Although there have been no reports of naturally occurring resistance to CIP or DOX in B. anthracis
, the in vitro
selection of resistant mutants and cloning of resistance genes are relatively simple laboratory procedures. Previous reports of strains engineered for resistance to tetracyclines (23
) or for multidrug resistance (MDR) (29
) have generated concerns about the use of such strains in a deliberate release of B. anthracis
. Also, the acquisition of resistance determinants by B. anthracis
has been documented in experiments with the organism in the rhizosphere of grass plants (26
). Therefore, the possibility of horizontal gene transfer from coexisting soil-dwelling bacteria that harbor many resistance genes cannot be excluded (9
), and rapid methods for detecting resistance are essential for public health preparedness and response.
The method described in this study is based on a method described previously by Rolain et al. (25
), in which a LightCycler assay (Roche Biochemicals, Mannheim, Germany) was used to estimate the growth and antibiotic susceptibilities of various Gram-positive and Gram-negative species. In agreement with that report, we found that a 4-h incubation time was sufficient for discrimination between the growth and inhibition of growth of B. anthracis
, as was reported for other rapidly growing Gram-positive organisms. However, efforts to adapt the described qPCR method to B. anthracis
presented numerous challenges.
The ideal antimicrobial susceptibility testing method for the rapid analysis of strains isolated during an outbreak of disease should facilitate the simultaneous processing of multiple samples. The purification of template DNA from cells in each well of the 12-well strip for each strain was considered to be too time-consuming, so the method was modified to use simple cell lysates. Also, since a comparison of target concentrations by qPCR will provide reliable, reproducible results only if efficient cell lysis is achieved for each sample, numerous lysis methods were investigated to optimize this critical step. The cell wall of B. anthracis
is known to be resistant to lysozyme and partially resistant to mutanolysin (39
). Heat lysis is routinely used to release DNA from B. anthracis
, and this lysis method is usually sufficient to provide enough DNA to detect the presence of B. anthracis
by PCR. However, we found that the heat method was not efficient, nor was it quantitatively reproducible, even for a single strain. The reproducible, complete lysis of B. anthracis
isolates was ultimately achieved with a purified Bacillus
phage lysin (27
). The optimized lysis procedure included three essential steps: (i) suspending B. anthracis
cells in a buffer containing Triton X-100, (ii) capture of the bacterial cells on a 0.1-μm Durapore PVDF filter, and (iii) enzymatic breach of the cell wall with a purified lysin. Triton X-100 was required to disrupt tenacious clumps of cells that prevented the access of the lysin enzyme to all cells. The removal of the culture medium while capturing the cells on the filter allowed efficient lysis in an optimized buffer. After the lysis reaction, the lysate was collected by centrifugation through the 0.1-μm Durapore filter. This step was essential to remove any spores that may have been present, ensuring that the lysate would be noninfectious and safe to handle in a biosafety level 2 (BSL-2) environment. Additional time was saved because each step could be performed with an 8- or 12-multichannel pipette using a 96-well plate format, including the use of a 0.1-μm PVDF filter plate that was developed by Millipore for this project.
Evaluation of the assay with numerous isolates of B. anthracis
was also essential. B. anthracis
is considered to be genetically monomorphic or clonal in nature (12
). However, we observed numerous variations in growth characteristics among the various strains tested. Initial tests indicated that cell lysis performed directly in Mueller-Hinton broth was efficient for the Sterne strain, but this was not the case for several wild-type strains of B. anthracis
that were tested. In addition, we observed an extended lag phase for several of the wild-type strains when colonies grown on tryptic soy agar (TSA) with sheep blood (SB) were used to prepare the inoculum for susceptibility tests in CAMHB. This lag time was detected as delayed qPCR signals from cell lysates prepared after 4 h of incubation in the no-drug control well (data not shown). Culture of the strains on Mueller-Hinton agar instead of TSA with SB eliminated the extended lag phase, suggesting that some strains of B. anthracis
require more time than others to adjust to a change in nutrient composition. Also noted among the wild-type strains were colony types that varied in color, from gray to white, and in size and shape and differences in tenacity when touched with an inoculating loop. Phenotypic variation among strains of B. anthracis
was reported previously (17
) and was even associated with differences in virulence (21
). However, the genetic basis of such variation remains to be determined. Such observations suggest that the use of surrogate strains or species to develop assays for B. anthracis
should be avoided.
Once the variables that affected optimal growth and lysis among the strains were identified and addressed, the evaluation of the assay was continued by using 14 geographically and genetically diverse strains of B. anthracis. When PCR results for these susceptible strains were compared with results for CIP-NS and DOX-NS derivatives of the Sterne strain, the observed CT values allowed us to define a ΔCT threshold (between the no-drug growth control and the antimicrobial drug dilution wells) that would consistently discriminate between susceptible and nonsusceptible isolates. Note that the ΔCT must be derived only from a comparison of samples within an individual susceptibility test. Slight variations in the numbers of cells in each inoculum preparation, in the growth rates of each strain, and in pipetting procedures will introduce differences in the total numbers of cells after incubation. The resulting differences in the final concentrations of DNA template in the cell lysates are amplified by the sensitivity of qPCR. Therefore, the mean CT value of either the no-drug control or samples from any of the antimicrobial drug dilution wells should not be compared to mean CT values outside that individual test.
An accurate interpretation of susceptibility test results requires following standardized procedures as well as testing appropriate antimicrobial agents and the correct range of concentrations of antimicrobial agents. Procedures established by the CLSI (8
) provide guidelines on specific parameters, such as the inoculum, incubation conditions, and composition of the medium, that are known to affect susceptibility test results. All guidelines specified for B. anthracis
(except incubation time) were therefore incorporated in the development of this rapid susceptibility test. The interpretation of the results was simplified by the absence of any overlap between the median ΔCT
values from susceptible and nonsusceptible strains at the breakpoint for susceptibility.
The qPCR method of susceptibility testing has several advantages. In addition to decreasing the time required to obtain results, the use of qPCR eliminates the issues involved with the visual reading of susceptibility test results in a BSL-3 environment. By definition, the endpoint of conventional MIC testing is based on visible growth. In a BSL-3 laboratory the small amount of growth at the bottom of the wells of clear plastic 96-well plates or strips of wells (JustOne strips) is difficult to observe in the biosafety cabinet. In addition to the distance from the observer, the growth must be viewed through both the glass front of the cabinet and the plastic face shield of a power-assisted personal respirator (PAPR). Even without these conditions, susceptibility testing requires trained, experienced personnel. qPCR provides an alternative method for susceptibility testing with quantitative results that are easily interpreted. qPCR results from this assay for B. anthracis can be used as a rapid screen by performing the qPCR on the no-drug control and samples from drug concentrations at and just above the breakpoint for susceptibility.
Alternative methods developed for rapid assays include real-time PCR to detect specific resistance genes. This approach requires primers that are specific for each gene. However, there are now more than 40 tetracycline resistance genes that have been described (6
), and most likely, there are many more that are as yet undiscovered. Without phenotypic data, the genetic approach has the potential to detect resistance genes that are not expressed, or, if expressed, the protein may not be functional. In either case the strain would be susceptible to DOX in spite of the positive PCR result. For ciprofloxacin, molecular analysis must be based on DNA sequence data to identify mutations in the DNA gyrase and topoisomerase IV genes.
Real-time PCR is used routinely by Laboratory Response Network (LRN) laboratories for the identification of biothreat agents. Therefore, the adaption of this PCR technology for susceptibility testing will provide a relatively simple assay using a technology that is both familiar and readily available in LRN reference laboratories. The use of the JustOne strips for the rapid susceptibility test also addresses the needs of LRN laboratories, as the strips are individually packaged and stable at room temperature for up to 2 years.
In this study, we have developed and evaluated an antimicrobial susceptibility test that has both conventional and molecular components. This test combines a broth microdilution susceptibility test (with a limited incubation time) and qPCR for the rapid analysis of growth (or inhibition of growth) at each concentration of the drug of interest. The method requires ≤6 h to complete, a relatively rapid time to results compared with conventional susceptibility tests for B. anthracis, which require 16 to 20 h of incubation time. This rapid method is now being adapted for use with other biothreat agents in ongoing efforts to reduce the time required to detect nonsusceptible strains of each species.