Murine Subcutaneous Infection Model Research Articles

Precisely Structured Nitric-Oxide-Releasing Copolymer Brush Defeats Broad-Spectrum Catheter-Associated Biofilm Infections In Vivo.

Gram-negative bacteria cannot be easily eradicated by antibiotics and are a major source of recalcitrant infections of indwelling medical devices. Among various device-associated infections, intravascular catheter infection is a leading cause of mortality. Prior approaches to surface modification, such as antibiotics impregnation, hydrophilization, unstructured NO-releasing, etc., have failed to achieve adequate infection-resistant coatings. We report a precision-structured diblock copolymer brush (H(N)-b-S) composed of a surface antifouling block of poly(sulfobetaine methacrylate) (S) and a subsurface bactericidal block (H(N)) of nitric-oxide-emitting functionalized poly(hydroxyethyl methacrylate) (H) covalently grafted from the inner and outer surfaces of a polyurethane catheter. The block copolymer architecture of the coating is important for achieving good broad-spectrum anti-biofilm activity with good biocompatibility and low fouling. The coating procedure is scalable to clinically useful catheter lengths. Only the block copolymer brush coating ((H(N)-b-S)) shows unprecedented, above 99.99%, in vitro biofilm inhibition of Gram-positive and Gram-negative bacteria, 100-fold better than previous coatings. It has negligible toxicity toward mammalian cells and excellent blood compatibility. In a murine subcutaneous infection model, it achieves >99.99% biofilm reduction of Gram-positive and Gram-negative bacteria compared with <90% for silver catheter, while in a porcine central venous catheter infection model, it achieves >99.99% reduction of MRSA with 5-day implantation. This precision coating is readily applicable for long-term biofilm-resistant and blood-compatible copolymer coatings covalently grafted from a wide range of medical devices.

DNA methylation from a Type I restriction modification system influences gene expression and virulence in Streptococcus pyogenes

DNA methylation is pervasive across all domains of life. In bacteria, the presence of N6-methyladenosine (m6A) has been detected among diverse species, yet the contribution of m6A to the regulation of gene expression is unclear in many organisms. Here we investigated the impact of DNA methylation on gene expression and virulence within the human pathogen Streptococcus pyogenes, or Group A Streptococcus. Single Molecule Real-Time sequencing and subsequent methylation analysis identified 412 putative m6A sites throughout the 1.8 Mb genome. Deletion of the Restriction, Specificity, and Methylation gene subunits (ΔRSM strain) of a putative Type I restriction modification system lost all detectable m6A at the recognition sites and failed to prevent transformation with foreign-methylated DNA. RNA-sequencing identified 20 genes out of 1,895 predicted coding regions with significantly different gene expression. All of the differentially expressed genes were down regulated in the ΔRSM strain relative to the parent strain. Importantly, we found that the presence of m6A DNA modifications affected expression of Mga, a master transcriptional regulator for multiple virulence genes, surface adhesins, and immune-evasion factors in S. pyogenes. Using a murine subcutaneous infection model, mice infected with the ΔRSM strain exhibited an enhanced host immune response with larger skin lesions and increased levels of pro-inflammatory cytokines compared to mice infected with the parent or complemented mutant strains, suggesting alterations in m6A methylation influence virulence. Further, we found that the ΔRSM strain showed poor survival within human neutrophils and reduced adherence to human epithelial cells. These results demonstrate that, in addition to restriction of foreign DNA, gram-positive bacteria also use restriction modification systems to regulate the expression of gene networks important for virulence.

A Biodegradable Polycationic Paint that Kills Bacteria in Vitro and in Vivo.

Bacterial colonization and subsequent formation of biofilms onto surfaces of medical devices and implants is a major source of nosocomial infections. Most antibacterial coatings to combat infections are either metal-based or nondegradable-polymer-based and hence limited by their nondegradability and unpredictable toxicity. Moreover, to combat infections effectively, the coatings are required to display simultaneous antibacterial and antibiofilm activity. Herein we report biocompatible and biodegradable coatings based on organo-soluble quaternary chitin polymers which were immobilized noncovalently onto surfaces as bactericidal paint. The polycationic paint was found to be active against both drug-sensitive and -resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and β-lactam-resistant Klebsiella pneumoniae. The cationic polymers were shown to interact with the negatively charged bacterial cell membrane and disrupt the membrane integrity, thereby causing leakage of intracellular constituents and cell death upon contact. Importantly, surfaces coated with the polymers inhibited formation of biofilms against both Gram-positive S. aureus and Gram-negative E. coli, two of the most clinically important bacteria that form biofilms. Surfaces coated with the polymers displayed negligible toxicity against human erythrocytes and embryo kidney cells. Notably, the polymers were shown to be susceptible toward lysozyme. Furthermore, subcutaneous implantation of polymer discs in rats led to 15-20% degradation in 4 weeks thereby displaying their biodegradability. In a murine model of subcutaneous infection, polymer-coated medical-grade catheter reduced MRSA burden by 3.7 log compared to that of noncoated catheter. Furthermore, no biofilm development was observed on the coated catheters under in vivo conditions. The polycationic materials thus developed herein represent a novel class of safe and effective coating agents for the prevention of device-associated infections.

Antimicrobial Salvage Therapy for Persistent Staphylococcal Bacteremia Using Daptomycin Plus Ceftaroline

PurposeGuidelines recommend daptomycin combination therapy as an option for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia after vancomycin failure. Recent data suggest that combining daptomycin with a β-lactam may have unique benefits; however, there are very limited clinical data regarding the use of ceftaroline with daptomycin. MethodsAll 26 cases from the 10 medical centers in which ceftaroline plus daptomycin was used for treatment of documented refractory staphylococcal bacteremia from March 2011 to November 2012 were included. In vitro (synergy studies, binding assays, cathelicidin LL-37 killing assays), and in vivo (virulence assays using a murine subcutaneous infection model) studies examining the effects of ceftaroline with daptomycin were also performed. FindingsDaptomycin plus ceftaroline was used in 26 cases of staphylococcal bacteremia (20 MRSA, 2 vancomycin-intermediate S aureus, 2 methicillin-susceptible S aureus [MSSA], 2 methicillin-resistant S epidermidis). Bacteremia persisted for a median of 10 days (range, 3–23 days) on previous antimicrobial therapy. After daptomycin plus ceftaroline was started, the median time to bacteremia clearance was 2 days (range, 1–6 days). In vitro studies showed ceftaroline synergy against MRSA and enhanced MRSA killing by cathelicidin LL-37 and neutrophils. Ceftaroline also induced daptomycin binding in MSSA and MRSA to a comparable degree as nafcillin. MRSA grown in subinhibitory concentrations of ceftaroline showed attenuated virulence in a murine subcutaneous infection model. ImplicationsCeftaroline plus daptomycin may be an option to hasten clearance of refractory staphylococcal bacteremia. Ceftaroline offers dual benefit via synergy with both daptomycin and sensitization to innate host defense peptide cathelicidin LL37, which could attenuate virulence of the pathogen.

Nafcillin enhances innate immune-mediated killing of methicillin-resistant Staphylococcus aureus

Based on in vitro synergy studies, the addition of nafcillin to daptomycin was used to treat refractory methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. Daptomycin is a de facto cationic antimicrobial peptide in vivo, with antistaphylococcal mechanisms reminiscent of innate host defense peptides (HDPs). In this study, the effects of nafcillin on HDP activity against MRSA were examined in vitro and in vivo. Exposures to β-lactam antimicrobials in general, and nafcillin in particular, significantly increased killing of S. aureus by selected HDPs from keratinocytes, neutrophils, and platelets. This finding correlated with enhanced killing of MRSA by whole blood, neutrophils, and keratinocytes after growth in nafcillin. Finally, nafcillin pretreatment ex vivo reduced MRSA virulence in a murine subcutaneous infection model. Despite the lack of direct activity against MRSA, these studies show potent, consistent, and generalized nafcillin-mediated "sensitization" to increased killing of MRSA by various components of the innate host response. The use of nafcillin as adjunctive therapy in MRSA bacteremia merits further study and should be considered in cases refractory to standard therapy. Nafcillin has been used as adjunctive therapy to clear persistent MRSA bacteremia. Nafcillin enhances killing of MRSA by a cadre of innate host defense peptides. Nafcillin increases binding of human cathelicidin LL-37 to the MRSA membrane. Nafcillin enhances killing of MRSA by neutrophils. Nafcillin reduces virulence of MRSA in a murine subcutaneous infection model.

Streptococcus pyogenes c-di-AMP Phosphodiesterase, GdpP, Influences SpeB Processing and Virulence

Small cyclic nucleotide derivatives are employed as second messengers by both prokaryotes and eukaryotes to regulate diverse cellular processes responding to various signals. In bacteria, c-di-AMP has been discovered most recently, and some Gram-positive pathogens including S. pyogenes use this cyclic nucleotide derivative as a second messenger instead of c-di-GMP, a well-studied important bacterial second messenger. GdpP, c-di-AMP phosphodiesterase, is responsible for degrading c-di-AMP inside cells, and the cellular role of GdpP in S. pyogenes has not been examined yet. To test the cellular role of GdpP, we created a strain with a nonpolar inframe deletion of the gdpP gene, and examined the properties of the strain including virulence. From this study, we demonstrated that GdpP influences the biogenesis of SpeB, the major secreted cysteine protease, at a post-translational level, susceptibility to the beta lactam antibiotic ampicillin, and is necessary for full virulence in a murine subcutaneous infection model.

Lipoprotein biosynthesis by prolipoprotein diacylglyceryl transferase is required for efficient spore germination and full virulence of Bacillus anthracis

Bacterial lipoproteins play a crucial role in virulence in some gram-positive bacteria. However, the role of lipoprotein biosynthesis in Bacillus anthracis is unknown. We created a B. anthracis mutant strain altered in lipoproteins by deleting the lgt gene encoding the enzyme prolipoprotein diacylglyceryl transferase, which attaches the lipid anchor to prolipoproteins. (14)C-palmitate labelling confirmed that the mutant strain lacked lipoproteins, and hydrocarbon partitioning showed it to have decreased surface hydrophobicity. The anthrax toxin proteins were secreted from the mutant strain at nearly the same levels as from the wild-type strain. The TLR2-dependent TNF-α response of macrophages to heat-killed lgt mutant bacteria was reduced. Spores of the lgt mutant germinated inefficiently in vitro and in mouse skin. As a result, in a murine subcutaneous infection model, lgt mutant spores had markedly attenuated virulence. In contrast, vegetative cells of the lgt mutant were as virulent as those of the wild-type strain. Thus, lipoprotein biosynthesis in B. anthracis is required for full virulence in a murine infection model.

Discriminating Virulence Mechanisms amongBacillus anthracisStrains by Using a Murine Subcutaneous Infection Model

Bacillus anthracis strains harboring virulence plasmid pXO1 that encodes the toxin protein protective antigen (PA), lethal factor, and edema factor and virulence plasmid pXO2 that encodes capsule biosynthetic enzymes exhibit different levels of virulence in certain animal models. In the murine model of pulmonary infection, B. anthracis virulence was capsule dependent but toxin independent. We examined the role of toxins in subcutaneous (s.c.) infections using two different genetically complete (pXO1(+) pXO2(+)) strains of B. anthracis, strains Ames and UT500. Similar to findings for the pulmonary model, toxin was not required for infection by the Ames strain, because the 50% lethal dose (LD(50)) of a PA-deficient (PA(-)) Ames mutant was identical to that of the parent Ames strain. However, PA was required for efficient s.c. infection by the UT500 strain, because the s.c. LD(50) of a UT500 PA(-) mutant was 10,000-fold higher than the LD(50) of the parent UT500 strain. This difference between the Ames strain and the UT500 strain could not be attributed to differences in spore coat properties or the rate of germination, because s.c. inoculation with the capsulated bacillus forms also required toxin synthesis by the UT500 strain to cause lethal infection. The toxin-dependent phenotype of the UT500 strain was host phagocyte dependent, because eliminating Gr-1(+) phagocytes restored virulence to the UT500 PA(-) mutant. These experiments demonstrate that the dominant virulence factors used to establish infection by B. anthracis depend on the route of inoculation and the bacterial strain.

The IL-8 Protease SpyCEP/ScpC of Group A Streptococcus Promotes Resistance to Neutrophil Killing

Interleukin-8 (IL-8) promotes neutrophil-mediated host defense through its chemoattractant and immunostimulatory activities. The Group A Streptococcus (GAS) protease SpyCEP (also called ScpC) cleaves IL-8, and SpyCEP expression is strongly upregulated in vivo in the M1T1 GAS strains associated with life-threatening systemic disease including necrotizing fasciitis. Coupling allelic replacement with heterologous gene expression, we show that SpyCEP is necessary and sufficient for IL-8 degradation. SpyCEP decreased IL-8-dependent neutrophil endothelial transmigration and bacterial killing, the latter by reducing neutrophil extracellular trap formation. The knockout mutant lacking SpyCEP was attenuated for virulence in murine infection models, and SpyCEP expression conferred protection to coinfecting bacteria. We also show that the zoonotic pathogen Streptococcus iniae possesses a functional homolog of SpyCEP (CepI) that cleaves IL-8, promotes neutrophil resistance, and contributes to virulence. By inactivating the multifunctional host defense peptide IL-8, the SpyCEP protease impairs neutrophil clearance mechanisms, contributing to the pathogenesis of invasive streptococcal infection.

Role for serine protease HtrA (DegP) of Streptococcus pyogenes in the biogenesis of virulence factors SpeB and the hemolysin streptolysin S.

The serine protease HtrA is involved in the folding and maturation of secreted proteins, as well as in the degradation of proteins that misfold during secretion. Depletion of HtrA has been shown to affect the sensitivity of many organisms to thermal and environmental stresses, as well as being essential for virulence in many pathogens. In the present study, we compared the behaviors of several different HtrA mutants of the gram-positive pathogen Streptococcus pyogenes (group A streptococcus). Consistent with prior reports, insertional inactivation of htrA, the gene that encodes HtrA, resulted in a mutant that grew poorly at 37 degrees C. However, an identical phenotype was observed when a similar polar insertion was placed immediately downstream of htrA in the streptococcal chromosome, suggesting that the growth defect of the insertion mutant was not a direct result of insertional inactivation of htrA. This conclusion was supported by the observation that a nonpolar deletion mutation of htrA did not produce the growth defect. However, this mutation did affect the production of several secreted virulence factors whose biogenesis requires extensive processing. For the SpeB cysteine protease, the loss of HtrA was associated with a failure to proteolytically process the zymogen to an active protease. For the streptolysin S hemolysin, a dramatic increase in hemolytic activity resulted from the depletion of HtrA. Interestingly, HtrA-deficient mutants were not attenuated in a murine model of subcutaneous infection. These data add to the growing body of information that implies an important role for HtrA in the biogenesis of secreted proteins in gram-positive bacteria.

Identification of a streptolysin S-associated gene cluster and its role in the pathogenesis of Streptococcus iniae disease.

Streptococcus iniae causes meningoencephalitis and death in cultured fish species and soft-tissue infection in humans. We recently reported that S. iniae is responsible for local tissue necrosis and bacteremia in a murine subcutaneous infection model. The ability to cause bacteremia in this model is associated with a genetic profile unique to strains responsible for disease in fish and humans (J. D. Fuller, D. J. Bast, V. Nizet, D. E. Low, and J. C. S. de Azavedo, Infect. Immun. 69:1994-2000, 2001). S. iniae produces a cytolysin that confers a hemolytic phenotype on blood agar media. In this study, we characterized the genomic region responsible for S. iniae cytolysin production and assessed its contribution to virulence. Transposon (Tn917) mutant libraries of commensal and disease-associated S. iniae strains were generated and screened for loss of hemolytic activity. Analysis of two nonhemolytic mutants identified a chromosomal locus comprising 9 genes with 73% homology to the group A streptococcus (GAS) sag operon for streptolysin S (SLS) biosynthesis. Confirmation that the S. iniae cytolysin is a functional homologue of SLS was achieved by PCR ligation mutagenesis, complementation of an SLS-negative GAS mutant, and use of the SLS inhibitor trypan blue. SLS-negative sagB mutants were compared to their wild-type S. iniae parent strains in the murine model and in human whole-blood killing assays. These studies demonstrated that S. iniae SLS expression is required for local tissue necrosis but does not contribute to the establishment of bacteremia or to resistance to phagocytic clearance.