Critical knowledge gaps of orthopedic infections pertain to bacterial colonization. The established dogma termed the Race for the Surface posits that contaminating bacteria compete with host cells ...for the implant post‐op, which remains unproven without real‐time in vivo evidence. Thus, we modified the murine longitudinal intravital imaging of the bone marrow (LIMB) system to allow real‐time quantification of green fluorescent protein (GFP+) host cells and enhanced cyan fluorescent protein (ECFP+) or red fluorescent protein (RFP+) methicillin‐resistant Staphylococcus aureus (MRSA) proximal to a transfemoral implant. Following inoculation with ~105 CFU, an L‐shaped metal implant was press‐fit through the lateral cortex at a 90° angle ~0.150 mm below a gradient refractive index (GRIN) lens. We empirically derived a volume of interest (VOI) = 0.0161 ± 0.000675 mm3 during each imaging session by aggregating the Z‐stacks between the first (superior) and last (inferior) in‐focus LIMB slice. LIMB postimplantation revealed very limited bacteria detection at 1 h, but by 3 h, 56.8% of the implant surface was covered by ECFP+ bacteria, and the rest were covered by GFP+ host cells. 3D volumetric rendering of the GFP+ and ECFP+ or RFP+ voxels demonstrated exponential MRSA growth between 3 and 6 h in the Z‐plane, which was validated with cross‐sectional ex vivo bacterial burden analyses demonstrating significant growth by ~2 × 104 CFU/h on the implant from 2 to 12 h post‐op (p < 0.05; r2 > 0.98). Collectively, these results show the competition at the surface is completed by 3 h in this model and demonstrate the potential of LIMB to elucidate mechanisms of bacterial colonization, the host immune response, and the efficacy of antimicrobials.
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
metabolically adapts through multiple strategies that are conserved in all CCs, including an
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
expression enhanced by NO stress and anaerobic growth. Furthermore, we demonstrate that
is regulated by MgrA and SrrAB, which modulate
virulence and hypoxic response. Transcriptome analysis of the
UAMS-1, UAMS-1 Δ
, and UAMS-1 Δ
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
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.
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
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
CC30 to NO and oxygen will guide the treatment of severe disease presentations.
Respiratory syncytial virus (RSV) infection results in millions of hospitalizations and thousands of deaths each year. Variations in the adaptive and innate immune response appear to be associated ...with RSV severity. To investigate the host response to RSV infection in infants, we performed a systems-level study of RSV pathophysiology, incorporating high-throughput measurements of the peripheral innate and adaptive immune systems and the airway epithelium and microbiota. We implemented a novel multi-omic data integration method based on multilayered principal component analysis, penalized regression, and feature weight back-propagation, which enabled us to identify cellular pathways associated with RSV severity. In both airway and immune cells, we found an association between RSV severity and activation of pathways controlling Th17 and acute phase response signaling, as well as inhibition of B cell receptor signaling. Dysregulation of both the humoral and mucosal response to RSV may play a critical role in determining illness severity.
The Ca2+‐sensing stromal interaction molecule (STIM) proteins are crucial Ca2+ signal coordinators. Cre‐lox technology was used to generate smooth muscle (sm)‐targeted STIM1‐, STIM2‐, and double ...STIM1/STIM2‐knockout (KO) mouse models, which reveal the essential role of STIM proteins in Ca2+ homeostasis and their crucial role in controlling function, growth, and development of smooth muscle cells (SMCs). Compared to Cre+/– littermates, sm‐STIM1‐KO mice showed high mortality (50% by 30 d) and reduced bodyweight. While sm‐STIM2‐KO was without detectable phenotype, the STIM1/STIM double‐KO was perinatally lethal, revealing an essential role of STIM1 partially rescued by STIM2. Vascular and intestinal smooth muscle tissues from sm‐STIM1‐KO mice developed abnormally with distended, thinned morphology. While depolarization‐induced aortic contraction was unchanged in sm‐STIM1‐KO mice, α1‐adrenergic‐mediated contraction was 26% reduced, and store‐dependent contraction almost eliminated. Neointimal formation induced by carotid artery ligation was suppressed by 54%, and in vitro PDGF‐induced proliferation was greatly reduced (79%) in sm‐STIM1‐KO. Notably, the Ca2+ store‐refilling rate in STIM1‐KO SMCs was substantially reduced, and sustained PDGF‐induced Ca2+ entry was abolished. This defective Ca2+ homeostasis prevents PDGF‐induced NFAT activation in both contractile and proliferating SMCs. We conclude that STIM1‐regulated Ca2+ homeostasis is crucial for NFAT‐mediated transcriptional control required for induction of SMC proliferation, development, and growth responses to injury.—Mancarella, S., Potireddy, S., Wang, Y., Gao, H., Gandhirajan, K., Autieri, M., Scalia, R., Cheng, Z., Wang, H., Madesh, M., Houser, S. R., Gill, D. L. Targeted STIM deletion impairs calcium homeostasis, NFAT activation, and growth of smooth muscle. FASEB J. 27, 893–906 (2013). www.fasebj.org
USA300, a clone of meticillin-resistant
Staphylococcus aureus, is a major source of community-acquired infections in the USA, Canada, and Europe. Our aim was to sequence its genome and compare it ...with those of other strains of
S aureus to try to identify genes responsible for its distinctive epidemiological and virulence properties.
We ascertained the genome sequence of FPR3757, a multidrug resistant USA300 strain, by random shotgun sequencing, then compared it with the sequences of ten other staphylococcal strains.
Compared with closely related
S aureus, we noted that almost all of the unique genes in USA300 clustered in novel allotypes of mobile genetic elements. Some of the unique genes are involved in pathogenesis, including Panton-Valentine leucocidin and molecular variants of enterotoxin Q and K. The most striking feature of the USA300 genome is the horizontal acquisition of a novel mobile genetic element that encodes an arginine deiminase pathway and an oligopeptide permease system that could contribute to growth and survival of USA300. We did not detect this element, termed arginine catabolic mobile element (ACME), in other
S aureus strains. We noted a high prevalence of ACME in
S epidermidis, suggesting not only that ACME transfers into USA300 from
S epidermidis, but also that this element confers a selective advantage to this ubiquitous commensal of the human skin.
USA300 has acquired mobile genetic elements that encode resistance and virulence determinants that could enhance fitness and pathogenicity.
Although it is well-established that fluorouracil- (FU-) based adjuvant therapy improves survival for patients with resected high-risk colon cancer, the magnitude of adjuvant therapy benefit across ...specific subgroups and for individual patients has been uncertain.
Using a pooled data set of 3,302 patients with stage II and III colon cancer from seven randomized trials comparing FU + leucovorin or FU + levamisole to surgery alone, we performed an analysis based on a Cox proportional hazards regression model. Treatment, age, sex, tumor location, T stage, nodal status, and grade were tested for both prognostic and predictive significance. Model derived estimates of 5-year disease-free survival and overall survival (OS) for surgery alone and surgery plus FU-based therapy were calculated for a range of patient subsets.
Nodal status, T stage, and grade were the only prognostic factors independently significant for both disease-free survival and OS. Age was significant only for OS. In a multivariate analysis, adjuvant therapy showed a beneficial treatment effect across all subsets. Treatment benefits were consistent across sex, location, age, T-stage, and grade. A significant stage by treatment interaction was present, with treatment benefiting stage III patients to a greater degree than stage II patients.
Patients with high-risk resected colon cancer obtain benefit from FU-based therapy across subsets of age, sex, location, T stage, nodal status, and grade. Model estimates of survival stratified by T stage, nodal status, grade, and age are available at http://www.mayoclinic.com/calcs. This information may improve patients' and physicians' understanding of the potential benefits of adjuvant therapy.
Leucovorin, fluorouracil, and oxaliplatin (FOLFOX) is the standard adjuvant therapy for resected stage III colon cancer. Adding cetuximab to FOLFOX benefits patients with metastatic wild-type KRAS ...but not mutated KRAS colon cancer.
To assess the potential benefit of cetuximab added to the modified sixth version of the FOLFOX regimen (mFOLFOX6) in patients with resected stage III wild-type KRAS colon cancer.
A randomized trial of 2686 patients aged 18 years or older at multiple institutions across North America enrolled following resection and informed consent between February 10, 2004, and November 25, 2009. The primary randomized comparison was 12 biweekly cycles of mFOLFOX6 with and without cetuximab. KRAS mutation status was centrally determined. The trial was halted after a planned interim analysis of 48% of predicted events (246/515) occurring in 1863 (of 2070 planned) patients with tumors having wild-type KRAS. A total of 717 patients with mutated KRAS and 106 with indeterminate KRAS were accrued. The 2070 patients with wild-type KRAS provided 90% power to detect a hazard ratio (HR) of 1.33 (2-sided α = .05), with planned interim efficacy analyses after 25%, 50%, and 75% of expected relapses.
Disease-free survival in patients with wild-type KRAS mutations. Secondary end points included overall survival and toxicity.
Median (range) follow-up was 28 (0-68) months. The trial demonstrated no benefit when adding cetuximab. Three-year disease-free survival for mFOLFOX6 alone was 74.6% vs 71.5% with the addition of cetuximab (HR, 1.21; 95% CI, 0.98-1.49; P = .08) in patients with wild-type KRAS, and 67.1% vs 65.0% (HR, 1.12; 95% CI, 0.86-1.46; P = .38) in patients with mutated KRAS, with no significant benefit in any subgroups assessed. Among all patients, grade 3 or higher adverse events (72.5% vs 52.3%; odds ratio OR, 2.4; 95% CI, 2.1-2.8; P < .001) and failure to complete 12 cycles (33% vs 23%; OR, 1.6; 95% CI, 1.4-1.9; P < .001) were significantly higher with cetuximab. Increased toxicity and greater detrimental differences in all outcomes were observed in patients aged 70 years or older.
Among patients with stage III resected colon cancer, the use of cetuximab with adjuvant mFOLFOX6 compared with mFOLFOX6 alone did not result in improved disease-free survival.
clinicaltrials.gov Identifier: NCT00079274.
Development of the intestinal microbiome in preterm infants has significant impact on infant health. Our objective was to determine if duration of antibiotics within the first 10 and 30 d after birth ...affects the intestinal microbiome.
Subjects were 24 0/7-31 6/7 wk of gestational age who received ≥ 50% breast milk and a total of ≥ 100 ml/kg of feeds by 10 d. Rectal (fecal) swabs were collected at 10 and 30 d and analyzed by 16S rRNA pyrosequencing. At both time points, we examined the rectal microbiome from infants who received only 2 d of antibiotics and those who received at least 7 d of antibiotics.
In the 29 infants enrolled in our study, we found a decrease in diversity index from 10 d samples in those who received more antibiotics. Such difference in diversity and richness was not as pronounced in 30 d samples. Firmicutes and Bacteroidetes were most abundant in the 10 d samples. While these two phyla remained dominant in 30 d samples, there was an increase in Proteobacteria and Actinobacteria.
Despite antibiotic therapy, neonates continued to acquire bacteria in the gastrointestinal tract. The process of bacterial acquisition is perturbed with the use of antibiotics.
Mycoplasma genitalium with 517 genes has the smallest gene complement of any independently replicating cell so far identified. Global transposon mutagenesis was used to identify nonessential genes in ...an effort to learn whether the naturally occurring gene complement is a true minimal genome under laboratory growth conditions. The positions of 2209 transposon insertions in the completely sequenced genomes of M. genitalium and its close relative M. pneumoniae were determined by sequencing across the junction of the transposon and the genomic DNA. These junctions defined 1354 distinct sites of insertion that were not lethal. The analysis suggests that 265 to 350 of the 480 protein-coding genes of M. genitalium are essential under laboratory growth conditions, including about 100 genes of unknown function.
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