Yvonne Benito

Staphylococcus aureus Panton-Valentine Leukocidin Causes Necrotizing Pneumonia

The Staphylococcus aureus Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by strains epidemiologically associated with the current outbreak of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and with the often-lethal necrotizing pneumonia. To investigate the role of PVL in pulmonary disease, we tested the pathogenicity of clinical isolates, isogenic PVL-negative and PVL-positive S. aureus strains, as well as purified PVL, in a mouse acute pneumonia model. Here we show that PVL is sufficient to cause pneumonia and that the expression of this leukotoxin induces global changes in transcriptional levels of genes encoding secreted and cell wall–anchored staphylococcal proteins, including the lung inflammatory factor staphylococcal protein A (Spa).

Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression

Staphylococcus aureus RNAIII is one of the largest regulatory RNAs, which controls several virulence genes encoding exoproteins and cell‐wall‐associated proteins. One of the RNAIII effects is the repression of spa gene (coding for the surface protein A) expression. Here, we show that spa repression occurs not only at the transcriptional level but also by RNAIII‐mediated inhibition of translation and degradation of the stable spa mRNA by the double‐strand‐specific endoribonuclease III (RNase III). The 3′ end domain of RNAIII, partially complementary to the 5′ part of spa mRNA, efficiently anneals to spa mRNA through an initial loop–loop interaction. Although this annealing is sufficient to inhibit in vitro the formation of the translation initiation complex, the coordinated action of RNase III is essential in vivo to degrade the mRNA and irreversibly arrest translation. Our results further suggest that RNase III is recruited for targeting the paired RNAs. These findings add further complexity to the expression of the S. aureus virulon.

Specific real-time polymerase chain reaction places Kingella kingae as the most common cause of osteoarticular infections in young children.

Background: The use of universal 16S rDNA polymerase chain reaction (PCR) has recently shown that the place of Kingella kingae in osteoarticular infections (OAI) in young children has been underestimated, but this technique is not the most sensitive or the most rapid method for molecular diagnosis. We developed a specific real-time PCR method to detect K. kingae DNA and applied it to the etiologic diagnosis of OAI. Patients and Methods: All children admitted to a pediatric unit for OAI between January 2004 and December 2005 were enrolled in this prospective study. Culture-negative osteoarticular specimens were tested by 16S rDNA PCR and by K. kingae-specific real-time PCR when sufficient sample remained. Results: By culture alone, a pathogen was identified in 45% of the 131 specimens tested (Staphylococcus aureus, n = 25; K. kingae, n = 17; others, n = 18). 16S rDNA PCR and K. kingae-specific PCR were both applied to 61 of the culture-negative samples. The combination of culture and 16S rDNA PCR identified a pathogen in 61% of cases (K. kingae DNA, n = 16; DNA of other microorganisms, n = 5). Specific real-time PCR identified a further 6 cases caused by K. kingae and confirmed all 16 universal PCR-positive cases, bringing the overall documentation rate to 66%. K. kingae was the leading cause of OAI in this pediatric series (n = 39, 45%), followed by S. aureus (n = 25, 29%) Conclusion: The K. kingae-specific real-time PCR places K. kingae as the leading cause of OAI in children at our hospital.

Molecular Diagnosis of Infective Endocarditis by PCR Amplification and Direct Sequencing of DNA from Valve Tissue

ABSTRACT We used broad-range eubacterial PCR amplification followed by direct sequencing to identify microbial pathogens in heart valve material from 29 patients with histologically confirmed infective endocarditis and 23 patients free of infective endocarditis. Microorganisms cultured by conventional techniques matched those identified by PCR in 21 cases. PCR alone identified the causative agent in three cases (Streptococcus bovis, Staphylococcus cohnii, and Coxiella burnetii), allowing better patient management. PCR corrected the initial bacteriological diagnosis in three cases (Streptococcus bovis, Streptococcus mutans, and Bartonella henselae). Among the 29 cases of histologically confirmed infective endocarditis, PCR findings were positive in 27 cases and were consistent with the bacterial morphology seen at Gram staining (26 cases) or with the results obtained by immunohistologic analysis with an anti-C. burnetii monoclonal antibody (one case). In two other cases of histologically confirmed infective endocarditis, PCR remained negative in a blood culture-negative case for which no bacteria were seen at histological analysis and in one case with visualization of cocci and blood cultures positive for Enterococcus faecalis. Ten clinical diagnoses of possible infective endocarditis were ruled out by histopathological analysis of the valves and subsequently by PCR. PCR was negative in 13 of the 14 patients in whom infective endocarditis was rejected on clinical grounds; the other patient was found to have Coxiella burnetii infective endocarditis on the basis of PCR and histopathological analysis and was subsequently included in the group of 29 definite cases. In total, PCR contributed to the diagnosis and management of infective endocarditis in 6 of 29 (20%) cases.

Effect of antibiotics on Staphylococcus aureus producing Panton-Valentine leukocidin.

ABSTRACT We examined the capacity of Staphylococcus aureus strains to release Panton-Valentine leukocidin (PVL) in the presence of antibiotics. No PVL was detected when S. aureus was incubated at inhibitory concentrations, while subinhibitory concentrations of oxacillin enhanced the PVL level; clindamycin, linezolid, and fusidic acid were inhibitory; and vancomycin had roughly no effect.

Contribution of a broad range polymerase chain reaction to the diagnosis of osteoarticular infections caused by Kingella kingae: description of twenty-four recent pediatric diagnoses.

Background: Microbiologic diagnosis of septic arthritis and osteomyelitis in children is hindered by the less than optimal yield of blood and osteoarticular fluid cultures. Patients and Methods: All patients admitted to a pediatric unit for osteoarticular infections (OAI) between January 2001 and February 2004 were enrolled in this prospective study. Osteoarticular fluid and biopsy samples that were negative by conventional culture were tested by polymerase chain reaction (PCR) with universal 16S ribosomal DNA primers. Results: We enrolled 171 children. Culture was positive in 64 cases (37.4%), yielding Kingella kingae in 9 cases. The 107 culture-negative specimens were tested by 16S ribosomal DNA PCR. Fifteen samples (14%) were positive, all for Kingella DNA sequences. K. kingae was the second cause of OAI in this population (30.4%), after Staphylococcus aureus (38%). Patients with Kingella infection diagnosed by culture (9 cases) did not differ from those diagnosed by PCR (15 cases) in terms of their clinical characteristics (including prior antibiotic therapy). The characteristics of the 24 children with arthritis (n = 17) or osteomyelitis (n = 7) were similar to those reported elsewhere. Fever (>38°C) and symptom onset shortly before hospitalization (median, 4.5 days) were significantly associated with arthritis. Conclusion: Use of molecular diagnostic methods increases the identification of K. kingae in osteoarticular infections.

Eubacterial PCR for bacterial detection and identification in 100 acute postcataract surgery endophthalmitis.

PURPOSE To evaluate eubacterial PCR compared with conventional cultures for detection and identification of bacterial agents in ocular samples from patients with acute postcataract endophthalmitis. METHODS Broad-range eubacterial PCR amplification was used, followed by direct DNA sequencing in ocular samples (aqueous humor, vitreous samples from tap or vitrectomy) from 100 consecutive patients presenting with acute postcataract endophthalmitis. Bacterial cultures were performed on the same ocular samples by using traditional methods (brain-heart infusion broth). RESULTS At the time of admission, the detection rate was not significantly different between cultures and PCR (38.2% for cultures versus 34.6% for PCR in aqueous humor samples; 54% versus 57% in vitreous from a vitreous tap). In contrast, in the vitreous obtained from vitrectomy, after intravitreous injection of antibiotics, PCR detected bacteria in 70% of the cases, compared with 9% in cultures. By combining PCR and cultures, bacterial identification was obtained in 47% of aqueous humor samples at admission, in 68% of vitreous samples from a vitreous tap at admission, and in 72% of vitreous samples from pars plana vitrectomy. Gram-positive bacteria predominated (94.3%). The concordance between cultures and PCR was 100%. The contamination rate was 2%. CONCLUSIONS Cultures and eubacterial PCR are complementary techniques for bacterial identification in eyes with acute postcataract endophthalmitis. PCR technique was needed for identification of the involved microbial pathogen in 25% of all the cases. Eubacterial PCR is more effective than cultures in detecting bacteria in vitreous samples from patients with previous intravitreous administration of antibiotics.

The staphylococcal toxins γ-haemolysin AB and CB differentially target phagocytes by employing specific chemokine receptors

Evasion of the host phagocyte response by Staphylococcus aureus is crucial to successful infection with the pathogen. γ-Hemolysin AB and CB (HlgAB, HlgCB) are bicomponent pore-forming toxins present in almost all human S. aureus isolates. Cellular tropism and contribution of the toxins to S. aureus pathophysiology are poorly understood. Here, we identify the chemokine receptors CXCR1, CXCR2 and CCR2 as targets for HlgAB, and the complement receptors C5aR and C5L2 as targets for HlgCB. The receptor expression patterns allow the toxins to efficiently and differentially target phagocytic cells. Murine neutrophils are resistant to HlgAB and HlgCB. CCR2 is the sole murine receptor orthologue compatible with γ-Hemolysin. In a murine peritonitis model, HlgAB contributes to S. aureus bacteremia in a CCR2-dependent manner. HlgAB-mediated targeting of CCR2+ cells highlights the involvement of inflammatory macrophages during S. aureus infection. Functional quantification identifies HlgAB and HlgCB as major secreted staphylococcal leukocidins.

Cross‐talk between Staphylococcus aureus leukocidins‐intoxicated macrophages and lung epithelial cells triggers chemokine secretion in an inflammasome‐dependent manner

Staphylococcus aureus is a major pathogen responsible for both nosocomial and community‐acquired infections. Central to its virulence is its ability to secrete haemolysins, pore‐forming toxins and cytolytic peptides. The large number of membrane‐damaging toxins and peptides produced during S. aureus infections has hindered a precise understanding of their specific roles in diseases. Here, we used comprehensive libraries of recombinant toxins and synthetic cytolytic peptides, of S. aureus mutants and clinical strains to investigate the role of these virulence factors in targeting human macrophages and triggering IL‐1β release. We found that the Panton Valentine leukocidin (PVL) is the major trigger of IL‐1β release and inflammasome activation in primary human macrophages. The cytolytic peptides, δ‐haemolysin and PSMα3; the pore‐forming toxins, γ‐haemolysin and LukDE; and β‐haemolysin synergize with PVL to amplify IL‐1β release, indicating that these factors cooperate with PVL to trigger inflammation. PVL+S. aureus causes necrotizing pneumonia in children and young adults. The severity of this disease is due to the massive recruitment of neutrophils that cause lung damage. Importantly, we demonstrate that PVL triggers IL‐1β release in human alveolar macrophages. Furthermore, IL‐1β released by PVL‐intoxicated macrophages stimulates the secretion of the neutrophil attracting chemokines, IL‐8 and monocyte chemotactic protein‐1, by lung epithelial cells. Finally, we show that PVL‐induced IL‐8/monocyte chemotactic protein‐1 release is abolished by the inclusion of IL‐1 receptor antagonist (IL‐1Ra) in a mixed culture of lung epithelial cells and macrophages. Together, our results identify PVL as the predominant S. aureus secreted factor for triggering inflammasome activation in human macrophages and demonstrate how PVL‐intoxicated macrophages orchestrate inflammation in the lung. Finally, our work suggests that anakinra, a synthetic IL‐1Ra, may be an effective therapeutic agent to reduce the massive neutrophils infiltration observed during necrotizing pneumonia and decrease the resulting host‐mediated lung injury.

PSMs of hypervirulent Staphylococcus aureus act as intracellular toxins that kill infected osteoblasts.

Epidemic community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is associated with more severe and acute forms of osteomyelitis than healthcare-associated (HA-) MRSA. Although S. aureus is now recognized as a facultative intracellular pathogen, the contribution of osteoblast invasion by CA-MRSA to the pathogenesis of osteomyelitis is unknown. Using an ex vivo model of intracellular infection of human osteoblasts, we demonstrated that CA-MRSA strains of diverse lineages share an enhanced ability to kill infected osteoblasts compared to HA-MRSA. Cytotoxicity comparisons of CA-MRSA isogenic deletion mutants revealed that phenol-soluble modulins (PSMs), a class of membrane-damaging exoproteins that are expressed at higher levels in CA-MRSA than in HA-MRSA, are involved in this osteoblast killing, whereas other major CA-MRSA virulence determinants, the Panton-Valentine leukocidin and alpha-toxin, are not involved. Similarly, functional agr and sarA regulators, which control the expression of PSMs and alpha-toxin, were required for the expression of the intracellular cytotoxic phenotype by CA-MRSA, whereas the saeRS regulator, which controls the expression of alpha-toxin but not PSMs, had no impact on cytotoxicity. Finally, PSM transcript levels determined by quantitative reverse-transcriptase PCR were significantly higher in CA-MRSA than in HA-MRSA strains and associated with cell damage in MRSA-infected osteoblasts. These findings provide new insights into the pathogenesis of severe CA-MRSA osteomyelitis and unravel a novel virulence strategy of CA-MRSA, based on the invasion and subsequent killing of osteoblasts by PSMs acting as intracellular toxins.