Catalytic antibody response to the Staphylococcus aureus virulence factor Efb

Staphylococcus aureus is a globally prevalent pathogen that can cause a wide variety of acute and chronic diseases in both adults and children, in both immune susceptible populations and healthy individuals. Its ability to cause persistent infections has been linked to multiple immune evasion strategies, including Efb-mediated complement inhibition. As new multi-drug-resistant strains emerge, therapeutic alternatives to traditional antibiotics must be developed. These experiments assessed the ability of healthy patient immunoglobulin to cleave Efb and disable the complement-inhibitory properties of Efb in vitro. Levels of immunoglobulin-mediated Efb catalysis varied both between immunoglobulin isoform/isotype and between individuals. Serum IgG showed the strongest catalytic activity of the immunoglobulin isotypes tested. Additionally, IgG hydrolyzed the virulence factor in a way that enabled only minimal binding to the complement component C3b, effectively blocking Efb-mediated inhibition of complement lysis. Salivary IgA and serum IgM did not block Efb-mediated inhibition of complement. Catalytic IgG selectively cleaved Efb and showed no cleavage of a variety of other proteins tested. Catalytic activity of IgG was inhibited by serine protease inhibitors, but not by other protease inhibitors, suggesting a serine-protease mechanism of catalysis. It is proposed that varying concentrations and activity levels of catalytic IgG between healthy individuals and those with current or recurrent S. aureus infections in both adult and pediatric populations be studied in order to assess the potential effectiveness of passive immunization therapy with catalytic monoclonal IgG. ^

Characterization of Staphylococcus aureus adhesins important for respiratory tract infection

Staphylococcus aureus is a leading cause of lower respiratory tract infections in both adult and pediatric populations. In the past two decades, reports have described emergent incidence of severe necrotizing pneumonia in previously healthy individuals, frequently caused by antibiotic resistant strains. Additionally, S. aureus remains the most common cause of ventilator-associated pneumonia, contributing morbidity and mortality in intensive care units. As treatment of infection is made more difficult by the resistance to multiple antibiotics including vancomycin, there is a pressing need for novel strategies to prevent and treat S. aureus infections. Targeting essential mechanisms that promote infection such as adhesion, colonization, invasion, evasion of immune system and signaling may lead to inhibition of pathogenic surge. Staphylococcal adhesins of the MSCRAMM family (microbial surface components recognizing adherent matrix molecules) represent viable targets for such investigations. Understanding the molecular mechanism of binding is the first step toward the development of such therapies. Analysis of bacterial strains isolated from patients with staphylococcal pneumonia show increased expression of protein A, SdrD, SdrC and ClfB, cell surface proteins members of the MSCRAMM family. In this study the interaction of these MSCRAMMs with candidate ligands has been examined. We found that SdrD mediates S. aureus adherence to the lung epithelial cell line A549. Consistently, bacteria expressing SdrD have increased persistence in the lungs of infected mice after bronchoalveolar lavage in comparison with bacteria lacking this protein. Inhibition studies revealed that bacterial attachment can be abolished using neutralizing antibodies against SdrD. Using phage display, neurexin β isoforms were identified as SdrC binding partners. Previous reports postulated that MSCRAMMS bind their ligands by a 'dock, lock and latch' mechanism of interaction. Our data suggested that ClfB, an MSCRAMM responsible for nasal colonization, binds cytokeratin 10 by a 'dock and lock' variant of this model, in which the 'latching' event is not necessary. In summary, we have characterized aspects of molecular interaction between several MSCRAMMS and host components. We hope that continued delineation of these interactions will lead to identification of novel therapeutic targets or preventive strategies against S. aureus infections. ^

Avaliação de oleuropeína e de sanitizantes químicos, isolados ou associados, para eliminação de biofilmes de Staphylococcus aureus, Listeria monocytogenes e Escherichia coli em superfícies inertes

Este estudo avaliou a eficiência da oleuropeína (OLE) (composto fenólico extraído das folhas de Oliveira) isolada e associada aos sanitizantes comerciais ácido peracético 2% (APA), hipoclorito de sódio 2% (HS), peróxido de hidrogênio 3% (PH), digluconato de clorexidina 2% (DC), cloreto de benzalcônio 1% (CB) e iodofor 2% (IO), para inativação de células em suspensão e biofilmes monoespécie e multiespécie formados em superfícies de aço inoxidável ou microplaca de poliestireno por Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 25923) e Escherichia coli (ATCC 25922), todas classificadas como fortes produtores de biofilmes. Os isolados foram semeados em caldo TSB (caldo tripticase soja), incubados (37°C/24h) e corrigidos a ~108células/mL (escala 0,5 McFarland). Para bactérias em suspensão, a resistência a sanitizantes foi determinada pela Concentração Inibitória Mínima (CIM) em tubos e pelo método de Disco Difusão em Ágar (DDA), no qual as bactérias foram plaqueadas em ágar TSA contendo discos de 6mm de papel filtro embebidos nos sanitizantes. Após a incubação, a medição dos halos de inibição foi feita com paquímetro. Para os ensaios de resistência dos biofilmes aos compostos sanitizantes, foram utilizadas microplacas de poliestireno 96 poços, as quais foram preparadas para incubação-fixação dos biofilmes e submetidas à leitura em espectrofotômetro de ELISA (600 nm). Em seguida, as placas foram lavadas com solução salina tamponada (PBS, pH 7.4) e os sanitizantes inseridos por 1 minuto. Após neutralização com tiossulfato de sódio (5 minutos), as placas foram lavadas com PBS e metanol, coradas com cristal violeta 1% e coradas com ácido acético glacial (33%) para nova leitura a 570nm. A eficácia da remoção do biofilme pelos sanitizantes foi comparada pelo índice de formação de biofilme (IFB). As imagens do aço inoxidável após tratamento com sanitizante foram feitas através de Microscopia Eletrônica de Varredura (MEV) e Microscopia Confocal, para visualizar a persistência dos biofilmes. Os valores de CIM (diluição 1:2) mostraram que OLE não teve atividade bactericida. No método DDA, L. monocytogenes, foi resistente à OLE, enquanto E. coli e S. aureus apresentaram resistência intermediária. Os sanitizantes comerciais apresentaram boa atividade bactericida nos ensaios de CIM e DDA, sendo que as associações de OLE aos sanitizantes comerciais aumentaram o efeito germicida. Nos ensaios com biofilmes em monoespécie, somente os sanitizantes comerciais, isolados ou associados com OLE, foram eficazes de reduzir o valor de BFI em microplaca de poliestireno. Em biofilmes multiespécie, OLE apresentou efeito antimicrobiano, sobretudo sobre a associação de L. monocytogenes + E. coli + S. aureus (redução: 91,49%). Nenhum dos compostos avaliados foi capaz de inativar completamente os biofilmes nas superfícies de aço inoxidável, uma vez que células viáveis foram observadas após os tratamentos com os sanitizantes, indicando persistência dos biofilmes. Os resultados indicam que a oleuropeína apresentou potencial para incrementar o efeito bactericida de sanitizantes comerciais para eliminação de biofilmes em superfícies inertes, sendo necessários estudos para compreender os mecanismos de ação dessas combinações.

The prevalence of genotypes that determine resistance to macrolides, lincosamides, and streptogramins B compared with spiramycin susceptibility among erythromycin-resistant Staphylococcus epidermidis

Coagulase-negative staphylococci, particularly Staphylococcus epidermidis , can be regarded as potential reservoirs of resistance genes for pathogenic strains, e.g., Staphylococcus aureus . The aim of this study was to assess the prevalence of different resistance phenotypes to macrolide, lincosamide, and streptogramins B (MLSB) antibiotics among erythromycin-resistant S. epidermidis, together with the evaluation of genes promoting the following different types of MLSB resistance: ermA, ermB, ermC, msrA, mphC, and linA/A’. Susceptibility to spiramycin was also examined. Among 75 erythromycin-resistant S. epidermidis isolates, the most frequent phenotypes were macrolides and streptogramins B (MSB) and constitutive MLSB (cMLSB). Moreover, all strains with the cMLSB phenotype and the majority of inducible MLSB (iMLSB) isolates were resistant to spiramycin, whereas strains with the MSB phenotype were sensitive to this antibiotic. The D-shape zone of inhibition around the clindamycin disc near the spiramycin disc was found for some spiramycin-resistant strains with the iMLSB phenotype, suggesting an induction of resistance to clindamycin by this 16-membered macrolide. The most frequently isolated gene was ermC, irrespective of the MLSB resistance phenotype, whereas the most often noted gene combination was ermC, mphC, linA/A’. The results obtained showed that the genes responsible for different mechanisms of MLSB resistance in S. epidermidis generally coexist, often without the phenotypic expression of each of them.

Phylogenetically distinct Staphylococcus aureus lineage prevalent among indigenous communities in northern Australia

The aim was to determine the evolutionary position of the Staphylococcus aureus clonal complex 75 (CC75) that is prevalent in tropical northern Australia. Sequencing of gap, rpoB, sodA, tuf, and hsp60 and the multilocus sequence typing loci revealed a clear separation between conventional S. aureus and CC75 and significant diversity within CC75.

Implementation Guide for Surveillance of Staphylococcus aureus Bacteraemia -- [Consultation Edition]

The Implementation Guide for the Hospital Surveillance of SAB has been produced by the Healthcare Associated Infection (HAI) Technical Working Group of the Australian Commission on Safety and Quality in Health Care (ACSQHC), and endorsed by the HAI Advisory Group. The Technical Working Group is made up of representatives invited from surveillance units and the ACSQHC, who have had input into the preparation of this Guide. The Guide has been developed to ensure consistency in reporting of SAB across public and private hospitals to enable accurate national reporting and benchmarking. It is intended to be used by Australian hospitals and organisations to support the implementation of healthcare associated Staphylococcus aureus bacteraemia(SAB) surveillance using the endorsed case definition1 in the box below and further detail in the Data Set Specification.

Changes in healthcare-associated Staphylococcus aureus bloodstream infections after the introduction of a national hand hygiene initiative

Background. Interventions that prevent healthcare-associated infection should lead to fewer deaths and shorter hospital stays. Cleaning hands (with soap or alcohol) is an effective way to prevent the transmission of organisms, but rates of compliance with hand hygiene are sometimes disappointingly low. The National Hand Hygiene Initiative in Australia aimed to improve hand hygiene compliance among healthcare workers, with the goal of reducing rates of healthcare-associated infection. Methods. We examined whether the introduction of the National Hand Hygiene Initiative was associated with a change in infection rates. Monthly infection rates for healthcare-associated Staphylococcus aureus bloodstream infections were examined in 38 Australian hospitals across 6 states. We used Poisson regression and examined 12 possible patterns of change, with the best fitting pattern chosen using the Akaike information criterion. Monthly bed-days were included to control for increased hospital use over time. Results. The National Hand Hygiene Initiative was associated with a reduction in infection rates in 4 of the 6 states studied. Two states showed an immediate reduction in rates of 17% and 28%, 2 states showed a linear decrease in rates of 8% and 11% per year, and 2 showed no change in infection rates. Conclusions. The intervention was associated with reduced infection rates in most states. The failure in 2 states may have been because those states already had effective initiatives before the national initiative’s introduction or because infection rates were already low and could not be further reduced.

Molecular characterization of endocarditis-associated Staphylococcus aureus

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted.

Changes in healthcare-associated infections after the introduction of a national hand hygiene initiative

Introduction: Interventions that prevent healthcare-associated infections should lead to fewer deaths and shorter hospital stays. Cleaning hands with soap and water or alcohol rub is an effectiveway to prevent the transmission of organisms, but compliance is sometimes low. The National Hand Hygiene Initiative in Australia aimed to improve hand hygiene compliance among healthcare workers, with the goal of reducing rates of healthcare-associated infections. Methods: We examined if the introduction of the National Hand Hygiene Initiative was associated with a change in infection rates. Monthly infection rates for six types of healthcare-associated infections were examined in 38 Australian hospitals across six states. Infection categories were: bloodstream infections, centralline associated bloodstream infections, methicillin-resistant and methicillin-sensitive Staphylococcus aureus, Staphylococcus aureus bacteraemia and surgical site infections. Results: The National Hand Hygiene Initiative was associated with a statistically significant reduction in infection rates in 11 out of 23 state and infection combinations studied. There was no change in infection rates for nine combinations, and there was an increase in three infection rates in South Australia. Conclusions: The intervention was associated with reduced infection rates in many cases. The lack of improvement in nine cases may have been because they already had effective initiatives before the national initiative’s introduction.

Choose wisely: Network, ontology and annotation resources for the analysis of Staphylococcus aureus omics data

Staphylococcus aureus (S. aureus) is a prominent human and livestock pathogen investigated widely using omic technologies. Critically, due to availability, low visibility or scattered resources, robust network and statistical contextualisation of the resulting data is generally under-represented. Here, we present novel meta-analyses of freely-accessible molecular network and gene ontology annotation information resources for S. aureus omics data interpretation. Furthermore, through the application of the gene ontology annotation resources we demonstrate their value and ability (or lack-there-of) to summarise and statistically interpret the emergent properties of gene expression and protein abundance changes using publically available data. This analysis provides simple metrics for network selection and demonstrates the availability and impact that gene ontology annotation selection can have on the contextualisation of bacterial omics data.

Epidemiology, treatment and outcome of Staphylococcus aureus bacteremia and endocarditis

Staphylococcus aureus is the second most common bloodstream isolate both in community- and hospital-acquired bacteremias. The clinical course of S. aureus bacteremia (SAB) is determined by its complications, particularly by the development of deep infections and thromboembolic events. Despite the progress of antimicrobial therapy, SAB is still associated with high mortality. However, injection drug users (IDUs) tend to have fewer complications and better prognosis than nonaddicts, especially in endocarditis. The present study was undertaken to investigate epidemiology, treatment and outcome of S. aureus bacteremia and endocarditis in Finland. In particular, differences in bacterial strains and their virulence factors, and host immune responses were compared between IDUs and nonaddicts. In Finland, 5045 SAB cases during 1995-2001 were included using the National Infectious Disease Register maintained by National Public Health Institute. The annual incidence of SAB increased, especially in elderly. While the increase in incidence may partly be explained by better reporting, it most likely reflects a growing population at risk, affected by such factors as age and/or severe comorbidity. Nosocomial infections accounted for 51% of cases, with no change in their proportion during the study period. The 28-day mortality was 17% and remained unchanged over time. A total of 381 patients with SAB were randomized to receive either standard antibiotic treatment or levofloxacin added to standard treatment. Levofloxacin combination therapy did not decrease the mortality, lower the incidence of deep infections, nor did it speed up the recovery during 3 months follow-up. However, patients with a deep infection appeared to benefit from combination therapy with rifampicin, as suggested also by experimental data. Deep infections were found in 84% of SAB patients within one week after randomization, and they appeared to be more common than previously reported. Endocarditis was observed in 74 of 430 patients (17%) with SAB, of whom 20 were IDUs and 54 nonaddicts. Right-sided involvement was diagnosed in 60% of addicts whereas 93% of nonaddicts had left-sided endocarditis. Unexpectedly, IDUs showed extracardiac deep infections, thromboembolic events and severe sepsis with the same frequency as nonaddicts. The prognosis of endocarditis was better among addicts due to their younger age and lack of underlying diseases in agreement with earlier reports. In total, all 44 IDUs with SAB were included and 20 of them had endocarditis. An equal number of nonaddicts with SAB were chosen as group matched controls. Serological tests were not helpful in identifying patients with a deep infection. No individual S. aureus strain dominated in endocarditis among addicts. Characterization of the virulence factors of bacterial strains did not reveal any significant differences in IDUs and nonaddicts.

Molecular Basis for the Role of Staphylococcus aureus Penicillin Binding Protein 4 in Antimicrobial Resistance

Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the D-Ala-D-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetyl-glucosamine and N-acetyl-muramic acid-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala. beta-Lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a beta-lactamase and is not trapped as an acyl intermediate with beta-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.

Bacteriophage therapy for Staphylococcus aureus bacteremia in streptozotocin-induced diabetic mice

The protective effect of bacteriophage was assessed against experimental Staphylococcus aureus lethal bacteremia in streptozotocin (STZ) induced-diabetic and non-diabetic mice. Intraperitoneal administrations of S. aureus (RCS21) of 2 x 10(8) CFU caused lethal bacteremia in both diabetic and non-diabetic mice. A single administration of a newly isolated lytic phage strain (GRCS) significantly protected diabetic and nondiabetic mice from lethal bacteremia (survival rate 90% and 100% for diabetic and non-diabetic bacteremic groups versus 0% for saline-treated groups). Comparison of phage therapy to oxacillin treatment showed a significant decrease in RCS21 of 5 and 3 log units in diabetic and nondiabetic bacteremic mice, respectively. The same protection efficiency of phage GRCS was attained even when the treatment was delayed up to 4 h in both diabetic and non-diabetic bacteremic mice. Inoculation of mice with a high dose (10(10) PFU) of phage GRCS alone produced no adverse effects attributable to the phage per se. These results suggest that phages could constitute valuable prophylaxis against S. aureus infections, especially in immunocompromised patients. (C) 2010 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.