Activity of Novel Benzoxazinorifamycins against Rifamycin-Resistant Streptococcus pyogenes

Serious gram-positive infections are frequently caused by Staphylococcus aureus or Streptococcus pyogenes. The rifamycin rifampin (RIF) is often used as part of the treatment for such infections, because it has potent bactericidal activity against both free-living and biofilm-associated gram-positive bacteria (13) and has favorable pharmacokinetics (2). Despite this, RIF's use as monotherapy has been limited because of treatment failure due to the emergence of rifamycin-resistant organisms (3, 4, 7, 8). Resistance results from amino acid substitutions within the conserved rifamycin resistance-determining region (RRDR) of the RpoB subunit of RNA polymerase, the target of rifamycins. Because of resistance emergence, RIF is used mainly as part of a multidrug treatment regimen in which the partner drug(s) is able to prevent outgrowth of resistant subpopulations.

A previous study described the activity of the benzoxazinorifamycins rifalazil (RFZ) and related new chemical entities (NCEs) against RIF-resistant S. aureus (10). The maximum MICs of the most potent NCEs were 2 to 4 μg/ml against the most rifamycin-resistant S. aureus strains. RIF and RFZ have MICs of >512 μg/ml against these same strains (10). Considering that a large proportion of serious gram-positive infections are caused by streptococci, we sought to determine in the present study whether NCEs had improved activity against rifamycin-resistant strains of S. pyogenes.

The two mutations conferring RIF resistance in clinical isolates of S. pyogenes (4, 7) and several mutations in other streptococci (1, 5, 6, 9, 12) all reside in the RRDR of RpoB in these organisms. S. pyogenes ATCC 19615 mutants with decreased susceptibility to RIF were selected for and characterized as described previously (10). The resistance frequencies of S. pyogenes for RIF were 7.0 × 10⁻⁸ (at 0.24 μg/ml), 6.0 × 10⁻⁸ (at 0.12 μg/ml), and 2.9 × 10⁻⁸ (at 0.06 μg/ml). Thirty-three colonies with decreased susceptibility to RIF were isolated, and oligonucleotide primers SPY-F and SPY-R (5′-AACCGTCGTATCCGTGCCGTTGGT-3′ and 5′-TGCCGTCGCAACAGCAACTACCTG-3′, respectively) were used to amplify and DNA sequence their RRDR regions, encoded by base pairs 1228 to 1884 (S. pyogenes MGAS315 coordinates; GenBank accession number NC 004070).

Seventeen discrete amino acid changes at 10 different positions within the S. pyogenes RRDR were identified (Table 1). Changes at H486, R489, and S491 correspond to positions that, when altered, confer the high levels of resistance to rifamycins in other bacteria (10). The H486N substitution was previously identified in a RIF-resistant clinical strain of S. pyogenes (7).

TABLE 1.

Susceptibility of S. pyogenes RIF-resistant mutants to RIF, RFZ, and select NCEs

S.pyogenes RpoB alterationa (no. isolated)	MIC (μg/ml)
	Rifampin	Rifalazil	NCE
			ABI-0043	ABI-0370	ABI-0418	ABI-0420
Noneb	0.031	0.001	0.000125	0.00025	0.00025	0.00025
S472P (1)	0.5	0.004	0.001	0.002	0.00025	0.004
D476Y (2)	2	0.031	0.016	0.031	0.002	0.002
D476G (1)	0.25	0.008	0.001	0.00025	0.002	0.0005
D476N (2)	0.5	0.004	0.00025	0.00025	0.00025	0.00025
H486N (2)	4	0.016	0.008	0.016	0.001	0.016
H486Y (2)	128	>128	0.063	0.031	0.031	0.031
R488H (2)	0.25	0.002	0.001	0.002	0.00025	0.002
R489H (2)	2	0.063	0.063	0.125	0.008	0.063
S491Y (2)	64	>128	0.25	0.125	1	0.25
S491F (1)	128	>128	0.25	0.25	1	0.5
A492P (1)	0.125	0.001	0.00025	0.00025	0.00025	0.00025
H514P (1)	0.125	0.0005	0.00025	0.00025	0.00025	0.00025
P524S (9)	0.25	0.002	0.0005	0.001	0.00025	0.002
P524T (1)	0.25	0.004	0.001	0.002	0.00025	0.002
P524L (1)	128	>128	0.031	0.031	0.004	0.031
I532L (2)	0.5	0.008	0.004	0.004	0.00025	0.004
I532F (1)	64	>128	0.125	0.125	0.063	0.125

^aThe changes denoted in boldface have been found in rifampin-resistant clinical strains of S. pyogenes (H486N only) and S. pneumoniae (all changes in boldface).

^bNo alterations were present compared to the sequence from MGAS315.

The MICs of RIF, RFZ, and NCEs for S. pyogenes ATCC 19615 and its rifamycin-resistant derivatives were determined using standard methods (11). S. pyogenes ATCC 19615 was 16- to 32-fold more susceptible to RFZ and NCEs than was S. aureus ATCC 29213 (Table 1) (10). MICs of the most potent NCEs (e.g., ABI-0043) against rifamycin-resistant S. pyogenes strains ranged from 0.00025 μg/ml to 0.25 μg/ml for the most resistant strain (Table 1) and 0.016 μg/ml to 4 μg/ml for the most rifamycin-resistant S. aureus strains (10). In fact, the MICs of NCEs against S. pyogenes mutants (Table 1) were consistently ≥8-fold lower than those for S. aureus mutants containing mutations in the corresponding codon of the rpoB gene (10).

It is anticipated that NCEs having in vivo efficacy against rifamycin-resistant S. aureus strains would be effective against the most resistant S. pyogenes mutants. Experimental infection models using resistant strains from both of these species should verify this assumption.