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.

Serogroups and genetic diversity of diarrheagenic strains of Escherichia coli: a retrospective study.

INTRODUCTION: Diverse serogroups of Escherichia coli cause sporadic cases and outbreaks of diarrhea among children. Our study aimed to evaluate the serogroups of diarrheagenic strains of E. coli that cause diarrheal disease in children under two years old, and clarify if the cases were sporadic or outbreaks. METHODOLOGY: The retrospective study included 130 strains of pathogenic E. coli, isolated from children who were less than two years of age, and had diarrheal disease, between May 2016 and July 2019. The study was conducted in the Bacteriology Laboratory (County Clinical Hospital, Mureș, Romania). The 130 strains were sero-grouped using polyvalent and monovalent O antisera. Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) was performed to evaluate the similarity between different E. coli strains, and a simplex polymerase chain reaction (PCR) was performed to detect the presence of the hlyA gene that is specific to the enterohemorrhagic strains. RESULTS: After agglutination with polyvalent O antisera, slightly more than half of the strains (50.77%) were sero-grouped as Shiga toxin-producing E. coli (STEC), and the rest of the strains belonged to the Enteropathogenic Escherichia coli (EPEC) serogroups. Serogroup O157 was the most frequently identified (16.51% of the total number of typeable strains), and one strain was positive for hlyA. ERIC-PCR revealed a high diversity of strains, with an overall 50% similarity. CONCLUSIONS: STEC serogroups were the most common strains causing diarrheal disease, and O-157 was the dominant serogroup identified. The strains included in our study presented high genetic diversity, suggesting that most of the cases were sporadic.

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.

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).

In Vitro Antifungal Activity of Silver Nanoparticles Biosynthesized with Beech Bark Extract.

Biosynthesis is a green method for the synthesis of silver nanoparticles (AgNPs). This study aimed to assess the antifungal activity of two silver nanoparticle solutions, synthesized using beech bark extract (BBE) and acetate and nitrate silver salts (AgNP Acetate BBE and AgNP Nitrate BBE), their influence on biofilm production, their potential synergistic effects with fluconazole, on different Candida spp., and their influence on virulence factors of C. albicans (germ tube production, gene expression for ALS3, SAP2, HSP70). Both the AgNP BBEs presented different minimum inhibitory concentrations for all the studied Candida spp., but biofilm production was inhibited only for C. albicans and C. guilliermondii. The growth rates of all the studied Candida spp. were inhibited in the presence of both AgNP BBEs, except for C. auris. Synergistic activity was observed for C. parapsilosis and C. guilliermondii, for different combinations of fluconazole with both the AgNP BBEs. The germ tube production of C. albicans was slightly inhibited by the AgNP BBEs. Only AgNP Acetate BBE was able to down-regulate the expression of SAP2. Overall, we can conclude that, even if more studies are necessary, AgNPs synthesized with beech bark extract might be an interesting alternative to classic antifungal treatments.

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.

Candida and Candidiasis-Opportunism Versus Pathogenicity: A Review of the Virulence Traits.

One of the most important questions in microbiology nowadays, is how apparently harmless, commensal yeasts like Candida spp. can cause a rising number of infections. The occurrence of the disease requires firstly the attachment to the host cells, followed by the invasion of the tissue. The adaptability translates into a rapid ability to respond to stress factors, to take up nutrients or to multiply under different conditions. By forming complex intracellular networks such as biofilms, Candida spp. become not only more refractive to antifungal therapies but also more prone to cause disease. The inter-microbial interactions can enhance the virulence of a strain. In vivo, the fungal cells face a multitude of challenges and, as a result, they develop complex strategies serving one ultimate goal: survival. This review presents the virulence factors of the most important Candida spp., contributing to a better understanding of the onset of candidiasis and raising awareness of the highly complex interspecies interactions that can change the outcome of the disease.

Antibacterial and Antioxidant Potential of Silver Nanoparticles Biosynthesized Using the Spruce Bark Extract.

Biosynthesized silver nanoparticles (AgNPs) are widely used in Pharmacy and Medicine. In particular, AgNPs synthesized and mediated by plant extracts have shown topossess several biological activities. In the present study, AgNPs were synthesized using Picea abies L. stem bark extract as reducing agent. Factors, such as metal ion solution, pH, and time, which play a role in the AgNPs synthesis, were assessed. The synthesized AgNPs were characterized by Ultraviolet-Visible Spectrometry, Fourier transform infrared spectroscopy, and Transmission Electron Microscopy (TEM). Further, the study has been extended to evaluate the antimicrobial and antioxidant activity of AgNPs. The broad peak obtained at 411-475 nm (UV-Vis spectroscopy), and the color change pattern, confirmed the synthesis of AgNPs. TEM results showed spherical or rarely polygonal AgNPs with an average size of 44 nm at pH = 9. The AgNPs showed antioxidant activity and antibacterial effect against human pathogenic Gram-positive and Gram-negative bacteria. The results show that spruce bark extract is suitable for obtaining AgNPs, with antibacterial and antioxidant activity.

Investigation of In Vitro Antioxidant and Antibacterial Potential of Silver Nanoparticles Obtained by Biosynthesis Using Beech Bark Extract.

Green synthesis is one of the rapid and best ways for silver nanoparticles (AgNP) synthesis. In the present study, synthesis and bioactivity of AgNPs has been demonstrated using water beech (Fagus sylvatica L.) bark extract. The physical and chemical factors such as time, metal ion solution, and pH, which play a vital role in the AgNPs synthesis, were assessed. The AgNPs were characterized by ultraviolet-visible (UV-Vis) spectrometry, Fourier transform infrared spectroscopy (FT-IR), and transmission electron microscopy (TEM). Antioxidant and antimicrobial activity of the obtained AgNPs was evaluated. AgNPs were characterized by color change pattern, and the broad peak obtained at 420-475 nm with UV-Vis confirmed the synthesis of AgNPs. FT-IR results confirmed that phenols and proteins of beech bark extract are mainly responsible for capping and stabilization of synthesized AgNPs. TEM micrographs showed spherical or rarely polygonal and triangular particles with an average size of 32 nm at pH = 9, and 62 nm at pH = 4. Furthermore, synthesized AgNPs were found to exhibit antioxidant activity and have antibacterial effect against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Pseudomonas aeruginosa. These results indicate that bark extract of F. sylvatica L. is suitable for synthesizing stable AgNPs, which act as an excellent antimicrobial agent.

New perspectives on the nutritional factors influencing growth rate of Candida albicans in diabetics. An in vitro study.

BACKGROUND: The link between Candida albicans and diabetes mellitus is well-acknowledged, but incompletely elucidated. OBJECTIVES: The purpose of this study is to assess the growth rate of C. albicans (CA) in the presence of different concentrations of glucose and fructose, two of the main pathophysiologic and nutritionally relevant sugars in diabetic patients, in order to obtain a better understanding of the nutrient acquisition strategy and its possible relation to the hyperglycemic status of diabetic patients. METHODS: The effects of different concentrations of glucose and fructose (1000 mg%, 500 mg%, 250 mg% and 100 mg% w/v) on the growth rate of CA have been studied by flow-cytometry. FINDINGS: We found that glucose concentration is directly related to CA growth, which may be linked to the frequent yeast infections that occur in non-controlled diabetic patients; we also show that fructose inhibits CA growth rate. MAIN CONCLUSIONS: As a consequence of our hypothesis, the study demonstrates that fructose-containing food may prevent the development of candidiasis, at least in oral sites.

New perspectives on the nutritional factors influencing growth rate of Candida albicans in diabetics. An in vitro study

BACKGROUND The link between Candida albicans and diabetes mellitus is well-acknowledged, but incompletely elucidated. OBJECTIVES The purpose of this study is to assess the growth rate of C. albicans (CA) in the presence of different concentrations of glucose and fructose, two of the main pathophysiologic and nutritionally relevant sugars in diabetic patients, in order to obtain a better understanding of the nutrient acquisition strategy and its possible relation to the hyperglycemic status of diabetic patients. METHODS The effects of different concentrations of glucose and fructose (1000 mg%, 500 mg%, 250 mg% and 100 mg% w/v) on the growth rate of CA have been studied by flow-cytometry. FINDINGS We found that glucose concentration is directly related to CA growth, which may be linked to the frequent yeast infections that occur in non-controlled diabetic patients; we also show that fructose inhibits CA growth rate. MAIN CONCLUSIONS As a consequence of our hypothesis, the study demonstrates that fructose-containing food may prevent the development of candidiasis, at least in oral sites.

Health Threats from Contamination of Spices Commercialized in Romania: Risks of Fungal and Bacterial Infections.

BACKGROUND: The study of fungal contamination in food and mycotoxicoses is a priority today, both internationally and nationally. The purpose of this study is to have a general view over the quality of the most common spices that are sold in Romanian markets, by assessing the degree of fungal, bacterial and mycotoxin contamination in pepper and chili powders. METHODS: We tested four types of spices: white pepper, black pepper, sweet and hot chili powders from 12 different distributing companies, summing a total of 35 sample types. The fungal and bacterial load was assessed by Standard Plate Count, while the mycotoxin content by High-performance liquid chromatography. Environmental conditions (humidity, pH) and the selling price for each product were also followed. RESULTS: Fungi were observed in 72.7% of black pepper samples, 33.3% in white pepper, 30% in sweet chili and 25% in hot chili products. The most common isolated fungus was Aspergillus spp., while Rhizopus, Mucor, Fusarium, Penicillium, Absidia species were found, in smaller percentage. Four producers (44.4%) presented fungal contamination of over 10^3 CFU/g and two producers (22.2%) presented no fungal contamination in their products. Bacterial contamination was found in 85.7% of the tested products, consisting mostly in Bacillus spp. Aflatoxin B1 was present in all the tested products, mostly in black pepper (mean value 126.3 ng/g); Ochratoxin A was present in sweet chili (mean value 328 ng/g) and Zearalenone in hot chili (mean value 604 ng/g) and sweet chili (mean value 382 ng/g). CONCLUSION: All spices presented either fungal contamination, mycotoxin contamination, or both. The high humidity and the high pH of spices represent favorable conditions for fungal growth. The selling price was partly related to the physic-chemical conditions and microbiological quality of the spices.

Active surveillance scheme in three Romanian hospitals reveals a high prevalence and variety of carbapenamase-producing Gram-negative bacteria: a pilot study, December 2014 to May 2015.

We report the findings of an active surveillance scheme for detection of asymptomatic carriers with carbapenemase-producing Gram-negative bacteria (CP-GNB) in Romanian hospitals. During a pilot study from December 2014 to May 2015, faecal cultures were screened in three hospitals (two large, one medium-size) for patients newly admitted to selected wards or inpatients transferred from other wards to an intensive-care unit. The study revealed a high prevalence of CP-GNB detected in 22/27 and 28/38 of the carbapenem non-susceptible isolates from Hospitals 1 and 3, respectively. CP-GNB identified through faecal screening included NDM-1-producing Serratia marcescens and Klebsiella pneumoniae, OXA-48-producing K. pneumoniae and OXA-23-producing Acinetobacter baumannii. The distribution of the CP-GNB varied between the hospitals, with NDM-1-producing S. marcescens and K. pneumoniae being prevalent in the north-central part of the country and OXA-23/24-producing A. baumannii, OXA-48-producing K.pneumoniae, Morganella morganii and VIM-2-producing Escherichia coli/Pseudomonas aeruginosa detected in the north-east of the country. Conjugation studies showed that carbapenem resistance was transferable and PCR-based replicon typing identified blaNDM-1 on IncFIIs in S. marcescens and K. pneumoniae from Hospital 1 and blaOXA-48 on IncL plasmids in all Klebsiella spp. isolates from Hospitals 1 and 3. Our findings underline the importance of active surveillance for detection of CP-GNB asymptomatic faecal carriers and suggest a likely endemic spread of CP-GNB in Romania.

Clinical and histological correlations in alveolar bone osteosynthesis using biological materials of cow origin.

Periodontitis is a set of inflammatory diseases affecting the periodontium, i.e., the tissues that surround and support the teeth. Periodontitis involves progressive loss of the alveolar bone around the teeth, and if left untreated, can lead to the loosening and subsequent loss of teeth and is one of the most common diseases worldwide. Modern techniques of treatment consist of guided bone regeneration, in cases of massive bone loss. We present a case of a middle age male with no risk factors, suffering from infected chronic marginal periodontitis with III/IV degree of mobility at the lower right canine and III degree of mobility at the lower incisors. X-ray exam reveals massive bone resorption in the anterior part of the mandible, especially in the right canine area. Because the buccal and lingual cortical bone were missing in the canine area, it was impossible for the bone to heal after the extraction of 4.3., without bone augmentation. The histopathological exam revealed the aspect of an organic protein matrix, partially calcified and the presence of some isolated cells with osteocytes like morphology. The difficulties of this procedure are to position and to close the flap without tension in order to facilitate the healing and bone cells formation - osteoblasts and osteocytes. The bone augmentation using biological material proved to be successful in treating massive bone defects in order to insert dental implants.

[Prevalence of tuberculosis in Mures County, Romania, 2006-2008]. / Prevalenta tuberculozei în judetul Mures si în tara în perioada 2006-2008.

UNLABELLED: The aim of this study is analysis of tuberculosis prevalence in Mures county for a period of three years (2006-2008). MATERIAL AND METHOD: We monitored the prevalence of tuberculosis cases in the Mures area recorded at Tg. Mures TB Dispensary, the new cases and readmitted cases, chronic or abandonment, separately for children and adults, evolving for 2006-2008, data compared with national registration. RESULTS: From 2006 to 2008, frequency of TB illness in our area decreased instead of the share of patients with positive results (BK+) which have increased, associated with an amplified risk. In our study the assessment during the three years showed a decrease of the total number of TB cases, instead of increased frequency of new cases of acute than those of chronic, increased cases of adults against children and cases of failed healing. Number of new cases of TB in Mures urban areas, by years, showed the same decreasing trend for cities monitored, except Tg. Mures where the number of new cases recorded in 2008 was higher than in 2006. CONCLUSION: TB control programs in order to be effective, must be conducted throughout the all country, to be permanent, to the satisfaction of the population and to integrated structure of community health.

[Bacterial etiology of the conditions associated with bacteremia/septicemia during the years 2006-2007]. / Etiologia bacteriana a afectiunilor asociate cu bacteriemie/septicemie in perioada 2006-2007.

PURPOSE: Evaluation of the etiology of bacterial diseases associated with bacteremia/ sepsis in the Emergency County Hospital of Tg. Mures during 2006-2007. MATERIALS AND METHODS: Blood samples of 684 patients hospitalized in Emergency County Hospital of Tg. Mures during January 2006 - December 2007 were analyzed. The isolated germs have been identified according to standard methods. The blood culture results were centralized and processed in electronic databases. RESULTS: In 2006 and 2007 the frequency of identified germs from blood cultures remained unchanged: the predominant germs are coagulase-negative staphylococci, followed by enterobacteria (Klebsiella spp prevailing), S. aureus (mostly methicillin-resistant (MRSA)) and other bacteria. CONCLUSIONS: The main bacteria isolated from blood culture are staphylococci and enterobacteria. In medical sections undertaking invasive procedures, the infections with methicillin-resistant staphylococci are common.

[Cumulative antibiogram--means of following local resistance phenomena based on material gathered in the laboratory of microbiology]. / Antibiograma cumulativa--posibilitate de urmarire a fenomenelor de rezistenta locale bazata pe materialul laboratorului de bacteiologie.

The cumulative antibiogram (CA) represents a report which summarizes the susceptibility of commonly isolated microorganisms to usual antibiotics in a defined period of time. We have evaluated the local resistance data during the year 2007 by generating CA from the bacteriology laboratory's database in Mures County Emergency Clinical Hospital for 3 categories of microorganisms: Gram-positive cocci, Gram-negative enteric rods and non-fermenters. A total of 1382 individual antibiotic susceptibility reports were analysed. Gram-positive cocci were susceptible 100% to glycopeptides and oxazolidinones. Fifty percent of S. aureus isolates were methicillin-resistant. More than 90% of S. aureus and MRSA (methicillin-resistant S. aureus) isolates were susceptible to trimethoprim-sulfamethoxazole. Gram-negative enteric rods were 100% susceptible to carbapenems. The susceptibility of Escherichia coli and Klebsiella pneumoniae to broad spectrum cephalosporins was 72% and 26%, respectively. The activity of carbapenems against Acinetobacter spp. and Pseudomonas aeruginosa was 43% and 35%, respectively. Colistin was active in 100% of the isolated nonfermenters. CA is a useful tool for evaluation of local resistance data but careful interpretation of generated data is required in order to avoid under- or overestimation of resistance.

[Evaluation of iron, fluoride and iodine concentrations in underground water in some counties in Romania]. / Evaluarea concentratiei de fier, fluor si iod din apa subterani din câteva judete ale României.

AIM: To compare the underground water mineral composition in iron, fluorine and iodine from different sources, with variable locations in Romania. METHODS: It is a lab study based on Hanna Instruments C99 colorimeter used for our tests, that follows the levels of mineral parameters from water samples from several underground water supplie sources from 10 different romanian counties. RESULTS: It were significant differences between sources from different counties, and also from different sources in the same county, regarding minerals levels in water and also comparing to admitted levels. The most various data were related to fluorine and iodine levels in water samples and risk of exposure by deficiency. CONCLUSION: It is important to know the importance of supply water monitoring, to evaluate both the drinking water quality parameters and mineral composition, in order to estimate the influence on health status of long shut consumer population.