Hybrids of Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC) Among Human and Animal Isolates in Finland.

Diarrhoeagenic Escherichia coli (DEC) cause serious foodborne infections in humans. Total of 450 Shigatoxigenic E. coli (STEC) strains isolated from humans, animals and environment in Finland were examined by multiplex PCR targeting the virulence genes of various DEC pathogroups simultaneously. One per cent (3/291) of the human STEC and 14% (22/159) of the animal and environmental STEC had genes typically present in enterotoxigenic E. coli (ETEC). The strains possessed genes encoding both Shiga toxin 1 and/or 2 (stx1 and/or stx2 ) and ETEC-specific heat-stable (ST) enterotoxin Ia (estIa). The identified stx subtypes were stx1a, stx1c, stx2a, stx2d and stx2g. The three human STEC/ETEC strains were isolated from the patients with haemolytic uraemic syndrome and diarrhoea and from an asymptomatic carrier. The animal STEC/ETEC strains were isolated from cattle and moose. The human and animal STEC/ETEC strains belonged to 11 serotypes, of which O2:H27, O15:H16, O101:H-, O128:H8 and O141:H8 have previously been described to be associated with human disease. Identification of multiple virulence genes offers further information for assessing the virulence potential of STEC and other DEC. The emergence of novel hybrid pathogens should be taken into account in the patient care and epidemiological surveillance.

Diarrhoeagenic Escherichia coli detected by 16-plex PCR in children with and without diarrhoea in Burkina Faso.

The importance of diarrhoeagenic Escherichia coli (DEC) in Africa is poorly understood, and is unknown in Burkina Faso. This study investigated the occurrence of five major DEC pathogroups in primary cultures of stool samples from 658 Burkinabe children under 5 years old using 16-plex PCR for virulence-associated genes. At least one DEC pathogroup was detected in 45% of 471 children with diarrhoea and in 29% of 187 children without diarrhoea (p <0.001). More than one DEC pathogroup was detected in 11% of children with and 1% of children without diarrhoea (p <0.001). Enteroaggregative E. coli was the most common pathogroup in both children with diarrhoea (26%) and children without diarrhoea (21%). Enteropathogenic E. coli and enterotoxigenic E. coli were detected significantly more often in children with diarrhoea (16% and 13%) than in children without diarrhoea (5% and 4%; p <0.001 for both pathogroups). Shiga toxin-producing E. coli and enteroinvasive E. coli were detected only in children with diarrhoea (2% and 1%, respectively). Diarrhoeagenic E. coli, especially enteropathogenic and enterotoxigenic, may be important, unrecognized causes of childhood diarrhoea in Burkina Faso.

Phenotype MicroArray in the metabolic characterisation of Salmonella serotypes Agona, Enteritidis, Give, Hvittingfoss, Infantis, Newport and Typhimurium.

The Phenotype MicroArray (PM) technology was used to study the metabolic characteristics of 29 Salmonella strains belonging to seven serotypes of S. enterica spp. enterica. Strains of serotypes Typhimurium (six strains among definite phage types DTs 1, 40 and 104) and Agona (two strains) were tested for 949 substrates, Enteritidis (six strains of phage type PT1), Give, Hvittingfoss, Infantis and Newport strains (two of each) were tested for 190 substrates and seven other Agona strains for 95 substrates. The strains represented 18 genotypes in pulsed-field gel electrophoresis (PFGE). Among 949 substrates, 18 were identified that could be used to differentiate between the strains of those seven serotypes or within a single serotype. Unique metabolic differences between the Finnish endemic Typhimurium DT1 and Agona strains were detected, for example, in the metabolism of D-tagatose, D-galactonic acid gamma-lactone and L-proline as a carbon source. Thus, the PM technique is a useful tool for identifying potential differential markers on a metabolic basis that could be used for epidemiological surveillance.

Yersinia enterocolitica and Y. enterocolitica-like species in clinical stool specimens of humans: identification and prevalence of bio/serotypes in Finland.

This study investigated the prevalence of Yersinia enterocolitica (YE) bio/serotypes and YE-like species in clinical stool specimens. The special aim was to find the best methods for accurate identification of YE species and, further, pathogenic strains among YE isolates. Of the 41,848 specimens cultured in ten laboratories during a 12-month period, 473 Yersinia strains were isolated from 462 patients. The strains were identified by 21 biochemical tests, serotyping, colony morphology, as well as by 16S rRNA and gyrB gene sequencing. The most prevalent Yersinia findings were YE biotype 1A (64% of the strains) and pathogenic bio/serotype 4/O:3 (16%). The cold-enrichment increased the number of all isolates, and 25% of the bio/serotype 4/O:3 and 2/O:9 strains were only found by cold-enrichment. In routine diagnostic laboratories, 50% of the YE-like species were identified as YE and in 26% the identification differed from that of the reference laboratory. The microscopic colony identification on CIN agar with positive CR-MOX test, combined with several biochemical tests, identified reliably the pathogenic YE bioserotypes and most YE BT 1A strains, but some strains of the YE-like species were so heterogenic that gene sequencing was the only way to identify them.

New 16-plex PCR method for rapid detection of diarrheagenic Escherichia coli directly from stool samples.

A rapid 16-plex polymerase chain reaction (PCR) suitable for routine diagnostics of diarrheagenic Escherichia coli (EHEC, EIEC, EAEC, ETEC, and EPEC) was developed, validated with control strains, and tested with 250 diarrhoeal stool samples. The specificity was 100% when tested with 289 control bacterial strains, and the analytical sensitivity of automated DNA extraction directly from stool samples was made by boiling the bacterial culture (10(4)-10(5) colony forming units/ml). The assay design starting directly from extraction of stool DNA allowed same day analysis without compromising sensitivity and specificity, which makes it superior compared to PCR after culturing the bacteria. The 16-plex PCR method demonstrated high prevalence of diarrheagenic E. coli in stool samples of patients returning from abroad (39.0%) in contrast to the patients with no travel history (8.7%; p < 0.001). The high prevalence of diarrheagenic E. coli suggests that their screening should be part of normal diarrhoea diagnostics, at least in the leading diagnostic laboratories.

Yersinia pseudotuberculosis causing a large outbreak associated with carrots in Finland, 2006.

A large outbreak of Yersinia pseudotuberculosis O:1 infection affected over 400 children from 23 schools and 5 day-care centres in two municipalities in southern Finland in August-September, 2006. A retrospective cohort study conducted in a large school centre showed that the outbreak was strongly associated with the consumption of grated carrots served at a school lunch. The risk of illness increased with the amount of carrots eaten. Poor quality carrots grown the previous year had been delivered to the school kitchens in the two municipalities affected. In the patients' samples and in the environmental samples collected from the carrot distributor's storage facility, identical serotypes and genotypes of Y. pseudotuberculosis were found, but the original source and the mechanism of the contamination of the carrots remained unclear. Outbreaks of Y. pseudotuberculosis linked to fresh produce have been detected repeatedly in Finland. To prevent future outbreaks, instructions in improved hygiene practices on the handling of raw carrots have been issued to farmers, vegetable-processing plants and institutional kitchens.

Emerging resistance to newer antimicrobial agents among Shigella isolated from Finnish foreign travellers.

In Finland, most cases of shigellosis are related to travel abroad. Antimicrobial drug resistance of 1814 Shigella strains isolated from Finnish patients during 1990-2005 was studied using discs of 12 antimicrobial agents. Since 2000, the E-test has been performed to determine ciprofloxacin minimum inhibitory concentrations of nalidixic acid-resistant isolates. The proportion of multi-resistant strains (resistant to >or =4 antimicrobials) was highest among isolates from China and India, but is increasing significantly in other parts of Asia. Resistance to nalidixic acid has become common among the strains from the Far East, and the first isolates also resistant to ciprofloxacin were detected during 2004-2005. All the ciprofloxacin-resistant isolates belonged to the S. flexneri 2a serotype. All the nalidixic acid-resistant S. flexneri strains had reduced susceptibility to ciprofloxacin, whereas 23% of the nalidixic acid-resistant S. sonnei strains were still completely susceptible to ciprofloxacin.

Diversity of toxic and nontoxic nodularia isolates (cyanobacteria) and filaments from the Baltic Sea.

Cyanobacteria of the genus Nodularia form toxic blooms in brackish waters worldwide. In addition, Nodularia spp. are found in benthic, periphytic, and soil habitats. The majority of the planktic isolates produce a pentapeptide hepatotoxin nodularin. We examined the morphologic, toxicologic, and molecular characters of 18 nodularin-producing and nontoxic Nodularia strains to find appropriate markers for distinguishing the toxic strains from the nontoxic ones in field samples. After classical taxonomy, the examined strains were identified as Nodularia sp., Nodularia spumigena, N. baltica, N. harveyana, and N. sphaerocarpa. Morphologic characters were ambiguous in terms of distinguishing between the toxic and the nontoxic strains. DNA sequences from the short 16S-23S rRNA internally transcribed spacer (ITS1-S) and from the phycocyanin operon intergenic spacer and its flanking regions (PC-IGS) were different for the toxic and the nontoxic strains. Phylogenetic analysis of the ITS1-S and PC-IGS sequences from strains identified as N. spumigena, and N. baltica, and N. litorea indicated that the division of the planktic Nodularia into the three species is not supported by the ITS1-S and PC-IGS sequences. However, the ITS1-S and PC-IGS sequences supported the separation of strains designated N. harveyana and N. sphaerocarpa from one another and the planktic strains. HaeIII digestion of PCR amplified PC-IGS regions of all examined 186 Nodularia filaments collected from the Baltic Sea produced a digestion pattern similar to that found in toxic isolates. Our results suggest that only one planktic Nodularia species is present in the Baltic Sea plankton and that it is nodularin producing.

Characterisation of rhizobia from African acacias and other tropical woody legumes using Biolog and partial 16S rRNA sequencing.

A Biolog (sole carbon source utilisation) user database of tropical and temperature rhizobial strains was created and used in conjunction with the partial 16S rRNA sequencing method to characterise 12 rhizobial isolates from African acacias and other tropical woody legumes. There was close agreement between the two methods but also some significant discrepancies. A high degree of diversity was shown in the relatively small sample of isolates, with 4 out of 5 of the currently proposed rhizobial genera represented. This is the first time Biolog has shown congruence with genotypic fingerprinting using a wide selection of rhizobial reference and test strains.

Biodiversity of rhizobia isolated from a wide range of forest legumes in Brazil.

Tropical forests have a high diversity of plant species; are they associated with a correspondingly rich microbial flora? We addressed this question by examining the symbiotic rhizobium bacteria that nodulate a diverse pool of forest legume species in Brazil. The 44 strains studied had been isolated from 29 legume tree species representing 13 tribes including all three subfamilies of the Leguminosae, and were chosen to represent major groups from a larger sample that had previously been characterized by SDS-PAGE of total proteins. Partial 16S rRNA gene sequence was determined, corresponding to positions 44-303 in the Escherichia coli sequence. Fifteen sequences were found, including six novel ones. However, all but one of them could be assigned to a genus because they grouped closely with sequences from previously described rhizobial species. Fast-growing strains had sequences similar to Rhizobium spp., Sinorhizobium spp. or Mesorhizobium spp., while the slow-growing strains had sequences similar to Bradyrhizobium spp. One strain with an intermediate growth rate had a unique sequence which indicated that the strain might belong to the genus Azorhizobium. Although the strains showed a variety of sequences, it was surprising that these strains isolated from taxonomically very diverse host plants in previously unexplored environments were mostly very similar to strains described previously, largely from agricultural systems.

Three phylogenetic groups of nodA and nifH genes in Sinorhizobium and Mesorhizobium isolates from leguminous trees growing in Africa and Latin America.

The diversity and phylogeny of nodA and nifH genes were studied by using 52 rhizobial isolates from Acacia senegal, Prosopis chilensis, and related leguminous trees growing in Africa and Latin America. All of the strains had similar host ranges and belonged to the genera Sinorhizobium and Mesorhizobium, as previously determined by 16S rRNA gene sequence analysis. The restriction patterns and a sequence analysis of the nodA and nifH genes divided the strains into the following three distinct groups: sinorhizobia from Africa, sinorhizobia from Latin America, and mesorhizobia from both regions. In a phylogenetic tree also containing previously published sequences, the nodA genes of our rhizobia formed a branch of their own, but within the branch no correlation between symbiotic genes and host trees was apparent. Within the large group of African sinorhizobia, similar symbiotic gene types were found in different chromosomal backgrounds, suggesting that transfer of symbiotic genes has occurred across species boundaries. Most strains had plasmids, and the presence of plasmid-borne nifH was demonstrated by hybridization for some examples. The nodA and nifH genes of Sinorhizobium teranga ORS1009T grouped with the nodA and nifH genes of the other African sinorhizobia, but Sinorhizobium saheli ORS609T had a totally different nodA sequence, although it was closely related based on the 16S rRNA gene and nifH data. This might be because this S. saheli strain was originally isolated from Sesbania sp., which belongs to a different cross-nodulation group than Acacia and Prosopis spp. The factors that appear to have influenced the evolution of rhizobial symbiotic genes vary in importance at different taxonomic levels.

Syphilis, homosexuality and legislation.

The proportion of cases with fresh syphilis in males contracted by homosexual contacts in Helsinki before and after the change of the criminal law in 1971 was studied. Since 1971, homosexuality is by law no longer a crime in Finland. In 1964, only 2% of males with fresh syphilis admitted a homosexual contact. The same figure was 8% in 1970, and increased to about 50% in 1974 and 1975. It was concluded that a change of legislation concerning homosexuality, probably by several different routes, changed the proportion of cases of detected homosexually transmitted early syphilis. This was thought to be of special importance for case finding and controlling of the spread of syphilis.