Hemolysis Production Research Articles

Serogroup identification, phenotypic detection of hemolysis and extended spectrum beta-lactamases of Escherichia coli isolated from psittacine of illegal wildlife trade in Fortaleza, Brazil

ABSTRACT This study aimed to identify serogroups of Escherichia coli important for human health in isolates from psittacine of illegal wildlife trade in Ceará State. In addition, hemolysis and production of Extended Spectrum Beta-Lactamases (ESBL) was assessed in the isolates. A total of 78 E. coli strains isolated from different Psittaciformes species from a wildlife rehabilitation center in Fortaleza, Brazil. The isolates used in this study were previously identified and stored. Serogroup identification was performed using polyvalent sera for EPEC (O55, O111, O119, O114, O125, O86, O126, O127, O128), EIEC (O136, O124) and EHEC (O157). ESBL detection was performed with double disk synergy method. For hemolysis detection, isolates were inoculated in blood agar base enriched with ovine blood. Only 31 (39.7%) isolates were seropositive and the most frequent were O127, O114, O128 and O111. There was no agglutination for serogroups O55, O124, O136 or O157. Considering both seropositive and seronegative isolates, 9 (11.5%) and 35 (44.9%) presented hemolysis and ESBL production, respectively. In conclusion, the investigated psittacine from illegal wildlife trade hosted ESBL-producing E. coli strains and some belong to important serogroups often linked to severe human infections.

Comparative prevalence and characterization of Vibrio spp. isolated from live and frozen white‐leg shrimp (Litopenaeus vannamei) in Korean markets

AbstractWe evaluated the prevalence and characteristics of Vibrio spp. in frozen and live samples of white‐leg shrimp (Litopenaeus vannamei) sold in Korea. The mean total bacterial counts of frozen and live white‐leg shrimp meat were approximately 2 ×109 and 1 ×1010 CFU/g, respectively. Eight V. parahaemolyticus isolates were recovered from frozen samples, whereas a total of 40 strains including 30 V. parahaemolyticus, 8 V. alginolyticus, 1 V. harveyi, and 1 V. fluvialis were isolated from live samples. The majority (≥92%) of the Vibrio spp. isolates were positive for six extracellular enzymatic tests, while 50 and 29% of the isolates were positive for hemolysis and slime production, respectively. In virulence gene profile, all V. parahaemolyticus isolates were positive for toxR and tlh genes, but negative for tdh and trh. V. alginolyticus isolates harbored collagenase, toxR, tlh, and VPI genes in 10, 75, 86, and 63%, respectively. Besides, V. fluvialis isolate was positive for toxR, vfh, and hupO genes. Moreover, 100, 91, 50, 45, and 27% of the isolates were resistant to ampicillin, piperacillin, colistin, vancomycin, and amikacin, respectively. These results implied that white‐leg shrimp‐borne Vibrio spp. are indicative of a potential consumer health risk.Practical applicationsVibrio is a well‐known group of seafoodborne pathogenic bacteria. Since white‐leg shrimp is the most preferred shrimp variety in Korea, we sought to evaluate the prevalence and characteristics of Vibrio spp. in both frozen and live white‐leg shrimp from several retail markets. A total of 48 isolates were presumptively identified by biochemical tests. Subsequently, the gyrB gene sequencing and the neighbor‐joining phylogenetic tree confirmed the prevalence of 4 and 1 Vibrio species in live and frozen samples, respectively. Prevalence of Vibrio in frozen samples was much lower than live samples. Putative virulence of the Vibrio spp. was examined by both pathogenicity tests and PCR gene detection. Majority of the isolates were positive for pathogenicity tests and a heterogeneity in virulence gene prevalence was detected among Vibrio spp. Antibiotic susceptibility was examined by disc diffusion test for 25 antibiotics belong to 13 classes. The Vibrio isolates were highly resistant to penicillin group, however, others had different susceptibilities. In addition, we examined the total bacterial count (TBC) of the white‐leg shrimp meat by aerobic plate count method to evaluate their total bacterial prevalence. The obtained TBCs were higher than the recommended value. With regards to the results, it is worth noting that the white‐leg shrimp in Korean markets are indicative of a potential public health risk.

L‐Arginine Prevents Heme‐Induced Acute Lung Injury (ALI) in a Rat Model of Trauma/Hemorrhage and Resuscitation with Transfusion

Resuscitation with long (> 28 days) stored packed red blood cells (PRBC) promotes acute lung injury (ALI) in our rat model of trauma/hemorrhage. Low nitric oxide (NO) production has been implicated in the development of ALI. Arginase can decrease NO production by decreasing L‐arginine availability for endothelial NO synthase (eNOS). We found that arginase, released from red blood cells (RBC) during prolonged storage, contributes to ALI in our rat model of trauma/hemorrhage. Prolonged storage also increases levels of free hemoglobin (Hb). Heme, a breakdown product of Hb, is metabolized by heme oxygenase to form carbon monoxide (CO). We were the first to report that heme‐derived CO inhibits endothelial nitric oxide (NO) synthase and promotes vasoconstriction in a manner that can be prevented by L‐arginine administration. This study tests the hypothesis that the hemolysis product, heme, promotes ALI in our rat model of trauma/hemorrhage and resuscitation with transfusion in a manner that is exacerbated by limited L‐arginine availability. Fresh PRBC were prepared from rat whole blood. Male Lewis rats were implanted with femoral arterial and venous catheters (trauma), subjected to a 30% hemorrhage and resuscitated with crystalloids and fresh PRBC containing heme alone or with L‐arginine or L‐lysine (competitive inhibitor of L‐arginine uptake). ALI was assessed by pulmonary protein leak (Evans blue dye ‐ EBD) and cytokine‐induced neutrophil chemoattractant‐1 (CINC‐1; rat homologue of human interleukin (IL)‐8) accumulation in bronchoalveolar lavage fluid (BALF). Heme alone caused a 5.6‐fold increase in pulmonary protein leak and 3.5‐fold increase in CINC‐1 accumulation. Co‐administration of L‐arginine decreased heme‐induced pulmonary protein leak by 77% and CINC‐1 accumulation by 32.4%. In contrast, L‐lysine, a competitive inhibitor of L‐arginine uptake, increased heme‐induced pulmonary protein leak by 102% and CINC‐1 accumulation by 106.4%. These data show that the Hb breakdown product, heme, promotes ALI in our rat model of trauma/hemorrhage and resuscitation with transfusion. L‐Arginine co‐administration protects against heme‐induced ALI, while blocking L‐arginine transport by L‐lysine exacerbates heme‐induced ALI. These results suggest that the other hemolysis product, heme, also contributes to ALI in a manner that is greatly exacerbated by decreased L‐arginine bioavailability. We conclude that hemolysis products arginase and heme work in concert to promote ALI during trauma/hemorrhage and resuscitation with transfusion.Support or Funding InformationSupported by grants AHA 0865241F (FKJ), NIH HL074966 (WD) and USAMRC DOD W81XWH‐07‐1‐0717 (RAJ, RMS).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of heme in lung bacterial infection after trauma hemorrhage and stored red blood cell transfusion: A preclinical experimental study.

BackgroundTrauma is the leading cause of death and disability in patients aged 1–46 y. Severely injured patients experience considerable blood loss and hemorrhagic shock requiring treatment with massive transfusion of red blood cells (RBCs). Preclinical and retrospective human studies in trauma patients have suggested that poorer therapeutic efficacy, increased severity of organ injury, and increased bacterial infection are associated with transfusion of large volumes of stored RBCs, although the mechanisms are not fully understood.Methods and findingsWe developed a murine model of trauma hemorrhage (TH) followed by resuscitation with plasma and leukoreduced RBCs (in a 1:1 ratio) that were banked for 0 (fresh) or 14 (stored) days. Two days later, lungs were infected with Pseudomonas aeruginosa K-strain (PAK). Resuscitation with stored RBCs significantly increased the severity of lung injury caused by P. aeruginosa, as demonstrated by higher mortality (median survival 35 h for fresh RBC group and 8 h for stored RBC group; p < 0.001), increased pulmonary edema (mean [95% CI] 106.4 μl [88.5–124.3] for fresh RBCs and 192.5 μl [140.9–244.0] for stored RBCs; p = 0.003), and higher bacterial numbers in the lung (mean [95% CI] 1.2 × 107 [−1.0 × 107 to 2.5 × 107] for fresh RBCs and 3.6 × 107 [2.5 × 107 to 4.7 × 107] for stored RBCs; p = 0.014). The mechanism underlying this increased infection susceptibility and severity was free-heme-dependent, as recombinant hemopexin or pharmacological inhibition or genetic deletion of toll-like receptor 4 (TLR4) during TH and resuscitation completely prevented P. aeruginosa–induced mortality after stored RBC transfusion (p < 0.001 for all groups relative to stored RBC group). Evidence from studies transfusing fresh and stored RBCs mixed with stored and fresh RBC supernatants, respectively, indicated that heme arising both during storage and from RBC hemolysis post-resuscitation plays a role in increased mortality after PAK (p < 0.001). Heme also increased endothelial permeability and inhibited macrophage-dependent phagocytosis in cultured cells. Stored RBCs also increased circulating high mobility group box 1 (HMGB1; mean [95% CI] 15.4 ng/ml [6.7–24.0] for fresh RBCs and 50.3 ng/ml [12.3–88.2] for stored RBCs), and anti-HMGB1 blocking antibody protected against PAK-induced mortality in vivo (p = 0.001) and restored macrophage-dependent phagocytosis of P. aeruginosa in vitro. Finally, we showed that TH patients, admitted to the University of Alabama at Birmingham ER between 1 January 2015 and 30 April 2016 (n = 50), received high micromolar–millimolar levels of heme proportional to the number of units transfused, sufficient to overwhelm endogenous hemopexin levels early after TH and resuscitation. Limitations of the study include lack of assessment of temporal changes in different products of hemolysis after resuscitation and the small sample size precluding testing of associations between heme levels and adverse outcomes in resuscitated TH patients.ConclusionsWe provide evidence that large volume resuscitation with stored blood, compared to fresh blood, in mice increases mortality from subsequent pneumonia, which occurs via mechanisms sensitive to hemopexin and TLR4 and HMGB1 inhibition.

RON Kinase Inhibition Reduces Renal Endothelial Injury in Sickle Cell Disease Mice

BACKGROUND: Patients with Sickle Cell Disease (SCD) have an approximately three-fold higher risk of developing chronic kidney disease (CKD) than the general population. Endothelial dysfunction and inflammation which are induced by hemolysis and lead to vasculopathy are major mechanisms in the development of CKD. Red blood cells (RBCs) hemolysis inside the renal tissue stimulates macrophages infiltration. Endocytosis of RBC hemolysis products by infiltrating macrophages activates to MT-SP1 protease which cleaves and activates non-active circulating Macrophage Stimulating Protein 1 (MSP1). MSP1 binds to and activates cell surface receptor tyrosine kinase, recepteur d'origine nantais (RON).OBJECTIVES: To determine the role of RON in the development of renal endothelial injury.METHODS: Human renal glomerular endothelial cells (HGEC) and a mouse model of SCD (Townes) were used. The animal protocol was approved by the Institutional Animal Care and Use Committee at the Children's National Health System. The SCD mice express human Hgb S and Hgb F. Control mice express human Hgb A1. Two-month-old mice were injected with 10 mg/kg of body weight of RONi (BSM-777607, Santa Cruz Scientific), an inhibitor of RON, subcutaneously daily for 14 consecutive days. Kidneys were collected, and paraffin-embedded sections were used for H&E and PAS staining, and immunostaining.RESULTS: Treatment of cultured human renal glomerular endothelial cells (HGEC) with MSP1 activated RON receptor and induced downstream signaling evidenced by increased phosphorylation of ERK and AKT kinases. RONi significantly reduced ERK and AKT phosphorylation. MSP1 treatment also increased motility of HGEC, and expression of F-actin and von Willebrand Factor (vWF). Renal disease in SCD mice was characterized by glomerular hypertrophy, capillary dilation and congestion, and significant endothelial injury demonstrated by H&E and PAS staining, and ICAM, vWF and CD34 immunostaining. We observed accumulation of MSP1 in the glomerular capillaries of SCD mice. Treatment of mice with RONi significantly reduced glomerular hypertrophy, capillary dilation and congestion, and also reduced endothelial injury (Figure 1).CONCLUSIONS: Renal glomerular accumulation of MSP1 and RON kinase activation contribute to the induction of renal endothelial injury in SCD mice. Inhibition of RON kinase activation significantly reduced endothelial injury and development of kidney disease.ACKNOWLEDGMENTS: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005 and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Myricetin protects Galleria mellonella against Staphylococcus aureus infection and inhibits multiple virulence factors

Staphylococcus aureus is an opportunistic pathogen related to a variety of life-threatening infections but for which antimicrobial resistance is liming the treatment options. We report here that myricetin, but not its glycosylated form, can remarkably decrease the production of several S. aureus virulence factors, including adhesion, biofilm formation, hemolysis and staphyloxanthin production, without interfering with growth. Myricetin affects both surface proteins and secreted proteins which indicate that its action is unrelated to inhibition of the agr quorum sensing system. Analysis of virulence related gene expression and computational simulations of pivotal proteins involved in pathogenesis demonstrate that myricetin downregulates the saeR global regulator and interacts with sortase A and α-hemolysin. Furthermore, Myr confers a significant degree of protection against staphylococcal infection in the Galleria mellonella model. The present findings reveal the potential of Myr as an alternative multi-target antivirulence candidate to control S. aureus pathogenicity.

Clonality, virulence determinants, and profiles of resistance of clinical Acinetobacter baumannii isolates obtained from a Spanish hospital.

IntroductionAcinetobacter baumannii is a nosocomial pathogen that is showing increasing rates of carbapenem resistance. Multi-Drug Resistant (MDR) International Clones (ICs), associated with certain oxacillinases, are being reported globally. This organism also harbors numerous virulence determinants. In this study, we aim at characterizing A. baumannii isolated from a Spanish hospital in terms of antimicrobial susceptibility, clonality, carbapenemase genes harbored, and virulence determinants expressed.Materials and methodsFifty nine clinical bloodstream isolates were obtained from 2009 until 2013. Antimicrobial Susceptibility Testing was performed according to the CLSI guidelines. PFGE and tri-locus PCR typing were then performed in order to determine local and international clonality. PCRs for the detection of common carbapenemases were also performed. Production of hemolysis, biofilms, siderophores, surface motility, and proteolysis were determined phenotypically. Doubling times for selected strains were also calculated. Finally, statistical analysis for detecting associations between these factors was conducted.Results and discussionCarbapenem non-susceptibility was 84.75%, suggesting the immediate need for intervention. PFGE showed the distribution of the majority of the isolates among 7 clusters. Although all three ICs were detected, IC II was predominant at 71.19%. blaOXA-24-like was the most prevalent carbapenemase (62.71%), followed by blaOXA-58-like (13.56%), and blaOXA-23-like (11.86%). Strains pertaining to IC II, and those harboring blaOXA-24-like, were positively associated with α-hemolysis, production of strong biofilms, and siderophore production. Harboring blaOXA-23-like and blaOXA-58-like was associated with attenuated virulence. These associations suggest that an interplay exists between these factors that could be locally exploited.ConclusionsAn alarmingly high rate of carbapenem non-susceptibility has been detected in this study. There was a predominance of IC II and blaOXA-24-like, and those factors were associated with heightened expression of virulence determinants. This association could be exploited for modifying treatment regimens and for improving on infection control protocols in this hospital.

Tea polyphenols inhibit the growth and virulence properties of Fusobacterium nucleatum

Fusobacterium nucleatum plays a key role in creating the pathogenic subgingival biofilm that initiates destructive periodontitis. It is also a common resident of the human gastrointestinal tract and has been associated with inflammatory bowel disease. The aim of the present study was to investigate the effects of green and black tea extracts as well as two of their bioactive components, EGCG and theaflavins, on the growth and virulence properties of F. nucleatum. The tea extracts and components displayed various degrees of antibacterial activity that may involve damage to the bacterial cell membrane and the chelation of iron. They also prevented biofilm formation by F. nucleatum at concentrations that did not interfere with bacterial growth. In addition, the treatment of a pre-formed F. nucleatum biofilm with the green tea extract and EGCG caused a time-dependent decrease in biofilm viability. The green and black tea extracts, EGCG, and theaflavins decreased the adherence of F. nucleatum to oral epithelial cells and matrix proteins. Moreover, these tea components also attenuated F. nucleatum-mediated hemolysis and hydrogen sulfide production, two other virulence factors expressed by this bacterium. In summary, this study showed that tea polyphenols may be of interest for treating F. nucleatum-associated disorders.

Intravascular hemolysis and the pathophysiology of sickle cell disease.

Hemolysis is a fundamental feature of sickle cell anemia that contributes to its pathophysiology and phenotypic variability. Decompartmentalized hemoglobin, arginase 1, asymmetric dimethylarginine, and adenine nucleotides are all products of hemolysis that promote vasomotor dysfunction, proliferative vasculopathy, and a multitude of clinical complications of pulmonary and systemic vasculopathy, including pulmonary hypertension, leg ulcers, priapism, chronic kidney disease, and large-artery ischemic stroke. Nitric oxide (NO) is inactivated by cell-free hemoglobin in a dioxygenation reaction that also oxidizes hemoglobin to methemoglobin, a non-oxygen-binding form of hemoglobin that readily loses heme. Circulating hemoglobin and heme represent erythrocytic danger-associated molecular pattern (eDAMP) molecules, which activate the innate immune system and endothelium to an inflammatory, proadhesive state that promotes sickle vaso-occlusion and acute lung injury in murine models of sickle cell disease. Intravascular hemolysis can impair NO bioavailability and cause oxidative stress, altering redox balance and amplifying physiological processes that govern blood flow, hemostasis, inflammation, and angiogenesis. These pathological responses promote regional vasoconstriction and subsequent blood vessel remodeling. Thus, intravascular hemolysis represents an intrinsic mechanism for human vascular disease that manifests clinical complications in sickle cell disease and other chronic hereditary or acquired hemolytic anemias.

Suppressor analysis of eepR mutant defects reveals coordinate regulation of secondary metabolites and serralysin biosynthesis by EepR and HexS.

The EepR transcription factor positively regulates secondary metabolites and tissue-damaging metalloproteases. To gain insight into mechanisms by which EepR regulates pigment and co-regulated factors, genetic suppressor analysis was performed. Suppressor mutations that restored pigment to the non-pigmented ∆eepR mutant mapped to the hexS ORF. Mutation of hexS also restored haemolysis, swarming motility and protease production to the eepR mutant. HexS is a known direct and negative regulator of secondary metabolites in Serratia marcescens and is a LysR family regulator and an orthologue of LrhA. Here, we demonstrate that HexS directly controls eepR and the serralysin gene prtS. EepR was shown to directly regulate eepR expression but indirectly regulate hexS expression. Together, these data indicate that EepR and HexS oppose each other in controlling stationary phase-associated molecules and enzymes.

Deferoxamine’s Influence on Iron Metabolism, the Rheological Properties of Blood, Endothelial Dysfunction in an Experimental Model of Ischemia/Reperfusion

Objectives. Prevention of violations of microcirculation should take into account the massive entry of hemolysis products into the bloodstream and the related subsequent activation of free radical oxidation. Results. By simulating ischemia/reperfusion on rats we demonstrated the positive effect of deferoxamine on iron metabolism parameters (higher levels of transferrin, less ferritin and free iron, which received the drug group), also observed a decrease in the concentration of von Willebrand factor, as a measure of endothelial dysfunction. Blood viscosity parameters in the deferoxamine group was the closest to the control group parameters. Conclusion. Thus, we can conclude about the positive effect of deferoxamine on the parameters of iron metabolism, microcirculation and blood rheology in critical conditions involving ischemia/reperfusion.

Cytotoxicity and genotoxicity of a trypanocidal drug quinapyramine sulfate loaded-sodium alginate nanoparticles in mammalian cells

We synthesized quinapyramine sulfate loaded-sodium alginate nanoparticles (QS-NPs) to reduce undesirable toxic effects of QS against the parasite Trypanosoma evansi, a causative agent of trypanosomosis. To determine the safety of the formulated nanoparticles, biocompatibility of QS-NPs was determined using Vero, Hela cell lines and horse erythrocytes in a dose-dependent manner. Our experiments unveiled a concentration-dependent safety/cytotoxicity (metabolic activity), genotoxicity (DNA damage, chromosomal aberrations), production of reactive oxygen species and hemolysis in QS-NPs treated cells. Annexin-V propidium iodide (PI) staining showed no massive apoptosis or necrosis. However, at very high doses (more than 300 times than the effective doses), we observed more toxicity in QS-NPs treated cells as compared to QS treated cells. QS-NPs were safe at effective trypanocidal doses and even at doses several times higher than the effective dose.

Evaluation of Virulence Factors of Candida Species Isolated from Superficial versus Systemic Candidiasis

Background: Various virulence factors are contributing to the colonization and pathogenicity of Candida during both superficial and systemic infections Objectives: The current study was conducted to investigate production of extracellular hydrolytic enzymes, hemolysis and biofilm formation among Candida species isolated from superficial versus systemic candidiasis and to compare the production of these virulence factors among albicans and non-albicans Candida species, in addition to comparing hemolysis and hydrolytic enzymes production under aerobic and anaerobic conditions. Methodology: A total of 51 Candida strains isolated from vulvovaginal candidiasis and 44 strains isolated from systemic candidiasis were included in the study. Germ tube test and Hi Crom Candida Differential Agar medium were used for species identification of the isolates. The Candida isolates were tested for expression of hemolytic activity, phospholipase, proteinase and esterase production under aerobic and anaerobic conditions, in addition to their ability of biofilm formation. Results: In both groups, 55 isolates were identified as Candida albicans (C. albicans), while 44 isolates were identified as non-albicans species. Hemolytic activity and biofilm formation were significantly higher among the systemic Candida isolates than superficial isolates, while expression of proteinase activity was significantly higher among superficial isolates. Expression of phospholipase and esterase was significantly higher among C. albicans isolates than non-albicans isolates. Expression of proteinase and esterase activities was significantly better under aerobic conditions than under anaerobic conditions. Conclusions: Hemolytic activity and bioflm formation were more evident among the systemic isolates while proteinase production appears to be more evident among the vaginal isolates. Phospholipase production is almost an exclusive characteristic feature of C. albicans, while C. albicans is the leading species in terms of esterase production. Aerobic conditions favor expression of proteinase and esterase activities.

Protective Effects of Ascorbic Acid and α-Tocopherol on the In Vitro Oxidation of Equine Erythrocytes Caused by Extracts of Wilted Red Maple Leaves

Red maple leaf toxicosis in horses is a debilitating condition. Horses which ingest wilted leaves of red maple, as well as those of silver and sugar maple, experience depression, lethargy, pale mucous membranes, and brown discoloration of plasma and urine due to severe oxidation of erythrocytes. The objective of this study was to evaluate the protective effect of equine dietary supplementation with the antioxidants ascorbic acid and α-tocopherol on hemolysis and methemoglobin production of erythrocytes when challenged by extracts of wilted red maple leaves in bioassays. Horses were assigned to one of four groups which received either (1) no dietary supplement, (2) ascorbic acid, (3) α-tocopherol, or (4) both ascorbic ascorbic acid and α-tocopherol for 12 weeks. At weeks 1, 12, and 24 after starting supplementation, blood samples were collected from horses and incubated with either 0 or 75 μL of leaf extract. Hemolysis and methemoglobin production were determined based on spectrophotometry. After 12 weeks of ascorbic acid supplementation, percentage hemolysis and methemoglobin production were significantly reduced in blood samples incubated with 75 μL of leaf extract. In contrast, only percentage hemolysis was significantly reduced after 12 weeks of α-tocopherol supplementation. After 24 weeks, values for percentage hemolysis and methemoglobin had returned to their baseline levels after termination of supplementation at week 12. The combination of both supplements did not significantly reduce hemolysis or methemoglobin production. These results suggest that daily dietary supplements of ascorbic acid (and to a lesser degree α-tocopherol) have the potential to lessen the effects of erythrocyte oxidation which occurs during red maple leaf toxicosis.

Antibiotic resistance, hemolysis and biogenic amine production assessments of Leuconostoc and Weissella isolates for kimchi starter development

We assessed the antibiotic resistance, hemolysis, and biogenic amine production of 23 Leuconostoc and 45 Weissella strains from kimchi. Antibiotic resistance was determined using the minimum inhibitory concentration (MIC) test and the disk diffusion method. The results from each method were in agreement, except in the case of penicillin G and gentamicin resistance. Based on the two test results, all strains were susceptible to ampicillin, chloramphenicol, erythromycin, and tetracycline, but resistant to streptomycin. The streptomycin resistance displayed by Weissella species may be attributed to intrinsic factors, as seen in other lactic acid bacteria. All strains were susceptible to penicillin G according to the MIC test results. The breakpoint value of the genera Leuconostoc and Weissella for penicillin G was 2 mg/L. Leuconostoc spp. were susceptible to gentamicin, while 11 of the Weissella isolates exhibited 2-fold MIC values compared with Leuconostoc strains, a finding that could not be ascribed to acquired resistance. We further demonstrated that biogenic amine production is strain-specific, and is more common among Weissella spp. than among Leuconostoc spp. Nine W. cibaria strains and two W. confusa strains exhibited α-hemolytic activity, and no β-hemolysis was observed in any of the isolates.

L-carnitine as a Potential Additive in Blood Storage Solutions: A Study on Erythrocytes

Erythrocytes undergo various changes during storage (storage lesion) that in turn reduces their functioning and survival. Oxidative stress plays a major role in the storage lesion and antioxidants can be used to combat this stress. This study elucidates the effects of l-carnitine (LC) on erythrocytes of stored blood. Blood was obtained from male Wistar rats and stored (4°C) for 20days in CPDA-1 (citrate phosphate dextrose adenine) solution. Samples were divided into-(i) controls (ii) LC 10 (l-carnitine at a concentration of 10mM) (iii) LC 30 (l-carnitine at a concentration of 30mM) and (iv) LC 60 (l-carnitine at a concentration of 60mM). Every fifth day, the biomarkers (haemoglobin, hemolysis, antioxidant enzymes, lipid peroxidation and protein oxidation products) were analysed in erythrocytes. Hemoglobin and protein sulfhydryls were insignificant during storage indicative of the maintenance of hemoglobin and sulfhydryls in all groups. Superoxide dismutase and malondialdehyde levels increased initially and decreased towards the end of storage. The levels of catalase and glutathione peroxidase were lower in experimentals than controls during storage. l-carnitine assisted the enzymes by scavenging the reactive oxygen species produced. Hemolysis increased in all groups with storage, elucidating that l-carnitine could not completely protect lipids and proteins from oxidative stress. Hence, this study opens up new avenues of using l-carnitine as a component of storage solutions with combinations of antioxidants in order to maintain efficacy of erythrocytes.

Incidence, Antibiotic Susceptibility, and Toxin Profiles of Bacillus cereus sensu lato Isolated from Korean Fermented Soybean Products.

Korean fermented soybean products, such as doenjang, kochujang, ssamjang, and cho-kochujang, can harbor foodborne pathogens such as Bacillus cereus sensu lato (B. cereus sensu lato). The aim of this study was to characterize the toxin gene profiles, biochemical characteristics, and antibiotic resistance patterns of B. cereus sensu lato strains isolated from Korean fermented soybean products. Eighty-eight samples of Korean fermented soybean products purchased from retails in Seoul were tested. Thirteen of 26 doenjang samples, 13 of 23 kochujang samples, 16 of 30 ssamjang samples, and 5 of 9 cho-kochujang samples were positive for B. cereus sensu lato strains. The contamination level of all positive samples did not exceed 4 log CFU/g of food (maximum levels of Korea Food Code). Eighty-seven B. cereus sensu lato strains were isolated from 47 positive samples, and all isolates carried at least one enterotoxin gene. The detection rates of hblCDA, nheABC, cytK, and entFM enterotoxin genes among all isolates were 34.5%, 98.9%, 57.5%, and 100%, respectively. Fifteen strains (17.2%) harbored the emetic toxin gene. Most strains tested positive for salicin fermentation (62.1%), starch hydrolysis (66.7%), hemolysis (98.9%), motility test (100%), and lecithinase production (96.6%). The B. cereus sensu lato strains were highly resistant to β-lactam antibiotics such as ampicillin, penicillin, cefepime, imipenem, and oxacillin. Although B. cereus sensu lato levels in Korean fermented soybean products did not exceed the maximum levels permitted in South Korea (<10(4) CFU/g), these results indicate that the bacterial isolates have the potential to cause diarrheal or emetic gastrointestinal diseases.

Nanotoxicity of silver nanoparticles to red blood cells: size dependent adsorption, uptake, and hemolytic activity.

Silver nanoparticles (AgNPs) are increasingly being used as antimicrobial agents and drug carriers in biomedical fields. However, toxicological information on their effects on red blood cells (RBCs) and the mechanisms involved remain sparse. In this article, we examined the size dependent nanotoxicity of AgNPs using three different characteristic sizes of 15 nm (AgNPs15), 50 nm (AgNPs50), and 100 nm (AgNPs100) against fish RBCs. Optical microscopy and transmission electron microscopy observations showed that AgNPs exhibited a size effect on their adsorption and uptake by RBCs. The middle sized AgNPs50, compared with the smaller or bigger ones, showed the highest level of adsorption and uptake by the RBCs, suggesting an optimal size of ∼50 nm for passive uptake by RBCs. The toxic effects determined based on the hemolysis, membrane injury, lipid peroxidation, and antioxidant enzyme production were fairly size and dose dependent. In particular, the smallest sized AgNPs15 displayed a greater ability to induce hemolysis and membrane damage than AgNPs50 and AgNPs100. Such cytotoxicity induced by AgNPs should be attributed to the direct interaction of the nanoparticle with the RBCs, resulting in the production of oxidative stress, membrane injury, and subsequently hemolysis. Overall, the results suggest that particle size is a critical factor influencing the interaction between AgNPs and the RBCs.

Pathobiology of Transfusion Reactions

Antibody-induced hemolytic transfusion reactions were first described over 300 years ago. Indeed, during its early evolution, transfusion medicine focused almost exclusively on issues in immunohematology to prevent such events. However, despite the best of efforts to avoid them, incompatible transfusions still occur, through both error and an inability to obtain compatible red blood cells for patients who are alloimmunized against multiple antigens. Because transfusing units of incompatible blood is potentially lethal, studies on human volunteers are not ethical. Thus, understanding of hemolytic transfusion reactions is generated through clinical cases, animal models, inference from related human pathologies, or studies using small volumes of transfused red blood cells. Over the past several decades, substantial new knowledge has been accumulated regarding the mechanisms of hemolysis, the metabolism of products of hemolysis, and the effects of both on recipient biology. Using these data sources, this article traces the historical generation of this knowledge and describes recent advances.

CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS ASSOCIATED WITH BOVINE MASTITIS

The present research was planned to study incidence, virulence properties and antibiogram of Staphylococcus aureus isolates recovered from bovine mastitis. A total 108 milk samples (48 cows and 60 buffaloes) from clinical and sub-clinical cases of bovine mastitis from different locations of Kolhapur and Sangali districts of Maharashtra state were collected during present investigation. Overall recovery of Staphylococcus spp. was 36 (33.33%) isolates (10 cows + 26 buffaloes) and 30 (27.77%) isolates were confirmed as S. aureus with an incidence rate of 16.66% in cows and 36.66% in buffaloes, whereas 6 (5.55%) isolates were other Staphylococcus spp. The incidence rate of S. aureus in subclinical and clinical mastitis was found to be 35.93% and 15.90% respectively. Conventional tube method for coagulase production, DNAse test and studies on haemolysis production of S. aureus isolates resulted in 9 (30%) CPS, 23 (76.66%) DNAse producing and 21 (70%) hemolytic strains. ABST of 30 S. aureus isolates by disc diffusion method indicated maximum resistance to Vancomycin (90%) followed by Ceftriaxone (80%), Penicillin-G (73.34%), Oxacillin (70%) and Amoxicillin (60%) whereas, the least resistance was observed to Methicillin (6.67%) and Chloramphenicol (10%). Multiple drug resistance was observed in 26 (86.66%) isolates. The PCR amplification of Coagulase gene (coa) of S. aureus isolates yielded 14 (46.66%) coagulase positive isolates with amplification sizes ranging from 600 to 850 bp. Four different coa genotypes were generated based on the sizes of amplicons viz. A, B, C and D. Out of 14 coa exhibiting strains of S. aureus, Genotype A (850 bp) was observed in 7.14% isolates, whereas genotype B (750 bp) in 14.28% isolates, 50% isolates were of genotype C (700 bp) and 28.57% of genotype D (600 bp). Amongst 14 coa positive isolates, 11 were from subclinical mastitis origin and only 3 were from clinical mastitis cases. All coagulase producing strains of S. aureus were haemolytic and DNAse producing with an exception of one DNAse negative strain. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Table Normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin-top:0in; mso-para-margin-right:0in; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0in; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi; mso-ansi-language:EN-IN; mso-fareast-language:EN-IN;}