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Broth Culture (broth + culture)
Selected AbstractsGrowth-induced changes in the proteome of Helicobacter pyloriELECTROPHORESIS, Issue 5-6 2006Christina Uwins Abstract Helicobacter pylori is a major human pathogen that is responsible for a number of gastrointestinal infections. We have used 2-DE to characterise protein synthesis in bacteria grown either on solid agar-based media or in each of two broth culture media (Brucella and brain heart infusion (BHI) broth). Significant differences were observed in the proteomes of bacteria grown either on agar-based or in broth media. Major changes in protein abundance were identified using principal component analysis (PCA), which delineated the profiles derived for the three key growth conditions (i.e. agar plates, Brucella and BHI broth). Proteins detected across the gel series were identified by peptide mass mapping and Edman sequencing. A number of proteins associated with protein synthesis in general as well as specific amino acid synthesis were depressed in broth-grown bacteria compared to plate-grown bacteria. A similar reduction was also observed in the abundance of proteins involved in detoxification. Two of the most abundant spots, identified as UreB and GroEL, in plate-grown bacteria showed a >140-fold drop in abundance in bacteria grown in Brucella broth compared to bacteria grown on agar plates. Two protein spots induced in bacteria grown in broth culture were both identified as glyceraldehyde 3-phosphate dehydrogenase based on their N -terminal amino acid sequences derived by Edman degradation. The underlying causes of the changes in the proteins abundance were not clear, but it was likely that a significant proportion of the changes were due to the alkaline pH of the broth culture media. [source] Disruption of nasopharyngeal epithelium by pneumococci is density-linkedEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 4 2003K. Lagrou Abstract Background The aim of this project was to study the influence of pneumococci on nasopharyngeal epithelial integrity as a function of time and pneumococcal density. Materials and methods Cell layers of an in vitro model of human nasopharyngeal epithelium were inoculated with different pneumococcal strains. The transepithelial electrical resistance (TEER), a measure of the integrity of the cell layers, and the pneumococcal concentration in the apical fluid on the epithelial cells were measured at different times after inoculation. Results Pneumococci caused a decrease in the TEER when a density of 1 × 107 CFU mL,1 was reached. The growth rate of pneumococci in our in vitro model differed between the strains tested and, for the same strain, between in vitro culture on the epithelial cells and broth culture. Differences in timing of the onset of decrease in the TEER between strains were the result of differences in growth rate on the epithelial cells. Antibiotic-induced lysis of pneumococci caused an immediate decrease in the TEER of the cell layers. Conclusion Pneumococci cause a decrease in the TEER at a density of 1 × 107 CFU mL,1. Our hypothesis is that this decrease in the TEER is the result of quorum-induced lysis of the pneumococci. [source] Detection of viable Escherichia coli O157:H7 by ethidium monoazide real-time PCRJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2009L. Wang Abstract Aims:, The aim of this study was to develop and optimize a novel method that combines ethidium bromide monoazide (EMA) staining with real-time PCR for the detection of viable Escherichia coli O157:H7 in ground beef. EMA can penetrate dead cells and bind to intracellular DNA, preventing its amplification via PCR. Methods and Results:, Samples were stained with EMA for 5 min, iced for 1 min and exposed to bright visible light for 10 min prior to DNA extraction, to allow EMA binding of the DNA from dead cells. DNA was then extracted and amplified by TaqMan® real-time PCR to detect only viable E. coli O157:H7 cells. The primers and TaqMan® probe used in this study target the uidA gene in E. coli O157:H7. An internal amplification control (IAC), consisting of 0·25 pg of plasmid pUC19, was added in each reaction to prevent the occurrence of false-negative results. Results showed a reproducible application of this technique to detect viable cells in both broth culture and ground beef. EMA, at a final concentration of 10 ,g ml,1, was demonstrated to effectively bind DNA from 108 CFU ml,1 dead cells, and the optimized method could detect as low as 104 CFU g,1 of viable E. coli O157:H7 cells in ground beef without interference from 108 CFU g,1 of dead cells. Conclusions:, EMA real-time PCR with IAC can effectively separate dead cells from viable E. coli O157:H7 and prevent amplification of DNA in the dead cells. Significance and Impact of the Study:, The EMA real-time PCR has the potential to be a highly sensitive quantitative detection technique to assess the contamination of viable E. coli O157:H7 in ground beef and other meat or food products. [source] Physicochemical properties of Shiga toxigenic Escherichia coliJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2005L. Rivas Abstract Aims:, To investigate the physicochemical surface properties, such as cellular surface charge, hydrophobicity and electron donor/acceptor potential of a selection of Shiga toxigenic Escherichia coli (STEC) isolates grown in broth and agar culture. Methods and Results:, Cellular surface charge was determined using zeta potential measurements. Hydrophobicity of the isolates was determined using bacterial adhesion to hydrocarbons assay, hydrophobic interaction chromatography and contact angle measurements. Microbial adhesion to solvents was used to determine the electron donor/acceptor characteristics. No differences of surface charge measurements were found between broth and agar grown cultures. Isolates belonging to serogroup O157 and serotypes O26:H11 and O111:H- were significantly (P < 0·05) less negatively charged than other STEC serotypes tested. All strains were hydrophilic with most methods and demonstrated a lower hydrophobicity in agar culture compared with broth culture. All strains demonstrated a strong microbial adhesion to chloroform indicating that STEC possess an electron donor and basic character. A relationship between serogroup O157 and other STEC serotypes was apparent using principal-component analysis (PCA). Conclusions:, Combining the results for physicochemical properties using PCA differentiated between strains belonging to the O157 serogroup and other STEC/non-STEC strains. PCA found similar results for broth and agar grown cultures. Significance and Impact of the Study:, Particular serotypes of STEC possess similar physicochemical properties which may play a role in their pathogenicity or potential attachment to various surfaces. [source] Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulenceMOLECULAR MICROBIOLOGY, Issue 2 2002Yaowei Kang Summary As reported previously for Ralstonia solanacearum strain GMI1000, wild-type strains AW1 and K60 were shown to produce Hrp pili. AW1 and K60 mutants lacking Hrp pili still exhibited twitching motility, which requires type 4 pili (Tfp), and electron microscopy revealed that they still made flexuous polar pili. Twitching-positive cells had an extracellular 17 kDa protein that was associated with piliation, and an internal 43-amino-acid sequence of this protein was typical of type 4 pilins. This amino acid sequence is encoded by an open reading frame, designated pilA, in the genomic sequence of GMI1000. PilA is 46% identical to a Pseudomonas aeruginosa type 4 pilin over its entire length and has all the conserved residues and motifs characteristic of type 4 group A pilins. pilA mutants did not make the 17 kDa PilA protein and did not exhibit twitching motility. When compared with its parent, an AW1 pilA mutant was reduced in virulence on tomato plants and in autoaggregation and biofilm formation in broth culture. Unlike AW1, a pilA mutant did not exhibit polar attachment to tobacco suspension culture cells or to tomato roots; it was also not naturally competent for transformation. We reported previously that twitching motility ceases in maturing AW1 colonies and that inactivation of PhcA, a global transcriptional regulator, results in colonies that continue to exhibit twitching motility. Similarly, in broth culture, expression of a pilA::lacZ fusion in AW1 decreased 10-fold at high cell density, but expression remained high in a phcA mutant. In addition, pilA::lacZ expression was positively regulated 10-fold by PehR, a response regulator that is known to be repressed by PhcA. This signal cascade is sufficient to explain why pilA expression, and thus twitching motility, decreases at high cell densities. [source] Pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in goatsAUSTRALIAN VETERINARY JOURNAL, Issue 6 2007GJ Eamens Objective, To evaluate pooled faecal culture for herd diagnosis of caprine Johne's disease and relate these findings to faecal shedding rates of Mycobacterium avium subsp paratuberculosis (Map). Design, Radiometric broth culture was applied to several pooling dilutions, and shedding rates were estimated from a regression equation based on bacterial growth rates and known processing losses during radiometric culture. Procedure, Sixteen faecal samples from goats naturally infected with sheep (n = 3) or cattle (n = 13) strains of Map, were diluted in normal goat faeces from 1 in 5 to 1 in 50. Cultures were confirmed by IS900 polymerase chain reaction and restriction endonuclease analysis, and mycobactin dependency. The numbers of viable Map in the culture inocula were determined by endpoint titration (most probable number) of nine samples and related to a cumulative growth index. Results, A pooling dilution of 1 in 25 with an incubation period of 10 weeks detected 13 of 16 culture positive goats, all shedding , 2 × 104 Map per gram of faeces. Two samples containing very low numbers of Map (< 2 × 103/g) were only culture positive from undiluted faeces. Thirteen of 16 goats were considered to be shedding low to moderate concentrations of Map (< 2 × 105/g faeces). Conclusions, These data support a pooling dilution of 1 in 25 for application of pooled faecal culture as a diagnostic tool in caprine Johne's disease control. A test based on this dilution would reduce laboratory costs of whole herd testing in goats by approximately 40% relative to serology and 75 to 90% relative to individual faecal culture. [source] Identification of an operon and inducing peptide involved in the production of lactacin B by Lactobacillus acidophilusJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2007A.E. Dobson Abstract Aim: To determine if a 9·5-kb region on the Lactobacillus acidophilus NCFM genome, encoded the genetic determinants for regulation and production of lactacin B, a class II bacteriocin. Methods: Transcriptional analysis was used to identify a 9·5-kb polycistronic region suspected of encoding the lab operon. The 12 putative open reading frames (LBA1803,LBA1791) were organized into three clusters: a production and regulation cluster encoding a putative two-component signal transduction system; an export cluster encoding a putative ABC transporter and a final cluster composed of three unknown proteins. Seven genes were typical of bacteriocins, encoding small, cationic peptides, each with an N-terminal double-glycine leader motif. Inactivation of a predicted ABC transporter completely abolished bacteriocin activity. When cloned and expressed together, LBA1803,LBA1800 resulted in markedly higher levels of lactacin B activity. The four peptides were chemically synthesized but exhibited no bacteriocin activity, alone or in combination. Only LBA1800 induced lactacin B production in broth cultures. Conclusions: Lactacin B production is encoded within the 9·5-kb lab operon of 12 genes that are transcribed in a single transcript. LBA1800 is an inducing peptide of bacteriocin production. Significance and Impact of the Study: A three-component regulatory system common to class II bacteriocins regulates the production of this bacteriocin by Lact. acidophilus. [source] Relevance of incubation temperature for Vibrio salmonicida vaccine productionJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2002D.J. Colquhoun Aims:,To investigate the relationships between water temperature, bacterial growth, virulence and antigen expression in Vibrio salmonicida, the causal agent of cold water vibriosis in Atlantic salmon (Salmo salar L.). Methods and Results:,The significance of sea temperature was investigated using historical clinical and oceanographic data. An upper threshold for disease of approx. 10°C was established. The effects of culture temperature and media type on bacterial growth were studied on solid and in liquid media. The highest rates of cell division were identified at 15°C on solid media and 10°C in liquid media. Outer membrane protein (OMP) expression and serological response in Atlantic salmon were studied using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. A novel 76-kDa OMP produced in unshaken cultures at 10°C was not found to stimulate a specific humoral response. Conclusions:,Diagnostic agar plate-based incubation of suspected V. salmonicida should be carried out at 15°C. High yield broth cultures for vaccine production should be incubated at 10°C or lower. Significance and Impact of the Study:,This study is, to the best of our knowledge, the first to identify different optimal temperatures in a bacterial species cultured on physically different types of media. The evidence presented suggests that V. salmonicida and possibly other bacteria destined for vaccine use in poikilothermic organisms should be cultured at temperatures consistent with that at which disease occurs. [source] Isolation and Characterization of Lactobacillus Species Having Potential for Use as Probiotic Cultures for DogsJOURNAL OF FOOD SCIENCE, Issue 3 2007S. McCoy ABSTRACT:, The need to control pathogenic microorganisms in the intestinal tract of dogs is a growing concern. There is interest in using probiotics such as species of Lactobacillus to help control canine intestinal infections. For successful use as a probiotic, the bacterial species should be of canine intestinal origin since these species exhibit host specificity. Serial dilutions of freshly voided dog feces were plated on Lactobacillus selection (LBS) agar to isolate the cultures. Isolates were identified based on Gram stain, catalase test, and fermentation patterns using API 50 CH kits. All potential isolates were compared for bile resistance based on relative ability to grow in broth containing 0.3% Oxgall, the ability to inhibit Salmonella Typhimurium in associative broth cultures, and the production of reuterin. Of the lactobacilli isolated, Lactobacillus reuteri was the dominant species. However, some cultures of L. acidophilus also were isolated. We found variations among the isolates of L. reuteri and L. acidophilus with respect to bile tolerance. In general, isolates of L. reuteri appeared to be more bile resistant than were isolates of L. acidophilus. There were also variations in the ability to inhibit growth of S. Typhimurium. Some isolates of L. reuteri produced reuterin while others did not. [source] Effect of High-Pressure Processing on Vibrio parahaemolyticus Strains in Pure Culture and Pacific OystersJOURNAL OF FOOD SCIENCE, Issue 4 2002H. Calik ABSTRACT Different strains of Vibrio parahaemolyticus (Vp) in broth cultures and Vp-inoculated live Pacific oysters (Crassostrea gigas) were subjected to high-pressure processing (HPP) at 241, 276, 310, and 345 MPa. Results showed Vp numbers were reduced by HPP in both pure culture and whole oysters. Vp inactivation was dependent on time and pressure. Optimum conditions for reducing Vp in pure culture and oysters to nondetectable levels were achieved at 345 MPa for 30 and 90 s, respectively. Resistance variations were detected between Vp in pure culture and in oysters. HPP proved to be an efficient means of reducing Vp in oysters. [source] | |||||||||||||