44 4 ART for Antibacterial Infection
These results indicated that bacteria can synthesize NO for adaptation to oxi-
dative stress circumstance, and ART can inhibit the synthesis of NO in bacteria,
hence representing a novel inhibitor of all kinds of NOS including bNOS. Thus,
we could expect that ART would sensitize those antibiotics that pose oxidative
stress to bNOS-dependent bacteria.
4.2.2.2 Attenuation of NO-Conferred Antibiotic
Tolerance by ART: In Vitro Study
We first tested whether bacterial NO burst under a hypoxic condition confers
B. licheniformis tolerance to RIF. After cultured for 12 h with RIF (20, 40, 60,
or 120 μg/ml), hypoxia-acclimatized bacteria proliferate at a higher rate, whereas
nonacclimatized bacteria exhibit no proliferation. Similarly, hypoxia-acclimatized
bacteria also show accelerated proliferation in the presence of cefotaxime (CEF)
(15, 25, or 50 μg/ml). These results demonstrated that hypoxia-induced NO allows
bacteria to thrive in exposure to RIF/CEF, suggesting that NO confers B. licheni-
formis tolerance to those antibiotics.
By attenuating the protective NO production in bacteria, ART was anticipated
to reverse NO-mediated protection of bacteria from antibiotics. Indeed, a com-
bination of ART (60 μg/ml) with RIF (20–120 μg/ml) leads to a lower rate of
bacterial growth than RIF alone, implying that ART potentates RIF by abrogat-
ing bacterial NO production. We observed that enhanced NO burst, at a maximal
level of 80 μM, occurs in bacteria in exposure to RIF (20–120 μg/ml), whereas a
decreased NO level (20 μM) or even a negligible NO level was measured in bacte-
ria cotreated by ART (60 μg/ml) with RIF (20–120 μg/ml).
As comparison, we also detected the NO level in bNOS-free E. coli, either with
or without CEF, but no correlation of bacterial proliferation with NO production
was established. The growth dynamics of E. coli under CEF (100 μg/ml) are simi-
lar with those under ART (60 μg/ml) + CEF (100 μg/ml). The maximal NO levels
are only 4–8 μM under CEF (100 μg/ml) or ART (60 μg/ml) + CEF (100 μg/ml).
From the extremely low level of NO in E. coli, we could infer that its own NO
may not be sufficient to detoxify antibiotics.
4.2.2.3 Bacterial Production of H 2 O 2 Due to CAT Inhibition by ART
As an antioxidant hemoprotein, CAT was expected to interact with ART and
to exhibit a lower enzymatic activity. If this is true, we could deduce that there
should be a similar mechanism by which ART binds to CAT and blocks the con-
version of toxic H 2 O 2 to nontoxic H 2 O or other detoxified compounds. To confirm
this deduction, we measured the CAT activity of B. licheniformis after incubation
with RIF (20–120 μg/ml) or RIF (20–120 μg/ml) + ART (60 μg/ml). The results
showed that CAT activity is considerably reduced once ART was included in the
culture (Fig. 4.2). For example, after 24 h, CAT activity in bacteria treated by RIF