this report is a result of a collaborative effort between the national microbiology laboratory ( nml ) , health canada , and the provincial public health laboratories ( pphls ) in canada .
the pphls represent every province in canada and also include the yukon and north west territories . all s. paratyphi b dt identified at the pphls
were forwarded to the nml for additional characterization . between 1998 and 2002 , 252 s. paratyphi b dt strains were submitted to the nml , of which 246 were from a human source .
incidence of s. paratyphi b dt has generally increased since 1998 ; the spike in s. paratyphi b dt in the third and fourth quarters of 1999 can be attributed to an outbreak in british columbia , alberta , and saskatchewan caused by contaminated alfalfa sprouts ( 7 ) .
in addition to this large outbreak , additional outbreaks were reported between 1998 and 2002 ; however , each outbreak was small , and most involved fewer than six persons .
total number of salmonella enterica serovar paratyphi b dt identified in canada ( gray bars ) and the number of multidrug - resistant s. paratyphi b dt identified over the same period ( black bars ) , by quarter .
antimicrobial susceptibility testing was performed on all strains by using the disk - diffusion method as described by the national committee for clinical laboratory standards ( 8) .
susceptibilities were determined for ampicillin ( a ) , chloramphenicol ( c ) , ciprofloxacin ( cp ) , streptomycin ( s ) , sulfamethoxazole ( su ) , tetracycline ( t ) , and trimethoprim ( tm ) .
of the 237 strains examined for susceptibility , 123 ( 52% ) were susceptible to all antimicrobial agents tested .
sixty - seven strains ( 28% ) displayed the pentaresistance phenotype ( acssut ) , and the second most prevalent resistance profile was su ( n = 41 , 17% ) .
single strains displayed the phenotypes a , t , cssu , asutm , assu , and acsuttm .
a significant increase was observed in acssut strains over the time period from 1998 to 2002 ( p < 0.001 ) .
rates for the years are as follows : 1998 , n = 2 ( 2% ) ; 1999 , n = 4 ( 18% ) ; 2000 , n = 2 ( 10.5% ) ; 2001 , n = 23 ( 39% ) ; and 2002 , n = 36 ( 58% ) .
no large outbreaks were reported during this time period . to determine if the pentaresistance phenotype was caused by sgi1 , polymerase chain reaction ( pcr ) was used to detect integrons and the left ( thdf - s001 ) and right ( s044-yidy ) junctions of sgi1 as previously described ( 2 ) .
of the 67 strains identified with the acssut phenotype , 63 contained 1.0-kb and 1.2-kb integrons and left and right junctions of sgi1 , which suggests that these strains contained sgi1 inserted into the same location on the chromosome as was described for dt104 ( 1 ) .
one strain with the acssut phenotype contained 1.0-kb and 1.2-kb integrons and gave a pcr product for the left junction amplification reaction but not the right junction , which suggests that a portion of sgi1 downstream of the integrons was missing .
three strains with the acssut phenotype did not give the characteristic size products for integron ( 0.7 kb , n = 2 ; 2.0 kb , n = 1 ) , and all were negative for the junction pcr , which suggests that these strains most likely harbored other resistance genes giving the acssut phenotype .
only four strains with the acssut phenotype were identified from a nonhuman source ( one poultry , three environmental ) .
although the three environmental isolates contained sgi1 , the acssut strain isolated from poultry did not ; instead , it contained a 2-kb integron .
of the human isolates for which source was reported for the acssut strains ( n = 53 ) , all were isolated from stool , with the exception of three that were isolated from blood . to ensure the sgi1 was intact ,
a selected number of isolates were subjected to additional pcr with primers representing all regions of the 44-kb sgi1 element ( table ) .
dna from all of these strains gave positive reactions for all primer sets described , which suggests sgi1 was intact in these strains .
s. paratyphi b dt recovered from 2000 to 2002 were subtyped by using pulsed - field gel electrophoresis ( pfge ) after dna extraction and digestion with blni according to the standardized salmonella protocol ( 9 ) .
pfge - generated dna profiles were entered into the bionumerics software program version 3.0 ( applied maths , st .
cluster analysis was performed by the unweighted pair - group method with arithmetic averages , and dna relatedness was calculated on the basis of the dice coefficient .
in addition , all pfge patterns were visually compared and assigned a number or letter identification ( 10 ) . a dendrogram depicting the s. paratyphi b dt blni macrorestriction patterns
of the 139 strains available to subtype ( total = 142 ) , visual comparison of the fingerprints revealed a total of 63 unique fingerprint patterns that grouped into 24 clusters .
analysis of the dendrogram revealed that all but three strains with the acssut phenotype were grouped into three closely related clusters named clusters 1 to 3 ( figure 2 ) .
the three strains that did not cluster with the other acssut strains did not harbor sgi1 as described above .
dendrogram depicting the dna fingerprints of salmonella enterica serovar paratyphi b dt identified from 2000 through 2002 .
cluster 2 contained 17 strains that represented 7 subtypes , all of which showed < 7 band differences between strains from cluster 1 .
cluster 3 contained 15 strains that represented 6 subtypes , all of which showed fewer than seven band differences between subtypes in cluster 2 , but had more than seven band differences between the cluster 1 fingerprints .
five were other acssut identified in 1998 ( n = 1 ) and 1999 ( n = 4 ) , and all were identified in cluster 2 .
in addition , one s. paratyphi b dt recently shown to harbor sgi1 that was isolated in singapore from a fish was grouped into cluster 3 ( 4 ) .
the canadian isolates in clusters 1 to 3 have been identified from multiple provinces , including british columbia , alberta , manitoba , ontario , and qubec , representing western and central regions of canada .
the emergence of multidrug - resistant s. paratyphi b dt was documented recently in the netherlands and scotland ( 11,12 ) .
some isolates had the acssut susceptibility pattern and did not harbor any plasmids , which suggests the resistance is of chromosomal origin ( 12 ) . however , in neither study was the presence of sgi1 or other resistance genes examined .
we demonstrate the rapid emergence of multidrug - resistant s. paratyphi b dt in canada .
the increase is due to three clusters , all of which contain the multidrug - resistant genomic island sgi1 . that three closely related clonal groups were present suggests sgi1
may have inserted into the genome of s. paratyphi b dt in three separate events , as shown by clusters 1 , 2 , and 3 , or the insertion may have occurred once , with strains diverging over time .
we also identified three strains with the acssut phenotype that did not contain sgi1 sequences , which emphasizes the need to monitor genotypic resistance factors and not just phenotypic resistance traits to understand the dissemination of antimicrobial resistance .
the emergence of multidrug - resistant enteric pathogens is a concern because of the lack of suitable antimicrobial agents available to treat invasive infections .
one organism that emerged in the 1990s is multidrug - resistant s. typhimurium dt104 , which harbors sgi1 ( 3 ) . along with the multidrug - resistant phenotype ,
however , in vitro studies have not shown any increase in invasiveness or survival in mammalian cells ( 15,16 ) .
whether multidrug - resistant dt104 is more virulent remains to be determined , the underlying question remains : why has this clone of dt104 emerged as a major pathogen ?
selective pressure , resulting from the widespread use of antimicrobial drugs in animals for growth promotion or prophylaxis , may have played a role in disseminating this organism .
however , this factor may not completely account for its prevalence , because other multidrug - resistant strains of dt104 have emerged but have not disseminated internationally .
for example , additional determinants in sgi1 may contribute to the fitness or virulence of salmonella strains harboring it . in the present study , three closely related clusters of s. paratyphi b dt carrying
sgi1 have emerged in canada and make up 46% ( 64 of 139 ) of all strains identified in 2001 and 2002 .
furthermore , three additional s. paratyphi b dt strains with the acssut phenotype were identified that do not harbor sgi1 and do not appear to be rapidly increasing in canada . in this hypothesis , sgi1 may predict the next emerging salmonella serotype .
other factors , such as processing food products and the structure of the food distribution system , could play a role in disseminating these organisms .
we continue to monitor this pathogen and are designing studies to improve understanding of the epidemiology of s. paratyphi b dt in canada .
 we document an increase in the number of multidrug - resistant salmonella enterica serovar paratyphi b dt+ identified in canada . 
 most of these strains harbor salmonella genomic island 1 ( sgi1 ) . 
 further studies are needed to determine factors contributing to the observed emergence of this multidrug - resistant strain . 
The Study
Conclusions