ABSTRACT
The activities of four oxadiazoles were investigated with 210 methicillin-resistant Staphylococcus aureus (MRSA) strains. MIC50 and MIC90 values of 1 to 2 and 4 μg/ml, respectively, were observed. We also evaluated the activity of oxadiazole ND-421 against other staphylococci and enterococci and in the presence of oxacillin for selected MRSA strains. The MIC for ND-421 is lowered severalfold in combination with oxacillin, as they synergize. The MIC90 of ND-421 against vancomycin-resistant enterococci is ≤1 μg/ml.
TEXT
Staphylococcus aureus is a leading cause of health care-related infections, with more than 80,000 severe methicillin-resistant S. aureus (MRSA) infections per year in the United States alone (1). The oxadiazoles are a new class of antibacterials with activity against Gram-positive bacteria; they are bactericidal and target cell-wall biosynthesis (2, 3). The oxadiazoles emerged from in silico screening against the structure of penicillin-binding protein (PBP) 2a. PBPs are involved in bacterial cell-wall biosynthesis. There are four PBPs (PBP1 to PBP4) in S. aureus, with MRSA having acquired the additional PBP2a, which confers resistance to β-lactam antibiotics (4, 5). The oxadiazoles bind to PBP2a, and likely to other PBPs, as activity is observed against S. aureus strains without PBP2a (2). The structure-activity relationship of the oxadiazoles revealed that phenol, indole, or pyrazole rings on the left-hand side of the molecule were tolerated (2, 6). The lead oxadiazole, ND-421, shows efficacy in murine models of MRSA infection, similar or superior to that of linezolid (3, 7). In the present report, we selected four oxadiazoles (Fig. 1), including ND-421 (compound 3), and investigated their activities with 210 MRSA strains (108 strains from the United States and 102 from Spain). This collection included 54 MRSA strains with relevant mechanisms of resistance (vancomycin-, linezolid-, and mecC-related) (Table 1). The Spanish collection of 102 strains was obtained from different institutions and origins, including different clonal complexes widely distributed in Spanish hospitals. The remaining 108 strains in the US were obtained from BEI resources, including different clones and staphylococcal cassette chromosome mec element (SCCmec) types.
Structures of the four oxadiazoles used in this study.
MIC values (in μg/ml) for antibacterial agents against 210 MRSA strains and S. aureus ATCC 29213 and for selected MRSA strains with different mechanisms of resistance
The MIC values of oxadiazoles 1 to 4 were determined by the microdilution method (8). S. aureus ATCC 29213 was used as the reference and quality control strain. Ceftaroline (9) and linezolid were included for comparison of antimicrobial activities with oxadiazoles. Table 1 shows the range of MIC values determined for the 210 MRSA strains (including the 54 strains with specific mechanisms of resistance), as well as the MIC50 and MIC90 values of these antibacterials. For oxadiazoles 1, 2, and 3, the MIC50 and MIC90 values were 2 and 4 μg/ml, respectively. Oxadiazole 1 contains a phenol ring, while 2 and 3 have the phenol group replaced by an indole ring (Fig. 1). Oxadiazole 4, containing a pyrazole ring, had MIC50 and MIC90 values of 1 and 4 μg/ml, respectively. A few strains showed higher MIC values for some oxadiazoles, as follows: 16 μg/ml (four strains), 32 (one strain), or >32 μg/ml (six strains). The MIC90 values of the oxadiazoles 1 to 4 were within 2-fold of those of linezolid and within 2- to 4-fold of that of ceftaroline (Table 1). Ceftaroline and the oxadiazoles are bactericidal, while linezolid is bacteriostatic. All of the mecC-mediated MRSA strains (which lack PBP2a but harbor PBP2c) (10), exhibited low MIC values for the oxadiazoles tested (MIC ≤ 4 μg/ml). For linezolid-resistant MRSA strains (LRSA), the MIC values for the oxadiazoles (2 to 8 μg/ml) were lower than those for linezolid (8 to >16 μg/ml). The MIC range for compound 3 (ND-421) in vancomycin-resistant MRSA isolates was 1 to 4 μg/ml, whereas vancomycin had an MIC of >32 μg/ml (Table 1).
The sequences of pbp1, pbp2, mecA, pbp3, and pbp4 genes in four selected MRSA isolates that showed high MIC values (>32 μg/ml) for oxadiazoles 2 and 4 were analyzed by PCR and sequencing (Table 2) (11, 12). The nucleotide sequences and their translation into amino-acid sequences were aligned and compared with reference sequences available from the NCBI database, namely, methicillin-susceptible Staphylococcus aureus (MSSA) strain ATCC 25923 for pbp1, pbp2, pbp3, and pbp4 (GenBank accession number CP009361.1) and MRSA strain N315 for mecA (GenBank accession number BA000018.3). The MRSA clonal complex 398 (CC398) strain (with an MIC of >32 μg/ml for compound 4) had four amino-acid changes in PBP1 (F465L, D480E, D662N, and S664T), four in PBP2 (D270E, T439V, D489E, and T691A), one in PBP2a (G246E), and one in PBP4 (E398A), but none in PBP3 (Table 2). No amino-acid changes in PBP1 and PBP2a were detected in the three MRSA CC5 strains (one strain with an MIC of >32 μg/ml for oxadiazole 4, and two strains for oxadiazole 2), although changes were identified in PBP2 (A285P ± A58V), PBP3 (K504R and D563E), and PBP4 (T189S ± A25T). The relationship between the mutations detected in S. aureus PBPs and oxadiazole resistance is not known; however, increased MIC is not seen for ND-421, indicating that replacement of the indole in ND-421 with the pyrazole ring in oxadiazole 4 and replacement of trifluoromethyl with fluorine in oxadiazole 2 increases resistance.
Mutations detected in PBPs in MRSA strains with high MIC values for oxadiazoles (>32 μg/ml)
ND-421 synergized in vitro with β-lactam antibiotics, as well as with oxacillin, in a murine MRSA infection model (7). In this study, the MIC values of ND-421 in the absence and presence of oxacillin at 1/2, 1/4, and 1/8 MIC were determined with 11 selected MRSA strains that displayed high MIC values for this oxadiazole (five isolates with MICs of 8 μg/ml and six isolates with MICs of 4 μg/ml), using the microdilution method (8). The MICs of oxacillin ranged from 32 to >512 μg/ml. In three isolates with very high oxacillin MICs (≥512 μg/ml), we tested ND-421 in the presence of oxacillin at 1/4 and 1/8 MIC (but not at 1/2 MIC). The addition of oxacillin lowered the MICs for ND-421 to ≤0.03 to 2 μg/ml (with 1/2 and 1/4 MIC of oxacillin) or to ≤0.03 to 4 μg/ml (with 1/8 MIC of oxacillin). The ND-421 MIC values decreased from 8 μg/ml to 0.25 to 1 μg/ml in the presence of 1/4 MIC of oxacillin (Table 3). In the presence of β-lactam antibiotics, cooperation between the transpeptidase domain of PBP2a (inhibited by the oxadiazoles) and the transglycosylase domain of PBP2 (inhibited by oxacillin) is required for cell-wall synthesis (13). We hypothesize that this could be the basis of synergy between ND-421 and oxacillin.
The effect of oxacillin on MIC values for ND-421 in selected MRSA strains
The susceptibility of ND-421 to 55 additional Gram-positive isolates (32 non-S. aureus staphylococci and 23 vancomycin-resistant enterococci [VRE]) was evaluated (Table 4). VRE strains results in 20,000 annual drug-resistant enterococcal infections in the United States alone, resulting in 1,300 deaths (1). The MIC values in all cases were ≤2 μg/ml, with the exception of five methicillin-resistant (MR) coagulase-negative staphylococci strains, which showed MICs of >8 μg/ml (MR-S. epidermidis and MR-S. haemolyticus), and one Enterococcus faecalis strain carrying the vanB2 gene, with an MIC of 4 μg/ml. The range of MICs for ND-421 exhibited by the 11 S. pseudintermedius strains was 0.25 to 2 μg/ml, and the range for most of the 15 vanA/vanB1/vanB2-carrying enterococci tested was ≤0.25 to 1 μg/ml (with only one exception, with an MIC of 4 μg/ml); the eight Enterococcus gallinarum and Enterococcus casseliflavus isolates with intrinsic vancomycin resistance (carrying vanC1 and vanC2 genes, respectively) also showed an MIC range of ≤0.25 to 1 μg/ml (Table 4). In particular, the activity of ND-421 against VRE is of note. Linezolid has been reported to show MIC90 values of 2 μg/ml for E. faecalis carrying vanA, E. faecium carrying vanA, and E. faecium carrying vanB (14). According to these results, ND-421, which was initially designed for PBP2a of MRSA, is also active in other Gram-positive bacteria, such as VRE, supporting the hypothesis that it targets other PBPs. In any case, regardless of the target, the result is the inhibition of cell-wall biosynthesis.
Range of MIC values for the oxadiazole ND-421 for Staphylococcus (non-S. aureus) and vancomycin-resistant Enterococcusa strains
In summary, we show that the oxadiazoles have MIC90 values of 4 μg/ml in 210 strains of MRSA. The MIC values for ND-421 are consistently lowered when used in combination with oxacillin. The compound ND-421 has shown an MIC of ≤1 μg/ml with most of VRE tested.
ACKNOWLEDGMENTS
The work with 102 MRSA and other Gram-positive strains from Spain (S. Ceballos and C. Torres) was funded by project SAF2016-76571-R from the Agencia Estatal de Investigación (AEI) of Spain and the Fondo Europeo de Desarrollo Regional (FEDER). The work with 108 MRSA strains from the United States (C. Kim, D. Ding, S. Mobashery, and M. Chang) was supported by a grant from the National Institutes of Health (AI90818). S. Ceballos has a predoctoral fellowship from the University of La Rioja (Spain).
FOOTNOTES
- Received 7 March 2018.
- Returned for modification 28 March 2018.
- Accepted 26 May 2018.
- Accepted manuscript posted online 4 June 2018.
- Copyright © 2018 American Society for Microbiology.