Annals of Microbiology, 56 (3) 201-205 (2006)
Identification of Enterococcus sp. from midgut of silkworm based on biochemical and 16S rDNA sequencing analysis Chen FEI1, 2, Xing-meng LU2*, Yong-hua QIAN1, Haiyan ZHANG1,2, Qaisar MAHMOOD3 1College 2College
of Animal Science and Technology, Northwest Agricultural and Forestry University, Yang Ling 712100, China; of Animal Sciences, Zhejiang University, 3College 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. Identification 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 Enterococcus 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 Nosema bombycis is the most intimidatory and damning disease for sericulture. It was the only quarantinable object of silkworm eggs production because it has the character of germinative infection. The prevention and cure of this disease is a very important technical problem and imperative to be solved for better sericulture production. Nosema bombycis spores germination in the silkworms’ intestines is the basic condition for its infection in silkworms (Iwano and Ishihar, 1989). A large number of enterococci were found in intestinal tract of silkworms (Takizawa and Iizuka, 1968). Enterococcus spp. is normally not pathogenic to silkworms (Lu et al., 1999) and, interestingly, they can inhibit germination of N. bombycis spores (Lu and Wang, 2002). There exists great physiological and biochemical diversity among the Enterococcus species (Lu et al., 1994, 1999). This antimicroaporidian property and polymorphism of enterococci make them an interesting candidate for biological control of silkworm diseases. Studies on Enterococcus sp. diversity are helpful to understand their phenotypic differences and their possible relationship with N. bombycis. The identification and characterization of Enterococcus from silkworms may have the practical meaning of prevention and cure of nosogenesis in the silk industry. In previous investigation two hundred strains of entero-
* Corresponding author. Phone: 0086-0571-86971305; E-mail:
[email protected]
cocci from the intestine of healthy, pebrine and bacterial flacherie diseased silkworms were isolated and characterized by using API 20 STREP (V.5.0) system (Lu et al., 1999, 2003). The traditional biochemical identification has however some disadvantages, especially concerning the instability of the phenotypic expression and a low sensitivity; moreover, it is a time consuming and tedious process. Especially, Enterococcus species show both species- and strain-specific biochemical diversities (Lu et al., 1994, 2003) that bring difficulty to identify Enterococcus through biochemical tests. Though the bacterial automating identification systems are suitable for the rapid identification, the model bacterial strains in database are limited. Moreover the classification of Enterococcus is changing frequently due to more recent discoveries. If database is not updated regularly to include recent discoveries, inaccurate results might be achieved. At present, the molecular methods for bacterial identification are based upon the sequences of 16S rDNA. Sequencing of 16S rDNA in conjunction with homology searching and constructing the phylogenic tree are powerful means to identify Enterococcus. Studying bacteria through the genetic and evolutionary point of view and classifying them on the molecular level not only can confirm the classification but also reveal the existing homology between wild and reference strains. Moreover, analysing the sequences of 16S rDNA can help us to find species-specific probes to be used for identification and practical purposes. The objectives of our study were to detect Enterococcus sp. having anti-microaporidian property. To this end, biochemical tests, phylogenetic analysis based on the 16S rDNA
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sequence and analysis of Enterococcus species-specific probes were carried out on one strain isolated from the midgut of healthy silkworms.
MATERIALS AND METHODS Strain source and culture conditions. The Enterococcus sp. was isolated from the midgut of healthy silkworms. To grow the isolate, decimally diluted digestive juices of healthy silkworms (0.5 ml) were mixed with the EF agar (Nissui, Tokyo, Japan) supplemented with 10 µg/mL vancomycin and were incubated at 35-37 °C for 48 h. The developed colonies were peach, brown or yellowish in colour. Single colony was selected from that EF plates, and shifted on to the Nutrient Broth. The same procedure was repeated 3 times. Liquid or solid Nutrient media were used for preserving the isolates for experimental purpose. Before DNA extraction, strains were grown aerobically in Brain Heart Infusion Broth overnight at 37 °C. Sequencing and analysis of 16S rDNA. Preparation of the DNA sample. The genomic DNA was extracted from the 50 ml liquid Brain Heart Infusion Broth medium according to protocols of Wang et al. (2005). PCR amplification of 16S rDNA. 27f and 1492r were selected from the universal primers shown in Table 1 (Stackebrant and Goodfellow, 1991). Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., China, synthesized the primers. The 16S rDNA fragment was amplified in a Perkin-Elmer DNA thermal cycler with the following program: 30 s at 94 °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 confirmed by electrophoresis analysing 5 µl PCR reaction mixtures 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. Company Japan, and sequence was submitted to GenBank.
teristics were analysed and phylogenetic tree was constructed by Mega 2 software. Analysis of the species-specific probes of Enterococcus species. The sequences of 16S rDNA of tested strain and species-specific probes of Enterococcus species were compared in order to find out the corresponding probe of this strain. Biochemical tests. The utilization of sugars under aerobic and anaerobic conditions was tested by the mellow test through bacterial minimum biochemical reaction tubes (Hangzhou Microbial Reagent Co., Ltd., China) in term of routine ways and means (Dong and Cai, 2001). The tests were conducted for sucrose, maltose, sorbitol, lactose, glucose, amylum, fructose, mannitol, mannitose, galactose, trehalose, raffinose, D-ribose, arabinose, rhamnose, aesculin and inulin.
RESULTS AND DISCUSSIONS Phylogenetic analysis and species-specific probe The results of the phylogenetic analysis are reported in Fig. 1 and Table 2. The tested strain was phylogenetically closely related to Enterococcus mundtii (100% sequence similarity), Enterococcus hirae (99%) and Enterococcus durans, Enterococcus faecium, Enterococcus azikeevi, Enterococcus villorum (98%). Modern bacterial taxonomy is inclined to use genotypic methods together with phenotype characteristics to decide the position of bacterial species within a phylogenetic tree, according to the 16S rDNA sequence. Presently the acceptable positional standard is that if the similarity of strain
Phylogenetic analysis. The 16S rDNA sequences were aligned and paralleled with the sequence of corresponding bacterium in GenBank by Blast software. The homologous charac-
TABLE 1 – List of primers Name
Sequence (5’-3’)a
27f
AGAGTTTGATCMTGGCTCAG
519r
GWATTACCGCGGCKGCTG
530f
GTGCCAGCMGCCGCGG
907r
CCGTCAATTCMTTTRAGTTT
926f
AAACTYAAAKGAATTGACGG
1492r
TACGGYTACCTTGTTACGACTT
a
M= C: A, Y=C: T, K=G:T, W=A:T (Stackebrant and Goodfellow,1991).
FIG. 1 – Phylogenetic tree based on 16S ribosomal DNA sequences Numbers in parentheses represent the sequences’ accession number in GenBank. Numbers in square indicate the clone number out of the total clones. The numbers next to the nodes represent the bootstrap values of 1000 replications, are given at branch points. The scale bar indicates 0.005 substitutions per nucleotide position.
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TABLE 2 – Similarity of Enterococcus spp. with other species on the basis of 16S rDNA Similarity (%)
Enteroccoccus hirae Enteroccoccus durans Enteroccoccus faecium Enteroccoccus azikeevi Enteroccoccus villorum Enteroccoccus pseudoavium
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
Enteroccoccus mundtii
Enteroccoccus hirae
Enteroccoccus sp.
Enteroccoccus mundtii
Enteroccoccus sp.
Strains
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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
under investigation and a reference strain sequences is 99100%, they are regarded as belonging to the same species while if similarity is 97-98%, they are regarded as belonging to the same genus (Drancourt et al., 2000; Janda and Abbott, 2002). According to the standard above, since the similarity of whole 16S rDNA sequences between strain under investigation and E. mundtii was 100% we can consider the tested strain as belonging to the species E. mundtii. However, since we found high similarity values also for other related Enterococcus species we analysed the 16S rDNA sequence of the strains with the aim to verify the presence of nucleotide fragments used as species- specific probes for E. mundtii. Indeed the use of probes for genes coding for ribosomal ribonucleic acid (rRNA) offers a great potential in microbiological identification. The rRNA molecules, in particular 16S
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rRNA and 23S rRNA, contain highly conserved regions common to all eubacteria as well as highly variable regions unique to a particular species. Thus, universal probes or primers that will anneal to the genes coding for rRNA of all eubacteria can be designed from the conserved regions of the genes for 16S rRNA. A newly developed set of probes targeting the 16S rDNA gene served as a confirmation method for a correct species identification (Manero and Blanch, 2002). Analysing the 16S rDNA sequence of Enterococcus sp., we found the same segment as the sequence of E. mundtii probe (5?-CACCGGGAAAAGAGGAGTGG-3?) (Stackebrant and Goodfellow, 1991; Manero and Blanch, 2002). So, according to the principle of DNA-DNA hybridisation, we can suppose that strain under investigation can hybridise with the species-specific probes of E. mundtii.
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TABLE 3 – The biochemical characteristics of isolated Enterococcus sp. Characteristics Sucrose
Results +
Characteristics
Results
Galactose
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Maltose
+
Trehalose
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Sorbitol
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Raffinose
–
Lactose
+
D-Ribose
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Glucose
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Arabinose
+
Amylum
–
Rhamnose
–
Fructose
+
Bile aesculin
+
Mannitol
+
Serum inulin
–
Mannitose
+
Biochemical tests Results of biochemical tests have been presented in Table 3. We used the most common biochemical tests used to characterize and identify the isolate in lour lab. The tested strain showed positive results for sucrose, maltose, sorbitol, lactose, glucose, fructose, mannitol, mannitose, galactose, trehalose, D-ribose, arabinose, aesculin, while there were negative results for amylum, raffinose, rhamnose and inulin. The results obtained by us for Enterococcus mundtii are in consistence with the Bergey’s Manual of Determinative Bacteriology (Holt and Krieg, 1994). Key reactions that facilitated differentiation of E. mundtii from E. faecalis and E. faecium were arabinose, sorbitol and raffinose utilization (Facklam and Colins, 1989). Our results were different from that of numerical identification by API 20 STREP (V.5.0, BioMerieux S.A.). Of the 17 tests in our experiment, the strain fermented sorbitol and arabinose, but didn’t ferment raffinose. We compared the results for the isolate with the characteristics of E. mundtii from Bergey’s Manual of Determinative Bacteriology (Holt and Krieg, 1994) and other reports (Collins et al., 1986; Kaufhold and Ferrieri, 1991; Manero and Blanch, 1999; Higashide et al., 2005). The isolate did not ferment raffinose, while the majority of the reported strains did. However, other biochemical test results for the isolate were highly consistent with those for other E. mundtii strains. The inability of API 20 Strep to identify the species E. mundtii could be related to the absence of this species in the API database (version 5.0). Misidentification by commercial biochemical assays, which also happened in our case, may account for the scarcity of reports on this organism (Kaufhold and Ferrieri, 1991). Relying on commercially available identification systems, whose databases do not include the more recent taxonomic changes, may lead to inaccurate identifications.
CONCLUSION Enterococcus mundtii was discovered in 1986 as nonmotile, yellow-pigmented bacterium isolated from cow teats, the hands of milkers, soil, and plants (Collins et al., 1986). Since then the species has rarely been isolated from environmental (Junco et al., 2001) or human sources (Facklam and Collins,1989). According to the sequences of 16S rDNA and species-specific probes of Enterococcus, we identified an isolated strain as E. mundtii. Because E. mundtii was isolat-
ed from plants, and silkworms living on leaves of mulberry, so we deduced those silkworms were infected through leaves of mulberry. Enterococcus is the main bacterial group (Takizawa and Iizuka, 1968) that can participate in the physiologic metabolism of hosts, and the important indispensable part of normal physiologic activities. It also was the main pathogen of silkworm bactericidal flacherie diseases (Lysenko, 1958). Lu et al. (1999, 2002, 2003) analysed and classified the enterococcal flora of the intestine of healthy, pebrine and bacterial flacherie diseased silkworms by using API 20 STREP (V.5.0) system based on numerical taxonomy; Wang et al. (2005) analysed the DNA of the enterococci isolated from the intestine of the silkworm by the random amplified polymorphic DNA (RAPD) technique, they all didn’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 supply the new microbial sources for prevention and cure of Nosema bombycis but also are very important to investigate the physiologic metabolism of silkworms and prevention and cure of bacterial flacherie diseases. Acknowledgements This work was supported by grants from National Natural Science Foundation of China (No. 30471311 and 30070578).
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