during a 12-month period ( 10/20019/2002 ) , 199 consecutive environmental specimens were submitted for possible detection of b. anthracis .
hemolysis types were determined by subculturing them on 5% horse , sheep , and human blood agar plates : - , - , and -hemolysis were defined , according to standard criteria ( 13 ) .
strong -hemolysis was characterized as hemolysis clearly extending the colony margin , and weak -hemolysis was characterized by a narrower hemolysis zone or slight hemolysis below colonies .
cultures were incubated at 35c for 1824 h in air ; blood agar plates were incubated for 48 h. motility testing was performed by using sterile h2o at time 0 , as well as trypticase soy broth ( tsb ) at time 0 and after a 2-h incubation at 35c .
part of a colony was dissolved in h2o and tsb and examined microscopically ( 2 ) .
media were supplied from bioprepare ( gerakas , greece ) , except for human blood agar plates prepared in house with red blood cell units obtained from blood banks and a blood agar base ( scharlau chemie , barcelona , spain ) .
specific pcr protocols amplifying a 152- , a 747- , and a 264-bp fragment of the chromosomal ba813 , the paga ( pxo1 ) , and the capc gene ( pxo2 ) sequences , respectively ( 5 ) . a pcr recommended by the world health organization , which targets a 639-bp sequence of the chromosomal b. anthracis s - layer gene ,
control strains included the nc08234 - 03 b. anthracis sterne strain ( pxo1/pxo2 ) , a b. anthracis strain isolated from the malignant pustule of an agricultural anthrax patient , and the bacillus subtilis eo-1 reference strain ( kindly provided by the hellenic agricultural ministry ) .
diverse hemolytic activity was demonstrated by the 72 bacilli on different blood agar plates . at 24 h ,
strong -hemolysis was produced by 55 ( 76% ) , 41 ( 57% ) , and 55 ( 76% ) strains on human , sheep , and horse blood agar plates , respectively , while 6 ( 8% ) , 10 ( 14% ) and 7 ( 10% ) strains demonstrated weak -hemolysis ( table ) .
prolongation of incubation to 48 h resulted in increased detection of strong -hemolysis on all media .
both b. anthracis strains produced -hemolysis on all media at 24 h , except for slight -hemolysis below areas of confluent growth on human blood agar plates .
after 48 h they remained -hemolytic on horse and sheep blood agar plates ; however , both produced strong -hemolysis on human blood agar plates . at time zero of incubation ,
motility was detected in 33 ( 46% ) and 45 strains ( 63% ) examined in h2o and tsb , respectively .
the 2-h incubation step in tsb detected another 13 motile strains ( total number of motile strains , 58 ; 81% ) and made recognition of motility much easier .
the capc and paga gene sequences were not amplified ; however , the ba813 sequence was amplified in seven strains ( 10% ; specificity 90% ) , and the s - layer sequence was amplified in another two strains ( 3% ; specificity 97% ) .
twenty - four isolates ( 33% ) grew on plet ; 21 of them were uniformly -hemolytic .
a positive correlation between the ability to grow on plet and ba813 pcr - positivity was detected .
five out of 24 strains ( 21% ) that grew on plet were positive by this pcr in comparison with 2 of 48 strains ( 4% ) that did not grow on plet ( fisher exact test , p = 0.037 ) .
although other genera are known to produce distinct hemolysis types on different blood agar plates ( 14 ) , comparative studies for bacilli have not been reported .
however , various blood agar plates manifested different abilities to support the expression of -hemolysis as well as to demonstrate weak and strong -hemolysis .
weak -hemolysis must thus be interpreted with caution ; isolates will be incubated for another 24 h or considered nonhemolytic strains .
-hemolysis was easier to recognize on all media after 48 h , but sheep blood agar plates were the least effective medium in detecting -hemolysis . finally , -hemolysis results obtained with horse and human blood agar plates , although not identical , were usually in agreement and differed from those obtained with sheep blood agar plates ( data not shown ) .
the production of strong -hemolysis on human blood agar plates by the b. anthracis strains was unexpected , as this organism has been considered traditionally nonhemolytic .
recently , however , the ability of b. anthracis to express -hemolysis was reported ( 15,16 ) .
broth culture supernatants possessed hemolytic activity against human and sheep erythrocytes , whereas richness of media affected hemolysis expression ( 15 ) .
another study demonstrated the induction of strong -hemolysis on human but not sheep blood agar plates under anaerobic conditions ( 16 ) .
in fact , a study conducted in 1957 reported 45 -hemolytic strains among 120 b. anthracis isolates that had been cultured on rabbit blood agar plates ( 17 )
. therefore , withholding the use of human blood agar plates would be prudent ; horse blood agar plates should be used as the most  informative  medium . according to our findings , the 2-h incubation of bacilli in tsb greatly improves recognition of motile strains .
increased motility detection using tsb rather than h2o has been demonstrated with 12 non  b .
apparently , motile bacilli become immobilized in distilled water , while the practice of incubating them in a rich broth until they reach exponential growth phase was actually recommended in the past ( 19 ) .
all bacilli tested negative for the presence of capc and paga sequences , but seven strains were positive for ba813 .
these results prompted us to assess the specificity of the s - layer pcr , which , to our knowledge , has not been evaluated before .
only two strains , different from the above seven , were positive : a motile , strongly -hemolytic strain and a nonmotile , -hemolytic strain . with the exception of the laboratory response network real - time pcr ( 9 ) ,
the specificity of other pcr protocols ( 48,20 ) has not been evaluated before by testing field isolates from suspected bioterrorism incidences .
false - positive results have been reported with other cap sequence pcr assays ( 4,6 ) , although efforts to establish a specific chromosomal assay have been frustrating ( 6,7,20 ) . as b. anthracis strains cured from one or
both plasmids exist naturally or may be obtained in vitro ( 3 ) , and false - negative results may be encountered with plasmid - specific pcr assays ( 4 ) , the availability of a chromosomal pcr is desirable .
our results indicate that to preserve the positive predictive value of the evaluated molecular tests , chromosomal assays should always be performed in conjunction with plasmid pcr . because environmental and , to a lesser extent , clinical samples may be heavily contaminated , a selective medium for b. anthracis would be useful .
plet is used in environmental investigations of agricultural anthrax outbreaks ( 3 ) , because it inhibits other bacilli and gram - negative rods ( 10 ) . in our study , the specificity of plet was low , but plet is still valuable , because by inhibiting two thirds of contaminating bacilli as well as other bacteria , background will decrease and isolating colonies will be easier and faster .
however , the characteristic colony morphology of the two b. anthracis strains on blood agar was not reproduced on plet .
examination of a large number of b. anthracis strains is required to confirm these observations .
a positive correlation was detected between the ability to grow on plet and ba813 pcr - positivity .
anthracis strains previously reported to be ba813 pcr  positive were isolated by using plet ( 20 ) .
ba813-positive bacilli may be very closely related to b. anthracis and demonstrate , therefore , phenotypic similarities like the ability to grow on plet . in conclusion ,
horse blood agar plates provide better recognition of -hemolysis , and testing after a 2-h - incubation in tsb improves motility detection .
the application of these tests along with plet agar will have a substantial impact on public health laboratories that process large numbers of specimens .
workload will decrease , and the presence of b. anthracis will be ruled out faster , leading to earlier termination of chemoprophylaxis and diminished anxiety of exposed persons .
standardization and validation of molecular assays as direct detection tests will further decrease turnaround time ; however , these methods only complement conventional testing . selective or differential media and further refinement of conventional techniques will still be needed .
 optimization of methods for ruling out bacillus anthracis leads to increased yields , faster turnaround times , and a lighter workload . 
 we used 72 environmental non  
 b . 
 anthracis bacilli to validate methods for ruling out b. anthracis . 
 most effective were horse blood agar , motility testing after a 2-h incubation in trypticase soy broth , and screening with a b. anthracis  selective agar . 
The Study
Conclusions