Multilocus sequence analysis reveals genetic diversity in Staphylococcus aureus isolate of goat with mastitis persistent after treatment with enrofloxacin

Staphylococcus aureus is one of the main bacterial agents responsible for cases of mastitis in ruminants, playing an important role in the persistence and chronicity of diseases treated with antimicrobials. Using the multilocus sequence typing technique, network approaches and study of the population diversity of microorganisms, we performed analyzes of S. aureus (ES-GPM) isolated from goats with persistent mastitis (GPM). The most strains of ES-GPM were categorically different phylogenetically from the others and could be divided into two lineages: one with a majority belonging to ES-GPM and the other to varied strains. These two lineages were separated by 27 nuclear polymorphisms. The 43 strains comprised 22 clonal complexes (CCs), of which the ES-GPM strains were present in CC133, CC5 and a new complex formed by the sequence type 4966. The genetic diversity of some alleles showed be greater diversity and polymorphism than others, such as of the aroE and yqiL genes less than glpF gene. In addition, the sequences ES-GPM to the arc gene and glpF alleles showed the greatest number of mutations for ES-GPM in relation to non-ES-GPM. Therefore, this study identified genetic polymorphisms characteristic of S. aureus isolated from milk of goats diagnosed with persistent mastitis after the failed treatment with the antibiotic enrofloxacin. This study may help in the future to identify and discriminate this agent in cases of mastitis, and with that, the most appropriate antibiotic treatment can be performed in advance of the appearance of persistent mastitis caused by the agent, reducing the chances of premature culling and animal suffering.

Biotechnological starter potential for cocoa fermentation from cabruca systems / Potencial biotecnológico de leveduras starter na fermentação do cacau de sistema cabruca

Cocoa is a fruit of great economic importance, being the main raw material in the manufacture of chocolate. Among the stages of pre-processing, the main and most important is the spontaneous fermentation of the cocoa pulp by microorganisms, especially the yeasts, which initiate the process and contribute to the death of the germ of the seed, releasing compounds that directly influence the quality of the final product (flavor and aroma). Poorly fermented almonds confer bitter and astringent taste on chocolate, so it is advantageous to select autochthonous yeasts with better performance in the fermentation (producing enzymes of interest in the process) to be used as inoculum starter when added in the spontaneous fermentation, where they can accelerate the fermentation and contribute to raising the quality of the product. Therefore, the objective of this work was to qualitatively determine the production of enzymes of biotechnological interest by yeasts for the fermentation of cocoa through the cup plate method, in order to select a candidate yeast inoculum and use molecular typing technique to evaluate the diversity. Many promising yeasts were identified for use as inoculum among the diverse yeast groups found.

A Short Tandem Repeat Assay for Studying Genetic Variation in Madurella mycetomatis (MmySTR)

Introduction: Madurella mycetomatis is the major causative agent of eumycetoma, a neglected tropical infection characterized by painless subcutaneous lesions, inflammation and grains draining from multiple sinuses. To study the epidemiology of mycetoma, a robust discriminatory typing technique is needed. We describe the use of a Short Tandem Repeats assay (MmySTR) for genotyping of M. mycetomatis isolates predominantly from Sudan. Methods: Eleven microsatellite markers (3 dinucleotides, 4 trinucleotide repeats and 4 tetranucleotide repeats) were selected from the M. mycetomatis MM55 genome using the Tandem Repeats Finder software. PCR amplification primers were designed for each microsatellite marker using primer3 software and amplified in a multicolor multiplex PCR approach. To establish the extent of genetic variation within the population, a collection of 120 clinical isolates from different regions was genotyped with this assay. Results: The 11 selected MmySTR markers showed a large genotypic heterogeneity. From a collection of 120 isolates, 108 different genotypes were obtained. The Simpsons’ diversity index (D) value for individual markers ranged from 0.081-0.881, the combined panel displayed an overall D value of 0.997. The MmySTR assay demonstrated high stability, reproducibility and specificity. Conclusion: The MmySTR assay is a promising new typing technique that can be used to genotype isolates of M. mycetomatis. Apart from the possible contribution of host factors, the genetic diversity observed among this group of isolates might contribute to the different clinical manifestations of mycetoma. We recommend the MmySTR assay to be used to establish a global reference database for future study of M. mycetomatis isolates.

OVERVIEW OF 3D PRINTING TECHNOLOGIES IN DENTISTRY

Today, in an age of high speeds and scientific discov-eries, people are trying with all their might to speed up the process of developing and publishing a new product. One of these ways is the use of rapid proto-typing technique (rapid creation of a prototype of the equipment being developed to conduct a series of tests on it or to refine it to the final product). In the begin-ning, classical methods (turning, milling, stamping) were used to make prototypes, but this was inconven-ient for a number of reasons (such production of blanks generates a large amount of waste, the work must be performed by qualified personnel). Therefore, in the 80s of the last century, an alternative appeared - the object is not made "at once" (from a workpiece by changing its shape), but layer by layer (moreover, rather unusual materials are used - metal and plastic powders, various types of clay, polymers sensitive to light), after which the layers are held together. This review focuses on common 3D printing technologies used in dentistry. Technologies and schemes of 3D printing are presented.

Association between gene polymorphisms of voltage-dependent Ca2+ channels and hypertension in the Dai people of China: a case-control study

Background : Abnormal calcium homeostasis related to the development of hypertension. As the key regulator of intracellular calcium concentration, voltage-gated calcium channels (VDCCs), the variations in these genes may have important effects on the development of hypertension. Here we evaluate VDCCs variability with respect to hypertension in the Dai ethnic group of China. Methods : A total of 1034 samples from Dai individuals were collected, of which 495 were used as cases, and 539 were used as controls. Blood pressure was measured using a standard mercury measurement method, three times with a rest for 5 min, and the average was used for analyses. Seventeen single nucleotide polymorphisms (SNPs) in the four protein-coding genes ( CACNA1A, CACNA1C, CACNA1S, CACNB2 ) of VDCCs were identified by multiplex PCR-SNP typing technique. Chi-square tests and regression models were used to analyse the associations of SNPs with hypertension. Results: The results of chi-square tests showed that the allele frequencies of 5 SNPs were significantly different between the case and the control groups (P<0.05), but the statistical significance was lost after Bonferroni’s correction. However, after adjusting for BMI, age, sex and other factors by logistic regression analyses, the results showed that 5 SNPs consistent with chi-square tests (rs2365293, rs17539088, rs16917217, rs61839222 and rs10425859) were still statistically positive. Conclusions: This finding suggested that the significant association of these SNPs with hypertension may be noteworthy in future studies.

Association between gene polymorphisms of voltage-dependent Ca2+ channels and hypertension in the Dai people of China: a case-control study

Background : Abnormal calcium homeostasis related to the development of hypertension. As the key regulator of intracellular calcium concentration, voltage-gated calcium channels (VDCCs), the variations in these genes may have important effects on the development of hypertension. Here we evaluate VDCCs variability with respect to hypertension in the Dai ethnic group of China. Methods : A total of 1034 samples from Dai individuals were collected, of which 495 were used as cases, and 539 were used as controls. Blood pressure was measured using a standard mercury measurement method, three times with a rest for 5 min, and the average was used for analyses. Seventeen single nucleotide polymorphisms (SNPs) in the four protein-coding genes ( CACNA1A, CACNA1C, CACNA1S, CACNB2 ) of VDCCs were identified by multiplex PCR-SNP typing technique. Chi-square tests and regression models were used to analyse the associations of SNPs with hypertension. Results: The results of chi-square tests showed that the allele frequencies of 5 SNPs were significantly different between the case and the control groups (P<0.05), but the statistical significance was lost after Bonferroni’s correction. However, after adjusting for BMI, age, sex and other factors by logistic regression analyses, the results showed that 5 SNPs consistent with chi-square tests (rs2365293, rs17539088, rs16917217, rs61839222 and rs10425859) were still statistically positive. Conclusions: This finding suggested that the significant association of these SNPs with hypertension may be noteworthy in future studies.

Association between gene polymorphisms of voltage-dependent Ca2+ channels andhypertension in the Dai people of China: a case-control study

Background: Abnormal calcium homeostasis related to the development of hypertension. As the key regulator of intracellular calcium concentration, voltage-gated calcium channels (VDCCs), the variations in these genes may have important effects on the development of hypertension. Here we evaluate VDCCs variability with respect to hypertension in the Dai ethnic group of China.Methods: A total of 1034 samples from Dai individuals were collected, of which 495 were used as cases, and 539 were used as controls. Blood pressure was measured using a standard mercury measurement method, three times with a rest for 5 min, and the average was used for analyses. Seventeen single nucleotide polymorphisms (SNPs) in the four protein-coding genes (CACNA1A, CACNA1C, CACNA1S, CACNB2) of VDCCs were identified by multiplex PCR-SNP typing technique. Chi-square tests and regression models were used to analyse the associations of SNPs with hypertension. Results: The results of chi-square tests showed that the allele frequencies of 5 SNPs were significantly different between the case and the control groups (P<0.05), but the statistical significance was lost after Bonferroni’s correction. However, after adjusting for BMI, age, sex and other factors by logistic regression analyses, the results showed that 5 SNPs consistent with chi-square tests (rs2365293, rs17539088, rs16917217, rs61839222 and rs10425859) were still statistically positive. Conclusions: This finding suggested that the significant association of these SNPs with hypertension may be noteworthy in future studies.

243. Transflection Fourier Transform Infrared Spectroscopy as a Real-Time Strain Typing Technique: A Vancomycin-Resistant Enterococcus faecium (VRE) Typing Prospective Study

Background Rapid bacterial strain typing for nosocomial outbreak surveillance is critical for timely outbreak detection and implementation of appropriate infection control protocols in hospitals. Pulsed-field gel electrophoresis (PFGE) remains the gold standard for strain typing, but it has the disadvantages of being time-consuming and costly. Transflection Fourier transform infrared (FTIR) spectroscopy is a nondestructive and reagent-free technique for rapid microbial identification and subspecies-level discrimination. The potential of employing transflection FTIR spectroscopy as a rapid, real-time typing technique was evaluated in the present study. Methods Transflection FTIR spectra were acquired from vancomycin-resistant Enterococcus faecium (VRE) isolates obtained from rectal swabs (n = 36) of patients in 6 units at a Montreal hospital over a 3-month period and from environmental screening samples (n = 2). Upon confirmation as VRE using a transflection FTIR spectral database previously developed in our laboratory, isolates were further typed by unsupervised hierarchical cluster analysis and principal component analysis of the FTIR spectral data with the use of a feature selection algorithm. Results Analysis of the FTIR data identified independent cases of VRE outbreak in 2 of 6 units; these outbreaks were confirmed retrospectively by PFGE. Based on the PFGE typing results for all 38 isolates included in this study, FTIR spectral analyses successfully identified 95% (n = 18) of isolates related to the outbreaks and 95% (n = 18) of non-outbreak-related isolates, resulting in a false-positive (n = 1), and a false-negative (n = 1), rate of 5%. Additionally, the two environmental isolates were identified as part of the outbreak from one of the outbreak-positive units. Conclusion The results in this study indicate that transflection FTIR spectroscopy-based typing can be considered as an alternative typing technique to PFGE, providing real-time results to track the spread of antibiotic-resistant pathogens within hospitals. Furthermore, when combined with the use of a transflection FTIR spectral database, both identification and typing of an isolate can be achieved from a single spectral measurement, thereby reducing the time and cost required for outbreak investigation. Disclosures All authors: No reported disclosures.

A Clinical TB Detection Method Based on Molecular Typing Technique with Quality Control

The gold standard for diagnosing pulmonary Mycobacterium tuberculosis (TB) is the detection of tubercle bacillus in patient sputum samples. However, current methods either require long waiting times to culture the bacteria or have a risk of getting false-positive results due to cross-contamination. In this study, a method to detect tubercle bacillus based on the molecular typing technique is presented. This method can detect genetic units, variable number of tandem repeat (VNTR), which are the characteristic of tuberculosis (TB), and performs quality control using a mathematical model, ensuring the reliability of the results. Compared to other methods, the proposed method was able to process and diagnose a large volume of samples in a run time of six hours, with high sensitivity and specificity. Our method is also in the pipeline for implementation in clinical testing. Reliable and confirmed results are stored into a database, and these data are used to further refine the model. As the volume of data processed from reliable samples increases, the diagnostic power of the model improves. In addition to improving the quality control scheme, the collected data can be also used to support other TB research, such as that regarding the evolution of the tubercle bacillus.