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Evaluation of the slomyco sensititre® panel for testing the antimicrobial susceptibility of mycobacterium marinum isolates

Chazel et al. Ann Clin Microbiol Antimicrob (2016) 15:30 Annals of Clinical Microbiology and Antimicrobials Open Access
Evaluation of the SLOMYCO Sensititre® panel for testing the antimicrobial susceptibility of Mycobacterium marinum isolatesMarion Chazel1, Hélène Marchandin1,2, Nicolas Keck3, Dominique Terru1, Christian Carrière1,5,6, Michael Ponsoda4, Véronique Jacomo4, Gilles Panteix4, Nicolas Bouzinbi1, Anne‑Laure Bañuls7, Marc Choisy7, Jérôme Solassol6,8,9, Alexandra Aubry10,11,12,13† and Sylvain Godreuil1,5,6*† Abstract
Background: The agar dilution method is currently considered as the reference method for Mycobacterium marinum
drug susceptibility testing (DST). As it is time‑consuming, alternative methods, such as the E‑test, were evaluated for
M. marinum DST, but without success. The SLOMYCO Sensititre® panel, recently commercialized by TREK Diagnostic
Systems (Cleveland, OH), can be used for DST in slow‑growing mycobacteria and for antimicrobial agents recom‑
mended by the Clinical and Laboratory Standards Institute (CLSI) for M. marinum DST. The main goal of this work was
to evaluate the SLOMYCO Sensititre® panel method for DST in M. marinum isolates from human patients and fish rela‑
tive to the reference agar dilution method.
Methods/Results: The reproducibility of the minimum inhibitory concentration (MIC) determination (±1 log2
dilution) was very good for both the agar dilution method and SLOMYCO Sensititre® panel (>90 % agreement). The
percentage essential agreement between methods varied, depending on the drug: between 97 and 75 % for cipro‑
floxacin, moxifloxacin, linezolid, isoniazid, clarithromycin, amikacin, rifabutin and rifampin, 74 % for trimethoprim, 72 %
for doxycycline, 70 % for sulfamethoxazole, 59 % for streptomycin, 33 % for ethambutol and only 2.2 % for ethiona‑
mide. When the agar dilution and SLOMYCO Sensititre® panel results were converted into interpretive criteria, the
category agreement was 100 % for amikacin, ciprofloxacin, clarithromycin, moxifloxacin, rifabutin, sulfamethoxazole
and trimethoprim, 98 % for ethambutol and 96 % for rifampin and no agreement for doxycycline.
Conclusions: The SLOMYCO Sensititre® panel method could provide a potential alternative to the reference agar
dilution method, when DST in M. marinum is required, except for doxycycline.
Keywords: Mycobacterium marinum, SLOMYCO Sensititre® panel, Agar dilution method, Antimicrobial susceptibility
testing, Human and fish isolates
in many fish species from cold or warm, fresh or salted Mycobacterium marinum, a slow-growing nontubercu- water, and also in many other aquatic animals, such as lous photochromogenic mycobacteria, is an ubiquitous amphibians, mammals and oysters []. In humans, waterborne organism [, ] that causes diseases M. marinum infection is commonly limited to the skin, but it can spread to deeper structures, resulting in teno- synovitis, arthritis and osteomyeliti, ely, in disseminated infection in immunocompromised †Alexandra Aubry and Sylvain Godreuil contributed equally to this work Department of Bacteriology‑Virology, INSERM U1058 "Pathogenesis and Control of Chronic Infections", Université Montpellier‑EFS, Mycobacterium marinum is naturally multi-drug resist- CHU Arnaud de Villeneuve 371 avenue du Doyen Gaston Giraud, ant and there is no standardized antimicrobial treat- 34295 Montpellier, France ment for M. marinum infections []. As the wild-type Full list of author information is available at the end of the article 2016 Chazel et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (mits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (o the data made available in this article, unless otherwise stated.
Chazel et al. Ann Clin Microbiol Antimicrob (2016) 15:30 susceptibility pattern of M. marinum is well known [] storage at −80 °C. To prior testing, each drug was freshly and acquired resistance has not been described so far, diluted in sterile deionized water. The concentration antimicrobial susceptibility testing is not recommended ranges of the tested antimicrobial agents are indicated in except in the case of treatment failure and relaps The Clinical and Laboratory Standards Institute (CLSI) recommends microdilution for M. marinum drug suscep- MIC determination by using the agar dilution method
tibility testing (DST) [], but the agar dilution method The agar dilution method was performed on Müller- is currently considered to be the reference method [, Hinton agar (Becton–Dickinson, France) supplemented As this method is time-consuming, alternative meth- with 5  % OADC according to CLSI [wofold dilu- ods, such as the E-test, were evaluated, but showed poor tions of the antibiotics to be tested were added to obtain agreement with the reference method and therefore are the required final concentrations. Confluent colonies of not suitable for DST in M. marinumcently, the different bacteria in LJ slants were swept with a loop the SLOMYCO Sensititre® panel was commercialized by and emulsified in sterile water and the concentration TREK Diagnostic Systems (Clevels is a was adjusted to the McFarland n. 1 standard turbidity. A standard-order broth microdilution panel that can be 1/100 dilution of each suspension adjusted to the McFar- used to evaluate the susceptibility of slow growing myco- land n. 1 standard was inoculated using a Steers replica- bacteria to 14 antimicrobial agents, including those rec- tor to deliver approximately 104 colony-forming units ommended by CLSI for M. marinum DST (CFU) per spot. Plates were incubated at 30 °C ]. The The main goal of this work was to evaluate the SLO- MICs (i.e., the lowest concentration of antibiotic result- MYCO Sensititre® panel method for DST in M. marinum ing in complete inhibition of growth) of the tested anti- strains from humans and fish relative to the reference biotics were determined after 7 and 14 days of growth of agar dilution method.
the different M. marinum isolates/controls.
MIC determination using the SLOMYCO Sensititre® panel
Bacterial strains and growth conditions
(broth micro‑dilution method)
The origin and other information concerning the tested Inocula for the SLOMYCO Sensititre® panel were pre- M. marinum isolates are described in Broutin et pared according to the CLSI and the manufacturer's The 35 M. marinum isolates from human patients and instrucLOMYCO plates were incubated in nine from fish were collected in France between 1995 and a non-CO2 incubator at 30 °C until the controls showed 2007. None was from patients who experienced treat- sufficient growth (7–14  days). The MICs were deter- ment failure or relapse. M. marinum identification was mined visually using an inverted mirror and read as the performed using GenoType Mycobacterium AS/CM, a lowest concentration of the antibiotic showing 100  % commercial multiplex line-probe assay (Hain Lifesci- growth inhibition.
ence GmbH, Nehren, Germany). Isolates were stored at
−80 °C in Middlebrook 7H9 broth (DIFCO, Detroit, MI, Analysis of the results
USA) containing 5 % OADC (DIFCO, Detroit, MI, USA) The reproducibility of both methods was evaluated by until determination of the Minimum inhibitory con- performing two independent tests for each method and centrations (MICs). Mycobacteria were then cultured for each of the 44 isolates (a total of 176 tests) and five in Löwenstein-Jensen (LJ) slants (bioMérieux, Marcy independent tests for the M. marinum ATCC 927 and l'Etoile, France) and in Middlebrook 7H10 agar (DIFCO, ATCC BAA-535/M strains (a total of 30 tests). Each test Detroit, MI, USA). The M. marinum ATCC 927 strain result was independently interpreted by two blinded (from fish) and the M. marinum ATCC BAA-535/M readers. The reproducibility value was defined as the per- strain isolated from an infected patient were used as con- centage of strains with the same MIC value ±1 log2 dilu- trols for MIC determination.
tion at each test. The essential agreement between the agar dilution and SLOMYCO Sensititre® panel results was expressed as the percentage of isolates that showed Amikacin, ciprofloxacin, clarithromycin, doxycycline, the same MIC value ±1 log2 dilution with the two meth- ethambutol, ethionamide, isoniazid, rifampin, rifabu- ods. Category agreement was evaluated using the break- tin, streptomycin and trimethoprim (Sigma-Aldrich, points for determining the susceptibility and resistance Lyon, France), linezolid (Pfizer, France) and moxifloxa- categories recommended by CLSor this study cin (Bayer, Wuppertal, Germany) were tested in this only very major errors (i.e., an isolate resistant [R] by study. Stock solutions of each drug were prepared using the reference method, but susceptible [S] by the tested the appropriate solvent and were filter-sterilized before method) and major errors (S by the reference method Chazel et al. Ann Clin Microbiol Antimicrob (2016) 15:30 Table 1 MIC (μg/mL) of the 14 antibiotics tested in 46 M. marinum isolates (35 clinical, 9 fish and 2 references strains),
determined by using the agar dilution method
Antimicrobial agent
Total (n = 46) Human (n = 35) Fish (n = 9) Total (n = 46) Human (n = 35) Fish (n = 9)
Amikacin (>32) Ciprofloxacin (>2) Clarithromycin (>16) Doxycycline (>4) Ethambutol (>4) Moxifloxacin (>2) Rifabutin (>2) Streptomycin (NA) Trimethoprim (>2) a Concentration range of the tested drugs by using the agar dilution method (ADM) and the SLOMYCO Sensititre® panel method (SSPM): amikacin (0.25–32 mg/L, ADM; 1–64 µg/mL, SSPM); ciprofloxacin (0.12–16 µg/mL, ADM, SSPM); clarithromycin (0.06–32 µg/mL, ADM, SSPM); doxycycline (0.12–16 µg/mL, ADM, SSPM); ethambutol (0.5–16 µg/mL, ADM, SSPM); ethionamide (0.3–20 µg/mL, ADM, SSPM); isoniazid (0.25–16 µg/mL, ADM; 0.25–8 µg/mL, SSPM); linezolid (0.12–32 µg/mL, ADM; SSPM, 1–64 µg/mL); moxifloxacin (0.06–8 µg/mL, ADM; 0.12–8 µg/mL, SSPM); rifabutin (0.06–16 µg/mL, ADM; 0.25–8 mg/L, SSPM); rifampin (0.12–8 mg/L, ADM; 0.25–8 µg/mL, SSPM); streptomycin (0.5–32 µg/mL, ADM; 0.5–64 µg/mL, SSPM); sulfamethoxazole (2.38–152 µg/mL, ADM, SSPM); trimethoprim (0.12–8 µg/mL, ADM, SSPM); NA not available and R by the test method) were considered. Minor errors (between 0.1 and 1 μg/mL) were lower than those of the (intermediate [I] by one method and S or R by the other other tested antibiotics.
method) were not considered because the CLSI break- The relatively high MIC90 of ethionamide (10 μg/mL), points allow only two characterization categories (R or S) isoniazid (8  μg/mL) and streptomycin (16  μg/mL), for which breakpoints are not available, suggests that they are not good candidates for the treatment of M. marinum infection. In contrast, linezolid (MIC90: 2  μg/mL) was The reproducibility of the results obtained with the very effective against the different M. marinum isolates agar dilution method (reference method) was very good with MIC values among the lowest in our study.
(Taor all antibiotics. The MICs of the 14 antimicro- The SLOMYCO Sensititre® panel method (tested bial agents determined by using the agar dilution method method) produced remarkably consistent and reproduc- were distributed in a narrow range (Table ). Comparison ible results (Table ). Reproducibility with this method of the MICs for the M. marinum isolates from infected was 100 % in the case of ethionamide, isoniazid, moxi- humans and fish did not reveal any difference.
floxacin, rifampin, trimethoprim and sulfamethoxazole, The MICs for the two reference M. marinum strains and between 98 and 87  % for amikacin, ciprofloxacin, (ATCC 927 and ATCC BAA-535/M) were within 0–1 clarithromycin, doxycycline, ethambutol, linezolid, dilution of the MICs of the 44 tested isolates and within rifabutin and streptomycin (Table  ercent- the expected range.
age essential agreement (±1 log2 dilution) between the The MICs at which 90  % of isolates were inhibited MICs obtained with the tested and the reference meth- (MIC90) of amikacin (4 μg/mL), clarithromycin (2 μg/mL) ods (Table greatly varied depending on the drug: from and rifabutin (0.12 μg/mL) were below the CSLI break- 98 % for ciprofloxacin and linezolid to 2.2 % for ethiona- point, whereas the MIC90 of ciprofloxacin (2  μg/mL), mide. Good agreement percentages were obtained for doxycycline (4 μg/mL), ethambutol (4 μg/mL), moxiflox- moxifloxacin (91.3 %), isoniazid (87 %) and clarithromy- acin (1 μg/mL), rifampin (1 μg/mL) and sulfamethoxazole cin (85 %); whereas, agreement was lower for amikacin (19 μg/mL) were close to the breakpoint []. The MIC90 (76.1  %), rifampin (76.1  %), rifampin (76  %), trimetho- of rifampin, rifabutin, moxifloxacin and trimethoprim prim (74  %), doxycycline (72  %), sulfamethoxazole Chazel et al. Ann Clin Microbiol Antimicrob (2016) 15:30 Table 2 Reproducibility of the results (i.e., MIC, expressed in µg/mL, of the tested antibiotics in the 46 M. marinum iso-
lates) obtained with the agar dilution method
No. of results within log2 concentration difference of
% agreement
Table 3 Reproducibility of the results (i.e., MIC, expressed in µg/mL, of the tested antibiotics in the 46 M. marinum iso-
lates) obtained with the SLOMYCO Sensititre® panel method
No. of results within log2 concentration difference of
% agreement
93.5 (81.1–98.3) (70 %), streptomycin (59 %) and particularly ethambutol (SMT-TMP), 98 % for ethambutol and 96 % for rifampin (33 %). In the case of ethambutol and ethionamide, the (Table wo very major discrepancies were observed SLOMYCO Sensititre® panel method underestimated for rifampin (2/46) and one for ethambutol (1/46). No the MICs by 2–3 dilutions compared with the refer- agreement was observed between the two methods for ence method. When the agar dilution and SLOMYCO doxycycline, with 46 (100  %) major discrepancies. The Sensititre® panel results were converted into inter- SLOMYCO Sensititre® method overestimated the dox- pretive categories (resistance/susceptibility) using the ycycline MIC by 1 dilution; however, with this method, CLSI breakpoints, the category agreement was 100  % doxycycline MIC90 (8  μg/mL) was close to the suscep- for amikacin, ciprofloxacin, clarithromycin, moxifloxa- tibility breakpoint obtained by using the agar dilution cin, rifabutin and sulfamethoxazole–trimethoprim method (4 μg/mL).
Chazel et al. Ann Clin Microbiol Antimicrob (2016) 15:30 Table 4 Comparison of the MIC values obtained by using the SLOMYCO Sensititre® panel and the agar dilution methods
for 46 M. marinum isolates
No. of results within log2 concentration difference of
% essential agreement
Table 5 Comparison of  the susceptibility testing results amenable to automation ompared to the time-
(resistance/susceptibility) and category errors (very major
consuming and cumbersome agar dilution method, our or major error) by  using the SLOMYCO Sensititre® panel objective was to compare the performance of these two
(SSPM) and  the agar dilution method (ADM) for  46 M.
marinum isolates following the CLSI guidelines
The lack of difference between the MICs for the M. % resistant % resistant Category
% agree‑
marinum isolates from infected humans and fish could errors (N°)
be explained by the fact that most of the clinical sam- ples were from patients who handled infected fish (from aquarium tanks or fish-related work) and, therefore, the antibiotic susceptibility profiles of the human isolates Ciprofloxacin (2) 0 reflected those of the fish isolates.
Our results show that the reproducibility of the SLO- MYCO Sensititre® results for the reference strains and the M. marinum isolates (from human and fish) was very good. All results of the independent tests were within the ±1 log2 dilution acceptable level of varia- tion. Moreover, the level of agreement (±1 log2 dilution) between the results (MICs and interpretive categories) obtained with the SLOMYCO Sensititre® panel and the agar dilution methods was good for most of the antibi- otics recommended by CLSI and AT] for the treat- ment of (rifampin, rifabutin, amikacin, clarithromycin and sulfamethoxazole–trimethoprim) or with therapeu- The manufacturer's guidelines (TREK Diagnostic Sys- tic potential (linezolid, isoniazid) for M. marinum infec- tems, Cleveland, OH) recommend the SLOMYCO Sen- tions. It should be noted that two very major errors were sititre® method for antibiotic susceptibility testing in M. observed for rifampin in isolates in which the MIC for marinum, but studies evaluating the concordance with rifampin was just above the breakpoint (2  μg/mL) and the reference agar dilution method have not been pub- that still belong to the wild type populalini- lished yet. As the SLOMYCO Sensititre® panel technique cal significance of these data is unclear. Moreover, the presents several advantages (commercial availability; MIC of the only rifampin-resistant M. marinum reported standardization; easy to set up and to interpret; and un] was clearly above the breakpoint (>16 µg/ Chazel et al. Ann Clin Microbiol Antimicrob (2016) 15:30 mL). Unfortunately, we could not evaluate the ability Conclusion
of the SLOMYCO Sensititre® panel to detect resistant In this study, we evaluated the SLOMYCO Sensititre® panel strains, because none of the isolates tested in this study method for susceptibility testing in 44 M. marinum iso- was considered as antibiotic-resistant.
lates (from humans and fish) relative to the reference agar Conversely, poor agreement was observed for etham- dilution method. Our results indicate that the SLOMYCO butol. The MICs for ethambutol obtained with the SLO- Sensititre® panel method could provide a potential alterna- MYCO Sensititre® panel were 2–3 dilutions lower than tive to the reference agar dilution method, when DST in M. those obtained with the reference method. However, this marinum is required, except for doxycycline. If doxycycline poor agreement resulted in only one very major error, susceptibility needs to be tested, the use of another method when MICs were converted into interpretive categories. (broth microdilution) is more appropriate.
This discrepancy corresponds to a strain with a MIC just above the breakpoint (16 µg/mL) (Table  In the case of doxycycline, no agreement was observed Written informed consent was obtained from the between methods resulting in 46 major errors. This could patients for the publication of this report.
be explained by (i) the overestimation of doxycycline MICs by the SLOMYCO Sensititre® panel that could be Authors' contributions
MC, HM, NK, DT, SG designed this study and did most of the writing, sup‑ due to the medium or pH, and (ii) the fact that the MIC50 ported by CC, MP, VJ, GP, NB, ALB, MC, JS, AA and SG who have been involved in drafting the manuscript and have made substantial contributions to data 90 were similar to the breakpoint value. Among the antibiotics recommended by CLSI and on the basis acquisition. All authors read and approved the final manuscript.
of in vitro susceptibility testing, our results confirm that Author details
rifampin, rifabutin, amikacin, clarithromycin and sul- 1 Département de Bactériologie‑Virologie, CHRU Montpellier, Montpellier, famethoxazole–trimethoprim are good options for the France. 2 UMR 5119 ECOSYM, Equipe Pathogènes et Environnements, Labo‑ ratoire de Bactériologie, U.F.R. des Sciences Pharmaceutiques et Biologiques, treatment of M. marinum infections, as reported in pre- Montpellier, France. 3 Laboratoire Départemental Vétérinaire de l'Hérault, vious studies [, , ulfamethoxazole and Montpellier, France. 4 Laboratoire Biomnis, Lyon, France. 5 Department trimethoprim showed good in  vitro activity against M. of Bacteriology‑Virology, INSERM U1058 "Pathogenesis and Control of Chronic Infections", Université Montpellier‑EFS, CHU Arnaud de Villeneuve 371 avenue marinum and could be considered as an alternative treat- du Doyen Gaston Giraud, 34295 Montpellier, France. 6 Université Montpellier, ment []. The present study brings new data on M. mari- Montpellier, France. 7 MIVEGEC (Laboratoire Maladies Infectieuses et Vecteurs, num susceptibility pattern to ethionamide, streptomycin Ecologie, Génétique, Evolution et Contrôle), UMR CNRS 5290/IRD 224, Univer‑ sité de Montpellier, Montpellier, France. 8 Department of Biopathology, CHRU and linezolid. The American Thoracic Society recom- Montpellier, Montpellier, France. 9 Department of Clinical Oncoproteomic, mendations for the treatment of some nontuberculous Montpellier Cancer Institute, Montpellier, France. 10 Centre National de Réfé‑ mycobacteria (NTM) infections include the use of strep- rence des Mycobactéries et de la Résistance aux Antituberculeux, Paris, France. 11 Laboratoire de Bactériologie‑Hygiène Paris, AP‑HP, Hôpital Pitié‑Salpêtrière, tomycin (for rifampin-resistant M. kansassii infection) 75013 Paris, France. 12 Centre for Immunology and Microbial Infections, team and ethionamide (for M. malmoense infection) [ 13, Sorbonne Universités, UPMC Univ Paris 06, U1135, 75013 Paris, France. However, the lack of clinical experience in M. marinum INSERM, U1135, Paris, France. infections and the absence of breakpoints for NTM sus- ceptibility and resistance to these two antibiotics did not This study was supported by the Montpellier University Teaching Hospital allow predicting their potential efficiency in M. marinum (CHU Montpellier, "Equipe Performante Recherche" contract). We would like to thank INSERM, IRD and CNRS for technical support. We thank Elisabetta Ander‑ infections [inezolid has been reported to be effective marcher for assistance in preparing and editing the manuscript, and Nicolas against mycobacteria (M. chelonae and M. marinum, Veziris for helpful discussions. The authors declare they have no conflict of ] and for treating skin and soft tissue infec, interest.
our study, linezolid was one of the most active antimicro- bial agents in agreement with the low MIC determined in The authors declare that they have no competing interests.
a previous study [espite the lack of breakpoint values Received: 4 December 2015 Accepted: 27 April 2016 for M. marinum, linezolid may be an interesting alterna- tive therapeutic agent due to its pharmacological proper- ties. This study confirmed that M. marinum is resistant to isoniazid and ethambuthe observed M. mari- num susceptibility pattern corresponded to the wild type References
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