International Journal of Antimicrobial Agents 43 (2014) 131–134

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Methodological agreement on the in vitro activity of ceftaroline against cefotaxime-susceptible and -resistant pneumococci David M. Livermore ∗ , Marina Warner, Shazad Mushtaq Antimicrobial Resistance & Healthcare-Associated Infections Reference Unit, Public Health England, 61 Colindale Avenue, London NW9 5EQ, UK

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Article history: Received 29 August 2013 Accepted 22 October 2013 Keywords: Etest Disc diffusion Streptococcus pneumoniae CLSI EUCAST

a b s t r a c t Ceftaroline reportedly has lower minimum inhibitory concentrations (MICs) than established cephalosporins for Streptococcus pneumoniae. We further evaluated this activity using 155 pneumococci chosen by serotype and cefotaxime MIC. MICs were determined by agar dilution on Mueller–Hinton agar and Iso-Sensitest agar and by Etest. Inhibition zones were measured for 5 ␮g and 30 ␮g ceftaroline discs using both CLSI/EUCAST and BSAC methodology. Ceftaroline was more active than cefotaxime, with MICs 2–8-fold lower for isolates with cefotaxime MICs of ≤1 mg/L and mostly in the range 0.125–0.5 mg/L for those with cefotaxime MICs of 2 mg/L to ≥16 mg/L. Twelve isolates belonging to serotypes 14 (n = 2), 19A (n = 6) and 19F (n = 4) were ceftaroline-resistant, with MICs of 0.5–1 mg/L. Essential agreement between MIC methods was excellent, with values on Iso-Sensitest agar and Mueller–Hinton agar identical ±1 doubling dilution in all cases, and with 154/155 values identical ±1 doubling dilution between agar dilution and Etest. Nevertheless, 5/11 isolates with agar dilution MICs of 0.5 mg/L (i.e. just resistant) ‘had’ MICs of 0.25 mg/L (just susceptible) by Etest. Inhibition zones also correlated with MICs, but discrimination around the breakpoint MICs was poor irrespective of method and disc type. In summary, the results confirm the good activity of ceftaroline against pneumococci, but susceptibility testing will present challenges in routine laboratories, with discs poorly discriminatory and with Etest prone to give susceptible results for isolates with MICs one doubling dilution above the breakpoint. © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

1. Introduction Ceftaroline reportedly has four- to eight-fold superior minimum inhibitory concentrations (MICs) to cefotaxime and ceftriaxone for pneumococci, including for strains with reduced susceptibility to penicillin and cephalosporins [1,2]. This behaviour correlates with strong affinity for the penicillin-binding proteins of pneumococci, even when these are modified by mutation and mosaic formation [3]. In this study, this activity was further characterised, testing ceftaroline by agar dilution, Etest and disc diffusion against a large panel of previously serotyped UK pneumococci, graded by cefotaxime MICs, seeking also to define agreement between methods. 2. Materials and methods 2.1. Streptococcus pneumoniae isolates Isolates (n = 155) were selected from among recent submissions to Public Health England for reference investigation and were

∗ Corresponding author. Present address: Norwich Medical School, University of East Anglia, Norwich, Norfolk NR4 7TJ, UK. Tel.: +44 1603 597 568. E-mail address: [email protected] (D.M. Livermore).

chosen to represent a wide range of cefotaxime MICs and serotypes (Table 1). Cefotaxime-resistant organisms, inevitably, were concentrated into a narrower range of serotypes, predominantly 6B, 9V, 14, 19A and 19F.

2.2. Antibiotics and susceptibility tests Ceftaroline powder was from AstraZeneca (Macclesfield, UK). MICs were measured both by the British Society for Antimicrobial Chemotherapy (BSAC) agar dilution method [4] [i.e. incubated for 18 h in a 5% CO2 atmosphere on Iso-Sensitest agar from Oxoid Ltd. (Basingstoke, UK) supplemented with 5% equine blood] and by an agar dilution method corresponding as closely as possible to Clinical and Laboratory Standards Institute (CLSI) broth microdilution criteria [5] using Mueller–Hinton agar (Oxoid Ltd.) supplemented with 5% ovine blood incubated in air for 20 h. Ceftaroline E-tests (bioMérieux, Lyos, France) were used in accordance with the manufacturer’s directions, i.e. on Mueller–Hinton agar supplemented with 5% ovine blood and incubated for 20 h in air. Disc diffusion tests were performed using 5 ␮g (Oxoid Ltd.) and 30 ␮g (Mast Group Ltd., Bootle, UK) discs. Both BSAC [4] and CLSI/European Committee on Antimicrobial Susceptibility Testing (EUCAST) [6] methodologies were followed, using Iso-Sensitest agar with 5% equine blood in a 5%

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D.M. Livermore et al. / International Journal of Antimicrobial Agents 43 (2014) 131–134

Table 1 Serotype distribution of isolates in relation to cefotaxime minimum inhibitory concentration (MIC). Serotype

No. of isolates with cefotaxime MIC (mg/L) 16

1

1 1

1 1 1

1

1 1 1 1 1

1 1 1

4

1 1 1 1

1 1 1

2

1

2

1

1

1

2

4

30

1

1 1 6

1

1 1

3

1

1 5

2 7 1

8 8

2 3 7

1 1

>16

1

0.03

5

1

0.06

8

5

4

5

16

18

11

2

1

9

18

6

2

5

6 1

0.25 mg/L breakpoint, whilst 11 were nonsusceptible on Iso-Sensitest agar. Thirty-nine serotypes were represented in the whole collection, but the ceftaroline-nonsusceptible isolates all belonged to serotypes 14 (n = 2), 19A (n = 6) and 19F (n = 4).

By Etest 0.008 0.015

31

1 1

0.03

4

2

0.06

1

12

1

3

44

1

7

34

5

1

6

0.125 0.25 0.5 1

1

a Dark grey shading indicates susceptible to ceftaroline by European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria; unshaded indicates resistant.

D.M. Livermore et al. / International Journal of Antimicrobial Agents 43 (2014) 131–134

(c) 45

50

40

45

Zome diameter (mm)

Zone diameter (mm)

(a)

133

35 30 25 20 15 10

0 0.008

30 25 20 15 5

0.016

0.031

0.063

0.125

0.250

0.500

0 0.002

1.000

MIC (mg/L)

0.004

0.008

0.016

45

50

40

45

35 30 25 20 15

0.125

0.250

0.500

1.000

0.125

0.250

0.500

1.000

20 15

0 0.002

1.000

0.500

25

5 0.063

0.250

30

10

0.031

0.125

35

5 0.016

0.063

40

10 0 0.008

0.031

MIC (mg/L)

(d)

Zone diameter (mm)

Zone diameter (mm)

35

10

5

(b)

40

0.004

0.008

0.016

MIC (mg/L)

0.031

0.063

MIC (mg/L)

Fig. 1. Minimum inhibitory concentration (MIC) versus inhibition zone distribution for ceftaroline: (a) CLSI/EUCAST methodology on Mueller–Hinton agar, 5 ␮g discs; (b) CLSI/EUCAST methodology on Mueller–Hinton agar, 30 ␮g discs; (c) BSAC methodology on Iso-Sensitest agar, 5 ␮g discs; and (d) BSAC methodology on Iso-Sensitest agar, 30 ␮g discs. All points are the mean of three zone measurements in different directions. CLSI, Clinical and Laboratory Standards Institute; EUCAST, European Committee on Antimicrobial Susceptibility Testing; BSAC, British Society for Antimicrobial Chemotherapy.

3.2. Agreement of ceftaroline minimum inhibitory concentrations by different methods The agreement between MICs of ceftaroline determined on Mueller–Hinton agar (CLSI) and Iso-Sensitest agar (BSAC) was excellent (Table 3): in 128/155 cases the results were identical; in 8/155 cases the values on Iso-Sensitest agar were two-fold higher, whereas in 19/155 cases the values on Iso-Sensitest agar were two-fold lower, with no greater disagreements, and no trend to increasing divergence at either extreme of the range. MICs by Etest on Mueller–Hinton agar (Table 3) were equal to those on the same medium by agar dilution in 133/155 cases, were two-fold lower in

9/155 and were two-fold higher in 12/155, with just one outlier where the Etest result was four-fold lower than by agar dilution. None the less, among 11 isolates with MICs of 0.5 mg/L on Mueller–Hinton agar (i.e. one doubling dilution above the breakpoint), 5 counted as susceptible by Etest with MICs of 0.25 mg/L. 3.3. Performance of disc susceptibility tests Zone diameter versus MIC plots are presented in Fig. 1, whilst Table 4 details mean zone diameters and standard deviations in relation to MICs. Whilst good correlation between MICs and zones was seen for all combinations of disc and method, there was

Table 4 Zone diameters for ceftaroline discs in relation to minimum inhibitory concentrations (MICs). MIC (mg/L) on corresponding medium

Mean ± S.D. zone diameter (mm) for isolates with indicated MIC 5 ␮g disc CLSI/EUCAST

0.004 0.008 0.015b 0.03 0.06 0.12 0.25 0.5–1c

– 35.56 ± 33.80 31.37 ± 29.26 ± 28.51 ± 26.84 ± 24.7

1.73a 2.67 1.64 1.78 2.54

30 ␮g disc BSAC 39.91 ± 40.84 ± 40.40 33.43 ± 32.32 ± 31.87 ± 29.87 ± 27.83 ±

1.85a 1.40 1.72 2.05 2.37 1.86 2.57

CLSI/EUCAST

BSAC

– 37.81 ± 42.60 35.05 ± 32.28 ± 31.07 ± 29.61 ± 27.41

42.62 ± 43.44 ± 43.10 37.25 ± 35.02 ± 34.68 ± 32.92 ± 30.96 ±

1.90a 1.79 1.29 1.81 1.65

1.84a 1.34 1.16 4.58 2.39 2.37 2.50

S.D., standard deviation; CLSI, Clinical and Laboratory Standards Institute; EUCAST, European Committee on Antimicrobial Susceptibility Testing; BSAC, British Society for Antimicrobial Chemotherapy. a Zone corresponding to MIC ≤ indicated value. b Only one isolate with a MIC of 0.015 mg/L on either medium, hence on valid S.D. can be calculated. c Only one isolate with a MIC of 1 mg/L on Mueller–Hinton agar, hence no valid S.D. can be calculated.

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considerable overlap of zone diameters for susceptible isolates with MICs of 0.125–0.25 mg/L and for resistant isolates with MICs of 0.5–1 mg/L. It follows that the ability of disc tests to discriminate effectively in the critical MIC range is limited. 4. Discussion These results confirm that ceftaroline has substantially lower MICs than cefotaxime for pneumococci: specifically, at EUCAST’s 0.25 mg/L breakpoint, ceftaroline retained activity against 67/79 isolates (85%) with intermediate or full resistance to cefotaxime. This gain was seen across all serotypes where cefotaxime resistance is prevalent. Results for different modes of susceptibility testing were in good general agreement. Nevertheless, and irrespective of method and disc type, diffusion tests discriminated poorly among isolates with the critical breakpoint-bounding MICs of 0.25–0.5 mg/L. Thus, EUCAST’s general advice for pneumococci and cephalosporins, i.e. to screen with oxacillin discs and to perform dilution tests on isolates found to be resistant [7], seems appropriate also for ceftaroline. The caveat is that diagnostic laboratories are likely to use gradient strips for this dilution test and that, although the general correlation of Etest and agar dilution MICs was excellent, these strips nevertheless found MICs of 0.25 mg/L (i.e. susceptible) for 5/11 isolates with agar dilution MICs of 0.5 mg/L (i.e. resistant). On this basis, we advocate caution when ‘borderline’ MICs of 0.25 mg/L are found by gradient methods, with especial attention paid to any microcolonies at fractionally higher dilutions along the strip. This caution extends beyond pneumococci, since the same issue was found in testing meticillin-resistant staphylococci, where ceftaroline Etests found MICs of 1 mg/L (i.e. susceptible) for around one-half of the meticillin-resistant Staphylococcus aureus with MICs of 2 mg/L (i.e. resistant) by agar dilution testing [8]. Despite excellent in vitro activity against penicillin- and cephalosporin-resistant pneumococci, further experience is urgently needed with ceftaroline in infections due to these pathogens, which were poorly represented in the phase 3 ‘FOCUS’ trials that supported licensing for community-acquired pneumonia [9]. This is reflected in the current European Medicines Agency Specification of Product Characteristics, which notes, ‘The available clinical data cannot substantiate efficacy against penicillin non-susceptible strains of S. pneumoniae.’ Acknowledgments The authors are grateful to Dr. Tim Harrison and colleagues of the Public Health England Respiratory and Vaccine-Preventable

Bacterial Infections Reference Unit for prior serotyping of the pneumococci used here. Funding: This study was supported by AstraZeneca UK. Competing interests: DML is partly self-employed and consults for numerous pharmaceutical and diagnostic companies, including Achaogen, Adenium, Allecra, Astellas, AstraZeneca, Bayer, Basilea, bioMérieux, Cubist, Curetis, Discuva, GSK, Kalidex, Merck, Meiji Seika, Pfizer, Roche, Tetraphase, VenatoRx and Wockhardt, holds grants from Basilea, Cubist, Meiji Seika, Merck and Wockhardt, has received lecture honoraria or travel reimbursement from AstraZeneca, Bruker, Curetis, GSK, J&J, Merck, Novartis, Pfizer and Tetraphase, and holds shares in Dechra, Eco Animal Health, GSK, Merck and Pfizer, collectively amounting to