The aim of this study was to analyse the genetic diversity among Clostridium perfringens isolates from Danish broiler chickens since both sick and presumably healthy animals were investigated. Isolates (n=279) collected from chickens from 25 farms were analysed by pulsed-field gel electrophoresis (PFGE) with the restriction enzyme SmaI. A high genetic diversity was found. Isolates with different PFGE types were toxin typed by PCR and all were found to be of type A. The results showed that healthy broiler chickens carried several different C. perfringens clones both within a flock and even within individual birds, whereas flocks suffering from necrotic enteritis (NE) or cholangio-hepatitis carried only one or two clones.
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.
Salmonella enterica, Campylobacter and Yersinia species, Shiga toxin-producing Escherichia coli (STEC), Listeria monocytogenes and Clostridium perfringens are the bacterial pathogens constituting the greatest burden of food-borne disease in Finland. Several molecular genetic methods have been applied to diagnose, discriminate and survey these bacteria. PCR, PCR-RFLP and PFGE are the most widely and successfully used. However, these methods are unable to replace conventional and internationally standardised phenotyping. Electronic database libraries of the different genomic profiles will enable continuous surveillance of infections and detection of possible infection clusters at an early stage. Furthermore, whole-genome sequence data have opened up new insights into epidemiological surveillance. Laboratory-based surveillance performed in a timely manner and exploiting adequate methods, and co-operation at local, national and international levels are among the key elements in preventing food-borne diseases. This paper reviews different applications of molecular genetic methods for investigating enteric bacterial pathogens and gives examples of the methods successfully used in diagnostics and epidemiological studies in Finland.
The aim of this study was to investigate the occurrence of Arcobacter species in raw milk in Finland. A total of 177 raw milk samples, each from a separate farm, were examined from June to August 2011. Arcobacter species were isolated using an enrichment and selective detection procedure. Overall, 26 (15 % ) of the 177 samples yielded Arcobacter spp. Samples from 25 farms were positive for Arcobacter butzleri and from 1 farm for Arcobacter cryaerophilus. Moreover, both Arcobacter butzleri and A. cryaerophilus were recovered from 1 positive sample. To evaluate a possible genetic variability, one strain of A. butzleri from each farm and the A. cryaerophilus sample were analyzed by pulsed-field gel electrophoresis. Genotyping revealed that Arcobacter spp. populations are heterogeneous, and no dominant clone has spread in the investigated samples. Our study is the first report on the isolation of both A. butzleri and A. cryaerophilus in raw milk in Finland. Based on our findings, the presence of Arcobacter species in raw milk may pose a potential hazard for human health, in particular for consumers who prefer drinking unpasteurized milk.
Clinical isolates of Bordetella pertussis collected during the year 2004 (n = 153) in eight European countries, Denmark, Finland, France, Germany, The Netherlands, Poland, Sweden, and United Kingdom, were analyzed by pulsed-field gel electrophoresis (PFGE), and their PFGE profiles were compared with those of isolates collected in 1999 (n = 102). The 255 isolates produced 59 distinct PFGE profiles. Among the 153 isolates from 2004, 36 profiles were found, while within the 102 isolates from 1999, 33 profiles were detected. One PFGE profile, BpSR11, was dominant (30% to 50%) in all countries except Denmark (10%) and Poland (0%). In comparison with 1999, there was an increase in BpSR11 prevalence in Finland in 2004 from 5% to 40%, coinciding with a major incidence peak. Some other PFGE profiles seemed to be associated with limited dissemination. Poland was the only country in which the most common actual European PFGE profiles were not found. In a dendrogram analysis, all common PFGE profiles were identified within PFGE group IV, and BpSR11 clustered together with PFGE subgroup IVbeta. Compared to the 1999 isolates, PFGE group V representative for pertactin variant prn3 strains had disappeared, and a new cluster was seen. In conclusion, some PFGE profiles, such as BpSR11, evidently have a higher capacity to spread, suggesting increased fitness to the present immunological environment. It is therefore of major interest to continue with surveillance programs of B. pertussis isolates, as both waning vaccine-derived immunity and strain variation may play a role in the persistence of pertussis.
Unlike most jurisdictions in the United States, Alaska performs pulsed-field gel electrophoresis (PFGE) characterization of all Campylobacter sp. isolates at the state public health laboratory--a practice that started in 2002. Moreover, in order to ensure early detection and response to campylobacteriosis outbreaks, the Alaska Section of Epidemiology has investigated all incident Campylobacter sp. case reports since 2004. This report summarizes the public health impact of routine incident case investigations and molecular characterization of all Campylobacter sp. isolates. In sum, we found that these efforts have contributed to better characterization of the epidemiology of campylobacteriosis in Alaska, and facilitated more rapid outbreak detection, more public health investigations, and earlier public health interventions.
On the basis of specially developed scheme for the isolation of Listeria strains comprising 2 enrichment stages and the use of growth inhibitors, 128 L. monocytogenes cultures were isolated from clinical material, foodstuffs and sewage water. Highly virulent L.monocytogenes strains isolated from clinical material belonged to serovar 4b (54%) and 1/2a (38%), while those isolated from foodstuffs and sewage water belonged to 4b (74%). The restriction analysis of the chromosomal DNA of the isolated cultures with the use of restrictase EcoR1 on the basis of pulsed-field gel electrophoresis (PFGE) made it possible to distinguish Listeria strains in accordance with 5 types of restrictograms. The restrictograms of highly virulent L. monocytogenes strains, serovar 4b, belonged to types 1 and 2, while those of L. monocytogenes strains, serovar 1/2a, belonged to types 2 and 3. The comparative use of different methods for typing L. monocytogenes (sero-, phago-, bio- and resistotyping, the analysis of plasmid composition and restriction analysis) revealed that the combination of serotyping and restriction analysis on the basis of PFGE proved to be most promising for the characterization of the isolated L. monocytogenes strains and the assessment of their epidemic importance.
Reliable molecular methods for determination of relatedness between bacterial isolates have become increasingly important to evaluate outbreaks and endemic situations with nosocomial pathogens. In the present study Simpson's index of diversity with calculated confidence intervals was used to compare amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoresis (PFGE) analysis of a hospital outbreak of ampicillin-resistant Enterococcus faecium and subsequent endemicity. The outbreak, in a Norwegian tertiary hospital, of infections caused by these enterococci started in 1995 and increased in 1996 after which the situation turned endemic. The purpose of this study was to compare the two methods in this setting and to determine the length of time during an outbreak that these methods are sufficiently valid to be of value for hospital infection control efforts. One hundred and sixty clinical isolates from urine specimens collected during the period 1995-1999 were included. The findings indicate that PFGE and AFLP are equally discriminative and could in this setting be used for typing purposes over the whole 5-year period.
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.
During 2000 and 2001, an outbreak of human salmonellosis occurred in Sweden and Norway, caused by Salmonella livingstone. In this study, the genotypic differences between three strains obtained from food sources during the outbreak, two human strains and 27 more or less unrelated strains were analysed, using the three methods; automated ribotyping, pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD). Each method was evaluated regarding its discriminatory ability, reproducibility and typeability. Simpson's discriminatory index calculated for each method was 0.556 for automated ribotyping, 0.766 for PFGE and 0.236 for RAPD. The reproducibility, defined as the minimum similarity between individual replicates in a cluster analysis, was 96% for automated ribotyping and PFGE, and 90% for RAPD. All the strains were typeable with each method. When combining results for the three genotyping methods, it was found that RAPD did not increase the discriminatory index and was therefore excluded from further analysis. Using a combination of the results obtained from ribotyping and PFGE (D=0.855), two strains that had been isolated from feed factories during 1998 were shown to be identical to the outbreak strain, indicating a possible route of contamination due to a clone of Salmonella livingstone persisting in feed producing facilities. No connection to poultry was established.