial, fibroblastlike, and lymphoid cells. Thin-section electron microscopy showed reticulate bodies dividing by binary fission and elementary bodies in the inclusions; mitochondria surrounded the inclusions. The inclusions were positive for periodic acid-Schiff stain but could not be stained by fluorescein-labeled anti-Chlamydia trachomatis major outer membrane protein monoclonal antibody. The bacterium was resistant to penicillin and streptomycin (MICs > 256 mg/L) but susceptible to tetracycline (MIC = 0.25 mg/L) and chloramphenicol (MIC = 0.5 mg/L). Sequence analysis of the 16SrRNA gene indicated that it was most closely related to 2 isolates of Waddlia chondrophila (94% and 96% identity). The 16S and 23S rRNA gene signatures were only 91% identical. We propose this novel bacterium be called W. malaysiensis.
3T3 Cells↗
Sequence divergence in type III secretion gene clusters of the Burkholderia cepacia complex.
The Burkholderia cepacia complex (BCC) comprises a group of bacteria associated with opportunistic infections, especially in cystic fibrosis patients. B. cenocepacia J2315, of the transmissible ET12 lineage, contains a type III secretion (TTS) gene cluster implicated in pathogenicity. PCR and hybridisation assays indicate that the TTS gene cluster is present in all members of the BCC except B. cepacia (formerly genomovar I). The TTS gene clusters of B. cenocepacia J2315 and B. multivorans are similar in organisation but have variable levels of gene identity. Nucleotide sequence data obtained for the equivalent region of the B. cepacia genome indicate the absence of TTS structural genes due to a rearrangement likely to involve more than one step.
Bacterial Proteins↗
Identification and expression of a Burkholderia pseudomallei collagenase in Escherichia coli.
Extracellular protein profiles were compared for broth-grown cultures of Burkholderia pseudomallei and its avirulent close relative Burkholderia thailandensis. A number of protein bands were present in the B. pseudomallei profile but absent or less abundant in the B. thailandensis profile. Four such prominent secreted proteins were identified by using N-terminal sequencing coupled to searches of the B. pseudomallei genome sequence database. The genes for two proteins with similarity to carbohydrate-binding proteins, and a further protein homologous to known bacterial collagenases, were present in both B. pseudomallei and B. thailandensis. The putative collagenase gene was cloned and expressed as a fusion protein in Escherichia coli. Cell lysates from Escherichia coli containing the recombinant protein exhibited detectable gelatinase and collagenase activities.
Amino Acid Sequence↗
Conservation of the opcL gene encoding the peptidoglycan-associated outer-membrane lipoprotein among representatives of the Burkholderia cepacia complex.
Members of the Burkholderia cepacia complex are Gram-negative beta-proteobacteria that are classified into nine genomic species or genomovars. Some representatives of this group of bacteria, such as Burkholderia multivorans (genomovar II) and Burkholderia cenocepacia (genomovar III), are considered to be dangerous pathogens for cystic fibrosis (CF) patients because of their capacity to colonize CF lungs. The opcL gene, which encodes the peptidoglycan-associated outer-membrane lipoprotein (PAL), was detected in the genome of Burkholderia sp. LB 400 by a similarity search that was based on the sequence of the Pseudomonas aeruginosa PAL, OprL. Primers that could amplify part of opcL from B. multivorans LMG 13010(T) were designed. This PCR fragment was used as a probe for screening of a B. multivorans genomic bank, allowing cloning of the complete opcL gene. The complete opcL gene could be PCR-amplified from DNA from all genomovars. The sequences of these opcL genes showed a high degree of conservation (> 95 %) among different species of the B. cepacia complex. OpcL protein that was purified from B. multivorans LMG 13010(T) was used to generate mouse polyclonal antisera against OpcL. The OpcL protein could be produced in Escherichia coli and detected in outer-membrane fractions by Western blot. Burkholderia cells were labelled by immunofluorescence staining using antibodies against OpcL, but only after treatment with EDTA and SDS. The opcL gene could be amplified directly from the sputa of 15 CF patients who were known to be colonized by B. cepacia; sequence data derived from the amplicons identified the colonizing strains as B. cenocepacia (genomovar III, n = 14) and B. multivorans (n = 1).
Amino Acid Sequence↗
Improved flagellin genotyping in the Burkholderia cepacia complex.
Oligonucleotide primers designed to N- and C-terminal sequences of Burkholderia cepacia complex fliC genes were used to amplify and sequence fliC genes from a strain of Burkholderia vietnamiensis and three isolates of Burkholderia multivorans with large fliC genes. Alignments incorporating the new sequences enabled the design of polymerase chain reaction (PCR) primers for extension of a published PCR/restriction fragment length polymorphism typing method, to include isolates that previously failed to yield fliC amplicons. Most B. vietnamiensis isolates and hitherto non-typable Burkholderia cenocepacia isolates contained much smaller fliC genes than previously reported. Although B. multivorans strains with larger fliC genes clustered together, relationships between strains based on fliC sequences did not generally correlate with genomovar status.
Burkholderia cepacia↗
Suppression-subtractive hybridisation reveals variations in gene distribution amongst the Burkholderia cepacia complex, including the presence in some strains of a genomic island containing putative polysaccharide production genes.
Some strains of the Burkholderia cepacia complex, including the ET12 lineage, have been implicated in epidemic spread amongst cystic fibrosis (CF) patients. Suppression-subtractive hybridisation was used to identify genomic regions within strain J2315 (ET12 lineage; genomovar IIIA) that were absent from a non-transmissible genomovar IIIB strain. Sequence data from 15 subtracted clones were used to interrogate the genome sequence of strain J2315 and identify genomic regions incorporating the subtracted sequences. Many of the genomic regions displayed abnormally low GC content and similarity to sequences implicated in gene transfer. The distribution of three subtracted regions amongst members of the B. cepacia complex varied. A large cluster of genes with strong sequence similarity to capsular production genes from Burkholderia mallei and other bacterial pathogens was identified. This genomic island was detected in some but not all representatives of genomovar IIIA, two out of four genomovar I strains, and one of two strains of Burkholderia multivorans, but was not detected in Burkholderia stabilis, Burkholderia vietnamiensis, genomovar VI or Burkholderia. ambifaria. The polysaccharide production gene cluster of strain J2315 carries an IS 407-like sequence within the gene similar to B. mallei wcbO that is lacking in other ET12 isolates. Genes from this cluster are expressed during exponential growth in broth.
Base Sequence↗
PCR-based detection of a cystic fibrosis epidemic strain of Pseudomonas Aeruginosa.
BACKGROUND: The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa is widespread among patients with cystic fibrosis (CF) in specialist centers around Liverpool and elsewhere in the UK. This study evaluates a new diagnostic PCR assay based on a unique DNA sequence (PS21) of LES, for its identification of colonies directly from sputum. METHODS: One hundred and fifty-eight sputum samples from 92 patients were cultured and P. aeruginosa isolates were typed by PS21 PCR and pulsed-field gel electrophoresis (PFGE). Subsequently, PS21 PCR was performed directly on sputum and the results were compared with culture, PFGE, and PS21 PCR typing. RESULTS: Eighty patients were colonized with P. aeruginosa, 63 by LES (79%). There was 100% concordance between PS21 PCR on colonies and PFGE typing. The sensitivity and specificity of PS21 PCR directly on sputum was 98.2% and 93.6%, respectively. CONCLUSIONS: This study shows that PS21 PCR can be used for simple and rapid screening of LES colonization in CF patients.
Base Sequence↗
Type III secretion homologs are present in Brucella melitensis, B. ovis, and B. suis biovars 1, 2, and 3.
Protein sequences from characterized type III secretion (TTS) systems were used as probes in silico to identify several TTS gene homologs in the genome sequence of Brucella suis biovar 1 strain 1330. Four of the genes, named flhB, fliP, fliR, and fliF on the basis of greatest homologies to known flagellar apparatus proteins, were targeted in PCR and hybridization assays to determine their distribution among other Brucella nomen species and biovars. The results indicated that flhB, fliP, fliR and fliF are present in Brucella melitensis, Brucella ovis, and Brucella suis biovars 1, 2 and 3. Similar homologos have been reported previously in Brucella abortus. Using RT-PCR assays, we were unable to detect any expression of these genes. It is not yet known whether the genes are the cryptic remnants of a flagellar system or are actively involved in a process contributing to pathogenicity or previously undetected motility, but they are distributed widely in Brucella and merit further study to determine their role.
Bacterial Proteins↗
Identification of type II and type III pyoverdine receptors from Pseudomonas aeruginosa.
Pseudomonas aeruginosa produces, under conditions of iron limitation, a high-affinity siderophore, pyoverdine (PVD), which is recognized at the level of the outer membrane by a specific TonB-dependent receptor, FpvA. So far, for P. aeruginosa, three different PVDs, differing in their peptide chain, have been described (types I-III), but only the FpvA receptor for type I is known. Two PVD-producing P. aeruginosa strains, one type II and one type III, were mutagenized by a mini-TnphoA3 transposon. In each case, one mutant unable to grow in the presence of the strong iron chelator ethylenediaminedihydroxyphenylacetic acid (EDDHA) and the cognate PVD was selected. The first mutant, which had an insertion in the pvdE gene, upstream of fpvA, was unable to take up type II PVD and showed resistance to pyocin S3, which is known to use type II FpvA as receptor. The second mutant was unable to take up type III PVD and had the transposon insertion in fpvA. Cosmid libraries of the respective type II and type III PVD wild-type strains were constructed and screened for clones restoring the capacity to grow in the presence of PVD. From the respective complementing genomic fragments, type II and type III fpvA sequences were determined. When in trans, type II and type III fpvA restored PVD production, uptake, growth in the presence of EDDHA and, in the case of type II fpvA, pyocin S3 sensitivity. Complementation of fpvA mutants obtained by allelic exchange was achieved by the presence of cognate fpvA in trans. All three receptors posses an N-terminal extension of about 70 amino acids, similar to FecA of Escherichia coli, but only FpvAI has a TAT export sequence at its N-terminal end.
Amino Acid Sequence↗
Molecular typing of, and distribution of genetic markers among, Burkholderia cepacia complex isolates from Brazil.
PCR tests were used to assign genomovar status to 39 non-cystic fibrosis (non-CF) and 11 CF Burkholderia cepacia complex isolates from patients in hospitals in Recife, Brazil. Non-CF isolates were assigned to genomovar IIIA (71.8%), genomovar I (15.4%), B. vietnamiensis (7.7%), and B. multivorans (5.1%). CF isolates were assigned to genomovar IIIA (18.2%), B. vietnamiensis (18.2%), and genomovar I (9.1%). Six CF isolates sharing recA PCR-restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) patterns could not be assigned to a genomovar. 16S rDNA sequence obtained from these isolates indicated a closest relationship to B. anthina, but the recA sequence was equally divergent from several genomovars. PCR screening indicated the presence of cblA in only two isolates, whereas the B. cepacia epidemic strain marker was found in 22 of 28 genomovar IIIA isolates. A type III secretion gene was detected in all but genomovar I isolates. RAPD and PCR-RFLP assays, targeting both recA and fliC, indicated a large amount of genetic variability among the isolates, with many novel patterns being observed. Nine genomovar IIIA isolates from different non-CF patients and clinical sources had identical genotypes, indicating the presence of a common clone.
Bacterial Typing Techniques↗