Parallel independent evolution of pathogenicity within the genus Yersinia.

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.

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

BACKGROUND: Y. 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. RESULTS: A 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%). CONCLUSIONS: The 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.

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.

BACKGROUND: We 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. RESULTS: MLVA 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. CONCLUSIONS: MLVA 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.

Alteration of flagellar phenotype of Escherichia coli strain P12b, the standard type strain for flagellar antigen H17, possessing a new non-fliC flagellin gene flnA, and possible loss of original flagellar phenotype and genotype in the course of subculturing through semisolid media.

A practically important phenomenon, resulting in the loss of the original flagellar phenotype (genotype) of bacteria, is described in the Escherichia coli H17 type strain P12b possessing two distinct genes for H17 and H4 flagellins, respectively. By PCR, sequencing, and phylogenetic investigation, the H17 gene (originally expressed) was considered a new non-fliC flagellin gene and assigned flnA, while the H4 gene (originally cryptic) was reaffirmed as fliC. H17 and H4 flagella differed morphologically. The phenomenon consisted in the replacement of H17 cells by H4 cells during subculturing through certain semisolid media and resulted from the excision of flnA (H17) entirely or in part. The substitution rate depended on the density and nutrient composition of media and reached 100% even after a single passage through 0.3% LB agar. Such phenomenon can lead to an unexpected loss of original H17 phenotype. Our review of the literature showed that the loss of the original flagellar genotype (phenotype) of P12b has occurred in some laboratories while the authors continued to consider their cultures H17. We showed how to distinguish these alternative flagellin genotypes using popular fliC primers. Attention was also paid to possible discrepancies between serological and molecular results in flagellar typing of E. coli.

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

BACKGROUND: Yersinia 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. METHODS: In 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. RESULTS: Strains 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. CONCLUSIONS: The 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.

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.

Strains of the cyanobacterial genera Calothrix and Rivularia isolated from the Baltic Sea display cryptic diversity and are distantly related to Gloeotrichia and Tolypothrix.

Strains of the cyanobacterial genus Calothrix display pronounced tapering filaments. These cyanobacteria are benthic, have a worldwide distribution and are among the most easily recognizable cyanobacterial genera. However, it is not clear whether cyanobacterial strains assigned to the genus Calothrix constitute a natural monophyletic group. We sequenced 16S rRNA genes from 42 cyanobacterial cultures and environmental samples belonging to the genus Calothrix, and the morphologically similar genera Rivularia, Gloeotrichia and Tolypothrix. Phylogenetic analysis of the 16S rRNA gene identified large sequence diversity among the Calothrix morphotype strains. Our results demonstrate that Calothrix, Gloeotrichia and Tolypothrix do not form a monophyletic group but instead display a high level of genetic diversity. The evolutionary distances between cyanobacteria, morphologically identified as Calothrix, suggest that they belong to at least five different genera. Our results also suggest that the genus Gloeotrichia is distantly related to the genus Calothrix. We found correlations between genetic grouping and morphology in redundancy analysis. However, morphology alone was not sufficiently reliable to distinguish strains from different 16S rRNA gene clusters. The high level of diversity that we observed confirms the hypothesis that the Rivulariaceae are species rich.

Bacterial diversity and function in the Baltic Sea with an emphasis on cyanobacteria.

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.

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.

Benthic cyanobacteria from the Baltic Sea contain cytotoxic Anabaena, Nodularia, and Nostoc strains and an apoptosis-inducing Phormidium strain.

Benthic cyanobacteria from aquatic environments have been reported to produce biologically active metabolites. However, the toxicity and other biological activities of benthic cyanobacteria from the Baltic Sea are not well known. We determined the biological activities of 21 Anabaena, Calothrix, Nodularia, Nostoc, and Phormidium strains isolated from benthic littoral habitats of the Baltic Sea. We studied whether benthic cyanobacterial extracts caused cytotoxicity by necrosis or induced apoptosis in two mammalian cell lines, a human leukemia cell line (HL-60) and a mouse fibroblast cell line (3T3 Swiss), and examined potential hepatotoxin (microcystin and nodularin) production. Five of the six benthic Anabaena strains, one of the two Nostoc strains, and two of the three Nodularia strains were highly cytotoxic to human leukemia cells. The Calothrix and Phormidium strains did not cause LDH leakage, but the extract of Phormidium strain BECID15 induced apoptosis in the HL-60 cells. Neither the microcystin synthetase E (mcyE) nor the nodularin synthetase F (ndaF) gene was amplified by PCR, and no microcystins or nodularins were detected by the protein phosphatase inhibition assay from the cyanobacterial strains included in this study. This indicates that benthic Baltic cyanobacteria contain potentially harmful cytotoxic compounds even though they do not produce microcystins or nodularins. These cytotoxic compounds remain to be characterized, and the mechanisms of cytotoxicity need to be studied further.