4 Beatriz Romero - jcm.asm.org 7/13/2011 آ  4 Beatriz Romero 1,2, Marأ­a-Isabel Morosini 1,2,...

download 4 Beatriz Romero - jcm.asm.org 7/13/2011 آ  4 Beatriz Romero 1,2, Marأ­a-Isabel Morosini 1,2, Elena

of 19

  • date post

  • Category


  • view

  • download


Embed Size (px)

Transcript of 4 Beatriz Romero - jcm.asm.org 7/13/2011 آ  4 Beatriz Romero 1,2, Marأ­a-Isabel Morosini 1,2,...

  • 1

    Re-Identification of Streptococcus bovis Isolates Causing Bacteraemia 1

    According with the New Taxonomy Criteria: Still an Issue? 2


    Beatriz Romero 1,2

    , María-Isabel Morosini 1,2

    , Elena Loza 1,2

    , 4

    Mercedes Rodríguez-Baños 1 , Enrique Navas

    3 , Rafael Cantón

    1,2 and Rosa del Campo

    1,2 * 5



    1 Servicio de Microbiología, Hospital Universitario Ramón y Cajal and IRYCIS; Unidad 8

    de Resistencia a Antibióticos y Virulencia Bacteriana asociada al Consejo Superior de 9

    Investigaciones Científicas (CSIC). 2 CIBER en Epidemiología y Salud Pública 10

    (CIBERESP). 3 Servicio de Enfermedades Infecciosas, Hospital Universitario Ramón y 11

    Cajal. Madrid, Spain 12


    *Corresponding author: Mailing address: Servicio de Microbiología, Hospital 14

    Universitario Ramón y Cajal. Ctra. Colmenar, Km 9,1. 28034 Madrid. Spain. 15

    Phone: 34-91 3368542; Fax: 34-91 3368809; e-mail: rosacampo@yahoo.com 16


    Short title: S. bovis re-identification. 18


    Key words: Streptococcus bovis, bacteraemia, 16S rDNA, sodA 20

    Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. J. Clin. Microbiol. doi:10.1128/JCM.00524-11 JCM Accepts, published online ahead of print on 13 July 2011

    on S eptem

    ber 27, 2020 by guest http://jcm

    .asm .org/

    D ow

    nloaded from


  • 2


    All Streptococcus bovis blood culture isolates recovered from Jan-2003 to Jan-2010 22

    (n=52) in our institution were re-identified using new taxonomic criteria based upon 23

    their genetic traits. Initial identification was performed by the semiautomatic WIDER 24

    System (Fco. Soria-Melguizo, Spain) and the API 20 Strep (BioMérieux, France). All 25

    isolates were re-identified by PCR amplification and sequencing of both 16S rDNA and 26

    sodA genes and by mass spectrometry using MALDI-TOF MS (Bruker, Germany). 27

    Results of 16s rDNA/sodA gene sequencing were as follows: Streptococcus gallolyticus 28

    subsp. gallolyticus 14/14, Streptococcus gallolyticus subsp. pasteurianus 24/24, 29

    Streptococcus spp. 7/0, Streptococcus infantarius subsp. infantarius 0/2, Streptococcus 30

    lutetiensis 0/5, Leuconostoc mesenteroides 4/0, and Lactococcus lactis 3/3. The 31

    MALDI-TOF MS identified 27 S. gallolyticus without sub-speciation, 4 L. 32

    mesenteroides, 3 L. lactis, 6 S. lutetiensis whereas 12 isolates rendered a “non reliable 33

    identification” result. Pulsed-field gel electrophoresis grouped all S. gallolyticus subsp. 34

    gallolyticus into 3 major clusters clearly different from those of the subsp. pasteurianus 35

    which, in turn, exhibited no clonal relationship. Resistance percentages of tested 36

    antimicrobials were: 38% erythromycin, 23% fosfomycin, 10% levofloxacin, 6% 37

    tetracycline, and 4% cotrimoxazole. The most frequent underlying diseases were 38

    hepatobiliar disorders (53%), endocarditis (17%) and malignancies (12%). We conclude 39

    that sequencing of sodA gene was the most discriminatory method, and that S. 40

    gallolyticus subsp. pasteurianus appear to have a higher genetic diversity than S. 41

    gallolyticus subsp. gallolyticus. 42

    on S eptem

    ber 27, 2020 by guest http://jcm

    .asm .org/

    D ow

    nloaded from


  • 3


    Streptoccocus bovis, a non-enterococcal group D Streptococcus, can be found as part of 44

    human gastrointestinal microbiota in 5-16% of individuals (17). However, it causes 45

    bacteraemia and endocarditis, particularly in men and in the elderly (7-8). S. bovis 46

    bacteraemia and colon tumours association was established in the late 1970s (14). 47

    Moreover, recent studies have described a frequent association between its isolation 48

    with chronic liver and biliary tract disorders (10). 49

    Streptococcal taxonomy has progressively changed according to the description of 50

    new species originally grouped as S. bovis. During the late 1990s and at the beginning 51

    of this decade, several authors re-named the S. bovis biotype I as Streptococcus 52

    gallolyticus subsp. gallolyticus (21); the biotype II/1 as Streptococcus lutetiensis (18) 53

    and finally the biotype II/2 as S. gallolyticus subsp. pasteurianus (20). 54

    Clinicians still remain unfamiliar with the new taxonomy of S. bovis species, 55

    mostly due to the complexity of current nomenclature and specific identification 56

    requirements based on molecular microbiology not available in routine clinical 57

    laboratories. Nevertheless, due to their specific disease association and their 58

    microbiology features, a proper identification of the S. bovis isolates is needed. The aim 59

    of this study was to review all S. bovis bacteraemic episodes documented along the last 60

    seven years in our hospital, focusing on the new taxonomy and the probable association 61

    of different subspecies and pathologies. 62



    Bacterial identification. All S. bovis isolates (n= 52, from 51 patients) causing 65

    bacteraemia recovered from blood cultures between January 2003 and January 2010 66

    were studied. Initially, S. bovis identification was routinely performed using the 67

    on S eptem

    ber 27, 2020 by guest http://jcm

    .asm .org/

    D ow

    nloaded from


  • 4

    commercial API 20 Strep gallery (bioMérieux, Marcy l´Etoile, France) and the 68

    semiautomated WIDER system (Fco. Soria Melguizo, Madrid, Spain) (5). The ability to 69

    grow on bile esculin agar (BD, New York, US) was determined after 24 h of incubation 70

    at 37ºC. Isolates were tested for the presence of Lancefield streptococcal antigen D by 71

    agglutination using the SLIDEX® Strepto Plus Kit (BioMérieux, Marcy l'Etoile, 72

    Francia). 73

    Subsequently, both 16S rDNA PCR with universal primers (16-F: 5’-AGGAT 74


    and sodA PCR with degenerate primers (dl: 5´-CCITAYICITAYGAYG 76


    performed (18). Amplicons (500 bp for 16S rDNA and 609 bp for sodA, respectively) 78

    were sequenced using an ABI prism 377 automated sequencer (PE, Norwalk, Conn, US) 79

    after ExoSAP-IT (Amersham, Bucks, UK) purification. Sequences were aligned using 80

    the ClustalW tool from the website www.ebi.ac.uk, and phylogenetic trees were 81

    constructed with the TreeView software (Michael Eisen, Stanford University, 82

    California, USA). 83

    Mass spectrometry using the Bruker Biotyper MALDI78 TOF MS system (Bruker 84

    Daltonics, Germany) was also performed as part of the bacterial re-identification 85

    scheme (3). 86

    Antimicrobial susceptibility testing. Susceptibility (microdilution method) was 87

    performed using the WIDER panels for Gram-positive organisms, and interpreted 88

    according to Clinical and Laboratory Standards Institute criteria (5-6).


    Genetic diversity. Clonal relatedness was determined by pulsed-field gel 90

    electrophoresis (PFGE) using a protocol initially described for Streptococcus suis 91

    on S eptem

    ber 27, 2020 by guest http://jcm

    .asm .org/

    D ow

    nloaded from


  • 5

    serotype 2 (15). A dendrogram was constructed using the Phoretix 5.0 software 92

    (Nonlinear Dynamics Ltd., United Kingdom) based on the Dice’s coefficient. 93

    Patients. Clinical charts of all patients were reviewed with the approval of the Ethic 94

    Committee of our institution to assess both demographic and clinical data as well as 95

    diagnostics and treatments. All patients in whom colonic pathologies were suspected 96

    were submitted to colonoscopy examination. A possible biliary source of bacteraemia 97

    was assigned if there was a clinical or surgical diagnosis of acute cholecystitis or 98

    cholangitis, after the exclusion of other possible focus of infection. 99


    RESULTS 101

    Isolates previously identified as Streptococcus bovis were re-identified using the current 102

    nomenclature. All 52 isolates, including the non-streptococcal ones, had a clear positive 103

    reaction with latex agglutination test for group D Streptococcus. API 20 Strep initial 104

    data were not available, and contemporary profiles corresponded to 29 S. bovis II/2 105

    (which included 24 S. gallolyticus subsp. pasteurianus and 5 S. lutetiensis isolates, 106

    according with the sodA identification), 14 S. bovis I, 2 S. bovis II/4, 3 L. lactis and 4 107

    Leuconostoc spp. 108

    Nucleotide sequences of 16S rDNA amplicons (500 bp) allowed the classification 109

    of the isolates as Streptococcus gallolyticus subsp. gallolyticus (n=14); Streptococcus 110

    gallolyticus subsp. pasteurianus (n=24); Streptococcus spp. (n=7); Lactococcus lactis 111

    (n=3) and Leuconostoc mesenteroides (n=4) (Figure 1