R2A Agar Plates Research Articles

Isolation of endophytic bacteria from bryophytes and study of their morphological, biochemical and biofilm formation properties

Aim: To isolate an endophytic bacterial community associated with bryophytes, which were collected from various locations in Mount Abu, Rajasthan, and to characterise their morphological, biochemical, and biofilm development properties. Methodology: Different bryophytes were selected from various regions of Mount Abu, Rajasthan. On an R2A agar plate, colonies with various morphologies were isolated. Biofilm development of bacterial isolates was done on a glass surface and visualised through scaning electron microscopy. Biochemical test, such as indole test, catalase test, urease test, MR and VP tests, citrate utilization test, gelatine and nitrate test, etc., were determined as per Berge’s Manual of Determinative Bacteriology for characterization of endophytic bacteria. Salkowski's technique was also used for estimating IAA production by endophytic bacteria. Results: The results confirmed that isolates belonged to two phyla groups, Firmicutes and γ-proteobacteria with predomination of Firmicutes. Scanning electron microscopy showed that among all the isolates, rod-shaped bacteria dominated. The metabolic properties of endophytic bacteria showed that most bacterial isolates metabolised glucose, maltose and sucrose, however, only few bacterial endophytes metabolized lactose. Some isolates produced IAA growth hormones, which were analysed through Salkowski's technique. Interpretation: The findings indicate that bryophytes have associated bacterial diversity. The morphological and biochemical characterization of bacteria revealed that they are members of the Firmicutes and γ-proteobacteria phyla. SEM confirmed that bacterial isolates can form biofilm on glass surfaces and produce a distinct amount of IAA in a tryptophan-supplemented medium, essential for the growth and development of bryophytes. Key words: Bryophytes, Biofilm, Endophytic bacteria

Arthrobacter hankyongi sp. nov., Isolated From Wet Land.

A Gram-staining-positive, catalase positive and oxidase negative, non-motile, non-flagellated, and oval-shaped bacterium, was designated as I2-34T, isolated from wetland in Soul South Korea. Colonies were round, entire, raised, and cream colored after two days of incubation on R2A agar plates at 25°C. Based on genomes (both 16S rRNA gene and draft genome) sequence analysis, strain I2-34T belongs to the genus Arthrobacter and was most closely related to Arthrobacter deserti YIM CS25T (98.0%). The strain I2-34T had a circular genome with length of 5,186,447 base pairs (67 contigs) and 4830 total genes. Out of 4696 were protein-coding genes, 54 tRNA and 4 rRNA genes. The chemotaxonomic analysis indicates iso-C16:0, anteiso-C15:0, and anteiso-C17:0 as major fatty acids, phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), and two unidentified glycolipids (GL1, GL2) as major polar lipids. The predominant quinone was MK-8(H2). The peptidoglycan type was A3α with an. L-Lys-L-Ala interpeptide bridge. Thus, the experimental data demonstrated here show that the novel isolate shares the similar major fatty acids, major polar lipid PG, DPG, and GLs, major and major quinone MK8-(H2) with the described members of the genus Arthrobacter. However, the low 16S rRNA gene sequence (98.0%), and some physiological and biochemical characteristics differentiate the I2-34T from its closest phylogenetic neighbors. As a result, the isolate represents a novel species in within the genus Arthrobacter and family Micrococcaceae for which the name Arthrobacter hankyongi sp. nov. is proposed. The type strain is I2-34T (= KACC 22217T, LMG 32197T).

Comparative Evaluation of Disinfection of Dental Water Unit Line Using Neem Extract and Sodium Hypochlorite: An In vitro Study

Background Dental Unit Water Lines DWU disinfection is important because of the risk of infection via aerosol to dental staff and patients particularly to those with comorbidities. Objectives To quantify microorganisms in dental water unit line prior to disinfection and to determine the effectiveness of Neem extract and Sodium hypochlorite in reducing the microbial count.Methods 50 mL of water samples were collected in sterile containers from the outlets of airwater syringe and high-speed hand piece of 20 dental chairs and the contamination of microorganisms in DWU total viable count were estimated. The samples were subjected to serial dilutions were inoculated onto R2A agar plates and the colony forming unitsmL were manually counted. The dental water unit lines were treated with either 5 Sodium hypochlorite or commercially available Neem extract after obtaining the baseline samples. Water samples were collected after 48 hours and were quantified again for total viable count. SPSS version 23 was used to analyse the data. Paired and unpaired ldquotrdquo tests were applied to analyse the data before and after the intervention.Results The difference between the two groups at baseline P gt0.05 was not statistically significant. In Group 1 Neem group the mean CFUmL at baseline was 128.70 and after the intervention it reduced to 51.90. In Group 2 Sodium hypochlorite group the mean CFUmL at baseline was 125.90 28.661 and after intervention it reduced to 0 both were found statistically significant P lt0.05.Conclusion Sodium hypochlorite was found to be significantly effective compared to Neem extract in disinfecting DUWL system.nbsp

Mucilaginibacter Phenanthrenivorans sp. nov., a Novel Phenanthrene Degradation Bacterium Isolated from Wetland Soil

BJC16-A38T, a Gram-negative, aerobic and non-motile rod-shaped strain was isolated from a permafrost wetland soil sample. BJC16-A38T was oxidase- and catalase-positive, and produced pale yellow colonies on modified R2A agar plates. The 16S rRNA gene sequence of BJC16-A38T shared the highest sequence similarity with those of Mucilaginibacter xinganensis BJC16-A31T (97.44%), Mucilaginibacter gotjawali SA3-7T (96.79%) and Mucilaginibacter frigoritolerans FT22T (96.14%). Phylogenetic analysis revealed that BJC16-A38T formed a separate lineage together with strain M. xinganensis BJC16-A31T in the genus Mucilaginibacter. BJC16-A38T contained menaquinone-7 (MK-7) as the predominant isoprenoid quinine. Major fatty acids in cells were iso-C15:0, summed feature 3 (16:1ω7c/16:1ω6c) and iso-C17:03-OH. BJC16-A38T contained phosphatidylethanolamine, two unknown polar lipids, six unidentified phospholipids and an unidentified aminolipid. The Genome of BJC16-A38T was sequenced using the Genome Analyzer IIx sequence platform and 38 contigs were produced in total with an average G + C percentage of 44.00%. The average nucleotide identity (ANI) of BJC16-A38T with respect to those of M. xinganensis BJC16-A31T, M. gotjawali SA3-7T and M. frigoritolerans FT22T were 79.60%, 77.24% and 77.58%, respectively. Digital DNA-DNA hybridization (DDH) values between BJC16-A38T and the tree reference strains were 21.30%, 19.60% and 19.70%, respectively. BJC16-A38T exhibited phenanthrene biodegradation activity that can degrade 88.02% phenanthrene in the MM medium after 7days cultivation. Phenotypic, chemotaxonomic, phylogenetic and genomic characteristics concluded that strain BJC16-A38T represents a novel species of the genus Mucilaginibacter. Hence, the name Mucilaginibacter phenanthrenivorans sp. nov. is proposed. The type strain is BJC16-A38T (= CGMCC 1.12693T = NBRC 110383T).

Swarming behavior of a novel strain of Brevibacillus thermoruber.

Brevibacillus thermoruber strain Nabari was isolated from compost and identified based on 16 S rRNA gene sequencing and DNA-DNA hybridization using B. thermoruber DSM 7064 T as the standard, despite some differences in their physiological and structural characteristics. When B. thermoruber Nabari was cultivated on various solid media containing 1.5% agar at 60°C, it rapidly propagated over the entire plate. In particular, on R2A-agar medium, it formed fine dendritic colonies. Macroscopic and microscopic observations of peripheral regions of the colonies indicated that the dendritic patterns were formed by bacterial swarming of some of the cells; large flows of bacterial cell populations were observed in the peripheral regions of the dendritic colonies. The cells were highly flagellated, but no extreme elongation of cells was observed. When B. thermoruber Nabari cells were cultivated at 37°C on R2A-agar plates, most colonies were nonmotile, but some colonies were motile. For example, a wandering colony moved on the plate and split into two, and then they collided to become one again. Additionally, a simple incubation system was devised to record the movement of colonies at high temperatures in this study while protecting the cameras from thermal damage.

ATP bioluminescence method as a rapid tool for assessment of cleanliness of commercial animal transport trailers

Animal transportation is widely recognized as a significant risk for disease transmission. At present, cleanliness of animal transport trailers is mostly assessed through subjective visual inspection (i.e., ‘white-glove’ test), which may sometimes be supplemented by microbiological testing with results obtained after at least 2-3 days. In this study, the feasibility of using adenosine triphosphate (ATP) bioluminescence method as a rapid tool for objectively assessing animal transport trailer cleanliness was investigated. Eighteen newly-cleaned transport trailers were tested using both ATP bioluminescence and microbiological techniques. In each trailer, six (6) locations including floors, walls, ramps, partitions and trailer exterior surfaces were sampled using an ATP meter, and MacConkey and R2A agar contact plates. From a total of more than 500 paired samples collected from all the sampled trailers, significant correlation was found between ATP bioluminescence method and standard microbiological technique using R2A agar (r = 0.206; p = 0.001) and MacConkey agar (r = 0.154; p = 0.002) plates. Using a threshold or ‘Pass’ limit of 390 RLU per 100 cm2 of trailer surface, ATP method was able to provide a good objective measure of the surface cleanliness, thus may be considered as an additional tool available for rapid and less costly assessment of trailer surface cleanliness.

Adhaeribacter radiodurans sp. nov., isolated from the rhizospheric soil of Elymus tsukushiensis, a plant native to the Dokdo Islands, Republic of Korea.

KUDC8001T was isolated from the rhizosperic soil of Elymus tsukushiensis in the Dokdo Islands, Republic of Korea. Strain KUDC8001T was Gram-stain-negative, non-motile and rod-shaped. KUDC8001T was catalase- and oxidase-positive. This strain is capable of growing at 4-37 °C and pH 7.0-8.0 and exhibited optimal growth at 25 °C and pH 7.0. It could be grown in R2A, nutrient agar and ISP2 agar plates. The cell width ranged from 0.7 to 1.0 µm, and length ranged from 2.5 to 5.5 µm. The genomic G+C content was 40.8 mol%. Phylogenetic analysis, based on 16S rRNA gene sequences, revealed that the strain KUDC8001T belongs to the genus Adhaeribacter, which is most closely related to the strain A. pallidiroseus HMF7616T (97.5%). The DNA relatedness of KUDC8001T with the type strains of A. pallidiroseus HMF7616T, A. swui 17 mud1-7T and A. arboris HMF7605T was ≤80.3 % based on average nucleotide identity calculations and ≤86.9 % based on average amino acid identity calculations. In silico DNA-DNA hybridization values of the strain KUDC8001T with the most closely related strains were 22.1, 24.0 and 24.4 %. Based on its phenotypic, phylogenetic, genetic and chemotaxonomic features, the strain KUDC8001T should be considered as a novel species in the genus Adhaeribacter, for which we have proposed the name Adhaeribacter radiodurans sp. nov. The type strain is KUDC8001T (=KCTC 82078T=CGMCC 1.18475T).

Caenimonas soli sp. nov., isolated from soil.

A non-motile, Gram-stain-negative, rod-shaped bacterium, designated strain S4T, was obtained from soil sampled at Wonju, Gyeonggi-do, Republic of Korea. Cells were white-coloured, aerobic, grew optimally at 25-32°C on R2A agar plate. A phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain S4T formed a lineage within the family Comamonadaceae. The closest members were Caenimonas terrae SGM1-15T (98.1% sequence similarity), Caenimonas koreensis EMB320T (97.5%) and Ramlibacter solisilvae 5-10T (97.8%). The sequence similarities of strain S4T with other members of the family Comamonadaceae were ≤ 97.5%. The sole respiratory quinone was ubiquinone-8 (Q-8) and the principal polar lipid was phosphatidylethanolamine. The predominant cellular fatty acids were summed feature 3 (iso-C15 :02-OH/C16 :1 ω7c), C16:0 and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The DNA G + C content was 65.1mol%. In addition, the average nucleotide identity (ANIu) and in silico DNA-DNA hybridization (dDDH) relatedness values between strain S4T and Caenimonas koreensis were 77.6 and 21%, respectively. Based on genomic, chemotaxonomic, phenotypic, and phylogenetic analyses, strain S4T represents a novel species in the genus Caenimonas, for which the name Caenimonas soli sp. nov. is proposed. The type strain is S4T (= KCTC 72742T = NBRC 114610T).

ASSESSMENT OF 16S rRNA SEQUENCE AND HETEROTROPHIC PLATE COUNT (HPC) METHODS OF IDENTIFYING BACTERIA FROM DRINKING WATER SYSTEMS IN BENIN CITY METROPOLIS

A variety of simple culture-based tests which are proposed to recover a wide range of microorganisms from water are collectively known as heterotrophic plate count (HPC) and used as an indirect indicator to give information about water quality. The aim of this study was to assess the heterotrophic plate count (HPC) culture-based dependent and 16S rRNA independent techniques of identifying bacteria. The HPC was conducted by incubating a filtered sample of water on R2A agar plates and enumerating the number of resultant bacterial colonies that grow on each plate. The molecular analysis was performed by extracting the deoxyribonucleic acid (DNA) from bacterial isolate and polymerase chain reaction (PCR) was done to obtain the amplicons (PCR products). Purified PCR products were sequenced by ABI V3.1 Big dye kit and the analysis of sequence was conducted by the basic local alignment search tool (BLAST) to identify their closest relatives. A total number of 17 isolates of Pseudomonas, Bacillus and Proteus were phenotypically identified, while the nucleotide sequence revealed the presence of 59 diverse strains with distribution (percentage) of Pseudomonas (25) 42.2 %, Bacillus 17 (28.8 %) and Proteus (17) 28.8 %. The investigated isolates when compared from gene data-base recorded genetic relatedness with similarity index of 61 to 100 %. The 16S rRNA gene sequences from DNA extractions showed microbial consortia in drinking water to comprise of a broad array of bacterial diversity, including those of critical concern to human health such as Pseudomonas and Bacillus. Finally, there was no significant correlation between the HPC test and 16S rRNA sequence analysis.

Contamination of a hospital plumbing system by persister cells of a copper-tolerant high-risk clone of Pseudomonas aeruginosa

BackgroundPseudomonas aeruginosa (PA) is an important opportunistic pathogen that thrives best in the distal elements of plumbing and waste-water systems. Although nosocomial outbreaks of PA have been associated with water sources, the role of the plumbing system of healthcare premises as a reservoir for this pathogen is still unclear. Materials and methodsWe collected water samples from 12 technical areas, distant from any medical activity, in a teaching hospital in France once a week for 11 weeks. We used a method that resuscitates persister cells because of the nutrient-poor conditions and the presence of inhibitors (e.g. chlorine and copper ions). Briefly, water was sampled in sterile bottles containing 100 μM of the copper-ion chelating agent diethyldithiocarbamate (DDTC). A portion of the samples was immediately filtered through 0.45-μm membranes, deposited on R2A agar plates, and incubated seven days at 22 °C (following European recommendations). The remaining water was incubated 14 days at 22 °C and then filtered and cultured on R2A, blood-, or cetrimide-containing agar plates. PA isolates were identified by MS MALDI-TOF, genotyped by PFGE and WGS, and tested for survival in a 150 μg/L copper (II) sulphate solution. ResultsAlthough the 12 water sampling points always tested negative with the recommended method, 67% were positive at least once for PA with the adapted method (i.e. with DDTC). The 14 PA persister isolates found throughout the plumbing system were clonal and belong to the high-risk clone ST308. Their genome harbours a 37-kb genomic island (GI-7) containing 13 genes linked to copper resistance. ST308 survived better in the copper solution than comparators that did not harbour GI-7 (P. aeruginosa strains PAO1, PA14, and ST235). The deletion of GI-7 in ST308 abrogated its tolerance to copper. The GI-7 nucleotide sequence shares 98% and 72% identity with sequences from the environmental species Pseudomonas putida and the phytopathogenic species Pseudomonas syringae, respectively. ConclusionWe report the contamination of the plumbing system of a healthcare premises by persister cells of the high-risk clone P. aeruginosa ST308. New recommendations for the monitoring of water contamination should consider persister cells. The genomic island GI-7, which confers tolerance to copper, probably originates from Pseudomonas species found in copper-contaminated soils and plants. Agricultural practices may have an unexpected consequence, allowing copper-tolerant pathogens to survive in the hospital environment and contaminate fragile patients.

Description of Variovorax humicola sp. nov., isolated from a forest topsoil.

Employing a modified cultivation method, we studied two bacterial strains, UC10 and UC38T, found on the Kyonggi University campus, Suwon in Gyeonggi-Do province, South Korea. These strains were non-spore-forming, Gram-stain-negative, motile and rod-shaped. Growth occurred in the presence of 0-2 % (w/v) NaCl, at pH 4-9 and a temperature range of 4-35 °C. On an R2A agar plate incubated for 5 days at 28 °C, irregular, raised and pale-yellowish colonies were observed. Comparative analysis of nearly full-length 16S rRNA gene sequences indicated that these strains were closely related to Variovorax guangxiensis GXGD002T, with 98.6 % similarity. Strains UC10 and UC38T were 98.0 % similar to V.ariovorax soli GH9-3T; 97.8 % to V.ariovorax dokdonensis DS-43T; 97.3-97.7 % to V.ariovorax ginsengisoli Gsoil 3165T; 97.7-98.0 % to V.ariovorax paradoxus IAM 12373T; 97.4-97.6 % to V.ariovorax defluvii 2C1-bT; and 97.3-97.4 % to V.ariovorax boronicumulans BAM-48T. The predominant ubiquinone was Q-8. The primary polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids were C16 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C17 : 0 cyclo. DNA-DNA hybridization assays indicated 89.2-91.4 % genomic DNA similarity between strains UC10 and UC38T. Moreover, genomic DNA similarity between these novel strains and reference strains of the genus Variovoraxwas less than the 70 %. Based on these results, strain UC38T was designated a representative of a novel species of the genus Variovorax, with the proposed name Variovorax humicola sp. nov. The type strain is UC38T (=KACC 18501T=NBRC 111520T).

Evaluation of Petrifilm™ aerobic count plates as an equivalent alternative to drop plating on R2A agar plates in a biofilm disinfectant efficacy test.

This paper compares Petrifilm™ aerobic count (AC) plates to drop plating on R2A agar plates as an alternative method for biofilm bacteria enumeration after application of a disinfectant. A Pseudomonas aeruginosa biofilm was grown in a Centers for Disease Control and Prevention biofilm reactor (ASTM E2562) and treated with 123 ppm sodium hypochlorite (as free chlorine) according to the Single Tube Method (ASTM E2871). Aliquots from the same dilution tubes were plated on Petrifilm™ AC plates and drop plated on R2A agar plates. The Petrifilm™ AC and R2A plates were incubated for 48 and 24 h, respectively, at 36 ± 1 °C. After nine experimental runs performed by two technicians, the mean difference in biofilm log densities [log biofilm density (LD) = log10(CFU/cm(2))] between the two methods for control coupons, treated coupons, and log reduction (LR) was 0.052 (p = 0.451), -0.102 (p = 0.303), and 0.152 (p = 0.313). Equivalence testing was used to assess equivalence of the two plating methods. The 90 % confidence intervals for the difference in control and treated mean LDs between methods were (-0.065, 0.170) and (-0.270, 0.064), both of which fall within a (-0.5, +0.5) equivalence criterion. The 90 % confidence interval for the mean LR difference (-0.113, 0.420) also falls within this equivalence criterion. Thus, Petrifilm™ AC plates were shown to be statistically equivalent to drop plating on R2A agar for the determination of control LDs, treated LDs, and LR values in an anti-biofilm efficacy test. These are the first published results that establish equivalency to a traditional plate counting technique for biofilms and for a disinfectant assay.

Use of sodium hypochlorite solutions to obtain axenic cultures of Nostoc strains (Cyanobacteria)

A method based on the treatment of akinetes using diluted solutions of sodium hypochlorite (SH solution) was developed to obtain axenic cultures of Nostoc strains (Nostoc Vaucher ex Bornet & Flahault, 1886:181). Three strains were independently grown on liquid BG-110 medium (BG-11 without a nitrogen source) until a massive differentiation of akinete cells took place. Samples of these akinete-rich cultures were homogenized and treated with diluted SH solutions (1, 2, and 3 %) for 10, 20, and 30 s. Subsequently, the treated akinetes were spread onto BG-110-agar plates and incubated under standard conditions for at least 2 weeks. Both the axenicity and the degree of contamination were monitored for each treatment by inoculating spring colonies in nutrient-broth or R2A-agar plates. Axenic cultures were obtained for the strains Nostoc sp. CCLFM I (with 1 % SH solution), Nostoc sp. CCLFM VIII (2 and 3 %) and Nostoc sp. CCLFM XXI (3 %), only applying 10 s of treatment exposure. This strategy was proven to be efficient for Nostoc cultures, as all of the tested strains became axenic. This method can be applied to virtually any strains that are capable of performing massive akinete differentiation; furthermore, it is a fast, inexpensive, and antibiotic-free approach.

Monitoring Dental-Unit-Water-Line Output Water by Current In-office Test Kits

The importance of monitoring contamination levels in the output water of dental-unit-water-lines (DUWLs) is essential as they are prone to developing biofilms that may contaminate water that is used to treat patients, with opportunistic pathogens such as species of Legionella, Pseudomonas and others. Dentists and practice staff are also at risk of being infected by means of cross-infection due to aerosols generated from DUWL water. The unit of measurement for the microbial contamination of water by aerobic mesophilic heterotrophic bacteria is the colony-forming unit per millilitre (cfu/ml) of water. The UK has its own guidelines set by the Department of Health for water discharged from DUWL to be between 100 and 200 cfu/ml of water. The benchmark or accepted standard laboratory test is by microbiological culture on R2A agar plates. However, this is costly and not convenient for routine testing in dental practices. A number of commercial indicator tests are used in dental surgeries, but they were not developed for the dental market and serve only to indicate gross levels of contamination when used outside of the manufacturer's recommended incubation period. The aim of this article is to briefly review the universal problem of DUWL contamination with microbial biofilms and to update dental professionals on the availability of currently available commercial in-office monitoring systems for aerobic mesophilic heterotrophic bacteria and to discuss their limitations for testing water samples in assuring compliance with recommended guidelines.

Flavobacterium panaciterrae sp. nov., a β-glucosidase producing bacterium with ginsenoside-converting activity isolated from the soil of a ginseng field.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DCY69(T) is JX233806. A Gram-reaction-negative, oxidase- and catalase-positive, non-gliding motile strain, designated strain DCY69(T), was isolated from the soil of a ginseng field in the Republic of Korea. Colonies of strain DCY69(T) were circular, 0.5-1.5 mm diameter, yellow, and convex on an R2A agar plate after 2 days. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain DCY69(T) belonged to the genus Flavobacterium with 90.5-98.3% gene sequence similarity. The major predominant quinone was MK-6. The major cellular fatty acids were iso-C15:0, iso-C17:0 3-OH, iso-C15:0 3-OH and summed feature 3 (containing C16:1ω7c and/or C16:1ω6c). The major polar lipids were phosphatidylethanolamine, one unidentified aminolipid and unidentified polar lipids (L1, L2). The genomic DNA G+C content of strain DCY69(T) was 35.0mol%. The strain DCY69(T) transformed ginsenoside Rb1 into Rd and F2. Based on the polyphasic taxonomic data, strain DCY69(T) is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium panaciterrae sp. nov. is proposed. The type strain is DCY69(T)(= KCTC 32392(T) = JCM 19161(T)), isolated from the soil of a ginseng field in the Republic of Korea.

The effect of hydrogen peroxide/colloidal silver on reducing the colonization and growth of heterotrophic bacteria in dental unit waterlines

The objective of the present study was to investigate reduction in the colonization and growth of heterotrophic bacteria in dental unit waterlines by using hydrogen peroxide/colloidal silver as disinfectant. Twenty-seven dental units were included; 6 units that were more than 20 years old and 7 units that had been in use for 2 years comprised the old and new treatment groups, respectively. Fourteen units served as controls. The treatment groups were disinfected continuously and every 4 weeks shock doses were applied over a 20-week period. Water samples were taken before treatment, 1 and 2 weeks after treatment, and thereafter every 4 weeks; then they were inoculated onto R2A agar plates. While 1-16-week results for the old treatment group showed total heterotrophic bacterial counts of higher than 1 × 10^5 cfu/mL, at 20 weeks they were below 7.5 × 10^2 cfu/mL. Only 2 units were able to reach levels of Keywords: Biofilm, aerobic heterotrophic bacterial count, dental unit waterlines, hydrogen peroxide/colloidal silver

Sphingomonas ginsengisoli sp. nov., isolated from soil of a ginseng field

A Gram-reaction-negative, oxidase- and catalase-positive, non-gliding motile bacteriun, designated DCY58(T), was isolated from a ginseng field in Pocheon Province, South Korea. Colonies of strain DCY58(T) were circular, 0.5-1.5 mm in diameter, yellow, and convex on an R2A agar plate after 2 days. The 16S rRNA gene sequence analysis revealed that strain DCY58(T) belongs to the family of the Sphingomonadaceae and is most closely related to the species of genus Sphingomonas with similarity levels of 95.7-96.4%. Strain DCY58(T) contained Q-10 as the predominant ubiquinone. The major cellular fatty acids included Summed Feature 8 (containing C(18:1ω)6c and/or C(18:1ω)7c), C(14:0) 2-OH and C(16:0). The DNA G+C content was 65.1 mol%. Furthermore, strain DCY58(T) differed from other related Sphingomonas species by a number of phenotypic characteristics. On the basis of the evidence presented in this study, strain DCY58(T) is described as a novel species of genus Sphingomonas, for which the name Sphingomonas ginsengisoli sp. nov., is proposed. The type strain is DCY58(T) (=KCTC 23762(T) =JCM 18016(T)).

Extracellular hydrolase enzyme production by soil fungi from King George Island, Antarctica

Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. In order to examine extracellular enzyme production in this chronically low-temperature environment, fungi were isolated from ornithogenic, pristine and human-impacted soils collected from the Fildes Peninsula, King George Island, Antarctica during the austral summer in February 2007. Twenty-eight isolates of psychrophilic and psychrotolerant fungi were obtained and screened at a culture temperature of 4°C for activity of extracellular hydrolase enzymes (amylase, cellulase, protease), using R2A agar plates supplemented with (a) starch for amylase activity, (b) carboxymethyl cellulose and trypan blue for cellulase activity or (c) skim milk for protease activity. Sixteen isolates showed activity for amylase, 23 for cellulase and 21 for protease. One isolate showed significant activity across all three enzyme types, and a further 10 isolates showed significant activity for at least two of the enzymes. No clear associations were apparent between the fungal taxa isolated and the type of source soil, or in the balance of production of different extracellular enzymes between the different soil habitats sampled. Investment in extracellular enzyme production is clearly an important element of the survival strategy of these fungi in maritime Antarctic soils.

Comparison of In-Office Dental Unit Waterline Test Kits

The authors conducted a study to determine the validity of two commercially available in-office dental unit waterline test kits compared to the gold standard, R2A agar. Samples were collected from the air/water syringes of dental units and cultured on HPC Samplers, Petrifilm AC Plates, and R2A agar plates. HPC Samplers and R2A agar plates were incubated for 7 days and counted manually using magnification. Petrifilm AC Plates were counted after incubation time of 5 and 7 days using an electronic-plate reader. Validity measurements were calculated using a cutoff value < or = 500 colony-forming units per milliliter. The accuracy for the HPC Sampler compared to R2A agar was 71%. The accuracy for the Petrifilm AC Plates at 5 and 7 days was 79% and 87% compared to R2A agar. The Petrifilm AC Plate (7-day incubation) demonstrated higher sensitivity, specificity, predictive values, and accuracy than the HPC Sampler kit.

Ornithinibacter aureus gen. nov., sp. nov., a novel member of the family Intrasporangiaceae

A novel strain of the class Actinobacteria was isolated from a seawater sample collected in the South China Sea using modified R2A agar plates. The strain was a Gram-stain-positive, nonmotile, non-spore-forming, catalase-positive, irregular rod-shaped bacterium. The strain grew at 4-45 °C and pH 5.0-10.2, and tolerated 5% (w/v) NaCl. Based on its 16S rRNA gene sequence, the organism was related phylogenetically to members of the genera Fodinibacter (96.7% similarity), Lapillicoccus (96.5 %), Knoellia (95.0-95.8 %), Oryzihumus (95.6 %) and Humibacillus (95.6 %). The cell-wall contained L-ornithine as the major diagnostic diamino acid in the peptidoglycan. MK-8(H₄) was the predominant menaquinone. Major cellular fatty acids were iso-C₁₈:₁ω9c, iso-C₁₆:₀, iso-C₁₅:₀ and C₁₇:₀. The G+C content of the DNA was 69.6 mol%. Phenotypic and phylogenetic data revealed that this strain represents a novel species in a new genus of the family Intrasporangiaceae, for which the name Ornithinibacter aureus gen. nov., sp. nov. is proposed; the type strain of Ornithinibacter aureus is HB09001(T) (=CGMCC 1.10341(T) =DSM 23364(T)).