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In vitro susceptibility of Burkholderia cepacia complex isolates: Comparison of disk diffusion, Etest®, agar dilution, and broth microdilution methods
In vitro Susceptibility of Burkholderia cepacia complex Isolates: Comparison of Disk Diffusion, Etest , Agar Dilution, and Broth Microdilution Methods Lorena Cristina Corrˆea Fehlberg, Adriana Gianinni Nicoletti, Ana Carolina Ramos, Fernanda Rodrigues-Costa, Adriana Pereira de Matos, Raquel Girardello, Elizabeth Andrade Marques, Ana Cristina Gales PII: DOI: Reference:
S0732-8893(16)30254-1 doi: 10.1016/j.diagmicrobio.2016.08.015 DMB 14169
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Revised date: Accepted date:
24 May 2016 17 August 2016 22 August 2016
Please cite this article as: Fehlberg Lorena Cristina Corrˆea, Nicoletti Adriana Gianinni, Ramos Ana Carolina, Rodrigues-Costa Fernanda, de Matos Adriana Pereira, Girardello Raquel, Marques Elizabeth Andrade, Gales Ana Cristina, In vitro Susceptibility of Burkholderia cepacia complex Isolates: Comparison of Disk Diffusion, Etest , Agar Dilution, and Broth Microdilution Methods, Diagnostic Microbiology and Infectious Disease (2016), doi: 10.1016/j.diagmicrobio.2016.08.015
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Fehlberg et al., 2016
In vitro Susceptibility of Burkholderia cepacia complex Isolates:
Microdilution Methods
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Comparison of Disk Diffusion, Etest®, Agar Dilution, and Broth
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Lorena Cristina Corrêa Fehlberg1§, Adriana Gianinni Nicoletti1, Ana Carolina Ramos1, Fernanda Rodrigues-Costa1, Adriana Pereira de Matos1, Raquel Girardello1, Elizabeth
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Andrade Marques2, Ana Cristina Gales1
Laboratório Alerta. Division of Infectious Diseases. Departament of Internal Medicine.
Escola Paulista de Medicina/Universidade Federal de São Paulo, São Paulo, Brazil. Departamento de Microbiologia, Imunologia e Parasitologia. Faculdade de Ciências
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2
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Médicas. Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil.
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Running title: Susceptibility of Burkholderia cepacia complex
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Words of the abstract: 149
Words of body of the text: 3132
Address correspondence to: §
Lorena C.C. Fehlberg
Laboratório Alerta, Departament of Internal Medicine, EPM/Unifesp. Rua Pedro de Toledo, 781 - 6º andar - Vila Clementino - São Paulo – 04039-042 - Brazil Phone/fax: 55 11 5576-4748 e-mail: [email protected]
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Abstract Broth microdilution, agar dilution, Etest® and disk diffusion techniques were compared to
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evaluate the susceptibility profile of 82 Bcc clinical isolates against six antimicrobials as recommended by CLSI. Broth microdilution was considered the “gold standard” method.
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The regression analysis was applied to determine the essential (EA) and categorical (CA) agreement rates. STX (MIC50, 1 mg/L) was the most potent antimicrobial tested against Bcc
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isolates. The worst in vitro activity was observed for chloramphenicol (MIC50, 16 mg/L) and ticarcillin-clavulanic acid (MIC50,>256 mg/L). The EA among broth microdilution and agar dilution results was good for the majority of antimicrobial tested. When comparing broth microdilution and Etest®, ceftazidime, SXT and chloramphenicol exhibited EA rates below
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90%. SXT showed an excellent CA (100%) when dilution methodologies were compared. However, a low CA rate was found for this agent between dilution and disk diffusion
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methodologies resulting in unacceptable very major and minor error rates.
Key-words: CLSI, EUCAST, antimicrobial resistance, trimethoprim-sulfamethoxazole,
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non-fermentative bacilli
Part of this study was presented at the 24th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), in Barcelona, Spain, May 10-13, 2014.
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1 Introduction The first report of B. cepacia, previously Pseudomonas cepacia, has occurred more
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than 60 years ago as a microorganism causing onion rot (Burkholder, 1950). In the last years, Burkholderia cepacia complex (Bcc) has emerged as an important pathogen causing
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outbreaks and a variety of infections especially in immunocompromised patients (Abbott, Peleg, 2015; Mahenthiralingam et al., 2008). Currently, Bcc comprises 20 genetically
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related species that show about 95% recA similarity. B. cenocepacia (genomovar III) and B. multivorans (genomovar II) are the most frequently species described in cystic fibrosis patients worldwide (DeSmet et al., 2015; Peeters et al., 2013; Vandamme, Dawyndt, 2011). Trimethoprim/sulfamethoxazole remains the first therapeutic option for Bcc
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infections. Ceftazidime and meropenem, either alone or in combination with aminoglycosides or minocycline, have been considered alternative therapeutic options
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(Abbott, Peleg, 2015; Avgeri et al., 2009; Gautam et al., 2015). However, the treatment of
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these infections is often problematic since Bcc isolates are intrinsically resistant to many antimicrobial agents such as polymyxins, aminoglycosides, and most beta-lactams,
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especially first- and second- generation cephalosporins (Abbott, Peleg, 2015; Avgeri et al., 2009). In addition, antimicrobial resistance has commonly developed during therapy (Gautam et al., 2015; Gilligan, 2014). Multiple mechanisms of antimicrobial resistance occur in Bcc like decreased permeability of the bacterial membrane structure, overexpression of RND efflux systems, alteration in the target site, and production of penicilinase PenA, an inducible Ambler class C beta-lactamase (Abbott, Peleg, 2015; Drevinek, Mahenthiralingam, 2010; Papp-Wallace et al., 2013). The production of dihydrofolate
reductase
enzyme
(dhfr)
has
also
contributed
for
trimethoprim/sulfamethoxazole resistance reported among Bcc isolates (Mahenthiralingam et al., 2005). 3
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Besides the difficulty in identifying Bcc at the species level (Vandamme, Dawynth, 2011), there is no consensus about the best method for predicting the antimicrobial
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susceptibility profile making even more complicated the selection of most appropriated therapy for treatment of Bcc infections (Horsley, Jones, 2012). The Clinical and Laboratory
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Standards Institute (CLSI) (CLSI, 2016a) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (EUCAST, 2013) show divergent recommendations for testing Bcc. According the CLSI guidelines (CLSI, 2016a), agar dilution and broth
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microdilution methods are recommended for testing seven antimicrobials (ceftazidime, chloramphenicol, levofloxacin, meropenem, minocycline, ticarcillin/clavulanate, and trimethoprim/sulfamethoxazole), while disk diffusion has been approved for predicting the susceptibility to
only
four
agents
(ceftazidime,
meropenem,
minocycline,
and
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trimethoprim/sulfamethoxazole). In contrast, no breakpoints have been proposed by
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EUCAST since there is a lack of clinical evidence correlating MICs with patient clinical outcomes (EUCAST, 2013).
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The aim of this study was to compare the antimicrobial susceptibility profile of Bcc
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clinical isolates obtained by four distinct methodologies against antimicrobial agents recommended for testing by CLSI guidelines (CLSI, 2016a).
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2 Materials and Methods 2.1 Clinical isolates. A total of 82 nonduplicated Bcc clinical isolates were included
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in this study. They were recovered from different source of infections of patients admitted at two Brazilian tertiary teaching hospitals between 1995 and 2010. The isolates were obtained
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from the following specimens: sputum (n= 47), bloodstream (n= 21), tracheal aspirate (n= 7), catheter tip (n= 2), urine (n= 2), and others (n= 3). All isolates recovered from sputum
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(n=47) were collected from cystic fibrosis patients while the remaining isolates were isolated from patients with other comorbidities. Each isolate was subcultured twice onto Burkholderia cepacia medium supplemented (Oxoid, Basingstoke, England) to ensure purity and viability. Bcc isolates were previously identified by recA sequencing as B. cenocepacia
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(n=44), B. multivorans (n=12), B. vietnamiensis (n=10), B. contaminans (n=9) and B. cepacia (n=7) (Fehlberg et al., 2013).
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2.2 Antimicrobial susceptibility testing (AST). The Bcc isolates were initially
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tested by CLSI broth microdilution using cation adjusted Müeller-Hinton broth (Oxoid, Basingstoke, England, batch number 1035177; val. April, 2016), which was considered the
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gold standard methodology (CLSI, 2016a). Antimicrobial susceptibility testing was also carried out by disk diffusion, Etest® (BioMerieux, Marcy I’Etoile, French), and agar dilution techniques using cation adjusted Müeller-Hinton agar poured plates (Oxoid, Basingstoke, England, batch number 1150501; val. February, 2017) (CLSI 2016a; CLSI 2015a; CLSI, 2015b). Determination of cation concentrations in the Müeller-Hinton broth and MüellerHinton agar was previously quantified by inductively coupled plasma optical emission spectrometry (ICP-OES) in a Perkin-Elmer 3000 DV as previously reported (Girardello et al., 2012). The antimicrobial agents tested were ceftazidime, chloramphenicol, levofloxacin, meropenem, minocycline, and trimethoprim/sulfamethoxazole. The antimicrobial powders were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). The antimicrobial disks 5
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were purchased from Oxoid (Basingstoke, England) and had the following concentrations: ceftazidime 30 µg, chloramphenicol 30 µg, levofloxacin 5 µg, meropenem 10 µg,
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minocycline 30 µg, and trimethoprim/sulfamethoxazole 1.25/23.75 µg. For all antimicrobials tested, the MICs and zone diameters were determined by
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complete inhibition of bacteria growth, excepted for trimethoprim/sulfamethoxazole, which slight growth (20% of the lawn of growth) was disregarded (CLSI, 2016a; CLSI, 2015a; CLSI 2015b). Inoculated AST plates were incubated for 20 h at 35oC in ambient air.
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American Type Culture Collection (ATCC) P. aeruginosa ATCC 27853, Escherichia coli ATCC 25922, E. coli ATCC 35218, Staphylococcus aureus ATCC 29213 were used as quality control for all methodologies while for disk diffusion we used the S. aureus ATCC 25923 as CLSI recommended. B. cepacia ATCC 25608 was tested as internal quality
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control. The AST results obtained by agar dilution, Etest® and disk diffusion were compared
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to those of broth microdilution. In addition, the AST results obtained by agar dilution were compared with those obtained by Etest® and disk diffusion. Moreover, the comparison
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between Etest® and disk diffusion results was also performed. The Etest® MICs were
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rounded up to the next log2 value when necessary. All AST results were read by three blinded observers. MICs and zone diameter criteria were interpreted according to the breakpoints established by CLSI M100-S26 document (CLSI, 2016a). Since there is no specific CLSI susceptibility breakpoints established for testing levofloxacin by disk diffusion against Bcc, these results were interpreted according to the CLSI breakpoints for P. aeruginosa (CLSI, 2016a). The discrepant results were confirmed by retesting all isolates simultaneously by the four AST methodologies. 2.3 Statistical analysis. The regression analysis was applied for comparison the antimicrobial susceptibility results. Essential and categorical agreements rates as well as error rates were calculated as recommended by CLSI M23-A4 document (CLSI, 2016b). 6
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Essential agreement was defined when the methodologies tested results agreed within MIC ±1-log2 dilution compared to those of the reference method. A result was determined to be
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discrepant if there was ±2-log2 or more dilution difference between test results. Categorical agreement was defined if the test results were within the same susceptibility category. The
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interpretative category errors were defined as follows: very major error, isolate resistant by the reference method but susceptible by the tested method (false susceptibility); major error, isolate susceptible by the reference method but resistant by the tested method (false
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resistance); and minor error, intermediate result by the methodologies tested and resistant or susceptible by the reference method, or vice-versa. Acceptable error levels were ≤1.5% for very major errors, ≤3.0% for major errors, and ≤10% for minor errors (CLSI, 2016b). All analysis was performed by SPSS for Windows 17.0 version (IBM Corporation, New York,
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USA, 2008).
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3 Results 3.1 Antimicrobial Susceptibility Testing
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In vitro susceptibility and MICs distribution for the six antimicrobial agents tested
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against 82 Bcc clinical isolates by each AST were summarized in Table 1 and Figure S1, respectively. According to the “gold-standard methodology” (broth microdilution), trimethoprim/sulfamethoxazole was the most in vitro potent antimicrobial tested against Bcc
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(MIC50, 1 mg/L; 97.6% susceptible) followed by minocycline (MIC50, 2 mg/L; 87.8% susceptible). Although levofloxacin and minocycline (MIC50, 2 mg/L) showed the same MIC50, only 50% Bcc were susceptible to levofloxacin. Meropenem (MIC50, 4 mg/L) and ceftazidime (MIC50, 4 mg/L) displayed very similar in vitro activity, inhibiting 87.8% and
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86.6% of Bcc isolates, respectively. Chloramphenicol (MIC50, 16 mg/L) also showed poor in
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vitro activity, with 30.5% isolates being inhibited at the susceptible breakpoints. In most cases, the MICs obtained by broth microdilution were 1-log2 dilution higher than those
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observed for other dilution techniques, except for chloramphenicol.
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In general, the susceptibility rates to all antimicrobial agents tested varied depending on the AST performed. The susceptibility rates to trimethoprim/sulfamethoxazole (97.6%) were identical independent of the dilution AST employed but were superior to that observed for disk diffusion (71.9%). In contrast, the susceptibility rates to ceftazidime (86.6%) and meropenem (87.8%) by broth microdilution were lower than those obtained by agar dilution (89.0%; 96.3%), Etest® (93.9%; 96.3%), and disk diffusion (93.9%; 94.0%), respectively. For minocycline, the highest susceptibility rate was observed by disk diffusion (94.0%) followed by Etest® and broth microdilution (87.8% for both methodologies), and agar dilution (74.4%). Chloramphenicol susceptibility rates varied from 30.5% (broth microdilution) to 43.7% (Etest®). The highest susceptibility rates to levofloxacin were 8
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detected by disk diffusion (96.3%), which were very superior to those of agar dilution (46.4%), broth microdilution (50.0%), and Etest® (58.5%).
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When evaluating the distinct Bcc species, the results obtained by broth microdilution showed that trimethoprim/sulfamethoxazole remained the most in vitro potent antimicrobial
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tested against B. cenocepacia (MIC50, 1 mg/L; 97.7% susceptible), B. multivorans (MIC50, 0.5 mg/L; 100% susceptible), B. vietnamiensis (MIC50, 1 mg/L; 100% susceptible), and B. (MIC50,
1
mg/L;
100%
susceptible).
Although
the
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contaminans
trimethoprim/sulfamethoxazole had shown a good in vitro activity against B. cepacia isolates (MIC50, 1 mg/L), only 85.7% B. cepacia were susceptible to this compound. The susceptibility rates to meropenem ranged from 75.0% for B. multivorans (MIC50, 4 mg/L) to
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93.2% for B. cenocepacia (MIC50, 4 mg/L) as shown in TableS1. Minocycline was four-fold more potent against B. multivorans (MIC50, 1 mg/L) than B. cepacia (MIC50, 4 mg/L) and B.
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cenocepacia (MIC50, 4 mg/L) isolates. The highest susceptibility rates to minocycline were
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also observed against B. multivorans (100.0%) followed by B. vietnamiensis (90.0%), B. contaminans (88.9%), and B. cenocepacia (88.6%). In contrast, the lowest susceptibility rate
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for minocycline was seen among B. cepacia (57.1%). Levofloxacin exhibited a good in vitro activity against B. multivorans (MIC50, 1 mg/L; 83.3% susceptible) and B. contaminans (MIC50, 2 mg/L; 88.9% susceptible) but it showed low activity against B. cenocepacia (MIC50, 4 mg/L; 34.1% susceptible), B. vietnamiensis (MIC50, 4 mg/L; 40.0% susceptible), and B. cepacia (MIC50, 2 mg/L; 57.1% susceptible). Independently of the individual Bcc species evaluated, chloramphenicol (MIC50, 8-32 mg/L) exhibited very poor activity (Table S1). 3.2 Essential and categorical agreements
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Essential agreement rates among the distinct AST methods against Bcc clinical isolates was shown in Table 2. In addition, the essential and categorical agreements were
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also calculated according to the Bcc species (Table S2-S4). Comparing broth microdilution results to those of agar dilution, the essential agreement rates were acceptable (>90.0%) for
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all antimicrobial agents tested, except for chloramphenicol (89.0%). In contrast, the essential agreement rates were below 90% for ceftazidime (78.0%), trimethoprim/sulfamethoxazole (80.5%), and chloramphenicol (85.3%), when broth microdilution results were compared to
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those of Etest®. Comparing agar dilution MICs to those obtained by Etest®, only 83.0% of the MICs varied within the ±1-log2 range.
The categorical agreement and errors rates encountered among broth microdilution
categorical
agreement
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and other AST methods were shown in Tables 3 and 4, respectively. An excellent (100%) was
observed for all
dilution methods
against
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trimethoprim/sulfamethoxazole with no errors detected. Surprisingly, when compared the
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disk diffusion results with those of dilution methodologies, the categorical agreement rates were poor for trimethoprim/sulfamethoxazole, ranging from 72.0% to 74.4%. In addition,
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unacceptable minor error (ranging from 23.1% to 24.3%) and major error (3.6%) rates were observed between dilution methodologies and disk diffusion for this agent. Good categorical agreement rates were observed for ceftazidime and meropenem when all AST methodologies were compared. The errors rates detected for these two antimicrobials were within the acceptable levels, except for ceftazidime, in which very major errors were observed when broth microdilution and Etest® (2.4%), and broth microdilution and disk diffusion (2.4%) results were compared. When comparing minocycline MICs obtained by broth microdilution with those of agar dilution, the categorical agreement rate was 83.0% resulting in high level of minor errors (17.0%). The categorical agreement rates were even lower when the minocycline 10
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agar dilution results were compared with those of Etest® (86.6%) or disk diffusion (79.2%) leading to unacceptable minor error rates (13.4% and 20.7%, respectively). The categorical
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agreement rates were very low for levofloxacin and chloramphenicol, independent of the techniques compared. The very major error rates encountered for levofloxacin were above
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the expected ranges when Etest® results were compared to those of with broth microdilution (2.4%) or agar dilution (2.4%). Conversely, high minor error rates were found when testing levofloxacin by broth microdilution versus agar dilution (12.2%), or by agar dilution and
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Etest® (14.6%). Discordant results were also noticed for chloramphenicol, especially when Etest® results were compared to those of broth microdilution (2.4% very major; 36.5% minor errors) or agar dilution (3.6% very major; 19.5% minor errors). Very high rates of minor errors were also observed when broth microdilution results were compared to those of
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agar dilution for this agent (26.8%).
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4 Discussion AST is one of the main tasks executed by routine microbiology labs because these
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tests are of crucial importance to guide antimicrobial therapy. In this manner, AST must be accurate, reproducible and timely. Non-fermentative Gram-negative bacilli represent an
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extra challenge to clinical labs since different AST have produced discordant results especially for Stenotrophomonas maltophilia, P. aeruginosa, and Acinetobacter baumannii
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(Nicodemo et al., 2004; Moodley et al., 2013; van der Heijden et al., 2007). In addition, CLSI and EUCAST have issued different recommendations for testing non-fermentative Gram-negative bacilli other than P. aeruginosa and Acinetobacter spp (CLSI 2016a; EUCAST 2013).
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Few studies have evaluated the performance of AST methodologies against
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Burkholderia spp. Most studies have tested B. pseudomallei, a species not grouped under the cepacia complex, against trimethoprim/sulfamethoxazole (Piliouras et al., 2002;
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Wuthiekanun et al., 2005). The treatment of Bcc infections, mainly in cystic fibrosis patients, is still based on the antimicrobial therapy combinations. Although, there is no
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strong evidence supporting the prescription of such therapeutic regimens, they have been prescribed based on previous clinical experience and results of in vitro studies (Gautam et al., 2015). However, in vitro techniques for predicting the susceptibility are not well standardized for Bcc. In addition, studies determining the susceptibility profile of Bcc isolates to distinct antimicrobial agents are scarce and mostly published prior to the last revision of Bcc taxonomy (Gautam et al., 2015; Lupo et al., 2015). In the current study, we evaluated the essential and categorical agreement of seven antimicrobials using four distinct AST techniques against a collection of 82 Bcc clinical isolates.
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This work demonstrated an excellent correlation between the results obtained by agar dilution and broth microdilution methodologies for all antimicrobial agents tested, except for
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chloramphenicol. The unacceptable minor error rates observed for levofloxacin, minocycline and chloramphenicol by dilution methodologies could be justified by the MIC values for
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these compounds that fell within the breakpoints.
Categorical susceptibility to trimethoprim/sulfamethoxazole was accurately predicted
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by the distinct methodologies; however, only two resistant Bcc isolates were found in our study. Overall, essential agreement rate for trimethoprim/sulfamethoxazole was lower than expected (80.5%) when Etest® results were compared to those of broth microdilution but it did not result in any miscategorizations. In contrast, the disk diffusion technique was not
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adequate for testing trimethoprim/sulfamethoxazole as well as other antimicrobial agents evaluated. In the attempt to verify if the time incubation could influence the size of
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inhibition zone disks, we randomly selected isolates and incubated them for 48 hours. No
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difference was observed in comparison to the initial results (20 hours incubation). An Australian study performed with 80 B. pseudomallei clinical isolates demonstrated high
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susceptibility rates of 90.0%, 92.5% and 97.5% for trimethoprim/sulfamethoxazole when agar dilution, MicroScan automated system and Etest® were tested, respectively, while only 41.3% of those isolates being susceptible to this agent by disk diffusion methodology (Piliouras et al., 2002). Lumbiganon and colleagues (Lumbiganon et al., 2000) also observed a significant discrepancy in predicting the trimethoprim/sulfamethoxazole susceptibility by broth microdilution (84.0%) and disk diffusion (53.5%) against 144 B. pseudomallei clinical isolates in Thailand. The agreement obtained between the two tests was very poor. Our results are in agreement with those previously reported indicating that the disk diffusion technique overestimates resistance. Thus, in our opinion, the susceptibility
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category of trimethoprim/sulfamethoxazole cannot be accurately estimated by this methodology.
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Levofloxacin has been considered an alternative treatment against Bcc infections since the treatment of these infections in any clinical setting is challenging owing to its Therefore, we also evaluated the agreement of
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innate antimicrobial resistance profile.
levofloxacin for all methodologies. Although the categorical agreement rate for levofloxacin
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was high, false susceptible results were observed for disk diffusion with unacceptable very major error rates. The unacceptable major and very major error rates detected in this study for levofloxavin by disk diffusion could be due to the breakpoints applied for interpretation of this test. Thus, our results corroborate CLSI recommendations regarding the prediction of
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levofloxacin susceptibility only by dilution methods.
In our study, we evaluated ticarcillin-clavulanic acid against Bcc isolates and 100%
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were resistant to this antimicrobial by all methodologies tested (data not showed). Our
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results are in agreement with previous studies that demonstrated high resistance rates to ticarcillin-clavulanic acid (Gautam et al., 2015, Livermore et al., 2014; Lupo et al., 2015). It
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should be noted that EUCAST (EUCAST, 2013) considers Bcc intrinsically resistant to ticarcillin and ticarcillin-clavulanic acid, while the CLSI recommended ticarcillin-clavulanic acid for in vitro testing by all techniques (Abbott, Peleg, 2015; CLSI, 2016a; Leclerq et al., 2013). Despite it, certain studies demonstrated that piperacillin/tazobactam seems to be an alternative beta-lactamase inhibitor for Bcc. A study conducted with 207 B. multivorans collected from non-cystic fibrosis patients in Czech Republic between 2011 and 2013 showed that piperacillin (MIC50, 1 mg/L) and piperacillin-tazobactam (MIC50, 1 mg/L) were active against this species. The authors also noted a reduction of ≥ 2log2-dilutions in the ECOFF value for piperacillin-tazobactam (four-fold) MIC in comparison to that of piperacillin alone (Hanulik et al., 2014). Another study performed with cystic fibrosis 14
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patients from Switzerland revealed that B. multivorans (n= 14; 92.9% susceptibility) was more susceptible to piperacillin/tazobactan than B. cenocepacia (n= 28; 60.7%
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susceptibility) (Lupo et al., 2015). However, this antimicrobial has not been recommended for testing by CLSI (CLSI, 2016a; Lupo et al., 2015; Livermore et al., 2014).
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Recently, the CLSI published that the antimicrobial breakpoints listed in Table 2B-3 could be also applied for categorization of Bcc. However, it is not clearly stated which Bcc
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species were included for testing when the breakpoints were standardized (CLSI, 2016a). Although the number of Bcc species tested was low, our results suggest that B. cenocepacia and B. cepacia seemed to be more resistant to the evaluated antimicrobials than other species tested in this study. However, at this moment, it is no possible to establish any
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relationship between Bcc identification at the species level and antimicrobial susceptibility profile.
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In summary, trimethoprim/sulfamethoxazole showed excellent in vitro activity
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against Bcc species by the dilution methodologies. Our results also are in agreement with EUCAST recommendations, demonstrating that disk diffusion is a poor reproducible
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technique in comparison with dilution tests (EUCAST, 2013). More studies are necessary for correct establishment of the valid breakpoints. In this way, antimicrobial therapy could be accurately guided.
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Funding This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo
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(FAPESP process #2012/04910-9).
Acknowledgments
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LCCF received a grant conceded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). ACG is a researcher from the National Council for Science and Technological Development (CNPq), Ministry of Science and Technology, Brazil (process
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#307816/2009-5).
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Transparency declarations
MSD.
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A.C.G. has recently received research funding and/or consultation fees AstraZeneca and
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Performance of MALDI-ToF MS for species identification of Burkholderia cepacia complex clinical isolates. Diagn Microbiol Infect Dis 2013; 77:126-128 (doi:
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10.1016/j.diagmicrobio.2013.06.011).
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Microbiology and Infectious Diseases, Barcelona, ES, 2014. Abstract P0239. European Society of Clinical Microbiology and Infectious Diseases, Basel, Switzerland; 2014.
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Thinkhamrop B. Comparison between the antimicrobial susceptibility of Burkholderia pseudomallei to trimethoprim-sulfamethoxazole by standard disk diffusion method and by
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minimal inhibitory concentration determination. J Med Assoc Thai 2000; 83:856-860. Lupo A, Isis E, Tinguely R, Endimiani A. Clonality and antimicrobial susceptibility of Burkholderia cepacia complex isolates collected from cystic fibrosis patients during 19982013 in Bern, Switzerland. New Microbiol 2015; 38:281-288. Mahenthiralingam E, Baldwin A, Dowson CG. Burkholderia cepacia complex bacteria: opportunistic pathogens with important natural biology. J Appl Microbiol 2008; 104:15391551 (doi:10.1111/j.1365-2672.2007.03706.x). Mahenthiralingam E, Urban TA, Goldberg JB. The multifarious, multireplicon Burkholderia cepacia complex. Nature Rev 2005; 3:145-156 (doi:10.1038/nrmicro1085). 19
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Acinetobacter
10.1128/JCM.03250-12).
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Nicodemo AC, Araujo MRE, Ruiz AS, Gales AC. In vitro susceptibility of Stenotrophomonas maltophilia isolates: comparison of disc diffusion, Etest® and agar
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resistance
determinants.
Biol
Chem
2013;
288:19090-19102
(doi:
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10.1074/jbc.M113.458315).
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Peeters C, Zlosnik JE, Spiker T, Hird TJ, LiPuma JJ, Vandamme P. Burkholderia
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pseudomultivorans sp. nov., a novel Burkholderia cepacia complex species from human
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respiratory samples and the rhizosphere. Syst Appl Microbiol 2013; 36:483-489 (doi: 10.1016/j.syapm.2013.06.003).
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Piliouras P, Ulett GC, Ashhurst-Smith C, Hirst RG, Norton RE. A comparison of antibiotic susceptibility testing methods for cotrimoxazole with Burkholderia pseudomallei. Inter J Antimicrob Agent 2002; 19:427-429 (doi.org/10.1016/S0924-8579(02)00016-X). van der Heijden IM, Levin AS, Pedri EH, Fung L, Rossi F, Duboc G, et al. Comparison of disc diffusion, Etest® and broth microdilution for testing susceptibility of carbapenemresistant Pseudomonas aeruginosa to polymyxins. Ann Clin Microbiol Antimicrob 2007; 6:8 (doi:10.1186/1476-0711-6-8).
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Vandamme P, Dawyndt P. Classification and identification of the Burkholderia cepacia complex: past, present and future. Syst Appl Microbiol 2011; 34:87-95 (doi:
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10.1016/j.syapm.2010.10.002). Wuthiekanun V, Cheng AC, Chierakul W, Amornchai P, Limmathurotsakul D,
Burkholderia
pseudomallei.
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2005;
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(doi:
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CE
PT
ED
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10.1093/jac/dki151).
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Chaowagul W, et al. Trimethoprim/sulfamethoxazole resistance in clinical isolates of
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Table 1. In vitro antimicrobial activity of six antimicrobials tested against Bcc clinical isolates according to the AST methodologies.
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* Gold standard method.
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NT, not tested.
The breakpoints for testing levofloxacin by disk diffusion were interpreted according to the
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CLSI breakpoints for P. aeruginosa (CLSI, 2016a).
22
ACCEPTED MANUSCRIPT Antimicrobial agents/
MIC50
MIC90
Range
Susceptible
Resistant
AST methodologies
(mg/L)
(mg/L)
(mg/L)
(%)
(%)
Broth microdilution*
4
16
1 - >256
86.6
7.3
Agar dilution
2
8
2 – 64
Etest®
2
4
1 - >256
Disk diffusion
-
-
-
Broth microdilution*
16
128
Agar dilution
16
64
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Fehlberg et al., 2016
Etest®
16
Disk diffusion
NT
89.0
6.1
93.9
3.7
93.9
3.7
2 - >256
30.5
35.4
8 – 256
36.6
37.8
64
4 - >256
47.6
30.5
NT
NT
NT
NT
8
0.25 - >32
50.0
17.1
4
1 - >32
46.4
15.9
2
Agar dilution
4
ED
Broth microdilution*
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Chloramphenicol
Levofloxacin
2
8
0.5 - >32
58.5
15.9
-
-
-
96.3
14.6
4
PT
4
0.03 – 16
87.8
1.2
CE
Etest®
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Ceftazidime
4
4
0.125 – 16
96.3
1.2
2
4
0.06 - >32
96.3
1.2
-
-
-
94.0
2.4
Broth microdilution*
2
8
0.5 – 32
87.8
3.7
Agar dilution
4
8
0.5 – 64
74.4
4.9
Etest®
4
8
1 - >256
87.8
4.9
Disk diffusion
-
-
-
94.0
2.4
1
2
0.125 – 32
97.6
2.4
Agar dilution
0.5
1
0.25 - >32
97.6
2.4
Etest®
0.5
1
0.125 - >32
97.6
2.4
-
-
-
71.9
4.9
Disk diffusion Meropenem Broth microdilution* Agar dilution Etest®
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Disk diffusion Minocycline
Trimethoprim/sulfamethoxazole Broth microdilution*
Disk diffusion
23
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Table 2. Comparison of essential agreement rates obtained by distinct AST techniques against Bcc clinical isolates.
minocycline; SXT, trimethoprim/sulfamethoxazole.
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CAZ, ceftazidime; CHL, chloramphenicol; LVX, levofloxacin; MEM, meropenem; MIN,
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Percentage of essential agreement (±1log2) was represented by the shaded area.
24
ACCEPTED MANUSCRIPT Percentage of isolates with log2 MIC variation
% of Essential
of
Agreement
Antimicrobial agents (n)
Fehlberg et al., 2016
≤ -3
-2
-1
0
1
2
≥3
0
0
6
76.9
14.6
0
2.4
97.5
CHL (82)
1.2
4.9
9.7
57.3
22
4.9
0
89.0
LVX (82)
1.2
3.6
17
66
11
1.2
0
93.9
MEM (82)
2.4
2.4
13.4
69.5
12.2
0
0
95.1
MIN (82)
0
2.4
42.7
46.3
8.5
0
0
97.5
SXT (82)
1.2
0
19.5
35.3
44
0
0
98.8
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CAZ (82)
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Broth microdilution vs. Agar dilution
Broth microdilution vs. Etest®
0
0
2.4
28
47.6
17
4.9
78.0
CHL (82)
1.2
3.6
14.6
35.4
35.4
7.3
2.4
85.3
LVX (82)
0
2.4
7.3
63.4
22
4.9
0
92.7
MEM (82)
0
0
6
48.8
44
1.2
0
98.8
MIN (82)
1.2
1.2
29.2
62.2
4.9
1.2
0
96.3
SXT (82)
1.2
1.2
9.7
35.4
35.4
15.9
1.2
80.5
CHL (82) LVX (82) MIN (82) SXT (82)
0
®
Agar dilution vs. Etest
1.2
34.1
47.6
14.6
2.4
83.0
2.4
3.6
26.9
52.4
11
3.6
0
90.2
1.2
1.2
7.3
63.4
22
2.4
2.4
92.7
0
0
1.2
52.5
41.1
2.4
2.4
95.1
0
1.2
12.2
61
25.6
0
0
98.8
0
2.4
4.9
56.1
30.5
4.9
1.2
91.4
AC
MEM (82)
PT
0
CE
CAZ (82)
ED
CAZ (82)
25
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Fehlberg et al., 2016
Table 3. Comparative categorical agreements rates among the distinct AST techniques against Bcc clinical isolates.
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CAZ, ceftazidime; CHL, chloramphenicol; LVX, levofloxacin; MEM, meropenem; MIN, minocycline; NT, no tested; SXT, trimethoprim-sulfamethoxazole.
P.
for
aeruginosa
(CLSI,
2016a).
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breakpoints
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CLSI
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The breakpoints for testing levofloxacin by disk diffusion were interpreted according to the
26
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Fehlberg et al., 2016
% Categorical Agreement
Agents
microdiluti
Broth
Agar
Broth
microdiluti dilutio microdiluti
Agar dilution
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Antimicrobial
Broth
vs. Disk
Etest® vs. Disk
on vs.
n vs.
on vs. Disk
dilution
Etest®
Etest®
diffusion
CAZ
97.5
91.4
94.0
91.4
94.0
100
CHL
72.0
60.9
76.8
NT
NT
NT
LVX
85.3
87.8
81.7
83.3
81.7
87.8
MEM
91.4
91.4
100
91.4
97.5
97.6
MIN
83.0
96.3
86.6
92.7
79.2
92.7
SXT
100
100
100
74.4
74.4
72.0
diffusio n
diffusio n
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on vs. Agar
27
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Table 4. Discordant results among the susceptibility methods: number and category of error for each antimicrobial agent tested against Bcc.
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CAZ, ceftazidime; CHL, chloramphenicol; LVX, levofloxacin; M, major error; MEM, meropenem; MI, minor error; MIN, minocycline; NT, no tested; SXT, trimethoprim-
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sulfamethoxazole; VM, very major error.
The bold values represent the unacceptable errors rates according to the CLSI M23-A4
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document parameters (CLSI, 2016b).
The breakpoints for testing levofloxacin by disk diffusion were interpreted according to the for
P.
aeruginosa
(CLSI,
2016a).
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breakpoints
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CLSI
28
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on vs.
dilution
Etest®
Agents
V
VM M
MI
CAZ
1.2 0.0
1.2
CHL
0.0 1.2 26.8
2
1.2 12.2
8.5
M MI
0.0 0.0
17
SXT
0.0 0.0
0.0
AC
MIN
V M
M
M I
Disk
diffusion
diffusion
VM
M
MI
V
M
M MI
diffusion V M
M
M I
4
0
2.
0. 36. 3. 0. 19 NT NT NT
N
N NT N N N
4
0
T
T
2.
0.
4
5
2 0
8
6 0 .5
2. 1. 14
0
9.7
0.
0.
0
0
0.
0.
0
0
0.
0.
0
0
8.5
3.6
0.0
4 2 .6
0. 0. 0. 0 0
0
0. 0. 13 0 0 .4 0. 0. 0. 0 0
0
2.4 0.0 6.1 1.2
3.6 0.0 13.4 2.4
0.0 1.2 7.3 0.0
1.2 0.0 7.3 0.0
0.0 2.4 23.1 0.0
0.
Disk
0.
6
1. 0. 4.
vs. Disk
Etest® vs.
2.
CE
MEM 0.0 0.0
vs. Etest®
PT
LVX
1’.
M
dilution
microdilution dilution vs.
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crobial
vs. Agar
Agar
Agar
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microdilution microdiluti
Broth
ED
Antimi
Broth
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Broth
Fehlberg et al., 2016
0
4.8
0. 0. 0. 0
0
0
T T T
0. 15. 2. 1. 8. 0 1. 2
8 1.2
4
2
5
0. 1. 1. 0
2
2
0. 20. 1. 0. 6. 0
7
2
0
1
2. 23. 0. 3. 24 4
1
0
6 .3
29
S0732-8893(16)30254-1 doi: 10.1016/j.diagmicrobio.2016.08.015 DMB 14169
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Revised date: Accepted date:
24 May 2016 17 August 2016 22 August 2016
Please cite this article as: Fehlberg Lorena Cristina Corrˆea, Nicoletti Adriana Gianinni, Ramos Ana Carolina, Rodrigues-Costa Fernanda, de Matos Adriana Pereira, Girardello Raquel, Marques Elizabeth Andrade, Gales Ana Cristina, In vitro Susceptibility of Burkholderia cepacia complex Isolates: Comparison of Disk Diffusion, Etest , Agar Dilution, and Broth Microdilution Methods, Diagnostic Microbiology and Infectious Disease (2016), doi: 10.1016/j.diagmicrobio.2016.08.015
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Fehlberg et al., 2016
In vitro Susceptibility of Burkholderia cepacia complex Isolates:
Microdilution Methods
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Comparison of Disk Diffusion, Etest®, Agar Dilution, and Broth
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Lorena Cristina Corrêa Fehlberg1§, Adriana Gianinni Nicoletti1, Ana Carolina Ramos1, Fernanda Rodrigues-Costa1, Adriana Pereira de Matos1, Raquel Girardello1, Elizabeth
1
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Andrade Marques2, Ana Cristina Gales1
Laboratório Alerta. Division of Infectious Diseases. Departament of Internal Medicine.
Escola Paulista de Medicina/Universidade Federal de São Paulo, São Paulo, Brazil. Departamento de Microbiologia, Imunologia e Parasitologia. Faculdade de Ciências
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2
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Médicas. Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil.
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Running title: Susceptibility of Burkholderia cepacia complex
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Words of the abstract: 149
Words of body of the text: 3132
Address correspondence to: §
Lorena C.C. Fehlberg
Laboratório Alerta, Departament of Internal Medicine, EPM/Unifesp. Rua Pedro de Toledo, 781 - 6º andar - Vila Clementino - São Paulo – 04039-042 - Brazil Phone/fax: 55 11 5576-4748 e-mail: [email protected]
1
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Fehlberg et al., 2016
Abstract Broth microdilution, agar dilution, Etest® and disk diffusion techniques were compared to
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evaluate the susceptibility profile of 82 Bcc clinical isolates against six antimicrobials as recommended by CLSI. Broth microdilution was considered the “gold standard” method.
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The regression analysis was applied to determine the essential (EA) and categorical (CA) agreement rates. STX (MIC50, 1 mg/L) was the most potent antimicrobial tested against Bcc
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isolates. The worst in vitro activity was observed for chloramphenicol (MIC50, 16 mg/L) and ticarcillin-clavulanic acid (MIC50,>256 mg/L). The EA among broth microdilution and agar dilution results was good for the majority of antimicrobial tested. When comparing broth microdilution and Etest®, ceftazidime, SXT and chloramphenicol exhibited EA rates below
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90%. SXT showed an excellent CA (100%) when dilution methodologies were compared. However, a low CA rate was found for this agent between dilution and disk diffusion
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methodologies resulting in unacceptable very major and minor error rates.
Key-words: CLSI, EUCAST, antimicrobial resistance, trimethoprim-sulfamethoxazole,
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non-fermentative bacilli
Part of this study was presented at the 24th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), in Barcelona, Spain, May 10-13, 2014.
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1 Introduction The first report of B. cepacia, previously Pseudomonas cepacia, has occurred more
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than 60 years ago as a microorganism causing onion rot (Burkholder, 1950). In the last years, Burkholderia cepacia complex (Bcc) has emerged as an important pathogen causing
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outbreaks and a variety of infections especially in immunocompromised patients (Abbott, Peleg, 2015; Mahenthiralingam et al., 2008). Currently, Bcc comprises 20 genetically
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related species that show about 95% recA similarity. B. cenocepacia (genomovar III) and B. multivorans (genomovar II) are the most frequently species described in cystic fibrosis patients worldwide (DeSmet et al., 2015; Peeters et al., 2013; Vandamme, Dawyndt, 2011). Trimethoprim/sulfamethoxazole remains the first therapeutic option for Bcc
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infections. Ceftazidime and meropenem, either alone or in combination with aminoglycosides or minocycline, have been considered alternative therapeutic options
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(Abbott, Peleg, 2015; Avgeri et al., 2009; Gautam et al., 2015). However, the treatment of
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these infections is often problematic since Bcc isolates are intrinsically resistant to many antimicrobial agents such as polymyxins, aminoglycosides, and most beta-lactams,
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especially first- and second- generation cephalosporins (Abbott, Peleg, 2015; Avgeri et al., 2009). In addition, antimicrobial resistance has commonly developed during therapy (Gautam et al., 2015; Gilligan, 2014). Multiple mechanisms of antimicrobial resistance occur in Bcc like decreased permeability of the bacterial membrane structure, overexpression of RND efflux systems, alteration in the target site, and production of penicilinase PenA, an inducible Ambler class C beta-lactamase (Abbott, Peleg, 2015; Drevinek, Mahenthiralingam, 2010; Papp-Wallace et al., 2013). The production of dihydrofolate
reductase
enzyme
(dhfr)
has
also
contributed
for
trimethoprim/sulfamethoxazole resistance reported among Bcc isolates (Mahenthiralingam et al., 2005). 3
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Fehlberg et al., 2016
Besides the difficulty in identifying Bcc at the species level (Vandamme, Dawynth, 2011), there is no consensus about the best method for predicting the antimicrobial
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susceptibility profile making even more complicated the selection of most appropriated therapy for treatment of Bcc infections (Horsley, Jones, 2012). The Clinical and Laboratory
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Standards Institute (CLSI) (CLSI, 2016a) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (EUCAST, 2013) show divergent recommendations for testing Bcc. According the CLSI guidelines (CLSI, 2016a), agar dilution and broth
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microdilution methods are recommended for testing seven antimicrobials (ceftazidime, chloramphenicol, levofloxacin, meropenem, minocycline, ticarcillin/clavulanate, and trimethoprim/sulfamethoxazole), while disk diffusion has been approved for predicting the susceptibility to
only
four
agents
(ceftazidime,
meropenem,
minocycline,
and
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trimethoprim/sulfamethoxazole). In contrast, no breakpoints have been proposed by
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EUCAST since there is a lack of clinical evidence correlating MICs with patient clinical outcomes (EUCAST, 2013).
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The aim of this study was to compare the antimicrobial susceptibility profile of Bcc
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clinical isolates obtained by four distinct methodologies against antimicrobial agents recommended for testing by CLSI guidelines (CLSI, 2016a).
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2 Materials and Methods 2.1 Clinical isolates. A total of 82 nonduplicated Bcc clinical isolates were included
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in this study. They were recovered from different source of infections of patients admitted at two Brazilian tertiary teaching hospitals between 1995 and 2010. The isolates were obtained
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from the following specimens: sputum (n= 47), bloodstream (n= 21), tracheal aspirate (n= 7), catheter tip (n= 2), urine (n= 2), and others (n= 3). All isolates recovered from sputum
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(n=47) were collected from cystic fibrosis patients while the remaining isolates were isolated from patients with other comorbidities. Each isolate was subcultured twice onto Burkholderia cepacia medium supplemented (Oxoid, Basingstoke, England) to ensure purity and viability. Bcc isolates were previously identified by recA sequencing as B. cenocepacia
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(n=44), B. multivorans (n=12), B. vietnamiensis (n=10), B. contaminans (n=9) and B. cepacia (n=7) (Fehlberg et al., 2013).
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2.2 Antimicrobial susceptibility testing (AST). The Bcc isolates were initially
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tested by CLSI broth microdilution using cation adjusted Müeller-Hinton broth (Oxoid, Basingstoke, England, batch number 1035177; val. April, 2016), which was considered the
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gold standard methodology (CLSI, 2016a). Antimicrobial susceptibility testing was also carried out by disk diffusion, Etest® (BioMerieux, Marcy I’Etoile, French), and agar dilution techniques using cation adjusted Müeller-Hinton agar poured plates (Oxoid, Basingstoke, England, batch number 1150501; val. February, 2017) (CLSI 2016a; CLSI 2015a; CLSI, 2015b). Determination of cation concentrations in the Müeller-Hinton broth and MüellerHinton agar was previously quantified by inductively coupled plasma optical emission spectrometry (ICP-OES) in a Perkin-Elmer 3000 DV as previously reported (Girardello et al., 2012). The antimicrobial agents tested were ceftazidime, chloramphenicol, levofloxacin, meropenem, minocycline, and trimethoprim/sulfamethoxazole. The antimicrobial powders were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). The antimicrobial disks 5
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Fehlberg et al., 2016
were purchased from Oxoid (Basingstoke, England) and had the following concentrations: ceftazidime 30 µg, chloramphenicol 30 µg, levofloxacin 5 µg, meropenem 10 µg,
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minocycline 30 µg, and trimethoprim/sulfamethoxazole 1.25/23.75 µg. For all antimicrobials tested, the MICs and zone diameters were determined by
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complete inhibition of bacteria growth, excepted for trimethoprim/sulfamethoxazole, which slight growth (20% of the lawn of growth) was disregarded (CLSI, 2016a; CLSI, 2015a; CLSI 2015b). Inoculated AST plates were incubated for 20 h at 35oC in ambient air.
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American Type Culture Collection (ATCC) P. aeruginosa ATCC 27853, Escherichia coli ATCC 25922, E. coli ATCC 35218, Staphylococcus aureus ATCC 29213 were used as quality control for all methodologies while for disk diffusion we used the S. aureus ATCC 25923 as CLSI recommended. B. cepacia ATCC 25608 was tested as internal quality
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control. The AST results obtained by agar dilution, Etest® and disk diffusion were compared
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to those of broth microdilution. In addition, the AST results obtained by agar dilution were compared with those obtained by Etest® and disk diffusion. Moreover, the comparison
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between Etest® and disk diffusion results was also performed. The Etest® MICs were
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rounded up to the next log2 value when necessary. All AST results were read by three blinded observers. MICs and zone diameter criteria were interpreted according to the breakpoints established by CLSI M100-S26 document (CLSI, 2016a). Since there is no specific CLSI susceptibility breakpoints established for testing levofloxacin by disk diffusion against Bcc, these results were interpreted according to the CLSI breakpoints for P. aeruginosa (CLSI, 2016a). The discrepant results were confirmed by retesting all isolates simultaneously by the four AST methodologies. 2.3 Statistical analysis. The regression analysis was applied for comparison the antimicrobial susceptibility results. Essential and categorical agreements rates as well as error rates were calculated as recommended by CLSI M23-A4 document (CLSI, 2016b). 6
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Essential agreement was defined when the methodologies tested results agreed within MIC ±1-log2 dilution compared to those of the reference method. A result was determined to be
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discrepant if there was ±2-log2 or more dilution difference between test results. Categorical agreement was defined if the test results were within the same susceptibility category. The
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interpretative category errors were defined as follows: very major error, isolate resistant by the reference method but susceptible by the tested method (false susceptibility); major error, isolate susceptible by the reference method but resistant by the tested method (false
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resistance); and minor error, intermediate result by the methodologies tested and resistant or susceptible by the reference method, or vice-versa. Acceptable error levels were ≤1.5% for very major errors, ≤3.0% for major errors, and ≤10% for minor errors (CLSI, 2016b). All analysis was performed by SPSS for Windows 17.0 version (IBM Corporation, New York,
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3 Results 3.1 Antimicrobial Susceptibility Testing
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In vitro susceptibility and MICs distribution for the six antimicrobial agents tested
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against 82 Bcc clinical isolates by each AST were summarized in Table 1 and Figure S1, respectively. According to the “gold-standard methodology” (broth microdilution), trimethoprim/sulfamethoxazole was the most in vitro potent antimicrobial tested against Bcc
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(MIC50, 1 mg/L; 97.6% susceptible) followed by minocycline (MIC50, 2 mg/L; 87.8% susceptible). Although levofloxacin and minocycline (MIC50, 2 mg/L) showed the same MIC50, only 50% Bcc were susceptible to levofloxacin. Meropenem (MIC50, 4 mg/L) and ceftazidime (MIC50, 4 mg/L) displayed very similar in vitro activity, inhibiting 87.8% and
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86.6% of Bcc isolates, respectively. Chloramphenicol (MIC50, 16 mg/L) also showed poor in
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vitro activity, with 30.5% isolates being inhibited at the susceptible breakpoints. In most cases, the MICs obtained by broth microdilution were 1-log2 dilution higher than those
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observed for other dilution techniques, except for chloramphenicol.
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In general, the susceptibility rates to all antimicrobial agents tested varied depending on the AST performed. The susceptibility rates to trimethoprim/sulfamethoxazole (97.6%) were identical independent of the dilution AST employed but were superior to that observed for disk diffusion (71.9%). In contrast, the susceptibility rates to ceftazidime (86.6%) and meropenem (87.8%) by broth microdilution were lower than those obtained by agar dilution (89.0%; 96.3%), Etest® (93.9%; 96.3%), and disk diffusion (93.9%; 94.0%), respectively. For minocycline, the highest susceptibility rate was observed by disk diffusion (94.0%) followed by Etest® and broth microdilution (87.8% for both methodologies), and agar dilution (74.4%). Chloramphenicol susceptibility rates varied from 30.5% (broth microdilution) to 43.7% (Etest®). The highest susceptibility rates to levofloxacin were 8
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detected by disk diffusion (96.3%), which were very superior to those of agar dilution (46.4%), broth microdilution (50.0%), and Etest® (58.5%).
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When evaluating the distinct Bcc species, the results obtained by broth microdilution showed that trimethoprim/sulfamethoxazole remained the most in vitro potent antimicrobial
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tested against B. cenocepacia (MIC50, 1 mg/L; 97.7% susceptible), B. multivorans (MIC50, 0.5 mg/L; 100% susceptible), B. vietnamiensis (MIC50, 1 mg/L; 100% susceptible), and B. (MIC50,
1
mg/L;
100%
susceptible).
Although
the
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contaminans
trimethoprim/sulfamethoxazole had shown a good in vitro activity against B. cepacia isolates (MIC50, 1 mg/L), only 85.7% B. cepacia were susceptible to this compound. The susceptibility rates to meropenem ranged from 75.0% for B. multivorans (MIC50, 4 mg/L) to
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93.2% for B. cenocepacia (MIC50, 4 mg/L) as shown in TableS1. Minocycline was four-fold more potent against B. multivorans (MIC50, 1 mg/L) than B. cepacia (MIC50, 4 mg/L) and B.
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cenocepacia (MIC50, 4 mg/L) isolates. The highest susceptibility rates to minocycline were
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also observed against B. multivorans (100.0%) followed by B. vietnamiensis (90.0%), B. contaminans (88.9%), and B. cenocepacia (88.6%). In contrast, the lowest susceptibility rate
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for minocycline was seen among B. cepacia (57.1%). Levofloxacin exhibited a good in vitro activity against B. multivorans (MIC50, 1 mg/L; 83.3% susceptible) and B. contaminans (MIC50, 2 mg/L; 88.9% susceptible) but it showed low activity against B. cenocepacia (MIC50, 4 mg/L; 34.1% susceptible), B. vietnamiensis (MIC50, 4 mg/L; 40.0% susceptible), and B. cepacia (MIC50, 2 mg/L; 57.1% susceptible). Independently of the individual Bcc species evaluated, chloramphenicol (MIC50, 8-32 mg/L) exhibited very poor activity (Table S1). 3.2 Essential and categorical agreements
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Essential agreement rates among the distinct AST methods against Bcc clinical isolates was shown in Table 2. In addition, the essential and categorical agreements were
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also calculated according to the Bcc species (Table S2-S4). Comparing broth microdilution results to those of agar dilution, the essential agreement rates were acceptable (>90.0%) for
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all antimicrobial agents tested, except for chloramphenicol (89.0%). In contrast, the essential agreement rates were below 90% for ceftazidime (78.0%), trimethoprim/sulfamethoxazole (80.5%), and chloramphenicol (85.3%), when broth microdilution results were compared to
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those of Etest®. Comparing agar dilution MICs to those obtained by Etest®, only 83.0% of the MICs varied within the ±1-log2 range.
The categorical agreement and errors rates encountered among broth microdilution
categorical
agreement
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and other AST methods were shown in Tables 3 and 4, respectively. An excellent (100%) was
observed for all
dilution methods
against
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trimethoprim/sulfamethoxazole with no errors detected. Surprisingly, when compared the
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disk diffusion results with those of dilution methodologies, the categorical agreement rates were poor for trimethoprim/sulfamethoxazole, ranging from 72.0% to 74.4%. In addition,
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unacceptable minor error (ranging from 23.1% to 24.3%) and major error (3.6%) rates were observed between dilution methodologies and disk diffusion for this agent. Good categorical agreement rates were observed for ceftazidime and meropenem when all AST methodologies were compared. The errors rates detected for these two antimicrobials were within the acceptable levels, except for ceftazidime, in which very major errors were observed when broth microdilution and Etest® (2.4%), and broth microdilution and disk diffusion (2.4%) results were compared. When comparing minocycline MICs obtained by broth microdilution with those of agar dilution, the categorical agreement rate was 83.0% resulting in high level of minor errors (17.0%). The categorical agreement rates were even lower when the minocycline 10
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agar dilution results were compared with those of Etest® (86.6%) or disk diffusion (79.2%) leading to unacceptable minor error rates (13.4% and 20.7%, respectively). The categorical
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agreement rates were very low for levofloxacin and chloramphenicol, independent of the techniques compared. The very major error rates encountered for levofloxacin were above
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the expected ranges when Etest® results were compared to those of with broth microdilution (2.4%) or agar dilution (2.4%). Conversely, high minor error rates were found when testing levofloxacin by broth microdilution versus agar dilution (12.2%), or by agar dilution and
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Etest® (14.6%). Discordant results were also noticed for chloramphenicol, especially when Etest® results were compared to those of broth microdilution (2.4% very major; 36.5% minor errors) or agar dilution (3.6% very major; 19.5% minor errors). Very high rates of minor errors were also observed when broth microdilution results were compared to those of
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agar dilution for this agent (26.8%).
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4 Discussion AST is one of the main tasks executed by routine microbiology labs because these
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tests are of crucial importance to guide antimicrobial therapy. In this manner, AST must be accurate, reproducible and timely. Non-fermentative Gram-negative bacilli represent an
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extra challenge to clinical labs since different AST have produced discordant results especially for Stenotrophomonas maltophilia, P. aeruginosa, and Acinetobacter baumannii
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(Nicodemo et al., 2004; Moodley et al., 2013; van der Heijden et al., 2007). In addition, CLSI and EUCAST have issued different recommendations for testing non-fermentative Gram-negative bacilli other than P. aeruginosa and Acinetobacter spp (CLSI 2016a; EUCAST 2013).
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Few studies have evaluated the performance of AST methodologies against
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Burkholderia spp. Most studies have tested B. pseudomallei, a species not grouped under the cepacia complex, against trimethoprim/sulfamethoxazole (Piliouras et al., 2002;
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Wuthiekanun et al., 2005). The treatment of Bcc infections, mainly in cystic fibrosis patients, is still based on the antimicrobial therapy combinations. Although, there is no
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strong evidence supporting the prescription of such therapeutic regimens, they have been prescribed based on previous clinical experience and results of in vitro studies (Gautam et al., 2015). However, in vitro techniques for predicting the susceptibility are not well standardized for Bcc. In addition, studies determining the susceptibility profile of Bcc isolates to distinct antimicrobial agents are scarce and mostly published prior to the last revision of Bcc taxonomy (Gautam et al., 2015; Lupo et al., 2015). In the current study, we evaluated the essential and categorical agreement of seven antimicrobials using four distinct AST techniques against a collection of 82 Bcc clinical isolates.
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This work demonstrated an excellent correlation between the results obtained by agar dilution and broth microdilution methodologies for all antimicrobial agents tested, except for
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chloramphenicol. The unacceptable minor error rates observed for levofloxacin, minocycline and chloramphenicol by dilution methodologies could be justified by the MIC values for
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these compounds that fell within the breakpoints.
Categorical susceptibility to trimethoprim/sulfamethoxazole was accurately predicted
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by the distinct methodologies; however, only two resistant Bcc isolates were found in our study. Overall, essential agreement rate for trimethoprim/sulfamethoxazole was lower than expected (80.5%) when Etest® results were compared to those of broth microdilution but it did not result in any miscategorizations. In contrast, the disk diffusion technique was not
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adequate for testing trimethoprim/sulfamethoxazole as well as other antimicrobial agents evaluated. In the attempt to verify if the time incubation could influence the size of
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inhibition zone disks, we randomly selected isolates and incubated them for 48 hours. No
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difference was observed in comparison to the initial results (20 hours incubation). An Australian study performed with 80 B. pseudomallei clinical isolates demonstrated high
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susceptibility rates of 90.0%, 92.5% and 97.5% for trimethoprim/sulfamethoxazole when agar dilution, MicroScan automated system and Etest® were tested, respectively, while only 41.3% of those isolates being susceptible to this agent by disk diffusion methodology (Piliouras et al., 2002). Lumbiganon and colleagues (Lumbiganon et al., 2000) also observed a significant discrepancy in predicting the trimethoprim/sulfamethoxazole susceptibility by broth microdilution (84.0%) and disk diffusion (53.5%) against 144 B. pseudomallei clinical isolates in Thailand. The agreement obtained between the two tests was very poor. Our results are in agreement with those previously reported indicating that the disk diffusion technique overestimates resistance. Thus, in our opinion, the susceptibility
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category of trimethoprim/sulfamethoxazole cannot be accurately estimated by this methodology.
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Levofloxacin has been considered an alternative treatment against Bcc infections since the treatment of these infections in any clinical setting is challenging owing to its Therefore, we also evaluated the agreement of
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innate antimicrobial resistance profile.
levofloxacin for all methodologies. Although the categorical agreement rate for levofloxacin
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was high, false susceptible results were observed for disk diffusion with unacceptable very major error rates. The unacceptable major and very major error rates detected in this study for levofloxavin by disk diffusion could be due to the breakpoints applied for interpretation of this test. Thus, our results corroborate CLSI recommendations regarding the prediction of
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levofloxacin susceptibility only by dilution methods.
In our study, we evaluated ticarcillin-clavulanic acid against Bcc isolates and 100%
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were resistant to this antimicrobial by all methodologies tested (data not showed). Our
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results are in agreement with previous studies that demonstrated high resistance rates to ticarcillin-clavulanic acid (Gautam et al., 2015, Livermore et al., 2014; Lupo et al., 2015). It
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should be noted that EUCAST (EUCAST, 2013) considers Bcc intrinsically resistant to ticarcillin and ticarcillin-clavulanic acid, while the CLSI recommended ticarcillin-clavulanic acid for in vitro testing by all techniques (Abbott, Peleg, 2015; CLSI, 2016a; Leclerq et al., 2013). Despite it, certain studies demonstrated that piperacillin/tazobactam seems to be an alternative beta-lactamase inhibitor for Bcc. A study conducted with 207 B. multivorans collected from non-cystic fibrosis patients in Czech Republic between 2011 and 2013 showed that piperacillin (MIC50, 1 mg/L) and piperacillin-tazobactam (MIC50, 1 mg/L) were active against this species. The authors also noted a reduction of ≥ 2log2-dilutions in the ECOFF value for piperacillin-tazobactam (four-fold) MIC in comparison to that of piperacillin alone (Hanulik et al., 2014). Another study performed with cystic fibrosis 14
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patients from Switzerland revealed that B. multivorans (n= 14; 92.9% susceptibility) was more susceptible to piperacillin/tazobactan than B. cenocepacia (n= 28; 60.7%
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susceptibility) (Lupo et al., 2015). However, this antimicrobial has not been recommended for testing by CLSI (CLSI, 2016a; Lupo et al., 2015; Livermore et al., 2014).
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Recently, the CLSI published that the antimicrobial breakpoints listed in Table 2B-3 could be also applied for categorization of Bcc. However, it is not clearly stated which Bcc
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species were included for testing when the breakpoints were standardized (CLSI, 2016a). Although the number of Bcc species tested was low, our results suggest that B. cenocepacia and B. cepacia seemed to be more resistant to the evaluated antimicrobials than other species tested in this study. However, at this moment, it is no possible to establish any
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relationship between Bcc identification at the species level and antimicrobial susceptibility profile.
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In summary, trimethoprim/sulfamethoxazole showed excellent in vitro activity
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against Bcc species by the dilution methodologies. Our results also are in agreement with EUCAST recommendations, demonstrating that disk diffusion is a poor reproducible
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technique in comparison with dilution tests (EUCAST, 2013). More studies are necessary for correct establishment of the valid breakpoints. In this way, antimicrobial therapy could be accurately guided.
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Funding This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo
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(FAPESP process #2012/04910-9).
Acknowledgments
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LCCF received a grant conceded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). ACG is a researcher from the National Council for Science and Technological Development (CNPq), Ministry of Science and Technology, Brazil (process
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#307816/2009-5).
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Transparency declarations
MSD.
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A.C.G. has recently received research funding and/or consultation fees AstraZeneca and
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Piliouras P, Ulett GC, Ashhurst-Smith C, Hirst RG, Norton RE. A comparison of antibiotic susceptibility testing methods for cotrimoxazole with Burkholderia pseudomallei. Inter J Antimicrob Agent 2002; 19:427-429 (doi.org/10.1016/S0924-8579(02)00016-X). van der Heijden IM, Levin AS, Pedri EH, Fung L, Rossi F, Duboc G, et al. Comparison of disc diffusion, Etest® and broth microdilution for testing susceptibility of carbapenemresistant Pseudomonas aeruginosa to polymyxins. Ann Clin Microbiol Antimicrob 2007; 6:8 (doi:10.1186/1476-0711-6-8).
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Vandamme P, Dawyndt P. Classification and identification of the Burkholderia cepacia complex: past, present and future. Syst Appl Microbiol 2011; 34:87-95 (doi:
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10.1016/j.syapm.2010.10.002). Wuthiekanun V, Cheng AC, Chierakul W, Amornchai P, Limmathurotsakul D,
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Chaowagul W, et al. Trimethoprim/sulfamethoxazole resistance in clinical isolates of
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Table 1. In vitro antimicrobial activity of six antimicrobials tested against Bcc clinical isolates according to the AST methodologies.
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* Gold standard method.
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NT, not tested.
The breakpoints for testing levofloxacin by disk diffusion were interpreted according to the
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CLSI breakpoints for P. aeruginosa (CLSI, 2016a).
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ACCEPTED MANUSCRIPT Antimicrobial agents/
MIC50
MIC90
Range
Susceptible
Resistant
AST methodologies
(mg/L)
(mg/L)
(mg/L)
(%)
(%)
Broth microdilution*
4
16
1 - >256
86.6
7.3
Agar dilution
2
8
2 – 64
Etest®
2
4
1 - >256
Disk diffusion
-
-
-
Broth microdilution*
16
128
Agar dilution
16
64
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Fehlberg et al., 2016
Etest®
16
Disk diffusion
NT
89.0
6.1
93.9
3.7
93.9
3.7
2 - >256
30.5
35.4
8 – 256
36.6
37.8
64
4 - >256
47.6
30.5
NT
NT
NT
NT
8
0.25 - >32
50.0
17.1
4
1 - >32
46.4
15.9
2
Agar dilution
4
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Broth microdilution*
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Chloramphenicol
Levofloxacin
2
8
0.5 - >32
58.5
15.9
-
-
-
96.3
14.6
4
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4
0.03 – 16
87.8
1.2
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Etest®
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Ceftazidime
4
4
0.125 – 16
96.3
1.2
2
4
0.06 - >32
96.3
1.2
-
-
-
94.0
2.4
Broth microdilution*
2
8
0.5 – 32
87.8
3.7
Agar dilution
4
8
0.5 – 64
74.4
4.9
Etest®
4
8
1 - >256
87.8
4.9
Disk diffusion
-
-
-
94.0
2.4
1
2
0.125 – 32
97.6
2.4
Agar dilution
0.5
1
0.25 - >32
97.6
2.4
Etest®
0.5
1
0.125 - >32
97.6
2.4
-
-
-
71.9
4.9
Disk diffusion Meropenem Broth microdilution* Agar dilution Etest®
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Disk diffusion Minocycline
Trimethoprim/sulfamethoxazole Broth microdilution*
Disk diffusion
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Table 2. Comparison of essential agreement rates obtained by distinct AST techniques against Bcc clinical isolates.
minocycline; SXT, trimethoprim/sulfamethoxazole.
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CAZ, ceftazidime; CHL, chloramphenicol; LVX, levofloxacin; MEM, meropenem; MIN,
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Percentage of essential agreement (±1log2) was represented by the shaded area.
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ACCEPTED MANUSCRIPT Percentage of isolates with log2 MIC variation
% of Essential
of
Agreement
Antimicrobial agents (n)
Fehlberg et al., 2016
≤ -3
-2
-1
0
1
2
≥3
0
0
6
76.9
14.6
0
2.4
97.5
CHL (82)
1.2
4.9
9.7
57.3
22
4.9
0
89.0
LVX (82)
1.2
3.6
17
66
11
1.2
0
93.9
MEM (82)
2.4
2.4
13.4
69.5
12.2
0
0
95.1
MIN (82)
0
2.4
42.7
46.3
8.5
0
0
97.5
SXT (82)
1.2
0
19.5
35.3
44
0
0
98.8
MA NU
SC R
CAZ (82)
IP T
Broth microdilution vs. Agar dilution
Broth microdilution vs. Etest®
0
0
2.4
28
47.6
17
4.9
78.0
CHL (82)
1.2
3.6
14.6
35.4
35.4
7.3
2.4
85.3
LVX (82)
0
2.4
7.3
63.4
22
4.9
0
92.7
MEM (82)
0
0
6
48.8
44
1.2
0
98.8
MIN (82)
1.2
1.2
29.2
62.2
4.9
1.2
0
96.3
SXT (82)
1.2
1.2
9.7
35.4
35.4
15.9
1.2
80.5
CHL (82) LVX (82) MIN (82) SXT (82)
0
®
Agar dilution vs. Etest
1.2
34.1
47.6
14.6
2.4
83.0
2.4
3.6
26.9
52.4
11
3.6
0
90.2
1.2
1.2
7.3
63.4
22
2.4
2.4
92.7
0
0
1.2
52.5
41.1
2.4
2.4
95.1
0
1.2
12.2
61
25.6
0
0
98.8
0
2.4
4.9
56.1
30.5
4.9
1.2
91.4
AC
MEM (82)
PT
0
CE
CAZ (82)
ED
CAZ (82)
25
ACCEPTED MANUSCRIPT
Fehlberg et al., 2016
Table 3. Comparative categorical agreements rates among the distinct AST techniques against Bcc clinical isolates.
IP T
CAZ, ceftazidime; CHL, chloramphenicol; LVX, levofloxacin; MEM, meropenem; MIN, minocycline; NT, no tested; SXT, trimethoprim-sulfamethoxazole.
P.
for
aeruginosa
(CLSI,
2016a).
CE
PT
ED
MA NU
breakpoints
AC
CLSI
SC R
The breakpoints for testing levofloxacin by disk diffusion were interpreted according to the
26
ACCEPTED MANUSCRIPT
Fehlberg et al., 2016
% Categorical Agreement
Agents
microdiluti
Broth
Agar
Broth
microdiluti dilutio microdiluti
Agar dilution
IP T
Antimicrobial
Broth
vs. Disk
Etest® vs. Disk
on vs.
n vs.
on vs. Disk
dilution
Etest®
Etest®
diffusion
CAZ
97.5
91.4
94.0
91.4
94.0
100
CHL
72.0
60.9
76.8
NT
NT
NT
LVX
85.3
87.8
81.7
83.3
81.7
87.8
MEM
91.4
91.4
100
91.4
97.5
97.6
MIN
83.0
96.3
86.6
92.7
79.2
92.7
SXT
100
100
100
74.4
74.4
72.0
diffusio n
diffusio n
AC
CE
PT
ED
MA NU
SC R
on vs. Agar
27
ACCEPTED MANUSCRIPT
Fehlberg et al., 2016
Table 4. Discordant results among the susceptibility methods: number and category of error for each antimicrobial agent tested against Bcc.
IP T
CAZ, ceftazidime; CHL, chloramphenicol; LVX, levofloxacin; M, major error; MEM, meropenem; MI, minor error; MIN, minocycline; NT, no tested; SXT, trimethoprim-
SC R
sulfamethoxazole; VM, very major error.
The bold values represent the unacceptable errors rates according to the CLSI M23-A4
MA NU
document parameters (CLSI, 2016b).
The breakpoints for testing levofloxacin by disk diffusion were interpreted according to the for
P.
aeruginosa
(CLSI,
2016a).
CE
PT
ED
breakpoints
AC
CLSI
28
ACCEPTED MANUSCRIPT
on vs.
dilution
Etest®
Agents
V
VM M
MI
CAZ
1.2 0.0
1.2
CHL
0.0 1.2 26.8
2
1.2 12.2
8.5
M MI
0.0 0.0
17
SXT
0.0 0.0
0.0
AC
MIN
V M
M
M I
Disk
diffusion
diffusion
VM
M
MI
V
M
M MI
diffusion V M
M
M I
4
0
2.
0. 36. 3. 0. 19 NT NT NT
N
N NT N N N
4
0
T
T
2.
0.
4
5
2 0
8
6 0 .5
2. 1. 14
0
9.7
0.
0.
0
0
0.
0.
0
0
0.
0.
0
0
8.5
3.6
0.0
4 2 .6
0. 0. 0. 0 0
0
0. 0. 13 0 0 .4 0. 0. 0. 0 0
0
2.4 0.0 6.1 1.2
3.6 0.0 13.4 2.4
0.0 1.2 7.3 0.0
1.2 0.0 7.3 0.0
0.0 2.4 23.1 0.0
0.
Disk
0.
6
1. 0. 4.
vs. Disk
Etest® vs.
2.
CE
MEM 0.0 0.0
vs. Etest®
PT
LVX
1’.
M
dilution
microdilution dilution vs.
MA NU
crobial
vs. Agar
Agar
Agar
IP T
microdilution microdiluti
Broth
ED
Antimi
Broth
SC R
Broth
Fehlberg et al., 2016
0
4.8
0. 0. 0. 0
0
0
T T T
0. 15. 2. 1. 8. 0 1. 2
8 1.2
4
2
5
0. 1. 1. 0
2
2
0. 20. 1. 0. 6. 0
7
2
0
1
2. 23. 0. 3. 24 4
1
0
6 .3
29