1. Printed by
F1-354
Chris Pillar, Ph.D.
Eurofins Medinet
14100 Park Meadow Dr., Suite 110
Chantilly, VA 20151
Tel: (703) 480-2535
Email: chris.pillar@eurofinsmedinet.com
Abstract
Background
Materials & Methods
Results
Activity Profile of CXA-101 against Gram-Positive and Gram-Negative
Pathogens by Broth and Agar Dilution
N.P. Brown1, C.M. Pillar1, D.C. Draghi1, M.K. Torres1, P. Grover1, V. Alluru1, C. Thornsberry1, D. F. Sahm1, Y. Ge2
1Eurofins Medinet, Chantilly, VA; 2Calixa Therapeutics, Inc., San Diego, CA
Background: CXA-101 (CXA) is a novel parenteral cephalosporin with excellent activity
against P. aeruginosa (PA). In this study, the in vitro antibacterial profile of CXA was
evaluated against Gram-negative (GN) and -positive (GP) organisms.
Methods: GP (n = 310; 70 S. aureus, 100 S. pneumoniae, 100 β-hemolytic streptococci,
40 enterococci) and GN (n = 785; 300 PA, 20 B. cepacia, 365 Enterobacteriaceae [EN],
50 M. catarrhalis, 50 H. influenzae) isolates collected from 2006-2007 were centrally
tested against CXA and comparators by broth microdilution (CLSI M7-A7) while a
selection of isolates were concurrently tested by agar dilution (CLSI M7-A7).
Results: CXA showed little activity against S. aureus and enterococci (MIC90 ≥64
µg/ml), but had activity by MIC50/MIC90 (µg/ml) similar to ceftazidime (CTZ) against S.
pneumoniae (≤0.12/4) and β-streptococci (Group A: ≤0.12/0.25; Group B: 0.5/1). CXA
had potent activity by MIC50/MIC90 (µg/ml) against EN overall (0.25/1), similar to CTZ
(0.12/2) and lower than cefepime (CEF [≤0.06/0.25]). Similar to other cephalosporins,
CXA was highly active by MIC50/MIC90 (µg/ml) against H. influenzae (≤0.12/0.25) and M.
catarrhalis (≤0.12/0.5). Against PA, the activity of CXA by MIC50/MIC90 (1/2) was
several-fold higher than CTZ (2/32), CEF (4/16), and imipenem (1/16). Both CXA and
CTZ had an MIC50/MIC90 of 4/32 µg/ml against B. cepacia, lower than that of CEF
(32/>32). 98.7% of PA tested had CXA MICs ≤8 µg/ml, the best agent among all anti-
pseudomonal drugs tested. Among evaluated GN and GP, broth microdilution results
correlated well with agar dilution results; MICs were within 1 doubling-dilution for 90% of
the tested isolates.
Conclusion: CXA was the most potent in vitro β-lactam evaluated against PA
highlighting its potential as an anti-pseudomonal β-lactam. It also displayed activity
similar to CTZ and CEF against other evaluated GN and streptococci. Good correlation
between broth and agar MICs illustrate that agar dilution is suitable for susceptibility
testing of CXA.
CXA-101 is a novel parenteral cephalosporin currently under development by
Calixa Therapeutics, Inc. The activity of CXA-101 against P. aeruginosa is of
particular interest due to its enhanced in vitro activity relative to that of other
cephalosporins (ceftazidime and cefepime). It also demonstrated good
activity against most drug resistant P. aeruginosa. It is important to
understand the spectrum of activity of this compound against a variety of
clinically relevant pathogens. Furthermore, it is important to understand how
the activity profile by broth microdilution compares to that observed with agar
dilution. In this study, the in vitro antibacterial profile of CXA-101 and
comparators was evaluated against gram-negative and -positive organisms by
both broth and agar dilution methods.
In total, 310 gram-positive organisms were studied, which included 70 S. aureus,
100 S. pneumoniae, 100 β-hemolytic streptococci, and 40 enterococci, and 785
gram-negative organisms, which included 300 P. aeruginosa, 20 Burkholderia
cepacia, 365 Enterobacteriaceae (80 Escherichia coli, 80 Klebsiella pneumoniae,
50 Citrobacter spp., 55 Enterobacter spp., 50 Proteus mirabilis, 50 Serratia
marcescens), 50 Moraxella catarrhalis, and 50 Haemophilus influenzae. All
isolates were tested in a single laboratory against CXA-101, ceftazidime,
cefepime, and other comparators by broth microdilution according to CLSI M7-A7.
A selection of 30 gram-positive (10 S. aureus and 20 S. pneumoniae) and 50
gram-negative (20 P. aeruginosa, 10 B. cepacia, 10 E. coli, and 10 K.
pneumoniae) isolates were concurrently tested by broth and agar dilution (CLSI
M7-A7). All isolates were clinical isolates collected during routine US
surveillance from 2006 to 2007.
Enterobacteriaceae (Table 3):
• CXA-101 had potent activity against Enterobacteriaceae
overall, with an MIC50 of 0.25 µg/ml and an MIC90 of 1
µg/ml. This activity was similar to that of ceftazidime,
which had an MIC50 of 0.12 µg/ml and an MIC90 of 2
µg/ml, and lower compared to cefepime (MIC50 of ≤0.06
µg/ml and an MIC90 of 0.25 µg/ml).
• MICs of CXA-101, ceftazidime and cefepime were
elevated against ceftazidime non-susceptible isolates
relative to ceftazidime-susceptible isolates; the CXA-
101, ceftazidime, and cefepime MIC50/MIC90s (µg/ml)
against ceftazidime non-susceptible isolates were 32/64,
>64/>64, 2/>32, respectively, and against ceftazidime-
susceptible isolates the MIC50/MIC90 (µg/ml) were
0.25/0.5, 0.12/0.5, ≤0.06/0.12, respectively.
• Similar to ceftazidime and cefepime, based on MIC90
(µg/ml), CXA-101 was most active against E. coli (0.25),
K. pneumoniae (0.5), P. mirabilis (0.5) and S.
marcescens (1) and less active against Enterobacter
spp. (64) and Citrobacter spp. (16).
H. influenzae and M. catarrhalis (Table 4):
• Similar to other cephalosporins, CXA-101 was highly active by MIC50/MIC90
(µg/ml) against H. influenzae (≤0.12/0.25) and M. catarrhalis (≤0.12/0.5),
regardless of β-lactamase status.
• Ceftazidime and imipenem had lower MIC90s of 0.12 and 0.06 µg/ml,
respectively, against M. catarrhalis than cefepime and CXA-101, which had
an MIC90 of 1 and 0.5 µg/ml, respectively.
Broth vs Agar (Table 6, Figure 2):
• Against S. aureus, CXA-101 MICs by agar dilution were equivalent to those by broth microdilution for all
isolates. For S. pneumoniae, 17 of 19 isolates had essential agreement for the two testing methods (agar MICs
at or within one doubling dilution of broth MIC).
• 8 of the 20 tested P. aeruginosa isolates had equivalent broth and agar MICs. In addition, 2/10 B. cepacia, 3/5
E. coli, 3/10 K. pneumoniae each had an equivalent broth and agar MIC; however, a large degree of essential
agreement was observed among the evaluated Gram-negative and Gram-positive with the agar MIC at or within
1 doubling-dilution the broth MIC for 90% of the tested isolates.
• Against the Gram-positive organisms overall, the regression line analysis showed an R-squared value of 0.9532
and an R-value of 0.9763 for CXA-101 (Figure 2a). The R-squared values of ceftazidime and cefepime were
0.8806 and 0.9534, respectively (data not shown).
• Against the Gram-negative organisms overall, the regression line analysis showed an R-squared value of
0.7354 and an R-value of 0.8576 for CXA-101 (Figure 2b). The R-squared values of ceftazidime and cefepime
were 0.9041 and 0.8393, respectively (data not shown).
Acknowledgement
This study was supported by Calixa Therapeutics, Inc.
P. aeruginosa and B. cepacia (Table 1 and Table 2):
• Against P. aeruginosa, the activity of CXA-101 by MIC50 and MIC90 were 1 µg/ml and 2 µg/ml, respectively, which were
multiple-fold higher than ceftazidime (MIC50/MIC90 of 2/32 µg/ml), cefepime (MIC50/MIC90 of 4/16 µg/ml), and imipenem
(MIC50/MIC90 of 1/16 µg/ml).
• Activity of CXA-101 was less affected by ceftazidime resistance among P. aeruginosa (MIC50/MIC90 of 1/1 µg/ml for
ceftazidime-susceptible strains and 2/8 µg/ml for ceftazidime non-susceptible strains) compared to cefepime (MIC50/MIC90
of 4/16 µg/ml for ceftazidime-susceptible strains and 16/>32 µg/ml for ceftazidime non-susceptible strains).
• 99% of P. aeruginosa tested had a CXA-101 MIC ≤8 µg/ml, compared to only 84% and 80% for ceftazidime and cefepime,
respectively, at the same concentration (Figure 1, Table 2). At 2, 4 and 8 µg/ml, CXA-101 inhibited more P. aeruginosa
isolates than other anti-pseudomonal antibiotics.
• Both CXA-101 and ceftazidime had an MIC50/MIC90 of 4/32 µg/ml against B. cepacia, similar to that of ceftazidime (4/>32)
and lower than that of cefepime (32/>32) and imipenem (16/64).
Gram-positives (Table 5):
• CXA-101 showed weak activity against S. aureus with an MIC50
and MIC90 (µg/ml) of 32 and 64, respectively.
• CXA-101 was active against S. pneumoniae, with an MIC50 of
≤0.12 µg/ml and an MIC90 of 4 µg/ml. The MIC90 of CXA-101
was one doubling dilution lower than that of ceftazidime (8 µg/ml),
but two doubling dilutions higher than that of cefepime (1 µg/ml).
• CXA-101 was also active against the β-hemolytic streptococci.
Against S. pyogenes the MIC50/MIC90 (µg/ml) was <0.12/0.25 and
against S. agalactiae the MIC50/MIC90 (µg/ml) was 0.5/1. This
activity was comparable to that of ceftazidime and less than that
of cefepime.
• CXA-101, ceftazidime, and cefepime each was inactive against E.
faecalis and E. faecium; each had an MIC50 and MIC90 of >32
µg/ml.
Conclusions
• CXA-101 was the most potent β-lactam against P. aeruginosa evaluated in this in vitro study. The activity of
CXA-101 against P. aeruginosa also included ceftazidime non-susceptible isolates, as evidenced by
inhibition of 99% of isolates at ≤ 8 µg/ml, including some multiple drug-resistant isolates, thus highlighting its
potential as an anti-pseudomonal β-lactam.
• CXA-101 also displayed activity similar to that of ceftazidime and cefepime against other evaluated Gram-
negatives and streptococci (minimal activity was observed for CXA-101 against evaluated S. aureus; no
appreciable activity was observed against enterococci).
• Susceptibility testing of CXA-101 by agar dilution correlated well with broth microdilution for S. aureus, E.
coli, K. pneumoniae, P. aeruginosa, and S. pneumoniae for which essential agreement between the two
methods (agar MIC within one-doubling dilution of broth MIC) was observed for 90% of the total evaluated
isolates. These results indicate that agar dilution is suitable for determining susceptibility of CXA-101 for
these pathogens.
Figure 2. Scatterplot of CXA-101 broth versus agar against all Gram-positive and Gram-negative
organisms
b. Gram-negative
Table 1. Antimicrobial susceptibility of CXA-101 and comparators
against P. aeruginosa and B. cepacia organisms by phenotype
Organism Antimicrobial agent Phenotype
a
Total n MIC50 MIC90 %S %I %R
Pseudomonas aeruginosa CXA-101 All 300 1 2 
b
 
CTZ S 253 1 1   
CTZ NS 47 2 8   
Ceftazidime All 300 2 32 84.3 3.7 12
CTZ S 253 2 4 100 0 0
CTZ NS 47 64 >64 0 23.4 76.6
Cefepime All 300 4 16 79.7 11.3 9
CTZ S 253 4 16 89.3 9.1 1.6
CTZ NS 47 16 >32 27.7 23.4 48.9
Imipenem All 300 1 16 80.7 5 14.3
CTZ S 253 1 8 86.6 4 9.5
CTZ NS 47 8 32 48.9 10.6 40.4
Burkholderia cepacia CXA-101 All 20 4 32 
b
 
CTZ S 12 4 8   
CTZ NS 8 NA
c
NA   
Ceftazidime All 20 4 32 60 10 30
CTZ S 12 4 4 100 0 0
CTZ NS 8 NA NA 0 25.0 75
Cefepime All 20 32 >32 20.0 10.0 70
CTZ S 12 16 >32 33.3 16.7 50
CTZ NS 8 NA NA 0 0 100
Imipenem All 20 16 64 20 20 60
CTZ S 12 8 32 33.3 25.0 41.7
CTZ NS 8 NA NA 0 12.5 87.5
a
CTZ, ceftazidime; S, susceptible; NS, non-susceptible
b
Dashed line indicates CLSI breakpoints unavailable for interpretation of susceptible (S), intermediate (I), and/or resistant (R )
(µg/ml)
Figure 1. MIC distributions of CXA-101 and
comparators against P. aeruginosa according to
ceftazidime phenotype
Table 2. Cumulative percentage of P. aeruginosa at MIC for all agents
Antimicrobial agent Total ≤0.25 0.25 0.5 1 2 4 8 16 32 ≥64
CXA-101 300 3.3 42.0 81.7 92.3 96.7 98.7 99.0 99.3 100
Ceftazidime 0.3 2.7 18.7 56.3 76.0 84.3 88.0 90.7 100
Cefepime 0.3 0.7 5.7 20.3 42.0 64.3 79.7 91.0 97.0 100
Imipenem 0.7 3.0 14.3 61.0 74.7 80.7 85.7 93.7 100
Meropenem 45.3 50.7 68.0 75.7 83.3 89.3 93.7 98.0 100
Piperacillin-tazobactam 1.7 4.0 6.7 16.3 44.7 66.3 81.0 85.0 100
Tobramycin 2.7 8.7 46.3 83.0 91.0 93.7 94.0 94.3 100
Aztreonam 0.7 4.3 6.3 7.3 24.3 55.7 71.0 82.3 100
MIC (µg/ml)
Table 4. Antimicrobial susceptibility of CXA-101 and comparators
against H. influenzae and M. catarrhalis
Organism Antimicrobial agent Phenotype Total n MIC50 MIC90 %S %I %R
Haemophilus influenzae CXA-101 All 50 ≤0.12 0.25 
a
 
β-lactamase negative 36 ≤0.12 0.25   
β-lactamase positive 14 ≤0.12 0.25   
Ceftazidime All 50 ≤0.06 ≤0.06 100  
β-lactamase negative 36 ≤0.06 0.12 100  
β-lactamase positive 14 ≤0.06 ≤0.06 100  
Cefepime All 50 ≤0.06 0.12 100  
β-lactamase negative 36 ≤0.06 0.12 100  
β-lactamase positive 14 ≤0.06 0.12 100  
Imipenem All 50 0.5 0.5 100  
β-lactamase negative 36 0.5 1 100  
β-lactamase positive 14 0.5 0.5 100  
Moraxella catarrhalis CXA-101 All (β-lactamase positive) 50 ≤0.12 0.5   
Ceftazidime All (β-lactamase positive) 50 ≤0.06 0.12 100  
Cefepime All (β-lactamase positive) 50 0.25 1   
Imipenem All (β-lactamase positive) 50 ≤0.03 0.06   
a
Dashed line indicates CLSI breakpoints unavailable for interpretation of susceptible (S), intermediate (I), and/or resistant (R )
(µg/ml)
Table 6. Agar dilution data and log2 fold differences between CXA-101 agar and broth
microdilution MICs
a. Gram-positive
a. All
0
10
20
30
40
50
60
70
80
90
100
≤0.06 0.12 0.25 0.5 1 2 4 8 16 32 >32
MIC (µg/ml)
%ofisolatesinhibitedatMIC
CXA-101
Ceftazidime
Cefepime
b. Ceftazidime-susceptible
0
10
20
30
40
50
60
70
80
90
100
≤0.06 0.12 0.25 0.5 1 2 4 8 16 32 >32
MIC (µg/ml)
%ofisolatesinhibitedatMIC
CXA-101
Ceftazidime
Cefepime
c. Ceftazidime non-susceptible
0
10
20
30
40
50
60
70
80
90
100
≤0.06 0.12 0.25 0.5 1 2 4 8 16 32 >32
MIC (µg/ml)
%ofisolatesinhibitedatMIC
CXA-101
Ceftazidim e
Cefepim e
Table 3. Antimicrobial susceptibility of CXA-101 and comparators
against Enterobacteriaceae by phenotype
Organism Antimicrobial agent Phenotype
a
Total n MIC50 MIC90 %S %I %R
Enterobacteriaceae CXA-101 All 365 0.25 1 
b
 
CTZ S 335 0.25 0.5   
CTZ NS 30 32 64   
Ceftazidime All 365 0.12 2 91.8 0.3 7.9
CTZ S 335 0.12 0.5 100 0 0
CTZ NS 30 >64 >64 0 3.3 96.7
Cefepime All 365 ≤0.06 0.25 97.3 1.1 1.6
CTZ S 335 ≤0.06 0.12 100 0 0
CTZ NS 30 2 >32 66.7 13.3 20
Escherichia coli CXA-101 All 80 ≤0.12 0.25   
Ceftazidime All 80 0.12 0.5 96.3 1.3 2.5
Cefepime All 80 ≤0.06 0.12 98.8 0 1.3
Klebsiella pneumoniae CXA-101 All 80 ≤0.12 0.5   
Ceftazidime All 80 0.12 0.25 98.8 0 1.3
Cefepime All 80 ≤0.06 ≤0.06 98.8 1.3 0
Citrobacter spp. CXA-101 All 50 0.5 16   
Ceftazidime All 50 0.5 >64 78 0 22
Cefepime All 50 ≤0.06 1 100 0 0
Enterobacter spp. CXA-101 All 55 0.5 64   
Ceftazidime All 55 0.5 >64 74.5 0 25.5
Cefepime All 55 0.12 16 85.5 5.5 9.1
Proteus mirabilis CXA-101 All 50 0.25 0.5   
Ceftazidime All 50 ≤0.06 ≤0.06 100 0 0
Cefepime All 50 ≤0.06 ≤0.06 100 0 0
Serratia marcescens CXA-101 All 50 0.5 1   
Ceftazidime All 50 0.12 0.5 98 0 2
Cefepime All 50 ≤0.06 0.12 100 0 0
a
CTZ, ceftazidime; S, susceptible; NS, non-susceptible
b
Dashed line indicates CLSI breakpoints unavailable for interpretation of susceptible (S), intermediate (I), and/or resistant (R )
c
NA, not applicable, total n <10 isolates
(µg/ml)
Table 5. Antimicrobial susceptibility of CXA-101 and comparators
against gram-positive organisms by phenotype
Organism Antimicrobial agent Phenotype
a
Total n MIC50 MIC90 %S %I %R
Staphylococcus aureus CXA-101 All 70 32 64 
b
 
OXA-S 50 32 32   
OXA-R 20 64 >64   
Ceftazidime All 70 8 32 68.6 5.7 25.7
OXA-S 50 8 8 96.0 4.0 0
OXA-R 20 32 >64 0 10.0 90.0
Cefepime All 70 2 16 88.6 4.3 7.1
OXA-S 50 2 2 100 0 0
OXA-R 20 8 32 60.0 15.0 25.0
Streptococcus pneumoniae CXA-101 All 100 ≤0.12 4   
PEN-S 62 ≤0.12 0.25   
PEN-I 26 0.5 4   
PEN-R 12 4 8   
Ceftazidime All 100 0.25 8   
PEN-S 62 0.12 0.5   
PEN-I 26 0.5 8   
PEN-R 12 8 16   
Cefepime All 100 ≤0.06 1   
PEN-S 62 ≤0.06 ≤0.06 100 0 0
PEN-I 26 0.12 1 100 0 0
PEN-R 12 1 2 75.0 25.0 0
Streptococcus pyogenes CXA-101 All 50 ≤0.12 0.25   
Ceftazidime All 50 0.12 0.12   
Cefepime All 50 ≤0.06 ≤0.06 100  
Streptococcus agalactiae CXA-101 All 50 0.5 1   
Ceftazidime All 50 0.5 0.5   
Cefepime All 50 0.12 0.12 100  
Enterococcus faecalis CXA-101 All 20 64 >64   
Ceftazidime All 20 >64 >64   
Cefepime All 20 >32 >32   
Enterococcus faecium CXA-101 All 20 >64 >64   
Ceftazidime All 20 >64 >64   
Cefepime All 20 >32 >32   
a
OXA, oxacillin; PEN, penicillin; S, susceptible; I, intermediate; R, resistant
b
Dashed line indicates CLSI breakpoints unavailable for interpretation of susceptible (S), intermediate (I), and/or resistant (R )
(µg/ml)
-2 -1 0 1 2
S. aureus 10 10
S. pneumoniae
c
19 1 12 5 1
P. aeruginosa 20 2 10 8
B. cepacia 10 3 4 2 1
E. coli 5 1 3 1
K. pneumoniae 10 4 3 3
Total 74 7 18 38 10 1
% of total 9.5 24.3 51.4 13.5 1.4
a
Negative number indicates that the broth microdilution MIC was lower than the agar dilution MIC, a positive number indicates that the broth
microdilution MIC was higher than the agar dilution MIC, and a zero indicates tat the broth microdilution MIC and the agar dilution MIC were equal
b
MIC values that were off-scale high (>) and that were off-scale low (≤) with no defined endpoint were excluded from the calculations
c
Agar dilution is not a CLSI accepted methodology for susceptibility testing of S. pneumoniae
Organism N
Log2 fold difference between agar and borth MICa, b