Heterogeneity spacers in 16S rDNA primers improve analysis of mouse gut microbiomes via greater nucleotide diversity.

Illumina-based amplicon sequencing suffers from the deleterious effects of highly homogenous nucleotide composition, limiting the number of high-quality reads generated per run. We attempted to alleviate this limitation by comparing the results obtained from 16S ribosomal DNA (16S rDNA) sequencing of mouse gut microbiomes using Illumina V3-V4 primers (Run 1) and custom primers that incorporate a heterogeneity spacer (0-7 nucleotides) upstream of the 16S priming region (Run 2). Overall, Run 2 had higher quality sequences, a more diverse microbial profile, and higher precision within, and variation between, experimental groups than Run 1. Our primer design offers a simple way to increase the quality of 16S rDNA sequencing and increases the number of useable reads generated per Illumina run.

Complete Eradication of Biofilm From Orthopedic Materials.

BACKGROUND: Interest exists in finding alternatives to current management strategies in periprosthetic joint infections, which typically include a 2-stage revision with placement of an antibiotic spacer and delayed placement of a new implant. We studied the efficacy of autoclaving, ultrasonication, and mechanical scrubbing for sterilization and biofilm eradication on infected cobalt-chrome discs. METHODS: Strains of Staphylococcus aureus MRSA252 or Staphylococcus epidermidis RP62A were grown on the discs. For each strain, discs were divided into 5 groups (5 discs each) and exposed to several sterilization and biofilm eradication treatments: (1) autoclave, (2) autoclave + sonication, (3) autoclave + saline scrub, (4) autoclave + 4% chlorhexidine (CHC) scrub, and (5) autoclave + sonication + CHC scrub. Sterilization and biofilm eradication were quantified with crystal violet assays and scanning electron microscopy. RESULTS: Relative to nontreated controls, autoclaving alone reduced biofilm load by 33.9% and 54.7% for MRSA252 and RP62A strains, respectively. Biofilm removal was maximized with the combined treatment of autoclaving and CHC scrub for MRSA252 (100%) and RP62A (99.5%). The addition of sonication between autoclaving and CHC scrubbing resulted in no statistically significant improvement in biofilm removal. High-resolution scanning electron microscopy revealed no cells or biofilm for this combined treatment. CONCLUSION: Using 2 commonly encountered bacterial strains in periprosthetic joint infection, infected cobalt-chrome discs were sterilized and eradicated of residual biofilm with a combination of autoclaving and CHC scrubbing.

Sibling sRNA RyfA1 Influences Shigella dysenteriae Pathogenesis.

Small regulatory RNAs (sRNAs) of Shigella dysenteriae and other pathogens are vital for the regulation of virulence-associated genes and processes. Here, we characterize RyfA1, one member of a sibling pair of sRNAs produced by S. dysenteriae. Unlike its nearly identical sibling molecule, RyfA2, predicted to be encoded almost exclusively by non-pathogenic species, the presence of a gene encoding RyfA1, or a RyfA1-like molecule, is strongly correlated with virulence in a variety of enteropathogens. In S. dysenteriae, the overproduction of RyfA1 negatively impacts the virulence-associated process of cell-to-cell spread as well as the expression of ompC, a gene encoding a major outer membrane protein important for the pathogenesis of Shigella. Interestingly, the production of RyfA1 is controlled by a second sRNA, here termed RyfB1, the first incidence of one regulatory small RNA controlling another in S. dysenteriae or any Shigella species.

Characterizing the transcriptional adaptation of Staphylococcus aureus to stationary phase growth.

Staphylococcus aureus is an important human pathogen that causes life-threatening infections, and is resistant to the majority of our antibiotic arsenal. This resistance is complicated by the observation that most antibacterial agents target actively growing cells, thus, proving ineffective against slow growing populations, such as cells within a biofilm or in stationary phase. Recently, our group generated updated genome annotation files for S. aureus that not only include protein-coding genes but also regulatory and small RNAs. As such, these annotation files were used to perform a transcriptomic analysis in order to understand the metabolic and physiological changes that occur during transition from active growth to stationary phase; with a focus on sRNAs. We observed ∼24% of protein-coding and 34% of sRNA genes displaying changes in expression by ≥3-fold. Collectively, this study adds to our understanding of S. aureus adaptation to nutrient-limiting conditions, and sheds new light onto the contribution of sRNAs to this process.

Genome-wide Annotation, Identification, and Global Transcriptomic Analysis of Regulatory or Small RNA Gene Expression in Staphylococcus aureus.

UNLABELLED: In Staphylococcus aureus, hundreds of small regulatory or small RNAs (sRNAs) have been identified, yet this class of molecule remains poorly understood and severely understudied. sRNA genes are typically absent from genome annotation files, and as a consequence, their existence is often overlooked, particularly in global transcriptomic studies. To facilitate improved detection and analysis of sRNAs in S. aureus, we generated updated GenBank files for three commonly used S. aureus strains (MRSA252, NCTC 8325, and USA300), in which we added annotations for >260 previously identified sRNAs. These files, the first to include genome-wide annotation of sRNAs in S. aureus, were then used as a foundation to identify novel sRNAs in the community-associated methicillin-resistant strain USA300. This analysis led to the discovery of 39 previously unidentified sRNAs. Investigating the genomic loci of the newly identified sRNAs revealed a surprising degree of inconsistency in genome annotation in S. aureus, which may be hindering the analysis and functional exploration of these elements. Finally, using our newly created annotation files as a reference, we perform a global analysis of sRNA gene expression in S. aureus and demonstrate that the newly identified tsr25 is the most highly upregulated sRNA in human serum. This study provides an invaluable resource to the S. aureus research community in the form of our newly generated annotation files, while at the same time presenting the first examination of differential sRNA expression in pathophysiologically relevant conditions. IMPORTANCE: Despite a large number of studies identifying regulatory or small RNA (sRNA) genes in Staphylococcus aureus, their annotation is notably lacking in available genome files. In addition to this, there has been a considerable lack of cross-referencing in the wealth of studies identifying these elements, often leading to the same sRNA being identified multiple times and bearing multiple names. In this work, we have consolidated and curated known sRNA genes from the literature and mapped them to their position on the S. aureus genome, creating new genome annotation files. These files can now be used by the scientific community at large in experiments to search for previously undiscovered sRNA genes and to monitor sRNA gene expression by transcriptome sequencing (RNA-seq). We demonstrate this application, identifying 39 new sRNAs and studying their expression during S. aureus growth in human serum.

Towards the complete small RNome of Acinetobacter baumannii.

In recent years, the Gram-negative bacterium Acinetobacter baumannii has garnered considerable attention for its unprecedented capacity to rapidly develop resistance to antibacterial therapeutics. This is coupled with the seemingly epidemic emergence of new hyper-virulent strains. Although strain-specific differences for A. baumannii isolates have been well described, these studies have primarily focused on proteinaceous factors. At present, only limited publications have investigated the presence and role of small regulatory RNA (sRNA) transcripts. Herein, we perform such an analysis, describing the RNA-seq-based identification of 78 A. baumannii sRNAs in the AB5075 background. Together with six previously identified elements, we include each of these in a new genome annotation file, which will serve as a tool to investigate regulatory events in this organism. Our work reveals that the sRNAs display high expression, accounting for >50 % of the 20 most strongly expressed genes. Through conservation analysis we identified six classes of similar sRNAs, with one found to be particularly abundant and homologous to regulatory, C4 antisense RNAs found in bacteriophages. These elements appear to be processed from larger transcripts in an analogous manner to the phage C4 molecule and are putatively controlled by two further sRNAs that are strongly antisense to them. Collectively, this study offers a detailed view of the sRNA content of A. baumannii, exposing sequence and structural conservation amongst these elements, and provides novel insight into the potential evolution, and role, of these understudied regulatory molecules.

Transcriptomic analysis of staphylococcal sRNAs: insights into species-specific adaption and the evolution of pathogenesis.

Next-generation sequencing technologies have dramatically increased the rate at which new genomes are sequenced. Accordingly, automated annotation programs have become adept at identifying and annotating protein coding regions, as well as common and conserved RNAs. Additionally, RNAseq techniques have advanced our ability to identify and annotate regulatory RNAs (sRNAs), which remain significantly understudied. Recently, our group catalogued and annotated all previously known and newly identified sRNAs in several Staphylococcus aureus strains. These complete annotation files now serve as tools to compare the sRNA content of S. aureus with other bacterial strains to investigate the conservation of their sRNomes. Accordingly, in this study we performed RNAseq on two staphylococcal species, Staphylococcus epidermidis and Staphylococcus carnosus, identifying 118 and 89 sRNAs in these organisms, respectively. The sRNA contents of all three species were then compared to elucidate their common and species-specific sRNA content, identifying a core set of between 53 and 36 sRNAs encoded in each organism. In addition, we determined that S. aureus has the largest set of unique sRNAs (137) while S. epidermidishas the fewest (25). Finally, we identify a highly conserved sequence and structural motif differentially represented within, yet common to, both S. aureus and S. epidermidis. Collectively, in this study, we uncover the sRNome common to three staphylococcal species, shedding light on sRNAs that are likely to be involved in basic physiological processes common to the genus. More significantly, we have identified species-specific sRNAs that are likely to influence the individual lifestyle and behaviour of these diverse staphylococcal strains.

VirF-independent regulation of Shigella virB transcription is mediated by the small RNA RyhB.

Infection of the human host by Shigella species requires the coordinated production of specific Shigella virulence factors, a process mediated largely by the VirF/VirB regulatory cascade. VirF promotes the transcription of virB, a gene encoding the transcriptional activator of several virulence-associated genes. This study reveals that transcription of virB is also regulated by the small RNA RyhB, and importantly, that this regulation is not achieved indirectly via modulation of VirF activity. These data are the first to demonstrate that the regulation of virB transcription can be uncoupled from the master regulator VirF. It is also established that efficient RyhB-dependent regulation of transcription is facilitated by specific nucleic acid sequences within virB. This study not only reveals RyhB-dependent regulation of virB transcription as a novel point of control in the central regulatory circuit modulating Shigella virulence, but also highlights the versatility of RyhB in controlling bacterial gene expression.