Effect of Ceftaroline, Ceftazidime/Avibactam, Ceftolozane/Tazobactam, and Meropenem/Vaborbactam on Establishment of Colonization by Vancomycin-Resistant Enterococci and Klebsiella pneumoniae in Mice.

Background: The potential for promotion of intestinal colonization with healthcare-associated pathogens by new antibiotics used to treat infections due to multidrug-resistant Gram-negative bacilli is unclear. Methods: Mice treated for 3 days with daily subcutaneous phosphate-buffered saline (control), ceftazidime/avibactam, ceftolozane/tazobactam, ceftaroline, and meropenem/vaborbactam were challenged with 10,000 colony-forming units (CFU) of vancomycin-resistant Enterococcus (VRE) resistant to each of the antibioics or carbapenemase-producing Klebsiella pneumoniae 1 day after the final treatment dose. The concentrations of VRE or K. pneumoniae in stool were measured on days 1, 3, 6, and 15 after challenge. Results: Control mice had transient low levels of VRE or K. pneumoniae (<3 log10 CFU/g) detected in stool with negative cultures on days 6 and 15 after challenge. In comparison to control mice, each of the antibiotics promoted establishment of high-density colonization with VRE (mean concentration, >8 log10 CFU/g of stool on day 1 after challenge) that persisted at >4 log10 CFU/g of stool through day 15 (P<0.01). In comparison to control mice, meropenem/vaborbactam and ceftaroline promoted high-density colonization with K. pneumoniae (peak concentration, >8 log10 CFU/g of stool) (P<0.01), ceftolozane/tazobactam promoted colonization to a lesser degree (peak concentration, >5 log10 CFU/g of stool), and ceftazidime/avibactam did not promote colonization (P>0.05). Conclusions: Our results suggest that several beta-lactam antibiotics recently developed for treatment of infections with resistant Gram-negative bacilli have the potential to promote colonization by healthcare-associated pathogens. Additional studies are needed to examine the impact of these agents in patients.

Staphylococcus aureus and Acinetobacter baumannii Inhibit Osseointegration of Orthopedic Implants.

Bacterial infections routinely cause inflammation and thereby impair osseointegration of orthopedic implants. Acinetobacter spp., which cause osteomyelitis following trauma, on or off the battlefield, were, however, reported to cause neither osteomyelitis nor osteolysis in rodents. We therefore compared the effects of Acinetobacter strain M2 to those of Staphylococcus aureus in a murine implant infection model. Sterile implants and implants with adherent bacteria were inserted in the femur of mice. Bacterial burden, levels of proinflammatory cytokines, and osseointegration were measured. All infections were localized to the implant site. Infection with either S. aureus or Acinetobacter strain M2 increased the levels of proinflammatory cytokines and the chemokine CCL2 in the surrounding femurs, inhibited bone formation around the implant, and caused loss of the surrounding cortical bone, leading to decreases in both histomorphometric and biomechanical measures of osseointegration. Genetic deletion of TLR2 and TLR4 from the mice partially reduced the effects of Acinetobacter strain M2 on osseointegration but did not alter the effects of S. aureus. This is the first report that Acinetobacter spp. impair osseointegration of orthopedic implants in mice, and the murine model developed for this study will be useful for future efforts to clarify the mechanism of implant failure due to Acinetobacter spp. and to assess novel diagnostic tools or therapeutic agents.

The master developmental regulator Jab1/Cops5/Csn5 is essential for proper bone growth and survival in mice.

Jab1, also known as Csn5/Cops5, is a key subunit of the COP9 Signalosome, a highly conserved macromolecular complex. We previously reported that the conditional knockout of Jab1 in mouse limb buds and chondrocytes results in severely shortened limbs and neonatal lethal chondrodysplasia, respectively. In this study, we further investigated the specific role of Jab1 in osteoblast differentiation and postnatal bone growth by characterizing a novel mouse model, the Osx-cre; Jab1flox/flox conditional knockout (Jab1 cKO) mouse, in which Jab1 is deleted in osteoblast precursor cells. Jab1 cKO mutant mice appeared normal at birth, but developed progressive dwarfism. Inevitably, all mutant mice died prior to weaning age. The histological and micro-computed tomography analysis of mutant long bones revealed severely altered bone microarchitecture, with a significant reduction in trabecular thickness. Moreover, Jab1 cKO mouse tibiae had a drastic decrease in mineralization near the epiphyseal growth plates, and Jab1 cKO mice also developed spontaneous fractures near the tibiofibular junction. Additionally, our cell culture studies demonstrated that Jab1 deletion in osteoblast precursors led to decreased mineralization and a reduced response to TGFß and BMP signaling. Moreover, an unbiased reporter screen also identified decreased TGFß activity in Jab1-knockdown osteoblasts. Thus, Jab1 is necessary for proper osteoblast differentiation and postnatal bone growth, likely in part through its positive regulation of the TGFß and BMP signaling pathways in osteoblast progenitor cells.

Age estimation of fragmented human dental remains by secondary dentin virtual analysis.

Unlike bones, teeth are remarkably resilient and can withstand severe trauma, making age assessment based on the dentition essential for forensic analysis. Modern techniques for age estimation focus on pulp-chamber volume measurements using radiographs and computerized tomography (CT); however, these are applicable only for complete teeth (i.e., with intact crown and root). In the current study, we developed a new approach using high-resolution micro-computerized tomography (µCT) to visualize the secondary dentin (SD), an inner layer surrounding the pulp which accumulates with age, thus facilitating age estimation of fragmented and broken teeth.The growth pattern of the SD with age was analyzed for 77 lower premolars from two anthropological collections. A comparison of SD virtual segmentation and histological measurement was highly correlative (ICC = 0.95). SD was measured per volume (mm3) of a 1 mm thick slice directly below the cemento-enamel junction. Regression analysis using SD measurements increased the success rates of age estimation (82%) compared with the "gold-standard" pulp/dentin method (54%) in the range of ± 10 years. The accuracy of age estimation based on SD analysis was improved to a range of 7-8 years.The SD method thus allows age estimation with greater prediction rates and better accuracy based on only a small fragment of a tooth in a non-invasive manner. This novel methodology is easy to use, accessible, and bears implications in various fields such as forensic sciences and anthropological research.

Reference gene identification for reverse transcription-quantitative polymerase chain reaction analysis in an ischemic wound-healing model.

Reference genes are often used in RT-quantitative PCR (qPCR) analysis to normalize gene expression levels to a gene that is expressed stably across study groups. They ultimately serve as a control in RT-qPCR analysis, producing more accurate interpretation of results. Whereas many reference genes have been used in various wound-healing studies, the most stable reference gene for ischemic wound-healing analysis has yet to be identified. The goal of this study was to determine systematically the most stable reference gene for studying gene expression in a rat ischemic wound-healing model using RT-qPCR. Twelve commonly used reference genes were analyzed using RT-qPCR and geNorm data analysis to determine stability across normal and ischemic skin tissue. It was ultimately determined that Ubiquitin C (UBC) and ß-2 Microglobulin (B2M) are the most stably conserved reference genes across normal and ischemic skin tissue. UBC and B2M represent reliable reference genes for RT-qPCR studies in the rat ischemic wound model and are unaffected by sustained tissue ischemia. The geometric mean of these two stable genes provides an accurate normalization factor. These results provide insight on dependence of reference-gene stability on experimental parameters and the importance of such reference-gene investigations.

Protein kinase inhibitor γ reciprocally regulates osteoblast and adipocyte differentiation by downregulating leukemia inhibitory factor.

The protein kinase inhibitor (Pki) gene family inactivates nuclear protein kinase A (PKA) and terminates PKA-induced gene expression. We previously showed that Pkig is the primary family member expressed in osteoblasts and that Pkig knockdown increases the effects of parathyroid hormone and isoproterenol on PKA activation, gene expression, and inhibition of apoptosis. Here, we determined whether endogenous levels of Pkig regulate osteoblast differentiation. Pkig is the primary family member in murine embryonic fibroblasts (MEFs), murine marrow-derived mesenchymal stem cells, and human mesenchymal stem cells. Pkig deletion increased forskolin-dependent nuclear PKA activation and gene expression and Pkig deletion or knockdown increased osteoblast differentiation. PKA signaling is known to stimulate adipogenesis; however, adipogenesis and osteogenesis are often reciprocally regulated. We found that the reciprocal regulation predominates over the direct effects of PKA since adipogenesis was decreased by Pkig deletion or knockdown. Pkig deletion or knockdown also simultaneously increased osteogenesis and decreased adipogenesis in mixed osteogenic/adipogenic medium. Pkig deletion increased PKA-induced expression of leukemia inhibitory factor (Lif) mRNA and LIF protein. LIF neutralizing antibodies inhibited the effects on osteogenesis and adipogenesis of either Pkig deletion in MEFs or PKIγ knockdown in both murine and human mesenchymal stem cells. Collectively, our results show that endogenous levels of Pkig reciprocally regulate osteoblast and adipocyte differentiation and that this reciprocal regulation is mediated in part by LIF. Stem Cells 2013;31:2789-2799.

Involvement of NH2 terminus of PKC-delta in binding to F-actin during activation of Calu-3 airway epithelial NKCC1.

Direct binding of nonmuscle F-actin and the C2-like domain of PKC-delta (deltaC2-like domain) is involved in hormone-mediated activation of epithelial Na-K-2Cl cotransporter isoform 1 (NKCC1) in a Calu-3 airway epithelial cell line. The goal of this study was to determine the site of actin binding on the 123-amino acid deltaC2-like domain. Truncations of the deltaC2-like domain were made by restriction digestion and confirmed by nucleotide sequencing. His6-tagged peptides were expressed in bacteria, purified, and analyzed with a Coomassie blue stain for predicted size and either a 6xHis protein tag stain or an INDIA His6 probe for expression of the His6 tag. Truncated peptides were tested for competitive inhibition of binding of activated, recombinant PKC-delta with nonmuscle F-actin. Peptides from the NH2-terminal region, but not the COOH-terminal region, of the deltaC2-like domain blocked binding of activated PKC-delta to F-actin. The deltaC2-like domain and three NH2-terminal truncated peptides of 17, 83, or 108 amino acids blocked binding, with IC50 values ranging from 1.2 to 2.2 nmol (6-11 microM). NH2-terminal deltaC2-like peptides also prevented methoxamine-stimulated NKCC1 activation and pulled down endogenous actin from Calu-3 cells. The proximal NH2 terminus of the deltaC2-like domain encodes a beta1-sheet region. The amino acid sequence of the actin-binding domain is distinct from actin-binding domains in other PKC isotypes and actin-binding proteins. Our results indicate that F-actin likely binds to the beta1-sheet region of the deltaC2-like domain in airway epithelial cells.