Isolation Of DNA Sequence Research Articles

Utilizing whole genome sequencing to delineate relapse and reinfection tuberculosis on the Canadian prairies

RATIONALE Recurrent tuberculosis (TB) accounts for 5% of the Canadian TB burden. Recurrence can occur from relapse or reinfection. Identifying reinfection has implications for developing control policies. OBJECTIVE The objectives of this study were to quantify reinfection in recurrent TB using whole genome phylogenetic and single nucleotide polymorphism (SNP) and compare with epidemiological and clinical parameters from the Canadian prairies. METHODS DNA sequences of Mycobacterium tuberculosis isolates from recurrent TB cases along with epidemiological and clinical parameters were collected from Alberta and Saskatchewan. Inclusion criteria were two or more culture positive notifications age ≥17 years. SNP and phylogenetic tree scale differences (TIP) were used to determine the relapse and reinfection categories. RESULTS Of 7,627 notifications of TB disease, 533 were recurrent. 93 pairs (180 cases) were culture positive from which 26 (50 cases) were available for sequencing. 19 cases with SNP and TIP values ≤25 and ≤.001 were classified relapse. Seven SNP and TIP values >160 and >.001 were classified reinfection. Five of seven reinfections were Indigenous cases from high TB incidence areas. The non-sequenced and sequenced pairs differed only in age. CONCLUSIONS Recurrent culture positive TB is uncommon on the Canadian prairies and is more likely to be relapse. Reinfection is more likely in Indigenous persons living in high TB incidence communities. A limitation was the risk of selection bias since only 28% of eligible cases were sequenced. Since we did not know the non-sequenced reinfection risk, we could only conclude that the non-sequenced and sequenced categories were similar.

First Reports of Cladosporium tenuissimum Causing Leaf spots on Hydrangea macrophylla in Anhui province in China.

Forever Summer Hydrangea (Hydrangea macrophylla) is a common flowering plant in the Yangtze River Valley area of China, and it is widely cultivated globally (Chen et al. 2015). In July 2023, H. macrophylla leaves exhibiting visible diseased lesions were reported in a nursery in Wuhu, Anhui Province, China. The incidence reached 40% in a 0.2 ha area. The primary disease symptom was multiple irregular necrotic spots (0.5 to 1 mm in diameter) appearing on the leaves. These spots on the leaves were faded yellow around the perimeter and grayish brown in the center.). 15 leaf samples were sterilized with 75% alcohol and rinsed three times in sterile distilled water, then transferred to antibiotic-added potato dextrose agar (PDA) for incubation at 27°C. The colonies were fluffy, flocculent, or hairy, dark green, gray-green to gray-brown in color, and spreading or protruding punctate with a colorless halo on PDA. The conidiophores were brown to dark brown, smooth or rough surface, mostly unbranched, clearly differentiated, erect or curved. The conidia displayed a light brown to brown hue, lemon shape, fusiform, elongated ellipsoid or others with obvious spore markings and spore umbilicus. Genomic DNA was extracted from fungal colonies on infected leaves of three collections separately (Braun et al. 2003) and the internal transcribed spacer regions (ITS), actin (ACT) genes and partial translation elongation factor-l-alpha (EF) were amplified and sequenced using the primers ITS1/4 (Yin et al. 2012), ACT-512F/ACT-783R and EF 1-728F/986R (Carbone and Kohn 1999), respectively. DNA sequences of isolates were identical and deposited in GenBank (accession no. OR362754 for ITS, OR611929 for ACT and PP209106 for EF). The consensus sequences from ITS, EF and ACT showed 100%, 98.98% and 100% identical to Cladosporium strains (accession no. OQ186140.1, MT154169.1 and OL322092.1), respectively. To confirm the pathogenicity of the isolates, hydrangeas were planted in 15-cm pots containing commercial potting mix (one plant/pot). Three healthy plants were inoculated at the five to eight leaf stage by spraying 50 μL of the isolate conidial suspension (4 × 106 spores/mL) on healthy leaves. Three plants treated with sterile distilled water were used as controls. After inoculation, all plants were placed in a humidity chamber (>95% relative humidity, 26°C) for 48 h and then transferred to a greenhouse at 22/27°C. All inoculated leaves exhibited symptoms similar to those observed in the nursery 10 days after inoculation, while no symptoms were observed for control leaves. The fungus was re-isolated and confirmed to be C. tenuissimum. Based on the above morphological characterization and molecular identification, the causal agent for this leaf spot disease was identified as C. tenuissimum. Although C. tenuissimum has been reported to cause disease on H. paniculata in northern China (Li et al.2021), this is the first time that C. tenuissimum has been found on H. macrophylla in southern China. This new disease of H. macrophylla caused by C. tenuissimum is a threat to urban greening and is worth further investigation.

Geno-Species Composition of Borrelia Circulating on the Black Sea Coast of the Krasnodar Territory

The aim of the study was to assess the current epidemiological situation on Ixodidae tick-borne borreliosis (ITBB) (2017–2022) on the Black Sea coast of the Krasnodar Territory, to study the species composition of Borrelia.Materials and methods. Ixodidae ticks, Ixodes ricinus, Haemaphysalis concinna, H. inermis, Rhipicephalus sanguineus, were examined for infection with tick-borne borreliosis pathogens, followed by DNA sequencing of isolates. Species identification of Borrelia was carried out based on analysis of the nucleotide sequence of the 16S rRNA gene fragment using BLAST algorithm. Additionally, the OspC genotype was determined for isolates of pathogenic genovariants through comparing the obtained sequences with reference ones in the MEGA 5 program. The data were processed applying the cartographic method using the QGIS 2.18 software.Results and discussion. Between 2017 and 2022, 101 cases were registered on the Black Sea coast of the Krasnodar Territory, which accounted for 1.6 % of all ITBB cases in the Krasnodar Territory. The number of complaints with tick bites in Sochi was up to 191.4 per 100 thousand population. During the study, the predominant Borrelia geno-species on the territory of the Black Sea coast of the Krasnodar Territory was determined using sequencing – Borrelia lusitaniae (78.1 %). The circulation of geno-species B. garinii (6.8 %), B. valasiana (5.7 %), B. afzelii (3.6 %), B. miyamotoi (2.6 %), B. tanukii, B. bissettii (1.6 % each) was also observed. For Borrelia pathogenic species, appurtenance to OspC geno-groups was ascertained: four isolates were assigned to invasive OspC geno-groups, of which three B. afzelii isolates were genogroup A8, one B. garinii isolate was G7. High rate of infection of ticks with Borrelia was recorded in the surveyed area (up to 94.5 %). Borrelia of pathogenic species account for only 10.4 % of the total number of isolates studied. Mostly, genovariants that rarely cause diseases in humans (B. lusitaniae, B. valasiana, B. bissettii) and non-pathogenic Borrelia (B. tanukii) were found, which indicates a low risk of infection with ITBB pathogens.

Isolation and molecular characterization of microbial isolates from Saryu river water

This study was aimed to investigate molecular characterization of microbial isolates likes bacteria, fungi using culture dependent approach and analyse the phylogenetic relationship between the isolates. The identification of bacteria and fungi was confirmed by DNA sequencing by PCR amplification using 16S rRNA and Internal Transcribed Spacer (ITS) genes, respectively. The DNA sequences of bacterial and fungal isolates were identified using Nucleotide Basic Alignment Search Tools (nBLAST) against the data available in GenBank. On the basis of retrieved DNA sequences from database phylogenetic trees were constructed and phylogenetic relationship among the bacterial and fungal isolates were computed.

Epidemiological study of antimicrobial-resistant bacteria in healthy free-ranging bantengs (Bos javanicus) and domestic cattle.

Antimicrobial-resistant microorganisms (ARMs) have been increasing among wild animals. Interactions occurring at the interface between wildlife, humans, and livestock can lead to the transmission of ARMs. Thus, the prevalence of ARMs in wild and domestic animals should be determined to address and prevent this issue. This study aimed to determine the resistance patterns of cefotaxime (CTX)-resistant Escherichia coli and identify the presence of extended-spectrum beta-lactamase (ESBL) genes in ESBL-producing E. coli among a population of wild banteng (Bos javanicus) and domestic cattle kept on farms located close to the Lam Pao non-hunting area, Kalasin province, Thailand. Forty-five fecal samples were taken from wild bantengs inhabiting the Lam Pao non-hunting area in Thailand, alongside 15 samples from domestic cattle. Bacterial culture, triple sugar iron, and motile indole lysine tests were conducted to identify E. coli. A polymerase chain reaction (PCR) was conducted for specific confirmation. MacConkey agar supplemented with 2 μg/mL of CTX was used to identify CTX-resistant E. coli, which would be used to identify ESBL production based on a double-disk synergy test. Extended-spectrum beta-lactamase-producing samples were subjected to disk diffusion tests to determine resistant patterns, and the sizes of PCR bands and DNA sequencing were used to differentiate ESBL gene types. All samples tested positive for E. coli. Forty-five isolates from 15 banteng samples and three isolates from one domestic cattle sample displayed CTX-resistant and ESBL-producing traits. The banteng and domestic cattle populations exhibited nine and three distinct resistant patterns, respectively. The PCR results indicated that the banteng isolates harbored the following genes: Cefotaxime-M1 (n = 38), CTX-M9 (n = 5), and the SHV group (n = 2). All three isolates from the domestic cattle sample contained the CTX-M1 gene. Classification of ESBL genes based on the DNA sequences of the banteng isolates showed the characteristics of CTX-M15 (n = 20), CTX-M55 (n = 6), CTX-M14 (n = 5), and CTX-M79 (n = 1). The three domestic cattle isolates exhibited the characteristics of CTX-M15, CTX-M55, and CTX-M79. Despite no previous antibiotic applications, approximately one-third of the banteng samples displayed CTX resistance, indicating ARM contamination within the ecosystem. The similarity in ESBL genes between the banteng and domestic cattle populations suggests potential gene transmissions between these animal groups. However, the initial source of ARMs remains unclear, as the banteng population exhibited more ESBL genes than the domestic cattle, suggesting the possibility of multiple ARM sources. These findings raise concerns because the banteng population inhabits an area that is an important source of freshwater and nourishes the entire north-east region of Thailand and other South-east Asian countries, including Laos, Cambodia, and Southern Vietnam.

Ability of the MeltPro MTB/PZA Assay to Detect Susceptibility to Pyrazinamide in Rifampin-Resistant Tuberculosis Patients.

Prediction of susceptibility to pyrazinamide (PZA) directly from sputum has been challenging. The MeltPro MTB/PZA assay, based on melting curve analysis, can simultaneously detect Mycobacterium tuberculosis and the resistance to PZA from sputum. We aimed to evaluate the MeltPro MTB/PZA assay to predict PZA resistance among rifampin-resistant tuberculosis (RR-TB) patients. We prospectively enrolled RR-TB patients in the registered trials, and their baseline sputum samples were obtained to perform the assay and culture. DNA sequencing of culture isolates was analyzed and used as the reference standard. Sanger sequencing was performed for samples with discrepant results between next-generation sequencing (NGS) and the investigational assay. The main analysis was conducted in the population of patients with interpretable results by both NGS and the assay. A total of 239 patients with RR-TB were screened, and 220 underwent the MeltPro MTB/PZA assay. The assay provided no information for 25 of 220 patients (11.4%). Among the remaining 195 patients, 13 had negative culture or insufficient raw NGS sequencing data, and 15 had indeterminate assay results. A total of 167 patients were included in the main analysis. Against DNA sequencing, the sensitivity, specificity, and negative predictive value of the assay for detecting resistance to PZA were 91.4% (95% confidence interval [CI], 87.1% to 95.6%), 89.9% (95% CI, 85.3% to 94.5%), and 95.2% (95% CI, 91.9% to 98.4%), respectively. In conclusion, the MeltPro MTB/PZA assay is a fast semiautomatic molecular platform to rapidly predict resistance to PZA from sputum and holds promise as a screening tool with satisfactory sensitivity. IMPORTANCE This study evaluated the accuracy of the MeltPro MTB/PZA assay at detecting the presence of PZA resistance through registered clinical trials. Compared to DNA sequencing, the assay had high sensitivity and negative predictive value, suggesting its potential utility as a screening tool in clinical practice. The assay could serve as an ideal primary screening tool in low PZA-resistant M. tuberculosis prevalence settings and could be used as an additional test to identify PZA resistance rapidly and initially in the RR-TB population.

Morphological and Molecular Analysis of Fungal Species Associated with Blast and Brown Spot Diseases of Oryza sativa.

Fungal diseases blast and brown spot in rice cause severe yield losses worldwide. Blast is caused by Magnaporthe oryzae, and Bipolaris oryzae is reported as the main causal organism of brown spot. Both diseases cause leaf lesions that are difficult differentiate until the later stages. Early detection and differentiation of the lesions would help the adoption of disease management strategies specific to the pathogens and prevent reductions in the quality and quantity of rice yields. This study was conducted in the Northern Province of Sri Lanka over five consecutive rice cultivating seasons to characterize the causal fungi of rice blast and brown spot diseases by morphological and molecular means and to develop a visual guide to differentiate the two diseases. Disease incidence was recorded in 114 fields from 2017 to 2019, and fungal isolates associated with the lesions of both diseases were cultured and subjected to morphological and molecular characterization. Competitive growth interactions between M. oryzae and the more common individual fungal isolates of the brown spot lesions were evaluated. Fungal metagenomic analysis was conducted for the fungal species isolated from brown spot lesions. A suppression of blast accompanied by an increased incidence of brown spot disease was observed during the study period. M. oryzae was confirmed to be the causal organism of the blast, and >20 species of fungi were identified to be associated with brown spot lesions through morphological and molecular studies and metagenomic analyses. Fungal internal transcribed spacer region sequencing revealed genetic variation in the highly conserved region of DNA sequences of blast and brown spot fungal isolates. B. oryzae, Curvularia, and Microdochium species were commonly isolated from brown spot lesions. In vitro competitive growth interactions between the fungal isolates revealed growth suppression of M. oryzae by the fungal isolates associated with brown spot lesions. Similarly, it can be speculated that the abundance and severity of blast in the field may have an influence on brown spot-associated fungi. A simple visual guide was developed to differentiate blast and brown spot lesions. The findings would be highly useful in the timely management of these major fungal diseases affecting rice.

Aflatoxin M1 reduction by microorganisms isolated from kefir grains

Aflatoxin M1 (AFM1) is a mycotoxin that often contaminates milk. Like other mycotoxins, it is thermostable and potentially carcinogenic. The present work was carried out to evaluate the ability of microorganisms isolated from Indonesian kefir grains to reduce AFM1 in contaminated phosphate buffer saline (PBS). Fourteen isolates of lactic acid bacteria, both aerobic (LAE) and anaerobic (LAN), and nine isolates of yeast (YEA) were used. The significantly highest AFM1 reduction percentage was shown by the isolate LAE7 (29.3 ± 0.6%) after 4 h incubation. DNA sequencing of LAE7 and YEA2 isolates showed that these isolates had homology (level of similarity) with species of Lactobacillus kefiri strain A/K and Saccharomyces cerevisiae NRRL Y-12632, respectively. The present work proved that isolates from Indonesian kefir grains could reduce AFM1 and have the potential for practical use.

DNA metabarcoding of the phytoplankton of Great Salt Lake’s Gilbert Bay: Spatiotemporal assemblage changes and comparisons to microscopy

The Great Salt Lake (GSL) is a unique hypersaline system with an understudied phytoplankton assemblage supporting a productive open water ecosystem in the largest embayment of the lake, Gilbert Bay. Determination of phytoplankton by microscopy has practical limitations that can constrain the scope of a study, but DNA metabarcoding may improve upon this through higher taxonomic resolution and the capacity to generate a large volume of assemblage data in comparatively little time. To determine if metabarcoding could replicate microscopy and expand the assessment of GSL phytoplankton, a 23S SSU rRNA metabarcoding and microscopy survey of Gilbert Bay was conducted in 2017 and 2018. Assemblage composition and relative abundances from each method were compared, and spatial and temporal assemblage changes from metabarcoding data were investigated using non-metric multidimensional scaling. Metabarcoding differed from microscopy in multiple taxonomic assignments and relative abundances, with poor correlation for most categories. Diatoms were overrepresented by metabarcoding relative to microscopy, and chlorophytes underrepresented. However, metabarcoding revealed seasonal and spatial patterns in assemblage, detected seasonal patterns within phytoplankton sequences of very low abundance, and detected potential cryptic speciation within the lake’s dominant Dunaliella viridis. Phylogenetic analysis revealed greater phytoplankton diversity than observed before in GSL, but demonstrated the need to improve taxonomic assignment of the resulting sequences, particularly within the diatoms. The expansion of detectable diversity and isolation of DNA sequences that can be traced through time and analyzed against environmental variables make metabarcoding a potentially effective tool for parallel use with microscopy in future GSL research.

Low Rate of Acquired Linezolid Resistance in Multidrug-Resistant Tuberculosis Treated With Bedaquiline-Linezolid Combination.

In this retrospective study in China, we aimed to: (1) determine the prevalence of linezolid (LZD) resistance among multidrug-resistant tuberculosis (MDR-TB)-infected patients; (2) monitor for dynamic LZD susceptibility changes during anti-TB treatment; and (3) explore molecular mechanisms conferring LZD resistance. A total of 277 MDR-TB patients receiving bedaquiline (BDQ)-containing regimens in 13 TB specialized hospitals across China were enrolled in the study. LZD and BDQ susceptibility rates were determined using the minimum inhibitory concentration (MIC) method, then DNA sequences of patient isolates were analyzed using Sanger sequencing to detect mutations conferring LZD resistance. Of 277 patients in our cohort, 115 (115/277, 41.5%) with prior LZD exposure yielded 19 (19/277, 6.9%) isolates exhibiting LZD resistance. The LZD resistance rate of LZD-exposed group isolates significantly exceeded the corresponding rate for non-exposed group isolates (P = 0.047). Genetic mutations were observed in 10 (52.6%, 10/19) LZD-resistant isolates, of which a Cys154Arg (36.8%, 7/19) substitution within ribosomal protein L3 was most prevalent. Analysis of sequential positive cultures obtained from 81 LZD-treated patients indicated that cultured organisms obtained from most patients (85.2%, 69/81) retained original LZD MIC values; however, organisms cultured later from two patients exhibited significantly increased MIC values that were attributed to the rplC substitution T460C. Overall, LZD resistance was detected in 6.9% of patients of an MDR-TB cohort in China. Low rate of acquired LZD resistance was noted in MDR-TB treated with BDQ-LZD combination.

Oomycetes in rhizosphere soil of ornamental plants from retail nurseries in Southern Sweden

AbstractTrade of herbaceous and woody ornamental plants is recognized as a principal pathway for the introduction of alien plant pathogens, such as soil‐borne Phytophthora and Pythium species (oomycetes). The rhizosphere soil of container‐grown plants obtained from 13 nurseries and garden centres in southern Sweden were sampled and the presence of Phytophthora and Pythium species determined using traditional baiting and isolation. DNA sequencing of isolates revealed five Phytophthora taxa including P. cinnamomi, P. citricola s.l., P. plurivora, P. undulata and a non‐identified Phytophthora sp., as well as Phytopythium cf. citrinum and several species of Pythium. Phytophthora cinnamomi was detected on two different ornamental plants at the same nursery and is a first record of P. cinnamomi in Sweden. The survey revealed that a diversity of potentially pathogenic oomycetes is associated with ornamental plants available for sale in Sweden and emphasizes the need to recognize potential risks if these organisms get introduced in nature.

Multi-year diagnosis of unpredictable fouling occurrences in a full-scale membrane bioreactor

The membrane bioreactor (MBR) at the Traverse City Regional Wastewater Treatment Plant has experienced sudden and unpredictable periods of substantial permeability decline since 2011. Early observations detected irregularly-shaped Gram-positive bacteria that correlated with plant upsets. Use of biomolecular techniques, such as DNA sequencing of laboratory isolates and the mixed liquor microbial community, and fluorescent in situ hybridization, identified the dispersed organisms as members of the genus Staphylococcus. However, Staphylococcus species were consistently present during normal operation and therefore were more likely to be an indicator of the upset, not the cause. The results suggest that these microorganisms are responding to specific influent wastewater constituents. We chemically analysed seven mixed liquor samples from periods of permeability decline in 2017 and 2018, and four samples from a period of normal operation. During upset conditions, the total carbohydrate content exceeded that of normal operation by 40%. Additionally, mixed liquor calcium concentrations were 65% above normal during the upset in 2017. It is hypothesized and supported through multivariate statistical analysis and estimation of specific resistance to filtration values that a calcium-intermediated polymer bridging mechanism with extracellular polymeric substance constituents is a major contributor to fouling and permeability disruptions in the Traverse City MBR.

Corneal ulcer/keratitis derived Aspergillus flavus &Aspergillus tamarii and their RAPD-PCR typing

The aim of the study was investigate the DNA polymorphisms of Aspergillus flavus and Aspergillus tamarii isolated from regional fungal keratitis cases based on random amplified polymorphic DNA (RAPD) analyses. Thirty-two A. flavus and six A. tamarii isolates were identified using conventional macroscopic and microscopic features. Five decamer primers were used to amplify the genomic DNA sequences of the test isolates, and 140 total RAPD patterns were generated among them. From each of the 38 isolates, a range of 15–30 RAPD types were revealed. All of these were isolated from male farmers from Tamil Nadu. In the future, RAPD analyses could be employed as a reliable and rapid technique to differentiate among keratitis isolates of fungal pathogens.

Deciphering Resistome and Virulome Diversity in a Porcine Slaughterhouse and Pork Products Through Its Production Chain.

We aimed to better understand resistome and virulome patterns on animal and process-area surfaces through a pig slaughterhouse to track possible contamination within the food production chain. Culture-dependent methods revealed high levels of microbial contamination, corresponding to mesophilic and pathogenic bacteria on both the animal and process-area surfaces mainly in the anesthesia (AA and AS) zone followed by “scorching and whip” (FA and FS) zone and also in the end products. To evaluate the potential risk of antibiotic resistance and virulence determinants, shotgun metagenomic DNA-sequencing of isolates from selected areas/products uncovered a high diversity and richness of antibiotic resistance genes (ARGs): 55–62 genes in the anesthesia area (AA and AS) and 35–40 in “animal-arrival zone” (MA and MS). The “scorching and whip” (FA and FS) area, however, exhibited lowered abundance of ARGs (1–6), indicating that the scalding and depilating process (an intermediate zone between “anesthesia” and “scorching and whip”) significantly decreased bacterial load by 1–3 log10 but also diminished the resistome. The high prevalence of antibiotic-inactivating enzyme genes in the “animal-arrival zone” (60–65%) and “anesthesia” area (56%) were mainly represented by those for aminoglycoside (46–51%) and lincosamide (14–19%) resistance, which did not reflect selective pressures by antibiotics most commonly used in pig therapy—tetracyclines and beta-lactams. Contrary to ARGs, greater number of virulence resistance genes were detected after evisceration in some products such as kidney, which reflected the poor hygienic practices. More than 19 general virulence features—mainly adherence, secretion system, chemotaxis and motility, invasion and motility were detected in some products. However, immune evasion determinants were detected in almost all samples analyzed from the beginning of the process, with highest amounts found from the anesthesia area. We conclude that there are two main sources of contamination in a pig slaughterhouse: the microorganisms carried on the animals’ hide, and those from the evisceration step. As such, focussing control measures, e.g., enhanced disinfection procedures, on these contamination-source areas may reduce risks to food safety and consumer health, since the antibiotic and virulence determinants may spread to end products and the environment; further, ARG and virulence traits can exacerbate pathogen treatments.

Should We Change EU Law to Disallow DNA Patents?

In the first half of the current decade, the US Supreme Court substantially restricted the patentability of isolated DNA sequence patents and methods of genetic diagnosis through its decisions in Mayo Collaborative Services v Prometheus Laboratories Inc in 2012, and in Association for Molecular Pathology v Myriad Genetics Inc in 2013. A key question for Europeans – and Britons – is whether European law should follow the legal directions in the US: should we also disallow DNA patents? Relatedly, should we disallow genetic diagnostics patents? This paper set the scene for an expert debate on this topic. It focusses on the patentability of DNA sequence patents. The debate was held on 18 November 2016 at the University of Cambridge before a general academic audience.

Arbuscular mycorrhizal fungi facilitate growth and competitive ability of an exotic species Flaveria bidentis

Understanding of mechanisms through which arbuscular mycorrhizal fungi (AMF) facilitate establishment and growth of exotic species is important in invasion ecology. We have poor understanding of mechanisms that mediate positive or negative impact of AMF to exotic or native species. We ask whether the quantitative and or qualitative differences in the AMF colonization to exotic Flaveria bidentis and native Setaria viridis provide competitive advantage to the invader over native species. Flaveria bidentis, a native of South America, is an aggressive invader in North China. Native S. viridis is widely distributed in farmlands, roadsides and wastelands, and also found in Flaveria bidentis invaded areas. We hypothesize that exotic species alter AMF composition, which facilitate the establishment and growth of exotic species and reduces the competitiveness of native species. Experiments were carried out to: (i) quantify AMF colonization in roots of field-grown F. bidentis or S. viridis, (ii) isolate DNA sequences of AMF species from roots and soils of exotic F. bidentis and native S. viridis to carry out phylogenetic studies, and (iii) study the effect of AMF Rhizophagus intraradices spores on the physiological performance traits of both exotic F. bidentis and native S. viridis. Our results suggest that certain AMF clones had higher colonization (85%) in roots of F. bidentis than S. viridis (21.1%). AMF-inoculation (130 spores per 100 g soil) enhanced the net photosynthetic rate in the native S. viridis when grown in monoculture but reduced its net photosynthetic rate when grown in competition with exotic F. bidentis. A lower corrected index of relative competition intensity in exotic F. bidentis, and higher levels of foliar nutrient accumulation was observed in response to AMF inoculation than native S. viridis. We show that higher AMF colonization in F. bidentis provides competitive advantage over native S. viridis. This study is important because it establishes the impact of AMF colonization on physiological and ecological performance of the invader.

Patterns of genetic screening for hereditary cancer syndromes: Effect of Supreme Court’s ruling invalidating single gene patent rights.

1580 Background: In 6/2013 the Supreme Court ruled that isolated DNA sequences found in nature could not be patented, resulting in rapid uptake of multigene panels. We sought to explore trends in genetic testing since this ruling. Methods: Results of all patients undergoing genetic testing and counseling at a single institution between 7/1/13 and 12/31/16 were reviewed. Associations between categorical variables were evaluated by chi-square tests or Fisher's exact tests as appropriate for category size. Results: 1663 patients underwent genetic testing over the study period. The median age was 49 years (range 18-86). Use of multigene panels versus targeted gene testing increased significantly in the years following the Supreme Court ruling (Table 1, P<0.001). While the percentage of patients found to have pathogenic mutations remained stable over the study period (9%), detection of variants of uncertain significance (VUS) increased significantly (Table 1, P<0.001). In 2013 BRCA1/2 mutations accounted for 91% of identified mutations; however this number decreased over time (2014-83%, 2015-70%, 2016-58%, P=0.01). Use of multigene panels detected 71% of mutations in non- BRCA1/2 genes such as CHEK(19), APC(44), MSH6(1), P53(1), and PTEN(1). Patients with a personal history of breast and/or ovarian cancer were more likely to have targeted testing than patients with other cancer types (590, 66% vs. 9, 33%, P=0.001). Conclusions: The uptake of multigene panels has increased since the 2013 Supreme Court ruling. While this technology allowed for the identification of many cancer-related genes that would be missed on targeted BRCA1/2 testing, it also resulted in a significantly increased detection of VUS, a finding with unknown clinical implications. [Table: see text]

A bacterium with close genetic identity to Pseudomonas mandelii associated with spring fish kills in wild bluegill Lepomis macrochirus Rafinesque and pumpkinseed sunfish Lepomis gibbosus (Linnaeus).

Pseudomonas fluorescens are known bacterial pathogens in fish. The P. fluorescens group contains at least nine different bacterial species, although species from fish have rarely been differentiated. Two isolated fish kills affecting wild bluegills, Lepomis macrochirus Rafinesque, and pumpkinseed sunfish, Lepomis gibbosus (Linnaeus), occurred in the spring of 2015 during cool water temperatures (12.5°C-15.5°C). Disease signs included severe bacteraemia with rare gross external signs. Pure bacterial cultures isolated from kidneys of all affected fish were identified as P. fluorescens using the API 20NE system, while no bacteria were isolated from asymptomatic fish. To further identify the species of bacterium within the P. fluorescens complex, genetic analysis of the 16S rRNA, rpoD and gyrB genes was conducted. DNA sequences of bacterial isolates from both mortality events were identical and had close identity (≥99.7%) to Pseudomonas mandelii. Although likely widespread in the aquatic environment, this is the first report of a bacterium closely resembling P. mandelii infecting and causing disease in fish. The bacterium grew at temperatures between 5°C and 30°C, but not at 37°C. It is possible that infections in fish were a result of immunosuppression associated with spring conditions combined with the psychrotrophic nature of the bacterium.

Formation of body appendages during caudal regeneration in Platynereis dumerilii: adaptation of conserved molecular toolsets.

BackgroundPlatynereis and other polychaete annelids with homonomous segmentation are regarded to closely resemble ancestral forms of bilateria. The head region comprises the prostomium, the peristomium, a variable number of cephalized body segments and several appendages, like cirri, antennae and palps. The trunk of such polychaetes shows numerous, nearly identical segments. Each segment bears a parapodium with species-specific morphology on either side. The posterior end of the trunk features a segment proliferation zone and a terminal pygidium with the anus and anal cirri. The removal of a substantial part of the posterior trunk is by no means lethal. Cells at the site of injury dedifferentiate and proliferate forming a blastema to regenerate both the pygidium and the proliferation zone. The pygidium forms new anal cirri, and the proliferation zone generates new segments at a rapid pace. The formation of body appendages like the cirri and the segmental parapodia can thus be studied in the caudal regenerate of Platynereis within only a few days.ResultsThe development of body appendages in Platynereis is regulated by a network of genes common to polychaetes but also shared by distant taxa. We isolated DNA sequences from P. dumerilii of five genes known to be involved in appendage formation within other groups: Meis/homothorax, Pbx1/extradenticle, Dlx/Distal-less, decapentaplegic and specificprotein1/buttonhead. Analyses of expression patterns during caudal regeneration by in situ hybridization reveal striking similarities related to expression in arthropods and vertebrates. All genes exhibit transient expression during differentiation and growth of segments. As was shown previously in other phyla Pdu-Meis/hth and Pdu-Pbx1/exd are co-expressed, although the expression is not limited to the proximal part of the parapodia. Pdu-Dll is prominent in parapodia but upregulated in the anal cirri. No direct dependence concerning Pdu-Dll and Pdu-sp/btd expression is observed in Platynereis. Pdu-dpp shows an expression pattern not comparable to its expression in other taxa.ConclusionsThe expression patterns observed suggest conserved roles of these genes during appendage formation across different clades, but the underlying mechanisms utilizing this toolset might not be identical. Some genes show broad expression along the proximodistal axis indicating a possible role in proximodistal patterning of body appendages. Other genes exhibit expression patterns limited to specific parts and tissues of the growing parapodia, thus presumably being involved in formation of taxon-specific morphological differences.Electronic supplementary materialThe online version of this article (doi:10.1186/s13227-016-0046-6) contains supplementary material, which is available to authorized users.

DevI is an evolutionarily young negative regulator of Myxococcus xanthus development.

During starvation-induced development of Myxococcus xanthus, thousands of rod-shaped cells form mounds in which they differentiate into spores. The dev locus includes eight genes followed by clustered regularly interspaced short palindromic repeats (CRISPRs), comprising a CRISPR-Cas system (Cas stands for CRISPR associated) typically involved in RNA interference. Mutations in devS or devR of a lab reference strain permit mound formation but impair sporulation. We report that natural isolates of M. xanthus capable of normal development are highly polymorphic in the promoter region of the dev operon. We show that the dev promoter is predicted to be nonfunctional in most natural isolates and is dispensable for development of a laboratory reference strain. Moreover, deletion of the dev promoter or the small gene immediately downstream of it, here designated devI (development inhibitor), suppressed the sporulation defect of devS or devR mutants in the lab strain. Complementation experiments and the result of introducing a premature stop codon in devI support a model in which DevRS proteins negatively autoregulate expression of devI, whose 40-residue protein product DevI inhibits sporulation if overexpressed. DevI appears to act in a cell-autonomous manner since experiments with conditioned medium and with cell mixtures gave no indication of extracellular effects. Strikingly, we report that devI is entirely absent from most M. xanthus natural isolates and was only recently integrated into the developmental programs of some lineages. These results provide important new insights into both the evolutionary history of the dev operon and its mechanistic role in M. xanthus sporulation. Certain mutations in the dev CRISPR-Cas (clustered regularly interspaced short palindromic repeat-associated) system of Myxococcus xanthus impair sporulation. The link between development and a CRISPR-Cas system has been a mystery. Surprisingly, DNA sequencing of natural isolates revealed that many appear to lack a functional dev promoter, yet these strains sporulate normally. Deletion of the dev promoter or the small gene downstream of it suppressed the sporulation defect of a lab strain with mutations in dev genes encoding Cas proteins. The results support a model in which the Cas proteins DevRS prevent overexpression of the small gene devI, which codes for an inhibitor of sporulation. Phylogenetic analysis of natural isolates suggests that devI and the dev promoter were only recently acquired in some lineages.