Transcriptomic fingerprint of bacterial infection in lower extremity ulcers.

Clinicians and researchers utilize subjective, clinical classification systems to stratify lower extremity ulcer infections for treatment and research. The purpose of this study was to examine whether these clinical classifications are reflected in the ulcer's transcriptome. RNA sequencing (RNA-seq) was performed on biopsies from clinically infected lower extremity ulcers (n = 44). Resulting sequences were aligned to the host reference genome to create a transcriptome profile. Differential gene expression analysis and gene ontology (GO) enrichment analysis were performed between ulcer severities as well as between sample groups identified by k-means clustering. Lastly, a support vector classifier was trained to estimate clinical infection score or k-means cluster based on a subset of genes. Clinical infection severity did not explain the major sources of variability among the samples and samples with the same clinical classification demonstrated high inter-sample variability. High proportions of bacterial RNA were identified in some samples, which resulted in a strong effect on transcription and increased expression of genes associated with immune response and inflammation. K-means clustering identified two clusters of samples, one of which contained all of the samples with high levels of bacterial RNA. A support vector classifier identified a fingerprint of 20 genes, including immune-associated genes such as CXCL8, GADD45B, and HILPDA, which accurately identified samples with signs of infection via cross-validation. This study identified a unique, host-transcriptome signature in the presence of infecting bacteria, often incongruent with clinical infection-severity classifications. This suggests that stratification of infection status based on a transcriptomic fingerprint may be useful as an objective classification method to classify infection severity, as well as a tool for studying host-pathogen interactions.

Analysis of proximal bone margins in diabetic foot osteomyelitis by conventional culture, DNA sequencing and microscopy.

Multiple approaches were employed to detect pathogens from bone margins associated with Diabetic Foot Osteomyelitis (DFO). Intra-operative bone specimens of 14 consecutive subjects with suspected DFO were collected over a six-month study period from Liverpool Hospital. Infected bone and a proximal bone margins presumed to be 'clean/non-infected' were collected. Bone material was subjected to conventional culture, DNA sequencing and microscopy. In total, eight of 14 (57%) proximal bone margins had no growth by conventional culture but were identified in all proximal bone specimens by DNA sequencing. Proximal margins had lower median total microbial counts than infected specimens, but these differences were not statistically significant. Pathogens identified by sequencing in infected specimens were identified in proximal margins and the microbiomes were similar (ANOSIM = 0.02, p = 0.59). Using a combination of SEM and/or PNA-FISH, we visualized the presence of microorganisms in infected bone specimens and their corresponding proximal margins of seven patients (50%) with DFO. We identify that bacteria can still reside in what seems to be proximal 'clean' margins. The significance and implications of clinical outcomes requires further analysis from a larger sample size that incorporates differences in surgical and post-operative approaches, correlating any outcomes back to culture-sequence findings.

Bacterial biofilms: a possible mechanism for chronic infection in patients with lumbar disc herniation - a prospective proof-of-concept study using fluorescence in situ hybridization.

A relationship has been suggested between lumbar disc herniation (LDH) and chronic bacterial infection frequently involving Propionibacterium acnes, which is known to cause chronic infection through the formation of biofilm aggregates. The objective of the study was to assess whether a disc infection involving biofilm formation is present in patients with LDH. A total of 51 LDH patients and 14 controls were included. Bacterial DNA was detected by real-time polymerase chain reaction (PCR) in 16/51 samples in the LDH group and 7/14 controls (p = 0.215). Sequencing identified bacteria in 9/16 and 6/7 PCR positive samples in the LDH and control groups, respectively. All samples were stained using fluorescence in situ hybridization (FISH) and examined by confocal laser scanning microscopy. Microscopy demonstrated tissue-embedded bacterial aggregates with host inflammatory cells in 7/51 LDH patients and no controls. The presence of both bacterial aggregates and inflammatory cells suggests a chronic infection in a subset of LDH patients. The finding of bacterial 16S rDNA in both LDH and control disc tissue highlights the importance of microscopic observation to discriminate infection vs contamination. Our findings may have therapeutic implications, as the treatment of biofilm infections is different and more challenging than traditional infections.