Isolations of aerobic bacteria from wild desert bighorn sheep (Ovis canadensis nelsoni and O. c. mexicana) in Arizona

Nasal, pharyngeal, cervical and vaginal swab specimens were obtained from 74 desert bighorn sheep for the purpose of investigating the normal aerobic bacterial flora of wild sheep. A total of 281 isolates was obtained and identified by standard microbiologic tests. One hundred seven of these isolates were gram positive and included Bacillus sp. (36%), Staphylococcus epidermidis (8%), S. aureus (4%), Corynebacterium sp. (diphtheroids, 4%), and Streptococcus sp. (48%). gram negative isolates totaled 174 and included Neisseria sp. (18%), Citrobacter sp. (3%), Enterobacter sp. (2%), Escherichia coli (2%), Proteus sp. (2%) and non-fermentative bacilli (NFB) (73%). Of the NFB isolates, Pseudomonas sp. (25%), Acinetobacter sp. (18%), Moraxella sp. (15%) were identified.     

Multiple approaches to enhance the cultivability of bacteria associated with the marine sponge Haliclona (gellius) sp

Three methods were examined to cultivate bacteria associated with the marine sponge Haliclona (gellius) sp.: agar plate cultures, liquid cultures, and floating filter cultures. A variety of oligotrophic media were employed, including media with aqueous and organic sponge extracts, bacterial signal molecules, and siderophores. More than 3,900 isolates were analyzed, and 205 operational taxonomic units (OTUs) were identified. Media containing low concentrations of mucin or a mixture of peptone and starch were most successful for the isolation of diversity, while the commonly used marine broth did not result in a high diversity among isolates. The addition of antibiotics generally led to a reduced diversity on plates but yielded different bacteria than other media. In addition, diversity patterns of isolates from agar plates, liquid cultures, and floating filters were significantly different. Almost 89% of all isolates were Alphaproteobacteria; however, members of phyla that are less commonly encountered in cultivation studies, such as Planctomycetes, Verrucomicrobia, and Deltaproteobacteria, were isolated as well. The sponge-associated bacteria were categorized into three different groups. The first group represented OTUs that were also obtained in a clone library from previously analyzed sponge tissue (group 1). Furthermore, we distinguished OTUs that were obtained from sponge tissue (in a previous study) but not from sponge isolates (group 2), and there were also OTUs that were not obtained from sponge tissue but were obtained from sponge isolates (group 3). The 17 OTUs categorized into group 1 represented 10 to 14% of all bacterial OTUs that were present in a large clone library previously generated from Haliclona (gellius) sp. sponge tissue, which is higher than previously reported cultivability scores for sponge-associated bacteria. Six of these 17 OTUs were not obtained from agar plates, which underlines that the use of multiple cultivation methods is worthwhile to increase the diversity of the cultivable microorganisms from sponges.     

Substantial alterations of the cutaneous bacterial biota in psoriatic lesions

For psoriasis, an idiopathic inflammatory disorder of the skin, the microbial biota has not been defined using cultivation-independent methods. We used broad-range 16S rDNA PCR for archaea and bacteria to examine the microbiota of normal and psoriatic skin. From 6 patients, 19 cutaneous samples (13 from diseased skin and 6 from normal skin) were obtained. Extracted DNA was subjected to the broad range PCR, and 1,925 cloned products were compared with 2,038 products previously reported from healthy persons. Using 98% sequence identity as a species boundary, 1,841 (95.6%) clones were similar to known bacterial 16S rDNA, representing 6 phyla, 86 genera, or 189 species-level operational taxonomic unit (SLOTU); 84 (4.4%) clones with <98% identity probably represented novel species. The most abundant and diverse phylum populating the psoriatic lesions was Firmicutes (46.2%), significantly (P<0.001) overrepresented, compared to the samples from uninvolved skin of the patients (39.0%) and healthy persons (24.4%). In contrast, Actinobacteria, the most prevalent and diverse phylum in normal skin samples from both healthy persons (47.6%) and the patients (47.8%), was significantly (P<0.01) underrepresented in the psoriatic lesion samples (37.3%). Representation of Propionibacterium species were lower in the psoriatic lesions (2.9+/-5.5%) than from normal persons (21.1+/-18.2%; P<0.001), whereas normal skin from the psoriatic patients showed intermediate levels (12.3+/-21.6%). We conclude that psoriasis is associated with substantial alteration in the composition and representation of the cutaneous bacterial biota.     

Bacteria and nematodes in the conjunctiva of mule deer from Wyoming and Utah

Swabs of conjunctiva were collected from 44 live and 226 hunter-harvested mule deer (Odocoileus hemionus) from Wyoming and Utah (USA). We identified 29 gram negative and 22 gram positive bacterial taxonomic categories, but many isolates from hunter-harvested animals were environmental contaminants. Staphylococcus spp. and Micrococcus spp. were the most common gram positive bacteria isolated, and Enterobacter spp., Escherichia coli, and Pseudomonas spp. were common gram negative bacteria isolated. Thelazia californiensis were found in 15% of hunter-harvested deer in Utah in 1994 and in 8% in 1995. Nematodes were found in 40% of live deer in 1995 and 66% in 1996. Three live animals showed clinical signs of infectious keratoconjunctivitis (IKC) in 1996, but pathogenic bacteria were not isolated from these individuals. Hemolytic, non-piliated Moraxella ovis was isolated from two clinically normal live deer in 1996 and isolates were similar to those cultured from IKC cases from Wyoming and Utah.     

The role of LncRNA MALAT-1 and MiRNA-9 in Psoriasis

BackgroundPsoriasis is a chronic skin disorder manifested by recurrent episodes of scaly, red, itchy skin patches that occur within apparently normal skin.ObjectivesThis study was performed to detect the expression of serum and tissue (lesion and non-lesion) LncRNA MALAT-1 and MiRNA-9 that might be used as biomarkers for psoriasis.MethodsBlood samples were obtained from 60 psoriasis patients and 40 controls, as well as 4 mm punch biopsy from lesional and non lesional skin of psoriatic patient and normal skin of healthy controls. Expression of LncRNA MALAT-1 and miRNNA-9 in serum and tissues was detected by real time qRT-PCR.Resultsa statistically significant increase in the expression of MALAT-1 in lesional and non-lesional skin and serum of psoriatic patients in comparison to controls were detected. Moreover, there was statistically significant increase in serum MiRNA-9 in patients in comparison to controls, while its tissue level was significantly lower in patients.ConclusionThis study highlights the dysregulation of LncRNA MALAT-1 and miRNA-9 in psoriasis. Elevated expression of MALAT-1 in lesional skin of psoriatic patients compared to non-lesional skin may possibly contribute to the development of psoriatic plaques.     

Wound fluid bacterial levels exceed tissue bacterial counts in controlled porcine partial-thickness burn infections

In the present study, an established controlled burn wound model was used to test the hypothesis that controlled surface contamination with is capable of generating a noninvasive method for the creation of a reproducible deep tissue burn wound infection. Using a liquid tight-wound chamber in Yorkshire pigs, partial-thickness burns were inoculated with saline-immersed for 24 hours. Noninoculated burns and unwounded skin immersed in normal saline served as controls. Bacterial cultures of wound fluid were performed daily, and tissue biopsies for bacteriological and histological evaluations were performed on days 1, 3, and 5. was only recovered from -inoculated wounds (tissue and fluid), whereas all controls contained endogenous only. The number of colony-forming units per gram of wound tissue did not correlate with the bacterial counts found in the overlying wound fluid for any wounds. Fluid counts were consistently higher than tissue counts by two logs. -inoculated wounds showed three times deeper tissue destruction than control wounds. Obtaining consistently deep tissue colonization without cross-contamination among wounds, this study introduces a noninvasive model for controlled burn wound infection suitable for future investigations regarding the efficacy of topical antibiotic wound treatment in experimental burns.     

Biodegradation of Crude Oil by Constructed Bacterial Consortia and the Constituent Single Bacteria Isolated From Malaysia

Three bacterial isolates identified as Pseudomonas aeruginosa (UKMP-8T), Rhodococcus sp. M15-2 (UKMP-5T), and Rhodococcus sp. ZH8 (UKMP-7T) based on biochemical, physiological, and morphological characteristics and on 16S rDNA sequences were isolated from groundwater of a crude oil refinery plant. From these three isolates, four bacterial consortia were designed by mixing the single bacterial cultures in the following ratios: (P. aeruginosa:Rhodococcus sp. M15-2, 1:1), (P. aeruginosa:Rhodococcus sp. ZH8, 1:1), (Rhodococcus sp. M15-2: Rhodococcus sp. ZH8, 1:1), and (P. aeruginosa: Rhodococcus sp. ZH8:Rhodococcus sp. M15-2, 1:1:1), respectively. Bacterial isolates and consortia showed differing preferences for nitrogen source (0.01% ammonium chloride, 0.10% yeast extract, or 0.50% peptone) to reach optimum growth. When fortified with the preferred nitrogen sources and grown in minimal salt medium, within 7 days all three single isolates and the four bacterial consortia biodegraded 97.6-99.9% of Tapis Massa oil without any significant differences.     

Analysis of the composition of bacterial communities in oil reservoirs from a southern offshore Brazilian basin

The aim of this study was to characterize and compare the bacterial community structure of two distinct oil samples from a petroleum field in Brazil by using both molecular, based on the construction of 16S rRNA gene libraries, and cultivation methods. Statistical comparisons of libraries based on Amplified Ribosomal DNA Restriction Analysis (ARDRA) data revealed no significant differences between the communities recovered in the non-biodegraded (NBD) and highly biodegraded oils (HBD). BlastN analysis of the 16S rRNA gene sequences representative of distinct ribotypes from both oils showed the presence of nine different bacterial genera in these samples, encompassing members of the genera Arcobacter, Halanaerobium, Marinobacter, Propionibacterium, Streptomyces, Leuconostoc, Acinetobacter, Bacillus and Streptococcus. Enrichments obtained using oil as inoculum and sole carbon source yielded bacterial isolates showing high 16S rRNA gene sequence similarity with Achromobacter xylosoxidans, Bacillus subtilis, Brevibacillus sp., Dietzia sp. and Methylobacterium sp. Comparison between the data obtained using cultivation-independent and enrichment cultures suggests that different selection of community members may occur when using distinct approaches. All the organisms found, except for Leuconostoc sp. and Streptococus sp., have been previously reported in the literature as hydrocarbon degraders and/or associated to oil field environments.     

Surface microflora of four smear-ripened cheeses

The microbial composition of smear-ripened cheeses is not very clear. A total of 194 bacterial isolates and 187 yeast isolates from the surfaces of four Irish farmhouse smear-ripened cheeses were identified at the midpoint of ripening using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR, and 16S rRNA gene sequencing for identifying and typing the bacteria and Fourier transform infrared spectroscopy and mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis for identifying and typing the yeast. The yeast microflora was very uniform, and Debaryomyces hansenii was the dominant species in the four cheeses. Yarrowia lipolytica was also isolated in low numbers from one cheese. The bacteria were highly diverse, and 14 different species, Corynebacterium casei, Corynebacterium variabile, Arthrobacter arilaitensis, Arthrobacter sp., Microbacterium gubbeenense, Agrococcus sp. nov., Brevibacterium linens, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus saprophyticus, Micrococcus luteus, Halomonas venusta, Vibrio sp., and Bacillus sp., were identified on the four cheeses. Each cheese had a more or less unique microflora with four to nine species on its surface. However, two bacteria, C. casei and A. arilaitensis, were found on each cheese. Diversity at the strain level was also observed, based on the different PFGE patterns and mtDNA RFLP profiles of the dominant bacterial and yeast species. None of the ripening cultures deliberately inoculated onto the surface were reisolated from the cheeses. This study confirms the importance of the adventitious, resident microflora in the ripening of smear cheeses.     

Isolation of bacterial strains that produce the endocrine disruptor,octylphenol diethoxylates,in paddy fields

Topsoil samples were collected from 36 different paddy fields in West Japan. Each soil sample was incubated with a basal salt-medium containing 0.2% OPPEO. Twelve samples possessed OPPEO-degrading activity, from which twelve cultures of OPPEO-degrading bacteria were isolated. The isolated bacteria grew on a medium containing 0.2% OPPEO as the sole carbon source, and OP2EO and OP3EO were accumulated in the medium under aerobic conditions. OP1EO and octylphenol, which have often been identified in surface water together with OP2EO, were not observed in this experiment. The bacterial isolates were gram negative and tentatively identified as Pseudomonas putida (10 isolates) and Burkholderia cepacia (one isolate) by BIOLOG and 16S rDNA RFLP analyses.     

The aerobic and anaerobic microbiology of pustular acne lesions

Specimens from 32 pustular acne lesions that were inoculated on media supportive for the growth of aerobic and anaerobic bacteria showed bacterial growth. Only aerobic or facultative bacteria were recovered in 15 (47%) specimens, only anaerobic bacteria in 11 (34%) specimens, and mixed aerobic and anaerobic bacteria in 6 (18%) specimens. A total of 57 isolates, 31 anaerobes (1.0 per specimen) and 26 aerobes (0.8 per specimen) were recovered. The predominant isolates were Staphylococcus sp. (19 isolates), Peptostreptococcus sp. (15), and Propionibacterium sp. (10). Twelve (37.5%) of the comedones yielded only one organism. This retrospective study highlighted the polymicrobial nature of over two-thirds of culture positive pustular acne lesions and suggests the potential for pathogenic role of aerobic and anaerobic organisms other than P. acnes and Staphylococcus sp. in acne vulgaris.     

The occurrence and comparative phenotypic characteristics of Staphylococcus spp. from healthy and diseased, household and shelter dogs, based on routine biochemical diagnostic methods

To determine the staphylococcal colonization pattern in healthy and diseased dogs, living in two particular environments, a number of microbiological samples were taken. Overall, twenty dogs, either healthy or with infected skin lesions, were examined. In each case bacterial swabs were collected from the nasal mucosa, ear, perineum, lumbo-sacralis triangle, and from the infection sites if such were present. A total number of 104 isolates representing different staphylococcal species were isolated and identified using routine biochemical methods applied in diagnostic laboratories. Among 17 isolated staphylococcal species, Staphylococcus intermedius was the most common species isolated from both healthy or diseased dogs living either in animal shelter or household environments. The pattern of Staphylococcus sp. colonization differs considerably for animals living in the two tested habitats. In particular, S. aureus MRSA and MSSA isolates were detected only in infected skin lesion samples from animals that dwelled in the animal shelter. As could be expected, S. intermedius was found to be a predominant causative agent in canine skin infections. In our study, we demonstrated that S. intermedius in its carrier-state, inhabits mainly the mucosal membrane of the nasal vestibule. It was also found in the samples taken from the skin, the lumbo-sacralis triangle and perineum, but was rarely isolated from the ears.     

Microflora of raw cacao beans

Whole non-moldy cacao beans from 14 sources cultured on Czapek's agar with six percent NaCl yielded species ofAspergillus glaucus group,Mucor pusillus and unidentified yeast and bacteria. The average number of bacterial colonies from the 14 sources of non-moldy cacao beans was 32.7 million per gram. The average number of colonies of filamentous fungi in dilution cultures was 57,000 colonies per gram. Yeast colonies were present in the amount of 15.8 million colonies per gram. The moldy beans analyzed contained fungi and bacterial counts in the billions; also, yeast was not isolated from the moldy samples.Paper Number 3170 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina 27607.     

Recovery of beta-lactamase producing bacteria in pediatric infections

The presence of beta-lactamase producing bacteria (beta LPB) was investigated in specimens obtained from 1469 children who presented with infections of the skin and soft tissue (648), upper respiratory tract (514), pulmonary sites (137), surgical sites (113), and other (57). Of 4989 bacterial isolates recovered, 910 (18%) were beta LPB, 492 (54%) aerobes, and 418 (46%) anaerobes. The beta LPB were recovered in 751 (51%) of the children. The most frequently recovered beta LPB was Staphylococcus aureus, which was recovered in 356 (47%) patients. Most isolates were recovered from patients with skin and soft-tissue infections (68% of patients), upper respiratory tract infections (49%), and pulmonary infections (35%). Bacteroides fragilis group was isolated in 35% of patients with beta LPB, mostly from surgical infections (98% of patients), pulmonary infections (36%), skin and soft-tissue infections (25%), and upper respiratory tract infections (20%). Twenty-five percent of the Bacteroides melaninogenicus group produced beta-lactamase. These organisms were recovered in 15% of patients with beta LPB. They were recovered in upper respiratory tract infections (38% of patients), pulmonary infections (22%), and skin and soft-tissue infections (7%). Other beta LPB were Pseudomonas aeruginosa (8% of total patients with beta LPB), Escherichia coli (4%), Bacteroides oralis (3%), Klebsiella pneumoniae (3%), Haemophilus influenzae (2%), Proteus (1%), and Branhamella catarrhalis (1%). The role of beta LPB in the failure of penicillin to eradicate many of the infections is discussed.     

Isolation of endophytic actinomycetes from selected plants and their antifungal activity

The isolation of endophytic actinomycetes from surface-sterilized tissues of 36 plant species was made using humic acid–vitamin (HV) agar as a selection medium. Of the 330 isolates recovered, 212 were from roots, 97 from leaves and 21 isolates from stems with a prevalence of 3.9, 1.7 and 0.3%, respectively. Identification of endophytic actinomycetes was based on their morphology and the amino acid composition of the whole-cell extract. Most isolates were classified as Streptomyces sp. (n = 277); with the remainder belonging to Microbispora sp. (n = 14), Nocardia sp. (n = 8) and Micromonospora sp. (n = 4). Four isolates were unclassified and 23 were lost during subculture. The most prevalent group of isolates were the Streptomyces sp. occurring in 6.4% of the tissue samples of Zingiber officinale. Scanning electron microscopy investigation of this plant revealed that 7.5% of the root and 5% of the leaf samples contained endophytes. Three of the Streptomyces sp. isolates strongly inhibited Colletotrichum musae, five were very active against Fusarium oxysporum and two strongly inhibited growth of both test fungi.     

非培养方法解析北京地区甘薯叶际细菌的群落结构

利用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术和磷脂脂肪酸(PLFA)分析方法,比较了北京通州、顺义、昌平、延庆地区甘薯叶际细菌的多样性和生物量,并调查了通州地区甘薯叶际细菌群落在不同生长季节的变化情况。PLFA分析结果发现所有检测样品中,革兰氏阳性细菌生物量均高于革兰氏阴性细菌生物量。PCR-DGGE方法与PLFA方法聚类分析结果较一致,甘薯叶际细菌群落结构受到时空因素、甘薯生理特性等的影响,不同地点、不同生长季节甘薯叶际细菌群落结构有较大差异,DGGE条带测序分析表明,Pseudomonas sp.在不同地点的甘薯叶际均为保守菌群,Bacillus sp.,Acinetobacter sp.,     

Yeast colonizing the intestine of rainbow trout (Salmo gairdneri) and turbot (Scophtalmus maximus)

Yeast were isolated from the intestine of farmed rainbow trout (Salmo gairdneri), turbot (Scophtalmus maximus), and free-living flat-fish (Pleuronectes platessa and P. flesus). The average number of viable yeasts recovered from farmed rainbow trout was 3.0 × 10³ and 0.5 × 10² cells per gram homogenized intestine for white and red-pigmented yeasts, respectively. The dominant species were Debaryomyces hansenii, Saccharomyces cerevisiae, Rhodotorula rubra, and R. glutinis. In 5 of 10 free-lving marine fish, > 100 viable yeast cells per gram intestinal mucus were recovered. Red-pigmented yeasts dominated and composed >90% of the isolates. Colonization experiments were performed by inoculating rainbow trout and turbot with fish-specific, isolated yeast strains and by examining the microbial intestinal colonization at intervals. Inoculation of experimental fish with pure cultures of R. glutinis and D. hansenii HF1 yielded colonization at a level several orders of magnitude higher than before the inoculation. Up to 3.8 × 10⁴, 3.1 × 10⁶, and 2.3 × 10⁹ viable yeast cells per gram intestine or feces were recovered in three separate colonization experiments. The high level of colonizing yeasts persisted for several weeks. The concentrations of yeasts in the tank water never exceeded 10³ viable cells per milliliter. No traces of fish sickness as a result of high yeast colonization were recorded during any of the colonization experiments. For periods of the experiments, the concentration of aerobic bacteria in the fish intestine was lower than the intestinal yeast concentration. Scanning electron microscopy studies demonstrated a close association of the yeasts with the intestinal mucosa. The mucosal colonization was further demonstrated by separating intestinal content, mucus, and tissue. All compartments were colonized by >10³ viable yeast cells per gram. No bacteria were detected on the micrographs, indicating that their affinity for the intestinal mucosa was less than that of the yeasts. Correspondence to: Thomas Andtid     

First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): new perspectives for an insect-bacteria association

We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.     

Ammonia-oxidizing bacteria along meadow-to-forest transects in the Oregon Cascade Mountains

Although nitrification has been well studied in coniferous forests of Western North America, communities of NH(3)-oxidizing bacteria in these forests have not been characterized. Studies were conducted along meadow-to-forest transects at two sites (Lookout and Carpenter) in the H. J. Andrews Experimental Forest, located in the Cascade Mountains of Oregon. Soil samples taken at 10- or 20-m intervals along the transects showed that several soil properties, including net nitrogen mineralization and nitrification potential rates changed significantly between vegetation zones. Nonetheless, terminal restriction fragment length polymorphism (T-RFLP) analysis of the PCR-amplified NH(3) monooxygenase subunit A gene (amoA) showed the same DNA fragments (TaqI [283 bp], CfoI [66 bp], and AluI [392 bp]) to dominate >/=45 of 47 soil samples recovered from both sites. Two fragments (491-bp AluI [AluI491] and CfoI135) were found more frequently in meadow and transition zone soil samples than in forest samples at both sites. At the Lookout site the combination AluI491-CfoI135 was found primarily in meadow samples expressing the highest N mineralization rates. Four unique amoA sequences were identified among 15 isolates recovered into pure culture from various transect locations. Six isolates possessed the most common T-RFLP amoA fingerprint of the soil samples (TaqI283-AluI392-CfoI66), and their amoA sequences shared 99.8% similarity with a cultured species, Nitrosospira sp. strain Ka4 (cluster 4). The other three amoA sequences were most similar to sequences of Nitrosospira sp. strain Nsp1 and Nitrosospira briensis (cluster 3). 16S ribosomal DNA sequence analysis confirmed the affiliation of these isolates with Nitrosospira clusters 3 and 4. Two amoA clone sequences matched T-RFLP fingerprints found in soil, but they were not found among the isolates.     

Isolation and phenotypical identification of spore-forming Bacillus species from Jordanian habitats with larvicidal activity against Drosophila melanogaster

Spore-forming Bacillus isolates were recovered from different Jordanian habitats. Of 37 samples, 187 colonies were selected. Forty-six (24·6%) of them were pathogenic to the third instar larvae of Drosophila melanogaster . Larvicidal activity of the isolates was from 0% (non-cultivated soil) to 83·3% (decomposed animal residues). The total spore count per gram weight varied from 0·1 × 10⁵–18 × 10⁵ among the 37 tested samples. Morphological and microscopical identification of the isolates showed the presence of 16 different Bacillus species. The pathogenic isolates were B. thuringiensis (44) and B. sphaericus (2).