A Reconsideration of the Taxonomic Position of Two Bacterial Strains Isolated from Flacherie-Diseased Silkworms in 1965

Journal of Insect Biotechnology and Sericology 86, 35-41 (2017)

A reconsideration of the taxonomic position of two bacterial strains isolated from flacherie-diseased silkworms in 1965

Kazuhiro Iiyama1＊, Mai Morishita1, 2, Jae Man Lee2, Hiroaki Mon2, Takahiro Kusakabe2, Kosuke Tashiro3, Taiki Akasaka4, Chisa Yasunaga-Aoki1 and Kazuhisa Miyamoto5

1 Laboratory of Insect Pathology and Microbial Control, Institute of Biological Control, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan 2 Laboratory of Insect Genome Science, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan 3 Laboratory of Molecular Gene Technology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan 4 Center for Advanced Instrumental and Educational Supports, Faculty of Agriculture, Kyushu University, Fukuoka, Japan 5 Insect-Microbe Research Unit, Division of Insect Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Ibaraki, Japan (Received September 30, 2016; Accepted November 21, 2016)

Recent advances in bacterial characterization methodologies have made taxonomic categorization significant- ly more accurate. Here, we re-evaluated the position of bacterial strains (532 and 652) belonging to the genus Hafnia, isolated from flacherie-diseased silkworms in 1965. Phylogenetic analysis based on the 16S rRNA gene sequences of these strains suggests that they belong to genus Enterobacter. Using multilocus sequence analysis (MLSA), these strains were further classified to MLSA group A, which is a “core” group of Enterobacter con- taining E. cloacae (the type species of the genus). Although these strains were closely related to E. mori, E. tabaci, and E. asburiae, they also had other MLSA characteristics that distinguished them from these neighbor- ing bacterial species. These data were supported by further biochemical analysis. Thus, it appears that the 532 and 652 strains isolated almost half a century ago belong to genus Enterobacter, and their unique characteristics strongly suggest that they are a novel bacterial species. Key words: genus Enterobacter, pathogenicity, multilocus sequence analysis, bacterial identification

sp. However, since these bacteria were not preserved, other INTRODUCTION strains isolated in 1965 were orally inoculated into silk- In 1965, several novel bacterial strains were isolated for worm larvae to test for pathogenicity in 1966. The bacteri- the first time from flacherie-diseased silkworms that had al strains tested were Hafnia (strains 532, 645, 652, 853, been inoculated with Streptococcus sp. (Ono et al., 1967). 889), Enterobacter (strains A, B, C), Serratia, Klebsiella, Notably, when the isolates were orally inoculated into and Streptococcus (strain 2). Notably, when inoculated silkworm larvae, the individuals in one of the experimen- larvae were maintained at 25°C, the Hafnia 532, 645, and tal plot areas showed distinctly different symptoms from 853 strains caused death, while the larvae inoculated with the others. In these larvae, disease progression was rela- other strains (including the other strains of Hafnia) did tively rapid and included symptoms such as diarrhea, an- not die. From these results, it was concluded that the orexia, and body transparency, which were noticeably flacherie disease of silkworm in 1965 was caused by these different from those typically observed in the silkworm three particular Hafnia sp. (Ono et al., 1967). Among after inoculation with known Streptococcus sp. strains. In these strains, 532 and 652 were preserved and stored as order to isolate the pathogenic bacteria again for further lyophilized cultures in glass ampoules, and are now testing, one loopful of contents from the midgut of the stocked at The Institute of Agrobiological Sciences, Na- diseased silkworms was streaked onto DHL medium, and tional Agriculture and Food Research Organization in Ja- 43 isolates were selected. These isolates were then classi- pan. In preliminary inoculation tests, the rehydrated Hafnia fied into four groups based on their biochemical charac- sp. strain 532 showed virulence in the silkworm larvae, teristics. Since bacteria belonging to group-1 were present while strain 652 was weakly virulent or avirulent. These in all of the diseased silkworms, the strains in this group results are similar to those described in the literature (Ono were tentatively considered the causal agents of the dis- et al., 1967) and indicate that the characteristics of these ease. Although the detailed identification procedure was bacteria in terms of their pathogenicity were not altered not described in the literature, the bacterial species in during storage. group-1 were categorized as Hafnia sp. and Enterobacter More than five decades have elapsed since these strains were first isolated. During this time, the technology used *To whom correspondence should be addressed. for bacterial identification has changed dramatically,- al Fax: +81-92-642-4421. Tel: +81-92-642-3033. lowing for more precise and accurate documentation of Email: [email protected] well-known and novel bacterial species. These technologi- 36 Iiyama et al. cal advances have greatly advanced the field and have Phylogenetic analyses provided an accumulation of knowledge concerning the Phylogenetic analyses of the nucleotide sequences for genetic and phenotypic characteristics used in bacterial the 16S rRNA gene and concatenated housekeeping genes taxonomy. In a preliminary experiment, we sequenced the (aptD-gyrB-infB-rpoB) were conducted using MEGA ver- 16S ribosomal RNA (rRNA) genes of the previously iso- sion 6.0 (Tamura et al., 2013). Phylogenetic trees were lated Hafnia sp. 532 and 652 strains in order to further constructed using the neighbor-joining method. Bootstrap classify these strains at the species level. Surprisingly, this confidence analysis was carried out with 1,000 -replica preliminary test suggested that the isolates be classified as tions in order to evaluate the robustness of the tree topol- members of the genus Enterobacter. Therefore, in the ogies. present study, we sought to more accurately establish the taxonomic position of these two bacterial strains based on Biochemical analyses their 16S rRNA and various housekeeping gene sequences A commercially available API 20E kit (SYSMEX as well as their biochemical characteristics. bioMerieux Co.) was used to investigate the biochemical characteristics of the bacterial strains. The tests were carried out according to the manufacturer’s instructions. In MATERIALS AND METHODS brief, inoculum was prepared using single colonies on the Genome extraction LB agar that were resuspended in 0.85% NaCl solution. Strains 532 and 652 were cultured in Luria-Bertani After inoculation, API plates were incubated at 37°C for broth (Lennox) at 30°C for 18 h. Genomic DNA was ex- 24 h, and then the reactions were judged accordingly. tracted from the cells using the cetyltrimethyl ammonium bromide method as previously described (Wilson, 1987). RESULTS AND DISCUSSION 16S rRNA gene sequencing Phylogenetic analysis based on 16S rRNA gene The 16S rRNA gene fragments were amplified from the sequences genomic DNA by LA Taq polymerase (TAKARA BIO In our analysis, almost all of the 16S rRNA genes of INC.) with the universal primers 27F (5’-GAGTTTGATC strains 532 and 652 were sequenced. These data have MTGGCTCAG-3’) and 1492R (5’-GGYTACCTTGTTAC been deposited in GenBank/EMBL/DDBJ under the ac- GACTT-3’). After electrophoresis, PCR fragments were cession numbers LC169757 and LC169758 for 532 and purified by using the Wizard SV Gel and PCR Clean-Up 652, respectively. Notably, the constructed phylogenetic System (Promega Co.). The fragments were sequenced tree revealed that strains 532 and 652 were more closely without cloning with an ABI Prism 377 sequencer (Ap- related with genus Enterobacter than with genus Hafnia plied Biosystems). (Fig. 1). Enterobacter species are often not further classified be- Genome sequencing yond the genus level, as their identification is difficult Paired-end (fragment size, 400 bp) libraries of the 532 (Paauw et al., 2008). Furthermore, the 16S rDNA se- and 652 strains were generated. Sequencing was performed quences of E. cloacae did not form a coherent cluster, but with MiSeq technology using MiSeq Reagent Kits v. 2 more closely resembled a patchy tree. In this tree, the (300-cycle kit; Illumina). After filtering with Trimmomatic clusters of E. cloacae strains interfused with those of E. v. 0.32 (Lohse et al., 2012), assembly was performed us- aerogenes, Escherichia coli, Citrobacter species, and Lecler- ing Velvet v. 1.2.03 in the DDBJ Read Annotation Pipe- cia species (Hoffmann and Roggenkamp, 2003; Tang et line using the specified parameters for strain 532 (k-mer, al., 1998). Moreover, biochemical and molecular studies 127; expected coverage, 70) and strain 652 (k-mer, 127; on E. cloacae also suggest genomic heterogeneity, resulting expected coverage, 100; coverage cutoff, 20) (Zerbino and in the categorization of six species: E. cloacae, E. asbur- Birney, 2008; Kaminuma et al., 2010). iae, E. hormaechei, E. kobei, E. ludwigii and E. nimipressuralis (synonym: Lelliottia nimipressuralis) (Mezzatesta Determination of aptD, gyrB, infB, and rpoB gene et al., 2012; Brady et al., 2013). This heterogeneity is re- sequences flected in the low bootstrap values in our phylogenetic Local BLAST was carried out to determine the aptD, tree (Fig. 1, values less than 50% are not shown in the gyrB, infB, and rpoB gene sequences for the 532 and 652 figure). Although identification at the species level was bacterial strains. The following sequence queries from E. not possible, the phylogenetic tree suggests that the strains cloacae subsp. cloacae LMG 2783T were used in the search: indeed belong to the genus Enterobacter. aptD (JX424847), gyrB (JX424977), infB (JX425106), and rpoB (JX425236). Reclassification of diseased silkworm bacterial strain 37

Fig. 1. Neighbor-joining phylogenetic tree obtained from the 16S rRNA gene sequences of the Enterobacteriaceae family. Bootstrap values (expressed as percentages of 1,000 replications) above 50% are shown at the branch points. Xenorhabdus nematophila ATCC 19061T is used as the outgroup.

Draft genome sequencing Phylogenetic analysis based on multilocus se- After filtering with Trimmomatic, 1,985,650 and quences 3,403,716 reads were assembled for the 532 and 652 We also further investigated the taxonomy of E. cloa- strains, respectively. The draft genome of 532 included 206 cae using multilocus sequence analysis (MLSA) based on contigs in 38 scaffolds with an N50 and N90 of 479,955 the aptD, gyrB, infB and rpoB sequences, which encode and 153,551 bp, respectively. The longest sequence was the ATP synthase beta subunit, DNA gyrase subunit B, 2,215,135 bp. The total estimated size of the whole ge- translation initiation factor 2, and RNA polymerase beta nome was 4,873,561 bp (mean depth of coverage, 79.57×) subunit, respectively. This analysis was previously used to with a GC content of 51.0%. Alternatively, the genome reclassify the genus Enterobacter to Enterobacter (MLSA analysis of 652 included 56 scaffolds, 109 contigs, and group A), Lelliottia (MLSA group B), Pluralibacter (MLSA

N50 and N90 lengths of 379,998 bp and 74,827 bp, respec- group C), Kosakonia (MLSA group C), and Cronobacter tively. The longest sequence size was 1,100,931 bp, while (MLSA group D) (Paauw et al., 2008; Brady et al., 2013). the total estimated size of the genome was 4,818,832 bp In our MLSA of strains 532 and 652 along with 78 with a coverage of 139.00× and a GC content of 51.1%. members of the Enterobacteriaceae family, strains 532 and The aptD, gyrB, infB, and rpoB sequences searched in 652 were classified to group A, which is positioned far our draft genome data have been deposited in GenBank/ from H. alvei (Fig. 2). Although these bacterial strains ap- EMBL/DDBJ under the accession numbers LC169759, pear to be closely related with E. mori, E. tabaci, and E. LC169761, LC169763, and LC169765 for 532 and asburiae, they were still classified as a separate branch in LC169760, LC169762, LC169764, and LC169766 for 652, the group A clade. respectively. Other genome sequences will be published elsewhere after annotation. 38 Iiyama et al.

Fig. 2. Neighbor-joining phylogenetic tree of the multilocus sequence analysis (MLSA) based on four concatenated housekeeping genes (atpD-gyrB-infB-ropB) of the Enterobacteriaceae family. Bootstrap values over 50% (1,000 replications) are shown at each node. Xenorhabdus nematophila ATCC 19061T is used as the outgroup.

Biochemical analyses itive for β-galactosidase, arginine dihydrolase, ornithine The biochemical characteristics of 532 and 652 were decarboxylase, and citrate utilization, but negative for ly- determined by API 20E (Table 1). These strains were pos- sine decarboxylase, H2S production, urease, tryptophan Reclassification of diseased silkworm bacterial strain 39

Table 1. Comparison of the phenotypic characteristics of strains 532, 652, related species, and multilocus sequence analysis (MLSA) groups 6) 6) 6) 6) 6) 5) 5) 5) 4) 3, 4) 3, Characteristic 2) 1) 1) Pluralibacter (MLSA group C) Kosakonia (MLSA group D) Cronobacter (MLSA group E) 532 652 [ Hafnia ] mori Enterobacter tabaci Enterobacter asburiae Enterobacter 1 biovar alvei Hafnia 2 biovar alvei Hafnia Enterobacter (MLSA group A) Lelliottia (MLSA group B) β-galactosidase + + nd nd + + (+) d nd nd nd nd nd Arginine dihydrolase + + nd + nd (−) − − + + − (+) d Lysine decarboxylase − − nd + nd − + + − − + (−) − Ornithine decarboxylase + + nd + nd + + + + + + d (+) Citrate utilization + + + + + (+) d − nd nd nd nd nd

H2S production − − − + + − − − −* −* nd nd −* Urease − − − nd nd − (−) − nd nd nd nd nd Tryptophan deaminase − − nd nd nd − − − nd nd nd nd nd Indole production − − − +/− + − − − − − − − (−) Voges-Proskauer − − + + + (−) d (−) (+)* +* +* +* v* Gelatinase − − − + nd − − − nd −* −* −* nd Carbon source utilization of: D-glucose + + + + + + + + v* +* +* +* +* D-mannitol + + + + + + + + +* +* +* +* +* inositol − − − + + (−) − − (+) − d (d) d D-sorbitol − − − +/− − + − − (+) d (−) + (d) L-rhamnose + + + + + − + − v* +* +* +* +* D-sucrose − − − + + + − − v* v* +* +* v* D-melibiose + − nd + − − − − v* +* v* v* +* amygdalin + + nd nd nd + (−) − nd nd nd nd nd L-arabinose + + + + + + + − +* +* +* +* +* Indexes are: +, 90-100%; (+), 70-89%; d, 50-69%; (d), 30-49%; (−), 10-29%; −, 0-9%; +/−, different results compared to the literature; nd, not determined or not described. When the positive ratio was not explained in the literature, the description from the text was used. In these cases, the indexes are: +*, positive; (+)*, majority positive; v*, variable; (−)*, majority negative; −*, negative. Data were obtained from: 1), this study; 2), Ono et al. (1967); 3), Zhu et al. (2011); 4), Duan et al. (2015); 5), API database; 6), Brady et al. (2013). Underlined characteristics are useful phenotypic traits for classification into MLSA groups (Brady et al., 2013). deaminase, indole production, Voges-Proskauer test, and contradictory to their inclusion in MLSA group A. These gelatinase production. D-glucose, D-mannitol, L-rhamnose, data corroborate our phylogenetic and genomic results. amygdalin, and L-arabinose appear to be the primary sourc- Moreover, in our MLSA, strains 532 and 652 appeared to es of carbon, whereas inositol, D-sorbitol, D-sucrose are be closely related with E. mori, E. tabaci, and E. asbur- not used. Notably, D-melibiose utilization was different iae. These strains could be distinguished from other relat- between the two strains (positive in 532; negative in 652). ed species by their lysine decarboxylase production, H2S In a previous study by Brady et al. (2013), 22 charac- production, indole production, Voges-Proskauer test, gela- teristics were reported to sufficiently distinguish between tinase production, and utilization of inositol, D-sorbitol, the MLSA groups. Among these, six tests were included L-rhamnose, and D-sucrose. It is important to note that in the API 20E methodology utilized in this study. Al- the results in the present study for the Voges-Proskauer though these tests were insufficient to fully classify the test differed from those previously reported (Ono et al., strains into distinct MLSA groups, the results were not 1967). It is likely that these discrepancies are due to dif- 40 Iiyama et al. ferences in media and analysis methodology. respectively, E. cowanii, E. radicincitans, E. oryzae and E. arachidis into Kosakonia gen. nov. as Kosakonia cowanii Taxonomic position of entomopathogenic bacte- comb. nov., Kosakonia radicincitans comb. nov., Kosakonia oryzae comb. nov. and Kosakonia arachidis comb. nov., re- ria isolated from the silkworm spectively, and E. turicensis, E. helveticus and E. pulveris The results presented in this study indicate that strains into Cronobacter as Cronobacter zurichensis nom. nov., 532 and 652 should be classified as members of the genus Cronobacter helveticus comb. nov. and Cronobacter pul- Enterobacter. Although these strains appear to be closely veris comb. nov., respectively, and emended description of related to E. mori, E. tabaci, and E. asburiae, 532 and the genera Enterobacter and Cronobacter. Syst. Appl. Mi- crobiol., 36, 309-319. 652 were distinguished from their neighbors in phyloge- Brenner, D.J., McWhorter, A.C., Kai, A., Steigerwalt, A.G. and netic tree based on their multilocus sequences (Fig. 2) and Farmer, J.J. 3rd (1986) Enterobacter asburiae sp. nov., a several biochemical characteristics (Table 1). Therefore, new species found in clinical specimens, and reassignment these strains should be described as a novel species in the of Erwinia dissolvens and Erwinia nimipressuralis to the future, and additional investigation concerning their physi- genus Enterobacter as Enterobacter dissolvens comb. nov. and Enterobacter nimipressuralis comb. nov. J. Clin. Micro- ological and other biochemical characteristics is warranted. biol., 23, 1114-1120. Interestingly, most strains of E. asburiae have been Duan, Y.Q., Zhou, X.K., Di-Yan, L., Li, Q.Q., Dang, L.Z., identified and characterized as opportunistic pathogens, Zhang, Y.G., Qiu, L.H., Nimaichand, S. and Li, W.J. 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