Identification of temporal shifts of oral bacteria in bone regeneration following mandibular bone defect injury and therapeutic surgery in a porcine model.

BACKGROUND: Considered the second largest and most diverse microbiome after the gut, the human oral ecosystem is complex with diverse and niche-specific microorganisms. Although evidence is growing for the importance of oral microbiome in supporting a healthy immune system and preventing local and systemic infections, the influence of craniomaxillofacial (CMF) trauma and routine reconstructive surgical treatments on community structure and function of oral resident microbes remains unknown. CMF injuries affect a large number of people, needing extensive rehabilitation with lasting morbidity and loss of human productivity. Treatment efficacy can be complicated by the overgrowth of opportunistic commensals or multidrug-resistant pathogens in the oral ecosystem due to weakened host immune function and reduced colonization resistance in a dysbiotic oral microbiome. AIMS: To understand the dynamics of microbiota's community structure during CMF injury and subsequent treatments, we induced supra-alveolar mandibular defect in Hanford miniature swine (n = 3) and compared therapeutic approaches of immediate mandibullar reconstructive (IMR) versus delayed mandibullar reconstructive (DMR) surgeries. METHODS: Using bacterial 16S ribosomal RNA gene marker sequencing, the composition and abundance of the bacterial community of the uninjured maxilla (control) and the injured left mandibula (lingual and buccal) treated by DMR were surveyed up to 70-day post-wounding. For the injured right mandibula receiving IMR treatment, the microbial composition and abundance were surveyed up to 14-day post-wounding. Moreover, we measured sera level of biochemical markers (e.g., osteocalcin) associated with bone regeneration and healing. Computed tomography was used to measure and compare mandibular bone characteristics such as trabecular thickness between sites receiving DMR and IMR therapeutic approaches until day 140, the end of study period. RESULTS: Independent of IMR versus DMR therapy, we observed similar dysbiosis and shifts of the mucosal bacteria residents after CMF injury and/or following treatment. There was an enrichment of Fusobacterium, Porphyromonadaceae, and Bacteroidales accompanied by a decline in Pasteurellaceae, Moraxella, and Neisseria relative abundance in days allotted for healing. We also observed a decline in species richness and abundance driven by reduction in temporal instability and inter-animal heterogeneity on days 0 and 56, with day 0 corresponding to injury in DMR group and day 56 corresponding to delayed treatment for DMR or injury and immediate treatment for the IMR group. Analysis of bone healing features showed comparable bone-healing profiles for IMR vs. DMR therapeutic approach.

Comparative Transcriptome Analysis of Superficial and Deep Partial-Thickness Burn Wounds in Yorkshire vs Red Duroc Pigs.

Hypertrophic scars are a common negative outcome of deep partial-thickness (DPT) burn wounds resulting in increased dermal thickness, wound area contracture, and inflammation of the affected area. The red Duroc and Yorkshire porcine breeds are common large animal models for studying dermal wounds due to their structural similarities to human skin; however, the porcine transcriptomic profiles of dermal burn wounds and healing process are not well known. In response, a longitudinal transcriptomic comparative study was conducted comparing red Duroc and Yorkshire superficial and DPT burn wounds to their respective control uninjured tissue. Using next-generation RNA sequencing, total RNAs were isolated from burn wound tissue harvested on 0, 3, 7, 15, 30, and 60 days postburn, and mRNA-seq and gene expression read counts were generated. Significant differentially expressed genes relative to uninjured tissue were defined, and active biological processes were determined using gene set enrichment analyses. Additionally, collagen deposition, α-smooth muscle actin (SMA) protein concentration, epidermal and dermal thickness measurements, and wound area changes in response to burn injury were characterized. Overall, the red Duroc pigs, in response to both burn wound types, elicited a more robust and prolonged inflammatory immune response, fibroblast migration, and proliferation, as well as heightened levels of extracellular matrix modulation relative to respective burn types in the Yorkshire pigs. Collectively, the red Duroc DPT burn wounds produce a greater degree of hypertrophic scar-like response compared with Yorkshire DPT burn wounds. These findings will facilitate future porcine burn studies down-selecting treatment targets and determining the effects of novel therapeutic strategies.

Draft Genome Sequences of Two Strains of Bifidobacterium dentium Isolated from a Crude Fecal Extract Used for Fecal Microbiota Transplantation in the Republic of Korea.

We present the draft genome sequences of two Bifidobacterium dentium strains isolated from a fecal extract for fecal microbiota transplantation at a hospital in the Republic of Korea. Phylogenetic and functional analyses were performed to understand the physiological characteristics and functions of Bifidobacterium spp. in the human intestine.

Identification of Metagenomics Structure and Function Associated With Temporal Changes in Rat (Rattus norvegicus) Skin Microbiome During Health and Cutaneous Burn.

The cutaneous skin microbiome is host to a vast ensemble of resident microbes that provide essential capabilities including protection of skin barrier integrity and modulation of the host immune response. Cutaneous burn-injury promotes alteration of cutaneous and systemic immune response that can affect both commensal and pathogenic microbes. A cross-sectional study of a limited number of burn patients revealed a difference in the bacteriome of burned versus control participants. Temporal changes of the skin microbiome during health and cutaneous burn-injury remains largely unknown. Furthermore, how this microbial shift relates to community function in the collective metagenome remain elusive. Due to cost considerations and reduced healing time, rodents are frequently used in burn research, despite inherent physiological differences between rodents and human skin. Using a rat burn model, a longitudinal study was conducted to characterize the rat skin bacterial residents and associated community functions in states of health (n = 30) (sham-burned) and when compromised by burn-injury (n = 24). To address the knowledge gap, traumatic thermal injury and disruption of cutaneous surface is associated with genus-level changes in the microbiota, reduced bacterial richness, and altered representation of bacterial genes and associated predicted functions across different skin microbial communities. These findings demonstrate that, upon burn-injury, there is a shift in diversity of the skin's organismal assemblages, yielding a core microbiome that is distinct at the genome and functional level. Moreover, deviations from the core community correlate with temporal changes post-injury and community transition from the state of cutaneous health to disease (burn-injury).

Temporal shifts in the mycobiome structure and network architecture associated with a rat (Rattus norvegicus) deep partial-thickness cutaneous burn.

With a diverse physiological interface to colonize, mammalian skin is the first line of defense against pathogen invasion and harbors a consortium of microbes integral in maintenance of epithelial barrier function and disease prevention. While the dynamic roles of skin bacterial residents are expansively studied, contributions of fungal constituents, the mycobiome, are largely overlooked. As a result, their influence during skin injury, such as disruption of skin integrity in burn injury and impairment of host immune defense system, is not clearly delineated. Burn patients experience a high risk of developing hard-to-treat fungal infections in comparison to other hospitalized patients. To discern the changes in the mycobiome profile and network assembly during cutaneous burn-injury, a rat scald burn model was used to survey the mycobiome in healthy (n = 30) (sham-burned) and burned (n = 24) skin over an 11-day period. The healthy skin demonstrated inter-animal heterogeneity over time, while the burned skin mycobiome transitioned toward a temporally stabile community with declining inter-animal variation starting at day 3 post-burn injury. Driven primarily by a significant increase in relative abundance of Candida, fungal species richness and abundance of the burned skin decreased, especially in days 7 and 11 post-burn. The network architecture of rat skin mycobiome displayed community reorganization toward increased network fragility and decreased stability compared to the healthy rat skin fungal network. This study provides the first account of the dynamic diversity observed in the rat skin mycobiome composition, structure, and network assembly associated with postcutaneous burn injury.

Clinical Utility of PNA-FISH for Burn Wound Diagnostics: A Noninvasive, Culture-Independent Technique for Rapid Identification of Pathogenic Organisms in Burn Wounds.

Burn injury results in an immediate compromised skin state, which puts the affected patient at an immediate risk for infection, including sepsis. For burn patients that develop infections, it is critical to rapidly identify the etiology so that an appropriate treatment can be administered. Current clinical standards rely heavily on culture-based methods for local and systemic infection testing, which can often take days to complete. While more advanced methods (ie, MALDI or NAAT) have improved turnaround times, they may still suffer from either the need for pure culture or sensitivity and specificity issues. Peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) offers a way to reduce this time from days to hours and provide species-specific identification. While PNA-FISH has had great utility in research, its use in clinical microbiology diagnostics has been minimal (including burn wound diagnostics). This work describes a nonculture-based identification technique using commercial available U.S. FDA-approved PNA-FISH probes for the identification of common clinical pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, present in burn wound infections. Additionally, calcofluor white was included for identification of Candida albicans. All three pathogens were identified from a tri-species infected deep-partial thickness rat burn wound model. These species were clearly identifiable in swab and tissue samples that were collected, with minimal autofluorescence from any species. Although autofluorescence of the tissue was present, it did not interfere or was otherwise minimized through sample preparation and analysis. The methodology developed was done so with patient care and diagnostic laboratories in mind that it might be easily transferred to the clinical setting.

Whole-Genome Sequence of Multidrug-Resistant Pseudomonas aeruginosa Strain BAMCPA07-48, Isolated from a Combat Injury Wound.

We report here the complete genome sequence of Pseudomonas aeruginosa strain BAMCPA07-48, isolated from a combat injury wound. The closed genome sequence of this isolate is a valuable resource for pathogenome characterization of P. aeruginosa associated with wounds, which will aid in the development of a higher-resolution phylogenomic framework for molecular-guided pathogen-surveillance.

Whole Genome Sequencing for Genomics-Guided Investigations of Escherichia coli O157:H7 Outbreaks.

Multi isolate whole genome sequencing (WGS) and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and long-term evolution and can complement currently employed typing schemes for outbreak ex- and inclusion, diagnostics, surveillance, and forensic studies.

Escherichia coli O157:H7 strains harbor at least three distinct sequence types of Shiga toxin 2a-converting phages.

BACKGROUND: Shiga toxin-producing Escherichia coli O157:H7 is a foodborne pathogen that causes severe human diseases including hemolytic uremic syndrome (HUS). The virulence factor that mediates HUS, Shiga toxin (Stx), is encoded within the genome of a lambdoid prophage. Although draft sequences are publicly available for a large number of E. coli O157:H7 strains, the high sequence similarity of stx-converting bacteriophages with other lambdoid prophages poses challenges to accurately assess the organization and plasticity among stx-converting phages due to assembly difficulties. METHODS: To further explore genome plasticity of stx-converting prophages, we enriched phage DNA from 45 ciprofloxacin-induced cultures for subsequent 454 pyrosequencing to facilitate assembly of the complete phage genomes. In total, 22 stx2a-converting phage genomes were closed. RESULTS: Comparison of the genomes distinguished nine distinct phage sequence types (PSTs) delineated by variation in obtained sequences, such as single nucleotide polymorphisms (SNPs) and insertion sequence element prevalence and location. These nine PSTs formed three distinct clusters, designated as PST1, PST2 and PST3. The PST2 cluster, identified in two clade 8 strains, was related to stx2a-converting phages previously identified in non-O157 Shiga-toxin producing E. coli (STEC) strains associated with a high incidence of HUS. The PST1 cluster contained phages related to those from E. coli O157:H7 strain Sakai (lineage I, clade 1), and PST3 contained a single phage that was distinct from the rest but most related to the phage from E. coli O157:H7 strain EC4115 (lineage I/II, clade 8). Five strains carried identical stx2a-converting phages (PST1-1) integrated at the same chromosomal locus, but these strains produced different levels of Stx2. CONCLUSION: The stx2a-converting phages of E. coli O157:H7 can be categorized into at least three phage types. Diversification within a phage type is mainly driven by IS629 and by a small number of SNPs. Polymorphisms between phage genomes may help explain differences in Stx2a production between strains, however our data indicates that genes encoded external to the phage affect toxin production as well.

Characterization of the pathogenome and phylogenomic classification of enteropathogenic Escherichia coli of the O157:non-H7 serotypes.

Escherichia coli of the O157 serogroup are comprised of a diverse collection of more than 100 O157:non-H7 serotypes that are found in the environment, animal reservoir and infected patients and some have been linked to severe outbreaks of human disease. Among these, the enteropathogenic E. coli O157:non-H7 serotypes carry virulence factors that are hallmarks of enterohemorrhagic E. coli, such as causing attaching and effacing lesions during human gastrointestinal tract infections. Given the shared virulence gene pool between O157:H7 and O157:non-H7 serotypes, our objective was to examine the prevalence of virulence traits of O157:non-H7 serotypes within and across their H-serotype and when compared to other E. coli pathovars. We sequenced six O157:non-H7 genomes complemented by four genomes from public repositories in an effort to determine their virulence state and genetic relatedness to the highly pathogenic enterohemorrhagic O157:H7 lineage and its ancestral O55:H7 serotype. Whole-genome-based phylogenomic analysis and molecular typing is indicative of a non-monophyletic origin of the heterogeneous O157:non-H7 serotypes that are only distantly related to the O157:H7 serotype. The availability of multiple genomes enables robust phylogenomic placement of these strains into their evolutionary context, and the assessment of the pathogenic potential of the O157:non-H7 strains in causing human disease.

Genomic epidemiology of the Haitian cholera outbreak: a single introduction followed by rapid, extensive, and continued spread characterized the onset of the epidemic.

UNLABELLED: For centuries, cholera has been one of the most feared diseases. The causative agent Vibrio cholerae is a waterborne Gram-negative enteric pathogen eliciting a severe watery diarrheal disease. In October 2010, the seventh pandemic reached Haiti, a country that had not experienced cholera for more than a century. By using whole-genome sequence typing and mapping strategies of 116 serotype O1 strains from global sources, including 44 Haitian genomes, we present a detailed reconstructed evolutionary history of the seventh pandemic with a focus on the Haitian outbreak. We catalogued subtle genomic alterations at the nucleotide level in the genome core and architectural rearrangements from whole-genome map comparisons. Isolates closely related to the Haitian isolates caused several recent outbreaks in southern Asia. This study provides evidence for a single-source introduction of cholera from Nepal into Haiti followed by rapid, extensive, and continued clonal expansion. The phylogeographic patterns in both southern Asia and Haiti argue for the rapid dissemination of V. cholerae across the landscape necessitating real-time surveillance efforts to complement the whole-genome epidemiological analysis. As eradication efforts move forward, phylogeographic knowledge will be important for identifying persistent sources and monitoring success at regional levels. The results of molecular and epidemiological analyses of this outbreak suggest that an indigenous Haitian source of V. cholerae is unlikely and that an indigenous source has not contributed to the genomic evolution of this clade. IMPORTANCE: In this genomic epidemiology study, we have applied high-resolution whole-genome-based sequence typing methodologies on a comprehensive set of genome sequences that have become available in the aftermath of the Haitian cholera epidemic. These sequence resources enabled us to reassess the degree of genomic heterogeneity within the Vibrio cholerae O1 serotype and to refine boundaries and evolutionary relationships. The established phylogenomic framework showed how outbreak isolates fit into the global phylogeographic patterns compared to a comprehensive globally and temporally diverse strain collection and provides strong molecular evidence that points to a nonindigenous source of the 2010 Haitian cholera outbreak and refines epidemiological standards used in outbreak investigations for outbreak inclusion/exclusion following the concept of genomic epidemiology. The generated phylogenomic data have major public health relevance in translating sequence-based information to assist in future diagnostic, epidemiological, surveillance, and forensic studies of cholera.

Genome Sequence of Escherichia coli O157:H7 Strain 2886-75, Associated with the First Reported Case of Human Infection in the United States.

First identified in 1982 as a human pathogen, enterohemorrhagic Escherichia coli of the O157:H7 serotype is a major cause of food-borne acquired human infections. Here, we report the genome sequence of the first known strain of this serotype isolated in the United States.