Members Of Microbiota Research Articles

Clonal dissemination of Acinetobacter radioresistens among Humboldt penguins (Spheniscus humboldti) inhabiting a barren northern Peruvian island.

Acinetobacter spp. are often isolated from natural sources, but knowledge about their presence in wild animals is fragmented and uncomplete. The present study aimed to characterize a series of Acinetobacter radioresistens isolated from Humboldt penguins (Spheniscus humboldti). Fifteen Humboldt penguins from an inhabited northern Peruvian island were sampled. Microorganisms were identified byMALDI-TOF MS. Antibiotic susceptibility to 12 antimicrobial agents was established, and clonal relationships were determined. A representative isolate was selected for whole genome sequencing (WGS). A. radioresistens were isolated from the feces of 12 (80%) Humboldt penguins, being susceptible to all the antimicrobial agents tested, except eight cefotaxime-intermediate isolates. AllA.radioresistens were clonally related. WGS showed that the isolate belonged to ST1972, the presence of two chromosomal encoded carbapenemases (blaOXA-23 and a putative subclass B3 metallo-β-lactamase), and a series of point mutations in antibiotic-resistance related chromosomal genes, which were considered as polymorphisms. In addition, a few virulence factors, including acapsule-encoding operon, superoxide dismutases, catalases, phospholipases and a siderophore receptor were identified. The present results suggest that A. radioresistens may be a common member of the gut microbiota of Humboldt penguins, but further studies in other geographical areas are needed to establish this finding.

Association Between Altered Microbiota Composition and Immune System-Related Genes in COVID-19 Infection.

Microbiota and immunity affect the host's susceptibility to SARS-CoV-2 infection and the severity of COVID-19. This study aimed to identify significant alterations in themicrobiota composition, immune signaling pathways, their potential association, and candidate microRNA in COVID-19 patients using an in silico study model. Enrichment online databases and Python programming were utilized to analyze GSE164805, GSE180594, and GSE182279, as well as NGS data of microbiota composition (PRJNA650244 and PRJNA660302) associated with COVID-19, employing amplicon-based/marker gene sequencing methods. C1, TNF, C2, IL1, and CFH genes were found to have a significant impact on immune signaling pathways. Additionally, we observed a notable decrease in Bacteroides spp. and Faecalibacterium sp., while Escherichia coli, Streptococcus spp., and Akkermansia muciniphila showed increased abundance in COVID-19. Notably, A. muciniphila demonstrated an association with immunity through C1 and TNF, while Faecalibacterium sp. was linked to C2 and IL1. The correlation between E. coli and CFH, as well as IL1 and Streptococcus spp. with C2, was identified. hsa-let-7b-5p was identified as a potential candidate that may be involved in the interaction between the microbiotacomposition, immune response, and COVID-19. In conclusion, integrative in silico analysis shows that these microbiota members are potentially crucial in the immune responses against COVID-19.

Prevalence and characteristics of ESBL-producing Escherichia coli in clinically healthy pigs: implications for antibiotic resistance spread in livestock.

This study aimed to compare and characterize the resistance profile and the presence of extended-spectrum beta-lactamase (ESBL) related genes in Escherichia coli isolated from healthy finishing pigs fed with or without antibiotics in their diets. A total of 27 ceftiofur-resistant E. coli isolates were obtained from 96 healthy pigs. The antibiotic resistance profile was tested, and all 27 isolates were classified as multidrug-resistant (MDR). A high proportion of isolates were resistant to cephalosporins, ampicillin, ciprofloxacin, and tetracyclines. The ESBL production was observed in 85% of isolates by double-disc synergy test. The MDR-E. coli isolates harbored ESBL genes, such as blaTEM, blaCTX-M-1, blaCTX-M-2, and blaCTX-M-8,25. In addition, other antibiotics resistance genes (ARGs) were also detected, such as sul2, ant(3″)-I, tetA, and mcr-1. The mobilization of the blaCTX-M gene was confirmed for nine E. coli isolates by conjugation assays. The presence of blaCTX-M on mobile genetic elements in these isolates was demonstrated by Southern blot hybridization, and the resistance to cephalosporins was confirmed in the transconjugants. Our results indicate the prevalence of CTX-M-producing E. coli strains harboring mobile genetic elements in the normal microbiota of healthy pigs. These findings highlight the significance of ESBL genes as a global health concern in livestock and the potential spread of antimicrobial resistance to other members of the gastrointestinal tract microbiota.

Neohesperidin dihydrochalcone can improve intestinal structure and microflora composition of diabetic zebrafish

This study focused on Neohesperidin dihydrochalcone (NHDC) and explored its effects on the intestinal tissue structure, short-chain fatty acid production, gut microbiota, and intestinal tissue oxidative stress in a diabetic zebrafish model with insulin resistance. By analyzing the relationship between gut microbiota changes and glucose metabolism, it revealed the mechanism through which NHDC improves insulin resistance by modulating the gut microbiota. The results indicate that NHDC intervention has a protective effect on the intestinal barrier in diabetic zebrafish, improving lipid metabolism disturbances, and enhancing intestinal antioxidant capacity. Regarding community diversity, NHDC treatment increased the diversity and richness of the gut microbiota in diabetic zebrafish. In terms of gut microbiota composition, NHDC treatment reduced the abundance of the inflammation-associated Proteobacteria while increasing the relative abundance of the Fusobacteria, thereby maintaining the stability of the zebrafish gut microbiota. Furthermore, NHDC treatment also increased the abundance of the Prevotella, which is beneficial for increasing glycogen storage and inhibiting liver gluconeogenesis. Therefore, we speculate that NHDC may regulate the production of SCFAs and reduce the expression levels of pro-inflammatory cytokines by reducing the abundance of harmful bacteria such as Proteobacteria, Plesiomonas, and Aeromonas, while increasing the abundance of Faecalibacterium and Fusobacteria. The changes in the abundance of these microbiota members are involved in processes such as inflammation stress, glycogen storage, and gluconeogenesis, collectively promoting the improvement of insulin resistance in diabetic zebrafish by NHDC.

Incomplete immunity in a natural animal-microbiota interaction selects for higher pathogen virulence

Incomplete immunity in recovered hosts is predicted to favor more virulent pathogens upon re-infection in the population.1 The microbiota colonizing animals can generate a similarly long-lasting, partial immune response, allowing for infection but dampened disease severity.2 We tracked the evolutionary trajectories of a widespread pathogen (Pseudomonas aeruginosa), experimentally passaged through populations of nematodes immune-primed by a natural microbiota member (P.berkeleyensis). This bacterium can induce genes regulated by a mitogen-activated protein kinase (MAPK) signaling pathway effective at conferring protection against pathogen-induced death despite infection.3 Across host populations, this incomplete immunity selected for pathogens more than twice as likely to kill as those evolved in non-primed (i.e., naive) or immune-compromised (mutants with a knockout of the MAPK ortholog) control populations. Despite the higher virulence, pathogen molecular evolution in immune-primed hosts was slow and constrained. In comparison, evolving pathogens in immune-compromised hosts were characterized by substantial genomic differentiation and attenuated virulence. These findings directly attribute the incomplete host immunity induced from microbiota as a significant force shaping the virulence and evolutionary dynamics of novel infectious diseases.

Dual membrane-spanning anti-sigma factors regulate vesiculation in Bacteroides thetaiotaomicron

Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are mediated in part by Outer Membrane Vesicles (OMVs). Here, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in Bacteroides thetaiotaomicron (Bt). We identified a family of Dual membrane-spanning anti-sigma factors (Dma) that control OMV biogenesis. We conducted molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, modulates OMV production by controlling the activity of the ECF21 family sigma factor, Das1, and its downstream regulon. Dma1 has a previously uncharacterized domain organization that enables Dma1 to span both the inner and outer membrane of Bt. Phylogenetic analyses reveal that this common feature of the Dma family is restricted to the phylum Bacteroidota. This study provides mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.

Inter-species interactions between two bacterial flower commensals and a floral pathogen reduce disease incidence and alter pathogen activity.

Fire blight, caused by Erwinia amylovora, is one of the most important plant diseases of pome fruits. Previous work largely suggested plant microbiota commensals suppressed disease by antagonizing pathogen growth. However, inter-species interactions of multiple flower commensals and their influence on pathogen activity and behavior have not been well studied. Here, we show that co-inoculating two bacterial strains that naturally colonize the apple flowers reduces disease incidence. We further demonstrate that the interactions between these two microbiota commensals and the floral pathogen led to the emergence of new gene expression patterns and a strong alteration of the external pH, factors that may modify the pathogen's behavior. Our findings emphasize the critical role of emergent properties mediated by inter-species interactions between plant microbiota and plant pathogens and their impact on plant health.

Bacteroides thetaiotaomicron metabolic activity decreases with polysaccharide molecular weight.

Polysaccharides are complex molecules that are commonly found in our diet. While humans lack the ability to degrade many polysaccharides, their intestinal microbiota contain bacterial commensals that are versatile polysaccharide utilizers. The gut commensal Bacteroides thetaiotaomicron dedicates roughly 20% of their genomes to the expression of polysaccharide utilization loci for the broad range utilization of polysaccharides. Although it is known that different polysaccharide utilization loci are dedicated to the degradation of specific polysaccharides with unique glycosidic linkages and monosaccharide compositions, it is often overlooked that specific polysaccharides may also exist in various molecular weights. These different physical attributes may impact their processability by starch utilization system-like systems, leading to differing growth rates and nutrient-sharing properties at the community level. Therefore, understanding how molecular weight impacts utilization by gut microbe may lead to the potential design of novel precision prebiotics.

Christensenella minuta interacts with multiple gut bacteria.

Gut microbes form complex networks that significantly influence host health and disease treatment. Interventions with the probiotic bacteria on the gut microbiota have been demonstrated to improve host well-being. As a representative of next-generation probiotics, Christensenella minuta (C. minuta) plays a critical role in regulating energy balance and metabolic homeostasis in human bodies, showing potential in treating metabolic disorders and reducing inflammation. However, interactions of C. minuta with the members of the networked gut microbiota have rarely been explored. In this study, we investigated the impact of C. minuta on fecal microbiota via metagenomic sequencing, focusing on retrieving bacterial strains and coculture assays of C. minuta with associated microbial partners. Our results showed that C. minuta intervention significantly reduced the diversity of fecal microorganisms, but specifically enhanced some groups of bacteria, such as Lactobacillaceae. C. minuta selectively enriched bacterial pathways that compensated for its metabolic defects on vitamin B1, B12, serine, and glutamate synthesis. Meanwhile, C. minuta cross-feeds Faecalibacterium prausnitzii and other bacteria via the production of arginine, branched-chain amino acids, fumaric acids and short-chain fatty acids (SCFAs), such as acetic. Both metagenomic data analysis and culture experiments revealed that C. minuta negatively correlated with Klebsiella pneumoniae and 14 other bacterial taxa, while positively correlated with F. prausnitzii. Our results advance our comprehension of C. minuta's in modulating the gut microbial network. C. minuta disrupts the composition of the fecal microbiota. This disturbance is manifested through cross-feeding, nutritional competition, and supplementation of its own metabolic deficiencies, resulting in the specific enrichment or inhibition of the growth of certain bacteria. This study will shed light on the application of C. minuta as a probiotic for effective interventions on gut microbiomes and improvement of host health.

Insights into gut microbiomes in stem cell transplantation by comprehensive shotgun long-read sequencing

The gut microbiome is a diverse ecosystem, dominated by bacteria; however, fungi, phages/viruses, archaea, and protozoa are also important members of the gut microbiota. Exploration of taxonomic compositions beyond bacteria as well as an understanding of the interaction between the bacteriome with the other members is limited using 16S rDNA sequencing. Here, we developed a pipeline enabling the simultaneous interrogation of the gut microbiome (bacteriome, mycobiome, archaeome, eukaryome, DNA virome) and of antibiotic resistance genes based on optimized long-read shotgun metagenomics protocols and custom bioinformatics. Using our pipeline we investigated the longitudinal composition of the gut microbiome in an exploratory clinical study in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT; n = 31). Pre-transplantation microbiomes exhibited a 3-cluster structure, characterized by Bacteroides spp. /Phocaeicola spp., mixed composition and Enterococcus abundances. We revealed substantial inter-individual and temporal variabilities of microbial domain compositions, human DNA, and antibiotic resistance genes during the course of alloHSCT. Interestingly, viruses and fungi accounted for substantial proportions of microbiome content in individual samples. In the course of HSCT, bacterial strains were stable or newly acquired. Our results demonstrate the disruptive potential of alloHSCTon the gut microbiome and pave the way for future comprehensive microbiome studies based on long-read metagenomics.

Divergent maturational patterns of the infant bacterial and fungal gut microbiome in the first year of life are associated with inter-kingdom community dynamics and infant nutrition

BackgroundThe gut microbiome undergoes primary ecological succession over the course of early life before achieving ecosystem stability around 3 years of age. These maturational patterns have been well-characterized for bacteria, but limited descriptions exist for other microbiota members, such as fungi. Further, our current understanding of the prevalence of different patterns of bacterial and fungal microbiome maturation and how inter-kingdom dynamics influence early-life microbiome establishment is limited.ResultsWe examined individual shifts in bacterial and fungal alpha diversity from 3 to 12 months of age in 100 infants from the CHILD Cohort Study. We identified divergent patterns of gut bacterial or fungal microbiome maturation in over 40% of infants, which were characterized by differences in community composition, inter-kingdom dynamics, and microbe-derived metabolites in urine, suggestive of alterations in the timing of ecosystem transitions. Known microbiome-modifying factors, such as formula feeding and delivery by C-section, were associated with atypical bacterial, but not fungal, microbiome maturation patterns. Instead, fungal microbiome maturation was influenced by prenatal exposure to artificially sweetened beverages and the bacterial microbiome, emphasizing the importance of inter-kingdom dynamics in early-life colonization patterns.ConclusionsThese findings highlight the ecological and environmental factors underlying atypical patterns of microbiome maturation in infants, and the need to incorporate multi-kingdom and individual-level perspectives in microbiome research to improve our understandings of gut microbiome maturation patterns in early life and how they relate to host health.5AtmDGbifCkZAT-p1B-BuFVideo

OPTIMIZATION OF EXPERIMENTAL MODEL SYSTEMS FOR EVALUATING RECIPROCAL INFLUENCE OF BIFIDOBACTERIUM ANIMALIS AND HUMAN BREAST CANCER CELLS IN VITRO.

The development of human breast cancer (BC) is known to be closely related to disturbances in the mammary gland microbiota. Bacteria of the genus Bifidobacterium are an important component of normal breast microbiota and exert antitumor activity. The molecular-biological mechanisms of interaction between BC cells and microbiota members remain poorly studied yet. The aim of this study was to develop and optimize an experimental model system for the co-cultivation of BC cells with Bifidobacterium animalis in vitro. Human ВС cells of the MCF-7, T47D, and MDA-MB-231 lines, as well as live and heat-inactivated bacteria of Bifidobacterium animalis subsp. lactis (B. animalis) were used as research objects. The growth kinetics and viability of B. animalis in the presence of different ВС cell lines and without them were determined by both the turbidimetry method and seeding on an elective nutrient medium. Glucose consumption and lactate production by bifidobacteria were assessed by biochemical methods. The viability of BC cells was determined by a standard colorimetric method. The growth kinetics of B. animalis in the complete DMEM nutrient medium showed standard patterns. The indicators of glucose consumption and lactate production of B. animalis confirm its physiological metabolic activity under the growth conditions. The presence of BC cells in the model system did not affect the duration of the growth phases of the B. animalis cells' population but contributed to the increase in their counts. A significant decrease in the number of live BC cells of all studied lines was observed only after 48 h of co-cultivation with live B. animalis. To achieve similar suppression of the BC cell viability, 10-30-fold higher counts of heatinactivated bacteria were required compared to live ones. The optimal conditions for co-cultivation of human BC cells and living B. animalis cells in vitro have been identified.

Bifidobacterium species viability in dairy-based probiotic foods: challenges and innovative approaches for accurate viability determination and monitoring of probiotic functionality.

Bifidobacterium species are essential members of a healthy human gut microbiota. Their presence in the gut is associated with numerous health outcomes such as protection against gastrointestinal tract infections, inflammation, and metabolic diseases. Regular intake of Bifidobacterium in foods is a sustainable way of maintaining the health benefits associated with its use as a probiotic. Owing to their global acceptance, fermented dairy products (particularly yogurt) are considered the ideal probiotic carrier foods. As envisioned in the definition of probiotics as "live organisms," the therapeutic functionalities of Bifidobacterium spp. depend on maintaining their viability in the foods up to the point of consumption. However, sustaining Bifidobacterium spp. viability during the manufacture and shelf-life of fermented dairy products remains challenging. Hence, this paper discusses the significance of viability as a prerequisite for Bifidobacterium spp. probiotic functionality. The paper focuses on the stress factors that influence Bifidobacterium spp. viability during the manufacture and shelf life of yogurt as an archetypical fermented dairy product that is widely accepted as a delivery vehicle for probiotics. It further expounds the Bifidobacterium spp. physiological and genetic stress response mechanisms as well as the methods for viability retention in yogurt, such as microencapsulation, use of oxygen scavenging lactic acid bacterial strains, and stress-protective agents. The report also explores the topic of viability determination as a critical factor in probiotic quality assurance, wherein, the limitations of culture-based enumeration methods, the challenges of species and strain resolution in the presence of lactic acid bacterial starter and probiotic species are discussed. Finally, new developments and potential applications of next-generation viability determination methods such as flow cytometry, propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR), next-generation sequencing, and single-cell Raman spectroscopy (SCRS) methods are examined.

Interactions Between Oral Microbiota and Cancers in the Aging Community: A Narrative Review.

The oral microbiome potentially wields significant influence in the development of cancer. Within the human oral cavity, an impressive diversity of more than 700 bacterial species resides, making it the second most varied microbiome in the body. This finely balanced oral microbiome ecosystem is vital for sustaining oral health. However, disruptions in this equilibrium, often brought about by dietary habits and inadequate oral hygiene, can result in various oral ailments like periodontitis, cavities, gingivitis, and even oral cancer. There is compelling evidence that the oral microbiome is linked to several types of cancer, including oral, pancreatic, colorectal, lung, gastric, and head and neck cancers. This review discussed the critical connections between cancer and members of the human oral microbiota. Extensive searches were conducted across the Web of Science, Scopus, and PubMed databases to provide an up-to-date overview of our understanding of the oral microbiota's role in various human cancers. By understanding the possible microbial origins of carcinogenesis, healthcare professionals can diagnose neoplastic diseases earlier and design treatments accordingly.

Recent advances in applying omic technologies for studying acetic acid bacteria in industrial vinegar production: A comprehensive review.

Vinegar and related bioproducts containing acetic acid as the main component are among the most appreciated fermented foodstuffs in numerous European and Asian countries because of their exceptional organoleptic and bio-healthy properties. Regarding the acetification process and obtaining of final products, there is still a lack of knowledge on fundamental aspects, especially those related to the study of biodiversity and metabolism of the present microbiota. In this context, omic technologies currently allow for the massive analysis of macromolecules and metabolites for the identification and characterization of these microorganisms working in their natural media without the need for isolation. This review approaches comprehensive research on the application of omic tools for the identification of vinegar microbiota, mainly acetic acid bacteria, with subsequent emphasis on the study of the microbial diversity, behavior, and key molecular strategies used by the predominant groups throughout acetification. The current omics tools are enabling both the finding of new vinegar microbiota members and exploring underlying strategies during the elaboration process. The species Komagataeibacter europaeus may be a model organism for present and future research in this industry; moreover, the development of integrated meta-omic analysis may facilitate the achievement of numerous of the proposed milestones. This work might provide useful guidance for the vinegar industry establishing the first steps towards the improvement of the acetification conditions and the development of new products with sensory and bio-healthy profiles adapted to the agri-food market.

Ecology- and genome-based identification of the Bifidobacterium adolescentis prototype of the healthy human gut microbiota.

Bifidobacteria are among the first microbial colonizers of the human gut, being frequently associated with human health-promoting activities. In the current study, an in silico methodology based on an ecological and phylogenomic-driven approach allowed the selection of a Bifidobacterium adolescentis prototype strain, i.e., B. adolescentis PRL2023, which best represents the overall genetic content and functional features of the B. adolescentis taxon. Such features were confirmed by in vitro experiments aimed at evaluating the ability of this strain to survive in the gastrointestinal tract of the host and its ability to interact with human intestinal cells and other microbial gut commensals. In this context, co-cultivation of B. adolescentis PRL2023 and several gut commensals revealed various microbe-microbe interactions and indicated co-metabolism of particular plant-derived glycans, such as xylan.IMPORTANCEThe use of appropriate bacterial strains in experimental research becomes imperative in order to investigate bacterial behavior while mimicking the natural environment. In the current study, through in silico and in vitro methodologies, we were able to identify the most representative strain of the Bifidobacterium adolescentis species. The ability of this strain, B. adolescentis PRL2023, to cope with the environmental challenges imposed by the gastrointestinal tract, together with its ability to switch its carbohydrate metabolism to compete with other gut microorganisms, makes it an ideal choice as a B. adolescentis prototype and a member of the healthy microbiota of adults. This strain possesses a genetic blueprint appropriate for its exploitation as a candidate for next-generation probiotics.

CRISPR-based screening of small RNA modulators of bile susceptibility in Bacteroides thetaiotaomicron

Microbiota-centric interventions are limited by our incomplete understanding of the gene functions of many of its constituent species. This applies in particular to small RNAs (sRNAs), which are emerging as important regulators in microbiota species yet tend to be missed by traditional functional genomics approaches. Here, we establish CRISPR interference (CRISPRi) in the abundant microbiota member Bacteroides thetaiotaomicron for genome-wide sRNA screens. By assessing the abundance of different protospacer-adjacent motifs, we identify the Prevotella bryantii B14 Cas12a as a suitable nuclease for CRISPR screens in these bacteria and generate an inducible Cas12a expression system. Using a luciferase reporter strain, we infer guide design rules and use this knowledge to assemble a computational pipeline for automated gRNA design. By subjecting the resulting guide library to a phenotypic screen, we uncover the sRNA BatR to increase susceptibility to bile salts through the regulation of genes involved in Bacteroides cell surface structure. Our study lays the groundwork for unlocking the genetic potential of these major human gut mutualists and, more generally, for identifying hidden functions of bacterial sRNAs.

Protective effect of Bifidobacterium animalis CGMCC25262 on HaCaT keratinocytes.

Bifidobacteria are the most prevalent members of the intestinal microbiota in mammals and other animals, and they play a significant role in promoting gut health through their probiotic effects. Recently, the potential applications of Bifidobacteria have been extended to skin health. However, the beneficial mechanism of Bifidobacteria on the skin barrier remains unclear. In this study, keratinocyte HaCaT cells were used as models to evaluate the protective effects of the cell-free supernatant (CFS), heat-inactivated bacteria, and bacterial lysate of Bifidobacterium animalis CGMCC25262 on the skin barrier and inflammatory cytokines. The results showed that all the tested samples were able to upregulate the transcription levels of biomarker genes associated with the skin barrier, such as hyaluronic acid synthetase (HAS) and aquaporins (AQPs). Notably, the transcription of the hyaluronic acid synthetase gene-2 (HAS-2) is upregulated by 3~4 times, and AQP3 increased by 2.5 times when the keratinocyte HaCaT cells were co-incubated with 0.8 to 1% CFS. In particular, the expression level of Filaggrin (FLG) in HaCaT cells increased by 1.7 to 2.7 times when incubated with Bifidobacterial samples, reaching its peak at a concentration of 0.8% CFS. Moreover, B. animalis CGMCC25262 also decreased the expression of the proinflammatory cytokine RANTES to one-tenth compared to the levels observed in HaCaT cells induced with tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). These results demonstrate the potential of B. animalis CGMCC25262 in protecting the skin barrier and reducing inflammatory response.

A Microcosm Experiment Reveals the Temperature-Sensitive Release of Mucochytrium quahogii (=QPX) from Hard Clams and Pallial Fluid as a Stable QPX Reservoir.

Mucochytrium quahogii, also known as QPX or Quahog Parasite Unknown, is the causative agent of QPX disease in the hard clam (Mercenaria mercenaria). Host-pathogen-environment interactions between M. quahogii, the hard clam, and temperature were explored in a microcosm experiment. Hard clams were housed in individual tanks with sterile seawater under two temperature regimes: low (13 °C) temperature, which is thought to be optimal for QPX disease development, and high (20 °C) temperature, which has been shown to promote "healing" of QPX-infected clams. Hard clam tissue, pallial fluid, seawater, and shell biofilms were collected and assayed for M. quahogii. The release of M. quahogii from naturally infected live hard clams into seawater was detected only in the low temperature treatment, suggesting that temperature influences the release of potentially infectious cells. M. quahogii was commonly found in hard clam pallial fluid, even after 9 weeks in the lab, suggesting pallial fluid is a stable reservoir of M. quahogii within its primary host and that M. quahogii is not a transient component of the hard clam microbiota. Overall, results support a host-specific relationship and that M. quahogii is a commensal member of the hard clam microbiota, supporting its classification as an opportunistic pathogen.

Molecular identification of Streptococcus mutans and Streptococcus sobrinus from un-stimulated saliva and their association with dental caries and orthodontic appliances

Background: Oral streptococci, particularly Streptococcus mutans (S. mutans), have been associated with several diseases affecting multiple anatomical sites. Streptococcus mutans and Streptococcus sobrinus (S. sobrinus), which belong to the Mutans streptococci group, are examples of Gram-positive bacteria that demonstrate facultative anaerobic growth characteristics. These bacteria are frequently seen as members of the native oral microbiota and are largely recognized as the primary causative agents of dental caries. Objective: To molecularly identify S. mutans and S. sobrinus using PCR and investigate their relationship with the caries status and orthodontia appliances. Method: The cross-sectional study, which was conducted in Baghdad from February 2021 to November 2022, involved 359 un-stimulated saliva samples from 340 participants were collected and processed immediately by culturing anaerobically (37°C/72 h) on Mitis Salivarius Bacitracin Agar (MSB-Agar). Morphological characteristics of the colonies, Gram stain were achieved for the bacterial growth. DNA extracted from cultured bacteria. S. mutans and S. sobrinus were identified molecularly by amplifying gtfB and gtfI, respectively, from DNA samples using conventional PCR. Results: From 279/ 336 (83.04%) bacterial DNA samples, 118/279 (42.29%) were positive for S. mutans gtfB and/or S. sobrinus gtfI; S. mutans 84/118 (71.2%), S. sobrinus 6/118 (5.1%) and mixed S. mutans/S. sobrinus 28/118 (23.7%). The results of association between molecular identification of S. mutans with dental caries and missing teeth features of individuals were statically not significant (0.068 and 0.323 > 0.05, respectively), while was significant with filling teeth and orthodontic appliance (0.020, 0.027 < 0.05, respectively). The relationship between molecular identification of S. sobrinus and dental caries, missing, filling teeth and orthodontic were statistically not significant (0.069, 0.975, 0.845 and 0.458 > 0.05), respectively. The relationship between molecular identification of mixed S. mutans/S. sobrinus with dental caries was statically significant (0.042 < 0.05). Conclusion: S. mutans was more prevalence; identification and discrimination for S. mutans/S. sobrinus (depending on gtfB and gtfI) by PCR was more efficacy. Mixed S. mutans/S. sobrinus in a reasonable percentage were related with the hard dental caries status. Identification by colony morphology alone was not achievable in this study. Mitis Salivarius agar with Bacitracin (MSB) is extremely selective for S. mutans and S. sobrinus but cannot distinguish them morphologically.