The impact of an antibody investigation algorithm emphasizing specificity on reducing potential false-positive warm autoantibody detection at a Canadian tertiary care centre.

BACKGROUND AND OBJECTIVES: To reduce potential false-positive warm autoantibody (WAA) by solid-phase red cell adherence assay (SPRCA), our centre implemented a new antibody investigation algorithm (AIA) by classifying cases with panreactive SPRCA but negative saline-indirect antiglobulin test as 'antibody of undetermined significance' (AUS) after excluding clinically significant antibodies. We assessed the effects of the new AIA and subsequent alloantibody formation in patients with AUS. MATERIALS AND METHODS: Samples from patients with positive SPRCA screens between 1 September 2017 and 31 August 2021 were selected for the study. Frequencies of antibodies classified by the old and new AIAs were compared using Fisher's exact test. Patient demographics, transfusion history and antibody formation in cases of AUS were collected. RESULTS: A significant reduction in potential WAA frequencies from 127/1167 (11%) to 53/854 (6%) was observed (p < 0.001) when compared between the old and new AIAs among 2021 positive SPRCA antibody screens. While no patients with AUS later transitioned to potential WAA using the new AIA, four patients developed alloantibodies, including anti-E, anti-C, both anti-C and anti-E, and anti-Wra . CONCLUSION: A significant reduction in the frequencies of potential false-positive WAA detection at our centre was observed after implementing the new AIA, leading to less resource and phenotypically matched red blood cell (RBC) use. Some patients still developed subsequent RBC alloimmunization, so clinically relevant alloantibodies should be carefully excluded before determining AUS, taking forming or evanescent antibodies into consideration.

Inhibitory activity of Limosilactobacillus reuteri isolated from camel milk against Helicobacter pylori effects in human gastric epithelial cells.

This study aimed to evaluate anti-Helicobacter pylori effects of Limosilactobacillus reuteri 2892 (L. reuteri 2892) isolated from camel milk in GC cell lines (AGS and MKN). From 15 camel milk samples, 132 microbial strains were isolated. Based on microbial and biochemical analysis, 11 potential probiotic candidates were selected. The potential probiotic candidates were assayed for anti-H. pylori activity, and the strain with the highest anti-H. pylori activity was identified genotypically. Based on 16S rDNA sequencing, the selected strain with the best activity against H. pylori (inhibition zone = 15.5 ± 0.8) belonged to the Lactobacillus reuteri strain 2892. Cell treatment with H. pylori HC-113 inhibits gene expression of Claudin-4, ZO-1, MUC5AC, and MUC2 in gastric cells, which are attenuated by L. reuteri 2892. The simulative effects of H. pylori HC-113 on the cell migration and invasion of gastric cells were lost when cells were cotreated with L. reuteri 2892. Cell treatment with H. pylori HC-113 promoted cell death, whereas cotreatment with L. reuteri 2892 markedly decreased necrotic and late apoptotic cells. The present study demonstrates that L. reuteri 2892 has potent anti-H. pylori effects and thus can be considered as an alternative protective agent against inflammatory effects of H. pylori in gastric cells.

Does decreasing the incubation period used in the antibody screen affect its sensitivity?

BACKGROUND: Pre-transfusion testing (PTT) encompasses a set of mandatory laboratory tests performed before red blood cell transfusion. The antibody screen, one component of PTT, commonly includes a 10-20 min incubation. The primary aim of this study was to determine if this period can be reduced when using current immunohematology methodologies. METHODS AND MATERIALS: Antibody screens were performed on reagent samples using Glass or Gel-based column agglutination technologies (CAT) and a solid phase red cell adherence (SPRCA) assay, with incubation periods of 1, 5, 10 and 15 min, and 20 min (SPRCA assay only). For each method, the shortest period producing a minimum of a 1+ reaction with all reagent samples was considered optimal. The sensitivity of each assay using the optimal period was calculated after performing antibody screens on 100 patient samples. RESULTS AND DISCUSSION: It was demonstrated that the incubation period in the SPRCA and Glass CAT systems can be reduced to 5 and 10 min, respectively, while achieving high assay sensitivity (98.9% in both). The incubation period in the Gel CAT system cannot be reduced from 15 min. Significant association between titre and reaction strength was observed for all three screening methods (p < 0.001 for both CAT methods, p = 0.041 for SPRCA). This study demonstrates that the incubation period used in the antibody screen can be reduced when using systems employing the Glass CAT and SPRCA methods, without affecting assay sensitivity. If confirmed, it could result in faster completion of PTT.

Evaluation of solid-phase panreactivity with negative direct antiglobulin testing.

Solid-phase red cell adherence (SPRCA) is a sensitive platform for antibody detection, but nonspecific reactions may occur. One pattern of apparent nonspecific reactivity is a panagglutinin with a negative direct antiglobulin test (DAT). The purpose of this study was to define the clinical characteristics of patients with these nonspecific reactions and their associated serologic findings. Twenty patients with panreactive SPRCA testing results were identified between November 2022 and May 2023. In addition to panagglutinins, these patients had (1) a negative polyethylene glycol (PEG) antibody detection test, (2) a negative PEG autocontrol, and (3) a negative DAT. The strength of SPRCA panreactivity and the results of eluate testing (by tube and SPRCA) were studied. Clinical characteristics of patients included age, sex, and primary diagnosis. Each patient was also assessed for evidence of hemolysis. Fourteen female and six male patients were evaluated (average age 44 years). Primary diagnoses included pregnancy (n = 10), acute bleeding (n = 4), orthopedic (n = 3), and other (n = 3). There was no clinical or laboratory evidence of hemolysis. The predominant strength of SPRCA panreactivity was evenly distributed across reaction grades (1+ to 3+). Fifty-five percent of the eluates tested in PEG showed panreactivity, consistent with warm-reactive autoantibodies, while 85 percent of eluates tested by SPRCA were panreactive. Six discrepant cases, in which PEG eluate testing was negative and solid-phase eluate testing showed panreactivity, were associated with weak solid-phase plasma panreactivity (1+). In addition, the reactivity strengths of the eluates tested by SPRCA were invariably more strongly reactive than those eluates tested in PEG. Panagglutination is a distinct SPRCA-only plasma reactivity pattern. Despite a negative PEG tube and DAT, most panagglutinins are warm-reactive autoantibodies. Fortunately, these "interfering" panagglutinins do not appear to be clinically significant and are easily managed by an alternative testing method such as PEG.

The Biological Activity of Fragmented Computer-Aided Design/Manufacturing Dental Materials before and after Exposure to Acidic Environment.

Three ceramic and composite computer-aided design/computer-aided manufacturing (CAD/CAM) materials from different manufacturers (Cerasmart (CS)-nanoceramic resin; Straumann Nice (SN)-glass ceramic and Tetric CAD (TC)-composite resin) were tested to investigate the biocompatibility and sustainability on human fibroblasts and keratinocytes cells. Each type of CAD/CAM blocks restorative materials with fine and rough surfaces was exposed to an acidic environment for one month. After that, various powders were obtained by milling. In parallel, powders were also prepared from each restorative material, which were not exposed to the acidic environment. The cytotoxic effects were investigated by means of MTT and LDH assays, as well as nitric oxide production on two human normal cell lines, namely, fibroblasts (BJ) and keratinocytes (HaCaT). In addition, the degree of adhesion of fibroblast cells to each CAD/CAM material was evaluated by scanning electron microscopy (SEM). The results showed that the two samples that were exposed to an acidic environment (CS and SN) induced a reduction of mitochondrial activity and plasma membrane damage as regards the fibroblast cells. A similar effect was observed in TC_fine-exposed material, which seemed to induce necrosis at the tested concentration of 1 mg/mL. No oxidative stress was observed in fibroblasts and keratinocytes treated with the CAD/CAM materials. Regarding the adhesion degree, it was found that the fibroblasts adhere to all the occlusal veneers tested, with the mention that the CS and SN materials have a weaker adhesion with fewer cytoplasmic extensions than TC material. With all of this considered, the CAD/CAM restorative materials tested are biocompatible and represent support for the attachment and dispersion of cells.

Development of a carboxyl-terminated indium tin oxide electrode for improving cell adhesion and facilitating low noise, real-time impedance measurements.

The working electrode's surface property is crucial to cell adhesion and signal collection in electric cell-substrate impedance sensing (ECIS). To date, the indium tin oxide (ITO)-based working electrode is of interest in ECIS study due to its high transparency and biocompatibility. Of great concern is the impedance signal loss, distortion, and data interpretation conflict profoundly created by the movement of multiple cells during ECIS study. Here, a carboxyl-terminated ITO substrate was prepared by stepwise surface amino silanization, with N-hydroxy succinimide (NHS) and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) treatment, respectively. We investigated the stepwise changes in the property of the treated ITO, cell-substrate adhesion, collective cell mobility, and time course of change in absolute impedance from multiple Chinese hamster ovary (CHO) cells [(Δt-Δ|Z|)CELLS]. The carboxyl-terminated ITO substrate with a surface roughness of 6.37 nm shows enhanced conductivity, 75% visible light transparency, improved cell adherence, reduced collective cell migration speed by approximately twofold, and diminished signal distortion in the [(Δt-Δ|Z|)CELLS]. Thus, our study provides an ITO surface-treatment strategy to reduce multiple cell movement effects and to obtain essential cell information from the ECIS study of multiple cells through undistorted (Δt-Δ|Z|)CELLS.

In Vitro Bioactivity and Biocompatibility of Bio-Inspired Ti-6Al-4V Alloy Surfaces Modified by Combined Laser Micro/Nano Structuring.

The bioactivity and biocompatibility play key roles in the success of dental and orthopaedic implants. Although most commercial implant systems use various surface microstructures, the ideal multi-scale topographies capable of controlling osteointegration have not yielded conclusive results. Inspired by both the isotropic adhesion of the skin structures in tree frog toe pads and the anisotropic adhesion of the corrugated ridges on the scales of Morpho butterfly wings, composite micro/nano-structures, including the array of micro-hexagons and oriented nano-ripples on titanium alloy implants, were respectively fabricated by microsecond laser direct writing and femtosecond laser-induced periodic surface structures, to improve cell adherence, alignment and proliferation on implants. The main differences in both the bioactivity in simulated body fluid and the biocompatibility in osteoblastic cell MC3T3 proliferation were measured and analyzed among Ti-6Al-4V samples with smooth surface, micro-hexagons and composite micro/nano-structures, respectively. Of note, bioinspired micro/nano-structures displayed the best bioactivity and biocompatibility after in vitro experiments, and meanwhile, the nano-ripples were able to induce cellular alignment within the micro-hexagons. The reasons for these differences were found in the topographical cues. An innovative functionalization strategy of controlling the osteointegration on titanium alloy implants is proposed using the composite micro/nano-structures, which is meaningful in various regenerative medicine applications and implant fields.

Biological dermal templates with native collagen scaffolds provide guiding ridges for invading cells and may promote structured dermal wound healing.

Dermal substitutes are of major importance in treating full thickness skin defects. They come in a variety of materials manufactured into various forms, such as films, hydrocolloids, hydrogels, sponges, membranes, and electrospun micro- and nanofibers. Bioactive dermal substitutes act in wound healing either by delivery of bioactive compounds or by being constructed from materials having endogenous activity. The healing success rate is highly determined by cellular and physiological processes at the host-biomaterial interface during crucial wound healing steps. Hence, it is important to design appropriate wound treatment strategies with the ability to work actively with tissues and cells to enhance healing. Therefore, in this study, we investigated biological dermal templates and their potential to stimulate natural cell adherence, guidance, and morphology. The most pronounced effect was observed in biomaterials with the highest content of native collagen networks. Cell attachment and proliferation were significantly enhanced on native collagen scaffolds. Cell morphology was more asymmetrical on such scaffolds, resembling native in vivo structures. Importantly, considerably lower expression of myofibroblast phenotype was observed on native collagen scaffolds. Our data suggest that this treatment strategy might be beneficial for the wound environment, with the potential to promote improved tissue regeneration and reduce abnormal scar formation.

Increased Intracellular Cyclic di-AMP Levels Sensitize Streptococcus gallolyticus subsp. gallolyticus to Osmotic Stress and Reduce Biofilm Formation and Adherence on Intestinal Cells.

Cyclic di-AMP is a recently identified second messenger exploited by a number of Gram-positive bacteria to regulate important biological processes. Here, we studied the phenotypic alterations induced by the increased intracellular c-di-AMP levels in Streptococcus gallolyticus, an opportunistic pathogen responsible for septicemia and endocarditis in the elderly. We report that an S. gallolyticus c-di-AMP phosphodiesterase gdpP knockout mutant, which displays a 1.5-fold higher intracellular c-di-AMP levels than the parental strain UCN34, is more sensitive to osmotic stress and is morphologically smaller than the parental strain. Unexpectedly, we found that a higher level of c-di-AMP reduced biofilm formation of S. gallolyticus on abiotic surfaces and reduced adherence and cell aggregation on human intestinal cells. A genome-wide transcriptomic analysis indicated that c-di-AMP regulates many biological processes in S. gallolyticus, including the expression of various ABC transporters and disease-associated genes encoding bacteriocin and Pil3 pilus. Complementation of the gdpP in-frame deletion mutant with a plasmid carrying gdpP in trans from its native promoter restored bacterial morphology, tolerance to osmotic stress, biofilm formation, adherence to intestinal cells, bacteriocin production, and Pil3 pilus expression. Our results indicate that c-di-AMP is a pleiotropic signaling molecule in S. gallolyticus that may be important for S. gallolyticus pathogenesis.IMPORTANCEStreptococcus gallolyticus is an opportunistic pathogen responsible for septicemia and endocarditis in the elderly and is also strongly associated with colorectal cancer. S. gallolyticus can form biofilms, express specific pili to colonize the host tissues, and produce a specific bacteriocin allowing killing of commensal bacteria in the murine colon. Nevertheless, how the expression of these colonization factors is regulated remains largely unknown. Here, we show that c-di-AMP plays pleiotropic roles in S. gallolyticus, controlling the tolerance to osmotic stress, cell size, biofilm formation on abiotic surfaces, adherence and cell aggregation on human intestinal cells, expression of Pil3 pilus, and production of bacteriocin. This study indicates that c-di-AMP may constitute a key regulatory molecule for S. gallolyticus host colonization and pathogenesis.

Thermal expansion of substrate may affect adhesion of Chinese hamster fibroblasts to surfaces during freezing.

Despite success in cryopreservation of cells in suspension, cryopreservation of cells in monolayers is still challenging. One of the major problems is detachment of the cells from the substrate which occurs during cryopreservation. We hypothesized that this detachment may be due to a mismatch in the coefficient of linear thermal expansion αL between glass and the frozen cell layer which manifests as residual stress and stress relaxation. This mismatch results in a difference between the thermal expansion of ice and glass as they undergo temperature changes. Rinzl plastic coverslips were selected as a possible substitute for glass because Rinzl has an αL (60 × 10-6/K) similar to that of ice (51 × 10-6/K) whereas glass has a much lower αL (5 × 10-6/K). V79-4 Chinese hamster fibroblasts were cultured on both glass and Rinzl coverslips until confluent and the area of coverage was measured before and after freezing at -9 °C. The glass coverslips showed significant loss of cells (coverage = 77.9 ±â€¯8.0%) compared with Rinzl (coverage = 97.9 ±â€¯1.4%). We concluded that Rinzl coverslips may improve cell attachment in future monolayer cryopreservation experiments.

Combined Effects of Gold Nanoparticles and Ionizing Radiation on Human Prostate and Lung Cancer Cell Migration.

The effect of 15 nm-sized gold nanoparticles (AuNPs) and/or ionizing radiation (IR) on the migration and adhesion of human prostate (DU145) and lung (A549) cancer cell lines was investigated. Cell migration was measured by observing the closing of a gap created by a pipette tip on cell monolayers grown in 6-well plates. The ratio of the gap areas at 0 h and 24 h were used to calculate the relative migration. The relative migration of cells irradiated with 5 Gy was found to be 89% and 86% for DU145 and A549 cells respectively. When the cells were treated with 1 mM AuNPs this fell to ~75% for both cell lines. However, when the cells were treated with both AuNPs and IR an additive effect was seen, as the relative migration rate fell to ~60%. Of interest was that when the cells were exposed to either 2 or 5 Gy IR, their ability to adhere to the surface of a polystyrene culture plate was significantly enhanced, unlike that seen for AuNPs. The delays in gap filling (cell migration) in cells treated with IR and/or AuNPs can be attributed to cellular changes which also may have altered cell motility. In addition, changes in the cytoskeleton of the cancer cells may have also affected adhesiveness and thus the cancer cell's motility response to IR.

Uterine Foxl2 regulates the adherence of the Trophectoderm cells to the endometrial epithelium.

BACKGROUND: Forkhead Transcription Factor L2 (FOXL2) is a member of the forkhead family with important roles in reproduction. Recent studies showed that FOXL2 is expressed in human and bovine endometrium and that its levels fluctuate during pregnancy. In this study, we aimed at evaluating the expression and function of FOXL2 in embryo implantation. METHODS: Mouse uteri at different days of pregnancy were isolated and analyzed for the expression and localization of FOXL2. A lentiviral strategy was further employed to either knockdown or overexpress FOXL2 in non-receptive human endometrial AN3-CA cells and in receptive Ishikawa cells, respectively. These genetically modified cells were compared to cells infected with a control lentivirus to determine the function of FOXL2 in trophectoderm cells adherence to Endometrial Epithelium was associated with the expression of genes known to be involved in acquisition of uterine receptivity. RESULTS: We report that FOXL2 is expressed in both, the luminal epithelium and the myometrium of the mouse uterus and that its expression declines prior to implantation. We found that endometrial cells expressing low FOXL2 levels, either endogenous or genetically manipulated, were associated with a higher attachment rate of mouse blastocysts or human Jeg3 spheroids and mouse blastocysts. In accordance, low-FOXL2 levels were associated with changes in the expression level of components of the Wnt/Fzd and apoptotic pathways, both of which are involved in uterine receptivity. Furthermore, FOXL2 expression was inversely correlated with G-protein signaling protein 2 (Rgs2) and cytokine expression. CONCLUSIONS: These results suggest that FOXL2 interferes with embryo attachment. Better understanding of the function of FOXL2 in the uterus would possibly suggest novel strategies for treatment of infertility attributed to repeated implantation failure.

Contribution of pilus type 2b to invasive disease caused by a Streptococcus agalactiae ST-17 strain.

BACKGROUND: Group B Streptococcus (GBS) is a major cause of invasive disease especially in neonates. In GBS three structurally distinct pilus polymers have been identified as important virulence factors and promising vaccine candidates. The vast majority of Group B Streptococci belonging to the hypervirulent serotype III ST-17 lineage bear pilus types 1 and 2b. The purpose of this study was to investigate the relative contribution of these two pilus types to the pathogenesis of a ST-17 strain. RESULTS: We performed in vivo and in vitro analysis of isogenic knockout mutants derived from the GBS COH1 ST-17 strain deprived of either pilus type 1 or 2b. We compared the two pilus mutants with the wild type strain in a mouse model of invasive disease, in vitro survival in macrophages, and adherence/invasion assays using human brain endothelial and lung epithelial cell lines. Significantly less of the pilus 2b mutant was recovered from the blood, lungs and brain tissue of infected mice compared to the wild-type and pilus 1 mutant strains. Further, while the pilus 2b mutant survived similarly in murine macrophages, it exhibited a lower capacity to adhere and invade human brain epithelial and lung endothelial cell lines. CONCLUSIONS: The data suggest an important role of pilus 2b in mediating GBS infection and host cell interaction of strains belonging to the hypervirulent GBS ST-17 lineage.

Basic leucine zipper (bZIP) domain transcription factor MBZ1 regulates cell wall integrity, spore adherence, and virulence in Metarhizium robertsii.

Transcription factors (TFs) containing the basic leucine zipper (bZIP) domain are widely distributed in eukaryotes and display an array of distinct functions. In this study, a bZIP-type TF gene (MBZ1) was deleted and functionally characterized in the insect pathogenic fungus Metarhizium robertsii. The deletion mutant (ΔMBZ1) showed defects in cell wall integrity, adhesion to hydrophobic surfaces, and topical infection of insects. Relative to the WT, ΔMBZ1 was also impaired in growth and conidiogenesis. Examination of putative target gene expression indicated that the genes involved in chitin biosynthesis were differentially transcribed in ΔMBZ1 compared with the WT, which led to the accumulation of a higher level of chitin in mutant cell walls. MBZ1 exhibited negative regulation of subtilisin proteases, but positive control of an adhesin gene, which is consistent with the observation of effects on cell autolysis and a reduction in spore adherence to hydrophobic surfaces in ΔMBZ1. Promoter binding assays indicated that MBZ1 can bind to different target genes and suggested the possibility of heterodimer formation to increase the diversity of the MBZ1 regulatory network. The results of this study advance our understanding of the divergence of bZIP-type TFs at both intra- and interspecific levels.

Binding of Streptococcus pneumoniae endopeptidase O (PepO) to complement component C1q modulates the complement attack and promotes host cell adherence.

The Gram-positive species Streptococcus pneumoniae is a human pathogen causing severe local and life-threatening invasive diseases associated with high mortality rates and death. We demonstrated recently that pneumococcal endopeptidase O (PepO) is a ubiquitously expressed, multifunctional plasminogen and fibronectin-binding protein facilitating host cell invasion and evasion of innate immunity. In this study, we found that PepO interacts directly with the complement C1q protein, thereby attenuating the classical complement pathway and facilitating pneumococcal complement escape. PepO binds both free C1q and C1 complex in a dose-dependent manner based on ionic interactions. Our results indicate that recombinant PepO specifically inhibits the classical pathway of complement activation in both hemolytic and complement deposition assays. This inhibition is due to direct interaction of PepO with C1q, leading to a strong activation of the classical complement pathway, and results in consumption of complement components. In addition, PepO binds the classical complement pathway inhibitor C4BP, thereby regulating downstream complement activation. Importantly, pneumococcal surface-exposed PepO-C1q interaction mediates bacterial adherence to host epithelial cells. Taken together, PepO facilitates C1q-mediated bacterial adherence, whereas its localized release consumes complement as a result of its activation following binding of C1q, thus representing an additional mechanism of human complement escape by this versatile pathogen.

Characterization of Lactobacillus isolated from dairy samples for probiotic properties.

In the present study twelve Lactobacillus isolates (LBS 1-LBS 12) were characterized for probiotic properties. Out of the twelve, eight isolates (LBS 1-6, 8 and 11) were bile resistant (survival > 50% at 0.3% bile salt w/v) and five isolates (LBS 1, 2, 5, 6 and 11) were found acid pH value resistant (survival > 50% at pH 3). All twelve isolates inhibited the growth of Staphylococcus aureus whereas isolate LBS 2 also inhibited the growth of Escherichia coli and Salmonella typhimurium. Antibiotic susceptibility testing of isolates was also performed and isolate LBS 2 was selected for further study based on its broad spectrum effect in clinical pathogen inhibition. LBS 2 was characterized phenotypically at Institute of Microbial Technology (IMTECH), Chandigarh, India and was confirmed as Lactobacillus rhamnosus by 16S rDNA sequencing and subsequent analysis using BLAST. The gene sequence was deposited in GenBank with accession number KJ562858. Scanning electron microscopy (SEM) study was used to study in vitro epithelial cell adherence and bile salt effect on isolate LBS 2. Epithelial cells adherence assay showed positive results and surface roughness of LBS 2 increased with increase in bile salt (0.15-0.45% w/v).

Correctly identifying the cells of origin is essential for tailoring treatment and understanding the emergence of cancer stem cells and late metastases.

Malignancy manifests itself by deregulated growth and the ability to invade surrounding tissues or metastasize to other organs. These properties are due to genetic and/or epigenetic changes, most often mutations. Many aspects of carcinogenesis are known, but the cell of origin has been insufficiently focused on, which is unfortunate since the regulation of its growth is essential to understand the carcinogenic process and guide treatment. Similarly, the concept of cancer stem cells as cells having the ability to stop proliferation and rest in a state of dormancy and being resistant to cytotoxic drugs before "waking up" and become a highly malignant tumor recurrence, is not fully understood. Some tumors may recur after decades, a phenomenon probably also connected to cancer stem cells. The present review shows that many of these questions are related to the cell of origin as differentiated cells being long-term stimulated to proliferation.

The interaction between bacterial enolase and plasminogen promotes adherence of Streptococcus pneumoniae to epithelial and endothelial cells.

Binding and conversion of the plasma protein plasminogen is an important pathogenesis mechanism of the human pathogen Streptococcus pneumoniae. Once converted into plasmin, the proteolytic activity of this major fibrinolysis component promotes degradation of extracellular matrix and the dissolution of fibrin clots. Here, we present the exploitation of plasminogen-binding as a further pivotal strategy of pneumococci facilitating adherence to eukaryotic cells. Flow cytometric measurements demonstrated the immobilization of plasminogen on host cell surfaces of human alveolar type II pneumocytes (A549), nasopharyngeal epithelium (Detroit 562) and brain-derived endothelial cells (HBMEC). These host-derived cells were employed in cell culture infection analyses followed by confocal microscopy to monitor the plasminogen-mediated adherence. Results of these studies revealed that host cell-bound plasminogen promotes pneumococcal adherence to human epithelial and endothelial cells in dose-dependent manner, whereas pneumococcal internalization was not enhanced. As an opposed effect pneumococcal-bound plasminogen reduced attachment to the epithelial and endothelial cells, and increased the interaction with neutrophil granulocytes. Moreover, the surface-displayed enolase, which serves as major pneumococcal plasminogen receptor, was identified as a key factor for plasminogen-mediated bacterial attachment in infection analyses with S. pneumoniae enolase mutants.

Techno-functional characterization of fecal lactobacilli isolates of Bos indicus calves for probiotic properties.

In this study, 105 bacterial colonies were isolated from the feces of newborn healthy Bos indicus calves and 37 isolates were confirmed using morphological, biochemical tests, and genus-specific PCR as lactobacilli. 11 isolates were then short-listed for in vitro probiotic testing based on their ability to dwell under acid and bile stress. Species-level identification using 16S rRNA gene sequencing revealed that they were Ligilactobacillus salivarius. These isolates flourished in 0.4% phenol, depicting resistance in adverse conditions encountered in the gastrointestinal tract. The results of cell surface hydrophobicity were found to be 74.50% for RBL12 and 62.62% for RBL09 in hexadecane and xylene, respectively, and that of auto-aggregation was highest in RBL26 (58.92%). These isolates also produced digestive enzymes like amylase, protease, and ß-galactosidase. Further assays reiterated their antimicrobial and coaggregation potential against diarrhea-causing pathogens like Escherichia coli ATCC-25922 and Salmonella arizonae ATCC-13314. Biosafety assessment revealed that none of the tested isolates were hemolytic and mucinolytic in nature. Furthermore, the antioxidant potential of the isolates was also confirmed using 1,1­diphenyl­2­picrylhydrazyl (DPPH) and ferric ion-reducing antioxidant power (FRAP) assay. Along with efficient utilization of inulin, isolates showed promising adhesion ability to the HT-29 cell line. The current findings hence conclude that these Lactobacillus isolates can be exploited as animal probiotics for potential application in young calves to foster gut health and immunity.

Detection and antibiotic resistance of diarrheagenic Escherichia coli from patients with diarrhea in Ulaanbaatar, Mongolia.

INTRODUCTION: Diarrheal diseases are common with worldwide distribution, and diarrheagenic Escherichia coli (DEC) strains are the main causative agents. The present study aimed to define the association of various pathotypes of E. coli from diarrheal patients in Mongolia. METHODOLOGY: A total of 341 E. coli strains were isolated from the stool of diarrheal patients. Bacterial susceptibility to antimicrobial agents was determined by the Kirby Bauer disk diffusion method. DEC isolates were identified by HEp-2 cell adherence assay and multiplex polymerase chain reaction (PCR). RESULTS: DEC pathogens were detected in 53.7% of 341 E. coli isolates. Enteroaggregative E. coli (EAEC) was the most common DEC pathotype identified by HEp-2 adherence assay and multiplex PCR methods in 97 samples (28.4%), followed by atypical enteropathogenic E. coli (EPEC) in 50 samples (14.7%), diffusely adherent E. coli (DAEC) in 25 samples (7.3%), enterohaemorrhagic E. coli (EHEC) in 6 samples (1.8%), enterotoxigenic E. coli (ETEC) in 4 samples (1.2%), and enteroinvasive E. coli (EIEC) in 1 sample (0.3%). DEC strains had > 50% antibiotic resistance against cephalothin, ampicillin, and trimethoprim/sulfamethoxazole. All tested DEC strains were susceptible to imipenem. Among the 183 DEC strains, 27 (14.8%) were extended spectrum beta-lactamase producing isolates, and 125 (68.3%) isolates were multiple drug resistant. CONCLUSIONS: We have identified six pathotypes of DEC from the clinical isolates tested and concluded that a high prevalence of antimicrobial resistance was observed in these pathotypes. EAEC was the most common pathotype identified and this is the first report of EHEC identification in Mongolia.