Antibiotic-resistant Colonies Research Articles

Echinocandin persistence directly impacts the evolution of resistance and survival of the pathogenic fungus Candida glabrata.

Candida glabrata is a prevalent fungal pathogen able to replicate inside macrophages and rapidly develop resistance against frontline antifungal echinocandins. Multiple studies have shown that echinocandin resistance is fueled by the survival of a small subpopulation of susceptible cells surviving lethal concentrations of echinocandins. Importantly, bacterial pathogens that exhibit high antibiotic persistence also impose a high burden and generate more antibiotic-resistant colonies. Nonetheless, the implications of echinocandin persistence (ECP) among the clinical isolates of C. glabrata have not been defined. Additionally, ECP level determination relies on a laborious and time-consuming method, which is prone to high variation. By exploiting in vivo systemic infection and ex vivo models, we showed that C. glabrata isolates with a higher ECP are associated with a higher burden and more likely develop echinocandin resistance upon micafungin treatment. Additionally, we developed an assay that reliably determines ECP levels in real time. Therefore, our study identified C. glabrata isolates displaying high ECP levels as important entities and provided a reliable and convenient tool for measuring echinocandin persistence, which is extendable to other fungal and bacterial pathogens.

Fluorescently Labeled DNA Interacts with Competence and Recombination Proteins and Is Integrated and Expressed Following Natural Transformation of Bacillus subtilis.

During competence, Bacillus subtilis is able to take up DNA from its environment through the process of transformation. We investigated the ability of B.subtilis to take up fluorescently labeled DNA and found that it is able to take up fluorescein-dUTP-, DyLight 550-dUTP-, and DyLight 650-dUTP-labeled DNA. Transformation with labeled DNA containing an antibiotic cassette resulted in uptake of the labeled DNA and also generated antibiotic-resistant colonies. DNA is primarily taken up at the pole, as it can be seen to colocalize with ComFC, which is a component of the competence machinery. The DNA is taken up rapidly and can be seen to localize with (the actively searching form of) RecA. Colocalization with a homologous locus on the chromosome increases over time. Using microfluidics, we observed replacement of the homologous locus and subsequent expression of the integrated labeled and unlabeled DNA, although whether the integrated DNA contains labeled nucleotides needs to be determined conclusively. Integrated DNA in cells with a doubling time of 60min is expressed on average 6h 45min after the addition of DNA and 4h 45min after the addition of fresh medium. We also found that the expression of the incoming DNA under these conditions can occur before cell division and, thus, before complete exit from the competence state. Because the competence machinery is conserved among naturally competent bacteria, this method of labeling is also suitable for studying transformation of other naturally competent bacteria.IMPORTANCE We used DNA that was covalently labeled with fluorescent nucleotides to investigate the transformation process of Bacillus subtilis at the molecular level. We show that the labeled DNA colocalizes with components of the competence machinery, the chromosome, and the recombination protein RecA. Using time-lapse microscopy and microfluidics, we visualized, in real-time, the uptake of fluorescently labeled DNA. We found that under these conditions, cell division is not required for the expression of integrated DNA. Because the competence machinery is conserved in naturally competent bacteria, this method can also be used to investigate the transformation process in many other bacterial species.

203 Genome Modifications by Sleeping Beauty Transposition and CRISPR/Cas9 to Improve Cow Milk Composition for Human Consumption

Genome manipulation of cattle represents a powerful tool to increase the nutritional value and reduce allergenicity of cow milk for human consumption. This could be accomplished by improving the amount of polyunsaturated fatty acids (ω-3 and ω-6) and simultaneously abolishing β-lactoglobulin (BLG), a potent allergen for predisposed humans. The aim of this study was to introduce the sequence for a desaturase construct (mFAT-2, from C. elegans), which is able to catalyse the synthesis of ω-3 and ω-6 fatty acids, into the bovine genome by Sleeping Beauty (SB) transposition, and simultaneously knocking out the bovine β-lactoglubulin gene using CRISPR/Cas9 system. The sgRNA (AAGTGCCTCCTGCTTGCCC) targeted to BLG exon 1 was synthesised as an oligo linker and cloned into the px459-Cas9. The mutation activity of the designed sgRNA at the target locus was determined by T7 endonuclease assay I (T7EI) mismatch detection assay. Briefly, bovine fetal fibroblasts (BFF) were seeded at 0.5 × 105 cells per well of a 24-well plate in triplicate, when the cells reached 80% confluence (12–24 h), cultures were transfected with 1 μg of px459-Cas9::BLG plasmid co-expressing Cas9 and sgRNA using polyethylenimine reagent (PEI; 3 ng μL−1). After 3 days of puromycin selection, genomic DNA from transfected cells were extracted and the sequence of interest was PCR-amplified and digested by T7EI restriction enzyme. Digestion products showed a mutation efficiency at the target locus of 29%. Subsequently, we chemically cotransfected 0.5 × 105 BFF with 0.5 μg of knockout vector (px459-Cas9::BLG) and 0.5 μg of SB plasmids (carrying mFAT-2 cDNA for mammary gland-specific expression) using 3 ng μL−1 PEI in triplicate. At 48 h post-transfection, cell cultures were subjected to 3 days of puromycin and 21 days of neomycin selection. PCR analysis of antibiotic resistant colonies revealed the presence of mFAT-2 transgene in almost 70% of the analysed cells lines. Genotyping of BLG exon 1 was performed by direct sequencing of PCR amplicons using primers flanking the target site. Despite the appreciable gene mutation activity of the sgRNA sequence previously determined by T7EI assay (29%), none of the cell lines analysed showed modification in the BLG target locus. We speculate that the SB vector might have disrupted the activity of targeting vector. We are currently performing additional experiments to accomplish gene addition (mFAT) and gene knockout (BGL) in one step using these highly efficient and precise transgenic tools. Genetically modified cells will be used as nuclear donor to produce transgenic cows by somatic cells nuclear transfer. The financial support of CONICET, UNRC and FONCYT is gratefully acknowledged.

In vivo selection of active deoxyribonucleoside kinase by a mutagenic nucleoside analog

A novel in vivo selection method for active deoxyribonucleoside kinase proteins is described here. A pool of randomly mutated genes for deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) was prepared and inserted into an expression vector. Enzymatically active mutants were selected by repeated cycles, including (i) introduction into Escherichia coli, (ii) treatment of the E. coli pool with a mutagenic deoxyribonucleoside (2-hydroxy-dA), and (iii) selection of antibiotic-resistant colonies resulting from mutations by phosphorylated 2-hydroxy-dA and the subsequent isolation of the plasmid DNAs. The ratio of the resistant colonies increased by two orders of magnitude from the first cycle to the fifth cycle, and then reached a plateau. Fifteen Dm-dNK mutants selected after the seventh and eighth evolution cycles were actually active in vivo. Moreover, one of the mutant proteins was as active as the wild-type protein in vitro. These results indicate that this novel in vivo evolution method was useful and that similar strategies would be applicable to other deoxyribonucleoside kinases. In addition, the distribution of mutated amino acids suggests important residues/regions in the Dm-dNK protein.

Structural basis for Zn 2+ -dependent intercellular adhesion in staphylococcal biofilms

Staphylococcal bacteria, including Staphylococcus epidermidis and Staphylococcus aureus, cause chronic biofilm-related infections. The homologous proteins Aap and SasG mediate biofilm formation in S. epidermidis and S. aureus, respectively. The self-association of these proteins in the presence of Zn(2+) leads to the formation of extensive adhesive contacts between cells. This study reports the crystal structure of a Zn(2+) -bound construct from the self-associating region of Aap. Several unusual structural features include elongated β-sheets that are solvent-exposed on both faces and the lack of a canonical hydrophobic core. Zn(2+)-dependent dimers are observed in three distinct crystal forms, formed via pleomorphic coordination of Zn(2+) in trans across the dimer interface. These structures illustrate how a long, flexible surface protein is able to form tight intercellular adhesion sites under adverse environmental conditions.

The effect of environmental pollutants and food processing on the development of antibiotic resistance

The purpose of this study was to determine how various compounds known to be positive mutagens, contribute to the development of mutations leading to ciprofloxacin resistance in Salmonella enterica subsp. enterica serotype Typhimurium. The molecular mechanism of ciprofloxacin resistance in treated strains was investigated. A modified version of the incorporation plate test was used for quantitative determination of ciprofloxacin resistant mutants and for assessment of the mutation frequency induced by the positive mutagens in different concentrations. An AS-PCR-RFLP for monitoring of gyrA mutations was applied. Mutation frequency, expressed as number of antibiotic resistant colonies per 10(8) viable cells, was much higher after exposure of bacterial cells to 3-(5-nitro-2-furyl) acrylic acid and 2-nitrofluorene. All isolated cultures retain decreased susceptibility to antibiotic after multiple passages in antibiotic-free medium. 2-nitrofluorene was the best inducer of mutations in gyrA and in regulation genes affecting suppression of synthesis of outer membrane porins. 3-(5-nitro-2-furyl) acrylic acid gives rise to overproduction of efflux pump. The data suggest that antibiotic resistance may not be only a consequence of misuse of antibiotics. A polluted environment as well as food processing could contribute to this unwanted process.

A Longitudinal Study of the Establishment and Proliferation ofEnterococcuson a Dairy Farm

Enterococci are Gram-positive, facultative anaerobic cocci. They are found in many environments (including milk and dairy products, vegetables, plants, cereals, and meats). Enterococci are considered commensal organisms, but can also be opportunistic pathogens associated with morbidity and mortality of humans and animals. A longitudinal study of antibiotic resistance of Enterococcus to ampicillin, erythromycin, and tetracycline was conducted on an academic teaching farm. Environmental samples were collected by drag swabs at select locations prior to and after the introduction of livestock. All samples were initially processed and screened with specialized media, and then replica plated on tryptic soy agar containing a predetermined amount of antibiotic. There was some variation in the quantity of bacterial and antibiotic-resistant colonies; however, resistance to tetracycline was extremely high. The increases of too numerous to count populations were not time-dependent and appeared consistently after the placement of cows. There is little information on the prevalence and epidemiology of antibiotic resistance of Enterococci outside of the hospital setting, including on dairy farms. Longitudinal studies are important in providing insight into the dynamics of establishment and proliferation of bacteria and of antibiotic resistance.

An investigation of horizontal transfer of feed introduced DNA to the aerobic microbiota of the gastrointestinal tract of rats

BackgroundHorizontal gene transfer through natural transformation of members of the microbiota of the lower gastrointestinal tract (GIT) of mammals has not yet been described. Insufficient DNA sequence similarity for homologous recombination to occur has been identified as the major barrier to interspecies transfer of chromosomal DNA in bacteria. In this study we determined if regions of high DNA similarity between the genomes of the indigenous bacteria in the GIT of rats and feed introduced DNA could lead to homologous recombination and acquisition of antibiotic resistance genes.ResultsPlasmid DNA with two resistance genes (nptI and aadA) and regions of high DNA similarity to 16S rRNA and 23S rRNA genes present in a broad range of bacterial species present in the GIT, were constructed and added to standard rat feed. Six rats, with a normal microbiota, were fed DNA containing pellets daily over four days before sampling of the microbiota from the different GI compartments (stomach, small intestine, cecum and colon). In addition, two rats were included as negative controls. Antibiotic resistant colonies growing on selective media were screened for recombination with feed introduced DNA by PCR targeting unique sites in the putatively recombined regions. No transformants were identified among 441 tested isolates.ConclusionsThe analyses showed that extensive ingestion of DNA (100 μg plasmid) per day did not lead to increased proportions of kanamycin resistant bacteria, nor did it produce detectable transformants among the aerobic microbiota examined for 6 rats (detection limit < 1 transformant per 1,1 × 108 cultured bacteria). The key methodological challenges to HGT detection in animal feedings trials are identified and discussed. This study is consistent with other studies suggesting natural transformation is not detectable in the GIT of mammals.

TRIM29 Functions as a Tumor Suppressor in Nontumorigenic Breast Cells and Invasive ER+ Breast Cancer

Tripartite motif-containing 29 (TRIM29) is a member of the TRIM protein family that has been implicated in hematologic and solid tumor cancers. We found that TRIM29 functions as a tumor suppressor in both the nontumorigenic MCF10A [estrogen receptor (ER)-/TRIM29+] breast cell line and the invasive MCF7 (ER+/TRIM29-) breast cell line. Silencing TRIM29 in MCF10A cells resulted in preneoplastic changes that included loss of polarity in three-dimensional culture, increased proliferation, anchorage-independent growth, and increased migration and invasion. Conversely, the introduction of TRIM29 into MCF7 cells caused reversion to a less aggressive phenotype by antagonizing the growth effect of 17β-estradiol. The interaction between TRIM29 and ER signaling in MCF7 cells was supported by a reduction in ERE binding in the presence of TRIM29 and suppression of ER-dependent gene expression of TFF1, FOS, and GREB1. By microarray analyses, we showed that younger women (<55 years of age) with early-stage, ER+ breast cancer who were given no adjuvant systemic therapy had a significantly lower risk of relapse when their tumor had high TRIM29 expression (P = 0.02). This effect was not observed in older women (>55 years of age) and thus may be due to menopause and loss of circulating estrogens. Our results suggest that loss of TRIM29 expression in normal breast luminal cells can contribute to malignant transformation and lead to progression of ER+ breast cancer in premenopausal women.

Detection of genetically modified microorganisms in soil using the most-probable-number method with multiplex PCR and DNA dot blot

The principal objective of this study was to detect genetically modified microorganisms (GMMs) that might be accidentally released into the environment from laboratories. Two methods [plate counting and most-probable-number (MPN)] coupled with either multiplex PCR or DNA dot blots were compared using genetically modified Escherichia coli, Pseudomonas putida, and Acinetobacter oleivorans harboring an antibiotic-resistance gene with additional gfp and lacZ genes as markers. Alignments of sequences collected from databases using the Perl scripting language (Perl API) and from denaturing gradient gel electrophoresis analysis revealed that the gfp, lacZ and antibiotic-resistance genes (kanamycin, tetracycline, and ampicillin) in GMMs differed from the counterpart genes in many sequenced genomes and in soil DNA. Thus, specific multiplex PCR primer sets for detection of plasmid-based gfp and lacZ antibiotic-resistance genes could be generated. In the plate counting method, many antibiotic-resistant bacteria from a soil microcosm grew as colonies on antibiotic-containing agar plates. The multiplex PCR verification of randomly selected antibiotic-resistant colonies with specific primers proved ineffective. The MPN-multiplex PCR method and antibiotic-resistant phenotype could be successfully used to detect GMMs, although this method is quite laborious. The MPN-DNA dot blot method screened more cells at a time in a microtiter plate containing the corresponding antibiotics, and was shown to be a more efficient method for the detection of GMMs in soil using specific probes in terms of labor and accuracy.

Epigenetic Regulatory Mechanisms Distinguish Retinoic Acid-mediated Transcriptional Responses in Stem Cells and Fibroblasts

Retinoic acid (RA), a vitamin A metabolite, regulates transcription by binding to RA receptor (RAR) and retinoid X receptor (RXR) heterodimers. This transcriptional response is determined by receptor interactions with transcriptional regulators and chromatin modifying proteins. We compared transcriptional responses of three RA target genes (Hoxa1, Cyp26a1, RARbeta(2)) in primary embryo fibroblasts (mouse embryonic fibroblasts), immortalized fibroblasts (Balb/c3T3), and F9 teratocarcinoma stem cells. Hoxa1 and Cyp26a1 transcripts are not expressed, but RARbeta(2) transcripts are induced by RA in mouse embryonic fibroblasts and Balb/c3T3 cells. Retinoid receptors (RARgamma, RXRalpha), coactivators (pCIP (NCOA3, SRC3)), and p300 and RNA polymerase II are recruited only to the RARbeta(2) RA response element (RARE) in Balb/c3T3, whereas these proteins are recruited to RAREs of all three genes by RA in F9 cells. In F9, RA reduces polycomb (PcG) protein Suz12 and the associated H3K27me3 repressive epigenetic modification at the RAREs of all three genes. In contrast, in Balb/c3T3 cells cultured in the +/-RA, Suz12 is not associated with the Hoxa1, RARbeta(2), and Cyp26a1 RAREs, whereas slow levels of the H3K27me3 mark are seen at these RAREs. Thus, Suz12 is not required for gene repression in the absence of RA. Even though the Hoxa1 RARE and proximal promoter show high levels of H3K9,K14 acetylation in Balb/c3T3, the Hoxa1 gene is not transcriptionally activated by RA. In Balb/c3T3, CpG islands are methylated in the Cyp26a1 promoter region but not in the Hoxa1 promoter or in these promoters in F9 cells. We have delineated the complex mechanisms that control RA-mediated transcription in fibroblasts versus stem cells.

Cloning and Targeted Disruption of Two Lipopolysaccharide Biosynthesis Genes, kdsA and waaG, of Pseudomonas aeruginosa PAO1 by Site-Directed Mutagenesis

The emergence of antibiotic resistance in bacterial pathogens poses a great challenge to public health and emphasizes the need for new antimicrobial targets. The recent development of microbial genomics and the availability of genome sequences allows for the identification of essential genes which could be novel and potential targets for antibacterial drugs. However, these predicted targets need experimental validation to confirm essentiality. Here, we report on experimental validation of a two potential targets in the lipopolysaccharide (LPS) biosynthesis pathway of the pathogen Pseudomonas aeruginosa PAO1 using insertion duplication. Two genes, kdsA and waaG, from LPS encoding proteins 2-dehydro-3-deoxyphosphooctonate aldolase and UDP-glucose (heptosyl) LPS α-1,3-glucosyltransferase were selected as putative target candidates for the gene disruption experiments using plasmid insertion mutagenesis to determine essentiality. The introduction of a selectable ampicillin and kanamycin resistance marker into the chromosome resulted in lack of recovery of antibiotic-resistant colonies suggesting the essentiality of these genes for the survival of P. aeruginosa. Several molecular analyses were carried out in order to confirm the essentiality of these genes. We propose that the above two validated drug targets are essential and can be screened for functional inhibitors for the discovery of novel therapeutic compounds against antibiotic-resistant opportunistic pathogen P. aeruginosa.

CCN3 (NOV) Is a Negative Regulator of CCN2 (CTGF) and a Novel Endogenous Inhibitor of the Fibrotic Pathway in an in Vitro Model of Renal Disease

Fibrosis is a major cause of end-stage renal disease, and although initiation factors have been elucidated, uncertainty concerning the downstream pathways has hampered the development of anti-fibrotic therapies. CCN2 (CTGF) functions downstream of transforming growth factor (TGF)-beta, driving increased extracellular matrix (ECM) accumulation and fibrosis. We examined the possibility that CCN3 (NOV), another CCN family member with reported biological activities that differ from CCN2, might act as an endogenous negative regulator of ECM and fibrosis. We show that cultured rat mesangial cells express CCN3 mRNA and protein, and that TGF-beta treatment reduced CCN3 expression levels while increasing CCN2 and collagen type I activities. Conversely, either the addition of CCN3 or CCN3 overexpression produced a marked down-regulation of CCN2 followed by virtual blockade of both collagen type I transcription and its accumulation. This finding occurred in both growth-arrested and CCN3-transfected cells under normal growth conditions after TGF-beta treatment. These effects were not attributable to altered cellular proliferation as determined by cell cycle analysis, nor were they attributable to interference of Smad signaling as shown by analysis of phosphorylated Smad3 levels. In conclusion, both CCN2 and CCN3 appear to act in a yin/yang manner to regulate ECM metabolism. CCN3, acting downstream of TGF-beta to block CCN2 and the up-regulation of ECM, may therefore serve to naturally limit fibrosis in vivo and provide opportunities for novel, endogenous-based therapeutic treatments.

Novel homologs of the multiple resistance regulator marA in antibiotic-contaminated environments

Antibiotics are commonly detected in the environment as contaminants. Exposure to antibiotics may induce antimicrobial-resistance, as well as the horizontal transfer of resistance genes in bacterial populations. We selected the resistance gene marA, mediating resistance to multiple antibiotics, and explored its distribution in sediment and water samples from surface and sewage treatment waters. Ciprofloxacin and ofloxacin (fluoroquinolones), sulphamethoxazole (sulphonamide), erythromycin, clarythromycin, and spiramycin (macrolides), lincomycin (lincosamide), and oxytetracycline (tetracycline) were measured in the same samples to determine antibiotic contamination. Bacterial populations from environmental samples were challenged with antibiotics to identify resistant isolates. The gene marA was found in almost all environmental samples and was confirmed by PCR amplification in antibiotic-resistant colonies. 16S rDNA sequencing revealed that the majority of resistant isolates belonged to the Gram-positive genus Bacillus, not previously known to possess the regulator marA. We assayed the incidence of marA in environmental bacterial populations of Escherichia coli and Bacillus by quantitative real-time PCR in correlation with the levels of antibiotics. Phylogenetic analysis indicated the possible lateral acquisition of marA by Bacillus from Gram-negative Enterobacteriaceae revealing a novel marA homolog in Bacillus. Quantitative PCR assays indicate that the frequency of this gene in antropised environments seems to be related to bacterial exposure to water-borne antibiotics.

Phosphorylation of β-Catenin by AKT Promotes β-Catenin Transcriptional Activity

Increased transcriptional activity of beta-catenin resulting from Wnt/Wingless-dependent or -independent signaling has been detected in many types of human cancer, but the underlying mechanism of Wnt-independent regulation is poorly understood. We have demonstrated that AKT, which is activated downstream from epidermal growth factor receptor signaling, phosphorylates beta-catenin at Ser552 in vitro and in vivo. AKT-mediated phosphorylation of beta-catenin causes its disassociation from cell-cell contacts and accumulation in both the cytosol and the nucleus and enhances its interaction with 14-3-3zeta via a binding motif containing Ser552. Phosphorylation of beta-catenin by AKT increases its transcriptional activity and promotes tumor cell invasion, indicating that AKT-dependent regulation of beta-catenin plays a critical role in tumor invasion and development.

Osteopontin Induction of Hyaluronan Synthase 2 Expression Promotes Breast Cancer Malignancy

Osteopontin (OPN) is a tumor-associated, secreted phosphoprotein that has been implicated in breast cancer progression and metastasis. Research concerning how OPN functions in tumor progression has led to the identification of a limited number of genes that contribute functionally to OPN-induced cellular behaviors. Recent microarray analysis, comparing 21NT breast cancer cells transfected to constitutively overexpress OPN with control cells, revealed hyaluronan synthase 2 (HAS2) to be a gene highly up-regulated in OPN-overexpressing cells. In this study, we further examined the relationship between OPN and HAS2. We show that 21NT OPN-transfected cells express high levels of HAS2, which is associated with increased HA production and matrix retention and is necessary for tumor cell adhesion to bone marrow endothelial cells and anchorage-independent growth. Finally, stable transfection of antisense HAS2 into 21NT cells overexpressing OPN resulted in a reduction in HAS2 expression, HA production, and pericellular retention. Antisense-mediated down-regulation of HAS2 also resulted in a significant decrease in cellular proliferation and colony growth in soft agar. To our knowledge, this is the first report of the ability of OPN to regulate HAS2 expression and HA production in breast cancer cells and further illustrates a unique functional relationship by which enhanced HA production facilitates OPN-mediated cell behaviors.

A Novel Diacylglycerol-lactone Shows Marked Selectivity in Vitro among C1 Domains of Protein Kinase C (PKC) Isoforms α and δ as Well as Selectivity for RasGRP Compared with PKCα

Although multiple natural products are potent ligands for the diacylglycerol binding C1 domain of protein kinase C (PKC), RasGRP, and related targets, the high conservation of C1 domains has impeded the development of selective ligands. We characterized here a diacylglycerol-lactone, 130C037, emerging from a combinatorial chemical synthetic strategy, which showed substantial selectivity. 130C037 gave shallow binding curves for PKC isoforms alpha, beta, gamma, delta, and epsilon, with apparent Ki values ranging from 340 nm for PKCalpha to 29 nm for PKCepsilon. When binding to isolated C1 domains of PKCalpha and -delta, 130C037 showed good affinity (Ki= 1.78 nm) only for deltaC1b, whereas phorbol 12,13-dibutyrate showed affinities within 10-fold for all. In LNCaP cells, 130C037 likewise selectively induced membrane translocation of deltaC1b. 130C037 bound intact RasGRP1 and RasGRP3 with Ki values of 3.5 and 3.8 nm, respectively, reflecting 8- and 90-fold selectivity relative to PKCepsilon and PKCalpha. By Western blot of Chinese hamster ovary cells, 130C037 selectively induced loss from the cytosol of RasGRP3 (ED50 = 286 nm), partial reduction of PKCepsilon (ED50 > 10 microm), and no effect on PKCalpha. As determined by confocal microscopy in LNCaP cells, 130C037 caused rapid translocation of RasGRP3, limited slow translocation of PKCepsilon, and no translocation of PKCalpha. Finally, 130C037 induced Erk phosphorylation in HEK-293 cells ectopically expressing RasGRP3 but not in control cells, whereas phorbol ester induced phosphorylation in both. The properties of 130C037 provide strong proof of principle for the feasibility of developing ligands with selectivity among C1 domain-containing therapeutic targets.

Development of Genetic Techniques for the Psychrotrophic Fish Pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum, a member of the Cytophaga-Flavobacterium-Bacteroides group, is an important pathogen of salmonid fish. Previous attempts to develop genetic techniques for this fastidious, psychrotrophic bacterium have met with failure. Here we describe the development of techniques for the genetic manipulation of F. psychrophilum and the identification of plasmids, selectable markers, a reporter system, and a transposon that function in several isolates of this fish pathogen. The antibiotic resistance genes ermF, cfxA, and tetQ function in F. psychrophilum. Cloning vectors based on the F. psychrophilum cryptic plasmid pCP1 which carried these selectable markers were introduced by conjugation from E. coli, resulting in antibiotic-resistant colonies of F. psychrophilum. Conjugative transfer of DNA into F. psychrophilum was strain dependent. Efficient transfer was observed for two of the seven strains tested (THC02-90 and THC04-90). E. coli lacZY functioned in F. psychrophilum when expressed from a pCP1 promoter, allowing its development as a reporter for studies of gene expression. Plasmids isolated from F. psychrophilum were efficiently introduced into F. psychrophilum by electroporation, but plasmids isolated from E. coli were not suitable for transfer by this route, suggesting the presence of a restriction barrier. DNA isolated from F. psychrophilum was resistant to digestion by Sau3AI and BamHI, indicating that a Sau3AI-like restriction modification system may constitute part of this barrier. Tn4351 was introduced into F. psychrophilum from E. coli and transposed with apparent randomness, resulting in erythromycin-resistant colonies. The techniques developed in this study allow for genetic manipulation and analysis of this important fish pathogen.

Approach for selection of individual enteric bacteria for competitive exclusion in turkey poults

The intentional early colonization of the intestinal tract with beneficial microflora, known as competitive exclusion, has been shown to successfully protect poultry from selected enteric pathogens. Although effective cultures have been produced and are available, an inexpensive, air-tolerant, and completely defined culture is needed. Presently, we developed an in vitro competition assay to select for individual facultative anaerobes of poultry enteric origin that could exclude Salmonella. Using this assay, 24 isolates were selected and stored individually. These 24 isolates were amplified in batch culture (tryptic soy broth, 4 h at 40 degrees C) and administered at final dilutions of 10, 100, or 1,000 cfu to day-of-hatch poults. Forty-eight hours later, poults were challenged with 100 to 1,000 cfu antibiotic-resistance-marked Salmonella enteritidis PT 13A by oral gavage. Five days later, all poults were killed, and cecal tonsils were aseptically removed for tetrathionate enrichment (24 h at 37 degrees C) followed by selective plating with marker antibiotics. Selected lactose-negative, antibiotic-resistant colonies typical of Salmonella were further confirmed by serogrouping. Treatment-related protection ranged from 0 to 100% in three experiments. Greatest protection was related to the lowest concentrations of the protective microflora in each experiment. These data suggest that effective combinations of competitive enteric microflora can be identified by appropriate in vitro selection methods.

Identification of 113 conserved essential genes using a high-throughput gene disruption system in Streptococcus pneumoniae.

The recent availability of bacterial genome sequence information permits the identification of conserved genes that are potential targets for novel antibiotic drug discovery. Using a coupled bioinformatic/experimental approach, a list of candidate conserved genes was generated using a Microbial Concordance bioinformatics tool followed by a targeted disruption campaign. Pneumococcal sequence data allowed for the design of precise PCR primers to clone the desired gene target fragments into the pEVP3 'suicide vector'. An insertion-duplication approach was employed that used the pEVP3 constructs and resulted in the introduction of a selectable chloramphenicol resistance marker into the chromosome. In the case of non-essential genes, cells can survive the disruption and form chloramphenicol-resistant colonies. A total of 347 candidate reading frames were subjected to disruption analysis, with 113 presumed to be essential due to lack of recovery of antibiotic-resistant colonies. In addition to essentiality determination, the same high-throughput methodology was used to overexpress gene products and to examine possible polarity effects for all essential genes.