BackgroundThe majority of in vitro studies of medically relevant biofilms involve the development of biofilm on an inanimate solid surface. However, infection in vivo consists of biofilm growth on, or suspended within, the semi-solid matrix of the tissue, whereby current models do not effectively simulate the nature of the in vivo environment. This paper describes development of an in vitro method for culturing wound associated microorganisms in a system that combines a semi-solid collagen gel matrix with continuous flow of simulated wound fluid. This enables culture of wound associated reproducible steady state biofilms under conditions that more closely simulate the dynamic wound environment. To demonstrate the use of this model the antimicrobial kinetics of ceftazidime, against both mature and developing Pseudomonas aeruginosa biofilms, was assessed. In addition, we have shown the potential application of this model system for investigating microbial metabolomics by employing selected ion flow tube mass spectrometry (SIFT-MS) to monitor ammonia and hydrogen cyanide production by Pseudomonas aeruginosa biofilms in real-time.ResultsThe collagen wound biofilm model facilitates growth of steady-state reproducible Pseudomonas aeruginosa biofilms under wound like conditions. A maximum biofilm density of 1010 cfu slide− 1 was achieved by 30 h of continuous culture and maintained throughout the remainder of the experiment. Treatment with ceftazidime at a clinically relevant dose resulted in a 1.2–1.6 log reduction in biofilm density at 72 h compared to untreated controls. Treatment resulted in loss of complex biofilm architecture and morphological changes to bacterial cells, visualised using confocal microscopy. When monitoring the biofilms using SIFT-MS, ammonia and hydrogen cyanide levels peaked at 12 h at 2273 ppb (±826.4) and 138 ppb (±49.1) respectively and were detectable throughout experimentation.ConclusionsThe collagen wound biofilm model has been developed to facilitate growth of reproducible biofilms under wound-like conditions. We have successfully used this method to: (1) evaluate antimicrobial efficacy and kinetics, clearly demonstrating the development of antimicrobial tolerance in biofilm cultures; (2) characterise volatile metabolite production by P. aeruginosa biofilms, demonstrating the potential use of this method in metabolomics studies.

Gram-negative, rod-shaped bacteria from the soil of white clover-ryegrass pastures were screened for their ability to nodulate white clover (Trifolium repens) cultivar Grasslands Huia and for DNA homology with genomic DNA from Rhizobium leguminosarum biovar trifolii ICMP2668 (NZP582). Of these strains, 3.2% were able to hybridize with strain ICMP2668 and nodulate white clover and approximately 19% hybridized but were unable to nodulate. Strains which nodulated but did not hybridize with strain ICMP2668 were not detected. DNA from R. leguminosarum biovar trifolii (strain PN165) cured of its symbiotic (Sym) plasmid and a specific nod probe were used to show that the relationship observed was usually due to chromosomal homology. Plasmid pPN1, a cointegrate of the broad-host-range plasmid R68.45 and a symbiotic plasmid pRtr5l4a, was transferred by conjugation to representative strains of nonnodulating, gram-negative, rod-shaped soil bacteria. Transconjugants which formed nodules were obtained from 6 of 18 (33%) strains whose DNA hybridized with that of PN165 and 1 of 9 (11%) strains containing DNA which did not hybridize with that of PN165. The presence and location of R68.45 and nod genes was confirmed in transconjugants from three of the strains which formed nodules. Similarly, a pLAFR1 cosmid containing nod genes from a derivative of R. leguminosarum biovar trifolii NZP514 formed nodules when transferred to soil bacteria. The genus Rhizobiuim is "characteristically able to invade

Aims The main aim of this study was to investigate the real‐time detection of volatile metabolites for the species‐level discrimination of pathogens associated with clinically relevant wound infection, when grown in a collagen wound biofilm model. Methods and Results This work shows that Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes produce a multitude of volatile compounds when grown as biofilms in a collagen‐based biofilm model. The real‐time detection of these complex volatile profiles using selected ion flow tube mass spectrometry and the use of multivariate statistical analysis on the resulting data can be used to successfully differentiate between the pathogens studied. Conclusions The range of bacterial volatile compounds detected between the species studied vary and are distinct. Discrimination between bacterial species using real‐time detection of volatile metabolites and multivariate statistical analysis was successfully demonstrated. Significance and Impact of the Study Development of rapid point‐of‐care diagnostics for wound infection would improve diagnosis and patient care. Such technological approaches would also facilitate the appropriate use of antimicrobials, minimizing the emergence of antimicrobial resistance. This study further develops the use of volatile metabolite detection as a new diagnostic approach for wound infection.

AimsTo determine if bacterial species responsible for clinically relevant wound infection produce specific volatile profiles that would allow their speciation. Methods and ResultsSelected-Ion Flow Tube-Mass Spectrometry in full mass scan mode was used to analyse headspace gases produced by wound-associated bacteria grown in vitro, so as to enable identification of bacterial volatile product ion profiles in the resulting mass spectra. Applying multivariate statistical analysis (hierarchical clustering and principal component analysis) to the resultant mass spectra enabled clear speciation. Moreover, bacterial volatile product ions could be detected from artificially contaminated wound dressing material, although the pattern of product ions detected was influenced by culture conditions. ConclusionsUsing selected product ions from the SIFT-MS mass spectra it is possible to discriminate wound-associated bacterial species grown under specific in vitro culture conditions. Significance and Impact of StudyThe results of this study have shown that wound associated bacteria can be discriminated using volatile analysis in vitro and that bacterial volatiles can be detected from wound dressing material. This indicates that volatile analysis of wounds or dressing material to identify infecting microbes has potential and warrants further study.

The fungus Chondrostereum purpureum, the causal agent of silverleaf, when grown in liquid culture or on agar, secreted extracellular proteinases into the medium. Proteinase activity in the mycelium itself was barely detectable. At least 95% of caseinolytic activity in the extracellular fluid (ECF) was inhibited by chelating agents. Fluid dialysed against EDTA was activated by metal ions, confirming the presence of metalloproteinases. Caseinolytic activity of ECF was also largely abolished by sulphydryl compounds and reagents. Superdex G75 chromatography and SDS-PAGE analysis of the proteinases present in concentrated ECF indicated at least four components with proteolytic activity. In addition to the major metalloproteinases, a very low level of a chymotrypsin-like activity was detected as well as high levels of the exopeptidase, leucine aminopeptidase. A wide range of caseinolytic activity levels was found when 29 C. purpureum isolates from New Zealand were grown in liquid culture, but much less variation was observed on agar. Proteinase activity was found throughout the infection zone of silverleaf-susceptible plants but could only be detected at the wounding site of resistant cultivars. The possible roles of these proteinases in infection and growth of the pathogen are discussed.