Survival and Competitiveness of Bradyrhizobium japonicum Strains 20 Years after Introduction into Field Locations in Poland

It was previously demonstrated that there are no indigenous strains of Bradyrhizobium japonicum forming nitrogen-fixing root nodule symbioses with soybean plants in arable field soils in Poland. However, bacteria currently classified within this species are present (together with Bradyrhizobium canariense) as indigenous populations of strains specific for nodulation of legumes in the Genisteae tribe. These rhizobia, infecting legumes such as lupins, are well established in Polish soils. The studies described here were based on soybean nodulation field experiments, established at the Poznań University of Life Sciences Experiment Station in Gorzyń, Poland, and initiated in the spring of 1994. Long-term research was then conducted in order to study the relation between B. japonicum USDA 110 and USDA 123, introduced together into the same location, where no soybean rhizobia were earlier detected, and nodulation and competitive success were followed over time. Here we report the extra-long-term saprophytic survival of B. japonicum strains nodulating soybeans that were introduced as inoculants 20 years earlier and where soybeans were not grown for the next 17 years. The strains remained viable and symbiotically competent, and molecular and immunochemical methods showed that the strains were undistinguishable from the original inoculum strains USDA 110 and USDA 123. We also show that the strains had balanced numbers and their mobility in soil was low. To our knowledge, this is the first report showing the extra-long-term persistence of soybean-nodulating strains introduced into Polish soils and the first analyzing the long-term competitive relations of USDA 110 and USDA 123 after the two strains, neither of which was native, were introduced into the environment almost 2 decades ago.     

New Hydrazides and Hydrazide‐Hydrazones of 2,3‐Dihalogen Substituted Propionic Acids: Synthesis and in vitro Antimicrobial Activity Evaluation

The main aim of this research was the synthesis, spectral identification and in vitro antimicrobial evaluation of new hydrazides and hydrazide‐hydrazones of 2,3‐dihalogen substituted propionic acids. New hydrazides were obtained by the substitution reaction of appropriate ethyl esters of 2,3‐dihalogen substituted propionic acids with hydrazine hydrate. Then obtained hydrazides were subjected to condensation reaction with various aldehydes which yielded with new hydrazide‐hydrazone derivatives. All obtained compounds were identified on the basis of spectral methods (¹H‐NMR, ¹³C‐NMR) and in vitro screened against a panel of bacterial and fungal strains according to EUCAST and CLSI guidelines.     

Polysaccharides and mucin 5AC (MUC5AC) expression in gallbladder mucosa of young patients with gallstones as evaluated by spatial visualization and quantification

The study aimed at examination of tissue expression of polysaccharides and secretory mucin 5AC (MUC5AC) in young patients (up to 25 years of age) with a symptomatic gallstones. For comparison, patients most frequently subjected to cholecystectomy were studied, i.e. patients of approximately 50 years of age with the same diagnosis. In quantitative studies on tissue expression of both mucus components, the modern technique of spatial visualization was applied for the first time. Application of the technique permitted to demonstrate significant positive relationships between expression of glycoproteins (immunocytochemical ABC technique for detection of MUC5AC) and expression of sugar components in mucus (PAS technique) and to confirm suitability of the technique for quantitative appraisal of both histochemical and immunocytochemical reactions. An even higher expression of polysaccharides in the entire mucosa and of MUC5AC was detected in gallbladder epithelium of 50-year-old patients, as compared to young patients with symptomatic gallstones. In the young patients, expression of polysaccharides correlated with inflammatory activity (grading), width of gallbladder wall and PLT level in peripheral blood. A significantly higher expression of polysaccharides in gallbladder epithelium was demonstrated in young patients admitted in the emergency mode to the hospital. These correlations in young patients may suggest a role of both mucus components in pathogenesis of cholelithiasis in this age group. A quantitative appraisal of mucus component expression in the two parts of gallbladder mucosa (epithelium vs. entire mucosa) using spatial visualization technique permitted to more accurately compare production of glycoproteins and of polysaccharides in patients with cholelithiasis and to demonstrate additional correlations of a potential clinical significance.     

Occurrence of Wolbachia in central European weevils: correlations with host systematics,ecology, and biology

We studied the occurrence of Wolbachia in relation to the systematics, ecology, and biology of 40 weevil species from central Europe. Identification of Wolbachia supergroups and phylogeny was performed on the basis of 16S rDNA, ftsZ, wsp, and hcpA sequences. Sixteen species (40%) were infected by Wolbachia. Six of these possess only supergroup A (15% of all studied species, 37.5% of the infected species), and four harbored only supergroup B (10 and 25%, respectively). Six species were infected by both supergroups A and B or their genomes harbored parts of these two supergroups (15 and 37.5%, respectively). No differences between Wolbachia supergroup frequencies were detected. There was almost no correlation between Wolbachia phylogeny and host systematics and phylogeny at the level of subfamily and tribe, because the representatives of both supergroups were detected in all the studied multi‐species tribes. Wolbachia strains were probably inherited from a common ancestor only in the case of the genus Strophosoma, where two of three analyzed species possessed bacteria which are genetically very close in all the studied genes. There was also only limited congruence between phylogenies obtained from the four studied genes. These results suggest horizontal transmission of Wolbachia strains between species and recombination events between different strains. A significant correlation was detected between infected and uninfected species in relation to mobility (flying species were 2× more frequently infected than non‐flying species), foraging (polyphagous species were 2.5× less frequently infected than mono‐ or oligophagous species), and reproductive mode (parthenogenetic weevils were infected nearly 2× as often as bisexuals). No differences were detected between mesophilous and xerothermophilous species, nor between those inhabiting open areas vs. arboreal species. However, these results might have been influenced by common ancestry among the studied weevils. Because weevils include many plant pests of economic importance, it is possible to use these data in developing alternative, biology‐based strategies for controlling them.     

Preventive antibiotic treatment of calves: emergence of dysbiosis causing propagation of obese state-associated and mobile multidrug resistance-carrying bacteria

In agriculture, antibiotics are used for the treatment and prevention of livestock disease. Antibiotics perturb the bacterial gut composition but the extent of these changes and potential consequences for animal and human health is still debated. Six calves were housed in a controlled environment. Three animals received an injection of the antibiotic florfenicol (Nuflor), and three received no treatment. Faecal samples were collected at 0, 3 and 7 days, and bacterial communities were profiled to assess the impact of a therapy on the gut microbiota. Phylogenetic analysis (16S-rDNA) established that at day 7, antibiotic-treated microbiota showed a 10-fold increase in facultative anaerobic Escherichia spp, a signature of imbalanced microbiota, dysbiosis. The antibiotic resistome showed a high background of antibiotic resistance genes, which did not significantly change in response to florfenicol. However, the maintenance of Escherichia coli plasmid-encoded quinolone, oqxB and propagation of mcr-2, and colistin resistance genes were observed and confirmed by Sanger sequencing. The microbiota of treated animals was enriched with energy harvesting bacteria, common to obese microbial communities. We propose that antibiotic treatment of healthy animals leads to unbalanced, disease- and obese-related microbiota that promotes growth of E. coli carrying resistance genes on mobile elements, potentially increasing the risk of transmission of antibiotic resistant bacteria to humans.     

High serum sCD163/sTWEAK ratio is associated with lower risk of digital ulcers but more severe skin disease in patients with systemic sclerosis

Introduction

Systemic sclerosis (SSc) is an autoimmune disease characterized by chronic inflammation, vascular injury and excessive fibrosis. CD163 is a scavenger receptor which affects inflammatory response and may contribute to connective tissue remodelling. It has recently been demonstrated that CD163 can bind and neutralize the TNF-like weak inducer of apoptosis (TWEAK), a multifunctional cytokine which regulates inflammation, angiogenesis and tissue remodelling. We aimed to investigate the relationships between serum levels of soluble CD163 (sCD163) and soluble TWEAK (sTWEAK) in relation to disease manifestations in SSc patients.

Methods

This study included 89 patients with SSc who had not received immunosuppressive drugs or steroids for at least 6 months and 48 age- and sex-matched healthy controls (HC) from four European centres. Serum concentrations of sTWEAK and sCD163 were measured using commercially available ELISA kits.

Results

The mean serum concentrations of sTWEAK were comparable between SSc patients (mean +/- SD: 270 +/- 171 pg/mL) and HC (294 +/- 147pg/mL, P >0.05). Concentration of sCD163 and sCD163/sTWEAK ratio were significantly greater in SSc patients (984 +/- 420 ng/mL and 4837 +/- 3103, respectively) as compared to HC (823 +/- 331 ng/mL and 3115 +/- 1346 respectively, P <0.05 for both). High sCD163 levels and a high sCD163/sTWEAK ratio (defined as > mean +2SD of HC) were both associated with a lower risk of digital ulcers in SSc patients (OR, 95%CI: 0.09; 0.01, 0.71, and 0.17; 0.06, 0.51, respectively). Accordingly, patients without digital ulcers had a significantly higher sCD163 concentration and sCD163/sTWEAK ratio as compared to SSc patients with digital ulcers (P <0.01 for both) and HC (P <0.05 for both). A high sCD163/sTWEAK ratio, but not high sCD163 levels, was associated with greater skin involvement.

Conclusions

The results of our study indicate that CD163-TWEAK interactions might play a role in the pathogenesis of SSc and that CD163 may protect against the development of digital ulcers in SSc. Further studies are required to reveal whether targeting of the CD163-TWEAK pathway might be a potential strategy for treating vascular disease and/or skin fibrosis in SSc.     

Suitability of antral follicle counts and computer-assisted analysis of ultrasonographic and magnetic resonance images for estimating follicular reserve in porcine,ovine and bovine ovaries ex situ

This study was conducted to determine if correlations exist between the numbers of microscopic follicles comprising ovarian follicular reserve (OFR) and antral follicle counts (AFCs), and to assess the usefulness of computerized analyses of ovarian ultrasonograms and magnetic resonance (MR) images for estimating OFR in excised porcine, ovine and bovine ovaries. As a pre-requisite to these analyses, we characterized and compared ovarian cortical histomorhpology and follicle populations in the three species varying in prolificacy and overall reproductive longevity, and hence the total number of microscopic and antral follicles. Ultrasonographic and MR images were obtained at the scanner settings optimized to provide opposing contrasts between antral follicles and the ovarian stroma. Commercially available ImageProPlus® analytical software was used to calculate numerical pixel values (NPVs) and pixel heterogeneity (standard deviation of the pixel values) along the computer-generated lines (4–6) placed in the area corresponding to the ovarian cortex. The numbers of primordial (r = 0.38, P < 0.01) and intermediate follicles (r = 0.37, P < 0.01) were correlated with the numbers of antral follicles in bovine ovarian sections. The numbers of primordial (r = 0.28, P < 0.05), intermediate (r = 0.31, P < 0.01) and primary follicles (r = 0.27, P < 0.05) correlated directly with mean NPVs of the ultrasonographic ovarian images in cattle. There was a negative correlation between primary follicle numbers and NPVs of MR images (3D FAST-SPOILED GRADIENT ECHO) of the porcine ovarian cortex (r = −0.31, P < 0.05). To summarize, the numbers of primordial and intermediate follicles could only be estimated from AFCs in cows. Using ultrasound NPVs, the numbers of primordial, intermediate and primary follicles could be directly estimated in bovine ovaries and the quantitative image attributes of MR images were useful for quantifying porcine primary follicles. The bovine ovarian model is compatible with human situation and hence future studies should be undertaken to ascertain the usefulness of AFCs and ultrasonographic image analyses for estimating OFR in women.     

TLR9 2848 GA Heterozygotic Status Possibly Predisposes Fetuses and Newborns to Congenital Infection with Human Cytomegalovirus

Background

Some single nucleotide polymorphisms (SNP), located in Toll-like receptor (TLR) genes, were reported to be associated with human cytomegalovirus (HCMV) infections. The study was aimed to assess the correlation of SNPs at TLR4 and TLR9 genes with the occurrence of congenital cytomegaly, based on available samples.

Methods

Reported case-control study included both HCMV infected and non-infected fetuses and newborns. The specimens were classified to the molecular analyses, based on serological features of the recent infection and HCMV DNAemia in body fluids. TLR SNPs were studied, using multiplex nested PCR-RFLP assay, and determined genotypes were confirmed by sequencing. Hardy-Weinberg equilibrium was assessed for the identified genotypes. The linkage disequilibrium was also estimated for TLR4 SNPs. A relationship between the status of TLR genotypes and congenital cytomegaly development was estimated, using a logistic regression model.

Results

Hardy Weinberg equilibrium was observed for almost all SNPs, both infected and non-infected patients, with exception of TLR4 896 A>G polymorphism in the control group (P≤0.050). TLR4 896 A>G and 1196 C>T SNPs were found in linkage disequilibrium in both study groups (P≤0.050). The CC genotype at TLR4 1196 SNP and the GA variant at TLR9 2848 G>A SNP were significantly associated with HCMV infection (P≤0.050). The risk of congenital cytomegaly was higher in heterozygotes at TLR9 SNP than in the carriers of other genotypic variants at the reported locus (OR 4.81; P≤0.050). The GC haplotype at TLR4 SNPs and GCA variants at TLR4 and TLR9 SNPs were significantly associated with HCMV infection (P≤0.0001). The ACA variants were more frequent among fetuses and neonates with symptomatic, rather than asymptomatic cytomegaly (P≤0.0001).

Conclusions

TLR4 and TLR9 polymorphisms may contribute to the development of congenital infection with HCMV in fetuses and neonates. The TLR9 2848 GA heterozygotic status possibly predisposes to HCMV infection, increasing the risk of congenital cytomegaly development.