Selective Plating Media Research Articles

Multifaceted Recognition of Vertebrate Rev1 by Translesion Polymerases ζ and κ

Translesion synthesis is a fundamental biological process that enables DNA replication across lesion sites to ensure timely duplication of genetic information at the cost of replication fidelity, and it is implicated in development of cancer drug resistance after chemotherapy. The eukaryotic Y-family polymerase Rev1 is an essential scaffolding protein in translesion synthesis. Its C-terminal domain (CTD), which interacts with translesion polymerase ζ through the Rev7 subunit and with polymerases κ, ι, and η in vertebrates through the Rev1-interacting region (RIR), is absolutely required for function. We report the first solution structures of the mouse Rev1 CTD and its complex with the Pol κ RIR, revealing an atypical four-helix bundle. Using yeast two-hybrid assays, we have identified a Rev7-binding surface centered at the α2-α3 loop and N-terminal half of α3 of the Rev1 CTD. Binding of the mouse Pol κ RIR to the Rev1 CTD induces folding of the disordered RIR peptide into a three-turn α-helix, with the helix stabilized by an N-terminal cap. RIR binding also induces folding of a disordered N-terminal loop of the Rev1 CTD into a β-hairpin that projects over the shallow α1-α2 surface and creates a deep hydrophobic cavity to interact with the essential FF residues juxtaposed on the same side of the RIR helix. Our combined structural and biochemical studies reveal two distinct surfaces of the Rev1 CTD that separately mediate the assembly of extension and insertion translesion polymerase complexes and provide a molecular framework for developing novel cancer therapeutics to inhibit translesion synthesis.

Comparison of 2 culture methods and PCR assays for Salmonella detection in poultry feces

The present work compared 2 culture methods and the combinations of pre-enrichment and enrichment culture methods with PCR assays [buffered peptone water-PCR and tetrathionate-PCR or modified semisolid Rappaport-Vassiliadis (MSRV)-PCR] for motile and nonmotile Salmonella strain detection using artificially contaminated poultry feces. The specificity and positive predictive values were equal to one in both culture methods. Specificity and positive predictive values, accuracy, sensitivity, and negative predictive values were higher for motile than nonmotile Salmonella strains in culture methods. Only Salmonellaenterica serovar Gallinarum was detected by the MSRV method with low accuracy, sensitivity, and negative predictive value. The detection level of motile strains was 2 ×100 to 22 × 102 cfu per 25 g for these methods, whereas it was 6.9 × 102 cfu per 25 g in culture methods for Salmonella Gallinarum. Extending the incubation time of the enrichment medium to 6 d in the TT method did not improve the isolation rates. In general, all selective plating media did not show any statistical differences in the parameters of performance studied. On the other hand, accuracy and sensitivity values were higher in MSRV-PCR and tetrathionate-PCR methods than in the buffered peptone water-PCR method. Specificity and positive predictive values were equal to one in most of the cases. In terms of detection limits, motile Salmonella strains were recovered from 5 × 100 cfu per 25 g in MSRV-PCR and tetrathionate-PCR methods, whereas the detection limit was better for nonmotile Salmonella in MSRV-PCR methods than in the tetrathionate-PCR method. Kappa coefficients showed that there was a very good agreement between tetrathionate and MSRV methods for motile Salmonella strains, whereas these methods did not show any concordance for nonmotile Salmonella strains. When buffered peptone water-PCR was compared with both tetrathionate-PCR and MSRV-PCR, agreement was poor for motile Salmonella strains and slight to fair for nonmotile Salmonella strains. The difference in isolation rate obtained with the methods used for motile and nonmotile Salmonella strains must be taken into account when a poultry fecal sample is considered negative for the presence of Salmonella.

人工湿地不同季节与单元之间根际微生物多样性

Selective plating medium and PCR-DGGE techniques were applied to analyze the seasonal changes in the number and diversity of micro-organisms in the rhizosphere of three sequenced cells in a vertical subsurface-flow wetland system constructed for municipal sewage treatment.The relationship between the community structures of the microorganisms and their functions in removal of the organic pollutants was also analyzed. The results obtained from the two applied methods showed that with the seasonal changes in the sequence of autumn-winter-spring-summer,the number of the micro-organisms displayed a general up-down-up pattern.The largest number of microorganisms from the rhizoshpere in three treatment cells detected by the selective plating medium method was in the autumn while that by the PCR-DGGE techniques was in the summer.Correlation analysis indicated that the seasonal change in temperature was the main factor that determined the observed pattern by affecting the growth of the microorganisms.In a given season,the number of micro-organisms obtained from the two applied methods displayed a different pattern in the three sequenced treatment cells.Towards the flow direction in the sewage treatment process,the number of micro-organisms increased from the first cell to the second cell and then decreased in the third cell,accordingly,with most of the organic pollutants being removed in the first two treatment cells. Based on the structure comparability of the microbial community among treatment cells,all soil samples collected in the same season,except for sample no.11,were clustered in the same branch,with all DGGE comparability values being higher than 20%,which can thus be classified into the same group.The DGGE spectrograms of the soil microorganisms from different seasons are different.The highest comparability value,40%,was found in the spring,while the lowest,about 25%,was in the summer,indicating that the level of the microbial diversity was higher in the summer season under the tested conditions. Four different sequences(YP1—YP 4)were obtained,and their closest known-origin groups were identified by BLASTX analysis as Enterobacter sp.,Vibrio sp.,Erwinia sp.and Klebsiella oxytoca,respectively.The BLASTX comparison tests showed that YP1 played an important role in decomposing organic matters;YP2 was fund to be related to the life activities of the Cerea Spore Bacilli with functions in phosphate dissolution;YP3 had functions similar to the apolipoprotein e(APOE) that involved in catabolism and metabolism of micro-organisms;and YP4 had an effect on nitrogen removal subject to microbial activities.

A comparative study of culture methods and polymerase chain reaction assay for Salmonella detection in poultry feed

The present work compared 2 culture methods and PCR assay for the detection of motile and non-motile Salmonella strains using artificially contaminated poultry feed. The specificity was 1 in all methods. The accuracy and sensitivity were between 0.5 and 1 for motile Salmonella strains, whereas these parameters were between 0 and 0.6 for non-motile Salmonella strains. The positive predictive value was 1 for tetrathionate (TT), PCR, and modified semisolid Rappaport-Vassiliadis (MSRV) methods in most of the strains studied. The negative predictive value of each method was very low for non-motile Salmonella strains. The detection level of motile strains was 8 to 20 cfu/25 g for all methods, whereas it was ≥10(4) cfu/25 g in culture methods for non-motile Salmonella strains. In general, the PCR method detected lower non-motile Salmonella contamination levels in feed than did culture methods. Extending incubation time of the enrichment medium to 6 d in the TT method did not improve the isolation rates. All selective plating media did not show any statistical differences in the parameters of performance studied. Kappa coefficients showed that there was good agreement between TT and MSRV methods, and MSRV and PCR methods for motile Salmonella strains in poultry feed samples. The agreement was fair between TT and PCR methods for these strains. For non-motile Salmonella strains, there was poor (TT and MSRV methods), slight (PCR and TT methods), and fair (MSRV and PCR methods) agreement. The TT, MSRV, and PCR methods are similar in terms of accuracy, sensitivity, specificity, positive predictive value, and negative predictive value for different motile Salmonella strains in poultry feed. For non-motile Salmonella strains, the use of the PCR method improves the same parameters, described before, in this matrix. The difference in detection levels obtained with the methods used for motile and nonmotile Salmonella strains and the difficulty to detect these last strains represent a potential problem, when a poultry feed sample is considered negative for the presence of Salmonella.

Development and Characterization of High Affinity Leptins and Leptin Antagonists

Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin.

Enterobacteriaceae Associated with Eggs of Podocnemis expansa and Podocnemis unifilis (Testudines: Chelonia) in Nonpolluted Sites of National Park of Araguaia Plains, Brazil

Fertile eggs of Podocnemis expansa and Podocnemis unifilis were investigated for the presence of enterobacteria, as these two endangered species have the potential for conservation measures that include egg transfer. Knowledge of normal microflora associated with turtles and turtle eggs would help effectively manage the transfer of these eggs among institutions. Thirty eggs of each species were collected, aseptically transferred, cracked inside plastic bags containing tetrathionate broth, and spread on selective media plates. Ten samples of sand and water were also examined for the presence of fecal coliforms using Colilert Quanti-Tray kits. Enterobacteriaceae were identified using an API 20E biochemical test kit. A majority of the bacteria isolated were potential pathogens. All egg samples were positive for Enterobacteriaceae, among which 15 eggs of P. expansa were positive for Shigella flexneri. Other isolates included Chromobacterium violaceum, Escherichia coli, and Aeromonas salmonicida subsp. salmonicida. Eggs of P. unifilis were most frequently positive for Shigella flexneri and C. violaceum, and less frequently positive for A. salmonicida subsp. salmonicida and Salmonella choleraesuis subsp. arizonae. The source of the bacteria in these eggs is unknown, but the nesting beaches were considered a source as water samples had low total coliform counts and E. coli was isolated from only one sand sample. This study demonstrated that Enterobacteriaceae are part of the indigenous microflora of chelonians.

Evaluation of Bacillus strains as model systems for the work on Bacillus anthracis spores

Available strain collections of Bacillus anthracis and Bacillus cereus were screened for B. cereus strains sharing major genotypic characteristics with B. anthracis. Based on the comparison of partial spoIIIAB sequences, whole genome sequences and MLST, a strain set representing different lineages including candidate model strains for B. anthracis was compiled. Spores from the selected strain set and two B. anthracis strains were prepared according to a newly optimized protocol transferable to biosafety level-3 (BSL3) conditions and phenotypic characteristics including scanning electron microscopy (SEM), heat inactivation, and germination were evaluated. Two B. cereus isolates were identified that were genetically related to B. anthracis and showed high similarity to B. anthracis spores in their heat inactivation profile and their response to the germinants l-alanine and inosine. In addition, these isolates were also mimicking B. anthracis on modified PLET, a selective plating medium for B. anthracis, and shared various other biochemical characteristics with B. anthracis. Therefore these two strains are not only appropriate models for B. anthracis in experiments based on spore characteristics but also in trials working with plating media. These two strains are now used within the BIOTRACER consortium as validated models for B. anthracis and will facilitate the development and optimization of tracing and detection systems for B. anthracis in the food and feed chain.

Catabolite repression of SOS-dependent and SOS-independent spontaneous mutagenesis in stationary-phase Escherichia coli

Previous work in our laboratory established that a spontaneous mutagenesis process operating in stationary-phase Escherichia coli cells undergoing selection is subject to regulation by the global regulatory mechanism known as catabolite repression (formerly also called glucose-repression). Here, we set out to determine the identity of this hitherto unknown catabolite-repressible spontaneous mutation generation mechanism(s). We used two different spontaneous mutation detection assays, reversion of a Lac(-) (lacI33OmegalacZ) frameshift marker and forward mutation to valine-resistance, and tested the effects of varying the nature of the carbon source(s) present in the selective plating medium on the mutability of bacterial cells carrying known defects in the recA, umuDC and dinB genes, three well-known SOS response genes, whose products are important for mutagenesis in E. coli. Consistent with the results of our previous Lac(-)-->Lac(+) assay using otherwise SOS-proficient bacterial cells, we found that the overall numbers of spontaneous Lac(+)E. coli revertants were highest when the selective medium contained lactose and lowest when it contained lactose plus the non-utilizable but strongly catabolite-repressing glucose analogue, methyl-alpha-d-glucopyranoside (alphaMG). In contrast, we found that the numbers of Lac(+) revertants appearing on the lactose and lactose+alphaMG selection plates were greatly diminished and not significantly different when the bacterial cells concerned carried either a DeltarecA or DeltadinB mutation. Furthermore, introducing the DeltadinB mutant allele into bacterial cells over-expressing the recA gene reduced the numbers of Lac(+) mutations to those being recovered with the DeltadinB cells. These results appear to suggest that (i) the DinB-dependent mutation generation pathway is alone responsible for spontaneous reversion of the lacI33OmegalacZ frameshift marker, and (ii) the varying numbers of Lac(+) colonies that we recover on the lactose and lactose+alphaMG plates provide a direct measure of the differential effects of these particular carbon compounds on the overall expression of the dinB gene. Interestingly, the yields of spontaneous Val mutations arising in wild-type, DeltarecA, DeltadinB and DeltaumuDC cells were found to be similar, but always tended to be highest when the medium contained only a non-repressing carbon source (glycerol) and lowest when it had been supplemented with a strong catabolite repressor such as glucose or alphaMG. Together, our results would seem to establish that stationary-phase E. coli cells exposed to strong selection pressures can accumulate spontaneous mutations via SOS-dependent and SOS-independent mutation generation pathways whose levels of expression are regulated by catabolite repression.

The study of pathogenic microbial communities in graywater using membrane bioreactor

Wastewater originating from any source in the residence except for the toilet is defined as graywater. If graywater is treated appropriately, it can be used as reused water. However, wastewater reclamation carries certain health risks, and hence this study is an analysis of pathogenic microorganism and microbial communities in treated graywater for the reuse of water treated by MBR. To reuse graywater, MBR system of a lab scale was constructed with sediment, anaerobic, anoxic and oxic reactors. In the oxic reactor, a submerged MF (pore size is 0.45 μm) membrane was installed to maintain activated sludge biomass. For the quantification of pathogenic organisms, a standard spread plate method was modified using the selective medium plates. Analysis of 16S rDNA was conducted to detect microbial community. Pathogenic microorganisms such as Escherichia coli, Coliform, Staphylococcus aureus and Salmonella were detected in effluent. According to analysis of phylum and class levels, species of microorganism become simplified through membrane. This suggests that the MF membrane in the MBR system could not perfectly remove microorganisms and further research in diverse pathogens is needed for wastewater reclamation.

Development of spoilage microbiota in beef stored in nisin activated packaging

The aim of this study was to assess the microbial populations causing the spoilage of chilled beef during storage and to evaluate the effect of the use of an antimicrobial packaging for the meat storage. A nisin activated antimicrobial packaging was developed by using a nisin, HCL and EDTA solution and used for the storage of beef cuts at 1 °C. The common spoilage related microbial groups were monitored during the storage of beef in activated and non activated plastic bags by using selective media. The use of the antimicrobial packaging caused an overall significant reduction of viable counts of Gram positive bacteria such as carnobacteria, lactic acid bacteria and Brochotrix thermosphacta whose development was inhibited for at least 11 days of storage compared to the control. Moreover, a 1–3 log cycles reduction of enterobacteria was also registered between 22 and 32 days of storage. The microbiota was assessed at species level by using Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of 16S rRNA gene of DNA extracted directly from meat and from bulk cells from selective media plates and showed that the species occurring within the targeted microbial groups did not change according to storage conditions. In conclusion, the use of the nisin activated packaging reduced the number of spoilage populations but did not affect the species diversity. Improved antimicrobial packaging is needed, possibly coupled with vacuum storage, to possibly achieve a simultaneous inhibition of more spoilage microbial groups and to preserve the microbiological quality of beef during chilled storage.

Protective effect of milk constituents and sublethal injuries limiting process effectiveness during PEF inactivation of Lb. rhamnosus

The inactivation of Lb. rhamnosus by pulsed electric field treatment (PEF) was studied in different fractions of raw milk and Ringer solution in order to evaluate the protective effect of nutrient rich media in comparison to aqueous buffer solutions. Apart from monitoring of culturability, analysis of the physiological fitness of Lb. rhamnosus was conducted aiming to identify sublethally damaged cells. Therefore, flow cytometry and a selective medium plating technique were used and compared to each other. The goal of the study was to apply three different parameters describing the physiological fitness of the model organism Lb. rhamnosus after PEF treatment such as culturability, membrane permeability and metabolic activity depending on treatment media and parameters. A concentration dependent protective effect of the milk protein fraction could be shown and allocated to micellar casein as the major milk protein. Increasing the concentration of whey proteins up to 2% showed a similar impact on limiting the PEF inactivation of Lb. rhamnosus. The evaluation of physiological fitness of cells was based on a determination of structural and functional characteristics by rapid cellular staining using carboxyfluorescein diacetate and propidium iodide. This approach showed good accordance to the conventional selective medium plating technique for the enumeration of sublethally-injured bacteria but flow cytometry provided additional information for the characterisation of this fraction. The extent of occurrence of dead, sublethal and vital fractions of cells was found dependent on the PEF treatment parameters such as electrical field strength and energy input as well as the different milk fractions used as treatment media.

An Approach for Mapping the Number and Distribution of Salmonella Contamination on the Poultry Carcass

Mapping the number and distribution of Salmonella on poultry carcasses will help guide better design of processing procedures to reduce or eliminate this human pathogen from poultry. A selective plating media with multiple antibiotics (xylose-lysine agar medium [XL] containing N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) and the antibiotics chloramphenicol, ampicillin, tetracycline, and streptomycin [XLH-CATS]) and a multiple-antibiotic-resistant strain (ATCC 700408) of Salmonella Typhimurium definitive phage type 104 (DT104) were used to develop an enumeration method for mapping the number and distribution of Salmonella Typhimurium DT104 on the carcasses of young chickens in the Cornish game hen class. The enumeration method was based on the concept that the time to detection by drop plating on XLH-CATS during incubation of whole chicken parts in buffered peptone water would be inversely related to the initial log number (N0)of Salmonella Typhimurium DT104 on the chicken part. The sampling plan for mapping involved dividing the chicken into 12 parts, which ranged in average size from 36 to 80 g. To develop the enumeration method, whole parts were spot inoculated with 0 to 6 log Salmonella Typhimurium DT104, incubated in 300 ml of buffered peptone water, and detected on XLH-CATS by drop plating. An inverse relationship between detection time on XLH-CATS and N0 was found (r =−0.984). The standard curve was similar for the individual chicken parts and therefore, a single standard curve for all 12 chicken parts was developed. The final standard curve, which contained a 95% prediction interval for providing stochastic results for N0, had high goodness of fit (r2 = 0.968) and was N0 (log) = 7.78±0.61 − (0.995 × detention time). Ninety-five percent of N0 were within ± 0.61 log of the standard curve. The enumeration method and sampling plan will be used in future studies to map changes in the number and distribution of Salmonella on carcasses of young chickens fed the DT104 strain used in standard curve development and subjected to different processing procedures.

Isolation and identification of Campylobacter spp. and Campylobacter coli from poultry carcasses by conventional culture method and multiplex PCR in Mashhad, Iran

Summary The genus Campylobacter is of great importance to public health because it includes several species that may cause diarrhoea. Poultry and poultry products are known as important sources of human campylobacteriosis. In this study, during the autumn months of 2005, a total of 100 samples from poultry carcasses, representing 20 broiler flocks were obtained by rinse test, after the chilling stage of processing. The samples were enriched in Preston broth, followed by streaking on selective media. Then, the suspected colonies were isolated on sheep blood agar and tested for morphology, motility and Gram-staining. Biochemical tests and hippurate hydrolysis activity were also performed. Concurrently, a multiplex PCR assay (m-PCR) with two sets of primers was employed for identification of Campylobacter genus and Campylobacter coli. The m-PCR assay was applied on bacterial cultures harvested from selective media plates. By conventional culture method, including hippurate hydrolysis test from suspected colonies, 76% of samples were positive for Campylobacter spp. and 2% for C. coli. In m-PCR assay 28% of the harvested cultures, were positive for Campylobacter genus but C. coli were not detected in any of the samples. According to this preliminary study, it seems that the contamination rate of poultry carcasses with other species of Campylobacter genus is higher than contamination with C. coli.

Recovery of Saccharomyces cerevisiae sublethally injured cells after Pulsed Electric Fields

The objective was to investigate the influence of the recovery liquid medium on the repair of Saccharomyces cerevisiae sublethally injured cells after Pulsed Electric Fields (PEF) in media of different pH. Sublethal injury was detected in the yeast S. cerevisiae after 50 pulses at 12.0 kV cm − 1 in both pH 4.0 and 7.0, by using a selective medium plating technique. PEF treatments cause a repairable sublethal injury in S. cerevisiae. Injured cells showed their maximum repair capacity when suspended in Sabouraud Broth compared to Peptone Water or citrate–phosphate buffer of pH 4.0. The extent to which cells repair their injuries depended on the treatment medium pH, and on the nature of the storage medium. No repair was detected when the recovery liquid medium was citrate–phosphate buffer of pH 7.0. Acid conditions favour repair and survival of PEF-treated S. cerevisiae cells. This work contributes to the description of the mechanisms of PEF injury and inactivation, which would be useful in defining adequate PEF treatments, alone or in combination with additional hurdles, to assure food stability.

Use of Human Lysozyme Transgenic Goat Milk in Cheese Making: Effects on Lactic Acid Bacteria Performance

Genetically engineered goats expressing elevated levels of the antimicrobial enzyme lysozyme in their milk were developed to improve udder health, product shelf life, and consumer well-being. The purpose of this study was to evaluate the effect of lysozyme on the development of lactic acid bacteria (LAB) throughout the cheese-making process. Raw and pasteurized milk from 7 lysozyme transgenic goats and 7 breed-, age-, and parity-matched nontransgenic controls was transformed into cheeses by using industry methods, and their microbiological load was evaluated. The numbers of colony-forming units of LAB were determined for raw and pasteurized goat milk, whey, and curd at d 2 and at d 6 or 7 of production. Selective plating media were used to enumerate lactococcal species separately from total LAB. Although differences in the mean number of colony-forming units between transgenic and control samples in raw milk, whey, and cheese curd were non-significant for both total LAB and lactococcal species from d 2 of production, a significant decrease was observed in both types of LAB among d 6 transgenic raw milk cheese samples. In pasteurized milk trials, a significant decrease in LAB was observed only in the raw milk of transgenic animals. These results indicate that lysozyme transgenic goat milk is not detrimental to LAB growth during the cheese-making process.

A collaborative study on a Nordic standard protocol for detection and enumeration of thermotolerant Campylobacter in food (NMKL 119, 3. Ed., 2007)

A Nordic standard protocol for detection and enumeration of thermotolerant Campylobacter in food has been elaborated (NMKL 119, 3. Ed., 2007). Performance and precision characteristics of this protocol were evaluated in a collaborative study with participation of 14 laboratories from seven European countries. The laboratories performed qualitative, semi-quantitative and quantitative analyses on samples of chicken meat, cut lettuce, and milk artificially inoculated with different concentrations including blank duplicates of one strain of Campylobacter coli (SLV-271) and one strain of Campylobacter jejuni (SLV-542). Expected concentrations (95% C.I.) (cfu g − 1 or ml − 1 ) of both strains in matrices were 0.6–1.4 and 23–60 for qualitative detection, and 0.6–1.4; 23–60; and 420–1200 for semi-quantitative detection. For quantitative determination, the expected concentrations of C. jejuni/ C. coli were 420–1200/580–1100; 2100–6000/6300–11,000; and 4100–11,000/53,000–97,000 cfu g − 1 or ml − 1 . The qualitative and semi-quantitative techniques resulted in comparable detection. The overall specificity and sensitivity of the detection techniques was 98.6% (95% C.I.: 95.1–99.8%) and 82.8% (C.I.: 78.4–86.6%), respectively. The sensitivity was not influenced by food matrix ( P = 0.359), but was significantly lower for C. coli compared to C. jejuni ( P = 0.007) and for concentrations below 1.4 cfu g − 1 ( P < 0.001). The detection techniques were therefore only considered satisfactory for detection of Campylobacter concentrations above approximately 25 cfu g − 1 for all matrices tested, which was supported by calculation of values for accordance, concordance, and concordance odds ratio. No statistical difference was found between enumerations obtained by the semi-quantitative and quantitative techniques for comparable concentrations of Campylobacter (420–1200 cfu g − 1 or cfu ml − 1 ) ( P = 0.104). Both techniques underestimated concentrations of thermotolerant Campylobacter in milk. The semi-quantitative technique estimated low inoculation levels of Campylobacter more correctly than the high inoculation levels. Counts obtained on the two selective plating media, Abeyta–Hunt–Bark agar added to it 0.1% triphenyl tetrazolium chloride and modified charcoal cephoperazone desoxycholate agar were not significantly different ( P = 0.143). Expressed as an absolute difference between log 10-transformed results, the overall values for repeatability ( r) and reproducibility ( R) were r = log 10 0.43 and R = log 10 1.99, respectively. By omitting results from laboratories with high level of variability in results, R was reduced to log 10 1.88. We suggest that the poor detection of low numbers, the underestimation in milk samples, and the large variation between laboratories can be explained by the general difficulties in handling Campylobacter. In conclusion, NMKL 119, 3. Ed., 2007, is regarded as an acceptable protocol for detection of thermotolerant Campylobacter at concentrations above 25 cfu g − 1 and also for enumeration of thermotolerant Campylobacter in chicken meat.

Shiga-toxigenic Escherichia coli O157 and non-Shiga-toxigenic E. coli O157 respond differently to culture and isolation from naturally contaminated bovine faeces

To quantify the effect of enrichment, immunomagnetic separation (IMS), and selective plating procedures on isolation of Shiga-toxigenic Escherichia coli O157 (STEC O157) and non-Shiga-toxigenic Escherichia coli O157 (non-STEC O157) from naturally contaminated bovine faeces. Two broth enrichment times, two IMS strategies, and two selective plating media were evaluated. STEC O157 and non-STEC O157 strains were often isolated from the same faecal specimen and responded differently to the isolation protocols. A large-volume IMS system was more sensitive than a conventional small-volume IMS method, but was also more expensive. STEC O157 was more frequently isolated from 6 h enriched broth and ChromAgar plates containing 0.63 mg l(-1) potassium tellurite (TCA). Non-STEC O157 was more frequently isolated from un-enriched broth and ChromAgar plates without tellurite (CA). The combination of 6-h enrichment in Gram-negative broth containing vancomycin, cefixime and cefsuludin, large volume IMS and selective plating on TCA maximized STEC O157 recovery from naturally contaminated cattle faecal specimens. The pairing of proper enrichment with a specific plating procedure is key for STEC O157 recovery from naturally contaminated bovine faeces. Incorporating tellurite into an E. coli O157 detection strategy may select for the subset of E. coli O157 that contains the Shiga-toxin genes.

A survey of the microbial and chemical composition of seven semi-ripened Provola dei Nebrodi Sicilian cheeses

The microbial and chemical composition of seven different semi-ripened (45 days) Provola dei Nebrodi Sicilian cheese samples were assessed in order to investigate the diversity of the microbial population in cheese made from different geographical areas throughout Sicily. The samples, which were obtained from seven different Provola dei Nebrodi manufacturers, were assessed using selective media. Interestingly, concentrations of presumptive lactobacilli represented over 90% of the total microbial population. In total, 105 presumptive Lactobacillus isolates were characterized to determine the relatedness of the isolates between the seven different cheeses. Randomly amplified polymorphic DNA polymerase chain reaction (RAPD PCR) analysis of the 105 presumptive lactobacilli indicated the presence of 22 distinct isolates. Further investigation of the isolates using pulsed field gel electrophoresis (PFGE) following restriction with the enzyme ApaI revealed the presence of 19 distinct macrorestriction patterns and the presence of between one and four distinct isolates per cheese sample (out of a total of 15 isolates per cheese randomly taken from Lactobacillus selective media plates). Analysis of the 16S rDNA sequence of each genetically distinct isolate demonstrated the dominance of the Lactobacillus casei species in all cheese samples assessed. Lactobacillus delbrueckii and Pediococcus pentosaceus species were also detected. The concentration of free amino acids, used to estimate the extent of proteolysis in each cheese, ranged from 59 to 433 mg 100 g(-1) cheese. Microbiological assessment of the cheeses demonstrated the dominance of Lactobacillus species after 45 days of ripening with levels ranging from 8.3 to 9.4 log CFU g(-1). This study provides new information on the diversity of lactobacilli within an artisanal Sicilian cheese, enabling the identification of 17 strains of Lact. casei, one strain of Lact. delbrueckii and Ped. pentosaceus through the combined use of RAPD PCR, PFGE and 16S rDNA sequencing.

TAK1 MAPK Kinase Kinase Mediates Transforming Growth Factor-β Signaling by Targeting SnoN Oncoprotein for Degradation

Transforming growth factor-beta (TGF-beta) regulates a variety of physiologic processes through essential intracellular mediators Smads. The SnoN oncoprotein is an inhibitor of TGF-beta signaling. SnoN recruits transcriptional repressor complex to block Smad-dependent transcriptional activation of TGF-beta-responsive genes. Following TGF-beta stimulation, SnoN is rapidly degraded, thereby allowing the activation of TGF-beta target genes. Here, we report the role of TAK1 as a SnoN protein kinase. TAK1 interacted with and phosphorylated SnoN, and this phosphorylation regulated the stability of SnoN. Inactivation of TAK1 prevented TGF-beta-induced SnoN degradation and impaired induction of the TGF-beta-responsive genes. These data suggest that TAK1 modulates TGF-beta-dependent cellular responses by targeting SnoN for degradation.

Prevalence of Yersinia enterocolitica in Different Phases of Production on Swine Farms

Swine have been identified as the primary reservoir of pathogenic Yersinia enterocolitica (YE), but little research has focused on the epidemiology of YE at the farm level. The objective of this study was to describe the prevalence of YE in different production phases on swine farms. In this cross-sectional study, individual pigs on eight swine operations were sampled for the presence of YE. On each farm, both feces and oral-pharyngeal swabs were collected from pigs in five different production phases: gestating, farrowing, suckling, nursery, and finishing. A pig was considered positive if either sample tested positive. Samples were cultured with cold enrichment followed by isolation on selective media plates. Presumptive isolates were confirmed as YE and assayed for the presence of ail with a multiplex PCR. Of the 2,349 pigs sampled, 120 (5.1%) tested positive, and of those, 51 were ail positive (42.5% of YE isolates). On all farms, there was a trend of increasing prevalence as pigs mature. Less than 1% of suckling piglets tested positive for YE. Only 1.4% (44.4% of which were ail positive) of nursery pigs tested positive, but 10.7% (48.1% of which were ail positive) of finishing pigs harbored YE. Interestingly, gestating sows had the second highest prevalence of YE at 9.1% (26.7% of which were ail positive), yet YE was never detected from the farrowing sows. These results represent the first on-farm description of YE in U.S. herds and provide the initial step for designing future studies of YE.