Etest® for antibiotic susceptibility testing of Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis: evaluation of a French collection

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Letters to the Editor / International Journal of Antimicrobial Agents 31 (2008) 484–504

Funding: Fund for Scientific Research—Flanders. Competing interests: None declared. Ethical approval: Local Ghent University Hospital Ethics Committee (project no 2004/233). References [1] Bhat S, Fujitani S, Potoski B, Capitano B, Linden PK, Shutt K, et al. Pseudomonas aeruginosa infections in the Intensive Care Unit: can the adequacy of empirical ␤-lactam antibiotic therapy be improved? Int J Antimicrob Agents 2007;30:458–62. [2] Blot S. Limiting the attributable mortality of nosocomial infection and multidrug resistance in intensive care units. Clin Microbiol Infect 2008;14:5–13. [3] Paterson DL, Rice LB. Empirical antibiotic choice for the seriously ill patient: are minimization of selection of resistant organisms and maximization of individual outcome mutually exclusive? Clin Infect Dis 2003;36:1006–12. [4] Depuydt P, Benoit D, Vogelaers D, Decruyenaere J, Vandijck D, Claeys G, et al. Systematic surveillance cultures as a tool to predict involvement of multidrug antibiotic bacteria in ventilator-associated pneumonia. Intensive Care Med December 8, 2007 [Epub ahead of print]. [5] Blot S, Depuydt P, Vogelaers D. Colonization status and appropriate antibiotic therapy in nosocomial bacteremia caused by antibioticresistant gram-negative bacteria in the ICU. Infect Control Hosp Epidemiol 2005;26:575–9. [6] Depuydt P, Blot S, Benoit D, Claeys G, Verschraegen G, Vandewoude K, et al. Antimicrobial resistance in nosocomial bloodstream infection associated with pneumonia and the value of systematic surveillance cultures in an adult intensive care unit. Crit Care Med 2006;34:653–9. [7] Depuydt P, Benoit D, Vogelaers D, Claeys G, Verschraegen G, Vandewoude K, et al. Outcome in bacteremia associated with nosocomial pneumonia and the impact of pathogen prediction by tracheal surveillance cultures. Intensive Care Med 2006;32:1773–81.

Pieter Depuydt ∗ Stijn Blot Dominique Benoit Johan Decruyenaere Department of Intensive Care, Ghent University Hospital, De Pintelaan 185, 9000 Gent, Belgium ∗ Corresponding author. Tel.: +32 9 3222808. E-mail addresses: [email protected], [email protected] (P. Depuydt) 24 December 2007 doi: 10.1016/j.ijantimicag.2008.01.001

Etest® for antibiotic susceptibility testing of Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis: evaluation of a French collection Sir, Owing to a high genetic relationship, Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis are proposed as members of a single species, Bacillus cereus sensu lato [1]. Bacillus anthracis has recently acquired notoriety related to its use as a biological weapon [2]. Bacillus cereus is an opportunistic human pathogen associated with

food poisoning, traumatic or war wound infections, endophthalmitis, pneumonia and bacteraemia in newborn children or in immunocompromised patients. Bacillus thuringiensis, an insect pathogen that is widely used in agriculture as a biopesticide, has been described as being responsible for gastrointestinal and wound infections. The potential of acquired resistance in clinical strains as well as utilisation of resistant strains of B. anthracis in bioterrorism underlines the importance of rapid determination of the susceptibility to antibiotics for each isolate. The goal of this study was to evaluate the accuracy of the Etest® for rapid minimum inhibitory concentration (MIC) determination of B. anthracis and other bacilli of the cereus group. A total of 130 bacterial strains were included. Thirty isolates of B. anthracis collected in France by the AFSSA/LERPAZ between 1984 and 2006 (29 bovine strains, 1 human strain) and the vaccine Delpy and Sterne strains were studied. Forty-eight clinical isolates of B. cereus isolated from soft tissue infections and war wounds were collected from 1991 to 2003 at the B´egin Military Hospital. Fifty strains of B. thuringiensis belonging to 50 different serotypes were provided by the World Health Organization Collaborating Centre for Entomopathogens at the Pasteur Institute of Paris (France). Seven mutants of B. cereus resistant to doxycycline (MICs of 8–64 mg/L compared with initial values of 0.06–0.125 mg/L) and five mutants of B. thuringiensis resistant to ciprofloxacin (MICs of 1–16 mg/L compared with initial values of 0.06–0.25 mg/L) obtained by in vitro selection following the method proposed by Szybalski and Bryson [3] were also included to assess the accuracy of the Etest® for diagnosis of acquired resistance. MICs were determined in parallel for nine clinically relevant antibiotics on Mueller–Hinton II medium by the agar dilution method [4] and the Etest® method, as described by the manufacturer (AB BIODISK, Solna, Sweden). All experiments were performed in a Biosafety Level 3 (BSL-3) laboratory. A variation of ± one dilution between the MICs obtained by Etest® and the agar dilution method was considered concordant. Table 1 shows the difference in dilutions between MICs determined by both methods as well as the percentage agreement. In this study, ␤-lactam resistance was expressed in 96% of B. cereus and B. thuringiensis strains. Only one isolate of B. anthracis was classified as resistant, with a MIC >64 mg/L for penicillin G and amoxicillin. All isolates were susceptible to ciprofloxacin, vancomycin and linezolid. Two strains of B. cereus were resistant to doxycycline (MICs of 2 mg/L and 16 mg/L), one to gentamicin and three to rifampicin. For B. anthracis there was no significant difference between MICs determined by Etest® and by agar dilution method for all antibiotics. Results of MICs determined by Etest® were easy to read because ellipse inhibition was well defined. For B. anthracis, discrepancies ranging from two to four dilutions for susceptible strains and as much as nine dilutions on a resistant strain had been highlighted for penicillin G when comparing Etest® and

Letters to the Editor / International Journal of Antimicrobial Agents 31 (2008) 484–504

491

Table 1 Comparison of the Etest® minimum inhibitory concentration (MIC) results with agar dilution MIC results for nine antimicrobial agents tested against 32 Bacillus anthracis, 55 Bacillus cereus and 55 Bacillus thuringiensis isolates Antibiotic

Species

No. of isolates with the following dilution difference in Etest® MICs compared with MICs obtained by the agar dilution reference method ≥−3

−2

−1

0

+1

+2

% Agreement

≥+3

Penicillin G

B. anthracis B. cereus B. thuringiensis

0 2 1

1 5 3

0 22 9

31 23 25

0 3 15

0 0 2

0 0 0

96.9 87.3 89.1

Amoxicillin

B. anthracis B. cereus B. thuringiensis

0 1 1

1 9 8

0 16 5

31 26 25

0 3 14

0 0 2

0 0 0

96.9 81.8 80.0

Ciprofloxacin

B. anthracis B. cereus B. thuringiensis

0 0 0

0 1 0

0 14 4

32 38 42

0 1 6

0 1 1

0 0 2

100 96.4 94.5

Gentamicin

B. anthracis B. cereus B. thuringiensis

0 0 1

0 7 1

28 27 12

4 21 40

0 0 0

0 0 1

0 0 0

100 87.3 94.5

Doxycycline

B. anthracis B. cereus B. thuringiensis

0 0 1

1 3 3

1 18 16

30 29 32

0 4 3

0 0 0

0 1 0

96.9 92.7 92.7

Clindamycin

B. anthracis B. cereus B. thuringiensis

1 0 1

0 2 1

20 24 6

10 27 40

1 2 6

0 0 1

0 0 0

96.9 96.4 94.5

Rifampicin

B. anthracis B. cereus B. thuringiensis

0 1 0

0 1 0

2 3 2

28 18 10

1 29 17

1 2 21

0 1 5

96.9 90.9 52.7

Vancomycin

B. anthracis B. cereus B. thuringiensis

0 0 0

0 0 1

0 0 0

5 12 3

27 40 33

0 3 17

0 0 1

100 94.5 65.5

Linezolid

B. anthracis B. cereus B. thuringiensis

0 1 0

1 5 1

30 33 9

1 16 44

0 0 1

0 0 0

0 0 0

96.9 89.1 98.2

broth microdilution methods [5]. These discrepancies have not been underlined by Turnbull et al. [6] or Luna et al. [7]. In our study, the agreement for penicillin G and amoxicillin was calculated at 96.9% for B. anthracis. A single penicillin-resistant B. anthracis strain was included, but penicillin G and amoxicillin MICs were 64 mg/L with both methods. With regard to B. cereus and B. thuringiensis, the correlation between both methods was lower, which could be explained by difficulties in determining the growth line intersecting MIC value on the Etest® strip owing to the presence of push back along the strip. However, the agreement was excellent for doxycycline and ciprofloxacin, including the few doxycycline-resistant strains and the in vitro selected mutants. For rifampicin and vancomycin, the MICs were higher with the Etest® but this did not modify the clinical categorisation according to Clinical and Laboratory Standard Institute (CLSI) recommendations for Staphylococcus aureus. For clindamycin, 40% of B. cereus strains exhibited one lower dilution MIC with Etest® , which modified the clinical categorisation because of the proximity between the MICs and the low breakpoint value chosen by the

CLSI. In conclusion, for ciprofloxacin, doxycycline and gentamicin, the major antibiotics for prophylaxis and treatment of B. cereus group infections, the Etest® and agar dilution method showed identical results. For B. anthracis, the excellent results for the nine antibiotics tested allow validation of the Etest® as a reliable and rapid method for MIC determination in BSL-3 laboratories involved in the diagnosis of anthrax.

Acknowledgments The authors would like to thank the French departmental veterinary laboratories for providing B. anthracis clinical isolates and the WHO Collaborating Center for Entomopathogens, Institut Pasteur, France. Funding: Grant of the Service de Sant´e des Arm´ees (France); grant of Etat-major des Arm´ees, France. Competing interests: None declared. Ethical approval: Not required.

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Letters to the Editor / International Journal of Antimicrobial Agents 31 (2008) 484–504

References [1] Helgason E, Okstad OA, Caugant DA, Johansen HA, Fouet A, Mock M, et al. Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis—one species on the basis of genetic evidence. Appl Environ Microbiol 2000;66:2627–30. [2] Inglesby TV, O’Toole T, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, et al. Working Group on Civilian Biodefense. Anthrax as a biological weapon, 2002: updated recommendations for management. JAMA 2002;287:2236–52 [Erratum: JAMA 2002;288:1849]. [3] Szybalski W, Bryson V. Genetic studies on microbial cross resistance to toxic agents. I. Cross resistance of Escherichia coli to fifteen antibiotics. J Bacteriol 1952;64:489–99. [4] Cavallo JD, Ramisse F, Girardet M, Vaissaire J, Mock M, Hernandez E. Antibiotic susceptibilities of 96 isolates of Bacillus anthracis isolated in France between 1994 and 2000. Antimicrob Agents Chemother 2002;46:2307–9. [5] Mohamed MJ, Marston CK, Popovic T, Weyant RS, Tenover F. Antimicrobial susceptibility testing of Bacillus anthracis: comparison of results obtained by using the National Committee for Clinical Laboratory Standards broth microdilution reference and Etest agar gradient diffusion methods. J Clin Microbiol 2002;40:1902–7. [6] Turnbull PCB, Sirianni NM, LeBron CI, Samaan MN, Sutton FN, Reyes AE, et al. MICs of selected antibiotics for Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides from a range of clinical and environmental sources as determined by the Etest. J Clin Microbiol 2004;42:3626–34. [7] Luna VA, King DS, Gulledge J, Cannons AC, Amuso PT, Cattani J. Susceptibility of Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides and Bacillus thuringiensis to 24 antimicrobials using Sensititre automated microbroth dilution and Etest agar gradient diffusion methods. J Antimicrob Chemother 2007;60:555–67.

A. M´erens a,b,∗ Service de Biologie M´edicale, Hˆopital d’Instruction des Arm´ees B´egin, 69 avenue de Paris, 94160 Saint-Mand´e, France b Ecole du Val-de-Grˆ ace, 1 place Alphonse Laveran, 75005 Paris, France a

J. Vaissaire ´ Laboratoire d’Etudes et de Recherches en Pathologie Animale et Zoonoses (LERPAZ), Agence Fran¸caise de S´ecurit´e Sanitaire des Aliments (AFSSA), 23 avenue du g´en´eral de Gaulle, 94700 Maisons-Alfort, France J.-D. Cavallo c,d de Biologie M´edicale, Hˆopital d’Instruction des Arm´ees B´egin, 69 avenue de Paris, 94160 Saint-Mand´e, France d Ecole du Val-de-Grˆ ace, 1 place Alphonse Laveran, 75005 Paris, France c Service

C. Le Doujet ´ Laboratoire d’Etudes et de Recherches en Pathologie Animale et Zoonoses (LERPAZ), Agence Fran¸caise de S´ecurit´e Sanitaire des Aliments (AFSSA), 23 avenue du g´en´eral de Gaulle, 94700 Maisons-Alfort, France C. Gros Service de Biologie M´edicale, Hˆopital d’Instruction des Arm´ees B´egin, 69 avenue de Paris, 94160 Saint-Mand´e, France

C. Bigaillon e,f de Biologie M´edicale, Hˆopital d’Instruction des Arm´ees B´egin, 69 avenue de Paris, 94160 Saint-Mand´e, France f Ecole du Val-de-Grˆ ace, 1 place Alphonse Laveran, 75005 Paris, France e Service

J.-C. Paucod Centre de Recherche du Service de Sant´e des Arm´ees, 38700, La Tronche, France F. Berger Ecole du Val-de-Grˆace, 1 place Alphonse Laveran, 75005 Paris, France E. Valade D. Vidal Centre de Recherche du Service de Sant´e des Arm´ees, 38700, La Tronche, France ∗ Corresponding

author. Tel.: +33 1 43 98 47 34; fax: +33 1 43 98 53 36. E-mail address: [email protected] (A. M´erens) doi: 10.1016/j.ijantimicag.2008.01.005

Experience with daptomycin in an infectious diseases service over 1 year: utility in an outpatient parenteral antibiotic programme Sir, Daptomycin is a novel cyclic lipopeptide with rapid cidal activity against Gram-positive organisms. In Glasgow hospitals, daptomycin is approved for restricted use, on the recommendation of an infectious diseases physician or microbiologist, for those patients with serious Grampositive infections (excluding primary pulmonary infections) when glycopeptides are failing or contraindicated. A retrospective review of our unit’s experience with daptomycin over 12 months was undertaken. Patients who had been managed through the infectious diseases inpatient unit or outpatient parenteral antibiotic therapy (OPAT) service from June 2006 (n = 1345) and who received at least one dose of daptomycin (n = 29) were identified and case records were reviewed. All patients were diagnosed with serious Gram-positive infections (Table 1). Eleven patients were bacteraemic (seven with Staphylococcus aureus), eight had methicillinresistant S. aureus infection, 13 had bone and joint infection and six had bacterial endocarditis. Twenty-one cases had received prior glycopeptide therapy and the indication was clinical/microbiological failure (15), allergy/intolerance (10), teicoplanin-resistant Staphylococcus epidermidis (1), to facilitate outpatient therapy (2) and empirical therapy in rapidly progressive soft tissue infection (1). Twenty patients received 6 mg/kg/day daptomycin and 17 received at least one additional agent with Gram-positive