Typical Colony Morphology Research Articles

Corona discharge development and its application to eliminate microorganism in raw milk

Raw cow’s milk or fresh milk is a natural food with a complete nutrients which provide many benefits for human health. Raw milk also provides almost ideal media for various microorganisms to grow rapidly that can harm human digestion, or make damages the milk itself. In attempt to investigate an appropriate processing technology as an alternative of thermal pasteurization, we developed non thermal corona discharge to eliminate total number of various microorganisms in milk. The generated corona discharge used 7 pins to plate surface configuration. With a distance 15 mm between the electrodes, the applied voltage of 6.48 kV by a homemade high voltage AC power supply yields in corona discharge that has temperature lower than 30°C after 8 minutes operation. The time dependent effects of the implementation of this discharge to raw milk were carried out at time interval 2, 4 and 8 minutes. It was found that there was three different types of bacteria colony morphology in the microorganism population of raw milk. The microorganism counts were decrease for 2 and 4 min corona treatments. However, when the corona treatment was done in 8 min, the total number of microorganism increased significantly. Although the corona discharge shows the potential benefits in milk decontamination processing at low temperature, more detailed study should be carried out to confirm the suitable in real application including the nutrient composition before and after treatment.

Bacterial Growth in Chloride and Perchlorate Brines: Halotolerances and Salt Stress Responses of Planococcus halocryophilus.

Extraterrestrial environments encompass physicochemical conditions and habitats that are unknown on Earth, such as perchlorate-rich brines that can be at least temporarily stable on the martian surface. To better understand the potential for life in these cold briny environments, we determined the maximum salt concentrations suitable for growth (MSCg) of six different chloride and perchlorate salts at 25°C and 4°C for the extremotolerant cold- and salt-adapted bacterial strain Planococcus halocryophilus. Growth was measured through colony-forming unit (CFU) counts, while cellular and colonial phenotypic stress responses were observed through visible light, fluorescence, and scanning electron microscopy. Our data show the following: (1) The tolerance to high salt concentrations can be increased through a stepwise inoculation toward higher concentrations. (2) Ion-specific factors are more relevant for the growth limitation of P. halocryophilus in saline solutions than single physicochemical parameters like ionic strength or water activity. (3) P. halocryophilus shows the highest microbial sodium perchlorate tolerance described so far. However, (4) MSCg values are higher for all chlorides compared to perchlorates. (5) The MSCg for calcium chloride was increased by lowering the temperature from 25°C to 4°C, while sodium- and magnesium-containing salts can be tolerated at 25°C to higher concentrations than at 4°C. (6) Depending on salt type and concentration, P. halocryophilus cells show distinct phenotypic stress responses such as novel types of colony morphology on agar plates and biofilm-like cell clustering, encrustation, and development of intercellular nanofilaments. This study, taken in context with previous work on the survival of extremophiles in Mars-like environments, suggests that high-concentrated perchlorate brines on Mars might not be habitable to any present organism on Earth, but extremophilic microorganisms might be able to evolve thriving in such environments.

Osteopetrotic induced pluripotent stem cells derived from patients with different disease-associated mutations by non-integrating reprogramming methods

BackgroundAutosomal recessive osteopetrosis is a genetically and phenotypically heterogeneous disease, caused by defects in osteoclast formation and function. The only available treatment is allogeneic stem cell transplantation that has still high morbidity and mortality. The goal of the present study was to generate iPSCs from bone marrow-derived MSCs of osteopetrosis patients with three most common mutations by using two different integration-free gene transfer methods and compare their efficiencies. The secondary objective was to select the most appropriate integration-free production method for our institutional iPSC bank using this rare disease as a prototype.MethodsTwo different integration-free gene transfer methods (episomal and Sendai viral vectors) were tested and compared on the same set of patient samples exhibiting three different mutations associated with osteopetrosis. Generated iPSCs were characterized by standard assays, including immunophenotyping, immunocytochemistry, RT-PCR, embryoid body, and teratoma assays. Karyotype analyses were performed to evaluate genetic stability.ResultsiPSC lines exhibiting typical ESC-like colony morphology were shown to express pluripotency markers by immunofluorescence staining. Over 90% of the cells were found positive for SSEA-4 and OCT3/4 and negative/weak positive for CD29 by flow cytometry. Immunohistochemical staining of teratoma and spontaneously differentiated embryoid body sections confirmed their trilineage differentiation potential. All iPSC lines expressed pluripotency-related genes. Karyotype analyses were found normal. Direct sequencing of PCR-amplified DNA showed that disease-related mutations were retained in the patient-specific iPSCs.ConclusionGeneration of iPSC using SeV and episomal DNA vectors have several advantages over other methods like the ease of production, reliability, high efficiency, and safety, which is required for translational research. Furthermore, owing to the pluripotency and self-renewal capacity, patient-specific iPSCs seem to be ideal cell source for the modeling of a rare genetic bone disease like osteopetrosis to identify osteoclast defects, leading to clinical heterogeneity in osteopetrosis patients, especially among those with different mutations in the same gene.

Phagosome Escape of Rough Mycobacterium abscessus Strains in Murine Macrophage via Phagosomal Rupture Can Lead to Type I Interferon Production and Their Cell-To-Cell Spread.

Mycobacterium abscessus complex (MAB) is a rapidly growing mycobacterium(RGM) whose clinical significance as an emerging human pathogen has been increasing worldwide. It has two types of colony morphology, a smooth (S) type, producing high glycopeptidolipid (GPL) content, and a rough (R) type, which produces low levels of GPLs and is associated with increased virulence. However, the mechanism responsible for their difference in virulence is poorly known. By ultrastructural examination of murine macrophages infected, we found that MAB-R strains could replicate more actively in the macrophage phagosome than the S variants and that they could escape into cytosol via phagosomal rupture. The cytosolic access of MAB-R strains via phagosomal rupture led to enhanced Type I interferon (IFN) production and cell death, which resulted in their cell-to-cell spreading. This behavior can provide an additional niche for the survival of MAB-R strains. In addition, we found that their enhancement of cell death mediated cell spreading are dependent on Type I IFN signaling via comparison of wild-type and IFNAR1 knockout mice. In conclusion, our data indicated that a transition of MAB-S strains into MAB-R variants increased their virulence via enhanced Type I IFN production, which led to enhanced survival in infected macrophage via cell death mediated cell-to-cell spreading. This result provides not only a novel insight into the difference in virulence between MAB-R and -S variants but also hints to their treatment strategy.

Quality control of batches of bacteriological examination kits «Selective differential-diagnostic medium for recovery of Bacillus anthracis and closely related bacilli of Bacillus cereus group»

Possibilities of quality control of experimental series of a set of reagents for bacteriological studies «Selective differential diagnostic environment for the isolation of the causative agent of anthrax and closely related bacilli of the Bacillus cereus group» is justified. Findings of the investigation of growth properties of the selective medium showed typical colonial morphology of virulent strains of Bacillus anthracis (B. anthracis 81/1, B. anthracis 1(CО), B. anthracis 1193, B. anthracis И-363, B. anthracis 1283) and its vaccine strains B. anthracis CTI-1, B. anthracis Ihtiman, B. anthracis Sterne 34 F2, B. anthracis 55, B. anthracis 71/12 - II Tsenkovsky’s vaccine). The colour of the batches of selective medium and Hottinger’s agar around the Bacillus anthracis colonies was unchanged. Whereas, the colour of selective medium around colonies of closely related saprophytes of genus Bacillus discoloured to bright-yellow, allowing to differentiate colonies of anthracic micro germs and closely related microorganisms. Presence of inhibiting properties of the selective medium was confirmed using heterologous germ cultures (Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Proteus vulgaris HX-19) which showed no growth in all series of the tested medium. Meanwhile, the typical colonial morphology of strains of these microorganisms was observed on Hottinger’s agar.

Bacteriological Investigation of S . Aureus in Urinary Tract Infection in South Port Said City

UTI is characterized by the evidence of uropathogens and pyuria and is accompanied by various clinical manifestations depending on the area of involvement. The aim of this study - was to isolate the bacterial infection to urinary tract infection in men and women at different ages and to investigate the susceptibility of bacteria implicated in urinary tract infection to different antimicrobial drugs (antibiotics).Also to determine minimum inhibitor concentration for resistant bacteria to antibiotic and molecular characterization of S. aureus associated . Materials and methods :One hundred urine specimens were received from patients admitted during January till December 2015. S. aureus isolates were confirmed by typical colonial morphology, and identified by differential tests as well as by the growth on characteristic agar, Susceptibility testing was carried out by disk diffusion method . Among the 100 isolates, 6 bacterial strain was isolated from the urine specimens of infected Patient admitted at Port-said area out-patient clinics, using agar medium and macCkonkey agar, This organism was characterized by biochemical tests and showed similarity with S. aureus . The genomic level confirmation done with 16S rDNA primer by submitting the genomic sequence to Gene Bank under acc.No-GU046545 after comparing, showed 98 % sequence similarity with S. aureus . Antibiotic susceptibility test revealed that Amikacin, Amoxicillin and Imepenem are the lowest resistant rate with percent of 100, 83 and 66%respectively and Ciprofloxacin showed the highest resistant rate with 84% . Pattern on antibiotic susceptibility test showed high resistant rate to some antibiotic that made it difficult for pregnant patients, although its frequency was low. The most prevalent Gram positive bacterial pathogen in Port-saed city was S. aureus and the most sensitive antibiotic against it is Amikacin.

Pathogenicity and inoculum concentration effects of Clavibacter michiganensis subsp. michiganensis on severity of bacterial canker of tomato

A total of 100 tomato plant samples exhibiting typical symptoms of bacterial canker were collected from tomato-growing areas of north-western areas of Pakistan. On isolation of the causal agent of this disease, 47 out of 100 samples yielded typical colony morphology of Clavibacter michiganensis subsp. michiganensis, when grown on Nutrient agar (NA) medium. 34 isolates out of the 47 exhibited Clavibacter michiganensis subsp. michiganensis like cultural characteristics when grown on Yeast extract-Dextrose-CaCO3 (YDC) medium. However, 27 isolates only out of these 34 were confirmed to be Gram positive. Pathogenicity of the 27 Clavibacter michiganensis subsp. michiganensis isolates were confirmed using cotyledon, young seedlings and old seedlings assays. Results showed that all these isolates were able to cause bacterial canker disease symptoms on tomato plants. Cotyledon assay was negative for isolates from Mansehra (MNS2); whereas, MNG1 isolate didn't produce any symptoms for all three tests. Old seedlings test was negative for four isolates mainly; MKD2, MNG1, DIK1 and NWS2. To investigate the hypothesis that appearance of symptoms depends on inoculum concentration of pathogen; two aggressive isolates of Clavibacter michiganensis subsp. michiganensis i.e., MKD1 (from Malakand) and KLM1 (from Kalam) were inoculated into tomato plants at different concentrations (i.e. 1×102 - 108 cells/ ml). Results reported that earlier and more severe disease symptoms of bacterial canker were produced by the more aggressive isolate (MKD1); compared to the moderately aggressive isolate (KLM1), which produced milder and delayed disease symptoms on tomato plants.

Association Between Loss of Type IV Pilus Synthesis Ability and Phenotypic Variation in the Cucurbit Pathogenic Bacterium Acidovorax citrulli.

Acidovorax citrulli is the causal agent of bacterial fruit blotch of cucurbits. We have shown that functional type IV pili (T4P) are required for full virulence of this bacterium. To identify A. citrulli genes required for T4P activity, we screened a library of about 10,000 transposon mutants of A. citrulli M6 for altered T4P-mediated twitching motility. This screen led to the identification of 50 mutants impaired in twitching ability due to transposon insertions into 20 different genes. Representative mutants with disruptions in these genes were further characterized. All mutants were compromised in their virulence in seed transmission and stem inoculation assays and had reduced biofilm formation ability relative to wild-type M6. When grown on nutrient agar, most mutants produced colonies with a translucent and fuzzy appearance, in contrast to the opaque and smooth appearance of wild-type colonies. The colony morphology of these mutants was identical to that of previously reported phenotypic variants of strain M6. The exceptions were M6 mutants disrupted in genes tonB, pilT, pilW, and pilX that exhibited typical wild-type colony morphology, although lacking twitching haloes surrounding the colony. Transmission electron microscopy revealed that most mutants lacked the ability to produce T4P. The exceptions were mutants with disruptions in tonB, pilT, pilW, and pilX genes that were shown to produce these appendages. These findings support the idea that colony phenotypic variation in A. citrulli is determined by the lack of ability to synthesize T4P but not by lack of T4P functionality.

Molecular Detection and Isolation of Mycoplasma capricolum subsp. capripneumoniae in Pashmina and Local Goats in Five Districts of Kashmir, India

Contagious caprine pleuropneumonia (CCPP) is caused by Mycoplasma capricolum subsp. caprip-neumoniae (Mccp), which belongs to Mycoplasma mycoides cluster, a group of closely related Mycoplasmas that are pathogenic to ruminants. The clinical lesions of CCPP are restricted to alveolar tissues of infected goats, which distinguish it from other respiratory diseases of small ruminants caused by members of the Mycoplasma mycoides cluster. The outbreaks of CCPP in Pashmina goats have been reported in cold arid regions of Kashmir province, such as Leh and Kargil districts and northern parts of the Kashmir valley. In the current study, a total of 55 lung samples and 70 nasal swabs were collected from clinically sick goats showing respiratory symptoms prior to death and processed for Mccp isolation and identification. The Mccp was detected in 33 (60.0%) and 24 (34.2%) samples, respectively by PCR. All the PCR positive samples for Mccp were further processed for isolation but only five isolates were obtained due to their fastidious nature which were confirmed by typical colony morphology and DNA sequencing of 548 bp 16SrRNA gene fragment. The five isolates were isolated from lung samples of Pashmina goats of Leh district. The results of the current study reveal that Pashmina goats of Ladakh region are highly vulnerable to CCPP and is the major cause of mortality and morbidity among them particularly during the winter season. Hence, there is an immediate need to isolate and characterise the organism for the development of autogenous vaccine to combat this economically important disease of goats. This communication records the first ever report on molecular detection and isolation of Mccp from Pashmina goats in India and is the first step towards development of vaccine against CCPP.

Isolation and Characteristics of Biotechnologically Important Antagonistic Thermophilic Bacteria from Rhizosphere of Haloxylon salicornicum.

Rhizobacteria are an active part of microbial population in the rhizosphere of plants. In this study, twenty rhizobacteria were isolated from the rhizosphere of a perennial grass, Haloxylon salicornicum, found in Cholistan desert, an arid landmass near Bahawalpur Pakistan, in one set of experimental conditions. Colony characteristics, biochemical and molecular analyses of these isolates were performed. All isolates were bacilli, gram positive with off-white colonies and exhibited typical bacilli colony morphology. None of the isolates was gelatinase, urease, indole, H2S and catalase producer. Eleven isolates were amylase producers and 8 isolates were acid producers. All isolates fermented glucose, 3 fermented lactose and 19 fermented fructose. Molecular data revealed that out of twenty isolates, 14 isolates showed 91-99% identity with Brevibacillus borstelensis, 4 with Bacillus subtilis (97-98%) and 2 with Bacillus licheniformis (94-99%) through BLAST analysis. All identified bacterial isolates cladded with their respective groups in the phylogenetic tree. Many (11-15 out of 20) of the isolates were more effective in inhibiting growth of the tested bacterial strains as compared to the positive control (Ampicillin 50 μg/disc). We conclude that bacilli are the predominant form populating rhizosphere of this desert grass. Among the isolated bacteria Brevibacillus borstelensis, Bacillus subtilis and Bacillus licheniformis are the most predominant species.

PM 7/60 (2) Pantoea stewartii subsp. stewartii

Specific scopeThis Standard describes a diagnostic protocol for Pantoea stewartii subsp. stewartii.Specific approval and amendmentApproved in 2005‐09.Revised in 2016‐04.

Emergence of undifferentiated colonies from mouse embryonic stem cells undergoing differentiation by retinoic acid treatment.

Retinoic acid (RA) is one of the most potent inducers of differentiation of mouse embryonic stem cells (ESCs). However, previous studies show that RA treatment of cells cultured in the presence of a leukemia inhibitory factor (LIF) also result in the upregulation of a gene called Zscan4, whose transient expression is a marker for undifferentiated ESCs. We explored the balance between these two seemingly antagonistic effects of RA. ESCs indeed differentiated in the presence of LIF after RA treatment, but colonies of undifferentiated ESCs eventually emerged from these differentiated cells - even in the presence of RA. These colonies, named secondary colonies, consist of three cell types: typical undifferentiated ESCs expressing pluripotency genes such as Pou5f1, Sox2, and Nanog; cells expressing Zscan4; and endodermal-like cells located at the periphery of the colony. The capacity to form secondary colonies was confirmed for all eight tested ESC lines. Cells from the secondary colonies - after transfer to the standard ESC medium - retained pluripotency, judged by their strong alkaline phosphatase (ALP) staining, typical colony morphology, gene expression profile, stable karyotype, capacity to differentiate into all three germ layers in embryoid body formation assays, and successful contribution to chimeras after injection into blastocysts. Based on flow cytometry analysis (FACS), the proportion of Zscan4-positive cells in secondary colonies was higher than in standard ESC colonies, which may explain the capacity of ESCs to resist the differentiating effects of RA and instead form secondary colonies of undifferentiated ESCs. This hypothesis is supported by cell-lineage tracing analysis, which showed that most cells in the secondary colonies were descendents of cells transiently expressing Zscan4.

Yap1 is dispensable for self-renewal but required for proper differentiation of mouse embryonic stem (ES) cells.

Yap1 is a transcriptional co-activator of the Hippo pathway. The importance of Yap1 in early cell fate decision during embryogenesis has been well established, though its role in embryonic stem (ES) cells remains elusive. Here, we report that Yap1 plays crucial roles in normal differentiation rather than self-renewal of ES cells. Yap1-depleted ES cells maintain undifferentiated state with a typical colony morphology as well as robust alkaline phosphatase activity. These cells also retain comparable levels of the core pluripotent factors, such as Pou5f1 and Sox2, to the levels in wild-type ES cells without significant alteration of lineage-specific marker genes. Conversely, overexpression of Yap1 in ES cells promotes nuclear translocation of Yap1, resulting in disruption of self-renewal and triggering differentiation by up-regulating lineage-specific genes. Moreover, Yap1-deficient ES cells show impaired induction of lineage markers during differentiation. Collectively, our data demonstrate that Yap1 is a required factor for proper differentiation of mouse ES cells, while remaining dispensable for self-renewal.

Genetic and Phenotypic characterization of Phytophthora colocasiae in Taro Growing Areas of India

Phenotypic and molecular methods were used for characterizing 40 Phytophthora colocasiae isolates obtained from Andhra Pradesh, Assam, Kerala, and Odisha regions of India over a period of five years. Phenotypic parameters such as virulence, colony morphology and mating type varied among isolates collected from different regions over the years. No correlation was observed between phenotypic parameters of the isolates and their geographical origins. Considerable inter and intra specific variation were detected by random amplified microsatellites (RAMS) analysis with 100% polymorphism among the isolates. Dendrogram constructed based on RAMS data using the unweighted pair group method with arithmetic mean (UPGMA) grouped the P. colocasiae isolates into two major clusters. No relationship was obtained between RAMS groups of the isolates and phenotypic characters/geographical origin. Population genetic analysis showed that P. colocasiae isolates were highly diverse among different regions. Analysis of molecular variance (AMOVA) showed that most of the genetic variability in P. colocasiae was confined to within a population (93.21%). These results indicate that P. colocasiae populations in India are highly diverse and care should be taken in developing disease management programmes or in breeding resistant cultivars.

SinR is a mutational target for fine-tuning biofilm formation in laboratory-evolved strains of Bacillus subtilis.

BackgroundBacteria often form multicellular, organized communities known as biofilms, which protect cells from a variety of environmental stresses. During biofilm formation, bacteria secrete a species-specific matrix; in Bacillus subtilis biofilms, the matrix consists of protein polymers and exopolysaccharide. Many domesticated strains of B. subtilis have a reduced ability to form biofilms, and we conducted a two-month evolution experiment to test whether laboratory culturing provides selective pressure against biofilm formation in B. subtilis.ResultsBacteria grown in two-month-long batch culture rapidly diversified their biofilm-forming characteristics, exhibiting highly diverse colony morphologies on LB plates in the initial ten days of culture. Generally, this diversity decreased over time; however, multiple types of colony morphology remained in our final two-month-old populations, both under shaking and static conditions. Notably, while our final populations featured cells that produce less biofilm matrix than did the ancestor, cells overproducing biofilm matrix were present as well. We took a candidate-gene approach to identify mutations in the strains that overproduced matrix and found point mutations in the biofilm-regulatory gene sinR. Introducing these mutations into the ancestral strain phenocopied or partially phenocopied the evolved biofilm phenotypes.ConclusionsOur data suggest that standard laboratory culturing conditions do not rapidly select against biofilm formation. Although biofilm matrix production is often reduced in domesticated bacterial strains, we found that matrix production may still have a fitness benefit in the laboratory. We suggest that adaptive specialization of biofilm-forming species can occur through mutations that modulate biofilm formation as in B. subtilis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0301-8) contains supplementary material, which is available to authorized users.

Characterization of single spore isolates of Agaricus bisporus (Lange) Imbach using conventional and molecular methods.

Strains A-15, S11, S-140, and U3 of Agaricus bisporus (Lange) Imbach, were used as parent strains for raising single spore homokaryotic isolates. Out of total 1,642 single spore isolates, only 36 single spore isolates were homokaryons and exhibited slow mycelial growth rate (≤2.0mm/day) and appressed colony morphology. All these SSIs failed to produce pinheads in Petri plates even after 65days of incubation, whereas the strandy slow growing SSIs along with parent strains were able to form the fructification in petriplates after 30days. Out of 24, six ISSR primers, exhibited scorable bands. In the ISSR fingerprints, single spore isolates, homokaryons, lacked amplification products at multiple loci; they grow slowly and all of them had appressed types of colony morphology. The study revealed losses of ISSR polymorphic patterns in non-fertile homokaryotic single spore isolates compared to the parental control or fertile heterokaryotic single spore isolates.

Evaluation of a Cost Effective Broth and Selective Agar Combination for the Detection of MRSA and Staphylococcus aureus from Surveillance Specimens Using Regular Workflow

To evaluate the use of selective agar and broth combination in a regular laboratory daily workflow. Swabs from 173 surveillance specimens were inoculated onto half of the Bio-Rad MRSASelect (M), SaSelect (S) and Sheep Blood agars (SBA) and the swab placed in the LIM broth. After overnight incubation, 10 microL of the LIM broth was inoculated onto the other half of the three agars and incubated overnight. All the and examined worked after agars were up approximate 14-18 hours of incubation for day one and two according to the regular workflow of the laboratory, without incubating for the full 24 hours for each incubation day. M agar and SBA were evaluated for Methicillin-Resistant Staphylococcus aureus (MRSA), while the S agar was evaluated for Staphylococcus aureus (SA) based on typical colony morphology development. Colonies on the SBA were picked and processed for definitive identification and cefoxitin susceptibility result. Trinity Medical Center, a community hospital with network hospitals Patient admitted to the hospital submitted swab for surveillance culture There were a total of 29 MRSA isolated in the study. On day one, both M agar and SBA detected 14 MRSA (48.3%) and on day two, M agar detected 10 (82.7%), while SBA detected 8 (75.8%) additional MRSA. LIM broth added 5 more MRSA to both agars on day 2, to give M agar a total of 29 (100%) and SBA agar a total of 27 (93.1%) of MRSA from the 173 specimens. There were a total of 62 SA isolated. Both the S agar and SBA isolated 34 (54.8%) on day one and 15 more (79%) on day two. The LIM broth added an additional 13 SA for both agars on day two. Using half of the agar plate for the initial swab and the other half for the broth creates an economic strategy for the detection of MRSA using the M agar and SA using the S agar. Both the M and S agars provided excellent identification and recovery of MRSA or SA based on color and colony morphology unless the colony was too young for color development. The color morphology from the M and S agars was distinguishable overnight after being subcultured from LIM broth. Working up the specimen according to the workflow of the laboratory without having to wait for each plate to incubate a full 24 hours, can still detect all the targeted organisms within 2 workdays using this cost effective strategy.

Mab_3168c, a Putative Acetyltransferase, Enhances Adherence, Intracellular Survival and Antimicrobial Resistance of Mycobacterium abscessus

Mycobacterium abscessus is a non-tuberculous mycobacterium. It can cause diseases in both immunosuppressed and immunocompetent patients and is highly resistant to multiple antimicrobial agents. M. abscessus displays two different colony morphology types: smooth and rough morphotypes. Cells with a rough morphotype are more virulent. The purpose of this study was to identify genes responsible for M. abscessus morphotype switching. With transposon mutagenesis, a mutant with a Tn5 inserted into the promoter region of the mab_3168c gene was found to switch its colonies from a rough to a smooth morphotype. This mutant had a higher sliding motility but a lower ability to form biofilms, aggregate in culture, and survive inside macrophages. Results of bioinformatic analyses suggest that the putative Mab_3168c protein is a member of the GCN5-related N-acetyltransferase superfamily. This prediction was supported by the demonstration that the mab_3168c gene conferred M. abscessus and M. smegmatis cells resistance to amikacin. The multiple roles of mab_3168c suggest that it could be a potential target for development of therapeutic regimens to treat diseases caused by M. abscessus.

The establishment of mouse embryonic stem cell cultures on 96-well plates for high-throughput screening.

Embryonic stem (ES) cells can be valuable for monitoring differentiation processes and for improving applications in basic developmental biology. The application of ES cells can be a useful tool for drug discovery and toxicology. Therefore, we suggest the high-throughput screening (HTS) system based on ES cells in this study. Firstly, we optimized the feeder-free condition and seeding cell number which can maintained for at least 7 days without over-confluency. We analyzed the system by cell viability, proliferation activity, RT-PCR and morphologic/immunohistochemical evaluations. The optimal cell seeding number was 30/well that was maintained the typical colonial morphology over 9 d with 1,000 U/ml LIF in the limited space. The cell in optimized condition expressed ALP, SSEA-1, Oct 4 and Nanog and the genetic expressions showed similar to protein expressions. The cell lineage marker expressions showed faint or none. The cell viability and proliferation activity were increased in time-dependent manner in our optimized HTS system. In conclusion, the novel HTS system using ES cells can by useful for developing models for drug discovery as well as toxicological screening in the near future.

Bisphosphonate related osteonecrosis of the jaws: clinico-pathological investigation and histomorphometric analysis

Clinico-pathological evaluation of bisphosphonate (BP)-related osteonecrosis of jaws, comparison between oral (PO) and intravenous (IV) BPs and histomorphometric analysis of associated actinomyces. Retrospective clinical and histomorphometric analysis. Data on background, clinical, treatment and follow-up were analyzed. Actinomyces colonies were identified by typical filamentous colony morphology, Gram, and PAS stains. Colony density and relative colony surface area were measured. Fifty-two patients were included, 37 (71.2%) IV, 15 (28.8%) PO BPs. with a mean duration of 2.33 and, 4.33 years respectively. One hundred percent of cases exhibited actinomyces colonization. The relative colony surface area was significantly higher in PO treatment (P = .024), with no differences in density, healing time or antibiotic treatment periods. High frequency of diabetes mellitus (60% PO cases) and corticosteroid treatment (25% of cases) were recorded. Infection with actinomyces is a common denominator in BP-related osteonecrosis of the jaws (BRONJ), regardless of administration route. Diabetes and corticosteroid treatment seem to be important contributing factors for BRONJ.