Cell-mediated immune responses in Staphylococcus aureus infections in mice.

Delayed hypersensitivity to staphylococcal antigens was shown in mice repeatedly infected with Staphylococcus aureus. It was characterized by footpad swelling at 48 hours with a mononuclear cell infiltrate and could be transferred to non-infected recipients by T lymphocytes from infected animals, but not by serum. Recipients of immune T cells produced very severe necrotic lesions when challenged with staphylococci. This was in contrast to the protection against necrosis in recipients afforded by serum from infected donors. When both serum and cells were transferred into the same mouse the humoral effects overshadowed or perhaps inhibited those mediated by cells with resultant protection against staphylococcal dermonecrosis.     

Secretory antibody responses in axenic mice to perorally administered Staphylococcus aureus

Topical oral immunization of axenic mice with non-replicating Staphylococcus aureus resulted in the production of both serum and salivary agglutinins. A latent period of approximately 5 days was noted before salivary antibodies were detected against both a carbohydrate (ChoA) and a protein (PrA) antigen isolated from this micro-organism. The local response to ChoA exhibited a linear increase until day 9 when the reciprocal titres reached a plateau [32 (16--64)]. Salivary antibodies to PrA peaked by day 13 at a mean reciprocal titre of 60 (32--128). The agglutinin response in saliva was found to be initially IgG; however, by day 9 of a 14-day-immunization regimen, IgA became the predominant class of exocrine anti-S. aureus antibodies. The serum agglutinin response followed that found in saliva by approximately 2--4 days. By day 14, all sera (6/6) contained PrA agglutinins, while 4/6 sera agglutinated sheep erythrocytes coated with ChoA. Serum antibodies to ChoA were exclusively IgM, in contrast to IgM and IgA agglutinins elicited by PrA. Absorption studies provided evidence of a specific local and systemic immune response to both ChoA and PrA antigens of perorally administered S. aureus.     

Antibody responses in patients with invasive Staphylococcus aureus infections

Correlation between antibody response and clinical outcome in Staphylococcus aureus bacteremia has yielded conflicting results. Immunization schedules have failed in clinical trials. Is the humoral response toward S. aureus of protective nature? A prospective study was performed in patients with invasive S. aureus (ISA) infections during the period 2003–2005. The antibody levels were determined at the beginning and at the end of treatment and one month later (n?=?96, n?=?71, and n?=?51, respectively). As controls, 115 healthy individuals were used. A quantitative enzyme-linked immunosorbent assay (ELISA) against eight purified antigens was performed. Bacterial isolates were grouped as to the production of alpha-toxin, agr type, and pulsed-field gel electrophoresis (PFGE) type. Large variations were seen in the antibody levels. The levels in the second sample were the highest. A correlation between agr group, PFGE group, alpha-toxin production, and initial antibody levels was observed. Patients with fatal outcome displayed lower initial antibody levels to all antigens and significantly so in regard to teichoic acid, lipase, enterotoxin A, and scalded skin syndrome toxin. In episodes with complicated bacteremia, initial significantly low levels to teichoic acid and lipase were registered. Low initial antibody levels against several antigens were associated with increased mortality and complicated bacteremia in invasive S. aureus infections. Bacterial properties, strain, and toxin production affected the antibody response.     

Rapid multiplex PCR assay for identification of USA300 community-associated methicillin-resistant Staphylococcus aureus isolates

Recent reports have noted a discernible increase in the number of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections in patients without traditional risk factors. In the United States, the most prominent CA-MRSA strain encodes Panton-Valentine leukocidin (PVL) cytotoxin genes, belongs to pulsed field gel electrophoresis type USA300 and multilocus sequence type 8, and carries staphylococcal cassette chromosome mec (SCCmec) type IV. At present, molecular characterization of MRSA strains, such as USA300, can be time-consuming and is often beyond the technical capability of many clinical laboratories, making routine identification difficult. We analyzed the chromosomal regions flanking the SCCmec element in 44 USA300 MRSA isolates and identified a signature "AT repeat" sequence within the conserved hypothetical gene SACOL0058 located 1.4 kb downstream of the 3' end of the J1-SCCmec chromosomal junction. Only USA300 isolates tested contained a sequence of > or =6 AT repeats in combination with PVL (e.g., related USA500 or Iberian strains had > or =6 AT repeats but were PVL negative). Using a locked nucleic acid primer specific for > or =6 AT repeats in combination with primers to detect PVL, we developed a multiplex PCR assay specific for the identification of USA300 strains. Multiplex results were 100% concordant with DNA sequencing, suggesting that the method has promise as a means of rapidly identifying USA300 isolates.     

Use of multiplex PCR to identify Staphylococcus aureus adhesins involved in human hematogenous infections

We have developed a multiplex PCR procedure to determine the distribution of nine adhesin genes in Staphylococcus aureus isolates. Only genes encoding bone sialoprotein binding protein and fibronectin binding protein B were significantly associated with hematogenous osteomyelitis/arthritis and native-valve endocarditis, respectively, suggesting their involvement in hematogenous tissue infections.     

Immunosuppressive effect of Staphylococcus aureus peptidoglycan on antibody response in mice

Immunosuppressive effect of an adjuvant, Staphylococcus aureus peptidoglycan, on the primary IgM antibody response in mice was studied with application of the hemolytic plaque assay. Peptidoglycan suppressed the IgM response to thymus-dependent sheep erythrocyte antigen when given intravenously before immunization. The effect of peptidoglycan and erythrocytes was both time and dose dependent. For the suppression, both the delay in the antibody response and overall decreased response were responsible. Peptidoglycan did not influence background counts of antibody-forming cells. Priming with subthreshold dose of erythrocytes did not overcome the suppression, although higher responses compared to unprimed animals were observed. Peptidoglycan did not suppress antibody response to thymus-independent antigens-lipopolysaccharide or high dose of erythrocytes. It is suggested that peptidoglycan-induced immunosuppression is mediated by a presently unidentified population of helper cells-macrophages or T lymphocytes.     

Rapid Staphylococcus aureus agr type determination by a novel multiplex real-time quantitative PCR assay

The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information.     

Staphylococcus aureus infections in psittacine birds

Staphylococcus aureus was isolated from internal organs of 13 different psittacine birds submitted for necropsy over a period of 6 years. The birds all had lesions consistent with septicaemia. S. aureus isolates included three different phage types. In seven of the 13 birds, concurrent infections with Chlamydophila species, Enterococcus hirae, Candida species, unidentified streptococci and coagulasenegative staphylococci were detected. One bird also had lesions of lymphoid leucosis. Few indications were found that staphylococcosis associated problems may spread epidemically. The present studies suggest that S. aureus is pathogenic for psittacine birds, although it does not seem to be a frequent cause of disease.     

Detection of antibody to Staphylococcus aureus teichoic acid by enzyme-linked immunosorbent assay.

A sensitive, specific, and rapid enzyme-linked immunosorbent assay has been developed for the detection of immunoglobulin G to Staphylococcus aureus teichoic acid in human sera. Detection of S. aureus teichoic acid antibody is at least 800 times more sensitive than a double diffusion in gel assay, and positive titers of 1:25,600 and greater were observed with this assay. Results with the enzyme-linked immunosorbent assay can be obtained within 3.5 h by using antigen-coated cuvettes. Quantitation of S. aureus teichoic acid antibody by this enzyme-linked immunosorbent assay may be useful in the initial as well as the follow-up diagnosis of serious S. aureus infections.     

Management of methicillin-resistant Staphylococcus aureus infections

This review addresses selected aspects of the management of severe healthcare-associated infections due to methicillin-resistant Staphylococcus aureus (MRSA), including the limitations of current therapy, potential alternative agents, new therapeutic options, clinical approaches to MRSA bacteraemia/endocarditis and ventilator-associated pneumonia, and strategies to improve outcomes in patients with severe MRSA infections.     

Methicillin resistance in Staphylococcus aureus infections among patients colonized with methicillin-susceptible Staphylococcus aureus

Objectives

We have noticed that patients colonized with methicillin-susceptible Staphylococcus aureus (MSSA) rarely get methicillin-resistant S. aureus (MRSA) infections. The purpose of this study was to compare the odds of a Staphylococcus aureus (SA) infection being an MRSA infection in MSSA carriers, MRSA carriers and non-carriers of SA.

Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct

Strains of Staphylococcus aureus were transformed with plasmid DNA containing a Photorhabdus luminescens lux operon (luxABCDE) that was genetically modified to be functional in both gram-positive and gram-negative bacteria. S. aureus cells containing this novel lux construct, downstream of an appropriate promoter sequence, are highly bioluminescent, allowing the detection of fewer than 100 CFU in vitro (direct detection of exponentially dividing cells in liquid culture). Furthermore, these bacteria produce light stably at 37 degrees C and do not require exogenous aldehyde substrate, thus allowing S. aureus infections in living animals to be monitored by bioluminescence. Two strains of S. aureus 8325-4 that produce high levels of constitutive bioluminescence were injected into the thigh muscles of mice, and the animals were then either treated with the antibiotic amoxicillin or left untreated. Bioluminescence from bacteria present in the thighs of the mice was monitored in vivo over a period of 24 h. The effectiveness of the antibiotic in the treated animals could be measured by a decrease in the light signal. At 8 h, the infection in both groups of treated animals had begun to clear, as judged by a decrease in bioluminescence, and by 24 h no light signal could be detected. In contrast, both groups of untreated mice had strong bioluminescent signals at 24 h. Quantification of CFU from bacteria extracted from the thigh muscles of the mice correlated well with the bioluminescence data. This paper shows for the first time that bioluminescence offers a method for monitoring S. aureus infections in vivo that is sensitive and noninvasive and requires fewer animals than conventional methodologies.     

Antiribitol-teichoic acid antibody (ARTA) in diagnosis of deep seated Staphylococcus aureus infections.

Antiribitol-teichoic acid antibody (ARTA) was detected in sera of 30 out of 50 patients (60%) with various acute deep seated Staphylococcus aureus infections and 5 out of 10 chronic osteomyelitis cases, whereas none of the sera from 50 patients with superficial Staphylococcus aureus infections as well from 50 patients without Staphylococcus aureus infections showed antibody response (p less than 0.01). This test is a definite advantage in diagnosis of deep seated staphylococcal infections like endocarditis, lung disease, meningitis and specially in osteomyelitis cases where organisms cannot be isolated and therefore helps in predicting the need for long term antimicrobial therapy.     

A comparative analysis of antibody repertoire against Staphylococcus aureus antigens in patients with deep-seated versus superficial staphylococcal infections

Immunoblot and an enzyme-linked immunosorbent assays were used to evaluate and compare IgG antibodies against S. aureus whole cell lysate, cell wall peptidoglycan and lipoteichoic acid to discriminate between deep-seated and superficial S. aureus infection. Serum samples were examined from patients with deep-seated (n = 25) and superficial (n = 25) S. aureus infections and 15 healthy controls. Patients with deep-seated infections exhibited a large number of immuno-reactive bands in their IgG immunoblot profile as compared to those with superficial infections and healthy controls. Anti-staphylococcal IgG antibodies that reacted with two antigens of apparent molecular weight 110 and 98 kDa were specifically present in 96% (24/25) of patients with deep-seated infections, and were absent in, superficial and control sera. Moreover other Gram-positive and Gram-negative bacteria did not share these two unique antigens. The ELISA assays revealed significantly elevated levels of IgG antibodies to peptidoglycan (PG) in 18 of 25 (72%) patients with deep infection and 15 of 25 (60%) patients with superficial staphylococcal infection. The elevated levels of IgG antibodies to teichoic acid (TA) antigen were detected in all (100%) deep-seated group patients and among 40% (10/25) patients with superficial infection. An increase in levels of antibodies to PG showed a positive correlation trend with levels of IgG antibodies to TA only in deep infection group. Thus immunoblot detection of these two unique antigens as well as detection of elevated antibodies against PG and TA may be useful for the discrimination of staphylococcal deep-seated and superficial infection in humans.     

Real-time nucleic acid sequence-based amplification assay for rapid detection and quantification of agr functionality in clinical Staphylococcus aureus isolates

Staphylococcus aureus infections are a significant cause of morbidity and mortality in health care settings. S. aureus clinical isolates vary in the function of the accessory gene regulator (agr), which governs the expression of virulence determinants, including surface and exoproteins, while agr activity has been correlated with patient outcome and treatment efficiency. Here we describe a duplex real-time nucleic acid sequence-based amplification (NASBA) detection and quantification platform for rapid determination of agr functionality in clinical isolates. Using the effector of agr response, RNAIII, as the assay target, and expression of the gyrase gene (gyrB) as a normalizer, we were able to accurately discriminate agr functionality in a single reaction. Time to positivity (TTP) ratios between gyrB and RNAIII showed very good correlation with the ratios of RNAIII versus gyrB RNA standard inputs and were therefore used as a simple readout to evaluate agr functionality. We validated the assay by characterizing 106 clinical S. aureus isolates, including strains with genetically characterized agr mutations. All isolates with dysfunctional agr activity exhibited a TTP ratio (TTP(gyrB)/TTP(RNAIII)) lower than 1.10, whereas agr-positive isolates had a TTP ratio higher than this value. The results showed that the assay was capable of determining target RNA ratios over 8 logs (10(-3) to 10(4)) with high sensitivity and specificity, suggesting the duplex NASBA assay may be useful for rapid determination of agr phenotypes and virulence potential in S. aureus clinical isolates.     

New epidemiology of Staphylococcus aureus infections in the Middle East

Staphylococcus aureus is a bacterial pathogen that is distributed worldwide and represents an increasing problem, both in hospitals and in the community. Global transmission of methicillin-resistant S. aureus (MRSA) has been the subject of many studies. Determining the incidence of colonization with community-acquired MRSA in hospitalized patients and outpatients has been the aim of several studies conducted in the Middle East (western Asia). The local epidemiology within countries in this region is changing, owing to the introduction of new strains with the intercontinental exchange of several clones. Sequence type 80-MRSA-IV is one common clone detected in different countries within the region showing country-based differences, and hence more likely to form clonal lineages. MRSA is endemic in this region, and the burden and the difficulty in detecting imported strains are increasing. This is also increasing the risk of domestic and global transmission. To counter the threat associated with the high incidence of MRSA carriage and infections, systematic surveillance of both hospital and community isolates is required, along with appropriate measures designed to limit their spread. Additionally, antibiotic stewardship is needed to contain the further development of the observed resistance and to help in preserving antibiotics as precious therapeutic resources. It is critical for countries in this region to establish both national and international initiatives to develop better measurements designed to limit and control the spread of infections. Finally, more sequence-based studies are needed to better understand the pathogenicity and epidemiology of these important pathogens.     

Enzyme-linked immunosorbent assay for detection of Staphylococcus aureus alpha-toxin.

A sandwich enzyme-linked immunosorbent assay was developed for measuring Staphylococcus aureus alpha-toxin. This assay was 500 to 1,000 times more sensitive than the commonly used hemolytic titration assay and was less variable. The binding of alpha-toxin to the adsorbed antibody was most effective after an overnight incubation at 27 degrees C. The toxin was detectable even at a log2 17 dilution of an S. aureus culture supernatant.     

Cellular assay for measuring anti-erythrocyte antibody responses

Splenocytes from mice immunized with sheep red cells (SRC) or donkey red cells (DRC) were able to lyse radiolabeled SRC or DRC respectively. The cytotoxic effect was mediated by the active supernatant which was released from the immune splenocytes and which was characterized as antibody.This cellular cytotoxic response was found to be a very reliable, efficient, rapid and objective assay for measuring anti-erythrocyte antibody responses.