Human intestinal organoids as models to study enteric bacteria and viruses.

Laboratory studies of host-microbe interactions have historically been carried out using transformed cell lines and animal models. Although much has been learned from these models, recent advances in the development of multicellular, physiologically active, human intestinal organoid (HIO) cultures are allowing unprecedented discoveries of host-microbe interactions. Here, we review recent literature using HIOs as models to investigate the pathogenesis of clinically important enteric bacteria and viruses and study commensal intestinal microbes. We also discuss limitations of current HIO culture systems and how technical advances and innovative engineering approaches are providing new directions to improve the model. The studies discussed here highlight the potential of HIOs for studying microbial pathogenesis, host-microbe interactions, and for preclinical development of therapeutics and vaccines.

Protocol of Detection of West Nile Virus in Clinical Samples.

West Nile virus (WNV) is one of the leading causes of arboviral encephalitis in the United States but is often underdiagnosed. Despite the wide breadth of WNV-induced clinical disease syndromes, many of the symptoms associated with WNV are nonspecific at the time of presentation; thus, choosing the right diagnostic tool is essential to not only understand the true burden of disease but also provide pathogen-directed interventions for WNV-infected patients. In this chapter, we briefly discuss the three most common types of diagnostic methods for WNV in human clinical samples: nucleic acid detection, enzyme-linked immunoassay (ELISA), and plaque reduction neutralization test (PRNT) and present the method for PRNT.

Group B Streptococcus CAMP Factor Does Not Contribute to Interactions with the Vaginal Epithelium and Is Dispensable for Vaginal Colonization in Mice.

The Gram-positive pathogen group B Streptococcus (GBS) is a leading cause of neonatal bacterial infections, preterm birth, and stillbirth. Although maternal GBS vaginal colonization is a risk factor for GBS-associated adverse birth outcomes, mechanisms promoting GBS vaginal persistence are not fully defined. GBS possesses a broadly conserved small molecule, CAMP factor, that is co-hemolytic in the presence of Staphylococcus aureus sphingomyelinase C. While this co-hemolytic reaction is commonly used by clinical laboratories to identify GBS, the contribution of CAMP factor to GBS vaginal persistence is unknown. Using in vitro biofilm, adherence and invasion assays with immortalized human vaginal epithelial VK2 cells, and a mouse model of GBS vaginal colonization, we tested the contribution of CAMP factor using GBS strain COH1 and its isogenic CAMP-deficient mutant (Δcfb). We found no evidence for CAMP factor involvement in GBS biofilm formation, or adherence, invasion, or cytotoxicity toward VK2 cells in the presence or absence of S. aureus. Additionally, there was no difference in vaginal burdens or persistence between COH1 and Δcfb strains in a murine colonization model. In summary, our results using in vitro human cell lines and murine models do not support a critical role for CAMP factor in promoting GBS vaginal colonization. IMPORTANCE Group B Streptococcus (GBS) remains a pervasive pathogen for pregnant women and their newborns. Maternal screening and intrapartum antibiotic prophylaxis to GBS-positive mothers have reduced, but not eliminated GBS neonatal disease, and have not impacted GBS-associated preterm birth or stillbirth. Additionally, this antibiotic exposure is associated with adverse effects on the maternal and neonatal microbiota. Identifying key GBS factors important for maternal vaginal colonization will foster development of more targeted, alternative therapies to antibiotic treatment. Here, we investigate the contribution of a broadly conserved GBS determinant, CAMP factor, to GBS vaginal colonization and find that CAMP factor is unlikely to be a biological target to control maternal GBS colonization.

Characterization of urinary microbiome in patients with bladder cancer: Results from a single-institution, feasibility study.

OBJECTIVES: The human microbiome has been linked to the development of several malignancies, but there is scarcity of data on the microbiome of bladder cancer patients. In this study, we analyzed microbial composition and diversity among patients with and without bladder cancer. MATERIAL AND METHODS: Samples were collected from 38 urothelial carcinoma (UC) patients and 10 noncancer controls from August 2018 to May 2019. DNA was extracted and processed for 16 S ribosomal RNA sequencing. Alpha diversity community characteristics including evenness and richness as well as beta diversity metrics were obtained. Linear discriminant analysis effect size was used to identify microbial components whose sequences were more abundant. Pairwise statistics provided quantitative assessment of significant distributions among groups. RESULTS: Thirty seven total samples contained high quality sequence data for subsequent analyses and divided into 3 cohorts: control (n = 10), muscle-invasive (n = 15) and superficial UC (n = 12). Control samples had significantly higher species evenness when compared to invasive (P = 0.031) and superficial tumors (P = 0.002). In addition, higher species richness was observed in noncancer versus cancer samples (Faith phylogenetic diversity, P < 0.05). Significantly enriched taxa were found in both control (Bacteroides, Lachnoclostridium, Burkholderiaceae) and cancer samples (Bacteroides and Faecalbacterium). CONCLUSION: Significantly decreased microbial community diversity was seen in the urine of patients with bladder cancer when compared to a noncancer group. Distinct taxa were noted suggesting unique microbial communities in the urine of bladder cancer patients.

Whole-Genome Sequences of Escherichia coli Isolates from Cocoa Beans Imported from Bolivia.

Four Escherichia coli isolates were obtained from a batch of cocoa beans imported from Bolivia. The cocoa beans were rejected by a U.S. chocolate manufacturer due to poor microbiological quality. The four isolates were whole-genome sequenced and the sequences analyzed to identify genotypes, serotypes, and virulence and antimicrobial resistance genes.

Bacterial Biomarkers of Marcellus Shale Activity in Pennsylvania.

Unconventional oil and gas (UOG) extraction, also known as hydraulic fracturing, is becoming more prevalent with the increasing use and demand for natural gas; however, the full extent of its environmental impacts is still unknown. Here we measured physicochemical properties and bacterial community composition of sediment samples taken from twenty-eight streams within the Marcellus shale formation in northeastern Pennsylvania differentially impacted by hydraulic fracturing activities. Fourteen of the streams were classified as UOG+, and thirteen were classified as UOG- based on the presence of UOG extraction in their respective watersheds. One stream was located in a watershed that previously had UOG extraction activities but was recently abandoned. We utilized high-throughput sequencing of the 16S rRNA gene to infer differences in sediment aquatic bacterial community structure between UOG+ and UOG- streams, as well as correlate bacterial community structure to physicochemical water parameters. Although overall alpha and beta diversity differences were not observed, there were a plethora of significantly enriched operational taxonomic units (OTUs) within UOG+ and UOG- samples. Our biomarker analysis revealed many of the bacterial taxa enriched in UOG+ streams can live in saline conditions, such as Rubrobacteraceae. In addition, several bacterial taxa capable of hydrocarbon degradation were also enriched in UOG+ samples, including Oceanospirillaceae. Methanotrophic taxa, such as Methylococcales, were significantly enriched as well. Several taxa that were identified as enriched in these samples were enriched in samples taken from different streams in 2014; moreover, partial least squares discriminant analysis (PLS-DA) revealed clustering between streams from the different studies based on the presence of hydraulic fracturing along the second axis. This study revealed significant differences between bacterial assemblages within stream sediments of UOG+ and UOG- streams and identified several potential biomarkers for evaluating and monitoring the response of autochthonous bacterial communities to potential hydraulic fracturing impacts.