Development of a fast and reliable method for the assessment of microbial colonization and growth on textiles by DNA quantification

There is a lack of relevant methods to assess the colonization of textiles by skin bacteria because present methods are mainly culture-based procedures. Therefore, the goal of this study was to develop a fast and sensitive culture-independent procedure for the quantification of microbial colonization and growth on textiles. We have established a suitable protocol to use DNA quantification as a reliable method for in vitroand in vivoinvestigations of textiles. For DNA extraction, a two-step procedure comprising treatment of the textile with a solution containing Triton X-100 and lysozyme for 1 h and a successive treatment by SDS and proteinase K for 2 h turned out to be most efficient. DNA extracted from textiles and fabrics was than quantified with the highly sensitive PicoGreen fluorescent dye. In vitrochallenge tests demonstrated a strong correlation between numbers of bacteria on textiles and amount of DNA extracted from textiles. Therefore, this method was used to compare different materials after in vivotrials for assessment of their susceptibility for microbial colonization and growth.     

Microbial Odor Profile of Polyester and Cotton Clothes after a Fitness Session

Clothing textiles protect our human body against external factors. These textiles are not sterile and can harbor high bacterial counts as sweat and bacteria are transmitted from the skin. We investigated the microbial growth and odor development in cotton and synthetic clothing fabrics. T-shirts were collected from 26 healthy individuals after an intensive bicycle spinning session and incubated for 28 h before analysis. A trained odor panel determined significant differences between polyester versus cotton fabrics for the hedonic value, the intensity, and five qualitative odor characteristics. The polyester T-shirts smelled significantly less pleasant and more intense, compared to the cotton T-shirts. A dissimilar bacterial growth was found in cotton versus synthetic clothing textiles. Micrococci were isolated in almost all synthetic shirts and were detected almost solely on synthetic shirts by means of denaturing gradient gel electrophoresis fingerprinting. A selective enrichment of micrococci in an in vitro growth experiment confirmed the presence of these species on polyester. Staphylococci were abundant on both cotton and synthetic fabrics. Corynebacteria were not enriched on any textile type. This research found that the composition of clothing fibers promotes differential growth of textile microbes and, as such, determines possible malodor generation.     

Deficiency of dermcidin-derived antimicrobial peptides in sweat of patients with atopic dermatitis correlates with an impaired innate defense of human skin in vivo

Antimicrobial peptides are an integral part of the epithelial innate defense system. Dermcidin (DCD) is a recently discovered antimicrobial peptide with a broad spectrum of activity. It is constitutively expressed in human eccrine sweat glands and secreted into sweat. Patients with atopic dermatitis (AD) have recurrent bacterial or viral skin infections and pronounced colonization with Staphylococcus aureus. We hypothesized that patients with AD have a reduced amount of DCD peptides in sweat contributing to the compromised constitutive innate skin defense. Therefore, we performed semiquantitative and quantitative analyses of DCD peptides in sweat of AD patients and healthy subjects using surface-enhanced laser desorption ionization time-of-flight mass spectrometry and ELISA. The data indicate that the amount of several DCD-derived peptides in sweat of patients with AD is significantly reduced. Furthermore, compared with atopic patients without previous infectious complications, AD patients with a history of bacterial and viral skin infections were found to have significantly less DCD-1 and DCD-1L in their sweat. To analyze whether the reduced amount of DCD in sweat of AD patients correlates with a decreased innate defense, we determined the antimicrobial activity of sweat in vivo. We showed that in healthy subjects, sweating leads to a reduction of viable bacteria on the skin surface, but this does not occur in patients with AD. These data indicate that reduced expression of DCD in sweat of patients with AD may contribute to the high susceptibility of these patients to skin infections and altered skin colonization.     

Differential attraction of malaria mosquitoes to volatile blends produced by human skin bacteria

The malaria mosquito Anopheles gambiae sensu stricto is mainly guided by human odour components to find its blood host. Skin bacteria play an important role in the production of human body odour and when grown in vitro, skin bacteria produce volatiles that are attractive to A. gambiae. The role of single skin bacterial species in the production of volatiles that mediate the host-seeking behaviour of mosquitoes has remained largely unknown and is the subject of the present study. Headspace samples were taken to identify volatiles that mediate this behaviour. These volatiles could be used as mosquito attractants or repellents. Five commonly occurring species of skin bacteria were tested in an olfactometer for the production of volatiles that attract A. gambiae. Odour blends produced by some bacterial species were more attractive than blends produced by other species. In contrast to odours from the other bacterial species tested, odours produced by Pseudomonas aeruginosa were not attractive to A. gambiae. Headspace analysis of bacterial volatiles in combination with behavioural assays led to the identification of six compounds that elicited a behavioural effect in A. gambiae. Our results provide, to our knowledge, the first evidence for a role of selected bacterial species, common on the human skin, in determining the attractiveness of humans to malaria mosquitoes. This information will be used in the further development of a blend of semiochemicals for the manipulation of mosquito behaviour.     

3-Methyl-3-sulfanylhexan-1-ol as a major descriptor for the human axilla-sweat odour profile

This study sets out to redress the lack of knowledge in the area of volatile sulfur compounds (VSCs) in axillary sweat malodour. Sterile odourless underarm sweat (500 ml) was collected from 30 male volunteers after excessive sweating. Five strains of bacteria, Corynebacterium tuberculostearicum, Corynebacterium minutissimum, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Bacillus licheniformis, were isolated and characterised for their ability to generate an authentic axillary odour from the sweat material collected. As expected, all of the five bacterial strains produced strong sweat odours. Surprisingly, after extensive olfactive evaluation, the strain of Staphylococcus haemolyticus produced the most sulfury sweat character. This strain was then chosen as the change agent for the 500 ml of odourless underarm sweat collected. After bacterial incubation, the 500-ml sample was further processed for GC-olfactometry (GC-O), GC/MS analysis. GC-O of an extract free of organic acids provided three zones of interest. The first was chicken-sulfury, the second zone was onion-like, and the third zone was sweat, clary sage-like. From the third zone, a new impact molecule, (R)- or (S)-3-methyl-3-sulfanylhexan-1-ol, was isolated and identified by GC/MS, MD-GC, and GC AED (atomic emission detector). (S)-3-methyl-3-sulfanylhexan-1-ol was sniff-evaluated upon elution from a chiral GC column and was described as sweat and onion-like; its opposite enantiomer, (R)-3-methyl-3-sulfanylhexan-1-ol, was described as fruity and grapefruit-like. The (S)-form was found to be the major enantiomer (75%).     

Possibilities of DNA Identification of Foreign Sweat and Grease Substance on Human Skin

Contact traces on a human body are a widespread object investigated in cases involving criminal acts against a person. In such cases, interpretation of the results of DNA analysis, as the most evident method, is accompanied by considerable difficulties. This paper shows the possibility of detecting genetic material in the sweat and grease deposits left by the donor on the skin surface of a living recipient. The comparative study of DNA amount in the sweat and grease substance on the surface of the skin of different areas of the body of men and women is carried out. Typing of microsatellite loci of autosomal DNA and Y chromosome in swabs from places of contact of donor males with integuments of recipients of both sexes is carried out. No statistically significant differences in the DNA amount of sweat and grease substance depending on its localization on human body are revealed. The DNA study of sweat and grease substance left by donor men on the skin of both men and women in half of cases reveals allelic combinations inherent in both the donor and the recipient. The results obtained indicate equal chances of detecting the DNA of contacting individuals.     

Exploration of hitherto-uncultured bacteria from the rhizosphere

The rhizosphere environment selects a particular microbial community that arises from the one present in bulk soil due to the release of particular compounds in exudates and different opportunities for microbial colonization. During plant–microorganism coevolution, microbial functions supporting plant health and productivity have developed, of which most are described in cultured plant-associated bacteria. This review discusses the state of the art concerning the ecology of the hitherto-uncultured bacteria of the rhizosphere environment, focusing on Acidobacteria, Verrucomicrobia and Planctomycetes . Furthermore, a strategy is proposed to recover bacterial isolates from these taxa from the rhizosphere environment.     

Microbial community dynamics and effect of environmental microbial reservoirs on red-backed salamanders (Plethodon cinereus)

Beneficial cutaneous bacteria on amphibians can protect against the lethal disease chytridiomycosis, which has devastated many amphibian species and is caused by the fungus Batrachochytrium dendrobatidis. We describe the diversity of bacteria on red-backed salamanders (Plethodon cinereus) in the wild and the stability of these communities through time in captivity using culture-independent Illumina 16S rRNA gene sequencing. After field sampling, salamanders were housed with soil from the field or sterile media. The captive conditions led to different trajectories of bacterial communities. Eight OTUs present on >90% of salamanders in the field, through time, and in both treatments were defined as the core community, suggesting that some bacteria are closely associated with the host and are independent of an environmental reservoir. One of these taxa, a Pseudomonas sp., was previously cultured from amphibians and found to be antifungal. As all host-associated bacteria were found in the soil reservoir, environmental microbes strongly influence host–microbial diversity and likely regulate the core community. Using PICRUSt, an exploratory bioinformatics tool to predict gene functions, we found that core skin bacteria provided similar gene functions to the entire community. We suggest that future experiments focus on testing whether core bacteria on salamander skin contribute to the observed resistance to chytridiomycosis in this species even under hygenic captive conditions. For disease-susceptible hosts, providing an environmental reservoir with defensive bacteria in captive-rearing programs may improve outcomes by increasing bacterial diversity on threatened amphibians or increasing the likelihood that defensive bacteria are available for colonization.     

Detection of bacterial cytotoxic activities from water-damaged ceiling tile material following incubation on blood agar

Samples of ceiling tiles with high levels of bacteria exhibited cytotoxic activities on a HEP-2 tissue culture assay. Ceiling tiles containing low levels of bacterial colonization did not show cytotoxic activities on the HEP-2 tissue culture assay. Using a spread plate procedure on blood agar plates, the levels of bacteria colonizing portions of cellulosic indoor ceiling tiles were easily identified. Levels of bacteria measured by this simple procedure may be a good indicator of microbial colonization of indoor building materials especially in the case of water damage. We suggest that bacterial levels above 150 CFU g⁻¹ of ceiling tile material indicate colonization has occurred. Received 24 December 1998/ Accepted in revised form 26 March 1999     

快速、高效的羊绒羊毛织品DNA提取方法的建立

目的：建立一种快速、高效的羊绒羊毛纺织品DNA提取的方法。方法：采用chelex-100法的3种处理、试剂盒法分别提取羊绒羊毛织品的DNA,用18S rDNA片段、山羊和绵羊源性成分PCR扩增结果来比较提取效果。结果：试剂盒法提取DNA的效果优于chelex-100法,整个提取过程约需2h。9种供试材料均提取到DNA,且含有山羊和/或绵羊源性成分,与显微镜观察结果的符合率为100%。结论：建立的试剂盒法是一种快速、高效的适用于羊绒羊毛织品DNA提取的方法,为应用分子生物学方法鉴别山羊绒和绵羊毛奠定了基础。     

Mechanism of bactericidal and fungicidal activities of textiles covalently modified with alkylated polyethylenimine

Our previous studies have led to a novel "nonrelease" approach to making materials bactericidal by covalently attaching certain moderately hydrophobic polycations to their surfaces. In the present work, this strategy is extended beyond the heretofore-used nonporous materials to include common woven textiles (cotton, wool, nylon, and polyester). Pieces of such cloths derivatized with N-hexylated+methylated high-molecular-weight polyethylenimine (PEI) are strongly bactericidal against several airborne Gram-positive and Gram-negative bacteria. In contrast, the immobilized and N-alkylated PEIs of low molecular weight have only a weak, if any, bactericidal activity. These findings support a mechanism of the antibacterial action whereby high-molecular-weight and hydrophobic polycationic chains penetrate bacterial cell membranes/walls and fatally damage them. The bactericidal textiles prepared herein are lethal not only to pathogenic bacteria but to fungi as well.     

Comparison of sampling techniques and different media for the enrichment and isolation of cellulolytic organisms from biogas fermenters

Biogas plants achieve its highest yield on plant biomass only with the most efficient hydrolysis of cellulose. This is driven by highly specialized hydrolytic microorganisms, which we have analyzed by investigating enrichment strategies for the isolation of cellulolytic bacteria out of a lab-scale biogas fermenter. We compared three different cultivation media as well as two different inoculation materials: Enrichment on filter paper in nylon bags (in sacco) or raw digestate. Next generation sequencing of the V3/V4 region of the bacterial 16S rRNA of metagenomic DNA from six different enrichment cultures, each in biological triplicates, revealed an average richness of 48 different OTU’s with an average evenness of 0.3 in each sample. β-Diversity of the bacterial community revealed significant differences between the two sampling techniques or the different media used. The isolation attempt of single cellulolytic organisms resulted in several clonal pure cultures. Regardless which medium or inoculation material, well-known cellulolytic key players such as Clostridium cellulosi, Herbinix hemicellulosilytica and Hungateiclostridium thermocellum were among the isolates. The inoculation material as well as the cultivation conditions are crucial to cultivate the representative cellulolytic organisms. Taking raw digestate as inoculation material and using the same material, filtered and sterilized, for supplementing media allowed to imitate the natural habitat. Pre-enrichment of cellulolytic organisms directly in their natural habitat led to significant advantages concerning high diversity and high abundance of unknown cellulolytic organisms, which is a key factor for the isolation of hitherto unknown species.     

Cooperative biodegradation of geosmin by a consortium comprising three gram-negative bacteria isolated from the biofilm of a sand filter column

AIMS: To isolate and identify bacteria from a sand filter column capable of degrading the taste and odour compound, geosmin. In doing so, to investigate if these organisms degrade geosmin either individually or if an alternative mechanism is utilized. METHODS AND RESULTS: Geosmin-degrading bacteria from a biologically active sand filter column were enriched by their growth in a minimal medium supplemented with geosmin as the sole carbon source. By day 51, 21.7 mg l(-1) of geosmin had been degraded as determined by solid-phase microextraction gas chromatography/mass spectrometry, and was accompanied by a 2.12 log(10) increase in active bacterial numbers as measured using the BacLight(TM) bacterial viability kit and flow cytometric enumeration. During the onset of geosmin degradation, the predominance of three bacteria, most similar to previously cultured species of Sphingopyxis alaskensis, Novosphingobium stygiae and Pseudomonas veronii based on 16S rRNA gene sequences was detected by denaturing gradient gel electrophoresis. Subsequent isolation of these organisms revealed that degradation of geosmin, when present as either the sole carbon source (ranging from 40 ng l(-1) to 20 mg l(-1)) or when spiked into sterile reservoir water (37 and 131 ng l(-1)), occurred only when all three isolates were present. None of the isolates was shown to be capable of degrading geosmin either individually or in any combination of two. CONCLUSIONS: This study has reported, for the first time, the cooperative degradation of geosmin by a consortium comprising three gram-negative bacteria isolated from a biologically active sand filter column. SIGNIFICANCE AND IMPACT OF THE STUDY: These results are important for researchers currently employing molecular-based approaches to further understand the biodegradation of geosmin by bacteria, as such studies may be complicated by the discovery of geosmin degradation occurring by a consortium. This study also advances the knowledge surrounding the types of bacteria capable of degrading the taste and odour compound, as investigations to date regarding this are limited.     

Heterotrophic bacterial guild structure: Relationship to biodegradative populations

Numerical taxonomic analysis of a freshwater bacterial guild demonstrated that the bacteria capable of growth on phenanthrene and polychlorinated biphenyl media were representative of the taxa obtained from low nutrient oligotrophic media. The diversity of heterotrophic bacteria and members of new taxa recovered from the guild followed a poisson distribution relative to the number of isolation media used. Moderately high nutrient, yeast extract peptone and glucose agar was found to be the most selective isolation medium relative to the total number of taxa recovered whereas low nutrient, lake water agar was the least selective medium used. Carbon source utilization patterns of the isolated taxa indicated that taxa within the guild had broad niche ranges and could potentially occupy many niches within a dynamic environment. The structure of the bacterial guild was dominated by mesophilic oligotrophs. The results of this investigation demonstrate that potential biodegradative populations are representative of the diverse taxa found in uncontaminated freshwater environments.     

Diversity of fungi, bacteria, and actinomycetes on leaves decomposing in a stream

Although fungi, bacteria, and specific bacterial taxa, such as the actinomycetes, have been studied extensively in various habitats, few studies have examined them simultaneously, especially on decomposing leaves in streams. In this study, sugar maple and white oak leaves were incubated in a stream in northeastern Ohio for 181 days during which samples were collected at regular intervals. Following DNA extraction, PCR-denaturing gradient gel electrophoresis (DGGE) was performed using fungus-, bacterium-, and actinomycete-specific primers. In addition, fungal and bacterial biomass was estimated. Fungal biomass differed on different days but not between leaves of the two species and was always greater than bacterial biomass. There were significant differences in bacterial biomass through time and between leaf types on some days. Generally, on the basis of DGGE, few differences in community structure were found for different leaf types. However, the ribotype richness of fungi was significantly greater than those of the bacteria and actinomycetes, which were similar to each other. Ribotype richness decreased toward the end of the study for each group except bacteria. Lack of differences between the two leaf types suggests that the microorganisms colonizing the leaf biofilm were primarily generalists that could exploit the resources of the leaves of either species equally well. Thus, we conclude that factors, such as the ecological role of the taxa (generalists versus specialists), stage of decay, and time of exposure, appeared to be more important determinants of microbial community structure than leaf quality.     

Diversity of Fungi, Bacteria, and Actinomycetes on Leaves Decomposing in a Stream

Although fungi, bacteria, and specific bacterial taxa, such as the actinomycetes, have been studied extensively in various habitats, few studies have examined them simultaneously, especially on decomposing leaves in streams. In this study, sugar maple and white oak leaves were incubated in a stream in northeastern Ohio for 181 days during which samples were collected at regular intervals. Following DNA extraction, PCR-denaturing gradient gel electrophoresis (DGGE) was performed using fungus-, bacterium-, and actinomycete-specific primers. In addition, fungal and bacterial biomass was estimated. Fungal biomass differed on different days but not between leaves of the two species and was always greater than bacterial biomass. There were significant differences in bacterial biomass through time and between leaf types on some days. Generally, on the basis of DGGE, few differences in community structure were found for different leaf types. However, the ribotype richness of fungi was significantly greater than those of the bacteria and actinomycetes, which were similar to each other. Ribotype richness decreased toward the end of the study for each group except bacteria. Lack of differences between the two leaf types suggests that the microorganisms colonizing the leaf biofilm were primarily generalists that could exploit the resources of the leaves of either species equally well. Thus, we conclude that factors, such as the ecological role of the taxa (generalists versus specialists), stage of decay, and time of exposure, appeared to be more important determinants of microbial community structure than leaf quality.     

Variation in host preferences of malaria mosquitoes is mediated by skin bacterial volatiles

The host preferences of the anthropophilic mosquito species in the Anopheles gambiae complex (Diptera: Culicidae) are mediated by skin bacterial volatiles. However, it is not known whether these mosquitoes respond differentially to skin bacterial volatiles from non‐human host species. In this study, the responses of two malaria mosquito species in the An. gambiae complex, Anopheles gambiae s.s. (hereafter, An. gambiae) and Anopheles arabiensis, with different host preferences, to volatiles released from skin bacteria were tested. Skin bacteria collected from human, cow and chicken skin significantly increased trap catches; traps containing bacteria collected from human skin caught the highest proportions of An. gambiae and An. arabiensis. Traps with bacteria of human origin caught a significantly higher proportion of An. gambiae than of An. arabiensis, whereas bacterial volatiles from the chicken attracted significantly higher numbers of An. arabiensis than of An. gambiae. Additionally, An. gambiae showed a specialized response to volatiles from four specific bacteria, whereas An. arabiensis responded equally to all species of bacteria tested. Skin bacterial volatiles may therefore play important roles in guiding mosquitoes with different host preferences. The identification of these bacterial volatiles can contribute to the development of an odour blend that attracts mosquitoes with different host preferences.     

Lineage-dependent ecological coherence in bacteria

Bacteria comprise an essential element of all ecosystems, including those present on and within the human body. Understanding bacterial diversity therefore offers enormous scientific and medical benefit, but significant questions remain regarding how best to characterize that diversity and organize it into biologically meaningful units. Bacterial communities are routinely characterized based on the relative abundances of taxa at the genus or even the phylum level, but the ecological coherence of these high-level taxonomic units is uncertain. Using human microbiota from the skin and gut as our model systems, we tested the ecological coherence of bacteria by investigating the habitat associations of bacteria at all levels of the taxonomic hierarchy. We observed four distinct taxonomic patterns of habitat association, reflecting different levels of ecological coherence among taxa. Our results support the hypothesis that deep-branch bacterial clades could be ecologically coherent and suggest that the phylogenetic depth of ecological coherence varies among the bacterial lineages and is an important factor to consider in studies of human microbiome associations.     

Use of the 'ex vivo' test to study long-term bacterial survival on human skin and their sensitivity to antisepsis

AIMS: To determine bacterial survival on human skin and their sensitivity to antisepsis. METHODS AND RESULTS: An 'ex vivo' protocol which uses human skin samples placed into diffusion cells, and electron microscopy (EM), were used to study the growth of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa inoculated onto skin samples over a 46-h incubation period at 32 degrees C. Concurrently variation in skin pH was evaluated at different time intervals during this period. In addition the antimicrobial activity of three antiseptics against the incubated micro-organisms was assessed quantitatively with the 'ex vivo' test, while their detrimental effects against bacteria were observed by EM. All three bacteria were still present in high number after 46 h inoculation on skin, although the concentration of E. coli and S. aureus were reduced by 2.74 and 1.58 log(10) reduction, respectively, over this period of time. Electron micrographs showed clear evidence of cell division and some bacteria appeared to be embedded into the skin layers. The antiseptics tested had some antibacterial activity against bacteria incubated on skin for 3 and 10 h, and EM evidence showed some morphological damages including cellular blebbing and the presence of fibrillar material around the cells. All micro-organisms had an acidifying effect on skin samples. CONCLUSIONS: Here, it was shown that bacterial pathogens can survive and grow when incubated on human skin. In addition, it is possible that they can penetrate the stratum corneum, which can provide some protection against antisepsis. SIGNIFICANCE AND IMPACT OF THE STUDY: The apparent low bactericidal activity of biocides attributed in part to bacterial protection from skin layers is particularly important to assess in order to ensure antisepsis efficacy.     

Clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle

Bacteria contaminate the uterus of most dairy cattle after parturition and endometritis causes infertility. An endometritis score can be ascribed based on the vaginal mucus character and odour but it is not clear if the clinical score reflects the number of uterine bacteria or the inflammatory response. The present study tested the hypothesis that clinical evaluation of endometritis reflects the number of bacteria present in the uterus, and the acute phase protein response. Swabs (n = 328) were collected from the uterine lumen of dairy cattle, 21 and 28 days postpartum, vaginal mucus was scored for character and odour, and blood samples collected for acute phase protein measurement. Bacteria were identified following aerobic and anaerobic culture, and the bacterial growth density was scored semi-quantitatively. When bacteria were categorised by their expected pathogenic potential in the uterus, purulent or fetid odour vaginal mucus was associated with the growth density of pathogenic bacteria but not opportunist contaminants. When bacteria were analysed independently, Arcanobacterium pyogenes, Proteus and Fusobacterium necrophorum growth densities were associated with mucopurulent or purulent vaginal mucus. The bacterial growth densities for A. pyogenes, Escherichia coli, non-hemolytic Streptococci, and Mannheimia haemolytica were associated with a fetid mucus odour. Peripheral plasma concentrations of alpha(1)-acid glycoprotein were higher if there was a fetid compared with a normal vaginal mucus odour (1.50 +/- 0.09 mg/mL versus 1.05 +/- 0.02 mg/mL, P < 0.001), but did not differ significantly between vaginal mucus character scores. The evaluation of the character and odour of vaginal mucus reflects the number of bacteria in the uterus, and the acute phase protein response.