Activity of cethromycin against prototype and efflux pump mutant strains
Since B. pseudomallei
wild-type (prototype) strains are generally resistant to macrolides because of AmrAB-OprA and BpeAB-OprB efflux pump expression,15,16
we initially tested the susceptibility of strain 1026b and several of its isogenetic efflux pump-proficient or -deficient strains. These experiments revealed that in contrast to the macrolides clarithromycin and erythromycin, cethromycin is a weak substrate for AmrAB-OprA and BpeAB-OprB, but not BpeEF-OprC (Table ). Time–kill curves with prototype strain 1026b revealed that cethromycin is bacteriostatic at the MIC and bactericidal above the MIC (Figure a). Similar observations were made with two clinical isolates, 2637a and 2660a, from Thailand that were less susceptible to cethromycin (measured MICs of 16 and 32 mg/L, respectively) (Figure b). As could be predicted by the MICs, the time–kill curves show that at 16 mg/L cethromycin was bactericidal for 2637a and bacteriostatic for 2660a.
Cethromycin and macrolide susceptibilities of Burkholderia pseudomallei wild-type and efflux pump mutant strains
Figure 2. Cethromycin time–kill curves for Burkholderia pseudomallei strains. At time 0, LB medium containing the indicated amounts of cethromycin were inoculated with ~106 to 107 cfu/mL of the strains listed below. The cultures were shaken at 37°C, (more ...)
Activity of cethromycin against a panel of B. pseudomallei clinical and environmental isolates
Because of the genetic diversity of B. pseudomallei
, it is important to evaluate the in vitro
activity of cethromycin against a larger number of strains from diverse sources, e.g. clinical versus environmental strains. Inclusion of environmental isolates is important, because patients in endemic regions acquire their infections from the environment,1,2,17
and environmental and clinical isolates are equally virulent (K. L. Propst, A. Goodyear, S. W. Dow and H. P. Schweizer, unpublished results). We assessed cethromycin activity against a panel of 60 (30 clinical and 30 environmental) isolates from Thailand. While there was some variability between MIC assays conducted at different times, especially with clinical isolates, the MIC range observed with clinical and environmental isolates was similar (4–64 mg/L with clinical and 4–32 mg/L with environmental isolates), and there was no indication of exceptionally susceptible or resistant strains. Overall, the MIC50
were 16 and 29.3 mg/L, respectively. Environmental isolates were consistently more susceptible (MIC50
13.3 mg/L and MIC90
21.3 mg/L) than clinical isolates (MIC50
17.3 mg/L and MIC90
High-level cethromycin resistance is due to AmrAB-OprA overexpression
To assess the propensity of B. pseudomallei for the development of cethromycin resistance and to elucidate possible resistance mechanisms, we selected spontaneous cethromycin-resistant mutants of 1026b. Two isolates that were resistant to ≥32 mg/L cethromycin were retained for further studies, and named Bp289 and Bp290. The two mutants exhibited the same MIC patterns and were multidrug resistant, including against macrolides and aminoglycosides (Table ). The drug resistance profiles were reminiscent of AmrAB-OprA-expressing strains. Strain 1026b constitutively expresses AmrAB-OprA at some level and is thus resistant to high levels of aminoglycosides and macrolides, yet is fairly susceptible to cethromycin. If this pump is indeed responsible for the high-level cethromycin resistance of Bp289 and Bp290, then these strains must be mutants that overexpress AmrAB-OprA. To assess the suspected role of AmrAB-OprA in the observed high-level cethromycin resistance, we therefore decided to: (i) assess amrB transcript levels in strain Bp289; (ii) check the amrR repressor gene and the amrR-amrA intergenic region for presence of mutations; and (iii) delete the amrAB-oprA operon from strain Bp289. Since Bp289 and Bp290 were derived from the same culture and exhibited the same resistance patterns, we decided to focus on Bp289.
Antimicrobial susceptibilities of prototype strain 1026b and its cethromycin-resistant derivatives
To determine whether AmrAB-OprA was overexpressed in Bp289 versus 1026b, we assessed amrB transcript levels in both isolates by qRT–PCR. These analyses showed that amrB transcripts were increased 4-fold in Bp289 when compared with the parental strain 1026b. This is more than sufficient to achieve the high resistance levels observed in this mutant.
Next, we analysed the amrR
repressor gene and the amrR-amrA
intergenic region for the presence of mutations that may have led to AmrAB-OprA expression in Bp289. The amrR
gene and the amrR-amrA
intergenic region were amplified from Bp289 genomic DNA, as described in the ‘Materials and methods’ section. The PCR yielded a 944 bp DNA fragment that contained the 672 bp amrR
gene, the 143 bp amrR-amrA
intergenic region and 26 bp of the amrA
gene. This DNA fragment was sequenced using the same primers used for PCR and aligned with the wild-type 1026b sequence. DNA sequence analysis of two separate PCR products obtained from Bp289 chromosomal DNA templates revealed no mutations in the amrR
coding sequence and the amrR-amrA
intergenic region. This is consistent with previously reported studies from our laboratory, which showed that constitutive amrAB-oprA
expression is not solely due to AmrR repressor mutations, but can also be achieved via a hitherto unknown mechanism(s).18
To demonstrate that the high-level cethromycin resistance of Bp289 was mainly due to AmrAB-OprA overexpression, we deleted the amrAB-oprA operon from this strain. Bp328, the resulting amrRAB-oprA deletion mutant, showed increased susceptibilities to cethromycin, aminoglycosides, macrolides and tetracycline compared with the parental strain Bp289 (Table ). Single-copy complementation with the amrAB-oprA operon from 1026b in strain Bp384 restored the resistance pattern to that seen in Bp289. A control strain, Bp383, carrying the empty mini-Tn7 cloning and gene integration vector exhibited the same resistance profile as that seen in the recipient strain Bp328. These data unequivocally show that AmrAB-OprA contributes to the high-level cethromycin resistance of Bp289 and is a major cethromycin resistance mechanism. However, Bp328 was more resistant to cethromycin, erythromycin and clarithromycin than Bp50, a Δ(amrRAB-oprA) derivative of 1026b. This indicates that the overexpression of AmrAB-oprA is not the only cethromycin resistance mechanism operating in strain Bp289. Since Bp289 was isolated by growing 1026b in LB broth containing cethromycin, multiple mutations might have occurred during the incubation period.
Other mechanisms contribute to cethromycin resistance
In an attempt to isolate mutants that became cethromycin resistant independent of AmrAB-OprA expression, cells of the Δ(amrRAB-oprA
) mutant strain Bp340 were plated on LB agar containing 4 mg/L (2× MIC) cethromycin. Of the nine colonies obtained with this selection procedure after a 100 h incubation at 37°C, eight were 4-fold more resistant to cethromycin (MIC
8 mg/L) than the parental strain Bp340 (MIC
2 mg/L). The MIC in one mutant was only marginally (2×) increased. The increased cethromycin MIC was accompanied by 2- to 4-fold increases in erythromycin MICs (from 8 mg/L in Bp340 to 16–32 mg/L in the mutants), but susceptibilities to other antimicrobial agents were unchanged. The simultaneously reduced susceptibilities to cethromycin and erythromycin may be indicative of a common macrolide/ketolide resistance mechanism, which is most likely not efflux as susceptibilities to unrelated drugs (e.g. chloramphenicol, tetracycline, gentamicin and norfloxacin) remained unchanged. The small number of colonies obtained after exposure to a low cethromycin concentration and their low-level resistance probably signifies that the underlying mechanism plays only a minor role in cethromycin resistance. It may, however, contribute to the previously observed residual cethromycin resistance of strain Bp328 (see preceding section).
Unlike macrolides, such as clarithromycin and erythromycin, which are readily effluxed by most B. pseudomallei
strains, cethromycin has significant activity against most B. pseudomallei
strains tested, with MICs ranging from 4 to 64 mg/L. The cethromycin MIC of prototype strain 1026b is 4–8 mg/L. For strain 1026b, this compares with MICs of 1.5, 4, 1 and 0.5 mg/L of amoxicillin
clavulanic acid, ceftazidime, doxycycline and trimethoprim
sulfamethoxazole, respectively, which are antibiotics currently used for melioidosis therapy. While cethromycin's anti-B. pseudomallei
activity is not as potent as that of many current melioidosis therapeutic agents, it approaches that of some of the β-lactam antibiotics (e.g. ceftazidime) currently used for acute-phase treatment. Only in vivo
animal experimentation will reveal whether cethromycin is effective for melioidosis therapy, either as a stand-alone drug or in combination with other antibiotics. The intrinsic resistance of B. pseudomallei
to many antibiotics is well documented.1,4,19
Moreover, chromosomal mutations can cause resistance to many clinically significant antibiotics. For example, mutations in the chromosomally encoded PenA β-lactamase can cause high-level ceftazidime resistance.20
Strains expressing the BpeEF-OprC efflux pump are resistant to chloramphenicol, tetracyclines and trimethoprim (T. Mima and H. P. Schweizer, unpublished results). Novel therapeutic agents that are not subject to these resistance mechanisms are therefore needed.