Antibiotic susceptibility of clinical Legionella pneumophila serogroup 1 strains isolated in Germany

Antibiotic susceptibility of clinical Legionella pneumophila serogroup 1 strains isolated in Germany Sir, Since the introduction of the first antibiotic agents into the therapy of bacterial diseases, the development of resistance to antibiotics in human bacterial pathogens has been extensively described. For Legionella, an environmental pathogen with a facultative intracellular lifestyle and the causative agent of a form of atypical pneumonia, selection of in vitro mutants against erythromycin, ciprofloxacin, norfloxacin and rifampicin has been described. No isolates resistant in vivo were detected for a long time.1,2 However, the first ciprofloxacin-resistant strain isolated from a clinical sample has been described recently.3 Furthermore, the possibility of selection for fluoroquinolone resistance by antibiotic treatment of Legionella pneumophila has been reported.4 Testing of L. pneumophila for antimicrobial susceptibility has not yet been standardized definitively because of the difficulty of growing bacteria on buffered yeast extract media containing charcoal, which is necessary for adequate growth of Legionella. This results in reduced activity of the most relevant antimicrobials and therefore leads to increasing MICs.5 The use of control strains to ensure that growth conditions are adequate is essential for accurate evaluation of the results. Furthermore, the MIC values for the control strains should be within those for the WT population.6 We performed antimicrobial susceptibility testing of a large set of clinical L. pneumophila isolates from Germany. The goal of this study was to collect data to survey the WT distribution of L. pneumophila in a defined geographical area. A total of 100 strains of L. pneumophila serogroup 1, which were isolated from clinical specimens during the period 2001–16, were chosen for testing of their susceptibility to eight antimicrobial agents: levofloxacin, moxifloxacin, ciprofloxacin, rifampicin, azithromycin, clarithromycin, erythromycin and doxycycline. The isolates were community acquired (61%), nosocomially acquired (18%) or travel associated (15%). The source of 6% of isolates could not be identified. The strains were subtyped using Dresden Panel monoclonal antibodies (MAbs) and sequence-based typing (SBT) according to the European Study Group for Legionella Infections (ESGLI) SBT protocol. The MICs were determined after incubation of the isolates on buffered charcoal yeast extract agar supplemented with α-ketoglutarate (BCYE-α) for 48 h by using the Etest (bioMérieux, France). The results were interpreted using published epidemiological cut-off values (ECOFFs) and recently determined tentative EUCAST breakpoints for L. pneumophila.5,6 In each experiment, L. pneumophila strain ATCC 33152 was used as a control strain and its MICs were always below the highest MICs for WT populations.5,6 The MAb typing showed the prevalence of MAb subtypes Knoxville (30%) and Philadelphia (26%) in the strains investigated in this study. The analysed isolates belonged to 39 different STs with ST1 the most prominent (17%). All isolates showed MIC values lower than or equal to the ECOFFs. In contrast, according to the tentative EUCAST breakpoints, nine isolates showed MICs outside the WT distribution for erythromycin (see Table 1). So far, we have not searched for mutations in genes encoding 23S rRNA (rrl) and L4 (rplD) and L22 (rplV) ribosomal proteins that result in decreased susceptibility to macrolides.7 Table 1 Distribution of the MICs for clinical isolates of L. pneumophila serogroup 1 from Germany (n = 100) Antimicrobial agent  Number of strains inhibited at indicated concentrations (mg/L)   0.008  0.016  0.032  0.064  0.125  0.25  0.5  1  2  4  8  Levofloxacin      1  5  43  30  21  0        Moxifloxacin            2  85  13        Ciprofloxacin            5  91  4  0      Rifampicin  11  69  20                  Azithromycin      4  10  43  26  4  13        Clarithromycin      1  3  40  30  26          Erythromycin        8  23  31  29  9        Doxycycline              33  52  13  2  0  Antimicrobial agent  Number of strains inhibited at indicated concentrations (mg/L)   0.008  0.016  0.032  0.064  0.125  0.25  0.5  1  2  4  8  Levofloxacin      1  5  43  30  21  0        Moxifloxacin            2  85  13        Ciprofloxacin            5  91  4  0      Rifampicin  11  69  20                  Azithromycin      4  10  43  26  4  13        Clarithromycin      1  3  40  30  26          Erythromycin        8  23  31  29  9        Doxycycline              33  52  13  2  0  Tentative EUCAST breakpoints6 are in bold. In particular, the MIC90 values ranged from 0.5 to 1.0 mg/L for fluoroquinolones (levofloxacin, moxifloxacin, ciprofloxacin). The MIC90 values obtained for macrolides (erythromycin, azithromycin, clarithromycin) also ranged from 0.5 to 1.0 mg/L. Interestingly, decreasing susceptibility to azithromycin was observed within the ST1 isolates in comparison with non-ST1 isolates. All ST1 isolates showed MICs from 0.25 to 1 mg/L while 96% of non-ST1 isolates did not have MICs >0.25 mg/L. These data correlate with the previously recognized relationship between STs and azithromycin susceptibility of clinical Legionella isolates.8 It was found that rifampicin is the most active drug against isolates with an MIC90 of 0.032 mg/L under in vitro conditions. In contrast, doxycycline was found to be the least active drug, with an MIC90 of 2.0 mg/L. In summary, the in vitro activity of the tested antibiotics against the German clinical isolates was in agreement with results from other studies.8–10 The introduction of routine susceptibility testing seems necessary for better understanding of the development of antibiotic resistance in vivo within the L. pneumophila population. The obtained MIC values can be used as a reference for defining ECOFFs and establishing the clinical breakpoints by EUCAST. Acknowledgements Part of this study was presented at the Annual Meeting of the European Study Group for Legionella Infections (ESGLI), Amsterdam, The Netherlands, 2016. Funding This work was supported by the Robert Koch Institute on behalf of the Federal Ministry of Health (grant 1369–351). Transparency declarations None to declare. References 1 Moffie BG, Mouton RP. Sensitivity and resistance of Legionella pneumophila to some antibiotics and combinations of antibiotics. J Antimicrob Chemother  1988; 22: 457– 62. Google Scholar CrossRef Search ADS PubMed  2 Nielsen K, Bangsborg JM, Hoiby N. Susceptibility of Legionella species to five antibiotics and development of resistance by exposure to erythromycin, ciprofloxacin, and rifampicin. Diagn Microbiol Infect Dis  2000; 36: 43– 8. Google Scholar CrossRef Search ADS PubMed  3 Bruin JP, Koshkolda T, IJzerman EP et al.   Isolation of ciprofloxacin-resistant Legionella pneumophila in a patient with severe pneumonia. J Antimicrob Chemother  2014; 69: 2869– 71. Google Scholar CrossRef Search ADS PubMed  4 Shadoud L, Almahmoud I, Jarraud S et al.   Hidden selection of bacterial resistance to fluoroquinolones in vivo: the case of Legionella pneumophila and humans. EBioMedicine  2015; 2: 1179– 85. Google Scholar CrossRef Search ADS PubMed  5 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  6 EUCAST. Antimicrobial Susceptibility Testing of Legionella pneumophila. http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/General_documents/Legionella_guidance_document_20160909.pdf. 7 Descours G, Ginevra C, Jacotin N et al.   Ribosomal mutations conferring macrolide resistance in Legionella pneumophila. Antimicrob Agents Chemother  2017; 61: e02188-16. Google Scholar CrossRef Search ADS PubMed  8 Mallegol J, Fernandes P, Melano RG et al.   Antimicrobial activity of solithromycin against clinical isolates of Legionella pneumophila serogroup 1. Antimicrob Agents Chemother  2014; 58: 909– 15. Google Scholar CrossRef Search ADS PubMed  9 Al-Matawah QA, Al-Zenki SF, Qasem JA et al.   Detection and quantification of Legionella pneumophila from water systems in Kuwait residential facilities. J Pathog  2012; 2012: 138389. Google Scholar CrossRef Search ADS PubMed  10 De Giglio O, Napoli C, Lovero G et al.   Antibiotic susceptibility of Legionella pneumophila strains isolated from hospital water systems in Southern Italy. Environ Res  2015; 142: 586– 90. Google Scholar CrossRef Search ADS PubMed  © The Author 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

Antibiotic susceptibility of clinical Legionella pneumophila serogroup 1 strains isolated in Germany

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Oxford University Press
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© The Author 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.
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0305-7453
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10.1093/jac/dkx386
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Abstract

Sir, Since the introduction of the first antibiotic agents into the therapy of bacterial diseases, the development of resistance to antibiotics in human bacterial pathogens has been extensively described. For Legionella, an environmental pathogen with a facultative intracellular lifestyle and the causative agent of a form of atypical pneumonia, selection of in vitro mutants against erythromycin, ciprofloxacin, norfloxacin and rifampicin has been described. No isolates resistant in vivo were detected for a long time.1,2 However, the first ciprofloxacin-resistant strain isolated from a clinical sample has been described recently.3 Furthermore, the possibility of selection for fluoroquinolone resistance by antibiotic treatment of Legionella pneumophila has been reported.4 Testing of L. pneumophila for antimicrobial susceptibility has not yet been standardized definitively because of the difficulty of growing bacteria on buffered yeast extract media containing charcoal, which is necessary for adequate growth of Legionella. This results in reduced activity of the most relevant antimicrobials and therefore leads to increasing MICs.5 The use of control strains to ensure that growth conditions are adequate is essential for accurate evaluation of the results. Furthermore, the MIC values for the control strains should be within those for the WT population.6 We performed antimicrobial susceptibility testing of a large set of clinical L. pneumophila isolates from Germany. The goal of this study was to collect data to survey the WT distribution of L. pneumophila in a defined geographical area. A total of 100 strains of L. pneumophila serogroup 1, which were isolated from clinical specimens during the period 2001–16, were chosen for testing of their susceptibility to eight antimicrobial agents: levofloxacin, moxifloxacin, ciprofloxacin, rifampicin, azithromycin, clarithromycin, erythromycin and doxycycline. The isolates were community acquired (61%), nosocomially acquired (18%) or travel associated (15%). The source of 6% of isolates could not be identified. The strains were subtyped using Dresden Panel monoclonal antibodies (MAbs) and sequence-based typing (SBT) according to the European Study Group for Legionella Infections (ESGLI) SBT protocol. The MICs were determined after incubation of the isolates on buffered charcoal yeast extract agar supplemented with α-ketoglutarate (BCYE-α) for 48 h by using the Etest (bioMérieux, France). The results were interpreted using published epidemiological cut-off values (ECOFFs) and recently determined tentative EUCAST breakpoints for L. pneumophila.5,6 In each experiment, L. pneumophila strain ATCC 33152 was used as a control strain and its MICs were always below the highest MICs for WT populations.5,6 The MAb typing showed the prevalence of MAb subtypes Knoxville (30%) and Philadelphia (26%) in the strains investigated in this study. The analysed isolates belonged to 39 different STs with ST1 the most prominent (17%). All isolates showed MIC values lower than or equal to the ECOFFs. In contrast, according to the tentative EUCAST breakpoints, nine isolates showed MICs outside the WT distribution for erythromycin (see Table 1). So far, we have not searched for mutations in genes encoding 23S rRNA (rrl) and L4 (rplD) and L22 (rplV) ribosomal proteins that result in decreased susceptibility to macrolides.7 Table 1 Distribution of the MICs for clinical isolates of L. pneumophila serogroup 1 from Germany (n = 100) Antimicrobial agent  Number of strains inhibited at indicated concentrations (mg/L)   0.008  0.016  0.032  0.064  0.125  0.25  0.5  1  2  4  8  Levofloxacin      1  5  43  30  21  0        Moxifloxacin            2  85  13        Ciprofloxacin            5  91  4  0      Rifampicin  11  69  20                  Azithromycin      4  10  43  26  4  13        Clarithromycin      1  3  40  30  26          Erythromycin        8  23  31  29  9        Doxycycline              33  52  13  2  0  Antimicrobial agent  Number of strains inhibited at indicated concentrations (mg/L)   0.008  0.016  0.032  0.064  0.125  0.25  0.5  1  2  4  8  Levofloxacin      1  5  43  30  21  0        Moxifloxacin            2  85  13        Ciprofloxacin            5  91  4  0      Rifampicin  11  69  20                  Azithromycin      4  10  43  26  4  13        Clarithromycin      1  3  40  30  26          Erythromycin        8  23  31  29  9        Doxycycline              33  52  13  2  0  Tentative EUCAST breakpoints6 are in bold. In particular, the MIC90 values ranged from 0.5 to 1.0 mg/L for fluoroquinolones (levofloxacin, moxifloxacin, ciprofloxacin). The MIC90 values obtained for macrolides (erythromycin, azithromycin, clarithromycin) also ranged from 0.5 to 1.0 mg/L. Interestingly, decreasing susceptibility to azithromycin was observed within the ST1 isolates in comparison with non-ST1 isolates. All ST1 isolates showed MICs from 0.25 to 1 mg/L while 96% of non-ST1 isolates did not have MICs >0.25 mg/L. These data correlate with the previously recognized relationship between STs and azithromycin susceptibility of clinical Legionella isolates.8 It was found that rifampicin is the most active drug against isolates with an MIC90 of 0.032 mg/L under in vitro conditions. In contrast, doxycycline was found to be the least active drug, with an MIC90 of 2.0 mg/L. In summary, the in vitro activity of the tested antibiotics against the German clinical isolates was in agreement with results from other studies.8–10 The introduction of routine susceptibility testing seems necessary for better understanding of the development of antibiotic resistance in vivo within the L. pneumophila population. The obtained MIC values can be used as a reference for defining ECOFFs and establishing the clinical breakpoints by EUCAST. Acknowledgements Part of this study was presented at the Annual Meeting of the European Study Group for Legionella Infections (ESGLI), Amsterdam, The Netherlands, 2016. Funding This work was supported by the Robert Koch Institute on behalf of the Federal Ministry of Health (grant 1369–351). Transparency declarations None to declare. References 1 Moffie BG, Mouton RP. Sensitivity and resistance of Legionella pneumophila to some antibiotics and combinations of antibiotics. J Antimicrob Chemother  1988; 22: 457– 62. Google Scholar CrossRef Search ADS PubMed  2 Nielsen K, Bangsborg JM, Hoiby N. Susceptibility of Legionella species to five antibiotics and development of resistance by exposure to erythromycin, ciprofloxacin, and rifampicin. Diagn Microbiol Infect Dis  2000; 36: 43– 8. Google Scholar CrossRef Search ADS PubMed  3 Bruin JP, Koshkolda T, IJzerman EP et al.   Isolation of ciprofloxacin-resistant Legionella pneumophila in a patient with severe pneumonia. J Antimicrob Chemother  2014; 69: 2869– 71. Google Scholar CrossRef Search ADS PubMed  4 Shadoud L, Almahmoud I, Jarraud S et al.   Hidden selection of bacterial resistance to fluoroquinolones in vivo: the case of Legionella pneumophila and humans. EBioMedicine  2015; 2: 1179– 85. Google Scholar CrossRef Search ADS PubMed  5 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  6 EUCAST. Antimicrobial Susceptibility Testing of Legionella pneumophila. http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/General_documents/Legionella_guidance_document_20160909.pdf. 7 Descours G, Ginevra C, Jacotin N et al.   Ribosomal mutations conferring macrolide resistance in Legionella pneumophila. Antimicrob Agents Chemother  2017; 61: e02188-16. Google Scholar CrossRef Search ADS PubMed  8 Mallegol J, Fernandes P, Melano RG et al.   Antimicrobial activity of solithromycin against clinical isolates of Legionella pneumophila serogroup 1. Antimicrob Agents Chemother  2014; 58: 909– 15. Google Scholar CrossRef Search ADS PubMed  9 Al-Matawah QA, Al-Zenki SF, Qasem JA et al.   Detection and quantification of Legionella pneumophila from water systems in Kuwait residential facilities. J Pathog  2012; 2012: 138389. Google Scholar CrossRef Search ADS PubMed  10 De Giglio O, Napoli C, Lovero G et al.   Antibiotic susceptibility of Legionella pneumophila strains isolated from hospital water systems in Southern Italy. Environ Res  2015; 142: 586– 90. Google Scholar CrossRef Search ADS PubMed  © The Author 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: Feb 1, 2018

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