AAC Accepted Manuscript Posted Online 6 April 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.00382-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Kibdelomycin is a bactericidal broad-spectrum aerobic antibacterial agent Sheo B. Singh,a,d,1 Priya Dayananth,a Carl J. Balibar,a Charles G. Garlisi,a Jun Lu,b Ryuta Kishii,c Masaya Takei,c Yasumichi Fukuda,c,e Sookhee Ha,a Katherine Younga a
Merck Research Laboratories, Kenilworth, NJ 07033 Merck Research Laboratories, West Point, PA 19486 c Kyorin Pharmaceutical Co., Ltd., 2399-1, Nogi, Nogi-machi, Shimotsuga-gun, Tochigi, 329-0114 Japan d current address: SBS Pharma Consulting LLC, Edison, NJ 08820 e current address: OP Bio Factory Co., Ltd., 5-8, Suzaki, Uruma, Okinawa, 904-2234 Japan b
1
To whom correspondence should be addressed. E-mail:
[email protected] 1
21 22 23 24 25
Abstract
26
discovery rate for new antibiotics declines. Kibdelomycin is a recently discovered natural
27
product antibiotic that inhibits bacterial growth by inhibiting bacterial DNA replication enzymes
28
DNA gyrase and topoisomerase IV. It was reported to be a broad-spectrum aerobic Gram-
29
positive agent with selective inhibition of the anaerobic bacterium Clostridium difficile. We
30
have extended the profiling of kibdelomycin using over 200 strains of Gram-positive and Gram-
31
negative aerobic pathogens recovered from worldwide patient populations. We report MIC50s,
32
MIC90s, and bactericidal activity of kibdelomycin. We confirm the Gram-positive spectrum and
33
report for the first time that kibdelomycin shows strong activity (MIC90 0.125 μg/ml) against
34
clinical strains of the Gram-negative non-fermenter Acinetobacter baumannii but shows only
35
weak activity against Pseudomonas aeruginosa. We confirm that kibdelomycin does not show
36
cross-resistance with quinolones and coumarin antibiotics in well-characterized resistant strains
37
of Staphylococcus aureus and Streptococcus pneumoniae. We also show that kibdelomycin is
38
not subject to efflux in Pseudomonas though it is in E. coli and it is generally affected by the
39
outer membrane permeability entry barrier in non-fermenters, P. aeruginosa and A. baumannii,
40
which may be addressable by structure-based chemical modification.
Bacterial resistance to antibiotics continues to grow and pose serious challenges while the
2
41
Bacterial resistance against antibiotics continues to emerge in clinical practice with a
42
frightening frequency, posing a serious health threat. Klevens et. al. (1) recently reported that
43
methicillin resistant Staphylococcus aureus (MRSA) infections alone are responsible for about
44
18,000 deaths per year in the United States. The dearth of new antibiotics exists due to lack of
45
discovery of novel chemical scaffolds that can be developed into clinically useful antibiotics,
46
making this problem dire particularly for the ESKAPE pathogens. (2) Most of the structural
47
leads that have been used for development of antibiotic drugs were discovered over five decades
48
ago, with the exception of linezolid and daptomycin, which were discovered in the 1980’s. Most
49
of these chemical leads originated from nature. Chemical modifications of the antibiotic lead
50
structures discovered decades ago have led to incrementally improved antibiotics that continue to
51
serve well and provide the current reservoir of clinical antibiotics (3; 4). However, the capacity
52
of such modifications is not limitless. As a result, additional improvements on existing chemical
53
scaffolds are proving increasingly challenging. The dearth of new antibiotics can be overcome by
54
discovery of new antibiotic scaffolds, with either known or novel modes of action that can be
55
developed as effective treatment options against drug-resistant bacteria.
56 57
We recently reported the discovery of a series of novel natural product antibiotics with
58
novel modes of action by application of antisense-based screening technology exemplified by
59
platensimycin, (5; 6) platencin (7; 8) and most recently kibdelomycin (9) and kibdelomycin A
60
(Figure 1) (10). The broad spectrum Gram-positive antibiotic, kibdelomycin, was reported in
61
2011 and is isolated from a Kibdelosporangium sp. Kibdelomycin exerts its activity by inhibiting
62
bacterial DNA synthesis through specific inhibition of the β subunits of DNA gyrase (GyrB) and
63
topoisomerase IV (ParE). Kibdelomycin has been shown to be a potent inhibitor of E. coli (IC50
64
60 nM) and S. aureus (IC50 9 nM) gyrase supercoiling activity and a less potent inhibitor of the
65
corresponding topoisomerase IV decatenating activity (E. coli, IC50 29000 nM; S. aureus, IC50
66
500 nM). Kibdelomycin potently inhibited the catalytic E. coli ATPase activity of gyrase B (IC50
67
11 nM) and topoisomerase IV (ParE) (IC50 900 nM) (9). Kibdelomycin A is a less potent
68
inhibitor of S. aureus gyrase supercoiling (IC50 400 nM) and topoisomerase IV catenation (ParE,
69
IC50 5000 nM) but has been shown to be a potent inhibitor of E. coli gyrase B ATPase activity
70
(IC50 9 nM) though a poor inhibitor of the E. coli ParE ATPase (IC50 6400 nM) (10). We
71
reported that kibdelomycin is a selective and potent growth inhibitor of Clostridium difficile 3
72
without significantly affecting anaerobic Gram-negative bacteria including Bacteroides species
73
(11). It showed potent in vivo activity against C. difficile infection without systemic exposure
74
(11). We recently reported an X-ray crystal structure of kibdelomycin bound to S. aureus and E.
75
coli GyrB and ParE (12). The crystal structure showed that kibdelomycin binds uniquely in a U-
76
shaped multi- contact binding mode occupying the ATP binding site with extension to another
77
part of the pocket (12). Kibdelomycin exhibits a low frequency of resistance and shows no cross
78
resistance to S. aureus strains resistant to other known gyrase inhibitors, such as novobiocin,
79
coumermycin and quinolones which is consistent with the novel dual arm U-shaped binding
80
mode described above. We describe here the kill kinetics of kibdelomycin against S. aureus and
81
activity of kibdelomycin against an expanded panel of wild-type and resistant strains of Gram-
82
positive and Gram-negative bacteria. We also studied the effect of efflux pumps and the
83
permeability barrier towards the susceptibility of kibdelomycin against key Gram-negative
84
pathogens. Interestingly, kibdelomycin demonstrates strong activity against geographically
85
diverse clinical strains of Acinetobacter baumannii and weak activity against Pseudomonas
86
aeruginosa.
87 88
Materials and Methods
89
Reagents. All reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise
90
indicated. Kibdelomycin and Kibdelomycin A were isolated from Kibdelosporangium sp. now
91
named Kibdelosporangium banguiesis, as described earlier (9; 10).
92
Bacterial strains. All the strains in Tables 1 and 2 were collected from clinical samples in Japan
93
or otherwise stated. The bacterial strains for Table 1 were obtained from ATCC and IID were
94
obtained from American type culture collection and the Japanese Society for
95
Bacteriology, respectively. The clinical strains in this study were isolated in Japan. Efflux
96
deficient E. coli was kindly provided from Okayama University (13). The quinolone resistant S.
97
aureus were selected sequentially from four clinical isolates (14). The quinolone resistant S.
98
pneumoniae strain was selected from IID553 (15). P. aeruginosa nfxB and nfxC, and nalB strains
99
were made from PAO4009 and PAO6006, respectively (16).
100
The strains (n = 200) in Table 3 were collected as a part of the Merck SMART surveillance
101
studies from (17) throughout the world represented by Belgium (n = 4), China (n = 10), Czech 4
102
Republic (n = 1), France (n = 5), Germany (n = 6), Hong Kong (n = 10), Ireland (n = 1), Italy (n
103
= 9), Japan (n = 8), Poland (n = 1), Portugal (n = 1), Singapore (n = 1), South Korea (n = 12),
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Spain (n = 6), Sweden (n = 2), Spain (n = 6), Sweden (n = 2), Taiwan (n =9), Thailand (n = 3),
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United Kingdom (n = 12), United States (n = 96). These samples were recovered from abscess,
106
blood, bronchial washings, ear, eye, nasopharynx/throat/nose, sinus, skin, sputum, tracheal
107
aspirate, urine, wound and soft tissue. The strains were selected randomly representing antibiotic
108
susceptible and resistant strains. The source of permeability and efflux deficient strains presented
109
in Table 4 are listed in the last column of the Table.
110
Determination of MICs. The MICs were determined either by broth dilution or agar dilution
111
methods recommended by the Clinical and Laboratory Standards Institute in Mueller-Hinton II
112
broth (Becton Dickinson, NJ) (18). The MIC was defined as the lowest concentration of an
113
antibacterial agent that inhibited visible growth after incubation.
114
Determination of time kill kinetics. Time kill kinetics of kibdelomycin were conducted using S.
115
aureus ATCC29213 at 1x, 2x 4x, and 8x of MIC (determined by microbroth dilution method)
116
corresponding to concentrations of 1, 2, 4, and 8 μg/ml, respectively by CLSI guidelines (19).
117
The MIC was determined at 5x105 CFU/ml inoculum prior to commencing the time kill
118
experiment. Briefly, a stock solution of 5.12 mg/ml of kibdelomycin was prepared in DMSO.
119
The inoculum suspension was prepared from growth on a TSA/5% sheep blood plate to equal the
120
turbidity of a 0.5 McFarland Standard in Mueller Hinton II Broth (MHBII, Becton Dikinson,
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Sparks, MD), diluted 1:3, and grown in fresh MHBII at 35°C and incubated while shaking at 150
122
rpm. After approximately 2 hr, the suspension was again adjusted to equal a 0.5 McFarland
123
Standard, was diluted 1:2 in fresh MHBII, and 1 ml was used to inoculate each 125 ml
124
Erlenmeyer flasks containing 8.75 ml of sterile MHBII. The final target cell density was
125
approximately 106 – 107 CFU/ml. Just prior to T0, 0.25 ml of the appropriate concentration of
126
kibdelomycin solution was added to each flask, gently mixed, followed by 1.0 ml of diluted
127
inoculum. Thus, test drug vessels contained 8.75 ml MHBII, 1.0 ml inoculum, and 0.25 ml drug
128
solution (40X). A total of 22 vessels were prepared in this fashion, immediately swirled to mix,
129
and 0.03 ml was removed from the initial 0.5 McFarland Standard tube for determination of the
130
viable count at baseline (T0) by serial ten-fold dilution in MHB II. All vessels were incubated at
131
35°C in a New Brunswick Scientific Series 25 Incubated Shaker rotating at 150 rpm to provide 5
132
gentle mixing. The vessels were sampled at 2 hr (T2), 4 hr (T4), 6 hr (T6), 8 hr (T8), and 24 hr
133
(T24) for determination of viable count. Viable counts were determined by removing 0.3 ml
134
from each vessel and serially diluting 10-fold in 0.27 ml of MHBII with the Biomek 2000, then
135
plating duplicate 10 μL samples onto TSA with 5% sheep blood plates using the track dilution
136
method (20); in brief, 10 μL aliquots were spotted in duplicate across the top of a square 100 mm x
137
100 mm TSA+ 5% sheep blood plate. The plates were then tilted at a 45o – 90o angle to allow the
138
10 μL aliquot to track across the agar surface to the opposite side of the plate. All plates were
139
incubated for 20-24 hr at 35°C. Colonies were counted manually, and the CFU/ml was
140
determined from the average count from the duplicate plates, followed by calculation of the
141
log10 CFU/ml. A bactericidal effect was defined as a 3 log10 CFU/ml decrease in viable count at
142
24 hr relative to the starting inoculum.
143
Deletion of E. coli tolC, mukB and mukF. EctolC was knocked out in strain BW25113 utilizing
144
the linear PCR product generated by amplification from pKD4 (21) with the primers P1 and P2
145
(Table 5). Transformation and selection on kanamycin were performed as described previously
146
(22), yielding strain BW25113ΔtolC::kan. Transformation with pFlp2 (23) allowed for
147
subsequent excision of the kanamycin resistance cassette through Flp mediation recombination
148
leaving an 84 nucleotide scar between the first and last six codons of EctolC, thus generating
149
strain BW25113ΔtolC.
150 151
EcmukB and EcmukF were deleted from strain BW25113ΔtolC using the aforementioned one-
152
step λ Red method in conjunction with the linear PCR products generated by amplification from
153
pKD4 (21) with the primers P3 and P4 (for EcmukB) and P5 and P6 (for EcmukF).
154
Transformation and selection on kanamycin were performed as described previously (22),
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yielding strains BW25113ΔtolCΔmukB::kan and BW25113ΔtolCΔmukF::kan, respectively. As
156
previously described, the mukB and mukF E. coli knockouts were temperature sensitive (24), so
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in order to facilitate downstream MIC testing, single-step temperature insensitive mutants were
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selected by plating on CAMHA containing 30 µg/ml kanamycin and isolating colonies which
159
grew overnight at 37°C.
160 161
Deletion of A. baumannii lpxA and lpxC. Unmarked deletions of lpxA and lpxC were
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constructed in NCIMB 12457 and ATCC 19606, respectively, using a version of the pEX18Ap
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suicide vector (23) in which the ampicillin resistance cassette was replaced with the kanamycin 6
164
resistance cassette from pKD4 (21). To create this pEX18Km vector, the pEX18Ap vector
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backbone was amplified with primers P7 and P8 and the kanamycin cassette was amplified in 2
166
sections to remove an internal PstI site using primers P9 and P10 for the 5’ portion and P11 and
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P12 for the 3’ portion. In-Fusion clonase (Clontech) was then used to fuse these three fragments
168
into the final pEX18Km vector.
169 170
Flanking regions of the AblpxA and AblpxC genes were fused together using splicing by overlap
171
extension. Primer pairs P13/P14 and P17/18 were used to amplify 500 bp regions upstream of
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lpxA and lpxC, respectively, and primer pairs P15/P16 and P19/P20 were used to amplify 500 bp
173
regions downstream of lpxA and lpxC, respectively. In a second round of PCR, up- and
174
downstream products were combined and amplified using primers P13/P16 for lpxA and
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P17/P20 for lpxC. These PCR products were subsequently digested with restriction enzymes
176
PstI/BamHI and ligated into a similarly digested pEX18Km vector. The resulting
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pEX18KmΔlpxA and pEX18KmΔlpxC vectors were mobilized into A. baumannii from RHO3
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E. coli (25) as described previously (22). To resolve cointegrants to double-crossover knockouts,
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single colonies were restreaked on CAMHA with 10% (w/v) sucrose and 10 µg/ml colistin to
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select for loss of sacB-containing vector backbone. This generated the final lpxA and lpxC
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knockout strains NCIMB12457ΔlpxA and ATCC19606ΔlpxC.
182 183 184
Results
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Kibdelomycin is a broad spectrum aerobic antibiotic
186
Kibdelomycin was shown to inhibit the growth of Gram-positive bacteria S. aureus
187
(wild-type and MRSA), Streptococcus pneumoniae and Enterococcus faecalis and a Gram-
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negative bacterium Haemophilus influenzae with MIC < 2 μg/ml tested by CLSI microbroth
189
dilution method (9). MIC measurements using the agar dilution method confirm these
190
antibacterial activities and help extend the prior observed antibacterial spectrum (Table 1). The
191
agar diffusion Kibdelomycin MICs of 0.25 μg/ml against S. aureus and S. pneumoniae strains
192
were 4-8 fold better than measured by the microbroth dilution method. Kibdelomycin inhibited
193
the growth of Enterococcus faecium with an agar MIC value of 1 μg/ml.
194
Kibdelomycin showed potent growth inhibition of the Gram-negative bacterium A.
195
baumannii (agar MIC 2 μg/ml), similar to that observed with H. influenzae, but unlike the case 7
196
of these two Gram-negative strains, kibdelomycin did not inhibit growth of E. coli nor that of
197
most P.aeruginosa PAO1 strains (MIC >16 μg/ml). A similar trend (up to16x vs. E. coli and 64x
198
vs. P. aeruginosa lower activity compared to A. baumannii) has been reported for a few of
199
NBTI’s (New Bacterial Topoisomerase Inhibitors) binding to Gyrase A/ParC, an entirely
200
different binding site than kibdelomycin.(26; 27) The desmethyl congener kibdelomycin A and
201
acetate analogs have been shown to be significantly less active in antibacterial assays (10).
202
In order to understand better whether the lack of Gram-negative activity of kibdelomycin
203
was due to weak target engagement or a lack of cellular accumulation caused either by efflux or
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poor permeability, we tested the activity of kibdelomycin on a series of hypersusceptible Gram-
205
negative mutants (Table 4). In E. coli, kibdelomycin activity was measured in a knockout of
206
tolC, the outer membrane protein component of the major efflux pump. Additionally, because it
207
has been reported that E. coli mutants of the chromosome partitioning complex MukBEF are
208
hypersusceptible to novobiocin (24), an inhibitor of the GyrB ATPase domain like kibdelomycin,
209
it was of interest to test kibdelomycin against knockouts of mukB and mukF. In Pseudomonas
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aeruginosa kibdelomycin was tested against strain ATCC35151, which has been described as
211
being hypersusceptible to most classes of antibiotics (28), presumably due to multiple defects in
212
membrane permeability. Finally, we tested kibdelomycin against two LPS deficient mutants of
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A. baumannii. Such mutants completely devoid of LPS biosynthesis have been described to be
214
exquisitely more permeable and abrogated for efflux (29), as the outer membrane protein
215
components of the efflux pumps no longer have the correct membrane composition in which to
216
fold and function.
217 218
In all cases, mutants were more susceptible to kibdelomycin than were wild-type parental
219
strains, indicating that the spectrum of activity is influenced by the ability to enter and
220
accumulate in the cell. Depending on the organism, kibdelomycin activity appears to be limited
221
by different factors. In E. coli, efflux appears to be a contributor to lack of activity as the
222
MB4902 lpxC mutant leading to permeability defects alone is not more susceptible to
223
kibdelomycin but tolC knockout strains (BW25113ΔtolC, MB5747, MB5746) are slightly more
224
susceptible. Activity of kibdelomycin can be improved further through mechanism-based
225
synergy as both mukB and mukF mutants are more susceptible, as is the case with novobiocin.
226
Conversely, in Pseudomonas aeruginosa, efflux does not appear to be the main contributor to 8
227
lack of activity as the MB5890 strain, in which 6 major efflux systems have been knocked out, is
228
not more susceptible to kibdelomycin; instead the hyperpermeable ATCC35151 strain is more
229
susceptible to inhibition. Finally, in A. baumannii, kibdelomycin activity is greatly enhanced in
230
both LPS deficient mutants (NCIMB12457ΔlpxA and ATCC19606ΔlpxC) by over 2 orders of
231
magnitude.
232 233
Kibdelomycin is not cross resistant with other gyrase inhibitors
234
We have shown earlier that kibdelomycin is still potently active against (no shift in MIC)
235
novobiocin-resistant S. aureus harboring a single mutation in GyrB (D89G) and only shows
236
moderate cross-resistance (4 fold MIC shift) to coumermycin-resistant S. aureus with three
237
mutations in GyrB (Q136E, I175T, L455I) (9). While the lack of cross-resistance against these
238
antibiotics is important for mechanistic analysis, cross-resistance analysis against quinolone-
239
resistant strains is more critical from a drug development perspective. Kibdelomycin was
240
evaluated against a variety of well-characterized quinolone-resistant S. aureus strains selected
241
through multiple rounds of mutations against quinolones. Mutant strains were sequenced to
242
determine nucleotide changes and mutations were mapped to gyrA, parC and grlA (Table 2).
243
The MIC (0.25-0.5 μg/ml) values of kibdelomycin were not affected by these mutations whereas
244
the MIC values of ciprofloxacin were shifted by 4->512 fold indicating a lack of cross-resistance
245
to kibdelomycin. A quinolone-resistant S. pneumoniae strain with mutations in parC and gyrA
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also retained wild-type level susceptibility to kibdelomycin similar to S. aureus (Table 2).
247 248
Bacterial profiling. Kibdelomycin was profiled against 200 bacterial strains collected from
249
various infection sites and tissue samples collected from patients throughout the world. These
250
strains were selected randomly and represented susceptible and antibiotic resistant strains. Only
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key Gram-positive and selected Gram-negative species that were susceptible to KBD were
252
selected for this larger study including A. baumannii. Linezolid, vancomycin, and levofloxacin
253
were used as comparators. The MIC50, MIC90 and MIC range of all four antibiotics against 200
254
diverse strains are presented in Table 3. Kibdelomycin showed strong activity against S. aureus
255
with MIC50 and MIC90 values of 1 and 2 μg/ml. Many strains showed susceptibility at 0.25
256
μg/ml as was observed in the agar dilution assay. Kibdelomycin MIC was unaffected in
257
linezolid, vancomycin and levofloxacin resistant strains. The activity of Kibdelomycin against a 9
258
small panel of S. epidermis and coagulase negative Staphylococcus was somewhat weaker.
259
Kibdelomycin showed good activity against S. pneumoniae with a narrow MIC range displaying
260
identical MIC50 and MIC90 (2 μg/ml). The activity against S. pyogenes was two fold lower than
261
S. pneumoniae with the same MIC50/MIC90 values of 4 μg/ml. It showed good activity against E.
262
faecalis with an MIC50 (0.5 μg/ml) and an MIC90 (2 μg/ml) and reasonable activity against E.
263
faecium with MIC50 and MIC90 values of 4 μg/ml. Most importantly the activity of
264
Kibdelomycin was indifferent to the vancomycin resistance status of E. faecalis and E. faecium
265
strains. Kibdelomycin showed potent activity against Gram-negative bacteria M. catarrhalis and
266
A. baumannii with MIC90 values of 0.5 and 0.125 μg/ml, respectively. Of the 19 strains of the
267
non-fermenter A. baumannii, only one strain collected from the United States demonstrated an
268
MIC value of >32 μg/ml and that strain also showed resistance to levofloxacin. A second strain
269
with an MIC value of 8 μg/ml was susceptible to levofloxacin (MIC 0.25 ug/ml). The
270
Kidelomycin MICs of the remaining 17 strains were < 0.12 μg/ml. The strains resistant to
271
levofloxacin in this group showed susceptibility to kibdelomycin. The differences in MIC of
272
kibdelomycin between clinical strains and laboratory strains of A. baumannii (Tables 1, 3, 4) are
273
perplexing. It may be due laboratory strain differences IID876 (Table 1) versus ATCC19606 and
274
NCIMB12457 (Table 4) and clinical strains (Table 3), compounded by the chelating tetramic
275
acid structural moiety of kibdelomycin affected by differences in agar (Table 1) and broth media
276
contents (Tables 3 and 4) and need further exploration. Kibdelomycin showed good activity
277
against H. influenzae with MIC50 of 2 μg/ml and MIC90 of 4 μg/ml. As expected, linezolid and
278
vancomycin did not show much activity against Gram-positive bacteria other than moderate
279
activity shown by linezolid against M. catarrhalis.
280 281
Time kill kinetics of kibdelomycin
282
Time kill kinetics of kibdelomycin were performed using the S. aureus ATCC 29213 strain at a
283
final cell density of 106-107 CFU/ml at 1x, 2x, 4x, and 8x of the MIC with incubations up to
284
24hr. Kibdelomycin showed slowly bactericidal activity at 4x and 8x MIC with a 3 log drops in
285
viable bacteria at 24hr (Figure 2). Regrowth was observed for cultures treated with kibdelomycin
286
at 2X MIC (no regrowth of cipro at 2X MIC). Although the culture at 24 hr was not tested for
287
increased MICs resistance would probably not have been selected under the kill curve conditions
288
since the frequency of resistance was previously determined to be 16 >16 >16 >16 >16 >16 >16 16 16 2
0.031 0.031 0.5 0.5 2 8 0.5 4 64 0.125
MIC was determined by CLSI agar diffusion method. All strains were obtained from ATCC or Japanese Society of Bacteriology. P. aeruginosa strains KH4013E (nfxB), KH4014a (nfxC), PAO6006 (nalB) were prepared from PAO4009 and PAO6006, respectively (16).
TABLE 2. In vitro antibacterial activity (minimum inhibitory concentration) of kibdelomycin and levofloxacin against quinolone-resistant Gram-positive strains Strains
Strain no.
Phenotype
Genotype
Kibdelomycin (MIC, μg/ml)
Levofloxacin (MIC, μg/ml)
S. aureus
MS5935
None
0.25
0.25
S. aureus S. aureus S. aureus
MS5935A MS5935B MS5935C
Wild-type parent 1st step mutant 2nd step mutant 3rd step mutant
0.5 0.5 0.5
1 16 64
S. aureus
MS5935D
4th step mutant
0.5
>128
S. pneumoniae
IID553
0.5
1
S. pneumoniae
NC9971
Wild-type parent Quinolone resistant
grlA (S80F) grlA (S80F), gyrA (S84L) grlA (S80F), gyrA (S84L), grlA (E84K) grlA (S80F), gyrA (S84L), grlA (E84K), gyrA (E88V) None parC (S79Y), gyrA (S81F)
0.5
32
MIC was determined by CLSI agar diffusion method. Similar results were obtained with quinolone resistant strains generated from parent S. aureus strains MS5952, MS5867 and 16
485 486 487 488 489 490 491
MR6009. The quinolone resistant S. aureus mutants were selected sequentially from clinical isolate MS5935 (14). The quinolone resistant S. pneumoniae strain was selected from IID553 (15). TABLE 3. Comparative in vitro activity of kibdelomycin, linezolid, vancomycin, and levofloxacin against clinical bacterial strains (n = 200) MIC (µg/ml) Organism type, (n) S. aureus (57)
Antimicrobial agent
MIC50
MIC90
Range
Kibdelomycin Linezolid Vancomycin Levofloxacin
1.00 2.00 1.00 8
2.00 16 4.00 >16
0.25 1.00 0.50 0.25
̶ 4.00 ̶ >16 ̶ >32 ̶ >16
Kibdelomycin Linezolid Vancomycin Levofloxacin
ND ND ND ND
ND ND ND ND
2.00 1.00 1.00 0.50
̶ 8.00 ̶ 2.00 ̶ 2.00 ̶ 8.00
Kibdelomycin Linezolid Vancomycin Levofloxacin
ND ND ND ND
ND ND ND ND
0.50 ̶ 1.00 ̶ 0.50 ̶ 0.125
Kibdelomycin Linezolid Vancomycin Levofloxacin
2.00 1.00 0.25 0.50
2.00 1.00 0.25 16
0.25 – 4.00 ≤ 0.25 ̶ 1.00 0.25 – 0.50 0.50 ̶ 16
Kibdelomycin Linezolid Vancomycin Levofloxacin
4.00 1.00 0.25 0.50
4.00 1.00 0.25 0.50
2.00 – 8.00 0.50 ̶ 1.00 0.25 – 0.50 0.25 ̶ 2.00
Kibdelomycin Linezolid Vancomycin Levofloxacin
0.50 1.00 2.00 >16
2.00 1.00 >32 >16
0.25 – 2.00 0.50 – 1.00 1.00 - >32 1.00 - >16
S. epidermidis (7)
Staphylococcus coagulase negative (8) 8.00 2.00 2.00 ̶ 16
S. pneumoniae (22)
S. pyogenes (15)
E. faecalis (20)
17
E. faecium (18) Kibdelomycin Linezolid Vancomycin Levofloxacin
4.00 2.00 >32 >16
4.00 2.00 >32 >16
2.00 – 4.00 1.00 – 2.00 0.50 - >32 1.00 - >16
Kibdelomycin Linezolid Vancomycin Levofloxacin
0.25 4.00 >32 1
0.50 4.00 >32 2
< 0.015 – 1.00 2.00 – 4.00 32 - >32 0.5 ̶ 4
Kibdelomycin Linezolid Vancomycin Levofloxacin
2.00 >16 >32 0.015
4.00 >16 >32 0.03
0.50 – 8.00 8.00 - >16 32 - >32 0.015 – 0.03
16 >32 8
0.125 >16 >32 >16
32 >16 >32 0.03 - >16
M. catarrhalis (12)
H. influenzae (17)
A. baumannii (19)
492 493 494 495 496 497
Kibdelomycin Linezolid Vancomycin Levofloxacin MIC was determined by CLSI micro dilution method.
TABLE 4. Minimum Inhibitory Concentration (MIC, μg/ml) against key Gram-negative bacteria with systematic changes in permeability and efflux Species
Strain
KBD
RIF
NOVO
CAM
ERY
IMI
E. coli
BW25113 WT
>128
16
>128
32
128
0.25
0.016
4
E. coli
BW25113 ΔtolC BW25113 ΔtolC ΔmukB::kan BW25113 ΔtolC ΔmukF::kan MB4903 WT
32
16
4
8
4
0.5
0.004
1
4
4
0.5
1
2
0.125
0.004
0.5
This study This study
E. coli E. coli E. coli E. coli E. coli E. coli P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa A. baumannii
CIPRO
TET
Source www.cgsc.biology.ya le.edu/ This study
16
8
1
2
4
0.5
0.008
1
>128
16
>128
16
128
0.25
0.031
>128
Young & Silver (30)
MB4902 lpxC101MB5747 tolC::tn10 MB5746 tolC::tn10 lpxC101MB5919 (CB0046) WT MB5890 (CB1101) Efflux mutant* ATCC12055 WT
>128
0.0625
64
4
2
0.063
0.008
64
Young & Silver (30)
128
8
2
2
4
0.5
0.004
64
Kodali et al (31)
32
0.5
2
2
0.25
0.5
0.156
128
Kodali et al (31)
32
32
>128
>128
128
8
1
32
Robertson et al (32)
64
32
128
2
16
1
0.004
0.5
This study
16
8
>128
128
128
1
0.125
16
ATCC.org
ATCC35151 hypersusceptible ATCC19606 WT
1
0.5
2
4
2
0.25
0.063
0.5
ATCC.org
16
2
8
128
64
0.25
1
4
ATCC.org
18
A. baumannii A. baumannii A. baumannii
498 499 500 501 502 503
ATCC19606 ΔlpxCNCIMB12457 WT
0.031 32
NCIMB12457 ΔlpxA-
0.125
0.0005
0.625
16
0.25
0.063
0.25
0.5
4
16
>128
32
0.5
0.5
8
0.0005
0.063
64
0.25
0.063
0.25
0.5
This study www.microbiologics. com This study
MIC was determined by CLSI micro broth dilution method. KBD (kibdelomycin), RIF (rifampicin), NOVO (novobiocin), CAM (chloramhenicol), ERY (ethythromycin), IMI (imipenem), CIPRO (ciprofloxacin), TET (tetracycline), *(mexAB-oprM) (mexCD-oprJ) (mexXY) (mexJKL) (mexHI-opmD) (opmH) TABLE 5. Primers used in this study Name Sequence* P1
tttacagtttgatcgcgctaaatactgcttcaccacaaggaatgcaaatggtgcaggctggagctgcttc
P2
cagacggggccgaagccccgtcgtcgtcatcagttacggaaagggttatgcatatgaatatcctccttag
P3
acgctgattaactggaacggcttttttgcccgaacttttgaccttgacgagtgcaggctggagctgcttc
P4
tggaagcgtttcagggagttgcggcgcaaatcctcgcaggccgacgacatcatatgaatatcctccttag
P5
ctggttgcctgggccagaaaaaatgacttctccatctcgctgccggtagagtgcaggctggagctgcttc
P6
ggctccgtaatcattaatcggctgccatttcgctggcagtccggtgaaatcatatgaatatcctccttag
P7
actcttcctttttcaatattattgaagc
P8
taactgtcagaccaagtttactcatatatac
P9
tgaaaaaggaagagtatgattgaacaagatggattg
P10
ttggtctgacagttagaagaactcgtcaagaaggcg
P11
tgccctgaatgaactgcaagacgaggcagcgc
P12
ttggtctgacagttagaagaactcgtcaagaaggcg
P13
ccaagcttgcatgcctgcagtttgcgtaaatagaatattatgaccg
P14
ccacgctctgattgttcaagaggtagaatggattaaatcgtg
P15
cacgatttaatccattctacctcttgaacaatcagagcgtgg
P16
cggtacccggggatcctctgagcagcagcctagcgcactaacaacc
P17
ccaagcttgcatgcctgcaggcgaaaggcgtattaattaacattac
P18
cacgtatggaattggacagtccacacgattgagagtacgctg
P19
cagcgtactctcaatcgtgtggactgtccaattccatacgtg
P20
cggtacccggggatcccataaaaacaggaaaccttacgtttctaac
504 505
19
O H 2N O
H 3C
O
O CH3 O
O
H 3C O
N
HO H 2C
O
CH 3 CH3 O CH 3
H
H H
O
CH 3 OH
HO H 3C
NH
Cl Cl
O HN
506 507 508
CH 3
Figure 1
20
509 510
Figure 2
21