Journal articles on the topic 'COMSTAT2'

Mathematical modeling holds great potential for quantitatively describing biofilm growth in presence or absence of chemical agents used to limit or promote biofilm growth. In this paper, we describe a general mathematical/statistical framework that allows for the characterization of complex data in terms of few parameters and the capability to (i) compare different experiments and exposures to different agents, (ii) test different hypotheses regarding biofilm growth and interaction with different agents, and (iii) simulate arbitrary administrations of agents. The mathematical framework is divided to submodels characterizing biofilm, including new models characterizing live biofilm growth and dead cell accumulation; the interaction with agents inhibiting or stimulating growth; the kinetics of the agents. The statistical framework can take into account measurement and interexperiment variation. We demonstrate the application of (some of) the models using confocal microscopy data obtained using the computer program COMSTAT.

Extracellular polymers can facilitate the non-specific attachment of bacteria to surfaces and hold together developing biofilms. This study was undertaken to qualitatively and quantitatively compare the architecture of biofilms produced by Pseudomonas aeruginosa strain PAO1 and its alginate-overproducing (mucA22) and alginate-defective (algD) variants in order to discern the role of alginate in biofilm formation. These strains, PAO1, Alg+ PAOmucA22 and Alg− PAOalgD, tagged with green fluorescent protein, were grown in a continuous flow cell system to characterize the developmental cycles of their biofilm formation using confocal laser scanning microscopy. Biofilm Image Processing (bip) and Community Statistics (comstat) software programs were used to provide quantitative measurements of the two-dimensional biofilm images. All three strains formed distinguishable biofilm architectures, indicating that the production of alginate is not critical for biofilm formation. Observation over a period of 5 days indicated a three-stage development pattern consisting of initiation, establishment and maturation. Furthermore, this study showed that phenotypically distinguishable biofilms can be quantitatively differentiated.

Plastic materials are widely used today in Paedodontics and Orthodontics for manufacturing preventive and therapeutic devices. Since these are worn for long times in the oral cavity biofilm forms on the smooth acrylic surfaces of those appliances. The biofilm must be removed not to destroy the oral microbiology. The aim of this study was to research the possibility of removing the microbial biofilm and disinfecting retainers using the photodynamic effect of toluidine blue O, Fotosan System (CMS Dental, Copenhagen, Denmark) in comparison to two products available on the market Corega Denture Cleanser Tablets (GlaxoSmithKline) and the Retainer Brite� Cleaning Tablets (DENTSPLY International Raintree Essix, FL, USA). The plastic material used in this experiment was the cold-cure acrylic Palapress� vario (Heraeus-Kulzer GmbH, Hanau, Germany). Images of the biofilm formed by Streptococcus pyogenes were obtained using a confocal laser scanning m icroscope. The images were analyzed using Comstat 2 software. The results showed that all the three investigated methods had a disinfectant effect. Corega Denture Cleanser Tablets reduced most of the biofilm formed on the plastic substrate.

Microbial biofilm formation can be influenced by many physiological and genetic factors. The conventional microtiter plate assay provides useful but limited information about biofilm formation. With the fast expansion of the biofilm research field, there are urgent needs for more informative techniques to quantify the major parameters of a biofilm, such as adhesive strength and total biomass. It would be even more ideal if these measurements could be conducted in a real-time, non-invasive manner. In this study, we used quartz crystal microbalance (QCM) and microjet impingement (MJI) to measure total biomass and adhesive strength, respectively, of S. mutans biofilms formed under different sucrose concentrations. In conjunction with confocal laser scanning microscopy (CLSM) and the COMSTAT software, we show that sucrose concentration affects the biofilm strength, total biomass, and architecture in both qualitative and quantitative manners. Our data correlate well with previous observations about the effect of sucrose on the adherence of S. mutans to the tooth surface, and demonstrate that QCM is a useful tool for studying the kinetics of biofilm formation in real time and that MJI is a sensitive, easy-to-use device to measure the adhesive strength of a biofilm.

ABSTRACTNeisseria meningitidisis a worldwide cause of meningitis and septicemia leading at least to 50,000 deaths every year. Nevertheless,N. meningitidisis also a commensal bacterium that asymptomatically colonizes the epithelial cells of the nasopharynx of 10 to 30% of healthy individuals. Occasionally,N. meningitidiscrosses the nasopharyngeal barrier and enters the bloodstream. During bacteremia,N. meningitidismay adhere to endothelial cells of brain vessels and invade meninges. To identify the genes required for meningococcal host colonization, we screened a signature-tagged transposon mutagenesis library using an innovativein vitrocolonization model in order to identify mutants displaying decreased capacity to colonize human epithelial cells. Approximately 1,600 defined insertion mutants of invasive serogroup C strain NEM8013 were screened. Candidate mutants were tested individually for quantification of bacterial biomass with confocal microscope and COMSTAT software. Five mutants were demonstrated to exhibit significantly decreased colonization ability. The identified genes, includingnarPandestD, appeared to be involved in adaptation to hypoxic conditions and stress resistance. Interestingly, the genesfadD1,nnrS, andNMV_2034(encoding a putative thioredoxin), prior to this study, had not been shown to be involved in colonization. Therefore, we provide here insights into the meningococcal functions necessary for the bacterium to adapt to growth on host cells.

ABSTRACT Four strains of Pseudomonas aeruginosa (wild type, ΔpilHIJK mutant, lasI mutant, and rpoS mutant) were genetically tagged with the green fluorescent protein, and the development of flow chamber-grown biofilms by each of them was investigated by confocal laser scanning microscopy. The structural developments of the biofilms were quantified by the computer program COMSTAT (A. Heydorn, A. T. Nielsen, M. Hentzer, C. Sternberg, M. Givskov, B. K. Ersbøll, and S. Molin, Microbiology 146:2395-2407, 2000). Two structural key variables, average thickness and roughness, formed the basis for an analysis of variance model comprising the four P. aeruginosa strains, five time points (55, 98, 146, 242, and 314 h), and three independent rounds of biofilm experiments. The results showed that the wild type, the ΔpilHIJK mutant, and the rpoS mutant display conspicuously different types of temporal biofilm development, whereas the lasI mutant was indistinguishable from the wild type at all time points. The wild type and the lasI mutant formed uniform, densely packed biofilms. The rpoS mutant formed densely packed biofilms that were significantly thicker than those of the wild type, whereas the ΔpilHIJK mutant formed distinct microcolonies that were regularly spaced and almost uniform in size. The results are discussed in relation to the current model of P. aeruginosa biofilm development.

The inhibitory kinetics of isooctyl 4-hydroxy-3-methoxycinnamate on the activity of monophenolase and diphenolase contained in tyrosinase was studied by enzymological kinetic method with Na2HPO4-NaH2PO4 buffer solution (pH=6.8) at 30 °C. Isooctyl 4-hydroxy-3-methoxycinnamate was found to inhibit the monophenolase and diphenolase activity of tyrosinase well. The isooctyl 4-hydroxy-3-methoxycinnamate concentrations leading to 50 % inhibitory rate (IC50) were 0.24 mmol/L for monophenolase and 0.45 mmol/L for diphenolase, much less than that of arbutin (IC50 =5.3 mmol/L for diphenolase activity). Isooctyl 4-hydroxy-3-methoxycinnamate could extend the lag time of tyrosinase for oxidation of L-tyrosine, 0.4 mmol/L of isooctyl 4-hydroxy-3-methoxycinnamate resulted in the extension of lag time from 1.1 min to 3.6 min. The inhibition kinetics of isooctyl 4-hydroxy-3-methoxycinnamate analyzed by Lineweaver-Burk plots demonstrated a competitive inhibitor for the oxidation of L-DOPA, the apparent Michaelis comstant, Km, and the inhibition constant for inhibitor binding with enzyme, KI, were determined to be 0.45 mmol/L and 0.20 mmol/L respectively.

ABSTRACT Indwelling prostheses and subcutaneous delivery devices are now routinely and indispensably employed in medical practice. However, these same devices often provide a highly suitable surface for bacterial adhesion and colonization, resulting in the formation of complex, differentiated, and structured communities known as biofilms. The University of Washington Engineered Biomaterials group has developed a novel drug delivery polymer matrix consisting of a poly(2-hydroxyethyl methacrylate) hydrogel coated with ordered methylene chains that form an ultrasound-responsive coating. This system was able to retain the drug ciprofloxacin inside the polymer in the absence of ultrasound but showed significant drug release when low-intensity ultrasound was applied. To assess the potential of this controlled drug delivery system for the targeting of infectious biofilms, we monitored the accumulation of Pseudomonas aeruginosa biofilms grown on hydrogels with and without ciprofloxacin and with and without exposure to ultrasound (a 43-kHz ultrasonic bath for 20 min daily) in an in vitro flow cell study. Biofilm accumulation from confocal images was quantified and statistically compared by using COMSTAT biofilm analysis software. Biofilm accumulation on ciprofloxacin-loaded hydrogels with ultrasound-induced drug delivery was significantly reduced compared to the accumulation of biofilms grown in control experiments. The results of these studies may ultimately facilitate the future development of medical devices sensitive to external ultrasonic impulses and capable of treating or preventing biofilm growth via “on-demand” drug release.

ABSTRACT The specific biofilm formation (SBF) assay, a technique based on crystal violet staining, was developed to locate plant essential oils and their components that affect biofilm formation. SBF analysis determined that cinnamon, cassia, and citronella oils differentially affected growth-normalized biofilm formation by Escherichia coli. Examination of the corresponding essential oil principal components by the SBF assay revealed that cinnamaldehyde decreased biofilm formation compared to biofilms grown in Luria-Bertani broth, eugenol did not result in a change, and citronellol increased the SBF. To evaluate these results, two microscopy-based assays were employed. First, confocal laser scanning microscopy (CLSM) was used to examine E. coli biofilms cultivated in flow cells, which were quantitatively analyzed by COMSTAT, an image analysis program. The overall trend for five parameters that characterize biofilm development corroborated the findings of the SBF assay. Second, the results of an assay measuring growth-normalized adhesion by direct microscopy concurred with the results of the SBF assay and CLSM imaging. Viability staining indicated that there was reduced toxicity of the essential oil components to cells in biofilms compared to the toxicity to planktonic cells but revealed morphological damage to E. coli after cinnamaldehyde exposure. Cinnamaldehyde also inhibited the swimming motility of E. coli. SBF analysis of three Pseudomonas species exposed to cinnamaldehyde, eugenol, or citronellol revealed diverse responses. The SBF assay could be useful as an initial step for finding plant essential oils and their components that affect biofilm formation and structure.

Semakin banyaknya kebutuhan data center maupun laboratorium komputer di Indonesia dipengaruhi oleh semakin banyaknya pengguna yang memanfaatkan komputer baik untuk bisnis maupun pendidikan. Salah satu kebutuhan utama yang tidak bisa dilepaskan dari pemakaian komputer adalah tempat penyimpanan baik berupa USB Flash Disk, HD Eksternal, HD Internal sampai HD untuk kebutuhan skala besar untuk komputer server yang berada di data center, laboratorium atau jaringan komputer. Ruang penyimpanan data atau data storage semakin berkembang dengan munculnya teknologi komputer jaringan yang memunculkan alternatif data storage berupa DAS, NAS, FC, FcoE dan iSCSI. iSCSI menggunakan standard TCP/IP protocol over Ethernet untuk menyediakan penyimpanan berbasis block. Saat ini ada 2 jenis multiprotocol SCSI Target utama di industri yaitu LIO dan COMSTAR yang menggantikan teknologi sebelumnya yaitu iET, SCST dan STGT. LIO (linux-iscsi.org) merupakan standard open source iSCSI Target untuk berbagi ruang penyimpanan di Linux. LIO mendukung storage fabrics, yaitu Fibre Channel (QLogic), FCoE, iEEE 1394, iSCSI, iSER (Mellanox InfiniBand), SRP (Mellanox InfiniBand), USB, vHost, dan lain-lain.

Three plants adopted by nomads at Tassili n’ajjer (south Algeria) in traditional medicine namely, Cymbopogon schoenanthus, Anabasis articulata and Salvia chudaei, were analysed for theirs antibacterial, antibiofilm and antioxidant properties. Total flavonoid and phenolic contents were measured with 2% AlCl3 and Folin-Ciocalteu’s reagent method, respectively. The antibacterial propertie was investigated by measurement of MIC of plants extract inhibing bacterial proliferation. The antibiofilm propertie was calculated by fluorescent quantization of the DAPI labeled bacterial biomass fixed on the surface and by COMSTAT analysis of confocal scanning laser microscopy (CSLM) images. DPPH radical scavenging and β-carotene/linoleate bleaching methods were used to determine the antioxidant activities of the plants. Total phenolic content was ranged from 21.98 to 2.51 (gallic acid equivalents mg/g methanolic extracts) and the total flavonoid content ranged from 19.27 to 1.65 (catechin equivalent mg/g methanolic extracts). Antibacterial activity against four Gram positive bacteria was shown with plants extracts. The biofilm inhibition concentration of extracts decreasing 50% of biofilm cell density (BIC50) for Salvia chudaei and Cymbopogon schoenanthus extracts ranges from 1 to 10 µg/mL. CSLM images analysis revealed that both surface covering by germ and three dimensional development of the biofilm were reduced with plants extracts. For antioxidant activities, the methanol extracts of the plants evaluated showed low antioxidant activity, with a IC50 between 1.94 and 6.16 mg/ml. Thus, Our systematic research showed that this three common plants of Sahara desert has diversified phytochemicals possessing satisfying extent of antimicrobial, antibiofilm and antioxidant activities.

ABSTRACT Bacterial biofilms cause serious problems, such as antibiotic resistance and medical device-related infections. To further understand bacterium-surface interactions and to develop efficient control strategies, self-assembled monolayers (SAMs) of alkanethiols presenting different functional groups on gold films were analyzed to determine their resistance to biofilm formation. Escherichia coli was labeled with green florescence protein, and its biofilm formation on SAM-modified surfaces was monitored by confocal laser scanning microscopy. The three-dimensional structures of biofilms were analyzed with the COMSTAT software to obtain information about biofilm thickness and surface coverage. SAMs presenting methyl, l-gulonamide (a sugar alcohol tethered with an amide bond), and tri(ethylene glycol) (TEG) groups were tested. Among these, the TEG-terminated SAM was the most resistant to E. coli biofilm formation; e.g., it repressed biofilm formation by E. coli DH5α by 99.5% ± 0.1% for 1 day compared to the biofilm formation on a bare gold surface. When surfaces were patterned with regions consisting of methyl-terminated SAMs surrounded by TEG-terminated SAMs, E. coli formed biofilms only on methyl-terminated patterns. Addition of TEG as a free molecule to growth medium at concentrations of 0.1 and 1.0% also inhibited biofilm formation, while TEG at concentrations up to 1.5% did not have any noticeable effects on cell growth. The results of this study suggest that the reduction in biofilm formation on surfaces modified with TEG-terminated SAMs is a result of multiple factors, including the solvent structure at the interface, the chemorepellent nature of TEG, and the inhibitory effect of TEG on cell motility.

ABSTRACTAcinetobacter baumanniibiofilms are difficult to eradicate. We investigated the effects of meropenem (2 mg/liter), imipenem (2 mg/liter), sulbactam (4 mg/liter), colistin (2 mg/liter), and tigecycline (2 mg/liter), alone or in combination, on biofilm-embedded carbapenem-resistant and carbapenem-susceptibleA. baumannii(CRAb and CSAb, respectively) cells, as well as on the architecture of the biofilms.A. baumanniiATCC 15151 (Ab15151) and its OXA-82-overproducing transformant, along with two clinical CSAb and two clinical CRAb isolates of differing clonalities, were used. The minimal bactericidal concentrations for biofilm-embedded cells of the six tested isolates were >50-fold those of their planktonic cells. When used individually, meropenem exhibited a higher killing effect than the other four antimicrobials on biofilm-embedded CSAb cells in the colony biofilm assay. For two clinical CRAb isolates, meropenem plus sulbactam or sulbactam plus tigecycline showed >100-fold the bactericidal effect exhibited by these agents used alone after 48 h of treatment. The effect of antimicrobials on the architecture of Ab15151 biofilm emitting green fluorescence was determined by confocal laser scanning microscopy using COMSTAT software. Significant decreases in the maximum biofilm thickness were observed after exposure to meropenem and imipenem. Meropenem plus sulbactam significantly decreased the biomass and mean thickness and increased the roughness coefficient of biofilms, but sulbactam plus tigecycline only decreased the maximum and mean biofilm thickness compared to any of these agents used alone. Meropenem was active against biofilm-embedded CSAb, whereas meropenem plus sulbactam exhibited synergism against biofilm-embedded CRAb and caused significantly more damage to the biofilm architecture than did any of the agents used alone.

The quorum-sensing (QS) systems control several virulence attributes of Pseudomonas aeruginosa. Five QS-deficient P. aeruginosa clinical isolates (CI) that were obtained from wound (CI-1), tracheal (CI-2, CI-3, CI-4) and urinary tract (CI-5) infections had previously been characterized. In this study, a flow-through continuous-culture system was utilized to examine in detail the biofilms formed by these isolates in comparison with the P. aeruginosa prototrophic strain PAO1. Analysis of the biofilms by confocal laser scanning microscopy and COMSTAT image analysis at 1 and 7 days post-inoculation showed that the isolates produced diverse biofilms. In comparison with PAO1, the CI produced biofilms that scarcely or partially covered the surface at day 1, although CI-1 produced larger microcolonies. At day 7, CI-2 and CI-4 produced mature biofilms denser than that produced by PAO1, while the biofilm formed by CI-1 changed very little from day 1. CI-1 was defective in both swarming and twitching motilities, and immunoblotting analysis confirmed that it produced a reduced level of PilA protein. The twitching-motility defect of CI-1 was not complemented by a plasmid carrying intact pilA. In the 48 h colony biofilm assay, the CI varied in susceptibility to imipenem, gentamicin and piperacillin/tazobactam. These results suggest that: (1) the isolates produced biofilms with different structures and densities from that of PAO1; (2) biofilm formation by the isolates was not influenced by either the isolation site or the QS deficiencies of the isolates; (3) the behaviour of CI-1 in the different biofilm systems may be due to its lack of swarming motility and type IV pilus-related twitching motility.

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is an extremely common airway commensal which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHI forms biofilm communities that promote persistence in vivo. For many bacterial species, density-dependent quorum-signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer 2 (AI-2) quorum signal pathway, increases NTHI virulence in the chinchilla model for otitis media infections. For example, bacterial counts in middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants compared with parental strains. As these phenotypes are consistent with those that we have observed for biofilm-defective NTHI mutants, we hypothesized that luxS may affect NTHI biofilms. A luxS mutant was generated using the well-characterized NTHI 86-028NP strain and tested to determine the effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of the biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHI 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHI 86-028NP supernatants. Analysis of lipooligosaccharide moieties using an enzyme-linked immunosorbent assay and immunoblotting showed decreased levels of biofilm-associated glycoforms in the NTHI 86-028NP luxS strain. Infection studies showed that NTHI 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we concluded that a luxS-dependent soluble mediator modulates the composition of the NTHI lipooligosaccharides, resulting in effects on biofilm maturation and bacterial persistence in vivo.

ABSTRACTTraditional therapeutic strategies to control chronic colonization in cystic fibrosis (CF) patients are based on the use of a single nebulized antibiotic. In this study, we evaluated the therapeutic efficacy and dynamics of antibiotic resistance inPseudomonas aeruginosabiofilms under sequential therapy with inhaled aztreonam (ATM) and tobramycin (TOB). Laboratory strains PAO1, PAOMS (hypermutable), PAOMA (mucoid), and PAOMSA (mucoid and hypermutable) and two hypermutable CF strains, 146-HSE (Liverpool epidemic strain [LES-1]) and 1089-HSE (ST1089), were used. Biofilms were developed using the flow cell system. Mature biofilms were challenged with peak and 1/10-peak concentrations of ATM (700 mg/liter and 70 mg/liter), TOB (1,000 mg/liter and 100 mg/liter), and their alternations (ATM/TOB/ATM and TOB/ATM/TOB) for 2 (t= 2), 4 (t= 4), and 6 days (t= 6). The numbers of viable cells (CFU) and resistant mutants were determined. Biofilm structural dynamics were monitored by confocal laser scanning microscopy and processed with COMSTAT and IMARIS software programs. TOB monotherapy produced an intense decrease in CFU that was not always correlated with a reduction in biomass and/or a bactericidal effect on biofilms, particularly for the CF strains. The ATM monotherapy bactericidal effect was lower, but effects on biofilm biomass and/or structure, including intense filamentation, were documented. The alternation of TOB and ATM led to an enhancement of the antibiofilm activity against laboratory and CF strains compared to that with the individual regimens, potentiating the bactericidal effect and/or the reduction in biomass, particularly at peak concentrations. Resistant mutants were not documented in any of the regimens at the peak concentrations and only anecdotally at the 1/10-peak concentrations. These results support the clinical evaluation of sequential regimens with inhaled antibiotics in CF, as opposed to the current maintenance treatments with just one antibiotic in monotherapy.

ABSTRACT After 13,000 samples of compounds purified from plants were screened, a new biofilm inhibitor, ursolic acid, has been discovered and identified. Using both 96-well microtiter plates and a continuous flow chamber with COMSTAT analysis, 10 μg of ursolic acid/ml inhibited Escherichia coli biofilm formation 6- to 20-fold when added upon inoculation and when added to a 24-h biofilm; however, ursolic acid was not toxic to E. coli, Pseudomonas aeruginosa, Vibrio harveyi, and hepatocytes. Similarly, 10 μg of ursolic acid/ml inhibited biofilm formation by >87% for P. aeruginosa in both complex and minimal medium and by 57% for V. harveyi in minimal medium. To investigate the mechanism of this nontoxic inhibition on a global genetic basis, DNA microarrays were used to study the gene expression profiles of E. coli K-12 grown with or without ursolic acid. Ursolic acid at 10 and 30 μg/ml induced significantly (P < 0.05) 32 and 61 genes, respectively, and 19 genes were consistently induced. The consistently induced genes have functions for chemotaxis and mobility (cheA, tap, tar, and motAB), heat shock response (hslSTV and mopAB), and unknown functions (such as b1566 and yrfHI). There were 31 and 17 genes repressed by 10 and 30 μg of ursolic acid/ml, respectively, and 12 genes were consistently repressed that have functions in cysteine synthesis (cysK) and sulfur metabolism (cysD), as well as unknown functions (such as hdeAB and yhaDFG). Ursolic acid inhibited biofilms without interfering with quorum sensing, as shown with the V. harveyi AI-1 and AI-2 reporter systems. As predicted by the differential gene expression, deleting motAB counteracts ursolic acid inhibition (the paralyzed cells no longer become too motile). Based on the differential gene expression, it was also discovered that sulfur metabolism (through cysB) affects biofilm formation (in the absence of ursolic acid).

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) causes chronic infections that feature the formation of biofilm communities. NTHI variants within biofilms have on their surfaces lipooligosaccharides containing sialic acid (NeuAc) and phosphorylcholine (PCho). Our work showed that NeuAc promotes biofilm formation, but we observed no defect in the initial stages of biofilm formation for mutants lacking PCho. In this study, we asked if alterations in NTHI PCho content affect later stages of biofilm maturation. Biofilm communities were compared for NTHI 2019 and isogenic mutants that either lacked PCho (NTHI 2019 licD) or were constitutively locked in the PCho-positive phase (NTHI 2019 lic ON ). Transformants expressing green fluorescent protein were cultured in continuous-flow biofilms and analyzed by confocal laser scanning microscopy. COMSTAT was used to quantify different biofilm parameters. PCho expression correlated significantly with increased biofilm thickness, surface coverage, and total biomass, as well as with a decrease in biofilm roughness. Comparable results were obtained by scanning electron microscopy. Analysis of thin sections of biofilms by transmission electron microscopy revealed shedding of outer membrane vesicles by NTHI bacteria within biofilms and staining of matrix material with ruthenium red in biofilms formed by NTHI 2019 lic ON . The biofilms of all three strains were comparable in viability, the presence of extracellular DNA, and the presence of sialylated moieties on or between bacteria. In vivo infection studies using the chinchilla model of otitis media showed a direct correlation between PCho expression and biofilm formation within the middle-ear chamber and an inverse relationship between PCho and persistence in the planktonic phase in middle-ear effusions. Collectively, these data show that PCho correlates with, and may promote, the maturation of NTHI biofilms. Further, this structure may be disadvantageous in the planktonic phase.

ABSTRACT We found that Acinetobacter baumannii contains a pgaABCD locus that encodes proteins that synthesize cell-associated poly-β-(1-6)-N-acetylglucosamine (PNAG). Both a mutant with an in-frame deletion of the pga locus (S1Δpga) and a transcomplemented strain (S1Δpga-c) of A. baumannii were constructed, and the PNAG production by these strains was compared using an immunoblot assay. Deleting the pga locus resulted in an A. baumannii strain without PNAG, and transcomplementation of the S1Δpga strain with the pgaABCD genes fully restored the wild-type PNAG phenotype. Heterologous expression of the A. baumannii pga locus in Escherichia coli led to synthesis of significant amounts of PNAG, while no polysaccharide was detected in E. coli cells harboring an empty vector. Nuclear magnetic resonance analysis of the extracellular polysaccharide material isolated from A. baumannii confirmed that it was PNAG, but notably only 60% of the glucosamine amino groups were acetylated. PCR analysis indicated that all 30 clinical A. baumannii isolates examined had the pga genes, and immunoblot assays indicated that 14 of the 30 strains strongly produced PNAG, 14 of the strains moderately to weakly produced PNAG, and 2 strains appeared to not produce PNAG. Deletion of the pga locus led to loss of the strong biofilm phenotype, which was restored by complementation. Confocal laser scanning microscopy studies combined with COMSTAT analysis demonstrated that the biovolume, mean thickness, and maximum thickness of 16-h and 48-h-old biofilms formed by wild-type and pga-complemented A. baumannii strains were significantly greater than the biovolume, mean thickness, and maximum thickness of 16-h and 48-h-old biofilms formed by the S1Δpga mutant strain. Biofilm-dependent production of PNAG could be an important virulence factor for this emerging pathogen that has few known virulence factors.

Abstract Background TOL and MEM possess in vitro activity against P. Aeruginosa grown in planktonic culture. Less is known about the activity of TOL and MEM on antibiotic-tolerant biofilms, such as those residing in the lungs of cystic fibrosis (CF) patients. The objective of this study is to compare the spatial and temporal activity of TOL and MEM on early and mature P. Aeruginosa biofilms grown in flow-chambers. Methods PAO1 and a clinical strain (strA) isolated from a child with CF were used. Isolates were cultivated in flow-chambers for 24 or 72 hours. Early (24-hours) biofilms were treated with the simulated human concentrations of 1 dose of either 2 g of TOL or MEM. Peak concentrations were 150 mg/L and 107.53 mg/L for TOL and MEM. Mature (72-hours) biofilms were treated with 3 doses of MEM or TOL administered every 8 hours. Images were acquired during antibiotic treatment using confocal laser scanning microscopy. Images were processed with Imaris and analyzed using COMSTAT 2.0. Pharmacodynamic modeling was performed with NONMEM. Results PAO1 and strA were susceptible to TOL and MEM per CLSI guidelines. Kill rates for TOL and MEM are shown below. TOL displayed smaller killing rates compared with MEM for early biofilms; TOL and MEM showed similar activity against the mature clinical strain (strA). MEM and TOL were less effective against strA compared with PAO1. Conclusion TOL and MEM displayed similar spatial activity against P. Aeruginosa biofilms grown in flow-chambers. Temporal differences in kill rates and bacterial recovery between TOL and MEM may inform future dosing strategies for biofilm-associated infections such as CF. Disclosures D. Verotta, Merck: Grant Investigator, Grant recipient. D. P. Nicolau, Merck: Investigator and Speaker’s Bureau, Research support. K. Yang, Merck: Grant Investigator, Grant recipient.

Nitrogen in plants is part of the green pigment chlorophyll, as well as proteins, nucleic acids, phytohormones and alkaloids that indicates the key role of this element in plant life. Chlorophyll is the most important pigment of the photosynthetic process determining the life of all heterotrophic organisms on the planet. The facts mentioned above presuppose close relationships between nitrogen and chlorophyll in plants. The nitrogen content in plants serves as a basis for adjusting their nitrogen nutrition and calculating fertilization rates for high yields. This causes comstant importance of studying the content of nitrogen and chlorophyll in plants, especially by means of novel techniques with involving remote sensing. This study was focused on relationship between 19 vegetation indices (VI) and biochemical characteristics of vegetation, in particular nitrogen and chlorophyll content. Study areas were located within production fields of two varieties of winter wheat grown for harvest in 2016 by the grain company Baryshivska. The test plots varied by phytopathological situation in the phase of milk ripeness. Fungal infection of Bogdana variety caused significant varietal differences in biochemical parameters that were calculated by Kjeldahl makro-method for total nitrogen and by aerial survey with UAV (drone) for chlorophyll content. Among 19 VIs calculated by ground spectrometry the major part (16 VIs) were consistent with changes in nitrogen and chlorophyll content in the cultivars. In particular, CI rededge , CI green , MTCI, RVI, D731 / D700 and D735 / D700 were more than doubled, and NDRE1 and D718 / D700 were almost 1.5 times higher in the Skagen variety compared to the Bogdan variety. Only 3 indices: NDVI, Green NDVI and NI had limits of fluctuations of the values within the same limits, as varietal differences of biochemical indicators.

Abstract Background BB-related infections are a major public health problem, as they are notoriously refractory to current treatments. One of the defining characteristics of BBs is the extracellular polymeric substance (EPS). Extracellular DNA and the bacterial DNABII family of proteins are key components of EPS and are crucial for BBs structural integrity. It is known that targeting DNABII proteins disrupts BBs. We hypothesized that HMGB1, a DNA-binding eukaryotic protein, could affect BBs as it binds to the same DNA structures as the DNABII proteins. HMGB1 is comprised of 3 domains, A Box, B Box, and C tail, all of which have different functions. We aimed to determine in vitro the effects of HMGB1 and its individual domains against BBs. Methods Klebsiella pneumoniae (KP), a common cause of nosocomial infections, was used for all BBs disruption assays. Human recombinant full-length HMGB1 (rHMGB1; 1–215), a C45S mutation variant (mHMGB1) and the HMGB1 domains A Box (1–89), B Box (90–176), AB Boxes (1–176), B-linker Box (80–179), and B-linker Box C106S were expressed (in E. coli) and purified to &gt;95%. To evaluate the effect of rHMGB1 and the various domains on established BBs, each protein species (200 nM) was added to preformed BBs at 24 hours. At 40 hours the BBs were washed, stained with LIVE/DEAD®, visualized via confocal laser scanning microscopy and images were analyzed by COMSTAT to calculate average thickness and biomass. Results Exogenous rHMGB1 and its individual domains, with the exception of A Box caused a significant reduction (P &lt; 0.05) in average thickness (AT) and biomass (BM) of KP biofilms when compared with untreated KP biofilms (% reduction mean ± SE in AT: 44% ± 0.33, 75% ± 0.04, 63% ± 0.1, 77% ± 0.03, 64% ± 0.08, 54% ± 0.15 and in BM: 61% ± 0.01, 80% ± 0.01, 68% ± 0.02, 67% ± 0.01, 73% ± 0.02, 56% ± 0.02 induced by rHMGB1, mHMGB1, B-Box, B-linker Box, AB Boxes, and B-linker Box C106S, respectively). Conclusion Full-length recombinant HMGB1 was able to significantly disrupt established KP biofilms as were all truncated HMGB1 forms containing the B Box domain and could potentially be used as a therapeutic treatment for BB-related infections. Disclosures J. Buzzo, ProclaRx: Collaborator, Research support. S. Goodman, ProclaRx: Collaborator and Scientific Advisor, Research support.

AbstractCandida albicans (C. albicans) is an opportunistic pathogen, which causes superficial infection and can lead to mortal systemic infections, especially in immunocompromised patients. The incidence of C. albicans infections is increasing and there are a limited number of antifungal drugs used in treatment. Therefore, there is an urgent need for new and alternative antifungal drugs. Pomegranate rind extract (PRE) is known for its broad-spectrum antimicrobial activities, including against C. albicans and recently, PRE and Zn (II) have been shown to induce synergistic antimicrobial activity against various microbes. In this study, the inhibitory activities of PRE, Zn (II) and PRE in combination with Zn (II) were evaluated against C. albicans. Antifungal activities of PRE and Zn (II) were evaluated using conventional microdilution methods and the interaction between these compounds was assessed by in vitro checkerboard and time kill assays in planktonic cultures. The anti-biofilm activities of PRE, Zn (II) and PRE in combination with Zn (II) were assessed using confocal laser scanning microscopy, with quantitative analysis of biofilm biomass and mean thickness analysed using COMSTAT2 analysis. In addition, antimicrobial interactions between PRE and Zn (II) were assayed in terms reactive oxygen species (ROS) production by C. albicans. PRE and Zn (II) showed a potent antifungal activity against C. albicans, with MIC values of 4 mg/mL and 1.8 mg/mL, respectively. PRE and Zn (II) in combination exerted a synergistic antifungal effect, as confirmed by the checkerboard and time kill assays. PRE, Zn (II) and PRE and Zn (II) in combination gave rise to significant reductions in biofilm biomass, although only PRE caused a significant reduction in mean biofilm thickness. The PRE and Zn (II) in combination caused the highest levels of ROS production by C. albicans, in both planktonic and biofilm forms. The induction of excess ROS accumulation in C. albicans may help explain the synergistic activity of PRE and Zn (II) in combination against C. albicans in both planktonic and biofilm forms. Moreover, the data support the potential of the PRE and Zn (II) combination as a novel potential anti-Candida therapeutic system.

Abstract As sessile cells of fungal biofilms are at least 500-fold more resistant to antifungal drugs than their planktonic counterparts, there is a requirement for new antifungal agents. Olygostyrylbenzenes (OSBs) are the first generation of poly(phenylene)vinylene dendrimers with a gram-positive antibacterial activity. Thus, this study aimed to investigate the antifungal activity of four OSBs (1, 2, 3, and 4) on planktonic cells and biofilms of Candida tropicalis. The minimum inhibitory concentration (MIC) for the planktonic population and the sessile minimum inhibitory concentrations (SMIC) were determined. Biofilm eradication was studied by crystal violet stain and light microscopy (LM), and confocal laser scanning microscopy (CLSM) was also utilized in conjunction with the image analysis software COMSTAT. Although all the OSBs studied had antifungal activity, the cationic OSBs were more effective than the anionic ones. A significant reduction of biofilms was observed at MIC and supraMIC50 (50 times higher than MIC) for compound 2, and at supraMIC50 with compound 3. Alterations in surface topography and the three-dimensional architecture of the biofilms were evident with LM and CLSM. The LM analysis revealed that the C. tropicalis strain produced a striking biofilm with oval blastospores, pseudohyphae, and true hyphae. CLSM images showed that a decrease occurred in the thickness of the mature biofilms treated with the OSBs at the most effective concentration for each one. The results obtained by microscopy were supported by those of the COMSTAT program. Our results revealed an antibiofilm activity, with compound 2 being a potential candidate for the treatment of C. tropicalis infections. Lay Summary This study aimed to investigate the antifungal activity of four OSBs (1, 2, 3, and 4) on planktonic cells and biofilms of Candida tropicalis. Our results revealed an antibiofilm activity, with compound 2 being a potential candidate for the treatment of C. tropicalis infections.

Abstract Als3 is a cell-surface glycoprotein of Candida albicans that plays essential roles in the processes of adherence and biofilm formation in vitro. In this study, we focused on the contribution of Als3 to the structure and drug susceptibility of biofilms. The C. albicans wild-type strain DAY185, the als3Δ/Δ null strain and the als3Δ/Δ + pALS3 complemented strain were used. Colony forming unit enumeration, crystal violet and cell surface hydrophobicity assays, scanning electron microscopy and confocal laser scanning microscopy coupled with analyses using COMSTAT software were performed to evaluate the biomass and architecture of the biofilms. The detailed architectural analysis showed a significant variation in the biofilm parameters of the als3Δ/Δ biofilms compared with those of the WT biofilms. Fluconazole, miconazole and amphotericin B were selected as the antifungal agents for the antimycotic susceptibility test, and increased susceptibility was found with the ALS3 deletion biofilms. A quantitative real-time polymerase chain reaction analysis showed downregulation of biofilm formation-related genes (ALS1, EFG1, HWP1, and CSH1) and drug resistance-related genes (ERG11, CDR1, CDR2, and MDR1) in the als3Δ/Δ biofilms. We concluded that Als3 contributes to biofilm formation by changing the biofilm architecture and is involved in the antifungal resistance of C. albicans biofilms.

AbstractIn a number of chronic respiratory diseases e.g. cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), the production of viscous mucin reduces pulmonary function and represents an effective barrier to diffusion of inhaled therapies e.g. antibiotics. Here, a 2-compartment Transwell model was developed to study impaired diffusion of the antibiotic colistin across an artificial sputum (AS) matrix/medium and to quantify its antimicrobial activity against Pseudomonas aeruginosa NH57388A biofilms (alone and in combination with mucolytic therapy). High-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) revealed that the presence of AS medium significantly reduced the rate of colistin diffusion (> 85% at 48 h; p < 0.05). Addition of alginate oligosaccharide (OligoG CF-5/20) significantly improved colistin diffusion by 3.7 times through mucin-rich AS medium (at 48 h; p < 0.05). Increased diffusion of colistin with OligoG CF-5/20 was shown (using confocal laser scanning microscopy and COMSTAT image analysis) to be associated with significantly increased bacterial killing (p < 0.05). These data support the use of this model to study drug and small molecule delivery across clinically-relevant diffusion barriers. The findings indicate the significant loss of colistin and reduced effectiveness that occurs with mucin binding, and support the use of mucolytics to improve antimicrobial efficacy and lower antibiotic exposure.

ABSTRACT Pseudomonas aeruginosa plays a major role in many chronic infections. Its ability to readily form biofilms contributes to its success as an opportunistic pathogen and its resistance/tolerance to antimicrobial/antibiotic therapy. A low-molecular-weight alginate oligomer (OligoG CF-5/20) derived from marine algae has previously been shown to impair motility in P. aeruginosa biofilms and disrupt pseudomonal biofilm assembly. As these bacterial phenotypes are regulated by quorum sensing (QS), we hypothesized that OligoG CF-5/20 may induce alterations in QS signaling in P. aeruginosa . QS regulation was studied by using Chromobacterium violaceum CV026 biosensor assays that showed a significant reduction in acyl homoserine lactone (AHL) production following OligoG CF-5/20 treatment (≥2%; P < 0.05). This effect was confirmed by liquid chromatography-mass spectrometry analysis of C 4 -AHL and 3-oxo-C 12 -AHL production (≥2%; P < 0.05). Moreover, quantitative PCR showed that reduced expression of both the las and rhl systems was induced following 24 h of treatment with OligoG CF-5/20 (≥0.2%; P < 0.05). Circular dichroism spectroscopy indicated that these alterations were not due to steric interaction between the AHL and OligoG CF-5/20. Confocal laser scanning microscopy (CLSM) and COMSTAT image analysis demonstrated that OligoG CF-5/20-treated biofilms had a dose-dependent decrease in biomass that was associated with inhibition of extracellular DNA synthesis (≥0.5%; P < 0.05). These changes correlated with alterations in the extracellular production of the pseudomonal virulence factors pyocyanin, rhamnolipids, elastase, and total protease ( P < 0.05). The ability of OligoG CF-5/20 to modify QS signaling in P. aeruginosa PAO1 may influence critical downstream functions such as virulence factor production and biofilm formation.

BackgroundPersister cells (PCs) make up a small fraction of microbial population, can survive lethal concentrations of antimicrobial agents. In recent years, Candida tropicalis has emerged as being a frequent fungal agent of medical devices subject to biofilm infections. However, PCs are still poorly understood.ObjectivesThis study aimed to investigate the relation of PCs on the redox status in C. tropicalis biofilms exposed to high doses of Amphotericin B (AmB), and alterations in surface topography and the architecture of biofilms.MethodsWe used an experimental model of two different C. tropicalis biofilms exposed to AmB at supra minimum inhibitory concentration (SMIC80), and the intra- and extracellular reactive oxygen species (iROS and eROS), reactive nitrogen species (RNS) and oxidative stress response were studied. Light microscopy (LM) and confocal laser scanning microscopy (CLSM) were also used in conjunction with the image analysis software COMSTAT.ResultsWe demonstrated that biofilms derived from the PC fraction (B2) showed a higher capacity to respond to the stress generated upon AmB treatment, compared with biofilms obtained from planktonic cells. In B2, a lower ROS and RNS accumulation was observed in concordance with higher activation of the antioxidant systems, resulting in an oxidative imbalance of a smaller magnitude compared to B1. LM analysis revealed that the AmB treatment provoked a marked decrease of biomass, showing a loss of cellular aggrupation, with the presence of mostly yeast cells. Moreover, significant structural changes in the biofilm architecture were noted between both biofilms by CLSM—COMSTAT analysis. For B1, the quantitative parameters bio-volume, average micro-colony volume, surface to bio-volume ratio and surface coverage showed reductions upon AmB treatment, whereas increases were observed in roughness coefficient and average diffusion distance. In addition, untreated B2 was substantially smaller than B1, with less biomass and thickness values. The analysis of the above-mentioned parameters also showed changes in B2 upon AmB exposure.ConclusionTo our knowledge, this is the first study that has attempted to correlate PCs of Candida biofilms with alterations in the prooxidant-antioxidant balance and the architecture of the biofilms. The finding of regular and PCs with different cellular stress status may help to solve the puzzle of biofilm resistance, with redox imbalance possibly being an important factor.

Abstract Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM The general feature of the biofilm is a densely packed microbial community with an extracellular matrix composed of polysaccharides and proteins. Candida glabrata was reported as the most biofilm-forming species responsible for causing virulent Candida infections among Candida non-albicans (CNA). Candida glabrata is the most azole-resistant species of all Candida species. The early and maturing biofilms enable the Candida cells to overcome the effects of azoles and exhibit higher drug resistance. Since simple azole mono-therapy rarely eradicates recalcitrant Candida biofilms, removal of the infected device becomes necessary for curing the infection. Objective The objective was to assess the biofilm inhibition effect of antifungal cyclic lipopeptide which has been purified by a multi-step process from the cell-free supernatant of Bacillus subtilis, and to analyze the synergistic activity of the purified lipopeptide lead compound designated as AF4 with the standard antifungal fluconazole using different concentrations. Methods Briefly, cell-free supernatant was extracted using a solvent mixture and silica gel-based adsorption chromatography. The potential antifungal cyclic lipopeptide variant AF4 was extracted from the soil isolate B. subtilis by a multi-step purification process that involves reversed-phase HPLC and exhibited a wide-spectrum of anti-Candida activity. The XTT reduction assay was used for metabolically active cells after treatment with a combination of drugs, and crystal violet (CV) assay was performed to quantify the cell biomass. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to analyze the biofilm morphology and architecture of the AF4 lipopeptide at (2xMIC) and fluconazole (8x-16xMIC)-treated biofilms. For confocal microscopy-based biofilm visualization, a combination of two different dyes was used. Extracellular polymeric substances were stained by concanavalin A-Alexa Fluor 488 conjugate and, the FUN- 1 fluorescent dye was used for staining the live biofilm-forming Candida cells. After capturing the Z-stack images in CLSM, the biomass (μm3/μm2), average thickness (μm), and roughness of the biofilm were analyzed by Comstat 2.1 software. Results The AFST analysis showed that the MIC of the lead antifungal lipopeptide was 8 μg/mL against the C. glabrata planktonic cells. Adding the antifungal compound at higher MICs such as by 2-folds exhibited strong antibiofilm effect that significantly enhanced the synergistic antifungal activity of the fluconazole against C. glabrata biofilm formation. Synergistic effect of fluconazole at 8x-16xMIC with 2xMIC of the lead compound AF4 on C. glabrata 24 h maturing biofilm had increased significantly as revealed by the XTT and CV assays. The results confirmed ˃ 50% inhibition of the biofilm in both 6 h developmental and 24 h maturing biofilms in 8xMIC fluconazole plus 2xMIC AF4 while compared with fluconazole alone at the same concentration. The results from the XTT and, CV assays and SEM and CLSM (Fig. 1) suggest that the combinatorial treatments of the lead antifungal lipopeptide and fluconazole at varying concentrations have the potential of eradicating or nearly eradicating the maturing biofilm of CAN. Conclusions The combinatorial study was found effective in combating the biofilm formation. A correlation between metabolic activity and antifungal resistance in maturing biofilms has been found.

In the contemporary urban environment of mass transit, it falls to a small group of public officers to keep large number of travellers safe. The small size of their force and the often limited powers they exert mean that these public safety ‘transit officers’ must project more authority and control than they really have. It is this ambience of authority and control which, in most situations they encounter and seek to influence, is enough to keep the public safe. This paper examines the ambience of a group of transit officers working on the railway lines of an Australian capital city. We seek to show how transit officers are both influenced by, and seek to influence, the ambience of their workplace and the public spaces they inhabit whilst on duty, and here we take ambience to apply to the surrounding atmosphere, the aura, and the emotional environment of a place or situation: the setting, tone, or mood. For these transit officers to keep the public safe, they must themselves remain safe. A transit officer who is disabled in a confrontation with a violent offender is unable to provide protection to his or her passengers. Thus, in the culture of the transit officers, their own workplace safety takes on a higher significance. It affects not just themselves. The ambience exuded by transit officers, and how transit officers see their relationship with the travelling public, their management and other organisational work groups, is an important determinant of their work group’s safety culture. Researching the Working Lives of Transit Officers in Perth Our discussion draws on an ethnographic study of the working lives and communication cultures of transit officers (TOs) employed by the Public Transport Authority (PTA) of Western Australia (WA). Transit officers have argued that to understand fully the challenges of their work it is necessary to spend time with them as they undertake their daily duties: roster in, roster out. To this end, the research team and the employer organisation secured an ARC Linkage Grant in partnership with the PTA to fund doctoral candidate and ethnographer Christine Teague to research the workers’ point of view, and the workers’ experiences within the organisation. The two-hundred TOs are unique in the PTA. Neither of the other groups who ride with them on the trains, the drivers and revenue protection staff (whose sole job is to sell and check tickets), experiences the combination of intense contact with passengers, danger of physical injury or group morale. The TOs of the PTA in Perth operate from a central location at the main train station and the end stations on each line. Here there are change lockers where they can lock up their uniforms and equipment such as handcuffs and batons when not on duty, an equipment room where they sign out their radios, and ticket-checking machines. At the main train station there is also a gym, a canteen and holding cells for offenders they detain. From these end stations and central location, the TOs fan out across the network to all suburbs where they either operate from stations or onboard the trains. The TOs also do ‘delta van’ duty providing rapid, mobile back-up support for their colleagues on stations or trains, and providing transport for arrested persons to the holding cell or police lock up. TOs are on duty whenever the trains are running–but the evenings and nights are when they are mainly rostered on. This is when trouble mostly occurs. The TOs’ work ends only after the final train has completed its run and all offenders who may require detaining and charging have been transferred into police custody. While the public perceive that security is the TOs’ most frequent role, much of the work involves non-confrontational activity such as assisting passengers, checking tickets and providing a reassuring presence. One way to deal with an ambiguous role is to claim an ambience of power and authority regardless. Various aspects of the TO role permit and hinder this, and the paper goes on to consider aspects of ambience in terms of fear and force, order and safety, and role confusion. An Ambience of Fear and Force The TOs are responsible for front-line security in WA’s urban railway network. Their role is to offer a feeling of security for passengers using the rail network after the bustle of the work day finishes, and is replaced by the mainly recreational travels of the after hours public. This is the time when some passengers find the prospect of evening travel on the public transport rail network unsettling–so unsettling that it was a 2001 WA government election promise (WA Legislative Council) that every train leaving the city centre after 7pm would have two TOs riding on it. Interestingly, recruitment levels have never been high enough for this promise to be fully kept. The working conditions of the TOs reflect the perception, and to an extent, the reality that some late night travel on public transport involves negotiating an edgy ambience with an element of risk, rubbing shoulders with people who may be loud, rowdy, travelling in a group, and or drug and alcohol affected. As Fred (all TO names are pseudonyms) comments: You’re not dealing with rational people, you’re not dealing with ‘people’: most of the people you’re dealing with are either drunk or under the influence of drugs, so they’re not rational, they don’t hear you, they don’t understand what you’re saying, they just have no sense of what’s right or wrong, you know? Especially being under the influence, so I mean, you can talk till you’re blue in the face with somebody who’s drunk or on drugs, I mean, all you have to say is one thing. ‘Oh, can I see your ticket please’, ‘oh, why do I need a fucking ticket’, you know? They just don’t get simple everyday messages. Dealing with violence and making arrest is a normal part of this job. Jo described an early experience in her working life as a TO:Within the first week of coming out of course I got smacked on the side of the head, but this lady had actually been certified, like, she was nuts. She was completely mental and we were just standing on the train talking and I’ve turned around to say something to my partner and she was fine, she was as calm as, and I turned around and talked to my partner and the next thing I know I ended up with her fist to the side of my head. And I went ‘what the hell was that’? And she went off, she went absolutely ballistic. I ended up arresting her because it was assault on an officer whether she was mental or not so I ended up arresting her.Although Jo here is describing how she experienced an unprovoked assault in the early days of her career as a TO, one of the most frequent precursors to a TO injury occurs when the TO is required to make an arrest. The injury may occur when the passenger to be arrested resists or flees, and the TO gives chase in dark or treacherous circumstances such as railway reserves and tunnels, or when other passengers, maybe friends or family of the original person of concern, involve themselves in an affray around the precipitating action of the arrest. In circumstances where capsicum spray is the primary way of enforcing compliance, with batons used as a defence tool, group members may feel that they can take on the two TOs with impunity, certainly in the first instance. Even though there are security cameras on trains and in stations, and these can be cued to cover the threatening or difficult situations confronting TOs, the conflict is located in the here-and-now of the exchanges between TOs and the travelling public. This means the longer term consequence of trouble in the future may hold less sway with unruly travellers than the temptation to try to escape from trouble in the present. In discussing the impact of remote communications, Rubert Murdoch commented that these technologies are “a powerful influence for civilised behaviour. If you are arranging a massacre, it will be useless to shoot the cameraman who has so inconveniently appeared on the scene. His picture will already be safe in the studio five thousand miles away and his final image may hang you” (Shawcross 242). Unfortunately, whether public aggression in these circumstances is useless or not, the daily experience of TOs is that the presence of closed circuit television (CCTV) does not prevent attacks upon them: nor is it a guarantee of ‘civilised behaviour’. This is possibly because many of the more argumentative and angry members of the public are dis-inhibited by alcohol or other drugs. Police officers can employ the threat or actual application of stun guns to control situations in which they are outnumbered, but in the case of TOs they can remain outnumbered and vulnerable until reinforcements arrive. Such reinforcements are available, but the situation has to be managed through the communication of authority until the point where the train arrives at a ‘manned’ station, or the staff on the delta vehicle are able to support their colleagues. An Ambience of Order and Safety Some public transport organisations take this responsibility to sustain an ambience of order more seriously than others. The TO ethnographer, Christine Teague, visited public transport organisations in the UK, USA and Canada which are recognised as setting world-class standards for injury rates of their staff. In the USA particularly, there is a commitment to what is called ‘the broken windows’ theory, where a train is withdrawn from service promptly if it is damaged or defaced (Kelling and Coles; Maple and Mitchell). According to Henry (117): The ‘Broken Windows’ theory suggests that there is both a high correlation and a causal link between community disorder and more serious crime: when community disorder is permitted to flourish or when disorderly conditions or problems are left untended, they actually cause more serious crime. ‘Broken windows’ are a metaphor for community disorder which, as Wilson and Kelling (1982) use the term, includes the violation of informal social norms for public behaviour as well as quality of life offenses such as littering, graffiti, playing loud radios, aggressive panhandling, and vandalism.This theory implies that the physical ambience of the train, and by extension the station, may be highly influential in terms of creating a safe working environment. In this case of ‘no broken window’ organisations, the TO role is to maintain a high ‘quality of life’ rather than being a role predominantly about restraining and bringing to justice those whose behaviour is offensive, dangerous or illegal. The TOs in Perth achieve this through personal means such as taking pride in their uniforms, presenting a good-natured demeanour to passengers and assisting in maintaining the high standard of train interiors. Such a priority, and its link to reduced workforce injury, suggests that a perception of order impacts upon safety. It has long been argued that the safety culture of an organisation affects the safety performance of that organisation (Pidgeon; Leplat); but it has been more recently established that different cultural groupings in an organisation conceive and construct their safety culture differently (Leith). The research on ‘safety culture’ raises a problematic which is rarely addressed in practice. That problematic is this: managers frequently engage with safety at the level of instituting systems, while workers engage with safety in terms of behaviour. When Glendon and Litherland comment that, contrary to expectations, they could find no relationship between safety culture and safety performance, they were drawing attention to the fact that much managerial safety culture is premised upon systems involving tick boxes and the filling in of report forms. The broken window approach combines the managerial tick box with managerial behaviour: a dis-ordered train is removed from service. To some extent a general lack of fit between safety culture and safety performance endorses Everett’s view that it is conceptually inadequate to conceive organisations as cultures: “the conceptual inadequacy stems from the failure to distinguish between culture and behavioural features of organizational life” (238). The general focus upon safety culture as a way of promoting improvements in safety performance assumes that compliance with a range of safety systems will guarantee a safe workplace. Such an assumption, however, risks positioning the injured worker as responsible for his or her own predicament and sets up an environment in which some management officials are wont to seek ways in which that injured worker’s behaviour failed to conform with safety rules or safety processes. Yet there are roles which place workers in harm’s way, including military duties, law enforcement and some emergency services. Here, the work becomes dangerous as it becomes disorderly. An Ambience of Roles and Confusion As the research reported here progressed, it became clear that the ambience around the presentation of the self in the role of a TO (Goffman) was an important part of how ‘safety’ was promoted and enacted in their work upon the PTA (WA) trains, face to face with the travelling public. Goffman’s view of all people, not specifically TOs, is that: Regardless of the particular objective which the individual has in mind and of his motive for having this objective, it will be in his interests to control the conduct of the others, especially their responsive treatment of him. This will largely be through influencing the perception and definition that others will come to formulate of him. He will influence them by expressing himself in such a way that the kind of impression given off will lead them to act voluntarily in accordance with his own plan. (3)This ‘influencing of perception’ is an important element of performing the role of a TO. This task of the TOs is made all the more difficult because of confusions about their role in relation to two other officers: police (who have more power to act in situations of public safety) and revenue project officers (who have less), as we now discuss. The aura of the TO role borrows somewhat from those quintessential law and order officers: the police. TOs work in pairs, like many police, to support each other. They have a range of legal powers including the power of arrest, and they carry handcuffs, a baton and capsicum spray as a means of helping ensure their safety and effectiveness in circumstances where they might be outnumbered. The tools of their trade are accessibly displayed on heavy leather belts around their waists and their uniforms have similarities with police uniforms. However, in some ways these similarities are problematic, because TOs are not afforded the same respect as police. This situation underlines of the ambiguities negotiated within the ambience of what it is to be a TO, and how it is to conduct oneself in that role. Notwithstanding the TOs’ law and order responsibilities, public perceptions of the role and some of the public’s responses to the officers can position these workers as “plastic cops” (Teague and Leith). The penultimate deterrent of police officers, the stun gun (Taser), is not available to TOs who are expected to control all incidents arising on duty through the fact that they operate in pairs, with capsicum spray available and, as a last resort, are authorised to use their batons in self defence. Furthermore, although TOs are the key security and enforcement staff in the PTA workforce, and are managed separately from related staff roles, they believe that the clarity of this distinction is compromised because of similarities in the look of Revenue Protection Officers (RPOs). RPOs work on the trains to check that passengers have tickets and have paid the correct fares, and obtain names and addresses to issue infringement notices when required. They are not PTA employees, but contracted staff from an outside company. They also work in pairs. Significantly, the RPO uniform is in many respects identical to that of the TO, and this appears to be a deliberate management choice to make the number of TOs seem greater than it is: extending the TO ambience through to the activities of the RPOs. However, in the event of a disturbance, TOs are required and trained to act, while RPOs are instructed not to get involved; even though the RPOs appear to the travelling public to be operating in the role of a law-and-order-keeper, RPOs are specifically instructed not to get involved in breaches of the peace or disruptive passenger behaviour. From the point of view of the travelling public, who observe the RPO waiting for TOs to arrive, it may seems as if a TO is passively standing by while a chaotic situation unravels. As Angus commented: I’ve spoken to quite a few members of public and received complaints from them about transit officers and talking more about the incident have found out that it was actually [RPOs] that are dealing with it. So it’s creating a bad image for us …. It’s Transits that are copping all the flak for it … It is dangerous for us and it’s a lot of bad publicity for us. It’s hard enough, the job that we do and the lack of respect that we do get from people, we don’t need other people adding to it and making it harder. Indeed, it is not only the travelling public who can mistake the two uniforms. Mike tells of an “incident where an officer [TO] has called for backup on a train and the guys have got off [the train at the next station] and just stood there, and he didn’t realise that they are actually [revenue protection] officers, so he effectively had no backup. He thought he did, but he didn’t.” The RPO uniform may confer an ambience of power borrowed from TOs and communicated visually, but the impact is to compromise the authority of the TO role. Unfortunately, what could be a complementary role to the TOs becomes one which, in the minds of the TO workforce, serves to undermine their presence. This effect of this role confusion is to dilute the aura of authority of the TOs. At one end of a power continuum the TO role is minimised by those who see it as a second-rate ‘Wannabe cop’ (Teague and Leith 2008), while its impact is diluted at the other end by an apparently deliberate confusion between the TO broader ‘law and order’ role, and the more limited RPO revenue collection activities. Postlude To the passengers of the PTA in Perth, the presence and actions of transit officers appear as unremarkable as the daily commute. In this ethnographic study of their workplace culture, however, the transit officers have revealed ways in which they influence the ambience of the workplace and the public spaces they inhabit whilst on duty, and how they are influenced by it. While this ambient inter-relationship is not documented in the organisation’s occupational safety and health management system, the TOs are aware that it is a factor in their level at safety at work, both positively and negatively. Clearly, an ethnography study is conducted at a certain point in time and place, and culture is a living and changing expression of human interaction. The Public Transport Authority of Western Australia is committed to continuous improvement in safety and to the investigation of all ways and means in which to support TOs in their daily activities. This is evident not only in their support of the research and their welcoming of the ethnographer into the workforce and onto the tracks, but also in their robust commitment to change as the findings of the research have progressed. In particular, changes in the ambient TO culture and in the training and daily practices of TOs have already resulted from this research or are under active consideration. Nonetheless, this project is a cogent indicator of the fact that a safety culture is critically dependent upon intangible but nonetheless important factors such as the ambience of the workplace and the way in which officers are able to communicate their authority to others. References Everett, James. “Organizational Culture and Ethnoecology in Public Relations Theory and Practice.” Public Relations Research Annual. Vol. 2. Eds. Larissa Grunig and James Grunig. Hillsdale, NJ, 1990. 235-251. 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