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Identification ofEnterococcus sp. from midgut of silkworm based on biochemical and 16S rDNA sequencing analysis

Identification ofEnterococcus sp. from midgut of silkworm based on biochemical and 16S rDNA... Annals of Microbiology, 56 (3) 201-205 (2006) Identification of Enterococcus sp. from midgut of silkworm based on biochemical and 16S rDNA sequencing analysis 1, 2 2 1 1,2 3 Chen FEI , Xing-meng LU *, Yong-hua QIAN , Haiyan ZHANG , Qaisar MAHMOOD College of Animal Science and Technology, Northwest Agricultural and Forestry University, Yang Ling 712100, China; 2 3 College of Animal Sciences, Zhejiang University, College of Environment and Resource, Zhejiang University, Hang Zhou 310029, China Received 24 February 2006 / Accepted 17 May 2006 Abstract - Enterococcus sp. was isolated from the midgut of silkworm against the germination of Nosema bombycis spores. Identifi- cation was based on the biochemical characteristics, 16S rDNA sequences analysis and species-specific probes of Enterococcus spp. The isolated strains fermented sorbitol and arabinose but did not ferment raffinose. Enterococcus sp. was clustered together with Ente- rococcus mundtii ATCC 43188 and 100% sequence homology was found by 16S rDNA sequences BLAST analysis and constructing the phylogenetic tree. Comparison of the sequences of the 16S rDNA species-specific probes of Enterococcus spp. with the 16S rDNA sequence of isolate revealed similar segment to the species-specific probe of E. mundtii. So, we can make conclusion the 16S rDNA segment of Enterococcus sp. can hybridise with species-specific probe of E. mundtii. Enterococcus mundtii was detected for the first time in the intestine of silkworm. Key words: Enterococcus mundtii, silkworm midgut, biochemical identification, 16S rDNA sequence analysis, species-specific DNA probes. INTRODUCTION cocci from the intestine of healthy, pebrine and bacterial flacherie diseased silkworms were isolated and characterized Nosema bombycis is the most intimidatory and damning dis- by using API 20 STREP (V.5.0) system (Lu et al., 1999, ease for sericulture. It was the only quarantinable object of 2003). The traditional biochemical identification has however silkworm eggs production because it has the character of ger- some disadvantages, especially concerning the instability of minative infection. The prevention and cure of this disease the phenotypic expression and a low sensitivity; moreover, is a very important technical problem and imperative to be it is a time consuming and tedious process. Especially, Ente- solved for better sericulture production. Nosema bombycis rococcus species show both species- and strain-specific bio- spores germination in the silkworms’ intestines is the basic chemical diversities (Lu et al., 1994, 2003) that bring diffi- condition for its infection in silkworms (Iwano and Ishihar, culty to identify Enterococcus through biochemical tests. 1989). A large number of enterococci were found in intes- Though the bacterial automating identification systems are tinal tract of silkworms (Takizawa and Iizuka, 1968). Ente- suitable for the rapid identification, the model bacterial rococcus spp. is normally not pathogenic to silkworms (Lu strains in database are limited. Moreover the classification et al., 1999) and, interestingly, they can inhibit germination of Enterococcus is changing frequently due to more recent of N. bombycis spores (Lu and Wang, 2002). There exists discoveries. If database is not updated regularly to include great physiological and biochemical diversity among the recent discoveries, inaccurate results might be achieved. Enterococcus species (Lu et al., 1994, 1999). This anti- At present, the molecular methods for bacterial identifi- microaporidian property and polymorphism of enterococci cation are based upon the sequences of 16S rDNA. Sequenc- make them an interesting candidate for biological control of ing of 16S rDNA in conjunction with homology searching and silkworm diseases. Studies on Enterococcus sp. diversity constructing the phylogenic tree are powerful means to iden- are helpful to understand their phenotypic differences and tify Enterococcus. Studying bacteria through the genetic their possible relationship with N. bombycis. The identifica- and evolutionary point of view and classifying them on the tion and characterization of Enterococcus from silkworms molecular level not only can confirm the classification but also may have the practical meaning of prevention and cure of reveal the existing homology between wild and reference nosogenesis in the silk industry. strains. Moreover, analysing the sequences of 16S rDNA can In previous investigation two hundred strains of entero- help us to find species-specific probes to be used for identi- fication and practical purposes. The objectives of our study were to detect Enterococcus sp. having anti-microaporidian property. To this end, bio- * Corresponding author. Phone: 0086-0571-86971305; E-mail: xmlu@zju.edu.cn chemical tests, phylogenetic analysis based on the 16S rDNA 202 C. Fei et al. sequence and analysis of Enterococcus species-specific teristics were analysed and phylogenetic tree was con- probes were carried out on one strain isolated from the structed by Mega 2 software. midgut of healthy silkworms. Analysis of the species-specific probes of Enterococcus species. The sequences of 16S rDNA of tested strain and MATERIALS AND METHODS species-specific probes of Enterococcus species were com- pared in order to find out the corresponding probe of this Strain source and culture conditions. The Enterococcus strain. sp. was isolated from the midgut of healthy silkworms. To grow the isolate, decimally diluted digestive juices of healthy Biochemical tests. The utilization of sugars under aerobic silkworms (0.5 ml) were mixed with the EF agar (Nissui, and anaerobic conditions was tested by the mellow test Tokyo, Japan) supplemented with 10 µg/mL vancomycin and through bacterial minimum biochemical reaction tubes were incubated at 35-37 °C for 48 h. The developed colonies (Hangzhou Microbial Reagent Co., Ltd., China) in term of rou- were peach, brown or yellowish in colour. Single colony was tine ways and means (Dong and Cai, 2001). The tests were selected from that EF plates, and shifted on to the Nutrient conducted for sucrose, maltose, sorbitol, lactose, glucose, Broth. The same procedure was repeated 3 times. amylum, fructose, mannitol, mannitose, galactose, trehalose, Liquid or solid Nutrient media were used for preserving raffinose, D-ribose, arabinose, rhamnose, aesculin and inulin. the isolates for experimental purpose. Before DNA extrac- tion, strains were grown aerobically in Brain Heart Infusion Broth overnight at 37 °C. RESULTS AND DISCUSSIONS Sequencing and analysis of 16S rDNA. Phylogenetic analysis and species-specific probe Preparation of the DNA sample. The genomic DNA was The results of the phylogenetic analysis are reported in Fig. extracted from the 50 ml liquid Brain Heart Infusion Broth 1 and Table 2. The tested strain was phylogenetically close- medium according to protocols of Wang et al. (2005). ly related to Enterococcus mundtii (100% sequence simi- larity), Enterococcus hirae (99%) and Enterococcus durans, PCR amplification of 16S rDNA. 27f and 1492r were select- Enterococcus faecium, Enterococcus azikeevi, Enterococcus ed from the universal primers shown in Table 1 (Stackebrant villorum (98%). and Goodfellow, 1991). Shanghai Sangon Biological Engi- Modern bacterial taxonomy is inclined to use genotypic neering Technology & Services Co., Ltd., China, synthesized methods together with phenotype characteristics to decide the primers. the position of bacterial species within a phylogenetic tree, The 16S rDNA fragment was amplified in a Perkin-Elmer according to the 16S rDNA sequence. Presently the accept- DNA thermal cycler with the following program: 30 s at 94 able positional standard is that if the similarity of strain °C, 30 s at 55 °C, and 1.5 min at 72 °C for 35 cycles. The PCR reactions were terminated at 72 °C for 7 min and, thereafter, cooled at 4 °C. To ascertain the specificity of the PCR amplification, negative control (PCR mix without DNA template) and positive control (PCR mix with Enterococcus spp. DNA template) were included. Amplification was con- firmed by electrophoresis analysing 5 µl PCR reaction mix- tures on a 1% agarose gel, and was screened by ChampGel gel image disposal system. The PCR product was purified using an EZ-10 Spin Column DNA Gel Extraction Kit (BBI), according to the manufacturer’s instructions. The 16S rDNA was sequenced by TaKaRa Biotechnology Co., Ltd. Compa- ny Japan, and sequence was submitted to GenBank. Phylogenetic analysis. The 16S rDNA sequences were aligned and paralleled with the sequence of corresponding bacteri- um in GenBank by Blast software. The homologous charac- TABLE 1 – List of primers Name Sequence (5’-3’) 27f AGAGTTTGATCMTGGCTCAG 519r GWATTACCGCGGCKGCTG 530f GTGCCAGCMGCCGCGG FIG. 1 – Phylogenetic tree based on 16S ribosomal DNA sequences 907r CCGTCAATTCMTTTRAGTTT Numbers in parentheses represent the sequences’ acces- 926f AAACTYAAAKGAATTGACGG sion number in GenBank. Numbers in square indicate the clone number out of the total clones. The numbers next 1492r TACGGYTACCTTGTTACGACTT to the nodes represent the bootstrap values of 1000 replications, are given at branch points. The scale bar indi- M= C: A, Y=C: T, K=G:T, W=A:T (Stackebrant and Goodfel- cates 0.005 substitutions per nucleotide position. low,1991). Ann. Microbiol., 56 (3), 201-205 (2006) 203 TABLE 2 – Similarity of Enterococcus spp. with other species on the basis of 16S rDNA Similarity (%) Strains Enteroccoccus sp. 100 99 98 98 98 98 97 97 97 97 97 95 95 95 95 96 96 95 94 95 96 97 96 Enteroccoccus mundtii 99 98 99 97 98 98 98 98 98 98 96 96 95 95 97 97 95 95 96 96 98 97 Enteroccoccus hirae 99 99 99 99 99 99 98 98 98 97 96 96 96 97 97 95 95 96 96 98 97 Enteroccoccus durans 99 98 99 99 99 98 98 98 97 96 96 96 97 97 95 95 96 97 98 97 Enteroccoccus faecium 99 98 98 99 99 98 98 97 96 96 96 97 97 95 95 96 97 97 98 Enteroccoccus azikeevi 98 98 98 98 98 97 97 96 95 96 97 97 95 95 96 96 97 97 Enteroccoccus villorum 98 98 98 98 98 96 96 96 95 97 97 96 95 96 97 97 97 Enteroccoccus pseudoavium 98 98 98 98 96 96 96 95 96 97 95 93 95 96 96 97 Enteroccoccus faecalis 98 97 98 96 96 96 95 97 97 95 95 95 96 97 97 Enteroccoccus ratti 97 97 96 96 96 95 96 97 95 95 95 96 97 97 Enteroccoccus raffinosus 99 96 95 95 95 98 98 96 96 96 97 99 99 Enteroccoccus devriesei 96 95 95 94 98 98 96 95 96 97 99 98 Enteroccoccus phoeniculicola 95 95 95 96 96 95 94 95 95 96 96 Enteroccoccus caccae 99 99 95 95 95 93 95 95 95 95 Enteroccoccus rotate 98 95 95 95 93 95 95 95 95 Enteroccoccus moraviensis 94 95 94 93 95 95 95 95 Enteroccoccus casseliflavus 99 92 94 92 93 89 96 Enteroccoccus gallinarum 96 96 97 97 98 98 Enteroccoccus sulfureus 92 93 92 88 95 Enteroccoccus cecorum 93 93 95 93 Enteroccoccus saccharolyticus 93 88 95 Enteroccoccus dispar 88 95 Enteroccoccus avium 97 under investigation and a reference strain sequences is 99- rRNA and 23S rRNA, contain highly conserved regions com- 100%, they are regarded as belonging to the same species mon to all eubacteria as well as highly variable regions while if similarity is 97-98%, they are regarded as belong- unique to a particular species. Thus, universal probes or ing to the same genus (Drancourt et al., 2000; Janda and primers that will anneal to the genes coding for rRNA of all Abbott, 2002). According to the standard above, since the eubacteria can be designed from the conserved regions of similarity of whole 16S rDNA sequences between strain the genes for 16S rRNA. A newly developed set of probes tar- under investigation and E. mundtii was 100% we can con- geting the 16S rDNA gene served as a confirmation method sider the tested strain as belonging to the species E. mundtii. for a correct species identification (Manero and Blanch, However, since we found high similarity values also for other 2002). related Enterococcus species we analysed the 16S rDNA Analysing the 16S rDNA sequence of Enterococcus sp., sequence of the strains with the aim to verify the presence we found the same segment as the sequence of E. mundtii of nucleotide fragments used as species- specific probes for probe (5?-CACCGGGAAAAGAGGAGTGG-3?) (Stackebrant E. mundtii. and Goodfellow, 1991; Manero and Blanch, 2002). So, Indeed the use of probes for genes coding for ribosomal according to the principle of DNA-DNA hybridisation, we can ribonucleic acid (rRNA) offers a great potential in microbio- suppose that strain under investigation can hybridise with the logical identification. The rRNA molecules, in particular 16S species-specific probes of E. mundtii. Enteroccoccus sp. Enteroccoccus mundtii Enteroccoccus hirae Enteroccoccus durans Enteroccoccus faecium Enteroccoccus azikeevi Enteroccoccus villorum Enteroccoccus.pseudoavium Enteroccoccus faecalis Enteroccoccus ratti Enteroccoccus raffinosus Enteroccoccus devriesei Enteroccoccus phoeniculicola Enteroccoccus caccae Enteroccoccus rotate Enteroccoccus moraviensis Enteroccoccus casseliflavus Enteroccoccus gallinarum Enteroccoccus sulfureus Enteroccoccus cecorum Enteroccoccus saccharolyticus Enteroccoccus dispar Enteroccoccus avium Enteroccoccus malodoratus 204 C. Fei et al. TABLE 3 – The biochemical characteristics of isolated Enterococcus sp. ed from plants, and silkworms living on leaves of mulberry, so we deduced those silkworms were infected through leaves Characteristics Results Characteristics Results of mulberry. Enterococcus is the main bacterial group (Tak- izawa and Iizuka, 1968) that can participate in the physio- Sucrose + Galactose + logic metabolism of hosts, and the important indispensable Maltose + Trehalose + part of normal physiologic activities. It also was the main Sorbitol + Raffinose – pathogen of silkworm bactericidal flacherie diseases Lactose + D-Ribose + (Lysenko, 1958). Lu et al. (1999, 2002, 2003) analysed and classified the enterococcal flora of the intestine of healthy, Glucose + Arabinose + pebrine and bacterial flacherie diseased silkworms by using Amylum – Rhamnose – API 20 STREP (V.5.0) system based on numerical taxonomy; Fructose + Bile aesculin + Wang et al. (2005) analysed the DNA of the enterococci iso- Mannitol + Serum inulin – lated from the intestine of the silkworm by the random amplified polymorphic DNA (RAPD) technique, they all did- Mannitose + n’t find E.mundtii in the intestine of silkworms. Therefore, this is the first report concerning the presence of E. mundtii in the intestine of silkworm. It complemented the research results for enterococcal flora of silkworms intestine, not only Biochemical tests supply the new microbial sources for prevention and cure of Results of biochemical tests have been presented in Table 3. Nosema bombycis but also are very important to investigate We used the most common biochemical tests used to char- the physiologic metabolism of silkworms and prevention and acterize and identify the isolate in lour lab. The tested strain cure of bacterial flacherie diseases. showed positive results for sucrose, maltose, sorbitol, lac- tose, glucose, fructose, mannitol, mannitose, galactose, tre- Acknowledgements halose, D-ribose, arabinose, aesculin, while there were neg- This work was supported by grants from National Natural Sci- ative results for amylum, raffinose, rhamnose and inulin. ence Foundation of China (No. 30471311 and 30070578). The results obtained by us for Enterococcus mundtii are in consistence with the Bergey’s Manual of Determinative REFERENCES Bacteriology (Holt and Krieg, 1994). Key reactions that facil- itated differentiation of E. mundtii from E. faecalis and E. fae- Collins M.D., John A., Farrow E., Jones D. (1986). Enterococcus cium were arabinose, sorbitol and raffinose utilization (Fack- mundtii. nov. Int. J. Syst. Bacteriol., 36 (1): 8-12. lam and Colins, 1989). Dong X., Cai M. (2001). Manual of System Determinative Com- Our results were different from that of numerical identi- mon Bacteriology. Science Press, Beijing, pp. 370-398. fication by API 20 STREP (V.5.0, BioMerieux S.A.). Of the 17 Drancourt M., Bollet C., Carlioz R., (2000). 16S ribosomal DNA tests in our experiment, the strain fermented sorbitol and sequence analysis of a large collection of environmental and arabinose, but didn’t ferment raffinose. We compared the clinical unidentifiable bacterial isolates. J. Clin. Microbiol., 38: 3623-3630. results for the isolate with the characteristics of E. mundtii from Bergey’s Manual of Determinative Bacteriology (Holt Facklam R.R., Collins M.D. (1989). Identification of Enterococcus species isolated from human infections by a conventional test and Krieg, 1994) and other reports (Collins et al., 1986; scheme. J. Clin. Microbiol., 27: 731-734. Kaufhold and Ferrieri, 1991; Manero and Blanch, 1999; Higashide T., Takahashi M., Kobayashi A. (2005). Endophthalmi- Higashide et al., 2005). The isolate did not ferment raffinose, tis caused by Enterococcus mundtii. J. Clin. Microbiol., 43 (3): while the majority of the reported strains did. However, 1475-1476. other biochemical test results for the isolate were highly con- Holt J.G., Krieg N.R. (1994). Bergey’s Manual of Determinative Bac- sistent with those for other E. mundtii strains. The inability th teriology. 9 edn., Williams & Wilkins, Baltimore. of API 20 Strep to identify the species E. mundtii could be Iwano H., Ishihara R. (1989). Intracellular germination of spores related to the absence of this species in the API database of a Nosema sp. immediately after their formation in cultured (version 5.0). Misidentification by commercial biochemical cell. J. Invertebr. Pathol., 57 (1): 125-127. assays, which also happened in our case, may account for Janda J.M., Abbott S.L. (2002). Bacterial identification for publi- the scarcity of reports on this organism (Kaufhold and Fer- cation: when is enough? J. Clin. Microbiol., 40: 1887-1891. rieri, 1991). Relying on commercially available identification Junco M.T.T., Martin M.G., Toledo M.L.P. (2001). Identification and systems, whose databases do not include the more recent antibiotic resistance of faecal enterococci isolated from water taxonomic changes, may lead to inaccurate identifications. samples. Int. J. Hyg. Environ. Health, 203: 363-368. Kaufhold A., Ferrieri P. (1991). Isolation of Enterococcus mundtii from normally sterile body sites in two patients. J. Clin. Micro- biol., 29 (5): 1075-1077. CONCLUSION Lysenko O. (1958). “Streptococcus bombycis”, its taxonomy and pathogenicity for silkworm caterpillars. J. Gen. Microbiol., 18: Enterococcus mundtii was discovered in 1986 as nonmotile, 774-781. yellow-pigmented bacterium isolated from cow teats, the Lu X., Hashimoto Y., Shimizu S. (1994). Taxonomical studies on hands of milkers, soil, and plants (Collins et al., 1986). Since enterococci isolated from the intestine of the silkworm, Bom- then the species has rarely been isolated from environmen- byx mori. Journal of Sericultural Science of Japan, 63: 481- tal (Junco et al., 2001) or human sources (Facklam and Collins,1989). According to the sequences of 16S rDNA and Lu X., Jin W., Qian Y. (1999). Distribution of the Enterococci iso- species-specific probes of Enterococcus, we identified an lated from the intestine of the silkworm, Bombyx mori. Acta isolated strain as E. mundtii. Because E. mundtii was isolat- Sericologica Sinica, 25 (3): 158-162. Ann. Microbiol., 56 (3), 201-205 (2006) 205 Lu X., Wang F. (2002). Inhibition of cultured supernatant of Ente- Stackebrant E., Goodfellow M. (1991). Nucleic Acid Techniques in rococci strains on germination of Nosema bombycis spores in Bacterial Systematics. Wiley, England. vitro. Acta Sericologica Sinica, 28 (2): 126-128. Takizawa Y., Iizuka T. (1968). The aerobic bacterial flora in the gut Lu X., Huang S., Wang F. (2003). Distribution of the Enterococci of larvae of the silkworm, Bombyx mori L. (I) The relation isolated from intestine of the pebrine infected silkworm. Acta between media and the numbers of living cells. Journal of Ser- Sericologica Sinica, 29 (2): 151-156. icultural Science of Japan, 37: 295-305. Manero A., Blanch A.R. (1999). Identification of Enterococcus spp. Wang Y., Lu X., Mu Z. (2005). RAPD analysis of the Enterococci with a biochemical key. Appl. Environ. Microbiol., 65 (10): isolated from the silkworm, Bombyx mori. Acta Sericologica 4425-4430. Sinica, 31 (1): 59-63. Manero A., Blanch A.R. (2002). Identification of Enterococcus spp. based on specific hybridisation with 16S rDNA probes. J. Micro- biol. Methods, 50 (2): 115-121. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Identification ofEnterococcus sp. from midgut of silkworm based on biochemical and 16S rDNA sequencing analysis

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Springer Journals
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Copyright © 2006 by University of Milan and Springer
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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Abstract

Annals of Microbiology, 56 (3) 201-205 (2006) Identification of Enterococcus sp. from midgut of silkworm based on biochemical and 16S rDNA sequencing analysis 1, 2 2 1 1,2 3 Chen FEI , Xing-meng LU *, Yong-hua QIAN , Haiyan ZHANG , Qaisar MAHMOOD College of Animal Science and Technology, Northwest Agricultural and Forestry University, Yang Ling 712100, China; 2 3 College of Animal Sciences, Zhejiang University, College of Environment and Resource, Zhejiang University, Hang Zhou 310029, China Received 24 February 2006 / Accepted 17 May 2006 Abstract - Enterococcus sp. was isolated from the midgut of silkworm against the germination of Nosema bombycis spores. Identifi- cation was based on the biochemical characteristics, 16S rDNA sequences analysis and species-specific probes of Enterococcus spp. The isolated strains fermented sorbitol and arabinose but did not ferment raffinose. Enterococcus sp. was clustered together with Ente- rococcus mundtii ATCC 43188 and 100% sequence homology was found by 16S rDNA sequences BLAST analysis and constructing the phylogenetic tree. Comparison of the sequences of the 16S rDNA species-specific probes of Enterococcus spp. with the 16S rDNA sequence of isolate revealed similar segment to the species-specific probe of E. mundtii. So, we can make conclusion the 16S rDNA segment of Enterococcus sp. can hybridise with species-specific probe of E. mundtii. Enterococcus mundtii was detected for the first time in the intestine of silkworm. Key words: Enterococcus mundtii, silkworm midgut, biochemical identification, 16S rDNA sequence analysis, species-specific DNA probes. INTRODUCTION cocci from the intestine of healthy, pebrine and bacterial flacherie diseased silkworms were isolated and characterized Nosema bombycis is the most intimidatory and damning dis- by using API 20 STREP (V.5.0) system (Lu et al., 1999, ease for sericulture. It was the only quarantinable object of 2003). The traditional biochemical identification has however silkworm eggs production because it has the character of ger- some disadvantages, especially concerning the instability of minative infection. The prevention and cure of this disease the phenotypic expression and a low sensitivity; moreover, is a very important technical problem and imperative to be it is a time consuming and tedious process. Especially, Ente- solved for better sericulture production. Nosema bombycis rococcus species show both species- and strain-specific bio- spores germination in the silkworms’ intestines is the basic chemical diversities (Lu et al., 1994, 2003) that bring diffi- condition for its infection in silkworms (Iwano and Ishihar, culty to identify Enterococcus through biochemical tests. 1989). A large number of enterococci were found in intes- Though the bacterial automating identification systems are tinal tract of silkworms (Takizawa and Iizuka, 1968). Ente- suitable for the rapid identification, the model bacterial rococcus spp. is normally not pathogenic to silkworms (Lu strains in database are limited. Moreover the classification et al., 1999) and, interestingly, they can inhibit germination of Enterococcus is changing frequently due to more recent of N. bombycis spores (Lu and Wang, 2002). There exists discoveries. If database is not updated regularly to include great physiological and biochemical diversity among the recent discoveries, inaccurate results might be achieved. Enterococcus species (Lu et al., 1994, 1999). This anti- At present, the molecular methods for bacterial identifi- microaporidian property and polymorphism of enterococci cation are based upon the sequences of 16S rDNA. Sequenc- make them an interesting candidate for biological control of ing of 16S rDNA in conjunction with homology searching and silkworm diseases. Studies on Enterococcus sp. diversity constructing the phylogenic tree are powerful means to iden- are helpful to understand their phenotypic differences and tify Enterococcus. Studying bacteria through the genetic their possible relationship with N. bombycis. The identifica- and evolutionary point of view and classifying them on the tion and characterization of Enterococcus from silkworms molecular level not only can confirm the classification but also may have the practical meaning of prevention and cure of reveal the existing homology between wild and reference nosogenesis in the silk industry. strains. Moreover, analysing the sequences of 16S rDNA can In previous investigation two hundred strains of entero- help us to find species-specific probes to be used for identi- fication and practical purposes. The objectives of our study were to detect Enterococcus sp. having anti-microaporidian property. To this end, bio- * Corresponding author. Phone: 0086-0571-86971305; E-mail: xmlu@zju.edu.cn chemical tests, phylogenetic analysis based on the 16S rDNA 202 C. Fei et al. sequence and analysis of Enterococcus species-specific teristics were analysed and phylogenetic tree was con- probes were carried out on one strain isolated from the structed by Mega 2 software. midgut of healthy silkworms. Analysis of the species-specific probes of Enterococcus species. The sequences of 16S rDNA of tested strain and MATERIALS AND METHODS species-specific probes of Enterococcus species were com- pared in order to find out the corresponding probe of this Strain source and culture conditions. The Enterococcus strain. sp. was isolated from the midgut of healthy silkworms. To grow the isolate, decimally diluted digestive juices of healthy Biochemical tests. The utilization of sugars under aerobic silkworms (0.5 ml) were mixed with the EF agar (Nissui, and anaerobic conditions was tested by the mellow test Tokyo, Japan) supplemented with 10 µg/mL vancomycin and through bacterial minimum biochemical reaction tubes were incubated at 35-37 °C for 48 h. The developed colonies (Hangzhou Microbial Reagent Co., Ltd., China) in term of rou- were peach, brown or yellowish in colour. Single colony was tine ways and means (Dong and Cai, 2001). The tests were selected from that EF plates, and shifted on to the Nutrient conducted for sucrose, maltose, sorbitol, lactose, glucose, Broth. The same procedure was repeated 3 times. amylum, fructose, mannitol, mannitose, galactose, trehalose, Liquid or solid Nutrient media were used for preserving raffinose, D-ribose, arabinose, rhamnose, aesculin and inulin. the isolates for experimental purpose. Before DNA extrac- tion, strains were grown aerobically in Brain Heart Infusion Broth overnight at 37 °C. RESULTS AND DISCUSSIONS Sequencing and analysis of 16S rDNA. Phylogenetic analysis and species-specific probe Preparation of the DNA sample. The genomic DNA was The results of the phylogenetic analysis are reported in Fig. extracted from the 50 ml liquid Brain Heart Infusion Broth 1 and Table 2. The tested strain was phylogenetically close- medium according to protocols of Wang et al. (2005). ly related to Enterococcus mundtii (100% sequence simi- larity), Enterococcus hirae (99%) and Enterococcus durans, PCR amplification of 16S rDNA. 27f and 1492r were select- Enterococcus faecium, Enterococcus azikeevi, Enterococcus ed from the universal primers shown in Table 1 (Stackebrant villorum (98%). and Goodfellow, 1991). Shanghai Sangon Biological Engi- Modern bacterial taxonomy is inclined to use genotypic neering Technology & Services Co., Ltd., China, synthesized methods together with phenotype characteristics to decide the primers. the position of bacterial species within a phylogenetic tree, The 16S rDNA fragment was amplified in a Perkin-Elmer according to the 16S rDNA sequence. Presently the accept- DNA thermal cycler with the following program: 30 s at 94 able positional standard is that if the similarity of strain °C, 30 s at 55 °C, and 1.5 min at 72 °C for 35 cycles. The PCR reactions were terminated at 72 °C for 7 min and, thereafter, cooled at 4 °C. To ascertain the specificity of the PCR amplification, negative control (PCR mix without DNA template) and positive control (PCR mix with Enterococcus spp. DNA template) were included. Amplification was con- firmed by electrophoresis analysing 5 µl PCR reaction mix- tures on a 1% agarose gel, and was screened by ChampGel gel image disposal system. The PCR product was purified using an EZ-10 Spin Column DNA Gel Extraction Kit (BBI), according to the manufacturer’s instructions. The 16S rDNA was sequenced by TaKaRa Biotechnology Co., Ltd. Compa- ny Japan, and sequence was submitted to GenBank. Phylogenetic analysis. The 16S rDNA sequences were aligned and paralleled with the sequence of corresponding bacteri- um in GenBank by Blast software. The homologous charac- TABLE 1 – List of primers Name Sequence (5’-3’) 27f AGAGTTTGATCMTGGCTCAG 519r GWATTACCGCGGCKGCTG 530f GTGCCAGCMGCCGCGG FIG. 1 – Phylogenetic tree based on 16S ribosomal DNA sequences 907r CCGTCAATTCMTTTRAGTTT Numbers in parentheses represent the sequences’ acces- 926f AAACTYAAAKGAATTGACGG sion number in GenBank. Numbers in square indicate the clone number out of the total clones. The numbers next 1492r TACGGYTACCTTGTTACGACTT to the nodes represent the bootstrap values of 1000 replications, are given at branch points. The scale bar indi- M= C: A, Y=C: T, K=G:T, W=A:T (Stackebrant and Goodfel- cates 0.005 substitutions per nucleotide position. low,1991). Ann. Microbiol., 56 (3), 201-205 (2006) 203 TABLE 2 – Similarity of Enterococcus spp. with other species on the basis of 16S rDNA Similarity (%) Strains Enteroccoccus sp. 100 99 98 98 98 98 97 97 97 97 97 95 95 95 95 96 96 95 94 95 96 97 96 Enteroccoccus mundtii 99 98 99 97 98 98 98 98 98 98 96 96 95 95 97 97 95 95 96 96 98 97 Enteroccoccus hirae 99 99 99 99 99 99 98 98 98 97 96 96 96 97 97 95 95 96 96 98 97 Enteroccoccus durans 99 98 99 99 99 98 98 98 97 96 96 96 97 97 95 95 96 97 98 97 Enteroccoccus faecium 99 98 98 99 99 98 98 97 96 96 96 97 97 95 95 96 97 97 98 Enteroccoccus azikeevi 98 98 98 98 98 97 97 96 95 96 97 97 95 95 96 96 97 97 Enteroccoccus villorum 98 98 98 98 98 96 96 96 95 97 97 96 95 96 97 97 97 Enteroccoccus pseudoavium 98 98 98 98 96 96 96 95 96 97 95 93 95 96 96 97 Enteroccoccus faecalis 98 97 98 96 96 96 95 97 97 95 95 95 96 97 97 Enteroccoccus ratti 97 97 96 96 96 95 96 97 95 95 95 96 97 97 Enteroccoccus raffinosus 99 96 95 95 95 98 98 96 96 96 97 99 99 Enteroccoccus devriesei 96 95 95 94 98 98 96 95 96 97 99 98 Enteroccoccus phoeniculicola 95 95 95 96 96 95 94 95 95 96 96 Enteroccoccus caccae 99 99 95 95 95 93 95 95 95 95 Enteroccoccus rotate 98 95 95 95 93 95 95 95 95 Enteroccoccus moraviensis 94 95 94 93 95 95 95 95 Enteroccoccus casseliflavus 99 92 94 92 93 89 96 Enteroccoccus gallinarum 96 96 97 97 98 98 Enteroccoccus sulfureus 92 93 92 88 95 Enteroccoccus cecorum 93 93 95 93 Enteroccoccus saccharolyticus 93 88 95 Enteroccoccus dispar 88 95 Enteroccoccus avium 97 under investigation and a reference strain sequences is 99- rRNA and 23S rRNA, contain highly conserved regions com- 100%, they are regarded as belonging to the same species mon to all eubacteria as well as highly variable regions while if similarity is 97-98%, they are regarded as belong- unique to a particular species. Thus, universal probes or ing to the same genus (Drancourt et al., 2000; Janda and primers that will anneal to the genes coding for rRNA of all Abbott, 2002). According to the standard above, since the eubacteria can be designed from the conserved regions of similarity of whole 16S rDNA sequences between strain the genes for 16S rRNA. A newly developed set of probes tar- under investigation and E. mundtii was 100% we can con- geting the 16S rDNA gene served as a confirmation method sider the tested strain as belonging to the species E. mundtii. for a correct species identification (Manero and Blanch, However, since we found high similarity values also for other 2002). related Enterococcus species we analysed the 16S rDNA Analysing the 16S rDNA sequence of Enterococcus sp., sequence of the strains with the aim to verify the presence we found the same segment as the sequence of E. mundtii of nucleotide fragments used as species- specific probes for probe (5?-CACCGGGAAAAGAGGAGTGG-3?) (Stackebrant E. mundtii. and Goodfellow, 1991; Manero and Blanch, 2002). So, Indeed the use of probes for genes coding for ribosomal according to the principle of DNA-DNA hybridisation, we can ribonucleic acid (rRNA) offers a great potential in microbio- suppose that strain under investigation can hybridise with the logical identification. The rRNA molecules, in particular 16S species-specific probes of E. mundtii. Enteroccoccus sp. Enteroccoccus mundtii Enteroccoccus hirae Enteroccoccus durans Enteroccoccus faecium Enteroccoccus azikeevi Enteroccoccus villorum Enteroccoccus.pseudoavium Enteroccoccus faecalis Enteroccoccus ratti Enteroccoccus raffinosus Enteroccoccus devriesei Enteroccoccus phoeniculicola Enteroccoccus caccae Enteroccoccus rotate Enteroccoccus moraviensis Enteroccoccus casseliflavus Enteroccoccus gallinarum Enteroccoccus sulfureus Enteroccoccus cecorum Enteroccoccus saccharolyticus Enteroccoccus dispar Enteroccoccus avium Enteroccoccus malodoratus 204 C. Fei et al. TABLE 3 – The biochemical characteristics of isolated Enterococcus sp. ed from plants, and silkworms living on leaves of mulberry, so we deduced those silkworms were infected through leaves Characteristics Results Characteristics Results of mulberry. Enterococcus is the main bacterial group (Tak- izawa and Iizuka, 1968) that can participate in the physio- Sucrose + Galactose + logic metabolism of hosts, and the important indispensable Maltose + Trehalose + part of normal physiologic activities. It also was the main Sorbitol + Raffinose – pathogen of silkworm bactericidal flacherie diseases Lactose + D-Ribose + (Lysenko, 1958). Lu et al. (1999, 2002, 2003) analysed and classified the enterococcal flora of the intestine of healthy, Glucose + Arabinose + pebrine and bacterial flacherie diseased silkworms by using Amylum – Rhamnose – API 20 STREP (V.5.0) system based on numerical taxonomy; Fructose + Bile aesculin + Wang et al. (2005) analysed the DNA of the enterococci iso- Mannitol + Serum inulin – lated from the intestine of the silkworm by the random amplified polymorphic DNA (RAPD) technique, they all did- Mannitose + n’t find E.mundtii in the intestine of silkworms. Therefore, this is the first report concerning the presence of E. mundtii in the intestine of silkworm. It complemented the research results for enterococcal flora of silkworms intestine, not only Biochemical tests supply the new microbial sources for prevention and cure of Results of biochemical tests have been presented in Table 3. Nosema bombycis but also are very important to investigate We used the most common biochemical tests used to char- the physiologic metabolism of silkworms and prevention and acterize and identify the isolate in lour lab. The tested strain cure of bacterial flacherie diseases. showed positive results for sucrose, maltose, sorbitol, lac- tose, glucose, fructose, mannitol, mannitose, galactose, tre- Acknowledgements halose, D-ribose, arabinose, aesculin, while there were neg- This work was supported by grants from National Natural Sci- ative results for amylum, raffinose, rhamnose and inulin. ence Foundation of China (No. 30471311 and 30070578). The results obtained by us for Enterococcus mundtii are in consistence with the Bergey’s Manual of Determinative REFERENCES Bacteriology (Holt and Krieg, 1994). Key reactions that facil- itated differentiation of E. mundtii from E. faecalis and E. fae- Collins M.D., John A., Farrow E., Jones D. (1986). Enterococcus cium were arabinose, sorbitol and raffinose utilization (Fack- mundtii. nov. Int. J. Syst. Bacteriol., 36 (1): 8-12. lam and Colins, 1989). Dong X., Cai M. (2001). Manual of System Determinative Com- Our results were different from that of numerical identi- mon Bacteriology. Science Press, Beijing, pp. 370-398. fication by API 20 STREP (V.5.0, BioMerieux S.A.). Of the 17 Drancourt M., Bollet C., Carlioz R., (2000). 16S ribosomal DNA tests in our experiment, the strain fermented sorbitol and sequence analysis of a large collection of environmental and arabinose, but didn’t ferment raffinose. We compared the clinical unidentifiable bacterial isolates. J. Clin. Microbiol., 38: 3623-3630. results for the isolate with the characteristics of E. mundtii from Bergey’s Manual of Determinative Bacteriology (Holt Facklam R.R., Collins M.D. (1989). Identification of Enterococcus species isolated from human infections by a conventional test and Krieg, 1994) and other reports (Collins et al., 1986; scheme. J. Clin. Microbiol., 27: 731-734. Kaufhold and Ferrieri, 1991; Manero and Blanch, 1999; Higashide T., Takahashi M., Kobayashi A. (2005). 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Annals of Microbiology – Springer Journals

Published: Nov 20, 2009

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