Clindamycin suppresses virulence expression in inducible clindamycin-resistant Staphylococcus aureus strains

Clindamycin is a protein synthesis inhibitory agent that has the ability to suppress the expression of virulence factors in Staphylococcus aureus. Recent guidelines recommend the use of clindamycin for the treatment of toxin-mediated infections. Clindamycin modulates virulence expression at sub-inhibitory concentrations (sub-MICs) in clindamycin-susceptible S. aureus strains but previous report shown that this effect was supressed for constitutive clindamycin resistant strains. However, no data are currently available on the impact of clindamycin at sub-MICs on the virulence of inducible clindamycin-resistant S. aureus strains. Here, we show that sub-MICs of clindamycin decrease Panton–Valentine leucocidin, toxic-shock-staphylococcal toxin (TSST-1) and alpha-haemolysin (Hla) expression in six inducible clindamycin-resistant isolates cultivated in vitro in CCY medium. These results suggest that the clindamycin anti-toxin effect is retained for inducible clindamycin-resistant S. aureus isolates; therefore, its usage should be considered within the treatment regimen of toxin related infections for inducible clindamycin-resistant S. aureus.


Introduction
Clindamycin is a protein synthesis inhibitory agent that has the ability to suppress the expression of virulence factors in Staphylococcus aureus at sub-inhibitory concentrations (sub-MICs). Indeed, several studies have reported the ability of clindamycin at sub-MICs to decrease the production of Panton-Valentine leucocidin (PVL), toxic-shock-staphylococcal toxin (TSST-1) or alpha-haemolysin (Hla) [1][2][3][4][5][6]. Therefore, recent guidelines recommend the use of clindamycin for the treatment of toxin-mediated infections (e.g., toxic shock syndrome and necrotizing pneumonia) [7]. This modulation of virulence expression by clindamycin occurs in clindamycin-susceptible S. aureus strains but is abolished in constitutive clindamycin-resistant strains [5].
Clindamycin resistance results from enzymatic methylation of the antibiotic binding site in the 50S ribosomal subunit (23S rRNA). The responsible methylase encoded by the erm gene can phenotypically result in a MLS B (macrolides, lincosamides, and group B streptogramins) resistance constitutive (in vitro resistance to all MLS B ) or inducible (a positive "D-test" in agar diffusion method: resistance to erythromycin, susceptible to lincosamides with a flattening of the inhibition zone in regard to erythromycin disc) [8]. Contrary to constitutive clindamycinresistant strains, no data are currently available on the impact of clindamycin at sub-MICs on the virulence of inducible clindamycin-resistant S. aureus strains. Here, we have shown in a selection of inducible clindamycinresistant S. aureus strains that clindamycin maintains its anti-toxin effect at sub-inhibitory concentrations.

Bacterial strain selection
One hundred eighty S. aureus strains from the French National Reference Centre of Staphylococci were genotyped by the S. aureus Genotyping Kit 2.0 (Clondiag Alere ® Jena, Germany), and the major macrolide resistance genes were recorded (ermA/B/C/T, linA, msrA, vatA/B, vgaA, vgb, and mbpB). The mecA gene responsible for methicillin resistance and the lukS-PVL and tst genes encoding for PVL and TSST-1, respectively, were also recorded. Macrolide phenotypic resistance was determined by the agar diffusion disk assay using antibiotic discs (erythromycin, lincomycin, and quinupristin-dalfopristin) (I2A, Montpellier, France) and Mueller-Hinton E agar medium (bioMérieux, Marcy l'Etoile, France) according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations [9]. Inducible MLS B resistance was detected phenotypically by an inhibition zone between the erythromycin disk and the lincomycin disks indicating a positive D-test [8]. S. aureus strains exhibiting inducible MLS B resistance were selected, and the minimal inhibitory concentrations (MIC) of clindamycin (provided by Pfizer, Amboise, France) were determined by micro-dilution method using casein hydrolysate-yeast extract medium (CCY) broth medium. The S. aureus strain ATCC 29213 was used for clindamycin MIC determination in both CCY (homemade) and Mueller-Hinton (bioMérieux, Marcyl'Etoile, France) broths.

Staphylococcus aureus culture conditions and toxin quantification
Since toxin production in Muller Hinton broth medium is very low, experiments were performed in CCY broth medium. Briefly, selected S. aureus strains were cultured overnight on Trypticase blood agar plates. Colonies were resuspended in CCY broth to a 0.5 McFarland adjusted turbidity. Cultures were performed at 37 °C with gyratory shaking (180 rpm). When the optical density reached a turbidity of a 2 McFarland (6 × 10 8 CFU/mL), clindamycin was added to the cultures to the final concentrations of 1/2 MIC, 1/4 MIC, and 1/8 MIC. Cultures with or without clindamycin (growth control) were incubated at 37 °C with shaking for 6 h (180 rpm) [3,10]. After the incubation time, culture supernatants were collected by centrifugation at 10,000g for 15 min and used for toxin quantification. The PVL, Hla and TSST-1 levels in the supernatant were quantified using a specific ELISA assay as previously described [3], using ELISA kit provided by GlaxoSmithKline, Brentford, United Kingdom, and TSST-1 antibodies provided by Toxin technology, Sarasota, FL, USA. Percentages of toxin release for each condition were calculated related to growth control without antibiotic according to formula: % of toxin release = test with antibiotic growth control without antibiotic × 100.

Inducible MLS B resistance in S. aureus strains and the MIC of clindamycin
Among the 180 strains tested, 78 were methicillin-susceptible S. aureus (MSSA), and 102 were methicillinresistant S. aureus (MRSA). Among these 180 strains, 112 strains harboured at least one gene encoded macrolide resistance and 68 strains harboured none. Ninety-two strains harboured only one macrolide resistance gene: 28 strains harboured ermA, 2 strains harboured ermB, 40 strains harboured ermC, 9 strains harboured ermT (all 9 belonging to the clonal complex 398), 9 strains harboured vgaA, 3 strains harboured msrA and one strain harboured linA. Twenty strains contained two resistance genes: 17 strains contained msrA and mbpB, one strain contained ermC and linA, one strain contained ermC and vgaA and one strain contained msrA and vgaA.

Effect of clindamycin on PVL expression and release in MLS B inducible S. aureus strains
According to culture condition, means of PVL measurements (two determinations) in growth control supernatant were equal to 682.43, 327.30 and 161.89 ng/ mL for ST2015-0940, ST2015-0934 and ST2017-0018 respectively.
Of the six S. aureus strains tested, three stains carried the lukS-PVL gene. For these three strains, the sub-MICs of clindamycin significantly decreased PVL release compared to that of the growth control (without antibiotics) (Fig. 1b). PVL concentrations, expressed as percentages of the growth control levels, in the supernatants treated with clindamycin at sub-MICs ranged from 10

Effect of clindamycin on TSST-1 expression and release in MLS B inducible S. aureus strains
Of the six S. aureus strains tested, two carried the tst gene and exhibited variable TSST-1 expression in comparison to that of the growth control: 251.04 ng/mL for ST2015-0773 and 2.49 ng/mL for ST2015-1098. The sub-MICs of clindamycin induced a dramatic decrease of TSST-1 release for ST2015-0773 with an undetectable TSST-1 expression at 1/2 MIC of clindamycin and an approximate 99% decrease at 1/8 MIC. For ST2015-1098, 1/2 MIC and 1/8 MIC of clindamycin resulted in an undetectable TSST-1 expression level (Fig. 1c).

Discussion and conclusions
Staphylococcus aureus produces many virulence factors that play an important role in the pathogenesis of infection, such as Hla [12], PVL [13,14] and TSST-1 [15]. Several studies have shown that clindamycin displays an anti-toxin in vitro effect at sub-inhibitory concentrations, inducing a decrease in toxin release [1][2][3][4][5][6]. Moreover, in a rabbit model of PVL+ CA-MRSA-induced necrotizing pneumonia, clindamycin was superior to vancomycin in reducing PVL induced tissue damage and the overall mortality rate, consistent with decreased PVL pulmonary concentrations [16]. Several case reports of necrotizing pneumonia and staphylococcal toxic shock highlight clindamycin's efficacy when used as an adjunctive antibiotic for the treatment of S. aureus related toxin infections [17]. Notably, a retrospective analysis of 92 cases of CA-MRSA necrotizing pneumonia, mainly due to PVLproducing strains, showed improved clinical outcomes in patients treated with antimicrobials inhibiting toxin production (linezolid or clindamycin) [18]. The underlying mechanism of clindamycin's anti-toxin effect is linked to the ribosome-blocking action in which the transcription of exoprotein and SaeRS global regulator system, are reduced [5,19]. This anti-toxin effect is abolished by the constitutive MLS B resistance mechanism [5] in macrolide and lincosamide-resistant strains.
Clindamycin resistance can be constitutive or inducible. S. aureus strains belonging to the latter category display, when tested, in vitro susceptibility to clindamycin. Nevertheless, in the first decade of the 21st century, treatment failure was reported in adult and paediatric populations when clindamycin was used for inducible MLS B S. aureus infections [8,20]. Consequently, the Clinical and Laboratory Standards Institute and EUCAST recommend searching and reporting for the inducible MLS B phenotype, but also stating that clindamycin may still be used for short-term therapy [9,20]. Nevertheless, no data are currently available on the anti-toxin effect of clindamycin in inducible MLS B -resistant S. aureus isolates. Here, we showed that sub-MICs of clindamycin efficiently resulted in a decrease in PVL, Hla and TSST-1 production for inducible MLS B S. aureus strains in vitro, regardless of the methicillin susceptibility. For one strain, ST2015-1098, displaying a very weak level of Hla expression in control condition (3.23 ng/mL), we detected suppressed Hla production only 1/8 MIC of clindamycin. We suspected a lack of sensitivity of the measure method which could have failed to detect Hla decreased expression at 1/2 and 1/4 MIC of clindamycin, explaining  6) to perform the t test to compare with growth control without antibiotic (= 100%) and calculate the 95% confidence interval of the average. *p < 0.05; **p < 0.01; ***p < 0.001. Lack of a chart bar means that the factor virulence release measure was below the detection limit (i.e., 0.75 ng/mL for Hla and 1.56 ng/mL for TSST-1)