[Our experience in laboratory diagnosis of rotaviruses].

OBJECTIVE: Group A rotavirus (RVA) is one of leading causes of gastroenteritis in children under five years of age and is also an important nosocomial pathogen. In Europe, the most prevalent genotypes of RVA are G1P[8], G2P[4], G3P[8], G4P[8], G9P[8] and G12P[8]. Severe dehydration is the most important complication of RVA gastroenteritis. Each year, rotavirus infection is responsible for 3,000 to 5,000 hospitalizations of children in the Czech Republic. The aim of this study was to detect rotaviruses in patients with suspected acute viral gastroenteritis. METHODS: A total of 1 566 stool samples were obtained from patients with acute gastroenteritis from March 2016 to December 2018. All samples were tested by the enzyme immunoassay, rapid immunochromatographic test and quantitative reverse transcription PCR assay to detect RVA. All RVA positive samples were G- and P-typed by Sanger sequencing. RESULTS AND CONCLUSION: RVA was detected in 13.7 % of the samples (214/1566). The incidence of RVA was 58.9 % (126/214) in males and 41.1 % (88/214) in females. The percentages of positivity ranged from 1 % to 33 % in different age groups. The highest proportion of positive patients was in the age group 4-5 years, 32.6 % (30/92). There was a significant difference in the incidence of rotaviruses between different age groups (p = 0.3946). The prevalent RVA genotypes were G1P[8], G9P[8], G3P[8], G2P[4] and G8P[8]. The detection of the G8P[8] genotype was unusual. The obtained results show that despite the possibility of vaccination, the incidence of RVA infection remains high in the Czech Republic.

Separation of methicillin-resistant from methicillin-susceptible Staphylococcus aureus by electrophoretic methods in fused silica capillaries etched with supercritical water.

Identification and prevention of Staphylococcus aureus-caused infections may benefit from a fast and dependable method to distinguish between the methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) S. aureus strains. The current methods involving polymerase chain reaction and/or other molecular tests are usually laborious and time-consuming. We describe here a fast and low-cost method employing capillary zone electrophoresis (CZE) to distinguish between MRSA and MSSA. The method makes use of a supercritical water-treated fused silica capillary, the inner surface of which has subsequently been modified with (3-glycidyloxypropyl)trimethoxysilane. With optimized proportions of suitable additives to the background electrolyte, a CZE separation of MRSA from MSSA may be completed within 12 min. The cells were baseline-resolved, and resolution was determined to be 3.61. The isoelectric points of MSSA and MRSA were found to be the same for both groups of these strains, pI = 3.4.

CIEF separation, UV detection, and quantification of ampholytic antibiotics and bacteria from different matrices.

The effect of antibiotics on the microbial cells and concentration of antibiotics in the human body is essential for the effective use of antimicrobial therapy. The capillary isoelectric focusing is a suitable technique for the separation and the detection of bacteria, and amphoteric substances from nature. However, the determination of isoelectric points of ampholytic antibiotics by conventional techniques is time consuming. For this reason, capillary isoelectric focusing seems to be appropriate as a simple and reliable way for establishing them. The separation conditions for the capillary isoelectric focusing of selected ampholytic antibiotics with known isoelectric points and pK as, ampicillin (pI 4.9), ciprofloxacin (pI 7.4), ofloxacin (pI 7.1), tetracycline (pI 5.4), tigecycline (pI 9.7), and vancomycin (pI 8.1), were found and optimized in the suitable pH ranges pH 2.0-5.3, 2.0-9.6, and 9.0-10.4. The established values of isoelectric points correspond with those found in the literature except tigecycline. Its pI was not found in the literature. As an example of a possible procedure for direct detection of both ampholytic antibiotics and bacteria, Staphylococcus epidermidis, in the presence of culture media or whole human blood, was found. The changes of the bacterial cells after their treatment with tetracycline were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Capillary isoelectric focusing allows the fast and simple determination of isoelectric points of relevant antibiotics, their quantification from the environment, as well as studying their effectiveness on microorganisms in biological samples.

Following the mechanisms of bacteriostatic versus bactericidal action using Raman spectroscopy.

Antibiotics cure infections by influencing bacterial growth or viability. Antibiotics can be divided to two groups on the basis of their effect on microbial cells through two main mechanisms, which are either bactericidal or bacteriostatic. Bactericidal antibiotics kill the bacteria and bacteriostatic antibiotics suppress the growth of bacteria (keep them in the stationary phase of growth). One of many factors to predict a favorable clinical outcome of the potential action of antimicrobial chemicals may be provided using in vitro bactericidal/bacteriostatic data (e.g., minimum inhibitory concentrations-MICs). Consequently, MICs are used in clinical situations mainly to confirm resistance, and to determine the in vitro activities of new antimicrobials. We report on the combination of data obtained from MICs with information on microorganisms' "fingerprint" (e.g., DNA/RNA, and proteins) provided by Raman spectroscopy. Thus, we could follow mechanisms of the bacteriostatic versus bactericidal action simply by detecting the Raman bands corresponding to DNA. The Raman spectra of Staphylococcus epidermidis treated with clindamycin (a bacteriostatic agent) indeed show little effect on DNA which is in contrast with the action of ciprofloxacin (a bactericidal agent), where the Raman spectra show a decrease in strength of the signal assigned to DNA, suggesting DNA fragmentation.

Antimicrobial effect of commercial phage preparation Stafal® on biofilm and planktonic forms of methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus may be a highly virulent human pathogen, especially when it is able to form a biofilm, and it is resistant to antibiotic. Infections caused by these bacteria significantly affect morbidity and mortality, primarily in hospitalized patients. Treatment becomes more expensive, more toxic, and prolonged. This is the reason why research on alternative therapies should be one of the main priorities of medicine and biotechnology. A promising alternative treatment approach is bacteriophage therapy. The effect of the anti-staphylococcal bacteriophage preparation Stafal® on biofilm reduction was assessed on nine S. aureus strains using both sonication with subsequent quantification of surviving cells on the catheter surface and evaluation of biofilm reduction in microtiter plates. It was demonstrated that the bacteriophages destroy planktonic cells very effectively. However, to destroy cells embedded in the biofilm effectively requires a concentration at least ten times higher than that provided by the commercial preparation. The catheter disc method (CDM) allowed easier comparison of the effect on planktonic cells and cells in a biofilm than the microtiter plate (MTP) method.

Molecular Techniques Complement Culture-Based Assessment of Bacteria Composition in Mixed Biofilms of Urinary Tract Catheter-Related Samples.

Urinary or ureteral catheter insertion remains one of the most common urological procedures, yet is considered a predisposing factor for urinary tract infection. Diverse bacterial consortia adhere to foreign body surfaces and create various difficult to treat biofilm structures. We analyzed 347 urinary catheter- and stent-related samples, treated with sonication, using both routine culture and broad-range 16S rDNA PCR followed by Denaturing Gradient Gel Electrophoresis and Sanger sequencing (PCR-DGGE-S). In 29 selected samples, 16S rRNA amplicon Illumina sequencing was performed. The results of all methods were compared. In 338 positive samples, from which 86.1% were polybacterial, 1,295 representatives of 153 unique OTUs were detected. Gram-positive microbes were found in 46.5 and 59.1% of catheter- and stent-related samples, respectively. PCR-DGGE-S was shown as a feasible method with higher overall specificity (95 vs. 85%, p < 0.01) though lower sensitivity (50 vs. 69%, p < 0.01) in comparison to standard culture. Molecular methods considerably widened a spectrum of microbes detected in biofilms, including the very prevalent emerging opportunistic pathogen Actinotignum schaalii. Using massive parallel sequencing as a reference method in selected specimens, culture combined with PCR-DGGE was shown to be an efficient and reliable tool for determining the composition of urinary catheter-related biofilms. This might be applicable particularly to immunocompromised patients, in whom catheter-colonizing bacteria may lead to severe infectious complications. For the first time, broad-range molecular detection sensitivity and specificity were evaluated in this setting. This study extends the knowledge of biofilm consortia composition by analyzing large urinary catheter and stent sample sets using both molecular and culture techniques, including the widest dataset of catheter-related samples characterized by 16S rRNA amplicon Illumina sequencing.