Reverse Transcriptase qPCR Research Articles

Mycobacterium leprae Survival Inside Acanthamoeba sp. Isolated from Water Source in Leprosy Endemic Area, Indonesia

Mycobacterium leprae is an acid-fast bacterium that causes leprosy diseases, which remains a problem worldwide. Even though leprosy prevalence in the world has decreased significantly, many endemic pocket areas continue reporting new cases and harbor M. leprae in the environment, including water and soil. The presence of obligate intracellular bacteria-M. leprae in the environment raises a question on how it survives. Free-living amoeba has been proposed as its reservoir host in the environment. The study was conducted to give evidence that M. leprae can survive inside free-living amoeba isolated from water sources of leprosy endemic areas. M. leprae from leprosy patients was cultured together with Acanthamoeba sp. isolated from the water source of the leprosy endemic area. Viability and duplication of M. leprae inside amoeba then observed at days 14 and 28 using reverse transcriptase PCR and qPCR. The results showed that M. leprae survived inside the amoeba until day 28, but no bacterial replication was observed. The study reveals in vitro evidence of viable M. leprae inside free-living amoeba of leprosy endemic area environment

The impact of functional feed on Atlantic salmon (Salmo salar) systemic immune response to high and low levels of sea lice infection (Lepeophtheirus salmonis) and co-infection with infectious salmon anemia virus

Due to the nature of open-pen farming, salmon are exposed to numerous pathogens shared by other farms and their wild counterparts. Industry must manage these outbreaks through vaccination, oral or bath treatments, and more recently through functional feed administration. Globally, the most important pathogens of salmon are sea lice (Lepeophtheirus salmonis, and Caligus rogercresseyi) not only due to their direct impacts on the host, but indirectly by enhancing host susceptibility to co-infection. This study aims to characterize molecular responses during a co-infection of L. salmonis and a salmon orthomyxovirus (infectious salmon anemia virus; ISAv) under administration of four functional feed diets: a control feed with a low 0.3 % EPA/DHA + high-ω6 (Ctrl), an EPA/DHA enriched pro-inflammatory diet 1 % EPA/DHA + high-ω6; FA + I), an EPA/DHA enriched anti-inflammatory diet (1 % EPA/DHA+high-ω3; FA-I), and a low EPA/DHA feed (0.3 %) with an immunostimulant added (0.3 % EPA/DHA + high-ω6 + immunostimulant (IS); Ctrl + IS). Atlantic salmon (40 fish per tank; 8 tanks per feed) were acclimated to one of the four experimental diets. Sea lice copepodids were introduced to all experimental tanks and 10 fish sampled from each tank at each time point (prior to infection [−3], and 11, 33, 47 days post infection). A high virulence ISAv isolate (ISAV-HPR4) was intraperitoneally injected into donor fish 6 days prior to their transfer into the experimental tanks for cohabitation (ca. 10–15 % of tank density; 4 tanks per feed group) to achieve peak shedding rates at time of stocking. Fatty acid enriched (FA + I and FA-I) diets had a significant impact on sea lice abundance on fish infected with lice only. Gene expression profiles measured by reverse transcriptase-qPCR showed significant upregulation in several antiviral genes (irf7b and mxb) associated with the interferon system in all but the fish fed the immunostimulating diet. An increase in transcript levels (irf7b, isg15a, mmp-9, mxb) accounted for high lice and high viral loads in diets FA + I and Ctrl + IS. As feeds successful at reducing lice were the least successful in survival of co-infected fish, there appears to be a trade-off for better anti-parasitic responses, which is enhanced through dietary supplementation. This will require further research to ensure careful selection of feed combinations and expected co-infection challenges.

Investigation of Azole Resistance Involving cyp51A and cyp51B Genes in Clinical Aspergillus flavus Isolates.

This study aimed to investigate azole resistance mechanisms in Aspergillus flavus, which involve cyp51A and cyp51B genes. Real-time Reverse Transcriptase qPCR method was applied to determine the overexpression of cyp51A and cyp51B genes for 34 A. flavus isolates. PCR sequencing of these two genes was used to detect the presence of gene mutations. Susceptibility test found sensitivity to voriconazole (VOR) in all strains. 14.7% and 8.8% of isolates were resistant to itraconazole (IT) and posaconazole (POS), respectively, with a cross-resistance in 5.8%. For the double resistant isolates (IT/POS), the expression of cyp51A was up to 17-fold higher. PCR sequencing showed the presence of 2 mutations in cyp51A: a synonymous point mutation (P61P) in eight isolates, which did not affect the structure of CYP51A protein, and another non synonymous mutation (G206L) for only the TN-33 strain (cross IT/POS resistance) causing an amino acid change in the protein sequence. However, we noted in cyp51B the presence of the only non-synonymous mutation (L177G) causing a change in amino acids in the protein sequence for the TN-31 strain, which exhibits IT/POS cross-resistance. A short single intron of 67 bp was identified in the cyp51A gene, whereas three short introns of 54, 53, and 160 bp were identified in the cyp51B gene. According to the models provided by PatchDock software, the presence of non-synonymous mutations did not affect the interaction of CYP51A and CYP51B proteins with antifungals. In our study, the overexpression of the cyp51A and cyp51B genes is the primary mechanism responsible for resistance in A. flavus collection. Nevertheless, other resistance mechanisms can be involved.

The impact of indole and mucin on sporulation, biofilm formation, and enterotoxin production in foodborne Clostridium perfringens.

Indole and mucin are compounds found in the host environment as they are produced by the host or by the host-associated microbiota. This study investigated whether indole and mucin impact Clostridium perfringens growth and sporulation, as well as enterotoxin production and biofilm formation. There was no impact on growth of Cl. perfringens for up to 400µM indole and 240mg/l mucin, and neither indole nor mucin affected sporulation. Reverse-transcriptase qPCR showed that mucin strongly upregulated the expression of Cl. perfringens enterotoxin (up to 121-fold increase), whereas indole had a much more modest effect (2-fold). This was also reflected in increased Cl. perfringens enterotoxin levels in mucin-treated Cl. perfringens (as assessed by a reversed passive latex agglutination assay). Finally, mucin and indole significantly increased biofilm formation of Cl. perfringens, although the effect size was relatively small (less than 1.5 fold). These results indicate that Cl. perfringens can sense its presence in a host environment by responding to mucin, and thereby markedly increased enterotoxin production.

Searching for homozygous haplotype deficiency in Manech Tête Rousse dairy sheep revealed a nonsense variant in the MMUT gene affecting newborn lamb viability

BackgroundRecessive deleterious variants are known to segregate in livestock populations, as in humans, and some may be lethal in the homozygous state.ResultsWe used phased 50 k single nucleotide polymorphism (SNP) genotypes and pedigree data to scan the genome of 6845 Manech Tête Rousse dairy sheep to search for deficiency in homozygous haplotypes (DHH). Five Manech Tête Rousse deficient homozygous haplotypes (MTRDHH1 to 5) were identified, with a homozygous deficiency ranging from 84 to 100%. These haplotypes are located on Ovis aries chromosome (OAR)1 (MTRDHH2 and 3), OAR10 (MTRDHH4), OAR13 (MTRDHH5), and OAR20 (MTRDHH1), and have carrier frequencies ranging from 7.8 to 16.6%. When comparing at-risk matings between DHH carriers to safe matings between non-carriers, two DHH (MTRDHH1 and 2) were linked with decreased insemination success and/or increased stillbirth incidence. We investigated the MTRDHH1 haplotype, which substantially increased stillbirth rate, and identified a single nucleotide variant (SNV) inducing a premature stop codon (p.Gln409*) in the methylmalonyl-CoA mutase (MMUT) gene by using a whole-genome sequencing approach. We generated homozygous lambs for the MMUT mutation by at-risk mating between heterozygous carriers, and most of them died within the first 24 h after birth without any obvious clinical symptoms. Reverse transcriptase-qPCR and western blotting on post-mortem liver and kidney biological samples showed a decreased expression of MMUT mRNA in the liver and absence of a full-length MMUT protein in the mutant homozygous lambs.ConclusionsWe identified five homozygous deficient haplotypes that are likely to harbor five independent deleterious recessive variants in sheep. One of these was detected in the MMUT gene, which is associated with lamb lethality in the homozygous state. A specific management of these haplotypes/variants in the MTR dairy sheep selection program would help enhance the overall fertility and lamb survival.

Mitigating the attachment of Salmonella Infantis on isolated poultry skin with cetylpyridinium chloride.

To provide the poultry industry with effective mitigation strategies, the effects of cetylpyridinium chloride (CPC) on the reduction of Salmonella Infantis, hilA expression, and chicken skin microbiota were evaluated. Chicken breast skins (4×4 cm; N = 100, n = 10, k = 5) were inoculated with Salmonella (Typhimurium or Infantis) at 4°C (30min) to obtain 108 CFU/g attachment. Skins were shaken (30s), with remaining bacteria being considered firmly attached. Treatments were applied as 30s dips in 50 mL: no inocula-no-treatment control (NINTC), no treatment control (NTC), tap water (TW), TW+600 ppm PAA (PAA), or TW+0.5% CPC (CPC). Excess fluid was shaken off (30s). Samples were homogenized in nBPW (1 min). Samples were discarded. Salmonella was enumerated and Log10 transformed. Reverse transcriptase-qPCR (rt-qPCR) was performed targeting hilA gene and normalized using the 2-ΔΔCt method. Data were analyzed using one-way ANOVA in RStudio with means separated by Tukey's HSD (P≤0.05). Genomic DNA of rinsates was extracted, 16S rRNA gene (V4) was sequenced (MiSeq), and data analyzed in QIIME2 (P≤0.05 and Q≤0.05). CPC and PAA affected Salmonella levels differently with CPC being effective against S. Infantis compared to TW (P<0.05). Treatment with CPC on S. Infantis-infected skin altered the hilA expression compared to TW (P<0.05). When inoculated with S. Typhimurium, there was no difference between the microbiota diversity of skins treated with PAA and CPC; however, when inoculated with S. Infantis, there was a difference in the Shannon's Entropy and Jaccard Dissimilarity between the two treatments (P<0.05). Using ANCOM at the genus level, Brochothrix was significant (W = 118) among skin inoculated with S. Typhimurium. Among S. Infantis inoculated, Yersiniaceae, Enterobacterales, Lachnospiraceae CHKCI001, Clostridia vadinBB60 group, Leuconostoc, Campylobacter, and bacteria were significant (40<W>8). CPC and PAA-treated skins had lowest relative abundance of the genera. In conclusion, CPC mitigated Salmonella Infantis, altered hilA expression, and influenced the chicken skin microbiota.

Reappraisal of intra-abdominal candidiasis: insights from peritoneal fluid analysis

BackgroundThe understanding of high mortality associated with intra-abdominal candidiasis (IAC) remains limited. While Candida is considered a harmless colonizer in the digestive tract, its role as a true pathogen in IAC is still debated. Evidence regarding Candida virulence in the human peritoneal fluid are lacking. We hypothesized that during IAC, Candida albicans develops virulence factors to survive to new environmental conditions. The objective of this observational exploratory monocentric study is to investigate the influence of peritoneal fluid (PF) on the expression of C. albicans virulence using a multimodal approach.Materials and methodsA standardized inoculum of a C. albicans (3.106 UFC/mL) reference strain (SC5314) was introduced in vitro into various PF samples obtained from critically ill patients with intra-abdominal infection. Ascitic fluids (AFs) and Sabouraud medium (SBD) were used as control groups. Optical microscopy and conventional culture techniques were employed to assess the morphological changes and growth of C. albicans. Reverse transcriptase qPCR was utilized to quantify the expression levels of five virulence genes. The metabolic production of C. albicans was measured using the calScreener™ technology.ResultsA total of 26 PF samples from patients with secondary peritonitis were included in the study. Critically ill patients were mostly male (73%) with a median age of 58 years admitted for urgent surgery (78%). Peritonitis was mostly hospital-acquired (81%), including 13 post-operative peritonitis (50%). The infected PF samples predominantly exhibited polymicrobial composition. The findings revealed substantial variability in C. albicans growth and morphological changes in the PF compared to ascitic fluid. Virulence gene expression and metabolic production were dependent on the specific PF sample and the presence of bacterial coinfection.ConclusionsThis study provides evidence of C. albicans virulence expression in the peritoneal fluid. The observed variability in virulence expression suggests that it is influenced by the composition of PF and the presence of bacterial coinfection. These findings contribute to a better understanding of the complex dynamics of intra-abdominal candidiasis and advocate for personalized approach for IAC patients.Trial registrationhttps://clinicaltrials.gov/ (NCT05264571; February 22, 2022)Graphical

Maintenance of adult stem cells from human minor salivary glands via the Wnt signaling pathway

BackgroundXerostomia is a salivary gland dysfunction that negatively impacts the life quality of patients; however, there is no effective treatment for xerostomia. Bioengineered organs, generated using stem cells obtained from newborn salivary glands and ligated injury models, are a new organ transplantation strategy that could be feasible for xerostomia treatment. Reconstruction of salivary gland organoids by seed cells obtained from human minor salivary glands will offer theoretical fundaments and technology support for clinical application and organ regeneration research. Herein, we aimed to propose a new method for culturing and enriching adult human minor salivary gland stem cells in vitro in a three-dimensional (3D) environment via Wnt signaling activation.MethodsObtained and characterized human minor salivary gland stem cells (hMSGSCs) with self-organization ability were 3D-cultured to generate organoids. We examined hMSGSCs proliferation and colony formation using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Telomerase reverse transcriptase staining, flow cytometry, immunofluorescence assay, RNA isolation, RT-PCR, and qPCR were performed to assess hMSGSCs structure and the function of reconstructive organoids in vitro.ResultshMSGSCs showed typical epithelial-like characteristics, such as positive for CD49f and cell KRT expression. hMSGSCs served as adult stem cells in salivary glands and could differentiate into acinar and duct cells. Upon the addition of Noggin, CHIR99021, and Wnt3A to the 3D culture system, hMSGSCs showed higher LGR5 expression and decreased AMY1B and MUC5B expression. Therefore, the Wnt and bone morphogenetic protein (BMP) pathways are important in regulating hMSGSCs self-organization and differentiation.ConclusionsWe showed that the stem cell properties of hMSGSCs in a 3D culture system can be maintained by activating the Wnt signaling pathway and inhibiting the BMP signaling pathway. Our findings contribute new insights on salivary gland organoid generation in vitro.

Prevalence of Different Courses of Feline Leukaemia Virus Infection in Four European Countries.

Prevalence of progressive feline leukaemia virus (FeLV) infection is known to still be high in cats in Europe, especially in Southern Europe, but the prevalence of other outcomes of FeLV infection has not been determined in most countries. The present study aimed to investigate the prevalence of progressive, regressive, abortive, and focal infection in four European countries, two with a high (Italy, Portugal) and two with a low expected prevalence (Germany, France). Blood samples of 934 cats (Italy: 269; Portugal: 240; France: 107; Germany: 318) were evaluated for the p27 antigen, as well as anti-whole virus, anti-SU, and anti-p15E antibodies by enzyme-linked immunosorbent assay (ELISA) in serum and for proviral DNA by quantitative polymerase chain reaction (qPCR) in whole blood. Positive p27 antigen ELISA results were confirmed by reverse transcriptase-qPCR (RT-qPCR) detecting viral RNA in saliva swabs and/or blood. The outcome of FeLV infection was categorised as progressive (antigen-positive, provirus-positive), regressive (antigen-negative, provirus-positive), abortive (antigen- and provirus-negative, antibody-positive), and focal (antigen-positive, provirus-negative) infection. Overall FeLV prevalence was 21.2% in Italy, 20.4% in Portugal, 9.5% in Germany, and 9.3% in France. Prevalence of progressive, regressive, abortive, and focal infection in Italy was 7.8%, 4.5%, 6.3%, and 2.6%; in Portugal 3.8%, 8.3%, 6.7%, and 1.7%; in Germany 1.9%, 1.3%, 3.5%, and 2.8%; in France 1.9%, 3.7%, 2.8%, and 0.9%, respectively. In conclusion, overall FeLV prevalence is still very high, especially in Southern European countries. Therefore, testing, separation of infected cats, and vaccination are still important measures to reduce the risk of FeLV infection.

The effect of synbiotic-fluoride therapy on multi-species biofilm

ObjectivesThe study objective was to examine the effect of synbiotic-fluoride (SF) therapy within a multi-species cariogenic biofilm model system comprising of S. mutans, S. sanguinis, and S. gordonii. MethodsThe SF therapy was prepared using 2% L-arginine (Arg), 0.2% NaF and probiotic L. rhamnosus GG (LRG). The 8 treatment groups were: Group 1: No treatment, Group 2: 2% Arg, Group 3: 0.2% NaF, Group 4: LRG, Group 5: 2% Arg+0.2% NaF, Group 6: 2% Arg+LRG, Group 7: 0.2% NaF+LRG, and Group 8: SF therapy (2% Arg+0.2% NaF +LRG). Multi-species biofilm model over 96 h comprising Streptococcus mutans, Streptococcus sanguinis, and Streptococcus gordonii was utilized. The biofilms received cariogenic challenge and SF therapy 2 × /day. The extracellular matrix components were analyzed for carbohydrates, proteins, and extra-cellular DNA (eDNA). The live/dead cells were imaged and quantified using confocal microscopy. The viable/dead bacterial concentrations were estimated using propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR). The gene expressions for gtfB, sagP, arcA, argG, and argH were measured using real-time reverse transcriptase qPCR. ResultsCarbohydrates and protein content with SF therapy were higher than non-LRG containing groups, while eDNA content was lower than other groups (p<0.05). Live bacterial proportions determined using confocal imaging with SF therapy were the lowest (p<0.05). The 2% Arg+LRG and SF therapy showed higher viable L. rhamnosus GG than 0.2% NaF+LRG (p<0.05). The dead S. mutans with SF therapy were higher than the other groups (p<0.05) with no difference from 2% Arg+0.2% NaF and 2% Arg+LRG (p>0.05). The SF therapy significantly downregulates gtfB and upregulates sagP, arcA, argG, argH gene expression (p<0.05). ConclusionSynbiotic-fluoride therapy effectuates multi-fold changes in the multi-species biofilm matrix and cellular components leading to superior ecological homeostasis than its individual contents, prebiotics (arginine), probiotic (L. rhamnosus GG), and fluorides (NaF). Clinical significanceThe ecological-based synbiotic-fluoride caries-preventive therapy aids in maintaining biofilm homeostasis to preempt/restore dysbiosis thereby sustaining dynamic-diverse health-associated microbial stability significant as a preventive regimen for high caries-risk patients.

SARS-CoV-2 Is More Efficient than HCoV-NL63 in Infecting a Small Subpopulation of ACE2+ Human Respiratory Epithelial Cells

Human coronavirus (HCoV)-NL63 is an important contributor to upper and lower respiratory tract infections, mainly in children, while severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can cause lower respiratory tract infections, and more severe, respiratory and systemic disease, which leads to fatal consequences in many cases. Using microscopy, immunohistochemistry (IHC), virus-binding assay, reverse transcriptase qPCR (RT-qPCR) assay, and flow cytometry, we compared the characteristics of the susceptibility, replication dynamics, and morphogenesis of HCoV-NL63 and SARS-CoV-2 in monolayer cultures of primary human respiratory epithelial cells (HRECs). Less than 10% HRECs expressed ACE2, and SARS-CoV-2 seemed much more efficient than HCoV-NL63 at infecting the very small proportion of HRECs expressing the ACE2 receptors. Furthermore, SARS-CoV-2 replicated more efficiently than HCoV-NL63 in HREC, which correlates with the cumulative evidence of the differences in their transmissibility.

A33 EFFECTS OF THE NEXT GENERATION PROBIOTIC, AKKERMANSIA MUCINIPHILA, ON INTESTINAL INFLAMMATION AND BARRIER FUNCTION

Abstract Background Inflammatory bowel disease (IBD), characterised by chronic intestinal inflammation, is hypothesised to arise from the interplay between susceptibility genes, the immune system, environmental factors, and gut microbiota. Akkermansia muciniphila is a symbiotic bacterium that accounts for 1-5% of the human fecal microbiota. This microbe has been hailed as a next-generation probiotic, principally with regard to its plethora of beneficial host interactions, including the ability to influence mucin secretion and strengthen the intestinal barrier. Purpose Though a clear-cut role and mechanism by which A. muciniphila influences inflammatory conditions is unknown, evidence indicates this microbe is depleted in IBD, suggesting it may have protective effects that are lost in these conditions. Here, we investigate the role and mechanism of A. muciniphila in intestinal inflammation and its influence on intestinal barrier function by utilizing barrier-disrupting models of colitis. Method Across several experimental models of intestinal inflammation including the chemically-induced dextran sulphate sodium (DSS) model, the parasitic-based model of Trichuris muris infection, and the spontaneous Muc2-/- model, A.muciniphila was administered by oral gavage. Disease activity index, macroscopic scoring and histological scoring were all performed to assess the severity of intestinal inflammation. Various pro- and anti-inflammatory cytokines were assessed within colonic tissue using commercially available ELISA kits.To investigate the effects that A. muciniphila has on barrier function in the context of colitis, reverse transcriptase qPCR was used to explore several factors, including several TJPs, AMPs, and mucins. To analyse the composition of the microbiota and changes in diversity with A. muciniphila supplementation, 16S rRNA sequencing of fecal samples was performed. Result(s) Though only minor benefits were derived from this microbe in germ-free mice, in specific pathogen-free (SPF) mice, administration of pasteurized A. muciniphila in a DSS recovery model ameliorated inflammation severity and promoted recovery compared to controls. When gavaged prior to DSS administration, both live and pasteurized A. muciniphila failed to diminish inflammatory markers indicating minimal preventative effects. T. muris-infected SPF mice treated with live A. muciniphila showed increased levels of Th2 and anti-inflammatory cytokines, decreased worm burden, and enhanced levels of the mucin, Muc5ac, compared with those receiving control broth or pasteurized bacteria. Further, both live and pasteurized A. muciniphila ameliorated the severity of inflammation in a mucin 2 deficient (Muc2-/-) mouse model of spontaneous colitis, indicating that these protective effects are Muc2-independent. Conclusion(s) These observations provide us not only with an enhanced understanding of the role A. muciniphila plays in the pathogenesis of intestinal inflammatory conditions but also may fuel novel avenues of treatment for those with IBD. Please acknowledge all funding agencies by checking the applicable boxes below CIHR Disclosure of Interest None Declared

Production and Quantification of Virulence Factors in Malassezia Species.

Seventy-seven strains of Malassezia were included in this study. Biofilm and hydrolytic enzyme production were studied by using specific solid media. The Real-Time reverse transcriptase qPCR method was applied to determine the overexpression of genes encoding the extracellular enzymes. All included Malassezia species produced biofilms. No statistically significant difference was observed between Malassezia species in biofilm formation (p = 0.567). All Malassezia species produced lipase, and 95% of Malassezia globosa showed a strong enzymatic activity (Pz = 0.55 ± 0.02). A statistically significant difference was observed between the mean keratinase indices of Malassezia slooffiae and the other Malassezia species (p = 0.005). The overexpression of one or more genes was observed in 100% of strains isolated from patients with folliculitis, 87.5% - with pityriasis versicolor, and 57.14% of the control group isolates. A statistically significant difference in the lipase gene expression (p = 0.042) was between the strains from patients with folliculitis and the control group. This investigation provides more information about the frequency of the production of the major enzymes considered virulence factors of Malassezia species. Interestingly, the overexpression of one or more genes was observed in strains isolated from patients with Malassezia disorders.

Influence of fecal microbiota transplantation on gut microbiota composition in transgenic mice modeling amyloid‐β plaques and neurofibrillary tangles

AbstractBackgroundThe gut microbiota, the aggregates of all microbial cells that inhabit the gut, bidirectionally communicates with the brain through cytokines, hormones, metabolites, and neurotransmitters via the gut microbiota‐brain axis. The gut microbiota is thought to contribute to the development of Alzheimer’s disease (AD), which is characterized by plaque deposition, neurofibrillary tangles, and neuroinflammation. We hypothesize that manipulation of the gut microbiota will alter development of AD pathologies and neuroinflammation via the gut microbiota‐brain axis.MethodWe performed fecal microbiota transplants (FMT) from aged (52‐64 weeks) 3xTg‐AD mice, which are modeling plaques and neurofibrillary tangles, to young 3xTg‐AD (n=5) or wild‐type mice (n=10) via oral gavage. Phosphate buffered saline (PBS) was gavaged into 3xTg‐AD (n=5) and wild‐type mice (n=10) as a control. At 8 weeks, mice were gavaged with FMT or PBS for 5 consecutive days, followed by fortnightly maintenance transplants for 24 weeks. The V4 region of the 16S rRNA gene was sequenced on the Illumina MiSeq. Data were analyzed using QIIME 2. Reverse transcriptase qPCR was used to assess microgliosis, astrocytosis, and Th1/Th2 inflammation in the hippocampus of the FMT cohort at 24 weeks of age.ResultWe observed a shift in microbiota composition of FMT‐treated mice when compared to control (PBS‐treated) mice. Bacteroides acidifaciens was increased in 3xTg‐AD and wild‐type mice receiving FMT. We demonstrate partial engraftment of the gut microbiota from aged 3xTg‐AD mice in all FMT‐treated mice, demonstrated by a Random Forest model, which correctly predicts treatment groups based on gut microbiota composition (Accuracy Ratio over baseline assignment: 2.6).ConclusionWe demonstrate the ability to transplant an aged gut microbiome into young mice. Future shallow shotgun metagenomic sequencing will be used to determine the species‐ and strains‐ that engraft in the GI tract. Additionally, targeted reverse transcription RT‐qPCR and immunostaining for plaques and hyperphosphorylated tau in the hippocampus will be used to assess how FMTs alter AD pathologies. These studies will contribute to our understanding of how features of the gut microbiota may contribute to AD development.

S1.4d Cryptococcus qPCR assays: the future for routine mycology labs and clinical trials dealing with cryptococcosis

S1.4 Fungal infections in Asia, bringing it out of the dark, September 21, 2022, 11:00 AM - 12:30 PMBackground: Routine laboratory testing for cryptococcal meningitis currently consists of Cryptococcal antigen (CrAg) testing in blood and cerebrospinal fluid (CSF), CSF India ink, and CSF fungal culture. Quantitative cryptococcal culture (QCC) is labor intensive and not feasible in most settings.ObjectivesWe evaluated quantitative (qPCR) and reverse transcriptase qPCR (RT-qPCR) assays to quantify cryptococcal load in CSF, plasma, and blood. We also investigated the dynamics of fungal DNA and RNA detection during antifungal treatment.MethodsWe developed a qPCR assay that can differentiate serotypes A, D, and B/C of Cryptococcus neoformans and C. gattii based on the amplification of a unique nuclear Quorum sensing protein 1 (QSP1) and a multicopy 28S rRNA gene and evaluated the assays on 205-patient samples from the AMBITION-cm trial in Botswana and Malawi (2018-2021). CSF, plasma, and whole blood samples were stored per patient and were sampled at day 0 (baseline), day 7 and 14 for CSF and at day 1, 3 and 7 for plasma and whole blood post antifungal treatment initiation. A Roche LightCycler480 and Graph pad prism were used for data analysis.ResultsA total of 205/209 stored patient samples (85 from Botswana; 124 from Malawi), were used. For QSP1 qPCR tested in CSF at D0, 138 (81.7%) were serotype A, 28 (16.6%) were serotype B/C and 3 (1.8%) were a mixed infection of serotype A and B/C. There was no amplification with 36 (17.6%) samples. There was no difference in fungal loads at D0, D7, and D14 between serotype A and B/C with the QSP1 qPCR assay, and QCC. QCC showed a good correlation with qPCR quantification with QSP1 qPCR (slope = 0.797, R2 = 0.73) and with 28S rRNA qPCR (Slope = 0.771, R2 = 0.778) assays. The fungal load at D0 was significantly higher in patients who died at week 2 (w2) and at week 10 (w10) as compared with patients who survived post-week 10 (P <.01), with no significant difference in initial fungal load in both treatment regimens (P >.05). Detection of Cryptococcus DNA (28S rRNA qPCR) in plasma or whole blood within the first 24 h of treatment was significantly associated with early mortality at w2 and mortality at w10 (P <.01). QSP1 RT-qPCR showed that detection of DNA was due to viable fungal cells as the quantification of QSP1 whole nucleic acids was systematically higher (X2 to 5) than that of DNA.ConclusionQuantification of C. neoformans and C. gattii load in CSF and plasma at D0 is useful in identifying patients at risk of death and may be a promising tool for monitoring treatment response in the future.

AcrAB-TolC Efflux Pump Mediated Resistance to Carbapenems among Clinical Isolates of Enterobacteriaceae

AcrAB-TolC is a resistance nodulation division type of efflux pump present in Enterobacteriaceae. It non-specifically effluxes antibiotics out of the bacterial cell, thus conferring drug resistance. Increase in the expression of the AcrAB-TolC efflux pump increases resistance to antibiotics. We aimed to study the expression levels of acrA and acrB that encodes AcrAB-TolC efflux pump, to understand efflux pump mediated resistance to carbapenem among clinical isolates of Enterobacteriaceae obtained from various clinical samples. Additionally, co -production of carbapenemase was also detected in the isolates demonstrating efflux pump mediated resistance to carbapenems. A total of 127 carbapenem resistant clinical isolates of Enterobacteriaceae, isolated from a tertiary care hospital were included in the study. An efflux pump inhibition (EPI) assay with reserpine, an efflux pump inhibitor, was performed to screen for isolates exhibiting efflux pump activity. Real Time Reverse Transcriptase qPCR was performed to detect the mRNA over expression levels of acrA and acrB that encodes AcrAB-TolC efflux pump. The control strains K. pneumoniae BAA2146 and E. coli AcrB were used. EPI assay with carbapenem showed that 56 /127(44%) isolates were screen positive indicating efflux pump activity. A total of 12 isolates showed 101 to 107 increase in the expression of both acrA and acrB when compared with the controls indicating a strong efflux pump activity, in addition to producing carbapenemase. The study highlights the role of efflux pump AcrAB-TolC in conferring resistance to carbapenem among clinical isolates of Enterobacteriaceae.

Multiplexed reverse-transcriptase quantitative polymerase chain reaction using plasmonic nanoparticles for point-of-care COVID-19 diagnosis.

Quantitative polymerase chain reaction (qPCR) offers the capabilities of real-time monitoring of amplified products, fast detection, and quantitation of infectious units, but poses technical hurdles for point-of-care miniaturization compared with end-point polymerase chain reaction. Here we demonstrate plasmonic thermocycling, in which rapid heating of the solution is achieved via infrared excitation of nanoparticles, successfully performing reverse-transcriptase qPCR (RT-qPCR) in a reaction vessel containing polymerase chain reaction chemistry, fluorescent probes and plasmonic nanoparticles. The method could rapidly detect SARS-CoV-2 RNA from human saliva and nasal specimens with 100% sensitivity and 100% specificity, as well as two distinct SARS-CoV-2 variants. The use of small optical components for both thermocycling and multiplexed fluorescence monitoring renders the instrument amenable to point-of-care use. Overall, this study demonstrates that plasmonic nanoparticles with compact optics can be used to achieve real-time and multiplexed RT-qPCR on clinical specimens, towards the goal of rapid and accurate molecular clinical diagnostics in decentralized settings.

Possible Antagonism between Cladosporium cladosporioides and Microcystis aeruginosa in a Freshwater Lake during Bloom Seasons.

To ensure drinking-water safety, it is necessary to understand the factors that regulate harmful cyanobacterial blooms (HCBs) and the toxins they produce. One controlling factor might be any relationship between fungi and the cyanobacteria. To test this possibility, water samples were obtained from Harsha Lake in southwestern Ohio during the 2015, 2016, and 2017 bloom seasons, i.e., late May through September. In each water sample, the concentration of the filamentous fungus Cladosporium cladosporioides was determined by quantitative PCR (qPCR) assay, and Microcystis aeruginosa microcystin-gene transcript copy number (McyG TCN) was quantified by reverse-transcriptase qPCR (RT-qPCR) analyses. The results showed that during each bloom season, the C. cladosporioides concentration and McyG TCN appeared to be interrelated. Therefore, C. cladosporioides concentrations were statistically evaluated via regression on McyG TCN in the water samples for lag times of 1 to 7 days. The regression equation developed to model the relationship demonstrated that a change in the C. cladosporioides concentration resulted in an opposing change in McyG TCN over an approximately 7-day interval. Although the interaction between C. cladosporioides and McyG TCN was observed in each bloom season, the magnitude of each component varied yearly. To better understand this possible interaction, outdoor Cladosporium spore-count data for the Harsha Lake region were obtained for late May through September of each year from the South West Ohio Air Quality Agency. The average Cladosporium spore count in the outdoor air samples was significantly greater in 2016 than in either 2015 or 2017, and the M. aeruginosa McyG TCN was significantly lower in Harsha Lake water samples in 2016 compared to 2015 or 2017. These results suggest that there might be a “balanced antagonism” between C. cladosporioides and M. aeruginosa during the bloom season.

The Effects of Sex on CB1 Expression in Male and Female Mice After Morphine Treatment

The endocannabinoid system (ECS) has recently gained interest as a promising therapeutic target for chronic pain treatment. An emerging body of research has revealed that the ECS interacts with the endogenous opioid system in a variety of complex processes. However, pain research has historically excluded female animals and failed to consider sex differences. This project aims to investigate whether morphine administration has a sexually dimorphic effect on type 1 cannabinoid receptors (CB1) in the ventral tegmental area (VTA) and amygdala of mice. These brain regions have been selected due to their role in both addiction and the emotional aspect of pain, which appear to be sexually dimorphic phenomena. Using reverse transcriptase qPCR, this study aims to determine (i) whether chronic morphine treatment impacts CB1 expression in the VTA and amygdala and (ii) whether this treatment differentially impacts male and female animals.

Orsellinic acid-loaded chitosan nanoparticles in gelatin/nanohydroxyapatite scaffolds for bone formation in vitro

AimOrsellinic acid (2,4-Dimethoxy-6-methylbenzoic acid) (OA) is a hydrophobic polyphenolic compound with therapeutic potential, but its impact on actuating osteogenesis remains unknown. The bioavailability of OA is hampered by its hydrophobic nature. This study aimed to fabricate nano-drug delivery system-based scaffolds for OA and test its potential for osteogenesis in vitro. Materials and methodsOA was loaded into chitosan nanoparticles (nCS + OA) using the ionic gelation technique at different concentrations. nCS + OA were incorporated onto the scaffolds containing gelatin (Gel) and nanohydroxyapatite (nHAp) by the lyophilization method. Biocomposite scaffolds were examined for their physicochemical and material characteristic properties. The effect of OA in the scaffolds for osteoblast differentiation was determined by alizarin red and von Kossa staining at the cellular level and by reverse transcriptase-qPCR and western blot analysis at the molecular level. Key findingsThe scaffolds showed excellent physiochemical and material characteristics and remained cyto-friendly to mouse mesenchymal stem cells (mMSCs, C3H10T1/2). The release of OA from Gel/nHAp/nCS scaffolds enhanced the differentiation of mMSCs towards osteoblasts, as observed through cellular and molecular studies. Moreover, the osteogenic potential of OA was mediated by the activation of FAK and ERK signaling pathways through integrins. SignificanceThe inclusion of OA into Gel/nHAp/nCS biocomposite scaffolds at 80 μM concentration promoted osteoblast differentiation via cell adhesion mediated signaling, compared with that shown by Gel/nHAp/nCS alone. Overall, this study identified the potential therapeutic OA containing Gel/nHAp/nCS scaffolds, accelerating its potential for clinical application towards bone regeneration.