JCM Accepts, published online ahead of print on June 009 J. Clin. Microbiol. doi:0./jcm.00-09 Copyright 009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. TITLE: Emergence of a clonal lineage of B. abortus biovar in clinical cases in Spain Authors: Sylvia Valdezate *, Ana Navarro, Virginia Rubio, Bruno Garin- Bastuji, David Albert, Purificación Hernandez, Pilar M. Alonso and Juan A. Saéz-Nieto. Laboratorio de Taxonomía, Servicio de Bacteriología. Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 0 Madrid, Spain. European Community and OIE/FAO Reference Laboratory for Brucellosis, 90 Maisons-Alfort, France. Servicio de Microbiología, Hospital de San Pedro Alcántara, Cáceres, Spain. Servicio de Microbiología, Hospital Xeral-Calde, Lugo, Spain. Corresponding author: Sylvia Valdezate Ph.D (svaldezate@isciii.es) Servicio de Bacteriología Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Crta. Majadahonda-Pozuelo km, 0 Madrid, Spain. Tel: + 9 Fax: + 9 09 9 Running title: Spanish isolates of B. abortus biovar Key words: B. abortus biovar, MLVA-genotypes, HOOF-genotypes, housekeeping genes, rpob, gyra, parc Downloaded from http://jcm.asm.org/ on May, 0 by guest
Brucella abortus biovar (BAb) is an uncommon cause of human brucellosis in Spain; where B. melitensis accounted for 9.% of all cases. BAb was detected from four patients in Cáceres (west-central Spain) with a febrile syndrome associated with arthralgia and myalgia, and from one patient in Lugo (northwestern Spain) with orchiepididymitis (March 00 and April 00). For tracing, these isolates were studied by: biotyping, S rdna, rpob, gyra, parc, housekeeping genes, multilocus variable-number tandem-repeat analysis (MLVA-), and hypervariable octameric oligonucleotide fingerprints (HOOFprints). Isolates were identified as BAb () by their CO -requirements, H S- production, agglutination in anti-a positive and anti-m negative mono-specific sera, and growth on thionin and basic fuchsin (0 µg/ml). They showed a single S rdna gene and rpob sequences (, 9). The latter gene harboured substitutions (-GAC, -ACT, and 0-GAA) respect to B. melitensis M AE009 (9). The replacement at codon is thought to be a B. abortus marker because of its presence in reference biovars. The rpob sequence showed a 00% match with the sequence of BAb strain / (DQ0) (9). The authenticity of BAb, however, has been questioned for many years (, ) Downloaded from http://jcm.asm.org/ on May, 0 by guest because the reference strain was a mixture of BAb and. Further work showed the obtained rpob differs at position 0 from the sequence of BAb
reference strain Tulya (AY0) (9). The Tulya strain, and other African BAb isolates, show distinct genetic patterns versus European isolates of the same biovar when analysed with different genetic markers. The division of BAb into two groups has been proposed: a, containing the Tulya and African field isolates; and b, containing the European isolates (including those we analysed) (0). Therefore, our five BAb isolate s analysis differed from those for the Tulya strain (9). The gyra/parc amplifications and sequencing were performed with the primers (Bru_gyrA+: - TGCAGCGGTCTTATCTTGATT- ; Bru_gyrA-9: -CAAACGAGGTCTGCAAAGG- ; Bru_parC+00: - CAAGCTGACCGAGCTTGAA- ; Bru_parC-: - CACGAAGGCCGTCAGTATATC- ) to provide polymorphisms related to the bacterial identification (). All showed one gyra (-T C) and one ParC [00-Ala(GCT) Val(GTT)] mutations compared to B. melitensis M. gyra change appears in BAb and in B. suis, while the parc mutation is only seen in BAb. The isolates showed the same sequence type, ST (gap-, aroa-, glk-, dnak-, gyrb-, trpe-, cobq-, omp-, int-hyp-), identical to the corresponding ST of BAb vaccine isolates S9 and RB. The Spanish isolates Downloaded from http://jcm.asm.org/ on May, 0 by guest differed at (glk, trpe) and (gap, aroa, glk, dnak, gyrb, trpe, cobq) of the 9
housekeeping genes to those of the BAb /9 and the Tulya strain, respectively (). Five MLVA- () (genetic similarity range, 0%-9%) and five HOOF-prints () (%-%) genotypes were obtained. Comparing those genotypes to other MLVA- genotypes (), the Spanish cluster split from other European and African BAb isolates (similarity index <0%), suggesting a different clonal lineage (Figure). In summary, we described the first reported cases of BAb causing human brucellosis in Spain. MLVA and HOOF-prints genotyping revealed a close genetic relationship between the detected emergent isolates; this is further supported by their identical S rdna, rpob, gyra, parc, and housekeeping gene sequences. GenBank assigned the following accession numbers: S rrna (EF90- ); gyra (EF0) parc (EF0), and rpob (EU9). This work was supported by a grant from the Instituto de Salud Carlos III to A.N, and by project MPY /0. The authors thank S. Allix and M. Thiébaud (Afssa) for assistance in biotyping, and Adrian Burton for checking the English version of the manuscript. Downloaded from http://jcm.asm.org/ on May, 0 by guest
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Figure. Dendrogram of clustered MLVA- genotypes of the Spanish B. abortus biovar human isolates (see Brucella 00 MLVA database: http://mlva.u-psud.fr), the European B. abortus biovar (b) isolates, the African B. abortus biovar (a) Tulya strain, and the B. abortus biovar RB and S9 vaccine isolates () using the categorical coefficient and UPGMA analysis (unweighted pair group method using arithmetic averages). Keys: genetic similarity (%), ID no. of strain, biotype, origin, location, MLVA genotypes, HOOF-prints genotypes and references (,). Footnote: a Bruce09 was not available for these previous studied strains (). Downloaded from http://jcm.asm.org/ on May, 0 by guest
Genetic similarity % ID. strain Biotype Origin Location MLVA- genotype a HOOF-prints Reference genotype Panel A Panel Panel B 0 0 0 0 0 0 90 00 CNM CNM9 CNM90 CNM9 CNM #RB(vacc.) #S9 (vacc.) #9- #Tulya #0 #0 #0 #0 #0 #0 #0 #0 #09 # # #0 #9- #9- #9- #9-9 #9-90 #9- #9- #0- b b b b b (r) b b b b b b b b b b b b b b b b b b b b b b b Cattle farmer dromedary unknown unknown Spain (Cacéres, /00) Spain (Cacéres, 0/00) Spain (Cacéres, 0/00) Spain (Lugo,0/00) Spain (Cacéres, 0/00) United States United States Sudan Uganda Germany (Uckermark) Germany (Rostock) Germany (Uckermark) Germany (Uckermark) Germany (Uckermark) France () Spain (Pamplona) France () France () Greece France () Italia (Sicilia) Turkey Bruce0 Bruce0 Bruce Bruce Bruce Bruce Bruce Bruce Bruce Bruce9 Bruce Bruce0 Bruce0 Bruce09 0 Bruce Bruce0,,,,,,0, This study,,,,,,, This study 9,,,,,,,0 This study,,,,,,, This study,,,,0,,,0 This study,,,,,,, (, ),,,,,,, (, ) () () () () () () () () () () () () () () () () () () () () () () Downloaded from http://jcm.asm.org/ on May, 0 by guest