Sub-inhibitory concentrations of oxacillin modify the expression of agr locus in Staphylococcus aureus clinical strains belonging to different clonal complexes

Staphylococcus aureus remains the major cause of endovascular infections with high morbidity and mortality rates that may reach 40% in case of infective endocarditis [1]. Even though most S. aureus genotypes exhibit the capacity to cause invasive disease, several studies have reported the association of particular clonal complexes (CC5, CC15 and CC30) with endovascular complications such as infective endocarditis [2‐4].

The ability of S. aureus to produce infection involves a wide variety of virulence determinants, such as exotoxins, adhesins and immune evasion genes, constituting a gene framework that is under the control of global regulators, most notably the quorum-sensing accessory gene regulator (agr). Under conditions of high cell density, agr is responsible for the increased expression of many toxins and degradative exoenzymes, and decreased expression of several colonization factors. This regulation is important for the timing of virulence factor expression during infection [5]. Thereby, when adhesion to host tissue is crucial, cell density and agr expression are low, resulting in an increase in the surface virulence factors that are required for the colonization process. Once infection is established, the increased cell density results in higher agr expression that leads to production of toxins which trigger the host inflammatory response [6, 7]. The agr system up-regulates the production of secreted virulence factors such as exotoxins, and down-regulates the production of cell associated virulence factors [5, 8‐10].

The agr locus consists of two divergent transcriptional loci, RNAII and RNAIII, driven by promoters P2 and P3, respectively [8, 9]. The P2 operon encodes four genes, agrB, agrD, agrC and agrA. The agrA and agrC genes constitute a two-component signaling system, of which agrC is the receptor, and agrA is the response regulator [11, 12]. Upon activation by agrC-dependent phosphorylation, agrA binds to the P2 promoter region for RNAII and the P3 promoter region for RNAIII, the effector of target gene regulation that also encodes delta-haemolysin, the expression of which serves as a surrogate for agr functionality [13, 14]. Moreover agrA directly up-regulates transcription of the phenol-soluble modulins operon (psmα and psmβ) whose role in the pro-inflammatory response has already been demonstrated [6, 7, 12, 15]. Several studies have reported that different mutations in the agr operon, mainly within the agrC or agrA genes, lead to decreased RNAIII expression and reduced virulence [9, 16‐18]. However, it is known that strains with dysfunctional agr are more likely to cause persistent infection and be associated with poor outcome [19‐21].

Despite the emergence of methicillin resistant S. aureus (MRSA), beta-lactams and specifically antistaphylococcal penicillins (nafcillin, oxacillin, cloxacillin and dicloxacillin), remain the usual treatment for the management of invasive infections caused by methicillin-susceptible S. aureus (MSSA). However, because of impaired diffusion and distribution of antibiotic, not all bacteria are exposed to the required lethal concentrations of bactericidal agents and therefore are likely subjected to sub-MIC antibiotic effects on the expression of virulence factors [7, 22‐30]. Thereby, the effect of subMICs oxacillin on phenol-soluble modulins and RNAIII have been studied [27, 28], showing a decrease in mRNA level in both virulence determinants in USA300 strain. However, increase in spa and lukE mRNA levels under exposure to subMICs of oxacillin, cephalotin and penicillin were reported by Subrt et al. [29]. Moreover, Rasigade et al. revealed that subMIC of oxacillin, moxifloxacin and linezolid led to increase fnbA/B mRNA levels [30]. Collectively all this studies add more evidence that, that suboptimal concentrations of different antibiotics modulate the expression of virulence factors in S. aureus, and therefore may exert an influence over the pathogenesis of infection [23]. Nevertheless, limited data exist showing the effect of oxacillin in MSSA clinical strains. Thereby, the aim of the present study was to investigate the impact of sub-inhibitory concentrations (subMICs) of oxacillin on the agr operon expression, as a key element of virulence regulatory network, in a collection of S. aureus clinical strains belonging to the main clonal complexes related with invasive diseases. In addition, we analyzed whether the presence of mutations in agr locus could be associated with differences in agr gene expression.

In the current study, we found that sub-lethal concentrations of oxacillin have an impact on the levels of agr gene expression in a selection of clinical MSSA and MRSA strains belonging to the main clonal complexes that were associated with invasive disease in Spanish hospitals, namely CC5, CC30, CC15, CC45 and CC8 [2‐4]. These differences between the presence and absence of oxacillin subMICs resulted in increases or decreases in the levels of gene expression that were strain-dependent. In addition, we observed that the strains generally shared different mutations in agr locus according to the CC and that these mutations had an impact on the expression values of agr.

A recent review has shown that sub-lethal concentrations of different antibiotics such as beta-lactams are able to modulate the expression of virulence genes [23]. Our study shows a strain-dependent effect of oxacillin subMICs on the agr operon expression measured as transcriptional changes in the RNAIII and /or agrA mRNA in the clinical strains included in this study. We observed an increase relative to the control condition without oxacillin in 11/14 S. aureus clinical strains being statically significant for 5 of them. Additionally, strains with low expression levels under exposure to oxacillin subMICs were also detected. Because of the agr operon plays an important role in S. aureus virulence the exposure to subMICs of oxacillin could have significant consequences on the pathogenesis of infection. The initiation of transcription of RNAIII strictly relies upon the expression of agrA, in a RNAII-dependent (agrBDCA) manner [8, 9, 11]. Our data support these observations since the transcription of agrA was observed in all strains with RNAIII expression. However, in the presence of oxacillin subMICs, most strains showed the same direction of change in expression values for both genes, except for 4 strains that showed divergent expression values in RNAIII and agrA, although not statistically significant. This unexpected result could be due to other factors affecting on agrA expression. It is important to take into account the wide network of global (sarA, sarU, sarX, codY) and other virulence regulators (mgrA, sigB, saeSR) that have a direct or indirect effect on the agr [35, 36], and specifically on the P2 and P3 promoters to which agrA binds [37], or on agrC-agrA interaction [38]. When we stratified the expression results by CC, we observed higher expression values of agr locus in presence of oxacillin subMICs than in absence of this antimicrobial agent for both CC15 and CC45. However, the relatively small number of strains affords a substantial chance for statistical bias and does not allow performing proper multivariate analyses. On the other hand, methicillin resistance has been associated with a decrease in RNAIII expression under exposure to suboptimal concentrations of different antibiotics [28, 34, 39]. Our results do not seem to support this hypothesis, and only one MRSA strain (SA_170006) showed a reduction in the expression values relative to the control. However, taking into account that from the 4 MRSA (mecA positive) strains, only one is defined as an MRSA according to CLSI breakpoints (SA_170006) [40]. This subtle difference could be impacting on the correlation MSSA/MRSA with RNAIII/agrA expression. Indeed, previous studies [28, 34] suggested that the expression of mecA and either its potential to subtly affect peptidoglycan structure or its interaction with other cell wall–associated proteins prevent the auto-inducing peptide (AIP) from being detected. These results in an unresponsive agr system that could be directly attributed to induced levels of mecA expression because of RNAIII expression was repressed as mecA expression increased.

Although the role of agr system for pathogenesis in animal model is well established [41, 42] and agr-defective mutants have shown an attenuated virulence and agr blocking agents exhibit anti-infective properties in experimental S. aureus infections [43], several studies have reported that agr defective mutants are frequently recovered from patients with bacteremia, where the mutants are associated with persistent infection and poor outcome [9, 19‐21]. According to previous reports we observed that 13/14 strains harbored both synonymous and non-synonymous mutations throughout the agr operon, especially in agrC and agrA [9, 17, 18]. Moreover, we have observed that while the genotypic analysis revealed similar mutations patterns accordingly to CC, the agr expression was clearly strain-dependent. Some of these mutations were associated to changes in agr gene expression in strains with and without a specific mutation. Deletions found in SA_180015 (CC8) and SA_80004 (CC5) in agrC were related with low expression levels and might be responsible for reduced gene expression in these strains. Both these deletions have previously been detected in strains with dysfunctional agr [9, 17, 18]. Strains of CC5 exhibited the most consistent agr gene expression although this group had the most divergent agr sequences within any CC, all mutations detected in this CC were associated with decreases expression values. Finally, our observations showed that CC30 exhibited low levels of agr gene expression with the presence of a previously identified mutation in agrC [18, 44]. We recognize that the attempt to correlate RNAIII / agrA expression with mutations within the agr operon without complementation experiments is problematic, since in most cases the sample size for any given mutation was one, making it difficult to draw strong correlations between mutations and agr expression. Even so, these results, collectively, indicate that mutations in agr have an impact on agr expression and could be correlated with levels of agr gene expression for strains belonging to the same CC. Nevertheless, although different studies have shown that mutations in agr-defective strains do not persist in natural populations and that inactivating mutations have a relatively short life, other limitation of this study is that we do not have the results from sequencing of the agr operon after exposure to antibiotic, and therefore, we do not know the influence that this selective pressure could exert on mutations in the agr locus. Furthermore, it is possible that mutations detected in the agr system may have an effect not only on the expression on the agr locus itself, but also on other virulence genes regulated by agr. However, although these results could be confirmed by complementation experiments with the wild type gene, we cannot ignore the potential effect that other regulators may also have on agr operon expression independently of the mutations detected [24, 29]. Our findings offer a valuable starting point for further studies that analyze gene expression changes induced by oxacillin subMICs from a whole transcriptomic approach would help to provide a more accurate overview of genes and regulators that compose the virulome framework in S. aureus and that may be affected under this condition.

We conclude that exposure to oxacillin subMICs have a stimulating or depressing effect on expression of the agr system for majority of the strains of S. aureus in our collection. In addition, the mutations we detected in the agr operon follow a common pattern among strains belonging to the same CC and could be responsible of changes in the agr expression. Consequently, the virulence mechanisms involved in the pathogenesis of infection could be affected. However, inpatient data supporting staphylococcal virulence modulation by antibiotics are rather frail, thus relevant clinical trials are necessary to extrapolate this finding to patient outcomes.