Functional characterization of obscure puffer ToNK-lysin: A novel immunomodulator possessing anti-bacterial and anti-inflammatory properties.

NK-lysins are one of the most abundant antimicrobial peptides produced by cytotoxic T lymphocytes (CTLs) and natural killer cells (NKs), and identified as a new class of intrinsically disordered proteins, playing critical roles in the cell-mediated cytotoxicity response, as well as immunomodulatory and antimicrobial activities upon a significant range of pathogens. In the present study, an NK-lysin was identified from Obscure puffer Takifugu obscurus (ToNK-lysin). The open reading frame of ToNK-lysin sequence spans 423 bp, encoding a peptide with 140 amino acids which shares a moderate residue identity (18%-60%) with NK-lysin of mammals and other teleost species. Phylogenetic analysis revealed that ToNK-lysin was most closely related to NK-lysins from the Pleuronectiformes (Bastard halibut Paralichthys olivaceus and Pacific halibut Hippoglossus stenolepis). Comprehensive computational analysis revealed that ToNK-lysin have substantial level of intrinsic disorder, which might be contribute to its multifunction. The transcripts of the ToNK-lysin were detected in multiple examined tissues and most abundant in gills. After bacterial and Poly I:C challenge, the transcriptional levels of ToNK-lysin were significantly up-regulated in the head kidney, liver and spleen at different time points. The recombinant ToNK-lysin showed significant antibacterial activity against Vibrio harveyi and Escherichia coli, and the ToNK-lysin treatment not only reduced the bacterial loads in liver and head kidney, but also alleviated the pathogen-mediated upregulation of immune-related genes. In addition, the co-incubation with rToNK-lysin protein remarkably degraded bacterial genomic DNA, suggesting the potential mechanism of ToNK-lysin against microbes. These results suggest that ToNK-lysin possess antibacterial and immunoregulatory function both in vivo and in vitro, which may allow it a potential applicability to the aquaculture industry.

Lights, fluorescence, action-Influencing wound treatment plans including debridement of bacteria and biofilms.

High bacterial loads within chronic wounds increase the risk of infection and complication. Detection and localization of bacterial loads through point-of-care fluorescence (FL) imaging can objectively inform and support bacterial treatment decisions. This single time-point, retrospective analysis describes the treatment decisions made on 1000 chronic wounds (DFUs, VLUs, PIs, surgical wounds, burns, and others) at 211 wound-care facilities across 36 US states. Clinical assessment findings and treatment plans derived from them, as well as subsequent FL-imaging (MolecuLight®) findings and any associated treatment plan changes, were recorded for analysis. FL signals indicating elevated bacterial loads were observed in 701 wounds (70.8%), while only 293 (29.6%) showed signs/symptoms of infection. After FL-imaging, treatment plans changed in 528 wounds as follows: more extensive debridement (18.7%), more extensive hygiene (17.2%), FL-targeted debridement (17.2%), new topical therapies (10.1%), new systemic antibiotic prescriptions (9.0%), FL-guided sampling for microbiological analysis (6.2%), and changes in dressing selection (3.2%). These real-world findings of asymptomatic bacterial load/biofilm incidence, and of the frequent treatment plan changes post-imaging, are in accordance with clinical trial findings using this technology. These data, from a range of wound types, facilities, and clinician skill sets, suggest that point-of-care FL-imaging information improves bacterial infection management.

Impact of 2nd wave of COVID-19-related lockdown on coastal water quality at Diu, western coast of India and role of total alkalinity on bacterial loads.

A detailed coastal water monitoring near Diu coast, western part of India was performed from October, 2020 to May, 2021 covering the 2nd lockdown time. Average monthly fluctuation from 7 different sampling stations of total 9 physico-chemical parameters such as pH, salinity, turbidity, nitrite (NO2), nitrate (NO3), ammonia (NH3), phosphate (PO4), total alkalinity and silicate were recorded. Initially, Mann-Kendall trend test for all the 9 parameters showed non-zero trend, which may be either linear or non-linear. During 2nd lockdown period, there was a fluctuation of value for parameters like pH, salinity, nitrate, nitrite and phosphate. Average total bacterial count and differential bacterial count also gradually decreased from March, 2021 sampling. Principal component analysis (PCA) plot covering all the physico-chemical parameters as well as the differential bacterial count showed a distinct cluster of all bacterial count with total alkalinity value. Subsequently, mathematical equation was formulated between total alkalinity value and all differential bacterial count. Upto our knowledge, this is the first report where mathematical equation was formulated to obtain value of different bacterial load based on the derived total alkalinity value of the coastal water samples near Diu, India.

Analysis of Gene Expression Profiles, Cytokines, and Bacterial Loads Relevant to Alcoholic Liver Disease Mice Infected With V. vulnificus.

Patients with liver disease are susceptible to infection with Vibrio vulnificus (V. vulnificus), but the specific reasons remain elusive. Through RNA-seq, we found that when mice with alcoholic liver disease (ALD) were infected with V. vulnificus by gavage, compared with the Pair group, the small intestinal genes affecting intestinal permeability were upregulated; and the number of differentially expressed genes related to immune functions (e.g., such as cell chemotaxis, leukocyte differentiation, and neutrophil degranulation) decreased in the liver, spleen, and blood. Further analysis showed that the number of white blood cells decreased in the Pair group, whereas those in the ALD mice did not change significantly. Interestingly, the blood bacterial load in the ALD mice was about 100 times higher than that of the Pair group. After the ALD mice were infected with V. vulnificus, the concentrations of T cell proliferation-promoting cytokines (IL-2, IL-23) decreased. Therefore, unlike the Pair group, ALD mice had weaker immune responses, lower T cell proliferation-promoting cytokines, and higher bacterial loads post-infection, possibly increasing their susceptibility to V. vulnificus infection. These new findings we presented here may help to advance the current understanding of the reasons why patients with liver disease are susceptible to V. vulnificus infection and provides potential targets for further investigation in the context of treatment options for V. vulnificus sepsis in liver disease patient.

Ten Thousand-Fold Higher than Acceptable Bacterial Loads Detected in Kenyan Hospital Environments: Targeted Approaches to Reduce Contamination Levels.

Microbial monitoring of hospital surfaces can help identify target areas for improved infection prevention and control (IPCs). This study aimed to determine the levels and variations in the bacterial contamination of high-touch surfaces in five Kenyan hospitals and identify the contributing modifiable risk factors. A total of 559 high-touch surfaces in four departments identified as high risk of hospital-acquired infections were sampled and examined for bacterial levels of contamination using standard bacteriological culture methods. Bacteria were detected in 536/559 (95.9%) surfaces. The median bacterial load on all sampled surfaces was 6.0 × 104 CFU/cm2 (interquartile range (IQR); 8.0 × 103-1.0 × 106). Only 55/559 (9.8%) of the sampled surfaces had acceptable bacterial loads, <5 CFU/cm². Cleaning practices, such as daily washing of patient sheets, incident rate ratio (IRR) = 0.10 [95% CI: 0.04-0.24], providing hand wash stations, IRR = 0.25 [95% CI: 0.02-0.30], having running water, IRR = 0.19 [95% CI: 0.08-0.47] and soap for handwashing IRR = 0.21 [95% CI: 0.12-0.39] each significantly lowered bacterial loads. Transporting dirty linen in a designated container, IRR = 72.11 [95% CI: 20.22-257.14], increased bacterial loads. The study hospitals can best reduce the bacterial loads by improving waste-handling protocols, cleaning high-touch surfaces five times a day and providing soap at the handwash stations.

Characterization of Brucella abortus S19 as a challenge strain for use in a mouse model of brucellosis.

The objective of this project was to conduct a feasibility study to determine whether the Brucella abortus S19 vaccine infects and persists in mice and determine whether S19 can be used as a challenge strain for vaccine trial studies. Groups of BALB/c mice were inoculated (intraperitoneally, subcutaneously, intranasally) and euthanized to determine colonization titers in the spleens and lungs. This study showed that S19 does infect and persist in the tissues of mice for 8 weeks and demonstrates that S19 can be used, safely and economically under BSL2 containment, as the challenge strain for future trials to evaluate vaccine efficacy.

Development of signature-tagged mutagenesis in Riemerella anatipestifer to identify genes essential for survival and pathogenesis.

Riemerella anatipestifer causes epizootic infectious disease in ducks, geese, turkeys and other birds, and serious economic losses especially to the duck industry. However, little is known about the molecular basis of its pathogenesis. In this study, signature-tagged transposon mutagenesis based on Tn4351 was developed in R. anatipestifer to identify genes essential for survival and pathogenesis. Seventeen tagged Tn4351 random mutation libraries of the R. anatipestifer strain WJ4 containing 5100 mutants were screened for survive using a duckling infection model. Twenty mutants that could not be recovered from the infected ducklings, were identified, and 17 mutated genes were identified by inverse PCR or genome-walking PCR. Of these genes, FIP52_03215, FIP52_04350 and FIP52_09345, were inserted into two mutant strains, and FIP52_03215 and FIP52_03175 were found exclusively on the chromosome of serotype 1 R. anatipestifer strains. Twelve out of 17 genes encoding for proteins were predicted to be involved in amino acid, nucleotide, coenzyme, or lipid transport and metabolism, one gene was predicted to be involved in signal transduction, one gene was predicted to be involved in DNA replication, recombination and repair, the other three genes had an unknown function. Animal experiments showed that the virulence of mutants 16-284, 7-295, 24-231, 9-232 and 19-214 were significantly attenuated compared to that of the wild-type WJ4. Moreover, the median lethal dose of mutant 16-284 was greater than 1010 CFU, and its virulence to ducklings was partially restored when it was complemented with the shuttle expression plasmid pRES-FIP52_09345. The results in this study will be helpful to further study the molecular mechanisms of the pathogenesis of R. anatipestifer infection.

Prevalence of Borrelia burgdorferi sensu lato and Borrelia miyamotoi in ixodid ticks in the Far East of Russia.

Borrelia burgdorferi sensu lato (s.l.) DNA was detected by PCR in Ixodes persulcatus Schulze, 1930, Haemaphysalis concinna Koch, 1844, Haemaphysalis japonica douglasi Nuttall et Warburton, 1915 and Dermacentor silvarum Olenev, 1932 ticks collected in the Amur region, the Jewish Autonomous region, the Sakhalin region and on the Khabarovsk territory. Infection rate of I. persulcatus with B. burgdorferi s.l. 10-69% exceeded the corresponding values of three other tick species in all examined regions during 1999-2014 despite different tick abundance and dominance structure. Bacterial loads estimated on the base of quantitative real time PCR varied from 102 to 109 genome-equivalents per a tick with maximal values for I. persulcatus and H. japonica. Phylogenetic analysis of 16S rRNA gene and 5S-23S rRNA intergenic spacer nucleotide sequences revealed two species: 1) Borrelia garinii of Asian type NT29 with several isolates of European type 20047; 2) Borrelia afzelii with identical sequences of the majority of studied isolates and VS461 reference strain in all regions except the Sakhalin Island where B. afzelii was not found. Borrelia miyamotoi of the relapsing fever group was detected as monoinfection or in combination with B. burgdorferi s.l. in 4.0 ±â€¯0.9% and 4.8 ±â€¯0.9% I. persulcatus ticks, respectively. Multiple locus sequence analysis of three fragments of 16S rRNA, glpQ and p66 genes proved that all the Far Eastern B. miyamotoi isolates belonged to the Asian type identical to FR64b strain (GenBank CP004217) from Japan. Wide distribution of Borrelia DNA in ticks, relative genetic homogeneity with similar sequences of the coding regions and the intergenic spacer of Borrelia wild isolates and temporal stability with high homology levels of the Far Eastern isolates of B. garinii, B. afzelii and B. miyamotoi with previously described spirochetes from the surrounding regions of Russia, China and Japan allowed us to suggest multiple ecological niches as the stability factor of the parasitic system.

The Chinese highly pathogenic porcine reproductive and respiratory syndrome virus infection suppresses Th17 cells response in vivo.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been shown to immunomodulate innate and adaptive immunity of pigs. The Chinese highly pathogenic PRRSV (HP-PRRSV) infection causes severe bacterial secondary infection in pigs. However, the mechanism in relation to the bacterial secondary infection induced by HP-PRRSV remains unknown. In the present study, Th17 cells response in peripheral blood, lungs, spleens and lymph nodes of piglets were analyzed, and bacterial loads in lungs of piglets were examined upon HP-PRRSV infection. Meanwhile the changes of CD4(+) and CD8(+) T cells in peripheral blood of the inoculated piglets were analyzed. The results showed that HP-PRRSV-inoculated piglets exhibited a suppressed Th17 cells response in peripheral blood and a reduced number of Th17 cells in lungs, and higher bacterial loads in lungs, compared with low pathogenic PRRSV. Moreover, HP-PRRSV obviously resulted in severe depletion of porcine T cells in peripheral blood at the early stage of infection. These findings indicate that HP-PRRSV infection suppresses the response of Th17 cells that play an important role in combating bacterial infections, suggesting a possible correlation between the suppression of Th17 cells response in vivo and bacterial secondary infection induced by HP-PRRSV. Our present study adds a novel insight into better understanding of the pathogenesis of the Chinese HP-PRRSV.

High nasopharyngeal pneumococcal density, increased by viral coinfection, is associated with invasive pneumococcal pneumonia.

BACKGROUND: We identified factors associated with pneumococcal colonization, high colonization density, and invasive pneumococcal pneumonia among patients hospitalized with acute lower respiratory tract infections (ALRTIs). METHODS: In 2010, 4025 cases were enrolled in surveillance in South Africa. A total of 969 of 4025 systematically selected nasopharyngeal-oropharyngeal specimens (24%) were tested for respiratory viruses and Streptococcus pneumoniae by real-time polymerase chain reaction. Of these, 749 (77%) had blood tested for S. pneumoniae. RESULTS: Pneumococcal colonization was detected in 55% of cases (534 of 969). On multivariable analysis that controlled for age and tuberculosis treatment, infection with influenza virus (adjusted odds ratio [OR], 2.2; 95% confidence interval [CI], 1.1-4.5), adenovirus (adjusted OR, 1.7; 95% CI, 1.1-2.7), rhinovirus (adjusted OR, 1.6; 95% CI, 1.1-2.3), and human immunodeficiency virus (HIV; adjusted OR, 1.6; 95% CI, 1.1-2.4) were associated with pneumococcal colonization. High colonization density was associated with respiratory virus coinfection (adjusted OR, 1.7; 95% CI, 1.1-2.6) and invasive pneumococcal pneumonia (adjusted OR, 2.3; 95% CI, 1.3-4.0), after adjustment for age and sex. Seven percent (52 of 749) had pneumococci detected in blood. On multivariable analysis among colonized cases, invasive pneumococcal pneumonia was associated with HIV (adjusted OR, 3.2; 95% CI, 1.4-7.5), influenza virus (adjusted OR, 8.2; 95% CI, 2.7-25.0), high colonization density (adjusted OR, 18.7; 95% CI, 2.3-155.1), and ≥5 days of hospitalization (adjusted OR, 3.7; 95% CI, 1.7-8.2). CONCLUSIONS: Respiratory virus infection was associated with elevated colonization density and, in turn, invasive pneumococcal pneumonia.