Pseudomonas aeruginosa cystic fibrosis isolates of similar RAPD genotype exhibit diversity in biofilm forming ability in vitro.

BACKGROUND: Pseudomonas aeruginosa is considered to grow in a biofilm in cystic fibrosis (CF) chronic lung infections. Bacterial cell motility is one of the main factors that have been connected with P. aeruginosa adherence to both biotic and abiotic surfaces. In this investigation, we employed molecular and microscopic methods to determine the presence or absence of motility in P. aeruginosa CF isolates, and statistically correlated this with their biofilm forming ability in vitro. RESULTS: Our investigations revealed a wide diversity in the production, architecture and control of biofilm formation. Of 96 isolates, 49% possessed swimming motility, 27% twitching and 52% swarming motility, while 47% were non-motile. Microtitre plate assays for biofilm formation showed a range of biofilm formation ability from biofilm deficient phenotypes to those that formed very thick biofilms. A comparison of the motility and adherence properties of individual strains demonstrated that the presence of swimming and twitching motility positively affected biofilm biomass. Crucially, however, motility was not an absolute requirement for biofilm formation, as 30 non-motile isolates actually formed thick biofilms, and three motile isolates that had both flagella and type IV pili attached only weakly. In addition, CLSM analysis showed that biofilm-forming strains of P. aeruginosa were in fact capable of entrapping non-biofilm forming strains, such that these 'non-biofilm forming' cells could be observed as part of the mature biofilm architecture. CONCLUSIONS: Clinical isolates that do not produce biofilms in the laboratory must have the ability to survive in the patient lung. We propose that a synergy exists between isolates in vivo, which allows "non biofilm-forming" isolates to be incorporated into the biofilm. Therefore, there is the potential for strains that are apparently non-biofilm forming in vitro to participate in biofilm-mediated pathogenesis in the CF lung.

Quantitative effects of carbohydrates and aromatic amino acids on Clostridium botulinum toxin gene expression using a rapid competitive RT/PCR assay.

A rapid competitive RT/PCR assay was developed to determine the effects of nutrients on Clostridium botulinum type E toxin gene expression. The type E strain (EVH) was grown in a nutrient-rich broth containing 1% glucose (base medium). Toxin gene expression was quantified at both mid and late exponential phases of growth. It was found that toxin encoding mRNA levels were highly growth phase dependent with elevated levels found in late exponential phase compared to mid exponential phase. Changing the carbohydrate source had a smaller effect on toxin encoding mRNA levels but as earlier results have suggested, toxin encoding mRNA levels show a strong correlation with type E growth rate. The results have important implications for the food industry whereby risk of type E botulism could be correlated to the nutrient composition of the contaminated food or assessed from C. botulinum growth rates in challenged foodstuffs.

The steady-state orgA specific mRNA levels in Salmonella enterica serovar Typhimurium are repressed by oxygen during logarithmic growth phase but not early-stationary phase.

The orgA gene from Salmonella enterica serovar Typhimurium is involved in promoting cellular invasion of the pathogen. Its exact role in virulence is still unclear mainly due to difficulties in understanding its complex regulation. In this study a novel competitive RT-PCR (cRT-PCR) system was developed to measure the steady-state orgA specific mRNA levels in cells under various growth parameters. Previous studies have been inconsistent regarding oxygen regulation of orgA. Using our system we found that oxygen repressed the copy levels 3.5-fold in cells grown only to logarithmic phase. Oxygen repression was not observed in cells grown to early-stationary phase, a parameter that has previously been demonstrated to be the most invasive stage of growth. The importance of NaCl in orgA gene regulation is also illustrated. Significant increases in copy numbers were observed after growth in high NaCl conditions. Measuring the steady-state mRNA levels using cRT-PCR provides an accurate insight into prokaryotic gene regulation prior to translation.

Quantification of toxin-encoding mRNA from Clostridium botulinum type E in media containing sorbic acid or sodium nitrite by competitive RT-PCR.

Competitive reverse transcription polymerase chain reaction (cRT-PCR) was used to quantify the toxin-encoding mRNA production of a Clostridium botulinum type E strain in media containing either sorbic acid or sodium nitrite. A 10-fold reduction in toxin mRNA production and a 25-fold reduction in the proportion of toxin mRNA to total RNA, was estimated when either 1 mg ml(-1) sorbic acid or 100 microg ml(-1) sodium nitrite were added to the medium at pH 7.0.