Acinetobacter baumannii as an oro-dental pathogen: a red alert!!

OBJECTIVES: This review highlights the existence and association of Acinetobacter baumannii with the oro-dental diseases, transforming this systemic pathogen into an oral pathogen. The review also hypothesizes possible reasons for the categorization of this pathogen as code blue due to its stealthy entry into the oral cavity. METHODOLOGY: Study data were retrieved from various search engines reporting specifically on the association of A. baumannii in dental diseases and tray set-ups. Articles were also examined regarding obtained outcomes on A. baumannii biofilm formation, iron acquisitions, magnitude of antimicrobial resistance, and its role in the oral cancers. RESULTS: A. baumannii is associated with the oro-dental diseases and various virulence factors attribute for the establishment and progression of oro-mucosal infections. Its presence in the oral cavity is frequent in oral microbiomes, conditions of impaired host immunity, age related illnesses, and hospitalized individuals. Many sources also contribute for its prevalence in the dental health care environment and the presence of drug resistant traits is also observed. Its association with oral cancers and oral squamous cell carcinoma is also evident. CONCLUSIONS: The review calls for awareness on the emergence of A. baumannii in dental clinics and for the need for educational programs to monitor and control the sudden outbreaks of such virulent and resistant traits in the dental health care settings.

Genetic Association of Toll-Like Receptor 4 (TLR4) Gene Polymorphism (rs4986790) With Oral Squamous Cell Carcinoma (OSCC): A Pilot Case-Control Study.

Introduction Oral squamous cell carcinoma (OSCC) is a highly prevalent and most common form of oral malignancy in the Indian population. Toll-like receptors belong to an important family of receptors that are involved in the process of pathogen recognition and mounting immune response. The expression of this receptor is dysregulated on the tumor cells as reported across several cancer types. The genetic variants in this gene could have a profound impact on the expression of the Toll-like receptor 4 (TLR4) gene.  Objective This study aimed to understand the association of TLR4 gene polymorphism (rs4986790) with OSCC. The objective of this study was to compare the allele and genotype frequencies between the two groups, viz., OSCC and normal healthy subjects, recruited in the study. Materials and methods The blood samples were collected from normal healthy subjects (N = 25) and OSCC patients (N = 25). Genomic DNA was isolated from all samples, and genotyping was performed for the TLR4 gene polymorphism (rs4986790) employing the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach. The frequency distribution of genotypes and alleles across the study groups was determined by the Chi-square test.  Results The allele frequency for TLR4 gene polymorphism (rs4986790) in the case group was found to be 60% (A allele) and 40% (G allele), respectively. The study population in both groups were found to agree with the Hardy-Weinberg equilibrium (HWE). The genotype frequency did not differ significantly among the two study groups which was evident from the p-value = 0.8285.  Conclusion The present study did not report any significant association of the TLR4 polymorphic marker rs4986790 with OSCC. Further investigations into the association of other polymorphic markers in the TLR4 gene, among the larger population of OSCC patients, could provide evidence of their association with OSCC.

Detection of the early putative biofilm marker pgaB among the MDR strains of A.baumannii.

Background: pgaB associated biofilm formation in Acinetobacter baumannii enhances the virulence in concert with the high propensity of drug resistance. This research is thus aimed to characterize the pgaB gene molecularly and to examine its co-occurrence with MDR. Methodology: MDR strains of A. baumannii (N = 73) were selected to detect the formation of biofilms. Genomic DNA was extracted further and screened for pgaB followed by amplicon sequencing from the representative strains. Frequency of its distribution in different groups of drug resistant strains at a significant p-value of <0.05 was further checked. Results: The biofilm assay showed high, low and negative biofilm formers in 58.9%, 31.5% and 0.9% of the strains respectively. The pgaB gene was detected in 14 strains of MDR A. baumannii (19.17%). Co-occurrence of pgaB gene was seen in all the strains that showed resistance to ß-lactam inhibitors, cephems, carbapenems, fluoroquinolones and folates followed by 96% for the aminoglycosides and 25% in the efflux pump groups. Conclusion: The study findings showed the occurrence of biofilms associated with pgaB in MDR A. baumannii strains. The results also suggest to track its role in varying the pattern of drug resistance with further experimentation.

Molecular Characterization of the glyA Gene From the Clinical Isolates of Tannerella forsythia.

BACKGROUND: The glyA gene in Tannerella forsythia is attributed for its virulence by producing the enzyme serine hydroxymethyltransferase (SHMT), which plays a vital role in bacterial cell metabolism. OBJECTIVES: The study is thus aimed to determine the frequency of the glyA gene from the clinical strains of T. forsythia isolated from periodontitis patients. MATERIALS AND METHODS: Forty-five patients with varying degrees of periodontitis were included in the study, and the plaque samples collected from them were anaerobically processed by inoculating onto sterile anaerobic blood agar plates using a gaspak system, with incubation at 37°C for 5-7 days. The DNA was extracted from the obtained isolated colony, and PCR was performed to confirm the presence of the glyA gene. RESULTS: In total, 46.6% (n = 7) of the cases in group III aggressive periodontitis (n = 15) and 6.66% (n = 1) in group II stage II periodontitis (n = 15) showed the presence of T. forsythia, and among them, 57.14% (n = 4) showed the presence of the glyA gene.  Conclusion: The findings of the study showed that the glyA gene may be associated with the pathogenesis of T. forsythia and could be thus a novel candidate for the future theragnostic approach to combat periodontitis.

Molecular Detection of Secreted Aspartyl Proteinases (Saps) From Dental Isolates of Candida albicans and Targeting With Psidium guajava Biocompounds: An In Vitro and In Silico Analysis.

Introduction Candida albicans (C. albicans) is an opportunistic yeast-like fungus and is considered a functional biome of the oral and gut microbiomes. The sap gene and its types play a vital role in the pathogenesis of C. albicans. The emergence of resistance traits is a major problem, and targeting the same with alternative medicines has sparked renewed interest in recent years. Objectives This study is thus aimed at detecting the frequency of sap gene types in the clinical isolates of C. albicans and evaluating the antifungal effect of the crude methanolic extract of Psidium guajava (P. guajava). Further in silico assessments will assess the inhibitory effect of six compounds of P. guajava against the Sap protein. Materials and methods C. albicans was characterized phenotypically in 20 patients with root caries, and the sap gene was detected by PCR. The crude methanolic extract was prepared, and its antifungal efficacy was evaluated by the agar well diffusion method. Auto-docking was performed to assess the best compound based on the docking and overall interactions. Results Six isolates were identified as C. albicans and sap gene types 1-3 were detected in the four strains. P. guajava methanolic extracts showed a promising antifungal effect at varying concentrations. In silico analysis showed myricetin possessing the maximum number of hydrogen bonds and high docking energy with one violation. Conclusion The study concludes that P. guajava has a promising inhibitory effect against C. albicans with myricetin as the best compound to target the sap gene of C. albicans. However, further experimental studies are to be considered for its effectiveness in treating the infections caused by C. albicans.

Prediction of Antigenic Vaccine Peptide Candidates From BfmRS Associated With Biofilm Formation in Acinetobacter baumannii.

INTRODUCTION:  A. baumannii is categorized as a priority pathogen due to its propensity for multi-drug resistance, exhibiting resistance against the last resort of antibiotics. It is also considered a potent nosocomial pathogen, so targeting the microbe using novel strategies would be the need of the hour. In this context, the in-silico computational approach would serve the best to design the possible epitope peptides, which may be further considered for the experimental trials for their immunological response.  Objective: To predict the immune-dominant epitope peptide candidates against the bfmR and bfmS proteins mediating the two-component system adaptation in the formation of biofilm in A. baumannii. MATERIALS AND METHODS: 11 different FASTA sequences of bfmR and bfmS from A. baumannii strains retrieved based on the blast-p similarity search tool were subjected to linear epitope B-cell epitope predictions under the IEDB B-cell epitope prediction server. Further analysis on antigenicity, allergenicity, and toxigenicity was achieved using the AntigenPro, Vaxijen, and AlgPred tools, with the physical and chemical properties evaluated using the Expasy Protparam server. Selection of the immunodominant peptides for T-cells was done through the databases under IEDB. The final assessment of protein-TLR2 interactions was done by MHC cluster servers. RESULTS: Four peptide sequences (E1-E4) were predicted for B-cell dominance, with E1, E2, and E4 as probable antigens. All were soluble and non-toxigenic. E1 and E3 were considered non-allergens. GRAVY values were negative for all the peptides, indicating the protein to be hydrophilic in nature. Analysis of the T-cell epitopes was promising, with 100% conservancy for class-I HLA alleles, high interaction scores for similarity with TLR2, and more hydrogen bonds for E2, followed by other epitope peptides. CONCLUSION: The promising four epitopes, as predicted for bfmR and bfmS in the present study, suggest their potent role as possible candidates for the design of vaccines targeting the TCS of A. baumannii, recommending further in vitro and in vivo experimental validation.

Prediction of Putative Epitope Peptides against BaeR Associated with TCS Adaptation in Acinetobacter baumannii Using an In Silico Approach.

Background and Objectives: The BaeR protein is involved in the adaptation system of A. baumannii and is associated with virulence factors responsible for systemic infections in hospitalized patients. This study was conducted to characterize putative epitope peptides for the design of vaccines against BaeR protein, using an immune-informatic approach. Materials and Methods: FASTA sequences of BaeR from five different strains of A. baumannii were retrieved from the UNIPROT database and evaluated for their antigenicity, allergenicity and vaccine properties using BepiPred, Vaxijen, AlgPred, AntigenPro and SolPro. Their physio-chemical properties were assessed using the Expasy Protparam server. Immuno-dominant B-cell and T-cell epitope peptides were predicted using the IEDB database and MHC cluster server with a final assessment of their interactions with TLR-2. Results: A final selection of two peptide sequences (36aa and 22aa) was made from the 38 antigenic peptides. E1 was considered a soluble, non-allergenic antigen, and possessed negative GRAVY values, substantiating the hydrophilic nature of the proteins. Further analysis on the T-cell epitopes, class I immunogenicity and HLA allele frequencies yielded T-cell immuno-dominant peptides. The protein-peptide interactions of the TLR-2 receptor showed good similarity scores in terms of the high number of hydrogen bonds compared to other protein-peptide interactions. Conclusions: The two epitopes predicted from BaeR in the present investigation are promising vaccine candidates for targeting the TCS of A. baumannii in systemic and nosocomial infections. This study also demonstrates an alternative strategy to tackling and mitigating MDR strains of A. baumannii and provides a useful reference for the design and construction of novel vaccine candidates against this bacteria.

Functional biomes beyond the bacteriome in the oral ecosystem.

Selective constraint and pressures upon the host tissues often signifies a beneficial microbiome in any species. In the context of oral microbiome this displays a healthy microbial cosmos resisting the colonization and helps in rendering protection. This review highlights the endeavors of the oral microbiome beyond the bacteriome encompassing virome, mycobiome, protozoa and archaeomes in maintaining the oral homeostasis in health and disease. Scientific data based on the peer-reviewed publications on the microbial communities of the oral microbiome were selected and collated from the scientific database collection sites of web of science (WOS), pubmed central, Inspec etc., from 2010 to 2021 using the search key words like oral microbiome, oral microbiota, oral virome, oral bacteriome, oral mycobiome and oral archaeome. Data excluded were from conference proceedings, abstracts and book chapters. The oral homeostasis in both the health and disease conditions, mostly is balanced by the unrevealed virome, mycobiome, oral protozoa and archaeome. The review documents the need to comprehend the diversity that prevails among the kingdoms in order to determine the specific role played by each domain. Oral microbiome is also a novel research arena to develop drug and targeted therapies to treat various oro-dental infections.

Biofilm-Associated Agr and Sar Quorum Sensing Systems of Staphylococcus aureus Are Inhibited by 3-Hydroxybenzoic Acid Derived from Illicium verum.

Biofilm-producing Staphylococcus aureus (S. aureus) is less sensitive to conventional antibiotics than free-living planktonic cells. Here, we evaluated the antibiofilm activity of Illicium verum (I. verum) and one of its constituent compounds 3-hydroxybenzoic acid (3-HBA) against multi-drug-resistant S. aureus. We performed gas chromatography-mass spectroscopy (GC-MS) to identify the major constituents in the methanolic extract of I. verum. Ligand-receptor interactions were studied by molecular docking, and in vitro investigations were performed using crystal violet assay, spreading assay, hemolysis, proteolytic activity, and growth curve analysis. The methanolic extract of I. verum inhibited S. aureus at 4.8 mg/mL, and GC-MS analysis revealed anethole, m-methoxybenzaldehyde, and 3-HBA as the major constituents. Molecular docking attributed the antibiofilm activity to an active ligand present in 3-HBA, which strongly interacted with the active site residues of AgrA and SarA of S. aureus. At a subinhibitory concentration of 2.4 mg/mL, the extract showed biofilm inhibition. Similarly, 3-HBA inhibited biofilm activity at 25 µg/mL (90.34%), 12.5 µg/mL (77.21%), and 6.25 µg/mL (62.69%) concentrations. Marked attrition in bacterial spreading was observed at 2.4 mg/mL (crude extract) and 25 µg/mL (3-HBA) concentrations. The methanol extract of I. verum and 3-HBA markedly inhibited ß-hemolytic and proteolytic activities of S. aureus. At the lowest concentration, the I. verum extract (2.4 mg/mL) and 3-HBA (25 µg/mL) did not inhibit bacterial growth. Optical microscopy and SEM analysis confirmed that I. verum and 3-HBA significantly reduced biofilm dispersion without disturbing bacterial growth. Together, we found that the antibiofilm activity of I. verum and 3-HBA strongly targeted the Agr and Sar systems of S. aureus.

Probiotic curd as antibacterial agent against pathogens causing oral deformities - in vitro microbiological study.

Major oral deformities fall under dental caries and periodontal diseases hence active prevention of these two diseases can contribute to good oral health and preserves oral hygiene. Streptococcus mutans, Staphylococcus aureus, Lactobacillus, Candida, and anaerobic organisms are the organisms responsible for causing oral cavity-related deformities. Probiotics which is the useful and nonpathogenic bacteria are added to food products which tend to be advantageous to human health. A wide range of studies indicates that these probiotics are useful against oral tissues. Hence, the primary goal of the study aims to determine the antibacterial potential of probiotic curd against pathogens causing various oral diseases and deformities. A laboratory-oriented in vitro microbiological study design was framed to detect the antimicrobial potential of the probiotic curd. Subgingival calculus specimens were collected and anaerobic organisms were isolated in thioglycollate broth. Lawn cultures were subjected to the surface of brain heart infusion agar and 100 µl of probiotic curd, normal curd, and filtrate were taken in a micropipette and inoculated over the specific wells. The culture plates were incubated anaerobically at 37°C for 24 h. The culture plates were monitored for the zone of inhibition to assess the antibacterial activity against the test pathogens. The results showed that there was no antibacterial activity against the anaerobic bacteria cultivated from subgingival calculus. However, further validation must be done on the same with purified components from the probiotic curd. Probiotic curd is normally considered a vital immune-boosting nutritional supplement. However, the antibacterial activity must be evaluated with care with the purified filtrates of the curd to substantiate its exact role against dental pathogens.

Delineating the potential targets of thymoquinone in ESKAPE pathogens using a computational approach.

The present study was designed to identify and analyze the targets of thymoquinone on drug resistant pathogens employing in silico tools. The target identification was performed using STITCH tool, followed by the functional analysis of protein targets by VICMPred. Further, VirulentPred was used to determine the nature of virulence of target proteins. The putative epitopes present on the virulent proteins were identified using BepiPred tool. The subcellular location of the virulent proteins was assessed using PSORTb. The results showed multiple targets of the pathogens being targeted. The nitric-oxide synthase-like protein of Staphylococcus aureus and acetyltransferase family protein, histone acetyltransferase HPA2, GNAT family acetyltransferase of Acinetobacter baumannii was found to be the virulent proteins interacting with thymoquinone. Molinspiration assessments showed zero violations suggesting the druggability of TQ. The study unveils the molecular mechanisms underlying the antimicrobial effect of thymoquinone as demonstrated by in silico procedures.

Hijacking of the Host's Immune Surveillance Radars by Burkholderia pseudomallei.

Burkholderia pseudomallei (B. pseudomallei) causes melioidosis, a potentially fatal disease for which no licensed vaccine is available thus far. The host-pathogen interactions in B. pseudomallei infection largely remain the tip of the iceberg. The pathological manifestations are protean ranging from acute to chronic involving one or more visceral organs leading to septic shock, especially in individuals with underlying conditions similar to COVID-19. Pathogenesis is attributed to the intracellular ability of the bacterium to 'step into' the host cell's cytoplasm from the endocytotic vacuole, where it appears to polymerize actin filaments to spread across cells in the closer vicinity. B. pseudomallei effectively evades the host's surveillance armory to remain latent for prolonged duration also causing relapses despite antimicrobial therapy. Therefore, eradication of intracellular B. pseudomallei is highly dependent on robust cellular immune responses. However, it remains ambiguous why certain individuals in endemic areas experience asymptomatic seroconversion, whereas others succumb to sepsis-associated sequelae. Here, we propose key insights on how the host's surveillance radars get commandeered by B. pseudomallei.

Molecular characterization and epitope-based vaccine predictions for ompA gene associated with biofilm formation in multidrug-resistant strains of A.baumannii.

The present study was conducted to molecularly characterize the biofilm associated ompA gene from the drug resistant strains of A. baumannii and its immuno-dominant vaccine epitope predictions through immuno-informatic approach. ompA was amplified by PCR from the genomic DNA and was sequenced. Using the ORF, ompA protein sequence was retrieved and was subjected for IEDB T cell and B cell epitope analysis for the selection of the epitope peptides. Selected peptides were evaluated using appropriate servers and tools to assess the propensity for its antigenicity, solubility, physico-chemical property, toxigenicity and class-I immunogenicity. MHC class I and II restriction of HLA alleles was also performed. 48% (n = 24) of the strains possessed ompA gene. Protein structure was successfully retrieved with the selection of two epitopes viz., E1- FDGVNRGTRGTSEEGTLGNA and E2-KLSEYPNATARIEGHTDNTGPRKL. Final docking with TLR-2, showed E2 as the best epitope candidate predicted with the highest number of hydrogen bonds.

Decoding The Genetic Alterations In Cytochrome P450 Family 3 Genes And Its Association With HNSCC.

INTRODUCTION: Cytochrome P450 (CYPs) are enzymes belonging to the family of heme-containing proteins, most commonly found in the endoplasmic reticulum and mitochondria. These enzymes catalyze a variety of functions including metabolism of steroids, fatty acids, natural compounds, drugs and carcinogenic chemicals. The inherent association of CYPs with disease conditions have turned the focus into the genetic alterations or variations associated with phenotypes such as drug responsiveness, chemical toxicity and bioconversion of procarcinogens to active carcinogens. RESULTS: A total of 8 genes of the CYP3 family were analyzed, among which 4 genes were found to harbour gross abnormalities and variations. The genes CYP3A4, CYP3A5, CYP3A7, CYP3A43 showed a common pattern of gene amplification in a group of patients. Truncating and missense variants were also identified of which rs199908125 of CYP3A4 and rs768530577 of CYP3A5 were reported in different populations. MATERIALS AND METHODS: The present observation study utilizes several computational tools to identify and predict the possible outcomes of gene alterations in CYP3 family of genes with head and neck squamous cell carcinoma (HNSCC). cBioportal hosts an exhaustive collection of datasets of various cancers which was the primary source of analysis. Oncoprint data obtained was further analysed using tools such as PROVEAN, I-Mutant and gnomAD. DISCUSSION: The gnomAD analysis revealed a few polymorphic rare variants with minor allele frequency less than 0.01, which could have a putative association with HNSCC. Five out of eight variants identified were found to be deleterious exhibiting decreased protein stability. CONCLUSION: Further screening of the genetic abnormalities through experimental validation in different populations are warranted to derive an association between the gene identifiers and disease phenotype.

Asymptomatic SARS-CoV-2 infection: is it all about being refractile to innate immune sensing of viral spare-parts?-Clues from exotic animal reservoirs.

A vast proportion of coronavirus disease 2019 (COVID-19) individuals remain asymptomatic and can shed severe acute respiratory syndrome (SARS-CoV) type 2 virus to transmit the infection, which also explains the exponential increase in the number of COVID-19 cases globally. Furthermore, the rate of recovery from clinical COVID-19 in certain pockets of the globe is surprisingly high. Based on published reports and available literature, here, we speculated a few immunovirological mechanisms as to why a vast majority of individuals remain asymptomatic similar to exotic animal (bats and pangolins) reservoirs that remain refractile to disease development despite carrying a huge load of diverse insidious viral species, and whether such evolutionary advantage would unveil therapeutic strategies against COVID-19 infection in humans. Understanding the unique mechanisms that exotic animal species employ to achieve viral control, as well as inflammatory regulation, appears to hold key clues to the development of therapeutic versatility against COVID-19.