leukotoxin and Staphylococcal-Infections

Staphylococcus aureus can cause superficial skin infections and, occasionally, deep-seated infections that entail spread through the blood stream. The organism expresses several factors that compromise the effectiveness of neutrophils and macrophages, the first line of defence against infection. S. aureus secretes proteins that inhibit complement activation and neutrophil chemotaxis or that lyse neutrophils, neutralizes antimicrobial defensin peptides, and its cell surface is modified to reduce their effectiveness. The organism can survive in phagosomes, express polysaccharides and proteins that inhibit opsonization by antibody and complement, and its cell wall is resistant to lysozyme. Furthermore, S. aureus expresses several types of superantigen that corrupt the normal humoral immune response, resulting in anergy and immunosuppression. In contrast, Staphylococcus epidermidis must rely primarily on cell-surface polymers and the ability to form a biolfilm to survive in the host.

Topics: Antibody Formation; Bacterial Toxins; Chemotaxis; Complement Activation; Defensins; Drug Resistance, Bacterial; Exotoxins; Humans; Immunity, Active; Immunity, Innate; Phagocytosis; Staphylococcal Infections; Staphylococcal Protein A; Staphylococcal Skin Infections; Staphylococcus aureus; Staphylococcus epidermidis; Virulence

Our previous studies identified a population of stem cell-like proliferating myeloid cells within inflamed tissues that could serve as a reservoir for tissue macrophages to adopt different activation states depending on the microenvironment. By lineage-tracing cells derived from CX3CR1+ precursors in mice during infection and profiling by single-cell RNA sequencing, in this study, we identify a cluster of BIRC5+ myeloid cells that expanded in the liver during chronic infection with either the parasite Schistosoma mansoni or the bacterial pathogen Staphylococcus aureus. In the absence of tissue-damaging toxins, S. aureus infection does not elicit these BIRC5+ cells. Moreover, deletion of BIRC5 from CX3CR1-expressing cells results in improved survival during S. aureus infection. Hence the combination of single-cell RNA sequencing and genetic fate-mapping CX3CR1+ cells revealed a toxin-dependent pathogenic role for BIRC5 in myeloid cells during S. aureus infection.

Topics: Animals; Mice; Myeloid Cells; Single-Cell Analysis; Staphylococcal Infections; Staphylococcus aureus

Staphylococcus aureus is among the most common zoonotic pathogens that cause foodborne illnesses worldwide. The main objectives of the current study were therefore to determine the antimicrobial susceptibility profiles of S. aureus isolated from goats in Korea and to investigate the molecular characteristics of identified methicillin-resistant S. aureus (MRSA). In the study, 481 S. aureus isolates (431 from the nasal cavity and 50 from carcass) were recovered from 1146 carcasses and nasal swabs between July 2018 and January 2019. Approximately 82% and 72.6% of nasal and carcass isolates, respectively, were resistant to at least one antimicrobial agent, with the highest rate of resistance to penicillin, followed by resistance to chloramphenicol and tetracycline. Relatively small proportions of the isolates were resistant to cefoxitin, clindamycin, and erythromycin. However, all S. aureus isolates were sensitive to linezolid, rifampin, and vancomycin. Six MRSA isolates were obtained, three each from the nasal cavity and carcass. MRSA isolates were of two sequence types (ST) (ST72 and ST398), three spa types (t664, t324, and t571), and two SCCmec types (IV and V). The ST72 MRSA isolates had identical PFGE profiles. In addition, ST72 MRSA-SCCmec IV isolates carried at least six staphylococcal leukotoxin- and enterotoxin-encoding genes (lukED, seg, sei, sem, sen, seo, and seq). The remaining ST398 isolate carried only the lukED gene and was additionally resistant to eight non-β-lactam antibiotics. To the best of our knowledge, this is the first report of MRSA from goats in Korea. There is a possibility of transmission of MRSA from goat to human or contamination of food products. Therefore, regular microbiological investigation in goats, farms, and slaughterhouses is critical to determine the existence of virulent and multi-drug resistant (MDR) S. aureus and to implement preventive strategies.

Topics: Animals; Anti-Bacterial Agents; Enterotoxins; Exotoxins; Farms; Food Microbiology; Foodborne Diseases; Goats; Humans; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Milk; Republic of Korea; Staphylococcal Infections; Staphylococcus aureus

We identified 199 Staphylococcus aureus isolates from quarter milk samples of 1,289 dairy cattle between 2014 and 2018. About 66% of the isolates were resistant to at least 1 antimicrobial agent; the highest rate of resistance was to penicillin, followed by resistance to ampicillin, erythromycin, and sulfadimethoxine. We obtained 30 methicillin-resistant S. aureus (MRSA) strains from 6 farms in 3 provinces. The MRSA strains exhibited a significantly higher resistance rate to most of the tested antimicrobials than the oxacillin-susceptible strains. The MRSA strains represented 5 genotypes: ST72-t324-SCCmec IV (n = 14), ST30-t1752-SCCmec IV (n = 8), ST188-t189-SCCmec NT (n = 6), ST188-t2284-SCCmec NT (n = 1), and NT-NT-SCCmec IV (n = 1). One of the ST188 MRSA strains represented a novel staphylococcal protein A (spa) type (t2284). In addition, 7 of the 8 ST30 MRSA strains were Panton-Valentine leukocidin (PVL)-positive and carried various staphylococcal enterotoxin encoding genes. This is the first report of PVL-positive ST30 MRSA-t1752-SCCmec IV from bovine mastitis in Korea. All of ST72-t324-SCCmec IV MRSA strains carried staphylococcal enterotoxin and leukotoxin encoding genes. They were also sensitive to most of the tested non-β-lactam antimicrobials. In contrast, ST188-t189 MRSA strains were resistant to multiple antimicrobials and predominantly carried the leukotoxin encoding gene. Taken together, these findings may indicate that dairy cows could be a major source for spreading MRSA strains, and contaminated milk could be a vehicle for transmission. Suitable hygienic measures should be established in dairy farms and processing plants to limit the likelihood of introducing MRSA into the food chain.

Topics: Animals; Bacterial Toxins; Cattle; Enterotoxins; Exotoxins; Female; Leukocidins; Mastitis, Bovine; Methicillin-Resistant Staphylococcus aureus; Milk; Republic of Korea; Staphylococcal Infections

Panton-Valentine Leukocidin (PVL) is a bicomponent leukotoxin produced by 3%-10% of clinical Staphylococcus aureus (SA) strains involved in the severity of hospital and community-acquired infections. Although PVL was long known as a pore-forming toxin, recent studies have challenged the formation of a pore at the plasma membrane, while its endocytosis and the exact mode of action remain to be defined. In vitro immunolabeling of human neutrophils shows that Neutrophil Extracellular Traps (NETosis) is triggered by the action of purified PVL, but not by Gamma hemolysin CB (HlgCB), a structurally similar SA leukotoxin. PVL causes the ejection of chromatin fibers (NETs) decorated with antibacterial peptides independently of the NADPH oxidase oxidative burst. Leukotoxin partially colocalizes with mitochondria and enhances the production of reactive oxygen species from these organelles, while showing an increased autophagy, which results unnecessary for NETs ejection. PVL NETosis is elicited through Ca

Topics: Adult; Bacterial Proteins; Bacterial Toxins; Cells, Cultured; Exotoxins; Extracellular Traps; Female; Healthy Volunteers; Hemolysin Proteins; Humans; Leukocidins; Male; Mitochondria; NADPH Oxidases; Neutrophils; Reactive Oxygen Species; Respiratory Burst; Staphylococcal Infections; Staphylococcus aureus

Recurrent

Topics: Animals; Disease Models, Animal; Exotoxins; H-2 Antigens; Host-Pathogen Interactions; Immune Tolerance; Immunodominant Epitopes; Major Histocompatibility Complex; Mice; Protein Binding; Staphylococcal Infections; Staphylococcus aureus; T-Lymphocytes; Vaccination

Topics: Animals; Antibodies, Bacterial; Antibodies, Neutralizing; Bacterial Proteins; Bacterial Toxins; Epitope Mapping; Epitopes, B-Lymphocyte; Exotoxins; Female; Hemolysin Proteins; Humans; Immunodominant Epitopes; Immunoglobulin G; Leukocidins; Mice; Mice, Inbred BALB C; Peptide Library; Staphylococcal Infections; Staphylococcus aureus; Virulence Factors

Staphylococcus aureus, which causes various infections, particularly suppurations, expresses many virulence factors. The resistance of S. aureus to methicillin (MRSA) which can spread to vancomycin constitutes a major challenge in infectiology. The search for virulence and resistance factors is therefore of interest to better understand the mechanisms of this pathogenicity. The objectives of this study were to determine the frequency of phenotypic and genotypic (mecA, vanB) resistances, the frequency of virulence genes (eta, etb, and lukS) and to investigate the resistant strains for the presence of virulence genes. On thirty-one strains isolated from infections at the Pasteur Institute of Côte d'Ivoire, the study of susceptibility to methicillin and vancomycin was carried out by phenotypic and molecular methods. We observed phenotypic and genotypic resistance to methicillin of 41.9% and 32.3% respectively. Despite a suspicion of very high vancomycin susceptibility reduced, 25.8% by phenotypic method, the vanB gene was only found in 3.2% of strains. The prevalence of virulence genes was high with the eta gene, 96.8%, and the lukS gene 45.2%. The mecA gene was present with an eta gene in 32.3% of strains and in 9.7% with the lukS gene, however the vanB gene was not present in any strain carrying virulence factors. These results should lead to the screening of other van genes for resistance to vancomycin.

Topics: Anti-Bacterial Agents; Cote d'Ivoire; Exfoliatins; Exotoxins; Humans; Methicillin Resistance; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Staphylococcal Infections; Staphylococcus aureus; Vancomycin

The bicomponent leukotoxins and the pyrogenic toxin superantigens (PTSAgs) are important virulence factors of Staphylococcus aureus. It is necessary to survey the prevalence and expression of these toxin-encoding genes for understanding the possible pathogenic capacity of S. aureus to cause disease.. Five leukotoxin genes and thirteen PTSAg determinants were detected for 177 S. aureus isolates from blood (n = 88) and wound (n = 89) infections by Polymerase Chain Reaction (PCR). The expression of leukotoxin ED (lukED) was determined by quantitative real-time PCR (qRT-PCR). The genetic backgrounds of isolates were analyzed by Staphylococcal Cassette Chromosome mec (SCCmec) typing (for methicillin-resistant S. aureus isolates), Pulsed-Field Gel Electrophoresis (PFGE), accessory gene regulator (agr) typing and Multilocus Sequence Typing (MLST, for representative isolates based on PFGE type) methods.. 99.4% (176/177) isolates contained at least one of leukotoxin genes. Among them, 94.9% (168/177), 81.4% (144/177) and 67.8% (120/177) isolates harbored hlgBC, lukED and lukAB, respectively. Compared to leukotoxin genes, there was a relatively lower overall prevalence of PTSAg genes [99.4% versus 72.9% (129/177), P < 0.001], and they were organized in 59 patterns, with the most common combination of the egc cluster with or without other PTSAg genes. Genetic analysis showed the distributions of certain toxin genes were associated with the genetic backgrounds of isolates. The egc cluster was a common feature of CC5 isolates, among which ST5 and ST764 isolates harbored more PTSAg genes. The lukED was not present in ST398 isolates, and its expression was quite different among isolates. No significant correlations were observed between the lukED expression levels of strains and the ST or agr types.. The present study elucidated the distribution of leukotoxin and PTSAg genes and the expression of lukED in blood and wound isolates, and analyzed the relationship between them with genetic characteristics of isolates. These data improve the current understanding of the possible pathogenicity of S. aureus.

Topics: Bacterial Toxins; China; Electrophoresis, Gel, Pulsed-Field; Exotoxins; Genetic Background; Humans; Methicillin-Resistant Staphylococcus aureus; Multilocus Sequence Typing; Polymerase Chain Reaction; Retrospective Studies; Staphylococcal Infections; Staphylococcus aureus; Superantigens; Virulence Factors; Wounds and Injuries

Bacterial infections from Staphylococcus pseudintermedius are the most common cause of skin infections (pyoderma) affecting dogs. Two component pore-forming leukocidins are a family of potent toxins secreted by staphylococci and consist of S (slow) and F (fast) components. They impair the innate immune system, the first line of defense against these pathogens. Seven different leukocidins have been characterized in Staphylococcus aureus, some of which are host and cell specific. Through genome sequencing and analysis of the S. pseudintermedius secretome using liquid chromatography mass spectrometry we identified two proteins, named "LukS-I" and "LukF-I", encoded on a degenerate prophage contained in the genome of S. pseudintermedius isolates. Phylogenetic analysis of LukS-I components in comparison to the rest of the leukocidin family showed that LukS-I was most closely related to S. intermedius LukS-I, S. aureus LukE and LukP, whereas LukF-I was most similar to S. intermedius LukF-I S. aureus gamma hemolysin subunit B. The killing effect of recombinant S. pseudintermedius LukS-I and LukF-I on canine polymorphonuclear leukocytes was determined using a flow cytometry cell permeability assay. The cytotoxic effect occurred only when the two recombinant proteins were combined. Engineered mutant versions of the two-component pore-forming leukocidins, produced through amino acids substitutions at selected points, were not cytotoxic. Anti-Luk-I produced in dogs against attenuated proteins reduced the cytotoxic effect of native canine leukotoxin which highlights the importance of Luk-I as a promising component in a vaccine against canine S. pseudintermedius infections.

Topics: Amino Acid Sequence; Animals; Antibodies, Bacterial; Bacterial Proteins; Cell Death; Dog Diseases; Dogs; Escherichia coli; Exotoxins; Genome, Bacterial; Leukocidins; Leukocytes; Mutation; Phylogeny; Recombinant Proteins; Staphylococcal Infections; Staphylococcus

Staphylococcal leukotoxins are a family of β-barrel, bicomponent, pore-forming toxins with membrane-damaging functions. These bacterial exotoxins share sequence and structural homology and target several host-cell types. Leukotoxin ED (LukED) is one of these bicomponent pore-forming toxins that Staphylococcus aureus produces in order to suppress the ability of the host to contain the infection. The recent delineation of the important role that LukED plays in S. aureus pathogenesis and the identification of its protein receptors, combined with its presence in S. aureus methicillin-resistant epidemic strains, establish this leukocidin as a possible target for the development of novel therapeutics. Here, the crystal structures of the water-soluble LukE and LukD components of LukED have been determined. The two structures illustrate the tertiary-structural variability with respect to the other leukotoxins while retaining the conservation of the residues involved in the interaction of the protomers in the bipartite leukotoxin in the pore complex.

Topics: Amino Acid Sequence; Bacterial Proteins; Exotoxins; Humans; Models, Molecular; Protein Conformation; Sequence Alignment; Staphylococcal Infections; Staphylococcus aureus

We set out to investigate the impact of common antibiotics on Panton-Valentine Leucocidin (PVL) expression by methicillin-sensitive Staphylococcus aureus (MSSA). PVL expression by methicillin-resistant S. aureus (MRSA) is reportedly enhanced by β-lactams, but inhibited by protein-synthesis inhibitors, a fact that has influenced management of infections associated with PVL. Although PVL is more frequently associated with MSSA than MRSA in the UK, the effect of antibiotics on PVL expression by MSSA has not been fully addressed.. MSSA was cultured in vitro with varying concentrations of flucloxacillin, clindamycin or linezolid and PVL expression measured by qRT-PCR and Western blotting. A murine MSSA abscess model was developed to measure leucocidin expression in vivo following antibiotic treatment.. 9% (27/314) of MSSA isolates from patients with uncomplicated community skin/soft tissue infections were positive for PVL genes (lukFS-PV). PVL expression by MSSA in broth was unaffected by varying concentrations of flucloxacillin, clindamycin or linezolid. In a murine abscess model, treatment with flucloxacillin did, however, enhance in vivo MSSA lukF-PV transcription and this was sustained even when flucloxacillin was combined with clindamycin, or clindamycin plus linezolid. Notwithstanding increased leucocidin transcription, functional leucotoxin activity was not enhanced. Treatment with flucloxacillin plus clindamycin significantly decreased leucotoxin activity, but the addition of a second protein synthesis inhibitor, linezolid, did not confer benefit.. Our results suggest flucloxacillin in combination with a single protein-synthesis inhibitor such as clindamycin would give the best treatment outcome.

Topics: Abscess; Animals; Anti-Bacterial Agents; Bacterial Toxins; Blotting, Western; Clindamycin; Disease Models, Animal; Exotoxins; Female; Humans; Leukocidins; Mice, Inbred BALB C; Microbial Sensitivity Tests; Real-Time Polymerase Chain Reaction; Soft Tissue Infections; Staphylococcal Infections; Staphylococcus aureus

The bicomponent leukotoxins produced by Staphylococcus aureus kill host immune cells through osmotic lysis by forming β-barrel pores in the host plasma membrane. The current model for bicomponent pore formation proposes that octameric pores, comprised of two separate secreted polypeptides (S and F subunits), are assembled from water-soluble monomers in the extracellular milieu and multimerize on target cell membranes. However, it has yet to be determined if all staphylococcal bicomponent leukotoxin family members exhibit these properties. In this study, we report that leukocidin A/B (LukAB), the most divergent member of the leukotoxin family, exists as a heterodimer in solution rather than two separate monomeric subunits. Notably, this property was found to be associated with enhanced toxin activity. LukAB also differs from the other bicomponent leukotoxins in that the S subunit (LukA) contains 33- and 10-amino-acid extensions at the N and C termini, respectively. Truncation mutagenesis revealed that deletion of the N terminus resulted in a modest increase in LukAB cytotoxicity, whereas the deletion of the C terminus rendered the toxin inactive. Within the C terminus of LukA, we identified a glutamic acid at position 323 that is critical for LukAB cytotoxicity. Furthermore, we discovered that this residue is conserved and required for the interaction between LukAB and its cellular target CD11b. Altogether, these findings provide an in-depth analysis of how LukAB targets neutrophils and identify novel targets suitable for the rational design of anti-LukAB inhibitors.

Topics: Amino Acid Substitution; Bacterial Proteins; CD11b Antigen; Cell Line, Tumor; Cell Membrane; Exotoxins; Glutamic Acid; HL-60 Cells; Humans; Leukocidins; Protein Binding; Staphylococcal Infections; Staphylococcus aureus

Leukotoxin M/F'-Panton Valentine (LukM/F'-PV), a beta pore-forming toxin secreted by Staphylococcus aureus, is a major virulence factor involved in the pathogenesis of bovine mastitis. The present study was aimed to determine immunogenicity of two recombinant subunits of LukM/F'-PV, rLukM (MW 38 kDa) and rLukF (MW 39 kDa), develop and validate an indirect enzyme linked immunosorbent assay (ELISA) using polyclonal antibodies raised in rabbits, and evaluate applicability of the assay to diagnose clinical and subclinical bovine mastitis. Additionally, in vitro assays were conducted to determine abilities of antibodies to neutralize cytotoxicity of the native leukotoxin. A total of 87 bovine milk samples (healthy, subclinical and clinical mastitis) were evaluated for the presence of toxin determinants. Receiver-operator characteristic curve for the experimental ELISA values statistically interpreted a cut-off score of >0.109 OD405, with an assay specificity of 100% and sensitivity in the range of 80-87.5%. In addition, area under curve of 0.93-0.98 revealed the test was accurate in categorizing samples from infected and non-infected bovine. The rLukF IgG-ELISA was more sensitive than rLukM IgG-ELISA. Furthermore, it was evident from MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium) dye reduction, indirect immunofluorescence and lactate dehydrogenase assays that anti-rLukM/rLukF antibodies, with high neutralizing titers, inhibited in vitro leukotoxic activity and protected bovine neutrophil membrane integrity from cytotoxicity of native leukotoxin. The findings demonstrated that antibodies produced from recombinant subunits contribute to specific and sensitive immunodiagnosis and may also have the potential to provide passive therapeutic benefit in the management of bovine mastitis.

Topics: Animals; Cattle; Enzyme-Linked Immunosorbent Assay; Exotoxins; Female; Mastitis, Bovine; Recombinant Proteins; Reproducibility of Results; Sensitivity and Specificity; Staphylococcal Infections; Staphylococcus aureus; Virulence Factors

Topics: Animals; Antibodies, Bacterial; Exotoxins; Host-Pathogen Interactions; Humans; Species Specificity; Staphylococcal Infections; Staphylococcal Vaccines; Staphylococcus aureus; Virulence Factors

Fatal exudative dermatitis (FED) is a recently described condition affecting red squirrels (Sciurus vulgaris) on the Isle of Wight and Jersey (Simpson et al., 2010a). Staphylococcus aureus strains isolated from skin lesions in cases of FED were characterised by molecular and phenotypic approaches. The strains were found to belong to a single MLST clonal complex (CC49) representing either ST49 or a novel single locus variant thereof (ST1957), were closely related by other molecular typing approaches, and all possessed the leukotoxin M encoding gene (lukM). In contrast S. aureus was either not isolated from none-FED cases or belonged to distinct and diverse molecular types that, with one exception, did not encode lukM. All isolates from FED cases were susceptible to all antimicrobials tested, including penicillin, and all proved negative for mecA and mecC as well as 14 other staphylococcal toxin genes. As all squirrels affected by FED were infected with S. aureus of the same lineage and encoded the lukM gene, it is possible that strains of this lineage may be involved in the pathogenesis of the dermatitis.

Topics: Animals; Bacterial Proteins; Dermatitis; Exotoxins; Microbial Sensitivity Tests; Molecular Typing; Multilocus Sequence Typing; Rodent Diseases; Rodentia; Sciuridae; Staphylococcal Infections; Staphylococcus aureus

Although Staphylococcus aureus is a major cause of outbreaks in neonatal intensive care units (NICUs), there are no studies on the epidemiology of S. aureus isolates responsible for infection in Portuguese NICUs. Between July 2005 and December 2007, a total of 54 methicillin susceptible S. aureus (MSSA) isolates were recovered from 16 infected infants, parents, health care workers (HCWs), and the environment in a level III NICU. Isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing. Virulence determinants were detected by multiplex polymerase chain reaction. Three major MSSA clones were endemic in the NICU, representing 70% (n=38) of the isolates: PFGE type A-ST5 (n=17); type B-ST30 (n=12); and type C-ST1 (n=9). Leukotoxins and hemolysins were present in all isolates, although none of them carried PVL. HCWs, plastic folders protecting clinical files, and mothers' nipples were identified as potential reservoirs and/or vehicles of dissemination of S. aureus. Consequently, additional infection control measures were implemented in this NICU.

Topics: Anti-Bacterial Agents; Cross Infection; Disease Reservoirs; Exotoxins; Female; Health Personnel; Hemolysin Proteins; Humans; Incidence; Infant; Infant, Newborn; Intensive Care Units, Neonatal; Male; Methicillin; Molecular Epidemiology; Population Surveillance; Portugal; Staphylococcal Infections; Staphylococcus aureus

Staphylococcus aureus produces numerous molecules that facilitate survival in the host. We recently identified a novel S. aureus leukotoxin (leukotoxin GH [LukGH]) using proteomics, but its role in virulence remains unclear. Here we investigated the role of LukGH in vivo.. We tested cytotoxicity of LukGH toward polymorphonuclear leukocytes (PMNs) from mice, rabbits, monkeys, and humans. LukGH was administered to mice, rabbits, and a cynomolgus monkey by subcutaneous or intradermal injection to assess cytotoxicity or host response in vivo. The effects of LukGH in vivo were compared with those of Panton-Valentine leukocidin (PVL), a well-characterized S. aureus leukotoxin. The contribution of LukGH to S. aureus infection was tested using mouse and rabbit infection models.. Susceptibility of PMNs to LukGH was similar between humans and cynomolgus monkeys, and was greater than that of rabbits, which in turn was greater than that of mice. LukGH or PVL caused skin inflammation in rabbits and a monkey, but deletion of neither lukGH nor lukGH and lukS/F-PV reduced severity of USA300 infections in rabbits or mice. Rather, some disease parameters (eg, rabbit abscess size) were increased following infection with a lukGH and lukS/F-PV deletion strain.. Our findings indicate that S. aureus leukotoxins enhance the host inflammatory response and influence the outcome of infection.

Topics: Animals; Disease Models, Animal; Exotoxins; Humans; Inflammation; Injections, Intradermal; Injections, Subcutaneous; Macaca fascicularis; Male; Mice; Neutrophils; Rabbits; Staphylococcal Infections; Staphylococcus aureus; Virulence Factors

Using different typing methods (MLST, spa-, SCCmec- and agr-typing), PFGE and DNA microarray-based chip analysis, we characterized 20 MRSA strains isolated from livestock and veterinarians. PFGE analysis after macrorestriction with EagI provided seven different band patterns, which could be grouped into four clusters. One cluster consisted of all MRSA ST398 strains isolated from pigs, calves, mastitis milk and two veterinarians. One strain of ST398 from a veterinarian and the two strains of ST1 and ST8 formed the three other clusters. Antimicrobial susceptibility testing showed that 15 of 20 strains were resistant to ampicillin, cefoxitin, clindamycin, erythromycin, oxacillin, penicillin and tetracycline. All strains were susceptible to rifampin and vancomycin, 19 were susceptible to ciprofloxacin and 18 were susceptible to sulphamethoxazole/trimethoprim. Genes encoding different enterotoxins, leukotoxins and haemolysins were found in certain strains.

Topics: Animals; Anti-Bacterial Agents; Bacterial Typing Techniques; Cattle; Cluster Analysis; Drug Resistance, Multiple, Bacterial; Enterotoxins; Exotoxins; Female; Genotype; Humans; Livestock; Mastitis, Bovine; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Milk; Oligonucleotide Array Sequence Analysis; Staphylococcal Infections; Swine; Switzerland; Veterinarians

We report a case of infection with coagulase-positive Staphylococcus pseudintermedius related to the implantation of a cardioverter-defribrillator device. This species is usually isolated from infected animals, and contact with a dog was the probable source of infection in this patient. This isolate produced a leukotoxin effective against human polymorphonuclear leukocytes.

Topics: Aged; Animals; Cell Survival; Coagulase; Dogs; Endocarditis, Bacterial; Exotoxins; Female; Humans; Neutrophils; Prosthesis-Related Infections; Staphylococcal Infections; Staphylococcus; Zoonoses

Staphylococcus aureus is a leading cause of bloodstream infections worldwide. In the United States, many of these infections are caused by a strain known as USA300. Although progress has been made, our understanding of the S. aureus molecules that promote survival in human blood and ultimately facilitate metastases is incomplete. To that end, we analyzed the USA300 transcriptome during culture in human blood, human serum, and trypticase soy broth (TSB), a standard laboratory culture media. Notably, genes encoding several cytolytic toxins were up-regulated in human blood over time, and hlgA, hlgB, and hlgC (encoding gamma-hemolysin subunits HlgA, HlgB, and HlgC) were among the most highly up-regulated genes at all time points. Compared to culture supernatants from a wild-type USA300 strain (LAC), those derived from an isogenic hlgABC-deletion strain (LACΔhlgABC) had significantly reduced capacity to form pores in human neutrophils and ultimately cause neutrophil lysis. Moreover, LACΔhlgABC had modestly reduced ability to cause mortality in a mouse bacteremia model. On the other hand, wild-type and LACΔhlgABC strains caused virtually identical abscesses in a mouse skin infection model, and bacterial survival and neutrophil lysis after phagocytosis in vitro was similar between these strains. Comparison of the cytolytic capacity of culture supernatants from wild-type and isogenic deletion strains lacking hlgABC, lukS/F-PV (encoding PVL), and/or lukDE revealed functional redundancy among two-component leukotoxins in vitro. These findings, along with a requirement of specific growth conditions for leukotoxin expression, may explain the apparent limited contribution of any single two-component leukotoxin to USA300 immune evasion and virulence.

Topics: Animals; Bacterial Proteins; Bacterial Toxins; Cell Membrane Permeability; Disease Models, Animal; Exotoxins; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Hemolysin Proteins; Humans; Mice; Microbial Viability; Neutrophils; Porosity; RNA, Messenger; Serum; Staphylococcal Infections; Staphylococcus aureus; Virulence

The effect of IL-1beta on Staphylococcus aureus was investigated in terms of mRNA expression profile of bicomponent leukotoxins (Luk ED, Luk PV, HlgA, and HlgCB) as well as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Upon exposure to higher concentrations of IL-1beta, S. aureus expressed significantly higher levels of MSCRAMMs mRNA [fibronectin-binding protein (FnBp), fibrinogen-binding protein or clumping factor (Clf), and collagen-binding protein (Cna)] and had significantly lower expression of mRNAs for bicomponent leukotoxins. Sequential in vitro passing of S. aureus in the absence of rhIL-1beta resulted in reduced binding to rhIL-1beta resulted in lack of significant changes in virulence gene expression upon exposure to low or high concentrations of rhIL-1beta. It is possible that IL-1beta modulates the pathogenic potential of S. aureus by altering its virulence gene expression to adapt to the host's inflammatory micromilieu. The ability to express higher levels of MSCRAMMs and low levels of leukotoxins might contribute towards the successful invasion and persistence of S. aureus in chronic inflammatory conditions. Determination of the mechanisms of IL-1-induced alterations in S. aureus gene expression may lead to the identification of novel therapeutic targets against this ever-evolving opportunistic pathogen.

Topics: Adhesins, Bacterial; Bacterial Proteins; Exotoxins; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Humans; Interleukin-1beta; Oligonucleotide Array Sequence Analysis; Recombinant Proteins; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus; Virulence; Virulence Factors

Leukotoxin family genes in Staphylococcus aureus isolated from domestic animals were examined by polymerase chain reaction. LukS and lukF genes were detected in all 48 avian and 72 porcine isolates of S. aureus. LukE and lukD genes, located in a putative staphylococcal pathogenicity island (Sapln3/Saplm3), were recognized in 44 (91.7%) of 48 avian isolates, but these genes were not detected in porcine isolates. In 297 bovine isolates collected from mastitic cow's milk and bulk milk from dairy farms in two regions, lukM and lukF-PV(P83) genes in addition to lukS-lukF and lukE-lukD genes were detected in 100 (62.5%) of the 160 isolates from Ishikawa and in118 (86.1%) of the 137 isolates from Hokkaido. When the lysogeny of S. aureus bovine isolates was examined by treatment with mitomycin C, clearing of the culture due to cell lysis was observed in 34 (91.9%) of 37 lukM-lukF-PV(P83) genes--positive isolates. In addition, we isolated a novel lukM-lukF-PV(P83)-carrying (designated phiLukM), and revealed that the lukM-lukF-PV(P83) genes were located very close to an amidase gene on the temperate phage genomes. These results suggest horizontal transmission of lukM-lukF-PV(P83) genes by temperate bacteriophages in S. aureus of bovine origin.

Topics: Animal Diseases; Animals; Animals, Domestic; Bacterial Proteins; Birds; Cattle; DNA, Bacterial; Exotoxins; Immunosuppressive Agents; Leukocidins; Polymerase Chain Reaction; Prevalence; Staphylococcal Infections; Staphylococcus aureus; Staphylococcus Phages; Swine

Well-characterized Staphylococcus aureus nasal and blood isolates (N = 429) were tested by polymerase chain reaction for the prevalence of genes that encode leukocidal toxins. The leukotoxin genes lukE+lukD were found at high prevalence, significantly more so in blood (82%) than in nasal isolates (60.5%). Although almost all isolates were positive for the gamma-hemolysin gene, none was positive for lukM. Genes encoding Panton-Valentine leukocidin (PVL) components were very rare in either nasal or blood isolates. The lukE+lukD-negative isolates were significantly more likely to be positive for the staphylococcal enterotoxin gene combination seg/sei (89.5%) and the toxic shock syndrome toxin-1 gene (39.3%) than lukE+lukD-positive isolates (41.7% and 12.7%, respectively). The lukE+lukD-negative isolates were also more likely to show positivity for the accessory gene regulatory locus agr III, but less likely to be positive for the agr II locus. The co-possession of different virulence factors and their probable synergy should receive more attention in order to better understand their role in pathogenicity.

Topics: Bacterial Proteins; Bacterial Toxins; Base Sequence; Confidence Intervals; DNA Fingerprinting; Exotoxins; Female; Gene Expression Regulation, Bacterial; Genotype; Humans; Logistic Models; Male; Molecular Sequence Data; Odds Ratio; Polymerase Chain Reaction; Prevalence; Probability; Sensitivity and Specificity; Staphylococcal Infections; Staphylococcus aureus

Staphylococcus intermedius isolates from dogs (n = 44) and pigeons (n = 62) were categorized into 12 types by intergenic ribosomal DNA spacer polymorphism analysis. All isolates from pigeons were lukS positive and all isolates from dogs were lukS and lukF positive by dot blot analysis. The mean leukotoxicity titer for dog isolates was at least 129-fold higher than that for pigeon isolates.

Topics: Animals; Bacterial Proteins; Bacterial Typing Techniques; Bird Diseases; Columbidae; Dog Diseases; Dogs; Exotoxins; Leukocidins; Molecular Sequence Data; Polymerase Chain Reaction; Prevalence; Sequence Analysis, DNA; Staphylococcal Infections; Staphylococcus

Among the toxins that Staphylococcus aureus is able to secrete, bi-component toxins named leukotoxins target specifically leukocytes, mainly phagocytic cells. Isolates from cows, goats and ewes with mastitis were selected on the basis of the presence or not of the genes encoding the recently described LukM/LukF'-PV leukotoxin. Of the 128 isolates tested, 126 had moderate to high leukotoxic activity to bovine polymorphonuclear cells (PMN). The supernatants of lukM-positive isolates were much more leukotoxic than the supernatants of lukM-negative isolates: mean leukotoxic titers were 122 versus 20 and 581 versus 26 for isolates of bovine and caprine origin, respectively. Among lukM/lukF'-PV positive isolates, those of caprine and ovine origins were more leukotoxic than were isolates of bovine origin (P < 0.01). The two most abundant proteins in the culture supernatant of a highly toxic isolate were purified and identified as the two components of LukM (LukM and LukF'-PV) on the basis of their molecular mass, N-terminal amino acid sequence, and high synergistic activity. LukM/LukF'-PV induced the flattening of bovine PMN at a concentration as low as 3.6 ng/ml (0.1 nM). A higher concentration (18 ng/ml) was necessary to produce the same effect on caprine or ovine PMN. Affinity-purified antibodies to LukM or to LukF'-PV neutralized the leukotoxic effect of all the culture supernatants. They neutralized with the same efficiency the toxic activity of supernatants from lukM/lukF'-PV positive or negative isolates. These results establish that LukM/LukF'-PV is very active on PMN of ruminants and suggest that this leukotoxin could be the most active leukotoxin produced by mastitis isolates. They prompt further studies to delineate the contribution of LukM/LukF'-PV to the pathogenesis of mastitis in ruminants and the protective effect of antibodies to this leukotoxin.

Topics: Animal Diseases; Animals; Antibodies, Bacterial; Cattle; Exotoxins; Female; Goat Diseases; Goats; Mastitis, Bovine; Sensitivity and Specificity; Sheep; Sheep Diseases; Species Specificity; Staphylococcal Infections; Staphylococcus aureus; Titrimetry; Virulence

Bacterial endophthalmitis is a serious complication of ocular surgery and of eye trauma; the leading causative organisms are Staphylococcus aureus strains. Tissue damage is due both to the host inflammatory response and to toxin synthesis by bacteria. Systemic treatment remains difficult because most antibiotics show poor ocular penetration. Moxifloxacin (MXF), a novel fluoroquinolone, was evaluated for its penetration into the vitreous of normal rabbit eyes and of eyes of rabbits infected for 24 h with methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA) following a single intravenous administration of 5 or 20 mg/kg. MXF penetration was rapid and efficient regardless of the dose, ranging from 28 to 52%. An inflammatory state of the vitreous significantly increased penetration after the 20-mg/kg dose, with penetration reaching 52%. Concentrations determined in the vitreous cavity following a 20-mg/kg administration showed a 3.5-fold decrease of the bacterial density within 5 h for MSSA (MIC, 0.125 micro g/ml) and a 1.6-fold decrease for MRSA (MIC, 4 micro g/ml) strains, respectively. By using a semiquantitative reverse transcription-PCR method, the expression of luk-PV and hlgCB, but not hlgA, encoding staphylococcal leukotoxins, was detected in the vitreous without MXF treatment. A slight decrease in the expression of leucotoxins and sarA, agr, and sigB virulence regulatory factors was observed 1 h following the administration of 5 mg of MXF per kg.

Topics: Animals; Anti-Infective Agents; Aza Compounds; Endophthalmitis; Exotoxins; Female; Fluoroquinolones; Male; Microbial Sensitivity Tests; Moxifloxacin; Quinolines; Rabbits; Reverse Transcriptase Polymerase Chain Reaction; Staphylococcal Infections; Staphylococcus aureus; Virulence Factors

Staphylococcus aureus is a major pathogen for cattle, causing various forms of subclinical and clinical mastitis. Two groups of virulence factors (leukotoxins and superantigens) are supposed to play an important role in the initiation and/or the exacerbation of this disease. In order to detect all known and putative members of leukotoxins and SAgs (superantigens), we tested secreted factors of different S. aureus isolates in flow cytometry-based assays. Isolates were sampled from 68 cows of different farms and cultured for 24h in vitro. Supernatants were then coincubated with purified polymorphonuclear granulocytes (PMN) or combinations of blood mononuclear cells (MNC) and PMN. Viable PMN and MNC were determined by quantitative flow cytometry. In addition, we recorded the proliferation-inducing potential of isolate supernatants for bovine MNC. Based on these criteria, the supernatants of S. aureus isolates fell in three groups. The first group (n=32), termed LT-SNs (leukotoxin-containing supernatants), killed purified granulocytes (neutrophils and eosinophils) in vitro. The second group of supernatants (n=20), termed SAg-SN (superantigen-containing supernatants), induced activation and proliferation of mononuclear cells (MNC) and, only in the presence of MNC, resulted in a selective depletion of neutrophils after 24h in vitro. The third group of supernatants (n=16) contained neither LTs or SAgs. Functionally, SAg-SNs behaved like purified staphylococcal enterotoxin A (SEA) or SEB tested in parallel. The absence of SAg-like activity in LT-SNs was confirmed by heat treatment of LT-SNs, which destroyed the leukocytotoxic activity, but did not reveal any MNC-activating potential. This study, therefore, suggests, that pathogenic S. aureus isolates either produce leukotoxins or superantigens and that both groups of virulence factors can easily be differentiated by the functional assays described. The prevalence of leukotoxin- or superantigen-producing isolates was comparable among cattle with subclinical (LT=41%; SAg=30.8%) mastitis. The higher frequency of LT-producing isolates in cases of clinical mastitis (LT=55.2%; SAg=27.6%) was not significant. At least, these findings argue against the dominant role of superantigens or leukotoxins in S. aureus-induced bovine mastitis.

Topics: Animals; Cattle; Dairying; Exotoxins; Female; Flow Cytometry; Leukocytes; Mastitis, Bovine; Milk; Staphylococcal Infections; Staphylococcus aureus; Superantigens; Virulence