key: cord-0754347-9fwdqu6n
authors: Bongiorno, Dafne; Lazzaro, Lorenzo Mattia; Stefani, Stefania; Campanile, Floriana
title: In Vitro Activity of Dalbavancin against Refractory Multidrug-Resistant (MDR) Staphylococcus aureus Isolates
date: 2020-12-03
journal: Antibiotics (Basel)
DOI: 10.3390/antibiotics9120865
sha: 49efb96e939b8c313b086388b908b54223954ba2
doc_id: 754347
cord_uid: 9fwdqu6n

The high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infections, always treated with vancomycin and daptomycin, has led to the emergence of vancomycin-intermediate (VISA), heteroresistant vancomycin-intermediate (hVISA) and daptomycin non-susceptible (DNS) S. aureus. Even if glycopeptides and daptomycin remain the keystone for treatment of resistant S. aureus, the need for alternative therapies that target MRSA has now become imperative. The in vitro antibacterial and bactericidal activity of dalbavancin was evaluated against clinically relevant S. aureus showing raised antibiotic resistance levels, from methicillin-susceptible to Multidrug-Resistant (MDR) MRSA, including hVISA, DNS and rifampicin-resistant (RIF-R) strains. A total of 124 S. aureus strains were tested for dalbavancin susceptibility, by the broth microdilution method. Two VISA and 2 hVISA reference strains, as well as a vancomycin-resistant (VRSA) reference strain and a methicillin-susceptible Staphylococcus aureus (MSSA) reference strain, were included as controls. Time–kill curves were assayed to assess bactericidal activity. Dalbavancin demonstrated excellent in vitro antibacterial and bactericidal activity against all S. aureus resistance classes, including hVISA and DNS isolates. The RIF-R strains showed the highest percentage of isolates with non-susceptibility, reflecting the correlation between rpoB mutations and VISA/hVISA emergence. Our observations suggest that dalbavancin can be considered as an effective alternative for the management of severe MRSA infections also sustained by refractory phenotypes.

Reduced susceptibility to glycopeptides in Staphylococcus aureus poses a great threat to antimicrobial chemotherapy worldwide, and particularly in methicillin-resistant S. aureus (MRSA), it is seriously challenging to the therapeutic field. Vancomycin-intermediate S. aureus strains with homogeneous (VISA) or heterogeneous (hVISA) phenotypes are increasingly being reported all over the world, exposing significant controversies on the present and future role of vancomycin and teicoplanin in the treatment of severe infections sustained by hVISA-MRSA isolates [1] . In these strains, often with vancomycin minimum inhibitory concentrations (MICs) in the 1-2 mg/L range, this reduced susceptibility has been attributed to various cell-wall abnormalities, evolving in a multistep fashion. Even if the genetic occurrence at the base of the hVISA phenotype has not yet been established, these strains often harbor modifications in graSR, vraSR and walKR two-component system (TCS) regulatory genes, and RNA polymerase beta subunit (rpoB) encoding genes [2] .

In this scenario, daptomycin is always used as an alternative option for the treatment of infections caused by S. aureus, with a potent bactericidal activity against MRSA, excluding VISA and hVISA The selected RIF-R strains showed the highest percentage of isolates with non-susceptibility to dalbavancin (n.9, 18%), although with MIC values between 0.25 and 0.5mg/L. These strains showed nearly all a hVISA phenotype and belonged to the most spread Italian clone ST228-SCCmec I-spa-type t001/t041 clone (7 out of 9), and to ST5-SCCmec II-spa-type t002 clone (1 out of 9), the same Antibiotics 2020, 9, 865 3 of 8 as the VISA/hVISA controls (Mu50/Mu3) included in the study, with which they share common characteristics including a thickened cell wall [10] . Only one RIF-R/VSSA strain, showing a one-fold higher dalbavancin MIC value, belonged to ST8-SCCmec IV/spa-type t008 clone usually spread in the community setting. In the interpretation of this result, which deserves further insights, it should be taken into account that the rifampicin-resistant phenotype of these strains occurred from different mutations in the gene encoding RNA-polymerase (rpoB), whose alteration has been associated with multiresistant daptomycin, vancomycin and beta-lactams phenotypes [8, 9] .

The results of time-kill curve assays provided a dynamic picture of the bactericidal activity against three model strains: the analyses were conducted with different concentrations of dalbavancin, equal to the MIC values 2, 4 and 8 times higher than the MIC value, respectively.

Dalbavancin exerted a potent bactericidal activity against the HA-MRSA/VSSA strain belonging to the E-MRSA15-ST22-SCCmec-IVh spa-type t223 clone after 8h from the starting inoculum at concentrations of 0.12 and 0.24 mg/L. Dalbavancin concentrations of 0.03 and 0.06 mg/L were not sufficiently bactericidal, therefore bacterial growth increased over time, miming the antibiotic-free control ( Figure 1 ). mutations in the gene encoding RNA-polymerase (rpoB), whose alteration has been associated with multiresistant daptomycin, vancomycin and beta-lactams phenotypes [8, 9] . The results of time-kill curve assays provided a dynamic picture of the bactericidal activity against three model strains: the analyses were conducted with different concentrations of dalbavancin, equal to the MIC values 2, 4 and 8 times higher than the MIC value, respectively.

Dalbavancin exerted a potent bactericidal activity against the HA-MRSA/VSSA strain belonging to the E-MRSA15-ST22-SCCmec-IVh spa-type t223 clone after 8h from the starting inoculum at concentrations of 0.12 and 0.24 mg/L. Dalbavancin concentrations of 0.03 and 0.06 mg/L were not sufficiently bactericidal, therefore bacterial growth increased over time, miming the antibiotic-free control ( Figure 1 ).

T2 T4 T8 T24 Free 3 × 10 6 1 × 10 6 2 × 10 7 2 × 10 9 2 × 10 9 DAL 0.03 mg/L (1×) 2 × 10 6 3 × 10 6 6.3 × 10 6 1.4 × 10 8 1 × 10 9 DAL 0.06 mg/L (2×) 2 × 10 6 1.2 × 10 6 3.5 × 10 6 6 × 10 7 1 × 10 8 DAL 0.12 mg/L (4×) 4 × 10 6 9 × 10 4 8.8 × 10 4 3 × 10 3 1.7 × 10 3 DAL 0.25 mg/L (8×) 2 × 10 6 1 × 10 4 1.3 × 10 4 2 × 10 3 2.5 × 10 3 Against the RIF-R/hVISA strain belonging to ST228-SCCmec I spa-type t041, dalbavancin bactericidal activity was exhibited at the higher concentration assayed of 4 mg/L (8X MIC) at 8 and 24 h intervals, and a non-bactericidal reduction of only 1 log 10 at lower concentrations (1 and 2 mg/L), at 24 h ( Figure 2 ). 

Clone characterization by means of: ST-Sequence Type; SCCmec-Staphylococcal Cassette Chromosome mec; spa type-staphylococcal protein A.

Antibiotics 2020, 9, x 4 of 8 Against the RIF-R/hVISA strain belonging to ST228-SCCmec I spa-type t041, dalbavancin bactericidal activity was exhibited at the higher concentration assayed of 4 mg/L (8X MIC) at 8 and 24 h intervals, and a non-bactericidal reduction of only 1 log10 at lower concentrations (1 and 2 mg/L), at 24 h ( Figure 2 ). 1 × 10 6 9.6 × 10 6 7.6 × 10 6 5 × 10 6 9.7 × 10 5 DAL 1 mg/L (2×) 1.1 × 10 6 8.3 × 10 6 6.9 × 10 6 5.3 × 10 6 2.5 × 10 5 DAL 2 mg/L (4×) 1.1 × 10 6 6.9 × 10 6 8.9 × 10 6 1.7 × 10 6 2 × 10 5 DAL 4 mg/L (8×) 1.2 × 10 6 8.5 × 10 6 1 × 10 5 3 × 10 3 2.2 × 10 3 The time-kill curve assay showed a stronger bactericidal activity of dalbavancin against the DNS strain belonging to ST1-SCCmec IV spa-type t386, at 24 h and at all the concentrations assayed ( Figure  3 ). The bacterial growth considerably decreased from 3-4 log10, in the presence of 1X-2X and 4X MIC, to 5 log10 with the highest dalbavancin concentration (16 mg/L-8X MIC). In this clone, the bactericidal activity was exhibited only after 24 h from the starting inoculum. These data therefore deserve an in-depth analysis, aimed at understanding if a correlation between daptomycin nonsusceptibility and delayed but conclusive bactericidal activity is conceivable [11, 12] . The time-kill curve assay showed a stronger bactericidal activity of dalbavancin against the DNS strain belonging to ST1-SCCmec IV spa-type t386, at 24 h and at all the concentrations assayed ( Figure 3 ). The bacterial growth considerably decreased from 3-4 log 10 , in the presence of 1X-2X and 4X MIC, to 5 log 10 with the highest dalbavancin concentration (16 mg/L-8X MIC). In this clone, the bactericidal activity was exhibited only after 24 h from the starting inoculum. These data therefore deserve an in-depth analysis, aimed at understanding if a correlation between daptomycin non-susceptibility and delayed but conclusive bactericidal activity is conceivable [11, 12] . 

A total of 124 strains of S. aureus selected from a large collection of isolates from 63 centers distributed throughout Italy-as part of the multicentre study CoSA-AMCLI 2012 [7, 8] -were tested for susceptibility to dalbavancin according to standard methods [13, 14] . Characterization of VSSA/hVISA/VISA phenotypes were also assessed by population analysis assay (PAP/AUC), following previously published procedures [15] . All isolates were already genetically characterized by PFGE, SCCmec-typing, Multilocus Sequence Typing (MLST-https://pubmlst.org/organisms), spatyping (https://spaserver.ridom.de), presence of pvl gene and evaluation of rpoB mutations responsible for the RIF-R phenotype, as previously published [7, 8] .

In particular, the sample consisted of n. 23 MSSA; n. 24 MRSA/VSSA; n. 22 MRSA/hVISA; n.5 DNS/MRSA and a selected sample n. 50 RIF-R/MRSA. Two VISA (Mu50 and NRS402) and two hVISA (Mu3 and NRS22) strains, a vancomycin-resistant (VRS1) strain and an MSSA (ATCC 29213) strain were included as controls.

Dalbavancin in vitro activity was tested by a microdilution method. For the preparation of dalbavancin, 100 mg of powder was completely dissolved in 10 mL Dimethyl sulfoxide (DMSO Sigma-Aldrich-Merck KGaA, Darmstadt, Germany). Microtiter plates were prepared with 100 µl of Mueller Hinton Broth, Cation-adjusted (CAMHB, NutriSelect™ Plus, Becton Dickinson, Franklin Lakes, NJ, USA), in which 100 µl of antibiotic were added at scalar concentrations starting from an initial concentration of 8 mg/L. For dalbavancin, 0.002% polysorbate-80 (Tween 80) (Merck, Darmstadt, Germany) was previously added to the broth CAMHB medium [13, 14] . A standard 1,00E+00 

A total of 124 strains of S. aureus selected from a large collection of isolates from 63 centers distributed throughout Italy-as part of the multicentre study CoSA-AMCLI 2012 [7, 8] -were tested for susceptibility to dalbavancin according to standard methods [13, 14] . Characterization of VSSA/hVISA/VISA phenotypes were also assessed by population analysis assay (PAP/AUC), following previously published procedures [15] . All isolates were already genetically characterized by PFGE, SCCmec-typing, Multilocus Sequence Typing (MLST-https://pubmlst.org/organisms), spa-typing (https://spaserver.ridom.de), presence of pvl gene and evaluation of rpoB mutations responsible for the RIF-R phenotype, as previously published [7, 8] .

In particular, the sample consisted of n. 23 MSSA; n. 24 MRSA/VSSA; n. 22 MRSA/hVISA; n. 5 DNS/MRSA and a selected sample n. 50 RIF-R/MRSA. Two VISA (Mu50 and NRS402) and two hVISA (Mu3 and NRS22) strains, a vancomycin-resistant (VRS1) strain and an MSSA (ATCC 29213) strain were included as controls.

Dalbavancin in vitro activity was tested by a microdilution method. For the preparation of dalbavancin, 100 mg of powder was completely dissolved in 10 mL Dimethyl sulfoxide (DMSO Sigma-Aldrich-Merck KGaA, Darmstadt, Germany). Microtiter plates were prepared with 100 µl of Mueller Hinton Broth, Cation-adjusted (CAMHB, NutriSelect™ Plus, Becton Dickinson, Franklin Lakes, NJ, USA), in which 100 µl of antibiotic were added at scalar concentrations starting from an initial concentration of 8 mg/L. For dalbavancin, 0.002% polysorbate-80 (Tween 80) (Merck, Darmstadt, Germany) was previously added to the broth CAMHB medium [13, 14] . A standard inoculum of 0.5 McFarland was used as described by the CLSI M07-A10 document [16] and the results interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoint criteria [10] .

The bactericidal activity of dalbavancin was evaluated by time-kill curves, according to standard procedures [17] . Briefly, the experiments were performed in duplicate in 20 mL tubes containing Cation-adjusted Mueller-Hinton broth (CAMHB), NutriSelect™ Plus, Becton Dickinson, Franklin Lakes, NJ, USA) using a starting inoculum of 10 5 -10 6 CFU/mL, with dalbavancin (1X, 2X, 4X and 8X MIC) supplemented with 0.002% Tween 80. Additionally, 100 µl serial dilutions were plated in Mueller Hinton Agar 2 (MH agar 2, NutriSelect™ Plus, Becton Dickinson, Franklin Lakes, NJ, USA), in different time intervals T0-T2-T4-T8 and T24 (0, 2, 4, 8 and 24 h) and after overnight incubation at 37 • C the grown colonies were counted. All experiments were repeated at least three times, and results of a representative experiment are presented. Killing curves were constructed by plotting the log 10 CFU ml−1 versus time over 24 h, and the change in bacterial concentration was determined. Data points are averages from duplicate viable count determinations (CFU/mL) within an experiment. Bactericidal activity was defined as a reduction of 99.9% (≥3 log10) of the total number of CFU/mL of the starting inoculum (10 5 -10 6 CFU/mL), after 24 h of exposure with the antibiotic. Bacteriostatic activity was defined as maintenance of the starting inoculum or a reduction of less than 99.9% (<3 log10) of the total number of CFU/mL of the starting inoculum [17].

Our study underlined the excellent in vitro antibacterial and bactericidal activity of dalbavancin against representative strains belonging to the major epidemiologically diffused phenotypes, including MRSA/hVISA, DNS and RIF-R strains, confirming the stability of its potency against S. aureus isolates [18] . MRSA strains showing heteroresistance to vancomycin (hVISA), often with vancomycin MICs in the 1-2 mg/L range, are increasingly being reported and a systematic review of the literature on hVISA reported that patients infected with these organisms had a 2.37-fold greater failure rate compared to those infected with fully susceptible (VSSA) organisms [19] . Consequently, significant controversy exists regarding the current and future roles of vancomycin and teicoplanin in the treatment of serious hVISA-MRSA infections. Our data corroborate with what has been recently reported by other authors, reinforcing the hypothesis that dalbavancin may be a valuable agent against problematic pathogens [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . The interpretation of the slightly higher rate of dalbavancin non-susceptibility among RIF-R/hVISA isolates needs further investigations, although it is possible to assume that the presence of rpoB mutations in these strains [8] , already associated with the emergence of vancomycin-intermediate resistance, may affect the antimicrobial activity. The major refractoriness of RIF-R/hVISA and DNS strains is also corroborated by other expression studies conducted on VISA and hVISA, in which the drastic change in the cell transcriptional profile was demonstrated to be mainly associated to rpoB mutations [21] . Nonetheless, it is to be mentioned that the dalbavancin MICs of these strains were only one/two dilutions above the EUCAST breakpoint, and that many in vitro and in vivo preclinical studies predicted that the pharmacokinetic/pharmacodynamic (PK/PD) profiles usually persist above the MIC level [22] . Our observations suggest that dalbavancin will be considered an excellent therapeutic alternative for the management of severe S. aureus infections sustained by MDR strains sharing diverse and increasing behaviors of antibiotic resistance, also belonging to most refractory MRSA phenotypes.

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The authors gratefully acknowledge Angelini S.p.A for the partial financial support to the project.

Antibiotics 2020, 9, 865 7 of 8

The authors declare no conflict of interest.