Campylobacter spp. is a gram-negative bacillus that causes infectious enteritis and consists of several species, including Campylobacter jejuni, Campylobacter coli, and Campylobacter fetus. Although C. jejuni and C. coli cause infectious enteritis primarily in immunocompetent hosts, C. fetus causes extraintestinal infections such as septicemia, meningitis, and perinatal infections in immunocompromised hosts, as well as myopericarditis in rare cases. Only a few cases of infectious myo(peri)carditis associated with C. coli in immunocompetent hosts have been reported. These studies concentrated on antecedent C. coli enterocolitis and never demonstrated a positive culture in the pericardial fluid.C. coli was identified from blood culture samples and blood-tinged pericardial fluid. He was successfully treated with antibacterial chemotherapy as well as pericardial fluid drainage and was discharged from the hospital with no complications.A 72-year-old Japanese man presented with a 2-week fever, cough, and vomiting lasting. He was on hemodialysis for polycystic kidney disease, as well as medication for diabetes and hypertension. A chest computed tomography (CT) scan and a transthoracic echocardiogram revealed bilateral pleural fluid and large pericardial fluid at the time of admission. C. coli in the pericardial fluid confirmed the diagnosis of C. coli pericarditis. C. coli may cause septic pericarditis in immunocompromised hosts, despite typically causing only enteritis.In this case, the presence of  Campylobacter spp. is well known as the causative agent of infectious enteritis. As of December 2014, Campylobacter had 26 species, two provisional species, and nine subspecies, including Campylobacter jejuni, Campylobacter coli, and Campylobacter fetus  of 178 patients) [C. coli was only found infrequently (16 [9%] of 178 patients) [C. coli is less prevalent than C. jejuni and C. fetus. There are few reports on C. coli-caused infectious diseases other than infectious enteritis.The bacteria emia  or pneumonia (33%) respectively [Salmonella and Shigella are uncommon but important gastrointestinal pathogens associated with myo(peri)carditis; however, the rising incidence of campylobacteriosis worldwide over the last decade has heightened interest in Campylobacter-associated myo(peri)carditis [C. fetus is almost always isolated from blood in cases where the pathogen has been linked to myopericarditis [C. fetus was isolated directly from pericardial fluid [C. coli-associated myo(peri)carditis has been described in a few case reports [C. coli enterocolitis, rather than C. coli growing from blood or pericardial fluid played a role. We present a case of infectious pericarditis caused by C. coli isolated from both pericardial fluid and blood in a patient who did not develop enterocolitis.Pericarditis is a pericardial inflammatory syndrome. Pericarditis and myocarditis have similar aetiologies, and overlapping forms are common in clinical practice. The majority of cases of acute pericarditis in developed countries are caused by viral infections or are autoreactive , and bacectively . Salmonecarditis . C. fetucarditis . In someneumonia 3% respecal fluid . C. coli reports , 11. TheKlebsiella pneumoniae, food poisoning caused by Salmonella spp. and oral candidiasis. He had no history of alcohol intake and was not infected with the human immunodeficiency virus.A 72-year-old Japanese man was admitted to the hospital with a fever and a large amount of pericardial fluid. He had been on hemodialysis for the past 19 years due to polycystic kidney disease. He had also been receiving treatment for hypertension and diabetes mellitus for the previous 32 years. His medical history included a urinary infection caused by He began coughing two weeks before his hospitalization. A chest X-ray and computed tomography (CT) scan one week before hospitalization revealed bilateral pleural fluid and pericardial fluid. An electrocardiogram taken three days before admission revealed flattening of the T wave and poor R wave progression Fig. . Hence, He was awake but vomited upon admission. There was no chest pain or enteritis in the patient. He had not eaten any raw or undercooked food prior to his admission. His physical exam revealed tachycardia, with a heart rate of 126 beats per minute, blood pressure of 172/84-mm Hg, and an axillary temperature of 38.6 °C. C-reactive protein , procalcitonin , hemoglobin A1c , and white blood cell  counts were all elevated. Creatinine phosphokinase , and lactate dehydrogenase  were, however, almost normal. Additional laboratory tests revealed that β-D-glucan levels were extremely high  and aspergillus antigen levels were mildly elevated . Anti-GPL-core IgA antibody, QuantiFERON TB-Gold plus, and cryptococcus antigen were all negative. A transthoracic echocardiogram revealed a significant amount of pericardial fluid. Spiral-shaped, gram-negative rods were cultured from admission blood (two sets) and on day 2 (two sets).C. coli or C. jejuni using Matrix-assisted laser desorption ionization time-of-flight analysis, but it could not tell the difference. Finally, using the Hippurate hydrolysis test, the organism was identified as C. coli. In the pericardial fluid culture, no mycobacteria or fungi grew.On day 2 of hospitalization, blood-tinged pericardial fluid was drained through pericardiocentesis. The pericardial fluid was examined and discovered to have pyogenic inflammation  as well as mildly elevated adenosine deaminase (ADA) levels (42.4 U/L). The pericardial pathological examination of the pericardial fluid revealed no bacterium, mycobacterium, fungus, or malignant findings. The spiral-shaped gram-negative rods grew in pericardial fluid culture, similar to the blood culture. Commercial automatic identification systems failed to recognize the organism. The organism was identified as C. coli bacteremic pericarditis was diagnosed.Drug susceptibility of the organism was evaluated. Minimum inhibitory concentrations against the organism were as follows: ampicillin, 1.0 mg/mL; imipenem, < 1.0 mg/mL; erythromycin, < 2.0 mg/mL; ciprofloxacin, < 0.25 mg/mL; and gentamicin, < 1.0 mg/mL. There stool was not microbiologically examined. Based on the findings, Meropenem  was administered intravenously for 44 days, followed by micafungin  for 15 days. On the sixth day of hospitalization, a transesophageal echocardiogram revealed no evidence of infective endocarditis. Furthermore, no organism grew from the blood on day 6 (two sets). He was discharged from the hospital on day 45 without any sequelae. His treatment was completed 14 days after he was discharged with oral clarithromycin . Even after 5 months after discharge, the patient never showed signs of disease relapse.Campylobacter-associated myo(peri)carditis [C. coli grew from blood and pericardial fluid. In the present case, it was unclear when, and where the patient became infected with C. coli in this case. He had no significant incidents, such as animal contact or eating raw meat. He is an elderly immunecompromised patient with diabetes and chronic renal failure on dialysis.Campylobacteriosis has become more common in the last decade, drawing attention to carditis . The obsCampylobacter causes myopericarditis is unknown, several intriguing putative mechanisms [C. fetus pericarditis appears to be the result of pericardium colonization following bacteremia in an immunocompromised host [C. fetus is almost always isolated from blood [C. fetus was isolated from blood in 90% (9/10) cases of C. fetus pericarditis. C. fetus myopericarditis could be caused by direct microbial invasion of the pericardium via the blood stream. On the other hand, C. jejuni causes myo(peri)carditis in immunocompetent hosts [C. jejuni was identified in 12 cases of C. jejuni-associated myo(peri)carditis through positive stool cultures and/or serological analyses, but it was never isolated from blood [C. jejun infection or a direct effect of Campylobacter spp. on the myocytes [C. coli has properties similar to C. jejuni and causes enterocolitis in immunocompetent people. C. coli-caused myo(peri)carditis in immunocompetent hosts with enteritis in the reported cases, and C. coli was identified from stool [C. coli caused pericarditis in an immunocompromised host who did not have enteritis, and C. coli was isolated from blood and pericardial fluid. Therefore, C. coli in the current case may directly invade the pericardium similar to C. fetus.Although the exact mechanism by which chanisms  have beesed host . When thom blood . C. fetuin 90% 9/ cases ofom stool . In the C. coli infections [C. coli is becoming increasingly resistant to them, according to recent reports [C. coli showed elevated minimum inhibitory concentrations to meropenem after long-term oral antibiotic treatment with tebipenem and faropenem for persistent infection, according to a case report [Quinolones and macrolides are frequently used in the treatment of fections . C. coli reports . In thise report . More reCandida or Aspergillus fungi were cultured from pericardial fluid, some cases of Candida pericarditis or aspergillus pericarditis were diagnosed [Laboratory testing revealed a highly elevated β-D-glucan (> 300 pg/mL) and a mildly elevated aspergillus antigen in this case (0.5 cutoff index). When β-D-glucan is high, it generally suggests the presence of fungal infection . When Caiagnosed –20. PhysThe pericardial fluid revealed a mildly elevated ADA (42.4 U/L) in the present case. High levels of ADA indicated tuberculous pericarditis. A recent meta-analysis of tuberculous pericarditis reported a pooled sensitivity of 0.90, specificity of 0.85, and negative possibility of 15% . HoweverC. coli-associated myo(peri)carditis is extremely rare; there have been a few reported cases without proof of positive culture in pericardial effusion. Because C. coli was isolated directly from the pericardial fluid in this case, the diagnosis was certain. C. coli has been suggested to cause septic pericarditis in immunocompromised hosts, although it typically causes enteritis.To summarize,


Clostridium perfringens is a spore-forming, Gram-positive anaerobic pathogen that causes several disorders in humans and animals. A multidrug-resistant Clostridium strain was isolated from the fecal sample of a patient who was clinically suspected of gastrointestinal infection and had a recent history of antibiotic exposure and diarrhea. The strain was identified by 16s rRNA sequencing as Clostridium perfringens. The strain’s pathogenesis was analyzed through its complete genome, specifically antimicrobial resistance-related genes. The Clostridium perfringens IRMC2505A genome contains 19  antibiotic-susceptible genetic species according to the k-mer-based detection of antimicrobial resistance genes. Genome mapping using CARD and VFDB databases revealed significant  genes with aligned reads against antibiotic-resistant genes or virulence factors, including phospholipase C, perfringolysin O, collagenase, hyaluronidase, alpha-clostripain, exo-alpha-sialidase, and sialidase activity. In conclusion, this is the first report on C. perfringens from Saudi Arabia that conducted whole genome sequencing of IRMC2505A and confirmed the strain as an MDR bacterium with several virulence factors. Developing control strategies requires a detailed understanding of the epidemiology of C. perfringens, its virulence factors, and regional antimicrobial resistance patterns. Clostridium perfringens (C. perfringens) is a spore-forming, Gram-positive bacterium. C. perfringens was first discovered in 1981 at the Johns Hopkins Hospital. It is a causative agent of gastroenteritis, myonecrosis, and enterotoxemia. Initially, it was known as Bacillus aerogenes capsulatus. Before the final name, Clostridium perfringens, this pathogen was called Bacillus welchii [C. perfringens can produce spores that are tolerant to environmental stress and have a wide variety of toxins involved in the pathogenesis of diseases. Spores of this pathogen can tolerate cooking temperatures and survive. As a result, C. perfringens can grow in foods that have been stored incorrectly. The most frequent causes of outbreaks are associated with incorrectly prepared or reheated food materials such as meats, poultry, or gravy and improper management of patients with C. perfringens infections before the exact diagnosis of causative agents [C. perfringens are caused by type A toxin. The pathophysiology of this pathogen is mostly associated with tissue necrosis caused by the toxin of C. perfringens. The majority of toxins from C. perfringens have pore-forming behaviors that open up, allowing water to flood in and cause swelling followed by cell death. The fermentation of glucose in this pathogen results in the generation of histotoxic gas, which is a defining feature of C. perfringens infection [C. perfringens [ welchii ,7,8,9,10nfection ,5,10. Thfringens ,3. Thesefringens . The patfringens .C. perfringens-associated diseases. It is a chromosomally encoded α-toxin produced by all strains of this pathogen [C. perfringens enterotoxin (cpe) is a clinically important enterotoxin gene in a few strains. This enterotoxin is the etiology of most gastrointestinal-associated diseases, including antibiotic-associated diarrhea, food-poisoning outbreaks, and nosocomial diarrheal disease. Outbreaks caused by C. perfringens are common, with severe illness or death reported. Current public health interventions reduce contamination at the food production source and cross-contamination in the food preparation environment [C. difficile and its genotype profile using the resolution tool provided by whole genome sequencing (WGS) to analyze genetic diversity and assess C. difficile strains associated with infection recurrences and hospital outbreaks. Moreover, novel strains can spread quickly across continents and nations, negatively affecting global health. Some strains are more contagious than others for various reasons, including sporulation, antibiotic resistance, and toxin production. Using a unified typing strategy such as WGS will dramatically change our understanding of epidemiology [C. perfringens’ worldwide distribution, limited information is available on the genome of this pathogen, particularly in Saudi Arabia. Genes associated with antimicrobial resistance in the genome of the clinical isolate C. perfringens IRMC2505A could account for multidrug resistance. We used the genome analysis of this clinical isolate to test this hypothesis and investigate antimicrobial resistance-related genes involved in different resistance mechanisms. The genome of C. perfringens IRMC2505A was completely sequenced and analyzed using bioinformatics.Phospholipase C (plc) is the main virulent factor in pathogen . C. perfironment ,7,8. Difemiology ,10. HoweClostridium > Clostridium perfringens. The Clostridium perfringens genome has 4159 protein-coding sequences (CDS), 80 transfer RNA (tRNA) genes, and 7 ribosomal RNA (rRNA) genes  and 3702 cross-genus protein families (PGFams).The assembled genome of IRMC2505A had 108 contigs with a total length of 4,322,225 bp and an average G + C content of 28.28% . This geA) genes . Figure  on type , functio on type , and the95103.10 . However95103.10 B. Protei95103.10 . The proAlr, Ddl, dxr, EF-G, EF-Tu, folA, Dfr, folP, gyrA, gyrB, Iso-tRNA, kasA, MurA, rho, rpoB, rpoC, S10p, and S12p) antibiotic targets in susceptible species were identified in the genome of Clostridium perfringens IRMC2505A using k-mer-based detection methods for AMR genes  involved in protein-altering cell wall charge conferring antibiotic resistance were also identified in the IRMC2505A genome. Only one gene in each category was associated with the resistant mechanism of the isolate Clostridium perfringens: antibiotic inactivation enzyme (NimB), antibiotic target protection protein (TetB(P)), antibiotic target replacement protein (fabV), efflux pump conferring antibiotic resistance (TetA(P)), gene conferring resistance via absence (gidB), and protein involved in antibiotic sequestration (FabK-like). The putative membrane protein (mprF) is similar to the genome of the comprehensive antibiotic resistance database . Tetracycline-resistant MFS efflux pump (TetA(P)) is similar to the genome reported in the NDARO, CARD, and PATRIC databases   genes with aligned reads against antibiotic-resistant genes or virulence factors, including phospholipase C, perfringolysin O, collagenase, hyaluronidase, alpha-clostripain, exo-alpha-sialidase, and sialidase activity (The CARD (comprehensive antibiotic resistance database), NDARO , and PATRIC (Pathosystems Resource Integration Center) databases revealed antimicrobial resistance genes . NineteeMR genes . Three g 10−234) . We comp 10−234) . SeventeA genome . More thactivity .Clostridium perfingens remains one of the only toxicogenic clostridial species identified by local investigations after an outbreak occurs. The bacterium is reported worldwide and has been listed among the leading species of foodborne and non-foodborne gastroenteritis [Although many food-borne diseases are surveyed and controlled, and several enteric organisms are monitored through databases, nteritis . Althougnteritis ,13.C. perfingens was isolated from a patient in Saudi Arabia’s Eastern Province. It would be beneficial to start a surveillance program and elucidate changes in the pathogen’s genome. This information is clinically significant since the antimicrobial resistance profile and genome characteristics of these isolates are still understudied in Saudi Arabia. Additionally, recent studies have identified hypervirulent clostridial spores in new environments and genomically linked them to new matrices other than hospital environments due to uncontrolled antimicrobial usage [This genomic study of al usage ,15. Our C. perfingens resistance mechanisms could decrease the misuse of broad-spectrum antibiotics and AMR genes’ potential transfer to other pathogens.We believe that a better understanding of C. perfingens ATCC 13124 195103.10, and C. perfringens str. 13 195102.6 species. The last species, C. perfringens str. 13, was sequenced for the first time in 2002 by Shimizu et al. It is a natural isolate found in the soil and classified as a type A strain [C. perfringens has the highest genome plasticity ) out of 51 other bacterial species. In other words, this bacterium has a highly open genome to acquire new adaptive genes for survival in hostile external environments. These properties are reflected by its low GC content (around 28%) and high 16S rRNA gene copy number [Phylogenetic analysis confirmed the phylotype and found 100% similarity between our isolate sequences, A strain . Genome y number .C. perfingens showed a multi-drug-resistant isolate involving nineteen resistance genes with different resistance mechanisms. Most of these genes were in the “antibiotic target in susceptible species” category. Many of these identical genes, with their antimicrobial resistance mechanisms, have been identified and described in different gram-negative bacteria where homologous combinations are found [Many research studies have indicated the emergence of multi-resistant strains of clostridial species and their significant risk to humans. In this study, the antimicrobial resistance profile of re found .tetA(P) and tetB(P), which are known to encode proteins that mediate active tetracycline efflux and ribosomal protection tetracycline-resistant mechanisms, respectively [mprF) reported codes for multiple peptide resistance factor proteins, which are involved in CAMP resistance [Predicting AMR genes based on NDARO/CARD/PATRIC databases showed tetracycline resistance (% query coverage and % identity > 97%) and defensin-like cationic antimicrobial peptides (CAMPs) (% query coverage = 100% and % identity = 95%). We detected two tetracycline-resistant genes, ectively . The antsistance . Moreovesistance .C. perfringens and its spores [C. perfringens should be investigated in many matrixes  [C. perfringens as a causative agent of antibiotic-associated diarrhea (AAD) in hospitalized patients [C. perfringens among patients. However, insufficient data were found in these areas. Consequently, this finding stresses the urgent need for a surveillance program to control AAD and guarantee efficient treatment.Antimicrobial agents are widely used in animals as growth promoters and can lead to the emergence of antimicrobial-resistant enteric microflora, including s spores . Hence, , soil…) . Motamedpatients . These sClostridium species found no evidence for the antimicrobial resistance patterns reported in KSA. Most of these limited studies used conventional methods  [C. perfringens in camel meat. They showed these bacteria’s high resistance to tetracycline and ceftiofur drugs and, to a lesser extent, other antimicrobial agents  [A similar situation was observed in Saudi Arabia, where a recent meta-analysis (2022) on the frequency of antibiotic-resistant  assays) . These rcomycin) ,26,27.C. perfringens is also known for its toxigenicity. Five toxinotypes (A–G) were historically identified based on the toxin produced by the strain . Afterward, two new toxinotypes (F and G) were added to the scheme, introducing enterotoxin CPE  and NetB toxin, respectively [C. perfringens is known to produce a wide range of degradative enzymes, including hydrolytic enzymes, membrane-damaging enzymes, pore-forming toxins, and intracellular toxins . The virulence analysis of our bacterial isolate showed a variable array of virulence genes and a 97% similarity to the C. perfringens genome. High query coverage sequences (≥97%) were distributed as follows: Sequences correlated to phospholipase C (plc) encoding the alpha toxin, sialidase genes , alpha-clostripain genes (cloSI), hyaluronidase genes , and collagenase genes (colA) encoding the κ-toxin. These genes are chromosomal and located specifically in the conserved region of the C. perfringens chromosome [C. perfringens in Saudi Arabia, including the Arabian Peninsula [ectively . Many viromosome . There heninsula .C. perfringens showed an MDR bacterium with several virulence factors. To our knowledge, our WGS study is one of the first studies conducted in Saudi Arabia and the Arabian Peninsula on C. perfringens, where national databases are still inexistent. To guarantee a complete understanding of C. perfringens’ epidemiology, a full study of this pathogen’s virulence factors, and antimicrobial resistance patterns is essential. Future studies will also be beneficial in developing an effective disease control strategy.In conclusion, this WGS study of This project was submitted for ethical approval to the ethical committee of Imam Abdulrahman Bin Faisal University (IRB-2022-01-398). All procedures were performed in accordance with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.A Clostridium perfringens strain was isolated using cycloserine, cefoxitin, and fructose agar medium from the fecal sample of a patient who was clinically suspected of gastrointestinal infection and had a recent history of antibiotic exposure and diarrhea. A positive sample, tested for both toxins A and B by GeneXpert, was cultured on cycloserine cefoxitin fructose agar selective medium (CCFA)  under anaerobic conditions at 37 °C for 24 h. The total DNA was extracted using the Gentra Puregene Yeast/Bact. Kit . The purity, quality, and quantity of genomic DNA were measured using Nanodrop 2000  as described in the manufacturer’s instructions. The isolate was PCR amplified, and 16S rRNA was sequenced to confirm the isolate.The whole genome of isolate IRMC2505A was sequenced using the Illumina platform. The paired reads were assembled and annotated using PATRIC (BV-BRC 3.28.5)  and the


Candida albicans biofilms are formed on indwelling medical devices and have an associationwith both oral and invasive candidiasis. This study aimed to compare the amount of adherent C. albicans and the biofilm formed on different denture base materials.The adhesion of C. albicans to denture base materials is widely recognized as the main reason for the development of denture stomatitis. The human mouth mucosal surface is colonized by indigenous microflora, which normally maintains an ecological balance among different species. However, certain environmental orbiological factors may disrupt this balance, leading to microbial diseases. C. albicans strains  biofilm were measured by the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)method and use of a microplate reader. Moreover, morphological alterations of C. albicans cells were investigated using scanning electron microscopy (SEM) In total, 56 polymethyl methacrylate (PMMA) acrylic resin disc-shaped samples were divided into four groups as follows: 1) chemically polymerized PMMA, 2) heat-polymerizedPMMA, 3) computer-aided design and computer-aided manufacturing (CAD/CAM) PMMA in high polish, and 4) CAD/CAM resins in glazed form. The adherent cells and formationof  The biofilm formation was significantly lower on CAD/CAM acrylic resins, compared to conventional denture base materials. The obtained results were confirmed by the SEM images of C. albicans biofilms. CAD/CAM PMMA-based polymers may be preferable to inhibit C. albicans biofilm formation and reduce Candida-associated denture stomatitis in long-term use. Based on the findings, the CAD/CAM technique can be used as an efficient technique for denture fabrication as it inhibits microbial accumulation, and consequently, microbial biofilm. Denture stomatitis is a multifactorial disease and can be caused by many predisposing factors. Some of these factors are related to underlying systemic and immune diseases,as well as a deficiency in salivary flow rate, while others are specific to denture wearers, such as poor denture hygiene. Moreover, it can be caused by roughness or thepresence of pores in the acrylic surface, which can result in denture-induced trauma. Denture hygiene and oral hygiene are very important factors in controlling thecolonization of microorganisms on the denture surface  - 3. AcCandida is a major part of the normal commensal flora in the oral cavity. A change in the oral microflora,followed by an increase in Candida colonization, which is the most common manifestation of oral candidiasis, can cause Candida-associated denture stomatitis in denture wearers , KCl ; pH: 7.4).Afterward, the washed C. albicans cells were re-suspended in RPMI-1640  media with 50 mmol−1 glucose. The cell densities were adjusted to 1.0×108 cells/mL and 0.15 opticaldensities at a wavelength of 530 nm. The four standard strains of et al. with some modification [ In this study, biofilm formation inhibition was determined using a technique previously described by Ramage ication  . In totC. albicans suspensions were diluted 1/1000 by RPMI-1640 medium; afterward, 500 μL of the standardized C. albicans suspensionswere added to each well of a 24-well tissue culture plate  containing the acrylic resin samples.Subsequently, the plates were incubated for 48 h at 37°C. In addition, 500 μL of the RPMI-1640 medium was used as the negative control (blank); however, the RPMI-1640 with theyeasts and without the acrylic resin samples were considered the positive controls. It should be mentioned that all experiments were performed in triplicate [ The resulting plicate  .C. albicans biofilms was calculated using a colorimetric assay. Biofilm formation was performed usinga 2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carbox-anilide (XTT)  reduction assay as an indicator of cell survival and proliferation,which was prepared in Ringers lactate solution (0.5 mg/mL). The metabolic activity of The solution was filter-sterilized (0.22μm-pore-size) and stored at -70°C. Prior to each assay, the XTT stock solution was mixed with menadione sodiumbisulfite . After 48 h of incubation, the discs were transferred to a new tissue culture plate and washed twice withsterile PBS. Subsequently, a 500 μL aliquot of XTT/menadione was added to each well of the 24-well plates. The plates were incubated at 37°C in a dark room for 3 h.Finally, the colorimetric changes were measured at 570 nm using a microplate reader . The biofilm formation was calculatedas follows: biofilm formation percent=(absorbance of test well/absorbance of growth control well)×100   . C. albicans, acrylic resin denture base samples were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4°C for 1 h.Scanning electron microscopy (SEM) was used to observe the biofilm that had formed on the surfaces. To examine the ultrastructural nature of grown biofilms and themorphological features of After being washed in buffer, the samples were postfixed in 1% osmium tetroxide in the same buffer for 30 min. The samples were dehydrated in the graded concentrationsof ethanol and critical point-dried in CO2 . They were then coated with colloidal gold and viewed under a Leo 435 VP SEM  at 15 kV   .C. albicans biofilm formation values for the conventional and CAD/CAM denture base materials.The quantitative data were presented as mean and standard deviation. A one-way ANOVA test was used to compare the biofilm formation of the four C. albicans strains betweenthese types of complete denture bases. It should be noted that a P-value of less than 0.05 was considered statistically significant. Descriptive statistics were used to describe the C. albicans biofilm formation was reported to be 0.255, 0.129, 0.124, and 0.086 in the studied groups, including 1) chemically-polymerizedPMMA, 2) heat-polymerized PMMA, 3) CAD/CAM resins with surface glaze, and 4) CAD/CAM resins with a high polish, respectively.C. albicans biofilm formation decreasedsignificantly in CAD/CAM polished acrylic denture samples (P<0.001) (According to P<0.001) .C. albicans after biofilm formation in thementioned group. As shown in C. albicans biofilm formation, compared to conventional acrylic resins. Fungal cells were found to a large extent on the surfaces of the conventional acrylic resins in SEM analysis.The morphology of the C. albicans biofilm showed that the CAD/CAM acrylic resins had an inhibitory effect on biofilm formation. The biofilm architecture wasseriously damaged in the CAD/CAM high-polished resin forms, and few C. albicans cells were observed on these acrylic resins.There was no significant difference between the biofilm formation of the heat-polymerized acrylic and CAD/CAM glazed denture samples. It is noteworthy that, in this study, significantly higher absorbance values were observed for the chemically polymerized PMMA acrylic samples incomparison with other types of acrylic resin. As shown in Despite the extreme increase in the use of dental implants, complete dentures are still considered a valuable treatment option for some edentulous patients who cannot afford or refuse implant placement  .Candida-associated denture stomatitis is a common disease in denture wearers, and various Candida strains have been isolated from them [ C. albicans is the most frequently isolated strain that can adhere and form biofilms on the oral mucosa and subsequentlycause denture stomatitis [ According to previous studies, om them  , 22. Amatitis  . Over tmatitis  , 15 .C. albicans exhibited differing adherence and biofilm formation abilities regarding the CAD/CAM and conventionally fabricated complete denture bases. There was a significant difference between biofilm formation on the conventional and CAD/CAM denture samples. Statistical analysis of the results revealed that CAD/CAM polished acrylic denture samples had the least amount of biofilm formation. These findings suggested that yeast cells were less likely to adhere to CAD/CAM denture bases than to conventional denture bases. The fact that surface roughness and porosity are important factors in microbial adhesion and colonization may explain the lower adherence [ C. albicans to the surface of the dentures. Based on the findings of this study,the acrylic resin manufactured by CAD/CAM technology, along with the polishing process had the least amount of C. albicans adherence, which is in line with theresults of previous studies [ In the present study, herence  . CAD/CAherence  , 25. Istudies  , 27 . In previous studies, the advantages of CAD/CAM technology with the polishing methods over conventionally fabricated complete dentures have been verified   which wIt can be caused by air trapped during mixing, insufficient mixing of polymer and monomer, the existence of residual monomer, and inadequate compression on the flask, which may affect the adherence of microorganisms on the surface of the denture and increase microorganism colonization  , 27. BC. albicans and other oral microorganisms to the dentures fabricated using the CAD/CAM technique are less thanthose fabricated by the conventional denture processing methods [ As a result, the CAD/CAM fabricated denture bases have a less porous and rough surface compared to conventional acrylic resin  . Therefmethods  . One remethods  , 29 . C. albicans adhesionand biofilm formation on the surfaces of the dentures. According to some previous studies, conventional complete denture bases have higher microbial colonization,and consequently, significant adhesion and biofilm formation of C. albicans in comparison with CAD/CAM complete dentures [ According to the results of the present and previous studies  , 30, tentures  , 31. Tet al. conducted an in vitro study in 2015 about the C. albicans biofilm formation on different types of denture base materials.The findings of the aforementioned study revealed that cobalt-chrome alloy is the least adhesive, while heat-cured acrylic resin and chemically cured acrylic resin haveshown the most adhesiveness, in that order [ Agraval t order  . Their et al. performed a review study to assess the advantages of the CAD/CAM method in denture manufacturing. Similar to this study,their results also indicated that the denture material used in CAD/CAM technology has better properties, compared to the common chemically and heat-cured materials [ Kattadiyil MT terials  .Candida and surface [ C. albicans biofilms appear mostlyin the fissures and pores of the chemically polymerized denture base. In the present research, the XTT assay was used to measure biofilm formation on acrylic resins since it has a high quantitative level and is more accurate incomparison with other methods used in previous studies  , 32. Ssurface  . Hence,C. albicans sincethey become a barrier against phagocytosis. These results were also in line with those of a study conducted by Gondim et al. [ As shown in the A and B images of  et al.  .C. albicans adherence and biofilm formation onthe denture base surfaces. Finally, according to the findings, CAD/CAM technology can be suggested as the first choice for denture fabrication since it helps prevent microbial infection in dental settings.Based on the results, complete dentures fabricated using CAD/CAM procedure showed hopeful potential for reducing the The authors wish to thank Mr. H. Argasi at the Research Consultation Center (RCC) of Shiraz University of Medical Sciences for his invaluable assistance in editing this manuscript.K. Z. and V. S. K. designed and managed the present research. Z. Z. performed the tests, analyzed the data, and wrote the first draft of the manuscript. V. L., A. A., and A. R. edited the final manuscript. R. G. and R. H. were project partners. All authors approved the final version of the manuscript. The authors declare that they have no conflicts of interest.This study was part of the Milad Larijani's thesis.


To investigate the relationship between aortic stiffness and postoperativeatrial fibrillation (POAF) in patients undergoing coronary artery bypassgrafting (CABG).This study included 110 patients undergoing elective isolated CABG. Aorticstiffness was measured using a noninvasive oscillometric sphygmomanometerbefore surgery. Characteristics of patients with and without POAF werecompared.vs. 58.3±8.4;P=0.014). Chronic obstructive pulmonary disease (COPD) wasmore common in patients with POAF , whereas the frequency of hypertension,diabetes mellitus, smoking, and previous coronary artery disease did notdiffer. C-reactive protein and cholesterol levels were similar betweenpatients with and without POAF. Left atrial diameter was greater in patientswith POAF . Peripheral (p) and central (c) systolicand diastolic blood pressures were also similar between the groups, whereasboth p and c pulse pressures (PP) were greater in patients with POAF . Pulse wave velocity (PWV) wassignificantly higher in POAF . PWV, pPR and COPD were independent predictors of POAF inmultivariate regression analysis. In receiver operating characteristicanalysis, PWV and pPP have similar accuracy for predicting POAF  .POAF developed in 32 (29.1%) patients. Patients with POAF were older(63.7±8.6 COPD, PWV, and PP are predictors of POAF. PP and PWV, easily measured inoffice conditions, might be useful for detecting patients with a higher riskof POAF. Numerous attempts, including medical and surgicaltherapies, have been made to prevent POAF. Despite promising results, POAF is stilla significant cause of morbidity and mortality, both in hospital stays and duringfollow-up. Therefore, recognizing patients with relatively higher risk POAFdevelopment has crucial importance.Postoperative atrial fibrillation (POAF) following coronary artery bypass grafting(CABG) was shown to be associated with long-term mortality for evaluating cardiovascular risk. Previous studies proposedPP and PWV as predictors of cardiovascular and all-cause mortality,5. Various studies,7 showed that PWV andPP may be related to atrial fibrillation (AF) development. We aimed to evaluate therelationship between aortic stiffness parameters and POAF in patients undergoingCABG.Aortic stiffness was reported to be associated with several cardiovascular outcomes.Brachial pulse pressure (PP) is an indirect evaluation of aortic stiffness and tendsto overestimate central hemodynamic. Pulse wave velocity (PWV) is solely dependenton central vascular functions, whereas PP is affected by both vascular andventricular functions. Therefore, PWV is accepted as the gold standard andrecommended by the current guidelinesThis study is prospective and observational. A total of 110 patients undergoingelective isolated CABG at our institution were included consecutively. Onlyon-pump CABG procedures were featured. Patients presenting with an acutecoronary syndrome (ACS) at index hospitalization were excluded. Patientsundergoing concomitant surgeries, such as valve repair/replacement,aneurysmectomy, and emergency procedures were also excluded. Patients with ahistory of AF  and a history of anyarrhythmia implying possible AF were also not considered. The same group ofcardiovascular surgeons and anaesthesiologists operated on patients using thesame techniques and myocardial protection. Demographic, laboratory, and clinicalvariables were recorded. All patients provided written informed consent and thestudy protocol was approved by the local ethics committee (14567952-050/924)following the Declaration of Helsinki and Good Clinical Practice guidelines.Patients were routinely followed by a heart rhythm monitor during intensive careunit stay. Daily electrocardiogram (ECG) was taken in the intensive care unit,as well as in the ward. Additional ECG was obtained in case the patient had anycomplaints such as pain, palpitation, lightheadedness, etc. POAF was defined asan occurrence of any episode of AF lasting > 30 seconds captured on ECG ormonitor.Measurements were performed at index hospitalization one to three days beforeCABG. Patients were asked to avoid intake of caffeinated beverages, alcoholicbeverages, and other stimulants within three hours of measurements. Patients hadto rest in the supine position for 10 minutes before measurement at roomtemperature between 08:00 and 10:00..Aortic stiffness was measured using a non-invasive oscillometricsphygmomanometer, Mobil-O-Graph® . PWV,augmentation index, peripheral (p), and central (c) systolic blood pressure(SBP), diastolic blood pressure (DBP), and PP were calculated by the softwaretool. The reliability of the Mobil-O-Graph® in estimating the PWV wasdemonstrated in previous studiest-test. Categorical variables between groups were assessedwith the Chi-square test or Fisher’s exact test, whichever was suitable.Logistic regression analysis was used to identify the independent predictors ofPOAF. Significant variables in univariate analysis were included in multivariateanalysis. Two separate models were constructed. In the first model, age and pPPwere excluded due to collinearity, whereas PWV was not included in the secondmodel. Receiver operating characteristic (ROC) curves and the area under thecurve (AUC) were obtained by plotting the sensitivity against the false-positiverate (1-specificity). ROC curves were compared according to DeLong etal.. The Youdenindex was used to determine the optimal cutoff values of PWV and pPP for theidentification of POAF. A P-value < 0.05 was considered statisticallysignificant.Statistical analysis was performed with IBM Corp. Released 2013, IBM SPSSStatistics for Windows, version 22.0, Armonk, NY: IBM Corp. and MedCalc bvbaversion 16 . The normality of the data was analyzed with theKolmogorov-Smirnov test. Continuous data were expressed as mean ±standard deviation and categorical data was expressed as percentages.Differences between patient subgroups were tested using Student’svs. 58.3±8.4;P=0.014). Chronic obstructive pulmonary disease (COPD) was morecommon in patients with POAF , whereas the frequency of hypertension (HT),diabetes mellitus, smoking, and previous coronary artery disease did not differ.C-reactive protein and cholesterol levels were similar between the two groups.Left atrial diameter (LAD) was greater in patients with POAF (35.9±1.6vs. 36.7±1.7 P<0.039). The useof medications was similar in the two groups. Baseline characteristics arepresented in vs. 50.3±11.6; P=0.018, cPP: 31.4±8.1vs. 36.2±8.9; P=0.008). PWV wassignificantly higher in POAF  (POAF developed in 32 (29.1%) patients. Patients with POAF were older(63.7±8.6 P=0.006) . Aortic PWV correlated strongly with age, moderately with pPP, and weakly with LAD. pPPcorrelated weakly with age and did not correlate with LAD .P=0.004), whereas pPP > 41 mmHg had 80.7%sensitivity and 48.7% specificity to predict POAF (AUC: 0.656,95% CI[0.558-0.744], P=0.007]. PWV and pPP had similar accuracy for predicting POAF(difference between AUC: 0,00517; 95% CI  - 0,129; zstatistic: 0.0818, P=0.94] (Age, COPD, LAD, pPP, cPP, and PWV were associated with POAF in univariatelogistic regression analysis .COPD an P=0.94] .. Although thefrequency of POAF varies depending on the description of POAF and researchmethodology, it seems that POAF is still a significant cause of morbidity. Thisstudy showed for the first time that PWV and PP are associated with POAF.POAF occurred in about one-third of patients in our study, which is compatible withthe literature. Therefore, PP is one of the most frequently studiedparameters related to vascular function, owing to the ease of measurement with asphygmomanometer. Since PP relies on both aortic and ventricular functions, in ourstudy we had the intention to investigate the effect of vascular function on POAFalone. Currently, PWV is the gold standard of noninvasive assessment of vascularstiffness. Initial methods, arterial catheterization, and tonometry-based methodsrequired more time, effort, and trained staff. Thankfully, the oscillometric methodsimplified the process, thus PWV is measured in a few minutes and incorporated intodaily routine examinations in many clinics.The sensitivity of PP seems better than PWV despite having less specificity.Unfortunately, individual accuracies for predicting AF do not favour one overanother. Nevertheless, these parameters may still aid the clinician as a fastbedside preoperative risk assessment. Historically, PP was proposed to be one of thesignificant determinants of cardiovascular risk showed thatincreased PP is a significant risk factor for new-onset AF in a largecommunity-based sample. A previous study showed an association between PP and atrial volume;however, we did not find any relationship in our study. PP may be related tosubclinical atrial dysfunction in patients with AF, even in patients with normalatrial size. On the otherhand, PWV in our study was related to atrial size, compatible with a previousstudy.Mitchell GF et al., which predisposes to AF.Fumagalli S et al. found thatvascular stiffness increasing with age is related to altered left ventricularperformance, which is evaluated with longitudinal strain in the elderly withpreserved ejection fraction. Therefore, subclinical left ventricular remodelingrelated to vascular stiffness might further cause atrial remodeling and,subsequently, AF. Furthermore, electrocardiographic studies demonstrated that increasedP-wave dispersion is associated with altered aortic elasticity, thus increasing therisk for AF in young prehypertensive patients.PWV and PP are also associated with left ventricular diastolic dysfunction. Oxidative stress and related inflammation might be othercauses triggering AF. Additionally, medications such as beta-agonists andanticholinergic drugs frequently used for COPD treatment cause AF. Pulmonary HT inCOPD also might induce atrial remodeling. Additional mechanisms, including altereddiastolic dysfunction and P-wave dispersion, seem to contribute to occurrences ofPOAF in COPD.COPD is the most consistent predictor of AF and POAF in numerous studies. Despite astrong relationship, the exact pathophysiology remained to be clarified. Hypoxia andhypercapnia were speculated to cause arrhythmia. The relationship between aortic stiffness and POAF might bedue to the similarity of the remodeling process in the atria and aorta. Inconclusion, PP and PWV might be useful for detecting patients with a susceptibilityto POAF.Central aortic hemodynamics seem more related to afterload owing to the proximity tothe heart. However, we opted for pPP since it is easily measured with a simple cuffand strongly correlated with cPR Aortic stiffness is a complex measurement and isnot fully understood yet. On the other hand, PWV serves as a holistic measure ofaortic stiffness. Although PWV is associated with HT and age, it is less affected byother conventional risk factorsThe small number of patients is the major limitation of this study. We excludedpatients undergoing emergency, off-pump, and concomitant valve surgeries, andpatients presenting with an ACS, which are daily routines of surgicalpractice.Aortic stiffness parameters of PWV and PP are associated with POAF. These easilyobtained measurements should be incorporated into the risk assessment of patientsundergoing CABG.


Tetraselmis and dinoflagellates within the family Symbiodiniaceae and the genus Amphidinium. The diversity of these microalgae has not been well surveyed. In this study, we used PCR and culture techniques to demonstrate the biodiversity of Tetraselmis and dinoflagellates in symbiosis with meiofaunal acoels. Here, 66 acoels were collected from seven localities around Okinawa, Ishigaki, and Kochi, Japan. While convolutids were heavily represented in this sampling, some acoels formed a clade outside Convolutidae and are potentially a new family of acoels harboring symbiotic microalgae. From the acoels collected, a total of 32 Tetraselmis and 26 Symbiodiniaceae cultures were established. Molecular phylogenies were constructed from cultured material  using the 18S rRNA gene (Tetraselmis) and 28S rRNA gene (dinoflagellates). The majority of Tetraselmis sequences grouped within the T. astigmatica clade but strains closely related to T. convolutae, T. marina, and T. gracilis were also observed. This is the first report of Tetraselmis species, other than T. convolutae, naturally associating with acoels. For dinoflagellates, members of Cladocopium and Miliolidium were observed, but most Symbiodiniaceae sequences formed clusters within Symbiodinium, grouping with S. natans, or sister to S. tridacnidorum. Several new Symbiodinium sequences from this study may represent novel species. This is the first molecular record of Miliolidium and Symbiodinium from acoels. Microalgal strains from this study will provide a necessary framework for future taxonomic studies and research on symbiotic relationships between acoels and microalgae.Acoels in the family Convolutidae are commonly found with microalgal symbionts. Convolutids can host green algal  Due to research  and deveresearch . Acoels research . A high research , as epipresearch , or liviresearch .Symsagittifera and Convolutriloba species harbor only Tetraselmis, a green algal chlorophyte  that harbor Tetraselmis and a dinoflagellate  includes 24 genera, 19 of which have images depicting an association with microalgal symbionts . Hosts torophyte , while Whidinium . The genagellate . KunihirTetraselmis  is globally distributed (Tetraselmis (~30 species) can be found free-living, mostly in marine and brackish environments , no research has been performed to clarify the diversity of Tetraselmis species naturally associating with acoels. To date, only T. convolutae from Symsagittifera roscoffensis has been identified to the species level  were isolated from these acoels and were investigated using PCR (18S rRNA and 28S rRNA genes) and culture techniques. Results from this work aim to better understand acoel-microalgal symbiosis, by providing novel insights into acoel and microalgal biodiversity; we also aim to establish several new strains of microalgae that will improve our current and future understanding of microalgal systematics and taxonomy.The goal of the present study is to investigate the biodiversity of endosymbiotic microalgae found in meiofaunal marine acoels. Here, acoels containing microalgal symbionts were collected in subtropical localities in Southern Shikoku (Kochi) and in the Ryukyu Islands (Okinawa and Ishigaki), Japan. Tetraselmis) or full-sized petri dishes (dinoflagellates). Microalgal cultures were maintained in half-concentration Daigo’s IMK Medium  and were transferred to new dishes with fresh culture media every month. Where possible, after host acoels were broken apart, host material was transferred into a 1.5 ml microcentrifuge tube for DNA extraction. Cultures were maintained at 25 °C  in Biotron Incubator LH-350 . Motile stages of Tetraselmis and Symbiodiniaceae were mounted on glass slides and imaged under a Zeiss Axioskop 2 Plus microscope  connected to a Canon EOS Kiss X8i digital camera .Macroalgal and sediment samples were collected from intertidal and subtidal zones at seven sites in Okinawa, Ishigaki, and Kochi  from AprGenomic DNA from cultured microalgae and whole-acoel isolates was extracted according to manufacturer protocols with one of the following kits: DNeasy Blood & Tissue Kit , QuickExtract FFPE DNA Extraction Kit , or MasterPure Complete DNA and RNA Purification Kit  .Tetraselmis culture (K19), established from acoels collected in 2019 from Ikenoura, was also sequenced. All PCR reactions were first amplified using the SR1–28-1483R primer pair and then nested. From both the whole-acoel isolates and microalgal cultures, the 18S rRNA gene  was amplified for Tetraselmis using SR1–18SRF and SR4–SR12 primer pairs, and a partial 28S rRNA gene  was amplified for Symbiodiniaceae using the D1RF1–28-1483R primer pair. Apart from general eukaryotic primers, extracted host DNA was also amplified using a specific primer pair, Tear F–Tear R, designed from the sequences obtained from cloning. Polymerases used in the PCRs and thermocycler conditions are listed in For acoel isolates, nested PCR was performed on extracted DNA using general eukaryotic primers  and S2, Following the nested reaction, PCR products were purified using Polyethylene Glycol (PEG) and directly sequenced or cloned. Acoel DNA inserts were screened by colony PCR using the S1512A–S1513A primer pair provided in the NEB cloning kit. Colony-PCR products were cut using a HAEIII restriction enzyme  following the manufacturer’s protocol. Digested products were checked on a 1% agarose gel. A minimum of four replicates of each banding pattern were selected. Select colony-PCR products were purified again with PEG, and then sequenced with BrilliantDye Terminator v3.1 , according to manufacturer protocol on a 3130 genetic analyzer . Occasionally, additional sequencing primers were used to join sequence fragments refer to .https://www.geneious.com) was used to process Sanger sequencing reads. Cloning primers and low-quality regions (HQ% ≤ 50) were manually trimmed and assembled with the “De Novo Assemble” tool. Tetraselmis and dinoflagellate sequences were identified with NCBI’s BLAST search tool, sorted into their respective data sets, and compiled with existing data from Genbank. Multiple Alignment using Fast Fourier Transform (MAFFT)   within GTetraselmis and Symbiodiniaceae, respectively. Bootstrap analysis was performed on 1,000 pseudo-replicates. Under the Bayesian Information Criterion (BIC), the general time reversible (GTR) substitution model was selected. MrBayes 3.2.7a (th generation. The rate variation across sites were drawn from a gamma distribution with a proportion being invariable (lset rates = invgamma). Other parameters were set to the default settings. Chain convergence was checked with Tracer v1.7 and the posterior probability values were obtained from the consensus tree after the first 25% was discarded as burn-in. Molecular Evolutionary Genetics Analysis version 11 (MEGA11) (Iqtree v1.6.12  was useds 3.2.7a  was used(MEGA11)  was usedSequences generated from this study were deposited to NCBI. Accession numbers are listed in Tetraselmis. These were distinguishable, based on the numerous blunt-ended colored rhabdoids seen only in the second morphotype. A third type of acoel harbored exclusively Symbiodiniaceae and did not have any visible ocelli or colored rhabdoids , and isolate 28 contained Symbiodinium, subclades AIII and AIV; all symbionts were cultured. In total, 32 Tetraselmis cultures, 26 Symbiodiniaceae cultures, and two Amphidinium cultures were established (and sequenced) from acoels , followed by T. convolutae , T. marina (two cultures), and T. gracilis (one culture). Deeper nodes in the Tetraselmis phylogenetic analyses were largely unresolved.Maximum-likelihood analysis of equences  formed fMiliolidium , Cladocopium (three cultures), and Symbiodinium . The Miliolidium clade included isolate 16 ; the genetic distance did not exceed 1%. The remaining sequences formed a sister group with either S. tridacnidorum (nine cultures), S. microdriaticum , or S. pilosum ; genetic distances between our sequences and sequences they grouped with ranged between 0.5–2%, 2–3%, and 4–6%, respectively.Maximum-Likelihood analysis of Symbiodiniaceae 28S sequences  formed solate 16  that groAmphidinium was also observed (isolates 19–21) and cultured (isolates 61 and K22) from acoels. Amphidinium cultures were identified to the genus level based on the presence of an operculum. The 28S sequences from these Amphidinium isolates, 61 and K22, clustered with A. gibbosum (A. gibbosum from the acoel Waminoa sp. (AB626894). Additionally, a Heterocapsa sequence (isolate 7) was sequenced, and was positioned sister to H. pseudotriquetra  were closely related to T. convolutae from Symsagittifera roscoffensis  are the only Tetraselmis to be formally described from acoels  is difficult to conclude based on 18S data alone. Additional morphological data are needed to make a distinction between these closely related strains.In this study, we found at least four distinct lineages of offensis . Tetrasem acoels . The novT. convolutae, we report members of the T. marina, T. gracilis, and T. astigmatica clades as symbionts of acoels for the first time. Specifically, isolate 5 and culture 57 from Kabira were closely related to T. verrucosa (KX904702), a free-living strain collected from Messolonghi, Greece  from Japan (T. gracilis (KU352751), a free-living strain collected from the Arabian Sea (unpublished NCBI data). However, another unpublished NCBI sequence, T. gracilis (KJ756816), was positioned in a separate clade and represents a strain from the North Sea . The strain CCMP880 (JN376804) is presumably the same strain used in the description of T. astigmatica , as well as ultrastructural data, is warranted to resolve what might be a species complex. Nonetheless, this study is the first to report lineages in T. marina, T. gracilis, and T. astigmatica as symbionts of acoels.The majority of ubclades , I—IV. Cigmatica . Both seve these . Of notecription . The mosTetraselmis species is based on the morphological characteristics of cells including symmetry, presence/position of the stigma, and the ultrastructure of the pyrenoid and flagella  have type strains for which both molecular and morphological data is available  appear in different clades, further clouding Tetraselmis taxonomy  data within this genus remains difficult. Classification of flagella . Currentvailable . Moreovetaxonomy . This stWaminoa) has been completely based on observational data and lacks corresponding molecular information  or possible background symbiosis. They have also not been established as a symbiont under experimental conditions (S. natans.Novel (28S) sequences generated in this study formed six clusters across iolidium . This stnditions . This isS. natans in cultures established from cnidarian or other metazoan hosts has been attributed to their abundance in the environment  or possible background symbiosis. They have also not been established as a symbiont under experimental conditions  of these Symbiodinium strains will have to be confirmed through sequencing of a more variable genetic region , and by morphological comparisons.The remaining three clusters, one in subclade AII and two in subclade AIV, could represent novel lineages. These latter three formed separate branches with moderate support. The presence of nditions . This isCladocopium sequences (isolate 27) grouped with other undescribed species, separate from C. goreaui and other described coral symbionts within that same clade. Our Cladocopium sequences (cultures 27.1–27.3) could not be compared with symbionts of Waminoa acoels—because different genes (ITS-2 and psbAncr) were utilized (Symbiodinium (culture 27.4) was also established, along with Cladocopium, from isolate 27. This potentially shows some adaptability in acoels, like corals that also host a variety of Symbiodiniaceae at the same time.utilized . It is iMiliolidium sequence was obtained from isolate 16. The genus Miliolidium has been reported as a symbiont of Foraminifera and Porifera, or as free-living . Still, the diversity of hosts that acquire Miliolidium make this genus an interesting target for understanding symbiosis between microalgae, metazoans, and protists.A e-living . It is uAmphidinium and Heterocapsa were also sequenced in this study. Amphidinium from isolate 61 (A. gibbosum based on morphology and molecular phylogenics (Amphidinium gibbosum has been reported from Waminoa litus and Heterochaerus langerhansi (Waminoa and Heterochaerus are relatively large acoels (measuring approximately 2 mm). As Amphidinium are much larger, in comparison to other acoel symbionts, they may occur more frequently in larger acoels. Future collections, targeting larger acoels, may uncover a higher diversity of Amphidinium. Heterocapsa species have never been reported as a symbiont of acoels. The single Heterocapsa sequence in our data likely represents a food, or other environmental contaminant, not a symbiont of acoels. Heterocapsa was neither abundant nor cultured in our experiments.olate 61  and thosolate 61  were idelogenics . Amphidigerhansi . Both WaTetraselmis-containing acoels were collected in this study. These are all believed to be Convolutidae, based on the presence of microalgae . This is similar to other Amphiscolops species.Fourty-four croalgae . We obsecroalgae  to 2B. Oody size . Only twmmersion . Isolatemmersion  and 1C wEven though a large portion of the acoels collected in this study were not sequenced, it is likely that these represent a variety of lineages in Convolutidae. For example, light micrographs of isolates 5 and 57  and 6L hSymbiodinium  and Miliolidium. This is potentially the first report of non-convolutids containing microalgal symbionts. A prominent diagnostic characteristic of the family Convolutidae is the presence of microalgal symbionts—no other acoel family is known to contain microalgal symbionts  with one of these symbionts. Acoels with and without colored rhabdoids all hosted Tetraselmis symbionts. However, isolate 33, a convolutid hosting Symbiodinium, was also observed. Isolate 33 was genetically identical to 13 other convolutids that all harbored Tetraselmis. This means that a single acoel species can contain either Tetraselmis or Symbiodiniaceae; but this was not observed concurrently within the same individual.Observations in the present study suggest that meiofaunal acoels prefer either Tetraselmis belonging to the T. marina clade. Similar observations were made with isolates 58 and 59, with symbionts that were closely related to T. convolutae. S. roscoffensis could associate with six other species of Tetraselmis, albeit with varying levels of abundance. For example, T. rubens and T. subcordiformis appeared less dense within the host acoel, relative to T. convolutae. A larger sample size, based on molecular data from acoel hosts, would be needed to substantiate further claims regarding host-symbiont preference. In our study, dinoflagellates (Amphidinium) and Tetraselmis appeared together in only one acoel, the putative Amphiscolops from Ikenoura. Tetraselmis from these isolates were closely related to S. roscoffensis   .Tetraselmis or Symbiodiniaceae. While seasonality cannot be completely disregarded as a driver of symbiotic preference, regular sampling at set locations targeting the same acoel species would have to be performed, to address such questions.Regarding seasonality, no clear patterns were observed with respect to either Tetraselmis and Symbiodiniaceae species. The majority of host acoels collected in this study were members of the family Convolutidae. We also introduce intriguing evidence that there may be a family outside of Convolutidae that harbors microalgal symbionts. Our survey of symbiotic microalgae found at least four lineages of Tetraselmis from acoels, including lineages closely related to T. convolutae, T. astigmatica, T. marina, and T. gracilis. This is the first report of Tetraselmis species, other than T. convolutae, as symbionts of acoels. Analysis of these sequences further highlights the need for taxonomic and systematic revision of Tetraselmis. Symbiodiniaceae from acoels included Symbiodinium lineages in subclades AII, AIII, and AIV, as well as Miliolidium and Cladocopium. Lineages from Symbiodinium and Miliolidium were identified for the first time as symbionts of acoels. We also were able to culture multiple lineages of Symbiodiniaceae from a single acoel. This shows that acoels containing multiple types of Symbiodiniaceae do exist, but their prevalence is unknown. Therefore, the true diversity of these acoel-dinoflagellate associations is likely still underestimated. Further genomic sequencing information would shed light on this issue. Strains of Tetraselmis and Symbiodiniaceae established in this study will be an important tool for taxonomic and experimental work. Future studies should focus on the identification of both acoels and symbionts using molecular data. This would be useful in mapping out the selectivity, seasonality, feeding ecology, and the symbiotic relationship between acoels and their microalgae.Meiofaunal marine acoels in Japan host a variety of 10.7717/peerj.16078/supp-1Supplemental Information 1Sequences generated in this study are highlighted in colors that denote sampling times: April (blue), May (green), July (pink), and October (orange). Maximum-likelihood bootstrap values <50 and Bayesian posterior probabilities <0.95 were omitted. Black dots indicate fully supported branches (100 ML/1.00 BPP). Long branches are shortened by multiples of the substitutions/site scale bar (indicated on the branch).Click here for additional data file.10.7717/peerj.16078/supp-2Supplemental Information 2Tetraselmis symbionts. (A) Tetraselmis established from isolate 57 collected from Kabira. (B)Tetraselmis established from isolate 35 collected from Kabira. (C) Tetraselmis established from isolate 40 collected from Kabira. (D) Tetraselmis established from isolate 31 collected from Kabira. (E) Tetraselmis established from isolate 58 collected from Shiraho. (F) Tetraselmis established from isolate K22 collected from Ikenoura. (G)Tetraselmis established from isolate 55 collected from Kabira. (H—K) Cultured Symbiodiniaceae symbionts. (H)Cladocopium established from isolate 27 collected from Kabira. (I) Symbiodinium established from isolate 66 collected from Shiraho. (J) Symbiodinium established from isolate 63 collected from Shiraho. (K) Symbiodinium established from isolate 33 collected from Kabira. Scale bars: 5 µm.(A–G) Cultured Click here for additional data file.10.7717/peerj.16078/supp-3Supplemental Information 3Sequences generated in this study are highlighted in blue; color is in reference to the month (April) in which the samples were collected. Maximum-likelihood bootstrap values <50 and Bayesian posterior probabilities <0.95 were omitted. Black dots indicate fully supported branches (100 ML/1.00 BPP). Long branches are shortened by multiples of the substitutions/site scale bar (indicated on the branch).Click here for additional data file.10.7717/peerj.16078/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.16078/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.16078/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.16078/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.16078/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.16078/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.16078/supp-10Supplemental Information 10Click here for additional data file.


Streptococcus pneumoniae (SPN) is the agent responsible for causing respiratory diseases, including pneumonia, which causes severe health hazards and child deaths globally. Antibiotics are used to treat SPN as a first-line treatment, but nowadays, SPN is showing resistance to several antibiotics. A vaccine can overcome this global problem by preventing this deadly pathogen. The conventional methods of wet-laboratory vaccine design and development are an intense, lengthy, and costly procedure. In contrast, epitope-based in silico vaccine designing can save time, money, and energy. In this study, pneumococcal surface protein A (PspA), one of the major virulence factors of SPN, is used to design a multi-epitope vaccine.For designing the vaccine, the sequence of PspA was retrieved, and then, phylogenetic analysis was performed. Several CTL epitopes, HTL epitopes, and LBL epitopes of PspA were all predicted by using several bioinformatics tools. After checking the antigenicity, allergenicity, and toxicity scores, the best epitopes were selected for the vaccine construction, and then, physicochemical and immunological properties were analyzed. Subsequently, vaccine 3D structure prediction, refinement, and validation were performed. Molecular docking, molecular dynamic simulation, and immune simulation were performed to ensure the binding between HLA and TLR4. Finally, codon adaptation and in silico cloning were performed to transfer into a suitable vector.The constructed multi-epitope vaccine showed a strong binding affinity with the receptor molecule TLR4. Analysis of molecular dynamic simulation, C-immune simulation, codon adaptation, and in silico cloning validated that our designed vaccine is a suitable candidate against SPN.S. pneumoniae. The designated vaccine can be further tested in the wet lab, and a novel vaccine can be developed.The in silico analysis has proven the vaccine as an alternative medication to combat against The online version contains supplementary material available at 10.1186/s43141-023-00506-9. Streptococcus pneumoniae (SPN) is the etiological agent of most of the community-acquired pneumonia and the reason behind millions of deaths worldwide [orldwide –3. This orldwide , 5. Thisorldwide . Howeverorldwide , 4, 6. Aorldwide . SPN is orldwide . In 2015orldwide . Accordiorldwide . Accordiorldwide .Though antibiotics are traditionally used as a first-line treatment for pneumococcal infections, several dispensable gene-mediated resistance to antibiotics is growing rapidly in some strains of SPN and therefore losing their reliability for the treatment of pneumococcal diseases , 2. The Currently, a number of vaccine development strategy is available. Inactivation or live attenuation of the pathogen is one of the traditional methods for vaccine development. Although vaccines developed in this process can show a strong immune response in the host, these can also produce undesirable allergic and toxic reactions in the host. Similarly, several recombinant vaccines including conjugated vaccines, subunit vaccines, and toxoid vaccines may also produce severe toxicity instead of eliciting desired immune response . In contS. pneumoniae which are currently known [Salmonella enteritidis flagellin as vaccine candidates [Several virulence factors of SPN may influence its spread in the host cell, escape the immune defense systems, and promote disease progression . Polysacly known , 6. Sevely known . In contndidates . Tarahomndidates . Dorostindidates . In anotndidates . Pneumocndidates .Pneumococcal surface protein A (PspA) is one of the important virulence factors which is found on the surface of SPN . Mutant The complete workflow and various tools used in this study for designing a multi-epitope vaccine by in silico processes are depicted in Fig. http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) [https://www.ddg-pharmfac.net/AllerTOP/) [The reference sequence of the PspA (accession no.: WP_001035315.1) was first retrieved from NCBI and then subjected to protein–protein blast (Blastp)  against en.html)  and AllelerTOP/)  server, https://services.healthtech.dtu.dk/service.php?NetCTL-1.2) [https://webs.iiitd.edu.in/raghava/toxinpred/multi_submit.php) [NetCTL v1.2 (CTL-1.2)  was usedmit.php) , and Allhttp://tools.iedb.org/mhcii/) [https://webs.iiitd.edu.in/raghava/ifnepitope) [γ response.Helper T lymphocytes (HTL) were identified by utilizing the MHC II search tool of the “Immune Epitope Database (IEDB)” (/mhcii/) . The NN-epitope)  was usedhttp://tools.iedb.org/bcell/). This tool identifies LBL by utilizing a semiempirical method for the prediction of antigenic determinants on protein antigens. The amino acid sequences of the selected protein were submitted in this tool, and best 3 epitopes were selected for vaccine construction.Linear B-cell lymphocyte (LBL) epitopes were predicted by using the IEDB Kolaskar and Tongaonkar antigenicity method , theoretical pI, and aliphatic and instability index were comprehended from this server. Subsequently, VaxiJen v2.0, MHC-I immunogenicity, AllerTop, ToxinPred, and SOLpro (http://scratch.proteomics.ics.uci.edu/) [The physiochemical properties of the vaccine were evaluated with the ExPasy ProtParam server , which cci.edu/)  servers http://scratch.proteomics.ics.uci.edu/) [http://galaxy.seoklab.org/refine) [https://saves.mbi.ucla.edu/) were utilized for structure validation.The three-dimensional structure of the vaccine was constructed by using SCRATCH server (ci.edu/) . This se/refine)  was used/refine)  and ERRA/refine)  from thehttps://cluspro.bu.edu/) [Molecular docking is the most powerful computational tool that predicts the interaction between protein–protein and protein ligand. To ensure the binding between HLA molecules TLR4 and our predicted vaccine, a docking study was performed using ClusPro 2.0 (bu.edu/) , an onlibu.edu/) . The recbu.edu/) . The intbu.edu/) . The intbu.edu/) .https://imods.iqfr.csic.es/) [To understand the residual impact of the vaccine with the receptor (TLR-4) consistently evoke an immune response against target cells. MD simulation was performed for TLR4-vaccine complex using iMods online server (sic.es/) . These thttps://kraken.iac.rm.cnr.it/C-IMMSIM/) [C-immune tool predictor (IMMSIM/)  positionhttps://www.ebi.ac.uk/Tools/st/emboss_backtranseq/) [http://www.jcat.de/) [Escherichia coli K12). GC content and CAI value were measured for the adapted and unadapted sequences. The presence of restriction sites in the vaccine construct was investigated in order to clone it to a suitable vector. Finally, the codon-optimized (adapted) DNA sequence of the vaccine was cloned into the E. coli pET28a( +) vector using the SnapGene® tool .EMBOSS backtranseq (ranseq/)  was usedcat.de/)  was usedProtein information of the top 10 protein sequences, obtained by BlastP against the nr database, is depicted in Supplementary Table Currently used vaccines mostly rely on B-cell immunity. Antigenic drift eventually allows any foreign substance to escape the antibody memory response. As a result, multi-epitope vaccines have been promoted because they can provide long-lasting protection when B cells, T-helper cells (CD4 +), and cytotoxic T cells (CD8 +) are combined. The host’s CD8 + T cells are capable of generating a potent immune response that targets the infected cell . Hence, Identifying helper T cells is essential during the design of a multi-epitope vaccine. T-helper cells (CD4 +) play a crucial role in inhibiting immunological response and controlling the efficient immune response to pathogens. In addition to these functions, T-helper cells also activate innate immune system cells, B lymphocytes, and cytotoxic T cells . Among 1Due to its capability to produce antibodies that interact with antigens, B-cell epitope identification in target antigens has gained immense interest among researchers during the development of multi-epitope-based vaccines. An effective and promising method for locating possible B-cell epitopes in a target vaccination candidate is to use in silico bioinformatics techniques . In totaEpitope prediction was followed by the formulation of vaccine. The epitopes for vaccine formulation were determined on the basis of checking the antigenic property of both B and T cells. Using VaxiJen v2.0, the ACC calculation of the peptide based on its physicochemical properties was performed. Three best epitopes that have the highest antigenic score and nonallergenic and nontoxic properties were screened from each set of the CTL, HTL, and LBL epitopes. For the formulation of the vaccine construct, 50 s ribosomal protein, which is used as an adjuvant, is attached with the CTL epitopes by “EAAAK” linker. The CTL epitopes are connected to each other by the “AAY” linker. As three CTL epitopes are used in this study, two “AAY” linkers have been used. CTL epitopes and HTL epitopes are connected by the “GPGPG” linker, and HTL epitopes are also linked among themselves with the “GPGPG” linker. For this reason, three “GPGPG” linkers are used. The next portion is LBL epitopes, which are connected to the HTL epitopes by the “KK” linker, and LBL epitopes themselves are also connected by the “KK” linker. So, three “KK” linkers are required to formulate the vaccine. The complete procedure of formulating the multi-epitope vaccine is illustrated in Fig. Physicochemical properties of the vaccine construct are depicted in Table A three-dimensional (3D) structure of the protein from vaccine sequence was obtained from SCRATCH server. GalaxyRefine server was subsequently utilized for the refinement of the protein 3D structure. GalaxyRefine server made the protein structure more stable and increased its quality score in SAVES server. GalaxyRefine server gave 5 refined models of the protein structure, and among these models, model 2 was selected as the best model on basis of its quality score. Ramachandran plot analysis has revealed that 95.4% residues of the 3D structure were in the most favored region for the refined structure  and the codon-optimized vaccine sequence, including Xhol and Ndel. Both the vaccine sequence and the vector were directionally cloned into their respective cloning sites. After cloning, the final length of the vector and the insert was found to be 6058 bp. The vaccine sequence inserted in the expression vector pET28a ( +) is represented in Fig. In silico cloning of the engineered construct was achieved for maximum expression in the The use of computational tools, particularly reverse vaccinology, is an appealing option for developing an epitomic vaccine for rapidly spreading disease. Multi-epitope-based vaccine design could be the best and most effective tool for disease control . RemarkaE. coli (0.918779). To produce better immunity in the host, a vaccine needs to be stable in its performance. The instability index was computed to be 20.34, which is significantly lower than the study of Bahadori et al. (2022) [E. coli K12 strain for codon optimization to enhance transcription and translation efficacy. The GC content of our vaccine is satisfactory as it falls in the optimal range of > 30 and < 70%, which is desirable for expression [Escherichia coli was performed to facilitate the path of further wet laboratory experiments by other researchers to produce an effective vaccine as well as to serve as a protocol for further in silico analysis of cloning experiments. In future, our team plans to use wet lab work and the E. coli expression system to produce this prototype vaccine.By utilizing several standard bioinformatics tools, the best antigenic protein and epitopes were identified to design a multi-epitope vaccine against the SPN. The vaccine was designed with three potential epitopes: CTL, HTL, and LBL. Linkers KK, CPGPG, and AAY joined these epitopes during the vaccine formulation to produce better and long-lasting protection. The critical problem of epitope vaccines is that they can quickly be broken down by proteinase in the body . To circ. 2022) , Dorosti. (2022) , and Dor , Dorost. (2022) , and the. (2022) , Dorosti. (2022) , and Tar. (2022) . The Ram. (2022) . FinallyStaphylococcus aureus [Vibrio harveyi [Neisseria gonorrhoeae [A large number of recent studies have used robust computational methods to select effective epitopes and design new vaccines against various pathogens, including s aureus , Vibrio  harveyi , Neisserorrhoeae , and SARorrhoeae . Based oStreptococcus pneumoniae by utilizing several bioinformatics tools. In this study, a multi-epitope-based vaccine was constructed using in silico tools, and its binding affinity with human cell receptor molecule (TLR4) was calculated. Analysis of molecular docking, molecular dynamic simulation, C-immune simulation, codon adaptation, and in silico cloning validated our designed compound as a suitable vaccine candidate. Our analysis has revealed that the designed vaccine in this study has the potential to generate higher immunogenicity in the host without any possible toxicity and allergenicity. Thus, our vaccine can be an attractive alternative to the conventional vaccines that are currently being used against pneumococcal disease and has several shortcomings.Drug targets and scientific approaches are involved in making effective and potentially lifesaving medications. Although many drugs are available in the market, vaccines play a significant role in preventing pathogen infection. Throughout this study, we investigated a novel therapeutic vaccine against the PspA of Additional file 1: Supplementary Table 1. Protein information of the 10 protein sequences obtained from NCBI by BlastP search. Supplementary Table 2. CTL Epitopes identified by NetCTL 1.2 server and their antigenic and allergenic properties predicted with VaxiJen and AllerTOP 2 server, respectively. Supplementary Table 3. HTL epitopes less than ic50 <250nm and interacting with 10 or more alleles. Supplementary Table 4. LBL epitopes predicted with IEDB B cell epitope prediction tool by utilizing Kolaskar & Tongaonkar Antigenicity method.