Microbiological assessment reveals that Salmonella, Shigella and Campylobacter infections are widespread in HIV infected and uninfected patients with diarrhea in Mozambique.

Diarrhea is an important cause of hospitalizations in Mozambique. However, little attention has been paid to the impact HIV infection on the prevalence or clinical manifestations of enteric bacterial infections. This study aimed to determine the prevalence of Salmonella spp., Shigella spp. and Campylobacter spp. in HIV-infected and HIV-uninfected patients with diarrhea, identify risk factors for infection, and explore the association between HIV status, viral load, and bacterial prevalence. We conducted a case-control study at the Centro de Saúde de Mavalane and Centro de Saúde 1° de Maio in Maputo, Mozambique, from November 2021 to May 2022. We recruited 300 patients, including 150 HIV-infected (cases) and 150 HIV-uninfected patients (controls), aged between 0-88 years, presenting with diarrhea. Stool samples were collected for bacterial isolation through culture, and for each HIV-infected patient, 4 ml of venous blood were obtained for viral load detection through PCR. A total of 129 patients (43.0%) had at least one bacterial infection. The prevalence of Salmonella spp., Shigella spp. and Campylobacter spp. was 33.0% (n = 99), 15.0% (n = 45) and 4.3% (n = 13), respectively. The prevalence of any bacterial infection did not differ significantly between HIV-infected (45.3%, n = 68) and HIV-uninfected patients (40.7%, = 61) (p = 0.414). Overall, having 2-3 symptoms of enteric disease (p = 0.008) and a basic education (p = 0.030) were factors associated with bacterial infection. Of the 148 patients for whom HIV-1 RNA levels were available, 115 had copy numbers ≤ 75. Another 13 had levels between 76 and 1,000 and the remaining 20 had an average of 327,218.45 copies/ml. Bivariate logistic regression found that Shigella spp. were associated with HIV (p = 0.038), although no association was found in the multivariate analysis. Enteric infections are common in both HIV-infected and -uninfected patients. Low schooling influences the occurrence of enteric infections, which highlights the need to raise awareness about their prevention.

Susceptibility antibiotic screening reveals high rates of multidrug resistance of Salmonella, Shigella and Campylobacter in HIV infected and uninfected patients from Mozambique.

BACKGROUND: Antibacterial resistance is a growing concern worldwide, including in Mozambique. Diarrhea is an important cause of mortality in Mozambique, yet few local studies have reported on the resistance of bacterial pathogens in this context. Therefore, this study aims to characterize antibiotic susceptibility patterns of Salmonella, Shigella and Campylobacter spp. among patients with diarrhea, including those who are HIV-infected and-uninfected. METHODS: We conducted antibiotic susceptibility testing on 157 stool isolates recovered from 129 patients aged between 0 and 80 years with diarrhea, including HIV infected (n = 68) and-uninfected individuals (n = 61), assisted at two health centers in Maputo city. The isolates comprised of 99 Salmonella, 45 Shigella and 13 Campylobacter strains. The Kirby-Bauer disk diffusion method was used on Mueller-Hinton II agar for Salmonella and Shigella spp., while Mueller-Hinton II agar with 5% defibrinated sheep blood was used for Campylobacter spp. We tested six antibiotics listed on the national essential medicines list, including ciprofloxacin, erythromycin, azithromycin, trimethoprim-sulfamethoxazole, gentamicin, and tetracycline. RESULTS: All isolates were resistant to at least one antibiotic. A high percentage of Salmonella spp. isolates were found to be resistant to trimethoprim-sulfamethoxazole (89.9%, n = 89), erythromycin (88.9%, n = 88) and tetracycline (76.8%, n = 76). In addition, 86.6% (n = 39) and 68.9% (n = 31) of Shigella isolates were resistant to trimethoprim-sulfamethoxazole and tetracycline, respectively. The majority of Campylobacter isolates (92.3%, n = 12) were resistant to erythromycin, azithromycin and tetracycline. Multidrug resistance (MDR) was observed in 79.8% of Salmonella spp., 76.9% of Campylobacter spp., and 57.8% of Shigella spp. Drug susceptibility profiles for Salmonella spp. and Campylobacter were similar in both HIV-1 infected and uninfected patients. However, Shigella spp. isolates obtained from patients without HIV infection were significantly more likely to be resistant to erythromycin, azithromycin or to exhibit multidrug resistance than those obtained from patients with HIV-1 infection (p < 0.05). All Shigella spp. and Campylobacter spp. isolates were susceptible to gentamicin. CONCLUSION: Our study highlights concerning rates of antibiotic resistance and MDR among diarrheal bacterial pathogens in Mozambique. Further research is needed to understand the impact of HIV, ART therapy and immunosuppression on antibiotic resistance. Urgent interventions are essential to prevent the spread of resistant strains.

Role of the microbiota-gut-brain axis in postacute COVID syndrome.

The COVID-19 pandemic has resulted in the infection of hundreds of millions of individuals over the past 3 years, coupled with millions of deaths. Along with these more acute impacts of infection, a large subset of patients has developed symptoms that collectively comprise "postacute sequelae of COVID-19" (PASC, also known as long COVID), which can persist for months and maybe even years. In this review, we outline the current knowledge on the role of impaired microbiota-gut-brain (MGB) axis signaling in the development of PASC and the potential mechanisms involved, which may lead to a better understanding of disease progression and treatment options in the future.

Potentially probiotic Limosilactobacillus reuteri from human milk strengthens the gut barrier in T84 cells and a murine enteroid model.

AIMS: At conception, the infant gut barrier is immature, gradually developing with regular intake of maternal milk. This study addressed whether the barrier-strengthening effect of breast feeding might be attributable, at least in part, to autochthonous beneficial human milk bacteria. METHODS AND RESULTS: Twelve bacterial strains from the breast milk of Pakistani mothers who underwent cesarean delivery (NPL-88, NPL-157, NPL-179, NPL-181, NPL-388 (Limosilactobacillus reuteri), NPL-76, NPL-495, NPL-504 (Limosilactobacillus fermentum), NPL-415 (Lactobacillus pentosus), NPL-412, NPL-416 (Lactiplantibacilllus plantarum) and NPL-374 (Bifidobacterium longum) were shortlisted based on their tolerance to acidic pH (2.8-4.2) and bile (0.1-0.3%). The effect of these bacteria on gut barrier function in the presence and absence of pathogens was assessed as changes in transepithelial electrical resistance (TEER) in the human T84 colonic epithelial cell line and in murine enteroid-derived monolayers (EDMs). The TEER of T84 cells monolayers rose in the presence of most of the human milk strains, being most pronounced in case of L. reuteri NPL-88 (34% within five h), exceeding the effect of the well-known probiotic L. acidophilus (20%). qRT-PCR, western blot and immunofluorescent staining associated the increase in TEER with enhanced expression of tight junction proteins. Pretreatment of murine EDMs with NPL-88 also largely prevented the ability of the pathogen, Salmonella, to decrease TEER (87 ± 1.50%; P < 0.0001, n = 4). CONCLUSIONS: Human milk lactic acid bacteria are potential probiotics that can strengthen gut barrier function and protect breastfed neonates against enteric infections.

Pentacyclic triterpenes modulate farnesoid X receptor expression in colonic epithelial cells: Implications for colonic secretory function.

The nuclear bile acid receptor, farnesoid X receptor (FXR), is an important regulator of intestinal and metabolic function. Previous studies suggest that pentacyclic triterpenes (PCTs), a class of plant-derived bioactive phytochemical, can modulate FXR activity and may therefore offer therapeutic benefits. Here, we investigated the effects of a prototypical PCT, hederagenin (HG), on FXR expression, activity, and antisecretory actions in colonic epithelial cells. T84 cells and murine enteroid-derived monolayers were employed to assess HG effects on FXR expression and activity in colonic epithelia. We measured mRNA levels by qRT-PCR and protein by ELISA and immunoblotting. Transepithelial Cl- secretion was assessed as changes in short circuit current in Ussing chambers. We determined HG treatment (5-10 µM) alone did not induce FXR activation but significantly increased expression of the receptor, both in T84 cells and murine enteroid-derived monolayers. This effect was accompanied by enhanced FXR activity, as assessed by FGF-15/19 induction in response to the synthetic, GW4064, or natural FXR agonist, chenodeoxycholic acid. Effects of HG on FXR expression and activity were mimicked by another PCT, oleanolic acid. Furthermore, we found FXR-induced downregulation of cystic fibrosis transmembrane conductance regulator Cl- channels and inhibition of transepithelial Cl- secretion were enhanced in HG-treated cells. These data demonstrate that dietary PCTs have the capacity to modulate FXR expression, activity, and antisecretory actions in colonic epithelial cells. Based on these data, we propose that plants rich in PCTs, or extracts thereof, have excellent potential for development as a new class of "FXR-targeted nutraceuticals".

Fostering Sustainable Biomedical Research Training in Mozambique: A Spin-Off of the Medical Education Partnership Initiative.

Background: The further development of research capacity in low- and middle-income countries is critical to the delivery of evidence-based healthcare, the design of sound health policy and effective resource allocation. Research capacity is also critical for the retention of highly skilled faculty and staff and for institutional internationalization. Objectives: We summarize the accomplishments, challenges and legacy of a five-year program to train biomedical researchers entitled "Enhanced Advanced Biomedical Research Training for Mozambique (EABRTM)". Methods: A program conducted from 2015-2021 built upon the Medical Education Partnership Initiative to develop research capacity at Eduardo Mondlane University (UEM) and allied institutions. The project included design and implementation of postgraduate training programs and bolstered physical and human research infrastructure. Findings: The program supported development and implementation of UEM's first doctoral (Bioscience and Public Health) and master (Biosciences) programs with 31 and 23 students enrolled to date, respectively. Three master programs were established at Lúrio University from which 176/202 (87.1%) and 107/202 (53.0%) students obtained a Postgraduate Diploma or master's degree, respectively. Scholarships were awarded to 39 biomedical researchers; 13 completed master degrees, one completed a PhD and five remain in doctoral studies. Thirteen administrative staff and four biomedical researchers were trained in research administration and in biostatistics, respectively. A total of 119 courses and seminars benefited 2,142 participants. Thirty-five manuscripts have been published to date in peer-reviewed international journals of which 77% are first-authored by Mozambicans and 44% last-authored by Africans. Sustainability was achieved through 59 research projects awarded by international agencies, totaling $16,363,656.42 and funds ($ 7,319,366.11) secured through 2025. Conclusions: The EABRTM program substantially increased research and mentorship capacity and trained a new generation of biostatisticians and research administrators. These programmatic outcomes significantly increased the confidence of early stage Mozambican researchers in their ability to successfully pursue their career goals.

The effect of a fennel seed extract on the STAT signaling and intestinal barrier function.

BACKGROUND: Foeniculum vulgare, F. vulgare, commonly known as fennel, is believed to be one of the world's oldest medicinal herbs and has been exploited by people for centuries as a nutritional aid for digestive disorders. In many southeast Asian countries, it is ingested as an after-meal snack, mukhvas, due to its breath-freshening and digestive aid properties. F. vulgare is used in some countries, such as Iran, as a complementary and alternative treatment for inflammatory bowel disease (IBD). METHODS: This study investigated the effects of fennel seed extract on intestinal epithelium barrier function and the Signal Transducer and Activator of Transcription (STAT) pathway. This pathway is active in inflammatory bowel disease. To study the protective effects of fennel seed extract in vitro, monolayers derived from the T84 colonic cell line were challenged with interferon-gamma (IFN-γ) and monitored with and without fennel seed extract. To complement our in vitro studies, the dextran sodium sulfate induced murine colitis model was employed to ascertain whether the protective effect of fennel seed extract can be recapitulated in vivo. RESULTS: Fennel seed extract was shown to exert a protective effect on transepithelial electrical resistance (TEER) in both T84 and murine models and showed increases in tight junction-associated mRNA in T84 cell monolayers. Both models demonstrated significant decreases in phosphorylated STAT1 (pSTAT1), indicating reduced activation of the STAT pathway. Additionally, mice treated with fennel seed showed significantly lower ulcer indices than control mice. CONCLUSIONS: We conclude barrier function of the gastrointestinal tract is improved by fennel seed extract, suggesting the potential utility of this agent as an alternative or adjunctive therapy in IBD.

Intestinal secretory mechanisms and diarrhea.

One of the primary functions of the intestinal epithelium is to transport fluid and electrolytes to and from the luminal contents. Under normal circumstances, absorptive and secretory processes are tightly regulated such that absorption predominates, thereby enabling conservation of the large volumes of water that pass through the intestine each day. However, in conditions of secretory diarrhea, this balance becomes dysregulated, so that fluid secretion, driven primarily by Cl- secretion, overwhelms absorptive capacity, leading to increased loss of water in the stool. Secretory diarrheas are common and include those induced by pathogenic bacteria and viruses, allergens, and disruptions to bile acid homeostasis, or as a side effect of many drugs. Here, we review the cellular and molecular mechanisms by which Cl- and fluid secretion in the intestine are regulated, how these mechanisms become dysregulated in conditions of secretory diarrhea, currently available and emerging therapeutic approaches, and how new strategies to exploit intestinal secretory mechanisms are successfully being used in the treatment of constipation.

Diarrhoeal pathogenesis in Salmonella infection may result from an imbalance in intestinal epithelial differentiation through reduced Notch signalling.

Infections with non-typhoidal Salmonella spp. represent the most burdensome foodborne illnesses worldwide, yet despite their prevalence, the mechanism through which Salmonella elicits diarrhoea is not entirely known. Intestinal ion transporters play important roles in fluid and electrolyte homeostasis in the intestine. We have previously shown that infection with Salmonella caused decreased colonic expression of the chloride/bicarbonate exchanger SLC26A3 (down-regulated in adenoma; DRA) in a mouse model. In this study, we focused on the mechanism of DRA downregulation during Salmonella infection, by using murine epithelial enteroid-derived monolayers (EDMs). The decrease in DRA expression caused by infection was recapitulated in EDMs and accompanied by increased expression of Atonal Homolog 1 (ATOH1), the goblet cell marker Muc2 and the enteroendocrine cell marker ChgA. This suggested biased epithelial differentiation towards the secretory, rather than absorptive phenotype. In addition, the downstream Notch effector, Notch intracellular domain (NICD) and Hes1 were decreased following Salmonella infection. The relevance of Notch signalling was further investigated using a γ-secretase inhibitor, which recapitulated the downregulation in Hes1 and DRA as well as upregulation in ATOH1 and Muc2 seen following infection. Our findings suggest that Salmonella infection may result in a shift from absorptive to secretory cell types through Notch inhibition, which explains why there is a decreased capacity for absorption and ultimately the accumulation of diarrhoeal fluid. Our work also shows the value of EDMs as a model to investigate mechanisms that might be targeted for therapy of diarrhoea caused by Salmonella infection. KEY POINTS: Salmonella is a leading foodborne pathogen known to cause high-chloride-content diarrhoea. Salmonella infection of murine enteroid-derived monolayers decreased DRA expression. Salmonella infection resulted in upregulation of the secretory epithelial marker ATOH1, the goblet cell marker Muc2 and the enteroendocrine cell marker ChgA. Downregulation of DRA may result from infection-induced Notch inhibition, as reflected by decreased expression of Notch intracellular domain and Hes1, as well as from decreased HNF1α signalling. The imbalance in intestinal epithelial differentiation favouring secretory over absorptive cell types is a possible mechanism by which Salmonella elicits diarrhoea and may be relevant therapeutically.

T cell protein tyrosine phosphatase protects intestinal barrier function by restricting epithelial tight junction remodeling.

Genome-wide association studies revealed that loss-of-function mutations in protein tyrosine phosphatase non-receptor type 2 (PTPN2) increase the risk of developing chronic immune diseases, such as inflammatory bowel disease (IBD) and celiac disease. These conditions are associated with increased intestinal permeability as an early etiological event. The aim of this study was to examine the consequences of deficient activity of the PTPN2 gene product, T cell protein tyrosine phosphatase (TCPTP), on intestinal barrier function and tight junction organization in vivo and in vitro. Here, we demonstrate that TCPTP protected against intestinal barrier dysfunction induced by the inflammatory cytokine IFN-γ by 2 mechanisms: it maintained localization of zonula occludens 1 and occludin at apical tight junctions and restricted both expression and insertion of the cation pore-forming transmembrane protein, claudin-2, at tight junctions through upregulation of the inhibitory cysteine protease, matriptase. We also confirmed that the loss-of-function PTPN2 rs1893217 SNP was associated with increased intestinal claudin-2 expression in patients with IBD. Moreover, elevated claudin-2 levels and paracellular electrolyte flux in TCPTP-deficient intestinal epithelial cells were normalized by recombinant matriptase. Our findings uncover distinct and critical roles for epithelial TCPTP in preserving intestinal barrier integrity, thereby proposing a mechanism by which PTPN2 mutations contribute to IBD.

Aberrant Epithelial Differentiation Contributes to Pathogenesis in a Murine Model of Congenital Tufting Enteropathy.

BACKGROUND & AIMS: Congenital tufting enteropathy (CTE) is an intractable diarrheal disease of infancy caused by mutations of epithelial cell adhesion molecule (EpCAM). The cellular and molecular basis of CTE pathology has been elusive. We hypothesized that the loss of EpCAM in CTE results in altered lineage differentiation and defects in absorptive enterocytes thereby contributing to CTE pathogenesis. METHODS: Intestine and colon from mice expressing a CTE-associated mutant form of EpCAM (mutant mice) were evaluated for specific markers by quantitative real-time polymerase chain reaction, Western blotting, and immunostaining. Body weight, blood glucose, and intestinal enzyme activity were also investigated. Enteroids derived from mutant mice were used to assess whether the decreased census of major secretory cells could be rescued. RESULTS: Mutant mice exhibited alterations in brush-border ultrastructure, function, disaccharidase activity, and glucose absorption, potentially contributing to nutrient malabsorption and impaired weight gain. Altered cell differentiation in mutant mice led to decreased enteroendocrine cells and increased numbers of nonsecretory cells, though the hypertrophied absorptive enterocytes lacked key features, causing brush border malfunction. Further, treatment with the Notch signaling inhibitor, DAPT, increased the numbers of major secretory cell types in mutant enteroids (graphical abstract 1). CONCLUSIONS: Alterations in intestinal epithelial cell differentiation in mutant mice favor an increase in absorptive cells at the expense of major secretory cells. Although the proportion of absorptive enterocytes is increased, they lack key functional properties. We conclude that these effects underlie pathogenic features of CTE such as malabsorption and diarrhea, and ultimately the failure to thrive seen in patients.

Gastrointestinal jabberwocky to bioengineering design: using function diagrams to teach physiology.

Function diagrams put the focus on physiology and physiological concepts rather than the associated anatomy. Function diagrams could potentially serve as an elaboration tool and memory aid (mnemonic) to improve learning and recall. The function diagram prototype of the gastrointestinal system can aid in the instruction of difficult gastrointestinal physiology topics using a sequential focus on fundamental gastrointestinal functions.

Claudin-2 pore causes leak that breaches the dam in intestinal inflammation.

The tight junction protein claudin-2 is upregulated in inflammatory bowel disease, and yet its deficit worsens infectious and chemical colitis. In this issue of the JCI, Raju and Shashikanth et al. examined the contribution of claudin-2 to immune-mediated colitis. The authors used transgenic mouse models to show that claudin-2 deficiency attenuated colitis progression as well as a leak barrier defect, albeit at the risk of intestinal obstruction. Further, inhibition of claudin-2 by targeting casein kinase 2 (CK2) also ameliorated colitis. The findings reveal unsuspected links between the pore and leak pathways of intestinal permeability and immune responses leading to colitis. They additionally suggest potential targets for therapeutic intervention in inflammatory bowel disease.

Potentiation of calcium-activated chloride secretion and barrier dysfunction may underlie EGF receptor tyrosine kinase inhibitor-induced diarrhea.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFr TKIs) are first-line therapies for various cancers, and cause dose-limiting severe diarrhea in many patients. We hypothesized that diarrhea caused by EGFr TKIs might reflect actions on epithelial transport, barrier function, or both, which we tested using cell cultures including murine and human enteroid-derived monolayers (EDMs), analyzed using electrophysiological and other relevant methods. EGFr TKIs (such as afatinib, erlotinib, and osimertinib) reversed the acute inhibitory effect of EGF on chloride secretion induced by carbachol (CCh) across T84 human colonic epithelial cells, which correlated with the diarrhea-inducing effect of each agent clinically. EGFr TKIs also reduced transepithelial electrical resistance (TEER), whereas co-treatment with CCh delayed the decrease in TEER compared with that of cells co-treated with EGF. Furthermore, afatinib and erlotinib prevented EGF- or CCh-induced EGFr phosphorylation. EGFr TKIs also suppressed phosphorylation of extracellular signal-regulated kinase (Erk)1/2 in response to EGF, whereas they had weaker effects on CCh-induced Erk1/2 phosphorylation. In human EDMs, EGF potentiated ion transport induced by CCh, whereas afatinib reversed this effect. The ability of EGFr TKIs to reverse the effects of EGF on calcium-dependent chloride secretion could contribute to the diarrheal side effects of these agents, and their disruption of epithelial barrier dysfunction is likely also pathophysiologically significant. CCh-activated Erk1/2 phosphorylation was relatively insensitive to EGFr TKIs and delayed the deleterious effects of EGFr TKIs on barrier function. These findings confirm and extend those of other authors, and may be relevant to designing strategies to overcome the diarrheal side effects of EGFr TKIs.

A prebiotic fructo-oligosaccharide promotes tight junction assembly in intestinal epithelial cells via an AMPK-dependent pathway.

Tight junctions play an important role in maintaining barrier integrity of intestinal epithelia. Activation of AMP-activated protein kinase (AMPK) promotes tight junction assembly in intestinal epithelial cells (IEC). Fructo-oligosaccharides (FOS), well-known prebiotics, have previously been shown to alleviate inflammation-associated intestinal epithelial disruption although the mechanisms were unclear. This study aimed to investigate any effect of FOS on AMPK activity and tight junction assembly under non-inflammatory and inflammatory conditions using T84 cells as an IEC model. As analyzed by western blot, FOS induced AMPK activation through a calcium sensing receptor (CaSR)-phospholipase C (PLC)- Ca2+/calmodulin-dependent protein kinase kinase-ß (CaMKKß) pathway. Calcium switch assays and immunofluorescence staining of zonula occludens-1 (ZO-1) revealed that FOS induced tight junction assembly via an CaMKKß-AMPK-dependent mechanism in IEC. Interestingly, FOS reversed the suppressive effect of lipopolysaccharide (LPS) on AMPK activity and tight junction assembly via a CaMKKß pathway. Taken together, these findings uncover a prebiotic-independent effect of FOS in promoting intestinal epithelial tight junction assembly through AMPK activation, which may have implications for the treatment of diseases whose pathogenesis involves impaired intestinal barrier function.