Structurally Designed Attenuated Subunit Vaccines for S. aureus LukS-PV and LukF-PV Confer Protection in a Mouse Bacteremia Model

Previous efforts towards S. aureus vaccine development have largely focused on cell surface antigens to induce opsonophagocytic killing aimed at providing sterile immunity, a concept successfully applied to other Gram-positive pathogens such as Streptococcus pneumoniae. However, these approaches have largely failed, possibly in part due to the remarkable diversity of the staphylococcal virulence factors such as secreted immunosuppressive and tissue destructive toxins. S. aureus produces several pore-forming toxins including the single subunit alpha hemolysin as well as bicomponent leukotoxins such as Panton-Valentine leukocidin (PVL), gamma hemolysins (Hlg), and LukED. Here we report the generation of highly attenuated mutants of PVL subunits LukS-PV and LukF-PV that were rationally designed, based on an octameric structural model of the toxin, to be deficient in oligomerization. The attenuated subunit vaccines were highly immunogenic and showed significant protection in a mouse model of S. aureus USA300 sepsis. Protection against sepsis was also demonstrated by passive transfer of rabbit immunoglobulin raised against LukS-PV. Antibodies to LukS-PV inhibited the homologous oligomerization of LukS-PV with LukF-PV as well heterologous oligomerization with HlgB. Importantly, immune sera from mice vaccinated with the LukS mutant not only inhibited the PMN lytic activity produced by the PVL-positive USA300 but also blocked PMN lysis induced by supernatants of PVL-negative strains suggesting a broad protective activity towards other bicomponent toxins. These findings strongly support the novel concept of an anti-virulence, toxin-based vaccine intended for prevention of clinical S. aureus invasive disease, rather than achieving sterile immunity. Such a multivalent vaccine may include attenuated leukotoxins, alpha hemolysin, and superantigens.     

Staphylococcus aureus Leukocidin A/B (LukAB) Kills Human Monocytes via Host NLRP3 and ASC when Extracellular,but Not Intracellular

Staphylococcus aureus infections are a growing health burden worldwide, and paramount to this bacterium’s pathogenesis is the production of virulence factors, including pore-forming leukotoxins. Leukocidin A/B (LukAB) is a recently discovered toxin that kills primary human phagocytes, though the underlying mechanism of cell death is not understood. We demonstrate here that LukAB is a major contributor to the death of human monocytes. Using a variety of in vitro and ex vivo intoxication and infection models, we found that LukAB activates Caspase 1, promotes IL-1β secretion and induces necrosis in human monocytes. Using THP1 cells as a model for human monocytes, we found that the inflammasome components NLRP3 and ASC are required for LukAB-mediated IL-1β secretion and necrotic cell death. S. aureus was shown to kill human monocytes in a LukAB dependent manner under both extracellular and intracellular ex vivo infection models. Although LukAB-mediated killing of THP1 monocytes from extracellular S. aureus requires ASC, NLRP3 and the LukAB-receptor CD11b, LukAB-mediated killing from phagocytosed S. aureus is independent of ASC or NLRP3, but dependent on CD11b. Altogether, this study provides insight into the nature of LukAB-mediated killing of human monocytes. The discovery that S. aureus LukAB provokes differential host responses in a manner dependent on the cellular contact site is critical for the development of anti-infective/anti-inflammatory therapies that target the NLRP3 inflammasome.     

Clostridium perfringens Delta Toxin Is Sequence Related to Beta Toxin,NetB, and Staphylococcus Pore-Forming Toxins,but Shows Functional Differences

Clostridium perfringens produces numerous toxins, which are responsible for severe diseases in man and animals. Delta toxin is one of the three hemolysins released by a number of C. perfringens type C and possibly type B strains. Delta toxin was characterized to be cytotoxic for cells expressing the ganglioside G_M2 in their membrane. Here we report the genetic characterization of Delta toxin and its pore forming activity in lipid bilayers. Delta toxin consists of 318 amino acids, its 28 N-terminal amino acids corresponding to a signal peptide. The secreted Delta toxin (290 amino acids; 32619 Da) is a basic protein (pI 9.1) which shows a significant homology with C. perfringens Beta toxin (43% identity), with C. perfringens NetB (40% identity) and, to a lesser extent, with Staphylococcus aureus alpha toxin and leukotoxins. Recombinant Delta toxin showed a preference for binding to G_M2, in contrast to Beta toxin, which did not bind to gangliosides. It is hemolytic for sheep red blood cells and cytotoxic for HeLa cells. In artificial diphytanoyl phosphatidylcholine membranes, Delta and Beta toxin formed channels. Conductance of the channels formed by Delta toxin, with a value of about 100 pS to more than 1 nS in 1 M KCl and a membrane potential of 20 mV, was higher than those formed by Beta toxin and their distribution was broader. The results of zero-current membrane potential measurements and single channel experiments suggest that Delta toxin forms slightly anion-selective channels, whereas the Beta toxin channels showed a preference for cations under the same conditions. C. perfringens Delta toxin shows a significant sequence homolgy with C. perfringens Beta and NetB toxins, as well as with S. aureus alpha hemolysin and leukotoxins, but exhibits different channel properties in lipid bilayers. In contrast to Beta toxin, Delta toxin recognizes G_M2 as receptor and forms anion-selective channels.     

Increased Susceptibility of Humanized NSG Mice to Panton-Valentine Leukocidin and Staphylococcus aureus Skin Infection

Staphylococcus aureus is a leading cause of skin and soft-tissue infections worldwide. Mice are the most commonly used animals for modeling human staphylococcal infections. However a supra-physiologic S. aureus inoculum is required to establish gross murine skin pathology. Moreover, many staphylococcal factors, including Panton-Valentine leukocidin (PVL) elaborated by community-associated methicillin-resistant S. aureus (CA-MRSA), exhibit selective human tropism and cannot be adequately studied in mice. To overcome these deficiencies, we investigated S. aureus infection in non-obese diabetic (NOD)/severe combined immune deficiency (SCID)/IL2rγ^null (NSG) mice engrafted with human CD34⁺ umbilical cord blood cells. These “humanized” NSG mice require one to two log lower inoculum to induce consistent skin lesions compared with control mice, and exhibit larger cutaneous lesions upon infection with PVL⁺ versus isogenic PVL^- S. aureus. Neutrophils appear important for PVL pathology as adoptive transfer of human neutrophils alone to NSG mice was sufficient to induce dermonecrosis following challenge with PVL⁺ S. aureus but not PVL^- S. aureus. PMX53, a human C5aR inhibitor, blocked PVL-induced cellular cytotoxicity in vitro and reduced the size difference of lesions induced by the PVL⁺ and PVL^- S. aureus, but PMX53 also reduced recruitment of neutrophils and exacerbated the infection. Overall, our findings establish humanized mice as an important translational tool for the study of S. aureus infection and provide strong evidence that PVL is a human virulence factor.     

Characterization of Staphylococcus aureus from Humans and a Comparison with ?solates of Animal Origin,in North Dakota,United States

Different clones of methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus have been found in humans as well as in animals and retail meat. However, more information about the genetic characteristics and similarities between strains is needed. The aim of this study was to identify and characterize Staphylococcus aureus from humans, and to compare their characteristics with isolates of animal origin. A total of 550 nasal swabs were taken from healthy humans, and S. aureus was isolated and identified. Positive S. aureus isolates were subjected to molecular typing and susceptibility testing. In addition, 108 MRSA isolates recovered from clinical patients in the state of North Dakota and 133 S. aureus isolates from animals and meat previously analyzed were included. The nasal carriage of S. aureus in healthy people was 7.6% and, in general, clones were genetically diverse. None of the S. aureus strains obtained from healthy people were mecA- or PVL-positive. A total of 105 (97.2%) MRSA isolates from clinical cases harbored the mecA gene and 11 (10.2%) isolated from blood stream infections harbored the PVL gene. The most common resistance profile among S. aureus from healthy people was penicillin, and from clinical cases were erythromycin-penicillin-ciprofloxacin. The rate of multidrug resistance (MDR) was 70% in humans. Most of the S. aureus harboring mecA and PVL genes were identified as ST5 and ST8, and exhibited MDR. However, S. aureus isolates of animal origin used for comparison exhibited a lower rate of MDR. The most common resistance profiles in isolates of animal origin were penicillin-tetracycline and penicillin-tetracycline-erythromycin, in animals and raw meat, respectively. The ST5 was also found in animals and meat, with ST9 and ST398 being the major clones. The genetic similarity between clones from humans and meat suggests the risk of spread of S. aureus in the food chain.     

Active Immunization with Extracellular Vesicles Derived from Staphylococcus aureus Effectively Protects against Staphylococcal Lung Infections,Mainly via Th1 Cell-Mediated Immunity

Staphylococcus aureus is an important pathogenic bacterium that causes various infectious diseases. Extracellular vesicles (EVs) released from S. aureus contain bacterial proteins, nucleic acids, and lipids. These EVs can induce immune responses leading to similar symptoms as during staphylococcal infection condition and have the potential as vaccination agent. Here, we show that active immunization (vaccination) with S. aureus-derived EVs induce adaptive immunity of antibody and T cell responses. In addition, these EVs have the vaccine adjuvant ability to induce protective immunity such as the up-regulation of co-stimulatory molecules and the expression of T cell polarizing cytokines in antigen-presenting cells. Moreover, vaccination with S. aureus EVs conferred protection against lethality induced by airway challenge with lethal dose of S. aureus and also pneumonia induced by the administration of sub-lethal dose of S. aureus. These protective effects were also found in mice that were adoptively transferred with splenic T cells isolated from S. aureus EV-immunized mice, but not in serum transferred mice. Furthermore, this protective effect of S. aureus EVs was significantly reduced by the absence of interferon-gamma, but not by the absence of interleukin-17. Together, the study herein suggests that S. aureus EVs are a novel vaccine candidate against S. aureus infections, mainly via Th1 cellular response.     

One Dose of Staphylococcus aureus 4C-Staph Vaccine Formulated with a Novel TLR7-Dependent Adjuvant Rapidly Protects Mice through Antibodies,Effector CD4+ T Cells,and IL-17A

A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4⁺ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4⁺ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.     

Silkworm Apolipophorin Protein Inhibits Hemolysin Gene Expression of Staphylococcus aureus via Binding to Cell Surface Lipoteichoic Acids

We previously reported that a silkworm hemolymph protein, apolipophorin (ApoLp), binds to the cell surface of Staphylococcus aureus and inhibits expression of the saePQRS operon encoding a two-component system, SaeRS, and hemolysin genes. In this study, we investigated the inhibitory mechanism of ApoLp on S. aureus hemolysin gene expression. ApoLp bound to lipoteichoic acids (LTA), an S. aureus cell surface component. The addition of purified LTA to liquid medium abolished the inhibitory effect of ApoLp against S. aureus hemolysin production. In an S. aureus knockdown mutant of ltaS encoding LTA synthetase, the inhibitory effects of ApoLp on saeQ expression and hemolysin production were attenuated. Furthermore, the addition of anti-LTA monoclonal antibody to liquid medium decreased the expression of S. aureus saeQ and hemolysin genes. In S. aureus strains expressing SaeS mutant proteins with a shortened extracellular domain, ApoLp did not decrease saeQ expression. These findings suggest that ApoLp binds to LTA on the S. aureus cell surface and inhibits S. aureus hemolysin gene expression via a two-component regulatory system, SaeRS.     

Monoclonal Antibody Targeting Staphylococcus aureus Surface Protein A (SasA) Protect Against Staphylococcus aureus Sepsis and Peritonitis in Mice

Epidemic methicillin-resistant Staphylococcus aureus (MRSA) imposes an increasing impact on public health. Due to multi-antibiotics resistance in MRSA strains, there is an urgent need to develop novel therapeutics such as effective monoclonal antibodies (mAbs) against MRSA infections. Staphylococcus aureus surface protein A (SasA), a large surface-located protein (~240 kDa), is one of MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) and a potential target for immunotherapeutic approaches against S. aureus infections. In the present study, we analyzed the sequence of SasA with bioinformatics tools and generated a protective monoclonal antibody (2H7) targeting the conserved domain of SasA. 2H7 was shown to recognize wild-type S. aureus and promote opsonophagocytic killing of S. aureus. In both sepsis and peritoneal infection models, prophylactic administration of 2H7 improved the survival of BALB/c mice challenged by S. aureus strain USA300 and ST239 (prevalent MRSA clones in North America and Asian countries, respectively) and enhanced bacterial clearance in kidneys. Additionally, 2H7 prophylaxis prevented the formation of intraperitoneal abscess in a murine model of peritoneal infection and therapeutic administration of 2H7 showed protective efficacy in a murine sepsis model. Our results presented here provide supporting evidences that an anti-SasA mAb might be a potential component in an antibody-based immunotherapeutic treatment of MRSA infections.     

High Prevalence of Exfoliative Toxins Among Carrier Isolates of Staphylococcus aureus from Healthy Individuals from Various Communities in Chennai, South India

Staphylococcus aureus causes infections both in community and hospital settings, nasal carriage is the important source of these infections. A total of 103 carrier isolates of S. aureus from 352 asymptomatic individuals were screened for methicillin-resistant S. aureus (MRSA) and exfoliative toxins (A, B and D) by two sets of multiplex PCRs. The overall nasal carriage of MRSA was found to be 13/352 (3.7 %), of which 4 were found to be positive for Panton valentine leucocidin (PVL). Twelve (11.65 %) strains were found to carry exfoliative toxins and belonged to one of the following spa types t159, t209 and t1515. High prevalence of exfoliative toxins, pvl and MRSA pose a major threat to public health, since the isolates were from the healthy in various community settings.     

A molecular modeling based screening for potential inhibitors to alpha hemolysin from Staphylococcus aureus

Staphylococcus aureus, a Gram-positive bacterium is pathogenic in nature. It is known that secreted toxins remain active after antibiotic treatment. The alpha hemolysin or alpha toxin damages cell membrane and induces apoptosis and degradation of DNA. The titer of alphahemolysin increases and causes hemostasis disturbances, thrombocytopenia, and pulmonary lesions during staphylococcal infection. Therefore, it is of interest to inhibit alpha hemolysin using novel compounds. We used the structure of alpha hemolysin(PDB: 7AHL) to screen structures for 100,000 compounds from the ZINC database using molecular docking with AutoDock VINA. Nine (9) successive hits were then subjected for pharmacokinetic and toxicity properties by PROTOX (a webserver for the prediction of oral toxicities of small molecules) and FAFDrugs (a tool for prediction of ADME and Toxicity). This exercise further identified hit #1 ({[3a-(Dihydroxymethyl)-6-hydroxy-2,2-dimethyl-1,3,4-trioxatetrahydro-2H-pentalen-5- yl]methyl}amino(9H-fluoren-9-yl)acetate with binding affinity: -10.3 kcal/mol) and hit #2 (6-(Dihydroxymethyl)-2-{2-[3- (methylamino)propyl]-2-azatricyclo[9.4.0.03,8]pentadeca-1(11),3,5,7,12,14-hexaen-6-yloxy}tetrahydro-2H-pyran-3,4,5-triol with binding affinity: -9.6 kcal/mol) with acceptable toxicity and ADME properties for potential predicted hemolysin inhibition. These compounds should then be evaluated in vitro using inhibitory studies.     

Immunisation With Immunodominant Linear B Cell Epitopes Vaccine of Manganese Transport Protein C Confers Protection against Staphylococcus aureus Infection

Vaccination strategies for Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA) infections have attracted much research attention. Recent efforts have been made to select manganese transport protein C, or manganese binding surface lipoprotein C (MntC), which is a metal ion associated with pathogen nutrition uptake, as potential candidates for an S. aureus vaccine. Although protective humoral immune responses to MntC are well-characterised, much less is known about detailed MntC-specific B cell epitope mapping and particularly epitope vaccines, which are less-time consuming and more convenient. In this study, we generated a recombinant protein rMntC which induced strong antibody response when used for immunisation with CFA/IFA adjuvant. On the basis of the results, linear B cell epitopes within MntC were finely mapped using a series of overlapping synthetic peptides. Further studies indicate that MntC113-136, MntC209-232, and MntC263-286 might be the original linear B-cell immune dominant epitope of MntC, furthermore, three-dimensional (3-d) crystal structure results indicate that the three immunodominant epitopes were displayed on the surface of the MntC antigen. On the basis of immunodominant MntC113-136, MntC209-232, and MntC263-286 peptides, the epitope vaccine for S. aureus induces a high antibody level which is biased to TH2 and provides effective immune protection and strong opsonophagocytic killing activity in vitro against MRSA infection. In summary, the study provides strong proof of the optimisation of MRSA B cell epitope vaccine designs and their use, which was based on the MntC antigen in the development of an MRSA vaccine.     

Molecular Characterization and Antimicrobial Susceptibility of Staphylococcus aureus Isolates from Clinical Infection and Asymptomatic Carriers in Southwest Nigeria

Few reports from Africa suggest that resistance pattern, virulence factors and genotypes differ between Staphylococcus aureus from nasal carriage and clinical infection. We therefore compared antimicrobial resistance, selected virulence factors and genotypes of S. aureus from nasal carriage and clinical infection in Southwest Nigeria. Non-duplicate S. aureus isolates were obtained from infection (n = 217) and asymptomatic carriers (n = 73) during a cross sectional study in Lagos and Ogun States, Nigeria from 2010–2011. Susceptibility testing was performed using Vitek automated systems. Selected virulence factors were detected by PCR. The population structure was assessed using spa typing. The spa clonal complexes (spa-CC) were deduced using the Based Upon Repeat Pattern algorithm (BURP). Resistance was higher for aminoglycosides in clinical isolates while resistances to quinolones and tetracycline were more prevalent in carrier isolates. The Panton-Valentine leukocidin (PVL) was more frequently detected in isolates from infection compared to carriage (80.2 vs 53.4%; p<0.001, chi²-test). Seven methicillin resistant S. aureus isolates were associated with spa types t002, t008, t064, t194, t8439, t8440 and t8441. The predominant spa types among the methicillin-susceptible S. aureus isolates were t084 (65.5%), t2304 (4.4%) and t8435 (4.1%). spa-CC 084 was predominant among isolates from infection (80.3%, n = 167) and was significantly associated with PVL (OR = 7.1, 95%CI: 3.9–13.2, p<0.001, chi^2- test). In conclusion, PVL positive isolates were more frequently detected among isolates from infection compared to carriage and are associated with spa-CC 084.     

Population structure of Staphylococcus aureus from remote African Babongo Pygmies

Background

Pandemic community-acquired methicillin-resistant Staphylococcus aureus isolates (CA-MRSA) predominantly encode the Panton-Valentine leukocidin (PVL), which can be associated with severe infections. Reports from non-indigenous Sub-Saharan African populations revealed a high prevalence of PVL-positive isolates. The objective of our study was to investigate the S. aureus carriage among a remote indigenous African population and to determine the molecular characteristics of the isolates, particularly those that were PVL-positive.

Methodology/Principal Findings

Nasal S. aureus carriage and risk factors of colonization were systematically assessed in remote Gabonese Babongo Pygmies. Susceptibility to antibiotics, possession of toxin-encoding genes (i.e., PVL, enterotoxins, and exfoliative toxins), S. aureus protein A (spa) types and multi-locus sequence types (MLST) were determined for each isolate. The carriage rate was 33%. No MRSA was detected, 61.8% of the isolates were susceptible to penicillin. Genes encoding PVL (55.9%), enterotoxin B (20.6%), exfoliative toxin D (11.7%) and the epidermal cell differentiation inhibitor B (11.7%) were highly prevalent. Thirteen spa types were detected and were associated with 10 STs predominated by ST15, ST30, ST72, ST80, and ST88.

Conclusions

The high prevalence of PVL-positive isolates among Babongo Pygmies demands our attention as PVL can be associated with necrotinzing infection and may increase the risk of severe infections in remote Pygmy populations. Many S. aureus isolates from Babongo Pygmies and pandemic CA-MRSA-clones have a common genetic background. Surveillance is needed to control the development of resistance to antibiotic drugs and to assess the impact of the high prevalence of PVL in indigenous populations.     

Th17 cell plasticity towards a T-bet-dependent Th1 phenotype is required for bacterial control in Staphylococcus aureus infection

Staphylococcus aureus is frequently detected in patients with sepsis and thus represents a major health burden worldwide. CD4⁺ T helper cells are involved in the immune response to S. aureus by supporting antibody production and phagocytosis. In particular, Th1 and Th17 cells secreting IFN-γ and IL-17A, are involved in the control of systemic S. aureus infections in humans and mice.To investigate the role of T cells in severe S. aureus infections, we established a mouse sepsis model in which the kidney was identified to be the organ with the highest bacterial load and abundance of Th17 cells. In this model, IL-17A but not IFN-γ was required for bacterial control. Using Il17aCre × R26YFP mice we could show that Th17 fate cells produce Th17 and Th1 cytokines, indicating a high degree of Th17 cell plasticity. Single cell RNA-sequencing of renal Th17 fate cells uncovered their heterogeneity and identified a cluster with a Th1 expression profile within the Th17 cell population, which was absent in mice with T-bet/Tbx21-deficiency in Th17 cells (Il17aCre x R26eYFP x Tbx21-flox). Blocking Th17 to Th1 transdifferentiation in Th17 fate cells in these mice resulted in increased S. aureus tissue loads.In summary, we highlight the impact of Th17 cells in controlling systemic S. aureus infections and show that T-bet expression by Th17 cells is required for bacterial clearance. While targeting the Th17 cell immune response is an important therapeutic option in autoimmunity, silencing Th17 cells might have detrimental effects in bacterial infections.     

Direct,Specific and Rapid Detection of Staphylococcal Proteins and Exotoxins Using a Multiplex Antibody Microarray

Background

S. aureus is a pathogen in humans and animals that harbors a wide variety of virulence factors and resistance genes. This bacterium can cause a wide range of mild to life-threatening diseases. In the latter case, fast diagnostic procedures are important. In routine diagnostic laboratories, several genotypic and phenotypic methods are available to identify S. aureus strains and determine their resistances. However, there is a demand for multiplex routine diagnostic tests to directly detect staphylococcal toxins and proteins.

Methods

In this study, an antibody microarray based assay was established and validated for the rapid detection of staphylococcal markers and exotoxins. The following targets were included: staphylococcal protein A, penicillin binding protein 2a, alpha- and beta-hemolysins, Panton Valentine leukocidin, toxic shock syndrome toxin, enterotoxins A and B as well as staphylokinase. All were detected simultaneously within a single experiment, starting from a clonal culture on standard media. The detection of bound proteins was performed using a new fluorescence reading device for microarrays.

Results

110 reference strains and clinical isolates were analyzed using this assay, with a DNA microarray for genotypic characterization performed in parallel. The results showed a general high concordance of genotypic and phenotypic data. However, genotypic analysis found the hla gene present in all S. aureus isolates but its expression under given conditions depended on the clonal complex affiliation of the actual isolate.

Conclusions

The multiplex antibody assay described herein allowed a rapid and reliable detection of clinically relevant staphylococcal toxins as well as resistance- and species-specific markers.     

Application of monoclonal antibodies generated against Panton-Valentine Leukocidin (PVL-S) toxin for specific identification of community acquired methicillin resistance Staphylococcus aureus

Panton-Valentine Leukocidin (PVL) produced by community acquired methicillin Staphylococcus aureus (CA-MRSA) involved in skin and soft-tissue infections and necrotizing pneumonia comprised of two fractions, namely PVL S and PVL F. In the present study, three monoclonal antibodies designated as MAb1, MAb9 and MAb10 were generated against recombinant PVL-S (35 kDa) protein of S. aureus. All the three MAbs specifically reacted to confirm PVL-S positive strains of S. aureus recovered from clinical samples in Western blot analysis. Similarly all the three MAbs did not show any binding to other tested 14 different pathogenic bacteria belonging to other genera and species in Western blot analysis. Furthermore, a simple dot-ELISA method was standardized for the identification of PVL-S toxin containing S. aureus strains. Initially in dot-ELISA, Protein A (Spa) of S. aureus posed background noise problems due to the non-specific binding of antibodies resulting in false positive reactions. With the addition of 10 mM diethylpyrocarbonate (DEPC) along with 5% milk in PBS in the blocking step prevented this non-specific binding of Spa to mouse anti-PVL monoclonal antibodies in dot-ELISA. Once standardized, this simple dot-ELISA was evaluated with nine PVL positive strains recovered from food, environmental and clinical samples and the results were compared with PCR assay for the presence of PVL toxin genes and also with Western blot analysis. A 100% correlation was found between dot-ELISA, PCR assay and Western blot analysis. Collectively our results suggest the newly developed simple dot-ELISA system can be of immense help in providing, rapid detection of the PVL toxin containing S. aureus strains at a relatively low cost and will be a valuable tool for the reliable identification of CA-MRSA.     

Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes

Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.     

Staphylococcus aureus Nasal Carriage among Beefpacking Workers in a Midwestern United States Slaughterhouse

Occupational contact with livestock is an established risk factor for exposure to livestock-associated methicillin-resistant Staphylococcus aureus (MRSA), particularly among industrial swine workers. While S. aureus is known to infect cattle, livestock-associated S. aureus carriage among workers in the beef production chain has received limited attention. Beefpacking workers, who slaughter, butcher and process cattle, have intensified exposure to potentially infectious animal materials and may be at risk of livestock-associated S. aureus exposure. We conducted a cross-sectional study of beefpacking workers (n = 137) at an industrial slaughterhouse in the Midwestern United States to evaluate prevalence and characteristics of S. aureus nasal colonization, specifically the absence of the scn gene to identify putative association with livestock, antibiotic susceptibility, presence of Panton-Valentin leukocidin (PVL) genes lukS-PV and lukF-PV, and spa type. Overall prevalence of S. aureus nasal carriage was 27.0%. No workers carried livestock-associated MRSA. Methicillin-sensitive S. aureus isolates (MSSA) recovered from five workers (3.6%) lacked the scn gene and were considered putative livestock-associated S. aureus (pLA-SA). Among pLA-SA isolates, spa types t338, t748, t1476 and t2379 were identified. To our knowledge, these spa types have not previously been identified as associated with livestock. Prevalence of human-adapted MRSA carriage in workers was 3.6%. MRSA isolates were identified as spa types t002, t008 and t024, and four of five MRSA isolates were PVL-positive. To date, this is the first study to indicate that industrial beefpacking workers in the United States may be exposed to livestock-associated S. aureus, notably MSSA, and to spa types not previously identified in livestock and livestock workers. Occupational exposure to livestock-associated S. aureus in the beef production chain requires further epidemiologic investigation.