Role of antimicrobial peptides in combating shigellosis and in antibiotic-associated diarrhea

Antimicrobial peptides (AMPs) constitute front-line components of innate immunity in multicellular organisms. AMPs are able to kill a wide range of pathogens and exhibit additional important functions such as chemotaxis, angiogenesis and wound healing. These peptides are constitutively expressed in immune and/or epithelial cells. However, their expression can also be induced or suppressed by different stimuli in a cell and tissue specific manner. In this thesis, the functional relevance of cathelicidins and defensins, two major families of AMPs in mammals, was studied in the context of enteric infectious diseases. Induction of these AMPs was explored in Shigella infection. The effect of antibiotics on the expression of AMPs in colonic epithelial cells was also investigated and the implication of this effect on Clostridium difficile associated diarrhea (CDAD) was assessed. In a rabbit model of shigellosis, cathelicidin CAP-18 was downregulated in the large intestinal epithelia. Oral treatment with sodium butyrate (NaB) counteracted this downregulation and led to the conversion of the proform of CAP-18 into its active form in the stool (paper I). These findings correlated with reduced Shigella load in the intestinal lumen and clinical as well as histopathological recovery. Association between CAP-18 induction and reduction of bacterial load was supported by partial blocking of the antimicrobial activity in stool extract of a butyrate treated rabbit with CAP-18 specific antibody along with in vitro shigellacidal activity of CAP-18 peptide. In patients with shigellosis, administration of NaB enema as adjunct to antibiotic therapy led to an early improvement of rectal histopathology along with early reduction of inflammatory cells and proinflammatory cytokines in the stool compared to placebo treated patients (paper II). NaB treatment also resulted in enhanced expression of the human cathelicidin LL-37 in the rectal epithelia and sustained secretion of LL-37 in the stool. Since all patients were treated with antibiotics, attenuation of Shigella load in stool and clinical symptoms occurred simultaneously in both group of patients. Sodium-4-phenyl-butyrate (PB), an analogue of butyrate, was demonstrated to exhibit a similar therapeutic efficiency as NaB in the rabbit model of shigellosis (paper III). Downregulation of CAP-18 expression was also observed in the epithelia of lung and trachea. This suppression could render the respiratory tract susceptible to secondary infections during shigellosis. After oral treatment with PB or NaB, CAP-18 reappeared in the lung epithelia, which might strengthen the immunity of the respiratory tract against opportunistic respiratory pathogens. Ciprofloxacin and clindamycin were found to suppress butyrate-induced expression of LL-37 in the colonic epithelial cell line, HT-29 (paper IV). In vivo inhibitory effect of ciprofloxacin was observed on CAP-18 expression in the rectal epithelia of Shigellainfected rabbits treated with NaB as well as of healthy rabbits. Induction of genes encoding HBD-3 and additional AMPs by NaB were also inhibited by ciprofloxacin. In vitro antibacterial activity of LL-37 against ciprofloxacin-resistant C. difficile indicates that the reduction of AMPs after antibiotic treatment may allow the overgrowth of C. difficile in the gut. In conclusion, induction of AMPs is a promising therapeutic strategy against shigellosis. Suppression of AMP expression by antibiotics may contribute to CDAD, which is classically known to occur through disruption of the intestinal microbiota after antibiotic treatment

Differential Host Immune Responses to Epidemic and Endemic Strains of Shigella dysenteriae Type 1

Shigella dysenteriae type 1 causes devastating epidemics in developing countries with high case-fatality rates in all age-groups. The aim of the study was to compare host immune responses to epidemic (T2218) and endemic strains of S. dysenteriae type 1. Shigellacidal activity of serum from rabbits immunized with epidemic or endemic strains, S. dysenteriae type 1-infected patients, and healthy adult controls from Shigella endemic and non-endemic regions was measured. Immunogenic cross-reactivity of antibodies against Shigella antigens was evaluated by Western blot analysis. Oxidative burst and phagocytic responses of monocytes and neutrophils to selected S. dysenteriae type 1 strains were assessed by flow cytometry. Rabbit antisera against epidemic strain were less effective in killing heterologous bacteria compared to endemic antisera (p=0.0002). Patients showed an increased serum shigellacidal response after two weeks of onset of diarrhoea compared to the acute stage (3-4 days after onset) against their respective homologous strains; the response against T2218 and heterologous endemic S. dysenteriae type 1 strains was not significant. The serum shigellacidal response against all the S. dysenteriae type 1 strains was similar among healthy controls from endemic and non-endemic regions and was comparable with the acute stage response by patients. Compared to endemic strains of S. dysenteriae type 1, T2218 was significantly resistant to phagocytosis by both monocytes and neutrophils. No obvious differences were obtained in the induction of oxidative burst activity and cathelicidin-mediated killing. Cross-reactivity of antibody against antigens present in the epidemic and endemic strains showed some differences in protein/peptide complexity and intensity by Western blot analysis. In summary, epidemic T2218 strain was more resistant to antibody-mediated defenses, namely phagocytosis and shigellacidal activity, compared to endemic S. dysenteriae type 1 strains. Part of this variation may be attributed to the differential complexity of protein/peptide antigens

Efficacy of sodium butyrate adjunct therapy in shigellosis: a randomized, double-blind, placebo-controlled clinical trial

BACKGROUND: Treatment of shigellosis in rabbits with butyrate reduces clinical severity and counteracts the downregulation of cathelicidin (CAP-18) in the large intestinal epithelia. Here, we aimed to evaluate whether butyrate can be used as an adjunct to antibiotics in the treatment of shigellosis in patients. METHODS: A randomized, double-blind, placebo-controlled, parallel-group designed clinical trial was conducted. Eighty adult patients with shigellosis were randomized to either the Intervention group (butyrate, n = 40) or the Placebo group (normal saline, n = 40). The Intervention group was given an enema containing sodium butyrate (80 mM), twice daily for 3 days, while the Placebo group received the same dose of normal saline. The primary endpoint of the trial was to assess the efficacy of butyrate in improving clinical, endoscopic and histological features of shigellosis. The secondary endpoint was to study the effect of butyrate on the induction of antimicrobial peptides in the rectum. Clinical outcomes were assessed and concentrations of antimicrobial peptides (LL-37, human beta defensin1 [HBD-1] and human beta defensin 3 [HBD-3]) and pro-inflammatory cytokines (interleukin-1β [IL-1β] and interleukin-8 [IL-8]) were measured in the stool. Sigmoidoscopic and histopathological analyses, and immunostaining of LL-37 in the rectal mucosa were performed in a subgroup of patients. RESULTS: Compared with placebo, butyrate therapy led to the early reduction of macrophages, pus cells, IL-8 and IL-1β in the stool and improvement in rectal histopathology. Butyrate treatment induced LL-37 expression in the rectal epithelia. Stool concentration of LL-37 remained significantly higher in the Intervention group on days 4 and 7. CONCLUSION: Adjunct therapy with butyrate during shigellosis led to early reduction of inflammation and enhanced LL-37 expression in the rectal epithelia with prolonged release of LL-37 in the stool. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00800930

Differential Host Immune Responses to Epidemic and Endemic Strains of Shigella dysenteriae Type 1

Shigella dysenteriae type 1 causes devastating epidemics in developing countries with high case-fatality rates in all age-groups. The aim of the study was to compare host immune responses to epidemic (T2218) and endemic strains of S. dysenteriae type 1. Shigellacidal activity of serum from rabbits immunized with epidemic or endemic strains, S. dysenteriae type 1-infected patients, and healthy adult controls from Shigellaendemic and non-endemic regions was measured. Immunogenic cross-reactivity of antibodies against Shigella antigens was evaluated by Western blot analysis. Oxidative burst and phagocytic responses of monocytes and neutrophils to selected S. dysenteriae type 1 strains were assessed by flow cytometry. Rabbit antisera against epidemic strain were less effective in killing heterologous bacteria compared to endemic antisera (p=0.0002). Patients showed an increased serum shigellacidal response after two weeks of onset of diarrhoea compared to the acute stage (3-4 days after onset) against their respective homologous strains; the response against T2218 and heterologous endemic S. dysenteriae type 1 strains was not significant. The serum shigellacidal response against all the S. dysenteriae type 1 strains was similar among healthy controls from endemic and non-endemic regions and was comparable with the acute stage response by patients. Compared to endemic strains of S. dysenteriae type 1, T2218 was significantly resistant to phagocytosis by both monocytes and neutrophils. No obvious differences were obtained in the induction of oxidative burst activity and cathelicidin-mediated killing. Cross-reactivity of antibody against antigens present in the epidemic and endemic strains showed some differences in protein/peptide complexity and intensity by Western blot analysis. In summary, epidemic T2218 strain was more resistant to antibody-mediated defenses, namely phagocytosis and shigellacidal activity, compared to endemic S. dysenteriae type 1 strains. Part of this variation may be attributed to the differential complexity of protein/peptide antigens

Seroprevalence of SARS-CoV-2 infection and associated factors among Bangladeshi slum and non-slum dwellers in pre-COVID-19 vaccination era: October 2020 to February 2021

Background Seroprevalence studies have been carried out in many developed and developing countries to evaluate ongoing and past infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Data on this infection in marginalized populations in urban slums are limited, which may offer crucial information to update prevention and mitigation policies and strategies. We aimed to determine the seroprevalence of SARS-CoV-2 infection and factors associated with seropositivity in slum and non-slum communities in two large cities in Bangladesh. Methods A cross-sectional study was carried out among the target population in Dhaka and Chattogram cities between October 2020 and February 2021. Questionnaire-based data, anthropometric and blood pressure measurements and blood were obtained. SARS-CoV-2 serology was assessed by Roche Elecsys® Anti-SARS-CoV-2 immunoassay. Results Among the 3220 participants (2444 adults, ≥18 years; 776 children, 10–17 years), the overall weighted seroprevalence was 67.3% (95% confidence intervals (CI) = 65.2, 69.3) with 71.0% in slum (95% CI = 68.7, 72.2) and 62.2% in non-slum (95% CI = 58.5, 65.8). The weighted seroprevalence was 72.9% in Dhaka and 54.2% in Chattogram. Seroprevalence was positively associated with limited years of formal education (adjusted odds ratio [aOR] = 1.61; 95% CI = 1.43, 1.82), lower income (aOR = 1.23; 95% CI = 1.03, 1.46), overweight (aOR = 1.2835; 95% CI = 1.26, 1.97), diabetes (aOR = 1.67; 95% CI = 1.21, 2.32) and heart disease (aOR = 1.38; 95% CI = 1.03, 1.86). Contrarily, negative associations were found between seropositivity and regular wearing of masks and washing hands, and prior BCG vaccination. About 63% of the population had asymptomatic infection; only 33% slum and 49% non-slum population showed symptomatic infection. Conclusion The estimated seroprevalence of SARS-CoV-2 was more prominent in impoverished informal settlements than in the adjacent middle-income non-slum areas. Additional factors associated with seropositivity included limited education, low income, overweight and pre-existing chronic conditions. Behavioral factors such as regular wearing of masks and washing hands were associated with lower probability of seropositivity.https://doi.org/10.1371/journal.pone.0268093pubpu

Phenylbutyrate Counteracts Shigella Mediated Downregulation of Cathelicidin in Rabbit Lung and Intestinal Epithelia: A Potential Therapeutic Strategy

BACKGROUND: Cathelicidins and defensins are endogenous antimicrobial peptides (AMPs) that are downregulated in the mucosal epithelia of the large intestine in shigellosis. Oral treatment of Shigella infected rabbits with sodium butyrate (NaB) reduces clinical severity and counteracts the downregulation of cathelicidin (CAP-18) in the large intestinal epithelia. AIMS: To develop novel regimen for treating infectious diseases by inducing innate immunity, we selected sodium 4-phenylbutyrate (PB), a registered drug for a metabolic disorder as a potential therapeutic candidate in a rabbit model of shigellosis. Since acute respiratory infections often cause secondary complications during shigellosis, the systemic effect of PB and NaB on CAP-18 expression in respiratory epithelia was also evaluated. METHODS: The readouts were clinical outcomes, CAP-18 expression in mucosa of colon, rectum, lung and trachea (immunohistochemistry and real-time PCR) and release of the CAP-18 peptide/protein in stool (Western blot). PRINCIPAL FINDINGS: Significant downregulation of CAP-18 expression in the epithelia of rectum and colon, the site of Shigella infection was confirmed. Interestingly, reduced expression of CAP-18 was also noticed in the epithelia of lung and trachea, indicating a systemic effect of the infection. This suggests a causative link to acute respiratory infections during shigellosis. Oral treatment with PB resulted in reduced clinical illness and upregulation of CAP-18 in the epithelium of rectum. Both PB and NaB counteracted the downregulation of CAP-18 in lung epithelium. The drug effect is suggested to be systemic as intravenous administration of NaB could also upregulate CAP-18 in the epithelia of lung, rectum and colon. CONCLUSION: Our results suggest that PB has treatment potential in human shigellosis. Enhancement of CAP-18 in the mucosal epithelia of the respiratory tract by PB or NaB is a novel discovery. This could mediate protection from secondary respiratory infections that frequently are the lethal causes in dysentery

Fatal Infection Caused by Chromobacterium violaceum: A Case Report from a Tertiary Care Hospital in Bangladesh

Chromobacterium violaceum is a Gram-negative bacterium, found in tropical and subtropical regions. C. violaceum infection rarely occurs, but once occurs, it is associated with significant mortality due to severe systemic infection. Since the first human case from Malaysia in 1927, >150 cases of C. violaceum infection have been reported worldwide. We have described here a fatal case of C. violaceum infection in a tertiary care hospital in Dhaka, Bangladesh. To the best of our knowledge, this is the first case of C. violaceum infection in Bangladesh

Tuning of regimes during two-phase flow through a cross-junction

We investigate the dynamics of two immiscible fluids in a cross-junction via three-dimensional numerical simulations using the volume of fluid approach to track the dispersed phase's evolution. Different regimes, namely the dripping, squeezing, and jetting dynamics, have been observed for different dimensionless parameters, and we unveil a transition in regimes due to the concomitant interplay of capillarity, viscosity, and wettability. Our results reveal that hydrophobic channel surfaces favor a transition from squeezing to dripping behavior at a lower value of the capillary number. Moreover, higher viscosity ratios advance the process of squeezing, necking, and breakage on hydrophobic surfaces. A wettability-capillarity regime map is also presented that will have significant implications regarding the choice of substrate wettability, fluid properties, and flow rate in droplet dispensing devices. © 2021 Author(s)

Persistence of Mucosal Mast Cells and Eosinophils in Shigella-Infected Children

Cells of the innate immune system and their mediators were studied at the single-cell level in the rectums of pediatric and adult patients with Shigella infection to better understand why children are at higher risk for severe infection. Adult patients had increased infiltration of mucosal mast cells (MMC) at the acute stage (3 to 5 days after the onset of diarrhea) and eosinophils in early convalescence (14 to 16 days after onset). Increased expression of stem cell factor and prostaglandin H synthase-1 (PGHS-1) was associated with increased tryptase-K(i)67-double-positive MMC in the acute stage and increased apoptosis of MMC, which led to a rapid decline in early convalescence. The eosinophils demonstrated increased expression of major basic protein (MBP), eotaxin, and CCR3, as well as increased necrotic death. The neutrophils showed enhanced α-defensin and lactoferrin expression in the acute phase. In contrast to adults, the pediatric patients demonstrated delayed accumulation of mast cells and eosinophils, while α-defensin expression persisted during convalescence. In contrast, neutrophil counts and lactoferrin expression were reduced in children compared to adults. The results suggest that children with shigellosis have a persistent activation of the innate immune response in the convalescent phase, indicating delayed elimination of Shigella antigens compared to adults