Modulation of Gut Microbiota Metabolism in Obesity-Related Type 2 Diabetes Reduces Osteomyelitis Severity.

Staphylococcus aureus is an opportunistic pathogen causing osteomyelitis through hematogenous seeding or contamination of implants and open wounds following orthopedic surgeries. The severity of S. aureus-mediated osteomyelitis is enhanced in obesity-related type 2 diabetes (obesity/T2D) due to chronic inflammation impairing both adaptive and innate immunity. Obesity-induced inflammation is linked to gut dysbiosis, with modification of the gut microbiota by high-fiber diets leading to a reduction in the symptoms and complications of obesity/T2D. However, our understanding of the mechanisms by which modifications of the gut microbiota alter host infection responses is limited. To address this gap, we monitored tibial S. aureus infections in obese/T2D mice treated with the inulin-like fructan fiber oligofructose. Treatment with oligofructose significantly decreased S. aureus colonization and lowered proinflammatory signaling postinfection in obese/T2D mice, as observed by decreased circulating inflammatory cytokines (tumor necrosis factor-α [TNF-α]) and chemokines (interferon-γ-induced protein 10 kDa [IP-10], keratinocyte-derived chemokine [KC], monokine induced by interferon-γ [MIG], monocyte chemoattractant protein-1 [MCP-1], and regulated upon activation, normal T cell expressed and presumably secreted [RANTES]), indicating partial reduction in inflammation. Oligofructose markedly shifted diversity in the gut microbiota of obese/T2D mice, with notable increases in the anti-inflammatory bacterium Bifidobacterium pseudolongum. Analysis of the cecum and plasma metabolome suggested that polyamine production was increased, specifically spermine and spermidine. Oral administration of these polyamines to obese/T2D mice resulted in reduced infection severity similar to oligofructose supplementation, suggesting that polyamines can mediate the beneficial effects of fiber on osteomyelitis severity. These results demonstrate the contribution of gut microbiota metabolites to the control of bacterial infections distal to the gut and polyamines as an adjunct therapeutic for osteomyelitis in obesity/T2D. IMPORTANCE Individuals with obesity-related type 2 diabetes (obesity/T2D) are at a five times increased risk for invasive Staphylococcus aureus osteomyelitis (bone infection) following orthopedic surgeries. With increasing antibiotic resistance and limited discoveries of novel antibiotics, it is imperative that we explore other avenues for therapeutics. In this study, we demonstrated that the dietary fiber oligofructose markedly reduced osteomyelitis severity and hyperinflammation following acute prosthetic joint infections in obese/T2D mice. Reduced infection severity was associated with changes in gut microbiota composition and metabolism, as indicated by increased production of natural polyamines in the gut and circulating plasma. This work identifies a novel role for the gut microbiome in mediating control of bacterial infections and polyamines as beneficial metabolites involved in improving the obesity/T2D host response to osteomyelitis. Understanding the impact of polyamines on host immunity and mechanisms behind decreasing susceptibility to severe implant-associated osteomyelitis is crucial to improving treatment strategies for this patient population.

Staphylococcus aureus Cell Wall Biosynthesis Modulates Bone Invasion and Osteomyelitis Pathogenesis.

Staphylococcus aureus invasion of the osteocyte lacuno-canalicular network (OLCN) is a novel mechanism of bacterial persistence and immune evasion in chronic osteomyelitis. Previous work highlighted S. aureus cell wall transpeptidase, penicillin binding protein 4 (PBP4), and surface adhesin, S. aureus surface protein C (SasC), as critical factors for bacterial deformation and propagation through nanopores in vitro, representative of the confined canaliculi in vivo. Given these findings, we hypothesized that cell wall synthesis machinery and surface adhesins enable durotaxis- and haptotaxis-guided invasion of the OLCN, respectively. Here, we investigated select S. aureus cell wall synthesis mutants (Δpbp3, Δatl, and ΔmreC) and surface adhesin mutants (ΔclfA and ΔsasC) for nanopore propagation in vitro and osteomyelitis pathogenesis in vivo. In vitro evaluation in the microfluidic silicon membrane-canalicular array (µSiM-CA) showed pbp3, atl, clfA, and sasC deletion reduced nanopore propagation. Using a murine model for implant-associated osteomyelitis, S. aureus cell wall synthesis proteins were found to be key modulators of S. aureus osteomyelitis pathogenesis, while surface adhesins had minimal effects. Specifically, deletion of pbp3 and atl decreased septic implant loosening and S. aureus abscess formation in the medullary cavity, while deletion of surface adhesins showed no significant differences. Further, peri-implant osteolysis, osteoclast activity, and receptor activator of nuclear factor kappa-B ligand (RANKL) production were decreased following pbp3 deletion. Most notably, transmission electron microscopy (TEM) imaging of infected bone showed that pbp3 was the only gene herein associated with decreased submicron invasion of canaliculi in vivo. Together, these results demonstrate that S. aureus cell wall synthesis enzymes are critical for OLCN invasion and osteomyelitis pathogenesis in vivo.

Staphylococcus aureus Tolerance and Genomic Response to Photodynamic Inactivation.

Staphylococcus aureus is an opportunistic pathogen with a clinical spectrum ranging from asymptomatic skin colonization to invasive infections. While traditional antibiotic therapies can be effective against S. aureus, the increasing prevalence of antibiotic-resistant strains results in treatment failures and high mortality rates. Photodynamic inactivation (PDI) is an innovative and promising alternative to antibiotics. While progress has been made in our understanding of the bacterial response to PDI, major gaps remain in our knowledge of PDI tolerance, the global cellular response, and adaptive genomic mutations acquired as a result of PDI. To address these gaps, S. aureus HG003 and isogenic mutants with mutations in agr, mutS, mutL, and mutY exposed to single or multiple doses of PDI were assessed for survival and tolerance and examined by global transcriptome and genome analyses to identify regulatory and genetic adaptations that contribute to tolerance. Pathways in inorganic ion transport, oxidative response, DNA replication recombination and repair, and cell wall and membrane biogenesis were identified in a global cellular response to PDI. Tolerance to PDI was associated with superoxide dismutase and the S. aureus global methylhydroquinone (MHQ)-quinone transcriptome network. Genome analysis of PDI-tolerant HG003 identified a nonsynonymous mutation in the quinone binding domain of the transcriptional repressor QsrR, which mediates quinone sensing and oxidant response. Acquisition of a heritable QsrR mutation through repeated PDI treatment demonstrates selective adaption of S. aureus to PDI. PDI tolerance of a qsrR gene deletion in HG003 confirmed that QsrR regulates the S. aureus response to PDI.IMPORTANCEStaphylococcus aureus can cause disease at most body sites, with illness ranging from asymptomatic infection to death. The increasing prevalence of antibiotic-resistant strains results in treatment failures and high mortality rates. S. aureus acquires resistance to antibiotics through multiple mechanisms, often by genetic variation that alters antimicrobial targets. Photodynamic inactivation (PDI), which employs a combination of a nontoxic dye and low-intensity visible light, is a promising alternative to antibiotics that effectively eradicates S. aureus in human infections when antibiotics are no longer effective. In this study, we demonstrate that repeated exposure to PDI results in resistance of S. aureus to further PDI treatment and identify the underlying bacterial mechanisms that contribute to resistance. This work supports further analysis of these mechanisms and refinement of this novel technology as an adjunctive treatment for S. aureus infections.

Identification of Penicillin Binding Protein 4 (PBP4) as a critical factor for Staphylococcus aureus bone invasion during osteomyelitis in mice.

Staphylococcus aureus infection of bone is challenging to treat because it colonizes the osteocyte lacuno-canalicular network (OLCN) of cortical bone. To elucidate factors involved in OLCN invasion and identify novel drug targets, we completed a hypothesis-driven screen of 24 S. aureus transposon insertion mutant strains for their ability to propagate through 0.5 µm-sized pores in the Microfluidic Silicon Membrane Canalicular Arrays (µSiM-CA), developed to model S. aureus invasion of the OLCN. This screen identified the uncanonical S. aureus transpeptidase, penicillin binding protein 4 (PBP4), as a necessary gene for S. aureus deformation and propagation through nanopores. In vivo studies revealed that Δpbp4 infected tibiae treated with vancomycin showed a significant 12-fold reduction in bacterial load compared to WT infected tibiae treated with vancomycin (p<0.05). Additionally, Δpbp4 infected tibiae displayed a remarkable decrease in pathogenic bone-loss at the implant site with and without vancomycin therapy. Most importantly, Δpbp4 S. aureus failed to invade and colonize the OLCN despite high bacterial loads on the implant and in adjacent tissues. Together, these results demonstrate that PBP4 is required for S. aureus colonization of the OLCN and suggest that inhibitors may be synergistic with standard of care antibiotics ineffective against bacteria within the OLCN.

Evolving concepts in bone infection: redefining "biofilm", "acute vs. chronic osteomyelitis", "the immune proteome" and "local antibiotic therapy".

Osteomyelitis is a devastating disease caused by microbial infection of bone. While the frequency of infection following elective orthopedic surgery is low, rates of reinfection are disturbingly high. Staphylococcus aureus is responsible for the majority of chronic osteomyelitis cases and is often considered to be incurable due to bacterial persistence deep within bone. Unfortunately, there is no consensus on clinical classifications of osteomyelitis and the ensuing treatment algorithm. Given the high patient morbidity, mortality, and economic burden caused by osteomyelitis, it is important to elucidate mechanisms of bone infection to inform novel strategies for prevention and curative treatment. Recent discoveries in this field have identified three distinct reservoirs of bacterial biofilm including: Staphylococcal abscess communities in the local soft tissue and bone marrow, glycocalyx formation on implant hardware and necrotic tissue, and colonization of the osteocyte-lacuno canalicular network (OLCN) of cortical bone. In contrast, S. aureus intracellular persistence in bone cells has not been substantiated in vivo, which challenges this mode of chronic osteomyelitis. There have also been major advances in our understanding of the immune proteome against S. aureus, from clinical studies of serum antibodies and media enriched for newly synthesized antibodies (MENSA), which may provide new opportunities for osteomyelitis diagnosis, prognosis, and vaccine development. Finally, novel therapies such as antimicrobial implant coatings and antibiotic impregnated 3D-printed scaffolds represent promising strategies for preventing and managing this devastating disease. Here, we review these recent advances and highlight translational opportunities towards a cure.

An in vitro platform for elucidating the molecular genetics of S. aureus invasion of the osteocyte lacuno-canalicular network during chronic osteomyelitis.

Staphylococcus aureus osteomyelitis is a devasting disease that often leads to amputation. Recent findings have shown that S. aureus is capable of invading the osteocyte lacuno-canalicular network (OLCN) of cortical bone during chronic osteomyelitis. Normally a 1 µm non-motile cocci, S. aureus deforms smaller than 0.5 µm in the sub-micron channels of the OLCN. Here we present the µSiM-CA (Microfluidic - Silicon Membrane - Canalicular Array) as an in vitro screening platform for the genetic mechanisms of S. aureus invasion. The µSiM-CA platform features an ultrathin silicon membrane with defined pores that mimic the openings of canaliculi. While we anticipated that S. aureus lacking the accessory gene regulator (agr) quorum-sensing system would not be capable of invading the OLCN, we found no differences in propagation compared to wild type in the µSiM-CA. However the µSiM-CA proved predictive as we also found that the agr mutant strain invaded the OLCN of murine tibiae.

The Response of nor and nos Contributes to Staphylococcus aureus Virulence and Metabolism.

Staphylococcus aureus causes a wide spectrum of disease, with the site and severity of infection dependent on virulence traits encoded within genetically distinct clonal complexes (CCs) and bacterial responses to host innate immunity. The production of nitric oxide (NO) by activated phagocytes is a major host response to which S. aureus metabolically adapts through multiple strategies that are conserved in all CCs, including an S. aureus nitric oxide synthase (Nos). Previous genome analysis of CC30, a lineage associated with chronic endocardial and osteoarticular infections, revealed a putative NO reductase (Nor) not found in other CCs that potentially contributes to NO resistance and clinical outcome. Here, we demonstrate that Nor has true nitric oxide reductase activity, with nor expression enhanced by NO stress and anaerobic growth. Furthermore, we demonstrate that nor is regulated by MgrA and SrrAB, which modulate S. aureus virulence and hypoxic response. Transcriptome analysis of the S. aureus UAMS-1, UAMS-1 Δnor, and UAMS-1 Δnos strains under NO stress and anaerobic growth demonstrates that Nor contributes to nucleotide metabolism and Nos to glycolysis. We demonstrate that Nor and Nos contribute to enhanced survival in the presence of human human polymorphonuclear cells and have organ-specific seeding in a tail vein infection model. Nor contributes to abscess formation in an osteological implant model. We also demonstrate that Nor has a role in S. aureus metabolism and virulence. The regulation overlap between Nor and Nos points to an intriguing link between regulation of intracellular NO, metabolic adaptation, and persistence in the CC30 lineage.IMPORTANCEStaphylococcus aureus can cause disease at most body sites, and illness spans asymptomatic infection to death. The variety of clinical presentations is due to the diversity of strains, which are grouped into distinct clonal complexes (CCs) based on genetic differences. The ability of S. aureus CC30 to cause chronic infections relies on its ability to evade the oxidative/nitrosative defenses of the immune system and survive under different environmental conditions, including differences in oxygen and nitric oxide concentrations. The significance of this work is the exploration of unique genes involved in resisting NO stress and anoxia. A better understanding of the functions that control the response of S. aureus CC30 to NO and oxygen will guide the treatment of severe disease presentations.

Enterococcus faecalis enhances cell proliferation through hydrogen peroxide-mediated epidermal growth factor receptor activation.

Enterococcus faecalis is a member of the intestinal and oral microbiota that may affect the etiology of colorectal and oral cancers. The mechanisms by which E. faecalis may contribute to the initiation and progression of these cancers remain uncertain. Epidermal growth factor receptor (EGFR) signaling is postulated to play a crucial role in oral carcinogenesis. A link between E. faecalis and EGFR signaling in oral cancer has not been elucidated. The present study aimed to evaluate the association between E. faecalis and oral cancer and to determine the underlying mechanisms that link E. faecalis to EGFR signaling. We report the high frequency of E. faecalis infection in oral tumors and the clinical association with EGFR activation. Using human oral cancer cells, we support the clinical findings and demonstrate that E. faecalis can induce EGFR activation and cell proliferation. E. faecalis activates EGFR through production of H(2)O(2), a signaling molecule that activates several signaling pathways. Inhibitors of H(2)O(2) (catalase) and EGFR (gefitinib) significantly blocked E. faecalis-induced EGFR activation and cell proliferation. Therefore, E. faecalis infection of oral tumor tissues suggests a possible association between E. faecalis infection and oral carcinogenesis. Interaction of E. faecalis with host cells and production of H(2)O(2) increase EGFR activation, thereby contributing to cell proliferation.

Later onset phenotypes of Krabbe disease: results of the world-wide registry.

The majority of newborns screening positive for Krabbe disease have not exhibited the expected early infantile phenotype, with most clinically normal despite low galactocerebrosidase activity and two mutations. Most are expected to develop the later onset phenotypes. The World-Wide Krabbe Registry was developed in part to expand our understanding of the natural history of these rare variants. As of June 2011, 122 patients were enrolled in the registry: 62% manifested early infantile onset (previously reported), 10% manifested onset at 7-12 months (late infantile), 22% manifested onset at 13 months to 10 years (later onset), and 5% manifested adolescent/adult onset. Data on disease course, galactocerebrosidase activity, DNA mutations, and results of neurodiagnostic studies were obtained from questionnaires and medical records. Initial signs (late infantile) included loss of milestones and poor feeding, whereas later onset and adolescent/adult phenotypes presented with changes in gait. Elevated cerebrospinal fluid protein and abnormal magnetic resonance imaging results were present in most, but not all, patients at diagnosis. Phenotypic variability occurred in four sibships. Five-year and 10-year survivals for all later onset phenotypes were at least 50%. The later onset Krabbe phenotypes differ from those with early infantile disease, but no specific predictor of phenotype was identified.

An Enterotoxin-Bearing Pathogenicity Island in Staphylococcus epidermidis.

Cocolonization of human mucosal surfaces causes frequent encounters between various staphylococcal species, creating opportunities for the horizontal acquisition of mobile genetic elements. The majority of Staphylococcus aureus toxins and virulence factors are encoded on S. aureus pathogenicity islands (SaPIs). Horizontal movement of SaPIs between S. aureus strains plays a role in the evolution of virulent clinical isolates. Although there have been reports of the production of toxic shock syndrome toxin 1 (TSST-1), enterotoxin, and other superantigens by coagulase-negative staphylococci, no associated pathogenicity islands have been found in the genome of Staphylococcus epidermidis, a generally less virulent relative of S. aureus. We show here the first evidence of a composite S. epidermidis pathogenicity island (SePI), the product of multiple insertions in the genome of a clinical isolate. The taxonomic placement of S. epidermidis strain FRI909 was confirmed by a number of biochemical tests and multilocus sequence typing. The genome sequence of this strain was analyzed for other unique gene clusters and their locations. This pathogenicity island encodes and expresses staphylococcal enterotoxin C3 (SEC3) and staphylococcal enterotoxin-like toxin L (SElL), as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and immunoblotting. We present here an initial characterization of this novel pathogenicity island, and we establish that it is stable, expresses enterotoxins, and is not obviously transmissible by phage transduction. We also describe the genome sequence, excision, replication, and packaging of a novel bacteriophage in S. epidermidis FRI909, as well as attempts to mobilize the SePI element by this phage.