49ART, or 200 μg/ml AMP + 60 μg/ml ART. In contrast, 30 or 3 colonies form on
the fecal plate that covers a layer of stool dilution prepared from E. coli-fed mice
injected with 100 or 200 μg/ml AMP. These results indicated that AMP with com-
bination with ART cannot allow colony growth, highlighting that ART is able to
potentate the antibacterial capacity of AMP in vivo.
Even AMP-resistant bacteria that carry Ampr plasmids were used for feeding,
only a few colonies are formed from the fece of mice injected with CEF (25 μg/
ml), whereas no colony appears from the fece of mice injected with CEF (25 μg/
ml) + ART (60 μg/ml). It was likely that ART can potentate CEF via mitigating
the NO-mediated protection, but we cannot, at this moment, definitely exclude a
possibility of ART exerting the direct cytotoxicity.
4.3.2.3 In Vivo Study in Mice Fed with B. Licheniformis
The daily feeding of mice with B. Licheniformis also elicits NO production in
mice, in which threefold increase of NO levels were observed after bacterial feed-
ing for three days. In control mice, the serum NO level is only 1.88 ± 0.242 μM,
but in the bacteria-fed mice, the serum NO level is 6.67 ± 2.42 μM. It was also
noted that 20 μg/ml RIF fails to decline the NO level (8.21 ± 2.42 μM), whereas
40 μg/ml RIF can decline the NO level (3.42 ± 2.42 μM). Interestingly, the NO
level (3.42 ± 2.42 μM) in bacteria-fed mice injected with RIF (40 μg/ml) is
equivalent to the NO level (3.93 ± 1.21 μM) in bacteria-fed mice injected with
RIF (20 μg/ml) + ART (60 μg/ml). These results suggested that ART can syner-
gize RIF for more effective antibacteria and decrease the dosage of RIF.
4.3.3 Discussion
One possibility of NO becoming cytotoxic is when it meets O 2 −. NO and O 2 −
interact to form ONOO−, one of the powerful RNS toxics to living cells.
Alternatively, peroxynitrous acid that also kills cells can form as a result of the
interaction between nitrite and H 2 O 2 under mildly acidic conditions (Kono et al.
1994 ). The induction of NOS in phagocytes is only one form of host defense reli-
ant upon NO (Vallance and Charles 1998 ).
Our in vivo tests ascertained that ART + AMP, ART + CEF, or ART + RIF can
suppress the bacterial infection occurring in mouse gastrointestinal tracts. During
the antibacterial process, a combination of ART with one kind of antibiotics can
remarkably decline the serum NO levels. Why NO is protective for bacteria? A
recent research might answer this question, in which NO has been described to
modulate bacterial biofilm formation through a multicomponent cyclic-di-GMP
signaling network (Plate and Marletta 2012 ).
4.3 In Vivo Evaluation on ART as a Synergist of Antibiotics ...