Impact of Oral Fidaxomicin Administration on the Intestinal Microbiota and Susceptibility to Clostridium difficile Colonization in Mice.

Clostridium difficile infection (CDI), a common cause of hospital-acquired infections, typically occurs after disruption of the normal gut microbiome by broad-spectrum antibiotics. Fidaxomicin is a narrow-spectrum antibiotic that demonstrates a reduced impact on the normal gut microbiota and is approved for the treatment of CDI. To further explore the benefits of this property, we used a murine model to examine the effects of fidaxomicin versus vancomycin on gut microbiota and susceptibility to C. difficile colonization while tracking microbiota recovery over time. Mice were exposed to fidaxomicin or vancomycin by oral gavage for 3 days and subsequently challenged with C. difficile spores at predetermined time points up to 21 days postexposure to antibiotics. Fecal samples were subsequently collected for analysis. Twenty-four hours postchallenge, mice were euthanized and the colon contents harvested. The microbiota was characterized using 16S rRNA gene sequencing. All fidaxomicin-exposed mice (except for one at day 8) were resistant to C. difficile colonization. However, 9 of 15 vancomycin-exposed mice were susceptible to C. difficile colonization until day 12. All vancomycin-exposed mice recovered colonization resistance by day 16. Bacterial diversity was similar prior to antibiotic exposure in both arms and decreased substantially after exposure. A shift in taxonomic structure and composition occurred after both exposures; however, the shift was greater in vancomycin-exposed than in fidaxomicin-exposed mice. In summary, compared with vancomycin, fidaxomicin exposure had less impact on microbiota composition, promoted faster microbial recovery, and had less impact on the loss of C. difficile colonization resistance.

The relationship between nasopharyngeal CCL5 and microbiota on disease severity among infants with bronchiolitis.

Emerging evidence suggests that the airway microbiota plays an important role in viral bronchiolitis pathobiology. However, little is known about the combined role of airway microbiota and CCL5 in infants with bronchiolitis. In this multicenter prospective cohort study of 1005 infants (age <1 year) hospitalized for bronchiolitis during 2011-2014, we observed statistically significant interactions between nasopharyngeal airway CCL5 levels and microbiota profiles with regard to the risk of both intensive care use (Pinteraction =.02) and hospital length-of-stay ≥3 days (Pinteraction =.03). Among infants with lower CCL5 levels, the Haemophilus-dominant microbiota profile was associated with a higher risk of intensive care use (OR, 3.20; 95%CI, 1.18-8.68; P=.02) and hospital length-of-stay ≥3 days (OR, 4.14; 95%CI, 2.08-8.24; P<.001) compared to the Moraxella-dominant profile. Conversely, among those with higher CCL5 levels, there were no significant associations between the microbiota profiles and these severity outcomes (all P≥.10).

The gut microbiota is associated with psychiatric symptom severity and treatment outcome among individuals with serious mental illness.

BACKGROUND: Emerging evidence implicates the gut microbiota in central nervous system functioning via its effects on inflammation, the hypothalamic-pituitary axis, and/or neurotransmission. Our understanding of the cellular underpinnings of the brain-gut relationship is based almost exclusively on animal models with some small-scale human studies. This study examined the relationship between the gut microbiota and psychiatric symptom severity and treatment response among inpatients with serious mental illness. METHOD: We collected data from adult inpatients (N = 111). Measures of diagnoses, suicide severity, trauma, depression, and anxiety were collected shortly after admission, while self-collected fecal swabs were collected early in the course of hospitalization and processed using 16S rRNA gene sequencing and whole genome shotgun sequencing methods. RESULTS: Results indicate that depression and anxiety severity shortly after admission were negatively associated with bacterial richness and alpha diversity. Additional analyses revealed a number of bacterial taxa associated with depression and anxiety severity. Gut microbiota richness and alpha diversity early in the course of hospitalization was a significant predictor of depression remission at discharge. CONCLUSIONS: This study is among the first to demonstrate a gut microbiota relationship with symptom severity among psychiatric inpatients as well as a relationship to remission of depression post-treatment. These findings are consistent with animal models and limited human studies as well as with the broader literature implicating inflammation in the pathophysiology of depression. These findings offer the foundation for further studies of novel therapeutic approaches to the treatment, prevention of, or recurrence of serious mental illness.

Challenges of Francisella classification exemplified by an atypical clinical isolate.

The accumulation of sequenced Francisella strains has made it increasingly apparent that the 16S rRNA gene alone is not enough to stratify the Francisella genus into precise and clinically useful classifications. Continued whole-genome sequencing of isolates will provide a larger base of knowledge for targeted approaches with broad applicability. Additionally, examination of genomic information on a case-by-case basis will help resolve outstanding questions regarding strain stratification. We report the complete genome sequence of a clinical isolate, designated here as F. novicida-like strain TCH2015, acquired from the lymph node of a 6-year-old male. Two features were atypical for F. novicida: exhibition of functional oxidase activity and additional gene content, including proposed virulence determinants. These differences, which could potentially impact virulence and clinical diagnosis, emphasize the need for more comprehensive methods to profile Francisella isolates. This study highlights the value of whole-genome sequencing, which will lead to a more robust database of environmental and clinical genomes and inform strategies to improve detection and classification of Francisella strains.

Isolation and concentration of bacteria from blood using microfluidic membraneless dialysis and dielectrophoresis.

A microfluidic system that combines membraneless microfluidic dialysis and dielectrophoresis to achieve label-free isolation and concentration of bacteria from whole blood is presented. Target bacteria and undesired blood cells are discriminated on the basis of their differential susceptibility to permeabilizing agents that alter the dielectrophoretic behavior of blood cells but not bacteria. The combined membraneless microdialysis and dielectrophoresis system isolated 79 ± 3% of Escherichia coli and 78 ± 2% of Staphylococcus aureus spiked into whole blood at a processing rate of 0.6 mL h-1. Collection efficiency was independent of the number of target bacteria up to 105 cells. Quantitative PCR analysis revealed that bacterial 16S rDNA levels were enriched more than 307-fold over human DNA in the fraction recovered from the isolation system compared with the original specimen. These data demonstrate feasibility for an instrument to accelerate the detection and analysis of bacteria in blood by first isolating and concentrating them in a microchamber.

Randomised clinical trial: faecal microbiota transplantation for recurrent Clostridum difficile infection - fresh, or frozen, or lyophilised microbiota from a small pool of healthy donors delivered by colonoscopy.

BACKGROUND: Faecal microbiota transplantation (FMT) has become routine in managing recurrent C. difficile infection (CDI) refractory to antibiotics. AIM: To compare clinical response and improvements in colonic microbiota diversity in subjects with recurrent CDI using different donor product. METHODS: Seventy-two subjects with ≥3 bouts of CDI were randomised in a double-blind study to receive fresh, frozen or lyophilised FMT product via colonoscopy from 50 g of stool per treatment from eight healthy donors. Recipients provided stools pre- and 7, 14 and 30 days post-FMT for C. difficile toxin and, in a subset, microbiome composition by 16S rRNA gene profiling. RESULTS: Overall resolution of CDI was 87% during 2 months of follow-up after FMT. Stool samples before FMT had significantly decreased bacterial diversity with a high proportion of Proteobacteria compared to donors. Cure rates were highest for the group receiving fresh product seen in 25/25 (100%), lowest for the lyophilised product 16/23 (78%; P = 0.022 vs. fresh and 0.255 vs. frozen) and intermediate for frozen product 20/24 (P = 0.233 vs. fresh). Microbial diversity was reconstituted by day 7 in the subjects receiving fresh or frozen product. Improvement in diversity was seen by day 7 in those randomised to lyophilised material with reconstitution by 30 days. CONCLUSIONS: Comparative efficacy in faecal microbiota transplantation was observed in subjects receiving fresh or frozen faecal product from the same donors. The lyophilised product had a slightly lowered efficacy compared with fresh product, but it resembled other treatments in microbial restoration 1 month after faecal microbiota transplantation.

Complete Genome Sequence of Turicibacter sp. Strain H121, Isolated from the Feces of a Contaminated Germ-Free Mouse.

Turicibacterbacteria are commonly detected in the gastrointestinal tracts and feces of humans and animals, but their phylogeny, ecological role, and pathogenic potential remain unclear. We present here the first complete genome sequence ofTuricibactersp. strain H121, which was isolated from the feces of a mouse line contaminated following germ-free derivation.

Transcriptional regulation of ALV bursal lymphomagenesis.

Avian leukosis virus (ALV) induces bursal lymphoma in chickens after rare integration of proviral long terminal repeat (LTR) sequences next to the c-myc proto-oncogene. Labile LTR-binding proteins are essential for c-myc hyperexpression, since LTR-enhanced transcription, as well as the activity of the LTR-binding proteins, is specifically decreased following protein synthesis inhibition. This lability is restricted to hematopoetic cells of ALV-susceptible chicken strains, suggesting that the labile proteins play an important role in susceptibility to lymphomagenesis. Five proteins that interact with the ALV LTR enhancer were identified from bursal lymphoma cells: two proteins are stable (b and c), while three proteins (a1, a3, and b*) are labile in pre-B cell types. Two cDNAs (a1/EBP and a3/EBP) encoding leucine zipper proteins that bind to the ALV LTR enhancer were cloned using a lambda gt11 cDNA expression library made from bursal lymphoma cells. We are presently studying the roles of a1/EBP and a3/EBP in labile regulation of LTR-enhanced c-myc transcription and susceptibility to bursal lymphomagenesis.

High-Quality Draft Genome Sequence of Francisella tularensis subsp. holarctica Strain OR96-0246.

The bacterial pathogen Francisella tularensis was recently renewed as a tier-one select agent. F. tularensis subsp. tularensis (type A) and holarctica (type B) are of clinical relevance. Here, we report the complete genome of a virulent F. tularensis type B strain and describe its usefulness in comparative genomics.

Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase.

Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant beta-lactamases have evolved with activity toward a variety of beta-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant beta-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 beta-lactamase are of interest. Previously, screens of random libraries encompassing 31 of 55 active site amino acid positions enabled the identification of the residues responsible for maintaining the substrate specificity of TEM-1 beta-lactamase. In addition to substitutions found in clinical isolates, many other specificity-altering mutations were also identified. Interestingly, many nonspecific substitutions in the N-terminal half of the active site omega loop were found to increase ceftazidime hydrolytic activity and decrease ampicillin hydrolytic activity. To complete the active sight study, eight additional random libraries were constructed and screened for specificity-altering mutations. All additional substitutions found to alter the substrate specificity were located in the C-terminal half of the active site loop. These mutants, much like the N-terminal omega loop mutants, appear to be less stable than the wild-type enzyme. Further analysis of a 165-YYG-167 triple mutant, selected for high levels of ceftazidime hydrolytic activity, provides an example of the correlation which exists between enzyme instability and increased ceftazidime hydrolytic activity in the ceftazidime-selected omega loop mutants.

Susceptibility of beta-lactamase to core amino acid substitutions.

To determine which amino acids in TEM-1 beta-lactamase are important for its structure and function, random libraries were previously constructed which systematically randomized the 263 codons of the mature enzyme. A comprehensive screening of these libraries identified several TEM-1 beta-lactamase core positions, including F66 and L76, which are strictly required for wild-type levels of hydrolytic activity. An examination of positions 66 and 76 in the class A beta-lactamase gene family shows that a phenylalanine at position 66 is strongly conserved while position 76 varies considerably among other beta-lactamases. It is possible that position 76 varies in the gene family because beta-lactamase mutants with non-conservative substitutions at position 76 retain partial function. In contrast, position 66 may remain unchanged in the gene family because non-conservative substitutions at this location are detrimental for enzyme structure and function. By determining the beta-lactam resistance levels of the 38 possible mutants at positions 66 and 76 in the TEM-1 enzyme, it was confirmed that position 76 is indeed more tolerant of non-conservative substitutions. An analysis of the Protein Data Bank files for three class A beta-lactamases indicates that volume constraints at position 66 are at least partly responsible for the low tolerance of substitutions at this position.