ANTIMICROBIAL SUSCEPTIBILITY TESTING FOR

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.
BMD vs. agar dilution N (%)
BMD vs. E-testTM N (%)
BMD vs. disk diffusion N (%)
Antimicrobials
N
MI*
M*
VM*
MI*
M*
VM*
MI*
M*
VM*
BMD, broth microdilution; TMP/SXT, trimethoprim/sulfamethoxazole.
*Category of error: MI, minor error; M, major error; VM, very major error.
a
Breakpoint criteria established against Enterobacteriaceae (CLSI, 2013).
b
Breakpoint criteria established against P. aeruginosa (CLSI, 2013).
REFERENCES
1.
2.
3.
4.
Clinical and Laboratory Standards Institute. Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters; Approved Guideline-Third Edition.
Document M23-A3. Wayne PA, 2008.
Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard; ninth edition.
Document M07-A9. Wayne, PA, 2012a.
Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility test; approved standard – eleventh edition. Document M02-A11.
Wayne, PA, 2012b.
Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twenty-three informational supplement. Document M100-S23.
Wayne, PA, 2013.
5. Drevinek P, E Mahenthiralingam. Burkholderia cepacia in cystic fibrosis: epidemiology and molecular mechanisms of virulence. Clin Microbiol Infect. 16: 821-30, 2010.
6. Fehlberg LCC, LHS Andrade, DM Assis, RHV Pereira, AC Gales, EA Marques. Performance of MALDI-ToF MS for species identification of Burkholderia cepacia complex
clinical isolates. Diagn Microbiol Infect Dis. 77: 126-128, 2013.
7. LiPuma JJ, BJ Currie, SJ Peacook, PAR Vandamme. Burkholderia, Stenotrophomonas, Ralstonia, Cupriavidus, Pandoraea, Brevundimonas, Comamonas, Delftia and
Acidovorax. In: Versalovic J, KC Carroll, G Funke, JH Jorgensen, ML Landry, DW Warnock. Manual of Clinical Microbiology. Washington DC, ASM Press, 10ªed., c. 41: 692-713,
2011.
8. Peeters C JE Zlosnik, T Spiker, TJ Hird, JJ LiPuma, P Vandamme. Burkholderia pseudomultivorans sp. nov., a novel Burkholderia cepacia complex species from human
respiratory samples and the rhizosphere. Syst Appl Microbiol. 36: 483-489, 2013.

Download Report