Pseudomonas aeruginosa in Dairy Goats: Genotypic and Phenotypic Comparison of Intramammary and Environmental Isolates

Following the identification of a case of severe clinical mastitis in a Saanen dairy goat (goat A), an average of 26 lactating goats in the herd was monitored over a period of 11 months. Milk microbiological analysis revealed the presence of Pseudomonas aeruginosa in 7 of the goats. Among these 7 does, only goat A showed clinical signs of mastitis. The 7 P. aeruginosa isolates from the goat milk and 26 P. aeruginosa isolates from environmental samples were clustered by RAPD-PCR and PFGE analyses in 3 genotypes (G1, G2, G3) and 4 clusters (A, B, C, D), respectively. PFGE clusters A and B correlated with the G1 genotype and included the 7 milk isolates. Although it was not possible to identify the infection source, these results strongly suggest a spreading of the infection from goat A. Clusters C and D overlapped with genotypes G2 and G3, respectively, and included only environmental isolates. The outcome of the antimicrobial susceptibility test performed on the isolates revealed 2 main patterns of multiple resistance to beta-lactam antibiotics and macrolides. Virulence related phenotypes were analyzed, such as swarming and swimming motility, production of biofilm and production of secreted virulence factors. The isolates had distinct phenotypic profiles, corresponding to genotypes G1, G2 and G3. Overall, correlation analysis showed a strong correlation between sampling source, RAPD genotype, PFGE clusters, and phenotypic clusters. The comparison of the levels of virulence related phenotypes did not indicate a higher pathogenic potential in the milk isolates as compared to the environmental isolates.     

Detection of Achromobacter xylosoxidans in Hospital,Domestic, and Outdoor Environmental Samples and Comparison with Human Clinical Isolates

Achromobacter xylosoxidans is an aerobic nonfermentative Gram-negative rod considered an important emerging pathogen among cystic fibrosis (CF) patients worldwide and among immunocompromised patients. This increased prevalence remains unexplained, and to date no environmental reservoir has been identified. The aim of this study was to identify potential reservoirs of A. xylosoxidans in hospital, domestic, and outdoor environments and to compare the isolates with clinical ones. From 2011 to 2012, 339 samples were collected in Dijon''s university hospital, in healthy volunteers'' homes in the Dijon area, and in the outdoor environment in Burgundy (soil, water, mud, and plants). We designed a protocol to detect A. xylosoxidans in environmental samples based on a selective medium: MCXVAA (MacConkey agar supplemented with xylose, vancomycin, aztreonam, and amphotericin B). Susceptibility testing, genotypic analysis by pulsed-field gel electrophoresis, and bla_OXA-114 sequencing were performed on the isolates. A total of 50 strains of A. xylosoxidans were detected in hospital (33 isolates), domestic (9 isolates), and outdoor (8 isolates) samples, mainly in hand washing sinks, showers, and water. Most of them were resistant to ciprofloxacin (49 strains). Genotypic analysis and bla_OXA-114 sequencing revealed a wide diversity among the isolates, with 35 pulsotypes and 18 variants of oxacillinases. Interestingly, 10 isolates from hospital environment were clonally related to clinical isolates previously recovered from hospitalized patients, and one domestic isolate was identical to one recovered from a CF patient. These results indicate that A. xylosoxidans is commonly distributed in various environments and therefore that CF patients or immunocompromised patients are surrounded by these reservoirs.     

Characterization and Pathogenic Relationships of Rhizoctonia solani Isolates in a Potato–Rice System and their Sensitivity to Fungicides

Potato is planted after rice in several parts of Punjab in India and both crops are attacked by Rhizoctonia solani Kühn. Potato tubers showing black scurf and rice plants affected by sheath blight were collected from different regions of the state and the isolates of R. solani so obtained were studied to determine their variability and to ascertain their cross-infectivity and response to fungicides. Potato isolates of R. solani did not infect rice plants but some rice isolates were weakly pathogenic on potato, the sclerotia being less firmly attached on tuber surface, indicating a possible unsuccessful attempt of rice isolates to infect potato. Rice isolates (66.6%) grew faster (>20 mm colony growth per 24 h) than those of the potato isolates (15–20 mm growth rate per 24 h). Hyphal width of isolates from both hosts varied from 7.2 to 12.1 μm. Colony growth of most potato isolates (61.2%) was appressed, whereas that of most rice isolates (53.3%) was fluffy. Rice isolates (73.3%) formed larger sclerotia (1.5–2.0 mm in diameter) than those of the potato isolates (0.5–1.0 mm in diameter). Anastomosis studies indicated that potato isolates belonged to AG-3 and AG-5 groups while rice isolates belonged to the AG-1-1-A group. Representative R. solani isolates from the two hosts showed significant variation in response to fungicides (i.e. carbendazim, carboxin, pencycuron, propiconazole and validamycin) based on their ED₅₀ and ED₉₀ values.     

Molecular Genetic and Functional Analysis of the Conjugation System of the pBS72 Plasmid from Bacillus subtilis Environmental Isolates

Microbiology - The conjugation system of the pBS72 plasmid, which is widespread among the extrachromosomal genetic elements of environmental Bacillus strains, is unique and may be assigned to a new...     

Immobilization of Nickel by Bacterial Isolates from the Indian Ridge System and the Chemical Nature of the Accumulated Metal

Bacterial isolates from Carlsberg-Ridge waters were tested for their ability to immobilize Ni. For this, test cultures were suspended for 60 d in seawater with and without added Ni at concentrations of 10 to 10,000 μ M. At an initial concentration of 100 μ M, isolates CR35 and CR48 caused a 89.8 and 6.95 μ M decrease in dissolved Ni at 28 2°C and a 14.75 and 6.38 μ M at 3 1°C. Analyses showed the contribution of hydroxyl, carbonyl, sulfide and phosphoryl groups in Ni binding. The study proves that Ni immobilization can occur even in the absence of Mn and suggests their plausible role in the ridge ecosystem.     

Longitudinal Study of Finnish Campylobacter jejuni and C. coli Isolates from Humans, Using Multilocus Sequence Typing, Including Comparison with Epidemiological Data and Isolates from Poultry and Cattle

We describe a study on the application of multilocus sequence typing for the analysis of Campylobacter jejuni and C. coli isolates from human domestically acquired infections in the Helsinki-Uusimaa area of Finland in 1996, 2002, and 2003. In addition, isolates from poultry meat and fecal samples of cattle from the seasonal peak (July to September) in 2003 were included in the study. In total, 361 Finnish C. jejuni and C. coli strains were typed. Sequence type 45 (ST-45) (45%), ST-21 (21%), and ST-677 (11%) clonal complexes were the most prevalent. The ST-45 and ST-677 complexes were overrepresented in comparison with previous studies. The longitudinal study revealed an association between C. coli (ST-828 complex) infection and elderly patients (≥60 years). Analysis of exposure factors, determined by a previous case-control study conducted during the seasonal peak in 2002, revealed that the ST-48 complex was significantly (P < 0.05) associated with the tasting or eating of raw minced meat. New and unassigned STs were associated with swimming in natural bodies of water, whereas the ST-677 complex was related to drinking nonchlorinated water from a small water plant or water from natural sources. The ST-45 complex was associated with contact with pet cats and dogs. In 2003, ST-45 occurrence was significantly associated with poultry whereas ST-50 was associated with isolates from humans. In contrast, ST-53, ST-58, ST-61, and ST-883 were significantly associated with isolates from cattle. Further studies are needed to reveal the significance of the observed associations.     

Comparison of Genotypes Between Environmental and Clinical Isolates of Cryptococcus neoformans var. grubii Based on Microsatellite Patterns

We applied multilocus microsatellite typing (MLMT) method to investigate the genetic relation between Cryptococcus neoformans var. grubii clinical and environmental isolates in S?o Paulo, Brazil. This MLMT method includes three functional gene sequences of C. neoformans var. grubii, which are dispersed on three chromosomes. In all, 89 strains (36 clinical and 53 environmental isolates) were analyzed. Of 36 clinical strains, 20 belonged to a major type of MLMT-13 (55.6%). They were mainly isolated from clinical specimens. About 52.8% of strains from the environment belong to a major type of MLMT-36, which are indigenous to environments and which were not isolated from clinical samples. Thus, we recognized two genotypes that distinguish majority of clinical and environmental strains. No differences were found in antifungal susceptibility and capsule size between major environmental and clinical MLMT types.     

International Comparison of Clinical, Bovine, and Environmental Escherichia coli O157 Isolates on the Basis of Shiga Toxin-Encoding Bacteriophage Insertion Site Genotypes

Escherichia coli O157:H7 genotypes in the bovine reservoir may differ in virulence. The proportion of clinical genotypes among cattle isolates was weakly (P = 0.054) related to the international incidence of E. coli O157:H7-associated hemolytic-uremic syndrome, varied among clinical isolates internationally, and also differed along the putative cattle-hamburger-clinical case transmission chain.     

Comparison of CpG Island Methylator Phenotype (CIMP) Frequency in Colon Cancer Using Different Probe- and Gene-Specific Scoring Alternatives on Recommended Multi-Gene Panels

Background

In colorectal cancer a distinct subgroup of tumours demonstrate the CpG island methylator phenotype (CIMP). However, a consensus of how to score CIMP is not reached, and variation in definition may influence the reported CIMP prevalence in tumours. Thus, we sought to compare currently suggested definitions and cut-offs for methylation markers and how they influence CIMP classification in colon cancer.

Methods

Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), with subsequent fragment analysis, was used to investigate methylation of tumour samples. In total, 31 CpG sites, located in 8 different genes (RUNX3, MLH1, NEUROG1, CDKN2A, IGF2, CRABP1, SOCS1 and CACNA1G) were investigated in 64 distinct colon cancers and 2 colon cancer cell lines. The Ogino gene panel includes all 8 genes, in addition to the Weisenberger panel of which only 5 of the 8 genes included were investigated. In total, 18 alternative combinations of scoring of CIMP positivity on probe-, gene-, and panel-level were analysed and compared.

Results

For 47 samples (71%), the CIMP status was constant and independent of criteria used for scoring; 34 samples were constantly scored as CIMP negative, and 13 (20%) consistently scored as CIMP positive. Only four of 31 probes (13%) investigated showed no difference in the numbers of positive samples using the different cut-offs. Within the panels a trend was observed that increasing the gene-level stringency resulted in a larger difference in CIMP positive samples than increasing the probe-level stringency. A significant difference between positive samples using ‘the most stringent’ as compared to ‘the least stringent’ criteria (20% vs 46%, respectively; p<0.005) was demonstrated.

Conclusions

A statistical significant variation in the frequency of CIMP depending on the cut-offs and genes included in a panel was found, with twice as many positives samples by least compared to most stringent definition used.     

Ribosomal DNA Sequencing and Phylogenetic Analysis of Environmental <Emphasis Type=Italic>Sporothrix schenckii</Emphasis> Strains: Comparison with Clinical Isolates

The authors reported the isolation and genetic characterization of Sporothrix schenckii strains from natural environmental samples and commercial amended and garden soils. Twenty-six isolates were recovered and identified as S. schenckii by using both phenotypic and molecular methods. The majority of the strains were isolated from commercial amended and garden soils, indicating that these products represent an important reservoir of the fungus. Sequencing and phylogenetic analysis of the D1-D2 region of the 28S rRNA gene of environmental isolates, including S. schenckii ATCC 10268 and two Italian clinical strains, revealed a degree of difference sufficient to justify the separation of the examined isolates in two principal groups (environmental and clinical). Such separation in two groups is further supported by two well-conserved nucleotide polymorphisms, caused by single-base transitions, in the D1-D2 domain of rDNA. In this study, the presence in nature and/or in commercial products of S. schenckii is discussed. To our knowledge, this is the first study that reports the environmental isolation of S. schenckii from southern Italy.     

Mechanical and Chemical Characterization of a TiC/C System Synthesized Using a Focus Plasma Arc

Titanium carbide-graphite (TiC/C) composite was successfully synthesized from Ti and C starting elemental powders using self-propagating high-temperature synthesis technique in an ultra-high plasma inert medium in a single stage. The TiC was exposed to a high-temperature inert medium to allow recrystallization. The product was then characterized using field emission scanning electron microscopy (FESEM) coupled with energy dispersive X-ray analysis (EDX), X-ray diffraction (XRD), Rietveld refinement, nanoindentation, and micro-hardness to determine the product’s properties. The recorded micro-hardness of the product was 3660 HV, which is a 14% enhancement and makes is comparable to TiC materials.     

琼脂糖和聚丙烯酰胺凝胶电泳技术检测小麦基因组DNA RAPD扩增产物的方法学比较

通过20%(wv)的琼脂糖凝胶和5%(wv)的聚丙烯酰胺凝胶电泳对小麦白粉病抗、感特性品种基因组DNA的RAPD检测结果表明:5%聚丙烯酰胺凝胶对线性DNA分子(01～20kb)和长度相差100bp以下的DNA分子的分离较20%的琼脂糖凝胶电泳效果好。因此,我们研究出了一项利用聚丙烯酰胺凝胶电泳检测小麦白粉病抗、感特性的新技术,在工作中建立了一种适合于检测小麦基因组DNA结构差异的电泳方法。该方法主要包括:(1)丙烯酰胺和亚甲基双丙烯酰胺的新配比;(2)分离DNA片段的最佳凝胶浓度;(3)电泳条件;(4)脱色、漂洗、银染、显色过程。实验发现,该技术对于小麦白粉病抗、感特性检测中的小片段和长度相差100bp以下的线性DNAPCR扩增结果的分辨效果较好。应用该技术在抗感品种间已经发现了DNA水平上的差异。     

Sensitivity of the Goldfish Motion Detection System Revealed by Incoherent Random Dot Stimuli: Comparison of Behavioural and Neuronal Data

Background

Global motion detection is one of the most important abilities in the animal kingdom to navigate through a 3-dimensional environment. In the visual system of teleost fish direction-selective neurons in the pretectal area (APT) are most important for global motion detection. As in all other vertebrates these neurons are involved in the control of slow phase eye movements during gaze stabilization. In contrast to mammals cortical pathways that might influence motion detection abilities of the optokinetic system are missing in teleost fish.

Results

To test global motion detection in goldfish we first measured the coherence threshold of random dot patterns to elicit horizontal slow phase eye movements. In addition, the coherence threshold of the optomotor response was determined by the same random dot patterns. In a second approach the coherence threshold to elicit a direction selective response in neurons of the APT was assessed from a neurometric function. Behavioural thresholds and neuronal thresholds to elicit slow phase eye movements were very similar, and ranged between 10% and 20% coherence. In contrast to these low thresholds for the optokinetic reaction and APT neurons the optomotor response could only be elicited by random dot patterns with coherences above 40%.

Conclusion

Our findings suggest a high sensitivity for global motion in the goldfish optokinetic system. Comparison of neuronal and behavioural thresholds implies a nearly one-to-one transformation of visual neuron performance to the visuo-motor output. In addition, we assume that the optomotor response is not mediated by the optokinetic system, but instead by other motion detection systems with higher coherence thresholds.     

Comparison of Species Richness Estimates Obtained Using Nearly Complete Fragments and Simulated Pyrosequencing-Generated Fragments in 16S rRNA Gene-Based Environmental Surveys

Pyrosequencing-based 16S rRNA gene surveys are increasingly utilized to study highly diverse bacterial communities, with special emphasis on utilizing the large number of sequences obtained (tens to hundreds of thousands) for species richness estimation. However, it is not yet clear how the number of operational taxonomic units (OTUs) and, hence, species richness estimates determined using shorter fragments at different taxonomic cutoffs correlates with the number of OTUs assigned using longer, nearly complete 16S rRNA gene fragments. We constructed a 16S rRNA clone library from an undisturbed tallgrass prairie soil (1,132 clones) and used it to compare species richness estimates obtained using eight pyrosequencing candidate fragments (99 to 361 bp in length) and the nearly full-length fragment. Fragments encompassing the V1 and V2 (V1+V2) region and the V6 region (generated using primer pairs 8F-338R and 967F-1046R) overestimated species richness; fragments encompassing the V3, V7, and V7+V8 hypervariable regions (generated using primer pairs 338F-530R, 1046F-1220R, and 1046F-1392R) underestimated species richness; and fragments encompassing the V4, V5+V6, and V6+V7 regions (generated using primer pairs 530F-805R, 805F-1046R, and 967F-1220R) provided estimates comparable to those obtained with the nearly full-length fragment. These patterns were observed regardless of the alignment method utilized or the parameter used to gauge comparative levels of species richness (number of OTUs observed, slope of scatter plots of pairwise distance values for short and nearly complete fragments, and nonparametric and parametric species richness estimates). Similar results were obtained when analyzing three other datasets derived from soil, adult Zebrafish gut, and basaltic formations in the East Pacific Rise. Regression analysis indicated that these observed discrepancies in species richness estimates within various regions could readily be explained by the proportions of hypervariable, variable, and conserved base pairs within an examined fragment.Culture-independent 16S rRNA gene surveys are now routinely utilized to examine the microbial diversity in various environmental habitats. However, in surveys of highly diverse ecosystems, the size of clone libraries typically constructed (100 to 500 clones) allows for the identification only of members of the community that are present in high abundance (2, 13, 14, 17, 24, 51). In addition to the failure to detect the rare members of the ecosystem, these relatively small datasets provide inaccurate estimates when used for computing species richness within an ecosystem. Regardless of the approach utilized to estimate species richness, the estimates obtained are highly dependent on sample size, and smaller datasets typically result in the underestimation of species richness (14, 44, 47, 55).The use of a pyrosequencing-based approach (40) in 16S gene-based diversity surveys promises to overcome both of the above-mentioned problems associated with inadequate sampling. The large number of 16S rRNA gene sequences produced (hundreds of thousands) allows access to rare members of the community (25; J. M. Tiedje, presented at the 108th General Meeting of the American Society for Microbiology, Boston, MA, 2008), as well as a relatively more accurate estimation of species richness. However, with the introduction of this new technology, it is necessary to correlate the results obtained from newer pyrosequencing-based surveys to the extensive collection of longer, capillary sequence-generated 16S rRNA gene sequences that has been deposited in public databases during the last 2 decades. Several recent studies have examined the utility of pyrosequencing fragments in providing an accurate survey of overall community structure (36) and investigated the ability of various fragments spanning the 16S rRNA gene to accurately predict the phylogenetic affiliation of pyrosequencing-generated fragments at various taxonomic cutoffs (35, 54). As such, these admirable efforts gave useful insights into the advantages and limitations of the pyrosequencing approach in 16S-based community surveys, pinpointed specific regions that provide better phylogenetic resolution than other pyrosequencing-generated regions, and provided a quantitative assessment of binning accuracy at various empirical cutoffs.However, while issues regarding correlating phylogenies of shorter and longer fragments are actively being addressed, efforts to calibrate species richness data obtained from various pyrosequencing fragments at various taxonomic cutoffs to estimates obtained using longer 16S rRNA gene fragments are still lacking. It is unclear how pairwise distances and, hence, operational taxonomic unit (OTU) assignments and species richness estimates computed using various shorter fragments spanning various regions of the 16S rRNA gene will correlate to pairwise distances computed using the nearly complete 16S rRNA gene. Elucidating such differences between shorter and nearly complete fragments, as well as between shorter fragments representing different regions in the 16S rRNA gene, is absolutely necessary for accurate meta-analysis of species richness in previously published and future datasets constructed using various sequencing approaches.Here, we constructed, sequenced, and analyzed a 16S rRNA library of 1,132 clones generated from an undisturbed tallgrass prairie soil in central Oklahoma and compared the numbers of OTUs and species richness values obtained using the full-length data sets (with and without the application of the Lane mask filter that excludes hypervariable regions from the phylogenetic analysis) (32) and fragments simulating pyrosequencing output generated by clipping where known conserved bacterial primers are encountered in the 16S rRNA gene. The lengths of the chosen simulated-pyrosequencing fragments represent amplicons that have been generated using the original GS20 pyrosequencing platform (≈100 bp) (25, 44, 48), similar to those currently being generated using the GS FLX pyrosequencing platform (≈250 bp) (1, 20, 35) or amplicons produced using the anticipated increase in the new GS XLR pyrosequencing platform (>250 bp). We show that the choice of the pyrosequenced fragment could indeed impact the number of OTUs calculated at different taxonomic cutoffs, with some fragments underestimating and others overestimating such parameters compared to the results with longer, nearly complete 16S rRNA gene fragments. We also show that even more marked differences could be encountered when comparing two pyrosequencing fragments within the same molecule. Further, we established a regression analysis that explains the nature of the observed discrepancies using the proportions of the hypervariable, variable, and conserved bases within fragments.     

Comparison of Methods of Extracting Salmonella enterica Serovar Enteritidis DNA from Environmental Substrates and Quantification of Organisms by Using a General Internal Procedural Control

This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of S. enterica serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 × 10³ to 1.8 × 10³ CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent S. enterica-specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR.     

In Vitro Comparison of Raypex 6 and Endopilot Using a Novel,Computer-Aided Measurement System,for Determining the Working Length

Introduction

Experimental evaluation of endometric devices usually relies on visual, subjective detection of the apical constriction to determine the accuracy of measurements. The aim of the present study was to analyze the accuracy of measurements of Raypex 6 and EndoPilot using a novel, objective image-analysis system.

Methods

Onehundred and twenty teeth were randomized and allocated to three groups: After coronal flaring, either Raypex 6 or EndoPilot were used to determine the endodontic working length during instrumentation using manual files (RPM and EPM group respectively). In addition, EndoPilot was used for continuous, automatic measurement during rotating instrumentation (EPA group). If the working length had been reached according to endometric results, the files were fixed in place. Tooth and file were then embedded and prepared for analysis. Subsequently, the distance between the tip of the file and the apical constriction (D_AC) or the apical foramen (D_AF) was calculated using trigonometric analysis and the position of the file relative to AC and AF was analyzed.

Results

Both inter- and intra-examiner-reliability of the trigonometric analysis were nearly perfect (ICC = 0.999, p<0.001). D_AC was not significantly different between groups (p>0.05, t-test). D_AF was significantly decreased when EPA had been used compared to EPM (p<0.05, Exact-test). EPA resulted in files being positioned beyond AF significantly more often than the other two methods (p<0.01).

Conclusions

All methods allowed reliable detection of AC. However, EPA significantly increased the risk of overpreparation. Objective, digital assessment based on image analysis was suitable to compare the accuracy of different endometric devices.     

Detection and Comparison of some Ghanaian Isolates of Cacao Swollen Shoot Virus (CSSV) by Enzyme-Linked Immunosorbent Assay (ELISA) and Immunoelectron Microscopy (IEM) Using an Antiserum to CSSV Strain 1A

Various isolates of Cacao Swollen Shoot Virus (CSSV) were detected without difficulty in leaves of Theobroma cacao L. by ELISA and immunosorbent electron microscopy (ISEM) using an antiserum to severe strain 1A. Many isolates were detected with relatively high values at dilutions of 1:30, whereas some other isolates were hardly or not at all detected at this dilution. Strain 1A was detected at dilutions of up to 1: 2560 of crude leaf extracts. All isolates yielding high reactions seem to be serologically closely related to strain 1A. Strains of the mottle-leaf type (AD 191, AD 196, AD 7, AD 36, AD 135, Kpeve) and others were poorly detected; their relationship to strain 1A is discussed. A close correlation was found between results obtained by ELISA and ISEM.