AAC Accepts, published online ahead of print on 17 March 2014 Antimicrob. Agents Chemother. doi:10.1128/AAC.02685-13 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Ceftaroline Heteroresistant Staphylococcus aureus
Stephanie N. Saravolatz, Hayley Martin, Joan Pawlak, BS, Leonard B. Johnson, MD, and Louis D. Saravolatz, MD St John Hospital and Medical Center and Wayne State University School of Medicine, Detroit, Michigan
Key words: Ceftaroline, Staphylococcus aureus, heteroresistance, Methicillin-resistant Staphylococcus aureus Running title: Ceftaroline Heteroresistant S.aureus Contact information: Louis D. Saravolatz, MD, MACP 19251 Mack Ave., Suite 335 Grosse Pointe Woods, Michigan, 48236 Phone :313-343-3362 Fax : 313-343-7784 E mail : [email protected]
35 36 37 38 39
1
40 41 42 43
Abstract
44
susceptible microorganisms, of subpopulations with a lesser susceptibility. Ceftaroline is
45
a novel cephalosporin with activity against MRSA. The aim of this study was to detect
46
the prevalence of ceftaroline heteroresistance in vitro in a select group of Staphylococcus
47
aureus (SA) strains. There were 57 isolates selected for evaluation: 20 MRSA, 20 VISA,
48
7 daptomycin non-susceptible SA (DNSSA), 6 linezolid non-susceptible SA (LNSSA)
49
and 4 hVISA. Minimal inhibitory concentrations and minimal bactericidal
50
concentrations were determined using broth microdilution method according to CLSI
51
guidelines. All isolates were analyzed by pulsed-field gel electrophoresis. Staphylococcal
52
cassette chromosome (SCC) mec types were determined by a multiplex PCR. Population
53
analysis profiles (PAP) were performed to determine heteroresistance on all the isolates
54
using plates made by adding varying amounts of ceftaroline to Brain Heart Infusion agar.
55
The frequencies of resistant subpopulations were one in 104 to 105 organisms. We
56
determined that 12 of the 57 (21%) isolates tested were ceftaroline heteroresistant SA
57
(CHSA). CHSA occurred among strains with reduced susceptibility to vancomycin,
58
daptomycin and linezolid but in none of the USA 300 isolates tested. Evaluation of the
59
heteroresistant strains demonstrated the phenotype was unstable. Further studies are
60
needed to determine whether CHSA has a role in clinical failures and the implications of
61
our study findings.
Heteroresistance refers to the presence, within a large population of antimicrobial
62 63 64 65
2
66 67 68
Ceftaroline is a new parenteral cephalosporin with antimicrobial activity against
69
Staphylococcus aureus (SA) strains with reduced susceptibility to methicillin and
70
vancomycin and has been approved for the treatment of acute bacterial skin and skin
71
structure infections and community acquired pneumonia. It is a novel agent because of
72
its avidity for penicillin-binding proteins (PBPs), including PBP2a, associated with
73
methicillin resistance (1). There are a few strains of S.aureus that are intermediate to
74
ceftaroline by the Clinical and Laboratory Standards Institute (CLSI) established
75
breakpoints (2, 3).
76 77
Heteroresistance refers to the presence, within a larger population of fully antimicrobial
78
susceptible microorganisms, of subpopulations with lesser susceptibility (4). Although
79
the clinical significance still is unclear, this has been reported among various
80
antimicrobial agents used against S.aureus including beta-lactams and vancomycin.
81
Typically the subpopulations with lesser susceptibility are present at a frequency of one
82
sub-clone in every 105 to 106 colonies. This is why it is difficult to detect these clones in
83
normal broth microdilution MIC testing using an inoculum of 5 x 104 CFU/well.
84
Population analysis profiles (PAP) which use a larger inoculum size is considered the
85
most reliable method to detect heteroresistant subpopulations. The aim of this study was
86
to detect the prevalence of ceftaroline heteroresistance in vitro in a select group of
87
Staphylococcus aureus strains.
88 89 90 91 92
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93 94 95 96 97 98 99
Material and Methods Isolates A collection of 57 isolates were selected for evaluation. Methicillin resistant SA (MRSA)
100
(n=20), heteroresistant vancomycin intermediate SA (hVISA) (n=4) and daptomycin non-
101
susceptible SA (DNSSA) (n=7) were isolated from patients admitted to St. John Hospital
102
and Medical Center Detroit, MI. VISA (N=20) and four of the linezolid non-susceptible
103
SA (LNSSA) were obtained through the Network on Antimicrobial Resistance in
104
Staphylococcus aureus (NARSA) program: supported under NIAID/NIH contract
105
#HHSN272200700055C. Two LNSSA isolates were obtained from Robinson Memorial
106
Hospital in Ohio. Ceftaroline was either not given to these patients or the exposure was
107
unknown.
108 109
Susceptibility Testing
110
MICs were determined using microdilution tests with cation-adjusted Mueller-Hinton
111
broth. MICs were determined in accordance with the CLSI guidelines. MICs were read
112
visually as the lowest drug concentration well with no visible bacterial growth. Minimal
113
bactericidal concentrations (MBC) were determined as the antibiotic concentration that
114
reduced the number of viable cells by ≥99% as determined by colony counts. We also
115
performed MICs using the E-test strip containing ceftaroline.
116 117 118
4
119 120
Molecular Testing
121
Staphylococcal cassette chromosome (SCC) mec types were determined by a multiplex
122
PCR method on all isolates (5). The isolates were analyzed by pulsed-field gel
123
electrophoresis (PFGE) using restriction enzyme SmaI. The PFGE gel patterns were
124
compared with the development of a dendrogram using GelCompar II software (Applied
125
Maths). Percent similarities were derived from the unweighted pair group method using
126
arithmetic averages (UPGMA) and based on Dice coefficients. Band position tolerance
127
was set at 1.25 and optimization was set at 0.5%. Isolates were determined to belong to
128
the PFGE strain group (USA-100 to USA-1100) if their similarity coefficient was
129
≥80% (6). The USA-100 to USA-1100 strains used for comparison were obtained from
130
NARSA.
131
Heteroresistance Testing Procedure
132
PAP assays to detect heteroresistance were performed as previously described with the
133
following modifications (7). Testing plates were prepared by adding ceftaroline to Brain
134
Heart Infusion (BHI) agar. BHI agar (Difco) was prepared according to the
135
manufacturers instructions. Ceftaroline (CPT) powder was reconstituted and added to the
136
BHI agar plates at concentrations of 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.0, and
137
4.0μg/ml. The isolates to be tested were grown overnight on blood agar plates (BAP).
138
The overnight BAP culture was suspended in saline and used to prepare samples at 107
139
CFU/ml and 104 CFU/ml. Aliquots of the samples containing 107 CFU/ml were than
140
spiral plated (Whitley Automatic Spiral Plater, Microbiology International) onto a drug
141
free BHI agar plate and on to BHI agar plates containing ceftaroline at all concentrations
5
142
to determine the presence of heteroresistance. Aliquots of the samples containing 104
143
CFU/ml were spiral plated onto the drug free BHI agar plates and plates containing 0.25,
144
0.5 and 0.75 μg/ml of ceftaroline. This was done to obtain an accurate determination of
145
CFU/ml. The plates were incubated at 35° C for 48 hours in ambient air. The colonies
146
were counted after 48 hrs using an automated colony counter (ProtoCOL
147
Frederick,MD,USA). PAP’s were generated by plotting log 10 CFU/ml against the
148
antibiotic concentration. The frequency of resistant subpopulations at the highest drug
149
concentration was calculated by dividing the number of colonies grown on an antibiotic
150
containing plate by the colony counts from the same bacterial inoculum plated onto
151
antibiotic free plates(8). We considered samples with a susceptible ceftaroline MIC and
152
growth in the intermediate or resistant range as heteroresistant. All samples were run
153
three separate times and the results were averaged for a final result. Colonies that grew
154
on the plates containing the highest concentration of ceftaroline were removed and
155
subcultured daily for seven days onto antibiotic-free media. The isolates were subcultured
156
and MICs were performed daily in order to determine if the resistance was stable (9).
157 158
Results
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Table 1a provides the data for PAP for the isolates that did not demonstrate
160
heteroresistance as there was no growth above 1 μg/ml. Among these 41 isolates there
161
were 3 hVISA and 14 VISA strains. There were four isolates (Table 1b) of VISA that
162
showed intermediate susceptibility to ceftaroline and did not demonstrate
163
heteroresistance to ceftaroline. PAP disclosed heteroresistance among 12 isolates tested
164
with growth at ceftaroline concentrations of 1.25 – 3 μg/ml (table 2). Ceftaroline
6
165
heteroresistance was seen among 2 of the 20 VISA strains tested which were not
166
significantly different than 5 of the 20 MRSA strains tested, (P=0.41, Fisher’s Exact
167
Test). The occurrence of ceftaroline heteroresistance also occurred in 2 of 6 LNSSA, 2
168
of 7 DNSSA and 1 of the 4 hVISA isolates. Of note is that although there were 9 USA
169
300 SCC mec type IVa isolates, none of these isolates demonstrated heteroresistance in
170
this study (Table 1a). Frequency of resistance populations ranged from 1.1x 10-5 to
171
2.2x10-4 (table2).
172 173
The heterogeneous growth in the presence of ceftaroline was determined to be unstable.
174
After daily passages onto drug free media, the MICs of the colonies obtained from the
175
plates with the highest concentration of ceftaroline returned to the MIC of the native
176
isolates.
177 178
Discussion
179 180
Among the 57 isolates tested 12 (21%) of the strains demonstrated
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heteroresistance by population analysis profiling method which is considered an
182
acceptable method for determining heteroresistance. Heteroresistance was found in
183
strains which were non-susceptible to daptomycin, resistant to linezolid, and intermediate
184
in susceptibility to vancomycin. Finally, we did identify one strain heteroresistant to
185
both ceftaroline and vancomycin. The rate of ceftaroline heteroresistance in this study is
186
higher than the rate of vancomycin heteroresistance reported to date which was 2.16%
7
187
among MRSA as reported by Liu and Chambers (10) who reviewed 14 studies. More
188
recently it was reported at a higher rate of 8.1% as noted by Khatib et al (11).
189
This rate of ceftaroline heteroresistance may be of concern if heteroresistance is a
190
precursor to resistant isolates. If the selective pressure of prolonged exposure to an
191
antimicrobial agent enhances the likelihood of the emergence of organisms resistant to
192
the therapeutic agent administered, we would expect to see an increasing number of
193
clinical cases resistant to ceftaroline with more widespread use of this agent. The
194
comparison of ceftaroline heteroresistance in this study to other studies reporting
195
vancomycin heteroresistance may not be valid with the selection bias used in identifying
196
our organisms as opposed to the random selection of MRSA isolates used in the
197
vancomycin heteroresistance studies.
198 199
The mechanism for ceftaroline heteroresistance is unknown. We know these strains are
200
virulent as they were isolated from clinical infections including bacteremia, pneumonia
201
and wound infections. We do not know if these strains are more or less virulent than
202
other ceftaroline susceptible strains. To date clinical cases of failure due to ceftaroline
203
heteroresistance have not been reported.
204 205
Vancomycin heteroresistance strains have demonstrated lower growth rates and thicker
206
cell walls than vancomycin susceptible strains (12). In addition these strains also produce
207
greater quantities of PBP 2 and PBP 2’ (13). Several isolates with high ceftaroline MICs
208
(MIC= 4) obtained from an antibiotic resistance surveillance system demonstrated
209
decreased PBP2a binding affinity due to alterations in the PBP2a (14). Although our
8
210
study did not evaluate strains for PBP production and cell wall thickness, we did not see a
211
correlation with ceftaroline heteroresistance and vancomycin heteroresistance. Recent in
212
vitro studies of ceftaroline activity against MRSA isolates with reduced vancomycin
213
susceptibility demonstrated increased activity compared with isolates with lower
214
vancomycin MICs (15), however, this observation was not seen among the isolates
215
evaluated in this study.
216 217
The clinical significance of ceftaroline heteroresistance is unclear. Heteroresistant strains
218
were mainly SCC mec type II (75%) and no strains were found among the SCC mec type
219
III or IVa. The small sample size of the CA-MRSA isolates in this study limits the
220
generalizability of these results. Studies evaluating the frequency of ceftaroline
221
heteroresistance among a larger set of CA-MRSA isolates should be performed to
222
confirm this finding.
223 224
The information in this study should suggest caution by clinicians using ceftaroline in
225
patients with resistance to other anti-MRSA agents, as heteroresistance was seen in
226
isolates demonstrating reduced susceptibility to daptomycin and linezolid and increased
227
ceftaroline MICs were noted in four VISA isolates. To date the occurrence of ceftaroline
228
heteroresistance has not been shown to be a risk factor for ceftaroline clinical failure and
229
the mechanism for the development of these strains is not known. Further work should
230
be done to evaluate the risk factors for and clinical significance of ceftaroline
231
heteroresistance.
232
9
233 234
Acknowledgement: Funding for the study was provided by Forest Laboratories, Inc.
235
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303
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304 305 306 307 308 309 310
311 312 313 314
Table 1a Results of samples that did not show growth above 1 μg/ml of ceftaroline on PAP plates. CPT MIC
CPT MBC
CPT E-test
PAP Results (log10 CFU/ml)
μg/ml
μg/ml
μg/ml
0
SA-11
0.5
0.5
SA-21 SA-23 SA-25 TX-O D-7 D-10
0.5 0.5 0.5 0.5 1 1
0.5 0.5 1 0.5 1 1
D-17 D-20 D-21 ND-10 ND-27 ND-29
1 0.5 1 0.5 0.5 1
ND-30 END-12 JhVISA-1 JhVISA-3 JhVISA-4 DNS-1
a
VAN MBC
μg/ml
μg/ml
L knee
1
1
MRSA
Buttock Thigh Unknown Blood Sputum Unknown
1 1 1 1 1 1
1 1 1 1 1 1
MRSA MRSA MRSA MRSA MRSA MRSA
Wound Blood Blood Respiratory Respiratory Respiratory
1 1 1 0.5 1 1
1 1 1 1 1 1
MRSA MRSA MRSA MRSA MRSA MRSA
USA-600 USA-100 No match USA-100 USA-300 USA-100
Respiratory Respiratory Blood Blood Blood Blood
1 1 1 1 2 1
1 1 1 1 2 2
MRSA MRSA hVISA hVISA hVISA DNSSA
III IVa II IVa II MSSA
No match USA-300 USA-100 USA-300 No match No match
Blood Blood Blood Blood Mu 50 Eye
2 1 2 2 8 8
2 2 2 2 8 16
DNSSA DNSSA DNSSA DNSSA VISA VISA
0 0 2.5 0 0 3.5
II II II IV d II II
USA-100 USA-100 No match USA-500 USA-100 No match
Blood Wound Blood Unknown Bone Unknown
8 4 4 4 4 8
8 8 4 4 4 8
VISA VISA VISA VISA VISA VISA
3.1 0 4.2 0 4.3 3.7
2.5 0 0 0 3.5 3
II MSSA III IV d II I
No match No match No match USA-500 USA-100 No match
Bile Bile Unknown Wound Blood Unknown
4 8 4 4 4 4
4 16 8 4 4 8
VISA VISA VISA VISA VISA VISA
2.4 4.8 0 4.4
0 4.2 0 0
II IV II II
USA-100 No match No match USA-100
Sputum Unknown Blood Blood
1 1 1 1
1 1 1 1
LNSSA LNSSA LNSSA LNSSA
0.25
0.5
0.75
0.5
7.2
7.1
4.9
3.2
0
IV a
USA-300
0.5 0.5 0.5 0.5 1 1
7.2 7 7.1 7.2 6.8 6.9
7.1 6.9 7 7.2 6.7 6.9
4.3 5.4 5 6.1 6.8 6.8
3.2 3.8 3.2 3.7 6 5.4
0 0 0 0 0 3.1
IV a IV a IV a IV a II II
USA-300 USA-300 USA-300 USA-300 No match USA-600
1 1 1 0.5 1 1
1 0.75 1 0.5 1 1
6.9 7.4 6.8 7.1 7.1 7
6.9 7.4 6.7 6.9 7.1 6.9
6.9 4.8 5.5 3.7 6.4 6.8
5.8 0 2.9 2.7 3.3 5.1
3.7 0 0 0 0 0
II IV a II IV II II
USA-600 USA-300 USA-600 No match USA-100 USA-600
1 0.5 1 0.5 0.5 0.5
1 1 1 1 1 1
1 1 1 1 0.75 1
6.9 6.6 6.7 6.8 7.1 7.1
6.9 6.6 6.7 6.9 7 7
6.8 6.3 6.6 6.7 5 6.9
5.5 4.5 4.9 5.6 4 6.2
4.4 2.5 0 2.7 3.7 2.5
II II II II IVa II
DNS-4 DNS-5 DNS-6 DNS-7 NRS-1 NRS-14
1 0.5 1 0.5 1 0.5
1 0.5 1 1 1 0.5
1 0.75 0.75 0.75 0.75 0.25
7 7.1 7 7 6.8 6.8
6.9 7 7 7 6.7 6.7
6.6 6.6 6.6 6.7 6.6 4.3
4.3 2.8 0 4.1 6.6 0
3.3 0 0 2.6 4.5 0
NRS-17 NRS-18 NRS-19 NRS-21 NRS-23 NRS-49
1 0.5 0.5 0.5 1 1
1 0.5 1 0.5 1 2
0.75 0.5 0.25 0.5 0.5 0.5
6.8 6.3 6.3 6.4 6.2 6.5
6.7 6.2 6.2 6.3 6 6.5
5.9 3.4 6.1 5.4 5.4 6.5
0 0 4.9 2.5 3.3 5.9
NRS-51 NRS-52 NRS-56 NRS-73 NRS-126 NRS-272
1 0.25 1 0.5 1 1
1 0.25 1 0.5 1 1
1 0.25 0.75 0.38 1 0.5
6.9 7.2 6.3 7.1 6.9 6.7
6.9 3 6.2 7.1 6.9 6.5
6.5 0 6.2 4.1 6.4 5
NRS-127 NRS-271 LNSSA-10 LNSSA-11 a
0.5 1 0.5 1
1 1 1 1
0.75 1 0.75 1
7 7.1 6.9 6.7
7.1 7.1 6.9 6.6
6.1 6.6 3.6 5.9
Sample
a
VAN MIC
a
a
a
1
SCC
Pulse
Source
Concentration of CPT in PAP plates (μg/ml) CPT=ceftaroline
VAN=vancomycin
14
Sample Type
315 316 317 318 319 320 321 322 323
324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351
Table 1b Results of ceftaroline intermediate organisms that did not show heteroresistance
CPT
CPT
MIC
MBC E-test
μg/ml
μg/ml
μg/ml
0
NRS-39
2
2
1.5
6.4
NRS-54
2
2
1
NRS-65
2
2
NRS-283
2
2
Sample
a b
CPT a
PAP Results (log10CFU/ml) a
a
0.75
6.3
6.2
5.4
6.8
6.8
6.6
2
6.3
6.3
1.5
6.9
6.9
0.25 0.5
a
a
1
a
a
a
2.5
2
a,b
1.25
1.5
SCC
4.2
3
2.4
0
0
I
6.2
4.6
4.3
4
3.7
0
III
6.3
6.2
6.1
5.2
4.6
4
0
III
6.8
6.8
6.5
5.2
4.1
2.8
2.5
II
Pulse No match No match No match USA200
VAN
VAN
MIC
MBC Sample μg/ml Type
Source
μg/ml
Urine
8
8
VISA
Unknown
4
4
VISA
Unknown
4
8
VISA
Unknown
4
4
VISA
Concentration of CPT in PAP plates (μg/ml) For all organisms there was no growth at CPT concentrations of 3μg/ml or 4μg/ml CPT=ceftaroline VAN=vancomycin
15
352 353 354 355 356 357 358 359 360
361 362 363 364 365 366 367 368 369 370 371 372 373
Table 2 Results of samples that were determined to be heteroresistant.
a
CPT MIC
CPT CPT MBC E-test
Sample
μg/ml
μg/ml
μg/ml
0
D-9
1
1
1
D-11
1
1
D-27
1
ND-14
a
PAP Results (log10 CFU/ml) a
0.25
a
0.5
7
6.9
1
6.9
2
1
1
1
ND-33
1
DNS-2
a
0.75
a
1
1.25
6.9
4.7
3.8
6.9
6.8
6.1
6.9
6.9
6.8
1
7
7
1
1
6.8
1
1
1
DNS-3
1
2
NRS-3
1
NRS-118
a
a
1.5
a
a
2
2.5
3.3
2.7
2.3
2.1
3.8
3.2
2.8
2.4
6.1
3
2.6
2.6
6.7
5.9
3.1
2.6
6.8
6.7
5.9
3.1
6.9
6.8
6.7
6.2
1.5
6.9
6.8
6.7
1
1.5
6.4
6.3
1
2
1.5
6.8
NRS-120
1
1
1
NRS-121
1
1
JhVISA-2
1
1
a
VAN VAN MIC MBC
a
Frequency
0
0
1.1x10
2
0
0
1.3x10
0
0
0
0
2.3
2.1
0
0
2.5
2.2
0
0
3.6
2.8
2.5
0
6.5
5.9
3.1
2.7
6.3
6.2
5.2
3
6.7
6.7
6.6
6.5
6.8
6.8
6.5
5.9
1
6.8
6.7
6.4
1
6.7
6.7
6.6
3
4
Source
Sample Type
SCC
Pulse
-5
II
USA-100
Blood
1
1
MRSA
-5
II
USA-600
Blood
1
1
MRSA
5.2x10
-5
II
USA-100
Wound
1
1
MRSA
0
1.1x10
-5
II
USA-100
Respiratory
1
1
MRSA
0
0
1.4x10
-5
II
No match
Respiratory
2
2
MRSA
0
0
0
4.8x10
-5
II
No match
Blood
2
2
DNSSA
2.4
0
0
0
3.8x10
-5
II
USA-100
Blood
2
2
DNSSA
2.6
2
0
0
0
3.8x10
-5
II
USA-100
Peritoneal
8
8
VISA
5.7
4.5
3.3
2.6
2.3
0
3.2x10
-5
I
No match
Respiratory
8
8
VISA
4.4
2.7
2.6
0
0
0
0
6.9x10
-5
IV d
USA-500
Unknown
2
2
LNSSA
5.9
4.8
3.1
0
0
0
0
0
2.2x10
-4
IV d
USA-500
Unknown
1
2
LNSSA
4.5
3.4
3.2
3
2.5
2
0
0
2.1x10
-5
II
USA-100
Blood
2
2
hVISA
μg/ml
μg/ml
Concentration of CPT in PAP plates (μg/ml) CPT=ceftaroline VAN=vancomycin
16
374
17
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Ceftaroline Heteroresistant Staphylococcus aureus
Stephanie N. Saravolatz, Hayley Martin, Joan Pawlak, BS, Leonard B. Johnson, MD, and Louis D. Saravolatz, MD St John Hospital and Medical Center and Wayne State University School of Medicine, Detroit, Michigan
Key words: Ceftaroline, Staphylococcus aureus, heteroresistance, Methicillin-resistant Staphylococcus aureus Running title: Ceftaroline Heteroresistant S.aureus Contact information: Louis D. Saravolatz, MD, MACP 19251 Mack Ave., Suite 335 Grosse Pointe Woods, Michigan, 48236 Phone :313-343-3362 Fax : 313-343-7784 E mail : [email protected]
35 36 37 38 39
1
40 41 42 43
Abstract
44
susceptible microorganisms, of subpopulations with a lesser susceptibility. Ceftaroline is
45
a novel cephalosporin with activity against MRSA. The aim of this study was to detect
46
the prevalence of ceftaroline heteroresistance in vitro in a select group of Staphylococcus
47
aureus (SA) strains. There were 57 isolates selected for evaluation: 20 MRSA, 20 VISA,
48
7 daptomycin non-susceptible SA (DNSSA), 6 linezolid non-susceptible SA (LNSSA)
49
and 4 hVISA. Minimal inhibitory concentrations and minimal bactericidal
50
concentrations were determined using broth microdilution method according to CLSI
51
guidelines. All isolates were analyzed by pulsed-field gel electrophoresis. Staphylococcal
52
cassette chromosome (SCC) mec types were determined by a multiplex PCR. Population
53
analysis profiles (PAP) were performed to determine heteroresistance on all the isolates
54
using plates made by adding varying amounts of ceftaroline to Brain Heart Infusion agar.
55
The frequencies of resistant subpopulations were one in 104 to 105 organisms. We
56
determined that 12 of the 57 (21%) isolates tested were ceftaroline heteroresistant SA
57
(CHSA). CHSA occurred among strains with reduced susceptibility to vancomycin,
58
daptomycin and linezolid but in none of the USA 300 isolates tested. Evaluation of the
59
heteroresistant strains demonstrated the phenotype was unstable. Further studies are
60
needed to determine whether CHSA has a role in clinical failures and the implications of
61
our study findings.
Heteroresistance refers to the presence, within a large population of antimicrobial
62 63 64 65
2
66 67 68
Ceftaroline is a new parenteral cephalosporin with antimicrobial activity against
69
Staphylococcus aureus (SA) strains with reduced susceptibility to methicillin and
70
vancomycin and has been approved for the treatment of acute bacterial skin and skin
71
structure infections and community acquired pneumonia. It is a novel agent because of
72
its avidity for penicillin-binding proteins (PBPs), including PBP2a, associated with
73
methicillin resistance (1). There are a few strains of S.aureus that are intermediate to
74
ceftaroline by the Clinical and Laboratory Standards Institute (CLSI) established
75
breakpoints (2, 3).
76 77
Heteroresistance refers to the presence, within a larger population of fully antimicrobial
78
susceptible microorganisms, of subpopulations with lesser susceptibility (4). Although
79
the clinical significance still is unclear, this has been reported among various
80
antimicrobial agents used against S.aureus including beta-lactams and vancomycin.
81
Typically the subpopulations with lesser susceptibility are present at a frequency of one
82
sub-clone in every 105 to 106 colonies. This is why it is difficult to detect these clones in
83
normal broth microdilution MIC testing using an inoculum of 5 x 104 CFU/well.
84
Population analysis profiles (PAP) which use a larger inoculum size is considered the
85
most reliable method to detect heteroresistant subpopulations. The aim of this study was
86
to detect the prevalence of ceftaroline heteroresistance in vitro in a select group of
87
Staphylococcus aureus strains.
88 89 90 91 92
3
93 94 95 96 97 98 99
Material and Methods Isolates A collection of 57 isolates were selected for evaluation. Methicillin resistant SA (MRSA)
100
(n=20), heteroresistant vancomycin intermediate SA (hVISA) (n=4) and daptomycin non-
101
susceptible SA (DNSSA) (n=7) were isolated from patients admitted to St. John Hospital
102
and Medical Center Detroit, MI. VISA (N=20) and four of the linezolid non-susceptible
103
SA (LNSSA) were obtained through the Network on Antimicrobial Resistance in
104
Staphylococcus aureus (NARSA) program: supported under NIAID/NIH contract
105
#HHSN272200700055C. Two LNSSA isolates were obtained from Robinson Memorial
106
Hospital in Ohio. Ceftaroline was either not given to these patients or the exposure was
107
unknown.
108 109
Susceptibility Testing
110
MICs were determined using microdilution tests with cation-adjusted Mueller-Hinton
111
broth. MICs were determined in accordance with the CLSI guidelines. MICs were read
112
visually as the lowest drug concentration well with no visible bacterial growth. Minimal
113
bactericidal concentrations (MBC) were determined as the antibiotic concentration that
114
reduced the number of viable cells by ≥99% as determined by colony counts. We also
115
performed MICs using the E-test strip containing ceftaroline.
116 117 118
4
119 120
Molecular Testing
121
Staphylococcal cassette chromosome (SCC) mec types were determined by a multiplex
122
PCR method on all isolates (5). The isolates were analyzed by pulsed-field gel
123
electrophoresis (PFGE) using restriction enzyme SmaI. The PFGE gel patterns were
124
compared with the development of a dendrogram using GelCompar II software (Applied
125
Maths). Percent similarities were derived from the unweighted pair group method using
126
arithmetic averages (UPGMA) and based on Dice coefficients. Band position tolerance
127
was set at 1.25 and optimization was set at 0.5%. Isolates were determined to belong to
128
the PFGE strain group (USA-100 to USA-1100) if their similarity coefficient was
129
≥80% (6). The USA-100 to USA-1100 strains used for comparison were obtained from
130
NARSA.
131
Heteroresistance Testing Procedure
132
PAP assays to detect heteroresistance were performed as previously described with the
133
following modifications (7). Testing plates were prepared by adding ceftaroline to Brain
134
Heart Infusion (BHI) agar. BHI agar (Difco) was prepared according to the
135
manufacturers instructions. Ceftaroline (CPT) powder was reconstituted and added to the
136
BHI agar plates at concentrations of 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.0, and
137
4.0μg/ml. The isolates to be tested were grown overnight on blood agar plates (BAP).
138
The overnight BAP culture was suspended in saline and used to prepare samples at 107
139
CFU/ml and 104 CFU/ml. Aliquots of the samples containing 107 CFU/ml were than
140
spiral plated (Whitley Automatic Spiral Plater, Microbiology International) onto a drug
141
free BHI agar plate and on to BHI agar plates containing ceftaroline at all concentrations
5
142
to determine the presence of heteroresistance. Aliquots of the samples containing 104
143
CFU/ml were spiral plated onto the drug free BHI agar plates and plates containing 0.25,
144
0.5 and 0.75 μg/ml of ceftaroline. This was done to obtain an accurate determination of
145
CFU/ml. The plates were incubated at 35° C for 48 hours in ambient air. The colonies
146
were counted after 48 hrs using an automated colony counter (ProtoCOL
147
Frederick,MD,USA). PAP’s were generated by plotting log 10 CFU/ml against the
148
antibiotic concentration. The frequency of resistant subpopulations at the highest drug
149
concentration was calculated by dividing the number of colonies grown on an antibiotic
150
containing plate by the colony counts from the same bacterial inoculum plated onto
151
antibiotic free plates(8). We considered samples with a susceptible ceftaroline MIC and
152
growth in the intermediate or resistant range as heteroresistant. All samples were run
153
three separate times and the results were averaged for a final result. Colonies that grew
154
on the plates containing the highest concentration of ceftaroline were removed and
155
subcultured daily for seven days onto antibiotic-free media. The isolates were subcultured
156
and MICs were performed daily in order to determine if the resistance was stable (9).
157 158
Results
159
Table 1a provides the data for PAP for the isolates that did not demonstrate
160
heteroresistance as there was no growth above 1 μg/ml. Among these 41 isolates there
161
were 3 hVISA and 14 VISA strains. There were four isolates (Table 1b) of VISA that
162
showed intermediate susceptibility to ceftaroline and did not demonstrate
163
heteroresistance to ceftaroline. PAP disclosed heteroresistance among 12 isolates tested
164
with growth at ceftaroline concentrations of 1.25 – 3 μg/ml (table 2). Ceftaroline
6
165
heteroresistance was seen among 2 of the 20 VISA strains tested which were not
166
significantly different than 5 of the 20 MRSA strains tested, (P=0.41, Fisher’s Exact
167
Test). The occurrence of ceftaroline heteroresistance also occurred in 2 of 6 LNSSA, 2
168
of 7 DNSSA and 1 of the 4 hVISA isolates. Of note is that although there were 9 USA
169
300 SCC mec type IVa isolates, none of these isolates demonstrated heteroresistance in
170
this study (Table 1a). Frequency of resistance populations ranged from 1.1x 10-5 to
171
2.2x10-4 (table2).
172 173
The heterogeneous growth in the presence of ceftaroline was determined to be unstable.
174
After daily passages onto drug free media, the MICs of the colonies obtained from the
175
plates with the highest concentration of ceftaroline returned to the MIC of the native
176
isolates.
177 178
Discussion
179 180
Among the 57 isolates tested 12 (21%) of the strains demonstrated
181
heteroresistance by population analysis profiling method which is considered an
182
acceptable method for determining heteroresistance. Heteroresistance was found in
183
strains which were non-susceptible to daptomycin, resistant to linezolid, and intermediate
184
in susceptibility to vancomycin. Finally, we did identify one strain heteroresistant to
185
both ceftaroline and vancomycin. The rate of ceftaroline heteroresistance in this study is
186
higher than the rate of vancomycin heteroresistance reported to date which was 2.16%
7
187
among MRSA as reported by Liu and Chambers (10) who reviewed 14 studies. More
188
recently it was reported at a higher rate of 8.1% as noted by Khatib et al (11).
189
This rate of ceftaroline heteroresistance may be of concern if heteroresistance is a
190
precursor to resistant isolates. If the selective pressure of prolonged exposure to an
191
antimicrobial agent enhances the likelihood of the emergence of organisms resistant to
192
the therapeutic agent administered, we would expect to see an increasing number of
193
clinical cases resistant to ceftaroline with more widespread use of this agent. The
194
comparison of ceftaroline heteroresistance in this study to other studies reporting
195
vancomycin heteroresistance may not be valid with the selection bias used in identifying
196
our organisms as opposed to the random selection of MRSA isolates used in the
197
vancomycin heteroresistance studies.
198 199
The mechanism for ceftaroline heteroresistance is unknown. We know these strains are
200
virulent as they were isolated from clinical infections including bacteremia, pneumonia
201
and wound infections. We do not know if these strains are more or less virulent than
202
other ceftaroline susceptible strains. To date clinical cases of failure due to ceftaroline
203
heteroresistance have not been reported.
204 205
Vancomycin heteroresistance strains have demonstrated lower growth rates and thicker
206
cell walls than vancomycin susceptible strains (12). In addition these strains also produce
207
greater quantities of PBP 2 and PBP 2’ (13). Several isolates with high ceftaroline MICs
208
(MIC= 4) obtained from an antibiotic resistance surveillance system demonstrated
209
decreased PBP2a binding affinity due to alterations in the PBP2a (14). Although our
8
210
study did not evaluate strains for PBP production and cell wall thickness, we did not see a
211
correlation with ceftaroline heteroresistance and vancomycin heteroresistance. Recent in
212
vitro studies of ceftaroline activity against MRSA isolates with reduced vancomycin
213
susceptibility demonstrated increased activity compared with isolates with lower
214
vancomycin MICs (15), however, this observation was not seen among the isolates
215
evaluated in this study.
216 217
The clinical significance of ceftaroline heteroresistance is unclear. Heteroresistant strains
218
were mainly SCC mec type II (75%) and no strains were found among the SCC mec type
219
III or IVa. The small sample size of the CA-MRSA isolates in this study limits the
220
generalizability of these results. Studies evaluating the frequency of ceftaroline
221
heteroresistance among a larger set of CA-MRSA isolates should be performed to
222
confirm this finding.
223 224
The information in this study should suggest caution by clinicians using ceftaroline in
225
patients with resistance to other anti-MRSA agents, as heteroresistance was seen in
226
isolates demonstrating reduced susceptibility to daptomycin and linezolid and increased
227
ceftaroline MICs were noted in four VISA isolates. To date the occurrence of ceftaroline
228
heteroresistance has not been shown to be a risk factor for ceftaroline clinical failure and
229
the mechanism for the development of these strains is not known. Further work should
230
be done to evaluate the risk factors for and clinical significance of ceftaroline
231
heteroresistance.
232
9
233 234
Acknowledgement: Funding for the study was provided by Forest Laboratories, Inc.
235
REFERENCES
236 237
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3. Saravolatz LD, Pawlak J, Johnson LB. 2012. In Vitro Susceptibilities and Molecular
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Analysis of Vancomycin-Intermediate and Vancomycin-Resistant Staphylococcus aureus
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7. Vidaillac C, Leonard SN, Rybak MJ. 2009. In Vitro Activity of Ceftaroline against
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9. Boyle-Vavra S, Berke SK, Lee JC, Daum RS. 2000. Reversion of the Glycopeptide
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10. Liu C, Chambers HF. 2003. Staphylococcus aureus with Heterogeneous Resistance
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Diagnostic Methods. Antimicrob. Agents Chemother. 47:3040-3045.
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11. Khatib R, Jinson J, Musta A, Sharma M, Fakih MG, Johnson LB, Riederer K,
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Shemes S. 2011. Relevance of vancomycin-intermediate susceptibility and
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J. Antimicrob.Chemother. 66:1594-1599.
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12. Smith TL, Pearson ML, Wilcox KR, Cruz C, Lancaster MV, Robinson-Dunn B,
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Tenover FC, Zervos MJ, Band JD,White E, Jarvis WR. 1999. Emergence of
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vancomycin resistance in Staphylococcus aureus. N. Engl. J. Med. 340:493-501.
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13. Hanaki H, Kuwahara-Arai K, Boyle-Vavra S, Daum RS, Labischinski H,
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Hiramatsu K. 1998. Activated cell-wall synthesis is associated with vancomycin
290
resistance in methicillin-resistant Staphylococcus aureus clinical strains Mu3 and Mu50.
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14. Mendes RE, Tsakris A, Sader HS, Jones RN, Biek D, McGhee P, Appelbaum
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PC, Kosowksa-Shick K. 2012. Characterization of methicillin-resistant Staphylococcus
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15. Werth BJ, Steed ME, Kaatz GW, Rybak MJ. 2013. Evaluation of Ceftaroline
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302
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303
13
304 305 306 307 308 309 310
311 312 313 314
Table 1a Results of samples that did not show growth above 1 μg/ml of ceftaroline on PAP plates. CPT MIC
CPT MBC
CPT E-test
PAP Results (log10 CFU/ml)
μg/ml
μg/ml
μg/ml
0
SA-11
0.5
0.5
SA-21 SA-23 SA-25 TX-O D-7 D-10
0.5 0.5 0.5 0.5 1 1
0.5 0.5 1 0.5 1 1
D-17 D-20 D-21 ND-10 ND-27 ND-29
1 0.5 1 0.5 0.5 1
ND-30 END-12 JhVISA-1 JhVISA-3 JhVISA-4 DNS-1
a
VAN MBC
μg/ml
μg/ml
L knee
1
1
MRSA
Buttock Thigh Unknown Blood Sputum Unknown
1 1 1 1 1 1
1 1 1 1 1 1
MRSA MRSA MRSA MRSA MRSA MRSA
Wound Blood Blood Respiratory Respiratory Respiratory
1 1 1 0.5 1 1
1 1 1 1 1 1
MRSA MRSA MRSA MRSA MRSA MRSA
USA-600 USA-100 No match USA-100 USA-300 USA-100
Respiratory Respiratory Blood Blood Blood Blood
1 1 1 1 2 1
1 1 1 1 2 2
MRSA MRSA hVISA hVISA hVISA DNSSA
III IVa II IVa II MSSA
No match USA-300 USA-100 USA-300 No match No match
Blood Blood Blood Blood Mu 50 Eye
2 1 2 2 8 8
2 2 2 2 8 16
DNSSA DNSSA DNSSA DNSSA VISA VISA
0 0 2.5 0 0 3.5
II II II IV d II II
USA-100 USA-100 No match USA-500 USA-100 No match
Blood Wound Blood Unknown Bone Unknown
8 4 4 4 4 8
8 8 4 4 4 8
VISA VISA VISA VISA VISA VISA
3.1 0 4.2 0 4.3 3.7
2.5 0 0 0 3.5 3
II MSSA III IV d II I
No match No match No match USA-500 USA-100 No match
Bile Bile Unknown Wound Blood Unknown
4 8 4 4 4 4
4 16 8 4 4 8
VISA VISA VISA VISA VISA VISA
2.4 4.8 0 4.4
0 4.2 0 0
II IV II II
USA-100 No match No match USA-100
Sputum Unknown Blood Blood
1 1 1 1
1 1 1 1
LNSSA LNSSA LNSSA LNSSA
0.25
0.5
0.75
0.5
7.2
7.1
4.9
3.2
0
IV a
USA-300
0.5 0.5 0.5 0.5 1 1
7.2 7 7.1 7.2 6.8 6.9
7.1 6.9 7 7.2 6.7 6.9
4.3 5.4 5 6.1 6.8 6.8
3.2 3.8 3.2 3.7 6 5.4
0 0 0 0 0 3.1
IV a IV a IV a IV a II II
USA-300 USA-300 USA-300 USA-300 No match USA-600
1 1 1 0.5 1 1
1 0.75 1 0.5 1 1
6.9 7.4 6.8 7.1 7.1 7
6.9 7.4 6.7 6.9 7.1 6.9
6.9 4.8 5.5 3.7 6.4 6.8
5.8 0 2.9 2.7 3.3 5.1
3.7 0 0 0 0 0
II IV a II IV II II
USA-600 USA-300 USA-600 No match USA-100 USA-600
1 0.5 1 0.5 0.5 0.5
1 1 1 1 1 1
1 1 1 1 0.75 1
6.9 6.6 6.7 6.8 7.1 7.1
6.9 6.6 6.7 6.9 7 7
6.8 6.3 6.6 6.7 5 6.9
5.5 4.5 4.9 5.6 4 6.2
4.4 2.5 0 2.7 3.7 2.5
II II II II IVa II
DNS-4 DNS-5 DNS-6 DNS-7 NRS-1 NRS-14
1 0.5 1 0.5 1 0.5
1 0.5 1 1 1 0.5
1 0.75 0.75 0.75 0.75 0.25
7 7.1 7 7 6.8 6.8
6.9 7 7 7 6.7 6.7
6.6 6.6 6.6 6.7 6.6 4.3
4.3 2.8 0 4.1 6.6 0
3.3 0 0 2.6 4.5 0
NRS-17 NRS-18 NRS-19 NRS-21 NRS-23 NRS-49
1 0.5 0.5 0.5 1 1
1 0.5 1 0.5 1 2
0.75 0.5 0.25 0.5 0.5 0.5
6.8 6.3 6.3 6.4 6.2 6.5
6.7 6.2 6.2 6.3 6 6.5
5.9 3.4 6.1 5.4 5.4 6.5
0 0 4.9 2.5 3.3 5.9
NRS-51 NRS-52 NRS-56 NRS-73 NRS-126 NRS-272
1 0.25 1 0.5 1 1
1 0.25 1 0.5 1 1
1 0.25 0.75 0.38 1 0.5
6.9 7.2 6.3 7.1 6.9 6.7
6.9 3 6.2 7.1 6.9 6.5
6.5 0 6.2 4.1 6.4 5
NRS-127 NRS-271 LNSSA-10 LNSSA-11 a
0.5 1 0.5 1
1 1 1 1
0.75 1 0.75 1
7 7.1 6.9 6.7
7.1 7.1 6.9 6.6
6.1 6.6 3.6 5.9
Sample
a
VAN MIC
a
a
a
1
SCC
Pulse
Source
Concentration of CPT in PAP plates (μg/ml) CPT=ceftaroline
VAN=vancomycin
14
Sample Type
315 316 317 318 319 320 321 322 323
324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351
Table 1b Results of ceftaroline intermediate organisms that did not show heteroresistance
CPT
CPT
MIC
MBC E-test
μg/ml
μg/ml
μg/ml
0
NRS-39
2
2
1.5
6.4
NRS-54
2
2
1
NRS-65
2
2
NRS-283
2
2
Sample
a b
CPT a
PAP Results (log10CFU/ml) a
a
0.75
6.3
6.2
5.4
6.8
6.8
6.6
2
6.3
6.3
1.5
6.9
6.9
0.25 0.5
a
a
1
a
a
a
2.5
2
a,b
1.25
1.5
SCC
4.2
3
2.4
0
0
I
6.2
4.6
4.3
4
3.7
0
III
6.3
6.2
6.1
5.2
4.6
4
0
III
6.8
6.8
6.5
5.2
4.1
2.8
2.5
II
Pulse No match No match No match USA200
VAN
VAN
MIC
MBC Sample μg/ml Type
Source
μg/ml
Urine
8
8
VISA
Unknown
4
4
VISA
Unknown
4
8
VISA
Unknown
4
4
VISA
Concentration of CPT in PAP plates (μg/ml) For all organisms there was no growth at CPT concentrations of 3μg/ml or 4μg/ml CPT=ceftaroline VAN=vancomycin
15
352 353 354 355 356 357 358 359 360
361 362 363 364 365 366 367 368 369 370 371 372 373
Table 2 Results of samples that were determined to be heteroresistant.
a
CPT MIC
CPT CPT MBC E-test
Sample
μg/ml
μg/ml
μg/ml
0
D-9
1
1
1
D-11
1
1
D-27
1
ND-14
a
PAP Results (log10 CFU/ml) a
0.25
a
0.5
7
6.9
1
6.9
2
1
1
1
ND-33
1
DNS-2
a
0.75
a
1
1.25
6.9
4.7
3.8
6.9
6.8
6.1
6.9
6.9
6.8
1
7
7
1
1
6.8
1
1
1
DNS-3
1
2
NRS-3
1
NRS-118
a
a
1.5
a
a
2
2.5
3.3
2.7
2.3
2.1
3.8
3.2
2.8
2.4
6.1
3
2.6
2.6
6.7
5.9
3.1
2.6
6.8
6.7
5.9
3.1
6.9
6.8
6.7
6.2
1.5
6.9
6.8
6.7
1
1.5
6.4
6.3
1
2
1.5
6.8
NRS-120
1
1
1
NRS-121
1
1
JhVISA-2
1
1
a
VAN VAN MIC MBC
a
Frequency
0
0
1.1x10
2
0
0
1.3x10
0
0
0
0
2.3
2.1
0
0
2.5
2.2
0
0
3.6
2.8
2.5
0
6.5
5.9
3.1
2.7
6.3
6.2
5.2
3
6.7
6.7
6.6
6.5
6.8
6.8
6.5
5.9
1
6.8
6.7
6.4
1
6.7
6.7
6.6
3
4
Source
Sample Type
SCC
Pulse
-5
II
USA-100
Blood
1
1
MRSA
-5
II
USA-600
Blood
1
1
MRSA
5.2x10
-5
II
USA-100
Wound
1
1
MRSA
0
1.1x10
-5
II
USA-100
Respiratory
1
1
MRSA
0
0
1.4x10
-5
II
No match
Respiratory
2
2
MRSA
0
0
0
4.8x10
-5
II
No match
Blood
2
2
DNSSA
2.4
0
0
0
3.8x10
-5
II
USA-100
Blood
2
2
DNSSA
2.6
2
0
0
0
3.8x10
-5
II
USA-100
Peritoneal
8
8
VISA
5.7
4.5
3.3
2.6
2.3
0
3.2x10
-5
I
No match
Respiratory
8
8
VISA
4.4
2.7
2.6
0
0
0
0
6.9x10
-5
IV d
USA-500
Unknown
2
2
LNSSA
5.9
4.8
3.1
0
0
0
0
0
2.2x10
-4
IV d
USA-500
Unknown
1
2
LNSSA
4.5
3.4
3.2
3
2.5
2
0
0
2.1x10
-5
II
USA-100
Blood
2
2
hVISA
μg/ml
μg/ml
Concentration of CPT in PAP plates (μg/ml) CPT=ceftaroline VAN=vancomycin
16
374
17
