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AIMS This study investigated the dose-effect of manganese (Mn) addition on wheat straw (WS) decomposition, and explored the potential mechanisms of Mn involved in the acceleration of WS decomposition in regards to the soil microbial communities and enzyme activities. METHODS AND RESULTS A 180-day incubation experiment was performed to examine the decomposition of WS under four Mn levels, that is, 0, 0.25, 1 and 2 mg g-1 . The effects of microbial communities and enzyme activities were evaluated using control (0 mg g-1 ) and Mn (0.25 mg g-1 ) treatments. Our results revealed that Mn (0.25 mg g-1 ) addition significantly increased WS decomposition, and enhanced the release of carbon and nitrogen. Optimal Mn addition (0.25 mg g-1 ) also caused significant increases in the activity of neutral xylanase (NEX), laccase (Lac), manganese peroxidase (MnP) and lignin peroxidase (LiP) within the incubation period. Mn (0.25 mg g-1 ) addition also enriched some operational taxonomic units (OTUs) that, in turn, had the potential ability to decompose crop straw, such as secreting lignocellulolytic enzymes. CONCLUSIONS Mn (0.25 mg g-1 ) could promote WS decomposition through enrichment of the microbial species involved in biomass decomposition, which enhanced the lignocellulose-degrading enzyme activity. SIGNIFICANCE AND IMPACT OF THE STUDY This study provides evidence for Mn to promote WS biodegradation after Mn application, opening new windows to improve the utilization efficiency of crop residues.

Streptococcus gordonii and Streptococcus sanguinis are primary colonizers of tooth surfaces and are generally associated with oral health, but can also cause infective endocarditis (IE). These species express “Siglec-like” adhesins that bind sialylated glycans on host glycoproteins, which can aid the formation of infected platelet–fibrin thrombi (vegetations) on cardiac valve surfaces. We previously determined that the ability of S. gordonii to bind sialyl T-antigen (sTa) increased pathogenicity, relative to recognition of sialylated core 2 O-glycan structures, in an animal model of IE. However, it is unclear when and where the sTa structure is displayed, and which sTa-modified host factors promote valve colonization. In this study, we identified sialylated glycoproteins in the aortic valve vegetations and plasma of rat and rabbit models of this disease. Glycoproteins that display sTa vs. core 2 O-glycan structures were identified by using recombinant forms of the streptococcal Siglec-like adhesins for lectin blotting and affinity capture, and the O-linked glycans were profiled by mass spectrometry. Proteoglycan 4 (PRG4), also known as lubricin, was a major carrier of sTa in the infected vegetations. Moreover, plasma PRG4 levels were significantly higher in animals with damaged or infected valves, as compared with healthy animals. The combined results demonstrate that, in addition to platelet GPIbα, PRG4 is a highly sialylated mucin-like glycoprotein found in aortic valve vegetations and may contribute to the persistence of oral streptococci in this protected endovascular niche. Moreover, plasma PRG4 could serve as a biomarker for endocardial injury and infection.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a significant clinical-therapeutic challenge. Of particular concern is antibiotic treatment failure in infections caused by MRSA that are “susceptible” to antibiotic in vitro. In the current study, we investigate specific purine biosynthetic pathways and stringent response mechanism(s) related to this life-threatening syndrome using genetic matched persistent and resolving MRSA clinical bacteremia isolates (PB and RB, respectively), and isogenic MRSA strain sets. We demonstrate that PB isolates (vs RB isolates) have significantly higher (p)ppGpp production, phenol-soluble-modulin expression, polymorphonuclear leukocyte lysis and survival, fibronectin/endothelial cell (EC) adherence, and EC damage. Importantly, an isogenic strain set, including JE2 parental, relP-mutant and relP-complemented strains, translated the above findings into significant outcome differences in an experimental endocarditis model. These observations indicate a significant regulation of purine biosynthesis on stringent response, and suggest the existence of a previously unknown adaptive genetic mechanism in persistent MRSA infection.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a significant clinically challenging subset of invasive, life-threatening S. aureus infections. We have recently demonstrated that purine biosynthesis plays an important role in such persistent infections. Cyclic di-AMP (c-di-AMP) is an essential and ubiquitous second messenger that regulates many cellular pathways in bacteria. However, whether there is a regulatory connection between the purine biosynthesis pathway and c-di-AMP impacting persistent outcomes was not known. Here, we demonstrated that the purine biosynthesis mutant MRSA strain, the ΔpurF strain (compared to its isogenic parental strain), exhibited the following significant differences in vitro: (i) lower ADP, ATP, and c-di-AMP levels; (ii) less biofilm formation with decreased extracellular DNA (eDNA) levels and Triton X-100-induced autolysis paralleling enhanced expressions of the biofilm formation-related two-component regulatory system lytSR and its downstream gene lrgB; (iii) increased vancomycin (VAN)-binding and VAN-induced lysis; and (iv) decreased wall teichoic acid (WTA) levels and expression of the WTA biosynthesis-related gene, tarH. Substantiating these data, the dacA (encoding diadenylate cyclase enzyme required for c-di-AMP synthesis) mutant strain (dacAG206S strain versus its isogenic wild-type MRSA and dacA-complemented strains) showed significantly decreased c-di-AMP levels, similar in vitro effects as seen above for the purF mutant and hypersusceptible to VAN treatment in an experimental biofilm-related MRSA endovascular infection model. These results reveal an important intersection between purine biosynthesis and c-di-AMP that contributes to biofilm-associated persistence in MRSA endovascular infections. This signaling pathway represents a logical therapeutic target against persistent MRSA infections. IMPORTANCE Persistent endovascular infections caused by MRSA, including vascular graft infection syndromes and infective endocarditis, are significant and growing public health threats. A particularly worrisome trend is that most MRSA isolates from these patients are “susceptible” in vitro to conventional anti-MRSA antibiotics, such as VAN and daptomycin (DAP), based on Clinical and Laboratory Standards Institute breakpoints. Yet, these antibiotics frequently fail to eliminate these infections in vivo. Therefore, the persistent outcomes in MRSA infections represent a unique and important variant of classic “antibiotic resistance” that is only disclosed during in vivo antibiotic treatment. Given the high morbidity and mortality associated with the persistent infection, there is an urgent need to understand the specific mechanism(s) of this syndrome. In the current study, we demonstrate that a functional intersection between purine biosynthesis and the second messenger c-di-AMP plays an important role in VAN persistence in experimental MRSA endocarditis. Targeting this pathway may represent a potentially novel and effective strategy for treating these life-threatening infections.

BackgroundMgrA is an important global virulence gene regulator in Staphylococcus aureus. In the present study, the role of mgrA in host-pathogen interactions related to virulence was explored in both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains.MethodsIn vitro susceptibilities to human defense peptides (HDPs), adherence to fibronectin (Fn) and endothelial cells (ECs), EC damage, α-toxin production, expression of global regulator (eg, agr RNAIII) and its downstream effectors (eg, α-toxin [hla] and Fn binding protein A [fnbA]), MgrA binding to fnbA promoter, and the effect on HDP-induced mprF and dltA expression were analyzed. The impact of mgrA on virulence was evaluated using a mouse bacteremia model.ResultsmgrA mutants displayed significantly higher susceptibility to HDPs, which might be related to the decreased HDP-induced mprF and dltA expression but decreased Fn and EC adherence, EC damage, α-toxin production, agr RNAIII, hla and fnbA expression, and attenuated virulence in the bacteremia model as compared to their respective parental and mgrA-complemented strains. Importantly, direct binding of MgrA to the fnbA promoter was observed.ConclusionsThese results suggest that mgrA mediates host-pathogen interactions and virulence and may provide a novel therapeutic target for invasive S. aureus infections.

GraS is a membrane sensor in Staphylococcus aureus that induces mprF and dltABCD expression to alter the surface positive charge upon exposure to cationic human defense peptides (HDPs). The sensing domain of GraS likely resides in the 9-residue extracellular loop (EL). In this study, we assessed a hospital-acquired methicillin-resistant S. aureus (HA-MRSA) strain (COL) for the specific role of two distinct EL mutations: F38G (bulk) and D/35/37/41K (charged inversion). Activation of mprF by polymyxin B (PMB) was reduced in the D35/37/41K mutant versus the D35/37/41G mutant, correlating with reduced surface positive charge; in contrast, these effects were less prominent in the F38G mutant but still lower than those in the parent. These data indicated that both electrostatic charge and steric bulk of the EL of GraS influence induction of genes impacting HDP resistance. Using mprF expression as a readout, we confirmed GraS signaling was pH dependent, increasing as pH was lowered (from pH 7.5 down to pH 5.5). In contrast to PMB activation, reduction of mprF was comparable at pH 5.5 between the P38G and D35/37/41K point mutants, indicating a mechanistic divergence between GraS activation by acidic pH versus cationic peptides. Survival assays in human blood and purified polymorphonuclear leukocytes (PMNs) revealed lower survival of the D35/37/41K mutant versus the F38G mutant, with both being lower than that of the parent. Virulence studies in the rabbit endocarditis model mirrored whole blood and PMN killing assay data described above. Collectively, these data confirmed the importance of specific residues within the EL of GraS in conferring essential bacterial responses for MRSA survival in infections.

Certain methicillin-resistant Staphylococcus aureus (MRSA) strains exhibit β-lactam susceptibility in vitro, ex vivo, and in vivo in the presence of NaHCO3 (NaHCO3-responsive MRSA). Here, we investigate the impact of NaHCO3 on factors required for PBP2a functionality., Certain methicillin-resistant Staphylococcus aureus (MRSA) strains exhibit β-lactam susceptibility in vitro, ex vivo, and in vivo in the presence of NaHCO3 (NaHCO3-responsive MRSA). Here, we investigate the impact of NaHCO3 on factors required for PBP2a functionality. Prototype NaHCO3-responsive and -nonresponsive MRSA strains (as defined in vitro) were assessed for the impact of NaHCO3 on the expression of genes involved in PBP2a production-maturation pathways (mecA, blaZ, pbp4, vraSR, prsA, sigB, and floA), membrane PBP2a and PrsA protein content, and membrane carotenoid content. Following NaHCO3 exposure in NaHCO3-responsive (versus nonresponsive) MRSA, there was significantly reduced expression of (i) mecA and blaZ, (ii) the vraSR-prsA gene axis, and (iii) pbp4. Carotenoid production was reduced while floA expression was increased by NaHCO3 exposure in all MRSA strains. This work underscores the distinct regulatory impact of NaHCO3 on a cadre of genes encoding factors required for the maintenance of the MRSA phenotype through PBP2a functionality and maturation.

Stem cells play a critical role in bone regeneration. Multiple populations of skeletal stem cells have been identified in long bone, while their identity and functions in alveolar bone remain unclear. Here, we identified a quiescent leptin receptor–expressing (LepR+) cell population that contributed to intramembranous bone formation. Interestingly, these LepR+ cells became activated in response to tooth extraction and generated the majority of the newly formed bone in extraction sockets. In addition, genetic ablation of LepR+ cells attenuated extraction socket healing. The parabiosis experiments revealed that the LepR+ cells in the healing sockets were derived from resident tissue rather than peripheral blood circulation. Further studies on the mechanism suggested that these LepR+ cells were responsive to parathyroid hormone/parathyroid hormone 1 receptor (PTH/PTH1R) signaling. Collectively, we demonstrate that LepR+ cells, a postnatal skeletal stem cell population, are essential for alveolar bone regeneration of extraction sockets.

Addition of sodium bicarbonate (NaHCO3) to standard antimicrobial susceptibility testing medium reveals certain methicillin-resistant Staphylococcus aureus (MRSA) strains to be highly susceptible to β-lactams. We investigated the prevalence of this phenotype (NaHCO3 responsiveness) to two β-lactams among 58 clinical MRSA bloodstream isolates. Of note, ∼75% and ∼36% of isolates displayed the NaHCO3 responsiveness phenotype to cefazolin (CFZ) and oxacillin (OXA), respectively., Addition of sodium bicarbonate (NaHCO3) to standard antimicrobial susceptibility testing medium reveals certain methicillin-resistant Staphylococcus aureus (MRSA) strains to be highly susceptible to β-lactams. We investigated the prevalence of this phenotype (NaHCO3 responsiveness) to two β-lactams among 58 clinical MRSA bloodstream isolates. Of note, ∼75% and ∼36% of isolates displayed the NaHCO3 responsiveness phenotype to cefazolin (CFZ) and oxacillin (OXA), respectively. Neither intrinsic β-lactam MICs in standard Mueller-Hinton broth (MHB) nor population analysis profiles were predictive of this phenotype. Several genotypic markers (clonal complex 8 [CC8]; agr I and spa t008) were associated with NaHCO3 responsiveness for OXA.

The cfr gene associated with linezolid resistance has attracted wide attention. However, little is known about its prevalence and mode of transmission in Enterococcus faecalis. In this study, we investigate the prevalence and genetic environment of the cfr gene in 91 E. faecalis isolates collected from swine faecal swabs in 30 farms in Guangdong Province, China in 2012. A relatively high prevalence of cfr was identified in E. faecalis isolates (11/91, 12.1%) by PCR. All the cfr-positive E. faecalis strains had a multidrug-resistance phenotype including erythrocin, tetracycline, gentamicin, kanamicin and ciprofloxacin, except vancomycin and linezolid. Molecular typing indicated that ST475 and ST16 were the most common types in cfr-positive E. faecalis strains. In addition, we demonstrated that all the cfr genes were located on plasmids by S1-PFGE and Southern blotting. A 12 kb cfr-positive plasmid (pE30) was identified in most (9/11) E. faecalis strains, but it couldn't mediate resistance to linezolid in the transconjugant. Sequence analysis showed that the pE30 was a pCPPF5-like plasmid and the region surrounding the cfr gene was the same as a cfr-carrying ISEnfa5-composite element in Streptococcus plasmid pStrcfr with 4 bp direct repeat (GTAT) on both sides. In conclusion, the cfr gene which had no linezolid resistance phenotype was present in multidrug-resistance E. faecalis strains, and the clonal spread of ST475 and ST16 strains and the horizontal transfer of the pCPPF5-like plasmids have contributed to the dissemination of cfr.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia (PB) represents an important subset of S. aureus endovascular infections. In this study, we investigated potential genetic mechanisms underlying the persistent outcomes. Compared with resolving bacteremia (RB) isolates (defined as isolates associated with negative results of blood cultures 2–4 days after initiation of therapy), PB strains (defined as isolates associated with positive results of blood cultures ≥7 days after initiation of therapy) had significantly earlier onset activation of key virulence regulons and structural genes (eg, sigB, sarA, sae, and cap5), higher expression of purine biosynthesis genes (eg, purF), and faster growth rates, with earlier entrance into stationary phase. Importantly, an isogenic strain set featuring a wild-type MRSA isolate, a purF mutant strain, and a purF-complemented strain and use of strategic purine biosynthesis inhibitors implicated a causal relationship between purine biosynthesis and the in vivo persistent outcomes. These observations suggest that purine biosynthesis plays a key role in the outcome of PB and may represent a new target for enhanced efficacy in treating life-threatening MRSA infections.

Bacteriophages are viruses that invade the bacterial host, disrupt bacterial metabolism, and cause the bacterium to lyse. Because of its remarkable antibacterial activity and unique advantages over antibiotics, for instance, bacteriophage is specific for one species of bacteria and resistance to phage is less common than resistance to antibiotics. Indeed, bacteriophage therapy for treating infections due to multidrug-resistant pathogens in humans has become a research hot spot. However, it is also worth considering that bacteriophages are transferable and could cotransfer host chromosomal genes, e.g., virulence and antimicrobial resistance genes, while lysogenizing and integrating into the bacterial chromosome (prophage), thus playing a role in bacterial evolution and virulence. In the current study, we identified a novel prophage, ϕSA169, from a clinical persistent MRSA bacteremia isolate, and we determined that ϕSA169 mediated well-defined in vitro and in vivo phenotypic and genotypic signatures related to the persistent outcome, which may represent a unique and important persistent mechanism(s)., Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections are life-threatening syndromes with few therapeutic options. The potential impact of bacteriophages on the persistent outcome has not been well studied. In this study, we investigated the role of a novel prophage (ϕSA169) in MRSA persistence by using a lysogen-free clinically resolving bacteremia (RB) isolate and comparing it to a derivative which was obtained by infecting the RB strain with ϕSA169, which has been lysogenized in a clinical persistent MRSA bacteremia (PB) isolate. Similar to the PB isolate, the ϕSA169-lysogenized RB strain exhibited well-defined in vitro and in vivo phenotypic and genotypic signatures related to the persistent outcome, including earlier activation of global regulators (i.e., sigB, sarA, agr RNAIII, and sae); higher expression of a critical purine biosynthesis gene, purF; and higher growth rates accompanied by lower ATP levels and vancomycin (VAN) susceptibility and stronger δ-hemolysin and biofilm formation versus its isogenic parental RB isolate. Notably, the contribution of ϕSA169 in persistent outcome with VAN treatment was confirmed in an experimental infective endocarditis model. Taken together, these results indicate the critical role of the prophage ϕSA169 in persistent MRSA endovascular infections. Further studies are needed to identify the mechanisms of ϕSA169 in mediating the persistence, as well as establishing the scope of impact, of this prophage in other PB strains. IMPORTANCE Bacteriophages are viruses that invade the bacterial host, disrupt bacterial metabolism, and cause the bacterium to lyse. Because of its remarkable antibacterial activity and unique advantages over antibiotics, for instance, bacteriophage is specific for one species of bacteria and resistance to phage is less common than resistance to antibiotics. Indeed, bacteriophage therapy for treating infections due to multidrug-resistant pathogens in humans has become a research hot spot. However, it is also worth considering that bacteriophages are transferable and could cotransfer host chromosomal genes, e.g., virulence and antimicrobial resistance genes, while lysogenizing and integrating into the bacterial chromosome (prophage), thus playing a role in bacterial evolution and virulence. In the current study, we identified a novel prophage, ϕSA169, from a clinical persistent MRSA bacteremia isolate, and we determined that ϕSA169 mediated well-defined in vitro and in vivo phenotypic and genotypic signatures related to the persistent outcome, which may represent a unique and important persistent mechanism(s).

Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia (PB) represents an important subset of S. aureus infection and correlates with poor clinical outcomes. MRSA isolates from patients with PB differ significantly from those of resolving bacteremia (RB) with regard to several in vitro phenotypic and genotypic profiles. For instance, PB strains exhibit less susceptibility to cationic host defense peptides and vancomycin (VAN) killing under in vivo-like conditions, greater damage to endothelial cells, thicker biofilm formation, altered growth rates, early activation of many global virulence regulons (e.g., sigB, sarA, sae and agr) and higher expression of purine biosynthesis genes (e.g., purF) than RB strains. Importantly, PB strains are significantly more resistant to VAN treatment in experimental infective endocarditis as compared to RB strains, despite similar VAN minimum inhibitory concentrations (MICs) in vitro. Here, we review relevant phenotypic and genotypic characteristics related to the PB outcome. These and future insights may improve our understanding of the specific mechanism(s) contributing to the PB outcome, and aid in the development of novel therapeutic and preventative measures against this life-threatening infection.

Numerous studies on methane (CH4) and nitrous oxide (N2O) emissions during the rice growth period have focused on paddy fields, but CH4 and N2O emissions from rice seedling nurseries are poorly documented. This holds especially true for South China, where the double rice cropping system dominates. Here, we report CH4 and N2O emissions from three types of rice seedling nurseries in South China: Seedlings raised on wetland soil beds (WSB) with continuous flooding, seedlings raised on dry land soil beds (DSB), and seedlings raised in wet conditions with moist irrigation on plastic trays (WPT). Moreover, global warming potential (GWP) for all the three seedling nursing systems were estimated over a 100-year horizon. The results show that the WSB method produced the highest CH4 with negligible N2O emissions. Among the three methods, the DSB plots emitted the highest N2O and thus the highest GWP. Relative to the WSB method, the WPT method significantly decreased total CH4 emissions by 45%, and the WPT method decreased GWPs by 28 and 68% compared to the WSB and DSB method, respectively. The GWP of CH4 and N2O of WSB, DSB, and WPT account 30, 68, and 22% of that for the whole transplanted early-rice growing season for an equal unit area, indicating that GHGs from seedling stage were not negligible. Moreover, the WPT method produced the best rice seedlings. Therefore, utilizing WPT instead of conventional WSB is proposed as a best method for mitigating the GWPs of CH4 and N2O while growing healthy seedlings in rice nurseries in the double rice cropping system in South China.

Background The global regulator sarA modulates virulence of methicillin-resistant Staphylococcus aureus (MRSA) via regulation of principal virulence factors (eg, adhesins and toxins) and biofilm formation. Resistance of S. aureus strains to β-lactam antibiotics (eg, oxacillin) depends on the production of penicillin-binding protein 2a (PBP2a), encoded by mecA METHODS: In the present study, we investigated the impact of sarA on the phenotypic and genotypic characteristics of oxacillin resistance both in vitro and in an experimental endocarditis model, using prototypic healthcare- and community-associated MRSA parental and their respective sarA mutant strain sets. Results All sarA mutants (vs respective MRSA parental controls) displayed significant reductions in oxacillin resistance and biofilm formation in vitro and oxacillin persistence in an experimental endocarditis model in vivo. These phenotypes corresponded to reduced mecA expression and PBP2a production and an interdependency of sarA and sigB regulators. Moreover, RNA sequencing analyses showed that sarA mutants exhibited significantly increased levels of primary extracellular proteases and suppressed pyrimidine biosynthetic pathway, argininosuccinate lyase-encoding, and ABC transporter-related genes as compared to the parental strain. Conclusions These results suggested that sarA regulates oxacillin resistance in mecA-positive MRSA. Thus, abrogation of this regulator represents an attractive and novel drug target to potentiate efficacy of existing antibiotic for MRSA therapy.

Background: Antibiotic-resistant H. pylori was increasingly found in infected individuals, which resulted in treatment failure and required alternative therapeutic strategies. Daphnetin, a coumarin-derivative compound, has multiple pharmacological activities. Methods: The mechanism of daphnetin on H. pylori was investigated focusing on its effect on cell morphologies, transcription of genes related to virulence, adhesion, and cytotoxicity to human gastric epithelial (GES-1) cell line. Results: Daphnetin showed good activities against multidrug resistant (MDR) H. pylori clinical isolates, with minimal inhibitory concentration (MIC) values ranging from 25 to 100 &mu, g/mL. In addition, daphnetin exposure resulted in H. pylori morphological changes. Moreover, daphnetin caused increased translocation of phosphatidylserine (PS), DNA damage, and recA expression, and RecA protein production vs. control group. Of great importance, daphnetin significantly decreased H. pylori adhesion to GES-1 cell line vs. control group, which may be related to the reduced expression of colonization related genes (e.g., babA and ureI). Conclusions: These results suggested that daphnetin has good activity against MDR H. pylori. The mechanism(s) of daphnetin against H. pylori were related to change of membrane structure, increase of DNA damage and PS translocation, and decrease of H. pylori attachment to GES-1 cells.

Endovascular infections caused by Staphylococcus aureus involve interactions with fibronectin present as extracellular matrix or surface ligand on host cells. We examined the expression, structure, and binding activity of the two major S. aureus fibronectin-binding proteins (FnBPA, FnBPB) in 10 distinct, methicillin-resistant clinical isolates from patients with either persistent or resolving bacteremia. The persistent bacteremia isolates ( n = 5) formed significantly stronger bonds with immobilized fibronectin as determined by dynamic binding measurements performed with atomic force microscopy. Several notable differences were also observed when the results were grouped by clonal complex 5 (CC5) strains ( n = 5) versus CC45 strains ( n = 5). Fibronectin-binding receptors on CC5 formed stronger bonds with immobilized fibronectin ( P < 0.001). The fnbA gene was expressed at higher levels in CC45, whereas fnbB was found in only CC5 isolates. The fnbB gene was not sequenced because all CC45 isolates lacked this gene. Instead, comparisons were made for fnbA , which was present in all 10 isolates. Sequencing of fnbA revealed discrete differences within high-affinity, fibronectin-binding repeats (FnBRs) of FnBPA that included (i) 5-amino-acid polymorphisms in FnBR-9, FnBR-10, and FnBR-11 involving charged or polar side chains, (ii) an extra, 38-amino-acid repeat inserted between FnBR-9 and FnBR-10 exclusively seen in CC45 isolates, and (iii) CC5 isolates had the SVDFEED epitope in FnBR-11 (a sequence shown to be essential for fibronectin binding), while this sequence was replaced in all CC45 isolates with GIDFVED (a motif known to favor host cell invasion at the cost of reduced fibronectin binding). These complementary sequence and binding data suggest that differences in fnbA and fnbB , particularly polymorphisms and duplications in FnBPA, give S. aureus two distinct advantages in human endovascular infections: (i) FnBPs similar to that of CC5 enhance ligand binding and foster initiation of disease, and (ii) CC45-like FnBPs promote cell invasion, a key attribute in persistent endovascular infections.

Objectives Persistent MRSA infections are especially relevant to endovascular infections and correlate with suboptimal outcomes. However, the virulence signatures of Staphylococcus aureus that drive such persistence outcomes are not well defined. In the current study, we investigated correlations between accessory gene regulator (agr) activation and the outcome of vancomycin treatment in an experimental model of infective endocarditis (IE) due to MRSA strains with different agr and clonal complex (CC) types. Methods Twelve isolates with the four most common MRSA CC and agr types (CC5-agr II, CC8-agr I, CC30-agr III and CC45-agr I) were evaluated for heterogeneous vancomycin-intermediate S. aureus (hVISA), agr function, agrA and RNAIII transcription, agr locus sequences, virulence and response to vancomycin in the IE model. Results Early agr RNAIII activation (beginning at 2 h of growth) in parallel with strong δ-haemolysin production correlated with persistent outcomes in the IE model following vancomycin therapy. Importantly, such treatment failures occurred across the range of CC/agr types studied. In addition, these MRSA strains: (i) were vancomycin susceptible in vitro; (ii) were not hVISA or vancomycin tolerant; and (iii) did not evolve hVISA phenotypes or perturbed δ-haemolysin activity in vivo following vancomycin therapy. Moreover, agr locus sequence analyses revealed no common point mutations that correlated with either temporal RNAIII transcription or vancomycin treatment outcomes, encompassing different CC and agr types. Conclusions These data suggest that temporal agr RNAIII activation and agr functional profiles may be useful biomarkers to predict the in vivo persistence of endovascular MRSA infections despite vancomycin therapy.

Invasive infections due to S. aureus, including methicillin-resistant S. aureus (MRSA) are prevalent and life-threatening. Combinations of antibiotic therapy have been employed in many clinical settings for improving therapeutic efficacy, reducing side effects of drugs, and development of antibiotic resistance. Pleuromutilins have a potential to be developed as a new class of antibiotics for systemic use in humans. In the current study, we investigated the relationship between pleuromutilins, including valnemulin, tiamulin and retapamulin, and 13 other antibiotics representing different mechanisms of action, against methicillin-susceptible and -resistant S. aureus both in vitro and in an experimental Galleria mellonella model. In vitro synergistic effects were observed in combination of all three study pleuromutilins with tetracycline by standard checkerboard and/or time-kill assays. In addition, the combination of pleuromutilins with ciprofloxacin or enrofloxacin showed antagonistic effects, while the rest combinations presented indifferent effects. Importantly, all study pleuromutilins in combination with tetracycline significantly enhanced survival rates as compared to the single drug treatment in the Galleria mellonella model caused by S. aureus strains. Taken together, these results demonstrated synergy effects between pleuromutilins and tetracycline against S. aureus both in vitro and in vivo.

Many serious bacterial infections are antibiotic refractory due to biofilm formation. A key structural component of biofilm is extracellular DNA, which is stabilized by bacterial proteins, including those from the DNABII family. TRL1068 is a high-affinity human monoclonal antibody against a DNABII epitope conserved across both Gram-positive and Gram-negative bacterial species. In the present study, the efficacy of TRL1068 for the disruption of biofilm was demonstrated in vitro in the absence of antibiotics by scanning electron microscopy. The in vivo efficacy of this antibody was investigated in a well-characterized catheter-induced aortic valve infective endocarditis model in rats infected with a methicillin-resistant Staphylococcus aureus (MRSA) strain with the ability to form thick biofilms, obtained from the blood of a patient with persistent clinical infection. Animals were treated with vancomycin alone or in combination with TRL1068. MRSA burdens in cardiac vegetations and within intracardiac catheters, kidneys, spleen, and liver showed significant reductions in the combination arm versus vancomycin alone ( P < 0.001). A trend toward mortality reduction was also observed ( P = 0.09). In parallel, the in vivo efficacy of TRL1068 against a multidrug-resistant clinical Acinetobacter baumannii isolate was explored by using an established mouse model of skin and soft tissue catheter-related biofilm infection. Catheter segments infected with A. baumannii were implanted subcutaneously into mice; animals were treated with imipenem alone or in combination with TRL1068. The combination showed a significant reduction of catheter-adherent bacteria versus the antibiotic alone ( P < 0.001). TRL1068 shows excellent promise as an adjunct to standard-of-care antibiotics for a broad range of difficult-to-treat bacterial infections.

Leaf senescence is a developmental programmed cell death (PCD) that occurs as a response to external and internal signals. Several factors, such as the environment, plant hormones, and senescence-associated genes, regulate leaf senescence. In Drosophila melanogaster, Will Die Slowly (WDS) is a WD-repeat protein, which is closely related to PCD. Phylogenetic analysis indicated that eight genes are highly homologous to D. melanogaster WDS (DmWDS) in Columbia ecotype (Col) of Arabidopsis thaliana. In this study, the expression patterns of three close homologues of DmWDS named WDS1, WDS2, and WDS3 were investigated. Real-time PCR revealed the spatio-temporal expression levels of these three genes. No tissue-specific expression of the three AtWDS genes was observed. These genes were expressed at every growth stage; the variation in their expression was similar: the expression of the three AtWDS reached the peak in leaves at the 37 days after sowing, at the time when the first pod initially appeared on the plant and the leaf 7 show 25 to 50% yellow; and the expression of the three AtWDS reached the peak in flowers at the 43.5 days after sowing, at the time when 50% of the flowers bloomed. In addition, the expression level of the three AtWDS peaked at the 48 h after the plants were treated with 0.5 mM salicylic acid (SA). WDS3 also exhibited a high expression at 24, 48, and 72 h. Taken together; these results suggest that AtWDS genes may be involved in plant PCD

Acinetobacter baumannii bacteremia represents a serious and increasing clinical problem due to the high mortality and treatment failures because of high rates of antibiotic resistance. Any additional new therapies for A. baumannii bacteremia would address a growing unmet medical need. ARV-1502 (designated as Chex1-Arg20 or A3-APO monomer in prior publications) is a designer proline-rich antimicrobial peptide chaperone protein inhibitor derived from insects and has demonstrated potent activity against multi-drug resistant (MDR) Gram-negative bacteria. In the current studies, we investigated the therapeutic efficacy of ARV-1502 administered intravenously (iv) alone and in combination with imipenem/cilastatin (IPM/CIL) in a mouse bacteremia model due to a MDR clinical A. baumannii strain, HUMC1. All ARV-1502 regimens (1.25, 2.5 and 5.0 mg/kg) significantly reduced bacterial density in the target tissues in a dose-dependent manner, as compared to the untreated control and IPM/CIL monotherapy (40 mg/kg) groups in the model. In addition, ARV-1502 treatment, even at the lowest dose, significantly improved survival vs. the control and IPM alone groups. As expected, IMP/CIL monotherapy had no therapeutic efficacy in the model, since the HUMC1 strain was resistant to IMP in vitro. However, the combination of ARV-1502 and IPM/CIL significantly enhanced the efficacy of ARV-1502, except the lowest dose of ARV-1502. The superior efficacy of ARV-1502 in the bacteremia model caused by MDR A. baumannii provides further support for studying this compound in severe infections caused by other MDR Gram-positive and -negative pathogens.

The surface of mesoporous TiO2 whiskers (MTw) was chemically modified with 3-aminopropyltriethoxysilane (APTES). The point of zero charge (pzc) of MTw and the modified material (MTwA) was 5.3 and 6.8, respectively. MTw and MTwA were then used as carrier to immobilize γ-glutamyltranspeptidase (GGT) from Bacillus subtilis (B. subtilis). Our results indicated that the loading capacity of MTwA for GTT was significantly superior to that of MTw. Although the optimum temperature and thermal stability of MTwA-GGT were slightly lower than those of MTw-GGT, its pH stability was greatly improved compared with either free enzyme or MTw-GGT. The affinity constant (K m ) of MTwA-GGT to γ-glutamyl-p-nitroanilide (GpNA) was 0.889 mM, higher than that of free enzyme but lower than that of MTw-GGT. In addition, MTwA-GGT displayed a good operational stability.

CD133 is one of the most common stem cell markers, and functional single nucleotide polymorphisms (SNPs) of CD133 may modulate its gene functions and thus cancer risk and patient survival. We hypothesized that potentially functional CD133 SNPs are associated with gastric cancer (GC) risk and survival. To test this hypothesis, we conducted a case-control study of 371 GC patients and 313 cancer-free controls frequency-matched by age, sex, and ethnicity. We genotyped four selected, potentially functional CD133 SNPs (rs2240688A>C, rs7686732C>G, rs10022537T>A, and rs3130C>T) and used logistic regression analysis for associations of these SNPs with GC risk and Cox hazards regression analysis for survival. We found that compared with the miRNA binding site rs2240688 AA genotype, AC + CC genotypes were associated with significantly increased GC risk (adjusted OR = 1.52, 95% CI = 1.09-2.13); for another miRNA binding site rs3130C>T SNP, the TT genotype was associated with significantly reduced GC risk (adjusted OR = 0.68, 95% CI = 0.48-0.97), compared with CC + CT genotypes. In all patients, the risk rs3130 TT variant genotype was significantly associated with overall survival (OS) (adjusted P(trend) = 0.016 and 0.007 under additive and recessive models, respectively). These findings suggest that these two CD133 miRNA binding site variants, rs2240688 and rs3130, may be potential biomarkers for genetic susceptibility to GC and possible predictors for survival in GC patients but require further validation by larger studies.

Objectives The underlying mechanisms of protective immunity of placental trophoblast cells against bacterial infection remain largely unknown. NOD1 are intracellular pattern recognition receptors that are activated by bacterial peptides and mediate innate immunity. This study aimed to investigate the expression and function of NOD1 in first trimester trophoblast cells, and evaluate the potential role of trophoblast cells in infection-associated inflammation. Study design Human extravillous trophoblast cell line HTR8 cells were stimulated with various concentrations of iE-DAP for various periods of time. NOD1 expression was detected by immunofluorescence, and the changes in NOD1 and RICK mRNA and protein in H8 cells were determined by real-time polymerase chain reaction and Western blot analysis. The concentrations of interleukin (IL)-8 and IL-6 secreted by H8 cells were examined by enzyme-linked immunosorbent assay. NF-κB transcription activity and P65 expression were detected by electrophoretic mobility shift assay and Western blot analysis. Results H8 cells expressed NOD1, and the effects of iE-DAP on NOD1 were dose- and time-dependent. The concentration of IL-8 increased gradually with increasing concentration of iE-DAP, and the levels of IL-8 and IL-6 were associated with the duration of exposure to iE-DAP. The dose of iE-DAP was significantly associated with expression of RICK and P65, and stimulation of H8 cells by iE-DAP altered NF-κB transcription activity. Conclusions NOD1 may have a role in mediating infection-associated inflammation. Once iE-DAP is recognized by NOD1, the inflammatory response may be induced via NOD1–RICK–NF-κB-mediated pathways.

Many host defense cationic antimicrobial peptides (HDPs) perturb the staphylococcal cell membrane (CM) and alter transmembrane potential (ΔΨ) as key parts of their lethal mechanism. Thus, a sense-response system for detecting and mediating adaptive responses to such stresses could impact organism survival; the Staphylococcus aureus LytSR two-component regulatory system (TCRS) may serve as such a ΔΨ sensor. One well-known target of this system is the lrgAB operon, which, along with the related cidABC operon, has been shown to be a regulator in the control of programmed cell death and lysis. We used an isogenic set of S. aureus strains: (i) UAMS-1, (ii) its isogenic Δ lytS and Δ lrgAB mutants, and (iii) plasmid-complemented Δ lytSR and Δ lrgAB mutants. The Δ lytS strain displayed significantly increased in vitro susceptibilities to all HDPs tested (neutrophil-derived human neutrophil peptide 1 [hNP-1], platelet-derived thrombin-induced platelet microbicidal proteins [tPMPs], and the tPMP-mimetic peptide RP-1), as well as to calcium-daptomycin (DAP), a cationic antimicrobial peptide (CAP). In contrast, the Δ lrgAB strain exhibited no significant changes in susceptibilities to these cationic peptides, indicating that although lytSR positively regulates transcription of lrgAB , increased HDP/CAP susceptibilities in the Δ lytS mutant were lrgAB independent. Further, parental UAMS-1 (but not the Δ lytS mutant) became more resistant to hNP-1 and DAP following pretreatment with carbonyl cyanide m -chlorophenylhydrazone (CCCP) (a CM-depolarizing agent). Of note, lytSR -dependent survival against CAP/HDP killing was not associated with changes in either surface positive charge, expression of mprF and dlt , or CM fluidity. The Δ lytS strain (but not the Δ lrgAB mutant) displayed a significant reduction in target tissue survival in an endocarditis model during DAP treatment. Collectively, these results suggest that the lytSR TCRS plays an important role in adaptive responses of S. aureus to CM-perturbing HDPs/CAPs, likely by functioning as a sense-response system for detecting subtle changes in ΔΨ.

Staphylococcus aureus produces numerous virulence factors but little is known about their in vivo regulation during an infection.The production of capsule and α-toxin, and the expression of their respective genes, cap5 and hla, were analyzed by comparing CYL11481 (derivative of Newman) and its isogenic regulatory mutants in vitro. The temporal expression of cap5 and hla and the regulatory genes in vivo was carried out using a rat infective endocarditis model.In vitro analyses showed that capsule was positively regulated by MgrA, Agr, Sae, ArlR, and ClpC, and negatively by CodY and SbcDC. The α-toxin was positively regulated by MgrA, Agr, Sae, ArlR, and SbcDC but negatively by ClpC and CodY. In vivo analyses showed that cap5 expression correlated best with mgrA expression, whereas hla expression correlated best with sae expression. Mutation in mgrA drastically reduced cap5 expression in vivo.Our results suggest that, in vitro, Agr is the most important regulator for capsule and α-toxin production, as well as for cap5 transcription, but SaeR is the most critical for hla transcription. However, in vivo, MgrA is the major transcriptional regulator of capsule, but not α-toxin, whereas saeR expression correlates best with hla expression.

Staphylococcus aureus is the most common cause of endovascular infections, including catheter sepsis and infective endocarditis (IE). Vancomycin (VAN) is the primary choice for treatment of methicillin-resistant S. aureus (MRSA) infections. However, high rates of VAN treatment failure in MRSA infections caused by VAN-susceptible strains have been increasingly reported. Biofilm-associated MRSA infections are especially prone to clinical antibiotic failure. The present studies examined potential relationships between MRSA susceptibility to VAN in biofilms in vitro and nonsusceptibility to VAN in endovascular infection in vivo . Using 10 “VAN-susceptible” MRSA bloodstream isolates previously investigated for VAN responsiveness in experimental IE, we studied the mechanism(s) of such in vivo VAN resistance, including: (i) VAN binding to MRSA organisms; (ii) the impact of VAN on biofilm formation and biofilm composition; (iii) VAN efficacy in an in vitro catheter-related biofilm model; (iv) effects on cell wall thickness. As a group, the five strains previously categorized as VAN nonresponders (non-Rsp) in the experimental IE model differed from the five responders (Rsp) in terms of lower VAN binding, increased biofilm formation, higher survival in the presence of VAN within biofilms in the presence or absence of catheters, and greater biofilm reduction upon proteinase K treatment. Interestingly, sub-MICs of VAN significantly promoted biofilm formation only in the non-Rsp isolates. Cell wall thickness was similar among all MRSA strains. These results suggest that sublethal VAN levels that induce biofilm formation and reduce efficacy of VAN in the in vitro catheter-associated biofilms may contribute to suboptimal treatment outcomes for endovascular infections caused by “VAN-susceptible” MRSA strains.

Mycoplasma gallisepticum is the most important pathogen in poultry among four pathogenic Mycoplasma species. Tiamulin is a pleuromutilin antibiotic that shows a great activity against M. gallisepticum and has been approved for use in veterinary medicine particularly for poultry. However, the pharmacokinetic/pharmacodynamics (PK/PD) profiles of tiamulin against M. gallisepticum are not well understood. Therefore, in the current studies, we investigated the in vivo PK/PD profiles of tiamulin using a well-established experimental intratracheal infection model of M. gallisepticum. The efficacy of tiamulin against M. gallisepticum was studied in 8-day-old chickens after intramuscular (i.m.) administration at 10 doses between 0-80 mg/kg. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to evaluate the PK parameters of tiamulin following i.m. administration at doses of 5, 40, and 80 mg/kg in Mycoplasma gallisepticum-infected neutropenic chickens. Real-time PCR (RT-PCR) was used for quantitative detection of M. gallisepticum. The MIC of tiamulin against M. gallisepticum strain S6 was 0.03 μg/mL. The PK/PD index, AUC24h/MIC, correlated well with the in vivo antibacterial efficacy. The in vivo data suggest that animal dosage regimens should supply AUC24h/MIC of tiamulin of 382.68 h for 2 log10 ccu equivalents M. gallisepticum reduction. To attain that goal, the administered dose is expected to be 45 mg/kg b.w. for treatment of M. gallisepticum infection with an MIC90 of 0.03 μg/mL.

The accessory gene regulator ( agr ) locus has been shown to be important for virulence in several animal models of Staphylococcus aureus infection. However, the role of agr in human infections, and specifically in antibiotic treatment, is controversial. Interestingly, agr dysfunction has been associated with reduced vancomycin responses. To systematically investigate the role of agr in virulence and treatment outcome in the context of endovascular infection, 10 well-characterized vancomycin-susceptible methicillin-resistant S. aureus (MRSA) bloodstream isolates (5 agr-I [clonal complex 45, or CC45] and 5 agr-II [CC5]) were studied for (i) agr function, (ii) RNAIII transcriptional profiles, (iii) agr locus sequences, (iv) intrinsic virulence and responses to vancomycin therapy in an experimental infective endocarditis (IE) model, and (v) in vivo RNAIII expression. Significant differences in agr function (determined by delta-hemolysin activity) correlated with the time point of RNAIII transcription (earlier RNAIII onset equals increased agr function). Unexpectedly, four MRSA strains with strong delta-hemolysin activities exhibited significant resistance to vancomycin treatment in experimental IE. In contrast, five of six MRSA strains with weak or no delta-hemolysin activity were highly susceptible to vancomycin therapy in the IE model. agr sequence analyses showed no common single-nucleotide polymorphism predictive of agr functionality. In vivo RNAIII expression in cardiac vegetations did not correlate with virulence or vancomycin treatment outcomes in the IE model. Inactivation of agr in two strains with strong delta-hemolysin activity did not affect virulence or the in vivo efficacy of vancomycin. Our findings suggest that agr dysfunction does not correlate with vancomycin treatment failures in this experimental IE model in two distinct MRSA genetic backgrounds.

Cationic antimicrobial peptides (CAPs) play important roles in host immune defenses. Plectasin is a defensin-like CAP isolated from the saprophytic fungus Pseudoplectania nigrella . NZ2114 is a novel variant of plectasin with potent activity against Gram-positive bacteria. In this study, we investigated (i) the in vivo pharmacokinetic and pharmacodynamic (PK/PD) characteristics of NZ2114 and (ii) the in vivo efficacy of NZ2114 in comparison with those of two conventional antibiotics, vancomycin or daptomycin, in an experimental rabbit infective endocarditis (IE) model due to a methicillin-resistant Staphylococcus aureus (MRSA) strain (ATCC 33591). All NZ2114 regimens (5, 10, and 20 mg/kg of body weight, intravenously [i.v.], twice daily for 3 days) significantly decreased MRSA densities in cardiac vegetations, kidneys, and spleen versus those in untreated controls, except in one scenario (5 mg/kg, splenic MRSA counts). The efficacy of NZ2114 was clearly dose dependent in all target tissues. At 20 mg/kg, NZ2114 showed a significantly greater efficacy than vancomycin ( P < 0.001) and an efficacy similar to that of daptomycin. Of importance, only NZ2114 (in 10- and 20-mg/kg regimens) prevented posttherapy relapse in cardiac vegetations, kidneys, and spleen, while bacterial counts in these target tissues continued to increase in vancomycin- and daptomycin-treated animals. These in vivo efficacies were equivalent and significantly correlated with three PK indices investigated: f C max /MIC (the maximum concentration of the free, unbound fraction of a drug in serum divided by the MIC), f AUC/MIC (where AUC is the area under the concentration-time curve), and f % T >MIC (% T >MIC is the cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions), as analyzed by a sigmoid maximum-effect ( E max ) model ( R 2 > 0.69). The superior efficacy of NZ2114 in this MRSA IE model suggests the potential for further development of this compound for treating serious MRSA infections.

Summary The pathogenesis of Staphylococcus aureus infective endocarditis (IE) is postulated to involve invasion and damage of endothelial cells (ECs). However, the precise relationships between S. aureus – EC interactions in vitro and IE virulence and treatment outcomes in vivo are poorly defined. Ten methicillin-resistant S. aureus (MRSA) clinical isolates previously tested for their virulence and vancomycin responsiveness in an experimental IE model were assessed in vitro for their hemolytic activity, protease production, and capacity to invade and damage ECs. There was a significant positive correlation between the in vitro EC damage caused by these MRSA strains and their virulence during experimental IE (in terms of bacterial densities in target tissues; P < 0.02). Importantly, higher EC damage was also significantly correlated with poor microbiologic response to vancomycin in the IE model (P < 0.001). Interestingly, the extent of EC damage was unrelated to a strain's ability to invade ECs, hemolytic activity and protease production, or β-toxin gene transcription. Inactivation of the agr locus in two MRSA strains caused ∼20% less damage as compared to the corresponding parental strains, indicating that a functional agr is required for maximal EC damage induction. Thus, MRSA-induced EC damage in vitro is a unique virulence phenotype that is independent of many other prototypical MRSA virulence factors, and may be a key biomarker for predicting MRSA virulence potential and antibiotic outcomes during endovascular infections.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia (PB) (positive blood cultures after ≥7 days of therapy) represents a clinically challenging subset of invasive MRSA infections. In this investigation, we examined the potential correlation of specific virulence signatures with PB versus resolving MRSA bacteremia (RB) (negative blood cultures within 2 to 4 days of therapy) strains. Thirty-six MRSA isolates from patients enrolled in a recent multinational clinical trial were studied for (i) susceptibility to host defense cationic peptides (HDPs) (i.e., thrombin-induced platelet microbicidal proteins [tPMPs] and human neutrophil peptide 1 [hNP-1]); (ii) adherence to host endovascular ligands (fibronectin) and cells (endothelial cells); and (iii) biofilm formation. We found that PB isolates exhibited significantly reduced susceptibilities to tPMPs and hNP-1 ( P < 0.001 and P = 0.023, respectively). There was no significant association between the PB outcome and fibronectin binding, endothelial cell binding, or biofilm formation ( P = 0.25, 0.97, and 0.064 versus RB strains, respectively). However, multiple logistic regression analysis revealed that the PB outcome was significantly associated with the combination of reduced susceptibilities to HDPs and extent of biofilm formation ( P < 0.0001). Similar results were obtained in a second analysis using days of bacteremia as a continuous outcome, showing that reduced HDP susceptibilities and increased biofilm formation cocontributed to predict the duration of bacteremia. Our data indicate that PB isolates have specific pathogenic signatures independent of conventional antimicrobial susceptibility. These combinatorial mosaics can be defined and used to prospectively distinguish PB from RB strains in advance and potentially to predict ultimate clinical outcomes.

Fibronectin-binding protein A plays an important role in Staphylococcus aureus endovascular infections. We characterized the sigB-saeRS-fnbA expression network with fibronectin binding in vitro and in an experimental infective endocarditis (IE) model using parental strains RN6390 and SH1000 and their respective isogenic saeRS mutants. In contrast to the in vitro data, there was no influence of saeRS on fnbA expression in the IE model, yet ex vivo fibronectin binding was reduced in saeRS mutants. Moreover, as opposed to the in vitro findings, sigB appeared to have a positive rather than a negative effect on saeRS expression within cardiac vegetations.

The persistent bacteremia (PB) syndrome is well represented in large clinical series of persons with methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection (PB prevalence, 20%–30%), and it is especially relevant to patients with endovascular infection [1, 2]. However, in up to one-third of cases, no readily identifiable cause of PB is identified, despite extensive clinical evaluation [3]. Therefore, understanding the molecular mechanisms and determinants of PB is essential to optimize prevention and therapy against life-threatening S. aureus infections. We hypothesize that S. aureus uses specific virulence determinants to persist in the bloodstream and cause PB in the context of endovascular infection. For example, the organism must avoid immediate killing by host-defense peptides liberated by platelets at sites of endovascular infection. Next, the organism must evade or survive phagocytosis and intracellular killing by neutrophil-associated oxidative and nonoxidative killing mechanisms (including those associated with antimicrobial peptides, such as α-defensins). During this phase, the pathogen must adhere to host cells and ligands to colonize vascular endothelium and avoid clearance by the reticuloendothelial system. Following adherence, the organism proliferates and invades tissues, creating reservoir foci. Finally, it deploys exotoxins and exoenzymes to reemerge from these reservoir sites, reenter the bloodstream, and hematogenously seed metastatic target organs (figure 1). Figure 1 Hypothesized life cycle of methicillin-resistant Staphylococcus aureus (SA) isolates that cause persistent bacteremia in the context of endovascular infection. AP, antimicrobial peptides; α-Tox, α-toxin; CM, cell membrane; FBG, fibrinogen; ... Because distinct infection foci are not synchronized, organisms are continuously reemerging into the bloodstream, accounting for persistent bacteremia. On the basis of this hypothesized life cycle of PB isolates, we investigated a cadre of in vitro phenotypic and genotypic characteristics considered to be involved in these phases of endovascular pathogenesis. In addition, we examined the in vivo virulence and antibiotic responsiveness of MRSA isolates associated with PB (hereafter, “PB isolates”) and those associated with resolving bacteremia (here-after, “RB isolates”) in a rabbit model of infective endocarditis.

Platelets (PLTs) act in antimicrobial host defense by releasing PLT microbicidal proteins (PMPs) or PLT kinocidins (PKs). Receptors mediating staphylocidal efficacy and PMP or PK release versus isogenic PMP-susceptible (ISP479C) and -resistant (ISP479R) Staphylococcus aureus strains were examined in vitro. Isolated PLTs were incubated with ISP479C or ISP479R (PLT/ S. aureus ratio range, 1:1 to 10,000:1) in the presence or absence of a panel of PLT inhibitors, including P2X and P2Y receptor antagonists of increasingly narrow specificity, and PLT adhesion receptors (CD41, CD42b, and CD62P). PLT-to- S. aureus exposure ratios of ≥10:1 yielded significant reductions in the viability of both strains. Results from reversed-phase high-performance liquid chromatography indicated that staphylocidal PLT releasates contained PMPs and PKs. At ratios below 10:1, the PLT antistaphylococcal efficacy relative to the intrinsic S. aureus PMP-susceptible or -resistant phenotype diminished. Apyrase (an agent of ADP degradation), suramin (a general P2 receptor antagonist), pyridoxal 5′-phosphonucleotide derivative (a specific P2X 1 antagonist), and cangrelor (a specific P2Y 12 antagonist) mitigated the PLT staphylocidal response against both strains, correlating with reduced levels of PMP and PK release. Specific inhibition occurred in the presence and absence of homologous plasma. The antagonism of the thromboxane A 2 , cyclooxygenase-1/cyclooxygenase-2, or phospholipase C pathway or the hindrance of surface adhesion receptors failed to impede PLT anti- S. aureus responses. These results suggest a multifactorial PLT anti- S. aureus response mechanism involving (i) a PLT-to- S. aureus ratio sufficient for activation; (ii) the ensuing degranulation of PMPs, PKs, ADP, and/or ATP; (iii) the activation of P2X 1 /P2Y 12 receptors on adjacent PLTs; and (iv) the recursive amplification of PMP and PK release from these PLTs.

The direct binding of bacteria to platelets is a central interaction in the pathogenesis of infective endocarditis. GspB is a serine-rich, cell wall glycoprotein of Streptococcus gordonii that mediates the binding of this organism to human platelets in vitro. To assess the contribution of this adhesin to the pathogenesis of endocarditis, we compared the virulence of S. gordonii M99 (which expresses GspB) with an isogenic, gspB mutant (PS846) in two rat models of endovascular infection. In the first group of experiments, animals were infected intravenously with M99 or PS846, and sacrificed 72 h later, to assess levels of bacteria within cardiac vegetations, kidneys, and spleens. When inoculated with 10(5)CFU, rats infected with PS846 had significantly lower densities of organisms within vegetations (mean: 3.84 log(10)CFU/g) as compared with M99-infected rats (6.67 log(10)CFU/g; P0.001). Marked differences were also seen in rats co-infected with M99 and PS846, at a 1:1 ratio. While M99 was found at high levels within vegetations, kidneys and spleens (mean log(10)CFU/g: 6.62, 5.07 and 4.18, respectively) PS846 was not detected within these tissues. Thus, platelet binding by GspB appears to be a major interaction in the pathogenesis of endocarditis due to S. gordonii.

Increasingly frequent reports have described the in vivo loss of daptomycin susceptibility in association with clinical treatment failures. The mechanism(s) of daptomycin resistance is not well understood. We studied an isogenic set of Staphylococcus aureus isolates from the bloodstream of a daptomycin-treated patient with recalcitrant endocarditis in which serial strains exhibited decreasing susceptibility to daptomycin. Since daptomycin is a membrane-targeting lipopeptide, we compared a number of membrane parameters in the initial blood isolate (parental) with those in subsequent daptomycin-resistant strains obtained during treatment. In comparison to the parental strain, resistant isolates demonstrated (i) enhanced membrane fluidity, (ii) increased translocation of the positively charged phospholipid lysyl-phosphotidylglycerol to the outer membrane leaflet, (iii) increased net positive surface charge ( P < 0.05 versus the parental strain), (iv) reduced susceptibility to daptomycin-induced depolarization, permeabilization, and autolysis ( P < 0.05 versus the parental strain), (v) significantly lower surface binding of daptomycin ( P < 0.05 versus the parental strain), and (vi) increased cross-resistance to the cationic antimicrobial host defense peptides human neutrophil peptide 1 (hNP-1) and thrombin-induced platelet microbicidal protein 1 (tPMP-1). These data link distinct changes in membrane structure and function with in vivo development of daptomycin resistance in S. aureus . Moreover, the cross-resistance to hNP-1 and tPMP-1 may also impact the capacity of these daptomycin-resistant organisms to be cleared from sites of infection, particularly endovascular foci.

Biofilm formations play an important role in Staphylococcus aureus pathogenesis and contribute to antibiotic treatment failures in biofilm-associated infections. The aim of this study was to evaluate the pharmacokinetic/pharmacodynamic (PK/PD) profiles of cefquinome against an experimental catheter-related biofilm model due to S. aureus, including three clinical isolates and one non-clinical isolate. The minimal inhibitory concentration (MIC), minimal biofilm inhibitory concentration (MBIC), biofilm bactericidal concentration (BBC), minimal biofilm eradication concentration (MBEC) and biofilm prevention concentration (BPC) and in vitro time-kill curves of cefquinome were studied in both planktonic and biofilm cells of study S. aureus strains. The in vivo post-antibiotic effects (PAEs), PK profiles and efficacy of cefquinome were performed in the catheter-related biofilm infection model in murine. A sigmoid E max model was utilized to determine the PK/PD index that best described the dose-response profiles in the model. The MICs and MBICs of cefquinome for the four S. aureus strains were 0.5 and 16 μg/mL, respectively. The BBCs (32-64 μg/mL) and MBECs (64-256 μg/mL) of these study strains were much higher than their corresponding BPC values (1-2 μg/mL). Cefquinome showed time-dependent killing both on planktonic and biofilm cells, but produced much shorter PAEs in biofilm infections. The best-correlated PK/PD parameters of cefquinome for planktonic and biofilm cells were the duration of time that the free drug level exceeded the MIC (fT > MIC, R (2) = 96.2%) and the MBIC (fT > MBIC, R (2) = 94.7%), respectively. In addition, the AUC24h/MBIC of cefquinome also significantly correlated with the anti-biofilm outcome in this model (R (2) = 93.1%). The values of AUC24h/MBIC for biofilm-static and 1-log10-unit biofilm-cidal activity were 22.8 and 35.6 h; respectively. These results indicate that the PK/PD profiles of cefquinome could be used as valuable guidance for effective dosing regimens treating S. aureus biofilm-related infections.

Thrombin-induced platelet microbicidal proteins (e.g. tPMP-1) are small cationic peptides released from mammalian platelets. As the cytoplasmic membrane (CM) is a primary target of tPMPs, distinct CM characteristics are likely to affect the cells' susceptibility profiles. In Staphylococcus aureus, CM surface charge and hydrophobicity are principally determined by the content and distribution of its three major phospholipid (PL) constituents: negatively charged phosphatidylglycerol (PG) and cardiolipin (CL) and positively charged lysyl-PG (LPG). PL composition profiles, and inner vs outer CM leaflet PL distributions, were compared in an isogenic tPMP-susceptible (tPMP(S)) and -resistant (tPMP(R)) S. aureus strain pair (ISP479C vs ISP479R respectively). All PLs were asymmetrically distributed between the outer and inner CM leaflets in both strains. However, in ISP479R, the outer CM leaflet content of LPG was significantly increased vs ISP479C (27.3+/-11.0 % vs 18.6+/-7.0 % respectively; P=0.05). This observation correlated with reduced binding of the cationic proteins cytochrome c, poly-L-lysine, tPMP-1 and the tPMP-1-mimetic peptide, RP1, to tPMP-1(R) whole cells and to model liposomal CMs with LPG content and distribution similar to that of tPMP-1(R) strains. Collectively, selected CM parameters correlated with reduced staphylocidal capacities of tPMP-1 against certain S. aureus strains, including relative increases in outer CM leaflet positive charge and reduced surface binding of cationic molecules. These findings offer new insights into mechanisms of antimicrobial peptide susceptibility and resistance in S. aureus.

Staphylococcus aureus is the leading cause of skin and skin structure infections (SSSI) in humans. Moreover, the high frequency of recurring SSSI due to S. aureus , particularly methicillin-resistant S. aureus (MRSA) strains, suggests that infection induces suboptimal anamnestic defenses. The present study addresses the hypothesis that interleukin-17A (IL-17A) and IL-22 play distinct roles in immunity to cutaneous and invasive MRSA infection in a mouse model of SSSI. Mice were treated with specific neutralizing antibodies against IL-17A and/or IL-22 and infected with MRSA, after which the severity of infection and host immune response were determined. Neutralization of either IL-17A or IL-22 reduced T cell and neutrophil infiltration and host defense peptide elaboration in lesions. These events corresponded with increased abscess severity, MRSA viability, and CFU density in skin. Interestingly, combined inhibition of IL-17A and IL-22 did not worsen abscesses but did increase gamma interferon (IFN-γ) expression at these sites. The inhibition of IL-22 led to a reduction in IL-17A expression, but not vice versa. These results suggest that the expression of IL-17A is at least partially dependent on IL-22 in this model. Inhibition of IL-17A but not IL-22 led to hematogenous dissemination to kidneys, which correlated with decreased T cell infiltration in renal tissue. Collectively, these findings indicate that IL-17A and IL-22 have complementary but nonredundant roles in host defense against cutaneous versus hematogenous infection. These insights may support targeted immune enhancement or other novel approaches to address the challenge of MRSA infection.

Staphylococcus aureus uses the two-component regulatory system GraRS to sense and respond to host defense peptides (HDPs). However, the mechanistic impact of GraS or its extracellular sensing loop (EL) on HDP resistance is essentially unexplored. Strains with null mutations in the GraS holoprotein (Δ graS ) or its EL (ΔEL) were compared for mechanisms of resistance to HDPs of relevant immune sources: neutrophil α-defensin (human neutrophil peptide 1 [hNP-1]), cutaneous β-defensin (human β-defensin 2 [hBD-2]), or the platelet kinocidin congener RP-1. Actions studied by flow cytometry included energetics (ENR); membrane permeabilization (PRM); annexin V binding (ANX), and cell death protease activation (CDP). Assay conditions simulated bloodstream (pH 7.5) or phagolysosomal (pH 5.5) pH contexts. S. aureus strains were more susceptible to HDPs at pH 7.5 than at pH 5.5, and each HDP exerted a distinct effect signature. The impacts of Δ graS and ΔΕL on HDP resistance were peptide and pH dependent. Both mutants exhibited defects in ANX response to hNP-1 or hBD-2 at pH 7.5, but only hNP-1 did so at pH 5.5. Both mutants exhibited hyper-PRM, -ANX, and -CDP responses to RP-1 at both pHs and hypo-ENR at pH 5.5. The actions correlated with Δ graS or ΔΕL hypersusceptibility to hNP-1 or RP-1 (but not hBD-2) at pH 7.5 and to all study HDPs at pH 5.5. An exogenous EL mimic protected mutant strains from hNP-1 and hBD-2 but not RP-1, indicating that GraS and its EL play nonredundant roles in S. aureus survival responses to specific HDPs. These findings suggest that GraS mediates specific resistance countermeasures to HDPs in immune contexts that are highly relevant to S. aureus pathogenesis in humans.

Valnemulin, a semisynthetic pleuromutilin antibiotic derivative, is greatly active against Mycoplasma . The objective of our study was to evaluate the effectiveness of valnemulin against Mycoplasma gallisepticum in a neutropenic intratracheal model in chickens using a pharmacokinetic/pharmacodynamic (PK-PD) method. The PK of valnemulin after intramuscular (i.m.) administration at doses of 1, 10, and 20 mg/kg of body weight in M. gallisepticum -infected neutropenic chickens was evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Real-time PCR (RT-PCR) was used for quantitative detection of M. gallisepticum . The ratio of the 24-h area under the concentration-time curve divided by the MIC (AUC 24 /MIC) correlated well with the in vivo antibacterial effectiveness of valnemulin ( R 2 = 0.9669). The AUC 24 /MIC ratios for mycoplasmastasis (a reduction of 0 log 10 color-changing unit [CCU] equivalents/ml), a reduction of 1 log 10 CCU equivalents/ml, and a reduction of 2.5 log 10 CCU equivalents/ml are 28,820, 38,030, and 56,256, respectively. In addition, we demonstrated that valnemulin at a dose of 6.5 mg/kg resulted in a reduction of 2.5 log 10 CCU equivalents/ml. These investigations provide a solid foundation for the usage of valnemulin in poultry with M. gallisepticum infections.

BACKGROUND The Staphylococcus aureus global regulators--agr, sarA, and sae--coordinately control alpha-toxin gene (hla) expression in vitro. However, their relative in vivo contributions to hla expression, particularly in endovascular infections, have not been defined. METHODS A plasmid-based hla-promoter:green fluorescent protein reporter system was constructed in 2 genetically related S. aureus strains: RN6390 (a natural sigma factor B [sigB]-deficient mutant), SH1000 (a sigB-repaired variant of RN6390 lineage), and their respective agr, sarA, agr/sarA, and sae mutants. These strain sets were used to quantify hla expression in vitro and in an experimental infective endocarditis (IE) model using flow cytometry. RESULTS In vitro, hla expression was positively modulated by all 3 regulons (sae > agr/sarA > agr and sarA) in both RN6390 and SH1000 backgrounds. In the IE model, hla expression in cardiac vegetations was lower in all single mutants than in the respective parental strains (P

gC1qR/p33 (gC1qR) is a ubiquitously expressed cellular protein that is also found in plasma and the extracellular matrix. In addition to its role in modulating the activation of complement and kinin cascades, gC1qR has been identified as a putative host ligand for endovascular pathogens, includingStaphylococcus aureus. The present study provides evidence of the ability of soluble gC1qR to enhanceS. aureus-fibrinogen interactions via simultaneously binding fibrinogen andS. aureus. This interaction was inhibited in vitro by two monoclonal antibodies (MAbs 74.5.2 and 60.11) recognizing distinct structural and functional domains of gC1qR. To evaluate the in vivo role of gC1qR, MAbs 74.5.2 and 60.11 were used in an experimental rat model ofS. aureusendocarditis. Each MAb (100 mg/kg of body weight, given intraperitoneally) reached sustained (>60 h) and high (100 to 200 μg/ml) serum levels. Prophylaxis with MAb 60.11 or 74.5.2 caused substantial reductions inS. aureuscolonization of aortic valves, kidneys, and the spleen compared to untreated controls. However, only MAb 74.5.2 prophylaxis therapy reached statistical significance, and only sera from animals protected with MAb 74.5.2 inhibited gC1qR-mediatedS. aureusinteractions with fibrinogen. Although not statistically significant, the reductions in bacterial colonization achieved with MAb 60.11 alone and in combination with MAb 74.5.2 (versus MAb 74.5.2 alone) suggest that there are effects of gC1qR blockade onS. aureusinfective endocarditis in addition to blocking gC1qR-mediatedS. aureusbinding to fibrinogen. Such impacts may include direct modulation of complement (MAb 60.11) and kinin cascades (MAb 74.5.2) and/or activation of immune and inflammatory responses via localized immune complex formation.

Begomovirus isolates were obtained from Stachytarpheta jamaicensis plants showing leaf curl and chlorosis symptoms collected in the Hainan province of China. The complete sequences of isolates Hn5-4, Hn6-1, Hn30 and Hn34 were determined to be 2748, 2751, 2748 and 2748 nucleotides long, respectively. The complete sequences of the four isolates share more than 94.9% nucleotide sequence identity, but all of them have less than 86% nucleotide sequence identity with other reported begomoviruses. The molecular data show that Hn5-4, Hn6-1, Hn30 and Hn34 are isolates of a distinct begomovirus species, for which the name Stachytarpheta leaf curl virus (StaLCV) is proposed. PCR and Southern blot analyses demonstrate that all the collected field samples are not associated with DNAbeta or DNA-B components. An infectious clone of StaLCV isolate Hn5-4 was constructed, and could efficiently infect Nicotiana benthamiana, N. tabacum Samsun, N. glutinosa, Lycopersicon esculentum and Petunia hybrida plants, inducing upward leaf roll and vein swelling symptoms. In addition, we illustrate that StaLCV can functionally interact with distinct DNAbeta molecules in plants.

Three viral isolates Hn8, Hn40 and Hn41 were obtained from Sida acuta showing yellow mosaic symptom in the Hainan province, China. Comparison of partial DNA-A sequences amplified with degenerate primers confirmed the existence of single type of Begomovirus. The complete nucleotide sequence of the DNA-A-like molecule of Hn8 was determined to be 2749 nucleotides, having a typical genetic organization of a Begomovirus. Hn8 DNA-A had the highest sequence identity (78%) with that of Ageratum yellow vein China virus-[G13] (AJ558120), and had less sequence identity with other begomoviruses. Based on the above molecular data, Hn8 was thus considered as a new Begomovirus species, for which the name Sida yellow mosaic China virus (SiYMCNV) is proposed. Satellite DNA-β molecules (Hn8-β, Hn40-β and Hn41-β) were found to be associated with Hn8, Hn40 and Hn41 and their complete nucleotide sequences were determined. Sequence analysis showed that Hn8-β, Hn40-β and Hn41-β shared more than 84% nucleotide sequence identity, and they were different from other characterized DNA-β, sharing the highest nucleotide sequence identity (47.8%) with DNA-β of Ageratum yellow vein virus.

Purpose To determine the response rate, time to disease progression, survival duration and rate, and toxicity with the combination of cetuximab and gemcitabine in patients with epidermal growth factor receptor (EGFR)-expressing advanced pancreatic cancer. Patients and Methods Patients with measurable locally advanced or metastatic pancreatic cancer who had never received chemotherapy for their advanced disease and had immunohistochemical evidence of EGFR expression were eligible for the multicenter phase II trial. Patients were treated with cetuximab at an initial dose of 400 mg/m2, followed by 250 mg/m2 weekly for 7 weeks. Gemcitabine was administered at 1,000 mg/m2 for 7 weeks, followed by 1 week of rest. In subsequent cycles, cetuximab was administered weekly, and gemcitabine was administered weekly for 3 weeks every 4 weeks. Results Sixty-one patients were screened for EGFR expression, 58 patients (95%) had at least 1+ staining, and 41 were enrolled onto the trial. Five patients (12.2%) achieved a partial response, and 26 (63.4%) had stable disease. The median time to disease progression was 3.8 months, and the median overall survival duration was 7.1 months. One-year progression-free survival and overall survival rates were 12% and 31.7%, respectively. The most frequently reported grade 3 or 4 adverse events were neutropenia (39.0%), asthenia (22.0%), abdominal pain (22.0%), and thrombocytopenia (17.1%). Conclusion Cetuximab in combination with gemcitabine showed promising activity against advanced pancreatic cancer. Further clinical investigation is warranted.

Expression of vascular endothelial growth factor (VEGF), a key angiogenic protein, has been linked with pancreatic cancer progression. However, the molecular basis for VEGF overexpression remains unclear. Immunohistochemical studies have indicated that VEGF overexpression coincides with elevated Stat3 activation in human pancreatic cancer specimens. In our study, more than 80% of the human pancreatic cancer cell lines used exhibited constitutively activated Stat3, with Stat3 activation correlated with the VEGF expression level. Blockade of activated Stat3 via ectopic expression of dominant-negative Stat3 significantly suppressed VEGF expression, angiogenesis, tumor growth, and metastasis in vivo. Furthermore, constitutively activated Stat3 directly activated the VEGF promoter, whereas dominant-negative Stat3 inhibited the VEGF promoter. A putative Stat3-responsive element on the VEGF promoter was identified using a protein-DNA binding assay and confirmed using a promoter mutagenesis assay. These results indicate that Stat3 directly regulates VEGF expression and hence angiogenesis, growth, and metastasis of human pancreatic cancer, suggesting that Stat3 signaling may be targeted for treatment of pancreatic cancer.