J Clin Pathol: Mol Pathol 2000;53:211–215 211 Identification by 16S ribosomal RNA gene

sequencing of an 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 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). Figure 2 Table 1 Oligonucleotides used for sequencing the 16S shows the base sequences of the purified band ribosomal RNA gene and the corresponding region in E coli K-12, S typhimurium, and S typhi. There was only Oligonucleotides Base sequence one base diVerence between the isolate and E LPW57 5'-AGTTTGATCCTGGCTCAG-3' coli K-12, but 48 and 47 base diVerences LPW58 5'-AGGCCCGGGAACGTATTCAC-3' between the isolate and S typhimurium (NCTC LPW69 5'-AGCACCGGCTAACTCCGT-3' 8391) and (St111), respectively, show- LPW70 5'-AGTTTTAACCTTGCGG-3' S typhi ing that the isolate was a strain of E coli.

www.molpath.com Enterobacteriaceae species in a bone marrow transplant recipient 213

Vancomycin resistant Staphylococcus aureus M 12 strains have been found in patients with

leukaemia, lung cancer, stomach cancer, myco- Mol Path: first published as 10.1136/mp.53.4.211 on 1 August 2000. Downloaded from sis fungoides, chronic renal failure, and diabetes mellitus17 18; and multiresistant species of pseudomonas and acinetobacter are usually found in immunocompromised patients in intensive care units.19 and human herpesvirus 8 were important emerging and re-emerging pathogens in patients infected with human immunodeficiency virus. Emerg- ing pathogens, such as Ochrobactrum interme- dium and parisiensis, have been 1380 bp isolated and identified using 16S rRNA sequencing in liver transplant recipients.20 21 Bone marrow transplantation is a situation where profound suppression of both the innate and adaptive immunity occurs, and the gastro- intestinal tracts of bone marrow transplant recipients contain numerous species of micro- organisms. Cytotoxic agents and other selective pressures will lead to a high frequency of Figure 1 DNA products from PCR of 16S ribosomal induced mutations in the microorganisms in gene. Lane M, molecular marker SPP1 EcoRI digest; lane the gastrointestinal tract of bone marrow 1, bacterial isolate from bone marrow transplant recipient; lane 2, negative control containing DNase I treated distilled transplant recipients and acquisition of genes water. from other microorganisms in the surround- ings that can result in a survival advantage for Discussion the bacteria. Immunocompromised hosts have always been We describe a strain of the Enterobacte- one of the most important sources of emerging riaceae family isolated from the gastro- pathogens and novel antimicrobial resistance. intestinal tract of a bone marrow transplant

Table 2 Biochemical profile and identification of the isolate from the BMT recipient by conventional biochemical tests, the Vitek GNI+ system, and the API 20E system

Biochemical reactions/enzymes Conventional Vitek GNI+ API 20E Motility +

Growth on MacConkey agar + http://mp.bmj.com/ Nitrate reduction + + Cytochrome oxidase – â-Galactosidase + + + Arginine dihydrolase – – – Lysine decarboxylase + + + Ornithine decarboxylase + + + Citrate utilisation – – – Malonate utilisatiion – – Acetamide utilisation – H S+++

2 on October 2, 2021 by guest. Protected copyright. Urease – – – Tryptophan deaminase – – – Indole + + Acetoin – – Gelatinase – – Glucose fermentation + + Glucose oxidation + + Fermentation/oxidation of Glucose + Mannitol + + + Inositol – – – Sorbitol + + + Rhamnose + + + Sucrose – – – Melibiose – – Amygdalin – Arabinose + + + Lactose – – Maltose + + Xylose + + RaYnose – – Adonitol – – Dulcitol – Salicin – Indoxyl-â-D-glucoside metabolism – Glucose fermentation in the presence of p-coumaric + Esculin hydrolysis – Polymyxin B resistance – 2,4,4'-Trichloro-2'-hydroxydiphenylether resistance – Identification Enterobacteriaceae Salmonella arizonae 73% 76% Salmonella spp 17% Salmonella arizonae 23%

BMT, bone marrow transplant.

www.molpath.com 214 Woo, Leung, Leung, et al

1 721 S typhimurium ------AGA GTTTGATCCT GGCTCAGATT GAACGCTGGC GGCAGGCCTA ACACATGCAA S typhimurium GGTGGCGAAG GCGGCCCCCT GGACAAAGAC TGACGCTCAG GTGCGAAAGC GTGGGGAGCA S typhi ------AGA GTTTGATCCT GGCTCAGATT GAACGCTGGC GGCAGGCCTA ACACATGCAA S typhi GGTGGCGAAG GCGGCCCCCT GGACAAAGAC TGACGCTCAG GTGCGAAAGC GTGGGGAGCA E coli K-12 AAATTGAAGA GTTTGATCAT GGCTCAGATT GAACGCTGGC GGCAGGCCTA ACACATGCAA E coli K-12 GGTGGCGAAG GCGGCCCCCT GGACGAAGAC TGACGCTCAG GTGCGAAAGC GTGGGGAGCA

Isolate ------A GTTTGATCAT GGCTCAGATT GAACGCTGGC GGCAGGCCTA ACACATGCAA Isolate GGTGGCGAAG GCGGCCCCCT GGACGAAGAC TGACGCTCAG GTGCGAAAGC GTGGGGAGCA Mol Path: first published as 10.1136/mp.53.4.211 on 1 August 2000. Downloaded from

61 781 S typhimurium GTCGAACGGT AACAGGAAGC AGCTTGCTGC TTTGCTGACG AGTGGCGGAC GGGTGAGTAA S typhimurium AACAGGATTA GATACCCTGG TAGTCCACGC CGTAAACGAT GTCTACTTGG AGGTTGTGCC S typhi GTCGAACGGT AACAGGAAGC AGCTTGCTGC TTTGCTGACG AGTGGCGGAC GGGTGAGTAA S typhi AACAGGATTA GATACCCTGG TAGTCCACGC CGTAAACGAT GTCTACTTGG AGGTTGTGCC E coli K-12 GTCGAACGGT AACAGGAAGA AGCTTGCTTC TTTGCTGACG AGTGGCGGAC GGGTGAGTAA E coli K-12 AACAGGATTA GATACCCTGG TAGTCCACGC CGTAAACGAT GTCGACTTGG AGGTTGTGCC Isolate GTCGAACGGT AACAGGAAGA AGCTTGCTGC TTTGCTGACG AGTGGCGGAC GGGTGAGTAA Isolate AACAGGATTA GATACCCTGG TAGTCCACGC CGTAAACGAT GTCGACTTGG AGGTTGTGCC

121 841 S typhimurium TGTCTGGGAA ACTGCCTGAT GGAGGGGGAT AACTACTGGA AACGGTGGCT AATACCGCAT S typhimurium CTTGAGGCGT GGCTTCCGGA GCTAACGCGT TAAGTAGACC GCCTGGGGAG TACGGCCGCA S typhi TGTCTGGGAA ACTGCCTGAT GGAGGGGGAT AACTACTGGA AACGGTGGCT AATACCGCAT S typhi CTTGAGGCGT GGCTTCCGGA GCTAACGCGT TAAGTAGACC GCCTGGGGAG TACGGCCGCA E coli K-12 TGTCTGGGAA ACTGCCTGAT GGAGGGGGAT AACTACTGGA AACGGTAGCT AATACCGCAT E coli K-12 CTTGAGGCGT GGCTTCCGGA GCTAACGCGT TAAGTCGACC GCCTGGGGAG TACGGCCGCA Isolate TGTCTGGGAA ACTGCCTGAT GGAGGGGGAT AACTACTGGA AACGGTAGCT AATACCGCAT Isolate CTTGAGGCGT GGCTTCCGGA GCTAACGCGT TAAGTCGACC GCCTGGGGAG TACGGCCGCA

181 901 S typhimurium AACGTCGCAA GACCAAAGAG GGGGACCTTC GGGCCTCTTG CCATCAGATG TGCCCAGATG S typhimurium AGGTTAAAAC TCAAATGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT GTGGTTTAAT S typhi AACGTCGCAA GACCAAAGAG GGGGACCTTC GGGCCTCTTG CCATCAGATG TGCCCAGATG S typhi AGGTTAAAAC TCAAATGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT GTGGTTTAAT E coli K-12 AACGTCGCAA GACCAAAGAG GGGGACCTTC GGGCCTCTTG CCATCGGATG TGCCCAGATG E coli K-12 AGGTTAAAAC TCAAATGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT GTGGTTTAAT Isolate AACGTCGCAA GACCAAAGAG GGGGACCTTC GGGCCTCTTG CCATCGGATG TGCCCAGATG Isolate AGGTTAAAAC TCAAATGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT GTGGTTTAAT

241 961 S typhimurium GGATTAGCTT GTTGGTGAGG TAACGGCTCA CCAAGGCGAC GATCCCTAGC TGGTCTGAGA S typhimurium TCGATGCAAC GCGAAGAACC TTACCTGGTC TTGACATCCA CAGAACTTTC CAGAGATGGA S typhi GGATTAGCTT GTTGGTGAGG TAACGGCTCA CCAAGGCGAC GATCCCTAGC TGGTCTGAGA S typhi TCGATGCAAC GCGAAGAACC TTACCTGGTC TTGACATCCA CAGAACTTTC CAGAGATGGA E coli K-12 GGATTAGCTA GTAGGTGGGG TAACGGCTCA CCTAGGCGAC GATCCCTAGC TGGTCTGAGA E coli K-12 TCGATGCAAC GCGAAGAACC TTACCTGGTC TTGACATCCA CGGAAGTTTT CAGAGATGAG Isolate GGATTAGCTA GTAGGTGGGG TAACGGCTCA CCTAGGCGAC GATCCCTAGC TGGTCTGAGA Isolate TCGATGCAAC GCGAAGAACC TTACCTGGTC TTGACATCCA CGGAAGTTTT CAGAGATGAG

301 1021 S typhimurium GGATGACCAG CCACACTGGA ACTGAGACAC GGTCCAGACT CCTACGGGAG GCAGCAGTGG S typhimurium TTGGTTCCTT CGGGAACTGT GAGACAGGTG CTGCATGGCT GTCGTCAGCT CGTGTTGTGA S typhi GGATGACCAG CCACACTGGA ACTGAGACAC GGTCCAGACT CCTACGGGAG GCAGCAGTGG S typhi TTGGTGCCTT CGGGAACTGT GAGACAGGTG CTGCATGGCT GTCGTCAGCT CGTGTTGTGA E coli K-12 GGATGACCAG CCACACTGGA ACTGAGACAC GGTCCAGACT CCTACGGGAG GCAGCAGTGG E coli K-12 AATGTGCCTT CGGGAACCGT GAGACAGGTG CTGCATGGCT GTCGTCAGCT CGTGTTGTGA Isolate GGATGACCAG CCACACTGGA ACTGAGACAC GGTCCAGACT CCTACGGGAG GCAGCAGTGG Isolate AATGTGCCTT CGGGAACCGT GAGACAGGTG CTGCATGGCT GTCGTCAGCT CGTGTTGTGA

361 1081 S typhimurium GGAATATTGC ACAATGGGCG CAAGCCTGAT GCAGCCATGC CGCGTGTATG AAGAAGGCCT S typhimurium AATGTCGGGT TAAGTCCCGC AACGAGCGCA ACCCTTATCC TTTGTTGCCA GCGGTTAGGC S typhi GGAATATTGC ACAATGGGCG CAAGCCTGAT GCAGCCATGC CGCGTGTATG AAGAAGGCCT S typhi AATGTCGGGT TAAGTCCCGC AACGAGCGCA ACCCTTATCC TTTGTTGCCA GCGGTCCGGC E coli K-12 GGAATATTGC ACAATGGGCG CAAGCCTGAT GCAGCCATGC CGCGTGTATG AAGAAGGCCT E coli K-12 AATGTTGGGT TAAGTCCCGC AACGAGCGCA ACCCTTATCC TTTGTTGCCA GCGGTCCGGC Isolate GGAATATTGC ACAATGGGCG CAAGCCTGAT GCAGCCATGC CGCGTGTATG AAGAAGGCCT Isolate AATGTTGGGT TAAGTCCCGC AACGAGCGCA ACCCTTATCC TTTGTTGCCA GCGGTCCGGC

421 1141 S typhimurium TCGGGTTGTA AAGTACTTTC AGCGGGGAGG AAGGTGTTGT GGTTAATAAC CGCAGCAATT S typhimurium CGGGAACTCA AAGGAGACTG CCAGTGATAA ACTGGAGGAA GGTGGGGATG ACGTCAAGTC S typhi TCGGGTTGTA AAGTACTTTC AGCGGGGAGG AAGGTGTTGT GGTTAATAAC CGCAGCAATT S typhi CGGGAACTCA AAGGAGACTG CCAGTGATAA ACTGGAGGAA GGTGGGGATG ACGTCAAGTC E coli K-12 TCGGGTTGTA AAGTACTTTC AGCGGGGAGG AAGGGAGTAA AGTTAATACC TTTGCTCATT E coli K-12 CGGGAACTCA AAGGAGACTG CCAGTGATAA ACTGGAGGAA GGTGGGGATG ACGTCAAGTC Isolate TCGGGTTGTA AAGTACTTTC AGCGGGGAGG AAGGGAGTAA AGTTAATACC TTTGCTCATT Isolate CGGGAACTCA AAGGAGACTG CCAGTGATAA ACTGGAGGAA GGTGGGGATG ACGTCAAGTC

481 1201 S typhimurium GACGTTACCC GCAGAAGAAG CACCGGCTAA CTCCGTGCCA GCAGCCGCGG TAATACGGAG S typhimurium ATCATGGCCC TTACGACCAG GGCTACACAC GTGCTACAAT GGCGCATACA AAGAGAAGCG S typhi GACGTTACCC GCAGAAGAAG CACCGGCTAA CTCCGTGCCA GCAGCCGCGG TAATACGGAG S typhi ATCATGGCCC TTACGACCAG GGCTACACAC GTGCTACAAT GGCGCATACA AAGAGAAGCG E coli K-12 GACGTTACCC GCAGAAGAAG CACCGGCTAA CTCCGTGCCA GCAGCCGCGG TAATACGGAG E coli K-12 ATCATGGCCC TTACGACCAG GGCTACACAC GTGCTACAAT GGCGCATACA AAGAGAAGCG Isolate GACGTTACCC GCAGAAGAAG CACCGGCTAA CTCCGTGCCA GCAGCCGCGG TAATACGGAG Isolate ATCATGGCCC TTACGACCAG GGCTACACAC GTGCTACAAT GGCGCATACA AAGAGAAGCG

541 1261 S typhimurium GGTGCAAGCG TTAATCGGAA TTACTGGGCG TAAAGCGCAC GCAGGCGCTC TGTCAAGTCG S typhimurium ACCTCGCGAG AGCAAGCGGA CCTCATAAAG TGCGTCGTAG TCCGGATTGG AGTCTGCAAC S typhi GGTGCAAGCG TTAATCGGAA TTACTGGGCG TGAAGCGCAC GCAGGCGGTC TGTCAAGTCG S typhi ACCTCGTGAG AGCAAGCGGA CCTCATAAAG TGCGTCGTAG TCCGGATTGG AGTCTGCAAC E coli K-12 GGTGCAAGCG TTAATCGGAA TTACTGGGCG TAAAGCGCAC GCAGGCGGTT TGTTAAGTCA E coli K-12 ACCTCGCGAG AGCAAGCGGA CCTCATAAAG TGCGTCGTAG TCCGGATTGG AGTCTGCAAC Isolate GGTGCAAGCG TTAATCGGAA TTACTGGGCG TAAAGCGCAC GCAGGCGGTT TGTTAAGTCA Isolate ACCTCGCGAG AGCAAGCGGA CCTCATAAAG TGCGTCGTAG TCCGGATTGG AGTCTGCAAC

601 1321 S typhimurium GATGTGAAAT CCCCGGGCTC AACCTGGGAA CTGCATTCGA AACTGGCAGG CTTGAGTCTT S typhimurium TCGACTCCAT GAAGTCGGAA TCGCTAGTAA TCGTGGATCA GAATGCCACG GTGAATACGT S typhi GATGTGAAGT CCCCGGGCTC AACCTGGGAA CTGCATTCGA AACTGGCAGG CTTGAGTCTT S typhi TCGACTCCAT GAAGTCGGAA TCGCTAGTAA TCGTGGATCA GAATGCCACG GTGAATACGT E coli K-12 GATGTGAAAT CCCCGGGCTC AACCTGGGAA CTGCATCTGA TACTGGCAAG CTTGAGTCTC E coli K-12 TCGACTCCAT GAAGTCGGAA TCGCTAGTAA TCGTGGATCA GAATGCCACG GTGAATACGT Isolate GATGTGAAAT CCCCGGGCTC AACCTGGGAA CTGCATCTGA TACTGGCAAG CTTGAGTCTC Isolate TCGACTCCAT GAAGTCGGAA TCGCTAGTAA TCGTGGATCA GAATGCCACG GTGAATACGT

661 1381 http://mp.bmj.com/ S typhimurium GTAGAGGGGG GTAGAATTCC AGGTGTAGCG GTGAAATGCG TAGAGATCTG GAGGAATACC S typhimurium TCCCGGGCCT S typhi GTAGAGGGGG GTAGAATTCC AGGTGTAGCG GTGAAATGCG TAGAGATCTG GAGGAATACC S typhi TCCCGGGCCT E coli K-12 GTAGAGGGGG GTAGAATTCC AGGTGTAGCG GTGAAATGCG TAGAGATCTG GAGGAATACC E coli K-12 TCCCGGGCCT Isolate GTAGAGGGGG GTAGAATTCC AGGTGTAGCG GTGAAATGCG TAGAGATCTG GAGGAATACC Isolate TCCCGGGCCT Figure 2 DNA sequences of the 16S rRNA gene of the isolate from the bone marrow transplant recipient, Escherichia coli K-12, Salmonella typhimurium (NCTC 8391), and Salmonella typhi (St111). The shaded bases represent those in the isolate that are diVerent from the corresponding ones in E coli K-12, S typhimurium (NCTC 8391), or S typhi (St111).

recipient. Conventional biochemical tests The identification of the organism in our on October 2, 2021 by guest. Protected copyright. failed to diVerentiate between species of E coli study was important because the management and salmonella. Reactions not favouring of the patient would be radically diVerent salmonella included metabolism of ortho- depending on which organism was identified. If nitrophenyl galactoside (ONPG) (2%) and the organism is a strain of E coli (as it was in our production of indole (1%). The only ONPG patient) no treatment is needed. This is positive salmonellae are S arizonae, S diarizo- because of evidence that, despite persistent nae, S bongori, and S indica, but the isolate was recovery of the bacteria in routine surveillance malonate positive (which was not in favour of cultures in the first and third weeks after bone S bongori and S indica), gelatinase negative marrow transplantation, the patient’s diarrhoea will subside spontaneously without any specific (which was not in favour of S arizonae and treatment. On the other hand, if the organism S diarizonae), and did not agglutinate with any turns out to be an ampicillin, cotrimoxazole, “individual” O or H salmonella antisera. Fur- and ciprofloxacin resistant species of salmo- thermore, none of the four salmonella species nella, ceftriaxone would have to be adminis- produces indole. Reactions not favouring tered to the patient to prevent the development E coli include the production of hydrogen sul- of infection in the gastrointestinal tract or inva- phide and the agglutination of poly O and poly sion into the systemic circulation. H salmonella antisera. Serotyping for EPEC 16S rRNA sequencing will continue to be and the Sereny test for ETEC were negative. the gold standard for bacterial identification. Furthermore, although is With the help of additional tests, the Vitek famous for crossreacting with poly O and poly GNI+ and API 20E systems were only able to H antisera, reactions that preclude it include identify 80.1–94.4% and 95.6–98.6%, respec- citrate utilisation, production of indole, and tively, of Enterobacteriaceae and common production of lysine decarboxylase. non-glucose fermenting Gram negative

www.molpath.com Enterobacteriaceae species in a bone marrow transplant recipient 215

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