S. Roland, C. Jolivalt et al.
MsrA), which contains the multicopy plasmid pUL5054 with
constitutive resistance to ERY.[12] Except 20, all silver–
NHCs were found to have significant activities against these
two resistant strains with MICs of 1–32 mgmLÀ1 (Table 1).
Complexes 11 and 21 (SIMesAgCl) are the most active with
MIC values of 1–4 mgmLÀ1, which is similar to the values
obtained against the susceptible strain. These two complexes
are therefore able to inhibit both sensitive and resistant
strains of S. aureus at clinically achievable concentrations.
The MICs of 11 and 21 against the susceptible S. aureus
strain (1 mgmLÀ1) are very close to those of CIP and ERY
(0.5 mgmLÀ1),[13] which are widely used antibiotics to cure S.
aureus infections. More interestingly, compounds 11 and 21
have MICs that are 128-fold lower than that of ERY against
the resistant strain S. aureus MsrA (128 mgmLÀ1) and 4- to
8-fold lower than that of CIP against S. aureus NorA
(16 mgmLÀ1).[13] Complex 13, which is active both against E.
Coli and S. aureus with MICs of 2–8 mgmLÀ1, is also inter-
esting for its broad spectrum of activity. Several complexes
are significantly more efficient than AgNO3 against the sus-
ceptible strain of S. aureus and the resistant strain S. aureus
NorA. In addition, AgNO3 is inactive against resistant S.
aureus MsrA, whereas MIC values reaching 1–4 mgmLÀ1
have been obtained with silver–NHCs. The lower activity
observed for the imidazolinium salt 22 by comparison with
the corresponding silver complex 21 demonstrates the major
role of silver in antibacterial activity.[14]
Multidrug combinations are increasingly important in
stamping out the spread of antibiotic resistance in bacterial
pathogens.[15] Enhanced antimicrobial activities have been
reported by combining antibiotics with silver-based com-
pounds, such as silver nanoparticles.[16] This led us to investi-
gate the activity of 8–21 against resistant S. aureus NorA in
the presence of CIP at subinhibitory concentration (MIC/8,
i.e., 2 mgmLÀ1). In several cases, an increased antibacterial
effect was observed (Table 1, right column). The most signif-
icant results were obtained with 10, 20, and 21, which gave
MICs that are four- to eightfold lower than those of the
silver complexes alone. A MIC as low as 0.5 mgmLÀ1 was
obtained with 21 under these conditions. Interestingly, the
presence of CIP at a subinhibitory concentration also poten-
tiates the antibacterial activity of salt 22 against S. aureus
NorA, whereas no enhanced activity was detected for
AgNO3. MIC determination in the presence of varying sub-
inhibitory concentrations of CIP and silver complex was
then undertaken for 20 and 21 to state more precisely the
effect of combining the two drugs. Complex 12 was also
studied for structural similarities to 20. An important syner-
gistic effect between CIP and 20 (SIPrAgCl) was evidenced
and clearly demonstrated by the isobole obtained (Figure 1).
As described by the Loewe theory, synergistic drug pairs
have a stronger than additive effect corresponding to an iso-
bole below the additive straight line.[17] This synergy allows
20 to be almost as efficient (MIC 2 mgmLÀ1) as 21 (MIC
0.5 mgmLÀ1) in the presence of 4 mgmLÀ1 of CIP, whereas
the MICs without CIP are 64 and 2 mgmLÀ1, respectively.
By comparison, synergistic effects between 12, 21, and CIP
Figure 1. Combination of silver–NHCs 12, 20, and 21 with CIP at varying
subinhibitory concentrations. MICs [mgmLÀ1
] determined against S.
aureus NorA. The lines indicate the drug pair concentrations required to
stop bacterial growth. Growth responses to one single compound alone
lie along each axis.
are less marked. Similar experiments carried out with com-
plexes 12, 21, and ERY showed no synergistic effect against
the resistant strain S. aureus MsrA, thus suggesting a specific
effect on S. aureus NorA. This could be ascribed to a specif-
ic inhibition of the NorA overexpressed efflux pump by
silver–NHCs, which thereby restore the antimicrobial activi-
ty of CIP. The development of efflux pump inhibitors (EPIs)
is an important strategy in combating MDR in S. aureus.[18]
However, the high intrinsic antimicrobial activity of 21 does
not allow any conclusions to be drawn on the possible mech-
anism of action as an EPI. This could be more accurately
proposed for 20 for which the activity against S. aureus
NorA in the absence of CIP is very low.
Previous in vitro studies carried out with commonly used
silver-based topical antimicrobial agents showed that kerat-
ACHUTNGRENiNUG nACTHUNGRTENoUNG cytes and fibroblasts are susceptible to lethal damage
when exposed to concentrations of silver that are lethal for
bacteria.[7] To evaluate the in vitro cytotoxicity of silver–
NHCs 8–21, we determined the inhibition percentage of
MRC5 (human noncancerous cells in rapid proliferation)
cell proliferation at concentrations of 50 and 10 mgmLÀ1.
For comparison, the activity of 22 was also examined. As
shown in Figure 2, silver–NHCs 8–21 as well as 22 inhibit
more than 90–95% of the cell growth at the highest concen-
tration. A high toxicity was also observed at 10 mgmLÀ1,
except for complex 9 and imidazolinium 22 (70 and 65% in-
hibition, respectively). This cytotoxicity is comparable to
that reported for AgNO3 on human fibroplasts at similar
concentrations.[19,7b]
Determination of IC50 values on MRC5 and EPC cell
lines for 11–13, 20, and 21 corroborated these preliminary
results (Table 2). These values are superior on the EPC qui-
escent cell line than on MRC5, suggesting a higher cytotox-
icity on cells in rapid proliferation. Values in the same range
were previously reported for 12, 20, and 21 against several
cancer cell lines.[2b] Compound 21 (SIMesAgCl) is the least
1444
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Chem. Eur. J. 2011, 17, 1442 – 1446