Modulation of the metabolite content of the unicellular rhodophyte Porphyridium purpureum using a 2-stage cultivation approach and chemical stressors.

There have been growing interests in microalgal biotechnology for the biorefining of bioactive compounds such as carotenoid pigments, ω-3 fatty acids, antioxidants or antimicrobials for sectoral applications in the pharmacology, nutraceutical and cosmetic fields. This study focused on the unicellular marine rhodophyte Porphyridium purpureum CCAP 1380/1 A, which was cultivated via a two-stage batch growth mode for 10 days using hydrogen peroxide (H2O2), the phytohormone methyl jasmonate (MJ) and three plant extracts (Passiflora incarnata, Panax ginseng and Valeriana officinalis). The microalgal biomass was then analysed for its protein, phycoerythtin, carbohydrate and pigment composition together with its pigment content and antioxidant activity. Of note, MJ increased the protein and phycoerythtin content (up to 225 µg BSA eq./mg DW and 15 mg/ml, respectively) while both the MJ and H2O2 treatments increased carotenoid pigment yields (ß-carotene and zeaxanthin, up to 5 and 4 mg/g, respectively). Carbohydrates were enhanced ∼10 fold by the Valeriana officinalis treatment (up 192 µg starch eq./mg). Overall, neutral lipids and antioxidants were mostly negatively affected by the plant extracts. The greatest antioxidant activity registered was obtained with the H2O2 treatment (15 µmol Trolox eq./g DW with TEAC assay). P. purpureum contains multiple valuable compounds of commercial interest. These results indicate that they can be favorably modulated using specific cultivation regimes and chemical enhancers, thereby facilitating the exploitation of the biomass by applying a suitable co-refinery pipeline.

Antibacterial Activity and Amphidinol Profiling of the Marine Dinoflagellate Amphidinium carterae (Subclade III).

Microalgae have received growing interest for their capacity to produce bioactive metabolites. This study aimed at characterising the antimicrobial potential of the marine dinoflagellate Amphidinium carterae strain LACW11, isolated from the west of Ireland. Amphidinolides have been identified as cytotoxic polyoxygenated polyketides produced by several Amphidinium species. Phylogenetic inference assigned our strain to Amphidinium carterae subclade III, along with isolates interspersed in different geographic regions. A two-stage extraction and fractionation process of the biomass was carried out. Extracts obtained after stage-1 were tested for bioactivity against bacterial ATCC strains of Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa. The stage-2 solid phase extraction provided 16 fractions, which were tested against S. aureus and E. faecalis. Fractions I, J and K yielded minimum inhibitory concentrations between 16 µg/mL and 256 µg/mL for both Gram-positive. A targeted metabolomic approach using UHPLC-HRMS/MS analysis applied on fractions G to J evidenced the presence of amphidinol type compounds AM-A, AM-B, AM-22 and a new derivative dehydroAM-A, with characteristic masses of m/z 1361, 1463, 1667 and 1343, respectively. Combining the results of the biological assays with the targeted metabolomic approach, we could conclude that AM-A and the new derivative dehydroAM-A are responsible for the detected antimicrobial bioactivity.

Comparative Response of Marine Microalgae to H2O2-Induced Oxidative Stress.

There have been growing interests in the biorefining of bioactive compounds from marine microalgae, including pigments, omega-3 fatty acids or antioxidants for use in the nutraceutical and cosmetic sectors. This study focused on the comparative responses of five marine microalgal species from different lineages, including the dinoflagellate Amphidinium carterae, chlorophyte Brachiomonas submarina, diatom Stauroneis sp., haptophyte Diacronema sp. and rhodophyte Rhodella violacea, to exposure during their batch growth to hydrogen peroxide (H2O2). A. carterae returned an enhanced signal with the DPPH assay (8.8 µmol Trolox eq/g DW) when exposed to H2O2, which was associated with reduced pigment yields and increased proportions in saturated C16 and C18 fatty acids. B. submarina showed enhanced antioxidant response upon exposure to H2O2 with the DPPH assay (10 µmol Trolox eq/g DW), a threefold decrease in lutein (from 2.3 to 0.8 mg/g) but a twofold increase in chlorophyll b (up to 30.0 mg/g). Stauroneis sp. showed a downward response for the antioxidant assays, but its pigment yields did not vary significantly from the control. Diacronema sp. showed reduced antioxidant response and fucoxanthin content (from 4.0 to 0.2 mg/g) when exposed to 0.5 mM H2O2. R. violacea exposed to H2O2 returned enhanced antioxidant activity and proportions of EPA but was not significantly impacted in terms of pigment content. Results indicate that H2O2 can be used to induce stress and initiate metabolic changes in microalgae. The responses were however species-specific, which would require further dosage optimisation to modulate the yields of specific metabolites in individual species.

The role of methyl jasmonate in enhancing biomass yields and bioactive metabolites in Stauroneis sp. (Bacillariophyceae) revealed by proteome and biochemical profiling.

The diatom Stauroneis sp. was previously identified as a promising source of fucoxanthin and omega-3 oils. Methyl jasmonate (MJ) supplementation is known to enhance metabolite yields in this species without impacting on growth or photosynthesis. Therefore, a label-free proteomics approach was undertaken to further evaluate the functional role of MJ on the diatom's physiology. Of the twenty cultivation regimes were screened, Uf/2 medium with green+white LED's induced the greatest metabolic response when exposed to 10 µM MJ treatment. These conditions significantly enhanced the pigment and total cellular lipids contents. The increase in fucoxanthin correlating with a 20% increase in Trolox reducing equivalent in the total antioxidant assay, indicating a non-enzymatic antioxidant role of fucoxanthin to mitigate the detrimental effects of a redox imbalance within chloroplasts. The proteomics identified 197 proteins up-regulated 48 h after MJ exposure including cell signalling cascades, photosynthetic processes, carbohydrate metabolism, lipid biosynthesis and chloroplast biogenesis. MJ strengthened the dark reactions of photosynthesis to support growth and metabolite fluxes. The MJ-induced ER stress protein triggered lipid body production, facilitating metabolite turnover and trafficking between cellular organelles. Plastid terminal oxidase and glutamate 1-semialdehyde 2,1-aminomutase may act as MJ-induced ROS responsive regulatory switch to support chloroplast biosynthesis. SIGNIFICANCE STATEMENT: Phytohormones represents a promising tool to enhance the high-value metabolite yields in plants and algae, however little is known of the role of methyl jasmonate in diatoms at a molecular level. A shotgun proteomics approach was undertaken to determine the influence of MJ on the diatom's cellular physiology in the marine diatom Stauroneis sp., revealing a signal transduction cascade leading to increased lipid and pigment content and identified promising targets for genetic engineering.

Marine mammals are natural hosts of Oceanivirga salmonicida, a bacterial pathogen of Atlantic salmon.

During 1992 and 1993, a bacterial disease occurred in a seawater Atlantic salmon Salmo salar farm, causing serious mortalities. The causative agent was subsequently named as Oceanivirga salmonicida, a member of the Leptotrichiaceae. Searches of 16S rRNA gene sequence databases have shown sequence similarities between O. salmonicida and uncultured bacterial clones from the digestive tracts of marine mammals. In the current study, oral samples were taken from stranded dolphins (common dolphin Delphinus delphis, striped dolphin Stenella coeruleoalba) and healthy harbour seals Phoca vitulina. A bacterium with growth characteristics consistent with O. salmonicida was isolated from a common dolphin. The isolate was confirmed as O. salmonicida, by comparisons to the type strain, using 16S rRNA gene, gyrB, groEL, and recA sequence analyses, average nucleotide identity analysis, and MALDI-TOF mass spectrometry. Metagenomic analysis indicated that the genus Oceanivirga represented a significant component of the oral bacterial microbiomes of the dolphins and seals. However, sequences consistent with O. salmonicida were only found in the dolphin samples. Analyses of marine mammal microbiome studies in the NCBI databases showed sequences consistent with O. salmonicida from the common dolphin, striped dolphin, bottlenose dolphin Tursiops truncatus, humpback whale Megaptera novaeangliae, and harbour seal. Sequences from marine environmental studies in the NCBI databases showed no sequences consistent with O. salmonicida. The findings suggest that several species of marine mammals are natural hosts of O. salmonicida.

Occupational Exposures to Organic Dust in Irish Bakeries and a Pizzeria Restaurant.

For decades, occupational exposure to flour dust has been linked to a range of respiratory diseases, including occupational asthma, thought to result from exposure to fungi present in the flour. Antifungal resistance is of increasing prevalence in clinical settings, and the role of occupational and environmental exposures, particularly for specific fungal species, is of concern. Occupational exposure to flour dust can occur in a range of occupational settings, however, few studies have focused on restaurant workers. The objective of this study was to measure occupational exposure to flour and microbial contamination, including azole resistance screening, in two small commercial bakeries and in a pizzeria. Personal full shift inhalable dust measurements were collected from workers, and were analyzed for inhalable dust and fungi, bacteria, azole resistance, and mycotoxins. Samples of settled dust were collected, and electrostatic dust cloths (EDC) were deployed and analyzed for microbial contamination, including azole resistance screening, and mycotoxins. Geometric mean exposures of 6.5 mg m-³ were calculated for inhalable dust, however, exposures of up to 18.30 mg m-³ were measured-70% of personal exposure measurements exceeded the occupational exposure limit for flour dust of 1.0 mg m-³. The air and EDC fungal counts were similar to those reported in previous studies for similar occupational environments. The fungi were dominated by Penicillium genera, however Aspergillus genera, including Fumigati and Flavi sections, were observed using culture-based methods, and the Fumigati section was also observed by molecular tools. Both Aspergillus sections were identified on the azole resistance screening. Mycotoxins were also detected in the settled dust samples, dominated by deoxynivalenol (DON). The role of environmental exposure in both the development of antimicrobial resistance and the total mycotoxin body burden is a growing concern; therefore, the presence of azole-resistant fungi and mycotoxin contamination, although low in magnitude, is of concern and warrants further investigation.

Influence of spectral intensity and quality of LED lighting on photoacclimation, carbon allocation and high-value pigments in microalgae.

Tailoring spectral quality during microalgal cultivation can provide a means to increase productivity and enhance biomass composition for downstream biorefinery. Five microalgae strains from three distinct lineages were cultivated under varying spectral intensities and qualities to establish their effects on pigments and carbon allocation. Light intensity significantly impacted pigment yields and carbon allocation in all strains, while the effects of spectral quality were mostly species-specific. High light conditions induced chlorophyll photoacclimation and resulted in an increase in xanthophyll cycle pigments in three of the five strains. High-intensity blue LEDs increased zeaxanthin tenfold in Rhodella sp. APOT_15 relative to medium or low light conditions. White light however was optimal for phycobiliprotein content (11.2 mg mL-1) for all tested light intensities in this strain. The highest xanthophyll pigment yields for the Chlorophyceae were associated with medium-intensity blue and green lights for Brachiomonas submarina APSW_11 (5.6 mg g-1 lutein and 2.0 mg g-1 zeaxanthin) and Kirchneriella aperta DMGFW_21 (1.5 mg g-1 lutein and 1 mg g-1 zeaxanthin), respectively. The highest fucoxanthin content in both Heterokontophyceae strains (2.0 mg g-1) was associated with medium and high white light for Stauroneis sp. LACW_24 and Phaeothamnion sp. LACW_34, respectively. This research provides insights into the application of LEDs to influence microalgal physiology, highlighting the roles of low light on lipid metabolism in Rhodella sp. APOT_15, of blue and green lights for carotenogenesis in Chlorophyceae and red light-induced photoacclimation in diatoms.

Continuous culture of Escherichia coli, under selective pressure by a novel antimicrobial complex, does not result in development of resistance.

We attempted to generate de novo resistance to a newly described biocidal complex, ITC (iodo-thiocyanate complex), and to levofloxacin (LVX) in Escherichia coli ATCC 25922, by means of selective chemostat culture. We measured resistance by determining the minimum inhibitory concentrations (MICs) for these agents. E. coli underwent 20-day parallel adaptive evolution routes under no antimicrobial selection, and gradually increasing ITC and LVX selection pressure. Long-term exposure of E. coli to ITC did not induce resistance to ITC, or cross-resistance to LVX. No distinct mutational pattern was evidenced from whole-genome sequence (WGS)-based comparisons of ITC-challenged and unchallenged bacterial populations. Moreover, the exposed E. coli population could not survive a 2 × MIC challenge of ITC. By contrast, resistance to LVX was rapidly induced (on day 1 the MIC had increased 16-fold), selected for (by day 14 the MIC had increased 64-fold) and enriched with a highly characteristic genome mutational pattern. WGS of this evolving population revealed that the majority of mutations appeared in the genes of LVX target proteins (GyrA, ParC, ParE) and drug influx (OmpF). This study suggests that the usage of ITC may not trigger the emergence of facile resistance or cross-resistance, in contrast to common antibiotics.

In vitro comparative cytotoxicity study of a novel biocidal iodo-thiocyanate complex.

Novel biocides, which avoid the induction of cross-resistance to antibiotics, are an urgent societal requirement. Here, we compared the cytotoxic and bactericidal effects of a new antimicrobial agent, the iodo-thiocyanate complex (ITC), with those of the common antiseptics, hydrogen peroxide (H2O2), povidone iodine (PVP-I) and Lugol's iodine (Lugol). The antimicrobials were co-incubated for 10 min with HeLa and Escherichia coli cells in the presence and absence of organic matter (Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum). The cytotoxic concentrations of ITC were equivalent to its bactericidal concentrations (7.8 µg ml-1). By contrast, cytotoxic effects of H2O2, PVP-I and Lugol were apparent at concentrations lower than their bactericidal concentrations (250, 250 and 125 µg ml-1, respectively). The cellular effects of ITC were not quenched by organic matter, unlike the other antiseptics. ITC, PVP-I and Lugol had hemolytic effect on horse erythrocytes at high concentrations, while H2O2 showed no hemolysis. ITC, at 30 or 300 µg ml-1, did not cause DNA breakage in HeLa cells as assessed by an in vitro comet assay in the absence of S9 metabolic activation, whereas H2O2 caused extensive single-strand DNA breaks. The pronounced antimicrobial potency of ITC and its favorable cytotoxicity profile suggests that ITC should be considered for antiseptic applications.

Fungal burden exposure assessment in podiatry clinics from Ireland.

Fungi are amongst the bioaerosols of most importance, as indicated by the growing interest in this field of research. The aim was to characterize the exposure to fungal burden in podiatry clinics using culture-based and molecular methods. METHODS: Airborne fungi were collected using an impaction air sampler and surface samples were also performed. Fourteen air samples were collected for direct detection of fungal DNA from filamentous fungi and dermatophytes. Overall, 63.6 % of the evening samples and 46 % of the morning samples surpassed the threshold values (150 CFU/m3). Molecular detection, by real time PCR, of the target fungal species/strains (Aspergillus and Stachybotrys species) was negative for all samples collected. Trichophyton rubrum was detected by PCR analysis in one DNA sample collected on day six. Results suggest the use of both culture-based and molecular methodologies are desirable for a complete evaluation of fungal burden in this particular health care setting.

Tandem Amplification of the Staphylococcal Cassette Chromosome mec Element Can Drive High-Level Methicillin Resistance in Methicillin-Resistant Staphylococcus aureus.

Hospital-associated methicillin-resistant Staphylococcus aureus (MRSA) strains typically express high-level, homogeneous (HoR) ß-lactam resistance, whereas community-associated MRSA (CA-MRSA) more commonly express low-level heterogeneous (HeR) resistance. Expression of the HoR phenotype typically requires both increased expression of the mecA gene, carried on the staphylococcal cassette chromosome mec element (SCCmec), and additional mutational event(s) elsewhere on the chromosome. Here the oxacillin concentration in a chemostat culture of the CA-MRSA strain USA300 was increased from 8 µg/ml to 130 µg/ml over 13 days to isolate highly oxacillin-resistant derivatives. A stable, small-colony variant, designated HoR34, which had become established in the chemostat culture was found to have acquired mutations in gdpP, clpX, guaA, and camS Closer inspection of the genome sequence data further revealed that reads covering SCCmec were ∼10 times overrepresented compared to other parts of the chromosome. Quantitative PCR (qPCR) confirmed >10-fold-higher levels of mecA DNA on the HoR34 chromosome, and MinION genome sequencing verified the presence of 10 tandem repeats of the SCCmec element. qPCR further demonstrated that subculture of HoR34 in various concentrations of oxacillin (0 to 100 µg/ml) was accompanied by accordion-like contraction and amplification of the SCCmec element. Although slower growing than strain USA300, HoR34 outcompeted the parent strain in the presence of subinhibitory oxacillin. These data identify tandem amplification of the SCCmec element as a new mechanism of high-level methicillin resistance in MRSA, which may provide a competitive advantage for MRSA under antibiotic selection.

A Novel Mechanism, Linked to Cell Density, Largely Controls Cell Division in Synechocystis.

Many studies have investigated the various genetic and environmental factors regulating cyanobacterial growth. Here, we investigated the growth and metabolism of Synechocystis sp. PCC 6803 under different nitrogen sources, light intensities, and CO2 concentrations. Cells grown on urea showed the highest growth rates. However, for all conditions tested, the daily growth rates in batch cultures decreased steadily over time, and stationary phase was obtained with similar cell densities. Unexpectedly, metabolic and physiological analyses showed that growth rates during log phase were not controlled primarily by the availability of photoassimilates. Further physiological investigations indicated that nutrient limitation, quorum sensing, light quality, and light intensity (self-shading) were not the main factors responsible for the decrease in the growth rate and the onset of the stationary phase. Moreover, cell division rates in fed-batch cultures were positively correlated with the dilution rates. Hence, not only light, CO2, and nutrients can affect growth but also a cell-cell interaction. Accordingly, we propose that cell-cell interaction may be a factor responsible for the gradual decrease of growth rates in batch cultures during log phase, culminating with the onset of stationary phase.

Antibacterial Potential of an Antimicrobial Agent Inspired by Peroxidase-Catalyzed Systems.

Antibiotic resistance is an increasingly serious threat to global health. Consequently, the development of non-antibiotic based therapies and disinfectants, which avoid induction of resistance, or cross-resistance, is of high priority. We report the synthesis of a biocidal complex, which is produced by the reaction between ionic oxidizable salts-iodide and thiocyanate-in the presence of hydrogen peroxide as an oxidation source. The reaction generates bactericidal reactive oxygen and iodine species. In this study, we report that the iodo-thiocyanate complex (ITC) is an effective bactericidal agent with activity against planktonic and biofilm cells of Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and methicillin-resistant S. aureus) bacteria. The minimum bactericidal concentrations and the minimum biofilm eradication concentrations of the biocidal composite were in the range of 7.8-31.3 and 31.3-250 µg ml-1, respectively. As a result, the complex was capable to cause a rapid cell death of planktonic test cultures at between 0.5 and 2 h, and complete eradication of dual and mono-species biofilms between 30 s and 10 min. Furthermore, the test bacteria, including a MRSA strain, exposed to the cocktail failed to develop resistance after serial passages. The antimicrobial activity of the ITC appears to derive from the combinational effect of the powerful species capable of oxidizing the essential biomolecules of bacteria. The use of this composition may provide an effective and efficient method for killing potential pathogens, as well as for disinfecting and removing biofilm contamination.

Evolution of the MIDTAL microarray: the adaption and testing of oligonucleotide 18S and 28S rDNA probes and evaluation of subsequent microarray generations with Prymnesium spp. cultures and field samples.

The toxic microalgal species Prymnesium parvum and Prymnesium polylepis are responsible for numerous fish kills causing economic stress on the aquaculture industry and, through the consumption of contaminated shellfish, can potentially impact on human health. Monitoring of toxic phytoplankton is traditionally carried out by light microscopy. However, molecular methods of identification and quantification are becoming more common place. This study documents the optimisation of the novel Microarrays for the Detection of Toxic Algae (MIDTAL) microarray from its initial stages to the final commercial version now available from Microbia Environnement (France). Existing oligonucleotide probes used in whole-cell fluorescent in situ hybridisation (FISH) for Prymnesium species from higher group probes to species-level probes were adapted and tested on the first-generation microarray. The combination and interaction of numerous other probes specific for a whole range of phytoplankton taxa also spotted on the chip surface caused high cross reactivity, resulting in false-positive results on the microarray. The probe sequences were extended for the subsequent second-generation microarray, and further adaptations of the hybridisation protocol and incubation temperatures significantly reduced false-positive readings from the first to the second-generation chip, thereby increasing the specificity of the MIDTAL microarray. Additional refinement of the subsequent third-generation microarray protocols with the addition of a poly-T amino linker to the 5' end of each probe further enhanced the microarray performance but also highlighted the importance of optimising RNA labelling efficiency when testing with natural seawater samples from Killary Harbour, Ireland.

An assessment of RNA content in Prymnesium parvum, Prymnesium polylepis, cf. Chattonella sp. and Karlodinium veneficum under varying environmental conditions for calibrating an RNA microarray for species detection.

Traditional methods of identification and enumeration can be somewhat ambiguous when identifying phytoplankton that requires electron microscopic examination to verify specific morphological features. Members of the genus Prymnesium (division Haptophyta), members of the Raphidophyceae and naked dinoflagellates are examples of such phytoplankton whose identification can be difficult. One alternative to traditional microscopy-based methods of identification is to use molecular protocols to detect target species. Methods that measure cellular DNA and RNA content can be used to estimate the number of cells present in a sample. This study investigated the variation of RNA yields in Prymnesium parvum, P. polylepis, cf. Chattonella sp. and Karlodinium veneficum cells grown under different light, temperature, salinity and inorganic nutrient conditions. This information was used to calibrate the signal intensity of a variety of oligonucleotide probes spotted onto the microarrays for the detection of toxic algae (MIDTAL), which is being developed to aid national monitoring agencies and to provide a faster means of identifying and quantifying harmful phytoplankton in water column samples.

Molecular fingerprinting of lacustrian cyanobacterial communities: regional patterns in summer diversity.

The assessment of lacustrian water quality is necessary to comply with environmental regulations. At the regional scale, difficulties reside in the selection of representative lakes. Given the risks towards water quality associated with phytoplankton blooms, a mesoscale survey was carried out in Irish lakes to identify patterns in the distribution and diversity of planktonic cyanobacteria. A stratified sampling strategy was carried out via geographic information systems (GIS) analysis of river catchment attributes due to the range of hydrogeomorphological features and the high number of lakes within the study area. 16S rRNA gene denaturing gradient gel electrophoresis analysis showed variation between the cyanobacterial communities sampled, with lower occurrence of cyanobacteria in August concomitant to increased wind and precipitation regimes. Multivariate analysis delineated three ecoregions based on land cover typology and revealed significant patterns in the distribution of cyanobacterial diversity. A majority of filamentous cyanobacteria genotypes occurred in larger lakes contained river catchments with substantial forest cover. In contrast, higher diversity of spherical cyanobacteria genotypes was observed in lakes of lesser trophic state. In the context of aquatic resource management, the combined use of GIS-based sampling strategy and molecular methods offers promising prospects for assessing microbial community structure at varying scales of space and time.

Contrasting responses to nutrient enrichment of prokaryotic communities collected from deep sea sites in the southern ocean.

Deep water samples (ca. 4,200 m) were taken from two hydrologically-similar sites around the Crozet islands with highly contrasting surface water productivities. Site M5 was characteristic of high productivity waters (high chlorophyll) whilst site M6 was subject to a low productivity regime (low chlorophyll) in the overlying waters. Samples were incubated for three weeks at 4 °C at in-situ and surface pressures, with and without added nutrients. Prokaryotic abundance increased by at least two-fold for all nutrient-supplemented incubations of water from M5 with little difference in abundance between incubations carried out at atmospheric and in-situ pressures. Abundance only increased for incubations of M6 waters (1.6-fold) when they were carried out at in-situ pressures and with added nutrients. Changes in community structure as a result of incubation and enrichment (as measured by DGGE banding profiles and phylogenetic analysis) showed that diversity increased for incubations of M5 waters but decreased for those with M6 waters. Moritella spp. came to dominate incubations carried out under in-situ pressure whilst the Archaeal community was dominated by Crenarchaea in all incubations. Comparisons between atmospheric and in situ pressure incubations demonstrated that community composition was significantly altered and community structure changes in unsuspplemented incubations at in situ pressure was indicative of the loss of functional taxa as a result of depressurisation during sampling. The use of enrichment incubations under in-situ conditions has contributed to understanding the different roles played by microorganisms in deep sea ecosystems in regions of low and high productivity.

Workplace exposure to bioaerosols in podiatry clinics.

OBJECTIVES: The aim of this study was to design and execute a pilot study to collect information on the personal exposure levels of podiatrists to microbial hazards in podiatry clinics and also to assess health and safety knowledge within the sector using a questionnaire survey. METHODS: A self-report quantitative questionnaire dealing with health and safety/health issues was issued to 250 podiatrist clinics. Fifteen podiatry clinics were randomly recruited to participate in the exposure study. Concentrations of airborne bacteria, fungi, yeasts, and moulds were assessed using a six-stage viable microbial cascade impactor. Personal samples of total inhalable dust and endotoxin were measured in the breathing zone of the podiatrist. RESULTS: A questionnaire response rate of 42% (N = 101) was achieved. Thirty-two per cent of respondents indicated that they had a respiratory condition; asthma was the most prevalent condition reported. The most frequently employed control measures reported were use of disposable gloves during patient treatments (73.3%), use of respiratory protective equipment (34.6%), use of protective aprons (16.8%), and eye protection (15.8%). A total of 15.8% of respondents used mechanical room ventilation, 47.5% used nail drills with local exhaust ventilation systems, and 11% used nail drills with water spray dust suppression. The geometric mean concentrations of bacteria, Staphylococci, fungi, and yeasts/moulds were 590, 190, 422, and 59 CFU m(-3), respectively. The geometric mean endotoxin exposure was 9.6 EU m(-3). A significant percentage of all the bioaerosols that were in the respirable fraction was representative of yeasts and moulds (65%) and Fungi (87%). CONCLUSIONS: Even if statistical analysis of data is limited by low sample numbers, this study showed that the frequency of cleaning and use of RPE varied between clinics sampled, and it is likely that refresher health and safety training focusing on health and safety hazards inherent in podiatry work and practical control measures is warranted.

Effect of subinhibitory concentrations of benzalkonium chloride on the competitiveness of Pseudomonas aeruginosa grown in continuous culture.

This study investigates the link between adaptation to biocides and antibiotics in Pseudomonas aeruginosa. An enrichment continuous culture of P. aeruginosa NCIMB 10421 (MIC 25 mg BKC l(-1)) was operated (D=0.04 h(-1), 792 h) with added benzalkonium chloride (BKC). A derivative, PA-29 (696 h), demonstrated a >12-fold decrease in sensitivity to the biocide (MIC >350 mg BKC l(-1)). The variant demonstrated a 256-fold increase in resistance to ciprofloxacin, with a mutation in the gyrA gene (Thr-83-->Ile). Similarly, culturing of the original strain in a continuous-culture system with ciprofloxacin selection pressure led to the evolution of BKC-adapted populations (MIC 100 mg BKC l(-1)). Efflux pump activity predominantly contributed to the developed phenotype of PA-29. An amino acid substitution (Val-51-->Ala) in nfxB, the Mex efflux system regulator gene, was observed for PA-29. Overexpression of both MexAB-OprM and MexCD-OprJ was recorded for PA-29. Similarly, mexR, a repressor of the Mex system, was downregulated. Competition studies were carried out in continuous culture between PA-29 and the original strain (in the presence of subinhibitory concentrations of BKC). The outcome of competition was influenced by the concentration of biocide used and the nature of limiting nutrient. The inclusion of 1 mg BKC l(-1) in the medium feed was sufficient to select (S=0.011) for the BKC-adapted strain in magnesium-limited culture. Conversely, the presence of 10 mg BKC l(-1) in the medium supply was insufficient to select for the same organism (S=-0.017) in the glucose-limited culture. These results indicate the importance of environmental conditions on selection and maintenance of biocide adaptation.