Research in a time of enteroids and organoids: how the human gut model has transformed the study of enteric bacterial pathogens

ABSTRACT

Enteric bacterial pathogens cause significant morbidity and mortality globally. Studies in tissue culture and animal models shaped our initial understanding of these host–pathogen interactions. However, intrinsic shortcomings in these models limit their application, especially in translational applications like drug screening and vaccine development. Human intestinal enteroid and organoid models overcome some limitations of existing models and advance the study of enteric pathogens. In this review, we detail the use of human enteroids and organoids to investigate the pathogenesis of invasive bacteria Shigella, Listeria, and Salmonella, and noninvasive bacteria pathogenic Escherichia coli, Clostridium difficile, and Vibrio cholerae. We highlight how these studies confirm previously identified mechanisms and, importantly, reveal novel ones. We also discuss the challenges for model advancement, including platform engineering to integrate environmental conditions, innate immune cells and the resident microbiome, and the potential for pre-clinical testing of recently developed antimicrobial drugs and vaccines.     

Vaccine development for bacterial pathogens: Advances,challenges and prospects

The advent and use of antimicrobials have played a key role in treating potentially life-threatening infectious diseases, improving health, and saving the lives of millions of people worldwide. However, the emergence of multidrug resistant (MDR) pathogens has been a significant health challenge that has compromised the ability to prevent and treat a wide range of infectious diseases that were once treatable. Vaccines offer potential as a promising alternative to fight against antimicrobial resistance (AMR) infectious diseases. Vaccine technologies include reverse vaccinology, structural biology methods, nucleic acid (DNA and mRNA) vaccines, generalised modules for membrane antigens, bioconjugates/glycoconjugates, nanomaterials and several other emerging technological advances that are offering a potential breakthrough in the development of efficient vaccines against pathogens. This review covers the opportunities and advancements in vaccine discovery and development targeting bacterial pathogens. We reflect on the impact of the already-developed vaccines targeting bacterial pathogens and the potential of those currently under different stages of preclinical and clinical trials. More importantly, we critically and comprehensively analyse the challenges while highlighting the key indices for future vaccine prospects. Finally, the issues and concerns of AMR for low-income countries (sub-Saharan Africa) and the challenges with vaccine integration, discovery and development in this region are critically evaluated.     

Dual role of phagocytic NADPH oxidase in bacterial killing

The classical model of bacterial killing by phagocytic cells has been recently challenged by questioning the toxic effect of oxygen products and attributing the fundamental role to K(+) ions in releasing antimicrobial proteins within the phagosome. In the present study we followed O(2)(*-) production, changes of membrane potential, K(+) efflux, and bacterial killing in the presence of increasing concentrations of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium. Efficiency of bacterial killing was assessed on the basis of bacterial survival measured by a new semiautomated method. Very low rates of O(2)(*-) production were accompanied by significant membrane depolarization and K(+) release and parallel improvement of bacterial killing. When O(2)(*-) production exceeded 20% of its maximal capacity, no further change was detected in the membrane potential and only minimal further K(+) efflux occurred, yet bacterial survival decreased parallel to the increase of O(2)(*-) production. The presented results indicate that both electrophysiological changes (depolarization and consequent ion movements) and the chemical effect of reactive oxygen species play a significant role in the killing of certain pathogens. The observation that an increase of membrane depolarization can compensate for decreased O(2)(*-) production may be important for potential therapeutic applications.     

The potential of human neutrophil peptides in tuberculosis therapy.

The problems of drug resistance and bacterial persistence in tuberculosis have prompted scientists to search for clues from the latest advances in microbiology and immunology. Recent research on human neutrophil peptides (HNPs) has highlighted their bactericidal action against Mycobacterium tuberculosis and suggested that neutrophils may play a more important defensive role in tuberculosis than previously thought. Human neutrophil peptides belong to a family of antimicrobial and cytotoxic peptides known as 'defensins'. Neutrophils use both oxidative and non-oxidative microbicidal mechanisms to provide the host with innate immunity against microbial infections. Defensins are most abundant among an array of oxygen-independent antimicrobial proteins and peptides in neutrophil granules. Defensins are effective against a wide spectrum of microbes including bacteria, viruses, fungi, spirochetes and mycobacteria. In addition to direct antimicrobial activity, HNPs can potentially influence the inflammatory or immune responses by modulating cytokine production or acting like opsonins or chemotactic factors. HNPs are active against M. tuberculosis grown in vitro or within macrophages. HNPs released by neutrophils recruited in the early lesion could attract monocytes to the site and macrophages may in vivo uptake the extracellular HNPs and kill the intracellular pathogens. As such, HNPs are potential therapeutic agents against tuberculosis. HNPs are also cytotoxic against a wide range of normal mammalian cells; however, there is evidence that defensins may not cause significant cytotoxicity at the therapeutic level. Finally, the clinical application of HNPs must be evaluated in the context of possible drug resistance, as some resistance-associated genes have been identified.     

Rifaximin--a novel antimicrobial for enteric infections

Rifaximin is a poorly absorbed rifamycin antimicrobial drug with in vitro activity against Gram-positive, Gram-negative and anaerobic bacteria. The minimal concentration that inhibits 90% of strains of bacterial pathogens (MIC90) ranges between 32 and 64 microg/ml. Less than 1% of the drug is absorbed after oral administration. After three days of therapy, the average fecal level of this drug is 8000 microg/g of stool. Selection of resistant mutants, a problem with the related rifampin, appears to be unusual with rifaximin. Rifaximin shortens the duration of travelers' diarrhea and non-dysenteric diarrheal illness due to enterotoxigenic, enteroaggregative E. coli and Shigella sonnei without major alteration of aerobic fecal flora and without important side effects. The drug has been successfully used in preliminary studies of small bowel bacterial overgrowth syndrome and hepatic encephalopathy. To explain the beneficial effect of the drug on bacterial diarrhea without change in colonic flora or high rates of pathogen eradication, rifaximin may be more active against pathogens in the small bowel rather than the colon and/or the drug may alter the virulence of enteric pathogens in addition to organism inhibition.     

The syntaxin SYP132 contributes to plant resistance against bacteria and secretion of pathogenesis-related protein 1

In contrast to many mammalian pathogens, potential bacterial pathogens of plants remain outside the host cell. The plant must, therefore, promote an active resistance mechanism to combat the extracellular infection. How this resistance against bacteria is manifested and whether similar processes mediate basal, gene-for-gene, and salicylate-associated defense, however, are poorly understood. Here, we identify a specific plasma membrane syntaxin, NbSYP132, as a component contributing to gene-for-gene resistance in Nicotiana benthamiana. Silencing NbSYP132 but not NbSYP121, the apparent orthologue of a syntaxin required for resistance to powdery mildew fungus, compromised AvrPto-Pto resistance. Because syntaxins may play a role in secretion of proteins to the extracellular space, we performed a limited proteomic analysis of the apoplastic fluid. We found that NbSYP132-silenced plants were impaired in the accumulation of at least a subset of pathogenesis-related (PR) proteins in the cell wall. These results were confirmed by both immunoblot analysis and imunolocalization of a PR protein, PR1a. These results implicate NbSYP132 as the cognate target soluble N-ethylmaleimide-sensitive factor attachment protein receptor for exocytosis of vesicles containing antimicrobial PR proteins. NbSYP132 also contributes to basal and salicylate-associated defense, indicating that SYP132-dependent secretion is a component of multiple forms of defense against bacterial pathogens in plants.     

Killing of African trypanosomes by antimicrobial peptides

Antimicrobial peptides are components of the innate immune systems of a wide variety of eukaryotic organisms and are being developed as antibiotics in the fight against bacterial and fungal infections. We explored the potential activities of antimicrobial peptides against the African trypanosome Trypanosoma brucei, a vector-borne protozoan parasite that is responsible for significant morbidity and mortality in both humans and animals. Three classes of mammalian antimicrobial peptides were tested: alpha-defensins, beta-defensins, and cathelicidins. Although members of all 3 classes of antimicrobial peptides showed activity, those derived from the cathelicidin class were most effective, killing both insect and bloodstream forms of the parasite. The mechanism of action of the cathelicidins against T. brucei involves disruption of surface membrane integrity. Administration of cathelicidin antimicrobial peptides to mice with late-stage T. brucei infection acutely decreased parasitemia and prolonged survival. These results highlight the potential use of antimicrobial peptides for the treatment of African trypanosomiasis.     

Additives Imparting Antimicrobial Properties to Acrylic Bone Cements

PMMA bone cements are mainly used to fix implanted prostheses and are introduced as a fluid mixture, which hardens over time. The problem of infected prosthesis could be solved due to the development of some new antibacterial bone cements. In this paper, we show the results obtained to develop four different modified PMMA bone cements by using antimicrobial additives, such as gentamicin, peppermint oil incorporated in hydroxyapatite, and silver nanoparticles incorporated in a ceramic glass matrix (2 and 4%). The structure and morphology of the modified bone cements were investigated by SEM and EDS. We perform experimental measurements on wettability, hydration degree, and degradation degree after immersion in simulated body fluid. The cytotoxicity was evaluated by MTT assay using the human MG-63 cell line. Antimicrobial properties were checked against standard strains Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. The addition of antimicrobial agents did not significantly affect the hydration and degradation degree. In terms of biocompatibility assessed by the MTT test, all experimental PMMA bone cements are biocompatible. The performance of bone cements with peppermint essential oil and silver nanoparticles against these two pathogens suggests that these antibacterial additives look promising to be used in clinical practice against bacterial infection.     

Mechanism of a prototypical synthetic membrane-active antimicrobial: Efficient hole-punching via interaction with negative intrinsic curvature lipids

Phenylene ethynylenes comprise a prototypical class of synthetic antimicrobial compounds that mimic antimicrobial peptides produced by eukaryotes and have broad-spectrum antimicrobial activity. We show unambiguously that bacterial membrane permeation by these antimicrobials depends on the presence of negative intrinsic curvature lipids, such as phosphatidylethanolamine (PE) lipids, found in high concentrations within bacterial membranes. Plate-killing assays indicate that a PE-knockout mutant strain of Escherichia coli drastically out-survives the wild type against the membrane-active phenylene ethynylene antimicrobials, whereas the opposite is true when challenged with traditional metabolic antibiotics. That the PE deletion is a lethal mutation in normative environments suggests that resistant bacterial strains do not evolve because a lethal mutation is required to gain immunity. PE lipids allow efficient generation of negative curvature required for the circumferential barrel of an induced membrane pore; an inverted hexagonal H_II phase, which consists of arrays of water channels, is induced by a small number of antimicrobial molecules. The estimated antimicrobial occupation in these water channels is nonlinear and jumps from ≈1 to 3 per 4 nm of induced water channel length as the global antimicrobial concentration is increased. By comparing to exactly solvable 1D spin models for magnetic systems, we quantify the cooperativity of these antimicrobials.     

Gammadelta intraepithelial lymphocytes are essential mediators of host-microbial homeostasis at the intestinal mucosal surface

The mammalian gastrointestinal tract harbors thousands of bacterial species that include symbionts as well as potential pathogens. The immune responses that limit access of these bacteria to underlying tissue remain poorly defined. Here we show that γδ intraepithelial lymphocytes (γδ IEL) of the small intestine produce innate antimicrobial factors in response to resident bacterial "pathobionts" that penetrate the intestinal epithelium. γδ IEL activation was dependent on epithelial cell-intrinsic MyD88, suggesting that epithelial cells supply microbe-dependent cues to γδ IEL. Finally, γδ T cells protect against invasion of intestinal tissues by resident bacteria specifically during the first few hours after bacterial encounter, indicating that γδ IEL occupy a unique temporal niche among intestinal immune defenses. Thus, γδ IEL detect the presence of invading bacteria through cross-talk with neighboring epithelial cells and are an essential component of the hierarchy of immune defenses that maintain homeostasis with the intestinal microbiota.     

Direct,Rapid Detection of Pathogens from Urine Samples

The problem of rapidly detecting pathogens directly from clinical samples poses significant analytical challenges. Addressing this issue in relation to urinary tract infections, we propose an effective protocol and related immunomagnetic test kits enabling versatile screening for the presence of pathogenic bacteria in unprocessed urine samples. To achieve this, the components of a typical immunomagnetic separation protocol were optimized towards the sensitive assessment of the aggregates formed out of immunomagnetically tagged target pathogens collected from clinical samples. Specifically, a dedicated immunomagnetic material was developed via the functionalization of standardized, micron-sized magnetic beads with generic antibodies against gram-specific bacterial constituents with mannan binding lectin. As such, we demonstrate efficient procedures for achieving the enhanced, specific, and pathogen-mediated cluster formation of these tailored affinity-coated magnetic beads in complex samples. We further show how cluster analysis, in conjunction with the use of nonspecific, inexpensive fluorescent dye, allows for a straightforward optical assessment of the bacterial load directly from urine samples. The optimized sensing protocol and related kits provide, in less than 60 min, qualitative (positive/negative) information on the bacterial load with 85% specificity and 96% sensitivity, which is appropriate to empower clinical microscopy with a new analytic dimension. The procedure is prone to automation, can be conveniently used in clinical microbiology laboratories and, since it preserves the viability of the captured bacteria, can be interfaced with downstream analyses and antimicrobial susceptibility testing. Moreover, the study emphasizes a suite of practical validation assays that are useful for bringing the tool-box of immunomagnetic materials outside the academic laboratory and into real-life applications.     

Resistance to trimethoprim-sulfamethoxazole.

Sulfonamides have a glorious history. In 1935, they were the first class of true antimicrobial agents with life-saving potency. Today, 66 years later, increased bacterial resistance to sulfonamides and to trimethoprim (TMP), a synthetic antimicrobial agent that is 30 years younger than sulfonamides, has limited their use to only a few indications. In the treatment and prophylaxis of patients with urinary tract infections, trimethoprim-sulfamethoxazole (TMP-SMZ) or TMP alone is still considered the first-line drug of choice, although increased bacterial resistance to these agents has been linked with treatment failure. TMP-SMZ has a possible role as a second- or third-line treatment for patients who have respiratory tract infections. In the developing world, where this inexpensive drug is widely used as first-line treatment, bacterial resistance has caused problems, especially with regard to the treatment of patients with severe respiratory tract infections. Use of TMP-SMZ as prophylaxis for Pneumocystis carinii infection has rapidly increased the multidrug resistance of bacterial pathogens found in human immunodeficiency virus-infected patients. Today, detailed and reliable knowledge on the resistance of bacterial pathogens to both TMP-SMZ and TMP is an essential requirement for the safe and effective use of these drugs in all clinical settings.     

Membrane potential is important for bacterial cell division

Many cell division-related proteins are located at specific positions in the bacterial cell, and this organized distribution of proteins requires energy. Here, we report that the proton motive force, or more specifically the (trans)membrane potential, is directly involved in protein localization. It emerged that the membrane potential modulates the distribution of several conserved cell division proteins such as MinD, FtsA, and the bacterial cytoskeletal protein MreB. We show for MinD that this is based on the membrane potential stimulated binding of its C-terminal amphipathic helix. This function of the membrane potential has implications for how these morphogenetic proteins work and provide an explanation for the effects observed with certain antimicrobial compounds.     

Complexation of peptidoglycan intermediates by the lipoglycodepsipeptide antibiotic ramoplanin: minimal structural requirements for intermolecular complexation and fibril formation

The peptide antibiotic ramoplanin inhibits bacterial peptidoglycan (PG) biosynthesis by interrupting late-stage membrane-associated glycosyltransferase reactions catalyzed by the transglycosylase and MurG enzymes. The mechanism of ramoplanin involves sequestration of lipid-anchored PG biosynthesis intermediates, physically occluding these substrates from proper utilization by these enzymes. In this report, we describe the first molecular-level details of the interaction of ramoplanin with PG biosynthesis intermediates. NMR analysis in conjunction with chemical dissection of the PG monomer revealed that the ramoplanin octapeptide D-Hpg-D-Orn-D-alloThr-Hpg-D-Hpg-alloThr-Phe-D-Orn recognizes MurNAc-Ala-gamma-D-Glu pyrophosphate, the minimum component of PG capable of high-affinity complexation and fibril formation. Ramoplanin therefore recognizes a PG binding locus different from the N-acyl-D-Ala-D-Ala moiety targeted by vancomycin. Because ramoplanin is structurally less complex than glycopeptide antibiotics such as vancomycin, peptidomimetic chemotherapeutics derived from this recognition sequence may find future use as antibiotics against vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, and related pathogens.     

Long-term prophylaxis of infection by selective decontamination in leukopenia and in mechanical ventilation

The autochthonous anaerobic bacterial flora in the mucosal layer of the gastrointestinal tract limits colonization by aerobic potential pathogens. This effect is called colonization resistance. Colonization of the digestive tract by potentially pathogenic microorganisms precedes infection in patients with leukopenia and in cases of mechanical ventilation and can be prevented by long-term administration of antimicrobial agents that spare the autochthonous anaerobic bacterial flora of the mucous membranes, a concept known as selective decontamination. Antimicrobial agents active against anaerobic flora reduce colonization resistance, permitting colonization by and overgrowth of potentially pathogenic microorganisms and possibly leading to infections with resistant microorganisms. A distinction can be made between antimicrobial agents that reduce colonization resistance and those that leave it intact by examining the effect of antimicrobial agents on aerobic intestinal flora of mice and humans.     

Targeting the host hemostatic system function in bacterial infection for antimicrobial therapies

The hemostatic system is an important player in host’s response to infection. It has been shown that host hemostatic factors as well as platelets, interact with various proteins from bacteria and play important roles in host defense against infections. This review summarizes studies of function of host hemostatic system in host defense against bacterial infections and efforts to target hemostatic system interaction with pathogens to develop potential antimicrobial therapies.     

呼吸机相关性肺炎病原学分析

目的 研究重症监护病房(intensive care unit,ICU)机械通气引发呼吸机相关性肺炎(ventilator associated pneumonia,VAP)院内感染病原体的构成及耐药情况.方法 对2006年1月至2007年12月我院ICU收治的96例机械通气引发的VAP患者的感染菌及其耐药情况进行回顾性调查分析.结果共检出感染菌161株,其中革兰阴性杆菌104株(64.6%),革兰阳性球菌32株(19.9%),真菌25株(15.5%),药敏结果 显示菌株多重耐药现象严重.结论 ICU机械通气VAP院内感染病原体以革兰阴性杆菌为主,药敏试验为多重耐药,l临床应重视ICU机械通气引发的院内感染,重视病原菌的培养鉴定,合理使用抗生素.     

Bacteriophages as an Alternative Method for Control of Zoonotic and Foodborne Pathogens

The global increase in multidrug-resistant infections caused by various pathogens has raised concerns in human and veterinary medicine. This has renewed interest in the development of alternative methods to antibiotics, including the use of bacteriophages for controlling bacterial infections. The aim of this review is to present potential uses of bacteriophages as an alternative to antibiotics in the control of bacterial infections caused by multidrug-resistant bacteria posing a risk to humans, with particular emphasis on foodborne and zoonotic pathogens. A varied therapeutic and immunomodulatory (activation or suppression) effect of bacteriophages on humoral and cellular immune response mechanisms has been demonstrated. The antibiotic resistance crisis caused by global antimicrobial resistance among bacteria creates a compelling need for alternative safe and selectively effective antibacterial agents. Bacteriophages have many properties indicating their potential suitability as therapeutic and/or prophylactic agents. In many cases, bacteriophages can also be used in food quality control against microorganisms such as Salmonella, Escherichia coli, Listeria, Campylobacter and others. Future research will provide potential alternative solutions using bacteriophages to treat infections caused by multidrug-resistant bacteria.     

Antimicrobial resistance and bacterial virulence

Hospitals are places with high selective pressure by antimicrobial agents. For this reason, bacteria producing nosocomial infections need to be not only virulent, but also resistant to antimicrobial agents. In the present review we analyse the effect of the acquisition of an antibiotic resistance phenotype in bacterial fitness and virulence. Besides that, we review as well the existence of common mechanisms for resistance to antimicrobial agents and bacterial virulence. In this line, we highlight the role of multidrug efflux pumps on bacterial virulence. Since opportunistic pathogens frequently have an environmental origin, we also discuss the role of natural ecosystems, as well as their potential contamination, on the selection of bacteria resistant to antimicrobial agents.