Phenomics and genomic features of Enterococcus avium IRMC1622a isolated from a clinical sample of hospitalized patient.

BACKGROUND: Enterococcus avium (E. avium) is a Gram-positive nosocomial pathogen that is commonly isolated from the alimentary tract. The objective of this functional genomics study was to identify the resistant genes by analyzing the genome of E. avium IRMC1622a, a type of bacteria found in feces collected from a patient at a Saudi Arabian tertiary hospital. METHODS: The bacterial strain IRMC1622a was identified by 16 S rRNA sequencing as Enterococcus sp. The resistance phenomics were performed using VITEK® 2, and morphological analysis was achieved using a scanning electron microscope (SEM). Finally, the whole bacterial genome of the bacterial strain IRMC1622a was subjected to sequencing during October 2023 using Oxford Nanopore long-read sequencing technology, and mining for resistant genes. RESULTS: The results of antimicrobial resistant phenomics indicated that the IRMC1622a strain was sensitive to all tested antimicrobial agents except for erythromycin, and the same result was confirmed by genomic analysis in addition to other classes of antibiotics. SEM showed E. avium IRMC1622a is ovoid shape, in single cells (L 1.2797 ± 0.1490 µm), in pairs (L 1.7333 ± 0.1054 µm), and in chains (L 2.44033 ± 0.1978 µm). The E. avium IRMC1622a genome has 14 (in CARD) antimicrobial resistance genes that were identified with several mechanisms of antimicrobial resistance, such as the efflux pump and conferring antibiotic resistance. The present study revealed that the E. avium IRMC1622a genome contains a high number of genes associated with virulence factors, and 14 matched pathogenic protein families and predicted as human pathogen (probability score 0.855). We report two (ISEnfa4 and ISEfa5) mobile genetic elements for the first time in the E. avium genome. CONCLUSIONS: The study concludes that E. avium IRMC1622a is susceptible to all tested antibacterials except erythromycin. The IRMC1622a has 14 genes encoding antimicrobial resistance mechanisms, including the efflux pump and conferring antibiotic resistance. This could indicate a potential rise in E. avium resistance in healthcare facilities. These observations may raise concerns regarding E. avium resistance in healthcare. We need more research to understand the pathophysiology of E. avium, which leads to hospital-acquired infections.

Evaluation of the antimicrobial efficacy of different nonsteroidal anti-inflammatory drugs alone or in combination with calcium hydroxide intracanal medicament: an in vitro study.

This study explored the antimicrobial effects of ketoprofen, piroxicam, and celecoxib alone or combined with calcium hydroxide (CH) against two strains of Enterococcus faecalis (E. faecalis) and assessed the influence of such combinations on the pH of CH. Minimum inhibitory concentrations (MICs) of the three tested NSAIDs were determined. Tested pastes were placed into wells punched in seeded agar plates and the bacterial inhibition zones were measured. Antibiofilm activity was assessed against 3 weeks of biofilm induced in bovine dentine blocks. The pH of the pastes was measured at four-time intervals. MIC values were 3.12, 25, and 25 mg/ml for ketoprofen, piroxicam, and celecoxib, respectively, and were similar for both bacterial strains except for celecoxib, which showed 8% growth at the highest tested concentration against vancomycin-resistant E. faecalis. Ketoprofen had the largest mean inhibition zone that was comparable to CH. None of the six tested pastes exhibited antibiofilm activity of a significant level in comparison to CH. A noticeable increase in the antibiofilm activity was found when 20% NSAIDs were added to CH while maintaining an alkaline pH. Ketoprofen was found to be the most effective among the tested NSAIDs. Although its effect was comparable to CH, adding ketoprofen at a ratio of 20% resulted in 50% higher antimicrobial action than CH alone. Accordingly, incorporating NSAIDs in inter-appointment dressing has the potential to utilize their anti-inflammatory, local analgesic, and antibacterial actions, which overcome the limitations of CH and improve the outcome of root canal treatment.

Genomic Landscape of Multidrug Resistance and Virulence in Enterococcus faecalis IRMC827A from a Long-Term Patient.

We report on a highly virulent, multidrug-resistant strain of Enterococcus faecalis IRMC827A that was found colonizing a long-term male patient at a tertiary hospital in Khobar, Saudi Arabia. The E. faecalis IRMC827A strain carries several antimicrobial drug resistance genes and harbours mobile genetic elements such as Tn6009, which is an integrative conjugative element that can transfer resistance genes between bacteria and ISS1N via an insertion sequence. Whole-genome-sequencing-based antimicrobial susceptibility testing on strains from faecal samples revealed that the isolate E. faecalis IRMC827A is highly resistant to a variety of antibiotics, including tetracycline, doxycycline, minocycline, dalfopristin, virginiamycin, pristinamycin, chloramphenicol, streptomycin, clindamycin, lincomycin, trimethoprim, nalidixic acid and ciprofloxacin. The isolate IRMC827A carries several virulence factors that are significantly associated with adherence, biofilm formation, sortase-assembled pili, manganese uptake, antiphagocytosis, and spreading factor of multidrug resistance. The isolate also encompasses two mutations (G2576T and G2505A) in the 23S rRNA gene associated with linezolid resistance and three more mutations (gyrA p.S83Y, gyrA p.D759N and parC p.S80I) of the antimicrobial resistance phenotype. The findings through next-generation sequencing on the resistome, mobilome and virulome of the isolate in the study highlight the significance of monitoring multidrug-resistant E. faecalis colonization and infection in hospitalized patients. As multidrug-resistant E. faecalis is a serious pathogen, it is particularly difficult to treat and can cause fatal infections. It is important to have quick and accurate diagnostic tests for multidrug-resistant E. faecalis, to track the spread of multidrug-resistant E. faecalis in healthcare settings, and to improve targeted interventions to stop its spread. Further research is necessary to develop novel antibiotics and treatment strategies for multidrug-resistant E. faecalis infections.

Genomic Insights into Virulence Factors and Multi-Drug Resistance in Clostridium perfringens IRMC2505A.

Clostridium perfringens is a spore-forming, Gram-positive anaerobic pathogen that causes several disorders in humans and animals. A multidrug-resistant Clostridium strain was isolated from the fecal sample of a patient who was clinically suspected of gastrointestinal infection and had a recent history of antibiotic exposure and diarrhea. The strain was identified by 16s rRNA sequencing as Clostridium perfringens. The strain's pathogenesis was analyzed through its complete genome, specifically antimicrobial resistance-related genes. The Clostridium perfringens IRMC2505A genome contains 19 (Alr, Ddl, dxr, EF-G, EF-Tu, folA, Dfr, folP, gyrA, gyrB, Iso-tRNA, kasA, MurA, rho, rpoB, rpoC, S10p, and S12p) antibiotic-susceptible genetic species according to the k-mer-based detection of antimicrobial resistance genes. Genome mapping using CARD and VFDB databases revealed significant (p-value = 1 × 10-26) genes with aligned reads against antibiotic-resistant genes or virulence factors, including phospholipase C, perfringolysin O, collagenase, hyaluronidase, alpha-clostripain, exo-alpha-sialidase, and sialidase activity. In conclusion, this is the first report on C. perfringens from Saudi Arabia that conducted whole genome sequencing of IRMC2505A and confirmed the strain as an MDR bacterium with several virulence factors. Developing control strategies requires a detailed understanding of the epidemiology of C. perfringens, its virulence factors, and regional antimicrobial resistance patterns.

Antibiofilm Activity of 3D-Printed Nanocomposite Resin: Impact of ZrO2 Nanoparticles.

Poly(methyl methacrylate) (PMMA) is a commonly used material, as it is biocompatible and relatively cheap. However, its mechanical properties and weak antibiofilm activity are major concerns. With the development of new technology, 3D-printed resins are emerging as replacements for PMMA. Few studies have investigated the antibiofilm activity of 3D-printed resins. Therefore, this study aimed to investigate the antibiofilm activity and surface roughness of a 3D-printed denture base resin modified with different concentrations of zirconium dioxide nanoparticles (ZrO2 NPs). A total of 60 resin disc specimens (15 × 2 mm) were fabricated and divided into six groups (n = 10). The groups comprised a heat-polymerized resin (PMMA) group, an unmodified 3D-printed resin (NextDent) group, and four 3D-printed resin groups that were modified with ZrO2 NPs at various concentrations (0.5 wt%, 1 wt%, 3 wt%, and 5 wt%). All specimens were polished using a conventional method and then placed in a thermocycler machine for 5000 cycles. Surface roughness (Ra, µm) was measured using a non-contact profilometer. The adhesion of Candida albicans (C. albicans) was measured using a fungal adhesion assay that consisted of a colony forming unit assay and a cell proliferation assay. The data were analyzed using Shapiro-Wilk and Kruskal-Wallis tests. A Mann-Whitney U test was used for pairwise comparison, and p-values of less than 0.05 were considered statistically significant. The lowest Ra value (0.88 ± 0.087 µm) was recorded for the PMMA group. In comparison to the PMMA group, the 3% ZrO2 NPs 3D-printed group showed a significant increase in Ra (p < 0.025). For the 3D-printed resins, significant differences were found between the groups with 0% vs. 3% ZrO2 NPs and 3% vs. 5% ZrO2 NPs (p < 0.025). The highest Ra value (0.96 ± 0.06 µm) was recorded for the 3% ZrO2 NPs group, and the lowest Ra values (0.91 ± 0.03 µm) were recorded for the 0.5% and 5% ZrO2 NPs groups. In terms of antifungal activity, the cell proliferation assay showed a significant decrease in the C. albicans count for the 0.5% ZrO2 NPs group when compared with PMMA and all other groups of 3D-printed resins. The group with the lowest concentration of ZrO2 NPs (0.5%) showed the lowest level of C. albicans adhesion of all the tested groups and showed the lowest Candida count (0.29 ± 0.03). The addition of ZrO2 NPs in low concentrations did not affect the surface roughness of the 3D-printed resins. These 3D-printed resins with low concentrations of nanocomposites could be used as possible materials for the prevention and treatment of denture stomatitis, due to their antibiofilm activities.

Potential Antigenic Candidates for the Development of Peptide-Based Vaccines to Induce Immunization against Helicobacter pylori Infection in BALB/c Mice.

Helicobacter pylori (H. pylori) has been identified as a group-1 definite carcinogen. As of yet, there is no available vaccine for this microorganism. Our study aimed to identify antigenic peptides in H. pylori using an in silico proteomic approach, and to evaluate their effectiveness as potential vaccine candidates. Four different peptide sequences were prioritized using the reverse vaccinology, namely, CagA1, CagA2, VacA, and SabA. Peptides emulsified with Freunde's adjuvant were used to immunize BALB/C mice. Subcutaneously immunized mice were challenged by oral administration of H. pylori. IgG, IgA, IL4, and IL17 were detected in mice sera. Histopathology of the dissected stomach of vaccinated and control mice were assessed using H&E stain. IgG was significantly higher in mice vaccinated with SabA. IL-4 was significantly increased in CagA1, CagA2, VacA, and SabA vaccinated mice compared to the adjuvant group. Additionally, histopathological examination of gastric tissue showed a protective effect in the vaccinated groups compared to adjuvant and PBS groups. Our findings indicate a promising effect of the tested epitopes, particularly the SabA antigen, to induce an immune response against H. pylori.

The Antifungal and Antibiofilm Activities of Caffeine against Candida albicans on Polymethyl Methacrylate Denture Base Material.

BACKGROUND: In this study, the effect of pure caffeine was established against Candida albicans (C. albicans) using different microbiological techniques. METHODS: Broth microdilution and colony forming units (CFUs) assays were used to detect the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). The Live/Dead fluorescent dyes were implemented to determine the yeast viability. Polymethyl methacrylate acrylic resin (PMMA) discs were prepared to evaluate caffeine's effects against adherent C. albicans using microplate reader, CFUs, and scanning electron microscope (SEM). RESULTS: caffeine's MIC was detected around 30 mg/mL, while the MFC was considered at 60 mg/mL. In an agar-well diffusion test, the inhibition zones were wider in caffeine groups. The Live/Dead viability test verified caffeine's antifungal effects. The optical density of the adherent C. albicans on PMMA discs were lower at 620 nm or 410 nm in caffeine groups. CFU count was also reduced by caffeine treatments. SEM revealed the lower adherent C. albicans count in caffeine groups. The effect of caffeine was dose-dependent at which the 60 mg/mL dose demonstrated the most prominent effect. CONCLUSION: The study reinforced caffeine's antifungal and antibiofilm properties and suggested it as an additive, or even an alternative, disinfectant solution for fungal biofilms on denture surfaces.

Silver Nanoparticles: A Promising Antifungal Agent against the Growth and Biofilm Formation of the Emergent Candida auris.

Candida auris is a globally-emerging pathogen that is correlated to nosocomial infections and high mortality rates, causing major outbreaks in hospitals and serious public health concerns worldwide. This study investigated the antifungal activity of silver nanoparticles (AgNPs) on clinical isolates of C. auris. A total of eight clinical isolates were collected from blood, urine, ear swab, and groin. C. auris was confirmed by MALDI-TOF MS, and gene sequencing. All isolates confirmed as C. auris were subjected to antimicrobial agents, including amphotericin B, fluconazole, caspofungin, voriconazole, micafungin, and flucytosine. A serial dilution of a silver nanoparticles solution was prepared to test antifungal susceptibility testing under planktonic conditions. Moreover, an antibiofilm activity assay was determined using a colony-forming assay and a cell viability assay by a live−dead yeast kit. Significant antifungal and antibiofilm activity of AgNPs was detected against all isolates; MIC was <6.25 µg/mL, the range of MFC was from 6.25 to 12.5 µg/mL for all isolates, and the highest value of IC50 was 3.2 µg/mL. Silver nanomaterials could represent a possible antimicrobial agent to prevent outbreaks caused by C. auris infections.

Predominance of non-Streptococcus mutans bacteria in dental biofilm and its relation to caries progression.

This study aims to assess differences in biofilm bacterial composition between patients with low and high caries. Patients without a medical problem and with no history of antibiotic use, mouth wash or fluoride application in the previous 3 months were recruited. Caries was recorded at cavitation level; score was calculated by a national mean (dmft of 4.8 and DMFT of 2.7). Pooled biofilm samples were collected from mesial, distal, buccal, lingual, and occlusal surfaces. Based on caries experience, individuals were classified into low and high caries and both groups were compared regarding bacteria identified using 16S rRNA gene sequencing, and molecular phylogenetic analysis of the isolates was performed. A total of twenty seven randomly selected samples with low (n = 13) and high (n = 14) caries. Identification of oral bacteria was performed using 16S rRNA sequence, Rothia mucilaginosa and R. aeria were identified in low caries individuals, while R. dentocariosa was detected in high caries individuals. Two Streptococcus spp. were identified only in low caries S. salivarius and S. gordonii whereas S. sanguinis, S. mitis, S. sinensis, S. rubneri, S. vestibularis, S. cristatus and S. massiliensis were identified only in individuals with high caries. This study revealed the absence of R. mucilaginosa in the high caries subjects and its coexistence with the low caries subjects. Streptococcus mutans was insignificant contributor of caries among samples, while, Streptococcus sanguinis was the main constituent of high caries Saudi patients.

Diagnostic deficiencies of C. difficile infection among patients in a tertiary hospital in Saudi Arabia: A laboratory-based case series.

Clostridioides difficile infection (CDI) has become a threatening public health problem in the developed world. In the kingdom of Saudi Arabia, prevalence of CDI is still unknown due to limited surveillance protocols and diagnostic resources. We used a two-step procedure to study and confirm C. difficile cases. We also studied toxin profiles of these isolates. Stool samples were collected from symptomatic patients and clinically suspected of CDI for almost 12 months. Isolates were confirmed by culture method followed by 16S rRNA sequencing. Multiplex PCR was performed for the identification of toxin A, toxin B and binary toxin genes and compared to Gene Expert results. Out of the 47 collected samples, 27 were successfully grown on culture media. 18 samples were confirmed as C. difficile by both culture and 16S rRNA sequencing. Interestingly, the rest of the isolates (9 species) belonged to different genera. Our results showed 95% of samples were positive for both toxin A and B (tcdA, tcdB) and all samples exhibited the toxin gene regulator tcdC. All samples were confirmed negative for the binary toxin gene ctdB and 11% of the isolates were positive for ctdA gene. Interestingly, one isolate harbored the binary toxin gene (cdtA +) and tested negative for both toxins A and B. We believe that combining the standard culture method with molecular techniques can make the detection of C. difficile more accurate.

Antifungal Activity of Denture Base Resin Containing Nanozirconia: In Vitro Assessment of Candida albicans Biofilm.

OBJECTIVE: To evaluate the antimicrobial effects of different concentrations of zirconium dioxide nanoparticles (nano-ZrO2) reinforcement of poly(methyl) methacrylate (PMMA) on surface roughness and C. albicans biofilm. METHODS: 20 heat-polymerized acrylic resin discs were conventionally made and divided into 4 groups (n = 5) according to nano-ZrO2 concentration: control (0% filler) and 3 experimental groups (2.5% (Z2.5), 5.0% (Z5.0), and 7.5% (Z7.5)). An optical profilometer was used for surface roughness evaluation, followed by Candida adherence assay. Specimens were sterilized, then immersed in cultured yeast (C. albicans), and incubated at 37°C for 48 hours. After that, discs were rinsed before extracting the clustered pellets of Candida. The attached C. albicans was counted using the direct method after spreading on agar media and incubating for 48 hours. Statistical analysis was performed using one-way ANOVA and Tukey's post hoc test at α = 0.05. RESULTS: Surface roughness was significantly increased with all modified groups compared with control (P < 0.01), which showed the lowest roughness value (0.027 ± 0.004 µm). There was no significant difference in the roughness value among reinforced groups (2.5, 5.0, and 7.5%) (P > 0.05), with Z7.5 showing the highest roughness value (0.042 ± 0.004 µm). Candida count was reduced as the nano-ZrO2 increased but not significantly (P=0.15). CONCLUSIONS: The addition of different concentrations of nano-ZrO2 particles to PMMA increased the surface roughness compared with control; in contrast, insignificant reduction of C. albicans biofilm was detected.

Drug Resistance-Associated Mutations in ERG11 of Multidrug-Resistant Candida auris in a Tertiary Care Hospital of Eastern Saudi Arabia.

Candida auris is an emerging multi-drug resistant pathogen with high mortality rate; nosocomial infections have been reported worldwide, causing a major challenge for clinicians and microbiological laboratories. The study aims to describe new cases of C. auris and detect drug resistance-associated mutations of C. auris by the sequencing of ERG11 and FKS1 genes. A total of six specimens were collected from blood, urine, ear swab, and groin screening samples. Isolates were incubated for 48 h on Sabouraud Dextrose agar (SDA) at 42 °C, then confirmed by MALDI-TOF MS. Furthermore, antifungal susceptibility testing was performed using the Vitek 2 system to detect Minimum Inhibitory Concentrations (MICs) of six antifungals. Sequences of 18S rRNA gene and ITS regions from isolates and phylogenetic analysis were performed. Gene sequencing was analysed to detect drug resistance-associated mutations by FKS1 and ERG11 genes sequencing. All C. auris isolates were confirmed by MALDI-TOF MS, and evolutionary analyses using sequences of 18S rRNA gene and ITS region. Antifungal susceptibility testing showed that all isolates were resistant to fluconazole. Sequencing of ERG11 and FKS1 genes from the isolates revealed the presence of two (F132Y and K143R) drug resistance-associated mutations in ERG11, however, FKS1 gene was devoid of mutations. The study sheds light on a public health threat of an emerging pathogen, and the hospital implemented strict contact screening and infection control precautions to prevent C. auris infection. Finally, there is a critical need to monitor the antifungal resistance in different geographical areas and implementation of efficient guidelines for treatment.