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.     

Comparative analysis of agr locus diversification and overall genetic variability among bovine and human Staphylococcus aureus isolates

The accessory gene regulator (agr) is a central system that controls the expression of Staphylococcus aureus virulence factors. In this study, the distribution of agr alleles, defined by agr restriction fragment length polymorphism within agr interference groups, among S. aureus isolates from bovine and human origin was measured with PCR-based techniques. Statistically highly significant associations of some agr alleles with the infection of a specific host were found. The genetic difference between the two S. aureus populations was further stressed after analysis of their genetic background by binary typing.     

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.     

Accessory gene regulator group polymorphisms in methicillin-resistant Staphylococcus aureus: an association with clinical significance

PURPOSE: Virulent gene expression in Staphylococcus aureus is controlled by regulators such as the accessory gene regulator (agr). Strains can be divided into four major agr groups (agr I-IV) on the basis of agrD and agrC polymorphisms. The purpose of this study was to define the proportion of agr I, II, and III polymorphisms and to compare the clinical characteristics between group I and non-group I polymorphisms of methicillin-resistant Staphylococcus aureus (MRSA) strains in a Korean tertiary care teaching hospital. MATERIALS AND METHODS: A total of 158 clinical isolates were evaluated by RFLPs (restriction fragment length polymorphisms). RESULTS: The mean age of the patients was 50.2 +/- 21.9 years old. There were 74 (49.3%), 66 (44.0%), 10 (6.7%), 7 (4.4%), and 1 (0.6%) strains in agr group I, II, III, I + II, and I + III polymorphisms, respectively. Only ear infections were a statistically significant clinical parameter according to univariate (p=0.001) and multivariate analysis (OR, 4.721 (1.273-17.508), p=0.020). CONCLUSION: This study suggests that agr group I is the most prevalent in Korea, and ear infections are correlated with the group I polymorphism, which is a different clinical trend from western countries. It can also be inferred that community-acquired MRSA correlates with agr group I.     

Differential abilities of capsulated and noncapsulated Staphylococcus aureus isolates from diverse agr groups to invade mammary epithelial cells

Staphylococcus aureus is the bacterium most frequently isolated from milk of bovines with mastitis. Four allelic groups, which interfere with the regulatory activities among the different groups, have been identified in the accessory gene regulator (agr) system. The aim of this study was to ascertain the prevalence of the different agr groups in capsulated and noncapsulated S. aureus bacteria isolated from mastitic bovines in Argentina and whether a given agr group was associated with MAC-T cell invasion and in vivo persistence. Eighty-eight percent of the bovine S. aureus strains were classified in agr group I. The remainder belonged in agr groups II, III, and IV (2, 8, and 2%, respectively). By restriction fragment length polymorphism analysis after PCR amplification of the agr locus variable region, six agr restriction types were identified. All agr group I strains presented a unique allele (A/1), whereas strains from groups II, III, and IV exhibited more diversity. Bovine S. aureus strains defined as being in agr group I (capsulated or noncapsulated) showed significantly increased abilities to be internalized within MAC-T cells, compared with isolates from agr groups II, III, and IV. agr group II or IV S. aureus strains were cleared more efficiently than agr group I strains from the murine mammary gland. The results suggest that agr group I S. aureus strains are more efficiently internalized within epithelial cells and can persist in higher numbers in mammary gland tissue than S. aureus strains classified in agr group II, III, or IV.     

Staphylococcal enterotoxin A gene (sea) expression is not affected by the accessory gene regulator (agr).

The goal of this work was to determine whether staphylococcal enterotoxin type A gene (sea) expression is regulated by an accessory gene regulator (agr). The Tn551 insertionally inactivated agr allele of Staphylococcus aureus ISP546 was transferred to three Sea+ S. aureus strains. Each of the Agr- strains produced as much staphylococcal enterotoxin A (SEA) as its parent strain. These results suggest that sea expression is regulated differently from that of seb, sec, and sed, which previously have been shown to require a functional agr system for maximal expression.     

Prevalence of agr specificity groups among methicilin resistant Staphylococcus aureus circulating at Charles Nicolle hospital of Tunis

Methicillin resistant Staphylococcus aureus (MRSA) is a major human pathogen with many clinical aspects. In S. aureus, the accessory gene regulator (agr) globally controls the production of virulence factors. There are four agr groups. Our study was done to define the agr specificity of MRSA circulating at Charles Nicolle hospital and to investigate a possible relationship between agr groups and human disease types. From January 2004 to June 2005, a total of 57 MRSA isolated from individual hospitalized patients were collected, representing 12% of the total S. aureus isolates. The isolates were identified by conventional methods. Methicillin resistance was detected by oxacillin and cefoxitin disks and confirmed by the amplification of mecA gene by PCR. The agr groups were identified by multiplex PCR. All the strains were recovered from different wards: medicine (57.8%) especially from dermatology (56.2%), surgery (28%) and pediatrics (7%). Cutaneous pus (36.84%) and blood culture (35.08%) represented the main specimens. The agr groups were distributed as follow nine (15.7%) belonged to group I, two (3.5%) belonged to group II and 23 (40.3%) belonged to group III. For 23 strains, the agr group was not identified. A relationship between agr group and type of disease was observed: agr group III strains were associated with non invasive infections (P=0.02) and agr group I strains with invasive infections especially bacteremia (P=0.002).     

Genetic variation among hospital isolates of methicillin-sensitive Staphylococcus aureus: evidence for horizontal transfer of virulence genes

Staphylococcus aureus strains often carry in their genomes virulence genes that are not found in all strains and that may be carried on discrete genetic elements. Strains also differ in that they carry one of four classes of an accessory gene regulator (agr) locus, an operon that regulates virulence factor expression and that has been proposed to be a therapeutic target. To look at their distribution among hospital strains, we investigated 38 methicillin-sensitive S. aureus isolates, classifying the isolates by agr class and screening them for the presence and restriction fragment length polymorphisms (RFLPs) of 12 core and 14 accessory virulence genes. Twenty-three (61%) were agr class I, 10 (26%) were agr class II, and 5 (13%) were agr class III. None were agr class IV. The S. aureus strains had distinguishable RFLP profiles, although clusters of isolates with clearly related core gene profiles were found among our strains, including all five agr class III strains, two sets of six strains within agr class I, and six strains within agr class II. Within these clusters there was evidence of horizontal acquisition and/or loss of multiple accessory virulence genes. Furthermore, two isolates from the same patient were identical except for the presence of the sea gene, indicating that movement of mobile elements may occur in vivo. Several strong correlations with the carriage of virulence genes between strains were seen, including a positive correlation between tst and agr class III and negative correlations between tst and lukE-splB and between lukE-splB and seg-sei. This suggests that the core genome or the presence of accessory genetic elements within a strain may influence acquisition and loss of other elements encoding virulence genes.     

Construction and characterization of an agr deletion mutant of Staphylococcus epidermidis

The physiological significance of the accessory gene regulator (agr) system of Staphylococcus epidermidis was investigated by construction of an agr deletion mutant via allelic replacement with a spectinomycin resistance cassette. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the protein pattern was strongly altered in the mutant; the amounts of most surface proteins were higher, whereas the amounts of most exoproteins were lower. The agr system of S. epidermidis thus appears to have an important impact on growth phase-dependent protein synthesis as has been shown for Staphylococcus aureus. The activity of the exoenzymes lipase and protease, assumed to be involved in staphylococcal pathogenicity, was investigated by agar diffusion assays and SDS-PAGE activity staining. A general reduction of these enzyme activities in the agr mutant was found. The difference in overall lipase activity was small, but zymographic analysis suggested a clear defect in lipase processing in the agr mutant.     

Staphylococcus quorum sensing in biofilm formation and infection

Cell population density-dependent regulation of gene expression is an important determinant of bacterial pathogenesis. Staphylococci have two quorum-sensing (QS) systems. The accessory gene regulator (agr) is genus specific and uses a post-translationally modified peptide as an autoinducing signal. In the pathogens Staphylococcus aureus and Staphylococcus epidermidis, agr controls the expression of a series of toxins and virulence factors and the interaction with the innate immune system. However, the role of agr during infection is controversial. A possible second QS system of staphylococci, luxS, is found in a variety of Gram-positive and Gram-negative bacteria. Importantly, unlike many QS systems described in Gram-negative bacteria, agr and luxS of staphylococci reduce rather than induce biofilm formation and virulence during biofilm-associated infection. agr enhances biofilm detachment by up-regulation of the expression of detergent-like peptides, whereas luxS reduces cell-to-cell adhesion by down-regulating expression of biofilm exopolysaccharide. Significant QS activity in staphylococci is observed for actively growing cells at a high cell density, such as during the initial stages of an infection and under optimal environmental conditions. In contrast, the metabolically quiescent biofilm mode of growth appears to be characterized by an overall low activity of the staphylococcal QS systems. It remains to be shown whether QS control in staphylococci represents a promising target for the development of novel antibacterial agents.     

The accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine arthritis model.

We have studied the role of the accessory gene regulator (agr) of Staphylococcus aureus as a virulence determinant in the pathogenesis of septic arthritis. At least 15 genes coding for potential virulence factors in Staphylococcus aureus are regulated by a putative multicomponent signal transduction system encoded by the agr/hld locus. agr and hld mutants show a decreased synthesis of extracellular toxins and enzymes, such as alpha-, beta-, and delta-hemolysin, leucocidin, lipase, hyaluronate lyase, and proteases, and at the same time an increased synthesis of coagulase and protein A as compared with the wild-type counterpart. We have used a recently described murine model of S. aureus-induced arthritis to study the virulence of S. aureus 8325-4 and two agr/hld mutants derived from it. Sixty percent of the mice injected with the wild-type strain developed arthritis, whereas agrA and hld mutants displayed joint involvement in only 10 and 30%, respectively. In addition, 40% of the mice inoculated with the wild-type strain displayed an erosive arthropathy; such changes were not detectable at all in mice inoculated with the agrA mutant. Serum levels of interleukin-6, a potent B-cell differentiation factor, were significantly higher (P < 0.001) in the mice inoculated with the wild-type strain than in those inoculated with the agrA mutant counterpart. Overall, our results suggest that the agr system of S. aureus is an important virulence determinant in the induction and progression of septic arthritis in mice.     

agr expression precedes escape of internalized Staphylococcus aureus from the host endosome

Staphylococcus aureus is a versatile pathogen capable of causing life-threatening infections. Many of its cell wall and exoproduct virulence determinants are controlled via the accessory gene regulator (agr). Although considered primarily as an extracellular pathogen, it is now recognized that S. aureus can be internalized by epithelial and endothelial cells. Traditional experimental approaches to investigate bacterial internalization are extremely time-consuming and notoriously irreproducible. We present here a new reporter gene method to assess intracellular growth of S. aureus in MAC-T cells that utilizes a gfp-luxABCDE reporter operon under the control of the Bacillus megaterium xylA promoter, which in S. aureus is expressed in a growth-dependent manner. This facilitates assessment of the growth of internalized bacteria in a nondestructive assay. The dual gfp-lux reporter cassette was also evaluated as a reporter of agr expression and used to monitor the temporal induction of agr during the MAC-T internalization process. The data obtained suggest that agr induction occurs prior to endosomal lysis and that agr-regulated exoproteins appear to be required prior to the release and replication of S. aureus within the infected MAC-T cells.