J Clin Pathol: Mol Pathol 2000;53:211–215 211 Identification by 16S ribosomal RNA gene sequencing of an Enterobacteriaceae species from Mol Path: first published as 10.1136/mp.53.4.211 on 1 August 2000. Downloaded from a bone marrow transplant recipient PCYWoo,PKLLeung, K W Leung, K Y Yuen Abstract with biochemical characteristics that do not fit Aims—To ascertain the clinical relevance into patterns of any known genus and species. of a strain of Enterobacteriaceae isolated Since the discovery of the polymerase chain from the stool of a bone marrow trans- reaction (PCR) and DNA sequencing, the plant recipient with diarrhoea. The isolate genomes of some bacteria have been se- could not be identified to the genus level quenced completely.1 A comparison of the by conventional phenotypic methods and genomic sequences of bacterial species showed required 16S ribosomal RNA (rRNA) gene that the 16S ribosomal RNA (rRNA) gene is sequencing for full identification. highly conserved within a species and among Methods—The isolate was investigated species of the same genus, and hence can be phenotypically by standard biochemical used as the new gold standard for the methods using conventional biochemical speciation of bacteria. Using this new standard, tests and two commercially available sys- phylogenetic trees based on base diVerences tems, the Vitek (GNI+) and API (20E) sys- between species are constructed; bacteria are tems. Genotypically, the 16S bacterial classified and re-classified into new genera;23 rRNA gene was amplified by the polymer- and classifications of non-cultivable micro- ase chain reaction (PCR) and sequenced. organisms are made possible.45 It can also be The sequence of the PCR product was useful in elucidating the relation of unknown compared with known 16S rRNA gene bacterial species to known ones, and new spe- sequences in the GenBank database by cies of bacteria such as Gemella sanguinis, multiple sequence alignment. Mycobacterium heidelbergense, and Massilis timo- Results—Conventional biochemical tests nae have been discovered using 16S rRNA did not reveal a pattern resembling any sequencing.6–8 Moreover, bacteria such as known member of the Enterobacteriaceae Mycobacterium celatum and Methylobacterium family. The isolate was identified as zatmanii, which were not known to cause Salmonella arizonae (73%) and Es- infections in humans have been identified in http://mp.bmj.com/ cherichia coli (76%) by the Vitek (GNI+) clinical specimens using this technique.910 and API (20E) systems, respectively. 16S Furthermore, bacteria that are diYcult to rRNA sequencing showed that there was identify were speciated successfully using this only one base diVerence between the technique.11 In our study, we report the isolate and E coli K-12, but 48 and 47 base application of such a technique to ascertain the diVerences between the isolate and S ty- clinical relevance of a strain of Enterobacte- phimurium (NCTC 8391) and S typhi riaceae isolated from the stool of a bone (St111), respectively, showing that it was marrow transplant recipient with diarrhoea. on October 2, 2021 by guest. Protected copyright. an E coli strain. The patient did not require any specific treatment and the Methods diarrhoea subsided spontaneously. PATIENT, SPECIMEN COLLECTION, AND Conclusions—16S rRNA gene sequencing MICROBIOLOGICAL METHODS was useful in ascertaining the clinical rel- Specimen collection and microbiological evance of the strain of Enterobacteriaceae methods were described in a previously pub- isolated from the stool of the bone marrow lished paper.12 Briefly, routine surveillance cul- Department of tures of throat and rectal swabs were performed Microbiology, The transplant recipient with diarrhoea. University of Hong (J Clin Pathol: Mol Pathol 2000;53:211–215) weekly in the first 30 days after bone marrow transplantation, and when diarrhoea occurred, Kong, University Keywords: 16S ribosomal RNA sequencing; bone Pathology Building, marrow transplantation stool was collected and cultured for potential Queen Mary Hospital, pathogens. All suspect colonies were identified Pokfulam Road, Hong by standard conventional biochemical Kong, The People’s The identification of bacteria in the clinical methods.13 In addition, the Vitek System Republic of China PCYWoo microbiology laboratory is performed tra- (bioMerieux Vitek, Hazelwood, Missouri, P K L Leung ditionally by isolating the organism and study- USA) and the API system (bioMerieux Vitek) K W Leung ing it phenotypically by means of Gram were used for the identification of the bacterial KYYuen staining, culture, and biochemical methods, isolate in our study. which have been the gold standard of bacterial Correspondence to: Dr Yuen identification. However, these methods of bac- EXTRACTION OF BACTERIAL DNA FOR 16S email: terial identification have two major drawbacks. RIBOSOMAL RNA GENE SEQUENCING [email protected] First, they cannot be used for non-cultivable Bacterial DNA extraction was modified from a 14 Accepted for publication organisms such as Tropheryma whippelii. Sec- published protocol. An aliquot of 80 µl of 20 April 2000 ond, we are occasionally faced with organisms NaOH (0.05 M) was added to 20 µl of www.molpath.com 212 Woo, Leung, Leung, et al bacterial cells suspended in distilled water and multiple sequence alignment using the CLUS- the mixture was incubated at 60°C for 45 min- TAL W program.16 utes, followed by the addition of 6 µl of Mol Path: first published as 10.1136/mp.53.4.211 on 1 August 2000. Downloaded from Tris/HCl (pH 7.0), achieving a final pH of 8.0. The resultant mixture was diluted 100 times Results and 5 µl of the diluted extract was used for PATIENT PCR. The 31 year old patient was diagnosed with acute myeloid leukaemia (M2) in March 1998. The disease was in first complete remission PCR, GEL ELECTROPHORESIS, AND 16S RIBOSOMAL after chemotherapy. She received a syngeneic RNA GENE SEQUENCING bone marrow transplant in July 1998 after con- PCR amplification of the 16S rRNA gene was 15 ditioning with busulfan/cyclophosphamide. modified from a published protocol. DNase I Three days before the bone marrow transplant treated distilled water and PCR master mix she developed diarrhoea. A strain of Entero- (which contains deoxynucleoside triphos- bacteriaceae that produced hydrogen sulphide phates (dNTPs), PCR buVer, and Taq and agglutinated with poly O and poly H polymerase) were used in all PCR reactions by salmonella antisera was isolated from the adding 1 U of DNase I (Pharmacia, Uppsala, patient’s stool on deoxycholate citrate agar and Sweden) to 40 µl of distilled water or PCR xylose lysine deoxycholate agar. The strain was master mix, incubating the mixture at 25°C for persistently recovered from the rectal swabs 15 minutes, and subsequently at 95°C for 10 taken in the first and third weeks after her bone minutes to inactivate the DNase I. The bacte- marrow transplant for surveillance culture, but rial DNA extract and control were amplified not in any other specimens. The marrow with 0.5 µM primers (LPW57, 5'-AGTTT engrafted on day 14 and the bone marrow GATCCTGGCTCAG-3'; and LPW58, 5'- transplant was uneventful. She was discharged AGGCCCGGGAACGTATTCAC-3') (Gibco on day 24. BRL, Rockville, Maryland, USA). The PCR mixture (50 µl) contained bacterial DNA, PCR buVer (10 mM Tris/HCl (pH 8.3), 50 mM IDENTIFICATION OF THE BACTERIAL STRAIN BY KCl, 3 mM MgCl2, and 0.01% gelatin), 200 CONVENTIONAL METHODS AND COMMERCIALLY µM of each dNTP, and 1.0 U Taq polymerase AVAILABLE SYSTEMS (Boehringer Mannheim, Mannheim, Ger- The bacterial strain was a Gram negative, fac- many). The mixtures were amplified for 40 ultative anaerobic rod. It grew on blood agar, cycles at 94°C for one minute, 55°C for one chocolate agar, and MacConkey agar to sizes of minute, and 72°C for two minutes, with a final 4 mm in diameter after 24 hours of incubation extension at 72°C for 10 minutes in an at 37°C in ambient air. It fermented glucose, automated thermal cycler (Perkin-Elmer reduced nitrate, and did not produce cyto- Cetus, Gouda, The Netherlands). DNase I chrome oxidase, typically a member of the treated distilled water was used as the negative Enterobacteriaceae family. Standard conven- http://mp.bmj.com/ control. An aliquot of 10 µl of each amplified tional or commercially available biochemical product was electrophoresed in 1.0% (wt/vol) tests did not reveal a pattern resembling any agarose gel, with a molecular size marker known member of the Enterobacteriaceae (SPP1 EcoRI digest; Boehringer Mannheim) family (table 2). The Vitek system (GNI+) in parallel. Electrophoresis in Tris-borate- showed that it was 73% Salmonella arizonae EDTA buVer was performed at 100 V for 1.5 and 17% Salmonella spp; whereas the API sys- hours. The gel was stained with ethidium bro- tem (20E) showed that it was 76% Escherichia mide (0.5 µg/ml) for 15 minutes, rinsed, and coli and 23% S arizonae. In addition, the isolate on October 2, 2021 by guest. Protected copyright. photographed under ultraviolet light illumina- was motile, and it agglutinated with poly O and tion. poly H salmonella antisera (Murex Biotech The PCR product was gel purified using the Ltd, Temple Hill, Dartford, UK), but did not QIAquick PCR purification kit (Qiagen, agglutinate with any individual O, H, or Vi sal- Hilden, Germany). Both strands of the PCR monella antisera. The strain was resistant to product were sequenced twice with an ABI 310 ampicillin, cephalothin, cefuroxime, gen- automated sequencer according to manufac- tamicin, cotrimoxazole, and ciprofloxacin; but turers’ instructions (Perkin-Elmer, Foster City, sensitive to cefoxitin, ceftriaxone, amoxicillin/ California, USA), using the PCR primers clavulanic acid, ceftazidime, amikacin, and (LPW57 and LPW58) and additional primers imipenem. designed from the sequencing data of the first round of the sequencing reaction (LPW69 and LPW70; table 1). The sequence of the PCR PCR AMPLIFICATION AND 16S RIBOSOMAL RNA product was compared with known 16S rRNA GENE SEQUENCING gene sequences in the GenBank database by PCR of the 16S rRNA gene of the bacteria showed a band at 1380 bp (fig 1).