Cold Atmospheric Pressure Microplasma Pipette for Disinfection of Methicillin-Resistant Staphylococcus aureus

Microbial infections should be controlled and prevented for successful wound healing and tissue regeneration. Various disinfection methods exist that use antibiotics, ultraviolet (UV), heat, radiation, or chemical disinfectants; however, cold atmospheric pressure plasma has exhibited a unique and effective antibacterial ability that is not affected by antibiotic resistance or pain. This study develops a cold atmospheric pressure microplasma pipette (CAPMP) that outputs an Ar plasma plume through a tube with an inner radius of 180 μm for disinfection in a small area. The CAPMP was evaluated using Staphylococcus aureus and methicillin-resistant Staphylococcus aureus diluted in liquid media, spread on solid agar, or covered by dressing gauze. An increase in the treatment time of CAPMP resulted in a decrease in the number of colonies of the grown microorganism (colony forming unit) and an increase in the disinfected area for both bacteria. The disinfection ability of CAPMP was observed when the bacteria were covered with dressing gauze and was dependent on the number of gauze layers.

Precursor-Directed Biosynthesis Mediated Amplification of Minor Aza Phenylpropanoid Piperazines in an Australian Marine Fish-Gut-Derived Fungus, Chrysosporium sp. CMB-F214

Chemical analysis of an M1 agar plate cultivation of a marine fish-gut-derived fungus, Chrysosporium sp. CMB-F214, revealed the known chrysosporazines A–D (11–14) in addition to a suite of very minor aza analogues 1–6. A microbioreactor (MATRIX) cultivation profiling analysis failed to deliver cultivation conditions that significantly improved the yields of 1–6; however, it did reveal that M2 agar cultivation produced the new natural product 15. A precursor-directed biosynthesis strategy adopting supplementation of a CMB-F214 M1 solid agar culture with sodium nicotinate enhanced production of otherwise inaccessible azachrysposorazines A1 (1), A2 (2), B1 (3), C1 (4), C2 (5) and D1 (6), in addition to four new chrysosporazines; chrysosporazines N–P (7–9) and spirochrysosporazine A (10). Structures inclusive of absolute configurations were assigned to 1–15 based on detailed spectroscopic and chemical analyses, and biosynthetic considerations. Non-cytotoxic to human carcinoma cells, azachrysosporazies 1–5 were capable of reversing doxorubicin resistance in P-glycoprotein (P-gp)-overexpressing human colon carcinoma cells (SW620 Ad300), with optimum activity exhibited by the C-2′ substituted analogues 3–5.

Gliding motility of a uranium tolerant Bacteroidetes bacterium Chryseobacterium sp. strain PMSZPI: Insights into the architecture of spreading colonies

Uranium tolerant soil bacterium Chryseobacterium sp. strain PMSZPI moved over solid agar surfaces by gliding motility thereby forming spreading colonies which is a hallmark of members of Bacteroidetes phylum. PMSZPI genome harbored orthologs of all the gld and spr genes considered as core bacteroidetes gliding motility genes of which gldK, gldL, gldM, and gldN were co-transcribed. Here, we present the intriguing interplay between gliding motility and cellular organization in PMSZPI spreading colonies. While nutrient deficiency enhanced colony spreading, high agar concentrations and presence of motility inhibitor like 5-hydroxyindole reduced the spreading. A detailed in situ structural analysis of spreading colonies revealed closely packed cells forming multiple layers at center of colony while the edges showed clusters of cells periodically arranged in hexagonal lattices interconnected with each other. The cell migration within the colony was visualized as branched structures wherein the cells were buried within extracellular matrix giving rise to fern like patterns. PMSZPI colonies exhibited strong iridescence that showed correlation with gliding motility. Presence of uranium reduced motility and iridescence and induced biofilm formation. This is a first report of gliding motility and iridescence in a bacterium from uranium enriched environment that could be of significant interest from an ecological perspective.

It Takes Two to Tango: A Bacterial Biofilm Provides Protection against a Fungus-Feeding Bacterial Predator

Fungus-bacterium interactions are widespread, encompass multiple interaction types from mutualism to parasitism, and have been frequent targets for microbial inoculant development. In this study, using in vitro systems combined with confocal laser scanning microscopy and real-time quantitative PCR, we test whether the nitrogen-fixing bacterium Kosakonia radicincitans can provide protection to the plant-beneficial fungus Serendipita indica, which inhabits the rhizosphere and colonizes plants as an endophyte, from the fungus-feeding bacterium Collimonas fungivorans. We show that K. radicincitans can protect fungal hyphae from bacterial feeding on solid agar medium, with probable mechanisms being quick hyphal colonization and biofilm formation. We furthermore find evidence for different feeding modes of K. radicincitans and C. fungivorans, namely “metabolite” and “hyphal feeding”, respectively. Overall, we demonstrate, to our knowledge, the first evidence for a bacterial, biofilm-based protection of fungal hyphae against attack by a fungus-feeding, bacterial predator on solid agar medium. Besides highlighting the importance of tripartite microbial interactions, we discuss implications of our results for the development and application of microbial consortium-based bioprotectants and biostimulants.

Rapid and real-time monitoring of bacterial growth against antibiotics in solid growth medium using a contactless planar microwave resonator sensor

Infection diagnosis and antibiotic susceptibility testing (AST) are pertinent clinical microbiology practices that are in dire need of improvement, due to the inadequacy of current standards in early detection of bacterial response to antibiotics and affordability of contemporarily used methods. This paper presents a novel way to conduct AST which hybridizes disk diffusion AST with microwave resonators for rapid, contactless, and non-invasive sensing and monitoring. In this research, the effect of antibiotic (erythromycin) concentrations on test bacterium, Escherichia coli (E. coli) cultured on solid agar medium (MH agar) are monitored through employing a microwave split-ring resonator. A one-port microwave resonator operating at a 1.76 GHz resonant frequency, featuring a 5 mm2 sensitive sensing region, was designed and optimized to perform this. Upon introducing uninhibited growth of the bacteria, the sensor measured 0.005 dB/hr, with a maximum change of 0.07 dB over the course of 15 hours. The amplitude change decreased to negligible values to signify inhibited growth of the bacteria at higher concentrations of antibiotics, such as a change of 0.005 dB in resonant amplitude variation while using 45 µg of antibiotic. Moreover, this sensor demonstrated decisive results of antibiotic susceptibility in under 6 hours and shows great promise to expand automation to the intricate AST workflow in clinical settings, while providing rapid, sensitive, and non-invasive detection capabilities.

Intravital imaging of Candida albicans identifies differential in vitro and in vivo filamentation phenotypes for transcription factor deletion mutants

Candida albicans is an important cause of human fungal infections. A widely studied virulence trait of C. albicans is its ability to undergo filamentation to hyphae and pseudohyphae. Although yeast, pseudohyphae and hyphae are present in pathological samples of infected mammalian tissue, it has been challenging to characterize the role of regulatory networks and specific genes during in vivo filamentation. In addition, the phenotypic heterogeneity of C. albicans clinical isolates is becoming increasingly recognized and correlating this heterogeneity with pathogenesis remains an important goal. Here, we describe the use of an intravital imaging approach to characterize C. albicans filamentation in a mammalian model of infection by taking advantage of the translucence of mouse pinna (ears). Using this model, we have found that the in vitro and in vivo filamentation phenotypes of different C. albicans isolates can vary significantly, particularly when in vivo filamentation is compared to solid agar-based assays. We also show that the well-characterized transcriptional regulators Efg1 and Brg1 appear to play important roles both in vivo and in vitro. In contrast, Ume6 is much more important in vitro than in vivo. Finally, strains that are dependent on Bcr1 for in vitro filamentation are able to form filaments in vivo. This intravital imaging approach provides a new approach to the systematic characterization of this important virulence trait during mammalian infection. Our initial studies provide support for the notion that the regulation and initiation of C. albicans filamentation in vivo is distinct from in vitro induction.

Assessing the efficacy of antibiotic treatment for the creation of axenic earthworms

Earthworms are an integral part of soil ecosystems, especially for their role in soil functions such as organic matter (OM) decomposition and nutrient cycling. Earthworms and microorganisms are interdependent, and a considerable portion of the contribution earthworms make to influencing OM fate is through interactions with microorganisms. However, the importance of the earthworm-associated microbiome is not fully understood, because it is difficult to separate the direct influence of the earthworms from the indirect influence of their microbiome. Here, we evaluated an antibiotic-based procedure for producing axenic ecologically-contrasting earthworm species (E. fetida, L. terrestris, A. chlorotica) as the first step towards soil studies aimed at understanding the importance of the earthworm microbiome for host health and function. Individual earthworms were exposed to antibiotics: cycloheximide (150 μg ml-1), ampicillin (100 μg ml-1), ciprofloxacin (50 μg ml-1), nalidixic acid (μg ml-1), and gentamicin (μg ml-1) either singly or in a cocktail via culture (96 h) in a semi-solid agar carrier. Compared to the non-antibiotic treated control, the cocktail (for all three species) and ciprofloxacin (for E. fetida and A. chlorotica) treatments significantly reduced (P<0.05) culturable microbial abundance on nutrient agar and potato dextrose agar. The microbial counts were reduced to below detection (<50 CFU individual-1) for E. fetida and A. chlorotica receiving the cocktail. Illumina 16S rDNA amplicon sequence analysis of culturable L. terrestris -associated bacteria showed that antibiotic treatment influenced community composition revealing putative sensitive (Comomonas, Kosakonia and Sphingobacterium) and insensitive (Aeromonas, Pseudochrobactrum) taxa. Overall, we report a rapid, with minimal earthworm- handling, process of creating axenic E. fetida and A. chlorotica individuals or L. terrestris with a suppressed microbiome as a tool to be used in future ecological studies of earthworm microbial interactions affecting host health and function.

Evaluation of the Lignite Biotransformation Capacity of Fusarium sp. NF01 Cultured on Different Growth Substrates

Screening and studying the lignite solubilization/degradation capacities of indigenous microorganisms are key to exploring the in-situ biotransformation of lignite. Here, a fungus was isolated from in-situ lignite samples and identified as Fusarium sp. NF01. This isolate was then cultured on four different carbon sources to evaluate its lignite transformation capacity. When cultured on a solid agar medium containing sodium gluconate or sodium glutamate, Fusarium sp. NF01 completely liquefied 0.5 g of lignite within six days, and when cultured in a liquid medium containing sodium gluconate, the weight of lignite decreased by 28.4% within seven days. Elemental analysis showed that the lignite biodegradation rate was inversely proportional to the C/O ratio of the residual lignite samples. Additionally, a 5.9% biodesulfurization rate was achieved when Fusarium sp. NF01 was cultured in the presence of sodium gluconate. Finally, Fourier-transform infrared analysis of the residual lignite samples revealed relatively weak signal intensities of the signature peaks representing the following: aromatic ring side chains; ether, ester, and alcohol bonds; aromatic ring carbon-carbon double bonds; and aliphatic methyl and methylene. The results showed that Fusarium sp. NF01 can degrade lignite in a carbon-dependent manner and could be thus used for the bioconversion of subsurface coalbeds.

Transcriptome differences between Cupriavidus necator NH9 grown with 3-chlorobenzoate and that grown with benzoate

RNA-seq analysis of Cupriavidus necator NH9, a 3-chlorobenzoate degradative bacterium, cultured with 3-chlorobenzaote and benzoate, revealed strong induction of genes encoding enzymes in degradation pathways of the respective compound, including the genes to convert 3-chlorobenzaote and benzoate to chlorocatechol and catechol, respectively, and the genes of chlorocatechol ortho-cleavage pathway for conversion to central metabolites. The genes encoding transporters, components of the stress response, flagellar proteins, and chemotaxis proteins showed altered expression patterns between 3-chlorobenzoate and benzoate. Gene Ontology enrichment analysis revealed that chemotaxis related terms were significantly upregulated by benzoate compared with 3-chlorobenzoate. Consistent with this, in semi-solid agar plate assays, NH9 cells showed stronger chemotaxis to benzoate than to 3-chlorobenzoate. These results, combined with the absence of genes related to uptake/chemotaxis for 3-chlorobenzoate located closely to the degradation genes of 3-chlorobenzoate, suggested that NH9 has not fully adapted to the utilization of chlorinated benzoate, unlike benzoate, in nature.

#89: Lytic Bacteriophage Against an Emergent Biofilm-Forming Enteroinvasive Escherichia coli

Background Bacteriophage are viruses that infect and parasitize bacteria. In the pre-antibiotic era, phage therapy was one of the few tools available against bacterial infection. After penicillin was discovered, phage therapy fell into disfavor in Western medicine, and its use was mostly limited to medical centers in the Soviet bloc. With current issues on multi-drug resistance, especially of Gram-negative bacteria, there is interest in revisiting phage therapy for use in medical and veterinary practice. This study focuses on the biofilm-forming enteroinvasive Escherichia coli (BF-EIEC) serotype O96:H19. Strain BF-EIEC 52.1 was isolated in a pediatric case–control diarrheal study in Bucaramanga, Colombia, in 2013–2014. This strain is genetically similar to a foodborne outbreak strain isolated in Italy in 2012, and in sporadic outbreaks in the UK, Spain, and Uruguay. BF-EIEC 52.1 produces biofilms in vitro, similarly to enteroaggregative E. coli, and yet invades host cells, similar to enteroinvasive E. coli and Salmonella enterica (J. Iqbal, O. Gómez-Duarte et al., manuscript in preparation), making this a potential emergent pathogen of concern. We hypothesized that lytic bacteriophage derived from wastewater sources are active against BF-EIEC 52.1. Methods Environmental wastewater samples were retrieved from the Amherst, New York, wastewater treatment facility. These were filtered against a 0.2 μm membrane and stored at 4°C. An attempt to isolate phage was done by direct plating over E. coli BF-EIEC 52.1 on solid Luria–Bertani (LB) agar. A second strategy involved enrichment for lytic phage by incubating sample water with BF-EIEC 52.1 in LB broth. Cloning was accomplished by re-filtering the preparation (to remove bacteria), then followed by serial limiting dilution of cell-free filtrate in liquid bacterial culture. Characterization of phage preparations included a demonstration of differential lytic activity against BF-EIEC 52.1 and two unrelated laboratory E. coli strains, DH5α and HB101, in both solid agar and liquid medium. Results Isolation of phage by direct plating was possible, but cloning phages proved difficult. With the alternative strategy, four of six wastewater specimens incubated with BF-EIEC 52.1 in liquid LB showed decreased turbidity compared with sterile water control. All positive specimens proved to have demonstrable lytic activity against host E. coli by plaque formation in solid medium, and in limiting dilution in liquid medium. Thirty-two putative phage preparations were cloned by repeated limiting dilution from an initial 16 independent wells. Of these, 27 preparations showed visible plaques against BF-EIEC 52.1 in solid agar, while 29 and 23 preparations showed plaques against HB101 and DH5α, respectively. All phage preparations inhibited the growth of low-inoculum BF-EIEC 52.1 in liquid medium for &gt;6 hours. Six virus preparations inhibited growth for &gt;72 hours. Conclusions Bacteriophage active against strain BF-EIEC 52.1 were isolated from urban wastewater samples, showing a range of activity against the parental host, as well as unrelated E. coli strains. Further study is needed to demonstrate activity in alternative clinical settings, such as in biofilm-associated infections or in animal models.