Ken-ichi Lee

Molecular phylogeny of the higher and lower taxonomy of the Fusarium genus and differences in the evolutionary histories of multiple genes

BackgroundSpecies of the Fusarium genus are important fungi which is associated with health hazards in human and animals. The taxonomy of this genus has been a subject of controversy for many years. Although many researchers have applied molecular phylogenetic analysis to examine the taxonomy of Fusarium species, their phylogenetic relationships remain unclear only few comprehensive phylogenetic analyses of the Fusarium genus and a lack of suitable nucleotides and amino acid substitution rates. A previous stugy with whole genome comparison among Fusairum species revealed the possibility that each gene in Fusarium genomes has a unique evolutionary history, and such gene may bring difficulty to the reconstruction of phylogenetic tree of Fusarium. There is a need not only to check substitution rates of genes but also to perform the exact evaluation of each gene-evolution.ResultsWe performed phylogenetic analyses based on the nucleotide sequences of the rDNA cluster region (rDNA cluster), and the β-tubulin gene (β-tub), the elongation factor 1α gene (EF-1α), and the aminoadipate reductase gene (lys2). Although incongruence of the tree topologies between lys2 and the other genes was detected, all genes supported the classification of Fusarium species into 7 major clades, I to VII. To obtain a reliable phylogeny for Fusarium species, we excluded the lys2 sequences from our dataset, and re-constructed a maximum likelihood (ML) tree based on the combined data of the rDNA cluster, β-tub, and EF-1α. Our ML tree indicated some interesting relationships in the higher and lower taxa of Fusarium species and related genera. Moreover, we observed a novel evolutionary history of lys2. We suggest that the unique tree topologies of lys2 are not due to an analytical artefact, but due to differences in the evolutionary history of genomes caused by positive selection of particular lineages.ConclusionThis study showed the reliable species tree of the higher and lower taxonomy in the lineage of the Fusarium genus. Our ML tree clearly indicated 7 major clades within the Fusarium genus. Furthermore, this study reported differences in the evolutionary histories among multiple genes within this genus for the first time.

Multivariate Analyses Revealed Distinctive Features Differentiating Human and Cattle Isolates of Shiga Toxin-Producing Escherichia coli O157 in Japan

ABSTRACT Genotypes of Shiga toxin-producing Escherichia coli (STEC) O157 isolated from humans and cattle were analyzed by uni- and multivariable logistic regression, and population structure methods, to gain insight into transmission and the nature of human infection. Eleven genotyping assays, including PCR typing of five virulence factors (stx 1, stx 2, stx 2c, eae, and ehxA) and a lineage-specific polymorphism assay using six markers (LSPA6), were considered in the analyses. The prevalence of the stx 1, stx 2, and stx 2c virulence factors was significantly different between human and cattle isolates. However, multivariable regression revealed that the presence of only the stx 2 gene was significantly associated with human isolates after controlling for confounding effects. LSPA6 typing demonstrated an apparent difference in the distribution of LSPA6 lineages between human and cattle isolates and a strong association between stx genotypes and LSPA6 genotypes. Population genetics tools identified three genetically distinct clusters of STEC O157. Each cluster was characterized by stx genotypes and LSPA6 genotypes. The human isolates typically comprised LSPA6 lineage I with stx 1 stx 2 strains and LSPA6 lineage I/II with stx 2 or stx 2 stx 2c strains. In contrast, the cattle isolates comprised LSPA6 lineage II strains with stx 2c or stx 2 stx 2c strains in addition to the clusters identified for the human isolates. Our analyses provide new evidence that the stx 2 gene is the most distinctive feature in human isolates compared to cattle isolates in Japan, and only a subset of the genetically diverse population isolated from cattle is involved in human illnesses. Our results may contribute to international comparisons and risk assessments of STEC O157.

Virulence Gene Profiles and Population Genetic Analysis for Exploration of Pathogenic Serogroups of Shiga Toxin-Producing Escherichia coli

ABSTRACT Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) is a serious public health concern, causing severe diarrhea and hemolytic-uremic syndrome. Patient symptoms are varied among STEC strains, possibly implying the presence of markers for STEC virulence other than Stx. To reveal the genotypic traits responsible for STEC virulence, we investigated 282 strains of various serogroups for the presence of 17 major virulence genes, i.e., stx 1, stx 2a, stx 2c, stx 2d, stx 2e, stx 2f, eae, tir, espB, espD, iha, saa, subA, ehxA, espP, katP, and stcE. Next, we examined the prevalence of virulence genes according to the seropathotypes in which serotypes were classified (seropathotypes A through E) based on the reported frequencies in human illness, as well as known associations with outbreaks and with severe disease. Our results demonstrate that the presence of both katP and stcE in STEC, in addition to the genes located in the locus of enterocyte effacement (LEE), including eae, tir, espB, and espD, may indicate the most pathogenic genotype of STEC. A population structure analysis of the profiles of virulence genes statistically supported the pathogenic genotype and, furthermore, revealed that there are serogroups with potentially higher pathogenicity than previously thought. Some strains in serogroups O26, O145, and O165 may have high virulence equivalent to that of serogroup O157. Several serogroups, including O14, O16, O45, O63, O74, 119, O128, and O untypeable, also may be potentially pathogenic, although rarely in humans.

Variation in Stress Resistance Patterns among stx Genotypes and Genetic Lineages of Shiga Toxin-Producing Escherichia coli O157

ABSTRACT To evaluate the relationship between bacterial genotypes and stress resistance patterns, we exposed 57 strains of Shiga toxin-producing Escherichia coli (STEC) O157 to acid, freeze-thaw, heat, osmotic, oxidative, and starvation stresses. Inactivation rates were calculated in each assay and subjected to univariate and multivariate analyses, including principal component analysis (PCA) and cluster analysis. The stx genotype was determined for each strain as was the lineage-specific polymorphism assay (LSPA6) genotype. In univariate analyses, strains of the stx 1 stx 2 genotype showed greater resistance to heat than strains of the stx 1 stx 2c genotype; moreover, strains of the stx 1 stx 2 genotype showed greater resistance to starvation than strains of the stx 2 or stx 2c genotypes. LSPA6 lineage I (LI) strains showed greater resistance to heat and starvation than LSPA6 lineage II (LII) strains. PCA revealed a general trend that a strain with greater resistance to one type of stress tended to have greater resistance to other types of stresses. In cluster analysis, STEC O157 strains were grouped into stress-resistant, stress-sensitive, and intermediate clusters. In stx genotypes, all strains of the stx 1 stx 2 genotype were grouped with the stress-resistant cluster, whereas 72.7% (8/11) of strains of the stx 1 stx 2c genotype grouped with the stress-sensitive cluster. In LI strains, 77.8% (14/18) of the strains were grouped with the stress-resistant cluster, whereas 64.7% (11/17) of LII strains were grouped with the stress-sensitive cluster. These results indicate that the genotypes of STEC O157 that are frequently associated with human illness, i.e., LI or the stx 1 stx 2 genotype, have greater multiple stress resistance than do strains of other genotypes.

Evaluation of genetic markers for identifying isolates of the species of the genus Fusarium

BACKGROUND Members of the genus Fusarium are well known as one of the most important plant pathogens causing food spoilage and loss worldwide. Moreover, they are associated with human and animal diseases through contaminated foods because they produce mycotoxins. To control fungal hazards of plants, animals and humans, there is a need for a rapid, easy and accurate identification system of Fusarium isolates with molecular methods. RESULTS To specify genes appropriate for identifying isolates of various Fusarium species, we sequenced the 18S rRNA gene (rDNA), internal transcribed spacer region 1, 5.8S rDNA, 28S rDNA, β-tubulin gene (β-tub), and aminoadipate reductase gene (lys2), and subsequently calculated the nucleotide sequence homology with pair-wise comparison of all tested strains and inferred the ratio of the nucleotide substitution rates of each gene. Inter-species nucleotide sequence homology of β-tub and lys2 ranged from 83.5 to 99.4% and 56.5 to 99.0%, respectively. The result indicated that sequence homologies of these genes against reference sequences in a database have a high possibility of identifying unknown Fusarium isolates when it is more than 99.0%, because these genes had no inter-species pair-wise combinations that had 100% homologies. Other markers often showed 100% homology in inter-species pair-wise combinations. The nucleotide substitution rate of lys2 was the highest among the six genes. CONCLUSION The lys2 is the most appropriate genetic marker with high resolution for identifying isolates of the genus Fusarium among the six genes we examined in this study.

Cooperative Recognition of Internationally Disseminated Ceftriaxone-Resistant Neisseriagonorrhoeae Strain

Ceftriaxone remains a first-line treatment for patients infected by Neisseria gonorrhoeae in most settings. We investigated the possible spread of a ceftriaxone-resistant FC428 N. gonorrhoeae clone in Japan after recent isolation of similar strains in Denmark (GK124) and Canada (47707). We report 2 instances of the FC428 clone in Australia in heterosexual men traveling from Asia. Our bioinformatic analyses included core single-nucleotide variation phylogeny and in silico molecular typing; phylogenetic analysis showed close genetic relatedness among all 5 isolates. Results showed multilocus sequence type 1903; N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) 233; and harboring of mosaic penA allele encoding alterations A311V and T483S (penA-60.001), associated with ceftriaxone resistance. Our results provide further evidence of international transmission of ceftriaxone-resistant N. gonorrhoeae. We recommend increasing awareness of international spread of this drug-resistant strain, strengthening surveillance to include identifying treatment failures and contacts, and strengthening international sharing of data.

Outbreak of Legionnaire’s Disease Caused by Legionella pneumophila Serogroups 1 and 13

In Japan, hot springs and public baths are the major sources of legionellosis. In 2015, an outbreak of Legionnaires’ disease occurred among 7 patients who had visited a spa house. Laboratory investigation indicated that L. pneumophila serogroup 1 and 13 strains caused the outbreak and that these strains were genetically related.

Penicillium camemberti and Penicillium roqueforti Enhance the Growth and Survival of Shiga Toxin-Producing Escherichia coli O157 under Mild Acidic Conditions

The effects of secondary starter molds of common mold-ripened cheeses on the Shiga toxin-producing Escherichia coli (STEC) O157 were assessed in 3 model systems. In the 1st model, 8 STEC O157 strains were incubated in the spent culture of Penicillium camemberti or Penicillium roqueforti under mild acidic conditions at 25 °C. In the spent cultures of the mold at pH 4.8 to 5.0, the lag times of STEC O157 growth were significantly shorter than those observed in fresh medium. Analyses of the spent culture of P. camemberti showed that the causative agents of the growth enhancement were produced by the mold in response to an acidic environment and were not fully inactivated in heat treatment. In the 2nd model, P. camemberti and STEC O157 were cocultured in acidified milk at 25 °C. The population of STEC O157 reached 10(8) CFU/mL in the presence of the mold, whereas the population steadily declined in the absence of the mold. Although this growth enhancement was partially attributable to alkalization by the mold, it was observed even when the pH of this model was stabilized. In the 3rd model, 2 STEC O157 strains were incubated in the spent cultures of molds at pH 4.5 at 10 °C. In the spent culture, proportions of injured cells were significantly lower and D values were significantly higher than those in control, except one STEC O157 strain in the spent culture of P. camemberti. These results showed that the molds could enhance the growth and survival of STEC O157 by changing the environment. Practical Application:  This study demonstrated that molds in foods can improve the growth and survival of the Shiga toxin-producing Escherichia coli O157. Because microbial interactions are ubiquitous in food, our results provide an important insight for understanding the behavior of microorganisms in food.

Spread and change in stress resistance of Shiga toxin‐producing Escherichia coli O157 on fungal colonies

To elucidate the effect of fungal hyphae on the behaviour of Shiga toxin‐producing Escherichia coli (STEC) O157, the spread and change in stress resistance of the bacterium were evaluated after coculture with 11 species of food‐related fungi including fermentation starters. Spread distances of STEC O157 varied depending on the co‐cultured fungal species, and the motile bacterial strain spread for longer distances than the non‐motile strain. The population of STEC O157 increased when co‐cultured on colonies of nine fungal species but decreased on colonies of Emericella nidulans and Aspergillus ochraceus. Confocal scanning microscopy visualization of green fluorescent protein‐tagged STEC O157 on fungal hyphae revealed that the bacterium colonized in the water film that existed on and between hyphae. To investigate the physiological changes in STEC O157 caused by co‐culturing with fungi, the bacterium was harvested after 7 days of co‐culturing and tested for acid resistance. After co‐culture with eight fungal species, STEC O157 showed greater acid resistance compared to those cultured without fungi. Our results indicate that fungal hyphae can spread the contamination of STEC O157 and can also enhance the stress resistance of the bacteria.