The RdRp genotyping of SARS-CoV-2 isolated from patients with different clinical spectrum of COVID-19.

BACKGROUND: The evolution of SARS-CoV-2 has been observed from the very beginning of the fight against COVID-19, some mutations are indicators of potentially dangerous variants of the virus. However, there is no clear association between the genetic variants of SARS-CoV-2 and the severity of COVID-19. We aimed to analyze the genetic variability of RdRp in correlation with different courses of COVID-19. RESULTS: The prospective study included 77 samples of SARS-CoV-2 isolated from outpatients (1st degree of severity) and hospitalized patients (2nd, 3rd and 4th degree of severity). The retrospective analyses included 15,898,266 cases of SARS-CoV-2 genome sequences deposited in the GISAID repository. Single-nucleotide variants were identified based on the four sequenced amplified fragments of SARS-CoV-2. The analysis of the results was performed using appropriate statistical methods, with p < 0.05, considered statistically significant. Additionally, logistic regression analysis was performed to predict the strongest determinants of the observed relationships. The number of mutations was positively correlated with the severity of the COVID-19, and older male patients. We detected four mutations that significantly increased the risk of hospitalization of COVID-19 patients (14676C > T, 14697C > T, 15096 T > C, and 15279C > T), while the 15240C > T mutation was common among strains isolated from outpatients. The selected mutations were searched worldwide in the GISAID database, their presence was correlated with the severity of COVID-19. CONCLUSION: Identified mutations have the potential to be used to assess the increased risk of hospitalization in COVID-19 positive patients. Experimental studies and extensive epidemiological data are needed to investigate the association between individual mutations and the severity of COVID-19.

Comparative study of virulence potential, phylogenetic origin, CRISPR-Cas regions and drug resistance of Escherichia coli isolates from urine and other clinical materials.

Introduction: Urinary tract infections (UTI), among which the main etiological factor is uropathogenic Escherichia coli (UPEC, E. coli), remain an important issue for clinicians. The aim of the study was to demonstrate clear differences in the pathogenic properties of urine-derived E. coli compared to other extraintestinal E. coli clinical isolates (derived from: blood, lower respiratory tracts, sputum, reproductive tract, body fluids, perianal pus, other pus, wound, postoperative wound and other sources). Methods: The collection of 784 E. coli isolates was collected from various materials of hospitalized patients. They were analyzed in terms of virulence-associated genes (papC, sfaD/sfaE, cnf1, usp., fimG/H, hlyA), belonging to phylogenetic groups and the presence of CRISPR-Cas regions using PCR. In addition, the epidemiological data and the antibiotic resistance profiles provided by the hospital's microbiology department were included for statistical analyses. Results: Urine-derived E. coli showed significantly greater virulence potential compared to other isolates, but they were generally unremarkable in terms of drug resistance. The isolates most often belonged to phylogenetic group B2. Drug resistance was negatively correlated with CRISPR 2 presence and high average virulence score, but positively correlated with CRISPR 4 presence. To the best of our knowledge, we are the first to report significant differences in sputum-derived isolates-they revealed the lowest virulence potential and, at the same time, the highest drug resistance. Discussion: In conclusion, we demonstrated significant differences of urinary-derived E. coli compared to other clinical E. coli isolates. We would like to suggest excluding penicillins from use in E. coli infection at this time and monitoring strains with a high pathogenicity potential.

Discriminating macromolecular interactions based on an impedimetric fingerprint supported by multivariate data analysis for rapid and label-free Escherichia coli recognition in human urine.

This manuscript presents a novel approach to address the challenges of electrode fouling and highly complex electrode nanoarchitecture, which are primary concerns for biosensors operating in real environments. The proposed approach utilizes multiparametric impedance discriminant analysis (MIDA) to obtain a fingerprint of the macromolecular interactions on flat glassy carbon surfaces, achieved through self-organized, drop-cast, receptor-functionalized Au nanocube (AuNC) patterns. Real-time monitoring is combined with singular value decomposition and partial least squares discriminant analysis, which enables selective identification of the analyte from raw impedance data, without the use of electric equivalent circuits. As a proof-of-concept, the authors demonstrate the ability to detect Escherichia coli in real human urine using an aptamer-based biosensor that targets RNA polymerase. This is significant, as uropathogenic E. coli is a difficult-to-treat pathogen that is responsible for the majority of hospital-acquired urinary tract infection cases. The proposed approach offers a limit of detection of 11.3 CFU/mL for the uropathogenic E. coli strain No. 57, an analytical range in all studied concentrations (up to 105 CFU/mL), without the use of antifouling strategies, yet not being specific vs other E.coli strain studied (BL21(DE3)). The MIDA approach allowed to identify negative overpotentials (-0.35 to -0.10 V vs Ag/AgCl) as most suitable for the analysis, offering over 80% sensitivity and accuracy, and the measurement was carried out in just 2 min. Moreover, this approach is scalable and can be applied to other biosensor platforms.

The Levels of Anti-SARS-CoV-2 Spike Protein IgG Antibodies Before and After the Third Dose of Vaccination Against COVID-19.

Purpose: The COVID-19 pandemic has been going on for almost three years, and so far, many preventive and therapeutic strategies have been developed. The issue of subsequent booster vaccinations is currently being discussed. We aimed to analyze how the third dose of vaccination against COVID-19 correlates with the dynamics of IgG anti-SARS-CoV-2 spike protein antibody levels in a group of healthy people. Patients and Methods: The prospective study included 93 participants before and after a second booster of COVID-19 vaccination, from whom 4 blood samples were collected at intervals. The levels of IgG anti-SARS-CoV-2 in serum were identified using the chemiluminescent immunoassay specific for the receptor-binding domain (RBD) of the S1 protein. The analysis of the results was performed using appropriate statistical methods, considering p <0.05 as a statistically significant value. Results: The IgG levels were significantly higher and less diverse after the same follow-up time from the second booster vaccination compared to the first booster. The antibody levels were positively correlated with female, healthcare workers, the elderly and participants with a negative COVID-19 history. Furthermore, the increase in IgG antibodies after the second booster vaccination correlated inversely with the baseline level of antibodies before the vaccination. The latest results showed that antibody levels dropped 1.5-fold after approx. 10 months from the second booster vaccination but still remained at a protective level. Conclusion: Booster vaccinations seem to better stimulate immune memory, and in the case of borderline IgG level induces the greatest increase in antibodies. It is worth considering the individual parameters of patients and measuring antibodies before vaccination.

Pathogenic Factors Correlate With Antimicrobial Resistance Among Clinical Proteus mirabilis Strains.

Proteus mirabilis is the third most common etiological factor of urinary tract infection. It produces urease, which contributes to the formation of a crystalline biofilm, considered to be one of the most important virulence factors of P. mirabilis strains, along with their ability to swarm on a solid surface. The aim of this study was to analyze the pathogenic properties of two selected groups of clinical P. mirabilis isolates, antimicrobial susceptible and multidrug resistant (MDR), collected from hospitals in different regions in Poland. The strains were examined based on virulence gene profiles, urease and hemolysin production, biofilm formation, and swarming properties. Additionally, the strains were characterized based on the Dienes test and antibiotic susceptibility patterns. It turned out that the MDR strains exhibited kinship more often than the susceptible ones. The strains which were able to form a stronger biofilm had broader antimicrobial resistance profiles. It was also found that the strongest swarming motility correlated with susceptibility to most antibiotics. The correlations described in this work encourage further investigation of the mechanisms of pathogenicity of P. mirabilis.

Fenugreek (Trigonella foenum-graecum L.) Seeds Dietary Supplementation Regulates Liver Antioxidant Defense Systems in Aging Mice.

Fenugreek seeds are widely used in Asia and other places of the world for their nutritive and medicinal properties. In Asia, fenugreek seeds are also recommended for geriatric populations. Here, we evaluated for the first time the effect of fenugreek seed feed supplementation on the liver antioxidant defense systems in aging mice. The study was conducted on 12-months aged mice which were given fenugreek seed dietary supplement. We evaluated the activities of various antioxidant defense enzymes such as superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx), and also estimated the phenolics and free radical scavenging properties in mice liver upon fenugreek supplementation. The estimation of SOD, GPx, and GR activities in aged mice liver revealed a significant (p < 0.01) difference among all the liver enzymes. Overall, this study reveals that fenugreek seed dietary supplementation has a positive effect on the activities of the hepatic antioxidant defense enzymes in the aged mice.

Mathematical Analysis of Induced Antibiotic Resistance Among Uropathogenic Escherichia coli Strains.

The aim of the study was a profound insight into the antibiotic resistance development in uropathogenic Escherichia coli strains with the use of some mathematical and statistical methods. During the previous study some antibiotics (amoxicillin, ciprofloxacin, gentamycin, and tobramycin) were applied to induce the resistance of E. coli strains, which led to the generation of ∼120 derivative strains with changed antibiotic susceptibility profiles. In this work, quantitative analysis was performed based on the strains defined as values of vectors of susceptibility for all the antibiotics' use. The Pearson correlation coefficient was used to define the correlation dissimilarity (distance) of the strains, which was further applied to hierarchical clustering. Analogously, the antibiotics were presented as vectors of susceptibility values of all the investigated strains. Correlation and cluster analysis were performed for antibiotics. The hclust method from the R system with the Ward method was used as a class agglomeration method. Mathematical analysis revealed two types of statistically relevant interactions-between antibiotics and derivative strains, as well as between the effect of individual antibiotics on the bacterial strains. These observed correlations can play a potential role for modeling uropathogenic E. coli (UPEC) resistant changes, based on the particular antibiotic used to initiate resistance development, or a model helping to predict drug resistance interactions in various UPEC strains. The obtained results can lead to development of much more sophisticated mathematical models, which, in turn, can be a potentially useful tool as a drug resistance trend predictor, both for clinicians and epidemiologists.

The hybrid allele 1 of carboxyl-ester lipase (CEL-HYB1) in Polish pediatric patients with chronic pancreatitis.

OBJECTIVES: It has previously been reported in a European case-control study with patients from Germany and France that CEL-HYB1, a hybrid allele of the carboxyl ester lipase (CEL) gene and its pseudogene CELP, increases susceptibility to chronic pancreatitis (CP). Here, we aimed to replicate this finding in Polish pediatric patients with CP. METHOD: The distribution of the CEL-HYB1 allele in a CP pediatric cohort (n = 147, median age at CP onset 7.6 years) with no history of alcohol/smoking abuse was compared with ethnically matched healthy controls (n = 500, median age 46 years). Screening was performed using long-range PCR followed by agarose gel-electrophoresis. RESULTS: We observed no significant difference in the carrier frequency of the CEL-HYB1 allele between CP patients (7/147, 4.8%) and controls (12/500, 2.4%; P = 0.16). CONCLUSIONS: This study found no statistically significant association between CEL-HYB1 and chronic pancreatitis in a cohort of Polish pediatric CP patients.

Ciprofloxacin, amoxicillin, and aminoglycosides stimulate genetic and phenotypic changes in uropathogenic Escherichia coli strains.

Antibiotic therapy and its consequences in bacterial and human aspects are widely investigated. Despite this, the emergence of new multidrug resistant bacteria is still a current problem. The scope of our work included the observation of changes among uropathogenic Escherichia coli strains after the treatment with a subinhibitory concentration of different antibiotics. The sensitive strains with or without virulence factors were incubated with amoxicillin, ciprofloxacin, gentamycin, or tobramycin. After each passage, the E. coli derivatives were compared to their wild types based on their susceptibility profiles, virulence genes, biofilm formations and the fingerprint profiles of PCR products amplified with using the (N)(6)(CGG)(4) primer. It turned out that antibiotics caused significant changes in the repertoire of bacterial virulence and biofilm formation, corresponding to acquired cross-resistance. The genomic changes among the studied bacteria were reflected in the changed profiles of the CGG-PCR products. In conclusion, the inappropriate application of antibiotics may cause a rapid rise of Multidrug Resistant (MDR) strains and give bacteria a chance to modulate their own pathogenicity. This phenomenon has been easily observed among uropathogenic E. coli strains and it is one of the main reasons for recurrent infections of the urinary tract.

Hydrogen peroxide stimulates uropathogenic Escherichia coli strains to cellulose production.

Reactive oxygen intermediates, such as hydrogen peroxide, are toxic molecules produced by immune cells in response to bacterial invasion into the host. Bacteria try to protect themselves against the immune system through specific properties such as biofilm formation. This phenomenon occurs also during urinary tract infections. Cellulose is an important factor of Escherichia coli biofilm and contributes to building a protective shield around bacterial cells upon the host immune response. In this study, we aimed to analyze the effect of hydrogen peroxide on the production of this biofilm component. To achieve this goal, 25 clinical E. coli strains isolated from patients with urinary tract infections were used. These bacterial strains were characterized based on their growth characteristics, their ability to form biofilm and their capacity to produce cellulose upon exposure to sub-lethal concentrations of hydrogen peroxide growth, and the biofilm formation of these strains was analyzed. Our results revealed that the analyzed uropathogenic E. coli strains slightly, but significantly, reduced growth and biofilm production upon hydrogen peroxide treatment. However, when separating these strains regarding their ability to produce cellulose, we found that general biofilm production was reduced but cellulose expression was induced upon peroxide treatment. This finding contributes to a better understanding of how bacterial biofilm formation is triggered and provides interesting insights into how uropathogenic E. coli protect themselves in an inhospitable environment.

The genetic background of antibiotic resistance among clinical uropathogenic Escherichia coli strains.

The spreading mechanisms of antibiotic resistance are related to many bacterial and environment factors. The overuse of antibiotics is leading to an unceasing emergence of new multidrug resistant strains. This problem also concerns uropathogenic Escherichia coli strains, which is the most common pathogen causing urinary tract infections. The aim of this study was the genetic analysis of antibiotic resistance in comparison to the phenotypic background of E. coli strains. The characterized collection of E. coli strains isolated 10 years ago from the urine samples of patients with urinary tract infections was used for antimicrobial susceptibility testing (the disc diffusion method) and analysis of antibiotic resistance genes (PCR reaction, sequencing). Additionally, the presence of ESBL strains was analyzed. Fourteen genes were associated with resistance to beta-lactams, aminoglycosides, sulfonamides and quinolones. The genetic analysis revealed that blaTEM-1 and sul2 were present in almost all of the studied strains. Other drug-resistance genes were very rare or non-existent. Otherwise, the phenotypic resistance to fluoroquinolones was well correlated with the genotypic background of the studied bacteria. The presence of particular genes and specific mutations indicate a high bacterial potential to multidrug resistance. On the other hand, it needs to be emphasized that the standard disk diffusion test for the routine antimicrobial susceptibility analysis is still the best way to estimate the current situation of bacterial drug-resistance.

A new look at the drug-resistance investigation of uropathogenic E. coli strains.

Bacterial drug resistance and uropathogenic tract infections are among the most important issues of current medicine. Uropathogenic Escherichia coli strains are the primary factor of this issue. This article is the continuation of the previous study, where we used Kohonen relations to predict the direction of drug resistance. The characterized collection of uropathogenic E. coli strains was used for microbiological (the disc diffusion method for antimicrobial susceptibility testing), chemical (ATR/FT-IR) and mathematical (artificial neural networks, Ward's hierarchical clustering method, the analysis of distributions of inhibition zone diameters for antibiotics, Cohen's kappa measure of agreement) analysis. This study presents other potential tools for the epidemiological differentiation of E. coli strains. It is noteworthy that ATR/FT-IR technique has turned out to be useful for the quick and simple identification of MDR strains. Also, diameter zones of resistance of this E. coli population were compared to the population of E. coli strains published by EUCAST. We observed the bacterial behaviors toward particular antibiotics in comparison to EUCAST bacterial collections. Additionally, we used Cohen's kappa to show which antibiotics from the same class are closely related to each other and which are not. The presented associations between antibiotics may be helpful in selecting the proper therapy directions. Here we present an adaptation of interdisciplinary studies of drug resistance of E. coli strains for epidemiological and clinical investigations. The obtained results may be some indication in deciding on antibiotic therapy.

Analysis of uropathogenic Escherichia coli biofilm formation under different growth conditions.

The ability to form different types of biofilm enables bacteria to survive in a harsh or toxic environment. Different structures of biofilms are related to different surfaces and environment of bacterial growth. The aim of this study was analysis of the biofilm formation of 115 clinical uropathogenic Escherichia coli strains under different growth conditions: surface for biofilm formation, medium composition and time of incubation. The biofilm formation after 24 h, 48 h, 72 h and 96 h was determined spectrophotometrically (A531) after crystal violet staining and it was correlated with bacterial growth (A600). The live and dead cells in biofilm structures was also observed on the glass surface by an epi-fluorescence microscope. Additionally, the presence of rpoS, sdiA and rscA genes was analyzed. The statistical significance was estimated by paired T-test. The observed biofilms were different for each particular strain. The biofilm formation was the highest in the rich medium (LB) after 24 h and its level hasn't changed in time. When biofilm level was compared to bacterial growth (relative biofilm) - it was higher in a minimal medium in comparison to enriched medium. These results suggest that most of the bacterial cells prefer to live in a biofilm community under the difficult environmental conditions. Moreover, biofilm formation on polyurethane surface did not correlate with biofilm formation on glass. It suggests that mechanisms of biofilm formation can be correlated with other bacterial properties. This phenomenon may explain different types of biofilm formation among one species and even one pathotype - uropathogenic Escherichia coli.

The effects of nickel(II) complexes with imidazole derivatives on pyocyanin and pyoverdine production by Pseudomonas aeruginosa strains isolated from cystic fibrosis.

Pseudomonas aeruginosa infection is problematic in patients with cystic fibrosis (CF). P. aeruginosa secretes a diversity of pigments, such as pyocyanin and pyoverdine. The aim of this study was to evaluate the effects of complexes of nickel(II) ([Ni(iaa)2(H2O)2]·H2O (iaa = imidazole-4-acetate anion), [Ni(1-allim)6](NO3)2 (1-allim = 1-allylimidazole) and NiCl2 on pyocyanin and pyoverdine production by 23 strains of P. aeruginosa isolated from cystic fibrosis under growth conditions specific for the CF respiratory system. The antibacterial effects and biophysical properties of the tested substances were measured by spectrofluorometric techniques, as well as by laser interferometry, confocal and atomic force microscopy. The cytotoxic properties of all compounds were measured by Annexin/IP assay against A549 cells. All tested compounds have no effect on pyocyanin production and decrease the pyoverdine secretion in about 40% of tested P. aeruginosa strains at non-cytotoxic range of concentrations. Imidazole-4-acetate anion and 1-allylimidazole have good diffusion properties in the mature P. aeruginosa PAO1 biofilm. In conclusion, the tested nickel(II) complexes do not have clinical implications in P. aeruginosa eradication in cystic fibrosis. The diffusion properties of 1-allylimidazole and imidazole-4-acetate and their lack of effect on A549 cells suggest that they might be considered for chemical synthesis with other transition metals.

Comparison of antibiotic resistance patterns in collections of Escherichia coli and Proteus mirabilis uropathogenic strains.

Escherichia coli and Proteus mirabilis are important urinary tract pathogens. The constant increase in the antibiotic resistance of clinical bacterial strains has become an important clinical problem. The aim of this study was to compare the antibiotic resistance of 141 clinical (Sweden and Poland) and 42 laboratory (Czech Republic) P. mirabilis strains and 129 clinical (Poland) uropathogenic E. coli strains. The proportion of unique versus diverse patterns in Swedish clinical and laboratory P. mirabilis strain collections was comparable. Notably, a similar proportion of unique versus diverse patterns was observed in Polish clinical P. mirabilis and E. coli strain collections. Mathematical models of the antibiotic resistance of E. coli and P. mirabilis strains based on Kohonen networks and association analysis are presented. In contrast to the three clinical strain collections, which revealed complex associations with the antibiotics tested, laboratory P. mirabilis strains provided simple antibiotic association diagrams. The monitoring of antibiotic resistance patterns of clinical E. coli and P. mirabilis strains plays an important role in the treatment procedures for urinary tract infections and is important in the context of the spreading drug resistance in uropathogenic strain populations. The adaptability and flexibility of the genomes of E. coli and P. mirabilis strains are discussed.

Microflora of the selected water reservoirs in Swietokrzyskie Voivodship.

One of the important environmental issues is the quality of surface waters in the world. Poland belongs to countries with a low quality of the inland waters. The sanitary condition of the five water reservoirs of south-east Poland was analyzed. Water and sediment samples were incubated on the selective and/or differential media. High concentrations of many common and pathogenic microbial indicators were shown in those samples. Those reservoirs are used by people, especially during summer. Because of the high epidemiological risk, detailed analysis of all inland waters should be performed routinely.

The use of infrared spectroscopy and artificial neural networks for detection of uropathogenic Escherichia coli strains' susceptibility to cephalothin.

BACKGROUND & AIMS: Infrared spectroscopy is an increasingly common method for bacterial strains' testing. For the analysis of bacterial IR spectra, advanced mathematical methods such as artificial neural networks must be used. The combination of these two methods has been used previously to analyze taxonomic affiliation of bacteria. The aim of this study was the classification of Escherichia coli strains in terms of susceptibility/resistance to cephalothin on the basis of their infrared spectra. The infrared spectra of 109 uropathogenic E. coli strains were measured. These data are used for classification of E. coli strains by using designed artificial neural networks. RESULTS: The most efficient artificial neural networks classify the E. coli sensitive/resistant strains with an error of 5%. CONCLUSIONS: Bacteria can be classified in terms of their antibiotic susceptibility by using infrared spectroscopy and artificial neural networks.

Trinucleotide repeat sequence-based PCR as a potential approach for genotyping Mycobacterium gordonae strains.

Diseases that are caused by non-tuberculous mycobacteria (NTM) continue to pose difficult clinical problems, and the epidemiological aspect of NTM-caused diseases is of great importance. In the case of Mycobacterium gordonae there is no adequate genotyping scheme. Here we present a potential rapid and reproducible genetic assay that uses trinucleotide repeat sequence-based PCR (TRS-PCR) for genotyping M. gordonae. The proposed method constitutes a useful single-primer PCR screen for genotyping this species. Among 10 TRS-containing primers, after applying (CAC)4-based PCR to 36 strains of M. gordonae, we found a discriminatory index of 0.975. The accuracy of this analysis was supported by a reasonable reproducibility of 92%. These results were compared with the Enterobacterial Repetitive Intergenic Consensus Sequences (ERIC)-PCR typing scheme which had lower discriminatory index of 0.93 and its reproducibility was only 86.3%.

(CGG)4-based PCR as a novel tool for discrimination of uropathogenic Escherichia coli strains: comparison with enterobacterial repetitive intergenic consensus-PCR.

Urinary tract infections are one of the most frequent bacterial diseases in humans, and Escherichia coli is most often the relevant pathogen. A specific pathotype of E. coli, known as uropathogenic E. coli (UPEC), often causes serious and difficult-to-treat infections of the urinary tract. We propose a new single-tube screening tool that uses an (N)(6)(CGG)(4) primer to generate fingerprint profiles that allow rapid discrimination and epidemiology of this group of bacteria. We found 71 different CGG-PCR profiles among 127 E. coli strains, while enterobacterial repetitive intergenic consensus (ERIC)-PCR of the same group yielded only 28 profiles. Additionally, the (CGG)(4)-based PCR test turned out to be very effective for clustering UPEC strains exhibiting multiple virulence genes and usually belonging to the B2 phylogenetic group, and it separated these strains from E. coli strains lacking most of the UPEC-specific virulence factors. Since the reproducibility of the CGG-PCR screen is higher than that of ERIC-PCR, our test should be a valuable means of increasing the discriminatory power of current UPEC typing schemes.