ANTIMICROBIAL SUSCEPTIBILITY TESTING FOR Burkholderia cepacia complex (BCC) CLINICAL ISOLATES: COMPARISON OF BROTH MICRODILUTION, AGAR DILUTION, E-TEST 1 1 TM P0240 AND DISK DIFFUSION METHODOLOGIES 1 1 1 Lorena C. C. Fehlberg 1 2 Laboratório ALERTA -Division of Infectious Diseases Universidade Federal de São Paulo, UNIFESP Rua Pedro de Toledo, 781, Vila Clementino São Paulo, Brazil [email protected] +55-11-5576-4748 1 Lorena C. C. Fehlberg , Ana Carolina R. Silva , Fernanda Rodrigues-Costa , Adriana G. Nicoletti , Adriana P. Matos , Rodrigo Cayô , Elizabeth A. Marques , Ana C. Gales , 1 2 Division of Infectious Diseases, Universidade Federal de São Paulo, UNIFESP, São Paulo, Brazil; Departament of Microbiology, Imunology and Parasitology of Universidade do Estado do Rio de Janeiro, UERJ, Rio de Janeiro, Brazil INTRODUCTION AND PURPOSE RESULTS Broth microdilution vs. agar dilution The Burkholderia cepacia complex (BCC) is a group of 18 genetically related bacteria. They are important opportunistic pathogens that - Essential agreement (MIC ±1log2) between broth microdilution and agar dilution was greater than 93% for all antimicrobials evaluated, except for chloramphenicol (89%). cause severe infections in patients with cystic fibrosis as well as in other vulnerable patients. The treatment of BCC infections is often problematic - An excellent essential and categorical agreement rates was observed by TMP/SXT (98.8% and 100%, respectively) (Table 1). since this species shows intrinsic resistance to many antimicrobial agents, including polymyxins, aminoglycosides and most beta-lactams. In addi- - Unacceptable minor errors rates were observed only for chloramphenicol (26.8%), minocycline (17%) and levofloxacin (12.2%) (Table 2). tion, emergence of antimicrobial resistance during the therapy has also been reported (5, 7-8). Broth microdilution vs. E-testTM - A good essential agreement (MIC ±1-log2) rates between broth microdilution and E-testTM were observed for levofloxacin (92.7%), minocycline (96.3%), ticarcillin/clavulanate A variety of laboratory methods can be used to measure the in vitro susceptibility of bacteria to antimicrobial agents. In many clinical mi- (97.5%) and meropenem (98.8%) (Table 1). crobiology laboratories, disk diffusion method is routinely used for testing of antimicrobial susceptibility (2). Although easy to perform, this method- - Despite showing an excellent categorical agreement rate (100%), the essential agreement rate between the reference methodology and E-testTM for TMP/SXT (80.4%) was ology is not able to predict the MIC. Since the successful therapy of BCC infections is difficult to be achieved (5), to date, no study comparatively lower than that of E-testTM vs. agar dilution (98.8%) (Figures 1 and 2). evaluated the performance of distinct methodologies to accurately predict the antimicrobial susceptibility of this bacterial group. The objective of - When comparing E-testTM results with those of broth microdilution, the poorest categorical agreement rate was observed for chloramphenicol (60.9%) followed by ceftazidime this study was to comparatively evaluate the performance of distinct methodologies, broth microdilution, agar dilution, E-testTM and disk diffusion, (78%). High unacceptable minor errors rates were also observed for chloramphenicol (36.5%) (Figures 2 and 3). - Unacceptable rates of very major errors were observed for ceftazidime (2.4%), chloramphenicol (2.4%) and levofloxacin (2.4%) (Table 2). to determine the susceptibility profile of BCC to seven antibiotics recommended by the Clinical and Laboratory Standards Institute (CLSI) for antimicrobial susceptibility testing against BCC. Figure 1. Percentage of essential agreement rates observed for broth microdilution (reference methodology) versus agar dilution and E-testTM for antimicrobials tested against BCC clinical isolates. METHODOLOGY CAZ, ceftazidime; CHL, chloramphenicol; LEV, levofloxacin; MER, meropenem; MIN, minocycline; TIC, ticarcillin/ clavulanate; TMP/SXT, trimethoprim/sulfamethoxazole. Clinical isolates. A total of 82 nonduplicated Bcc clinical isolates were recovered from different body sites infections of patients attended at two Broth microdilution vs. disk diffusion Figure 2. Percentage of categorical agreement between the broth microdilution (reference methodology) and agar dilution, E-testTM or disk diffusion for antimicrobials tested against BCC clinical isolates. CAZ, ceftazidime; CHL, chloramphenicol; LEV, levofloxacin; MER, meropenem; MIN, minocycline; TIC, ticarcillin/clavulanate; TMP/SXT, trimethoprim/sulfamethoxazole. - Categorical agreement rates between the disk diffusion and broth microdilution were greater than 91% for ceftazidime (91.4%), meropenem (91.4%), minocycline (94%) and ticarcillin/clavulanate (97.5%) (Table 1). - Surprisingly, a low percentage of categorical agreement was observed for TMP/SXT (74.4%) (Figure 4). High rates of minor (23.1%) and major (2.4%) errors were detected for Brazilian tertiary hospitals between 1995 and 2010. Only one isolate per patient was included in this study. All isolates were properly stored at -80ºC this antimicrobial (Table 2). and were subcultured on Burkholderia cepacia medium, supplemented with polymyxin 75.000 IU, gentamicin 2.5 mg and ticarcillin 50 mg (Oxoid, Basingstoke, England). They were identified by recA sequencing (6) as B. cenocepacia (n=44), B. multivorans (n=12), B. vietnamiensis (n=10), B. CONCLUSIONS contaminans (n=9) and B. cepacia (n=7). - In general, the agar dilution results were more concordant with those of the broth microdilution, reference methodology, than with those of E-testTM. Antimicrobial susceptibility testing (AST). AST was carried out for seven antimicrobial agents standardized for testing by CLSI M100-S23 document (4): ceftazidime, chloramphenicol, levofloxacin, meropenem, minocycline, ticarcillin/clavulanate and trimethoprim/sulfamethoxazole (TMP/ - The emergence of multi-drug resistant Gram-negative bacilli has resuscitated the use of older antimicrobial compounds like chloramphenicol. In our study, the worst essential and SXT). The antimicrobial powders and disks were acquired from Sigma-Aldrich (St. Loius, Missouri, USA) and Oxoid (Basingstoke, England) categorical agreement rates were observed for this agent, independently of antimicrobial susceptibility technique applied. Thus, our results suggest that the breakpoints applied manufacturers, respectively. The CLSI broth microdilution (2) using the cation adjusted Müller-Hinton broth (Oxoid, Basingstoke, England, batch were probably not adequate and indicate that chloramphenicol breakpoints must be revaluated. number 1035177; val. Apr. 2016) was considered as the gold standard method. The AST results obtained by agar dilution, E-testTM (BioMerieux, - Low categorical agreement and unacceptable very major errors rates were observed for TMP/SXT combination against BCC when disk diffusion results were compared to those of Marcy I’Etoile, França) and disk diffusion were compared to those of broth microdilution (2-3). All methods were read by three blinded observers. MICs Figure 3. Scattergram results for chloramphenicol comparing MICs determined by broth microdilution with those of agar dilution (A), E-testTM (B) and disk diffusion (C) against 82 BCC clinical isolates. The and zone diameter criteria were interpreted according to the breakpoints established by CLSI M100-S23 document (4). Since there are no specific and B). Horizontal and vertical lines indicate the susceptible (green) and resistant (red) breakpoints. CLSI susceptibility breakpoints established for testing levofloxacin, ticarcillin/clavulanate, and chloramphenicol by disk diffusion against BCC, these Table 1. Essential and categorical agreements rates between broth microdilution (reference test) versus agar dilution, E-testTM, or disk diffusion against BCC clinical isolates. results were interpreted according to the CLSI breakpoints for Pseudomonas aeruginosa and Enterobacteriaceae, respectively (4). For all Essential agreement MIC ± 1log2 N (%) antimicrobials tested, the MICs and zone diameter were determined by complete inhibition of bacteria growth, excepted for TMP/SXT, that the Antimicrobials N measure was done regarding the slight growth (20% of the lawn of growth) (2, 4). ATCCs P. aeruginosa 27853, Escherichia coli 25922, E. coli 35218, Staphylococcus aureus 29213, S. aureus 25923 and B. cepacia 25608 were tested as quality control. Ceftazidime TM Statistical analysis. The regression analysis was applied for comparison the MICs results between broth microdilution, agar dilution, or E-test broth microdilution. It indicates that probably it is not possible to correctly predict the susceptibility category of TMP/SXT by disk diffusion. diagonal blue line represents complete agreement, and the numbers represent the occurrences observed at each point. The broken lines (gray) represent ±1log2 MIC agreement limits between test results (A and 82 BMD vs. Agar Dilution 80 (97.5) BMD vs. E-testTM 64 (78) Categorical agreement N (%) BMD vs. Agar Dilution 80 (97.5) BMD vs. E-testTM 75 (91.4) BMD vs. Disk Diffusion 82 73 (89) 70 (85.3) 59 (72) 50 (60.9) 43 (52.4) Levofloxacinb 82 77 (93.9) 76 (92.7) 70 (85.3) 72 (87.8) 68 (83.3) Meropenem 82 79 (96.3) 81 (98.8) 75 (91.4) 75 (91.4) 75 (91.4) test results. Categorical agreement was defined if the test results were within the same susceptibility category. Errors were ranked as follows: very Minocycline 82 80 (97.5) 79 (96.3) 68 (83) 79 (96.3) 77 (92.7) major error, false-susceptible result by agar dilution, E-testTM or disk diffusion; major error, false-resistant result by agar dilution, E-testTM and disk Ticarcillin/clavulanateb 82 80 (97.5) 80 (97.5) 80 (97.5) 80 (97.5) 80 (97.5) diffusion; and minor error, intermediate result by agar dilution, E-testTM or disk diffusion methods and resistant or susceptible category by broth TMP/SXT 82 81 (98.8) 66 (80.4) 82 (100) 82 (100) 61 (74.4) size of the disk diffusion zones of inhibition. Essential and categorical agreements rates as well as error rates were calculated as recommended by CLSI M23-A3 document (1). Essential agreement was defined as when the agar dilution and E-test results agreed within MIC ±1-log2 dilution compared to those of the reference broth microdilution test. A result was determined to be discrepant if there was ±2-log2 dilution difference between Figure 4. Scattergram results for trimethoprim/sulfamethoxazole comparing disk diffusion results with the microdilution test. Acceptable error levels were ≤1.5% for very major errors, ≤3.0% for major errors, and ≤10% for minor errors (1). All analysis was performed by SPSS for Windows 17.0 version (IBM Corporation, New York, USA, 2008). reference broth microdilution method against 82 BCC clinical isolates. The numbers represent the occurrences observed at each point. Horizontal and vertical lines indicate the susceptible (green) and resistant (red) breakpoints. BMD, broth microdilution; TMP/SXT, trimethoprim/sulfamethoxazole. a Breakpoint criteria established against Enterobacteriaceae (CLSI, 2013). b Breakpoint criteria established against P. aeruginosa (CLSI, 2013). Ceftazidime 82 1 (1.2) 0 (0.0) 1 (1.2) 5 (6) 0 (0.0) 2 (2.4) 5 (6.1) 0 (0.0) 2 (2.4) Chloramphenicola 82 22 (26.8) 1 (1.2) 0 (0.0) 30 (36.5) 0 (0.0) 2 (2.4) 28 (34.1) 1 (1.2) 10 (12.2) Levofloxacinb 82 10 (12.2) 1 (1.2) 1 (1.2) 8 (9.7) 0 (0.0) 2 (2.4) 11 (13.4) 0 (0.0) 3 (3.6) Meropenem 82 7 (8.5) 0 (0.0) 0 (0.0) 7 (8.5) 0 (0.0) 0 (0.0) 6 (7.3) 1 (1.2) 0 (0.0) Minocycline 82 14 (17) 0 (0.0) 0 (0.0) 3 (3.6) 0 (0.0) 0 (0.0) 6 (7.3) 0 (0.0) 1 (1.2) Ticarcillin/clavulanateb 82 1 (1.2) 1 (1.2) 0 (0.0) 1 (1.2) 1 (1.2) 0 (0.0) 1 (1.2) 1 (1.2) 0 (0.0) TMP/SXT 82 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 19 (23.1) 2 (2.4) 0 (0.0) 75 (91.4) Chloramphenicola TM Table 2. Categorical errors observed for the seven antimicrobial agents tested by distinct antimicrobial susceptibility techniques against BCC clinical isolates. 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