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.

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.

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.

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.

Candida auris and other phylogenetically related species - a mini-review of the literature.

The lesser-known non-albicans Candida species are often overlooked and difficult to diagnose in most microbiology laboratories. Candida auris, a relatively newly discovered species, is responsible for outbreaks in various geographical regions. Because of its increased resistance to antifungal drugs, C. auris is responsible for hard-to-treat infections and its pathogenicity is still incompletely elucidated. Non-albicans species phylogenetically related to C. auris, like the C. haemulonii complex might also play a role in human pathology. The current summary of the literature presents the emergence, virulence, laboratory identification, and molecular mechanisms responsible for antifungal resistance of emergent rare non-albicans Candida species.

Candida Spp. in Lower Respiratory Tract Secretions - A Ten Years Retrospective Study.

INTRODUCTION: Lower respiratory tract secretions (LRTS) like sputum and tracheal aspirates are frequently sent to the microbiology laboratory from patients with various respiratory pathologies. Improper collection techniques can lead to false-positive results, resulting in improper therapy. AIM OF THE STUDY: To determine the percentage of contaminated samples sent to the microbiology laboratory, to establish the prevalence of Candida spp. in non-contaminated samples and therefore, the presence of Candida spp. originating in lower respiratory tract infections. Material and. METHODS: A 10-year data survey was conducted to assess the differences in Candida prevalence from contaminated versus non-contaminated samples, assessed and categorised by Bartlett grading system, and to emphasise the importance of quality control for potentially contaminated samples. The data were analysed according to gender, age, referring departments, and the species of Candida. For the statistical analysis, Kruskal-Wallis and Fisher tests were used, and the alpha value was set for 0.5. RESULTS: The prevalence of Candida spp. in all analysed samples was 31.60%. After excluding the contaminated samples, the actual prevalence was 27.66%. Of all sputum samples, 31.6% were contaminated. Patients aged more than 40 years old were more prone to provide contaminated sputum samples. C. albicans is more prevalent in non-contaminated sputum samples. In both sputum and tracheal aspirates, the chances of identifying a single species are higher than the chances of identifying multiple species. CONCLUSIONS: The study emphasises the importance of assessing the quality of sputum samples because of the high number of improperly collected samples sent to the microbiology laboratory.

Hemolysin-Producing Strains among Diarrheagenic Escherichia coli Isolated from Children under 2 Years Old with Diarrheal Disease.

Even if serotyping based on O antigens is still routinely used by most laboratories for the detection of diarrheagenic Escherichia coli, this method can provide false-positive reactions, due to the high diversity of O antigens. Molecular methods represent a valuable tool that clarifies these situations. In the Bacteriology Laboratory of Mureș County Hospital, between May 2016 and July 2019, 160 diarrheagenic E. coli strains were isolated from children under 2 years old with diarrheic disease. The strains were identified as Shiga toxin-producing E. coli (STEC)/enteropathogenic Escherichia coli (EPEC) via agglutination with polyvalent sera. STEC strains were serotyped using monovalent sera for serogroup O157. Simplex PCR was performed on the strains to determine the presence of the hlyA gene, and, for the positive ones, the hemolytic activity was tested. Antibiotic susceptibility of the identified diarrheagenic E. coli strains was also investigated. STEC strains were the most frequently identified (49.1%), followed by EPEC (40.2%). The hlyA gene was identified in 12 cases, representing 18.2% of the STEC strains. Even if the extended-spectrum ß-lactamase (ESBL)-producing strains represented only 10%, a relevant percentage of multidrug-resistant (MDR) strains (24%) was identified.