Thioglycollate Medium Research Articles

Detecting Perkinsus-like organisms and Perkinsus marinus (Myzozoa: Perkinsidae) within new bivalve hosts in the southeastern Gulf of California.

Perkinsus marinus, a parasite myzozoa native from the Atlantic Ocean, has spread across various bivalve species in the Pacific Ocean. In the Gulf of California, clams like Larkinia grandis and Leukoma grata have become a secondary fishery, experiencing demand. During annual sampling (August 2017 - July 2018, n = 30/month), the Ray's fluid thioglycollate medium (RFTM) revealed presumptive hypnospores of Perkinsus sp. in L. grandis, with a low parasite prevalence (4.16%) and density (≤ 3.14 hypnospores g-1 tissue). From summer 2020 to summer 2021, P. marinus was seasonally identified in L. grata via staining and PCR tests, showing mean prevalence and density of 48.33% and 48 hypnospores g-1 tissue, respectively. The infection level was light in both clams. This parasite's presence in these new hosts within the southeastern Gulf of California confirms its ability to expand into non-native places, signaling a need for intervention strategies in managing and transporting mollusk species within the region.

Synergistic Effect of Metronidazole and Chlorhexidine against Porphyromonas gingivalis Growth: An In Vitro Study.

Aim: To evaluate the potential synergistic activity of metronidazole (MTZ) and chlorhexidine (CHX) against Porphyromonas. gingivalis (P. gingivalis) growth. Methods: Antimicrobial susceptibility tests of P. gingivalis to MTZ and CHX were performed on in vitro serial 2-fold dilutions of MTZ (from 1 mg/mL to 0.015 mg/mL) and CHX (from 1 mg/mL to 0.03 mg/mL) in thioglycollate medium broth in a 96-well plate. The turbidity of each sample was analyzed by absorbance spectrophotometry at 450 nm wavelengths by using an enzyme-linked immunosorbent assay (ELISA) reader. The MIC50 (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) were assessed. To investigate the potential synergism between MTZ and CHX, bacterial cells were treated with MTZ or CHX, as described above, either alone or in combination. Results: The MIC50 of MTZ was 0.03 mg/mL while that of CHX ranged from 0.12 to 0.06 mg/mL. MTZ and CHX exerted a significant inhibitory effect on P. gingivalis growth in a dose-dependent manner. MTZ at a low and ineffective concentration of 0.015 mg/mL, associated with a suboptimal concentration of CHX (0.03 mg/mL), exhibited a significant synergistic inhibitory effect on bacterial growth (50% inhibition vs. control) (p < 0.001), and the effect was more remarkable with 0.06 mg/mL CHX (75% inhibition vs. control). Conclusions: CHX and MTZ showed a significant synergistic effect against P. gingivalis growth. A non-effective concentration of MTZ (0.015 mg/mL) combined with suboptimal concentrations of CHX (0.03 mg/mL and 0.06 mg/mL) were related to a 50% growth in the inhibition and 99.99% death of P. gingivalis, respectively. The applicability of the clinical use of these concentrations should be tested in randomized controlled trials.

Can pre-analytical procedures improve microbiological culture yield in patients with periprosthetic infections?

BackgroundThe detection of causative pathogens plays a crucial role in the diagnosis and targeted treatment of periprosthetic joint infections (PJI). While there have been improvements in analytic methods in the past, pre-analytical procedures have not yet been sufficiently investigated. The objective of this study was to compare the culture yield of four different pre-analytical procedures.MethodsPatients with perioperative diagnosis of PJI were included in a single center cross-sectional study (2021–2022). Tissue samples (n = 20) of each patient were randomly and equally distributed to each of the four study arms. Tissue samples were either send to the laboratory without culture medium (group A) or were transported in thioglycolate medium immediately after sampling at three different temperatures (room temperature, 4 °C, 37° for 24 h; group B-D). Culture media were investigated for growth on days 1, 3, 7, 12, 14. All organisms, the number of positive samples and the time to positivity were recorded and compared between the study arms. Single positive cultures were considered as contamination.ResultsIn total, 71 patients were included. The proportions of culture negative samples (10–15%) and polymicrobial infections (51–54%) were comparable between the four arms. Seven patients (10%) were culture-negative in group A, but showed growth in thioglycolate media (group B-D). Furthermore, 13% of patients showed growth in all groups, but additional organisms were cultured in thioglycolate. There was growth beyond day 7 of culturing only in thioglycolate, but not in group A. A storage temperature of 4 °C showed a longer time to positivity compared to the other groups (p < 0.001).ConclusionsPre-analytical storage of tissue samples in thioglycolate broth did not improve the culture yield and did not detect additional cases of infection compared to the standard (pre-analytical storage in sterile containers). However, including a thioglycolate medium to the sampling algorithm reduced the rate of culture-negative infections and helped to identify additional organisms.

Survey on Perkinsus species in two economic mussels (Mytilus coruscus and M. galloprovincialis) along the coast of the East China Sea and the Yellow Sea.

Perkinsus, a parasitic pathogen of marine bivalves, is widely distributed among various mollusks in numerous countries. However, the prevalence and diversity of Perkinsus species in the two economically important mussels, Mytilus coruscus and M. galloprovincialis, in China remain unknown. The presence of the Perkinsus species was identified in the two mussels sampled along the coast of the East China Sea and the Yellow Sea, using both the alternative Ray's fluid thioglycolate medium (ARFTM) and conventional polymerase chain reaction (PCR). The ARFTM test indicated the presence of Perkinsus-like hypnospores in the two mussels. The diameter of the hypnospores in M. coruscus was significantly smaller than that in M. galloprovincialis. The prevalence of Perkinsus in M. galloprovincialis and M. coruscus ranged from 0 to 37.5% and 0 to 25%, respectively. The mean intensity of Perkinsus in M. galloprovincialis and M. coruscus ranged from 0 to 5.14 and 0 to 4.92, respectively. The PCR assay showed that the prevalence of Perkinsus spp. in M. galloprovincialis and M. coruscus was 0 to 25.0% and 0 to 12.5%, respectively. The homology analysis of the newly obtained internal transcribed spacer (ITS) sequences of Perkinsus revealed the highest identity of 100% with P. beihaiensis. The phylogenetic analysis indicated that the Perkinsus isolates from the two mussels were clustered with P. beihaiensis. The results of the molecular biology indicated that only P. beihaiensis was detected in the two mussels. The highest prevalence of P. beihaiensis was observed in Liaoning province (Dalian, 20.83%), followed by Shandong province, Zhejiang province and Fujian province. Consequently, it is recommended that surveillance should be conducted in Dalian, where the prevalence and mean intensity of P. beihaiensis in M. galloprovincialis are the highest.

Recommendations for the development of sterility testing procedures for biological medicinal products based on pharmacopoeial methods

INTRODUCTION. The distinctive nature and composition of biological medicinal products present certain challenges for the detection of microbial contamination, which affects the reliability of sterility control results. The main challenges associated with testing biologicals include changes in the appearance and physical properties of culture media or samples, an increased risk of contamination or false-positive results in direct inoculation tests, filtration issues, etc. When drafting product specification files and analytical method validation reports in dossiers for biologicals, manufacturers need adequate, reproducible, and pharmacopoeia-based sterility testing procedures for specific medicinal products, including live and inactivated, viral and bacterial, single- and multi-component vaccines, bacteriophages and interferons, sera and immunoglobulins. These challenges can be addressed through providing recommendations for developing analytical procedures based on compendial methods of direct inoculation and membrane filtration.AIM. This study aimed to offer key recommendations for the development of sterility testing procedures for biologicals based on pharmacopoeial methods.DISCUSSION. This article presents a retrospective analysis of the results of reviewing regulatory documentation for biological medicinal products submitted to the testing centre of the Scientific Centre for Expert Evaluation of Medicinal Products in 2021–2023. Having considered major issues with the analytical procedures described in the Sterility section of regulatory submissions, the authors offer the following recommendations for addressing these issues. The authors advise against using low-pH Sabouraud culture media to test blood products by direct inoculation. Biologicals containing mercury preservatives should be tested using a thioglycollate medium at two temperature settings. When testing various biologicals, analysts should refrain from plugging vials with cotton gauze. Instead, they should pour culture media into glass vials with rubber stoppers and crimps. Biologicals in large primary packaging (≥10 mL) should be tested by membrane filtration. Membrane filtration tests for biologicals should use solvents and rinsing agents selected in validation studies. It is also recommended to set the maximum volume of a biological medicinal product that can be filtered through a membrane filter. A reduction in the filtration speed is recommended to minimise foaming when testing blood products.CONCLUSION. The recommendations presented in this article can guide the development of sterility testing procedures for inclusion in the relevant section of regulatory submissions for biologicals. Manufacturers, control laboratories, and regulatory authorities may implement these recommendations to harmonise their approach to sterility testing of biologicals.

First report of Perkinsus marinus occurrence associated with wild Pacific oysters Crassostrea gigas from the west coast of Korea

This study reports the occurrence of Perkinsus marinus associated with wild Pacific oyster (Crassostrea gigas) specimens collected along the west coast of Korea. Confirmation of P. marinus presence was achieved by conventional PCR using World Organization of Animal Health (WOAH)-recommended primers that specifically targeted regions of the rDNA locus (ITS1, 5.8S, and ITS2). Sequencing of 10 samples revealed two distinct sequences differing by a single base pair, indicating potential haplotype variability. One sequence closely resembled the P. marinus strain found in Maryland, USA, whereas the other exhibited divergence, indicative of species diversity in the Korean strain, as was evident from the haplotype network analysis. Further validation involved the Ray’s Fluid Thioglycollate Medium (RFTM) assay, which initially yielded inconclusive results, possibly due to low infection intensity. Subsequently, RFTM and 2 M NaOH assays conducted on the isolates in the present study, cultured P. marinus cells in standard DMEM/F12 medium, and a positive P. marinus strain (ATCC 50509), revealed characteristic hypnospores of P. marinus upon Lugol’s iodine staining. These comprehensive investigations underscore the conclusive confirmation of P. marinus in Korean waters and mark a significant milestone in our understanding of the distribution and characteristics of this parasite in previously unreported regions.

Protective Effect of Silibinin on Lipopolysaccharide-Induced Endotoxemia by Inhibiting Caspase-11-Dependent Cell Pyroptosis.

To explore the protective effect and the underlying mechanism of silibinin (SIB), one of the active compounds from Silybum marianum (L.) Gaertn in endotoxemia. Mouse peritoneal macrophage were isolated via intraperitoneally injection of BALB/c mice with thioglycolate medium. Cell viability was assessed using the cell counting kit-8, while cytotoxicity was determined through lactate dehydrogenase cytotoxicity assay. The protein expressions of interleukin (IL)-1 α, IL-1 β, and IL-18 were determined by enzyme-linked immunosorbent assay. Intracellular lipopolysaccharide (LPS) levels were measured by employing both the limulus amoebocyte lysate assay and flow cytometry. Additionally, proximity ligation assay was employed for the LPS and caspase-11 interaction. Mice were divided into 4 groups: the control, LPS, high-dose-SIB (100 mg/kg), and low-dose-SIB (100 mg/kg) groups (n=8). Zebrafish were divided into 4 groups: the control, LPS, high-dose-SIB (200 εmol/L), and low-dose-SIB (100 εmol/L) groups (n=30 for survival experiment and n=10 for gene expression analysis). The expression of caspase-11, gasdermin D (GSDMD), and N-GSDMD was determined by Western blot and the expressions of caspy2, gsdmeb, and IL-1 β were detected using quantitative real-time PCR. Histopathological observation was performed through hematoxylineosin staining, and protein levels in bronchoalveolar lavage fluid were quantified using the bicinchoninicacid protein assay. SIB noticeably decreased caspase-11 and GSDMD-mediated pyroptosis and suppressed the secretion of IL-1 α, IL-1 β, and IL-18 induced by LPS (P<0.05). Moreover, SIB inhibited the translocation of LPS into the cytoplasm and the binding of caspase-11 and intracellular LPS (P<0.05). SIB also attenuated the expression of caspase-11 and N-terminal fragments of GSDMD, inhibited the relative cytokines, prolonged the survival time, and up-regulated the survival rate in the endotoxemia models (P<0.05). SIB can inhibit pyroptosis in the LPS-mediated endotoxemia model, at least in part, by inhibiting the caspase-11-mediated cleavage of GSDMD. Additionally, SIB inhibits the interaction of LPS and caspase-11 and inhibits the LPS-mediated up-regulation of caspase-11 expression, which relieves caspase-11-dependent cell pyroptosis and consequently attenuates LPS-mediated lethality.

Volatile biomarkers of Gram-positive bacteria of clinical relevance as a tool for infection diagnosis.

Volatile organic compounds (VOCs) are being studied as potential biomarkers in many infections. Therefore, this study aimed to analyze the volatile profile of three Gram-positive bacteria of clinical relevance to identify potential volatile biomarkers that allow their differentiation. L. monocytogenes, S. aureus, and E. faecalis clinical isolates were inoculated in a thioglycollate medium until grown. Then, VOCs were extracted by solid-phase microextraction, and the data obtained were subjected to multivariate analysis. According to our results, there was a high production of aldehydes in E. faecalis. In the case of alcohols, they only increased in L. monocytogenes, while ketones were produced significantly in all three bacteria, mainly due to acetoin. Acids were produced significantly in E. faecalis and L. monocytogenes. Potential biomarkers of L. monocytogenes could be 1-butanol and 2-methylbutanoic acid. In the case of E. faecalis, the VOC most related to its presence was nonanal. Lastly, potential biomarkers of S. aureus could be isoamyl butanoate and methionol, although some pyrazines have also been associated with this bacterium. The identification of potential biomarkers of these clinically relevant bacteria could open the way for the diagnosis of these infections through the analysis of volatile compounds.

Characterisation of common hypothetical surface peptides between protozoan parasites (Perkinsus olseni) originating from different geographical locations.

Perkinsus olseni and P. marinus are classified as notifiable pathogens by the World Organisation for Animal Health and are known to cause perkinsosis in a variety of molluscs globally. Mass mortalities due to these parasites in farms and in the wild have been a recurrent issue. Diagnosis for these protozoans is currently done using Ray's fluid thioglycollate medium method followed by optical microscopy or molecular assays. Both require a high level of skill and are time-consuming. An immunoassay method would make the diagnosis of perkinsosis quicker and cheaper. The present study used mass spectrometry-based proteomics to investigate common hypothetical surface peptides between different geographical isolates of P. olseni, which could be used to develop immunoassays in the future. Two peptides were identified: POLS_08089, which is a 42.7 kDa peptide corresponding to the 60S ribosomal subunit protein L4; and POLS_15916, which is a conserved hypothetical protein of 55.6 kDa. The identification of peptides may allow the development of immunoassays through a more targeted approach.

Occurrence and seasonal variation of Perkinsus sp. Infection in wild mollusk populations from coastal waters of Qingdao, northern China

Perkinsosis has been recognized as one of the major threats to natural and farmed bivalve populations, many of which are of commercial as well as environmental significance. Three Perkinsus species have been identified in China, and the Manila clam (Ruditapes philippinarum) was the most frequently infected species in northern China. Although the occurrence and seasonal variation of Perkinsus spp. have previously been examined, the pathological characteristics of these infections in wild Manila clams and sympatric species in China have seldom been reported. In the present study, the prevalence and intensity of Perkinsus infection in wild populations of Manila clams and 10 sympatric species from three sites were investigated by Ray’s fluid thioglycolate medium (RFTM) assay seasonally across a single year. Perkinsus infection was only identified in Manila clams, with a high prevalence (274/284 = 96.48 %) and low intensity (89.8 % with a Mackin value ≤ 2, suggesting generally low-intensity infections) throughout the year. Heavily infected clams were mainly identified in Tianheng in January, which displayed no macroscopic signs of disease. An overview of the whole visceral mass section showed that the trophozoites mostly aggregated in gills and connective tissue of the digestive tract, to a lesser extent in the mantle and foot, and even less frequently in adductor muscle and connective tissues of the gonad. PCR and ITS-5.8S rRNA sequencing of 93 representative RFTM-positive samples revealed a 99.69 to 100 % DNA sequence identity to Perkinsus olseni. Unexpectedly, significantly higher infection intensities were usually identified in January and April when the Condition Index (CI) was relatively high. We propose that factors associated with the anthropogenic harvesting pressure and irregular disturbances should be responsible for the uncommon seasonal infection dynamics of perkinsosis observed in the present study.

Microflora of atherosclerotic plaques and blood of patients with atherosclerosis

Background. Over the past decades, the role of the microbial factor in the development of atherosclerosis has been discussed. To date, data on the composition of the microflora of atherosclerotic plaques have been accumulated, but in these studies the blood microflora in atherosclerosis was not studied.&#x0D; Aim. To determine the contamination frequency of biopsy samples of atherosclerotic plaques and peripheral blood with cultured microorganisms in patients with atherosclerosis of the brachiocephalic arteries.&#x0D; Material and methods. A cross-sectional study in 35 patients with atherosclerosis of the carotid arteries; the average age of the patients was 58.2±8.3 years, was performed. Blood and atherosclerotic plaque samples from 23 men (mean age 58.6±10.3 years) and 12 women (mean age 57.3±14.3 years) were examined. Anaerobic flasks for blood cultivation were incubated at 35 °C for up to 6 months, from which they were regularly inoculated onto solid nutrient media to obtain culture growth. Cultures of tissues of atherosclerotic plaques of the carotid arteries in test tubes with thioglycollate medium were thermostated until visible growth of the culture was obtained; observation was carried out for up to 2 months. Identification of crops was carried out using commercial kits. For statistical analysis of the results obtained, the equality of the average values in two samples was checked using the Student’s statistical reliability test. Correlation analysis of the tightness of the connection was carried out using the Spearman rank correlation method.&#x0D; Results. The frequency of detection of microorganisms in samples of atherosclerotic plaques was 48.6% (17/35), including Cutibacterium acnes — in 31.4% (11/35), the genus Staphylococcus — in 22.9% (8/35), associations of these microorganisms — in 5.7% (2/35) of samples. C. acnes was cultured in the blood of 11.4% (4/35) of patients. In 5.7% (2/35) of patients, cultures of C. acnes bacteria were isolated from both atherosclerotic plaque biopsies and blood. Bacterial cultures exhibited slow growth on nutrient media. Correlation analysis of the closeness of the connection according to the Chaddock scale demonstrated the presence of a high and very high closeness of connection between the amount of high-density lipoproteins and the daily growth of C. acnes in atherosclerotic plaques (p=0.812) and blood (rxy=–0.969), the content of leukocytes showed a very high closeness of connection with days of growth of C. acnes in the blood (rxy=–0.957); Student's t-test revealed a statistically significant relationship with the fact of detection of C. acnes cultures in atherosclerotic plaques (p=0.013).&#x0D; Conclusion. Bacterial cultures isolated from blood samples and atherosclerotic plaques of patients with atherosclerosis had slow growth on nutrient media, and the period of their growth or the fact of detection correlated with the number of leukocytes and the level of high-density lipoproteins in the blood of patients.

8-Oxoguanine DNA Glycosylase 1 Inhibition Suppresses Inflammatory Responses in Sickle Cell Disease

Background: The continuous production of reactive oxygen species (ROS) and oxidative stress contribute to ongoing inflammation and the pathology of sickle cell disease (SCD). ROS are known to mediate DNA damage through oxidation of nucleotide bases with guanine being the primary target. Although guanine base lesions vary according to the nature of the oxidants, 7,8-dihydro-8-oxoguanine (8-oxoG) is the most frequent oxidation product of DNA, which is repaired by 8-Oxoguanine-DNA glycosylase-1 (OGG1) via the DNA base excision repair pathway (OGG1-BER). The release of the 8-oxoG base from the genome by OGG1-BER is a prerequisite for increasing expression of pro-inflammatory cytokines and immune response. OGG1 inhibition has been shown to attenuate proinflammatory responses via the mitochondrial DNA-cGAS-STING axis in mice (Qin et al, 2020), a pathway also involved in the activation of neutrophils in SCD (Tumburu et al, 2021). Here, we evaluate the effect of OGG1 inhibition on expression of pro-inflammatory cytokines and immune response in SCD. Methods: Blood samples were obtained from ethnic-matched healthy controls (HbAA) and patients with SCD (HbSS) enrolled under protocols NCT03049475 and NCT00047996. CD14+ monocytes were isolated from PBMC of HbAA and HbSS subjects using EasySep™ Human Monocyte Isolation Kit (STEMCELL Technologies). 8-oxoG was measured by staining CD14+ monocytes with anti-8-oxoG antibody; OGG1 and pro-inflammatory cytokine levels were evaluated by qRT-PCR and western blotting. After differentiation, macrophages derived from CD14+ monocytes were treated with or without 10 µM SU0268 (OGG1 inhibitor) for 1 hour and then incubated with plasma from healthy controls or HbSS patients in steady or crisis status, followed by qRT-PCR quantitation of IL-1ꞵ, IL-6, TNF-α, and OGG1 mRNA levels. These measurements were repeated after OGG1 knockdown using OGG1 siRNA in macrophages derived from CD14+ monocytes. We repeated the study on peritoneal macrophages (PMs) from Townes HbAA and HbSS mice that were collected 4 days after a 3% Brewer's thioglycolate medium intraperitoneal injection. The level of 8-oxoG was measured by staining PMs with anti-8-oxoG antibody. The mRNA and protein level of OGG1 and pro-inflammatory cytokines were evaluated by qRT-PCR and western blotting. PMs from HbAA mice were treated with 10 µM SU0268 (OGG1 inhibitor) or vehicle for 1 hour, before incubation with plasma from HbAA or HbSS mice, the mRNA levels of IL-1ꞵ, IL-6, TNF-α, and OGG1 were measured by qRT-PCR. Results: CD14+ monocytes isolated from human HbSS PBMC showed increased levels of 8-oxoG nuclear staining when compared with human HbAA CD14+ monocytes, consistent with an increased oxidative burden in SCD patients (Fig 1A). The mRNA and protein levels of OGG1 were also increased in HbSS monocytes, suggestive of a potential therapeutic target of OGG1. Consistent with previous studies, mRNA levels of pro-inflammatory cytokines IL-1ꞵ, IL-6, and TNF-α were increased in SS compared with AA monocytes. To investigate the role of OGG1 in regulating cytokine expression, we treated the macrophages derived from CD14+ monocytes with SU0268, a selective OGG1 inhibitor (Tahara et al, 2018). Macrophages treated with plasma from HbSS patients (steady and crisis status) showed increased expression of OGG1, IL-1ꞵ, IL-6, and TNF-α compared with cells treated with normal plasma, and the increase was largely blocked by SU0268 pretreatment (Fig1B). Further, siRNA knockdown of OGG1 decreased the expression of IL-1ꞵ and TNF-α, confirming the role of OGG1 in regulating pro-inflammatory cytokine expression. We then evaluated the effect of OGG1 in regulating cytokines expression in cells form SCD mice. The levels of 8-oxoG and OGG1, as well as cytokines (IL-1ꞵ, IL-6, and TNF-α), were increased in PMs obtained from HbSS mice compared with macrophages obtained from HbAA mice. Similar to the results obtained from human macrophages, murine cells treated with plasma from SS mice showed increased expression of IL-1ꞵ, IL-6, TNF-α and OGG1 compared with cells treated with plasma from HbAA mice, and pretreatment with SU0268 largely blocked their increased expression. Conclusion: OGG1 has a regulatory role in the expression of pro-inflammatory cytokines in SCD. OGG1 inhibition could be a potential therapeutic approach to control sickle cell inflammation.

A rapid and simple sterility test method based on solid culture medium containing blood

Current sterility test performed for most biological products takes 14 days. We evaluated solid medium, containing 5% blood for use in the membrane filtration (MF) and direct inoculation (DI) sterility test. Representative microorganisms prepared in a sample matrix at approximately 0.1, 1, 10 and 100 colony forming units were tested for growth by compendial MF sterility test using fluid thioglycolate medium and tryptic soy broth and also on the Schaedler blood agar (SBA). Sterility test performed on SBA was significantly more sensitive and faster in detecting various microorganisms than the compendial method, particularly for sample matrix containing 0.01% thimerosal (p < 0.05). SBA detected all microorganisms within 7 days. To implement solid medium in the DI sterility test, multiple BA plates were inoculated with the sample. All representative microorganisms were detected within 5 days. The sterility test using solid medium required 3 different incubation conditions, 30–35 °C aerobically and anaerobically to detect bacteria, and at 20–25 °C aerobically to detect mold and yeast. To eliminate aerobic incubation of solid medium at 20–25 °C, we evaluated representative species of mold and yeast for their growth at 30–35 °C and 20–25 °C in the sterility test performed on solid medium. Penicillium chrysogenum could not be detected at 30–35 °C consistently within 7 days. Sterility test performed on solid medium without any additional technology could be completed in 7 days, as compared to the 14 days required for the current compendial method.

METHODS OF STERILIZATION OF MEDICAL POLYMER COMPOSITES BASED ON POLYHYDROXYBUTYRATE WITH ELASTOMERIC ADDITIVE

Introduction. Polyhydroxybutyrate is a biodegradable and completely biocompatible component, and in combination with various modifying additives can be suitable for the manufacture of medical products used in surgical practice as bone implants or their parts. There implants have a number of advantages over traditional metal products, but for their integration into the body they require thorough sterilization cleaning, which in the case of poly-mer compositions has a number of limitations associated with the possible destruction of the material structure during various cleaning stages. Purpose of the study. Find optimal methods for sterilization and disinfection of materials based on polyhydroxybutyrate (PHB) and an elastomeric additive – butadiene-nitrile rubber (NBR-28). Material and methods. Two-component PHB-NBR compositions with PHB content from 30 to 90% were studied. Four methods of sterilization and disinfection were used: autoclaving, air sterilization, disinfection with chlorine solution and ethanol solution. Determined mechanical characteristics are strength and elongation of the material at break. Sterility control – by the method of washings with subsequent observation of the growth of bacteria and fungi in Sabouraud's medium and thioglycollate medium. Results. Sterilized and disinfected samples showed no microbial growth in both culture media. No change in mechanical characteristics was detected for samples subjected to solution cleaning methods. High temperature cleaning reduced the mechanical properties of samples by 20–80% depending on the sterilization mode. Conclusions. The data obtained show that for sterilization and disinfection of PCM based on the biodegradable polymer PHB, solution methods are suitable without restrictions and the autoclave sterilization method is suitable with minor restrictions, while air sterilization leads to the destruction of PCM. The work was financially supported by the Ministry of Science and Higher Education of Russian Federation (Research theme state registration number 122041300207-2).

Endodontic operative field asepsis: a comparison between general dentists and specialists

Objective The aim was to evaluate the establishment of an aseptic endodontic operative field in general dentistry by assessing general dentists’ ability to reduce the amount of contamination to a non-cultivable level, and to compare the operative field asepsis at a general dentistry clinic with that at an endodontic specialist clinic. Materials and Methods A total of 353 teeth were included in the study (153 in general dentistry, 200 at the specialist clinic). After isolation, control samples were taken, the operative fields disinfected with 30% hydrogen peroxide (1 min) followed by 5% iodine tincture or .5% chlorhexidine solution. Samples were collected from the access cavity area and buccal area, placed in a fluid thioglycolate medium, incubated (37°, 7 d), evaluated for growth/non-growth. Results Significantly more contamination was observed at the general dentistry clinic (31.6%, 95/301), than at the endodontic specialist clinic (7.0%, 27/386) (p <.001). In general dentistry, significantly more positive samples were collected in the buccal area than in the occlusal area. Significantly more positive samples were collected when the chlorhexidine protocol had been used, both in general dentistry (p <.001) and at the specialist clinic (p =.028). Conclusions The result from this study shows insufficient endodontic aseptic control in general dentistry. At the specialist clinic, both disinfection protocols were able to reduce the amount of microorganisms to a non-cultivable level. The observed difference between the protocols may not reflect a true difference in the effectiveness of the antimicrobial solutions, as confounding factors may have contributed to the result.

Conjunctival Microbiota and Blepharitis Symptom Scores in Patients With Ocular Rosacea.

Investigation of the relationship between blepharitis-related symptom scores, tear film functions, and conjunctival microbiota in patients with ocular rosacea (OR) compared with healthy controls was aimed. Consecutive 33 eyes of 33 patients with OR who were admitted from the dermatology clinic and age-matched and gender-matched 30 healthy controls were included in the study. Tear breakup time (TBUT), Schirmer score, and blepharitis symptom score (BLISS) were recorded for each patient. For the bacteriological examination, bacterial culture was obtained by inoculating the samples on chocolate agar, blood agar, and fluid thioglycollate medium. The growth of different colonies of bacteria was identified using matrix-assisted laser desorption or ionization time-of-flight mass spectrometry. Bacterial culture positivity was observed in 13 eyes (39.4%) in the patients with OR and 10 eyes (33.3%) in the controls ( P =0.618). Patients with OR showed worse TBUT and Schirmer scores, and higher BLISSs ( P =0.005, P =0.007, and P =0.001, respectively). Patients with OR with conjunctival culture-positive results showed higher BLISSs (8.0±4.7) compared with those with negative results (4.7±2.3; P <0.001). The most frequent bacteria was Micrococcus luteus (18.2%) in patients with OR and Staphylococcus epidermidis (20.0%) in the controls. This pilot study showed that patients with OR had similar conjunctival culture positivity compared with healthy controls. However, the observation of different dominant bacterial species in conjunctival microbiota and the observation of worse BLISSs in patients with OR with positive culture might suggest a potential role of conjunctival microbiota in the pathogenesis of OR.

First record of Perkinsus marinus infecting Crassostrea sp. in Rio Grande do Norte, Brazil, using real-time PCR

A pathogen with high virulence potential in some host species, Perkinsus marinus remains a challenge for the ecological integrity of marine ecosystems and the health of bivalve molluscs. This study investigates the occurrence of P. marinus in Crassostrea sp. in estuaries of the Potengi River and the Guaraíras lagoon in Rio Grande do Norte, Brazil. A total of 203 oyster samples that tested positive for Perkinsus sp. in Ray's fluid thioglycollate medium (RFTM) were subjected to species-specific quantitiative PCR, where 61 animals (30.05 %) presented amplification graphs with a melting temperature of 80.1 ± 0.6 °C matching the positive control. This was the first record of P. marinus in oysters in these estuaries using qPCR as a diagnostic tool.

The endoparasite Perkinsus olseni affecting the Mediterranean mussels (Mytilus galloprovincialis) in the Italian and Spanish waters: A new possible threat for mussel aquaculture and wild animal population

Dinoflagellates belonging to the Perkinsus genus are OIE (World Organization for animal Health)-listed pathogens extremely virulent for clams and oysters in many marine ecosystems throughout the world. During the monitoring activities of the Mediterranean mussel (Mytilus galloprovincialis) in Campania region (Italy), the presence of typical trophozoites of Perkinsus sp. was observed in mussels from farms and natural banks. Simultaneously, following mussel mortality in the Spanish waters of Catalonia, histopathological studies revealed the presence of the same parasite. Although perkinsosis is an endemic disease in clams in Italy (with prevalence from 40 to 80%), there are no reports to date of its presence in Mediterranean mussels and of the effect on this species. For this study, histopathology, Ray’s Fluid Thioglycollate Medium (RFTM), and molecular diagnostics with conventional Polimerase Chain Reaction (PCR) and qPCR were performed. In samples from Italy, histopathology in the mussel from one farm revealed a prevalence of 26% in February 2019, 40% in February 2020, 16% in November 2020, and 23% in April 2021. In a natural bank, Perkinsus was also detected in May 2020 but in lower prevalence. In Spain, in July 2020, the presence of the parasite was 20% in one site and 10% in a second site and related to animal mortality. In both areas, Perkinsus sp. elicited multiple inflammatory capsules of different size or infiltrates at the level of the digestive gland and gonad. Molecular diagnostics of the Internal Transcriber Spacer (ITS) region of the rDNA (ITS1, 5.8S, and ITS2) showed a 97% similarity of P. olseni from Italy with samples from New Zealand, Australia, and Uruguay and in bivalves such as Pitar rostrata, Astrovenus sp., and Haliotis sp., whereas in Spain the identity was 99% samples from South Korean venerids such as Anadara granosa. Phylogenetic analysis group together P. olseni from Italian and Spanish mussels but place them distant from other P. olseni described in the clams from Europe (Italy, France, and Spain). Direct impact of transboundary animal diseases in aquaculture constitutes a serious consequence for export living animals and their products, as well for international trade. This compromises food security, also causing a high socioeconomic impact on aquaculture exporting nations. P. olseni is a generalist pathogen able to infect different bivalve species, possibly passing from clams to oysters and mussels. Recognized international organizations should take this into account in the view of possible cross-infection. Other studies are needed to define pathogen virulence in this species.

A STUDY ON BACTERIOLOGICAL IDENTIFICATION OF SUB-GINGIVAL PLAQUES IN PERIODONTITIS PATIENTS

Objective: The current study's objectives were to isolate and characterize the periodontal pathogens from subgingival plaque obtained from patients with chronic periodontitis using conventional microbiological techniques.&#x0D; Materials: In the Department of Microbiology at a Tertiary Care Teaching Hospital was where this study was carried out. Over the course of six months, from January to June 2019, samples were taken from subgingival pockets in patients with chronic periodontitis who visited the periodontology outpatient department at our institute of dental sciences. There were 21 cases in the research. For the purpose of preventing saliva contamination, tooth surfaces were dried using sterile gauze. Subgingival plaque samples were taken from the majority of diseased sites using a sterile periodontal Gracy curette, put in a test tube with fluid thioglycollate medium, and taken to the microbiology lab where they were processed right away using conventional microbiological procedures.&#x0D; Results: E. coli grew in the majority of samples (9), followed by Pseudomonas species and Staphylococci in 7 samples. Only sample number 7 out of 21 showed no growth for Candida albicans; the others all exhibited development. Samples like Sample 1, 5, 6, 8, 10, 11, 12, 13, 15, 16, 17, 18, 19, and 20 produced isolates that were multidrug resistant.&#x0D; Conclusion: Therefore, while developing a treatment plan for adult patients with periodontitis, the microbial diversity discovered in the current study should be taken into account. Variations in the quantities and species of cultivable bacteria have been found in investigations of periodontitis patients from different geographic regions, including developed and developing countries.

Возможная роль представителей кишечного микробиома в биотрансформации арил-, пиридилсодержащих фосфинов и их производных (по данным спектроскопии ЯМР 31Р)

Organophosphorus compounds (OPCs) are used in many areas of human activity. Unique physicochemical properties and high biological activity not only determine the applied value of OPCs but also give them the properties of xenobiotics that are harmful to human health, which largely depend on the state of the endogenous microbiota. The purpose of this work is to assess the effect of pyridyl- and aryl-containing phosphines, their oxides, and sulfides on the growth of Bifidobacterium bifidum and Escherichia coli and to identify possible patterns of biotransformation of OPCs using 31P NMR spectroscopy. Bacteria were cultivated in thioglycollate medium containing the tested phosphorus compounds. At the stationary growth phase was determined the cell concentration and were recorded spectra 31P NMR. It was shown that the most reliable (p&lt;0,05) and opposite effect on the growth dynamics of both species of bacteria was exhibited by phosphine sulfides 1 and 3: PS1 (pyridyl) reduced the population reproduction rate by 35–40%, and PS3 (aryl) increased it by 45–60%. At the same time, OPCs themselves were most likely not metabolized. Phosphine oxides reduced the average titer of bacteria compared to the control, and PO3 caused complete cell elimination after 24 hours cultivation. In the medium with PO2 was identified PS2 appeared, which may be caused by the biologically mediated process of oxide-to-sulfide conversion. All phosphines are chemically labile and they were oxidized abiotically to oxides and sulfides. Formed sulfides could be the reason for a significant increase (to 35%) in the growth rate bacteria population both species. Five new unidentified organophosphorus compounds (UPC) were recorded on medias with B. bifidum, some of which may belong to the products of biotransformation of the original OPCs. Some organic phosphates, homologous to the phosphine oxides tested, have a δp value neighboring to the taken NMR spectra. This may indicate the way of their biological transformation: oxidation of the CP bond in the phosphine oxides to an ester bond of organic phosphates. This ability of bifidobacteria to metabolize OPCs will make it possible to develop a therapeutic strategy based on the use of B. bifidum as a microorganism that degrades phosphorus-containing xenobiotics.