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

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To whom correspondence should be addressed. E-mail: [email protected]

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21 22 23 24 25

Abstract

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discovery rate for new antibiotics declines. Kibdelomycin is a recently discovered natural

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product antibiotic that inhibits bacterial growth by inhibiting bacterial DNA replication enzymes

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DNA gyrase and topoisomerase IV. It was reported to be a broad-spectrum aerobic Gram-

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positive agent with selective inhibition of the anaerobic bacterium Clostridium difficile. We

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have extended the profiling of kibdelomycin using over 200 strains of Gram-positive and Gram-

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negative aerobic pathogens recovered from worldwide patient populations. We report MIC50s,

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MIC90s, and bactericidal activity of kibdelomycin. We confirm the Gram-positive spectrum and

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report for the first time that kibdelomycin shows strong activity (MIC90 0.125 μg/ml) against

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clinical strains of the Gram-negative non-fermenter Acinetobacter baumannii but shows only

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weak activity against Pseudomonas aeruginosa. We confirm that kibdelomycin does not show

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cross-resistance with quinolones and coumarin antibiotics in well-characterized resistant strains

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of Staphylococcus aureus and Streptococcus pneumoniae. We also show that kibdelomycin is

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not subject to efflux in Pseudomonas though it is in E. coli and it is generally affected by the

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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

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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

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methicillin resistant Staphylococcus aureus (MRSA) infections alone are responsible for about

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18,000 deaths per year in the United States. The dearth of new antibiotics exists due to lack of

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discovery of novel chemical scaffolds that can be developed into clinically useful antibiotics,

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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

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of these chemical leads originated from nature. Chemical modifications of the antibiotic lead

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structures discovered decades ago have led to incrementally improved antibiotics that continue to

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serve well and provide the current reservoir of clinical antibiotics (3; 4). However, the capacity

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of such modifications is not limitless. As a result, additional improvements on existing chemical

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scaffolds are proving increasingly challenging. The dearth of new antibiotics can be overcome by

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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

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platensimycin, (5; 6) platencin (7; 8) and most recently kibdelomycin (9) and kibdelomycin A

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(Figure 1) (10). The broad spectrum Gram-positive antibiotic, kibdelomycin, was reported in

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2011 and is isolated from a Kibdelosporangium sp. Kibdelomycin exerts its activity by inhibiting

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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

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60 nM) and S. aureus (IC50 9 nM) gyrase supercoiling activity and a less potent inhibitor of the

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corresponding topoisomerase IV decatenating activity (E. coli, IC50 29000 nM; S. aureus, IC50

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500 nM). Kibdelomycin potently inhibited the catalytic E. coli ATPase activity of gyrase B (IC50

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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

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(IC50 9 nM) though a poor inhibitor of the E. coli ParE ATPase (IC50 6400 nM) (10). We

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reported that kibdelomycin is a selective and potent growth inhibitor of Clostridium difficile 3

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without significantly affecting anaerobic Gram-negative bacteria including Bacteroides species

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(11). It showed potent in vivo activity against C. difficile infection without systemic exposure

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(11). We recently reported an X-ray crystal structure of kibdelomycin bound to S. aureus and E.

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coli GyrB and ParE (12). The crystal structure showed that kibdelomycin binds uniquely in a U-

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shaped multi- contact binding mode occupying the ATP binding site with extension to another

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part of the pocket (12). Kibdelomycin exhibits a low frequency of resistance and shows no cross

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resistance to S. aureus strains resistant to other known gyrase inhibitors, such as novobiocin,

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coumermycin and quinolones which is consistent with the novel dual arm U-shaped binding

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mode described above. We describe here the kill kinetics of kibdelomycin against S. aureus and

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activity of kibdelomycin against an expanded panel of wild-type and resistant strains of Gram-

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positive and Gram-negative bacteria. We also studied the effect of efflux pumps and the

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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

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aeruginosa.

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Materials and Methods

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Reagents. All reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise

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indicated. Kibdelomycin and Kibdelomycin A were isolated from Kibdelosporangium sp. now

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named Kibdelosporangium banguiesis, as described earlier (9; 10).

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Bacterial strains. All the strains in Tables 1 and 2 were collected from clinical samples in Japan

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or otherwise stated. The bacterial strains for Table 1 were obtained from ATCC and IID were

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obtained from American type culture collection and the Japanese Society for

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Bacteriology, respectively. The clinical strains in this study were isolated in Japan. Efflux

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deficient E. coli was kindly provided from Okayama University (13). The quinolone resistant S.

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aureus were selected sequentially from four clinical isolates (14). The quinolone resistant S.

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pneumoniae strain was selected from IID553 (15). P. aeruginosa nfxB and nfxC, and nalB strains

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were made from PAO4009 and PAO6006, respectively (16).

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The strains (n = 200) in Table 3 were collected as a part of the Merck SMART surveillance

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studies from (17) throughout the world represented by Belgium (n = 4), China (n = 10), Czech 4

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Republic (n = 1), France (n = 5), Germany (n = 6), Hong Kong (n = 10), Ireland (n = 1), Italy (n

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= 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,

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blood, bronchial washings, ear, eye, nasopharynx/throat/nose, sinus, skin, sputum, tracheal

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aspirate, urine, wound and soft tissue. The strains were selected randomly representing antibiotic

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susceptible and resistant strains. The source of permeability and efflux deficient strains presented

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in Table 4 are listed in the last column of the Table.

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Determination of MICs. The MICs were determined either by broth dilution or agar dilution

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methods recommended by the Clinical and Laboratory Standards Institute in Mueller-Hinton II

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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.

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aureus ATCC29213 at 1x, 2x 4x, and 8x of MIC (determined by microbroth dilution method)

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corresponding to concentrations of 1, 2, 4, and 8 μg/ml, respectively by CLSI guidelines (19).

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The MIC was determined at 5x105 CFU/ml inoculum prior to commencing the time kill

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experiment. Briefly, a stock solution of 5.12 mg/ml of kibdelomycin was prepared in DMSO.

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The inoculum suspension was prepared from growth on a TSA/5% sheep blood plate to equal the

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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

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rpm. After approximately 2 hr, the suspension was again adjusted to equal a 0.5 McFarland

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Standard, was diluted 1:2 in fresh MHBII, and 1 ml was used to inoculate each 125 ml

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Erlenmeyer flasks containing 8.75 ml of sterile MHBII. The final target cell density was

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approximately 106 – 107 CFU/ml. Just prior to T0, 0.25 ml of the appropriate concentration of

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kibdelomycin solution was added to each flask, gently mixed, followed by 1.0 ml of diluted

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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,

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and 0.03 ml was removed from the initial 0.5 McFarland Standard tube for determination of the

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viable count at baseline (T0) by serial ten-fold dilution in MHB II. All vessels were incubated at

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35°C in a New Brunswick Scientific Series 25 Incubated Shaker rotating at 150 rpm to provide 5

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gentle mixing. The vessels were sampled at 2 hr (T2), 4 hr (T4), 6 hr (T6), 8 hr (T8), and 24 hr

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(T24) for determination of viable count. Viable counts were determined by removing 0.3 ml

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from each vessel and serially diluting 10-fold in 0.27 ml of MHBII with the Biomek 2000, then

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plating duplicate 10 μL samples onto TSA with 5% sheep blood plates using the track dilution

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method (20); in brief, 10 μL aliquots were spotted in duplicate across the top of a square 100 mm x

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100 mm TSA+ 5% sheep blood plate. The plates were then tilted at a 45o – 90o angle to allow the

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10 μL aliquot to track across the agar surface to the opposite side of the plate. All plates were

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incubated for 20-24 hr at 35°C. Colonies were counted manually, and the CFU/ml was

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determined from the average count from the duplicate plates, followed by calculation of the

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log10 CFU/ml. A bactericidal effect was defined as a 3 log10 CFU/ml decrease in viable count at

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24 hr relative to the starting inoculum.

143

Deletion of E. coli tolC, mukB and mukF. EctolC was knocked out in strain BW25113 utilizing

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the linear PCR product generated by amplification from pKD4 (21) with the primers P1 and P2

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(Table 5). Transformation and selection on kanamycin were performed as described previously

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(22), yielding strain BW25113ΔtolC::kan. Transformation with pFlp2 (23) allowed for

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subsequent excision of the kanamycin resistance cassette through Flp mediation recombination

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leaving an 84 nucleotide scar between the first and last six codons of EctolC, thus generating

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strain BW25113ΔtolC.

150 151

EcmukB and EcmukF were deleted from strain BW25113ΔtolC using the aforementioned one-

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step λ Red method in conjunction with the linear PCR products generated by amplification from

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pKD4 (21) with the primers P3 and P4 (for EcmukB) and P5 and P6 (for EcmukF).

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Transformation and selection on kanamycin were performed as described previously (22),

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yielding strains BW25113ΔtolCΔmukB::kan and BW25113ΔtolCΔmukF::kan, respectively. As

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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

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grew overnight at 37°C.

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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

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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

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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

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into the final pEX18Km vector.

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Flanking regions of the AblpxA and AblpxC genes were fused together using splicing by overlap

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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

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regions downstream of lpxA and lpxC, respectively. In a second round of PCR, up- and

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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

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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.

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Results

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Kibdelomycin is a broad spectrum aerobic antibiotic

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Kibdelomycin was shown to inhibit the growth of Gram-positive bacteria S. aureus

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(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

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dilution method (9). MIC measurements using the agar dilution method confirm these

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antibacterial activities and help extend the prior observed antibacterial spectrum (Table 1). The

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agar diffusion Kibdelomycin MICs of 0.25 μg/ml against S. aureus and S. pneumoniae strains

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were 4-8 fold better than measured by the microbroth dilution method. Kibdelomycin inhibited

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the growth of Enterococcus faecium with an agar MIC value of 1 μg/ml.

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Kibdelomycin showed potent growth inhibition of the Gram-negative bacterium A.

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baumannii (agar MIC 2 μg/ml), similar to that observed with H. influenzae, but unlike the case 7

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of these two Gram-negative strains, kibdelomycin did not inhibit growth of E. coli nor that of

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most P.aeruginosa PAO1 strains (MIC >16 μg/ml). A similar trend (up to16x vs. E. coli and 64x

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vs. P. aeruginosa lower activity compared to A. baumannii) has been reported for a few of

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NBTI’s (New Bacterial Topoisomerase Inhibitors) binding to Gyrase A/ParC, an entirely

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different binding site than kibdelomycin.(26; 27) The desmethyl congener kibdelomycin A and

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acetate analogs have been shown to be significantly less active in antibacterial assays (10).

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In order to understand better whether the lack of Gram-negative activity of kibdelomycin

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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-

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negative mutants (Table 4). In E. coli, kibdelomycin activity was measured in a knockout of

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tolC, the outer membrane protein component of the major efflux pump. Additionally, because it

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has been reported that E. coli mutants of the chromosome partitioning complex MukBEF are

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hypersusceptible to novobiocin (24), an inhibitor of the GyrB ATPase domain like kibdelomycin,

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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

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being hypersusceptible to most classes of antibiotics (28), presumably due to multiple defects in

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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

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exquisitely more permeable and abrogated for efflux (29), as the outer membrane protein

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components of the efflux pumps no longer have the correct membrane composition in which to

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fold and function.

217 218

In all cases, mutants were more susceptible to kibdelomycin than were wild-type parental

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strains, indicating that the spectrum of activity is influenced by the ability to enter and

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accumulate in the cell. Depending on the organism, kibdelomycin activity appears to be limited

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by different factors. In E. coli, efflux appears to be a contributor to lack of activity as the

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MB4902 lpxC mutant leading to permeability defects alone is not more susceptible to

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kibdelomycin but tolC knockout strains (BW25113ΔtolC, MB5747, MB5746) are slightly more

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susceptible. Activity of kibdelomycin can be improved further through mechanism-based

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synergy as both mukB and mukF mutants are more susceptible, as is the case with novobiocin.

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Conversely, in Pseudomonas aeruginosa, efflux does not appear to be the main contributor to 8

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lack of activity as the MB5890 strain, in which 6 major efflux systems have been knocked out, is

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not more susceptible to kibdelomycin; instead the hyperpermeable ATCC35151 strain is more

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susceptible to inhibition. Finally, in A. baumannii, kibdelomycin activity is greatly enhanced in

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both LPS deficient mutants (NCIMB12457ΔlpxA and ATCC19606ΔlpxC) by over 2 orders of

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magnitude.

232 233

Kibdelomycin is not cross resistant with other gyrase inhibitors

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We have shown earlier that kibdelomycin is still potently active against (no shift in MIC)

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novobiocin-resistant S. aureus harboring a single mutation in GyrB (D89G) and only shows

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moderate cross-resistance (4 fold MIC shift) to coumermycin-resistant S. aureus with three

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mutations in GyrB (Q136E, I175T, L455I) (9). While the lack of cross-resistance against these

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antibiotics is important for mechanistic analysis, cross-resistance analysis against quinolone-

239

resistant strains is more critical from a drug development perspective. Kibdelomycin was

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evaluated against a variety of well-characterized quinolone-resistant S. aureus strains selected

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through multiple rounds of mutations against quinolones. Mutant strains were sequenced to

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determine nucleotide changes and mutations were mapped to gyrA, parC and grlA (Table 2).

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The MIC (0.25-0.5 μg/ml) values of kibdelomycin were not affected by these mutations whereas

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the MIC values of ciprofloxacin were shifted by 4->512 fold indicating a lack of cross-resistance

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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

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various infection sites and tissue samples collected from patients throughout the world. These

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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

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selected for this larger study including A. baumannii. Linezolid, vancomycin, and levofloxacin

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were used as comparators. The MIC50, MIC90 and MIC range of all four antibiotics against 200

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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

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linezolid, vancomycin and levofloxacin resistant strains. The activity of Kibdelomycin against a 9

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small panel of S. epidermis and coagulase negative Staphylococcus was somewhat weaker.

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Kibdelomycin showed good activity against S. pneumoniae with a narrow MIC range displaying

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identical MIC50 and MIC90 (2 μg/ml). The activity against S. pyogenes was two fold lower than

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S. pneumoniae with the same MIC50/MIC90 values of 4 μg/ml. It showed good activity against E.

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faecalis with an MIC50 (0.5 μg/ml) and an MIC90 (2 μg/ml) and reasonable activity against E.

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faecium with MIC50 and MIC90 values of 4 μg/ml. Most importantly the activity of

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Kibdelomycin was indifferent to the vancomycin resistance status of E. faecalis and E. faecium

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strains. Kibdelomycin showed potent activity against Gram-negative bacteria M. catarrhalis and

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A. baumannii with MIC90 values of 0.5 and 0.125 μg/ml, respectively. Of the 19 strains of the

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non-fermenter A. baumannii, only one strain collected from the United States demonstrated an

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MIC value of >32 μg/ml and that strain also showed resistance to levofloxacin. A second strain

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with an MIC value of 8 μg/ml was susceptible to levofloxacin (MIC 0.25 ug/ml). The

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Kidelomycin MICs of the remaining 17 strains were < 0.12 μg/ml. The strains resistant to

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levofloxacin in this group showed susceptibility to kibdelomycin. The differences in MIC of

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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

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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

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contents (Tables 3 and 4) and need further exploration. Kibdelomycin showed good activity

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against H. influenzae with MIC50 of 2 μg/ml and MIC90 of 4 μg/ml. As expected, linezolid and

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vancomycin did not show much activity against Gram-positive bacteria other than moderate

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activity shown by linezolid against M. catarrhalis.

280 281

Time kill kinetics of kibdelomycin

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Time kill kinetics of kibdelomycin were performed using the S. aureus ATCC 29213 strain at a

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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

Kibdelomycin is a bactericidal broad-spectrum aerobic antibacterial agent.

Bacterial resistance to antibiotics continues to grow and pose serious challenges, while the discovery rate for new antibiotics declines. Kibdelomycin...
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