Invasive fungal pathogens from the tropical and temperate areas - a challenge in pathology and diagnosis.

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.

Biofilm Formation and Antibiotic Resistance Profiles in Carbapenemase-Producing Gram-Negative Rods-A Comparative Analysis between Screening and Pathological Isolates.

(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 blaKPC, blaOXA-48-like, and blaNDM 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.

Fusarium spp. in Human Disease: Exploring the Boundaries between Commensalism and Pathogenesis.

Fusarium is a large fungal genus that is widely distributed in the environment, mostly known for its plant pathogenicity. Rarely, it is involved in human pathology, where the type of infection caused is highly dependent upon the portal of entry and the immune status of the host. The study at hand aims to summarize routine methods used in diagnosing such infections as well as more advanced molecular diagnostic methods, techniques that can play a huge role in differentiating between colonization and infection when trying to decide the therapeutic outcome. Consequently, to further support our findings, two different strains (one isolated from corneal scrapings and one isolated from purulent discharge) were analyzed in a clinical context and thoroughly tested using classical and modern diagnostic methods: identification by macroscopical and microscopical examinations of the culture and mass spectrometry, completed by molecular methods such as PCR for trichothecene and ERIC-PCR for genetic fingerprinting. Isolation of a clinically relevant Fusarium spp. from a sample still remains a diagnostic challenge for both the clinician and the microbiologist, because differentiating between colonization and infection is very strenuous, but can make a difference in the treatment that is administered to the patient.

Filamentous Fungi Infections: Yet Another Victim of COVID-19?

Filamentous fungi have always been a matter of concern in the medical field, but nowadays, due to an increase in the risk factors and the added infections with the SARS-CoV-2 virus, they are slowly but surely emerging as a dangerous health threat worldwide. Our study aims to estimate the incidence of mold infections in central Romania, as well as assess the impact the pandemic had on them while evaluating other parameters such as age, associated bacterial and fungal infections and comorbidities. Purulent discharge and respiratory secretion specimens were collected and analyzed over a period of 10 years. A total of 68 samples tested positive for molds, with an increased number of positive samples during the pandemic. The highest number of specimens came from the outpatient department, followed by medical wards, with the most common filamentous fungus being Aspergillus section Flavi. Associated diseases included affections of the respiratory system, followed by the cardiovascular system and people who suffered from a viral infection with SARS-CoV-2, and they were mostly present in seniors. The most common associated infections were with Staphylococcus aureus and Candida nonalbicans. A statistically significant correlation was found between the association of mold infection and SARS-CoV-2 and an increase in mortality.

Uncovering the Resistance Mechanisms in Extended-Drug-Resistant Pseudomonas aeruginosa Clinical Isolates: Insights from Gene Expression and Phenotypic Tests.

(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 (blaKPC, blaOXA48-like, blaNDM, blaGES-2, blaSPM, blaIMP, blaVIM) 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 blaGES-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.

Antibacterial Effect of 16 Essential Oils and Modulation of mex Efflux Pumps Gene Expression on Multidrug-Resistant Pseudomonas aeruginosa Clinical Isolates: Is Cinnamon a Good Fighter?

The purpose of the study was to describe the antimicrobial activity of 16 common essential oils (EOs) on multidrug-resistant (MDR) Pseudomonas aeruginosa clinical isolates, including the determination of the effects on mex efflux pumps gene expression. Seventy-two clinical isolates of P. aeruginosa collected between 2020-2022 were screened for susceptibility to EOs using Kirby-Bauer disk diffusion to identify potential candidates for future alternative therapies. The minimal inhibitory concentration (MIC) was further determined for the EO that proved antibacterial activity following the disk diffusion screening. Positive and negative controls were also used for method validation. Since cinnamon EO exhibited the best antimicrobial activity, it was further used to evaluate its influence on mex A, B, C, E, and X efflux pumps gene expression using real-time RT-PCR. Cinnamon EO inhibited all P. aeruginosa strains, followed by thyme EO (37.5%, n = 27) and lavender EO (12.5%, n = 9). The other EOs were less efficient. The MIC detection showed that cinnamon at a concentration of 0.05% v/v inhibited all MDR P. aeruginosa isolates. Thyme, turmeric, peppermint, basil, clove, and lavender EOs presented various results, most of them having activity at concentrations higher than 12.5% v/v. By studying the activity of cinnamon EO on mex efflux pumps, it was found that mexA and mexB (66.5%) were generally under-expressed. The remarkable results produced using the very low concentrations of cinnamon EO, with 100% antimicrobial activity against multi-, extended-, and pan- drug-resistant (MDR, XDR, PDR) P. aeruginosa clinical isolates, completed with the severe alteration of the RNA messaging system, supports its potential to be used as adjuvant treatment, with impact on therapeutic results.

Antifungal and anti-virulence activity of six essential oils against important Candida species - a preliminary study.

Opportunistic infections with Candida species are becoming more problematic, considering their increasing virulence and resistance to antifungal drugs. Aim: To assess the antifungal and anti-virulence activity of basil, cinnamon, clove, melaleuca, oregano and thyme essential oils (EOs) on five Candida species (C. albicans, C. auris, C. krusei, C. parapsilosis and C. guillermondii). Methods: The MIC, growth rate, antibiofilm activity, regulation of gene expression (ALS3, SAP2, HSP70) and germ-tube formation were evaluated by specific methods. Results: Most EOs inhibited Candida species growth and reduced the expression of some virulence factors. Cinnamon and clove EO showed the most significant inhibitory effects. Conclusions: The tested EOs are promising agents for facilitating the management of some Candida infections.

Epidemiology, Evolution of Antimicrobial Profile and Genomic Fingerprints of Pseudomonas aeruginosa before and during COVID-19: Transition from Resistance to Susceptibility.

BACKGROUND: The purpose of the study was to describe the epidemiological implication of Pseudomonas aeruginosa between 2017-2022 in a tertiary hospital from Romania, including the molecular fingerprinting of similar phenotypic strains (multidrug-resistant isolates), which would have an important health impact. The study also describes the resistance profile of P. aeruginosa before and during COVID-19, which might bring new information regarding the management of antibiotic treatments. MATERIALS AND METHODS: Information regarding wards, specimen types, species, and antibiotic resistance profile of 1994 strains of Pseudomonas spp. Isolated over a period of 6 years in Mures Clinical County Hospital, Romania, was collected from the WHONET database. From 50 multidrug-resistant isolates, molecular fingerprinting was performed by Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) to prove the potential clonal distribution. RESULTS: A number of 1994 Pseudomonas spp. were isolated between 2017-2022, from which P. aeruginosa was the most frequent species, 97.39% (n = 1942). P. aeruginosa was most frequently isolated in 2017 (n = 538), with the dermatology department as the main source, mainly from pus secretion. A drop in the harvesting rate was noted in 2020 due to COVID-19 restrictions. Regarding the resistance profile, there are a few modifications. The susceptibility of P. aeruginosa to carbapenems, piperacillin-tazobactam, and amikacin suffered alterations before and during COVID-19. The molecular fingerprinting showed three P. aeruginosa clusters, including strains with 80-99% similarity.

Silver Nanoparticles Biosynthesized with Spruce Bark Extract-A Molecular Aggregate with Antifungal Activity against Candida Species.

Due to their high content of biomolecules, combined with silver's well known antimicrobial potential, silver nanoparticles biosynthesized using spruce bark (AgNP SBEs) demonstrate antibacterial and antioxidant activity, making them a versatile option for developing new antimicrobial agents that might be used for medical treatment or as adjuvants for the classical agents. This study aims to analyze if silver nanoparticles (AgNPs) mediated by spruce bark extract (SBE) and silver salts (AgNP SBE Acetate, AgNP SBE Nitrate) presents antifungal activity against five different Candida spp., synergistic activity with fluconazole, and if they interact with some virulence factors of C. albicans. AgNP SBEs presented MICs (minimum inhibitory concentrations) for all the five tested Candida spp. AgNP SBEs inhibited the growth of C. parapsilosis, C. krusei, and C. guilliermondii, exerted synergistic activity with fluconazole for C. parapsilosis and C. guilliermondii, and inhibited biofilm production for C. albicans, C. auris, and C. guilliermondii. MICs of AgNP SBE Acetate significantly inhibited the production of germ tubes of C. albicans. The expression of C. albicans SAP2 gene was down-regulated by the short-time treatment with MICs of AgNP SBE Acetate, while ALS3 and HSP70 genes were up-regulated by the AgNPs MICs. These results emphasize the potential of using the AgNP SBEs as treatments/adjuvants options, not only against the redundant C. albicans but also for the non-albicans Candida species (which are not as frequently involved in human pathologies, but, sometimes, can be more aggressive).