A Decade of Burkholderia cenocepacia Virulence Determinant Research

The Burkholderia cepacia complex (Bcc) is a group of genetically related environmental bacteria that can cause chronic opportunistic infections in patients with cystic fibrosis (CF) and other underlying diseases. These infections are difficult to treat due to the inherent resistance of the bacteria to antibiotics. Bacteria can spread between CF patients through social contact and sometimes cause cepacia syndrome, a fatal pneumonia accompanied by septicemia. Burkholderia cenocepacia has been the focus of attention because initially it was the most common Bcc species isolated from patients with CF in North America and Europe. Today, B. cenocepacia, along with Burkholderia multivorans, is the most prevalent Bcc species in patients with CF. Given the progress that has been made in our understanding of B. cenocepacia over the past decade, we thought that it was an appropriate time to review our knowledge of the pathogenesis of B. cenocepacia, paying particular attention to the characterization of virulence determinants and the new tools that have been developed to study them. A common theme emerging from these studies is that B. cenocepacia establishes chronic infections in immuno-compromised patients, which depend more on determinants mediating host niche adaptation than those involved directly in host cells and tissue damage.

Efficacy of Tenofovir 1% Vaginal Gel in Reducing the Risk of HIV-1 and HSV-2 Infection

Human Immunodeficiency Virus (HIV) is a retrovirus that can result in rare opportunistic infections occurring in humans. The onset of these infections is known as Acquired Immune Deficiency Syndrome (AIDS). Sexual transmission is responsible for the majority of infections 1, resulting in transmission of HIV due to infected semen or vaginal and cervical secretions containing infected lymphocytes. HIV microbicides are formulations of chemical or biological agents that can be applied to the vagina or rectum with the intention of reducing the acquisition of HIV. Tenofovir is an NRTI that is phosphorylated by adenylate kinase to tenofovir diphosphate, which in turn competes with deoxyadeosine 5’-triphosphate for incorporation into newly synthesized HIV DNA. Once incorporated, tenofovir diphosphate results in chain termination, thus inhibiting viral replication. Tenofovir has been formulated into a range of vaginal formulations, such as rings, tablets gels and films. It has been shown to safe and effective in numerous animal models, while demonstrating safety and acceptability in numerous human trials. The most encouraging results came from the CAPRISA 004 clinical trial which demonstrated that a 1% Tenofovir vaginal gel reduced HIV infection by approximately 39%.

Disinfection of Burkholderia cepacia complex from non-touch taps in a neonatal nursery

Burkholderia cepacia complex (Bcc) comprises nine closely related species or genomovars. It is an important causative agent of opportunistic infections and waterborne nosocomial infections. B. cepacia (formerly genomovar I) was identified from the blood culture of a baby in our neonatal unit (NU) in March 2005. B. cepacia was isolated four times from clinical specimens since the introduction of non-touch taps in the NU from 2000 to 2005 and only once from 1994 to 2000. Environmental samples were collected from the NU, including tap water from non-touch taps. Clinical and environmental isolates of Bcc were characterized using molecular identification and strain typing. A literature review was undertaken to delineate a method for eradication of Bcc. Several variations for hot water eradication of the organism from the taps were attempted. Genotyping and molecular analysis revealed that tap water isolates were B. cenocepacia which was a different species from the B. cepacia isolated from blood cultures of the neonate. However, B. cenocepacia has been known to cause nosocomial outbreaks and it was eventually eradicated from the NU by using repeated thermal shock (hot water at 65 degrees C for 10 min), changing taps and decolonizing sinks with hypochlorite. Molecular typing is useful in assisting the investigation of Bcc nosocomial infections.

Burkholderia cepacia complex epidemiology in persons with cystic fibrosis from Australia and New Zealand

The Burkholderia cepacia complex (Bcc) is a group of significant opportunistic respiratory pathogens which affect people with cystic fibrosis. In this study, we sought to ascertain the epidemiology and geographic species distribution of 116 Bcc isolates collected from people with CF in Australia and New Zealand. We performed a combination of recA-based PCR, amplified rDNA restriction analysis (ARDRA), pulsed-field gel electrophoresis and repetitive extragenic palindromic PCR on each isolate. Each Burkholderia cenocepacia isolate was also screened by PCR for the presence of the B. cepacia epidemic strain marker. One hundred and fourteen isolates were assigned to a species using recA-based PCR and ARDRA. B. cenocepacia, B. multivorans and B. cepacia accounted for 45.7%, 29.3% and 11.2% of the isolates, respectively. Strain analysis of B. cenocepacia revealed that 85.3% of the isolates were unrelated. One related B. cenocepacia strain was identified amongst 15 people. Whilst full details of person-to-person contact was not available, all patients attended CF centres in Queensland (Qld) and New South Wales (NSW). Although person-to-person transmission of B. cenocepacia strains has occurred in Australia, the majority of CF-related Bcc infections in Australia and New Zealand are most likely acquired from the environment.

A two-tier model of polymyxin B resistance in Burkholderia cenocepacia

P>Burkholderia cenocepacia is an environmental bacterium causing serious human opportunistic infections and is extremely resistant to multiple antibiotics including antimicrobial peptides, such as polymyxin B (PmB). Extreme antibiotic resistance is attributed to outer membrane impermeability ('intrinsic' resistance). Previous work showed that production of full-length lipopolysaccharide (LPS) prevents surface binding of PmB. We hypothesized that two tiers of resistance mechanisms rendering different thresholds of PmB resistance exist in B. cenocepacia. To test this notion, candidate genes were mutated in two isogenic strains expressing full-length LPS or truncated LPS devoid of heptose ('heptoseless LPS') respectively. We uncovered various proteins required for PmB resistance only in the strain with heptoseless LPS. These proteins are not involved in preventing PmB binding to whole cells or permeabilization of the outer membrane. Our results support a two-tier model of PmB resistance in B. cenocepacia. One tier sets a very high threshold mediated by the LPS and the outer membrane permeability barrier. The second tier sets a lower threshold that may play a role in PmB resistance only when outer membrane permeability is compromised. This model may be of general applicability to understanding the high antimicrobial peptide resistance of environmental opportunistic pathogens.

A system for the construction of targeted unmarked gene deletions in the genus Burkholderia

Burkholderia species are extremely multidrug resistant, environmental bacteria with extraordinary bioremediation and biocontrol properties. At the same time, these bacteria cause serious opportunistic infections in vulnerable patient populations while some species can potentially be used as bioweapons. The complete DNA sequence of more than 10 Burkholderia genomes provides an opportunity to apply functional genomics to a collection of widely adaptable environmental bacteria thriving in diverse niches and establishing both symbiotic and pathogenic associations with many different organisms. However, extreme multidrug resistance hampers genetic manipulations in Burkholderia. We have developed and evaluated a mutagenesis system based on the homing endonuclease I-SceI to construct targeted, non-polar unmarked gene deletions in Burkholderia. Using the cystic fibrosis pathogen Burkholderia cenocepacia K56-2 as a model strain, we demonstrate this system allows for clean deletions of one or more genes within an operon and also the introduction of multiple deletions in the same strain. We anticipate this tool will have widespread environmental and biomedical applications, facilitating functional genomic studies and construction of safe strains for bioremediation and biocontrol, as well as clinical applications such as live vaccines for Burkholderia and other Gram-negative bacterial species.

Size-fitting of Intravaginal Rings for Macaques and in vitro Release Kinetics of Zinc Finger Inhibitors

Background: Small molecule inhibitors of the zinc finger domain (ZFI) in the nucleocapsid protein (NCp7) of HIV-1 are potent inhibitors of HIV and SIV
replication and may have utility as topical products to prevent infection. Furthermore, intravaginal rings (IVRs) were developed as coitally-independent,
sustained release devices which could be used for administration of HIV microbicides. The aims of these studies were to demonstrate that IVRs sized for
macaques are practical and compatible with the current generation of thioester-based NCp7 inhibitors.

Methods: Non-medicated silicone elastomer vaginal rings of various sizes thought to be applicable for macaques were prepared and tested for vaginal fit in Pigtailed and Chinese Rhesus macaques. Macaques were monitored for 8 weeks for mucosal disruption by colposcopy and proinflammatory cytokine markers in cervical vaginal lavages (CVL) using Luminex bead-based technology. Three different ZFIs (compounds 52, 89 and 122, each derived from an N-substituted S-acyl-2-mercaptobenzamide thioester scaffold) were loaded at 50 mg into an optimal matrix-type ring design. In vitro continuous release studies were then conducted over 28 days and analyzed by HPLC. Rate of release was determined by linear regression analysis.

Results: Qualitative evaluation at the time of ring insertion suggested that the 25 mm ring provided optimal fit in both macaque species. All rings remained in
place during the study period (2 to 4 weeks), and the animals did not attempt to remove the rings. No tissue irritation was observed, and no signs of physical
discomfort were noted. Also, no significant induction of cervicovaginal proinflammatory markers was observed during the 8-week period during and following ring insertion. One Pigtailed macaque showed elevated IL-8 levels in the CVL during the period when the ring was in place; however, these levels were comparable to those observed in two control macaques. In vitro release of the ZFIs peaked at day 1 and then continually declined to near steady-state rates between 20-30 mcg/day. The percent release after 14 days was 2.9, 2.0 and 0.9 for ZFI 89, 52 and 122, respectively.

Conclusions: IVRs of 25mm diameter, determined to be the optimal size for macaques, were well tolerated and did not induce inflammation. Release of all ZFI compounds followed t 0.5 kinetics. These findings suggest that efficacy testing in primate models is warranted to fully evaluate the potential to prevent
transmission.

Isolation of Alternaria alternata from an emollient cream:implications for public health

A specimen of emollient cream, which was observed to be contaminated peripherally with a filamentous fungus was examined for the presence of fungi and the resulting fungal colonies were examined phenotypically and genotypically. Subsequent DNA extraction and PCR amplification of the large internal transcribed spacer region [ITS1-5.8S-ITS2] yielded an amplicon of 512 bp. Sequence analysis identified this as Alternaria alternata at the 100% homology level with all 512/512 bases called. This organism has been previously reported as a cause of opportunistic infections involving skin and immunocompromised patients. This is the first report of an emollient cream as a source of this organism. It highlights the need for proper management of such preparations in order to minimize the potential spread of fungi to susceptible patient populations.

Autologous scleral patch graft in tube shunt surgery

Purpose . To investigate alternative surgical method to cover subconjunctival portion of the tube in tube shunt surgery in glaucoma Methods. In thirteen eyes of twelve patients who underwent tube shunt surgery at WEH between July 1991 to November 1996 a "rotating" autologous scleral lamellar graft was created to cover the subconjuctival course of the tube. These patients were followed for clinical signs of conjuctival breakdown, graft melt, tube erosion, anterior staphylomas, graft related inflammation, infections or graft related complications. Results. The patients were followed up for a mean of 16 5 months( range 1-60 months). All eyes tolerated the autologous graft well without any clinical evidence of tube erosions, graft melting , graft related or intraocular inflammation Conclusions The use of autologous sciera, to cover the tube in a tube shunt procedure appears to be effective and without inappropriate short term risk. It offers the advantages of availability , lower cost, and eliminates the potential risk of transmission of infectious disease( i.e. slow-virus, HIV etc.).

Burkholderia cenocepacia Lipopolysaccharide Modification and Flagellin Glycosylation Affect Virulence but Not Innate Immune Recognition in Plants

Burkholderia cenocepacia causes opportunistic infections in plants, insects, animals, and humans, suggesting that “virulence” depends on the host and its innate susceptibility to infection. We hypothesized that modifications in key bacterial molecules recognized by the innate immune system modulate host responses to B. cenocepacia. Indeed, modification of lipo- polysaccharide (LPS) with 4-amino-4-deoxy-L-arabinose and flagellin glycosylation attenuates B. cenocepacia infection in Arabi- dopsis thaliana and Galleria mellonella insect larvae. However, B. cenocepacia LPS and flagellin triggered rapid bursts of nitric oxide and reactive oxygen species in A. thaliana leading to activation of the PR-1 defense gene. These responses were drastically reduced in plants with fls2 (flagellin FLS2 host receptor kinase), Atnoa1 (nitric oxide-associated protein 1), and dnd1-1 (reduced production of nitric oxide) null mutations. Together, our results indicate that LPS modification and flagellin glycosylation do not affect recognition by plant receptors but are required for bacteria to establish overt infection.

Phenotypic characterization of an international Pseudomonas aeruginosa reference panel: strains of cystic fibrosis (CF) origin show less in vivo virulence than non-CF strains

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF) and acute opportunistic infections in people without CF. Forty two P. aeruginosa strains from a range of clinical and environmental sources were collated into a single reference strain panel to harmonise research on this diverse opportunistic pathogen. To facilitate further harmonized and comparable research on P. aeruginosa, we characterised the panel strains for growth rates, motility, virulence in the Galleria mellonella infection model, pyocyanin and alginate production, mucoid phenotype, lipopolysaccharide (LPS) pattern, biofilm formation, urease activity, antimicrobial and phage susceptibilities. Phenotypic diversity across the P. aeruginosa panel was apparent for all phenotypes examined agreeing with the marked variability seen in this species. However, except for growth rate, the phenotypic diversity among strains from CF versus non-CF sources was comparable. CF strains were less virulent in the G. mellonella model than non-CF strains (p=0.037). Transmissible CF strains generally lacked O antigen, produced less pyocyanin, and had low virulence in G. mellonella. Further, in the three sets of sequential CF strains, virulence, O-antigen expression and pyocyanin production were higher in the earlier isolate compared to the isolate obtained later in infection. Overall, full phenotypic characterization of the defined panel of P. aeruginosa strains increases our understanding of the virulence and pathogenesis of P. aeruginosa and may provide a valuable resource for the testing of novel therapies against this problematic pathogen.

Detection of the icaADBC gene cluster and biofilm formation in Staphylococcus epidermidis isolates from catheter-related urinary tract infections.

Cho SH, Naber K, Hacker J, Ziebuhr W. Institut für Molekulare Infektionsbiologie, Röntgenring 11, D-97070 Würzburg, Germany. Biofilm production in Staphylococcus epidermidis is an important virulence factor that is mediated by the expression of the icaADBC operon. In this study 41 S. epidermidis isolates obtained from catheter-related urinary tract infections were analyzed for the presence of the icaADBC operon and biofilm formation. Eighteen of 41 isolates (44%) were shown to carry ica-specific DNA, but only 11 isolates (27%) produced biofilms spontaneously under normal growth conditions. Upon induction by external stress or antibiotics, biofilm formation could be stimulated in five of seven ica-positive, biofilm-negative isolates, indicating that the icaADBC expression was down-regulated in these strains. Genetic analyses of the ica gene clusters of the remaining two ica-positive, biofilm-negative strains revealed a spontaneous ICAC::IS256 insertion in one strain. Insertion of the element caused a target site duplication of seven base pairs and a biofilm-negative phenotype. After repeated passages the insertion mutant was able to revert to a biofilm-forming phenotype which was due to the precise excision of IS256 from the icaC gene. The data show that icaC::IS256 integrations occur during S. epidermidis polymer-related infections and the results highlight the biological relevance of the IS256-mediated phase variation of biofilm production in S. epidermidis during an infection.