In vitro susceptibility testing of ceftobiprole against 880 European respiratory tract infection isolates of methicillin-resistant Staphylococcus aureus followed by whole genome sequencing of ceftobiprole-resistant isolates

Journal Pre-proof In vitro susceptibility testing of ceftobiprole against 880 European respiratory tract infection isolates of methicillin-resistant Staphylococcus aureus followed by whole genome sequencing of ceftobiprole-resistant isolates

Stephen Hawser, Nimmi Kothari, James A. Karlowsky, Tatiana Wiktorowicz, Kamal Hamed PII:

S0732-8893(19)31059-4

DOI:

https://doi.org/10.1016/j.diagmicrobio.2019.114978

Reference:

DMB 114978

To appear in:

Diagnostic Microbiology & Infectious Disease

Received date:

24 October 2019

Revised date:

14 December 2019

Accepted date:

18 December 2019

Please cite this article as: S. Hawser, N. Kothari, J.A. Karlowsky, et al., In vitro susceptibility testing of ceftobiprole against 880 European respiratory tract infection isolates of methicillin-resistant Staphylococcus aureus followed by whole genome sequencing of ceftobiprole-resistant isolates, Diagnostic Microbiology & Infectious Disease(2019), https://doi.org/10.1016/j.diagmicrobio.2019.114978

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© 2019 Published by Elsevier.

Journal Pre-proof DMID-2019-899 R1

In vitro susceptibility testing of ceftobiprole against 880 European respiratory tract infection isolates of methicillin-resistant Staphylococcus aureus followed by whole genome sequencing of ceftobiprole-resistant isolates

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Stephen Hawser,a Nimmi Kothari,a James A. Karlowsky,a Tatiana Wiktorowicz,b and Kamal

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Hamedb,*

IHMA Europe Sàrl, Monthey, Switzerland

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Basilea Pharmaceutica International Ltd., Basel, Switzerland

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Running title: Ceftobiprole susceptibility of European MRSA

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Keywords: ceftobiprole, Europe, MRSA, Staphylococcus aureus, whole genome sequencing

*Address correspondence to Kamal Hamed, [email protected]. Mailing address: Grenzacherstrasse 487, 4005 Basel, Switzerland. Phone: +41 79 378 9888.

Abstract word count: 69 words Text word count: 1,160 words

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Journal Pre-proof ABSTRACT Ceftobiprole was active (MIC, ≤2 mg/L) against most isolates (99.7%; 877/880) of methicillinresistant Staphylococcus aureus from respiratory tract infections collected in 14 European countries during 2016-2017. Whole-genome sequence analysis showed that two of the three ceftobiprole-resistant (MIC, 4 mg/L) isolates identified were clonal complex 8 (CC8) and one was CC5, and that different mutations were present in genes encoding penicillin-binding

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proteins, mecA, and other proteins in each ceftobiprole-resistant isolate.

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Journal Pre-proof Ceftobiprole is an advanced-generation, broad-spectrum parenteral cephalosporin approved in 17 European and eight non-European countries for the treatment of adults with community-acquired pneumonia (CAP) and hospital-acquired pneumonia (HAP) (excluding ventilator-associated pneumonia) caused by susceptible Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), and Gram-negative pathogens [1]. Ceftobiprole is active against methicillin-resistant staphylococci due to its high affinity for

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common penicillin-binding proteins (PBPs) as well as for variant PBPs, including PBP2a [2, 3].

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Ceftobiprole demonstrates relative stability to many types of β-lactamase and a low propensity to

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develop resistance [4].

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CAP is the most common presentation of pneumonia and is an important cause of morbidity and mortality worldwide. Inpatient therapy for CAP is generally initiated with

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intravenous antimicrobial agents, followed by step-down to an oral course of therapy [5]. The

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standard regimen per treatment guidelines for inpatients with CAP is a β-lactam plus a macrolide or a respiratory fluoroquinolone, with MRSA coverage added in cases of prior respiratory

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isolation of MRSA or for patients with a recent hospitalization during which they received

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parenteral antimicrobials and locally validated risk factors for MRSA exist [5, 6]. HAP is among the commonest nosocomial infections, with an incidence of 5-10 cases per 1,000 hospital admissions in the United States, approximately one-quarter of which are acquired in intensive care units [7, 8]. Empirical antimicrobial therapy for HAP targets both Gram-positive and Gramnegative pathogens and considers the types and susceptibility patterns of local pathogens as well as variables such as duration of previous hospital stays, recent antimicrobial therapy, and the presence of comorbidities [8-11]. MRSA coverage is added in cases of increasing likelihood of MRSA and high risk of mortality or receipt of intravenous antimicrobials during the previous 90

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Journal Pre-proof days. The use of ceftobiprole in therapy for patients with pneumonia has recently been reviewed [12]. The current study investigated the in vitro activities of ceftobiprole and nine comparator agents against MRSA isolated from samples collected from patients with respiratory tract infections (RTIs). RTI isolates of MRSA (n=880) were collected from clinical laboratories in 14 European countries during 2016-2017 as part of an ongoing post-marketing surveillance study.

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Most isolates (n=459, 52.2%) were from sputum followed by endotracheal aspirate (n=181,

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20.6%), bronchiolar lavage (n=87, 9.9%), bronchial brushing (n=77, 8.8%), and other sources

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(n=76, 8.6%). MICs were determined using EUCAST broth microdilution methodology [13] and interpreted using 2019 EUCAST breakpoints [14]. The EUCAST susceptible MIC

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breakpoint for ceftobiprole tested against S. aureus (including MRSA) is ≤2 mg/L [14].

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Ceftobiprole-resistant isolates (MIC >2 mg/L) [14] were subjected to whole genome sequencing

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(WGS) to attempt to decipher mechanisms of resistance. DNA for WGS was prepared using Illumina TruSeq libraries (Illumina Inc., San Diego, CA, USA; www.illumina.com) and WGS

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was performed on an Illumina NextSeq 500 system using 2x150 bp PE v2.5 sequencing

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chemistry. WGS data analysis was accomplished using open-source as well as proprietary software and databases (Ares Genetics GmbH, www.ares-genetics.com). Multilocus sequence typing (MLST) was performed using WGS data [15]. The results of antimicrobial susceptibility testing of ceftobiprole and comparator antimicrobial agents are shown in Table 1. Susceptibility rates of the MRSA isolates for ceftobiprole were 100% (North), 99.7% (South), 99.4% (East), and 99.3% (West) across the four regions of Europe (Table 1). Daptomycin (100% susceptible), vancomycin (100%), linezolid (99.3-100%), and trimethoprim-sulfamethoxazole (97.7-99.4%) also demonstrated high rates of

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Journal Pre-proof susceptibility. Gentamicin and tetracycline susceptibilities were >80% in all four regions while susceptibilities to clindamycin (36.3-86.1% susceptible), erythromycin (8.9-40.5%), and levofloxacin (13.5-25.6%) were lower. Ceftobiprole MIC50 and MIC90 values were 1 mg/L and 1-2 mg/L, respectively, for isolates of MRSA for all countries analyzed (Table 2). The lowest rates of susceptibility to ceftobiprole were observed for isolates from Russia (95.0%), although the number of isolates

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tested was relatively small (n=20), France (98.8%) and Italy (99.2%). Isolates from the other 11

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countries were 100% susceptible to ceftobiprole. Three ceftobiprole-resistant isolates (MIC, 4

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mg/L) [14] were identified, one each from France (sputum), Italy (sputum) and Russia

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(endotracheal aspirate). All three ceftobiprole-resistant isolates were confirmed with repeat testing.

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WGS analysis showed that the ceftobiprole-resistant isolates of MRSA from France and

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Russia belonged to clonal complex 8 (CC8) and the isolate from Italy to CC5 (Table 3). No DNA sequence changes in the genes encoding PBPs were identified in the French isolate;

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however, an E239K mutation in mecA and a H682Y mutation in acrB were detected. The Italian

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isolate had mutations in PBP1 (N118D), PBP3 (T438S), and PBP4 (T189S, T409A) as well as a N146K mutation in mecA and changes in gdpP (cyclic-di-AMP phosphodiesterase) and acrB (a transmembrane transporter). The Russian isolate had a single change in PBP2 (R262C) and a mutation in gdpP. Ceftobiprole resistance in MRSA has been described previously. A single Italian hospital reported 12% (12/102 isolates) ceftobiprole resistance in a 2017-2018 collection of MRSA where CC5 predominated (9/12 ceftobiprole-resistant isolates were CC5); the remaining three isolates were CC8, CC22, and CC59 [16]. The Italian isolate in our study was a CC5 (ST228) but did

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Journal Pre-proof not have the same changes in PBP genes as described by Morroni et al [16]. Another study that investigated HAP isolates of S. aureus from 13 laboratories distributed across Italy, identified three ceftobiprole-resistant MRSA isolates (3/66; 4.5% of MRSA were ceftobiprole-resistant); all three isolates were CC5 (ST228) [17]. Published studies to compare to our French and Russian ceftobiprole-resistant isolates could not be identified. Bongiorno et al., previously studied 10 isolates of MRSA with low-level resistance (≤8

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mg/L) to ceftaroline and ceftobiprole and reported four different mutations in mecA (N146K,

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N204K, T235I, E239K) present in its non-penicillin-binding domain and that these changes were

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correlated with small decreases in ceftaroline and ceftobiprole susceptibility [18]. We identified

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the same E239K and N146K mutations in two of our three ceftobiprole-resistant isolates (Table 3). The non-penicillin-binding domain has been hypothesized to be functionally important for

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cell-wall biogenesis [18]. Bongiorno et al., also identified the same mutations we observed in

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gdpP (D105N, P392S) in one of our ceftobiprole-resistant isolates [18]. Other reports have described ceftobiprole resistance among MRSA associated with missense mutations in PBP2a

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[19-21]. In the current study, ceftobiprole MICs were only 4 mg/L for all three ceftobiprole-

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resistant MRSA isolates (i.e., no high-level resistance). The low-level resistance observed was consistent with the lack of mutations identified in the mecA binding site and suggests that the resistance may be due, at least in part, to substitutions in the non-penicillin-binding domain of mecA [18]. In conclusion, data from the present study confirm the continued high susceptibility (99.3-100%) of MRSA isolated from RTIs to ceftobiprole. The three ceftobiprole-resistant isolates identified (~0.3% of all MRSA tested) each had an MIC of 4 mg/L, one doubling dilution above the ceftobiprole-susceptible breakpoint of ≤2 mg/L. Data in the current study

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Journal Pre-proof align with an earlier large-scale surveillance study testing ceftobiprole against 4,147 European isolates of MRSA (MIC90, 2 mg/L; 98.3% susceptible) [22] and with a recent study from the United States in which >99% of bacteremic S. aureus (813 MSSA and 558 MRSA) were ceftobiprole-susceptible [23]. Continued surveillance to monitor activity and susceptibility/resistance trends for ceftobiprole is warranted, and a greater understanding of

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resistance lineages and clonality is needed.

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Journal Pre-proof Funding The present study was funded by a grant from Basilea Pharmaceutica International Ltd.

Transparency Declaration SH, NK and JAK are employees of IHMA Europe. KH and TW are employees of Basilea

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Pharmaceutica International Ltd.

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Acknowledgement

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The authors wish to thank the technical staff at IHMA Europe for their support.

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hospital acquired pneumonia in adults: summary of NICE guidance. BMJ 2014; 349: g6722. 12. Giacobbe DR, De Rosa FG, Del Bono V, Grossi PA, Pea F, Petrosillo N, Rossolini GM, Tascini C, Tumbarello M, Viale P, Bassetti M. Ceftobiprole: drug evaluation and place in therapy. Expert Rev Anti-Infect Ther 2019; doi.org/10.1080/14787210.2019.1667229. 13. International Organization for Standardization, 2006. Clinical laboratory testing and in vitro diagnostic test systems - susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices - Part 1: Reference Method for

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bloodstream infections in the United States (2016-2017). Diagn Microbiol Infect Dis

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Journal Pre-proof Table 1 In vitro activity of ceftobiprole and comparator agents against 880 respiratory tract infection isolates of MRSA from 14 European countries.

South (386)

West (283)

East (168)

99.7

99.3

99.4

81.6

57.2

36.3

100

100

100

31.1

40.6

8.9

93.0

86.3

90.5

90.5

25.6

13.5

23.7

24.4

100

99.7

99.3

100

Ceftobiprole

≤2

100

Clindamycin Daptomycin

≤0.25 ≤1

86.1

Erythromycin

≤1

Gentamicin

≤1

Levofloxacin

≤1

Linezolid

≤4

100

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Antimicrobial agent

Susceptible MIC breakpoint (mg/L)

North (43)

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% of isolates susceptible European regionb (n)

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25.6

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83.7 90.9 81.3 85.1 Tetracycline ≤1 Trimethoprim97.7 99.2 99.3 99.4 sulfamethoxazolea ≤2 100 100 100 100 Vancomycin ≤2 a MIC breakpoint expressed as trimethoprim component of combination. b European regions: North = United Kingdom; West = Austria, Belgium, France, Germany, Netherlands; East = Czech Republic, Hungary, Romania, Russia; South = Greece, Italy, Portugal, Spain.

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Tot al (88 0) 99. 5 65. 3 100 29. 7 88. 8 19. 4 99. 7 86. 4 99. 2 100

Journal Pre-proof Table 2. In vitro susceptibility of 880 respiratory tract infection isolates of MRSA from 14 European countries to ceftobiprole.

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Ceftobiprole MIC (mg/L) Minimum Maximum MIC90 MIC MIC 1 0.5 2 1 0.5 2 2 0.5 2 2 0.25 4 2 0.5 2 2 0.5 2 2 0.5 2 2 0.5 4 NA 0.25 0.25 2 0.5 2 NA 1 1 2 0.5 4 2 0.5 2 2 0.5 2

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% of isolates susceptible to Country n MIC50 ceftobiprole Austria 16 100 1 Belgium 70 100 1 Czech Republic 90 100 1 France 87 98.8 1 Germany 109 100 1 Greece 53 100 1 Hungary 50 100 1 Italy 127 99.2 1 Netherlands 1 100 NAa Portugal 109 100 1 Romania 8 100 NA Russia 20 95.0 1 Spain 97 100 1 United Kingdom 43 100 1 a NA = not applicable because <10 isolates were tested.

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Journal Pre-proof Table 3. Whole genome sequencing analysis of three ceftobiprole-resistant isolates of MRSA. Mutations identifieda MLS Ceftobiprole MIC Isolate Specimen T (mg/L) pbp1 pbp2 pbp3 pbp4 mecA gdpP acrB 247 15803 (CC8 E239 H682 43 France Sputum ) 4 K Y 228 D105 16914 (CC5 N118 T438 T189S, N146 N, A677 66 Italy Sputum ) 4 D S T409A K P392S V ~347 0 16122 Endotracheal (CC8 R262 07 Russia aspirate ) 4 C I52V a The following reference sequences (genome NCTC 8325, NC_007795.1) were used for gene comparisons: pbp1 | Locus name: SAOUHSC_01145 | UniProtKB ID: Q2FZ94; pbp2 | Locus name: SAOUHSC_01467| UniProtKB ID: Q2FYI0; pbp3 | Locus name: SAOUHSC_01652| UniProtKB ID: Q2FY21; pbp4 | Locus name: SAOUHSC_00646| UniProtKB ID: Q2G2X6; mecA | Locus name: (not in reference genome) | UniProtKB ID: E2D9B8; gdpP | Locus name: SAOUHSC_00015| UniProtKB ID: Q2G2T6; and acrB | Locus name: SAOUHSC_02525| UniProtKB ID: Q2FVZ5.

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Count ry

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