Gut microbiota promotes stem cell differentiation through macrophage and mesenchymal niches in early postnatal development.

Intestinal stem cell maturation and development coincide with gut microbiota exposure after birth. Here, we investigated how early life microbial exposure, and disruption of this process, impacts the intestinal stem cell niche and development. Single-cell transcriptional analysis revealed impaired stem cell differentiation into Paneth cells and macrophage specification upon antibiotic treatment in early life. Mouse genetic and organoid co-culture experiments demonstrated that a CD206+ subset of intestinal macrophages secreted Wnt ligands, which maintained the mesenchymal niche cells important for Paneth cell differentiation. Antibiotics and reduced numbers of Paneth cells are associated with the deadly infant disease, necrotizing enterocolitis (NEC). We showed that colonization with Lactobacillus or transfer of CD206+ macrophages promoted Paneth cell differentiation and reduced NEC severity. Together, our work defines the gut microbiota-mediated regulation of stem cell niches during early postnatal development.

Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies.

Whether the human fetus and the prenatal intrauterine environment (amniotic fluid and placenta) are stably colonized by microbial communities in a healthy pregnancy remains a subject of debate. Here we evaluate recent studies that characterized microbial populations in human fetuses from the perspectives of reproductive biology, microbial ecology, bioinformatics, immunology, clinical microbiology and gnotobiology, and assess possible mechanisms by which the fetus might interact with microorganisms. Our analysis indicates that the detected microbial signals are likely the result of contamination during the clinical procedures to obtain fetal samples or during DNA extraction and DNA sequencing. Furthermore, the existence of live and replicating microbial populations in healthy fetal tissues is not compatible with fundamental concepts of immunology, clinical microbiology and the derivation of germ-free mammals. These conclusions are important to our understanding of human immune development and illustrate common pitfalls in the microbial analyses of many other low-biomass environments. The pursuit of a fetal microbiome serves as a cautionary example of the challenges of sequence-based microbiome studies when biomass is low or absent, and emphasizes the need for a trans-disciplinary approach that goes beyond contamination controls by also incorporating biological, ecological and mechanistic concepts.

Deep learning-guided discovery of an antibiotic targeting Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial Gram-negative pathogen that often displays multidrug resistance. Discovering new antibiotics against A. baumannii has proven challenging through conventional screening approaches. Fortunately, machine learning methods allow for the rapid exploration of chemical space, increasing the probability of discovering new antibacterial molecules. Here we screened ~7,500 molecules for those that inhibited the growth of A. baumannii in vitro. We trained a neural network with this growth inhibition dataset and performed in silico predictions for structurally new molecules with activity against A. baumannii. Through this approach, we discovered abaucin, an antibacterial compound with narrow-spectrum activity against A. baumannii. Further investigations revealed that abaucin perturbs lipoprotein trafficking through a mechanism involving LolE. Moreover, abaucin could control an A. baumannii infection in a mouse wound model. This work highlights the utility of machine learning in antibiotic discovery and describes a promising lead with targeted activity against a challenging Gram-negative pathogen.

Human milk oligosaccharides promote intestinal epithelium regeneration independent of the microbiota during necrotizing enterocolitis.

PURPOSE: Necrotizing enterocolitis (NEC) is a severe intestinal disease primarily affecting premature infants, marked by impaired epithelial regeneration. Breastfed infants are less susceptible to NEC than formula-fed ones, and human milk oligosaccharides (HMO) found in breast milk have prebiotic properties that can protect against NEC. However, it is unclear how HMOs influence intestinal epithelium regeneration in relation to the gut microbiota. METHODS: Broad-spectrum antibiotics were administered to pregnant dams to reduce the microbiota in offspring. NEC was induced through administration of hyperosmolar formula, lipopolysaccharide, and hypoxia from postnatal days (p) 5-9. Intestinal epithelial organoids were derived from p9 mice. HMOs were isolated from human donor breast milk and then solubilized in the formula for each feed or culture media for organoids. RESULTS: HMOs did not alter the microbiota profile in the presence of a normal or reduced microbiota. In the reduced microbiota, HMO treatment decreased NEC intestinal injury, and increased proliferation and stem cell activity. Additionally, in the complete absence of the microbiota, HMOs stimulated intestinal organoid growth. CONCLUSION: This study demonstrates that HMOs promoted intestinal epithelial regeneration independent of the gut microbiota. These findings provide further insight into the various benefits HMOs may have in the protection against NEC.

Human gut microbiota after bariatric surgery alters intestinal morphology and glucose absorption in mice independently of obesity.

OBJECTIVE: Bariatric surgery is an effective treatment for type 2 diabetes (T2D) that changes gut microbial composition. We determined whether the gut microbiota in humans after restrictive or malabsorptive bariatric surgery was sufficient to lower blood glucose. DESIGN: Women with obesity and T2D had biliopancreatic diversion with duodenal switch (BPD-DS) or laparoscopic sleeve gastrectomy (LSG). Faecal samples from the same patient before and after each surgery were used to colonise rodents, and determinants of blood glucose control were assessed. RESULTS: Glucose tolerance was improved in germ-free mice orally colonised for 7 weeks with human microbiota after either BPD-DS or LSG, whereas food intake, fat mass, insulin resistance, secretion and clearance were unchanged. Mice colonised with microbiota post-BPD-DS had lower villus height/width and crypt depth in the distal jejunum and lower intestinal glucose absorption. Inhibition of sodium-glucose cotransporter (Sglt)1 abrogated microbiota-transmissible improvements in blood glucose control in mice. In specific pathogen-free (SPF) rats, intrajejunal colonisation for 4 weeks with microbiota post-BPD-DS was sufficient to improve blood glucose control, which was negated after intrajejunal Sglt-1 inhibition. Higher Parabacteroides and lower Blautia coincided with improvements in blood glucose control after colonisation with human bacteria post-BPD-DS and LSG. CONCLUSION: Exposure of rodents to human gut microbiota after restrictive or malabsorptive bariatric surgery improves glycaemic control. The gut microbiota after bariatric surgery is a standalone factor that alters upper gut intestinal morphology and lowers Sglt1-mediated intestinal glucose absorption, which improves blood glucose control independently from changes in obesity, insulin or insulin resistance.

Sensitivity to Neutralizing Antibodies and Resistance to Type I Interferons in SARS-CoV-2 R.1 Lineage Variants, Canada.

Isolating and characterizing emerging SARS-CoV-2 variants is key to understanding virus pathogenesis. In this study, we isolated samples of the SARS-CoV-2 R.1 lineage, categorized as a variant under monitoring by the World Health Organization, and evaluated their sensitivity to neutralizing antibodies and type I interferons. We used convalescent serum samples from persons in Canada infected either with ancestral virus (wave 1) or the B.1.1.7 (Alpha) variant of concern (wave 3) for testing neutralization sensitivity. The R.1 isolates were potently neutralized by both the wave 1 and wave 3 convalescent serum samples, unlike the B.1.351 (Beta) variant of concern. Of note, the R.1 variant was significantly more resistant to type I interferons (IFN-α/ß) than was the ancestral isolate. Our study demonstrates that the R.1 variant retained sensitivity to neutralizing antibodies but evolved resistance to type I interferons. This critical driving force will influence the trajectory of the pandemic.

Biogeographic Variation and Functional Pathways of the Gut Microbiota in Celiac Disease.

BACKGROUND & AIMS: Genes and gluten are necessary but insufficient to cause celiac disease (CeD). Altered gut microbiota has been implicated as an additional risk factor. Variability in sampling site may confound interpretation and mechanistic insight, as CeD primarily affects the small intestine. Thus, we characterized CeD microbiota along the duodenum and in feces and verified functional impact in gnotobiotic mice. METHODS: We used 16S rRNA gene sequencing (Illumina) and predicted gene function (PICRUSt2) in duodenal biopsies (D1, D2 and D3), aspirates, and stool from patients with active CeD and controls. CeD alleles were determined in consented participants. A subset of duodenal samples stratified according to similar CeD risk genotypes (controls DQ2-/- or DQ2+/- and CeD DQ2+/-) were used for further analysis and to colonize germ-free mice for gluten metabolism studies. RESULTS: Microbiota composition and predicted function in CeD was largely determined by intestinal location. In the duodenum, but not stool, there was higher abundance of Escherichia coli (D1), Prevotella salivae (D2), and Neisseria (D3) in CeD vs controls. Predicted bacterial protease and peptidase genes were altered in CeD and impaired gluten degradation was detected only in mice colonized with CeD microbiota. CONCLUSIONS: Our results showed luminal and mucosal microbial niches along the gut in CeD. We identified novel microbial proteolytic pathways involved in gluten detoxification that are impaired in CeD but not in controls carrying DQ2, suggesting an association with active duodenal inflammation. Sampling site should be considered a confounding factor in microbiome studies in CeD.

Fecal prevalence of Clostridium innocuum DNA in healthy horses and horses with colitis.

This study compared the prevalence of C. innocuum DNA in the feces of healthy horses and horses with acute colitis. C. innocuum was identified in 22% (15/68) of colitis cases and 18% (12/68) of healthy horses (p = 0.416).

Novel Fecal Biomarkers That Precede Clinical Diagnosis of Ulcerative Colitis.

BACKGROUND & AIMS: Altered gut microbiota composition and function have been associated with inflammatory bowel diseases, including ulcerative colitis (UC), but the causality and mechanisms remain unknown. METHODS: We applied 16S ribosomal RNA gene sequencing, shotgun metagenomic sequencing, in vitro functional assays, and gnotobiotic colonizations to define the microbial composition and function in fecal samples obtained from a cohort of healthy individuals at risk for inflammatory bowel diseases (pre-UC) who later developed UC (post-UC) and matched healthy control individuals (HCs). RESULTS: Microbiota composition of post-UC samples was different from HC and pre-UC samples; however, functional analysis showed increased fecal proteolytic and elastase activity before UC onset. Metagenomics identified more than 22,000 gene families that were significantly different between HC, pre-UC, and post-UC samples. Of these, 237 related to proteases and peptidases, suggesting a bacterial component to the pre-UC proteolytic signature. Elastase activity inversely correlated with the relative abundance of Adlercreutzia and other potentially beneficial taxa and directly correlated with known proteolytic taxa, such as Bacteroides vulgatus. High elastase activity was confirmed in Bacteroides isolates from fecal samples. The bacterial contribution and functional significance of the proteolytic signature were investigated in germ-free adult mice and in dams colonized with HC, pre-UC, or post-UC microbiota. Mice colonized with or born from pre-UC-colonized dams developed higher fecal proteolytic activity and an inflammatory immune tone compared with HC-colonized mice. CONCLUSIONS: We have identified increased fecal proteolytic activity that precedes the clinical diagnosis of UC and associates with gut microbiota changes. This proteolytic signature may constitute a noninvasive biomarker of inflammation to monitor at-risk populations that can be targeted therapeutically with antiproteases.

Human Gut Metagenomes Encode Diverse GH156 Sialidases.

The intestinal lining is protected by a mucous barrier composed predominantly of complex carbohydrates. Gut microbes employ diverse glycoside hydrolases (GHs) to liberate mucosal sugars as a nutrient source to facilitate host colonization. Intensive catabolism of mucosal glycans, however, may contribute to barrier erosion, pathogen encroachment, and inflammation. Sialic acid is an acidic sugar featured at terminal positions of host glycans. Characterized sialidases from the microbiome belong to the GH33 family, according to CAZy (Carbohydrate-Active enZYmes Database). In 2018 a functional metagenomics screen using thermal spring DNA uncovered the founding member of the GH156 sialidase family, the presence of which has yet to be reported in the context of the human microbiome. A subset of GH156 sequences from the CAZy database containing key sialidase residues was used to build a hidden Markov model. HMMsearch against public databases revealed ~10× more putative GH156 sialidases than currently cataloged by CAZy. Represented phyla include Bacteroidota, Verrucomicrobiota, and Firmicutes_A from human microbiomes, all of which play notable roles in carbohydrate fermentation. Analyses of metagenomic data sets revealed that GH156s are frequently encoded in metagenomes, with a greater variety and abundance of GH156 genes observed in traditional hunter-gatherer or agriculturalist societies than in industrialized societies, particularly relative to individuals with inflammatory bowel disease (IBD). Nineteen GH156s were recombinantly expressed and assayed for sialidase activity. The five GH156 sialidases identified here share limited sequence identity to each other or the founding GH156 family member and are representative of a large subset of the family. IMPORTANCE Sialic acids occupy terminal positions of human glycans where they act as receptors for microbes, toxins, and immune signaling molecules. Microbial enzymes that remove sialic acids, sialidases, are abundant in the human microbiome where they may contribute to shaping the microbiota community structure or contribute to pathology. Furthermore, sialidases have proven to hold therapeutic potential for cancer therapy. Here, we examined the sequence space of a sialidase family of enzymes, GH156, previously unknown in the human gut environment. Our analyses suggest that human populations with disparate dietary practices harbor distinct varieties and abundances of GH156-encoding genes. Furthermore, we demonstrate the sialidase activity of 5 gut-derived GH156s. These results expand the diversity of sialidases that may contribute to host glycan degradation, and these sequences may have biotechnological or clinical utility.

Colitis-associated microbiota drives changes in behaviour in male mice in the absence of inflammation.

Inflammatory bowel diseases (IBD) are chronic inflammatory conditions of the gastrointestinal tract. IBD are associated with a high prevalence of cognitive, behavioural and emotional comorbidities, including anxiety and depression. The link between IBD and the development of behavioural comorbidities is poorly understood. As the intestinal microbiota profoundly influences host behaviour, we sought to determine whether the altered gut microbiota associated with intestinal inflammation contributes to the development of behavioural abnormalities. Using the dextran sulphate sodium (DSS) model of colitis, we characterized intestinal inflammation, behaviour (elevated plus maze and tail suspension test) and the composition of the microbiota in male mice. Cecal contents from colitic mice were transferred into germ-free (GF) or antibiotic (Abx)-treated mice, and behaviour was characterized in recipient mice. Gene expression was measured using qPCR. DSS colitis was characterized by a significant reduction in body weight and an increase in colonic inflammatory markers. These changes were accompanied by increased anxiety-like behaviour, an altered gut microbiota composition, and increased central Tnf expression. Transfer of the cecal matter from colitic mice induced similar behavioural changes in both GF and Abx-treated recipient mice, with no signs of colonic or neuroinflammation. Upon characterization of the microbiota in donor and recipient mice, specific taxa were found to be associated with behavioural changes, notably members of the Lachnospiraceae family. Behavioural abnormalities associated with intestinal inflammation are transmissible via transfer of cecal matter, suggesting that alterations in the composition of the gut microbiota play a key role in driving behavioural changes in colitis.

Effects of municipal wastewater effluents on the digestive gland microbiome of wild freshwater mussels (Lasmigona costata).

Gut microbial communities are vital for maintaining host health, and are sensitive to diet, environment, and chemical exposures. Wastewater treatment plants (WWTPs) release effluents containing antimicrobials, pharmaceuticals, and other contaminants that may negatively affect the gut microbiome of downstream organisms. This study investigated changes in the diversity and composition of the digestive gland microbiome of flutedshell mussels (Lasmigona costata) from upstream and downstream of two large (service >100,000) WWTPs. Mussel digestive gland microbiome was analyzed following the extraction, PCR amplification, and sequencing of bacterial DNA using the V3-V4 hypervariable regions of the 16 S rRNA gene. Bacterial alpha diversity decreased at sites downstream of the second WWTP and these sites were dissimilar in beta diversity from sites upstream and downstream of the first upstream WWTP. The microbiomes of mussels collected downstream of the first WWTP had increased relative abundances of Actinobacteria, Bacteroidetes, and Firmicutes, with a decrease in Cyanobacteria, compared to upstream mussels. Meanwhile, those collected downstream of the second WWTP increased in Proteobacteria and decreased in Actinobacteria, Bacteroidetes, and Tenericutes. Increased Proteobacteria has been linked to adverse effects in mammals, but their functions in mussels is currently unknown. Finally, effluent-derived bacteria were found in the microbiome of mussels downstream of both WWTPs but not in those from upstream. Overall, results show that the digestive gland microbiome of mussels collected upstream and downstream of WWTPs differed, which has implications for altered host health and the transport of WWTP-derived bacteria through aquatic ecosystems.

Exposure to high fructose corn syrup during adolescence in the mouse alters hepatic metabolism and the microbiome in a sex-specific manner.

KEY POINTS: The prevalence of obesity and non-alcoholic fatty liver disease in children is dramatically increasing at the same time as consumption of foods with a high sugar content. Intake of high fructose corn syrup (HFCS) is a possible aetiology as it is thought to be more lipogenic than glucose. In a mouse model, HFCS intake during adolescence increased fat mass and hepatic lipid levels in male and female mice. However, only males showed impaired glucose tolerance. Multiple metabolites including lipids, bile acids, carbohydrates and amino acids were altered in liver in a sex-specific manner at 6 weeks of age. Some of these changes were also present in adulthood even though HFCS exposure ended at 6 weeks. HFCS significantly altered the gut microbiome, which was associated with changes in key microbial metabolites. These results suggest that HFCS intake during adolescence has profound metabolic changes that are linked to changes in the microbiome and these changes are sex-specific. ABSTRACT: The rapid increase in obesity, diabetes and fatty liver disease in children over the past 20 years has been linked to increased consumption of high fructose corn syrup (HFCS), making it essential to determine the short- and long-term effects of HFCS during this vulnerable developmental window. We hypothesized that HFCS exposure during adolescence significantly impairs hepatic metabolic signalling pathways and alters gut microbial composition, contributing to changes in energy metabolism with sex-specific effects. C57bl/6J mice with free access to HFCS during adolescence (3-6 weeks of age) underwent glucose tolerance and body composition testing and hepatic metabolomics, gene expression and triglyceride content analysis at 6 and 30 weeks of age (n = 6-8 per sex). At 6 weeks HFCS-exposed mice had significant increases in fat mass, glucose intolerance, hepatic triglycerides (females) and de novo lipogenesis gene expression (ACC, DGAT, FAS, ChREBP, SCD, SREBP, CPT and PPARα) with sex-specific effects. At 30 weeks, HFCS-exposed mice also had abnormalities in glucose tolerance (males) and fat mass (females). HFCS exposure enriched carbohydrate, amino acid, long chain fatty acid and secondary bile acid metabolism at 6 weeks with changes in secondary bile metabolism at 6 and 30 weeks. Microbiome studies performed immediately before and after HFCS exposure identified profound shifts of microbial species in male mice only. In summary, short-term HFCS exposure during adolescence induces fatty liver, alters important metabolic pathways, some of which continue to be altered in adulthood, and changes the microbiome in a sex-specific manner.

Potential Contributions of Anaerobes in Cystic Fibrosis Airways.

Cystic fibrosis (CF) is the most common, lethal genetic disease among the Caucasian population. The leading cause of mortality is recurrent acute exacerbations resulting in chronic airway inflammation and subsequent downward progression of pulmonary function. Traditionally, these periods of clinical deterioration have been associated with several principal pathogens. However, a growing body of literature has demonstrated a polymicrobial lower respiratory community compromised of facultative and obligate anaerobes. Despite the understanding of a complex bacterial milieu in CF patient airways, specific roles of anaerobes in disease progression have not been established. In this paper, we first present a brief review of the anaerobic microorganisms that have been identified within CF lower respiratory airways. Next, we discuss the potential contribution of these organisms to CF disease progression, in part by pathogenic potential and also through synergistic interaction with principal pathogens. Finally, we propose a variety of clinical scenarios in which these anaerobic organisms indirectly facilitate principal CF pathogens by modulating host defense and contribute to treatment failure by antibiotic inactivation. These mechanisms may affect patient clinical outcomes and contribute to further disease progression.

Azithromycin and the microbiota of cystic fibrosis sputum.

BACKGROUND: Azithromycin is commonly prescribed drug for individuals with cystic fibrosis (CF), with demonstrated benefits in reducing lung function decline, exacerbation occurrence and improving nutrition. As azithromycin has antimicrobial activity against components of the uncultured microbiome and increasingly the CF microbiome is implicated in disease pathogenesis - we postulated azithromycin may act through its manipulation. Herein we sought to determine if the CF microbiome changed following azithromycin use and if clinical benefit observed during azithromycin use associated with baseline community structure. RESULTS: Drawing from a prospectively collected biobank we identified patients with sputum samples prior to, during and after initiating azithromycin and determined the composition of the CF microbial community by sequencing the V3-V4 region of the 16S rRNA gene. We categorized patients as responders if their rate of lung function decline improved after azithromycin initiation. Thirty-eight adults comprised our cohort, nine who had not utilized azithromycin in at least 3 years, and 29 who were completely naïve. We did not observe a major impact in the microbial community structure of CF sputum in the 2 years following azithromycin usage in either alpha or beta-diversity metrics. Seventeen patients (45%) were classified as Responders - demonstrating reduced lung function decline after azithromycin. Responders who were naïve to azithromycin had a modest clustering effect distinguishing them from those who were non-Responders, and had communities enriched with several organisms including Stenotrophomonas, but not Pseudomonas. CONCLUSIONS: Azithromycin treatment did not associate with subsequent large changes in the CF microbiome structure. However, we found that baseline community structure associated with subsequent azithromycin response in CF adults.

TLR2 Plays a Pivotal Role in Mediating Mucosal Serotonin Production in the Gut.

Serotonin (5-hydroxytryptamine [5-HT]) is a key enteric signaling molecule that mediates various physiological processes in the gut. Enterochromaffin (EC) cells in the mucosal layer of the gut are the main source of 5-HT in the body and are situated in close proximity to the gut microbiota. In this study, we identify a pivotal role of TLR2 in 5-HT production in the gut. Antibiotic treatment reduces EC cell numbers and 5-HT levels in naive C57BL/6 mice, which is associated with downregulation of TLR2 expression but not TLR1 or TLR4. TLR2-deficient (Tlr2 -/-) and Myd88 -/- mice express lower EC cell numbers and 5-HT levels, whereas treatment with TLR2/1 agonist upregulates 5-HT production in irradiated C57BL/6 mice, which are reconstituted with Tlr2 -/- bone marrow cells, and in germ-free mice. Human EC cell line (BON-1 cells) release higher 5-HT upon TLR2/1 agonist via NF-κB pathway. Tlr2 -/- mice and anti-TLR2 Ab-treated mice infected with enteric parasite, Trichuris muris, exhibited attenuated 5-HT production, compared with infected wild-type mice. Moreover, excretory-secretory products from T. muris induce higher 5-HT production in BON-1 cells via TLR2 in a dose-dependent manner, whereby the effect of excretory-secretory products is abrogated by TLR2 antagonist. These findings not only suggest an important role of TLR2 in mucosal 5-HT production in the gut by resident microbiota as well as by a nematode parasite but also provide, to our knowledge, novel information on the potential benefits of targeting TLR2 in various gut disorders that exhibit aberrant 5-HT signaling.

Characterizing the gut microbiota in adults with bipolar disorder: a pilot study.

Background: Convergent evidence implicates gut microbiota in human health and disease. Hitherto, relatively few studies have evaluated the gut microbiota profile in individuals with bipolar disorder (BD) relative to healthy controls (HC). Methods: Fecal samples were collected from subjects (aged 18-65) meeting DSM-5-defined criteria for BD and age- and sex-matched HC without current or past history of mental or major medical disorders. Samples were sequenced using Illumina sequencing and association of specific taxa and co-occurrence of taxa with sample groups including the effect of diet was assessed using cluster analysis and analysis of communities of microorganisms (ANCOM). Nutritional composition was evaluated using the Dietary Questionnaire for Epidemiological Studies (DQES v2) Food Frequency Questionnaire. Results: Forty-six subjects were enrolled (n=23 BD, n=23 HC). Cluster analyses did not identify any significant differences between BD and HC (p=0.38). Lower microbiota diversity was observed among BD subjects relative to HC (p=0.04). A greater abundance of a Clostridiaceae OTU was observed among BD subjects when compared to HC and of Collinsella among BD-II subjects relative to BD-I. Cluster analysis revealed that neither diagnosis (p=0.38) nor diet (p=0.43) had a significant effect on overall gut microbiota composition. Limitations: This study has a small sample size and insufficient control for some potential moderating factors (e.g. psychotropic medication and smoking). Conclusion: This study suggests that individuals with BD may have a distinct gut microbiota profile compared to healthy controls, with a greater abundance of Clostridiaceae and Collinsella. These findings need to be replicated in future studies with larger sample sizes.

Completion of draft bacterial genomes by long-read sequencing of synthetic genomic pools.

BACKGROUND: Illumina technology currently dominates bacterial genomics due to its high read accuracy and low sequencing cost. However, the incompleteness of draft genomes generated by Illumina reads limits their application in comprehensive genomics analyses. Alternatively, hybrid assembly using both Illumina short reads and long reads generated by single molecule sequencing technologies can enable assembly of complete bacterial genomes, yet the high per-genome cost of long-read sequencing limits the widespread use of this approach in bacterial genomics. Here we developed a protocol for hybrid assembly of complete bacterial genomes using miniaturized multiplexed Illumina sequencing and non-barcoded PacBio sequencing of a synthetic genomic pool (SGP), thus significantly decreasing the overall per-genome cost of sequencing. RESULTS: We evaluated the performance of SGP hybrid assembly on the genomes of 20 bacterial isolates with different genome sizes, a wide range of GC contents, and varying levels of phylogenetic relatedness. By improving the contiguity of Illumina assemblies, SGP hybrid assembly generated 17 complete and 3 nearly complete bacterial genomes. Increased contiguity of SGP hybrid assemblies resulted in considerable improvement in gene prediction and annotation. In addition, SGP hybrid assembly was able to resolve repeat elements and identify intragenomic heterogeneities, e.g. different copies of 16S rRNA genes, that would otherwise go undetected by short-read-only assembly. Comprehensive comparison of SGP hybrid assemblies with those generated using multiplexed PacBio long reads (long-read-only assembly) also revealed the relative advantage of SGP hybrid assembly in terms of assembly quality. In particular, we observed that SGP hybrid assemblies were completely devoid of both small (i.e. single base substitutions) and large assembly errors. Finally, we show the ability of SGP hybrid assembly to differentiate genomes of closely related bacterial isolates, suggesting its potential application in comparative genomics and pangenome analysis. CONCLUSION: Our results indicate the superiority of SGP hybrid assembly over both short-read and long-read assemblies with respect to completeness, contiguity, accuracy, and recovery of small replicons. By lowering the per-genome cost of sequencing, our parallel sequencing and hybrid assembly pipeline could serve as a cost effective and high throughput approach for completing high-quality bacterial genomes.