Three Bordetella pertussis typing methods, pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and multi-locus variable number tandem repeat analysis (MLVA) were compared using a collection of Swedish strains. Of the three typing methods used, PFGE was found to be the most discriminatory. MLVA and MLST were less discriminatory, but may be valuable for strain discrimination when culture is not possible as they are based on PCR. The combination of MLVA/MLST was found to be equally discriminatory as PFGE and should therefore also be considered. The relationship between predominant lineages in Sweden and The Netherlands, characterized by the PFGE type BpSR11 and the allele for the pertussis toxin promoter ptxP3, respectively, was investigated. Linkage was found between the PFGE type BpSR11 and ptxP3 in that all BpSR11 strains carried ptxP3. On the other hand ptxP3 was found in several other PFGE-types. The presence of the ptxP3 allele in different genetic backgrounds may indicate horizontal gene transfer within B. pertussis or homoplasy. Alternatively, this observation may be due to convergence of PFGE types.
This study investigated the possible intrafamilial similarity of mutans streptococcal strains in some families with a child with Down syndrome using chromosomal DNA fingerprinting. The isolates were genotyped using arbitrarily primed polymerase chain reaction with the OPA 02 and OPA 03 primers. The results showed that five children with Down syndrome harbored mutans streptococci genotypes different from those of their mothers. A matching of genotypes was observed within the control pair (mother/child without Down syndrome). After six months, new samples were collected from all participants. Analysis showed that samples from children with Down syndrome were colonized by a new strain of Streptococcus mutans that did not match the previously collected one. The results suggest the S. mutans indigenous bacteria change more than once in children with Down syndrome.
BACKGROUND: An increasing incidence of penicillin-resistant Streptococcus pneumoniae (PRP) was detected in Malmo in 1994. OBJECTIVE: To evaluate clonality and factors facilitating the spread of PRP among children in day-care centers (DCCs). METHODS: We used phenotypic and DNA-fingerprinting methods in conjunction with epidemiologic data from the South Swedish Pneumococcal Intervention Project's investigation of 63 DCCs during a 3-year period (1995 to 1997) in the Malmo region. RESULTS: A questionnaire about building and hygiene standards disclosed no statistically significant risk factor for carriage of pneumococci. However, age younger than the mean age at the DCC or in the child group was positively associated with carriage. Contrary to expectations no association with the number of children, either at the DCC or in the individual groups, was found. Of 2912 investigated children 1224 (42%) were carriers of S. pneumoniae, and 373 (12.8%) were PRP carriers (MIC > or = 0.1 microg/ml). Among isolates with MIC > or = 0.5 microg/ml 9 serogroups and 30 genetic types were found. Two clones in serogroups 9 (33%) and 19 (24%) were dominant in most municipality districts, and dominance was sustained during the whole study period. The previously internationally recognized serotype 9V clone seemed to be very stable, with a single DNA type and resistance pattern during the study period. In contrast the serogroup 19 isolates and other serogroups had diverse DNA types and resistance patterns, supporting the hypothesis that DCCs have a unique microenvironment facilitating the recombination of penicillin-binding protein genes among streptococci. In five DCCs we found PRP isolates with two different serogroups but an identical genetic type, indicating that serotype shift may be a common phenomenon in DCCs. CONCLUSION: Multivariate logistic regression of risk factors disclosed that young age of the children in the child groups was a significant risk factor for carriage of S. pneumoniae.
Thirty urogenital Chlamydia trachomatis isolates collected in Moscow in 2005 were typed using newly developed molecular typing approaches: (1) multilocus sequence typing (MLST(7)) based on sequences of seven housekeeping genes (http://pubmlst.org/chlamydiales/), (2) MLST(5) based on the investigation of five target regions of the chlamydial genome and (3) ompA gene sequencing supplemented with three variable number tandem repeat (VNTR) loci of the genome. ompA typing divided all isolates into 11 groups with E serotype dominating, while MLST7, MLST5 and VNTR analysis divided them into eight, 20 and 18 groups, respectively. The discriminatory power of each method calculated using the Hunter-Gaston discriminatory index was found to be 0.83 for the ompA typing scheme, 0.82 for MLST(7) and 0.95 for MLST(5). A novel sequence type combining 13% of all strains was discovered, as well as new alleles of genes. This is the first study characterizing the genetic diversity of the urogenital C. trachomatis population in Central Russia using MLST. We conclude that the MLST(7) scheme is the best possible choice for global epidemiological purposes, whereas MLST(5) is more appropriate for tracing local outbreaks.
Bureau of Microbial Hazards, Health Products and Food Branch, Food Directorate, Health Canada, Tunney's Pasture, PL 2204A2, Ottawa, Ontario, Canada K1A 0L2.
Pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) analysis, and automated ribotyping were compared for epidemiological typing of Clostridium botulinum type E using clinical and food isolates associated with four botulism outbreaks occurring in the Canadian Arctic. All type E strains previously untypeable by PFGE, even with the use of a formaldehyde fixation step, could be typed by the addition of 50 microM thiourea to the electrophoresis running buffer. Digestion with SmaI or XhoI followed by PFGE was used to link food and clinical isolates from four different type E botulism outbreaks and differentiate them from among 39 group II strains. Strain differentiation was unsuccessful with the automated ribotyping system, producing a single characteristic EcoRI fingerprint common to all group II strains. RAPD analysis of C. botulinum group II strains was not consistently reproducible with primer OPJ-6 or OPJ-13, apparently discriminating between epidemiologically related strains. A modified PFGE protocol was judged to be the most useful method for typing epidemiologically related C. botulinum type E strains, based on its ability to type all strains reproducibly and with an adequate level of discrimination.
Campylobacter spp. are a leading cause of bacterial gastroenteritis worldwide. The need for molecular subtyping methods with enhanced discrimination in the context of surveillance- and outbreak-based epidemiologic investigations of Campylobacter spp. is critical to our understanding of sources and routes of transmission and the development of mitigation strategies to reduce the incidence of campylobacteriosis. We describe the development and validation of a rapid and high-resolution comparative genomic fingerprinting (CGF) method for C. jejuni. A total of 412 isolates from agricultural, environmental, retail, and human clinical sources obtained from the Canadian national integrated enteric pathogen surveillance program (C-EnterNet) were analyzed using a 40-gene assay (CGF40) and multilocus sequence typing (MLST). The significantly higher Simpson's index of diversity (ID) obtained with CGF40 (ID = 0.994) suggests that it has a higher discriminatory power than MLST at both the level of clonal complex (ID = 0.873) and sequence type (ID = 0.935). High Wallace coefficients obtained when CGF40 was used as the primary typing method suggest that CGF and MLST are highly concordant, and we show that isolates with identical MLST profiles are comprised of isolates with distinct but highly similar CGF profiles. The high concordance with MLST coupled with the ability to discriminate between closely related isolates suggests that CFG40 is useful in differentiating highly prevalent sequence types, such as ST21 and ST45. CGF40 is a high-resolution comparative genomics-based method for C. jejuni subtyping with high discriminatory power that is also rapid, low cost, and easily deployable for routine epidemiologic surveillance and outbreak investigations.
Notes
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Using multilocus DNA fingerprinting with phage M13 DNA as a probe, we have investigated a heterogeneous group of four human populations from Eastern Europe and Northeastern Asia. These populations belong to two language families: Indo-European (Eastern Slavonic branch: Russians, Belarussians) and Altaian (Turkic branch: Yakuts). The experimental results were treated by different statistical techniques: cluster analysis, multidimensional scaling, and multiple correspondence analysis. Coefficients of genetic differentiation were estimated using similarity indices and heterozygosities. The results of our study demonstrated similarity of Belarussian populations and significant differences between the group of Slavonic populations and Yakuts.
We used DNA fingerprinting with M13 phage DNA as a probe to estimate the degree of genomic variability and genetic relationships in a heterogeneous group of 13 populations from Eastern Europe and Siberia. The popultaions belong to three language families: Indo-European (Slavonic: Russians, Byelorussians), Uralic (Finno-Ugric: Maris, Mordvinians, Udmurts), and Altaic (Turkic: Bashkirs, Tatars, Chuvashes, Yakuts). Multivariate statistical analyses were used (multidimensional scaling, cluster, and multiple correspondence analyses), and coefficients of gene differentiation ( Gst') were evaluated. The level of interpopulation subdivision in the various ethnic groups appeared to be different: the Byelorussian populations revealed no regional differences, in contrast to the Bashkir populations, which formed a heterogeneous group. The populations subdivided into three general clusters: Slavonic populations formed a separate tight cluster characterized by a minimal level of interpopulation diversity, Bashkir and Yakut populations formed the second cluster, and the Finno-Ugric and several populations of the Turkic linguistic groups formed the third cluster. The robustness of these results obtained by different statistical data treatments reveals that multilocus DNA fingerprinting can be reliably used for population studies.
The aim of the present investigation was to compare DNA fingerprinting and serotyping (heat-stabile and heat-labile antigens) of isolates from epidemic outbreaks as well as of solitary isolates. Campylobacter jejuni isolates from two epidemic outbreaks in Sweden, one milkborne (35 isolates) and one waterborne (17 isolates), and one waterborne outbreak in Norway (11 isolates), as well as 30 solitary isolates from Swedish patients with gastroenteritis, were analyzed. A total of 93 isolates were analyzed. In the waterborne outbreak in Norway, only one serotype with one DNA pattern was found. In the milkborne outbreak in Sweden, two serotypes (HS2:HL4 and HSNT:HL4) with two different DNA patterns were found. The isolates from the waterborne outbreak in Sweden were different serotypes. For two isolates of the same serotype, different DNA patterns were seen. This was also recorded for isolates from solitary cases. It was concluded that serotyping is a useful tool in most epidemiological situations but sometimes lacks sufficient discriminatory power. DNA fingerprinting can add valuable epidemiological information to that supplied by serotyping and can in some situations provide sufficient epidemiological information when used alone.
