In vitro antibacterial properties of cefiderocol, a novel siderophore cephalosporin, against Gram

42 Pages·2017·1.11 MB·English

AAC Accepted Manuscript Posted Online 23 October 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.01454-17
Copyright © 2017 Ito et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
In vitro antibacterial properties of cefiderocol, a novel
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siderophore cephalosporin, against Gram-negative
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bacteria
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5 Akinobu Ito#, Takafumi Sato, Merime Ota, Miki Takemura, Toru Nishikawa, Shinsuke n
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6 Toba, Naoki Kohira, Satoshi Miyagawa, Naoki Ishibashi, Shuhei Matsumoto, Rio d
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7 Nakamura, Masakatsu Tsuji, and Yoshinori Yamano f
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9 Shionogi & Co., Ltd., Toyonaka, Osaka 561-0825, Japan //
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12 Drug Discovery & Disease Research Laboratory, Shionogi & Co., Ltd. o
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13 Shionogi Pharmaceutical Research Center, Futaba-cho 3-1-1, Toyonaka, Osaka p
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14 561-0825, Japan. 2
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15 Phone: [phone], FAX: [phone], e-mail: [email protected] 1
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17 Running title: Antibacterial properties of cefiderocol t
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19 Key words: Cefiderocol; Time kill; penicillin binding protein (PBP); Morphology;
20 iron-transporter; efflux pump; porin
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Abstract
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22 Cefiderocol (CFDC, S-649266), a novel parenteral siderophore cephalosporin
23 conjugated with a catechol moiety, has a characteristic antibacterial spectrum with a
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24 potent activity against a broad range of aerobic Gram-negative bacterial species
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25 including carbapenem-resistant strains of Enterobacteriaceae and non-fermenting lo
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26 bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii. Cefiderocol
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27 has affinity mainly for penicillin-binding protein 3 (PBP 3) of Enterobacteriaceae and h
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28 non-fermenting bacteria similar to ceftazidime. Deficiency of iron-transporter of PiuA a
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29 in P. aeruginosa or both CirA and Fiu in Escherichia coli caused 16-fold increases in s
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30 cefiderocol MICs, suggesting that these iron transporters contribute to the permeation of /

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31 cefiderocol across outer membrane. Deficiency of OmpK35/36 in Klebsiella
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32 pneumoniae and overproduction of efflux pump MexA-MexB-OprM in P. aeruginosa ,
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33 showed no significant impact on the activity of cefiderocol.
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Introduction
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35 Nosocomial infections caused by Gram-negative bacteria are increasingly
36 difficult to treat due to the global spread of multi-drug resistant (MDR) strains which
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37 are resistant to several antibiotics such as carbapenems, cephalosporins,
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38 aminoglycosides and quinolones (1). The WHO has listed carbapenem-resistant lo
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39 Enterobacteriaceae (CRE), carbapenem-resistant Pseudomonas aeruginosa and
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40 carbapenem-resistant Acinetobacter baumannii, as the pathogens against which urgent h
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41 development of new antibiotics are needed, since the emergence of these resistant a
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42 pathogens poses serious public health issues due to the limited number of treatment s
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43 options (2-3). /

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44 Since the 1980s, numerous attempts to conjugate iron-binding functional
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45 groups onto β-lactams have been designed to hijack the iron uptake systems of ,
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46 Gram-negative bacteria and circumvent the outer membrane barriers (4-6). However,
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47 none of the molecules have been approved for clinical use for various reasons such as e
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48 lack of correlation between in vitro and in vivo efficacy (7-9). Cefiderocol (CFDC,
49 S-649266), a novel catechol-substituted siderophore cephalosporin, is structurally
50 different from other recently developed hydroxypyridone-substituted siderophore
51 monobactam antibiotics such as BAL30072, MB-1, and MC-1, and has been reported to
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52 have the potent antibacterial activity against MDR Gram-negative pathogens including
53 carbapenem-resistant strains of Enterobacteriaceae, P. aeruginosa and A. baumannii as
54 well as potent in vivo efficacy against multiple clinical strains of Gram-negative bacteria
55 in mouse lung infection models (10-16). This is the first report of cefiderocol evaluating
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56 the in vitro features including antibacterial spectrum against Gram-negative and n
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57 Gram-positive bacteria and mode of action such as PBP binding affinity and e
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58 morphological changes as well as impacts of various β-lactamases, efflux pump m

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59 overexpression, and deficiency of porin or iron-transporter on the in vitro activity. :/
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Results
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61 Antibacterial activity against Gram-negative and Gram-positive bacteria
62 MICs of cefiderocol were ≤ 2 μg/mL against a broad range of Gram-negative
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63 bacterial strains including Enterobacteriaceae such as Enterobacter spp., Escherichia
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64 coli, Klebsiella spp., Proteus spp., Providencia spp., Salmonella spp. and Yersinia spp. lo
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65 as well as Vibrio species (Table 1). Cefiderocol showed in vitro activity against
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66 non-fermenting bacteria such as Acinetobacter spp., Pseudomonas spp. and h
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67 Burkholderia spp., and cefiderocol also showed in vitro activity against intrinsic a
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68 multidrug-resistant bacteria of Stenotrophomonas maltophilia and Elizabethkingia s
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69 meningoseptica as well as the causative pathogens for respiratory tract infections such /

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70 as Haemophilus spp., Moraxella catarrhalis, and Bordetella parapertussis. On the other
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71 hand, MIC of cefiderocol against Campylobacter jejuni ATCC 33560 and 794009 as ,
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72 well as ceftriaxone-resistant Neisseria gonorrhoeae 868339 were relatively high (MIC
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73 was >4 μg/mL) among the Gram-negative bacteria tested, though MICs of cefiderocol e
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74 against other two N. gonorrhoeae including azithromycin-resistant strain were ≤0.5
75 μg/mL. MICs of cefiderocol against aerobic Gram-positive bacteria growing in aerobic
76 or microaerophilic atmosphere were ≥4 μg/mL except for S. pneumoniae ATCC 49619,
77 S. pyogenes ATCC 10389 and Micrococcus luteus ATCC 9341, against which MICs of
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78 cefiderocol were 2, 1 and 4 μg/mL, respectively. The activities against these strains of
79 Gram-positive bacteria were weaker than other tested β-lactam compounds. MICs of
80 cefiderocol against anaerobic Gram-negative and Gram-positive bacteria showed
81 variation within genera and were higher than those of cefepime or meropenem with the
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82 cefiderocol MICs of 0.5 to >32 μg/mL except for Fusobacterium necrophorum, against n
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83 which MIC of cefiderocol was 0.031 g/mL (Table 2). Although cefiderocol showed e
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84 activity against some ATCC strains of Bacteroides spp., Prevotella spp. and Clostridium m

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85 spp. with MICs of 1 to 2 g/mL, cefiderocol did not show the potent activity against :/
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86 multiple clinical isolates of Bacteroides spp., Prevotella spp. and Clostridium difficile, .a
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87 of which MIC50s were 32 μg/mL or higher (Table 3). r
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88
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89 Antibacterial activity against Gram-negative bacteria harboring various il 2
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90 β-lactamases 1
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91 Cefiderocol exhibited potent in vitro activity against 33 strains of g
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92 Gram-negative bacteria harboring various kinds of β-lactamases including ESBLs and
93 carbapenemases (Table 4). The MICs of cefiderocol were 8 µg/mL or lower against all
94 the test strains including carbapenemase-producers such as Klebsiella pneumoniae
95 harboring NDM-1, KPC or GES-4, P. aeruginosa harboring VIM or IMP-1, and A.
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96 baumannii harboring OXA-23 and/or OXA-51-like, and OXA-58. Other tested
97 antibiotics including various classes of β-lactams, amikacin and ciprofloxacin showed
98 less activity against most of these carbapenemase-producers, with the MICs of 16
99 µg/mL or more, while colistin showed the MICs of 1 µg/mL or lower.
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100 n
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101 Affinity for penicillin binding proteins, morphological changes, and time ed

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102 kill m

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103 Affinity (IC50s) of cefiderocol for PBPs of E. coli NIHJ JC-2, K. pneumoniae SR22291, ://
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104 P. aeruginosa ATCC 27853, and A. baumannii ATCC 17978 were determined (Table 5). .a
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105 IC50s of cefiderocol against PBP3 of E. coli NIHJ JC-2, K. pneumoniae SR22291, P. r
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106 aeruginosa ATCC 27853, and A. baumannii ATCC 17978 were 0.04, 0.062, 0.06, and
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107 0.67 µg/mL, respectively, which were lower than those of ceftazidime, indicating the il 2
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108 higher affinity of cefiderocol for PBP3 compared to those of ceftazidime. Other than the 1
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109 affinity for PBP3, cefiderocol had the affinity for PBP2 of K. pneumoniae SR22291 as g
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110 ceftazidime showed (IC s of cefiderocol and ceftazidime were 0.063 and 0.41 µg/mL,
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111 respectively), and cefiderocol also had the affinity for PBP1a of P. aeruginosa ATCC
112 27853 (IC s of cefiderocol and ceftazidime were 0.85 and 3.62 µg/mL, respectively).
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113 Morphological changes of these four bacteria were examined by phase-contrast
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114 microscopy after exposure to cefiderocol (Figure S1 and S2). Filamentous cells were
115 observed in all the test strains after exposure to cefiderocol, similar to those observed
116 after exposure to ceftazidime. In the time kill study with the four strains of E. coli NIHJ
117 JC-2, K. pneumoniae SR22291, P. aeruginosa ATCC 27853, and A. baumannii ATCC
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118 17978, cefiderocol reduced the bacterial counts in a similar manner to ceftazidime after n
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119 exposed at 1×, 4×, or 16×MIC, and the killing rates were similar between 4× and e
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120 16×MIC (Figure 1 and 2). m

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121 :/
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122 Effects of transposon insertion into or deletions of genes relating to outer .a
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123 membrane permeability on the activities or
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124 The effects of the deficiency of iron transporters were examined using P.
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125 aeruginosa and E. coli (Table 6 and 7). The MICs of cefiderocol against all the test il 2
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126 strains of P. aeruginosa PAO1 derivative mutant strains which have transposon (Tn) 1
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127 insertion into the genes of iron-transporters including major siderophore receptors for g
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128 pyoverdine (fpvA and fpvB), pyochelin (fptA), or enterobactin (pirA) were from 0.063 to
129 0.125 μg/mL, equivalent to the MIC against parent strain PAO1, with the exception
130 against the strains having Tn insertion into the probable iron transport receptor piuA
131 (Table 6). The MIC of cefiderocol increased to 2 μg/mL (PW8599) by Tn insertion into
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132 piuA, which was complemented by the introduction of wild PiuA (SR-L00252). MICs
133 of ceftazidime ranged from 1 to 2 μg/mL against all the tested strains with Tn insertion
134 into iron-transporter related genes as well as the parent strain of PAO1. MIC of
135 cefiderocol against E. coli BW25113 with deletion of iron-transporters cirA and/or fiu
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136 were 0.063 or 0.125 μg/mL, within 2-fold compared to that against the parent strain, n
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137 whereas the MIC of cefiderocol increased 16-fold by the double knock out of cirA and e
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138 fiu (Table 7). m

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139 MICs of cefiderocol against PAO1 derivative mutant strains which have Tn :/
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140 insertion into the genes of multidrug efflux pump MexAB-OprM, its transcriptional .a
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141 regulator and porin OprD, which are involved in the β-lactam resistance, were examined r
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142 (Table 6). MICs of aztreonam against the strains with Tn insertion into either mexB
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143 (PW1781) or oprM (PW1783), which lost the function of MexAB-OprM efflux pump, il 2
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144 were 16-fold lower than that against PAO1, while the decreases in cefiderocol MICs by 1
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145 Tn insertion were 2- or 4-fold. MIC of cefiderocol was also determined against PW1781 g
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146 or PW1783 in CAMHB which contains ferric iron, and the decreases in cefiderocol
147 MICs were also 2- or 4-fold compared to that against PAO1, respectively. MICs of
148 ceftazidime, aztreonam, and ciprofloxacin against the strains with Tn insertion into
149 either mexR (PW1776) or nalD (PW7066), which lead the overexpression of
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150 MexAB-OprM efflux pump, were 4-fold higher than that against PAO1, while the
151 increases in cefiderocol MICs by Tn insertion were within 2-fold. MIC of cefiderocol
152 was also determined against PW1776 or PW7066 in CAMHB, and the increases in
153 cefiderocol MICs were also 2-fold compared to that against PAO1. Against the strain
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154 with Tn insertion into oprD, imipenem showed 8-fold higher MIC compared to the n
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155 parent strain, while the increases in MIC of cefiderocol and other test antibiotics were e
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156 within 2-fold. m

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157 MIC of cefiderocol was determined against K. pneumoniae NVT2001S and its :/
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158 derivative mutant strains which were deficient in porin ompK35 and/or ompK36, which .a
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159 involved in the resistance to various classes of β-lactam antibiotics including r
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160 carbapenems (Table 7). MIC of meropenem against the double deletion mutant strain
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161 was 8-fold higher than that against the parental strain NVT2001S, whereas the increases il 2
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162 in MICs of cefiderocol and ceftazidime by the deletion of ompK35 and/or ompK36 were 1
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163 2- to 4-fold compared to that against parental strain. g
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