Anca Delia Mare

Essential oils (EOs) are complex plant-derived products known for their broad-spectrum antibacterial activity. This study aims to evaluate the chemical composition of eight essential oils-EOs (Caryophylli aetheroleum, Menthae aetheroleum, Origani aetheroleum, Rosmarini aetheroleum, Salviae aetheroleum, Melaleucae aetheroleum, Limonis aetheroleum, and Curcumae aetheroleum) and to evaluate their antibacterial and antibiofilm activity against five opportunistic pathogens with biofilm-forming potential (methicillin-susceptible and methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli, and Klebsiella pneumoniae). GC-MS was used to determine the chemical composition of the EOs, and antibacterial activity was evaluated using broth microdilution to determine the minimum inhibitory concentration and minimum bactericidal concentration. Biofilm inhibition was assessed by a crystal violet assay. Oxygenated monoterpenes and phenolic compounds were dominant in Origani, Menthae, Rosmarinus, Melaleucae, and Caryophylli aetheroleum. Potent inhibitory effects against the tested bacterial strains were observed for clove, tea tree, oregano, and rosemary EOs. The antimicrobial efficacy of EOs is closely linked to their chemical composition. Tea tree and oregano EOs exhibited the broadest spectrum of antimicrobial activity, while peppermint and curcuma oils were the least potent. Cytotoxicity thresholds from the literature suggest that some effective EO concentrations exceed safe mucosal limits, particularly in continuous high-dose applications, but short-contact delivery systems or adjunctive use with different agents may mitigate safety concerns. These findings support further investigation into their therapeutic applications in oral health products.

Backgrunod/Objectives: Routine identification of common bacterial pathogens is typically efficient, utilizing standardized, cost-effective methods. However, the diagnostic process becomes significantly more complex when dealing with rare or unexpected microorganisms, especially as they can be considered colonizers in many cases. Methods: This study presents diagnostic details of an uncommon pathogen, Vibrio alginolyticus, isolated from auricular discharge in a patient with non-Hodgkin lymphoma diagnosed with persistent otitis externa and explores its identification through both conventional and modern laboratory approaches. Sequential ear discharge cultures resulted in phenotypically similar but genomically different Vibrio alginolyticus isolates. We complemented classical methods like conventional culture (on Columbia agar and CLED agar), Vitek2 Compact identification, and EUCAST disk diffusion antimicrobial susceptibility testing (following the EUCAST version 12.0 guidelines) with MALDI-TOF mass spectrometry and Illumina/Nanopore whole genome sequencing. Comparative analysis of the genomes was performed with the PeGAS pipeline, Unicycler, and 1928Diagnostics SNP analysis. Results: The Vitek2 analysis identified both isolates as V. alginolyticus with 99% confidence, and this was supported by the MALDI-TOF MS results. The first isolate (A) was fully susceptible to the antibiotics tested, while the second (B) showed resistance to ciprofloxacin. Whole genome sequencing revealed 99.23% and 98.60% nucleotide identity to the V. alginolyticus reference genome for isolates A and B, respectively, with a 99.8% match between them. Isolate B acquired a gyrA (c.1870C>T) mutation that correlates with the ciprofloxacin resistance (MIC > 0.5 mg/L). Both genomes carry hlyA (hemolysin), toxR (cholera toxin regulator), genes involved in biofilm formation (rpoN, relA, spoT, opp), luxS (motility), proA, vacB (virulence factors), and tet(34) (oxytetracycline resistance). A core genome SNP distance of V. alginolyticus and documented host resistance evolution, with a virulence repertoire that could explain the clinical evolution. Conclusions: This case highlights the utility of molecular methods in confirming species identity, detecting resistance markers, characterizing virulence determinants, and differentiating a pathogen from a colonizer, supporting targeted clinical management.

Background/Objectives: Glutamic acid (GLA) is an essential amino acid with a key role in human metabolism. A potential involvement in anticancer therapy and possible antibacterial and anti-biofilm effects were also observed. Glutamine (GLN) and monosodium glutamate (MSG) are GLA structural derivatives for which the last two effects were evaluated, with contradictory results. Therefore, this study aimed to assess the antibacterial activity and the influence on the biofilm formation of GLA, GLN, MSG, and glutamic acid diethyl ester (GLADE) on clinically relevant bacteria. Methods: Gram-positive and Gram-negative bacterial reference strains were used to test the antibacterial and anti-biofilm effects of GLA, GLN, MSG, and GLADE. The antibacterial properties were assessed by detecting the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The influence on biofilm formation was assessed by the crystal violet method, reading the optical densities (ODs) by spectrophotometry. Results: GLN did not demonstrate an inhibitory capacity at the maximum tested concentration (2.86 mg/mL); GLA showed inhibitory activity at 1.76 mg/mL and 0.88 mg/mL; MSG inhibited the growth of all bacterial strains at 112 mg/mL; GLADE had the most promising results on all bacterial strains (MICs of 12.75 mg/mL and 25.5 mg/mL). GLADE showed satisfactory MBC values on all bacterial strains (at 51 mg/mL and 25.5 mg/mL). Conclusions: GLA and some structural analogues are attractive options for possible antibacterial activity; optimizing GLADE to increase its antibacterial activity could be a new approach.

Intercellular communication helps regulate the cell density and the virulence traits in yeasts and bacteria. The study aims to identify the effects of quorum sensing molecules (QSMs) like farnesol and tyrosol on Candida spp. virulence traits. The effects of farnesol and tyrosol were studied on the growth rate of Candida albicans, Candida parapsilosis, Candida krusei, Candida auris, and Candida guilliermondii at different time points, on a 48 hours incubation period. The growth rate was assessed spectrophotometrically. The biofilm formation abilities of Candida spp. were assessed by crystal violet staining technique. Moreover, the expression of C. albicans virulence genes (ALS3, HSP70, SAP2) was analyzed as a response to 100 μM farnesol and tyrosol, by RT-PCR. Generally, farnesol was found to inhibit the growth rate and biofilm formation mostly in non-albicans species, while tyrosol exerted a non-consistent response on the different Candida species. The expression of ALS3 and HSP70 in C. albicans was upregulated by the QSMs. Both farnesol and tyrosol are involved in the regulation of Candida spp. virulence mechanisms, dependent on the used concentration and exposure time and in a species-dependent manner.

Background: The outbreak of the COVID-19 pandemic caught healthcare systems in many countries unprepared. Shortages of personnel, medicines, disinfectants, and intensive care unit (ICU) capacities, combined with inadvertent use of antibiotics and emergence of drug-resistant secondary infections, led to a surge in COVID-19-related mortality. Objective: We aimed to evaluate the prevalence of secondary bacterial infections and the associated antibiotic resistance in a temporary established ICU dedicated to COVID-19 patients. We also assessed the utility of clinical and routine laboratory data as predictors of secondary infections and mortality in these patients. Methods: We examined the medical records of 243 patients admitted to the COVID-19 Medical Support Unit of Târgu Mures, Romania, between 1 August 2020 and 31 January 2021. Results: Among the 243 patients admitted to the COVID-19 Medical Support Unit of Târgu Mures between 1 August 2020 and 31 January 2021, 59 (24.3%) presented secondary infections. Acinetobacter baumannii and Klebsiella pneumoniae were the most frequent isolates (31.1% and 18.9%, respectively), most of them multidrug resistant. Chronic obstructive pulmonary disease had a higher prevalence in patients who developed secondary infections (p = 0.012). Secondary infections were associated with longer stay in the ICU and with higher mortality (p = 0.006 and p = 0.038, respectively). Conclusions: Early identification of secondary infections and proper use of antibiotics are necessary to limit the spread of multidrug-resistant microorganisms in COVID-19 patients admitted in the ICU.

With approximately half a billion events per year, lower respiratory tract infections (LRTIs) represent a major challenge for the global public health. Among LRTI cases, those caused by Gram-negative bacteria (GNB) are associated with a poorer prognostic. Standard-of-care etiologic diagnostics is lengthy and difficult to establish, with more than half of cases remaining microbiologically undocumented. Recently, syndromic molecular diagnostic panels became available, enabling simultaneous detection of tens of pathogen-related and antimicrobial-resistance genetic markers within a few hours. In this narrative review, we summarize the available data on the performance of molecular diagnostics in GNB pneumonia, highlighting the main strengths and limitations of these assays, as well as the main factors influencing their clinical utility. We searched MEDLINE and Web of Science databases for relevant English-language articles. Molecular assays have higher analytical sensitivity than cultural methods, and show good agreement with standard-of-care diagnostics regarding detection of respiratory pathogens, including GNB, and identification of frequent patterns of resistance to antibiotics. Clinical trials reported encouraging results on the usefulness of molecular assays in antibiotic stewardship. By providing early information on the presence of pathogens and their probable resistance phenotypes, these assays assist in the choice of targeted therapy, in shortening the time from sample collection to appropriate antimicrobial treatment, and in reducing unnecessary antibiotic use.

(1) Background: Carbapenem-resistant (CR) bacteria pose a significant global public health challenge due to their ability to evade treatment with beta-lactam antibiotics, including carbapenems. This study investigates the biofilm-forming capabilities of CR clinical bacterial isolates and examines the impact of serum on biofilm formation. Additionally, the study evaluates the resistance profiles and genetic markers for carbapenemase production. (2) Methods: Bacterial isolates were collected from the microbiology laboratory of Mures County Clinical Hospital between October 2022 and September 2023. Pharyngeal and rectal swabs were screened for carbapenem-resistant bacteria using selective media. Lower respiratory tract samples were also analyzed for CR Gram-negative bacteria. The isolates were tested for their ability to form biofilms in the presence and absence of fetal bovine serum at 24 and 48 h. Carbapenemase production was detected phenotypically and confirmed via PCR for relevant genes. (3) Results: Out of 846 screened samples, 4.25% from pharyngeal swabs and 6.38% from rectal swabs tested positive for CR bacteria. Acinetobacter baumannii and Klebsiella pneumoniae were the most common species isolated. Biofilm formation varied significantly between clinical isolates and standard strains, with clinical isolates generally showing higher biofilm production. The presence of serum had no significant effect on biofilm formation in Klebsiella spp., but stimulated biofilm formation for Acinetobacter spp. Carbapenemase genes bla_KPC, bla_OXA-48-like, and bla_NDM were detected in various isolates, predominantly in Klebsiella spp., but were not the main determinants of carbapenem resistance, at least in screening isolates. (4) Conclusions: This study highlights the variability in biofilm formation among CR clinical isolates and underscores the differences between the bacteria found as carriage versus infection. Both bacterial species and environmental factors variably influence biofilm formation. These insights are crucial for the development of effective treatment and infection control strategies in clinical settings.

Fungi play a vital role in ensuring a physiological balance in the surrounding environments, interacting closely with humans, plants, and animals. While most of the time their contribution is beneficial, occasionally, they can become harmful, especially in patients with weakened immune systems. The work at hand aims to present the most common fungal pathogens involved in invasive infections, focusing on fungi that are present in the tropical and temperate areas of the world. While in the former, due to the humid climate, most fungal infections are caused by dimorphic fungi such as Coccidioides spp., Blastomyces spp., Histoplasma spp., Emergomyces spp. and Paracoccidioides spp., in the latter, after Candida spp., the most frequent fungi that are involved in disseminated mycosis are Aspergillus spp., Fusarium spp. and species from the order Mucorales. Nowadays, the etiology, severity, and number of cases of fungal diseases are starting to rise significantly. There are no exact reasons reported for this increase, but several factors are thought to be incriminated: the expansion of the range of medical conditions that constitute risk factors for developing the disease, an improvement in the available diagnostic methods, the commodity offered by modern traveling services associated with the lack of an available vaccine against fungal infections, as well as climatic influences. All the above-mentioned aspects consequently caused infections that used to be endemic to be spread worldwide. Therefore, it is of critical importance to understand the epidemiology, clinical manifestations of fungi induced diseases, virulence factors, and diagnosis for each of those pathogens.

A healthy, sustainable diet contributes massively to the general well-being of a population. Nowadays, people have started to significantly improve their diet by reducing processed products, as well as including a higher number of fruits, vegetables, cereals, and spices to flavor their food. However, making the right food choice, without any harmful pathogens that pose a risk to human health, can remain quite challenging. Despite prioritizing food safety in its processing, production, and distribution, food contamination remains a prevalent and undesirable occurrence. This study aims to detect the fungal load of commonly used green leaf spices commercialized in Romania and the impact of those microorganisms and their secondary metabolites on consumers. Six (28.57%) out of the twenty-one tested samples showed different degrees of fungal contamination, mostly with Aspergillus section Flavi, followed by Aspergillus section Nigri and other fungi in lower amounts. Three main fungal secondary metabolites with potential impact on consumers, namely mycotoxins, were determined with high-performance liquid chromatography (HPLC): Aflatoxin B1, Ochratoxin A, and Zearalenone. Moreover, their legal limits (5 μg/kg, 15 μg/kg, and 50 μg/kg, respectively) were exceeded by 95.24%, 100%, and 85.71%, respectively. Environmental factors that affect the processing and packaging of these spices did not show any relation to fungal contamination, conversely to price, which significantly correlates with the mycological quality of the products.

(1) Background: The purpose of the study was to describe the activity of mex efflux pumps in Multidrug-Resistant (MDR) clinical isolates of Pseudomonas aeruginosa and to compare the carbapenem-resistance identification tests with PCR; (2) Methods: Sixty MDR P. aeruginosa were analyzed for detection of carbapenemase by disk diffusion inhibitory method, carbapenem inactivation method and Modified Hodge Test. Endpoint PCR was used to detect 7 carbapenemase genes (bla_KPC, bla_OXA48-like, bla_NDM, bla_GES-2, bla_SPM, bla_IMP, bla_VIM) and mcr-1 for colistin resistance. The expression of mexA, mexB, mexC, mexE and mexX genes corresponding to the four main efflux pumps was also evaluated; (3) Results: From the tested strains, 71.66% presented at least one carbapenemase gene, with bla_GES-2 as the most occurring gene (63.3%). Compared with the PCR, the accuracy of phenotypic tests did not exceed 25% for P. aeruginosa. The efflux pump genes were present in all strains except one. In 85% of the isolates, an overactivity of mexA, mexB and mostly mexC was detected. Previous treatment with ceftriaxone increased the activity of mexC by more than 160 times; (4) Conclusions: In our MDR P. aeruginosa clinical isolates, the carbapenem resistance is not accurately detected by phenotypic tests, due to the overexpression of mex efflux pumps and in a lesser amount, due to carbapenemase production.