Antibacterial Efficacy of AgNPs-PVP and Unprotected AgNPs
As discussed previously, S. pneumoniae and NTHi are the two most
common bacteria pathogens causing
OM,
accounting for 58% of the total OM episodes in the
US4,5. The antibacterial efficacy of AgNPs-PVP and
unprotected AgNPs was thus tested using these two pathogens. The minimum
inhibitory concentration (MIC) and half maximal inhibitory concentration
(IC50) were used to quantify the antibacterial efficacy.
MIC was obtained via broth microdilution assay, where MIC corresponds to
the lowest concentration of antimicrobials that prevented bacterial
growth. To better characterize the affected bacterial growth before
complete eradication (i.e., MIC) was achieved, IC50 was
calculated, defined as the concentration of antimicrobials that led to a
stationary OD600 that was half of that without
antimicrobials. For antimicrobials with the same MIC, lower
IC50 indicates higher antimicrobial effect.
The antimicrobial efficacy of AgNPs against NTHi showed an average
IC50 of 28.72 µM (~4.88 µg/mL) and an
average MIC of 50 µM (~8.5 µg/mL) (Figure 4A). The
average values of IC50 and MIC were much reduced in the
presence of PVP, which became 9.45 µM (~1.61 µg/mL) and
12.5 µM (~2.13 µg/mL) respectively (Figure 4A). The
greater antimicrobial efficacy of AgNPs-PVP than unprotected AgNPs was
likely a result of the stabilized particulates (Figure 3) with greater
surface-to-volume ratio than aggregates that led to enhanced
interactions with pathogens.
The antibacterial efficacy of AgNPs-PVP was comparable to tradition
antibiotics against NTHi, such as Amoxicillin (with MIC of 0.5 - 2
µg/mL), Clarithromycin (with MIC of 2 - 8 µg/mL), and Azithromycin (with
MIC of 0.25 - 2 µg/mL)62. Contrary to those
small-molecule antibiotics, efficacy of AgNPs and AgNPs-PVP againstS. pneumoniae was better than that against NTHi (Figure 4B), with
IC50 values of 17.88 µM (~2.99 µg/mL)
and 4.14 µM (~0.70 µg/mL) for AgNPs and AgNPs-PVP
respective and MIC values of 25 µM (~4.18 µg/mL) and
6.25 µM (~1.04 µg/mL) for AgNPs and AgNPs-PVP
respective. The stronger antimicrobial effects of AgNPs and AgNPs-PVP
against S. pneumoniae than that against NTHi could be due to
their gram types or the known production of
H2O2 by S. pneumoniae (at levels
around 0.1 – 0.71 mM as a mechanism for competitive survival during
coinfections)63. To better understand this
differential efficacy of AgNPs-PVP, Streptococcus mutans (S.
mutans) , a gram-positive pathogen (same as S. pneumoniae ) with
much lower activity of H2O2 production
(at levels around 0 - 0.06 mM64,65) was tested.
S. mutans is one of the microorganisms inhabiting the oral
cavity, which has been studied for its etiology of dental caries and
infective endocarditis66. Interestingly, the
colonization of S. mutans in mouth has been shown to be
correlated with the colonization of S. pneumonia in the
nasopharynx67, thus making S. mutans a pathogen
of interest for OM treatment. Indeed, the MIC values of AgNPs and
AgNPs-PVP against S. mutans were 50 µM (~8.35
µg/mL) and 12.5 µM (~2.09 µg/mL) respectively, which
were comparable to NTHi and greater than S. pneumoniae (Figure
4C). Similarly, the IC50 value of AgNPs and
AgNPs-PVP against S. mutans were 32.03 µM (~5.35
µg/mL) comparable to that of NTHi. The IC50 value of
AgNPs-PVP, 4.87 µM (~0.81 µg/mL), was smaller than that
of NTHi (9.45 µM (~1.58 µg/mL)) and close to that ofS. pneumoniae (4.14 µM (~0.70 µg/mL)), which
could be explained by the bacteriostatic effect of low levels of
H2O2 on S.
mutans 68. Taken together, the MIC and
IC50 values of AgNPs and AgNPs-PVP against S.
mutans confirmed that their greater efficacy against S.
pneumoniae was likely a result of the
H2O2-producing capability and not gram
types, as gram-positive S. mutans and gram-negative NTHi
demonstrated comparable values.