Streptomyces Californicus Research Articles

Antimicrobial Activities and Painting Application of Pigmented-Producing Actinobacteria Isolated from Rhizospheric Soils of Mosses (Taxithelium nepalense (Schwägr.) Broth. and Barbula indica (Hook.) Spreng.)

In the survey of biodiversity of actinobacteria associated with mosses (Taxithelium nepalense (Schwägr.) Broth. and Barbula indica (Hook.) Spreng.), certain strains of pigment producing actinobacteria were isolated and purified on SCA and incubated at 30°C for 1 week. Based on deep-shade color of actinobacterial pigments, 4 strains were collected and used for painting color preparation. To evaluate the antimicrobial activities, the crude extracts were prepared from 4 actinobacterial strains and tested with Escherichia coli PSRU-01 and Staphylococcus aureus PSRU-01. The results indicated that the crude extracts of C7, C13, C15 and D13 could not inhibit growth of E. coli PSRU-01, but S. aureus PSRU-01 was inhibited. Two fungal testers, including Colletotrichum sp. PSRU-01 and Fusarium sp. PSRU-01, were completely inhibited by the crude pigment extracts of C13, C15 and D13. Based on phylogenetic results, the actinobacterial strains were closely related to Streptomyces californicus (C7, 100% identity), Streptomyces bungoensis (C13, 99.8% similarity), Streptomyces humi (C15, 99.9% similarity), and Streptomyces rectiverticillatus (D13, 99.8% similarity). They also shared phenotypic characteristics with Streptomyces. The cultivated cells of actinobacteria on broken-milled rice were used for pigment extraction and followed by determination of the extracted pigments for mixing with acrylic color in the shade violet, green, orange and pink colors. Application of actinobacterial pigments in painting is the first report and it is an innovative utilization of actinobacterial pigments in non-scientific field in Thailand.

Isolation, Identification, and Application of Pigment-Producing Actinobacteria from Stingless Bee Hives for Handicraft Production

Actinobacteria are distributed in natural habitats and produce a vast variety of natural pigments that are different in colour shades and applied in textile industry and others. The extracted actinobacterial pigments are used as eco-friendly natural dyes and colour and non-toxic to living organisms and environments compared with chemical or synthetic colours. In order to produce actinobacterial pigments for handicraft making, eight strains of the pigmented producing actinobacteria were isolated from stingless bee (Tetragonilla collina Smith, 1857) hives. Based on deep colour shades presented, 4 actinobacterial strains were chosen to prepare various colour for handicrafts, including yellow, violet, green, and pink. To identify actinobacterial strains selected, phylogenetic identification was carried out. The phylogenetic results indicated that strain C2 phylogenetically shared the 16S rDNA sequence 99.6 % similarity with its closest species, namely Streptomyces cellulosae. Strain C4 had the phylogenetic relationship close to Streptomyces californicus (99.9% similarity of 16S rDNA). Strain E1 was a closest member belonged to Streptomyces chartreusis (99.9% 16S rDNA sequence similarity), and strain E2 was phylogenetically closely related to Streptomyces aureoversilis with 99.2% 16S rDNA sequence similarity. To produce the handicraft colour, we cultivated four actinobacterial strains on broken-milled rice, and actinobacterial pigments were extracted using ethyl acetate. The crude extracts obtained were mixed with white flower clay that was used for handicraft making, and the artificial clay flowers were made. This study is the first to report the use of natural colours obtained from pigment-producing actinobacteria in handicrafts in Thailand and other countries. Future research will explore the application of actinobacterial pigments in various fields, including fine arts, the ceramic industry, and others.

Endophytic actinomycetes from mangrove plant \(\textit{Avicennia marina}\) in Quang Ninh province, Vietnam: distribution, cytotoxicity, and antioxidant activities

Mangrove endophytes have recently gained considerable attention due to their diversity and abundance of novel bioactive secondary metabolites. Despite the fact that Streptomyces species are producers of more than 75% of commercialized antibiotics, Streptomyces associated with the extremely widespread mangrove plant Avicennia marina remain poorly characterized. In this study, nine actinomycetes were isolated from A. marina growing in a mangrove forest, as yet unexplored, of Quang Ninh province, Vietnam. Phylogenetic analysis of actinomycetes-specific 16S rRNA sequences indicated that they were subjected to five Streptomyces species including Streptomyces cacaoi, Streptomyces californicus, Streptomyces enissocaesillis, Streptomyces coelicoflavus, and Streptomyces variabilis, which have not been previously reported in mangrove plants. Among them, S. cacaoi AM1 showed strong inhibition effects against six tested pathogenic bacteria with inhibitory zones ranging from 7.5−22.3 mm. Using standard 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, 100 µg/mL ethyl acetate extract of AM1 showed potent cytotoxicity against breast cancer MCF-7 and lung cancer A549 cell lines with cell viability of 16.5 ± 1.28% and 17.69 ± 2.3%, respectively. As for antioxidant activities, AM1 extract exhibited strong antioxidant activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical (92.4 ± 0.004%) and superoxide anion radical (42.4 ± 0.019%), which were positively correlated to high polyphenol (84.3 ± 3.4 µg GAE/g FW) and flavonoid (34.9 ± 4.8 µg QE/g FW) contents. These findings indicated that S. cacaoi AM1 could be a promising reservoir of antibacterial, anticancer, and antioxidant agents. This is the first report of mangrove endophytic Streptomyces derived from A. marina.

Isolation and Characterization of a New Endophytic Actinobacterium Streptomyces californicus Strain ADR1 as a Promising Source of Anti-Bacterial, Anti-Biofilm and Antioxidant Metabolites.

In view of the fast depleting armamentarium of drugs against significant pathogens, like methicillin-resistant Staphylococcus aureus (MRSA) and others due to rapidly emerging drug-resistance, the discovery and development of new drugs need urgent action. In this endeavor, a new strain of endophytic actinobacterium was isolated from the plant Datura metel, which produced secondary metabolites with potent anti-infective activities. The isolate was identified as Streptomyces californicus strain ADR1 based on 16S rRNA gene sequence analysis. Metabolites produced by the isolate had been investigated for their antibacterial attributes against important pathogens: S. aureus, MRSA, S. epidermis, Enterococcus faecium and E. faecalis. Minimum inhibitory concentration (MIC90) values against these pathogens varied from 0.23 ± 0.01 to 5.68 ± 0.20 μg/mL. The metabolites inhibited biofilm formation by the strains of S. aureus and MRSA (Biofilm inhibitory concentration [BIC90] values: 0.74 ± 0.08–4.92 ± 0.49 μg/mL). The BIC90 values increased in the case of pre-formed biofilms. Additionally, the metabolites possessed good antioxidant properties, with an inhibitory concentration (IC90) value of 217.24 ± 6.77 µg/mL for 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging. An insight into different classes of compounds produced by the strain ADR1 was obtained by chemical profiling and GC-MS analysis, wherein several therapeutic classes, for example, alkaloids, phenolics, terpenes, terpenoids and glycosides, were discovered.

Borrelidin - An Antiangiogenic Compound

Borrelidin is identified from other Streptomyces speciessuch as S. parvulus, S. albovinaceous, S.Griseusand an unidentified Streptomyces species. It is isolated from Streptomyces californicus. Apart from microbial sources, borrelidin also can be synthesized chemically which has similar structure and functions. The present study is an effort in the direction of isolating an antibiotic capable of acting on clinical resistant strains of infectious organisms.

Streptomyces sundarbansensis sp. nov., an actinomycete that produces 2-allyloxyphenol

A novel actinomycete producing 2-allyloxyphenol, designated strain MS1/7(T), was isolated from sediments of the Sundarbans mangrove forest, India. Growth on International Streptomyces Project (ISP) media 2, 3, 4, 5 and 7 produced olive green to grey aerial hyphae that carried smooth-surfaced spores in a flexuous (Rectiflexibiles) arrangement. The strain contained ll-diaminopimelic acid, but no diagnostic sugars in whole-cell hydrolysates. Hexa-, octa- and a minor amount of tetra-hydrogenated menaquinones with nine isoprene units [MK-9 (H(4), H(6), H(8) and H(10))] were present as isoprene analogues. Diagnostic phospholipids were phosphatidylethanolamine and diphosphatidylglycerol. The predominant fatty acids were anteiso-C(15 : 0) (34.80 %), iso-C(16 : 0) (16.45 %), C(16) (10.53 %) and anteiso-C(17 : 0) (10.92 %). The strain showed greater than 99 % 16S rRNA gene sequence similarity to the type strains of several recognized species of the genus Streptomyces, but in the phylogenetic tree based on 16S rRNA gene sequences it formed a distinct phyletic line and demonstrated closest relationships to viomycin-producers (Streptomyces californicus NRRL B-1221(T), Streptomyces floridae MTCC 2534(T) and Streptomyces puniceus NRRL B-2895(T)). However, strain MS1/7(T) could be distinguished from these and other closely related species based on low levels of DNA-DNA relatedness (<44 %) and disparate physiological features, principally amino acid utilization and growth in NaCl. Strain MS1/7(T) is therefore suggested to represent a novel species of the genus Streptomyces, for which the name Streptomyces sundarbansensis sp. nov. is proposed. The type strain is MS1/7(T) ( = MTCC 10621(T) = DSM 42019(T)).

The biocontaminants and complexity of damp indoor spaces: more than what meets the eyes

Nine types of biocontaminants in damp indoor environments from microbial growth are discussed: (1) indicator molds; (2) Gram negative and positive bacteria; (3) microbial particulates; (4) mycotoxins; (5) volatile organic compounds, both microbial (MVOCs) and non-microbial (VOCs); (6) proteins; (7) galactomannans; (8) 1-3-beta-D-glucans (glucans) and (9) lipopolysaccharides (LPS--endotoxins). When mold species exceed those outdoors contamination is deduced. Gram negative bacterial endotoxins, LPS in indoor environments, synergize with mycotoxins. The gram positive Bacillus species, Actinomycetes (Streptomyces, Nocardia and Mycobacterium), produce exotoxins. The Actinomycetes are associated with hypersensitivity pneumonitis, lung and invasive infections. Mycobacterial mycobacterium infections not from M. tuberculosis are increasing in immunocompetent individuals. In animal models, LPS enhance the toxicity of roridin A, satratoxins G and aflatoxin B1 to damage the olfactory epithelium, tract and bulbs (roridin A, satratoxin G) and liver (aflatoxin B1). Aflatoxin B1 and probably trichothecenes are transported along the olfactory tract to the temporal lobe. Co-cultured Streptomyces californicus and Stachybotrys chartarum produce a cytotoxin similar to doxorubicin and actinomycin D (chemotherapeutic agents). Trichothecenes, aflatoxins, gliotoxin and other mycotoxins are found in dust, bulk samples, air and ventilation systems of infested buildings. Macrocyclic trichothecenes are present in airborne particles <2 microm. Trichothecenes and stachylysin are present in the sera of individuals exposed to S. chartarum in contaminated indoor environments. Haemolysins are produced by S. chartarum, Memnoniella echinata and several species of Aspergillus and Penicillium. Galactomannans, glucans and LPS are upper and lower respiratory tract irritants. Gliotoxin, an immunosuppressive mycotoxin, was identified in the lung secretions and sera of cancer patients with aspergillosis produced by A. fumigatus, A. terreus, A. niger and A. flavus.

Effects of co‐culture of amoebae with indoor microbes on their cytotoxic and proinflammatory potential

Free-living amoebae are ubiquitous environmental protozoa found in both natural and man-made environments, including moisture-damaged buildings. Furthermore, the interaction between amoebae and bacteria has been shown to enhance the virulence and pathogenicity of some bacteria. While the inhabitants of moisture damaged buildings are known to be at risk of suffering adverse health effects, the exact causative agents and mechanisms are still obscure. To examine the possible role of amoebae in the health effects associated with moisture damages, the effects of amoebae on the cytotoxicity and proinflammatory potential of nonpathogenic microbes common in moisture-damaged buildings were investigated. First, two bacterial and three fungal strains were cultured both individually and in coculture with Acanthamoeba polyphaga. Then, mouse RAW264.7 macrophages were exposed to the cocultures as well as the individually grown bacteria, fungi, and amoebae. Finally, cell viability and production of proinflammatory mediators, i.e., nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), and interleukin 6 (IL-6), were measured in macrophages after the exposure. The results revealed that cocultivation with amoebae increased the cytotoxicity of the bacterium Streptomyces californicus and the fungus Penicillium spinulosum. Moreover, the macrophages produced up to 10 times higher concentrations of NO after the exposure to these cocultures than after the exposure to individually grown microbes. Finally, the production of the cytokines was up to two orders of magnitude higher (IL-6) and up to four times higher (TNF-alpha) after exposure to the cocultures when compared to individually grown microbes. We conclude that amoebae are able to potentiate the cytotoxicity and proinflammatory properties of certain microbes associated with moisture damages.

DNA damage and p53 in RAW264.7 cells induced by the spores of co-cultivated Streptomyces californicus and Stachybotrys chartarum

Our recent studies have revealed that the co-cultivation of environmental microbes, Streptomyces californicus and Stachybotrys chartarum, potentiates the immunotoxic properties of the spores. In the present study, the spore-induced genotoxic potential of these microbes was investigated. Dose related differences in genotoxic and cytotoxic effects and in p53 level in mouse RAW264.7 macrophages were studied after 24 h exposure to the spores of separately cultivated Streptomyces californicus or Stachybotrys chartarum alone, a simple spore-mixture of these microbes as well as to the spores of co-cultivated microbes. The genotoxic effect of the exposures was determined by the Comet assay and p53 level was analyzed by immunoblotting. Cytotoxicity was assessed by using flow cytometric analysis and also by the MTT test. The results revealed that the spores of co-cultivated microbes evoked DNA damage, p53 accumulation and cytotoxicity at a lower dose than the other exposures, and at the highest dose there was a 2.5-fold increase in DNA damage compared to control. In addition, the spores of Streptomyces californicus alone induced a 1.5-fold increase in DNA damage compared to control, dose dependent p53 accumulation and also extensive cytotoxicity. In contrast, the mixture of separately cultivated spores or the spores of Stachybotrys chartarum alone did not induce DNA damage with any tested dose although they triggered significant cytotoxicity and a slightly increased p53 level. Our results suggest that the detected genotoxic responses are the result of DNA damage in RAW264.7 cells by some genotoxically active metabolite(s) and the production of this compound was stimulated in Streptomyces californicus when it was co-cultivated with Stachybotrys chartarum.

Co-cultivation of Streptomyces californicus and Stachybotrys chartarum stimulates the production of cytostatic compound(s) with immunotoxic properties

We have recently shown that the actinobacterium Streptomyces californicus and the fungus Stachybotrys chartarum originating from moisture damaged buildings possess both immunotoxic and immunostimulatory characteristics, which are synergistically potentiated by microbial interaction. In the search for the causative agent(s) behind the immunotoxicity, the cytostatic effects of the co-cultivated spores of S. californicus and S. chartarum were compared to those caused by widely used cytostatic agents produced by streptomycetes. The RAW264.7 macrophages were exposed to four doses of doxorubicin (DOX), actinomycin D (AMD), mitomycin C (MMC) or phleomycin (PHLEO) for 24 h. Kinetics of the spores of the co-cultivated and the separately cultivated microbes (1x10(6) spores/ml) was compared to DOX (0.15 muM). Apoptotic responses were analyzed by measuring DNA content and mitochondria membrane depolarization with flow cytometer, and by the fluorometric caspase-3 assay. The present data indicate that interactions during co-cultivation of S. californicus and S. chartarum stimulate the production of an unidentified cytostatic compound(s) capable of inducing mitochondria mediated apoptosis and cell cycle arrest at S-G(2)/M. The spores of co-cultivated microbes caused a 4-fold collapse of mitochondrial membrane potential and an almost 6-fold caspase-3 activation and DNA fragmentation when compared to control. Similar responses were induced by DNA cleaving compounds, especially DOX and AMD, at the relatively low concentrations, but not the spores of the same microbes when they were grown separately. These data suggest that when growing in the same habitat, interactions between S. californicus and S. chartarum stimulates the production of an unknown cytostatic compound(s) which evoke immunotoxic effects similar to those by chemotherapeutic drugs.

Effects of amoebae on the growth of microbes isolated from moisture-damaged buildings

Dampness, moisture, and mold in buildings are associated with adverse health outcomes. In addition to fungi and bacteria, amoebae have been found in moisture-damaged building materials. Amoebae and a growing list of bacteria have been shown to have mutual effects on each other's growth, but the interactions between amoebae and microbes common in moisture-damaged buildings have not been reported. We co-cultivated the amoeba Acanthamoeba polyphaga with bacteria and fungi isolated from moisture-damaged buildings in laboratory conditions for up to 28 days. The microbes selected were the bacteria Streptomyces californicus, Bacillus cereus, and Pseudomonas fluorescens, and the fungi Stachybotrys chartarum, Aspergillus versicolor, and Penicillium spinulosum. Fungi and bacteria generally benefited from the presence of the amoebae, whereas the growth of amoebae was hindered by Streptomyces californicus, Stachybotrys chartarum, and Bacillus cereus. Pseudomonas fluorescens slightly enhanced amoebae viability. Amoebae were indifferent to the presence of Aspergillus versicolor and Penicillium spinulosum. Thus, our results show that amoebae can alter the survival and growth of some microbes in moisture-damaged buildings.

Bacterial strains from moldy buildings are highly potent inducers of inflammatory and cytotoxic effects

We aimed to identify inflammatory and cytotoxic potential of individual indoor air bacterial and fungal strains, as well as extracts of indoor air filter samples containing bacteria and fungi. Mouse RAW264.7 macrophages were exposed in vitro to four bacterial strains; Streptomyces californicus, Mycobacterium terrae, Bacillus cereus and Pseudomonas fluorescens, and three fungal strains; Penicillium spinulosum, Aspergillus versicolor and Stachybotrys chartarum. Furthermore, RAW264.7 macrophages were exposed to indoor air filter sample extracts representing 'low' (n = 21) and 'high' (n = 20) exposure to viable fungi or bacteria. Production of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) as well as cell viability were measured after 24 h exposure. The results show that the bacterial strains induce more profound production of NO, TNF-alpha and IL-6 than the studied fungal strains. They also decrease the viability of mouse macrophages. Similarly, the indoor air filter samples with high concentration of bacteria induced a statistically significant increase in TNF-alpha and IL-6 production as well as a decrease in cell viability. Altogether, these results suggest that indoor air bacterial strains are potent inducers of inflammatory responses and thus possibly related to adverse health effects of the inhabitants. There is abundant documentation of the association between building dampness and mold and adverse health effects on occupants, but the causal agents of the effects are still unclear. In order to reveal these causal links, experimental studies with in vitro and in vivo methods are needed. The present findings shed new light on studies of the microbial constituents of indoor air in moldy buildings responsible for adverse health effects. These results imply that bacteria should also be monitored in cases of suspected microbial contamination of indoor air.

The Proportions of Streptomyces californicus and Stachybotrys chartarum in Simultaneous Exposure Affect Inflammatory Responses in Mouse RAW264.7 Macrophages

Adverse health outcomes associated with moisture-damaged buildings originate from an exposure consisting of complex interactions between various microbial species and other indoor pollutants. The concentrations and proportions of microbial components in such environments can vary greatly with the growth conditions. In this study, we aimed to evaluate the effects of simultaneous exposure with modified proportions of actinobacteria Streptomyces californicus and fungi Stachybotrys chartarum on inflammatory responses (cytokines macrophage inflammatory protein 2 [MIP2], interleukin 6 [IL-6] and tumor necrosis factor α [TNFα]; nitric oxide) and cytotoxicity (MTT-test and DNA content analysis) in mouse RAW264.7 macrophage cell line. Five different proportions of microbial spores were studied (Str. californicus: S. chartarum 10:1; 5:1; 1:1; 1:5; 1:10). RAW264.7 cells were coexposed to the total dose of 3 × 105 spores/ml for 24 h and also both of these microbial spores on their own at the respective doses. At least the 1.5-fold synergistic increase in cytokine production of RAW264.7 macrophages was detected when coexposure contained an equal amount or more fungal spores (S. chartarum) than bacterial spores (Str. californicus) compared to the sum response caused by these microbial spores separately. On the contrary, NO production after coexposure was nearly 40% less than the sum response induced by the microbial spores separately, when coexposure contains 5 times more bacterial than fungal spores. In addition, coexposure slightly changed the cytotoxic potency of the spores. The present results revealed that mutual proportions of fungal and bacterial spores in simultaneous exposure affect the nature of their interactions leading to increased or suppressed production of inflammatory mediators in RAW264.7 macrophages.

Interactions between Streptomyces californicus and Stachybotrys chartarum can induce apoptosis and cell cycle arrest in mouse RAW264.7 macrophages

Exposure to complex mixtures of bacteria and fungi in moisture-damaged buildings is a potential cause of inflammatory related symptoms among occupants. The present study assessed interactions between two characteristic moldy house microbes Streptomyces californicus and Stachybotrys chartarum. Differences in cytotoxic and inflammatory responses in mouse (RAW264.7) macrophages were studied after exposure to the spores of co-cultivated microbes, the mixture of separately cultivated spores, and the spores of either of these microbes cultivated alone. The RAW264.7 cells were exposed to six doses (1 × 10 4 to 3 × 10 6 spores/ml) for 24 h, and the time course of the induced responses was evaluated after 4, 8, 16, and 24 h of exposure (1 × 10 6 spores/ml). The cytotoxic potential of the spores was characterized by the MTT test, DNA content analysis, and enzyme assay for caspase-3 activity. The production of cytokines (IL-1β, IL-6, IL-10, TNFα, and MIP2) was measured immunochemically and nitric oxide by the Griess method. Co-cultivation increased the ability of the spores to cause apoptosis by more than 4-fold and the proportion of RAW264.7 cells at the G 2/M stage increased nearly 2-fold when compared to the response induced by the mixture of spores. In contrast, co-cultivation decreased significantly the ability of the spores to trigger the production of NO and IL-6 in RAW264.7 cells. In conclusion, these data suggest that co-culture of S. californicus and S. chartarum can result in microbial interactions that significantly potentiate the ability of the spores to cause apoptosis and cell cycle arrest in mammalian cells.

Synergistic interaction in simultaneous exposure to Streptomyces californicus and Stachybotrys chartarum.

The microbial exposure associated with health complaints in moldy houses consists of a heterogeneous group of components, including both living and dead bacteria, fungi, and their metabolites and active compounds. However, little is known about the interactions between different microbes and their metabolites, although the cytotoxicity and inflammatory potential of certain individual microbes have been reported. In this study, we investigated the inflammatory responses of mouse RAW264.7 macrophages after exposure to six indoor air microbes (Aspergillus versicolor, Penicillium spinulosum, Stachybotrys chartarum, Bacillus cereus, Mycobacterium terrae, and Pseudomonas fluorescens) alone and together with the actinomycete Streptomyces californicus. The production of nitric oxide, levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6), and cytotoxicity were measured. The coexposure to Sta. chartarum and Str. californicus caused a synergistic increase in the production of IL-6 but not other cytokines. In further experiments, the metabolites from Sta. chartarum or from closely related fungi (atranones B and E, satratoxin G, trichodermin, 7-alpha-hydroxytrichodermol, staplabin, and SMTP-7) and the known fungal toxins sterigmatocystin, citrinin, and ochratoxin A were each tested with Str. californicus. The testing revealed a synergistic response in TNF-alpha and IL-6 production after coexposure to Str. californicus with both trichodermin and 7-alpha-hydroxytrichodermol. Finally, the synergistic inflammatory response caused by Str. californicus and trichodermin together was studied by analyzing for the presence of nuclear factor-kappa-B (NF-kappa-B) in nuclear extracts of the exposed cells. The exposure to Str. californicus induced the binding of NF-kappa-B proteins to the NF-kappa-B consensus sequence as well as to the natural NF-kappa-B site of the IL-6 promoter. Adding trichodermin to the exposure did not increase the DNA binding.

Starch in plasterboard sustains Streptomyces californicus growth and bioactivity of spores.

The effects of plasterboard composition on Streptomyces californicus growth and bioactivity of spores were studied. Streptomyces californicus was grown on 13 modified plasterboards under saturated humidity conditions. The total content of fatty acid methyl esters was used for quantifying S. californicus biomass, while the spore-induced cytotoxicity and production of nitric oxide (NO), tumour necrosis factor-alpha, and interleukine-6 (IL-6) in mouse macrophages was used to assess the bioactivity of spores. Removal of starch completely from the plasterboard or only from the core reduced significantly the biomass production and the biological activity of spores in comparison with reference board. The biocide added into the core or on the liner decreased the growth markedly and inhibited the sporulation totally. The biomass production correlated positively with the spore number, cytotoxicity, and production of NO and IL-6. Streptomyces californicus grew under nutrient limitation on all studied plasterboards. The starch is the major factor enabling S. californicus to grow and to produce biologically active metabolites on plasterboard. The composition of building material has an impact on microbial growth and bioactivity of spores which may be involved in complex mechanisms leading to respiratory symptoms in the occupants in moisture damaged buildings.

The relation between growth of four microbes on six different plasterboards and biological activity of spores.

Microbial growth on water-damaged building materials is commonly associated with adverse health effects in the occupants. We examined the growth of Stachybotrys chartarum, Aspergillus versicolor, Penicillium spinulosum, and Streptomyces californicus, isolated from water-damaged buildings, on six different brands of plasterboards. The microbial growth was compared with the biological activity of the spores, that is the potential to induce cytotoxicity and proinflammatory mediators in RAW264.7 macrophages. These results showed that the microbial growth on plasterboard depended on both the microbial strain and the brand of plasterboard used. The biological activity of spores appeared to be regulated by different growth conditions on plasterboards so that good microbial growth was associated with a low bioactivity of the spores, whereas the spores collected from plasterboard supporting only weak growth usually were biologically active. Cytotoxicity of either S. chartarum or A. versicolor did not correlate with any particular growth conditions or induced inflammatory responses. Instead, there were positive correlations between cytotoxicity and levels of induced proinflammatory cytokines for P. spinulosum and S. californicus. These data suggest that both the microbial growth on plasterboard and the resulting bioactivity of spores vary and might be affected by changing the growth conditions provided by the plasterboards.

EFFECT OF LINER AND CORE MATERIALS OF PLASTERBOARD ON MICROBIAL GROWTH, SPORE-INDUCED INFLAMMATORY RESPONSES, AND CYTOTOXICITY IN MACROPHAGES

Microorganisms, when grown on wetted plasterboards, can produce bioactive compounds capable of inducing inflammatory and toxic reactions in mammalian cells. The paper liner of plasterboard is commonly regarded as the major substrate for microbial growth. In this study, we cultured Stachybotrys chartarum, Aspergillus versicolor, Penicillium spinulosum, and Streptomyces californicus on liners and cores of plasterboards in order to examine the role of these main plasterboard components on microbial growth and the resulting bioactivity, which was assessed as the ability of microbial spores to induce inflammatory responses and to evoke cytotoxicity in mouse macrophages. The microbes, isolated from mold problem buildings, were grown under saturated humidity conditions on wetted liners and cores of six different plasterboards. The spores were collected, applied to RAW264.7 macrophages at different doses, and evaluated 24 h after exposure for their ability to evoke cytotoxicity and to stimulate production of nitric oxide (NO), tumor necrosis factor alpha (TNFα), and interleukin-6 (IL-6). In general, microbial growth was better on the cores than on the liners. All of the studied microbes collected from cores induced a dose-dependent production of TNFα in macrophages. The TNFα production stimulated by spores of Stachybotrys, Aspergillus, and Streptomyces paralleled their cytotoxicity. Spores of Streptomyces and Aspergillus collected from liners were among the most potent inducers of NO and IL-6. Good growth of Stachybotrys on cores was associated with high cytotoxicity. Penicillium grew only on cores, but it did not induce major inflammatory mediator productions, nor was it significantly cytotoxic. These results indicate that previously reported microbial growth on plasterboards and spore-induced production of important inflammatory mediators and cell death in macrophages is not only due to the paper liner of plasterboard, but the core material also has a crucial role.

Inflammatory Responses in Mice after Intratracheal Instillation of Spores of Streptomyces californicus Isolated from Indoor Air of a Moldy Building

Microbialgrowth in buildings is associated with respiratory symptoms in the occupants. However, the specific effects of the microbes and the way they provoke clinical manifestations are poorly understood. In the current study, mice were exposed via intratracheal instillation to single doses of the spores of Streptomyces californicus, isolated from indoor air of a moisture-damaged building (2.2 × 107, 1.1 × 108, and 3.3 × 108 spores), or lipopolysaccharide (50 μg). Inflammation and toxicity in lungs were evaluated 24 h later. The time course of the effects was explored with the dose of 1.1 × 108 spores for up to 7 days. The microbial spores elevated proinflammatory cytokine (i.e., TNFα and IL-6) levels in bronchoalveolar lavage fluid (BALF) and in serum in a dose- and time-dependent manner and evoked expression of inducible nitric oxide synthase in BAL cells. Both TNFα and IL-6 responses peaked at 6 h after instillation, but TNFα leveled off more quickly than IL-6. The cytokine surge was followed by inflammatory cell recruitment into airways. Moreover, the spores increased dose- and time-dependently total protein, albumin, hemoglobin, and lactate dehydrogenase concentrations in BALF during the first 24 h. Histopathological examination of lungs confirmed the inflammatory changes. With the exception of macrophage and lymphocyte numbers, all parameters returned to control level at 7 days. In summary, these observations indicate that the spores of S. californicus are capable of provoking an acute inflammation in mouse lungs and can cause cytotoxicity. Thus, S. californicus can be considered as a species with potential to cause adverse health effects in occupants of moisture-damaged buildings.

Induction of Cytotoxicity and Production of Inflammatory Mediators in RAW264.7 Macrophages by Spores Grown on Six Different Plasterboards

Dampness and microbial growth in buildings are associated with respiratory symptoms in the occupants, but details of the phenomenon are not sufficiently understood. The current study examined the effects of growth conditions provided by six plasterboards on cytotoxicity and inflammatory potential of the spores of Streptomyces californicus, Penicillium spinulosum, Aspergillus versicolor, and Stachybotrys chartarum. The microbes were isolated from mold problem buildings and thereafter grown on six different plasterboards. The spores were harvested, applied to RAW264.7 macrophages (104, 105, 106 spores/106