Impact of Aries River Contaminants on Algae and Plants.

The Aries River (Western Romania) represents one of the most important affluents of the Mures River, with great significance in the Mures Tisza basin. The environmental quality of the Aries basin is significantly affected by both historic mining activities and contemporary impacts. Thus, an evaluation of the effects of the main contaminants found in water (organochlorine pesticides-OCPs, monocyclic aromatic hydrocarbons-MAHs, polycyclic aromatic hydrocarbons-PAHs, and metals) on cyanobacteria and plants was performed. Among OCPs, hexachlorocyclohexane isomers, dichlorodiphenyltrichloroethane, and derivatives were detected in plants while admissible concentrations were detected in water. Among MAHs, high levels of benzene were detected both in water and in plants. The levels of PAHs exceeded the allowable values in all samples. Increased concentrations of metals in water were found only at Baia de Aries, but in plants, all metal concentrations were high. The pH, nitrates, nitrites, and phosphates, as well as metals, pesticides, and aromatic hydrocarbons, influenced the physiological characteristics of algae, test plants, and aquatic plants exposed to various compounds dissolved in water. Considering that the Aries River basin is the site of intense past mining activities, these data provide information about the impact on water quality as a consequence of pollution events.

Phytochemical Characterization and Antimicrobial Activity of Several Allium Extracts.

Microbial infections affect both the human population and animals. The appearance of more and more microbial strains resistant to classical treatments led to the need to develop new treatments. Allium plants are known for their antimicrobial properties due to their high content of thiosulfinates, especially allicin, polyphenols or flavonoids. The hydroalcoholic extracts of six Allium species obtained by cold percolation were analyzed regarding their phytochemical compounds and antimicrobial activity. Among the six extracts, Allium sativum L. and Allium ursinum L. have similar contents of thiosulfinates (approx. 300 µg allicin equivalents/g), and the contents of polyphenols and flavonoids were different between the tested species. The HPLC-DAD method was used to detail the phytochemical composition of species rich in thiosulfinates. A. sativum is richer in allicin (280 µg/g) than A. ursinum (130 µg/g). The antimicrobial activity of A. sativum and A. ursinum extracts against Escherichia coli, Staphylococcus aureus, Candida albicans and Candida parapsilosis can be correlated with the presence of large amounts of thiosulfinates. Both extracts have shown results against Candida species (inhibition zones of 20-35 mm) and against Gram-positive bacteria, Staphylococcus aureus (inhibition zones of 15-25 mm). These results demonstrate the antimicrobial effect of the extracts and suggest their use as an adjuvant treatment for microbial infections.

Double-Network Chitosan-Based Hydrogels with Improved Mechanical, Conductive, Antimicrobial, and Antibiofouling Properties.

In recent years, the antimicrobial activity of chitosan-based hydrogels has been at the forefront of research in wound healing and the prevention of medical device contamination. Anti-infective therapy is a serious challenge given the increasing prevalence of bacterial resistance to antibiotics as well as their ability to form biofilms. Unfortunately, hydrogel resistance and biocompatibility do not always meet the demands of biomedical applications. As a result, the development of double-network hydrogels could be a solution to these issues. This review discusses the most recent techniques for creating double-network chitosan-based hydrogels with improved structural and functional properties. The applications of these hydrogels are also discussed in terms of tissue recovery after injuries, wound infection prevention, and biofouling of medical devices and surfaces for pharmaceutical and medical applications.

Correlation between CRISPR Loci Diversity in Three Enterobacterial Taxa.

CRISPR-Cas is an adaptive immunity system of prokaryotes, composed of CRISPR arrays and the associated proteins. The successive addition of spacer sequences in the CRISPR array has made the system a valuable molecular marker, with multiple applications. Due to the high degree of polymorphism of the CRISPR loci, their comparison in bacteria from various sources may provide insights into the evolution and spread of the CRISPR-Cas systems. The aim of this study was to establish a correlation between the enterobacterial CRISPR loci, the sequence of direct repeats (DR), and the number of spacer units, along with the geographical origin and collection source. For this purpose, 3474 genomes containing CRISPR loci from the CRISPRCasdb of Salmonella enterica, Escherichia coli, and Klebsiella pneumoniae were analyzed, and the information regarding the isolates was recorded from the NCBI database. The most prevalent was the I-E CRISPR-Cas system in all three studied taxa. E. coli also presents the I-F type, but in a much lesser percentage. The systems found in K. pneumoniae can be classified into I-E and I-E*. The I-E and I-F systems have two CRISPR loci, while I-E* has only one locus upstream of the Cas cluster. PCR primers have been developed in this study for each CRISPR locus. Distinct clustering was not evident, but statistically significant relationships occurred between the different CRISPR loci and the number of spacer units. For each of the queried taxa, the number of spacers was significantly different (p < 0.01) by origin (Africa, Asia, Australia and Oceania, Europe, North America, and South America) but was not linked to the isolation source type (human, animal, plant, food, or laboratory strains).

Molecular Typing Reveals Environmental Dispersion of Antibiotic-Resistant Enterococci under Anthropogenic Pressure.

As a consequence of global demographic challenges, both the artificial and the natural environment are increasingly impacted by contaminants of emerging concern, such as bacterial pathogens and their antibiotic resistance genes (ARGs). The aim of this study was to determine the extent to which anthropogenic contamination contributes to the spread of antibiotic resistant enterococci in aquatic compartments and to explore genetic relationships among Enterococcus strains. Antimicrobial susceptibility testing (ampicillin, imipenem, norfloxacin, gentamycin, vancomycin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole) of 574 isolates showed different rates of phenotypic resistance in bacteria from wastewaters (91.9-94.4%), hospital effluents (73.9%), surface waters (8.2-55.3%) and groundwater (35.1-59.1%). The level of multidrug resistance reached 44.6% in enterococci from hospital effluents. In all samples, except for hospital sewage, the predominant species were E. faecium and E. faecalis. In addition, E. avium, E. durans, E. gallinarum, E. aquimarinus and E. casseliflavus were identified. Enterococcus faecium strains carried the greatest variety of ARGs (blaTEM-1, aac(6')-Ie-aph(2″), aac(6')-Im, vanA, vanB, ermB, mefA, tetB, tetC, tetL, tetM, sul1), while E. avium displayed the highest ARG frequency. Molecular typing using the ERIC2 primer revealed substantial genetic heterogeneity, but also clusters of enterococci from different aquatic compartments. Enterococcal migration under anthropogenic pressure leads to the dispersion of clinically relevant strains into the natural environment and water resources. In conclusion, ERIC-PCR fingerprinting in conjunction with ARG profiling is a useful tool for the molecular typing of clinical and environmental Enterococcus species. These results underline the need of safeguarding water quality as a strategy to limit the expansion and progression of the impending antibiotic-resistance crisis.

Descriptive Analysis of Circulating Antimicrobial Resistance Genes in Vancomycin-Resistant Enterococcus (VRE) during the COVID-19 Pandemic.

COVID-19 offers ideal premises for bacteria to develop antimicrobial resistance. In this study, we evaluated the presence of several antimicrobial resistance genes (ARG) in vancomycin-resistant Enterococcus (VRE) isolated from rectal swabs from patients at a hospital in Cluj-Napoca, Romania. Rectal swabs were cultivated on CHROMID® VRE (bioMérieux, Marcy-l' Étoile, France) and positive isolates were identified using MALDI-TOF Mass Spectrometry (Bruker Daltonics, Bremen, Germany) and further analyzed using the PCR technique for the presence of the following ARGs: van A, van B, tet(M), tet(L), ermB, msrA, mefA, aac(6')-Im, aph(2)-Ib, ant(4')-Ia, sul1, sul2, sul3, and NDM1. We isolated and identified 68 isolates of Enterococcus faecium and 11 isolates of Enterococcus faecalis. The molecular analysis showed 66 isolates positive for the vanA gene and eight positive for vanB. The most frequent association of ARG in VRE was vanA-tet(M)-ermB. There was no statistically significant difference between Enterococcus faecium and Enterococcus faecalis regarding ARGs. Our work proves that during the COVID-19 pandemic, highly resistant isolates of Enterococcus were present in patients in the intensive care unit; thus, better healthcare policies should be implemented for the management and control of these highly resistant isolates in the future.

Inherent and Composite Hydrogels as Promising Materials to Limit Antimicrobial Resistance.

Antibiotic resistance has increased significantly in the recent years, and has become a global problem for human health and the environment. As a result, several technologies for the controlling of health-care associated infections have been developed over the years. Thus, the most recent findings in hydrogel fabrication, particularly antimicrobial hydrogels, could offer valuable solutions for these biomedical challenges. In this review, we discuss the most promising strategies in the development of antimicrobial hydrogels and the application of hydrogels in the treatment of microbial infections. The latest advances in the development of inherently and composite antimicrobial hydrogels will be discussed, as well as hydrogels as carriers of antimicrobials, with a focus on antibiotics, metal nanoparticles, antimicrobial peptides, and biological extracts. The emergence of CRISR-Cas9 technology for removing the antimicrobial resistance has led the necessity of new and performant carriers for delivery of the CRISPR-Cas9 system. Different delivery systems, such as composite hydrogels and many types of nanoparticles, attracted a great deal of attention and will be also discussed in this review.

CRISPR-Cas System: The Powerful Modulator of Accessory Genomes in Prokaryotes.

Being frequently exposed to foreign nucleic acids, bacteria and archaea have developed an ingenious adaptive defense system, called CRISPR-Cas. The system is composed of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) array, together with CRISPR (cas)-associated genes. This system consists of a complex machinery that integrates fragments of foreign nucleic acids from viruses and mobile genetic elements (MGEs), into CRISPR arrays. The inserted segments (spacers) are transcribed and then used by cas proteins as guide RNAs for recognition and inactivation of the targets. Different types and families of CRISPR-Cas systems consist of distinct adaptation and effector modules with evolutionary trajectories, partially independent. The origin of the effector modules and the mechanism of spacer integration/deletion is far less clear. A review of the most recent data regarding the structure, ecology, and evolution of CRISPR-Cas systems and their role in the modulation of accessory genomes in prokaryotes is proposed in this article. The CRISPR-Cas system's impact on the physiology and ecology of prokaryotes, modulation of horizontal gene transfer events, is also discussed here. This system gained popularity after it was proposed as a tool for plant and animal embryo editing, in cancer therapy, as antimicrobial against pathogenic bacteria, and even for combating the novel coronavirus - SARS-CoV-2; thus, the newest and promising applications are reviewed as well.

Antibiotic Resistance in Pseudomonas spp. Through the Urban Water Cycle.

Selection and dissemination of resistant bacteria and antibiotic resistance genes (ARGs) require a deeper understanding since antibiotics are permanently released to the environment. The objective of this paper was to evaluate the phenotypic resistance of 499 isolates of Pseudomonas spp. from urban water sources, and the prevalence of 20 ARGs within those isolates. Resistance to penicillins, cephalosporins, carbapenems, quinolones, macrolides, and tetracyclines was mainly observed in the hospital effluent, municipal wastewater and river water downstream the city. Resistant strains were frequently identified as P. aeruginosa and P. putida. P. aeruginosa isolates were mostly resistant to cefepime, ceftazidime, imipenem, and gentamycin, while P. putida strains were especially resistant to piperacillin-tazobactam. ARGs such as blaTEM-1, blaSHV-1, blaPER-1, blaAmpC, blaVIM-1, PstS, qnrA, qnrB, ermB, tetA, tetB and tetC have been detected. The blaAmpC gene was found in P. aeruginosa, while blaTEM-1 and blaPER-1 genes were found in P. putida. Class 1 integron integrase gene was found in 6.81% of the Pseudomonas isolates.

Antibiotic resistance profiling of pathogenic Enterobacteriaceae from Cluj-Napoca, Romania.

INTRODUCTION: Members of the family Enterobacteriaceae are commonly identified in the clinical laboratory, being responsible for a substantial range of infections. This study aimed to investigate phenotypic and genotypic resistance traits in pathogenic Enterobacteriaceae isolated from outpatients in Cluj-Napoca, Romania. METHODS: Pathogenic Enterobacteriaceae were isolated from urinary tract infections, wound infections and persistent diarrhea in a private laboratory from Cluj-Napoca, Romania. Bacterial strains were biochemically identified and subjected to antimicrobial susceptibility testing by disk diffusion. The carriage of antibiotic resistance genes and of class 1 integron were assessed by PCR. RESULTS: E. coli and Enterobacter spp. were the most prevalent pathogens. High levels of resistance were observed against folate pathway inhibitors (74%), fluoroquinolones (49%) and penicillins (44%). The incidence of carbapenem resistance was 3%. The strains displaying phenotypic resistance were able to produce ß-lactamase enzymes encoded by bla TEM, bla TEM-1, bla SHV-1 and bla CTX-M, aminoglycoside modifying enzymes due to the carriage of aac(3)-IIIa, aac(6')-II and aac(6')-Ie-aph(2"), to possess fluoroquinolones resistance due to qnrS DNA gyrase protection proteins and resistance to folate pathway inhibitors due to dihydropteroate synthases encoded by sul1, sul2 and sul3 genes. The high frequency of intI1 integrase was associated to sulphonamide resistance (r=0.48; p<0.001) and also to fluoroquinolone resistance (r=0.27; p=0.011), but no significant associations in the co-occurrence of specific antibiotic resistance genes and intI1 were found in pathogenic Enterobacteriaceae. CONCLUSIONS: An important proportion of pathogenic Enterobacteriaceae were multidrug resistant, due to a wide diversity of mechanisms encoding genetic resistance.

Exploring the Role of Coliform Bacteria in Class 1 Integron Carriage and Biofilm Formation During Drinking Water Treatment.

This study investigates the role of coliforms in the carriage of class 1 integron and biocide resistance genes in a drinking water treatment plant and explores the relationship between the carriage of such genes and the biofouling abilities of the strain. The high incidence of class 1 integron and biocide resistance genes (33.3 % of the isolates) highlights the inherent risk of genetic contamination posed by coliform populations during drinking water treatment. The association between the presence of intI1 gene and qac gene cassettes, especially qacH, was greater in biofilm cells. In coliforms recovered from biofilms, a higher frequency of class 1 integron elements and higher diversity of genetic patterns occurred, compared to planktonic cells. The coliform isolates under the study proved to mostly carry non-classical class 1 integrons lacking the typical qacEΔ1/sul1 genes or a complete tni module, but bearing the qacH gene. No link was found between the carriage of integron genes and the biofouling degree of the strain, neither in aerobic or in anaerobic conditions. Coliform bacteria isolated from established biofilms rather adhere in oxygen depleted environments, while the colonization ability of planktonic cells is not significantly affected by oxygen availability.

Microbiological contamination and resistance genes in biofilms occurring during the drinking water treatment process.

Biofilms are the predominant mode of microbial growth in drinking water systems. A dynamic exchange of individuals occurs between the attached and planktonic populations, while lateral gene transfer mediates genetic exchange in these bacterial communities. Integrons are important vectors for the spread of antimicrobial resistance. The presence of class 1 integrons (intI1, qac and sul genes) was assessed in biofilms occurring throughout the drinking water treatment process. Isolates from general and specific culture media, covering a wide range of environmental bacteria, fecal indicators and opportunistic pathogens were tested. From 96 isolates tested, 9.37% were found to possess genetic determinants of putative antimicrobial resistance, and these occurred in both Gram-positive and Gram-negative bacteria. Class 1 integron integrase gene was present in 8.33% of bacteria, all positive for the qacEΔ1 gene. The sul1 gene was present in 3.12% of total isolates, representing 37.5% of the class 1 integron positive cells. The present study shows that biofilm communities in a drinking water treatment plant are a reservoir of class 1 integrons, mainly in bacteria that may be associated with microbiological contamination. Eight out of nine integron bearing strains (88.8%) were identified based on 16S rRNA gene sequencing as either enteric bacteria or species that may be connected to animal and anthropogenic disturbance.

Poly-ß-hydroxybutyrate accumulation in bacterial consortia from different environments.

The aim of the present study was to examine soil samples from various vegetation zones in terms of physicochemical properties, microbial communities, and isolation and identification (by polymerase chain reaction and transmission electron microscopy) of bacteria producing poly-ß-hydroxybutyrates (PHBs). Soil samples were analysed originating from zones with heterogeneous environmental conditions from the Romanian Carpathian Mountains (mountain zone with alpine meadow, karstic zone with limestone meadow, hill zone with xerophilous meadow, and flood plain zone with hygrophilic meadow). Different bacterial groups involved in the nitrogen cycle (aerobic mesophilic heterotrophs, ammonifiers, denitrifiers, nitrifiers, and free nitrogen-fixing bacteria from Azotobacter genus) were analysed. Soil biological quality was assessed by the bacterial indicator of soil quality, which varied between 4.3 and 4.7. A colony polymerase chain reaction technique was used for screening PHB producers. With different primers, specific bands were obtained in all the soil samples. Some wild types of Azotobacter species were isolated from the 4 studied sites. Biodegradable polymers of PHB were assessed by negative staining in transmission electron microscopy. The maximum PHB granules density was obtained in the strains isolated from the xerophilous meadow (10-18 granules/cell), which was the most stressful environment from all the studied sites, as the physicochemical and microbiological tests proved.