Measuring the activity and temperature of rats is commonly required in biomedical research. Conventional approaches necessitate single housing, which affects their behavior and wellbeing. We have ...used a subcutaneous radiofrequency identification (RFID) transponder to measure ambulatory activity and temperature of individual rats when group-housed in conventional, rack-mounted home cages. The transponder location and temperature is detected by a matrix of antennae in a baseplate under the cage. An infrared high-definition camera acquires side-view video of the cage and also enables automated detection of vertical activity. Validation studies showed that baseplate-derived ambulatory activity correlated well with manual tracking and with side-view whole-cage video pixel movement. This technology enables individual behavioral and temperature data to be acquired continuously from group-housed rats in their familiar, home cage environment. We demonstrate its ability to reliably detect naturally occurring behavioral effects, extending beyond the capabilities of routine observational tests and conventional monitoring equipment. It has numerous potential applications including safety pharmacology, toxicology, circadian biology, disease models and drug discovery.
The use of manuka honey for the topical treatment of wounds has increased worldwide owing to its broad spectrum of activity towards bacteria in both planktonic and biofilm growth modes. Despite this, ...the potential consequences of bacterial exposure to manuka honey, as may occur during the treatment of chronic wounds, are not fully understood. Here, we describe changes in antimicrobial susceptibility and virulence in a panel of bacteria, including wound isolates, following repeated exposure (ten passages) to sub-inhibitory concentrations of a manuka honey based wound gel. Changes in antibiotic sensitivity above 4-fold were predominantly related to increased vancomycin sensitivity in the staphylococci. Interestingly,
Staphylococcus epidermidis
displayed phenotypic resistance to erythromycin following passaging, with susceptibility profiles returning to baseline in the absence of further honey exposure. Changes in susceptibility to the tested wound gel were moderate (≤ 1-fold) when compared to the respective parent strain. In sessile communities, increased biofilm eradication concentrations over 4-fold occurred in a wound isolate of
Pseudomonas aeruginosa
(WIBG 2.2) as evidenced by a 7-fold reduction in gentamicin sensitivity following passaging. With regards to pathogenesis, 4/8 bacteria exhibited enhanced virulence following honey wound gel exposure. In the pseudomonads and
S. epidermidis
, this occurred in conjunction with increased haemolysis and biofilm formation, whilst
P. aeruginosa
also exhibited increased pyocyanin production. Where virulence attenuation was noted in a passaged wound isolate of
S. aureus
(WIBG 1.6), this was concomitant to delayed coagulation and reduced haemolytic potential. Overall, passaging in the presence of a manuka honey wound gel led to changes in antimicrobial sensitivity and virulence that varied between test bacteria.
Pseudomonas aeruginosa can cause ocular surface infections such as keratitis, a sight threatening inflammation of the cornea, particularly in contact lens wearers. To effectively model ocular surface ...infections, is important to first understand interactions between components of the ocular surface environment such as the cornea, tear film, commensal microbiota, invading pathogens, and factors such as contact lens wear. Investigations presented in this doctoral thesis represent a series of related but distinct studies designed to contribute to the further understanding of these interactions, with the ultimate aim of developing a representative in vitro model of the cornea and ocular surface for infection studies. To characterise the microbiome of contact lens wearers and non contact lens wearers, tear fluid was collected using a microcapillary technique and analysed by 16S ribosomal RNA (rRNA) sequencing. Genera such as Staphylococcus, Corynebacterium and Propionibacterium were found to have the highest relative abundance across samples. Pseudomonas had higher relative abundance in contact lens wearers, with a specific operational taxonomic unit (OTU) found to be significantly higher relative abundance following DESeq2 analysis. Following the characterisation of the microbiome, the effects of antimicrobial tear proteins, such as lysozyme and lactoferrin, on the growth and virulence phenotype of novel clinical isolates of P. aeruginosa were investigated. The addition of proteins to cultures in complex media significantly decreased carrying capacity (P<0.0001). Growth in a complex salt medium that was compositionally similar to tears was associated with a significant decrease in the carrying capacity and growth rate of the culture and a significant increase in generation time. This is likely due to nutrient limitation, suggesting a further feature of the tear film in conferring protection against infection, in addition to direct antimicrobial functions. When investigating the effect of tear proteins on virulence phenotypes, protease and pyocyanin production were unaffected following exposure of isolates to lysozyme and lactoferrin, with haemolytic activity significantly increased in all but one test bacteria (P<0.0001). Swimming motility was significantly increased in three organisms (P<0.05) and swarming motility was significantly decreased in one (P<0.05). This suggests that these particular tear innate immune factors have various effects on virulence phenotype which may be due to the upregulation of virulence factor production by the associated quorum sensing signalling pathways. Experiments to assess the effect of contact lens materials on biofilm formation indicated that silicone hydrogel materials such as senofilcon A and comfilcon A support biofilm growth more favourably than hydrogel materials such as etafilcon A and omafilcon A. This is important since silicone hydrogels are used in extended lens wear, with users at higher risk of P. aeruginosa keratitis. To understand the interaction between lenses and the tear film, lenses were soaked in lysozyme solution before inoculation with the test bacteria. A significant reduction in P. aeruginosa attachment to etafilcon A (P<0.05-0.01) was observed, suggesting this hydrogel material adsorbs lysozyme more readily. To build on these findings, the final aim of this doctoral study worked towards developing an in vitro model of the ocular surface that could be translated to infection research. Primary human stromal cells and corneal epithelial cells were successfully cultured from donor corneas and incorporated into and onto the surface of a collagen gel. Culturing epithelial cells in a high Ca2+medium was found to provide the most integral epithelial multilayer. The addition of stromal cells and a fibronectin basal lamina substitute to the collagen gel were found to further improve epithelial growth. To conclude, this model could be further developed for use in studying ocular surface infections by incorporating an antimicrobial tear film and other ocular surface components and could be used in conjunction with bacteria and contact lenses to further understand the complex interactions investigated in this thesis.
Diathermy is used in most theatres for every case but how many people think about the contents of diathermy plume or surgical smoke? Surgical smoke is an unpleasant smell that leaves many considering ...whether surgical masks reduce the risk of inhaling its contents. This article suggests that practitioners should be more aware of its contents and take action to lessen the risk to themselves and their colleagues.
Background: Antimicrobials are used to directly control bacterial infections in pet (ornamental) fish and are routinely added to the water these fish are shipped in to suppress the growth of ...potential pathogens during transport. Methodology/Principal Findings: To assess the potential effects of this sustained selection pressure, 127 Aeromonas spp. isolated from warm and cold water ornamental fish species were screened for tolerance to 34 antimicrobials. Representative isolates were also examined for the presence of 54 resistance genes by a combination of miniaturized microarray and conventional PCR. Forty-seven of 94 Aeromonas spp. isolates recovered from tropical ornamental fish and their carriage water were tolerant to > or = 15 antibiotics, representing seven or more different classes of antimicrobial. The quinolone and fluoroquinolone resistance gene, qnrS2, was detected at high frequency (37% tested recent isolates were positive by PCR). Class 1 integrons, IncA/C broad host range plasmids and a range of other antibiotic resistance genes, including floR, blaTEM-1, tet(A), tet(D), tet(E), qacE2, sul1, and a number of different dihydrofolate reductase and aminoglycoside transferase coding genes were also detected in carriage water samples and bacterial isolates. Conclusions: These data suggest that ornamental fish and their carriage water act as a reservoir for both multi-resistant bacteria and resistance genes.
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