In order to learn if induction of PmefE
by LL-37 has biological significance, we tested whether induction of PmefE-lacZ
with LL-37 confers resistance to LL-37 or erythromycin using microdilution assays optimized for MIC determination of CAMPs (28
). Cultures were grown to an optical density at 600 nm (OD600
) of 0.4, split, and grown with or without addition of subinhibitory concentrations of LL-37 or erythromycin for 1 h. The subinhibitory concentrations used (Table ) did not appear to affect growth of the bacteria, as assessed by determination of the CFU, but were sufficiently high to induce PmefE-lacZ
in the reporter strain (data not shown). A final concentration of 2 × 105
bacteria/ml adjusted in 5-fold-diluted TH medium supplemented with 0.5% yeast extract was added to serial dilutions of LL-37 or erythromycin. After 1 h at 37°C, samples were plated on TSA II blood agar plates (BD), and after 16 h at 37°C with 5% CO2
, the MICs and the minimal bactericidal concentrations (MBCs) were determined.
MICs and MBCs of wild-type, mega, and mefE-mel mutant strains for LL-37 and erythromycine
Wild-type strain GA17457 and the PmefE-lacZ reporter derivative XZ7042 showed high levels of resistance to LL-37 (Table ), which were further increased upon growth in subinhibitory concentrations of LL-37 (Table ). Concomitantly, the MIC for erythromycin increased by 2- to 16-fold. Growth in the presence of erythromycin caused an increase in LL-37 resistance, as well as a 4-fold increase in erythromycin resistance. Taken together, independent of the nature of the inducer, induction increased resistance toward both compounds. In regard to the MBCs, LL-37 induction of resistance was less pronounced than that with erythromycin, consistent with the weaker induction observed with LL-37 in the plate diffusion assay (Fig. ).
To determine whether the increased resistance to LL-37 and erythromycin relied on the mega element, two mega deletion mutants were constructed. By allelic replacement, the 5.5-kb mega region (bp 1 to 5,532; GenBank accession number AF274302) was replaced in GA17457 and XZ7042 with the spectinomycin resistance cassette (containing the aad9
gene) from pUC-spc (12
). The resulting mutants XZ8006 and XZ8004, respectively, were less resistant to LL-37 than their parental strains and were susceptible to erythromycin, confirming that mega was required for resistance (Table ). In addition, growth in the presence of subinhibitory concentrations of LL-37 did not induce increased resistance to LL-37 or erythromycin (Table ); hence, inducible resistance in the wild-type strains required the mega element.
We analyzed whether pneumococcal resistance to LL-37 required the MefE/Mel efflux pump encoded on mega. A mefE
deletion mutant, XZ8009, was constructed by replacing the region containing the mefE
genes (from bp 83 in mefE
to bp 3,858 in mel
) with the kanamycin resistance cassette (containing the aph3
gene) of pSF191 (33
) in GA17457. XZ8009 showed the same levels of resistance to LL-37 as the mega-deletion mutants under all tested conditions (Table ), demonstrating that the MefE/Mel efflux pump mediated the resistance to LL-37 and erythromycin.
Induction of MefE/Mel-mediated resistance by LL-37 was an unexpected finding, especially after a rather narrow spectrum of 14- and 15-membered macrolides had been described as inducers/substrates of this efflux pump (32
). The potential clinical implications of this finding are 2-fold, as follows: (i) failure of macrolide-based treatment of mega-containing strains due to host-mediated induction of MefE/Mel by LL-37, resulting in a higher level of resistance, and (ii) underestimation of macrolide resistance of mega-containing strains, based on in vitro
MICs determined under noninducing conditions. To assess the in vivo
induction of MefE/Mel by LL-37, further studies are required to determine whether local concentrations of LL-37 in the host are sufficiently high to cause induction. Serum concentrations of LL-37 are reported to be in the μg/ml range and can increase dramatically during acute inflammation; however, the local concentrations may vary greatly (27
). Daneman et al. have described that the risk for macrolide treatment failures increased with MICs of ≥1 μg/ml but did not increase further with higher MICs (4
). Their finding would be consistent with low-level in vitro
MICs, as observed for mega-containing isolates, that increase upon in vivo
induction of a resistance determinant, resulting in MICs that exceed the threshold established by existing guidelines (MICs ≥ 16 μg/ml) (25
) beyond which macrolides should no longer be used. Taken together, induction of MefE/Mel by LL-37 provides an intriguing, possible explanation for the observed macrolide treatment failures in disease caused by low-level-resistant pneumococci (3
is naturally resistant to high levels of CAMPs, which has been attributed to mechanisms altering the surface charge, thereby decreasing CAMP affinity, and to several other factors that await further characterization (18
). Recently, Majchrzykiewicz et al. (24
) have shown that incubation with LL-37 altered expression of ~10% of the genome in S. pneumoniae
, including the genes of known and putative regulatory proteins, suggesting the possibility of an indirect induction of Mef/Mel by LL-37. Our data suggest that the MefE/Mel efflux pump can further contribute to cathelicidin resistance and may thereby contribute to increased survival in the human host. Due to the limitations of our in vitro
assay system, which required high concentrations of LL-37 at or beyond the soluble concentration of LL-37 (~2,000 μg/ml), this important observation needs to be further evaluated for biological significance with in vivo
experiments. Actual efflux-mediated cathelicidin resistance has been demonstrated for the MtrCDE efflux pump of Neisseria
). This additional function of MefE/Mel could account for an increasing number of pneumococcal isolates that contain both macrolide resistance determinants ErmB and MefE/Mel (15
). Due to the high-resistance levels associated with ErmB, MefE/Mel does not contribute to macrolide resistance, but it may help protect MefE/Mel-containing strains from antimicrobial host defenses.