Extraction of Aerosol-Deposited Yersinia pestis from Indoor Surfaces To Determine Bacterial Environmental Decay.

UNLABELLED: Public health and decontamination decisions following an event that causes indoor contamination with a biological agent require knowledge of the environmental persistence of the agent. The goals of this study were to develop methods for experimentally depositing bacteria onto indoor surfaces via aerosol, evaluate methods for sampling and enumerating the agent on surfaces, and use these methods to determine bacterial surface decay. A specialized aerosol deposition chamber was constructed, and methods were established for reproducible and uniform aerosol deposition of bacteria onto four coupon types. The deposition chamber facilitated the control of relative humidity (RH; 10 to 70%) following particle deposition to mimic the conditions of indoor environments, as RH is not controlled by standard heating, ventilation, and air conditioning (HVAC) systems. Extraction and culture-based enumeration methods to quantify the viable bacteria on coupons were shown to be highly sensitive and reproducible. To demonstrate the usefulness of the system for decay studies,Yersinia pestis persistence as a function of surface type at 21 °C and 40% RH was determined to be >40%/min for all surfaces. Based upon these results, at typical indoor temperature and RH, a 6-log reduction in titer would expected to be achieved within 1 h as the result of environmental decay on surfaces without active decontamination. The developed approach will facilitate future persistence and decontamination studies with a broad range of biological agents and surfaces, providing agent decay data to inform both assessments of risk to personnel entering a contaminated site and decontamination decisions following biological contamination of an indoor environment. IMPORTANCE: Public health and decontamination decisions following contamination of an indoor environment with a biological agent require knowledge of the environmental persistence of the agent. Previous studies on Y. pestis persistence have utilized large liquid droplet deposition to provide persistence data. As a result, methods were developed to deposit aerosols containing bacteria onto indoor surfaces, reproducibly enumerate bacteria harvested from coupons, and determine surface decay utilizing Y. pestis The results of this study provide foundational methods required to evaluate surface decay of bacteria and potentially other biological agents, such as viruses, in aerosol particles as a function of surface type and environment. Integrating the data from both aerosol and liquid deposition surface decay studies will provide medical and public health personnel with a more complete understanding of agent persistence on surfaces in contaminated areas for assessment of health risks and to inform decontamination decisions.

Complete Genome Sequence of the Quality Control Strain Staphylococcus aureus subsp. aureus ATCC 25923.

Staphylococcus aureus subsp. aureus ATCC 25923 is commonly used as a control strain for susceptibility testing to antibiotics and as a quality control strain for commercial products. We present the completed genome sequence for the strain, consisting of the chromosome and a 27.5-kb plasmid.

STING ligand c-di-GMP improves cancer vaccination against metastatic breast cancer.

Cancer vaccination may be our best and most benign option for preventing or treating metastatic cancer. However, breakthroughs are hampered by immune suppression in the tumor microenvironment. In this study, we analyzed whether cyclic diguanylate (c-di-GMP), a ligand for stimulator of interferon genes (STING), could overcome immune suppression and improve vaccination against metastatic breast cancer. Mice with metastatic breast cancer (4T1 model) were therapeutically immunized with an attenuated Listeria monocytogenes (LM)-based vaccine, expressing tumor-associated antigen Mage-b (LM-Mb), followed by multiple low doses of c-di-GMP (0.2 µmol/L). This treatment resulted in a striking and near elimination of all metastases. Experiments revealed that c-di-GMP targets myeloid-derived suppressor cells (MDSC) and tumor cells. Low doses of c-di-GMP significantly increased the production of IL12 by MDSCs, in correlation with improved T-cell responses to Mage-b, whereas a high dose of c-di-GMP (range, 0.3-3 mmol/L) activated caspase-3 in the 4T1 tumor cells and killed the tumor cells directly. On the basis of these results, we tested one administration of high-dose c-di-GMP (3 mmol/L) followed by repeated administrations of low-dose c-di-GMP (0.2 µmol/L) in the 4T1 model, and found equal efficacy compared with the combination of LM-Mb and c-di-GMP. This finding correlated with a mechanism of improved CD8 T-cell responses to tumor-associated antigens (TAA) Mage-b and Survivin, most likely through cross-presentation of these TAAs from c-di-GMP-killed 4T1 tumor cells, and through c-di-GMP-activated TAA-specific T cells. Our results demonstrate that activation of STING-dependent pathways by c-di-GMP is highly attractive for cancer immunotherapy.

c-di-GMP as a vaccine adjuvant enhances protection against systemic methicillin-resistant Staphylococcus aureus (MRSA) infection.

Cyclic diguanylate (c-di-GMP) is a novel immunomodulator and immune enhancer that triggers a protective host innate immune response. The protective effect of c-di-GMP as a vaccine adjuvant against Staphylococcus aureus infection was investigated by subcutaneous (s.c.) vaccination with two different S. aureus antigens, clumping factor A (ClfA) and a nontoxic mutant staphylococcal enterotoxin C (mSEC), then intravenous (i.v.) challenge with viable methicillin-resistant S. aureus (MRSA) in a systemic infection model. Mice immunized with c-di-GMP plus mSEC or c-di-GMP plus ClfA vaccines then challenged with MRSA produced strong antigen-specific antibody responses demonstrating immunogenicity of the vaccines. Bacterial counts in the spleen and liver of c-di-GMP plus mSEC and c-di-GMP plus ClfA-immunized mice were significantly lower than those of control mice (P<0.001). Mice immunized with c-di-GMP plus mSEC or c-di-GMP plus ClfA showed significantly higher survival rates at day 7 (87.5%) than those of the non-immunized control mice (33.3%) (P<0.05). Furthermore, immunization of mice with c-di-GMP plus mSEC or c-di-GMP plus ClfA induced not only very high titers of immunoglobulin G1 (IgG1), but c-di-GMP plus mSEC also induced significantly higher levels of IgG2a, IgG2b and IgG3 compared to alum adjuvant (P<0.01 and P<0.001, respectively) and c-di-GMP plus ClfA induced significantly higher levels of IgG2a, IgG2b and IgG3 compared to alum adjuvant (P<0.001). Our results show that c-di-GMP should be developed as an adjuvant and immunotherapeutic to provide protection against systemic infection caused by S. aureus (MRSA).

c-di-GMP is an effective immunomodulator and vaccine adjuvant against pneumococcal infection.

Cyclic diguanylate (c-di-GMP) is a unique bacterial intracellular signaling molecule capable of stimulating enhanced protective innate immunity against various bacterial infections. The effects of intranasal pretreatment with c-di-GMP, or intraperitoneal coadministration of c-di-GMP with the pneumolysin toxoid (PdB) or pneumococcal surface protein A (PspA) before pneumococcal challenge, were investigated in mice. We found that c-di-GMP had no significant direct short-term effect on the growth rate of Streptococcus pneumoniae either in vitro or in vivo. However, intranasal pretreatment of mice with c-di-GMP resulted in a significant decrease in bacterial load in lungs and blood after serotypes 2 and 3 challenge, and a significant decrease in lung titers after serotype 4 challenge. Potential cellular mediators of these enhanced protective responses were identified in lungs and draining lymph nodes. Intraperitoneal coadministration of c-di-GMP with PdB or PspA before challenge resulted in significantly higher antigen-specific antibody titers and increased survival of mice, compared to that obtained with alum adjuvant. These findings demonstrate that local or systemic c-di-GMP administration stimulates innate and adaptive immunity against invasive pneumococcal disease. We propose that c-di-GMP can be used as an effective broad spectrum immunomodulator and vaccine adjuvant to prevent infectious diseases.

Cyclic di-GMP stimulates protective innate immunity in bacterial pneumonia.

Innate immunity is the primary mechanism by which extracellular bacterial pathogens are effectively cleared from the lung. We have previously shown that cyclic di-GMP (c-di-GMP [c-diguanylate]) is a novel small molecule immunomodulator and immunostimulatory agent that triggers protective host innate immune responses. Using a murine model of bacterial pneumonia, we show that local intranasal (i.n.) or systemic subcutaneous (s.c.) administration of c-di-GMP prior to intratracheal (i.t.) challenge with Klebsiella pneumoniae stimulates protective immunity against infection. Specifically, i.n. or s.c. administration of c-di-GMP 48 and 24 h prior to i.t. K. pneumoniae challenge resulted in significantly increased survival. Pretreatment with c-di-GMP resulted in a 5-fold reduction in bacterial CFU in the lung (P < 0.05) and an impressive >1,000-fold decrease in CFU in the blood (P < 0.01). c-di-GMP administration stimulated a robust innate response to bacterial challenge, characterized by enhanced accumulation of neutrophils and alphabeta T cells, as well as activated NK and alphabeta T lymphocytes, which was associated with earlier and more vigorous expression of chemokines and type I cytokines. Moreover, lung macrophages recovered from Klebsiella-infected mice pretreated with c-di-GMP expressed greater quantities of inducible nitric oxide synthase and nitric oxide ex vivo than did macrophages isolated from infected mice pretreated with the control, c-GMP. These findings demonstrate that c-di-GMP delivered in either a compartmentalized or systemic fashion stimulates protective innate immunity in the lung and protects mice against bacterial invasion. We propose that the cyclic dinucleotide c-di-GMP may be used clinically as an effective immunomodulator, immune enhancer, and vaccine adjuvant to protect against respiratory infection and pneumonia in humans and animals.

Bacterial c-di-GMP is an immunostimulatory molecule.

Cyclic diguanylate (c-di-GMP) is a bacterial intracellular signaling molecule. We have shown that treatment with exogenous c-di-GMP inhibits Staphylococcus aureus infection in a mouse model. We now report that c-di-GMP is an immodulator and immunostimulatory molecule. Intramammary treatment of mice with c-di-GMP 12 and 6 h before S. aureus challenge gave a protective effect and a 10,000-fold reduction in CFUs in tissues (p < 0.001). Intramuscular vaccination of mice with c-di-GMP coinjected with S. aureus clumping factor A (ClfA) Ag produced serum with significantly higher anti-ClfA IgG Ab titers (p < 0.001) compared with ClfA alone. Intraperitoneal injection of mice with c-di-GMP activated monocyte and granulocyte recruitment. Human immature dendritic cells (DCs) cultured in the presence of c-di-GMP showed increased expression of costimulatory molecules CD80/CD86 and maturation marker CD83, increased MHC class II and cytokines and chemokines such as IL-12, IFN-gamma, IL-8, MCP-1, IFN-gamma-inducible protein 10, and RANTES, and altered expression of chemokine receptors including CCR1, CCR7, and CXCR4. c-di-GMP-matured DCs demonstrated enhanced T cell stimulatory activity. c-di-GMP activated p38 MAPK in human DCs and ERK phosphorylation in human macrophages. c-di-GMP is stable in human serum. We propose that cyclic dinucleotides like c-di-GMP can be used clinically in humans and animals as an immunomodulator, immune enhancer, immunotherapeutic, immunoprophylactic, or vaccine adjuvant.

3',5'-cyclic diguanylic acid reduces the virulence of biofilm-forming Staphylococcus aureus strains in a mouse model of mastitis infection.

The cyclic dinucleotide 3',5'-cyclic diguanylic acid (c-di-GMP) is a naturally occurring small molecule that regulates important signaling systems in bacteria. We have recently shown that c-di-GMP inhibits Staphylococcus aureus biofilm formation in vitro and its adherence to HeLa cells. We now report that c-di-GMP treatment has an antimicrobial and antipathogenic activity in vivo and reduces, in a dose-dependent manner, bacterial colonization by biofilm-forming S. aureus strains in a mouse model of mastitis infection. Intramammary injections of 5 and 50 nmol of c-di-GMP decreased colonization (bacterial CFU per gram of gland) by 0.79 (P > 0.05) and 1.44 (P < 0.01) logs, respectively, whereas 200-nmol doses allowed clearance of the bacteria below the detection limit with a reduction of more than 4 logs (P < 0.001) compared to the untreated control groups. These results indicate that cyclic dinucleotides potentially represent an attractive and novel drug platform which could be used alone or in combination with other agents or drugs in the prevention, treatment, or control of infection.

c-di-GMP (3'-5'-cyclic diguanylic acid) inhibits Staphylococcus aureus cell-cell interactions and biofilm formation.

Staphylococcus aureus is an important pathogen of humans and animals, and antibiotic resistance is a public health concern. Biofilm formation is essential in virulence and pathogenesis, and the ability to resist antibiotic treatment results in difficult-to-treat and persistent infections. As such, novel antimicrobial approaches are of great interest to the scientific, medical, and agriculture communities. We recently proposed that modulating levels of the cyclic dinucleotide signaling molecule, c-di-GMP (cyclic diguanylate [3',5'-cyclic diguanylic acid], cGpGp), has utility in regulating phenotypes of prokaryotes. We report that extracellular c-di-GMP shows activity against human clinical and bovine intramammary mastitis isolates of S. aureus, including methicillin-resistant S. aureus (MRSA) isolates. We show that chemically synthesized c-di-GMP is soluble and stable in water and physiological saline and stable following boiling and exposure to acid and alkali. Treatment of S. aureus with extracellular c-di-GMP inhibited cell-to-cell (intercellular) adhesive interactions in liquid medium and reduced (>50%) biofilm formation in human and bovine isolates compared to untreated controls. c-di-GMP inhibited the adherence of S. aureus to human epithelial HeLa cells. The cyclic nucleotide analogs cyclic GMP and cyclic AMP had a lesser inhibitory effect on biofilms, while 5'-GMP had no major effect. We propose that cyclic dinucleotides such as c-di-GMP, used either alone or in combination with other antimicrobial agents, represent a novel and attractive approach in the development of intervention strategies for the prevention of biofilms and the control and treatment of infection.

3',5'-Cyclic diguanylic acid (c-di-GMP) inhibits basal and growth factor-stimulated human colon cancer cell proliferation.

The novel cyclic dinucleotide, 3',5'-cyclic diguanylic acid, cGpGp (c-di-GMP), is a naturally occurring small molecule that regulates important signaling mechanisms in prokaryotes. Recently, we showed that c-di-GMP has "drug-like" properties and that c-di-GMP treatment might be a useful antimicrobial approach to attenuate the virulence and pathogenesis of Staphylococcus aureus and prevent or treat infection. In the present communication, we report that c-di-GMP (50 microM) has striking properties regarding inhibition of cancer cell proliferation in vitro. c-di-GMP inhibits both basal and growth factor (acetylcholine and epidermal growth factor)-induced cell proliferation of human colon cancer (H508) cells. Toxicity studies revealed that exposure of normal rat kidney cells and human neuroblastoma cells to c-di-GMP at biologically relevant doses showed no lethal cytotoxicity. Cyclic dinucleotides, such as c-di-GMP, represent an attractive and novel "drug-platform technology" that can be used not only to develop new antimicrobial agents, but also to develop novel therapeutic agents to prevent or treat cancer.

Chemical behavior of bis(3'-5')diguanylic acid in aqueous solutions.

This paper describes unique behavior of bis(3'-5')diguanylic acid (c-di-GMP) under some conditions. Thus, c-di-GMP exists as the monomer in aprotic organic solvents such as DMSO. By contrast, c-di-GMP smoothly aggregates in water and in low-concentration aqueous solutions of some salts, such as sodium chloride and ammonium acetate, to give a mixture of many aggregates. The resulting multiple aggregates converge to the single compound (provably the monomer) in a >154 mM (0.9%) sodium chloride aqueous solution, in a >100 mM ammonium acetate buffer, and in a >100 mM phosphate buffer.

Transcriptional analysis and operon structure of the tagA-orf2-orf3-mop-tagD region on the Vibrio pathogenicity island in epidemic V. cholerae.

The Vibrio pathogenicity island (VPI) in epidemic Vibrio cholerae is an essential virulence gene cluster. The VPI can excise from the chromosome and form extrachromosomal circular excision products. The VPI is 41.2-kb in size and encodes 29 potential proteins, several of which have no known function and whose regulation is not well understood. To determine the transcriptional organization of the tagA-orf2-orf3-mop-tagD region located at the 5'-(left) end of the VPI, we used reverse-transcriptase-PCR (RT-PCR), Northern blot analysis and DNA sequencing. RT-PCR primers were designed to transcribe and amplify regions spanning two or more open reading frames so as to establish the transcriptional organization. RT-PCR and Northern blot results demonstrated that the tagA-tagD region is transcribed as a polycistronic message and organized into several potential operons including tagA-orf2, orf3-mop, orf3-mop-tagD and tagD alone. Transcriptional lacZ fusions supported the existence of a promoter upstream of orf3 that was toxT-dependent. Interestingly, our data suggests that the orf3 promoter can drive the expression of either a long transcript (orf3-mop-tagD) or a short transcript (orf3-mop) without tagD. Our data also suggests that tagD can be expressed from two different promoters and that tagD is either transcribed alone or co-expressed with orf3-mop under certain conditions. These studies provide new insight into the genetic structure, transcriptional organization and regulation of a cluster of virulence genes on the VPI of epidemic V. cholerae.

Role of exopolysaccharide, the rugose phenotype and VpsR in the pathogenesis of epidemic Vibrio cholerae.

Vibrio cholerae, the causative agent of cholera can produce an exopolysaccharide (EPS). Some strains can also phenotypically switch from a smooth to a 'rugose' phenotype characterized by small wrinkled colonies, overproduction of EPS, increased biofilm formation in vitro and increased resistance to various stressful conditions. High frequency switching to the rugose phenotype is more common in epidemic strains than in non-pathogenic strains, suggesting EPS production and the rugose phenotype are important in cholera epidemiology. VpsR up-regulates Vibrio polysaccharide (VPS) genes and the synthesis of extracellular EPS (VPS). However, the function of VPS, the rugose phenotype and VpsR in pathogenesis is not well understood. We report that rugose strains of both classical and El Tor biotypes of epidemic V. cholerae are defective in the in vitro production of extracellular collagenase activity. In vivo studies in rabbit ileal loops suggest that VpsR mutants are attenuated in reactogenicity. Intestinal colonization studies in infant mice suggest that VPS production, the rugose phenotype and VpsR have a role in pathogenesis. Our results indicate that regulated VPS production is important for promoting in vivo biofilm formation and pathogenesis. Additionally, VpsR might regulate genes with roles in virulence. Rugose strains appear to be a subpopulation of cells that might act as a 'helper' phenotype promoting the pathogenesis of certain strains. Our studies provide new insight into the potential role of VPS, the rugose phenotype and VpsR in the pathogenesis of epidemic V. cholerae.

Identification of genes involved in the switch between the smooth and rugose phenotypes of Vibrio cholerae.

Vibrio cholerae can switch to a 'rugose' phenotype characterized by an exopolysaccharide (EPS) matrix, wrinkled colony morphology, increased biofilm formation and increased survival under specific conditions. The vps gene cluster responsible for the biosynthesis of the rugose EPS (rEPS) is positively regulated by VpsR. We recently identified media (APW#3) promoting EPS production and the rugose phenotype and found epidemic strains switch at a higher frequency than non-pathogenic strains, suggesting this switch and the rugose phenotype are important in cholera epidemiology. In this study, transposon mutagenesis on a smooth V. cholerae strain was used to identify mutants that were unable to shift to the rugose phenotype under inducing conditions to better understand the molecular basis of the switch. We identified vpsR, galE and vps previously associated with the rugose phenotype, and also identified genes not previously associated with the phenotype, including rfbD and rfbE having roles in LPS (lipopolysaccharide) synthesis and aroB and aroK with roles in aromatic amino acid synthesis. Additionally, a mutation in amiB encoding N-acetylmuramoyl-L-alanine amidase caused defects in the switch, motility and cell morphology. We also found that a gene encoding a novel regulatory protein we termed RocS (regulation of cell signaling) containing a GGDEF and EAL domains and associated with c-di-GMP levels is important for the rugose phenotype, EPS, biofilm formation and motility. We propose that modulation of cyclic dinucleotide (e.g. c-di-GMP) levels might have application in regulating various phenotypes of prokaryotes. Our study shows the molecular complexity of the switch between the smooth and rugose phenotypes of V. cholerae and may be relevant to similar phenotypes in other species.

Analysis of the Vibrio pathogenicity island-encoded Mop protein suggests a pleiotropic role in the virulence of epidemic Vibrio cholerae.

Epidemic Vibrio cholerae contain a large essential virulence gene cluster called the Vibrio pathogenicity island (VPI). We recently reported that no in vitro difference in virulence was found in El Tor strain N16961 containing a mutation in the VPI-encoded mop gene but this mutant was hypervirulent and reactogenic in rabbit ileal loops. In this paper, we report in vitro studies showing that independent Mop mutants of strain 3083 are significantly attenuated (approximately 40-fold) in cholera toxin (CT) production and have significantly increased motility and biofilm forming ability but appear to be unaffected in TcpA, hemagglutinin protease and hemolysin compared to their parent. The 3083 Mop mutant showed a 100-fold decrease in its in vivo intestinal colonization ability in the infant mouse competition assays. While reverse transcription polymerase chain reaction and phenotypic studies of a mop plasmid in both mutant and wild-type backgrounds suggest Mop is expressed by the plasmid, the differences in CT and biofilm formation could not be restored in any of the mutants. The inability to complement the Mop mutants in trans may be due either to the selection of secondary mutations or to mop possibly being part of an operon. Our findings that Mop is associated with CT, motility, biofilm formation and intestinal colonization support a hypothesis in which Mop has a pleiotropic role in the pathogenesis and persistence of epidemic V. cholerae.

The vibrio pathogenicity island-encoded mop protein modulates the pathogenesis and reactogenicity of epidemic vibrio cholerae.

Epidemic Vibrio cholerae possess the VPI (Vibrio pathogenicity island) essential virulence gene cluster. The VPI is 41.2 kb in size and encodes 29 potential proteins, several of which have no known function. We show that the VPI-encoded Orf4 is a predicted 34-kDa periplasmic protein containing a zinc metalloprotease motif. V. cholerae seventh-pandemic (El Tor) strain N16961 carrying an orf4 mutation showed no obvious difference relative to its parent in the production of cholera toxin and the toxin-coregulated pilus, motility, azocasein digestion, and colonization of infant mice. However, analysis of rabbit ileal loops revealed that the N16961 orf4 mutant is hypervirulent, causing increased serosal hemorrhage and reactogenicity compared to its parent. Histology revealed a widening of submucosa, with an increase in inflammatory cells, diffuse lymphatic vessel dilatation, edema, endothelial cell hypertrophy of blood vessels, blunting of villi, and lacteal dilatation with lymphocytes and polymorphonuclear leukocytes. The mutant could be complemented in vivo with an orf4 gene on a plasmid but not with an orf4 gene containing a site-directed mutation in the putative zinc metalloprotease motif. Although its mechanism of its action is being studied further, our results suggest that the Orf4 protein is a zinc metalloprotease that modulates the pathogenesis and reactogenicity of epidemic V. cholerae. Based on our findings, we name this VPI-encoded protein Mop (for modulation of pathogenesis).

High-frequency rugose exopolysaccharide production by Vibrio cholerae.

Vibrio cholerae can shift to a "rugose" phenotype, thereby producing copious exopolysaccharide (EPS), which promotes its environmental survival and persistence. We report conditions that promote high-frequency rugose EPS production (HFRP), whereby cells switch at high frequency (up to 80%) to rugose EPS production. HFRP appeared to be more common in clinical strains, as HFRP was found in 6 of 19 clinical strains (32%) (including classical, El Tor, and non-O1 strains) but in only 1 of 16 environmental strains (6%). Differences were found between strains in rugose colony morphology, conditions promoting HFRP, the frequency of rugose-to-smooth (R-S) cell reversion, and biofilm formation. We propose that rugose EPS and HFRP provide an evolutionary and adaptive advantage to specific epidemic V. cholerae strains for increased persistence in the environment.

Prevalence of virulence-associated genes in clinical and environmental Vibrio cholerae strains isolated in Brazil between 1991 and 1999.

Genes located on the CTX element and the Vibrio cholerae pathogenicity island (VPI) were investigated in 297 clinical V. cholerae O1 and 76 environmental O1 and non-O1 isolates from Brazil between 1991 and 1999. RAPD analysis suggested that serogroup O1 strains regardless of clinical or environmental source were clonal while non-O1 strains showed greater diversity. PCR analysis showed that 71% of O1 clinical isolates had a complete set of CTX element target genes (ctxA, ctxB, zot and ace) and 68% a complete set of the VPI genes studied (orf1, aldA, tagA, tcpA, toxT and int genes). The results also showed that 72.4% of environmental O1 isolates possessed ctxA, ctxB, zot and ace genes while environmental non-O1 strains rarely possessed virulence genes. Our data are consistent with the hypothesis that the CTX element and the VPI can have a mosaic structure in some V. cholerae strains, genotype diversity is due to the circulation of virulence genes which are more commonly found in O1 strains in Brazil. This study also shows that the aquatic environment is a potential source for virulence genes and toxigenic V. cholerae during epidemic periods.