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Arthrospira platensis(commonly known as spirulina) is a photosynthetic cyanobacterium1. It is a highly nutritious food that has been consumed for decades in the US, and even longer by indigenous cultures2. Its widespread use as a safe food source and proven scalability have driven frequent attempts to convert it into a biomanufacturing platform. But these were repeatedly frustrated by spirulina’s genetic intractability. We report here efficient and versatile genetic engineering methodology for spirulina that allows stable expression of bioactive protein therapeutics at high levels. We further describe large-scale, indoor cultivation and downstream processing methods appropriate for the manufacturing of biopharmaceuticals in spirulina. The potential of the platform is illustrated by pre-clinical development and human testing of an orally delivered antibody therapeutic against campylobacter, a major cause of infant mortality in the developing world and a growing antibiotic resistance threat3,4. This integrated development and manufacturing platform blends the safety of food-based biotechnology with the ease of genetic manipulation, rapid growth rates and high productivity characteristic of microbial platforms. These features combine for exceptionally low-cost production of biopharmaceuticals to address medical needs that are unfeasible with current biotechnology platforms.

Background Clostridium difficile is the most common cause of hospital-acquired infections, responsible for >450000 infections annually in the USA. Probiotics provide a promising, well-tolerated adjunct therapy to standard C. difficile infection (CDI) treatment regimens, but there is a paucity of data regarding their effectiveness for the treatment of an initial CDI. Objectives We conducted a pilot randomized controlled trial of 33 participants from February 2013 to February 2015 to determine the feasibility and health outcomes of adjunct probiotic use in patients with an initial mild to moderate CDI. Methods The intervention was a 28 day, once-daily course of a four-strain oral probiotic capsule containing Lactobacillus acidophilus NCFM, Lactobacillus paracasei Lpc-37, Bifidobacterium lactis Bi-07 and B. lactis Bl-04. The control placebo was identical in taste and appearance. Registered at clinicaltrials.gov: trial registration number = NCT01680874. Results Probiotic adjunct therapy was associated with a significant improvement in diarrhoea outcomes. The primary duration of diarrhoea outcome (0.0 versus 1.0 days; P = 0.039) and two exploratory outcomes, total diarrhoea days (3.5 versus 12.0 days; P = 0.005) and rate of diarrhoea (0.1 versus 0.3 days of diarrhoea/stool diary days submitted; P = 0.009), all decreased in participants with probiotic use compared with placebo. There was no significant difference in the rate of CDI recurrence or functional improvement over time between treatment groups. Conclusions Probiotics are a promising adjunct therapy for treatment of an initial CDI and should be further explored in a larger randomized controlled trial.

Campylobacterinfections are among the leading bacterial causes of diarrhea and of ‘environmental enteropathy’ (EE) and growth failure worldwide. However, the lack of an inexpensive small animal model of enteric disease withCampylobacterhas been a major limitation for understanding its pathogenesis, interventions or vaccine development. We describe a robust standard mouse model that can exhibit reproducible bloody diarrhea or growth failure, depending on the zinc or protein deficient diet and on antibiotic alteration of normal microbiota prior to infection. Zinc deficiency and the use of antibiotics create a niche forCampylobacterinfection to establish by narrowing the metabolic flexibility of these mice for pathogen clearance and by promoting intestinal and systemic inflammation. Several biomarkers and intestinal pathology in this model also mimic those seen in human disease. This model provides a novel tool to testing specific hypotheses regarding disease pathogenesis as well as vaccine development that is currently in progress.Author SummaryCampylobacter jejunihas been identified as one of the leading causes of enteropathy and diarrhea. In developing countries, these repeated enteric infections often result in growth deficits and cognitive impairment. There is a lack of small animal models ofCampylobacterinfection. This is a major hurdle in understanding the pathogenesis ofCampylobacterinfection in order to lead to therapeutic treatments and vaccines. We have developed a highly reproducible mouse model ofCampylobacterinfection that has clinical outcomes that match those of malnourished children. We hope that these insights intoCampylobactersusceptibility will lead to the development of treatments against this major cause of diarrheal illness.

BACKGROUND: Environmental enteric dysfunction (EED) is widespread throughout the tropics and in children is associated with stunting and other adverse health outcomes. One of the hallmarks of EED is villus damage. In children with severe acute malnutrition (SAM) the severity of enteropathy is greater and short term mortality is high, but the metabolic consequences of enteropathy are unknown. Here, we characterize the urinary metabolic alterations associated with villus health, classic enteropathy biomarkers and anthropometric measurements in severely malnourished children in Zambia.METHODS/PRINCIPAL FINDINGS: We analysed 20 hospitalised children with acute malnutrition aged 6 to 23 months in Zambia. Small intestinal biopsies were assessed histologically (n = 15), anthropometric and gut function measurements were collected and the metabolic phenotypes were characterized by 1H nuclear magnetic resonance (NMR) spectroscopy. Endoscopy could not be performed on community controls children. Growth parameters were inversely correlated with enteropathy biomarkers (p = 0.011) and parameters of villus health were inversely correlated with translocation and permeability biomarkers (p = 0.000 and p = 0.015). Shorter villus height was associated with reduced abundance of metabolites related to gut microbial metabolism, energy and muscle metabolism (p = 0.034). Villus blunting was also related to increased sucrose excretion (p = 0.013).CONCLUSIONS/SIGNIFICANCE: Intestinal villus blunting is associated with several metabolic perturbations in hospitalized children with severe undernutrition. Such alterations include altered muscle metabolism, reinforcing the link between EED and growth faltering, and a disruption in the biochemical exchange between the gut microbiota and host. These findings extend our understanding on the downstream consequences of villus blunting and provide novel non-invasive biomarkers of enteropathy dysfunction. The major limitations of this study are the lack of comparative control group and gut microbiota characterization.

Molecular characterization of virulence and antimicrobial resistance profiles were determined for Shigella species isolated from children with diarrhea in Fortaleza, Brazil. Fecal specimens were collected along with socioeconomic and clinical data from children with moderate to severe diarrhea requiring emergency care. Shigella spp. were isolated by standard microbiological techniques, and we developed 4 multiplex polymerase chain reaction assays to detect 16 virulence-related genes (VRGs). Antimicrobial susceptibility tests were performed using disk diffusion assays. S. flexneri and S. sonnei were the predominant serogroups. S. flexneri was associated with low monthly incomes; more severe disease; higher number of VRGs; and presence of pic, set, and sepA genes. The SepA gene was associated with more intense abdominal pain. S. flexneri was correlated with resistance to ampicillin and chloramphenicol, whereas S. sonnei was associated with resistance to azithromycin. Strains harboring higher numbers of VRGs were associated with resistance to more antimicrobials. We highlight the correlation between presence of S. flexneri and sepA, and increased virulence and suggest a link to socioeconomic change in northeastern Brazil. Additionally, antimicrobial resistance was associated with serogroup specificity in Shigella spp. and increased bacterial VRGs.

Enteroaggregative Escherichia coli (EAEC) is a common cause of infectious diarrhea, especially in children living in poor-resource countries. In this article, we present a SYBR Green-based real-time polymerase chain reaction (qPCR) method for quantitative detection of EAEC in DNA directly extracted from human stool samples. To test the proposed qPCR system, we examined specificity, sensitivity, repeatability, and also the degree of DNA extraction efficiency using EAEC strain 042 spiked into EAEC-free stool sample. The specificity of this assay was proved using six strains of EAEC, seven strains of other E. coli types, and one strain of Shigella. The detection limit of qPCR was 67 CFU/reaction. In naturally infected stool samples, we found EAEC in quantities varying from 6.7 × 10(5) to 2 × 10(9 ) CFU/g of feces. We could not detect any reduction after stool DNA extraction for the amounts of 10(7) and 10(6) CFU/mL of spiked EAEC. This qPCR assay is simple, rapid, reproducible, sensitive, specific, and allows rapid EAEC quantification to be used in a variety of further EAEC studies. This new quantitative method provides a relatively simple means to quantify EAEC, which will likely be key to understanding the pathophysiology and impact of EAEC infection.

This study determined the prevalence of Campylobacter jejuni/coli and its relation with nutritional status in children from Northeastern Brazil. This was a case-control study design. Stool samples were evaluated for hipO (C. jejuni), ask (C. coli), and cdtABC (C. jejuni's cytolethal distending toxin) genes. The nutritional status from these children was assessed by anthropometric measures and z-scores. C. jejuni and C. coli were detected in 9.6% (8/83) and 6.0% (5/83) in the diarrhea group and in 7.2% (6/83) and 1.2% (1/83) of the nondiarrhea group, respectively. Children with positive molecular detection of C. jejuni showed significantly lower z-scores than children without C. jejuni. The cdtABC operon was found in 57% of hipO(+) samples. C. jejuni/coli prevalence was similar in diarrhea and nondiarrhea groups. There was a significant association of C. jejuni infection with lower nutritional status.

Studies of microbial pathogens and the toxins they produce are important for determining the mechanisms by which they cause disease and spread throughout a population. Some bacteria produce secretory enterotoxins (such as cholera toxin or the heat-labile or stable enterotoxins produced by Escherichia coli) that invade cells directly. Others invade cells or produce cytotoxins (such as those produced by Shigella, enteroinvasive E coli, or Clostridium difficile) that damage cells or trigger host responses that cause small or large bowel diseases (such as enteroaggregative or enteropathogenic E coli or Salmonella). Viruses (such as noroviruses and rotaviruses) and protozoa (such as Cryptosporidium, Giardia, or Entamoeba histolytica) disrupt cell functions and cause short- or long-term disease. Much epidemiologic data about these pathogens have been collected from community- and hospital-acquired settings, as well as from patients with traveler's or persistent diarrhea. These studies have led to practical approaches for prevention, diagnosis, and treatment.

“Norwalk-like viruses” (NLVs) are a common cause of epidemic gastroenteritis in adults and children in developed countries. However, little is known about the role of NLVs in endemic pediatric gastroenteritis in developing countries. We sequenced Genogroup I and II NLV reverse transcription-polymerase chain reaction (RT-PCR) products from an 81-nucleotide region of the viral RNA polymerase gene to examine the molecular epidemiology of NLV infection in children younger than 5 years of age in Forteleza, Ceara, Brazil. NLV-positive PCR products were obtained from stool specimens collected over a 16-month period (1990–1991) from diarrhea cases and controls in a cohort of 120 children in an urban shantytown and from a study in the same city of hospitalized children with persistent diarrhea. Eight unique strains were detected in 15 specimens from 10 cohort children and in two hospital specimens. Nucleotide identity between the strains (5 Genogroup I, 3 Genogroup II) ranged from 63% to 88%. We designated these strains BraV1–8, for Brazil virus 1–8. The degree of genomic diversity of NLV strains we identified in this cohort during a short time period suggests multiple foci of infection within the community. Furthermore, sequence analysis of strains from two children with multiple symptomatic NLV infections indicates that infection with one strain was not protective against subsequent infection with a different strain in the same genogroup. These findings have implications for vaccine development and the prevention of pediatric gastroenteritis in developing countries. J. Med. Virol. 58:426–434, 1999. © 1999 Wiley-Liss, Inc.

Enteroaggregative Escherichia coli (EAEC) is an important agent that causes endemic and epidemic diarrhoeal diseases worldwide. Several EAEC virulence-related genes (VRGs) have been described but their role in the clinical outcome of infection is not completely defined. This study investigated the prevalence of EAEC and potential associations of its VRGs with risk of or protection from diarrhoeal diseases in children from urban communities in north-eastern Brazil. The case–control study included 166 children, who had their stools evaluated for the EAEC diagnostic genes (aaiC and aatA) using PCR. Positive samples were further analysed by multiplex PCR and identified 18 VRGs. EAEC was found in the same proportion in both groups (41 %). The plasmid-borne gene encoding a hexosyltransferase homologue (capU) was the most frequently detected (89.6 %), followed by dispersin protein (aap, 58.2 %) and EAEC HilA homologue (eilA, 57.8 %). The AAF/III fimbrial subunit (agg3A) gene was observed at lower frequency (1.5 %). Plasmid-encoded toxin (pet) or AAF/II fimbrial subunit (aafA) was associated significantly with disease. AAF/IV fimbrial subunit (agg4A) or hypothetical plasmid-encoded haemolysin (orf61) was detected significantly more in controls than in children with diarrhoea. In addition, one set of genes in combination, aaiC and agg3/4C but lacking agg4A and orf61, was associated with diarrhoea cases; and another one, orf61 in the absence of pet and aafA, was correlated with control children. These data confirm a high prevalence, endemicity and heterogeneity of EAEC strains in the developing urban areas of north-eastern Brazil. Statistical correlation between cases and controls was seen with either isolated or combined sets of genes, suggesting that the pathophysiology of EAEC infection involves a complex and dynamic modulation of several VRGs.

Toxin A (TxA) is able to induce most of the classical features of Clostridium difficile -associated disease in animal models. The objective of this study was to determine the effect of an inhibitor of adenosine deaminase, EHNA [erythro-9-(2-hydroxy-3-nonyl)-adenine], on TxA-induced enteritis in C57BL6 mice and on the gene expression of adenosine receptors. EHNA (90 μmol/kg) or phosphate-buffered saline (PBS) was injected intraperitoneally (i.p.) 30 min prior to TxA (50 μg) or PBS injection into the ileal loop. A 2A adenosine receptor agonist (ATL313; 5 nM) was injected in the ileal loop immediately before TxA (50 μg) in mice pretreated with EHNA. The animals were euthanized 3 h later. The changes in the tissue were assessed by the evaluation of ileal loop weight/length and secretion volume/length ratios, histological analysis, myeloperoxidase assay (MPO), the local expression of inducible nitric oxide synthase (NOS2), pentraxin 3 (PTX3), NF-κB, tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β) by immunohistochemistry and/or quantitative reverse transcription-PCR (qRT-PCR). The gene expression profiles of A 1 , A 2A , A 2B , and A 3 adenosine receptors also were evaluated by qRT-PCR. Adenosine deaminase inhibition, by EHNA, reduced tissue injury, neutrophil infiltration, and the levels of proinflammatory cytokines (TNF-α and IL-1β) as well as the expression of NOS2, NF-κB, and PTX3 in the ileum of mice injected with TxA. ATL313 had no additional effect on EHNA action. TxA increased the gene expression of A 1 and A 2A adenosine receptors. Our findings show that the inhibition of adenosine deaminase by EHNA can prevent Clostridium difficile TxA-induced damage and inflammation possibly through the A 2A adenosine receptor, suggesting that the modulation of adenosine/adenosine deaminase represents an important tool in the management of C. difficile- induced disease.

Introduction Long-term care facilities (LTCF) residents have been estimated to have the highest incidence of diarrheal illness among adults living in the developed world. This study describes undiagnosed diarrhea, intestinal inflammation, and Clostridium difficile colonization in a LTC population and explores whether these are associated with functional decline, as defined by weight loss or a change in cognitive or ADL status. Methods An observational study of a convenience sampling of residents in a 180-bed LTCF was obtained; evaluation of stool and medical records was done. Stool specimens were evaluated for consistency, gross blood, inflammation (via quantitative fecal lactoferrin, IBD-SCAN), and C difficile (via PCR for gdh). SPSS and STATA were used and significance was set at P Results There were 46 stools collected; 13 of the subjects were male, 28 were older than 65 years, and 35 were prescribed 5 to 15 medications. Twenty-six of the 46 stools collected had elevated quantitative fecal lactoferrin levels. Although only 5 subjects were reported to have diarrhea (4 with elevated lactoferrin), 28 stool specimens were observed to be liquid or semi-solid (19 with elevated lactoferrin), and these liquid/ semisolid stools were significantly correlated with lactoferrin positivity (P = .017). In analysis of functional status, there was no statistically significant association between change in ADL (n = 17) or cognitive status (n = 5) and elevated lactoferrin. However, all 3 subjects who had significant weight loss had elevated lactoferrin, although the mean fecal lactoferrin was not statistically different from those without weight loss. Of the 2 samples with C difficile, both were liquid and, when compared with all other liquid stools (n = 22), the mean lactoferrin was statistically higher (134.1 versus 28.8 μg/mL, P = .008). These 2 subjects had neither weight loss nor change in cognitive status, but 1 had a change in ADL status. Discussion and conclusions Diarrhea in LTCF residents is underdiagnosed. Diarrhea and the presence of C difficile in the stool are associated with intestinal inflammation, as detected by fecal lactoferrin. With our small numbers, we were not able to identify a specific link; however, we were able to identify a correlation between weight loss and intestinal inflammation, but, with just 2 samples, not C difficile colonization. This relationship highlights the importance of larger studies to further examine the rate of diarrhea in LTCF; the effect of diarrhea and intestinal inflammation on weight loss; and the interaction of C difficile colonization with weight loss, malnutrition, and functional decline.

Immunocompromised patients are susceptible to a variety of serious gastrointestinal infections. Among the most common or most severe are Mycobacterium avium-intracellulare, herpes simplex, candidiasis, cryptosporidiosis, and Salmonellosis. Some of these infections are difficult or impossible to treat successfully. The correct diagnosis often can only be established if it is first suspected and the appropriate studies are performed.