Summary
Population-level genetic tools to study the epidemiology of potato cyst nematodes (PCN) are needed to evaluate their adaptive potential and validate management strategies. The effector gene ...vap1 is essential in early infection by PCN. We expected high allelic variation due to diversifying selection. A technique was developed specifically to amplify vap1 fragments of PCN, and electrophoretically separate variants in a denaturing gradient (DGGE). Sequenced gene variants differed in intron as well as exon regions. Siblings were more similar in vap1 pattern than juveniles from different cysts. Nearly all juveniles analysed had unique patterns with up to ten or five vap1 variants for Globodera pallida or G. rostochiensis, respectively. Patterns differed significantly among populations, with similarities increasing from between-species, within-species to within-pathotype comparisons. In conclusion, the method is suitable to determine the similarity of heterogeneous PCN populations for studies on their temporal change and spread.
ABSTRACT
BACKGROUND:
Klebsiella pneumoniae
causes both nosocomial and community-associated infections. Hypervirulent
K. pneumoniae
(hvKP), new variant of
K. pneumoniae
, can cause invasive infections ...in young healthy individuals as well as in the immunocompromised population. Hypervirulent strains frequently belong to capsular serotypes K1 or K2. Emergence of antimicrobial resistance in hvKP is a cause for concern.
AIM AND OBJECTIVE:
The present study was done to detect the K1 and K2 serotypes among clinical isolates of
K. pneumoniae
, spectrum of infections caused by them and presence of common beta-lactamases encoding genes in them.
MATERIALS AND METHODS:
A total of 370 isolates of
K. pneumoniae
, isolated from various clinical samples over a period of 1 year was included in this study. Antibiotic susceptibility testing to various classes of antimicrobials was done as per Clinical and Laboratory Standard Institute guidelines. The presence of
K2A
(specific to serotype K2), magA (specific to serotype K1), and
rmpA
genes was detected by multiplex polymerase chain reaction (PCR). Extended-spectrum beta-lactamases (TEM, SHV, and CTX-M), plasmid-mediated AmpCs (MOX, CIT, DHA, ACC, EBC, and FOX), and carbapenemase genes (IMP, VIM, NDM, KPC, and OXA-48) were also determined by PCR.
RESULTS:
Among the 370 isolates, 8 harbored
K2A
gene and one harbored
magA. rmpA
gene was detected in three isolates along with K1 or K2 serotypes. Seven
K2A
-positive isolates were resistant to one or more classes of antimicrobials. The studied ESBL genes were present in four isolates. Two isolates harbored carbapenemase genes (NDM-1, OXA-48) along with ESBLs.
Conclusions:
K2 serotype is more prevalent among hvKP isolates. They can harbor ESBLs and Carbapenemase genes. K1 serotype is rather uncommon in
K. pneumoniae
. Acquisition of multidrug-resistant genes by these strains adds to their virulence and limits the treatment options.
Indoor air pollution caused by fungal contamination is suspected to have a public health impact. Monitoring of the composition of the indoor airborne fungal contaminants is therefore important. To ...avoid problems linked to culture-dependent protocols, molecular methods are increasingly being proposed as an alternative. Among these molecular methods, the polymerase chain reaction (PCR) and the real-time PCR are the most frequently used tools for indoor fungal detection. However, even if these tools have demonstrated their appropriate performance, some of them are not able to discriminate between species which are genetically close. A solution to this could be the use of a post-qPCR high resolution melting (HRM) analysis, which would allow the discrimination of these species based on the highly accurate determination of the difference in melting temperature of the obtained amplicon. In this study, we provide a proof-of-concept for this approach, using a dye adapted version of our previously developed qPCR SYBR®Green method to detect Aspergillus versicolor in indoor air, an important airborne fungus in terms of occurrence and cause of health problems. Despite the good performance observed for that qPCR method, no discrimination could previously be made between A. versicolor, Aspergillus creber and Aspergillus sydowii.
In this study, we developed and evaluated an HRM assay for the discrimination between A. versicolor, Aspergillus creber and Aspergillus sydowii.
Using HRM analysis, the discrimination of the 3 Aspergillus species could be made. No false positive, nor false negatives were observed during the performance assessment including 20 strains of Aspergillus. The limit of detection was determined for each species i.e., 0.5 pg of gDNA for A. creber and A. sydowii, and 0.1 pg of gDNA for A. versicolor. The HRM analysis was also successfully tested on environmental samples.
We reported the development of HRM tools for the discrimination of A. versicolor, A. creber and A. sydowii. However, this study could be considered as a study case demonstrating that HRM based on existing qPCR assays, allows a more accurate identification of indoor air contaminants. This contributes to an improved insight in the diversity of indoor airborne fungi and hence, eventually in the causal link with health problems.
Fusarium species, which can produce mycotoxins, are the predominant pathogens causing maize ear rot, a disease that results in severe economic losses and serves as a potential health risk for humans ...and animals. A survey was conducted in 2012 to investigate the contamination of maize by Fusarium species and fumonisins B
1
and B
2
. A total of 250 maize samples were randomly collected from nine provinces (Hebei, Shanxi, Inner Mongolia, Yunnan, Sichuan, Guizhou, Heilongjiang, Liaoning and Ningxia) in China. Fusarium species were isolated and identified using morphological (electron microscope) and molecular methods (polymerase chain reaction (PCR) and sequencing). Fumonisins B
1
and B
2
were analysed using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) with OPA (2-Mercaptoethanol, o-phthaldialdehyde) post-column derivatisation. A total of 2321 Fusarium isolates (20.7%) were obtained from all the samples. These isolates included nine Fusarium species, namely, F. graminearum, F. verticillioides, F. subglutinans, F. proliferatum, F. temperatum, F. oxysporum, F. equiseti, F. meridionale and F. chlamydosporum. The incidence of occurrence of Fusarium species in Guizhou was the highest, while in Inner Mongolia it was the lowest. F. verticillioides was the dominant species of maize ear rot in Liaoning, Sichuan, Hebei and Ningxia. F. graminearum was the dominant species in Yunnan, Guizhou and Shanxi. F. subglutinans was the dominant species in Heilongjiang. F. verticillioides and F. graminearum percentages were the same in Inner Mongolia. The incidence of fumonisins in Liaoning was high (up to 81.0%) and in Heilongjiang low (up to 10.3%). Except Shanxi, more than 50% of maize samples from other provinces were contaminated with fumonisins, with concentrations less than 500 ng g
−1
. About 33% of maize samples from Yunnan were contaminated with high levels of fumonisins, and average of fumonisin levels were 5191 ng g
−1
. Fusarium species causing maize ear rot in different areas in China were highly diverse and such areas with exposure to high levels of fumonisin contamination have a potential health risk for human and animals.
Exophiala jeanselmei is an opportunistic pathogenic black yeast growing in humid environments such as water reservoirs of air-conditioning systems. Because this fungal contaminant could be vaporized ...into the air and subsequently cause health problems, its monitoring is recommended. Currently, this monitoring is based on culture and microscopic identification which are complex, sometimes ambiguous and time-demanding, i.e., up to 21 days. Therefore, molecular, culture-independent methods could be more advantageous for the monitoring of E. jeanselmei. In this study, we developed a SYBR®green real-time PCR assay based on the internal transcribed spacer 2 from the 18S ribosomal DNA complex for the specific detection of E. jeanselmei. The selectivity (100 %), PCR efficiency (95.5 %), dynamic range and repeatability of this qPCR assay were subsequently evaluated. The limit of detection for this qPCR assay was determined to be 1 copy of genomic DNA of E. jeanselmei. Finally, water samples collected from cooling reservoirs were analyzed using this qPCR assay to deliver a proof of concept for the molecular detection of E. jeanselmei in environmental samples. The results obtained by molecular analysis were compared with those of classical methods (i.e., culture and microscopic identification) used in routine analysis and were 100 % matching. This comparison demonstrated that this SYBR®green qPCR assay can be used as a molecular alternative for monitoring and routine investigation of samples contaminated by E. jeanselmei, while eliminating the need for culturing and thereby considerably decreasing the required analysis time to 2 days.
The phototrophic dinoflagellate Biecheleriopsis adriatica is a small suessioid species characterized by a fragile thin wall. Although the morphology of this dinoflagellate is well established, there ...is currently little information available on its distribution and the environmental factors that influence this distribution. Thus, to investigate the spatial and seasonal distributions of the vegetative cells of B. adriatica in Korean waters, surface water samples were collected on a seasonal basis from 28 stations in the East, West, and South Sea of Korea and Jeju Island from April 2015 to October 2018, and abundances of the vegetative cells of B. adriatica were quantifled using quantitative real-time polymerase chain reactions, for which we developed the species-speciflc primer and probe set. Simultaneously, major environmental parameters, including temperature, salinity, nutrient concentrations, and dissolved oxygen concentrations were measured. The vegetative cells of B. adriatica were detected at 20 of the 28 sampling stations: 19 stations in summer and 6 in autumn, although from no stations in either spring or winter. The ranges of water temperature and salinity at sites where this species was detected were 17.7-26.4°C and 9.9-34.3, respectively, whereas those of nitrate and phosphate concentrations were not detectable-96.2 and 0.18-2.66 pM, respectively. Thus, the sites at which this species is found are characterized by a narrow range of temperature, but wide ranges of salinity and concentrations of nitrate and phosphate. The highest abundance of the vegetative cells of B. adriatica was 41.7 cells mL-1, which was recorded in Jinhae Bay in July 2018. In Jinhae Bay, the abundance of vegetative cells was significantly positively correlated with the concentration of nitrate, but was negatively correlated with salinity. On the basis of these flndings, it appears that the abundance of B. adriatica vegetative cells shows strong seasonality, and in Jinhae Bay, could be affected by the concentrations of nitrate.
Background: In recent years, use of powdered infant formula (PIF) milk for neonates feed is increasing; therefore, the quality control (QC) of PIF products is very important. The aim of present study ...was detection of toxigenic Bacillus cereus species in PIF milk using PCR assay. Methods: The cross-sectional study was carried out on 125 samples of powdered infant formula milk (PIF) purchased between March 2015 and April 2016 in Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. Briefly, 0.1 dilutions were prepared and inoculated on Bacillus cereus selective media (MYP) and incubated at 30 °C for 24 hours. The suspicious colonies were verified using biochemical tests based on standard methods. Final confirmation of studied isolates was carried out by ITS gene detection using polymerase chain reaction (PCR) assay. Presence of nonhemolytic enterotoxin (NHE) (linked to diarrhoea syndrome) and emetic toxin (EM) (linked to emetic syndrome) virulence genes were investigated using polymerase chain reaction assay. Results: In this study, of 125 PIF samples, 84 (67.2%) were contaminated. Of various recovered bacteria from these samples, 110 bacterial isolates were suspected to be Bacillus spp. using phenotypic methods. The ITS PCR results showed that 91.8% of the isolates were B. cereus. Respectively, 53.63 and 79% of B. cereus isolates possessed NHE and EM virulence genes. Conclusion: Our data revealed that near 80% of Bacillus cereus isolates have emetic toxin (EM) gene, as result virulence potency of this isolates is very high. However, the low number of this organisms in foods is very important and food safety protocols for these opportunistic toxigenic bacteria should be revised. Since the pasteurization process is ineffective on B. cereus spores; therefore, spores can remain in PIF milk and the vegetative bacterial cells can cause food poisoning in neonates. Therefore, modification of foods quality control protocols is essential in order to identify virulence genes in this bacterium.
DNA microarrays are useful for the simultaneous detection of microorganisms in water samples. Specific probes targeting waterborne pathogens are selected with bioinformatics tools, synthesized and ...spotted onto a DNA array. Here, the construction of a DNA chip for waterborne pathogen detection is described, including the processes of probe in silico selection, synthesis, validation, and data analysis.
The pseudo-molecular method is employed to obtain analytical expressions for the elastic constants of an ensemble of anisotropic particles, in both disc-like and rod-like geometries. These particles ...interact via a phenomenological pair potential constructed from the non-spherical correction to the dispersion forces between two identical molecules. The molecular shape appears in the calculations of the elastic constants in two different cases. The first case considers a molecular volume of ellipsoidal shape continuously deformed from a positive (prolate spheroid, rod-like molecule) to large negative (oblate spheroid, disc-like molecule) values of a parameter describing some kind of eccentricity. The second one considers a molecular volume shape continuously deformed from a cylinder (calamitic molecule) to a plane disc by changing the ratio between the diameter of the cylinder and its long axis. The particular cases of Maier-Saupe and Nehring-Saupe interactions are obtained as simple limiting cases of the general pair potential interaction. These general results may be helpful to understand the limits of the pseudo-molecular method, and to understand the origin of elastic constants in discotic liquid crystals from a molecular perspective.
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