Bacterial Load In Blood Research Articles

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.

Improvement of pneumococcal pneumonia diagnosis using quantitative real-time PCR targeting lytA in adult patients: a prospective cohort study

ObjectivesThe aim of the study was to assess the performance of real-time PCR targeting the lytA gene (rtPCR-lytA) in plasma, urine and nasopharyngeal (NP) samples for the diagnosis of pneumococcal community-acquired pneumonia (P-CAP). MethodsProspective observational study including all consecutive adults with CAP from November 2015 to May 2017. P-CAP was defined if pneumococcus was identified using conventional methods (CM) and/or a positive rtPCR-lytA was detected in blood, urine or NP samples (NP cut-off ≥8000 copies/mL). Diagnostic performance of each test was calculated. ResultsA total of 133 individuals with CAP were included. Of these, P-CAP was diagnosed in 62 (46.6%). The proportion of P-CAP diagnosed by rtPCR-lytA methods was significantly higher than that diagnosed by CM (87.1% versus 59.7%, p 0.005). The rtPCR-lytA identified Streptococcus pneumoniae in 25 patients (40.3% of all individuals with P-CAP) whose diagnosis would have been missed by CM. NP-rtPCR-lytA allowed diagnosis of 62.3% of P-CAP. A nasopharyngeal colonization density ≥2351 copies/mL predicted P-CAP diagnosis (area under the curve = 0.82, sensitivity 83.3%, specificity 80.9%). There was a positive correlation between increasing bacterial load in blood and CURB-65 score (Spearman correlation coefficient r = 0.4, p 0.001), pneumonia severity index (r = 0.3, p 0.02) and time to clinical stability (r = 0.33, p 0.01). Median bacterial load in blood was higher in P-CAP patients with bacteraemia (0.65 × 103 versus 0 × 103 copies/mL, p 0.002), intensive care unit admission (0.68 × 103 versus 0 × 103 copies/mL, p 0.04) or mechanical ventilation (7.45 × 103 versus 0 × 103 copies/mL, p 0.04). ConclusionsThe use of rtPCR-lytA methods significantly increased the diagnosis of P-CAP compared with CM. Nasopharyngeal swabs rtPCR-lytA detection, with an accurate cut-off value, was the most promising among molecular methods for the diagnosis of P-CAP.

Long-Term Anti-Bacterial Immunity against Systemic Infection by Salmonella enterica Serovar Typhimurium Elicited by a GMMA-Based Vaccine.

Salmonella Typhimurium (STm) represents the most prevalent cause of invasive non-typhoidal Salmonella (iNTS) disease, and currently no licensed vaccine is available. In this work we characterized the long-term anti-bacterial immunity elicited by a STm vaccine based on Generalized Modules of Membrane Antigens (GMMA) delivering O:4,5 antigen, using a murine model of systemic infection. Subcutaneous immunization of mice with STmGMMA/Alhydrogel elicited rapid, high, and persistent antigen-specific serum IgG and IgM responses. The serum was bactericidal in vitro. O:4,5-specific IgG were also detected in fecal samples after immunization and positively correlated with IgG observed in intestinal washes. Long-lived plasma cells and O:4,5-specific memory B cells were detected in spleen and bone marrow. After systemic STm challenge, a significant reduction of bacterial load in blood, spleen, and liver, as well as a reduction of circulating neutrophils and G-CSF glycoprotein was observed in STmGMMA/Alhydrogel immunized mice compared to untreated animals. Taken together, these data support the development of a GMMA-based vaccine for prevention of iNTS disease.

Effectiveness of boiled Chrysicthys nigrodigitatus against rats-induced Salmonella Typhi infection

The decline in efficiency and the frequent toxicity effects observed with antibiotics has urge researchers to turn to food as alternative. The present work aimed at valorizing boiled Chrysicthys nigrodigitatus against Salmonella Typhi infections. Flesh of C. nigrodigitatus was boiled in 4.7 L with a ratio of 1/1.5 (Kg/V) for 17 min at approximately 100 °C. Oil extraction was done using Bligh and Dyer method. In vivo activity of different diets formulated with boiled fish oil, oil + amoxicillin and boiled fish was evaluated on rat-induced Salmonella Typhi infection. The bacterial load in blood, dietary intake and weight gain of treated rats were used to monitor the disease progression. The hematological and lipid parameters were evaluated after treatment. Diets formulated with oil and flesh of C. nigrodigitatus healed the infected rats between the 16th and 18th day. Moreover, the fish-based diet corrected the anorexia involved in the infection, increased the body weight of infected rats and normalizing the levels of red blood (6.01 ± 0.58 × 106 cells/μL) and white blood cells (22.85 ± 1.20 × 103 cells/μL). This diet also lowered total cholesterol (76.44 ± 3.57 mg/dL), LDL-cholesterol (24.32 ± 4.60 mg/dL), atherogenic index (0.850 ± 0.367 mg/dL) and increased HDL-cholesterol level (41.62 ± 3.41 mg/dL). The overall results indicated that boiled C. nigrodigitatus can be used alone or associated to amoxicillin to overcome typhoid fever and regulate the levels of red blood cells, white blood cells, total cholesterol, LDL-cholesterol, atherogenic index and HDL-cholesterol associated in S. Typhi infection.

Biological relevance of Granzymes A and K during E. coli sepsis.

Aims: Recent in vitro findings suggest that the serine protease Granzyme K (GzmK) may act as a proinflammatory mediator. However, its role in sepsis is unknown. Here we aim to understand the role of GzmK in a mouse model of bacterial sepsis and compare it to the biological relevance of Granzyme A (GzmA).Methods: Sepsis was induced in WT, GzmA-/- and GzmK-/- mice by an intraperitoneal injection of 2x108 CFU from E. coli. Mouse survival was monitored during 5 days. Levels of IL-1α, IL-1β, TNFα and IL-6 in plasma were measured and bacterial load in blood, liver and spleen was analyzed. Finally, profile of cellular expression of GzmA and GzmK was analyzed by FACS.Results: GzmA and GzmK are not involved in the control of bacterial infection. However, GzmA and GzmK deficient mice showed a lower sepsis score in comparison with WT mice, although only GzmA deficient mice exhibited increased survival. GzmA deficient mice also showed reduced expression of some proinflammatory cytokines like IL1-α, IL-β and IL-6. A similar result was found when extracellular GzmA was therapeutically inhibited in WT mice using serpinb6b, which improved survival and reduced IL-6 expression. Mechanistically, active extracellular GzmA induces the production of IL-6 in macrophages by a mechanism dependent on TLR4 and MyD88.Conclusions: These results suggest that although both proteases contribute to the clinical signs of E. coli-induced sepsis, inhibition of GzmA is sufficient to reduce inflammation and improve survival irrespectively of the presence of other inflammatory granzymes, like GzmK.

Granzyme A inhibition reduces inflammation and increases survival during abdominal sepsis

Aims: Peritonitis is one of the most common causes of sepsis, a serious syndrome characterized by a dysregulated systemic inflammatory response. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed by NK and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role and the therapeutic potential of GzmA in the pathogenesis of peritoneal sepsis.Methods: The level of extracellular GzmA as well as GzmA activity were analyzed in serum from healthy volunteers and patients with confirmed peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (WT) and GzmA-/- mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics alone or in combination serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days, levels of some proinflammatory cytokines were measured in serum and bacterial load and diversity was analyzed in blood and spleen at different times.Results: Clinically, elevated GzmA was observed in serum from patients with abdominal sepsis suggesting that GzmA plays an important role in this pathology. In the CLP model GzmA deficient mice, or WT mice treated with an extracellular GzmA inhibitor, showed increased survival, which correlated with a reduction in proinflammatory markers in both serum and peritoneal lavage fluid. GzmA deficiency did not influence bacterial load in blood and spleen and GzmA did not affect bacterial replication in macrophages in vitro, indicating that GzmA has no role in bacterial control. Analysis of GzmA in lymphoid cells following CLP showed that it was mainly expressed by NK cells. Mechanistically, we found that extracellular active GzmA acts as a proinflammatory mediator in macrophages by inducing the TLR4-dependent expression of IL-6 and TNFα.Conclusions: Our findings implicate GzmA as a key regulator of the inflammatory response during abdominal sepsis and provide solid evidences about its therapeutic potential for the treatment of this severe pathology.

152. rapid Ultra-high Enrichment of Bacterial Pathogens at Low Concentration from Blood for Species ID and AMR Prediction Using Nanopore Sequencing

BackgroundEach year in the United States there are over 1.7 million cases of sepsis that account for a third of hospital deaths. A key to reducing morbidity and mortality rates is early, appropriate antibiotic therapy. Most new diagnostic approaches still suffer from insufficient sensitivity to low bacterial loads in blood and limited sets of detection targets for bacterial species identification (ID) and antimicrobial resistance (AMR) determination. As such, blood culture remains the gold standard for diagnosing bacteremia despite limitations such as > 2-day turnaround time (TAT), incompatibility with fastidious organisms, and frequent inability to recover causative pathogens.Methods31 clinically relevant bacterial pathogens, made up of 17 gram-positive and 14 gram-negative bacterial species, were spiked into 2 to 4 healthy donor blood samples at 1 to 5 CFU/mL. The samples were run through our proprietary Blood2Bac™ pipeline, sequenced on a nanopore platform, and data were passed through Keynome®, our proprietary machine learning algorithm to determine species ID and AMR.ResultsBy assessing the efficiency of pathogen DNA enrichment and genome coverage post sequencing, we report high performance of 3 CFU/mL for 3 bacterial species and ≤ 2 CFU/mL for the 28 remaining species, which includes S. aureus, E. coli, and Streptococcus spp., three of the leading causes of sepsis.For all 31 bacterial species tested, Keynome called species ID with 100% accuracy. In addition, Keynome also predicted the AMR profile of pathogens with 100% accuracy for 19 drug/species AMR combinations, including ciprofloxacin for E. coli, clindamycin for S. aureus, and aztreonam for K. pneumoniae.ConclusionBlood2Bac is able to enrich a wide range of bacterial pathogens directly from blood and enable bacterial whole genome sequencing with an estimated TAT of 12 hours. When coupled with Keynome, our process provides accurate species ID and AMR calls for key BSI pathogens even at single-digit CFU/mL concentrations. Our species-agnostic and culture-free process enables detection of a diverse range of bacterial species with high sensitivity, providing a robust and comprehensive diagnostic.DisclosuresChiahao Tsui, n/a, Day Zero Diagnostics (Employee, Shareholder) Lisa S. Cunden, PhD, Day Zero Diagnostics (Shareholder) Nicole Billings, PhD, Day Zero Diagnostics (Employee) Imaly A. Nanayakkara, PhD, Day Zero Diagnostics (Employee, Shareholder) Ian Herriott, BS, Day Zero Diagnostics (Employee, Shareholder) Rachel R. Martin, n/a, Day Zero DIagnostics (Employee) Michelle Chen, MS, Day Zero Diagnostics (Employee, Shareholder) Febriana Pangestu, n/a, Day Zero Diagnostics (Employee, Shareholder) Paul Knysh, PhD, Day Zero Diagnostics (Employee) Cabell Maddux, n/a, Day Zero Diagnostics (Employee, Shareholder) Zachary Munro, n/a, Day Zero Diagnostics Inc. (Employee, Shareholder) Miriam Huntley, PhD, Day Zero Diagnostics (Employee, Shareholder)

Orf6 and orf10 in Prophage phiv142-3 Enhance the Iron-Acquisition Ability and Resistance of Avian Pathogenic Escherichia coli Strain DE142 to Serum

Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli (ExPEC), is the causative agent of avian colibacillosis, a disease that causes huge economic losses in the poultry industry and is characterized by infection through respiratory tract colonization followed by bacteraemia. A previous study in our lab demonstrated that phiv142-3 enhanced the survival ability of APEC strain DE142 in chickens serum. However, the mechanism of this affect has not been completely revealed. Here, we analyzed the transcriptional level of the prophage phiv142-3 region in DE142 when grown in chicken serum. Several upregulated genes attracted our attention, and a series of mutants were constructed. Deletion of orf6 or orf10 from phiv142-3 led to lower yields compared with WT after cultivation in serum for 10 h (P < 0.05). Furthermore, avian infection assays showed that compared with WT, the bacterial loads in blood and heart tissue of chickens challenged with DE142Δorf6 were decreased to 3.9 and 13%, while the bacterial burden in blood and heart from chickens infected with DE142Δorf10 was decreased to 7.2 and 8%, respectively (P < 0.05). DE142Δorf6 showed an obviously attenuated growth rate in the logarithmic phase when cultured in iron-deficient medium, and the transcription level of the iutA gene decreased to 43% (P < 0.05). The bactericidal assays showed that the survival of the mutant DE142Δorf10 was ~60% compared with WT in 50% chicken serum. The K1 capsule-related genes (kpsF, kpsE, kpsC, and kpsM) were down-regulated nearly 2-fold in DE142Δorf10 (P < 0.01). Together, these results suggested that orf6 affects growth by contributing to the uptake ability of iron, while orf10 increases resistance to serum by upregulating K1 capsule-related genes.

Growth Performance, Hematological Disorder and Bacterial Challenge on Nile Tilapia (Oreochromis Niloticus) Using Moringa Oleifera Plant Leaf as Feed Supplement

Tilapia (Oreochromis niloticus) has been gaining ground in cultivation and sale in Bangladesh but due to price hike of fish feed the culture of this fish faced many challenges. In the present study, the optimal level of dietary moringa (Moringa oleifera) leaf powder was used as feed supplement to determine the growth performances, some selective blood parameters and bacterial load in Nile tilapia, Oreochromis niloticus. Commercial diet containing moringa leaf powder at 0g/kg, 100g/kg and 200 g/kg diet was used. The diet containing 100g moringa leaf/kg diet resulted in increased weight gain, average daily gain, specific growth rate, condition factor and survival rate. A significant (p&lt;0.05) reduction in RBC and WBC were found as the moringa leaf inclusion increased in the diets but no significant (p&lt;0.05) differences were observed among the three different rearing conditions. In most of the cases, the diet containing 100g moringa leaf powder/kg diet displayed lower amount of bacterial count but no significant improvement was found in bacterial load among the different treatment conditions. The results suggest that dietary moringa leaf powder could be included in the diet of Nile tilapia at possibly not over 100 g/kg diet for the betterment of growth, survival, immune response and microbial challenge.&#x0D; Bangladesh J. Zool. 48(1): 151-166, 2020

Effect of inhibiting the activity of double-stranded RNA-dependent protein kinase in sepsis mice

Objective: To observe the effects of 2-aminopurine (2-AP), a double-stranded RNA-dependent protein kinase (PKR) inhibitor, on organ function, plasma inflammatory factor expression and 7 days mortality in sepsis mice induced by cecal ligation puncture (CLP). Methods: Forty specific specific pathogen free C57BL/6 mice were randomly divided into sham group (n=10), CLP group (n=10), CLP+2-AP group (n=10) and 2-AP group (n=10). CLP was used to establish sepsis mice models.Peripheral blood serum was collected 24 hours after operation, alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatinine (Cr), blood urea nitrogen (BUN) and inflammatory factors (IL-1β, IL-10 and TNF-α) were detected; peripheral blood and peritoneal lavage fluid were taken for bacterial clearance detection. Another 60 C57BL/6 mice were selected to observe the 7-day survival rate according to the above groups (n=15). Independent sample t test was used to compare the measurement data between groups. Results: The levels of ALT, AST, Cr and BUN in CLP Group and CLP+2-AP group were significantly higher than those in sham group (all P<0.001). The levels of ALT and AST in CLP+2-AP group were significantly lower than those in CLP Group (t=27.88, 11.33, both P<0.001); the levels of Cr and BUN in CLP+2-AP group were significantly lower than those in CLP Group (t=11.02, 7.15, bothP<0.001). Compared with sham group, the levels of pro-inflammatory (IL-1β and TNF-α) and anti-inflammatory (IL-10) cytokines in CLP group were significantly higher (all P<0.001); the levels of IL-1β and IL-10 in CLP+2-AP group were significantly lower (all P<0.001), but the levels of TNF-α in CLP+2-AP group were not significantly lower (P=0.33). The 7-day survival rate was 100% in sham group, 13.3% in CLP+2-AP group, 86.7% in 2-AP group and 20.0% in CLP+2-AP group. Inhibition of PKR activation slightly improved the trend of 7-days survival rate of CLP model mice (analysis by mantel Cox test, χ(2)=0.0012, P=0.97). Conclusion: In sepsis mice model, inhibition of PKR activity can reduce the expression of inflammatory factors in plasma, decrease bacterial load in blood and abdominal cavity, and protect organ function, which could suggest that inhibition of PKR activity has potential application in sepsis treatment.

Effect of deletion of SUMOylation on dendritic cell function in septic mice and its role in sepsis.

To investigate the effect of deletion of small ubiquitin-like modifier (SUMO) on the function of dendritic cells (DC) in septic mice and its role in sepsis. Septic models of DC-specific ubiquitin-conjugating enzyme 9 (UBC9) deficient (UBC9ΔDC) mice and wild type (WT) mice with cecal ligation and puncture (CLP) were established. The differences in 7-day mortality of the mice were analyzed. Bacteria loads of blood, liver, and spleen were tested. ELISA was used to detect the levels of IL-1β, IL-6, IL-18, and TNF-α in plasma and culture medium of bone marrow-derived dendritic cells (BMDC). The levels of cytokine IFN-γ and IL-4 in supernatant of spleen mononuclear cells were detected by ELISA.The expressions of MHC II, CD54, and CD80 on the cell surface of DC were analyzed by flow cytometry. The percentages of Th1, and Th2 cells in spleen mononuclear cells were analyzed by flow cytometry. Compared with the WT septic mice, the 7-day mortality of UBC9ΔDC septic mice was higher (P<0.05). Bacterial loads in blood (P<0.01), liver (P<0.01), and spleen (P<0.05) were significantly increased in UBC9ΔDC septic mice. Levels of IL-1β and IL-18 in plasma and culture supernatant of BMDC were also significantly increased in UBC9ΔDC septic mice (all P<0.01). There was no significant difference in the number of DC and the expression of cell surface molecules in DC of UBC9ΔDC septic mice (all P>0.05).The percentage of Th2 cells was significantly increased in UBC9ΔDC septic mice (P<0.05). The ratio of Th1 to Th2 was decreased in UBC9ΔDC septic mice but the difference was not significant (P>0.05). Levels of IFN-γ and IL-4 were increased in UBC9ΔDC septic mice, and the ratio of IFN-γ to IL-4 were significantly decreased in UBC9ΔDC septic mice (all P<0.05). Deletion of SUMOylation may increase the mortality of mice with sepsis through regulating the release of inflammatory factors from DC and abnormal activation of T cells by DC.

Sleep enhances numbers and function of monocytes and improves bacterial infection outcome in mice

Sleep strongly impacts both humoral and cellular immunity; however, its acute effects on the innate immune defense against pathogens are unclear. Here, we elucidated in mice whether sleep affects the numbers and functions of innate immune cells and their defense against systemic bacterial infection. Sleep significantly increased numbers of classical monocytes in blood and spleen of mice that were allowed to sleep for six hours at the beginning of the normal resting phase compared to mice kept awake for the same time. The sleep-induced effect on classical monocytes was neither caused by alterations in corticosterone nor myelopoiesis, bone marrow egress or death of monocytes and did only partially involve Gαi-protein coupled receptors like chemokine receptor 2 (CCR2), but not the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) or lymphocyte function-associated antigen 1 (LFA-1). Notably, sleep suppressed the expression of the clock gene Arntl in splenic monocytes and the sleep-induced increase in circulating classical monocytes was abrogated in Arntl-deficient animals, indicating that sleep is a prerequisite for clock-gene driven rhythmic trafficking of classical monocytes. Sleep also enhanced the production of reactive oxygen species by monocytes and neutrophils. Moreover, sleep profoundly reduced bacterial load in blood and spleen of mice that were allowed to sleep before systemic bacterial infection and consequently increased survival upon infection. These data provide the first evidence that sleep enhances numbers and function of innate immune cells and therewith strengthens early defense against bacterial pathogens.

Intestinal permeability modulation through a polyphenol-rich dietary pattern in older subjects: MaPLE project outcomes and perspectives

AbstractIn recent years, research has been focusing on strategies to counteract inflammatory processes and age-related diseases(1). During ageing, a low-grade systemic inflammation is often associated to an altered intestinal permeability (IP) a condition that has been shown to promote inflammation possibly through the translocation of dietary and bacterial factors into the blood stream that activates the immune system(2).In this regard, dietary pattern and environmental factors could play a fundamental role because of their potential ability to modulate inflammation, IP and the gut microbial ecosystem (GME). Moreover, it has been hypothesized that bioactive compounds such as polyphenols may affect IP and GME(3).The MaPLE project (Microbiome mAnipulation through Polyphenols for managing gut Leakiness in the Elderly) aimed to investigate the hypothesis that a polyphenol-rich diet can improve IP condition in a target population with beneficial changes at intestine and systemic level. To this aim, a randomised, controlled, cross-over dietary intervention study (8-week polyphenol-rich diet versus 8-week control diet, separated by a wash-out period) was carried out in a group of older subjects (&gt; 60 years) living in a well-controlled setting (i.e. nursing home). Markers related with IP, inflammation, oxidative stress, vascular function and intestinal microbial ecosystem were investigated in serum, urine and/or fecal samples. Moreover, blood bacteria DNAemia, and serum/urine metabolomics has been assessed. Moreover, a consistent nutritional evaluation of the standard menu (provided by the nursing home) and of weighed food diaries was performed, providing also data on actual polyphenol intake during the intervention. The results show there were higher levels of IP in the older subjects, and that the polyphenol-enriched diet changed the levels of serum zonulin, a marker of IP. In addition, an association between zonulin and blood bacterial load was demonstrated. Ongoing in vitro and in vivo experiments are exploring the potential effects of different polyphenols on IP and the mechanisms involved. The MaPLE project will generate new data to improve the understanding on the role of polyphenols in the modulation of intestinal microbiome and its interactions with the host.

Impact of Intermittent Hypoxia on Sepsis Outcomes in a Murine Model

Sleep apnea has been associated with a variety of diseases, but its impact on sepsis outcome remains unclear. This study investigated the effect of intermittent hypoxia [IH]–the principal feature of sleep apnea–on murine sepsis. 5-week-old male C57BL6 mice were assigned to groups receiving severe IH (O2 fluctuating from room air to an O2 nadir of 5.7% with a cycle length of 90 seconds), mild IH (room air to 12%, 4 minutes/cycle), or room air for 3 weeks. Sepsis was induced by cecal ligation and puncture and survival was monitored. Sepsis severity was evaluated by murine sepsis scores, blood bacterial load, plasma tumor necrosis factor-α [TNF-α]/interleukin-6 [IL-6] levels and histopathology of vital organs. Compared with normoxic controls, mice subjected to severe IH had earlier mortality, a lower leukocyte count, higher blood bacterial load, higher plasma TNF-α and IL-6 levels, more severe inflammatory changes in the lung, spleen and small intestine. Mice subjected to mild IH did not differ from normoxic controls, except a higher IL-6 level after sepsis induced. The adverse impact of severe IH was reversed following a 10-day normoxic recovery. In conclusion, severe IH, not mild IH, contributed to poorer outcomes in a murine sepsis model.

Cas9 regulated gene expression and pathogenicity in Riemerella anatipestifer

Riemerellosis, a Riemerella anatipestifer infection, can cause meningitis, pericarditis, parahepatitis, and airsacculitis in ducks, leading to serious economic losses in the duck meat industry. However, the molecular mechanism of the pathogenesis and virulence factors of this infection are poorly understood. In the present study, we created a mutant strain RA-YMΔCas9 using trans-conjugation. Bacterial virulence tests indicated that the median lethal dose (LD50) of RA-YMΔCas9 was 5.01 × 107 CFU, significantly lower than that of the RA-YM strain, which was 1.58 × 105 CFU. The distribution and blood bacterial load from the infection groups showed no significant difference in the brain between the RA-YMΔCas9 mutant and the wild-type RA-YM strains, however, the number of mutant strains were significantly reduced in the liver, heart, and blood. Animal immunization experiments demonstrated that the intranasal administration of RA-YMΔCas9 in ducklings provided 80% protection after challenge with the wild-type strain, showing potential use as a live mucosal vaccine. RNAseq analysis indicated that Cas9 protein played a regulatory role in gene expression. This study is the first to report on the involvement of Cas9 in the regulation and pathogenesis of R. anatipestifer, and provides a theoretical basis for the development of relevant genetic engineering vaccines.

Cecropin-like antimicrobial peptide protects mice from lethal E.coli infection.

Resistance of pathogenic bacteria to standard antibiotics is an issue of great concern, and new treatments for bacterial infections are needed. Antimicrobial peptides (AMPs) are small, cationic, and amphipathic molecules expressed by metazoans that kill pathogens. They are a key part of the innate immune system in both vertebrates and invertebrates. Due to their low toxicity and broad antimicrobial activities, there has been increasing attention to their therapeutic usage. Our previous research demonstrated that four peptides—DAN1, DAN2, HOLO1 and LOUDEF1—derived from recently sequenced arthropod genomes exhibited potent antimicrobial effects in-vitro. In this study, we show that DAN2 protected 100% of mice when it was administered at a concentration of 20 mg/kg thirty minutes after the inoculation of a lethal dose of E. coli intraperitoneally. Lower concentrations of DAN2—10mg/kg and 5mg/kg protected more than 2/3s of the mice. All three dose levels reduced bacterial loads in blood and peritoneal fluid by 10-fold or more when counted six hours after bacterial challenge. We determined that DAN2 acts by compromising the integrity of the E. coli membrane. This study supports the potential of DAN2 peptide as a therapeutic agent for treating antibiotic resistant Gram-negative bacterial infections.

Enhancing Autophagy Protects Against Sepsis-Induced Neuromuscular Dysfunction Associated with Qualitative Changes to Acetylcholine Receptors.

Sepsis-induced myopathy is a heavy burden for patients during respiratory failure as well as after discharge, which could be characterized with qualitative changes to nAChR in a rat model of sepsis, regulated by NRG-1. Autophagy is an innate immune defense mechanism against microbial challenges. We found neuromuscular dysfunction in anterior tibial muscle of male Sprague-Dawley rats 24 h after cecal ligation and puncture (CLP). CLP resulted in increased systemic and local inflammation in anterior tibial muscle tissue. The start-up phase of autophagy, as measured by LC3II, was activated immediately after CLP and continued until 24 h; the degradation phase was suppressed until 24 h, after a brief increase at 4 h (revealed by p62). NRG-1 first increased, and then decreased to a level lower than that in the sham group. Meanwhile, expression of γ- and α7- acetylcholine receptors was detected at 8 and 16 h after CLP; levels increased continuously until 24 h. Subsequently, we investigated the significance of autophagy in CLP-induced neuromuscular dysfunction by treatment with rapamycin or 3-methyladenine, which were classical pharmaceuticals for enhancing or suppressing autophagy. Rapamycin activated autophagy, limited the CLP-induced systemic pro-inflammatory response and blood bacterial load without affecting local inflammatory response, upregulated NRG-1, downregulated γ- and α7-acetylcholine receptors, and improved 7-day neuromuscular function and survival rate. In contrast, 3-methyladenine enhanced local inflammatory response, suppressed autophagy, worsened 7-day neuromuscular function. We conclude that impaired autophagy may contribute to sepsis-induced neuromuscular dysfunction in young male rats. Enhancing autophagy with rapamycin alleviated qualitative changes to acetylcholine receptors without triggering local anti-inflammatory response and improved anterior tibial muscle function in septic early phase (24 h) as well as in septic chronic phase (7d). Enhancing autophagy soon after sepsis is a potential strategy for treatment of sepsis-induced myopathy.

Deletion of the Riemerella anatipestifer type IX secretion system gene sprA results in differential expression of outer membrane proteins and virulence

ABSTRACTRiemerella anatipestifer (RA), the causative agent of infectious serositis that targets ducklings and other poultry, secretes protein via the type IX secretion system (T9SS). The proteins transported by T9SS are located on the bacterial cell surface or secreted into the extracellular milieu. In this study, a sprA deletion mutant was constructed encoding a core protein of T9SS to investigate its influence on outer membrane protein expression and its role in virulence. Compared with the wild-type RA-YM strain, the deletion mutant ΔsprA failed to digest gelatin, showed the same growth rate in the logarithmic phase and exhibited greater sensitivity to the bactericidal activity of duck sera, whereas the complemented strain restored these phenotypes. The outer membrane proteome of RA-YM and the ΔsprA mutant were analyzed by Tandem Mass Tags, which revealed 198 proteins with predicted localization to the cell envelope. Sixty-three of these proteins were differentially expressed in the outer membrane, with 43 up-regulated and 20 down-regulated. Among the twelve outer membrane proteins which were secreted by T9SS, four proteins were up-regulated and one protein was down-regulated. Animal experiments demonstrated that the median lethal dose of the mutant strain ΔsprA was about 500 times higher than that of the wild-type RA-YM strain, and bacterial loads in blood, brain, heart, liver and spleen of the ΔsprA-infected ducks were significantly reduced. Our results indicate that the SprA is a virulence-associated factor of RA, and its absence results in altered abundance of outer membrane proteins, and secretion disorders associated with some of the T9SS effector proteins.

Antibody Dependent Enhancement of Acinetobacter baumannii Infection in a Mouse Pneumonia Model.

Acinetobacter baumannii has become a pathogen of increasing medical importance because of the emergence of multidrug-resistant strains and the high rate of mortality of infected patients. Promising animal study results have been reported recently with active and passive immunization against A. baumannii virulence factors. In the present study, a monoclonal IgG3 antibody, 8E3, was developed with specificity for the K2 capsular polysaccharide of A. baumannii, and its therapeutic potential was assessed. 8E3 enhanced macrophage-mediated bactericidal activity against the A. baumannii clinical strain AB899. However, 8E3 treatment (passive immunization) of AB899-infected mice led to a substantial increase in mortality and to substantial increases in bacterial load in blood, lung, and in splenic samples. In vitro investigations showed a large binding capacity in the supernatant of bacterial cultures, suggesting that shed capsule components act as a binding sink for 8E3. Investigations of 8E3 pharmacokinetics in mice demonstrated that unbound concentrations of the antibody dropped below detection limits within 24 hours after a 200 mg/kg dose. However, total concentrations of antibody declined slowly, with an apparent terminal half-life (t 1/2) of 6.7-8.0 days, suggesting that the vast majority of 8E3 in blood is bound (e.g., with soluble capsule components in blood). We hypothesize that high concentrations of 8E3-capsule immune complexes act to inhibit bacterial clearance in vivo. To the best of our knowledge, this is the first demonstration of antibody-dependent enhancement of A. baumannii infection, and these observations highlight the complexity of antibody-based therapy for A. baumannii infections.

The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis

In this study, the Riemerella anatipestifer mutant strain RA1062 was obtained by screening a random Tn4351 transposon mutant library. The mutant strain was unreactive with the anti-CH3 lipopolysaccharide monoclonal antibody, as demonstrated with an enzyme-linked immunosorbent assay, and its M949_RS01035 gene was inactivated. When cultured in trypticase soy broth, the late stage growth of the mutant RA1062 was significantly decreased. The mutant RA1062 was stained with crystal violet and presented a rough lipopolysaccharide phenotype, which differed from that of the wild-type strain CH3, suggesting that deletion of the M949_RS01035 gene resulted in defective lipopolysaccharide. Silver staining and Western blot analyses further confirmed that the RA1062 lipopolysaccharide had a deficiency in ladder-like binding pattern, as compared to lipopolysaccharide of the wild-type CH3 strain. In addition, the mutant RA1062 showed a higher susceptibility to complement-dependent killing, increased bacterial adhesion and invasion capacities to Vero cells, decreased blood bacterial loads, and attenuated virulence in infected ducks, when compared to the wild-type strain CH3. Moreover, RNA-Seq and real-time polymerase chain reaction analyses indicated that two genes were up-regulated and two were down-regulated in the mutant RA1062 genome. Furthermore, an animal protection experiment showed that immunization of ducks with inactivated RA1062 bacterin conferred effective cross-protection against challenge with the virulent R. anatipestifer serotypes 1, 2, and 10. This study presents evidence that the M949_RS01035 gene is involved in bacterial phenotype, virulence, and gene regulation in R. anatipestifer. The mutant strain RA1062 could be used as a cross-protective vaccine candidate.