Evidence of persistence of Prevotella spp. in the cystic fibrosis lung.

Purpose. Prevotella spp. represent a diverse genus of bacteria, frequently identified by both culture and molecular methods in the lungs of patients with chronic respiratory infection. However, their role in the pathogenesis of chronic lung infection is unclear; therefore, a more complete understanding of their molecular epidemiology is required.Methodology. Pulsed Field Gel Electrophoresis (PFGE) and Random Amplified Polymorphic DNA (RAPD) assays were developed and used to determine the degree of similarity between sequential isolates (n=42) from cystic fibrosis (CF) patients during periods of clinical stability and exacerbation.Results. A wide diversity of PFGE and RAPD banding patterns were observed, demonstrating considerable within-genus heterogeneity. In 8/12 (66.7 %) cases, where the same species was identified at sequential time points, pre- and post-antibiotic treatment of an exacerbation, PFGE/RAPD profiles were highly similar or identical. Congruence was observed between PFGE and RAPD (adjusted Rand coefficient, 0.200; adjusted Wallace RAPD->PFGE 0.459, PFGE->RAPD 0.128). Furthermore, some isolates could not be adequately assigned a species name on the basis of 16S rRNA analysis: these isolates had identical PFGE/RAPD profiles to Prevotella histicola.Conclusion. The similarity in PFGE and RAPD banding patterns observed in sequential CF Prevotella isolates may be indicative of the persistence of this genus in the CF lung. Further work is required to determine the clinical significance of this finding, and to more accurately distinguish differences in pathogenicity between species.

Community dynamics and the lower airway microbiota in stable chronic obstructive pulmonary disease, smokers and healthy non-smokers.

RATIONALE: The role bacteria play in the progression of COPD has increasingly been highlighted in recent years. However, the microbial community complexity in the lower airways of patients with COPD is poorly characterised. OBJECTIVES: To compare the lower airway microbiota in patients with COPD, smokers and non-smokers. METHODS: Bronchial wash samples from adults with COPD (n=18), smokers with no airways disease (n=8) and healthy individuals (n=11) were analysed by extended-culture and culture-independent Illumina MiSeq sequencing. We determined aerobic and anaerobic microbiota load and evaluated differences in bacteria associated with the three cohorts. Culture-independent analysis was used to determine differences in microbiota between comparison groups including taxonomic richness, diversity, relative abundance, 'core' microbiota and co-occurrence. MEASUREMENT AND MAIN RESULTS: Extended-culture showed no difference in total load of aerobic and anaerobic bacteria between the three cohorts. Culture-independent analysis revealed that the prevalence of members of Pseudomonas spp. was greater in the lower airways of patients with COPD; however, the majority of the sequence reads for this taxa were attributed to three patients. Furthermore, members of Bacteroidetes, such as Prevotella spp., were observed to be greater in the 'healthy' comparison groups. Community diversity (α and ß) was significantly less in COPD compared with healthy groups. Co-occurrence of bacterial taxa and the observation of a putative 'core' community within the lower airways were also observed. CONCLUSIONS: Microbial community composition in the lower airways of patients with COPD is significantly different to that found in smokers and non-smokers, indicating that a component of the disease is associated with changes in microbiological status.

Inflammatory and immunological biomarkers are not related to survival in adults with Cystic Fibrosis.

BACKGROUND: Chronic Pseudomonas aeruginosa pulmonary infection is associated with a decline in lung function and reduced survival in people with Cystic Fibrosis (CF). Damaging inflammatory and immunological mediators released in the lungs can be used as markers of chronic infection, inflammation and lung tissue damage. METHODS: Clinical samples were collected from CF patients and healthy controls. Serum IgG and IgA anti-Pseudomonas antibodies, sputum IL-8 and TNFα, plasma IL-6 and urine TNFr1 were measured by ELISA. Sputum neutrophil elastase (NE), cathepsin S and cathepsin B were measured by spectrophotometric and fluorogenic assays. The relationship between IgG and IgA, inflammatory mediators and long-term survival was determined. RESULTS: IgG and IL-6 positively correlated with mortality. However, multivariate analysis demonstrated that after adjusting for FEV(1), IgG was not independently related to mortality. A relationship was observed between IgG and IL-6, TNFα, TNFr1 and between IgA and IL8, cathepsin S and cathepsin B. CONCLUSIONS: These data indicate that biomarkers of inflammation are not independent predictors of survival in people with CF.

Use of culture and molecular analysis to determine the effect of antibiotic treatment on microbial community diversity and abundance during exacerbation in patients with cystic fibrosis.

BACKGROUND: Anaerobic bacteria are increasingly regarded as important in cystic fibrosis (CF) pulmonary infection. The aim of this study was to determine the effect of antibiotic treatment on aerobic and anaerobic microbial community diversity and abundance during exacerbations in patients with CF. METHODS: Sputum was collected at the start and completion of antibiotic treatment of exacerbations and when clinically stable. Bacteria were quantified and identified following culture, and community composition was also examined using culture-independent methods. RESULTS: Pseudomonas aeruginosa or Burkholderia cepacia complex were detected by culture in 24/26 samples at the start of treatment, 22/26 samples at completion of treatment and 11/13 stable samples. Anaerobic bacteria were detected in all start of treatment and stable samples and in 23/26 completion of treatment samples. Molecular analysis showed greater bacterial diversity within sputum samples than was detected by culture; there was reasonably good agreement between the methods for the presence or absence of aerobic bacteria such as P aeruginosa (κ=0.74) and B cepacia complex (κ=0.92), but agreement was poorer for anaerobes. Both methods showed that the composition of the bacterial community varied between patients but remained relatively stable in most individuals despite treatment. Bacterial abundance decreased transiently following treatment, with this effect more evident for aerobes (median decrease in total viable count 2.3×10(7) cfu/g, p=0.005) than for anaerobes (median decrease in total viable count 3×10(6) cfu/g, p=0.046). CONCLUSION: Antibiotic treatment targeted against aerobes had a minimal effect on abundance of anaerobes and community composition, with both culture and molecular detection methods required for comprehensive characterisation of the microbial community in the CF lung. Further studies are required to determine the clinical significance of and optimal treatment for these newly identified bacteria.

Changes in antibiotic susceptibility in staphylococci habituated to sub-lethal concentrations of tea tree oil (Melaleuca alternifolia).

AIMS: To investigate the effect of sub-lethal challenge with tea tree oil (TTO) on the antibiotic resistance profiles of staphylococci. METHODS AND RESULTS: Isolates of methicillin-resistant/-sensitive Staphylococcus aureus (MRSA and MSSA) and coagulase-negative staphylococci (CONS) were habituated to sub-lethal concentrations of TTO (72 h). Following habituation, the minimum inhibitory concentrations (MIC) of antibiotics and TTO were determined. Habituated MRSA/MSSA cultures had higher (P < 0.05) MIC values than control cultures for the examined antibiotics. Habituated MRSA/MSSA cultures also displayed decreased susceptibility to TTO. Although the MIC of habituated MRSA/MSSA for the examined antibiotics reverted to control values after subsequent culture in the absence of TTO, the increased MIC against TTO were maintained. When compared with control cultures, habituated CoNS cultures had higher (P < 0.05) MIC values against three-fifths of the antibiotics examined; no changes in TTO MIC were observed. CONCLUSIONS: TTO habituation 'stress-hardens' MRSA and MSSA, evidenced by transient decreased antibiotic susceptibility and stable decreased TTO susceptibility. Although TTO habituation did not decrease susceptibility of CoNS to TTO, such cultures showed transient decreased antibiotic susceptibility. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of TTO at sub-lethal concentrations may reduce the efficacy of topical antibiotics used with TTO in combination therapies.

Incorporation of chitosan in acrylic bone cement: effect on antibiotic release, bacterial biofilm formation and mechanical properties.

Bacterial infection remains a significant problem following total joint replacement. Efforts to prevent recurrent implant infection, including the use of antibiotic-loaded bone cement for implant fixation at the time of revision surgery, are not always successful. In this in vitro study, we investigated whether the addition of chitosan to gentamicin-loaded Palacos R bone cement increased antibiotic release and prevented bacterial adherence and biofilm formation by Staphylococcus spp. clinical isolates. Furthermore, mechanical tests were performed as a function of time post-polymerisation in pseudo-physiological conditions. The addition of chitosan to gentamicin-loaded Palacos R bone cement significantly decreased gentamicin release and did not increase the efficacy of the bone cement at preventing bacterial colonisation and biofilm formation. Moreover, the mechanical performance of cement containing chitosan was significantly reduced after 28 days of saline degradation with the compressive and bending strengths not in compliance with the minimum requirements as stipulated by the ISO standard for PMMA bone cement. Therefore, incorporating chitosan into gentamicin-loaded Palacos R bone cement for use in revision surgery has no clinical antimicrobial benefit and the detrimental effect on mechanical properties could adversely affect the longevity of the prosthetic joint.

Comparison of the cidal activity of tea tree oil and terpinen-4-ol against clinical bacterial skin isolates and human fibroblast cells.

AIMS: The aim of this study was to compare both the antimicrobial activity of terpinen-4-ol and tea tree oil (TTO) against clinical skin isolates of meticillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) and their toxicity against human fibroblast cells. METHODS AND RESULTS: Antimicrobial activity was compared by using broth microdilution and quantitative in vitro time-kill test methods. Terpinen-4-ol exhibited significantly greater bacteriostatic and bactericidal activity, as measured by minimum inhibitory and bactericidal concentrations, respectively, than TTO against both MRSA and CoNS isolates. Although not statistically significant, time-kill studies also clearly showed that terpinen-4-ol exhibited greater antimicrobial activity than TTO. Comparison of the toxicity of terpinen-4-ol and TTO against human fibroblasts revealed that neither agent, at the concentrations tested, were toxic over the 24-h test period. CONCLUSIONS: Terpinen-4-ol is a more potent antibacterial agent against MRSA and CoNS isolates than TTO with neither agent exhibiting toxicity to fibroblast cells at the concentrations tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Terpinen-4-ol should be considered for inclusion as a single agent in products formulated for topical treatment of MRSA infection. However, further work would initially be required to ensure that resistance would not develop with the use of terpinen-4-ol as a single agent.

Effect of pH on the antimicrobial susceptibility of planktonic and biofilm-grown clinical Pseudomonas aeruginosa isolates.

The pH at the site of infection is one of a number of factors that may significantly influence the in vivo activity of an antibiotic prescribed for treatment of infection and it may be of particular importance in the treatment of cystic fibrosis (CF) pulmonary infection, as acidification of the airways in CF patients has been reported. As Pseudomonas aeruginosa is the most frequent causative pathogen of CF pulmonary infection, this study determines the effect that growth at a reduced pH, as may be experienced by P. aeruginosa during infection of the CF lung, has on the susceptibility of clinical P. aeruginosa isolates, grown planktonically and as biofilms, to tobramycin and ceftazidime. Time-kill assays revealed a clear loss of tobramycin bactericidal activity when the isolates were grown under acidic conditions. MIC and MBC determinations also showed decreased tobramycin activity under acidic conditions, but this effect was not observed for all isolates tested. In contrast, growth of the isolates at a reduced pH had no adverse effect on the bacteriostatic and bactericidal activity of ceftazidime. When the isolates were grown as biofilms, the pH at which the biofilms were formed did not affect the bactericidal activity of either tobramycin or ceftazidime, with neither antibiotic capable of eradicating biofilms formed by the isolates at each pH. This was in spite of the fact that the concentrations of both antibiotics used were much higher than the concentrations required to kill the isolates growing planktonically. These results show that growth in an acidic environment may reduce the susceptibility of clinical P. aeruginosa isolates to tobramycin.

Sputum antibiotic concentrations: implications for treatment of cystic fibrosis lung infection.

BACKGROUND: The success of antibiotic therapy may be predicted based on the achievement of pharmacodynamic indices (PDIs), which are determined by the susceptibility of the infecting bacteria and the concentrations of antibiotics achieved at the site of infection. The aim of this study was to determine whether PDIs associated with clinical effectiveness for ceftazidime and tobramycin were achieved at the site of infection in the lungs of cystic fibrosis (CF) patients following intravenous administration during treatment of an acute exacerbation. METHODS: Serum and sputum samples were collected from 14 CF patients and the concentration of both antibiotics in the samples determined. The susceptibility of bacteria cultured from sputum samples to both antibiotics alone and in combination was also determined. RESULTS: A total of 22 Pseudomonas aeruginosa isolates and 4 Burkholderia cepacia complex isolates were cultured from sputum samples with 55% and 4% of isolates susceptible to ceftazidime and tobramycin, respectively. Target PDIs for ceftazidime and tobramycin, an AUC/MIC ratio of 100 and a C(max)/MIC ratio of 10, respectively, were not achieved in serum or sputum simultaneously or even individually for any patient. Although the combination of ceftazidime and tobramycin was synergistic against 20 of the 26 isolates cultured, the concentrations of both antibiotics required for synergy were achieved simultaneously in only 38% of serum and 14% of sputum samples. CONCLUSION: Key PDIs associated with clinical effectiveness for ceftazidime and tobramycin were not achieved at the site of infection in the lungs of CF patients.

Biofilm formation by bacteria isolated from retrieved failed prosthetic hip implants in an in vitro model of hip arthroplasty antibiotic prophylaxis.

Bacterial infection primarily with Staphylococcus spp. and Propionibacterium acnes remains a significant complication following total hip replacement. In this in vitro study, we investigated the efficacy of gentamicin loading of bone cement and pre- and postoperative administration of cefuroxime in the prevention of biofilm formation by clinical isolates. High and low initial inocula, representative of the number of bacteria that may be present at the operative site as a result of overt infection and skin contamination, respectively, were used. When a high initial inoculum was used, gentamicin loading of the cement did not prevent biofilm formation by the 10 Staphylococcus spp. and the 10 P. acnes isolates tested. Similarly, the use of cefuroxime in the fluid phase with gentamicin-loaded cement did not prevent biofilm formation by four Staphylococcus spp. and four P. acnes isolates tested. However, when a low bacterial inoculum was used, a combination of both gentamicin-loaded cement and cefuroxime prevented biofilm formation by these eight isolates. Our results indicate that this antibiotic combination may protect against infection after intra-operative challenge with bacteria present in low numbers as a result of contamination from the skin but would not protect against bacteria present in high numbers as a result of overt infection of an existing implant.

Effect of reduced pH on inorganic polyphosphate accumulation by Burkholderia cepacia complex isolates.

AIMS: Burkholderia cepacia complex (Bcc) isolates causing pulmonary infection in cystic fibrosis (CF) patients grow within an acidic environment in the lung. As exposure to acid pH has been shown to increase intracellular inorganic polyphosphate (polyP) formation in some bacteria, we investigated the inter-relationship between acidic pH and polyP accumulation in Bcc isolates. METHODS AND RESULTS: The formation of polyP by one Burkholderia cenocepacia clinical isolate was initially examined at a range of pH values by measuring total intracellular polyP accumulation and phosphate uptake. The pattern of polyP accumulation corresponded with the pattern of phosphate uptake with the maximum for both occurring at pH 5.5. Phosphate uptake and formation of polyP by this isolate was further determined over 48 h at pH 5.5, 6.5 and 7.5; formation of polyP was maximal at pH 5.5 at all time points studied. Sixteen of 17 additional clinical and environmental Bcc isolates examined also exhibited maximum phosphate uptake at pH 5.5. CONCLUSIONS: Both clinical and environmental Bcc isolates, of five genomovars, show enhanced formation of polyP in an acidic environment. Given both the speculated role of polyP in pathogenesis, cell signalling and biofilm formation and the acidic nature of the CF lung, this may be of considerable clinical importance. SIGNIFICANCE AND IMPACT OF THE STUDY: Growth of Bcc in an acidic environment, such as that found in the lungs of CF patients may be influenced in part by polyP accumulation.

Effect of oxygen limitation on the in vitro antimicrobial susceptibility of clinical isolates of Pseudomonas aeruginosa grown planktonically and as biofilms.

Pseudomonas aeruginosa, the predominant causative pathogen of chronic lung infection in patients with cystic fibrosis, may grow under anaerobic conditions as biofilms in the lungs of cystic fibrosis patients. To determine if growth under anaerobic conditions affects the antimicrobial susceptibility of P. aeruginosa, the susceptibility of clinical isolates of P. aeruginosa grown planktonically and as biofilms to a range of antibiotics was determined. Growth under anaerobic conditions did not reduce the ability of ceftazidime, meropenem, aztreonam, piperacillin, or piperacillin/tazobactam to inhibit planktonic growth, with MIC50 values for these antibiotics remaining unchanged or decreasing. However, tobramycin was less effective at inhibiting planktonic bacterial growth under anaerobic conditions, with the MIC50 of tobramycin increasing twofold. Growth under anaerobic conditions also decreased the bactericidal activity of tobramycin, with the MBC50 of tobramycin increasing fourfold. The killing kinetics of tobramycin was also examined under aerobic and anaerobic conditions for selected isolates. When isolates 6A and 12A were grown aerobically, concentration-dependent decreases in total viable count were apparent with tobramycin. In contrast, when these isolates were grown anaerobically, tobramycin at the same concentrations did not decrease the total viable count. When isolates were grown as biofilms under both aerobic and anaerobic conditions, isolate- and concentration-dependent differences in killing of the biofilms by tobramycin were apparent. However, tobramycin at concentrations up to 128 mg/l was unable to eradicate biofilms of any of the isolates tested, whether biofilms were grown aerobically or anaerobically. These results show that oxygen limitation may reduce, in a strain-dependent manner, the susceptibility to tobramycin of P. aeruginosa grown planktonically and as biofilms.

Evaluation of a poly(vinyl pyrollidone)-coated biomaterial for urological use.

The associated problems of bacterial biofilm formation and encrustation that may cause obstruction or blockage of urethral catheters and ureteral stents often hinders the effective use of biomaterials within the urinary tract. In this in vitro study, we have investigated the surface properties of a hydrophilic poly(vinyl pyrollidone) (PVP)-coating applied to polyurethane and determined its suitability for use as a urinary tract biomaterial by comparing its lubricity and ability to resist bacterial adherence and encrustation with that of uncoated polyurethane and silicone. The PVP-coated polyurethane was significantly more hydrophilic and more lubricious than either uncoated polyurethane or silicone. Adherence of a hydrophilic Escherichia coli isolate to PVP-coated polyurethane and uncoated polyurethane was similar but significantly less than adherence to silicone. Adherence of a hydrophobic Enterococcus faecalis isolate to PVP-coated polyurethane and silicone was similar but was significantly less than adherence to uncoated polyurethane. Struvite encrustation was similar on the PVP-coated polyurethane and silicone but significantly less than on uncoated polyurethane. Furthermore, hydroxyapatite encrustation was significantly less on the PVP-coated polyurethane than on either uncoated polyurethane or silicone. The results suggest that the PVP-coating could be useful in preventing complications caused by bacterial biofilm formation and the deposition of encrustation on biomaterials implanted in the urinary tract and, therefore, warrants further evaluation.

Detection of prosthetic hip infection at revision arthroplasty by immunofluorescence microscopy and PCR amplification of the bacterial 16S rRNA gene.

In this study the detection rates of bacterial infection of hip prostheses by culture and nonculture methods were compared for 120 patients with total hip revision surgery. By use of strict anaerobic bacteriological practice during the processing of samples and without enrichment, the incidence of infection by culture of material dislodged from retrieved prostheses after ultrasonication (sonicate) was 22%. Bacteria were observed by immunofluorescence microscopy in 63% of sonicate samples with a monoclonal antibody specific for Propionibacterium acnes and polyclonal antiserum specific for Staphylococcus spp. The bacteria were present either as single cells or in aggregates of up to 300 bacterial cells. These aggregates were not observed without sonication to dislodge the biofilm. Bacteria were observed in all of the culture-positive samples, and in some cases in which only one type of bacterium was identified by culture, both coccoid and coryneform bacteria were observed by immunofluorescence microscopy. Bacteria from skin-flake contamination were readily distinguishable from infecting bacteria by immunofluorescence microscopy. Examination of skin scrapings did not reveal large aggregates of bacteria but did reveal skin cells. These were not observed in the sonicates. Bacterial DNA was detected in 72% of sonicate samples by PCR amplification of a region of the bacterial 16S rRNA gene with universal primers. All of the culture-positive samples were also positive for bacterial DNA. Evidence of high-level infiltration either of neutrophils or of lymphocytes or macrophages into associated tissue was observed in 73% of patients. Our results indicate that the incidence of prosthetic joint infection is grossly underestimated by current culture detection methods. It is therefore imperative that current clinical practice with regard to the detection and subsequent treatment of prosthetic joint infection be reassessed in the light of these results.

Antimicrobial susceptibility of bacteria isolated from orthopedic implants following revision hip surgery.

The susceptibilities of 49 isolates recovered from orthopedic implants to seven antimicrobial agents were evaluated by the broth microdilution method. Ciprofloxacin and vancomycin were more active than gentamicin, representing aminoglycosides which are routinely incorporated into bone cement, and also more active than the peroperative antimicrobial agents cefamandole and erythromycin. The use of ciprofloxacin and vancomycin in vivo, therefore, warrants further evaluation.

Improved detection of infection in hip replacements. A currently underestimated problem.

Our aim was to determine if the detection rate of infection of total hip replacements could be improved by examining the removed prostheses. Immediate transfer of prostheses to an anaerobic atmosphere, followed by mild ultrasonication to dislodge adherent bacteria, resulted in the culture of quantifiable numbers of bacteria, from 26 of the 120 implants examined. The same bacterial species were cultured by routine microbiological techniques from only five corresponding tissue samples. Tissue removed from 18 of the culture-positive implants was suitable for quantitative tissue pathology and inflammatory cells were present in all samples. Furthermore, inflammatory cells were present in 87% of tissue samples taken from patients whose implants were culture-negative. This suggests that these implants may have been infected by bacteria which were not isolated by the techniques of culture used. The increased detection of bacteria from prostheses by culture has improved postoperative antibiotic therapy and should reduce the need for further revision.

Characterization and assessment of a novel poly(ethylene oxide)/polyurethane composite hydrogel (Aquavene) as a ureteral stent biomaterial.

The effective long-term use of indwelling ureteral stents is often hindered by the formation of encrusting deposits which may cause obstruction and blockage of the stent. Development of improved ureteral stent biomaterials capable of preventing or reducing encrustation is therefore particularly desirable. In this study, the suitability as a ureteral stent biomaterial of Aquavene, a novel poly(ethylene oxide)/polyurethane composite hydrogel was compared with that of silicone and polyurethane, two materials widely employed in ureteral stent manufacture. Examination of Aquavene in dry and hydrated states by confocal laser scanning microscopy, scanning electron microscopy, and atomic force microscopy showed the presence of numerous channels within a cellular matrix structure. The channel size increased considerably to as much as 10 microm in diameter in the hydrated state. Aquavene provided superior resistance to encrustation and intraluminal blockage over a 24-week period in a simulated urine flow model. Unobstructed urine flow continued with Aquavene at 24 weeks, whereas silicone and polyurethane stents became blocked with encrustation at 8 and 10 weeks, respectively. Weight loss within Aquavene on the order of 9% (w/w) over the 24-week flow period indicates that extraction of the noncrosslinked poly(ethylene oxide) hydrogel may be responsible for the prevention of encrustation blockage of this biomaterial. In the dry state, Aquavene was significantly harder than either silicone or polyurethane, as shown by Young's modulus, and rapidly became soft on hydration. These additional properties of Aquavene would facilitate ease of stent insertion in the dry state past obstructions in the ureter and provide improved patient comfort on subsequent biomaterial hydration in situ. Aquavene is a promising candidate for use in the urinary tract, as it is probable that effective long-term urine drainage would be maintained in vivo. Further evaluation of this novel biomaterial is therefore warranted.