Leila M. Sihvonen

Parallel independent evolution of pathogenicity within the genus Yersinia.

Significance Our past understanding of pathogen evolution has been fragmented because of tendencies to study human clinical isolates. To understand the evolutionary trends of pathogenic bacteria though, we need the context of their nonpathogenic relatives. Our unique and detailed dataset allows description of the parallel evolution of two key human pathogens: the causative agents of plague and Yersinia diarrhea. The analysis reveals an emerging pattern where few virulence-related functions are found in all pathogenic lineages, representing key “foothold” moments that mark the emergence of these pathogens. Functional gene loss and metabolic streamlining are equally complementing the evolution of Yersinia across the pathogenic spectrum. The genus Yersinia has been used as a model system to study pathogen evolution. Using whole-genome sequencing of all Yersinia species, we delineate the gene complement of the whole genus and define patterns of virulence evolution. Multiple distinct ecological specializations appear to have split pathogenic strains from environmental, nonpathogenic lineages. This split demonstrates that contrary to hypotheses that all pathogenic Yersinia species share a recent common pathogenic ancestor, they have evolved independently but followed parallel evolutionary paths in acquiring the same virulence determinants as well as becoming progressively more limited metabolically. Shared virulence determinants are limited to the virulence plasmid pYV and the attachment invasion locus ail. These acquisitions, together with genomic variations in metabolic pathways, have resulted in the parallel emergence of related pathogens displaying an increasingly specialized lifestyle with a spectrum of virulence potential, an emerging theme in the evolution of other important human pathogens.

Symptoms and sources of Yersinia enterocolitica-infection: a case-control study

BackgroundYersinia enterocolitica (YE) is the causative agent of yersiniosis. YE encompass strains of diverse pathogenicity: YE biotypes 1B and 2-5 are considered pathogenic, whereas biotype 1A is in general considered nonvirulent. Also YE-like species, which can sometimes be misidentified as YE, are considered nonvirulent.MethodsIn order to study differences in clinical picture caused by different YE types and their possible sources a case-control study was conducted in 2006. In this case-control study, 295 case-patients with YE or YE-like finding and their 758 controls responded to the questionnaire about symptoms and possible sources of infection.ResultsStrains of pathogenic YE bio/serotypes 3-4/O:3 or 2/O:9 were found in 18%, YE biotype 1A in 65% and YE -like strains of 17% of the patients. Patients infected with the strains of pathogenic YE bio/serotypes were younger and had fever more often than those with BT 1A who suffered more from vomiting. Symptoms of reactive arthritis were reported by 10% of pathogenic YE infections, 3% of YE BT 1A, and 0.3% of the controls. Eating or tasting raw or medium done pork was a significant risk factor for pathogenic YE bio/serotype infection (OR 6.6; 95% CI 1.7-24.9) as well as eating in a canteen (OR 3.5; 95% CI 1.6-7.9). Imported fruits and berries were associated with increased risk of YE BT 1A finding.ConclusionsThe symptoms of the patients with YE BT 1A differed from yersiniosis caused by the classic pathogenic YE bio/serotypes. In addition, the patients with YE BT 1A had more protracted gastrointestinal disorders and unspecific complaints. Small children were overrepresented in classic pathogenic bio/serotypes while in BT 1A or YE-like species were not found among children younger than two years. This suggests the lacking virulence of the BT 1A strains. We can not, however, rule out the possibility that some strains of genetically heterogeneous group of BT 1A may cause an illness.

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.

Multilocus Variable-Number Tandem-Repeat Analysis, Pulsed-Field Gel Electrophoresis, and Antimicrobial Susceptibility Patterns in Discrimination of Sporadic and Outbreak-Related Strains of Yersinia enterocolitica

BackgroundWe assessed the potential of multilocus variable-number tandem-repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing for discriminating 104 sporadic and outbreak-related Yersinia enterocolitica (YE) bio/serotype 3-4/O:3 and 2/O:9 isolates. MLVA using six VNTR markers was performed in two separate multiplex PCRs, and the fluorescently labeled PCR products were accurately sized on an automated DNA sequencer.ResultsMLVA discriminated 82 sporadic YE 3-4/O:3 and 2/O:9 strains into 77 types, whereas PFGE with the restriction enzyme NotI discriminated the strains into 23 different PFGE pulsotypes. The discriminatory index for a sporadic strain was 0.862 for PFGE and 0.999 for MLVA. MLVA confirmed that a foodborne outbreak in the city of Kotka, Finland in 2003 had been caused by a multiresistant YE 4/O:3 strain that was distinctly different from those of epidemiologically unrelated strains with an identical PFGE pulsotype. The multiresistance of Y. enterocolitica strains (19% of the sporadic strains) correlated significantly (p = 0.002) with travel abroad. All of the multiresistant Y. enterocolitica strains belonged to four PFGE pulsotypes that did not contain any susceptible strains. Resistance to nalidixic acid was related to changes in codons 83 or 87 that stemmed from mutations in the gyrA gene. The conjugation experiments demonstrated that resistance to CHL, STR, and SUL was carried by a conjugative plasmid.ConclusionsMLVA using six loci had better discriminatory power than PFGE with the NotI enzyme. MLVA was also a less labor-intensive method than PFGE and the results were easier to analyze. The conjugation experiments demonstrated that a resistance plasmid can easily be transferred between Y. enterocolitica strains. Antimicrobial multiresistance of Y. enterocolitica strains was significantly associated with travel abroad.

The ail Gene Is Present in Some Yersinia enterocolitica Biotype 1A Strains

One chromosomal virulence marker of Yersinia is the gene ail, which encodes Ail, an outer membrane protein that promotes attachment and invasion. A high correlation has been found between the ail gene and the virulence of Yersinia. Here, we report two Yersinia enterocolitica biotype 1A strains that are usually nonpathogenic and carry the ail gene. The ail gene sequences of biotype 1A strains displayed similarity to the bioserotype 1B/O:8 strain 8081. The finding suggests that ail-based detection methods for Y. enterocolitica alone are insufficient to detect real pathogenic strains.

Genetic diversity in strains of the genus Anabaena isolated from planktonic and benthic habitats of the Gulf of Finland (Baltic Sea)

Late summer cyanobacterial blooms in the Baltic Sea contain Anabaena sp. together with Nodularia spumigena and Aphanizomenon flos-aquae. Although Anabaena is common especially in the Gulf of Finland, very little is known about its genetic diversity. Here we undertook a molecular phylogenetic study of 68 Anabaena strains isolated from the brackish Gulf of Finland. We sequenced the 16S rRNA genes from 54 planktonic and 14 benthic Anabaena strains, and rbcL and rpoC1 genes from a subset of these strains. Phylogenetic trees showed that Anabaena strains, from both planktonic and benthic habitats, were genetically diverse. Although the Anabaena strains were morphologically diverse, in our study only one genetically valid species was found to exist in the plankton. Evolutionary distances between benthic Anabaena strains were greater than between planktonic strains, suggesting that benthic habitats allow for the maintenance of greater genetic diversity than planktonic habitats. A number of novel lineages containing only sequences obtained in this study were compiled in the phylogenetical analyses. Thus, it seemed that novel lineages of the genus Anabaena may be present in the Baltic Sea. Our results demonstrate that the Baltic Sea Anabaena strains show surprisingly high genetic diversity.

Bacterial Diversity and Function in the Baltic Sea with an Emphasis on Cyanobacteria

Abstract In this article we summarize the current knowledge of Baltic Sea cyanobacteria, focusing on diversity, toxicity, and nitrogen fixation in the filamentous heterocystous taxa. We also review the recent results of our microbial diversity studies in planktonic and benthic habitats in the Baltic Sea. Based on molecular analyses, we have improved the understanding of cyanobacterial population structure by assessing genetic diversity within species that are morphologically inseparable. Moreover, we have studied microbial functions such as toxin production and nitrogen fixation in situ under different environmental conditions. Phosphorus limitation of bloom-forming, nitrogen-fixing cyanobacteria was clearly verified, emphasizing the importance of continuous efforts to reduce this element in the Baltic Sea. We have designed a rapid and reliable detection method for the toxic cyanobacterium Nodularia spumigena, which can be used to study bloom formation of this important toxin producer.

Bacteria Contribute to Sediment Nutrient Release and Reflect Progressed Eutrophication-Driven Hypoxia in an Organic-Rich Continental Sea

In the sedimental organic matter of eutrophic continental seas, such as the largest dead zone in the world, the Baltic Sea, bacteria may directly participate in nutrient release by mineralizing organic matter or indirectly by altering the sediment’s ability to retain nutrients. Here, we present a case study of a hypoxic sea, which receives riverine nutrient loading and in which microbe-mediated vicious cycles of nutrients prevail. We showed that bacterial communities changed along the horizontal loading and vertical mineralization gradients in the Gulf of Finland of the Baltic Sea, using multivariate statistics of terminal restriction fragments and sediment chemical, spatial and other properties of the sampling sites. The change was mainly explained by concentrations of organic carbon, nitrogen and phosphorus, which showed strong positive correlation with Flavobacteria, Sphingobacteria, Alphaproteobacteria and Gammaproteobacteria. These bacteria predominated in the most organic-rich coastal surface sediments overlain by oxic bottom water, whereas sulphate-reducing bacteria, particularly the genus Desulfobacula, prevailed in the reduced organic-rich surface sediments in the open sea. They correlated positively with organic nitrogen and phosphorus, as well as manganese oxides. These relationships suggest that the bacterial groups participated in the aerobic and anaerobic degradation of organic matter and contributed to nutrient cycling. The high abundance of sulphate reducers in the surficial sediment layers reflects the persistence of eutrophication-induced hypoxia causing ecosystem-level changes in the Baltic Sea. The sulphate reducers began to decrease below depths of 20 cm, where members of the family Anaerolineaceae (phylum Chloroflexi) increased, possibly taking part in terminal mineralization processes. Our study provides valuable information on how organic loading affects sediment bacterial community compositions, which consequently may maintain active nutrient recycling. This information is needed to improve our understanding on nutrient cycling in shallow seas where the dead zones are continuously spreading worldwide.

Clinical isolates of Yersinia enterocolitica Biotype 1A represent two phylogenetic lineages with differing pathogenicity-related properties

BackgroundY. enterocolitica biotype (BT) 1A strains are often isolated from human clinical samples but their contribution to disease has remained a controversial topic. Variation and the population structure among the clinical Y. enterocolitica BT 1A isolates have been poorly characterized. We used multi-locus sequence typing (MLST), 16S rRNA gene sequencing, PCR for ystA and ystB, lipopolysaccharide analysis, phage typing, human serum complement killing assay and analysis of the symptoms of the patients to characterize 298 clinical Y. enterocolitica BT 1A isolates in order to evaluate their relatedness and pathogenic potential.ResultsA subset of 71 BT 1A strains, selected based on their varying LPS patterns, were subjected to detailed genetic analyses. The MLST on seven house-keeping genes (adk, argA, aroA, glnA, gyrB, thrA, trpE) conducted on 43 of the strains discriminated them into 39 MLST-types. By Bayesian analysis of the population structure (BAPS) the strains clustered conclusively into two distinct lineages, i.e. Genetic groups 1 and 2. The strains of Genetic group 1 were more closely related (97% similarity) to the pathogenic bio/serotype 4/O:3 strains than Genetic group 2 strains (95% similarity). Further comparison of the 16S rRNA genes of the BT 1A strains indicated that altogether 17 of the 71 strains belong to Genetic group 2. On the 16S rRNA analysis, these 17 strains were only 98% similar to the previously identified subspecies of Y. enterocolitica. The strains of Genetic group 2 were uniform in their pathogenecity-related properties: they lacked the ystB gene, belonged to the same LPS subtype or were of rough type, were all resistant to the five tested yersiniophages, were largely resistant to serum complement and did not ferment fucose. The 54 strains in Genetic group 1 showed much more variation in these properties. The most commonly detected LPS types were similar to the LPS types of reference strains with serotypes O:6,30 and O:6,31 (37%), O:7,8 (19%) and O:5 (15%).ConclusionsThe results of the present study strengthen the assertion that strains classified as Y. enterocolitica BT 1A represent more than one subspecies. Especially the BT 1A strains in our Genetic group 2 commonly showed resistance to human serum complement killing, which may indicate pathogenic potential for these strains. However, their virulence mechanisms remain unknown.

Phosphorus Chemistry and Bacterial Community Composition Interact in Brackish Sediments Receiving Agricultural Discharges

Background External nutrient discharges have caused eutrophication in many estuaries and coastal seas such as the Baltic Sea. The sedimented nutrients can affect bacterial communities which, in turn, are widely believed to contribute to release of nutrients such as phosphorus from the sediment. Methods We investigated relationships between bacterial communities and chemical forms of phosphorus as well as elements involved in its cycling in brackish sediments using up-to-date multivariate statistical methods. Bacterial community composition was determined by terminal restriction fragment length polymorphism and cloning of the 16S rRNA gene. Results and Conclusions The bacterial community composition differed along gradients of nutrients, especially of different phosphorus forms, from the estuary receiving agricultural phosphorus loading to the open sea. This suggests that the chemical composition of sediment phosphorus, which has been affected by riverine phosphorus loading, influenced on bacterial communities. Chemical and spatial parameters explained 25% and 11% of the variation in bacterial communities. Deltaproteobacteria, presumptively sulphate and sulphur/iron reducing, were strongly associated to chemical parameters, also when spatial autocorrelation was taken into account. Sulphate reducers correlated positively with labile organic phosphorus and total nitrogen in the open sea sediments. Sulphur/iron reducers and sulphate reducers linked to iron reduction correlated positively with aluminium- and iron-bound phosphorus, and total iron in the estuary. The sulphate and sulphur/iron reducing bacteria can thus have an important role both in the mineralization and mobilization of nutrients from sediment. Significance Novelty in our study is that relationships between bacterial community composition and different phosphorus forms, instead of total phosphorus, were investigated. Total phosphorus does not necessarily bring out interactions between bacteria and phosphorus chemistry since proportions of easily usable mobile (reactive) phosphorus and immobile phosphorus forms in different sediments can vary. Our study suggested possible feedbacks between different forms of phosphorus and bacterial community composition.