De novo engineering of a bacterial lifestyle program.

Synthetic biology has shown remarkable potential to program living microorganisms for applications. However, a notable discrepancy exists between the current engineering practice-which focuses predominantly on planktonic cells-and the ubiquitous observation of microbes in nature that constantly alternate their lifestyles on environmental variations. Here we present the de novo construction of a synthetic genetic program that regulates bacterial life cycle and enables phase-specific gene expression. The program is orthogonal, harnessing an engineered protein from 45 candidates as the biofilm matrix building block. It is also highly controllable, allowing directed biofilm assembly and decomposition as well as responsive autonomous planktonic-biofilm phase transition. Coupling to synthesis modules, it is further programmable for various functional realizations that conjugate phase-specific biomolecular production with lifestyle alteration. This work establishes a versatile platform for microbial engineering across physiological regimes, thereby shedding light on a promising path for gene circuit applications in complex contexts.

Search for a Shared Genetic or Biochemical Basis for Biofilm Tolerance to Antibiotics across Bacterial Species.

Is there a universal genetically programmed defense providing tolerance to antibiotics when bacteria grow as biofilms? A comparison between biofilms of three different bacterial species by transcriptomic and metabolomic approaches uncovered no evidence of one. Single-species biofilms of three bacterial species (Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii) were grown in vitro for 3 days and then challenged with respective antibiotics (ciprofloxacin, daptomycin, and tigecycline) for an additional 24 h. All three microorganisms displayed reduced susceptibility in biofilms compared to planktonic cultures. Global transcriptomic profiling of gene expression comparing biofilm to planktonic and antibiotic-treated biofilm to untreated biofilm was performed. Extracellular metabolites were measured to characterize the utilization of carbon sources between biofilms, treated biofilms, and planktonic cells. While all three bacteria exhibited a species-specific signature of stationary phase, no conserved gene, gene set, or common functional pathway could be identified that changed consistently across the three microorganisms. Across the three species, glucose consumption was increased in biofilms compared to planktonic cells, and alanine and aspartic acid utilization were decreased in biofilms compared to planktonic cells. The reasons for these changes were not readily apparent in the transcriptomes. No common shift in the utilization pattern of carbon sources was discerned when comparing untreated to antibiotic-exposed biofilms. Overall, our measurements do not support the existence of a common genetic or biochemical basis for biofilm tolerance against antibiotics. Rather, there are likely myriad genes, proteins, and metabolic pathways that influence the physiological state of individual microorganisms in biofilms and contribute to antibiotic tolerance.

Level of preparedness of the residential building industry in Australia to climate change adaptation: a case of residential building companies in Brisbane, Queensland.

The consequences of climate change are profound for the residential building industry and, unless appropriate adaptation strategies are implemented, will increase exponentially. The consequences of climate change, such as increased repair costs, can be reduced if buildings are designed and built to be adaptive to climate change risks. This research investigates the preparedness of the Australian residential building sector to adapt to such risks, with a view to informing the next review of the National Construction Code (2022), which at present does not include provisions for climate change adaptation. Twelve semi-structured interviews were conducted with construction managers from residential building companies in Brisbane, Queensland to understand their level of preparedness to adapt with climate change risks. Three aspects of preparedness were investigated: participant's awareness of climate change risks, their company's capacity to include climate change information in planning, and actions taken to address climate change risks. Participants were also asked about climate change adaptation policies and what they thought the path towards increased preparedness in the residential construction industry to climate change risks might involve. Qualitative analysis of interview data was undertaken using NVivo software, and illustrative examples and direct quotes from this data are included in the results. The results indicate a low level of preparedness of the residential building industry to adapt with climate risks. Levels of awareness of managing the consequences of climate change risks, analytical capacity, and the actions taken to address climate change were all found to be low. Legislating climate adaptation practices and increasing the adaptation awareness of the residential constructors are some of the recommendations to enhance the preparedness of the residential construction industry in Australia to adapt with climate change risks.

Probing the Role of bba30, a Highly Conserved Gene of the Lyme Disease Spirochete, Throughout the Mouse-Tick Infectious Cycle.

Borrelia burgdorferi, the causative agent of Lyme disease, has a complex and segmented genome consisting of a small linear chromosome and up to 21 linear and circular plasmids. Some of these plasmids are essential as they carry genes that are critical during the life cycle of the Lyme disease spirochete. Among these is a highly conserved linear plasmid, lp54, which is crucial for the mouse-tick infectious cycle of B. burgdorferi. However, the functions of most lp54-encoded open reading frames (ORFs) remain unknown. In this study, we investigate the contribution of a previously uncharacterized lp54 gene during the infectious cycle of B. burgdorferi. This gene, bba30, is conserved in the Borrelia genus but lacks any identified homologs outside the genus. Homology modeling of BBA30 ORF indicated the presence of a nucleic acid binding motif, helix-turn-helix (HTH), near the amino terminus of the protein, suggesting a putative regulatory function. A previous study reported that spirochetes with a transposon insertion in bba30 exhibited a noninfectious phenotype in mice. In the current study, however, we demonstrate that the highly conserved bba30 gene is not required by the Lyme disease spirochete at any stage of the experimental mouse-tick infectious cycle. We conclude that the undefined circumstances under which bba30 potentially confers a fitness advantage in the natural life cycle of B. burgdorferi are not factors of the experimental infectious cycle that we employ.

Bisphosphonate use in children with cerebral palsy.

BACKGROUND: Cerebral palsy (CP) is a heterogeneous group of non-progressive disorders of posture or movement, caused by a lesion of the developing brain. Osteoporosis is common in children with cerebral palsy, particularly in children with reduced gross motor function, and leads to an increased risk of fractures. Gross motor function in children with CP can be categorised using a tool called the Gross Motor Function Classification System (GMFCS). Bisphosphonate increases bone mineral density (BMD) and reduces fracture rates. Bisphosphonate is used widely in the treatment of adult osteoporosis. However, the use of bisphosphonate in children with CP remains controversial, due to a paucity of evidence and a lack of recent trials examining the efficacy and safety of bisphosphonate use in this population. OBJECTIVES: To examine the efficacy and safety of bisphosphonate therapy in the treatment of low BMD or secondary osteoporosis (or both) in children with cerebral palsy (GMFCS Levels III to V) who are under 18 years of age. SEARCH METHODS: In September 2020, we searched CENTRAL, MEDLINE, Embase, six other databases, and two trial registers for relevant studies. We also searched the reference lists of relevant systematic reviews, trials, and case studies identified by the search, and contacted the authors of relevant studies in an attempt to identify unpublished literature. SELECTION CRITERIA: All relevant randomised controlled trials (RCTs), and quasi-RCTs, comparing at least one bisphosphonate (given at any dose, orally or intravenously) with placebo or no drug, for the treatment of low BMD or osteoporosis in children up to 18 years old, with cerebral palsy (GMFCS Levels III to V). DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by Cochrane. We were unable to conduct any meta-analyses due to insufficient data, and therefore provide a narrative assessment of the results. MAIN RESULTS: We found two relevant RCTs (34 participants). Both studies included participants with non-ambulatory CP or CP and osteoporosis. Participants in both studies were similar in severity of CP, age distribution, and sex distribution. The two trials used different bisphosphonate medications and different intervention durations, but further comparison of the interventions was not possible due to a lack of published data from one trial. One trial received funding and support from research, academic, and hospital foundations, with pharmaceutical companies providing components of the calcium and vitamin supplement; the other trial did not report sources of funding. We judged one study at an overall high risk of bias; the other as overall unclear risk of bias. PRIMARY OUTCOME: Compared to placebo or no treatment, both studies provided very low certainty evidence of improved BMD at least four months post-intervention in children treated with bisphosphonate. Only one study (12 participants) provided sufficient detail to assess a measure of the effect, and reported an improvement at six months post-intervention in lumbar spine z-score (mean difference (MD) 18%, 95% confidence interval (CI) 6.57 to 29.43; very low certainty evidence). SECONDARY OUTCOMES: Very low certainty evidence from one study found that bisphosphonate reduced serum N-telopeptides (NTX) more than placebo; the other study reported that both bisphosphonate plus alfacalcidol and alfacalcidol alone reduced NTX, but did not compare groups. One study reported inconclusive results between groups for serum bone-specific alkaline phosphatase (BAP). The other study reported that both bisphosphonate plus alfacalcidol and alfacalcidol alone reduced BAP, but did not compare groups. Neither study reported data for the effect of bisphosphonate treatment on changes in volumetric BMD in the distal radius or tibia, changes in fracture frequency, bone pain, or quality of life. One study reported that two participants had febrile events noted during their first dosing schedule, but no further adverse events were reported in either relevant study. AUTHORS' CONCLUSIONS: Based on the available evidence, there is very low certainty evidence that bisphosphonate treatment may improve bone health in children with cerebral palsy. We could only include one study with 14 participants in the assessment of the effect size; therefore, the precision of the effect estimate is low. We could only evaluate one planned primary outcome, as there was insufficient detail reported in the relevant studies. Further research from RCTs on the effect and safety of bisphosphonate to improve bone health in children with cerebral palsy is required. These studies should clarify the optimal standard treatment regarding weight-bearing exercises, vitamin D and calcium supplementation, and should include fracture frequency as a primary outcome.

The Lyme disease spirochete's BpuR DNA/RNA-binding protein is differentially expressed during the mammal-tick infectious cycle, which affects translation of the SodA superoxide dismutase.

When the Lyme disease spirochete, Borrelia burgdorferi, transfers from a feeding tick into a human or other vertebrate host, the bacterium produces vertebrate-specific proteins and represses factors needed for arthropod colonization. Previous studies determined that the B. burgdorferi BpuR protein binds to its own mRNA and autoregulates its translation, and also serves as co-repressor of erp transcription. Here, we demonstrate that B. burgdorferi controls transcription of bpuR, expressing high levels of bpuR during tick colonization but significantly less during mammalian infection. The master regulator of chromosomal replication, DnaA, was found to bind specifically to a DNA sequence that overlaps the bpuR promoter. Cultured B. burgdorferi that were genetically manipulated to produce elevated levels of BpuR exhibited altered levels of several proteins, although BpuR did not impact mRNA levels. Among these was the SodA superoxide dismutase, which is essential for mammalian infection. BpuR bound to sodA mRNA in live B. burgdorferi, and a specific BpuR-binding site was mapped 5' of the sodA open reading frame. Recognition of posttranscriptional regulation of protein levels by BpuR adds another layer to our understanding of the B. burgdorferi regulome, and provides further evidence that bacterial protein levels do not always correlate directly with mRNA levels.

Microbial growth rates and local external mass transfer coefficients in a porous bed biofilm system measured by 19 F magnetic resonance imaging of structure, oxygen concentration, and flow velocity.

19 F nuclear magnetic resonance (NMR) oximetry and 1 H NMR velocimetry were used to noninvasively map oxygen concentrations and hydrodynamics in space and time in a model packed bed biofilm system in the presence and absence of flow. The development of a local oxygen sink associated with a single gel bead inoculated with respiring Escherichia coli was analyzed with a phenomenological model to determine the specific growth rate of the bacteria in situ, returning a value (0.66 hr-1 ) that was close to that measured independently in planktonic culture (0.62 hr-1 ). The decay of oxygen concentration in and around the microbiologically active bead was delayed and slower in experiments conducted under continuous flow in comparison to no-flow experiments. Concentration boundary layer thicknesses were determined and Sherwood numbers calculated to quantify external mass transfer resistance. Boundary layers were thicker in no-flow experiments compared to experiments with flow. Whereas the oxygen concentration profile across a reactive biofilm particle was symmetric in no-flow experiments, it was asymmetric with respect to flow direction in flow experiments with Sherwood numbers on the leading edge (Sh = 7) being larger than the trailing edge (Sh = 3.5). The magnitude of the experimental Sh was comparable to values predicted by a variety of correlations. These spatially resolved measurements of oxygen distribution in a geometrically complex model reveal in innovative detail the local coupling between microbial growth, oxygen consumption, and external mass transfer.

The zone model: A conceptual model for understanding the microenvironment of chronic wound infection.

In 2008, two articles in Wound Repair and Regeneration changed the clinical perspective on chronic wounds. They stated that chronic wounds that do not heal contain bacterial biofilms and that these biofilms may be one of the reasons for the nonhealing properties of the wounds. However, we still do not understand the exact role biofilms play in the halted healing process, and we are not able to successfully treat them. The reason for this could be that in vivo biofilms differ substantially from in vitro biofilms, and that most of the knowledge about biofilms originates from in vitro research. In this article, we introduce the zone model as a concept for understanding bacterial behavior and the impact of the microenvironment on both the host and the bacteria. Until now, identification of bacteria, gene expression, and postscript regulation have been looking at a bulk of bacteria and averaging the behavior of all the bacteria. As the zone model dictates that every single bacterium reacts to its own microenvironment, the model may facilitate the planning of future research with improved clinical relevance. The zone model integrates physiology and biology from single cells, microbial aggregates, local host response, surrounding tissue, and the systemic context of the whole host. Understanding the mechanisms behind the actions and reactions by a single bacterium when interacting with other neighboring bacteria cells, other microorganisms, and the host will help us overcome the detrimental effects of bacteria in chronic wounds. Furthermore, we propose use of the terminology "bacterial phenotype" when describing the actions and reactions of bacteria, and the term "biofilms" to describe the morphology of the bacterial community.

Conceptual Model of Biofilm Antibiotic Tolerance That Integrates Phenomena of Diffusion, Metabolism, Gene Expression, and Physiology.

Transcriptomic, metabolomic, physiological, and computational modeling approaches were integrated to gain insight into the mechanisms of antibiotic tolerance in an in vitro biofilm system. Pseudomonas aeruginosa biofilms were grown in drip flow reactors on a medium composed to mimic the exudate from a chronic wound. After 4 days, the biofilm was 114 µm thick with 9.45 log10 CFU cm-2 These biofilms exhibited tolerance, relative to exponential-phase planktonic cells, to subsequent treatment with ciprofloxacin. The specific growth rate of the biofilm was estimated via elemental balances to be approximately 0.37 h-1 and with a reaction-diffusion model to be 0.32 h-1, or one-third of the maximum specific growth rate for planktonic cells. Global analysis of gene expression indicated lower transcription of ribosomal genes and genes for other anabolic functions in biofilms than in exponential-phase planktonic cells and revealed the induction of multiple stress responses in biofilm cells, including those associated with growth arrest, zinc limitation, hypoxia, and acyl-homoserine lactone quorum sensing. Metabolic pathways for phenazine biosynthesis and denitrification were transcriptionally activated in biofilms. A customized reaction-diffusion model predicted that steep oxygen concentration gradients will form when these biofilms are thicker than about 40 µm. Mutant strains that were deficient in Psl polysaccharide synthesis, the stringent response, the stationary-phase response, and the membrane stress response exhibited increased ciprofloxacin susceptibility when cultured in biofilms. These results support a sequence of phenomena leading to biofilm antibiotic tolerance, involving oxygen limitation, electron acceptor starvation and growth arrest, induction of associated stress responses, and differentiation into protected cell states.IMPORTANCE Bacteria in biofilms are protected from killing by antibiotics, and this reduced susceptibility contributes to the persistence of infections such as those in the cystic fibrosis lung and chronic wounds. A generalized conceptual model of biofilm antimicrobial tolerance with the following mechanistic steps is proposed: (i) establishment of concentration gradients in metabolic substrates and products; (ii) active biological responses to these changes in the local chemical microenvironment; (iii) entry of biofilm cells into a spectrum of states involving alternative metabolisms, stress responses, slow growth, cessation of growth, or dormancy (all prior to antibiotic treatment); (iv) adaptive responses to antibiotic exposure; and (v) reduced susceptibility of microbial cells to antimicrobial challenges in some of the physiological states accessed through these changes.

Direct Microscopic Observation of Human Neutrophil-Staphylococcus aureus Interaction In Vitro Suggests a Potential Mechanism for Initiation of Biofilm Infection on an Implanted Medical Device.

The ability of human neutrophils to clear newly attached Staphylococcus aureus bacteria from a serum-coated glass surface was examined in vitro using time-lapse confocal scanning laser microscopy. Quantitative image analysis was used to measure the temporal change in bacterial biomass, neutrophil motility, and fraction of the surface area policed by neutrophils. In control experiments in which the surface was inoculated with bacteria but no neutrophils were added, prolific bacterial growth was observed. Neutrophils were able to control bacterial growth but only consistently when the neutrophil/bacterium number ratio exceeded approximately 1. When preattached bacteria were given a head start and allowed to grow for 3 h prior to neutrophil addition, neutrophils were unable to maintain control of the nascent biofilm. In these head-start experiments, aggregates of bacterial biofilm with areas of 50 µm2 or larger formed, and the growth of such aggregates continued even when multiple neutrophils attacked a cluster. These results suggest a model for the initiation of a biofilm infection in which a delay in neutrophil recruitment to an abiotic surface allows surface-attached bacteria time to grow and form aggregates that become protected from neutrophil clearance. Results from a computational model of the neutrophil-biofilm surface contest supported this conceptual model and highlighted the stochastic nature of the interaction. Additionally, we observed that both neutrophil motility and clearance of bacteria were impaired when oxygen tension was reduced to 0% or 2% O2.

A widely conserved bacterial cytoskeletal component influences unique helical shape and motility of the spirochete Leptospira biflexa.

Leptospires and other members of the evolutionarily ancient phylum of Spirochaetes are bacteria often characterized by long, highly motile spiral- or wave-shaped cells. Morphology and motility are critical factors in spirochete physiology, contributing to the ability of these bacteria to successfully colonize diverse environments. However, the mechanisms conferring the helical structure of Leptospira spp. have yet to be fully elucidated. We have identified five Leptospira biflexa bactofilin proteins, a recently characterized protein family with cytoskeletal properties. These five bactofilins are conserved in all species of the Leptospiraceae, indicating that these proteins arose early in the evolution of this family. One member of this protein family, LbbD, confers the optimal pitch distance in the helical structure of L. biflexa. Mutants lacking lbbD display a unique compressed helical morphology, a reduced motility and a decreased ability to tolerate cell wall stressors. The change in the helical spacing, combined with the motility and cell wall integrity defects, showcases the intimate relationship and coevolution between shape and motility in these spirochetes.

Measuring Antimicrobial Efficacy against Biofilms: a Meta-analysis.

Through a statistical meta-analysis of published data on antimicrobial efficacy against biofilms formed by two common bacterial species, it was concluded that the particular experimental method used is the most important factor determining the outcome of the test. An expected dose-response relationship (greater killing with higher doses or longer treatment times) was observed for data sets derived from a single method but was not observed when data from multiple studies using diverse methods were pooled. Method-specific properties such as the surface area/volume ratio, areal biofilm cell density, and microbial species were shown to influence quantitative measurements of biofilm killing. A better appreciation of the method characteristics that affect antibiofilm efficacy tests could aid decision-making related to investment in research and development and regulatory approvals for biofilm control strategies. The following recommendations are offered to those working in research and development related to biofilm control: (i) report the log reduction, surface area/volume ratio, and biofilm areal cell density; (ii) include data for a benchmark agent, making sure that this agent performs competitively at the dose tested; (iii) measure the dose-response relationship, i.e., make measurements at multiple treatment concentrations or dose durations; and (iv) use a standardized method in addition to research methods.

Nisin penetration and efficacy against Staphylococcus aureus biofilms under continuous-flow conditions.

Biofilms may enhance the tolerance of bacterial pathogens to disinfectants, biocides and other stressors by restricting the penetration of antimicrobials into the matrix-enclosed cell aggregates, which contributes to the recalcitrance of biofilm-associated infections. In this work, we performed real-time monitoring of the penetration of nisin into the interior of Staphylococcus aureus biofilms under continuous flow and compared the efficacy of this lantibiotic against planktonic and sessile cells of S. aureus. Biofilms were grown in Center for Disease Control (CDC) reactors and the spatial and temporal effects of nisin action on S. aureus cells were monitored by real-time confocal microscopy. Under continuous flow, nisin caused loss of membrane integrity of sessile cells and reached the bottom of the biofilms within ~20 min of exposure. Viability analysis using propidium iodide staining indicated that nisin was bactericidal against S. aureus biofilm cells. Time-kill assays showed that S. aureus viability reduced 6.71 and 1.64 log c.f.u. ml-1 for homogenized planktonic cells in exponential and stationary phase, respectively. For the homogenized and intact S. aureus CDC biofilms, mean viability decreased 1.25 and 0.50 log c.f.u. ml-1, respectively. Our results demonstrate the kinetics of biofilm killing by nisin under continuous-flow conditions, and shows that alterations in the physiology of S. aureus cells contribute to variations in sensitivity to the lantibiotic. The approach developed here could be useful to evaluate the antibiofilm efficacy of other bacteriocins either independently or in combination with other antimicrobials.

Non-invasive imaging of oxygen concentration in a complex in vitro biofilm infection model using 19 F MRI: Persistence of an oxygen sink despite prolonged antibiotic therapy.

PURPOSE: Oxygen availability is a critical determinant of microbial biofilm activity and antibiotic susceptibility. However, measuring oxygen gradients in these systems remains difficult, with the standard microelectrode approach being both invasive and limited to single-point measurement. The goal of the study was to develop a 19 F MRI approach for 2D oxygen mapping in biofilm systems and to visualize oxygen consumption behavior in real time during antibiotic therapy. METHODS: Oxygen-sensing beads were created by encapsulating an emulsion of oxygen-sensing fluorocarbon into alginate gel. Escherichia coli biofilms were grown in and on the alginate matrix, which was contained inside a packed bed column subjected to nutrient flow, mimicking the complex porous structure of human wound tissue, and subjected to antibiotic challenge. RESULTS: The linear relationship between 19 F spin-lattice relaxation rate R1 and local oxygen concentration permitted noninvasive spatial mapping of oxygen distribution in real time over the course of biofilm growth and subsequent antibiotic challenge. This technique was used to visualize persistence of microbial oxygen respiration during continuous gentamicin administration, providing a time series of complete spatial maps detailing the continued bacterial utilization of oxygen during prolonged chemotherapy in an in vitro biofilm model with complex spatial structure. CONCLUSIONS: Antibiotic exposure temporarily causes oxygen consumption to enter a pseudosteady state wherein oxygen distribution becomes fixed; oxygen sink expansion resumes quickly after antibiotic clearance. This technique may provide valuable information for future investigations of biofilms by permitting the study of complex geometries (typical of in vivo biofilms) and facilitating noninvasive oxygen measurement.

Physiologic and Genetic Factors Influencing the Zoonotic Cycle of Borrelia burgdorferi.

Borrelia burgdorferi is a symbiont of ticks of the Ixodes ricinus complex. These ticks serve as vectors to disseminate the spirochete to a variety of susceptible vertebrate hosts, which, in turn, act as reservoirs for naïve ticks to become infected, perpetuating the infectious life cycle of B. burgdorferi. The pivotal role of ticks in this life cycle and tick-spirochete interactions are the focus of this chapter. Here, we describe the challenging physiological environment that spirochetes encounter within Ixodes ticks, and the genetic factors that B. burgdorferi uses to successfully infect, persist, and be transmitted from the vector.

Borrelia burgdorferi SpoVG DNA- and RNA-Binding Protein Modulates the Physiology of the Lyme Disease Spirochete.

The SpoVG protein of Borrelia burgdorferi, the Lyme disease spirochete, binds to specific sites of DNA and RNA. The bacterium regulates transcription of spoVG during the natural tick-mammal infectious cycle and in response to some changes in culture conditions. Bacterial levels of spoVG mRNA and SpoVG protein did not necessarily correlate, suggesting that posttranscriptional mechanisms also control protein levels. Consistent with this, SpoVG binds to its own mRNA, adjacent to the ribosome-binding site. SpoVG also binds to two DNA sites in the glpFKD operon and to two RNA sites in glpFKD mRNA; that operon encodes genes necessary for glycerol catabolism and is important for colonization in ticks. In addition, spirochetes engineered to dysregulate spoVG exhibited physiological alterations.IMPORTANCEB. burgdorferi persists in nature by cycling between ticks and vertebrates. Little is known about how the bacterium senses and adapts to each niche of the cycle. The present studies indicate that B. burgdorferi controls production of SpoVG and that this protein binds to specific sites of DNA and RNA in the genome and transcriptome, respectively. Altered expression of spoVG exerts effects on bacterial replication and other aspects of the spirochete's physiology.

Analysis of Clostridium difficile biofilms: imaging and antimicrobial treatment.

BACKGROUND: Clostridium difficile, a spore-forming Gram-positive anaerobic bacillus, is the most common causative agent of healthcare-associated diarrhoea. Formation of biofilms may protect C. difficile against antibiotics, potentially leading to treatment failure. Furthermore, bacterial spores or vegetative cells may linger in biofilms in the gut causing C. difficile infection recurrence. OBJECTIVES: In this study, we evaluated and compared the efficacy of four antibiotics (fidaxomicin, surotomycin, vancomycin and metronidazole) in penetrating C. difficile biofilms and killing vegetative cells. METHODS: C. difficile biofilms grown initially for 48 or 72 h using the colony biofilm model were then treated with antibiotics at a concentration of 25 × MIC for 24 h. Vegetative cells and spores were enumerated. The effect of treatment on biofilm structure was studied by scanning electron microscopy (SEM). The ability of fidaxomicin and surotomycin to penetrate biofilms was studied using fluorescently tagged antibiotics. RESULTS: Both surotomycin and fidaxomicin were significantly more effective than vancomycin or metronidazole (P < 0.001) at killing vegetative cells in established biofilms. Fidaxomicin was more effective than metronidazole at reducing viable spore counts in biofilms (P < 0.05). Fluorescently labelled surotomycin and fidaxomicin penetrated C. difficile biofilms in < 1 h. After 24 h of treatment, SEM demonstrated that both fidaxomicin and surotomycin disrupted the biofilm structure, while metronidazole had no observable effect. CONCLUSIONS: Fidaxomicin is effective in disrupting C. difficile biofilms, killing vegetative cells and decreasing spore counts.

Multi-omic Analyses of Extensively Decayed Pinus contorta Reveal Expression of a Diverse Array of Lignocellulose-Degrading Enzymes.

Fungi play a key role cycling nutrients in forest ecosystems, but the mechanisms remain uncertain. To clarify the enzymatic processes involved in wood decomposition, the metatranscriptomics and metaproteomics of extensively decayed lodgepole pine were examined by RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. Following de novo metatranscriptome assembly, 52,011 contigs were searched for functional domains and homology to database entries. Contigs similar to basidiomycete transcripts dominated, and many of these were most closely related to ligninolytic white rot fungi or cellulolytic brown rot fungi. A diverse array of carbohydrate-active enzymes (CAZymes) representing a total of 132 families or subfamilies were identified. Among these were 672 glycoside hydrolases, including highly expressed cellulases or hemicellulases. The CAZymes also included 162 predicted redox enzymes classified within auxiliary activity (AA) families. Eighteen of these were manganese peroxidases, which are key components of ligninolytic white rot fungi. The expression of other redox enzymes supported the working of hydroquinone reduction cycles capable of generating reactive hydroxyl radicals. These have been implicated as diffusible oxidants responsible for cellulose depolymerization by brown rot fungi. Thus, enzyme diversity and the coexistence of brown and white rot fungi suggest complex interactions of fungal species and degradative strategies during the decay of lodgepole pine.IMPORTANCE The deconstruction of recalcitrant woody substrates is a central component of carbon cycling and forest health. Laboratory investigations have contributed substantially toward understanding the mechanisms employed by model wood decay fungi, but few studies have examined the physiological processes in natural environments. Herein, we identify the functional genes present in field samples of extensively decayed lodgepole pine (Pinus contorta), a major species distributed throughout the North American Rocky Mountains. The classified transcripts and proteins revealed a diverse array of oxidative and hydrolytic enzymes involved in the degradation of lignocellulose. The evidence also strongly supports simultaneous attack by fungal species employing different enzymatic strategies.

Gel-Entrapped Staphylococcus aureus Bacteria as Models of Biofilm Infection Exhibit Growth in Dense Aggregates, Oxygen Limitation, Antibiotic Tolerance, and Heterogeneous Gene Expression.

An experimental model that mimicked the structure and characteristics of in vivo biofilm infections, such as those occurring in the lung or in dermal wounds where no biomaterial surface is present, was developed. In these infections, microbial biofilm forms as cell aggregates interspersed in a layer of mucus or host matrix material. This structure was modeled by filling glass capillary tubes with an agarose gel that had been seeded with Staphylococcus aureus bacteria and then incubating the gel biofilm in medium for up to 30 h. Confocal microscopy showed that the bacteria formed in discrete pockets distributed throughout the gel matrix. These aggregates enlarged over time and also developed a size gradient, with the clusters being larger near the nutrient- and oxygen-supplied interface and smaller at greater depths. Bacteria entrapped in gels for 24 h grew slowly (specific growth rate, 0.06 h(-1)) and were much less susceptible to oxacillin, minocycline, or ciprofloxacin than planktonic cells. Microelectrode measurements showed that the oxygen concentration decreased with depth into the gel biofilm, falling to values less than 3% of air saturation at depths of 500 µm. An anaerobiosis-responsive green fluorescent protein reporter gene for lactate dehydrogenase was induced in the region of the gel where the measured oxygen concentrations were low, confirming biologically relevant hypoxia. These results show that the gel biofilm model captures key features of biofilm infection in mucus or compromised tissue: formation of dense, distinct aggregates, reduced specific growth rates, local hypoxia, and antibiotic tolerance.

Transcriptome and Secretome Analyses of the Wood Decay Fungus Wolfiporia cocos Support Alternative Mechanisms of Lignocellulose Conversion.

UNLABELLED: Certain wood decay basidiomycetes, collectively referred to as brown rot fungi, rapidly depolymerize cellulose while leaving behind the bulk of cell wall lignin as a modified residue. The mechanism(s) employed is unclear, but considerable evidence implicates the involvement of diffusible oxidants generated via Fenton-like chemistry. Toward a better understanding of this process, we have examined the transcriptome and secretome of Wolfiporia cocos when cultivated on media containing glucose, purified crystalline cellulose, aspen (Populus grandidentata), or lodgepole pine (Pinus contorta) as the sole carbon source. Compared to the results obtained with glucose, 30, 183, and 207 genes exhibited 4-fold increases in transcript levels in cellulose, aspen, and lodgepole pine, respectively. Mass spectrometry identified peptides corresponding to 64 glycoside hydrolase (GH) proteins, and of these, 17 corresponded to transcripts upregulated on one or both woody substrates. Most of these genes were broadly categorized as hemicellulases or chitinases. Consistent with an important role for hydroxyl radical in cellulose depolymerization, high transcript levels and upregulation were observed for genes involved in iron homeostasis, iron reduction, and extracellular peroxide generation. These patterns of regulation differ markedly from those of the closely related brown rot fungus Postia placenta and expand the number of enzymes potentially involved in the oxidative depolymerization of cellulose. IMPORTANCE: The decomposition of wood is an essential component of nutrient cycling in forest ecosystems. Few microbes have the capacity to efficiently degrade woody substrates, and the mechanism(s) is poorly understood. Toward a better understanding of these processes, we show that when grown on wood as a sole carbon source the brown rot fungus W. cocos expresses a unique repertoire of genes involved in oxidative and hydrolytic conversions of cell walls.