Antimicrobial susceptibilities of clinical Legionella longbeachae isolates

Antimicrobial susceptibilities of clinical Legionella longbeachae isolates Sir, Legionella spp., particularly Legionella pneumophila, are an important cause of community-acquired pneumonia.1 Standard treatment involves intracellularly active agents, supported by in vitro and clinical outcome data.1,Legionella spp. are fastidious, requiring charcoal-containing culture media, buffered charcoal yeast extract (BCYE), which binds inhibitory substances and facilitates growth.1 However, charcoal also binds antimicrobials,2,3 and attempts to remove charcoal from media yield poor growth.3 Despite this, MICs have been determined using BCYE by broth dilution, agar dilution and Etest methods,2–4 with the Etest method comparable to other methods.4 In New Zealand, Legionella longbeachae predominates,5 and our centre has a large collection of clinical isolates following the introduction of mandatory testing of all respiratory samples of patients with suspected pneumonia.6 We sought to define WT susceptibilities to commonly used antimicrobials [azithromycin (Sigma PHR1088), clarithromycin (Sigma PHR1038), ciprofloxacin (Sigma 17850), moxifloxacin (Bayer), rifampicin (Sigma R3501) and tetracycline (Sigma T-3383)] using our archived isolates. We established broth dilution MIC ranges and compared them with those obtained using the Etest method. Sixty-one archived L. longbeachae clinical isolates cultured from respiratory samples of patients admitted to Christchurch Hospital, Christchurch, New Zealand between 2005 and 2014 were studied. Isolates were confirmed as L. longbeachae by standard phenotypic testing and MALDI-TOF MS analysis and were subcultured twice on BCYE media supplemented with α-ketoglutarate at 35 °C. Colonies were suspended in sterile saline to a turbidity equivalent to that of a 0.5 McFarland standard. Broth dilutions were performed as recommended by CLSI M100-S227 using 2-fold serial dilutions of the antibiotics in buffered yeast extract (BYE) broth. The broth dilution MIC plates were incubated at 35 °C in ambient air and inhibitory concentrations were determined at 72 h. Etests were performed by the inoculation of the organism (turbidity equivalent to that of a 0.5 McFarland standard) on to BCYE plates (Fort Richard Laboratories, Auckland, New Zealand). Etest gradient strips (AB Biodisk) were applied for azithromycin, clarithromycin, ciprofloxacin, moxifloxacin, rifampicin and tetracycline. Plates were incubated at 35 °C and read at 48 h. Etest strips were read by eyepiece according to the manufacturer’s instructions. MIC50 and MIC90 values and ranges after 48 h of incubation are shown in Table 1 for both Etest- and broth dilution-derived MICs. Table 1 MIC (mg/L) values for L. longbeachae by broth dilution and Etest, with L. pneumophila (Etest on BCYE) for comparison9 Antibiotic  Etest (BCYE)   Broth dilution (BYE)   L. pneumophila Etest MIC90  MIC50  MIC90  range  MIC50  MIC90  range  Azithromycin  0.125  0.19  0.064–0.25  0.062  0.125  0.032–0.25  0.25  Ciprofloxacin  0.5  0.5  0.38–0.75  0.015  0.031  ≤0.008–0.064  0.5  Clarithromycin  0.25  0.5  0.094–0.5  0.031  0.125  ≤0.008–0.25  0.25  Moxifloxacin  0.5  0.5  0.25–0.75  0.015  0.031  ≤0.008–0.064  0.5  Rifampicin  0.125  0.38  0.064–1  0.007  0.031  ≤0.008–0.064  0.032  Tetracycline  16  32  8–32  32  64  8–64  8  Antibiotic  Etest (BCYE)   Broth dilution (BYE)   L. pneumophila Etest MIC90  MIC50  MIC90  range  MIC50  MIC90  range  Azithromycin  0.125  0.19  0.064–0.25  0.062  0.125  0.032–0.25  0.25  Ciprofloxacin  0.5  0.5  0.38–0.75  0.015  0.031  ≤0.008–0.064  0.5  Clarithromycin  0.25  0.5  0.094–0.5  0.031  0.125  ≤0.008–0.25  0.25  Moxifloxacin  0.5  0.5  0.25–0.75  0.015  0.031  ≤0.008–0.064  0.5  Rifampicin  0.125  0.38  0.064–1  0.007  0.031  ≤0.008–0.064  0.032  Tetracycline  16  32  8–32  32  64  8–64  8  To evaluate the potential effect of charcoal-binding antimicrobials, susceptibilities of the ATCC strain 29213 of Staphylococcus aureus were determined using both Mueller–Hinton and BCYE media, using the same methodology and antimicrobials. MIC values fell within the CLSI range on Mueller–Hinton agar, but MIC values were markedly elevated on BCYE media for all antimicrobials. Rifampicin was most affected, with nine dilutions difference between the MICs on the different agars. To the best of our knowledge, this is the first study determining the antimicrobial susceptibility patterns of clinical L. longbeachae isolates. All antimicrobials tested except for tetracycline had low MICs, with the lowest MIC90 (0.031 mg/L) of ciprofloxacin, rifampicin and moxifloxacin by broth dilution. Broth dilution-derived MICs were lower than Etest values, particularly for the quinolones, with seven dilutions difference between their MIC90 values. Compared with the other antimicrobials, tetracycline had markedly elevated MIC90 values by broth dilution and Etest (64 and 32 mg/L). This may be due to poor diffusion of tetracycline into the media or variations in electrolyte content of the media leading to chelation of tetracycline. Additionally, tetracycline reductase enzymes have been identified in soil-derived L. longbeachae specimens, which have been shown to inactivate tetracycline in vivo, but as yet have not been identified in L. pneumophila.8 Our results are similar to MICs for L. pneumophila determined by the same methodology4,9 (Table 1), with higher Etest-derived MIC90 values of rifampicin (0.38 versus 0.032 mg/L) and tetracycline (32 versus 8 mg/L). The reason for this difference is unclear and may represent a genuine difference in susceptibility between the two Legionella species. A major limitation is the effect of charcoal-binding antimicrobials as illustrated by the S. aureus control and the lower broth dilution-derived MIC values using BYE. The effect of charcoal binding has previously been explored; reducing the charcoal concentration of BCYE by 75% yielded 4–8-fold lower MIC90 values of ciprofloxacin and gemifloxacin, although levofloxacin, erythromycin and clarithromycin MIC values were unaffected.2 The difference in MIC persisted in comparison with BYE media, confirming the difference in MIC is due to charcoal-binding free drug. Despite this, non-L. pneumophila species have been found to grow poorly on charcoal-free media3 and adequate growth is necessary for accurate MIC derivation. Accordingly, EUCAST recommend BCYE for L. pneumophila susceptibility testing.10 These findings may not translate to in vivo efficacy and more data are needed to define further the WT susceptibilities. However, they support use of the Etest method on BCYE media for MIC derivation and reinforce the use of quinolones in clinical disease. Our results are derived from clinical isolates in one geographical area of New Zealand only and may not be representative of isolates in other regions or environmental isolates. Acknowledgements We thank Anja Werno, Clinical Director of Microbiology at Canterbury Health Laboratories. Funding This study was supported by internal funding. Transparency declarations None to declare. References 1 Fields BS, Benson RF, Besser RE. Legionella and Legionnaires’ disease: 25 years of investigation. Clin Microbiol Rev  2002; 15: 506– 26. Google Scholar CrossRef Search ADS PubMed  2 García MT, Pelaz C, Giménez MJ et al.   In vitro activities of gemifloxacin versus five quinolones and two macrolides against 271 Spanish isolates of Legionella pneumophila: influence of charcoal on susceptibility test results. Antimicrob Agents Chemother  2000; 44: 2176– 8. Google Scholar CrossRef Search ADS PubMed  3 Pendland SL, Martin SJ, Chen C et al.   Comparison of charcoal- and starch-based media for testing susceptibilities of Legionella species to macrolides, azalides, and fluoroquinolones. J Clin Microbiol  1997; 35: 3004– 6. Google Scholar PubMed  4 Rhomberg PR, Bale MJ, Jones RN. Application of the Etest to antimicrobial susceptibility testing of Legionella spp. Diagn Microbiol Infect Dis  1994; 19: 175– 8. Google Scholar CrossRef Search ADS PubMed  5 Notifiable Diseases in New Zealand Annual Report 2016. https://surv.esr.cri.nz/PDF_surveillance/AnnualRpt/AnnualSurv/2016/2016AnnualNDReportFinal.pdf. 6 Murdoch DR, Podmore RG, Anderson TP et al.   Impact of routine systematic polymerase chain reaction testing on case finding for Legionnaires' disease: a pre-post comparison study. Clin Infect Dis  2013; 57: 1275– 81. Google Scholar CrossRef Search ADS PubMed  7 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Second Informational Supplement M100-S22 . CLSI, Wayne, PA, USA, 2012. 8 Forsberg KJ, Patel S, Wencewicz TA et al.   The tetracycline destructases: a novel family of tetracycline-inactivating enzymes. Chem Biol  2015; 22: 888– 97. Google Scholar CrossRef Search ADS PubMed  9 Bruin JP, Ijzerman EP, den Boer JW et al.   Wild-type MIC distribution and epidemiological cut-off values in clinical Legionella pneumophila serogroup 1 isolates. Diagn Microbiol Infect Dis  2012; 72: 103– 8. Google Scholar CrossRef Search ADS PubMed  10 EUCAST. Antimicrobial Susceptibility Testing of Legionella pneumophila. http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/General_documents/Legionella_guidance_document_20160909.pdf. © The Author(s) 2017. 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Antimicrobial susceptibilities of clinical Legionella longbeachae isolates

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Oxford University Press
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0305-7453
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1460-2091
D.O.I.
10.1093/jac/dkx484
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Abstract

Sir, Legionella spp., particularly Legionella pneumophila, are an important cause of community-acquired pneumonia.1 Standard treatment involves intracellularly active agents, supported by in vitro and clinical outcome data.1,Legionella spp. are fastidious, requiring charcoal-containing culture media, buffered charcoal yeast extract (BCYE), which binds inhibitory substances and facilitates growth.1 However, charcoal also binds antimicrobials,2,3 and attempts to remove charcoal from media yield poor growth.3 Despite this, MICs have been determined using BCYE by broth dilution, agar dilution and Etest methods,2–4 with the Etest method comparable to other methods.4 In New Zealand, Legionella longbeachae predominates,5 and our centre has a large collection of clinical isolates following the introduction of mandatory testing of all respiratory samples of patients with suspected pneumonia.6 We sought to define WT susceptibilities to commonly used antimicrobials [azithromycin (Sigma PHR1088), clarithromycin (Sigma PHR1038), ciprofloxacin (Sigma 17850), moxifloxacin (Bayer), rifampicin (Sigma R3501) and tetracycline (Sigma T-3383)] using our archived isolates. We established broth dilution MIC ranges and compared them with those obtained using the Etest method. Sixty-one archived L. longbeachae clinical isolates cultured from respiratory samples of patients admitted to Christchurch Hospital, Christchurch, New Zealand between 2005 and 2014 were studied. Isolates were confirmed as L. longbeachae by standard phenotypic testing and MALDI-TOF MS analysis and were subcultured twice on BCYE media supplemented with α-ketoglutarate at 35 °C. Colonies were suspended in sterile saline to a turbidity equivalent to that of a 0.5 McFarland standard. Broth dilutions were performed as recommended by CLSI M100-S227 using 2-fold serial dilutions of the antibiotics in buffered yeast extract (BYE) broth. The broth dilution MIC plates were incubated at 35 °C in ambient air and inhibitory concentrations were determined at 72 h. Etests were performed by the inoculation of the organism (turbidity equivalent to that of a 0.5 McFarland standard) on to BCYE plates (Fort Richard Laboratories, Auckland, New Zealand). Etest gradient strips (AB Biodisk) were applied for azithromycin, clarithromycin, ciprofloxacin, moxifloxacin, rifampicin and tetracycline. Plates were incubated at 35 °C and read at 48 h. Etest strips were read by eyepiece according to the manufacturer’s instructions. MIC50 and MIC90 values and ranges after 48 h of incubation are shown in Table 1 for both Etest- and broth dilution-derived MICs. Table 1 MIC (mg/L) values for L. longbeachae by broth dilution and Etest, with L. pneumophila (Etest on BCYE) for comparison9 Antibiotic  Etest (BCYE)   Broth dilution (BYE)   L. pneumophila Etest MIC90  MIC50  MIC90  range  MIC50  MIC90  range  Azithromycin  0.125  0.19  0.064–0.25  0.062  0.125  0.032–0.25  0.25  Ciprofloxacin  0.5  0.5  0.38–0.75  0.015  0.031  ≤0.008–0.064  0.5  Clarithromycin  0.25  0.5  0.094–0.5  0.031  0.125  ≤0.008–0.25  0.25  Moxifloxacin  0.5  0.5  0.25–0.75  0.015  0.031  ≤0.008–0.064  0.5  Rifampicin  0.125  0.38  0.064–1  0.007  0.031  ≤0.008–0.064  0.032  Tetracycline  16  32  8–32  32  64  8–64  8  Antibiotic  Etest (BCYE)   Broth dilution (BYE)   L. pneumophila Etest MIC90  MIC50  MIC90  range  MIC50  MIC90  range  Azithromycin  0.125  0.19  0.064–0.25  0.062  0.125  0.032–0.25  0.25  Ciprofloxacin  0.5  0.5  0.38–0.75  0.015  0.031  ≤0.008–0.064  0.5  Clarithromycin  0.25  0.5  0.094–0.5  0.031  0.125  ≤0.008–0.25  0.25  Moxifloxacin  0.5  0.5  0.25–0.75  0.015  0.031  ≤0.008–0.064  0.5  Rifampicin  0.125  0.38  0.064–1  0.007  0.031  ≤0.008–0.064  0.032  Tetracycline  16  32  8–32  32  64  8–64  8  To evaluate the potential effect of charcoal-binding antimicrobials, susceptibilities of the ATCC strain 29213 of Staphylococcus aureus were determined using both Mueller–Hinton and BCYE media, using the same methodology and antimicrobials. MIC values fell within the CLSI range on Mueller–Hinton agar, but MIC values were markedly elevated on BCYE media for all antimicrobials. Rifampicin was most affected, with nine dilutions difference between the MICs on the different agars. To the best of our knowledge, this is the first study determining the antimicrobial susceptibility patterns of clinical L. longbeachae isolates. All antimicrobials tested except for tetracycline had low MICs, with the lowest MIC90 (0.031 mg/L) of ciprofloxacin, rifampicin and moxifloxacin by broth dilution. Broth dilution-derived MICs were lower than Etest values, particularly for the quinolones, with seven dilutions difference between their MIC90 values. Compared with the other antimicrobials, tetracycline had markedly elevated MIC90 values by broth dilution and Etest (64 and 32 mg/L). This may be due to poor diffusion of tetracycline into the media or variations in electrolyte content of the media leading to chelation of tetracycline. Additionally, tetracycline reductase enzymes have been identified in soil-derived L. longbeachae specimens, which have been shown to inactivate tetracycline in vivo, but as yet have not been identified in L. pneumophila.8 Our results are similar to MICs for L. pneumophila determined by the same methodology4,9 (Table 1), with higher Etest-derived MIC90 values of rifampicin (0.38 versus 0.032 mg/L) and tetracycline (32 versus 8 mg/L). The reason for this difference is unclear and may represent a genuine difference in susceptibility between the two Legionella species. A major limitation is the effect of charcoal-binding antimicrobials as illustrated by the S. aureus control and the lower broth dilution-derived MIC values using BYE. The effect of charcoal binding has previously been explored; reducing the charcoal concentration of BCYE by 75% yielded 4–8-fold lower MIC90 values of ciprofloxacin and gemifloxacin, although levofloxacin, erythromycin and clarithromycin MIC values were unaffected.2 The difference in MIC persisted in comparison with BYE media, confirming the difference in MIC is due to charcoal-binding free drug. Despite this, non-L. pneumophila species have been found to grow poorly on charcoal-free media3 and adequate growth is necessary for accurate MIC derivation. Accordingly, EUCAST recommend BCYE for L. pneumophila susceptibility testing.10 These findings may not translate to in vivo efficacy and more data are needed to define further the WT susceptibilities. However, they support use of the Etest method on BCYE media for MIC derivation and reinforce the use of quinolones in clinical disease. Our results are derived from clinical isolates in one geographical area of New Zealand only and may not be representative of isolates in other regions or environmental isolates. Acknowledgements We thank Anja Werno, Clinical Director of Microbiology at Canterbury Health Laboratories. Funding This study was supported by internal funding. Transparency declarations None to declare. References 1 Fields BS, Benson RF, Besser RE. Legionella and Legionnaires’ disease: 25 years of investigation. Clin Microbiol Rev  2002; 15: 506– 26. Google Scholar CrossRef Search ADS PubMed  2 García MT, Pelaz C, Giménez MJ et al.   In vitro activities of gemifloxacin versus five quinolones and two macrolides against 271 Spanish isolates of Legionella pneumophila: influence of charcoal on susceptibility test results. Antimicrob Agents Chemother  2000; 44: 2176– 8. Google Scholar CrossRef Search ADS PubMed  3 Pendland SL, Martin SJ, Chen C et al.   Comparison of charcoal- and starch-based media for testing susceptibilities of Legionella species to macrolides, azalides, and fluoroquinolones. J Clin Microbiol  1997; 35: 3004– 6. Google Scholar PubMed  4 Rhomberg PR, Bale MJ, Jones RN. Application of the Etest to antimicrobial susceptibility testing of Legionella spp. Diagn Microbiol Infect Dis  1994; 19: 175– 8. Google Scholar CrossRef Search ADS PubMed  5 Notifiable Diseases in New Zealand Annual Report 2016. https://surv.esr.cri.nz/PDF_surveillance/AnnualRpt/AnnualSurv/2016/2016AnnualNDReportFinal.pdf. 6 Murdoch DR, Podmore RG, Anderson TP et al.   Impact of routine systematic polymerase chain reaction testing on case finding for Legionnaires' disease: a pre-post comparison study. Clin Infect Dis  2013; 57: 1275– 81. Google Scholar CrossRef Search ADS PubMed  7 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Second Informational Supplement M100-S22 . CLSI, Wayne, PA, USA, 2012. 8 Forsberg KJ, Patel S, Wencewicz TA et al.   The tetracycline destructases: a novel family of tetracycline-inactivating enzymes. Chem Biol  2015; 22: 888– 97. Google Scholar CrossRef Search ADS PubMed  9 Bruin JP, Ijzerman EP, den Boer JW et al.   Wild-type MIC distribution and epidemiological cut-off values in clinical Legionella pneumophila serogroup 1 isolates. Diagn Microbiol Infect Dis  2012; 72: 103– 8. Google Scholar CrossRef Search ADS PubMed  10 EUCAST. Antimicrobial Susceptibility Testing of Legionella pneumophila. http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/General_documents/Legionella_guidance_document_20160909.pdf. © The Author(s) 2017. 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.

Journal

Journal of Antimicrobial ChemotherapyOxford University Press

Published: Apr 1, 2018

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