Rapid detection of ceftazidime/avibactam resistance by MALDI-TOF MS

Rapid detection of ceftazidime/avibactam resistance by MALDI-TOF MS Sir, Over the last decades, MDR Gram-negative bacilli have emerged as an important aetiological agent of nosocomial-acquired infections worldwide. The combination of β-lactam and new β-lactamases inhibitors has become an option for treating those infections with fewer side effects than old compounds such as polymyxins. Avibactam is a diazabicyclooctane β-lactamase inhibitor that demonstrates excellent inhibitory properties against class A, class C and some class D β-lactamases (basically OXA-48). In contrast, it does not possess activity against most MBL-producing isolates.1 Recently, ceftazidime/avibactam was approved for the treatment of patients with complicated intra-abdominal infections, complicated urinary tract infections and hospital-acquired pneumonia.2 To reduce the development of drug-resistant bacteria and preserve the susceptibility of ceftazidime/avibactam, it should be used only to treat infections that are proven or strongly suspected to be caused by susceptible bacteria. However, susceptibility testing of new drugs in routine laboratories is restricted to disc and/or gradient diffusion methodologies until its inclusion on commercial panels of susceptibility automated systems. According to a recent evaluation, ceftazidime/avibactam susceptibility testing using Etest and disc diffusion methods presented very major and major errors, respectively, compared to broth microdilution, and those tests should be interpreted cautiously in clinical practice.3 MALDI-TOF MS has been applied for the detection of carbapenems hydrolysis.4 As susceptibility testing to ceftazidime/avibactam might be tricky, herein, we evaluated the performance of MALDI-TOF MS for rapid detection of ceftazidime/avibactam resistance by testing against 118 previous molecular characterized bacterial clinical isolates, including a range of different β-lactamase-producing Enterobacteriaceae and Pseudomonas aeruginosa (Table 1).5,6 In addition, 21 non-carbapenemase-producing isolates were also studied. Species identification was confirmed by MALDI-TOF MS (Biotyper 3.3 version; Bruker Daltonics, Bremen, Germany). Table 1. Molecular characterization of bacterial isolates evaluated by MALDI-TOF MS ceftazidime/avibactam hydrolysis assay Microorganism (n) β-Lactam resistance determinant β-Lactamase molecular class (Ambler) Ceftazidime/avibactam MIC range (mg/L) MALDI-TOF MS ceftazidime/avibactam hydrolysis assaya Klebsiella pneumoniae (44), Serratia marcescens (4), Citrobacter spp. (3), Escherichia coli (2), Rauoltella planticola (1), Klebsiella oxytoca (1), Enterobacter cloacae complex (1), Proteus mirabilis (1), Morganella morganii (1) KPC-2 class A: carbapenemase <0.03–2 negative K. pneumoniae (2) BKC-1 0.5 negative K. pneumoniae (1) GES-5 0.06 negative S. marcescens (1) GES-16 4 negative S. marcescens (1) SME-1 0.06 negative K. pneumoniae (1), E. coli (1) CTX-M-16 class A: ESBL 0.125–0.25 negative K. pneumoniae (1) CTX-M-8 0.25 negative K. pneumoniae (1) CTX-M-2/ΔOmpK36b 0.06 negative K. pneumoniae (4) SHV-11/ΔOmpK36b <0.03–2 negative K. pneumoniae (1) TEM-11 /ΔOmpK36b <0.03 negative P. aeruginosa (1) GES-1 16 negative E. cloacae complex (1) PER-2 2 negative E. cloacae complex (1) KPC-2/NDM-1 class A and class B: carbapenemase >64 positive P. mirabilis (6), K. pneumoniae (2), E. cloacae complex (1), S. marcescens (1) IMP-1 class B: carbapenemase 64 to >64 positive (6); negative (4)c P. aeruginosa (1) IMP-18 >64 positive K. pneumoniae (6), E. cloacae complex (1), Enterobacter hormaechei (1), Citrobacter freundii (1) NDM-1 >64 positive P. aeruginosa (7) SPM-1 >64 positive E. cloacae complex (1), P. aeruginosa (1) VIM-1 >64 positive E. coli (2) MIR-1 class C: cephalosporinase 0.125–0.25 negative E. coli (1) CMY-2 0.25 negative K. pneumoniae (1) FOX-5 <0.03 negative Acinetobacter baumannii (2) OXA-72 class D: carbapenemase >64 negative A. baumannii (1) OXA-58 1 negative K. pneumoniae (1) OXA-48 1 negative Salmonella spp. (1) – multi-susceptible 0.5 negative E. coli (1) – 0.5 negative K. pneumoniae (1) – 0.25 negative E. coli ATCC 25922 – 0.25 negative P. aeruginosa ATCC 27853 – 2 negative Microorganism (n) β-Lactam resistance determinant β-Lactamase molecular class (Ambler) Ceftazidime/avibactam MIC range (mg/L) MALDI-TOF MS ceftazidime/avibactam hydrolysis assaya Klebsiella pneumoniae (44), Serratia marcescens (4), Citrobacter spp. (3), Escherichia coli (2), Rauoltella planticola (1), Klebsiella oxytoca (1), Enterobacter cloacae complex (1), Proteus mirabilis (1), Morganella morganii (1) KPC-2 class A: carbapenemase <0.03–2 negative K. pneumoniae (2) BKC-1 0.5 negative K. pneumoniae (1) GES-5 0.06 negative S. marcescens (1) GES-16 4 negative S. marcescens (1) SME-1 0.06 negative K. pneumoniae (1), E. coli (1) CTX-M-16 class A: ESBL 0.125–0.25 negative K. pneumoniae (1) CTX-M-8 0.25 negative K. pneumoniae (1) CTX-M-2/ΔOmpK36b 0.06 negative K. pneumoniae (4) SHV-11/ΔOmpK36b <0.03–2 negative K. pneumoniae (1) TEM-11 /ΔOmpK36b <0.03 negative P. aeruginosa (1) GES-1 16 negative E. cloacae complex (1) PER-2 2 negative E. cloacae complex (1) KPC-2/NDM-1 class A and class B: carbapenemase >64 positive P. mirabilis (6), K. pneumoniae (2), E. cloacae complex (1), S. marcescens (1) IMP-1 class B: carbapenemase 64 to >64 positive (6); negative (4)c P. aeruginosa (1) IMP-18 >64 positive K. pneumoniae (6), E. cloacae complex (1), Enterobacter hormaechei (1), Citrobacter freundii (1) NDM-1 >64 positive P. aeruginosa (7) SPM-1 >64 positive E. cloacae complex (1), P. aeruginosa (1) VIM-1 >64 positive E. coli (2) MIR-1 class C: cephalosporinase 0.125–0.25 negative E. coli (1) CMY-2 0.25 negative K. pneumoniae (1) FOX-5 <0.03 negative Acinetobacter baumannii (2) OXA-72 class D: carbapenemase >64 negative A. baumannii (1) OXA-58 1 negative K. pneumoniae (1) OXA-48 1 negative Salmonella spp. (1) – multi-susceptible 0.5 negative E. coli (1) – 0.5 negative K. pneumoniae (1) – 0.25 negative E. coli ATCC 25922 – 0.25 negative P. aeruginosa ATCC 27853 – 2 negative a A positive result indicates that a ceftazidime hydrolysis MS profile was detected and a negative result indicates that a ceftazidime intact molecule MS profile was observed. b Outer membrane protein study was performed as previously described.12 c Ceftazidime/avibactam hydrolysis was detected by extending the incubation period. Table 1. Molecular characterization of bacterial isolates evaluated by MALDI-TOF MS ceftazidime/avibactam hydrolysis assay Microorganism (n) β-Lactam resistance determinant β-Lactamase molecular class (Ambler) Ceftazidime/avibactam MIC range (mg/L) MALDI-TOF MS ceftazidime/avibactam hydrolysis assaya Klebsiella pneumoniae (44), Serratia marcescens (4), Citrobacter spp. (3), Escherichia coli (2), Rauoltella planticola (1), Klebsiella oxytoca (1), Enterobacter cloacae complex (1), Proteus mirabilis (1), Morganella morganii (1) KPC-2 class A: carbapenemase <0.03–2 negative K. pneumoniae (2) BKC-1 0.5 negative K. pneumoniae (1) GES-5 0.06 negative S. marcescens (1) GES-16 4 negative S. marcescens (1) SME-1 0.06 negative K. pneumoniae (1), E. coli (1) CTX-M-16 class A: ESBL 0.125–0.25 negative K. pneumoniae (1) CTX-M-8 0.25 negative K. pneumoniae (1) CTX-M-2/ΔOmpK36b 0.06 negative K. pneumoniae (4) SHV-11/ΔOmpK36b <0.03–2 negative K. pneumoniae (1) TEM-11 /ΔOmpK36b <0.03 negative P. aeruginosa (1) GES-1 16 negative E. cloacae complex (1) PER-2 2 negative E. cloacae complex (1) KPC-2/NDM-1 class A and class B: carbapenemase >64 positive P. mirabilis (6), K. pneumoniae (2), E. cloacae complex (1), S. marcescens (1) IMP-1 class B: carbapenemase 64 to >64 positive (6); negative (4)c P. aeruginosa (1) IMP-18 >64 positive K. pneumoniae (6), E. cloacae complex (1), Enterobacter hormaechei (1), Citrobacter freundii (1) NDM-1 >64 positive P. aeruginosa (7) SPM-1 >64 positive E. cloacae complex (1), P. aeruginosa (1) VIM-1 >64 positive E. coli (2) MIR-1 class C: cephalosporinase 0.125–0.25 negative E. coli (1) CMY-2 0.25 negative K. pneumoniae (1) FOX-5 <0.03 negative Acinetobacter baumannii (2) OXA-72 class D: carbapenemase >64 negative A. baumannii (1) OXA-58 1 negative K. pneumoniae (1) OXA-48 1 negative Salmonella spp. (1) – multi-susceptible 0.5 negative E. coli (1) – 0.5 negative K. pneumoniae (1) – 0.25 negative E. coli ATCC 25922 – 0.25 negative P. aeruginosa ATCC 27853 – 2 negative Microorganism (n) β-Lactam resistance determinant β-Lactamase molecular class (Ambler) Ceftazidime/avibactam MIC range (mg/L) MALDI-TOF MS ceftazidime/avibactam hydrolysis assaya Klebsiella pneumoniae (44), Serratia marcescens (4), Citrobacter spp. (3), Escherichia coli (2), Rauoltella planticola (1), Klebsiella oxytoca (1), Enterobacter cloacae complex (1), Proteus mirabilis (1), Morganella morganii (1) KPC-2 class A: carbapenemase <0.03–2 negative K. pneumoniae (2) BKC-1 0.5 negative K. pneumoniae (1) GES-5 0.06 negative S. marcescens (1) GES-16 4 negative S. marcescens (1) SME-1 0.06 negative K. pneumoniae (1), E. coli (1) CTX-M-16 class A: ESBL 0.125–0.25 negative K. pneumoniae (1) CTX-M-8 0.25 negative K. pneumoniae (1) CTX-M-2/ΔOmpK36b 0.06 negative K. pneumoniae (4) SHV-11/ΔOmpK36b <0.03–2 negative K. pneumoniae (1) TEM-11 /ΔOmpK36b <0.03 negative P. aeruginosa (1) GES-1 16 negative E. cloacae complex (1) PER-2 2 negative E. cloacae complex (1) KPC-2/NDM-1 class A and class B: carbapenemase >64 positive P. mirabilis (6), K. pneumoniae (2), E. cloacae complex (1), S. marcescens (1) IMP-1 class B: carbapenemase 64 to >64 positive (6); negative (4)c P. aeruginosa (1) IMP-18 >64 positive K. pneumoniae (6), E. cloacae complex (1), Enterobacter hormaechei (1), Citrobacter freundii (1) NDM-1 >64 positive P. aeruginosa (7) SPM-1 >64 positive E. cloacae complex (1), P. aeruginosa (1) VIM-1 >64 positive E. coli (2) MIR-1 class C: cephalosporinase 0.125–0.25 negative E. coli (1) CMY-2 0.25 negative K. pneumoniae (1) FOX-5 <0.03 negative Acinetobacter baumannii (2) OXA-72 class D: carbapenemase >64 negative A. baumannii (1) OXA-58 1 negative K. pneumoniae (1) OXA-48 1 negative Salmonella spp. (1) – multi-susceptible 0.5 negative E. coli (1) – 0.5 negative K. pneumoniae (1) – 0.25 negative E. coli ATCC 25922 – 0.25 negative P. aeruginosa ATCC 27853 – 2 negative a A positive result indicates that a ceftazidime hydrolysis MS profile was detected and a negative result indicates that a ceftazidime intact molecule MS profile was observed. b Outer membrane protein study was performed as previously described.12 c Ceftazidime/avibactam hydrolysis was detected by extending the incubation period. The MALDI-TOF MS hydrolysis assay was performed as previously reported with a few modifications.7 The isolates were grown overnight on Mueller–Hinton agar plates (OxoidTM, Basingstoke, UK) at 37°C. A 1 μL loop of each organism was inoculated into 100 μL of adjusted solution (20 mM Tris-HCl, pH 6.8) with ceftazidime/avibactam (0.20 and 0.05 g/L, respectively; Allergan, Pittsburgh, PA, USA). The reaction tubes were incubated at 37°C for 2 h. After the incubation period, each sample tube was centrifuged at 13 000 rpm for 2 min. One microlitre of the supernatant was spotted onto a MALDI-TOF MS target plate, followed by the addition of 1 μL of α-cyano-4-hydroxycinnamic acid matrix solution (10 g/L; diluted into 49.9% water, 50% acetonitrile and 0.1% trifluoroacetic acid). The mass spectrum was obtained using a Bruker Daltonics Microflex LT instrument, using the Flex Control 3.4 software in the m/z range of 400–600 Da. Avibactam restored the ceftazidime activity when the intact molecular mass peak of ceftzidime ([M + H]+ 547.6 Da) and its pyridine-eliminated molecular mass peak (468.7 Da) were present. In contrast, a resistant phenotype was detected if a mass peak corresponding to pyridine-eliminated hydrolysed form was observed (442.6 Da).7 All isolates were submitted to ceftazidime/avibactam agar dilution susceptibility testing and confirmed by microdilution following the EUCAST guideline.8 In addition, PCR and sequencing experiments were repeated and confirmed the presence of β-lactamase-encoding genes in isolates that showed discordant results (unexpected profile according to β-lactamase content).6,7 In this study, MALDI-TOF MS showed good results for the detection of a ceftazidime/avibactam category of susceptibility compared with those of antimicrobial susceptibility testing (94% agreement, 111 of 118) and molecular methods (97% agreement, 114 of 118) (Table 1). Ceftazidime/avibactam was active against all class A and C β-lactamase-producing isolates tested by the MALDI-TOF MS assay. Carbapenem-hydrolysing class D β-lactamases produced by Acinetobacter spp. commonly do not recognize ceftazidime as a good substrate. In addition, it has been demonstrated that avibactam inhibits mainly OXA-48. In our study, ceftazidime/avibactam hydrolysis was not detected in OXA-48-, OXA-58- and OXA-72-producing isolates. Interestingly, an important reduction of ceftazidime MICs was observed for OXA-48-producing (from 16 to 1 mg/L) and OXA-58-producing (from 64 to 1 mg/L) isolates, when it was combined with avibactam. In contrast, both OXA-72-producing isolates showed high ceftazidime (MICs >64 mg/L) and ceftazidime/avibactam MICs (>64 mg/L), suggesting that other mechanisms of resistance such as hyperexpression of AdeABC systems are probably present in these isolates.9 Interestingly, although avibactam inhibits GES variants, high ceftazidime/avibactam MICs were observed for isolates producing GES-1 and GES-16. These isolates also produce AmpC, so co-production of both class A and class C enzymes may impair the avibactam inhibitory activity. As expected, the majority of MBL-producing isolates (86.2%) were not inhibited by avibactam, because ceftazidime’s intact molecule disappeared and the pyridine-eliminated hydrolysed form was observed. Four IMP-1-producing isolates, two Proteus mirabilis and two Klebsiella pneumoniae, initially presented the intact molecular mass peak of ceftazidime detectable. However, ceftazidime hydrolysis was detected in these isolates, when the incubation period was extended to 4 h. These results are in accordance to the ceftazidime/avibactam susceptibility results. Previous studies have also reported in vivo activity of ceftazidime/avibactam against NDM-1-producing Enterobaceriaceae.10,11 Thus, the possible activity of ceftazidime/avibactam against some MBL-producing Enterobacteriaceae should be confirmed by further studies. As ceftazidime/avibactam has been prescribed for treating infections caused by MDR pathogens, which have few or no therapeutic alternatives, the early and accurate detection of ceftazidime/avibactam in vitro activity against those isolates would be useful to guide antimicrobial therapy. In this study, we applied a ceftazidime/avibactam assay for rapid detection of bacterial resistance to this combination. According to our findings, in addition to rapid microorganism identification, MALDI-TOF MS proved to be an excellent tool for screening ceftazidime/avibactam activity against Enterobacteriaceae isolates, particularly those that do not produce AmpC. To help clinicians to prescribe correctly the ceftazidime/avibactam, before the susceptibility testing results become available, routine laboratories could easily apply this MALDI-TOF MS assay. However, if an isolate shows both peaks corresponding to intact and hydrolysed molecules even after extending the incubation period to 4 h, a non-interpretable result must be reported, advising physicians to wait for final antimicrobial susceptibility results. Acknowledgements We thank Ana Clara Narciso Barbosa for her valuable support during the supplementary tests. Funding The study was carried out as part of our routine work. A. C. G. is a researcher for the National Council for Science and Technological Development (CNPq), Ministry of Science and Technology, Brazil (Process number: 307816/2009–5). M. A. J. is a researcher for the São Paulo Research Foundation (FAPESP; grant no. 12/50191–4). Transparency declarations C. G. C. and A. C. G. have recently received research funding and/or consultation fees from MSD and Pfizer. All other authors: none to declare. References 1 King DT , King AM , Lal SM et al. Molecular mechanism of avibactam-mediated β-lactamase inhibition . ACS Infect Dis 2015 ; 1 : 175 – 84 . Google Scholar CrossRef Search ADS PubMed 2 EMA . ZaviceftaTM (Ceftazidime/Avibactam); Summary of Opinion. http://www.ema.europa.eu/docs/en_GB/document_library/Summary_of_opinion_-_Initial_authorisation/human/004027/WC500205395.pdf. 3 Shields RK , Clancy CJ , Pasculle AW et al. Verification of ceftazidime/avibactam and ceftolozane/tazobactam susceptibility testing methods against carbapenem-resistant Enterobacteriaceae and Pseudomonas aeruginosa . J Clin Microbiol 2018 ; 56 : e01093-17 . Google Scholar CrossRef Search ADS PubMed 4 Carvalhaes CG , Cayô R , Visconde MF et al. Detection of carbapenemase activity directly from blood culture vials using MALDI-TOF MS: a quick answer for the right decision . J Antimicrob Chemother 2014 ; 69 : 2132 – 6 . Google Scholar CrossRef Search ADS PubMed 5 Ramos AC , Cayô R , Carvalhaes CG et al. Dissemination of multidrug-resistant Proteus mirabilis clones carrying a novel integron-borne blaIMP-1 in a tertiary hospital . Antimicrob Agents Chemother 2018 ; 62 : e01321-17 . Google Scholar CrossRef Search ADS PubMed 6 Carvalhaes CG , da Silva AC , Streling AP et al. Detection of carbapenemase activity using VITEK MS: interplay of carbapenemase type and period of incubation . J Med Microbiol 2015 ; 64 : 946 – 7 . Google Scholar CrossRef Search ADS PubMed 7 Sparbier K , Schubert S , Weller U et al. Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based functional assay for rapid detection of resistance against β-lactam antibiotics . J Clin Microbiol 2012 ; 50 : 927 – 37 . Google Scholar CrossRef Search ADS PubMed 8 EUCAST . Breakpoint Tables for Interpretation of MICs and Zone Diameters, Version 8.0, 2018 . http://www.eucast.org. 9 Peleg AY , Adams J , Paterson DL. Tigecycline efflux as a mechanism for nonsusceptibility in Acinetobacter baumannii . Antimicrob Agents Chemother 2007 ; 51 : 2065 – 9 . Google Scholar CrossRef Search ADS PubMed 10 Monogue ML , Abbo LM , Rosa R et al. In vitro discordance with in vivo activity: humanized exposures of ceftazidime/avibactam, aztreonam, and tigecycline alone and in combination against New Delhi metallo-β-lactamase-producing Klebsiella pneumoniae in a murine lung infection model . Antimicrob Agents Chemother 2017 ; 61 : e00486-17 . Google Scholar CrossRef Search ADS PubMed 11 MacVane SH , Crandon JL , Nichols WW et al. Unexpected in vivo activity of ceftazidime alone and in combination with avibactam against New Delhi metallo β-lactamase-producing Enterobacteriaceae in a murine thigh infection model . Antimicrob Agents Chemother 2014 ; 58 : 7007 – 9 . Google Scholar CrossRef Search ADS PubMed 12 Carvalhaes CG , Picão RC , Nicoletti AG et al. Cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results . J Antimicrob Chemother 2010 ; 65 : 249 – 51 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. 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Abstract

Sir, Over the last decades, MDR Gram-negative bacilli have emerged as an important aetiological agent of nosocomial-acquired infections worldwide. The combination of β-lactam and new β-lactamases inhibitors has become an option for treating those infections with fewer side effects than old compounds such as polymyxins. Avibactam is a diazabicyclooctane β-lactamase inhibitor that demonstrates excellent inhibitory properties against class A, class C and some class D β-lactamases (basically OXA-48). In contrast, it does not possess activity against most MBL-producing isolates.1 Recently, ceftazidime/avibactam was approved for the treatment of patients with complicated intra-abdominal infections, complicated urinary tract infections and hospital-acquired pneumonia.2 To reduce the development of drug-resistant bacteria and preserve the susceptibility of ceftazidime/avibactam, it should be used only to treat infections that are proven or strongly suspected to be caused by susceptible bacteria. However, susceptibility testing of new drugs in routine laboratories is restricted to disc and/or gradient diffusion methodologies until its inclusion on commercial panels of susceptibility automated systems. According to a recent evaluation, ceftazidime/avibactam susceptibility testing using Etest and disc diffusion methods presented very major and major errors, respectively, compared to broth microdilution, and those tests should be interpreted cautiously in clinical practice.3 MALDI-TOF MS has been applied for the detection of carbapenems hydrolysis.4 As susceptibility testing to ceftazidime/avibactam might be tricky, herein, we evaluated the performance of MALDI-TOF MS for rapid detection of ceftazidime/avibactam resistance by testing against 118 previous molecular characterized bacterial clinical isolates, including a range of different β-lactamase-producing Enterobacteriaceae and Pseudomonas aeruginosa (Table 1).5,6 In addition, 21 non-carbapenemase-producing isolates were also studied. Species identification was confirmed by MALDI-TOF MS (Biotyper 3.3 version; Bruker Daltonics, Bremen, Germany). Table 1. Molecular characterization of bacterial isolates evaluated by MALDI-TOF MS ceftazidime/avibactam hydrolysis assay Microorganism (n) β-Lactam resistance determinant β-Lactamase molecular class (Ambler) Ceftazidime/avibactam MIC range (mg/L) MALDI-TOF MS ceftazidime/avibactam hydrolysis assaya Klebsiella pneumoniae (44), Serratia marcescens (4), Citrobacter spp. (3), Escherichia coli (2), Rauoltella planticola (1), Klebsiella oxytoca (1), Enterobacter cloacae complex (1), Proteus mirabilis (1), Morganella morganii (1) KPC-2 class A: carbapenemase <0.03–2 negative K. pneumoniae (2) BKC-1 0.5 negative K. pneumoniae (1) GES-5 0.06 negative S. marcescens (1) GES-16 4 negative S. marcescens (1) SME-1 0.06 negative K. pneumoniae (1), E. coli (1) CTX-M-16 class A: ESBL 0.125–0.25 negative K. pneumoniae (1) CTX-M-8 0.25 negative K. pneumoniae (1) CTX-M-2/ΔOmpK36b 0.06 negative K. pneumoniae (4) SHV-11/ΔOmpK36b <0.03–2 negative K. pneumoniae (1) TEM-11 /ΔOmpK36b <0.03 negative P. aeruginosa (1) GES-1 16 negative E. cloacae complex (1) PER-2 2 negative E. cloacae complex (1) KPC-2/NDM-1 class A and class B: carbapenemase >64 positive P. mirabilis (6), K. pneumoniae (2), E. cloacae complex (1), S. marcescens (1) IMP-1 class B: carbapenemase 64 to >64 positive (6); negative (4)c P. aeruginosa (1) IMP-18 >64 positive K. pneumoniae (6), E. cloacae complex (1), Enterobacter hormaechei (1), Citrobacter freundii (1) NDM-1 >64 positive P. aeruginosa (7) SPM-1 >64 positive E. cloacae complex (1), P. aeruginosa (1) VIM-1 >64 positive E. coli (2) MIR-1 class C: cephalosporinase 0.125–0.25 negative E. coli (1) CMY-2 0.25 negative K. pneumoniae (1) FOX-5 <0.03 negative Acinetobacter baumannii (2) OXA-72 class D: carbapenemase >64 negative A. baumannii (1) OXA-58 1 negative K. pneumoniae (1) OXA-48 1 negative Salmonella spp. (1) – multi-susceptible 0.5 negative E. coli (1) – 0.5 negative K. pneumoniae (1) – 0.25 negative E. coli ATCC 25922 – 0.25 negative P. aeruginosa ATCC 27853 – 2 negative Microorganism (n) β-Lactam resistance determinant β-Lactamase molecular class (Ambler) Ceftazidime/avibactam MIC range (mg/L) MALDI-TOF MS ceftazidime/avibactam hydrolysis assaya Klebsiella pneumoniae (44), Serratia marcescens (4), Citrobacter spp. (3), Escherichia coli (2), Rauoltella planticola (1), Klebsiella oxytoca (1), Enterobacter cloacae complex (1), Proteus mirabilis (1), Morganella morganii (1) KPC-2 class A: carbapenemase <0.03–2 negative K. pneumoniae (2) BKC-1 0.5 negative K. pneumoniae (1) GES-5 0.06 negative S. marcescens (1) GES-16 4 negative S. marcescens (1) SME-1 0.06 negative K. pneumoniae (1), E. coli (1) CTX-M-16 class A: ESBL 0.125–0.25 negative K. pneumoniae (1) CTX-M-8 0.25 negative K. pneumoniae (1) CTX-M-2/ΔOmpK36b 0.06 negative K. pneumoniae (4) SHV-11/ΔOmpK36b <0.03–2 negative K. pneumoniae (1) TEM-11 /ΔOmpK36b <0.03 negative P. aeruginosa (1) GES-1 16 negative E. cloacae complex (1) PER-2 2 negative E. cloacae complex (1) KPC-2/NDM-1 class A and class B: carbapenemase >64 positive P. mirabilis (6), K. pneumoniae (2), E. cloacae complex (1), S. marcescens (1) IMP-1 class B: carbapenemase 64 to >64 positive (6); negative (4)c P. aeruginosa (1) IMP-18 >64 positive K. pneumoniae (6), E. cloacae complex (1), Enterobacter hormaechei (1), Citrobacter freundii (1) NDM-1 >64 positive P. aeruginosa (7) SPM-1 >64 positive E. cloacae complex (1), P. aeruginosa (1) VIM-1 >64 positive E. coli (2) MIR-1 class C: cephalosporinase 0.125–0.25 negative E. coli (1) CMY-2 0.25 negative K. pneumoniae (1) FOX-5 <0.03 negative Acinetobacter baumannii (2) OXA-72 class D: carbapenemase >64 negative A. baumannii (1) OXA-58 1 negative K. pneumoniae (1) OXA-48 1 negative Salmonella spp. (1) – multi-susceptible 0.5 negative E. coli (1) – 0.5 negative K. pneumoniae (1) – 0.25 negative E. coli ATCC 25922 – 0.25 negative P. aeruginosa ATCC 27853 – 2 negative a A positive result indicates that a ceftazidime hydrolysis MS profile was detected and a negative result indicates that a ceftazidime intact molecule MS profile was observed. b Outer membrane protein study was performed as previously described.12 c Ceftazidime/avibactam hydrolysis was detected by extending the incubation period. Table 1. Molecular characterization of bacterial isolates evaluated by MALDI-TOF MS ceftazidime/avibactam hydrolysis assay Microorganism (n) β-Lactam resistance determinant β-Lactamase molecular class (Ambler) Ceftazidime/avibactam MIC range (mg/L) MALDI-TOF MS ceftazidime/avibactam hydrolysis assaya Klebsiella pneumoniae (44), Serratia marcescens (4), Citrobacter spp. (3), Escherichia coli (2), Rauoltella planticola (1), Klebsiella oxytoca (1), Enterobacter cloacae complex (1), Proteus mirabilis (1), Morganella morganii (1) KPC-2 class A: carbapenemase <0.03–2 negative K. pneumoniae (2) BKC-1 0.5 negative K. pneumoniae (1) GES-5 0.06 negative S. marcescens (1) GES-16 4 negative S. marcescens (1) SME-1 0.06 negative K. pneumoniae (1), E. coli (1) CTX-M-16 class A: ESBL 0.125–0.25 negative K. pneumoniae (1) CTX-M-8 0.25 negative K. pneumoniae (1) CTX-M-2/ΔOmpK36b 0.06 negative K. pneumoniae (4) SHV-11/ΔOmpK36b <0.03–2 negative K. pneumoniae (1) TEM-11 /ΔOmpK36b <0.03 negative P. aeruginosa (1) GES-1 16 negative E. cloacae complex (1) PER-2 2 negative E. cloacae complex (1) KPC-2/NDM-1 class A and class B: carbapenemase >64 positive P. mirabilis (6), K. pneumoniae (2), E. cloacae complex (1), S. marcescens (1) IMP-1 class B: carbapenemase 64 to >64 positive (6); negative (4)c P. aeruginosa (1) IMP-18 >64 positive K. pneumoniae (6), E. cloacae complex (1), Enterobacter hormaechei (1), Citrobacter freundii (1) NDM-1 >64 positive P. aeruginosa (7) SPM-1 >64 positive E. cloacae complex (1), P. aeruginosa (1) VIM-1 >64 positive E. coli (2) MIR-1 class C: cephalosporinase 0.125–0.25 negative E. coli (1) CMY-2 0.25 negative K. pneumoniae (1) FOX-5 <0.03 negative Acinetobacter baumannii (2) OXA-72 class D: carbapenemase >64 negative A. baumannii (1) OXA-58 1 negative K. pneumoniae (1) OXA-48 1 negative Salmonella spp. (1) – multi-susceptible 0.5 negative E. coli (1) – 0.5 negative K. pneumoniae (1) – 0.25 negative E. coli ATCC 25922 – 0.25 negative P. aeruginosa ATCC 27853 – 2 negative Microorganism (n) β-Lactam resistance determinant β-Lactamase molecular class (Ambler) Ceftazidime/avibactam MIC range (mg/L) MALDI-TOF MS ceftazidime/avibactam hydrolysis assaya Klebsiella pneumoniae (44), Serratia marcescens (4), Citrobacter spp. (3), Escherichia coli (2), Rauoltella planticola (1), Klebsiella oxytoca (1), Enterobacter cloacae complex (1), Proteus mirabilis (1), Morganella morganii (1) KPC-2 class A: carbapenemase <0.03–2 negative K. pneumoniae (2) BKC-1 0.5 negative K. pneumoniae (1) GES-5 0.06 negative S. marcescens (1) GES-16 4 negative S. marcescens (1) SME-1 0.06 negative K. pneumoniae (1), E. coli (1) CTX-M-16 class A: ESBL 0.125–0.25 negative K. pneumoniae (1) CTX-M-8 0.25 negative K. pneumoniae (1) CTX-M-2/ΔOmpK36b 0.06 negative K. pneumoniae (4) SHV-11/ΔOmpK36b <0.03–2 negative K. pneumoniae (1) TEM-11 /ΔOmpK36b <0.03 negative P. aeruginosa (1) GES-1 16 negative E. cloacae complex (1) PER-2 2 negative E. cloacae complex (1) KPC-2/NDM-1 class A and class B: carbapenemase >64 positive P. mirabilis (6), K. pneumoniae (2), E. cloacae complex (1), S. marcescens (1) IMP-1 class B: carbapenemase 64 to >64 positive (6); negative (4)c P. aeruginosa (1) IMP-18 >64 positive K. pneumoniae (6), E. cloacae complex (1), Enterobacter hormaechei (1), Citrobacter freundii (1) NDM-1 >64 positive P. aeruginosa (7) SPM-1 >64 positive E. cloacae complex (1), P. aeruginosa (1) VIM-1 >64 positive E. coli (2) MIR-1 class C: cephalosporinase 0.125–0.25 negative E. coli (1) CMY-2 0.25 negative K. pneumoniae (1) FOX-5 <0.03 negative Acinetobacter baumannii (2) OXA-72 class D: carbapenemase >64 negative A. baumannii (1) OXA-58 1 negative K. pneumoniae (1) OXA-48 1 negative Salmonella spp. (1) – multi-susceptible 0.5 negative E. coli (1) – 0.5 negative K. pneumoniae (1) – 0.25 negative E. coli ATCC 25922 – 0.25 negative P. aeruginosa ATCC 27853 – 2 negative a A positive result indicates that a ceftazidime hydrolysis MS profile was detected and a negative result indicates that a ceftazidime intact molecule MS profile was observed. b Outer membrane protein study was performed as previously described.12 c Ceftazidime/avibactam hydrolysis was detected by extending the incubation period. The MALDI-TOF MS hydrolysis assay was performed as previously reported with a few modifications.7 The isolates were grown overnight on Mueller–Hinton agar plates (OxoidTM, Basingstoke, UK) at 37°C. A 1 μL loop of each organism was inoculated into 100 μL of adjusted solution (20 mM Tris-HCl, pH 6.8) with ceftazidime/avibactam (0.20 and 0.05 g/L, respectively; Allergan, Pittsburgh, PA, USA). The reaction tubes were incubated at 37°C for 2 h. After the incubation period, each sample tube was centrifuged at 13 000 rpm for 2 min. One microlitre of the supernatant was spotted onto a MALDI-TOF MS target plate, followed by the addition of 1 μL of α-cyano-4-hydroxycinnamic acid matrix solution (10 g/L; diluted into 49.9% water, 50% acetonitrile and 0.1% trifluoroacetic acid). The mass spectrum was obtained using a Bruker Daltonics Microflex LT instrument, using the Flex Control 3.4 software in the m/z range of 400–600 Da. Avibactam restored the ceftazidime activity when the intact molecular mass peak of ceftzidime ([M + H]+ 547.6 Da) and its pyridine-eliminated molecular mass peak (468.7 Da) were present. In contrast, a resistant phenotype was detected if a mass peak corresponding to pyridine-eliminated hydrolysed form was observed (442.6 Da).7 All isolates were submitted to ceftazidime/avibactam agar dilution susceptibility testing and confirmed by microdilution following the EUCAST guideline.8 In addition, PCR and sequencing experiments were repeated and confirmed the presence of β-lactamase-encoding genes in isolates that showed discordant results (unexpected profile according to β-lactamase content).6,7 In this study, MALDI-TOF MS showed good results for the detection of a ceftazidime/avibactam category of susceptibility compared with those of antimicrobial susceptibility testing (94% agreement, 111 of 118) and molecular methods (97% agreement, 114 of 118) (Table 1). Ceftazidime/avibactam was active against all class A and C β-lactamase-producing isolates tested by the MALDI-TOF MS assay. Carbapenem-hydrolysing class D β-lactamases produced by Acinetobacter spp. commonly do not recognize ceftazidime as a good substrate. In addition, it has been demonstrated that avibactam inhibits mainly OXA-48. In our study, ceftazidime/avibactam hydrolysis was not detected in OXA-48-, OXA-58- and OXA-72-producing isolates. Interestingly, an important reduction of ceftazidime MICs was observed for OXA-48-producing (from 16 to 1 mg/L) and OXA-58-producing (from 64 to 1 mg/L) isolates, when it was combined with avibactam. In contrast, both OXA-72-producing isolates showed high ceftazidime (MICs >64 mg/L) and ceftazidime/avibactam MICs (>64 mg/L), suggesting that other mechanisms of resistance such as hyperexpression of AdeABC systems are probably present in these isolates.9 Interestingly, although avibactam inhibits GES variants, high ceftazidime/avibactam MICs were observed for isolates producing GES-1 and GES-16. These isolates also produce AmpC, so co-production of both class A and class C enzymes may impair the avibactam inhibitory activity. As expected, the majority of MBL-producing isolates (86.2%) were not inhibited by avibactam, because ceftazidime’s intact molecule disappeared and the pyridine-eliminated hydrolysed form was observed. Four IMP-1-producing isolates, two Proteus mirabilis and two Klebsiella pneumoniae, initially presented the intact molecular mass peak of ceftazidime detectable. However, ceftazidime hydrolysis was detected in these isolates, when the incubation period was extended to 4 h. These results are in accordance to the ceftazidime/avibactam susceptibility results. Previous studies have also reported in vivo activity of ceftazidime/avibactam against NDM-1-producing Enterobaceriaceae.10,11 Thus, the possible activity of ceftazidime/avibactam against some MBL-producing Enterobacteriaceae should be confirmed by further studies. As ceftazidime/avibactam has been prescribed for treating infections caused by MDR pathogens, which have few or no therapeutic alternatives, the early and accurate detection of ceftazidime/avibactam in vitro activity against those isolates would be useful to guide antimicrobial therapy. In this study, we applied a ceftazidime/avibactam assay for rapid detection of bacterial resistance to this combination. According to our findings, in addition to rapid microorganism identification, MALDI-TOF MS proved to be an excellent tool for screening ceftazidime/avibactam activity against Enterobacteriaceae isolates, particularly those that do not produce AmpC. To help clinicians to prescribe correctly the ceftazidime/avibactam, before the susceptibility testing results become available, routine laboratories could easily apply this MALDI-TOF MS assay. However, if an isolate shows both peaks corresponding to intact and hydrolysed molecules even after extending the incubation period to 4 h, a non-interpretable result must be reported, advising physicians to wait for final antimicrobial susceptibility results. Acknowledgements We thank Ana Clara Narciso Barbosa for her valuable support during the supplementary tests. Funding The study was carried out as part of our routine work. A. C. G. is a researcher for the National Council for Science and Technological Development (CNPq), Ministry of Science and Technology, Brazil (Process number: 307816/2009–5). M. A. J. is a researcher for the São Paulo Research Foundation (FAPESP; grant no. 12/50191–4). Transparency declarations C. G. C. and A. C. G. have recently received research funding and/or consultation fees from MSD and Pfizer. All other authors: none to declare. References 1 King DT , King AM , Lal SM et al. Molecular mechanism of avibactam-mediated β-lactamase inhibition . ACS Infect Dis 2015 ; 1 : 175 – 84 . Google Scholar CrossRef Search ADS PubMed 2 EMA . ZaviceftaTM (Ceftazidime/Avibactam); Summary of Opinion. http://www.ema.europa.eu/docs/en_GB/document_library/Summary_of_opinion_-_Initial_authorisation/human/004027/WC500205395.pdf. 3 Shields RK , Clancy CJ , Pasculle AW et al. Verification of ceftazidime/avibactam and ceftolozane/tazobactam susceptibility testing methods against carbapenem-resistant Enterobacteriaceae and Pseudomonas aeruginosa . J Clin Microbiol 2018 ; 56 : e01093-17 . Google Scholar CrossRef Search ADS PubMed 4 Carvalhaes CG , Cayô R , Visconde MF et al. Detection of carbapenemase activity directly from blood culture vials using MALDI-TOF MS: a quick answer for the right decision . J Antimicrob Chemother 2014 ; 69 : 2132 – 6 . Google Scholar CrossRef Search ADS PubMed 5 Ramos AC , Cayô R , Carvalhaes CG et al. Dissemination of multidrug-resistant Proteus mirabilis clones carrying a novel integron-borne blaIMP-1 in a tertiary hospital . Antimicrob Agents Chemother 2018 ; 62 : e01321-17 . Google Scholar CrossRef Search ADS PubMed 6 Carvalhaes CG , da Silva AC , Streling AP et al. Detection of carbapenemase activity using VITEK MS: interplay of carbapenemase type and period of incubation . J Med Microbiol 2015 ; 64 : 946 – 7 . Google Scholar CrossRef Search ADS PubMed 7 Sparbier K , Schubert S , Weller U et al. Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based functional assay for rapid detection of resistance against β-lactam antibiotics . J Clin Microbiol 2012 ; 50 : 927 – 37 . Google Scholar CrossRef Search ADS PubMed 8 EUCAST . Breakpoint Tables for Interpretation of MICs and Zone Diameters, Version 8.0, 2018 . http://www.eucast.org. 9 Peleg AY , Adams J , Paterson DL. Tigecycline efflux as a mechanism for nonsusceptibility in Acinetobacter baumannii . Antimicrob Agents Chemother 2007 ; 51 : 2065 – 9 . Google Scholar CrossRef Search ADS PubMed 10 Monogue ML , Abbo LM , Rosa R et al. In vitro discordance with in vivo activity: humanized exposures of ceftazidime/avibactam, aztreonam, and tigecycline alone and in combination against New Delhi metallo-β-lactamase-producing Klebsiella pneumoniae in a murine lung infection model . Antimicrob Agents Chemother 2017 ; 61 : e00486-17 . Google Scholar CrossRef Search ADS PubMed 11 MacVane SH , Crandon JL , Nichols WW et al. Unexpected in vivo activity of ceftazidime alone and in combination with avibactam against New Delhi metallo β-lactamase-producing Enterobacteriaceae in a murine thigh infection model . Antimicrob Agents Chemother 2014 ; 58 : 7007 – 9 . Google Scholar CrossRef Search ADS PubMed 12 Carvalhaes CG , Picão RC , Nicoletti AG et al. Cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results . J Antimicrob Chemother 2010 ; 65 : 249 – 51 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Jun 6, 2018

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