Chronic Rhinosinusitis-Microbiological Etiology, Potential Genetic Markers, and Diagnosis.

Chronic rhinosinusitis (CRS) is a significant public health problem. Bacterial colonization and impaired mucociliary clearance play a significant role in the inflammatory process. Several inflammatory pathways and host defense elements are altered in CRS, which may contribute to observed differences in the microbiome. To date, researching CRS has been difficult due to limited access to the studied tissue and a lack of available biomarkers. Ongoing scientific research is increasingly based on simple and objective analytical methods, including sensors, detection with PCR, and sequencing. Future research on microbiota and human factors should also include genomics, transcriptomics, and metabolomics approaches. This report analyzes the changes that occur in the paranasal sinuses of people with acute and chronic rhinosinusitis, the composition of the microbiota, the human genetic markers that may shed light on the predisposition to CRS, and the advantages and disadvantages of classical and molecular diagnostic methods, as well as addressing the difficulties of sinusitis treatment.

Asymptomatic Bacteriuria in Kidney Transplant Recipients-A Narrative Review.

Urinary tract infections (UTIs) are the most prevalent complications in kidney transplant (KTx) recipients. The most frequent finding in this group of patients is asymptomatic bacteriuria (ASB). Here, we provide an overview of the available evidence regarding ASB in KTx recipients, including its etiopathology, clinical impact and management. There is a growing body of evidence from clinical trials that screening for and treating ASB is not beneficial in most KTx recipients. However, there are insufficient data to recommend or discourage the use of a "screen-and-treat strategy" for ASB during the first 1-2 months post-transplant or in the case of an indwelling urinary catheter. Despite its frequency, ASB after KTx is still an understudied phenomenon.

May Staphylococcus lugdunensis Be an Etiological Factor of Chronic Maxillary Sinuses Infection?

Staphylococcus lugdunensis is an opportunistic pathogen found in the healthy human skin microbiome bacterial community that is able to cause infections of diverse localization, manifestation, and course, including laryngological infections, such as necrotizing sinusitis. Chronic maxillary sinusitis is a disease present in up to one third of European and American populations, and its etiology is not fully described. Within this study, we aimed to characterize 18 S. lugdunensis strains recovered from maxillary sinuses and evaluate them as etiological agents of chronic disease. We performed MLST analysis, the complex analysis of both phenotypic and genetic virulence factors, antibiotic susceptibility profiles, and biofilm formation assay for the detection of biofilm-associated genes. Altogether, S. lugdunensis strains were clustered into eight different STs, and we demonstrated several virulence factors associated with the chronic disease. All tested strains were able to produce biofilm in vitro with numerous strains with a very strong ability, and overall, they were mostly susceptible to antibiotics, although we found resistance to fosfomycin, erythromycin, and clindamycin in several strains. We believe that further in-depth analysis of S. lugdunensis strains from different niches, including the nasal one, should be performed in the future in order to reduce infection rate and broaden the knowledge about this opportunistic pathogen that is gaining attention.

X-ray and UV Radiation Damage of dsDNA/Protein Complexes.

Radiation and photodynamic therapies are used for cancer treatment by targeting DNA. However, efficiency is limited due to physico-chemical processes and the insensitivity of native nucleobases to damage. Thus, incorporation of radio- and photosensitizers into these therapies should increase both efficacy and the yield of DNA damage. To date, studies of sensitization processes have been performed on simple model systems, e.g., buffered solutions of dsDNA or sensitizers alone. To fully understand the sensitization processes and to be able to develop new efficient sensitizers in the future, well established model systems are necessary. In the cell environment, DNA tightly interacts with proteins and incorporating this interaction is necessary to fully understand the DNA sensitization process. In this work, we used dsDNA/protein complexes labeled with photo- and radiosensitizers and investigated degradation pathways using LC-MS and HPLC after X-ray or UV radiation.

Fusion of DNA-binding domain of Pyrococcus furiosus ligase with TaqStoffel DNA polymerase as a useful tool in PCR with difficult targets.

The DNA coding sequence of TaqStoffel polymerase was fused with the DNA-binding domain of Pyrococcus furiosus ligase. The resulting novel recombinant gene was cloned and expressed in E. coli. The recombinant enzyme was purified and its enzymatic features were studied. The fusion protein (PfuDBDlig-TaqS) was found to have enhanced processivity as a result of the conversion of the TaqDNA polymerase from a relatively low processive to a highly processive enzyme. The abovementioned processivity enhancement was about threefold as compared to the recombinant TaqStoffel DNA polymerase (TaqS), and the recombinant fusion protein was more thermostable. It had a half-life of 23 min at 99 °C as compared to 10 min for TaqS. The fusion protein also showed a significantly higher resistance to PCR inhibitors such as heparin or lactoferrin and the fusion polymerase-amplified GC-rich templates much more efficiently and was efficient even with 78% GC pairs.

A new assay for the simultaneous identification and differentiation of Klebsiella oxytoca strains.

Klebsiella oxytoca is the second most frequently identified species of Klebsiella isolated from hospitalized patients. Klebsiella spp. is difficult to identify using conventional methods and is often misclassified in clinical microbiology laboratories. K. oxytoca is responsible for an increasing number of multi-resistant infections in hospitals because of insufficient detection and identification. In this study, we propose a new simple method called pehX-LM PCR/XbaI, which simultaneously indicates K. oxytoca species and genotype by the fingerprint pattern. The pehX-LM PCR/XbaI is a combination of the following two methods: species-specific amplification of pehX gene and non-specific amplification of short restriction fragments by the LM PCR method. The specificity and the discrimination power of the pehX-LM PCR/XbaI method were determined by typing 209 K. oxytoca strains (included 9 reference strains), 28 K. pneumoniae, and other 25 strains belonging to the Enterobacteriaceae. The typing results were confirmed by the PCR melting profile method. Unlike the known fingerprinting methods, the pehX-LM PCR/XbaI leads to a clear pattern (approx. 3-5 bands) with a sufficient, relatively high discriminatory power. As a result, the time and cost of a single analysis are lower. The method can be used both in clinical and environmental research.

New PCR test for detection of Candida glabrata based on the molecular target chosen by the RAPD technique.

Rapid, reliable diagnosis is a necessary condition for the successful treatment of infections. Such diagnostic assays are continually being developed. The paper presents a method for selecting the molecular target for PCR-based diagnostics based on the comparison of RAPD patterns. A sequence encoding Candida glabrata CBS138 hypothetical protein was selected. The limit of detection for PCR and real-time PCR reactions with DNA extracted from blood samples spiked with Candida glabrata was estimated at 1 CFU/ml. The application of the assays developed in this study would thus seem to be promising as a complementary method in the diagnostics of C. glabrata infections.

Optical method supported by machine learning for urinary tract infection detection and urosepsis risk assessment.

The study presents an optical method supported by machine learning for discriminating urinary tract infections from an infection capable of causing urosepsis. The method comprises spectra of spectroscopy measurement of artificial urine samples with bacteria from solid cultures of clinical E. coli strains. To provide a reliable classification of results assistance of 27 algorithms was tested. We proved that is possible to obtain up to 97% accuracy of the measurement method with the use of use of machine learning. The method was validated on urine samples from 241 patients. The advantages of the proposed solution are the simplicity of the sensor, mobility, versatility, and low cost of the test.

Urinary Tract Infections Caused by K. pneumoniae in Kidney Transplant Recipients - Epidemiology, Virulence and Antibiotic Resistance.

Urinary tract infections are the most common complication in kidney transplant recipients, possibly resulting in the deterioration of a long-term kidney allograft function and an increased risk of recipient's death. K. pneumoniae has emerged as one of the most prevalent etiologic agents in the context of recurrent urinary tract infections, especially with multidrug resistant strains. This paper discusses the epidemiology and risk factors associated with urinary tract infections in kidney transplant recipients, multi-drug resistance of K. pneumoniae (ESBL, KPC, NDM), treatment and pathogenesis of K. pneumoniae infections, and possible causes of recurrent UTIs. It also addresses the issue of colonization/becoming a carrier of K. pneumoniae in the gastrointestinal tract and asymptomatic bacteriuria in relation to a symptomatic UTI development and epidemiology.

Antibiotic resistance, virulence, and phylogenetic analysis of Escherichia coli strains isolated from free-living birds in human habitats.

Wild birds can be colonized by bacteria, which are often resistant to antibiotics and have various virulence profiles. The aim of this study was to analyze antibiotic resistance mechanisms and virulence profiles in relation to the phylogenetic group of E. coli strains that were isolated from the GI tract of wildfowl. Out of 241 faecal samples, presence of E. coli resistant to a cephalosporin (ESBL/AmpC) was estimated for 33 isolates (13,7%). Based on the analysis of the coexistence of 4 genes encoding ESBLs/AmpC (blaCTX-M, blaTEM, blaSHV, blaAmpC) and class 1 and 2 integrons genes (intI1, intI2) a subset of two resistance profiles was observed among the investigated E. coli isolates carrying blaAmpC, blaSHV, and blaCTX-M, blaTEM, class 1 and 2 integrons, respectively. The E. coli isolates were categorized into 4 phylogenetic groups A (39.4%), B2 (24.25%), D (24.25%) and B1 (12.1%). The pathogenic B2 and D groups were mainly typical for the Laridae family. Among the 28 virulence factors (Vfs) detected in pathogenic phylogenetic groups B2 and D, 7 were exclusively found in those groups (sfa, vat, tosA, tosB, hly, usp, cnf), while 4 VFs (fecA, fyuA, irp2, kspMTII) showed a statistically significant association (P≤0.05) with phylogroups A and B1. Our results indicated that strains belonging to commensal phylogroups A/B1 possess extensive iron acquisition systems (93,9%) and autotransporters (60,6%), typical for pathogens, hence we suggest that these strains evolve towards higher levels of virulence. This study, which is a point assessment of the virulence and drug resistance potential of wild birds, confirms the importance of taking wild birds as a reservoir of strains that pose a growing threat to humans. The E. coli analyzed in our study derive from different phylogenetic groups and possess an arsenal of antibiotic resistance genes and virulence factors that contribute to their ability to cause diseases.

RecA proteins from Deinococcus geothermalis and Deinococcus murrayi--cloning, purification and biochemical characterisation.

BACKGROUND: Escherichia coli RecA plays a crucial role in recombinational processes, the induction of SOS responses and mutagenic lesion bypasses. It has also been demonstrated that RecA protein is indispensable when it comes to the reassembly of shattered chromosomes in γ-irradiated Deinococcus radiodurans, one of the most radiation-resistant organisms known. Moreover, some functional differences between E. coli and D. radiodurans RecA proteins have also been shown. RESULTS: In this study, recA genes from Deinococcus geothermalis and Deinococcus murrayi, bacteria that are slightly thermophilic and extremely γ-radiation resistant, were isolated, cloned and expressed in E. coli. After production and purification, the biochemical properties of DgeRecA and DmuRecA proteins were determined. Both proteins continued to exist in the solutions as heterogenous populations of oligomeric forms. The DNA binding by DgeRecA and DmuRecA proteins is stimulated by Mg2+ ions. Furthermore, both proteins bind more readily to ssDNA when ssDNA and dsDNA are in the same reaction mixture. Both proteins are slightly thermostable and were completely inactivated in 10 s at 80°C. Both proteins hydrolyze ATP and dATP in the presence of ssDNA or complementary ssDNA and dsDNA, but not in the absence of DNA or in the presence of dsDNA only, and dATP was hydrolyzed more rapidly than ATP. They were also able to promote DNA strand exchange reactions by a pathway common for other RecA proteins. However, we did not obtain DNA strand exchange products when reactions were performed on an inverse pathway, characteristic for RecA of D. radiodurans. CONCLUSIONS: The characterization of DgeRecA and DmuRecA proteins made in this study indicates that the unique properties of D. radiodurans RecA are probably not common among RecA proteins from Deinococcus sp.

Evaluating the antibacterial activity of muramyl dipeptide derivatives, retro-tuftsin derivatives, and anthraquinone oligopeptides against a range of pathogenic bacteria.

Search for new and efficient antibiotic is crucial because of microbial drug resistance and problems with side effects of the administered medication. In this study, we evaluate the in vitro microbiological activity of muramyl dipeptide derivatives, retro-tuftsin derivatives (i.e., tuftsin with reversed amino acid sequences), and combinations of retro-tuftsin derivatives with substituted anthraquinones. The potency of the investigated derivatives towards methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae ESBL (extended-spectrum ß-lactamases) was compared based on the spectroscopically-measured minimal inhibitory concentrations (MIC values). The bacterial growth have also been studied with different concentrations of compounds. Statistical analysis of the results revealed that certain modifications lead to promising activity against S. aureus (anthraquinone analogue - 3c and retro-tuftsin derivative - 2b), while other derivatives exhibit activity against P. aeruginosa (muramyl dipeptide derivative - 1d and retro-tuftsin derivative - 2b). The obtained results of microbiological activity indicate that the structure of the tested compounds may be the basis for further modifications.

The Many Faces of Enterococcus spp.-Commensal, Probiotic and Opportunistic Pathogen.

Enterococcus spp. are Gram-positive, facultative, anaerobic cocci, which are found in the intestinal flora and, less frequently, in the vagina or mouth. Enterococcus faecalis and Enterococcus faecium are the most common species found in humans. As commensals, enterococci colonize the digestive system and participate in the modulation of the immune system in humans and animals. For many years reference enterococcal strains have been used as probiotic food additives or have been recommended as supplements for the treatment of intestinal dysbiosis and other conditions. The use of Enterococcus strains as probiotics has recently become controversial due to the ease of acquiring different virulence factors and resistance to various classes of antibiotics. Enterococci are also seen as opportunistic pathogens. This problem is especially relevant in hospital environments, where enterococcal outbreaks often occur. Their ability to translocate from the gastro-intestinal tract to various tissues and organs as well as their virulence and antibiotic resistance are risk factors that hinder eradication. Due to numerous reports on the plasticity of the enterococcal genome and the acquisition of pathogenic microbial features, we ask ourselves, how far is this commensal genus from acquiring pathogenicity? This paper discusses both the beneficial properties of these microorganisms and the risk factors related to their evolution towards pathogenicity.

Genetic Background and Antibiotic Resistance Profiles of K. pneumoniae NDM-1 Strains Isolated from UTI, ABU, and the GI Tract, from One Hospital in Poland, in Relation to Strains Nationally and Worldwide.

In recent years, there has been an observed increase in infections caused by carbapenem-resistant Klebsiella pneumonia (Kp) strains. The aim of this study was the phenotypic and genotypic analysis of eight K. pneumoniae NDM (Kp NDM) isolates, recovered in Poland during the years 2016 and 2018 from seven patients with urinary tract infections (UTIs), asymptomatic bacteriuria (ABU), or colonization of the gut. PCR melting profile genotyping indicated a close relationship between the strains derived from 2018, which were not related to the strain isolated in 2016. WGS results were analyzed in relation to international Kp isolates. Clonal and phylogenetic analyses were performed based on multilocus sequence typing (MLST) and single nucleotide polymorphisms (SNPs) of the core genome. The metallo-ß-lactamase was assigned to the NDM-1 type and the sequence was identified as ST11. Eleven antimicrobial resistance genes were detected, mostly from plasmid contigs. Unprecedented profiles of plasmid replicons were described with the IncFII/pKPX-1 dominant replicon. In terms of the KL24 and O2v1 capsular antigen profiles, these isolates corresponded to Greek strains. Strains isolated from UTI, ABU, and colonization GI tract patients were not carrying environment-specific virulence genes. Based on the assessment of strain relationships at the genome level and their direction of evolution, the international character of the sublines was demonstrated, with a documented epidemic potential in Poland and Greece. In conclusion, some groups of patients, e.g., renal transplant recipients or those with complicated UTIs, who are frequently hospitalized and undergoing antibiotic therapy, should be monitored not only for the risk of UTI, but also for colonization by Kp NDM strains.

The New Klebsiellapneumoniae ST152 Variants with Hypermucoviscous Phenotype Isolated from Renal Transplant Recipients with Asymptomatic Bacteriuria-Genetic Characteristics by WGS.

Klebsiellapneumoniae (Kp) is one of the most important etiological factors of urinary tract infections in renal transplant (RTx) recipients. We described the antimicrobial susceptibility phenotypes and genomic features of two hypermucoviscous (HM) Kp isolates recovered from RTx recipients with asymptomatic bacteriuria (ABU). Using whole genome sequencing (WGS) data, we showed that the strains belong to the ST152 lineage with the KL149 capsular serotype, but without rmpA/magA genes, which is typical for HM+ hypervirulent Kp. These new strains carried virulence-associated genes that predispose for urinary tract infections (UTIs). Likewise, both strains carried the ecp gene encoding pilus common for extended-spectrum ß-lactamase (ESBL) Escherichiacoli. Although the two ST152 isolates were closely related and differed by only nine single nucleotide polymorphisms (SNPs) in their chromosomes, they had different plasmid compositions and chromosomal elements, with isolate KP28872 carrying an ESBL plasmid and an integrative conjugative element. These two isolates are an example of the high plasticity of the K. pneumoniae accessory genome. The identification of patients with ABU matched with the correct epidemiological profiling of isolates could facilitate interventions to prevent or rapidly treat K. pneumoniae infections.

Escherichia coli Strains with Virulent Factors Typical for Uropathogens were Isolated from Sinuses from Patients with Chronic Rhinosinusitis-Case Report.

Escherichia coli were isolated from three patients with chronic rhinosinusitis (CRS) by intraoperative sinus tissue biopsy. Taking into account the unusual replicative niche and previous treatment failures, it was decided to focus on the virulence and drug resistance of these bacteria. The strains turned out to be multi-sensitive, but the rich virulence factors profile of bacteria typical for phylogenetic group B2 deserved attention. Tests were carried out for the presence of 32 genes using the PCR method. Particularly noteworthy are the toxins Cnf-1, HlyA, Usp-an extensive iron uptake system (enterobactin, salmochelin, yersiniabactin and outer membrane hemin receptor ChuA)-SPATE autotransporters such as vat and pic, Ag43 autoaggregative protein-important for biofilm formation-and TosA/B which enhance the fitness of E.coli. All these virulence factors are identified predominantly in UPEC strains and provide a fitness advantage during colonization of the sinuses. Patients with CRS should be asked for past or present UTI. The specific virulence factors of E. coli that facilitate the colonization of the GI tract and urinary tract may also favor the colonization of a new ecological niche (sinuses) as a result of microbial imbalance or dysbiosis.

Linezolid-resistant Enterococcus faecium strains isolated from one hospital in Poland -commensals or hospital-adapted pathogens?

One of the most pressing problems of enterococci infections is occurring resistance to linezolid, which is an antibiotic used in the treatment of infections caused by vancomycin-resistant strains (VRE). The main objective of our research was to investigate the relationship of 19 linezolid-resistant E. faecium isolates from 18 patients hospitalized at Clinical Hospital in Gdansk (Poland). One of the LZDREF was isolated in 2003 (K2003), and another 18 were collected from 2013 to 2017. Genotyping with PCR MP method indicated 14 main unrelated genetic profiles and no association with K2003 strain. Two isolates with the same genotype and genetically closely related two sub-types (2 isolates for each sub-type) were hospital-derived colonizations of patients. The other unrelated genotypes were discussed in the context of colonization, nosocomial infections, and commensal origin, taking into account prior exposure to linezolid. We determined the presence of a point mutation G2576T in six loci of 23S rDNA. There was also a significant correlation (p<0.0015) between the presence of MIC>32 value and the presence of G2576T point mutation on the sixth rrn. We also detected 5 virulence genes for all isolates: gelE, cylA, asa1, hyl, esp. Correlation (p≤0.0001) was observed between the presence of gelE gene encoding gelatinase and two other genes: cylA and asa1 encoding cytolysin and collagen binding protein responsible for aggregation of bacterial cells, respectively. Significant correlation was also observed between asa1 and cfr genes encoding 23S rRNA rybonuclease responsible for resistance to PhLOPSA antibiotics (p = 0.0004). The multidimensional analysis has also shown the correlation between cfr gene and GI-tract (p = 0, 0491), which suggests horizontal gene transfer inside the gut microbiota and the risk of colonization with linezolid-resistant strains without previously being treated with the antibiotic. The patient could have been colonized with LZDRVREF strains which in the absence of competitive microbiota quickly settle in ecological niches favourable for them and pose a risk for the patient.

PCR melting profile (PCR MP)--a new tool for differentiation of Candida albicans strains.

BACKGROUND: We have previously reported the use of PCR Melting Profile (PCR MP) technique based on using low denaturation temperatures during ligation mediated PCR (LM PCR) for bacterial strain differentiation. The aim of the current study was to evaluate this method for intra-species differentiation of Candida albicans strains. METHODS: In total 123 Candida albicans strains (including 7 reference, 11 clinical unrelated, and 105 isolates from patients of two hospitals in Poland) were examined using three genotyping methods: PCR MP, macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE) and RAPD techniques. RESULTS: The genotyping results of the PCR MP were compared with results from REA-PFGE and RAPD techniques giving 27, 26 and 25 unique types, respectively. The results showed that the PCR MP technique has at least the same discriminatory power as REA-PFGE and RAPD. CONCLUSION: Data presented here show for the first time the evaluation of PCR MP technique for candidial strains differentiation and we propose that this can be used as a relatively simple and cheap technique for epidemiological studies in short period of time in hospital.

Usefulness of PCR melting profile method for genotyping analysis of Klebsiella oxytoca isolates from patients of a single hospital unit.

The development of rapid and simple typing methods is required in order to identify possible sources of human exposure to opportunistic pathogens. Klebsiella spp. belongs to a group of bacteria that are opportunistic pathogens responsible for an increasing number of multiresistant infections in hospitals. Recently, we showed the high genetic diversity of K. oxytoca using a large collection of strains isolated from the patients of several hospitals in Poland over a 50-year period. Our results showed that the internal transcribed spacer polymerase chain reaction method (ITS-PCR) is useful for the phylogenetic delineation of genetic groups in K. oxytoca and the high discriminatory power of the PCR melting profiles (PCR MP) method can be useful for epidemiological studies of K. oxytoca. In the present study the usefulness of PCR MP was tested on two sets of strains isolated from a single unit over a short period of time. The results revealed that PCR MP has a high discriminatory power and can be useful for epidemiological studies of closely related strains of K. oxytoca isolated from a single unit over a short period of time to identify the source, reservoirs and the tract of infection spread. The advantage of PCR MP for the above application was shown by using the procedure at increasing denaturation temperature during PCR to confirm genotyping results. Considering this feature and the high discriminatory power of PCR MP, as shown in this report for determination of the genetic similarities of consecutive K. oxytoca strains, we propose that PCR MP is one of the best techniques for short-term epidemiology analysis.

Host and pathogen factors in Klebsiella pneumoniae upper urinary tract infections in renal transplant patients.

PURPOSE: To analyse the role of virulence factors (VFs) and host in Klebsiella pneumoniae upper urinary tract infections (UTIs) in renal transplant (RTx) recipients. METHODOLOGY: Clinical and demographic data were registered prospectively. Phylogenetic background of K. pneumoniae isolates was analysed by PCR melting profiles (MP) and the following VFs genes: fimH-1, uge, kpn, ycfM, mrkD, rmpA, magA, hlyA, cnf-1, irp-1, irp-2, fyuA, entB, iutA, iroN by PCR. RESULTS: We studied urine cultures and clinical data from 61 episodes of K. pneumoniae UTI in 54 RTx recipients. There were 32 cases of AB (53%), 10 cases of lower UTI (16%), 19 cases of AGPN (31%), including six cases of bacteraemia. In total, 74 % of strains were extended-spectrum beta-lactamase+, and there were two carbapenemase-producing strains. PCR MP typing showed a diverse population with 52 different genetic profiles of K. pneumoniae. Analysis of the DNA profiles indicated 45 unrelated, unique genotypes and 7 related (16 isolates from 15 patients) genotypes. Urine flow impairment emerged as an independent predictor of K. pneumoniae upper UTIs (OR 14.28, CI 2.7-75.56, P 0.002), while we did not find any association between the profile of VFs and developing upper UTIs. The prevalence of the uge gene was lower in RTx patients on everolimus when compared to isolates from patients not receiving mTOR inhibitors (33.3 % vs 82.8 % P<0.05). CONCLUSIONS: K. pneumoniae upper UTI may be a marker of urine flow impairment. Bacterial VFs could not discriminate between upper and lower UTIs. However, immunosuppression may influence the selection of particular VFs.