Susceptibility testing of Bacillus species

J. M. Andrews,* and R. Wise

Department of Microbiology, City Hospital NHS Trust, Birmingham, UK

Sir,

Recent events have highlighted the need for diagnostic laboratories to be aware that isolates of Bacillus species require identification. Should the isolate prove to be Bacillus anthracis, the guidelines issued by the PHLS–CDSC1 suggest that organisms should be tested for antibiotic susceptibility as soon as possible, although no specific recommendations are given.

In this study, conventional agar incorporation MICs2 for Bacillus spp. were determined on Iso-Sensitest agar (ISA) (Oxoid, Basingstoke, UK) and Mueller–Hinton agar (MH) (Oxoid) at an inoculum of 104 cfu/spot, and by a commercial gradient method (Etest; Cambridge Diagnostics, Cambridge, UK) following the manufacturer’s instructions on MH, and modified using ISA. For the gradient method an inoculum equivalent to a 0.5 McFarland standard was swabbed on to the surface of the agar before application of the strip. The antibiotics studied were benzyl penicillin, tetracycline, doxycycline and ciprofloxacin, and incubation was at 35–37°C in air for 18–20 h. As there were no archive isolates of B. anthracis from human sources in our laboratory collection (eight strains are available from the NCTC in the UK, but only to holders of a DEFRA licence in respect to the Animal Pathogens Order 1998), Bacillus cereus, a closely related species with similar colonial appearance and growth requirements, was chosen as a surrogate for B. anthracis. As B. cereus is generally resistant to penicillin, penicillin-sensitive isolates of Bacillus subtilis were also included. Five isolates of B. cereus, including control strains NCTC 9945 and ATCC 13061, five isolates of B. subtilis, including control strains NCTC 8236 and NCTC 10400, three isolates of Bacillus spp. and Staphylococcus aureus controls 6571 and ATCC 29213 were studied. MICs for each isolate were determined by each method in triplicate and interpreted using BSAC breakpoints.3 Detection of ß-lactamase was initially undertaken by two commercial methods, Nitrocefin (Oxoid) and Intralactam strips (Mast Diagnostics, Merseyside, UK), on growth taken from a purity plate and also an area of growth around the penicillin gradient strip.

Generally, for the gradient method, growth of all isolates was visually more luxuriant on MH compared with ISA; this was less apparent with the conventional agar MIC determinations. Gradient tests for Bacillus spp. were unreadable on 36/312 (11.5%) occasions, due either to an irregular ellipse or to tiny colonies at the base of the ellipse (Table 1); all endpoints could be read by the conventional agar methods. MICs for the control S. aureus strains were within the acceptable ranges for penicillin, tetracycline and ciprofloxacin2 by all methods (acceptable ranges are not available for doxycycline). A summary of interpretations by each of the four methods is shown in Table 1. There was general agreement in the interpretation of conventional MICs determined on ISA and MH, except for three isolates (two B. cereus and one B. subtilis) with tetracycline MICs of 1–2 mg/L, where differences in interpretation reflected acceptable MIC variations that crossed the breakpoint. For the gradient method a comparison was difficult because of the number of unreadable results. Seven isolates were deemed resistant to tetracycline yet susceptible to doxycycline, which suggests that caution must be used when interpreting susceptibility to tetracycline analogues and that a re-evaluation of breakpoints for this group may be necessary.


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Table 1.  Comparison of susceptibilities of 13 isolates of Bacillus spp. determined by four methods
 
There was poor correlation between penicillin resistance and detection of ß-lactamase. All five isolates of B. cereus were resistant to penicillin (MICs >= 16 mg/L), but only control strain NCTC 9945 and one other isolate gave a positive reaction with nitrocefin and intralactam even when isolates were exposed to penicillin before testing, or when incubation was extended for 1 h at 35–37°C. Detection was subsequently repeated using an adaptation of the double disc method.4 Briefly, 1 mL of an overnight broth culture of S. aureus NCTC 6571 was added to 100 mL of Brain Heart Infusion agar (Oxoid), and 5 mL of this suspension were poured into a 90 mm sterile petri dish. A 25 µg ampicillin disc was placed on the surface of the agar, and a sterile 6 mm blotting paper disc was placed 20 mm from it (disc centre to disc centre) and dosed with 23 µL of a suspension of test organism with an optical density equivalent to a 2 McFarland standard. After incubation at 35–37°C for 48 h, zones of inhibition were examined for growth of S. aureus in the area around the disc containing the test strain. All of the organisms studied, except one isolate of B. subtilis (Z104 MIC range 0.004–0.016 mg/L), gave a positive result with this method of detection-inducible ß-lactamases. These data were consistent with penicillin MICs, except for one isolate of B. subtilis that was susceptible by conventional methods (Z34; MIC on ISA and MH 0.015 and 0.008 mg/L, respectively), but for which results were unreadable by the gradient method.

Difficulty was reported previously when reading gradient MICs for 50 isolates of B. anthracis; penicillin MICs determined by the gradient method on MH were lower than those determined by a microdilution technique in MH broth, and there were concerns that methods for detecting ß-lactamase, such as the nitrocefin test, were unreliable.5 Although further work is needed, conventional broth or agar MIC determinations currently appear to be the most reliable methods for determining susceptibility of B. anthracis. As testing would be performed in category three facilities, a breakpoint or abbreviated MIC method using BSAC-recommended breakpoint concentrations would be appropriate in the UK. Detection of ß-lactamase production by a double disc method is more reliable than nitrocefin or intralactam.

Footnotes

* Corresponding author. Tel: +44-121-507-5693;Fax: +44-121-551-7763; E-mail: jenny.andrews{at}cityhospbham.wmids.nhs.uk Back

References

1 . Public Health Laboratory Service–CDSC. (2001). Provisional guidelines for action in the event of a deliberate release of anthrax. CDSC 1.2.

2 . Working Party on Antibiotic Sensitivity Testing of the British Society for Antimicrobial Chemotherapy. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 5–16.[Abstract/Free Full Text]

3 . Working Party on Antibiotic Sensitivity Testing of the British Society for Antimicrobial Chemotherapy. (2001). Establishing MIC breakpoints and the interpretation of in vitro susceptibility testing. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 17–28.[Abstract/Free Full Text]

4 . Bryan, L. E. and Godfrey, A. J. (1991). ß-Lactam antibiotics: mode of action and bacterial resistance. In Antibiotics in Laboratory Medicine (Lorian, V., Ed.), p. 648. Williams and Wilkins, Baltimore, MD.

5 . Tenover, F. C. (2001). An approach to testing organisms not found in the NCCLS Guidelines. In Abstracts of the Forty-first Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, USA, 2001. Abstract 1075, American Society for Microbiology, Washington, DC.