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Multiple-Locus Variable-Number Tandem-Repeat Analysis Genotyping of Human Brucella Isolates from Turkey

Selçuk Kiliç, Ivan N. Ivanov, Riza Durmaz, Mehmet Refik Bayraktar, Ergin Ayaslioglu, M. Hamidullah Uyanik, Hikmet Aliskan, Ekrem Yasar, Gülçin Bayramoglu, Ahmet Arslantürk, Gilles Vergnaud and Todor V. Kantardjiev J. Clin. Microbiol. 2011, 49(9):3276. DOI: 10.1128/JCM.02538-10. Published Ahead of Print 27 July 2011. Downloaded from on March 20, 2012 by guest

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with a geographical surface of 783. and Central and South America (19. Turkey. Turkey4. 3276–3283 0095-1137/11/$12. and Bruce21.1.000 cases annually. mainly in the Mediterranean littoral. Its prevalence varies widely from region to region due to several factors. Ankara.kilic@rshm. Department of Microbiology and Clinical Microbiology. investigation of outbreaks.563 cases in 2004 to 9.12 and Todor V. indicated that Turkish human Brucella melitensis isolates were most closely related to the neighboring countries’ isolates included in the East Mediterranean group. Kirikkale. Kirikkale University. socioeconomic status. E-mail: selcuk.03/100. A number of MLVA genotypes appeared to be partially restricted to some geographic areas and displayed no annual variation. Rapid and accurate typing procedures are crucial for epidemiologic surveillance. 14. France (MLVA-16Orsay) including eight minisatellite (panel 1) and eight microsatellite (panel 2. This multiple-locus variable-number tandem-repeat analysis (MLVA) genotyping system. p. with more than 500.000 human brucellosis cases are reported annually. Baskent University. Bagneux. 70% of which lives in cities and 30% in rural areas. control of animal movements.74. Ivanov. and 32 included two to eight nomadism. Turkey10. Recently. Department of Infectious Diseases and Clinical Microbiology.11. The isolates were genotyped by using an MLVA assay developed in Orsay. Brucellosis is the most common anthropozoonosis. Microbiology Laboratory. Kantardjiev2 ¨ Refik Saydam National Public Health Agency.562 km2.asm.9 ¸ ¸ ¨¸ ˘ Ahmet Arslanturk.818 cases in 2008 (17). Mission pour la ´ ´ Recherche et l’Innovation Scientifique. 9. Turkey8. American Society for Microbiology. Department of Microbiology and Clinical Microbiology. and education induced a decline in the number of annually reported human cases. 3276 milk processing methods. Sanliurfa. climatic conditions. Ataturk University. Turkey3. Mailing address: Refik Saydam National Public Health Agency. from 18.asm. Bacterial Zoonoses Research and Reference Laboratory (National Brucellosis Reference Laboratory). a selection of 16 variable-number tandem repeats has been proposed for fingerprinting Brucella isolates (7. Department of Microbiology and Clinical Microbiology. Bruce18. No. Children’s Hospital. Adana.000). MLVA-16Orsay. France11. Many molecular typing methods commonly used for the subtyping of isolates of other bacterial species are not appropriate for routine typing of Brucella strains. comprised eight minisatellite markers (panel 1. State Hospital. 9 Multiple-Locus Variable-Number Tandem-Repeat Analysis Genotyping of Human Brucella Isolates from Turkey † Selcuk Kılıc. Turkey9. Universite Paris Sud 11. The reported incidence is 150 cases per 1 million inhabitants (24). Panel 2B displayed a very high discriminatory power. panel . and follow-up of a control program. This study. * Corresponding author.1* Ivan N. 73 were represented by a unique isolate. two of these represented 85% of the strains. and environmental conditions. Erzurum. Livestock vaccination. Sofia.000 population) until 2004 (25. Karadeniz Technical University. and comprises seven regions. Panels 1 and 2A distinguish 14 genotypes. Bruce19. MLVA-16Orsay yielded 105 genotypes. 06100 Sihhiye Ankara.6 Hikmet Alıskan. Department of Microbiology and Clinical Microbiology.4 Ergin Ayaslıoglu. Cemal Gursel Street 18. possibly reflecting persistence of genotypes in certain areas for a time span of at least a decade. Turkey7. It has a population of 72 million. National Centre of Infectious and Parasitic Diseases.3 Mehmet Refik Bayraktar. Africa. Malatya.1128/JCM. the Middle East.02538-10 Copyright © 2011. Brucellosis is endemic. and none has proven to be fully satisfactory for epidemiological trace-back investigations of brucellosis (1. Microbiology Laboratory. Asia. subdivided into 2A and 2B) markers.8 Gulcin Bayramoglu. Institut de ´ Genetique et Microbiologie. Department of Communicable Diseases Research. Bruce42. representing the first molecular typing results of human Brucella isolates from Turkey. it remains a significant public health concern.00 doi:10. elimination of infected animals. † Supplemental material for this article may be found at http://jcm .10 Gilles Vergnaud. Bulgaria2. Turkey6. 2011. Inonu University. Bruce45.7 Ekrem Yasar. 26). and DGA/MRIS. Diyarbakir. Bruce11. 25). Department of Microbiology and Clinical Microbiology. Trabzon. the Arabian Peninsula.JOURNAL OF CLINICAL MICROBIOLOGY. husbandry practices. Sept. Hamidullah Uyanık. Bruce08. Bruce43. the Indian subcontinent. Three loci from panel 2B had diversity index values higher than 0. Fax: 00 90 312 458 24 04. and approximately 10. The isolates from different patients within the same outbreak or from the same patient before first-line therapy and after relapse showed identical genotypes. social customs. 49. including food Turkey is a relatively large country in the eastern Mediterranean region. 2012 by guest A multiple-locus variable-number tandem-repeat analysis (MLVA) was applied to investigate the epidemiological relationship and genetic diversity among 162 human Brucella isolates collected from all geographic regions of Turkey in an 8-year period (2001 to 2008).org/ on March 20. CNRS. While the disease was eradicated in the vast majority of industrialized regions around the world.2 Rıza Durmaz. All Rights Reserved.5 ¸ ¸ ¸ ˘ M. France12 Received 15 December 2010/Returned for modification 13 February 2011/Accepted 25 April 2011 Downloaded from http://jcm.65/100. A steady increase of reported human cases was observed from 1986 (3. Turkey5. Turkey1.1. Orsay. Bruce12. Published ahead of print on 27 July 2011. 25). Phone: 00 90 312 458 21 69. Kutahya. Bruce06. Harran University. and Bruce55) for species identification and eight complementary microsatellite markers (panel 2A. UMR 8621.

16 M.000 g for 10 min to obtain a clear nucleic acid-containing supernatant. The cluster analysis was performed using the UPGMA (unweighted pair group method with arithmetic mean) algorithm and the categorical (or Hamming’s) dPCR4.5. 2.4 M Bruce55. four (2. 0 to 85 years).5%) presented with acute brucellosis ( 2 months of illness).6 years. Two microliters of the supernatant was used as the template in the PCR assays. panel 1 genotypes 42 (27 strains) and 43 (109 strains) are the most common genotypes (see Table S1 in the supplemental material). Data analysis. A total of 162 presumptive Brucella isolates from 159 patients (isolates BRU-S001 to BRU-S162) submitted to the Refik Saydam National Public Health Agency for a precise identification at the species and biovar levels were enrolled in this study. range. Azerbeijan. Genetic diversity (Hunter-Gaston diversity index [HGDI]) and confidence intervals were calculated using online tools at www. Baku. Bruce07. 3 (161 isolates) and B. MLVA-16Orsay genotyping results.778.0.788 in panel 2B. lysis by Tbilisi phage. 0. an increased incidence from late spring (May) to midsummer (peak in June or July). and two (1.182 in panel 2A and from 0. The MLVA-16Orsay genotypes of B. and two isolates were obtained from each of these patients. and 2 l of thermolysate solution. melitensis bv. The seasonal distribution of isolates in the present collection was in agreement with the global epidemiology of Brucella in Turkey.asm. The genetic diversity of Brucella strains isolated from human and animal infection has not yet been investigated in Turkey. Bruce09. melitensis reference strains (biovar [bv] 1. In brief. melitensis strains. i.6%) case had acute and subacute phases of a single illness episode. 16. 2 mM MgCl2.1%) had subacute. One (0. PCR amplification was performed in a total volume of 25 l containing 1 Gold buffer. 24 from central Anatolia. bv. and extension at 70°C for 30 s. The number of Brucella isolates analyzed from each region (64 isolates from eastern Anatolia.4 M Bruce11.2 years (range. and Bruce30) for further subspecies differentiation. panel 1 primers were combined and run into four duplex PCR (dPCR). Loci Bruce06. and Bruce30 had the highest variability ( on March 20. 3. 2011 BRUCELLA MLVA GENOTYPES FROM TURKEY 3277 2B. 38. The primer multiplexing was arranged in a manner that avoids overlapping of the resulting PCR fragments according to published allele size ranges (14) and ensures unambiguous interpretation. range. Three B. 14. A total of 162 Brucella isolates were identified as B.. whereas more recently isolated strains were stored at 80°C in 10% skim milk. 0. melitensis isolates were compared to the corresponding data obtained for the reference strains and field isolates investigated previously (15). and for trace-back analyses (1. The seven others (numbers 42. dPCR2. (This study was presented in part at the 3rd Eurasia Congress of Infectious Diseases [formerly ICCAID].org.740. primer annealing at 60°C for 30 s. 62.45 M Bruce12. MLVA-16Orsay genotyping. The initial denaturation step (96°C for 5 min) was followed by 30 cycles of denaturation at 96°C for 30 s. see Table 1). Panels 1 and 2A showed limited diversity. Primer concentrations were adjusted as follows: dPCR1. 14) with a slight modification. and 121) were not previously observed. abortus bv. ATCC 23458) and Brucella abortus bv. Two patients experienced relapse episodes. ether. In particular. Electrophoretic separation was performed by applying the M500 method of the QIAxcel capillary electrophoresis system coupled with a high-resolution cartridge (Qiagen. 0. urease activity. 57.0 to 0. S1 in the supplemental material and confirms the species identification deduced from biotyping.4 M Bruce08.hpa-bioinformatics. Bruce16.4 M Bruce42.778. Chi-square analysis was used to correlate patient characteristics with genotype. bv.16 mg/ml bovine serum albumin..5% dimethyl sulfoxide. ATCC 23448) as well as Brucella melitensis Rev-1 vaccine strain (BRU-S163) were included as control strains. Belgium) as a character data set. Clustering analysis with previously published typing data from more than 500 isolates (15) is shown in Fig. 0. A loop of bacterial colony was suspended into 200 l TE buffer (10 mM Tris [pH 8. 6 of which (numbers 100. The amplification was run in a QB-96 cycler (Quanta Biotech Ltd. 1 reference strain (544. H2S production. 119.17. 17 from the Mediterranean. sensitivity to the fuchsin and thionin dyes (20 and 40 g/ml). 3 (one isolate). Female patients (mean age.329) at Bruce42.5%) had chronic. and five (3.05 (Qiagen. MLVA-11Orsay (composed of the eight panel 1 and three panel 2A loci) discriminated 14 genotypes. 0.6 years. A total of 160 of these isolates were collected over an 8-year period (from 2001 to 2008) at various tertiary care centers in Turkey. 11. 0. This method has been reported to be highly discriminatory to distinguish strains within a local outbreak. 18. 2 to 79 years) were slightly older than male patients (mean age.u-psud. 0. These characteristics confirm the representativity of the present strain collection. whereas panel 2B displayed a very high discriminatory power. 0. 23) and typing data from several hundred isolates can be queried and accessed via the Internet (http://mlva. The HGDI values ranged from 0. and 85) observed in a single strain. the MLVA16Orsay assay was applied to investigate epidemiological relationships among human brucellosis isolates collected from all regions of Turkey and to determine the most common genotypes among Brucella strains in Turkey.e. PCR amplification products were obtained for all 162 isolates. 1 U AmpliTaq Gold (Applied Biosystems). 49. Turbidity was adjusted to a McFarland standard of approximately 0.0]. 61.3%) had relapse brucellosis. 30. Brucella isolates. 63/9.35 M Bruce45. The isolates were identified at the genus level by conventional microbiological methods and biotyped as previously described based on requirement of CO2 for growth. respectively. 2009. and dPCR3. 63) were previously observed. The PCR was performed as previously described (1. Bruce11. ATCC 23457. The HGDI in panel 1 was highest (0. Brucella DNA samples were prepared by a simple thermolysate procedure. 84. United Kingdom). 14). Bruce04. 43. for discriminating isolates originating from restricted geographic sources at the subspecies level. and agglutination with monospecific antiserum for A and M antigens (2). and the ratio of males (n 86) to females (n 73) was 1. 26 from southeastern Anatolia. with a gradual decrease in autumn and winter. 44. and to some extent phylogenetically relevant (1. One isolate of cerebrospinal fluid in 1998 (BRU-S131) and one from the blood culture of a preterm baby with congenital brucellosis in 2009 (BRU-S130) were also included in the The mean age of the 159 patients was 34.165 to 0.4 M Bruce43. and 0. 2. In the present study. Values of the HGDI can range from 0 (no polymorphism) to 1 (all samples are different). In panel 2B. 0. 1 mM EDTA). and Bruce45 showed only one allele (HGDI 0). Germany). Bruce04. 113. Bacterial suspensions were heated at 100°C for 10 min and then centrifuged at 13. appropriate concentrations of each flanking primer. Panel 1 loci gave 10 different genotypes among 161 B. 2012 by guest RESULTS Patient characteristics. and fragment sizes converted to repeat unit numbers were imported into BioNumerics (Applied Maths.VOL. Bruce16.2 M Bruce06. The MLVA-16Orsay assay has been shown to be an appropriate method for species identification in the Brucella 92. 0. Three of those were new genotypes (numbers 83.) MATERIALS AND METHODS Brucella strains. 102. Downloaded from http://jcm. Analysis of electrophoresis patterns was carried out with the BioCalculator software version 3. Most patients (n 147. 6 from Marmara) was roughly in proportion to brucellosis incidence. Eighty-five percent of the isolates belong to . (ii) PCR amplification. Brucella strains isolated in early years were recovered from freeze-dried stocks. Germany). with a final extension step at 70°C for 5 min. 14 from the Black Sea. 0.25 mM deoxynucleoside triphosphate (dNTP) mix. 109. 11 from the Aegean. ATCC 23456. 0 to 85 years). (i) DNA sample preparation.

The remaining 32 genotypes included the 88 clustered strains (clustering rate was Downloaded from http://jcm.000–0. throughout the study period) years.asm. 6. 6. 3. 5. 3 2.000–0.000 0.000 0. 11 8 0. The isolates in the largest MLVA-11Orsay genotype group (genotype 125) were isolated in all age groups.) .991 0. The bar size is proportional to the number of isolates. the isolates in the second largest genotype group (genotype 116) were not isolated from adults between the ages of 20 and 30 (weak significance.1% versus 45. 20. 7.369–0. of alleles Tandem repeat copy no. MLVA-11 genotype 125 is observed all over the country. 4. MLVA-16Orsay yielded a total of 105 genotypes. 7. 8.0768–0.0019–0. all strains analyzed clustered within the Eastern Mediterranean group (Fig. whereas genotype 116 was isolated mainly in the central Anatolia region.702–0. 7. 2012 by guest FIG. 6.000 0. 3). 3. respectively.012 0. TABLE 1.096 0.544 0. with permission of Basarsoft Ltd. S1 in the supplemental material). 5.018–0.270 0. 9.036 0. 7. In the minimum spanning tree clustering using previously published B. Genotypes 116 and 125 were isolated during.3278 KILIÇ ET AL. Panel 1 Bruce06 Bruce08 Bruce11 Bruce12 Bruce42 Bruce43 Bruce45 Bruce55 Total Panel 2A Bruce18 Bruce19 Bruce21 Total (panels 1 and 2A) Panel 2B Bruce04 Bruce07 Bruce09 Bruce16 Bruce30 MLVA-16Orsay a 1 3 1 3 3 3 1 2 10 3 4 1 14 1 3. 5. P 0. MICROBIOL.000 7 6 8 8 6 105 on March 20.96).000–0.14.814 0. 21.255 0. Numbers of alleles and HGDI values of 161 B. abortus bv. abortus genotypes (see Fig..049 0. 13.165 0. 7.basarsoft. 5.115–0.037 4.24). However. (Adapted from a map available at www. 14 1. 4 3 melitensis typing data. 6. 4.084 0. 23 8 0. abortus strain BRU-S093 was not identical to the previously described B. Each color corresponds to a different MLVA-11Orsay genotype. The distribution of the main genotypes was not associated with a specific period of time. 6. 73 of which were represented by a unique strain.142 0. The relative frequencies of the two most frequent genotypes were essentially identical in male and female patients (54. B. 3.097–0.329 0. 2 0. 5 3 12.000–0. chi-square 0.740 0.778 0.806 0. six and eight (i. There was also no significant difference between the spectrum of genotypes isolated from children and those isolated from adults.096 0. 4.026–0.778 0. 14 8.465 0. The genotype of B.049 0. and P 0. MLVA-11 genotypes 116 (27 isolates) or 125 (109 isolates). 4. and the genotype 104 was essentially found in patients from the Aegean region (Fig.777 HGDI. 9. chi-square 4.182 0. 4. 4. 5.522 0.2522–0.743–0.7513–0. 3.437 0.9%. Hunter-Gaston diversity index. 5. 2). melitensis isolates from Turkey Locus No. 3.044 0.222 0. Distribution of MLVA-11Orsay genotypes showed variation in different geographical regions. The isolates with the genotypes 103 and 104 were primarily observed in the Black Sea region. CLIN. melitensis genotypes.000 0. 6 18.000–0. 1. Geographic distribution of panel 1 and 2A genotypes (genotype 69.161 0. 1). 9 12 8.000 0.002.406 0. HGDIa Confidence interval J. Cluster analysis for Turkish B.

melitensis strains. These results reveal that human brucellosis in Turkey seems to be related more to ovicaprine than to cattle infection. melitensis. two isolates cultured from one patient’s blood samples during the acute and subacute phases of a single illness episode and one isolate corresponding to a laboratoryacquired infection from this patient’s isolates showed identical MLVA-16Orsay patterns (Fig. in the setting of a local outbreak investigation. 29. and B. The MLVA-16Orsay typing assay also showed very high concordance with available epidemiological data. or Bruce30 (Fig. 29 (BRU-S066. Panel 2B markers in MLVA-16Orsay loci displayed very high discriminatory power. The outbreak genotypes typically comprised family members and patients who were presumed to have contracted brucellosis from a common point source (consumption of homemade cheese). 3). MLVA genotypes did not show significant differences among gender or different age groups. Genotypes 22. accounting for 99% of the total cases. 49. while panels 1 and 2A showed limited diversity. BRU-S157. Genotypes 21 to 25. genotype 30. Additionally. There was good correlation between molecular typing results and epidemiological data. and -55) loci. The size of the shapes indicates the number of strains described in the genotype. such as the financial compensation of owners of slaughtered seropositive cattle. may play a significant role. epidemiological. brucellosis control measures. Color codes are associated with the main B. The numbers represent the 14 MLVA-11Orsay genotypes found in this study. 2012 by guest FIG. The seven strains in the second most frequent genotype were isolated from five separate provinces in two geographic regions between 2004 and 2008. BRU-S082. genotypes 22 (BRU-S080 and BRU-S081). melitensis outbreak observed in June 2003 in a small village in the Kirikkale province from the central Anatolia region yielded six genotypes. 3. The data obtained in Turkey are consistent with the results obtained in the Mediterranean region (1. Previous studies conducted in different regions of Turkey found that human brucellosis was almost exclusively caused by B. BRU-S158. which were representing 14 MLVA-11Orsay genotypes. 21. 43 (BRU-S094 to BRU-S097). Minimum spanning tree analysis of published B. (15). These findings showed that the genotypic variation of Turkish isolates was mostly associated with the highly variable panel 2B loci and to a much lesser extent panel 2A (locus Bruce19) and panel 1 (loci on March 20. and 30 differ only by 1 repeat unit at one or two of the most variable loci. melitensis bv. and epidemiologically related isolates were of identical or very closely related genotypes. genotype 35. melitensis isolates (green). a total of 162 human Brucella isolates collected from different parts of Turkey during an 8-year period was evaluated by bacteriological. In addition. DISCUSSION In the present study. 22). 3). 12. melitensis isolates using the MLVA-11Orsay data. BRU-S078). This may reflect microevolution via a stepwise mutational event of the most variable loci from a very limited number of ancestors. Although the discrimination power of MLVA-8Orsay and MLVA-11Orsay is very low for evaluation of the cross-transmis- Downloaded from http://jcm. Each of the circles showing white and green colors included the Turkish genotype (white) and the genotypes found in Eastern Mediterranean B. BRU-S083) obtained from family members who did not share the same MLVA-16Orsay genotype may either represent persistent circulating strains causing sporadic infections or be the result of mutation events in the course of the outbreak. melitensis strains were recovered from the compilation by Maquart et al. and 100 (BRU-S145 and BRUS146) were each recovered from patients of the same family that contracted brucellosis from unpasteurized dairy products. 26). The 161 Turkish B. 11).asm. whereas the added panel 2B increased the number of genotypes to 105. and genotype 29 was shared by three isolates. In agreement with previous molecular studies (1.6%) (Fig. the highly polymorphic panel 2B might be sufficient for a rapid and low-cost result. and 25 each comprised two isolates. The genotype 71 patients (isolates BRU-S058 and BRU-S059) have a common history of occupational exposure to infected animals. 3 was the biovar most frequently isolated in humans (4–6. Bruce16. Bruce04. The MLVA-16Orsay genotypes of the isolates obtained from two patients (genotype 24 and genotype 27) during the acute phase and after relapse were identical. 3). melitensis MLVA clusters. 16. 23. and molecular typing characteristics.VOL. are associated with the green East Mediterranean B. Turkish isolates are shown in white. 2011 BRUCELLA MLVA GENOTYPES FROM TURKEY 3279 Application of the MLVA panel to 11 isolates from an epidemiologically linked B. Relevance of MLVA-16Orsay genotyping for patient management and source identification. BRU-S075. -42. The most frequently observed genotype comprised eight isolates obtained from five separate provinces in three geographic regions over 7 years (from 2002 to 2009). BRU-S159). MLVA-16Orsay yielded a total of 105 genotypes. 2. melitensis (bv. melitensis isolates. All isolates but one were B. Two isolates (genotype 21. 54. 13. The published data for B. The frequency of different MLVA genotypes varied among the seven geographical regions. 8. . For example. 54 (BRU-S126 and BRU-S127). which may be partly attributed to the virulence of the organism. No such measure exists for sheep or goats. MLVA-11Orsay (combined panel 1 and 2A markers) yielded 14 genotypes.

2012 by guest FIG.Downloaded from http://jcm. 3. strain ID. isolation date (year). and MLVA-11Orsay (panels 1 and 2A) genotypes corresponding to each isolate in the database for each set of on March 20. and 2B are shown the individual MLVA-16Orsay loci and the numbers of tandem-repeat units for each isolate.asm. strain. and the specimen source. A total of 105 genotypes were observed. MLVA-8Orsay (panel 1). 2A. 3280 . the following data are indicated: genotype. The color code reflects the MLVA-11Orsay genotype and is identical to the Fig. In the columns. Under panels 1. Cluster analysis for 162 human isolates of Brucella and Rev1 vaccine strain based on the data set of MLVA-16Orsay. 1 color code. geographic region.

2012 by guest FIG. —Continued. 3. 2011 BRUCELLA MLVA GENOTYPES FROM TURKEY 3281 Downloaded from http://jcm.VOL.asm. on March 20. .

. Reference deleted. 35:337–338. BMC Microbiol. 11. Agents 23:405–407. 2008. 2007. 2007.). M. eastern. Downloaded from http://jcm. those of the typical West Mediterranean family. 23. Microbiol. 16. S. Evaluation of a multilocus variable-number tandem-repeat analysis scheme for typing human Brucella isolates in a region of brucellosis endemicity. Turkey.. Alton. including the consumption of homemade cheese and cream. N. Brucella “HOOFPrints”: strain typing by multilocus analysis of variable number tandem repeats (VNTRs). Dokuzoguz. Istanbul. D. et al. Turkey. Paris. 24.. In E. D. and S. Ministry of Health. Antimicrob. For such purposes. 47:3147– 3155. 2005. M. This observation might reflect homoplasy and convergent evolution. Italy. 2. some isolates recovered from separate re- gions and with no known direct epidemiological links displayed identical MLVA-16Orsay profiles (genotypes 7. M. 11. 2003. Bovine brucellosis: in relation to epidemiology and human infection. M. some of the associated isolates may result from either the lack of control of animal movements between regions or the circulation of improperly processed milk products or household products in the market. Nockler. et al. 55. We detailed a small outbreak in a village where a major part of the population was occupied with agriculture and/or livestock farming. Le Fleche. P. 34–36. B. Ankara. 6:9. Brucella and brucellosis in man and animals. melitensis versus B. we have planned a project to characterize the genotypes circulating in livestock. J. the most common genotypes (42 and 43) found in the current study were also observed in other parts of the world (1. MICROBIOL. and Y. The data produced in this investigation can be queried in the Brucella MLVA database release (starting from the Brucella 2010 release) at http://mlva. These six genotypes were very closely related and differ by single repeat unit differences at one or two of the most variable loci. M. 1:41–45.3282 KILIÇ ET AL. which are particularly common in rural areas or farmland people residing in the southeastern. Baykam. 19. Tumbay. In J. MLVA-16Orsay divided the 11 cases investigated here into six genotypes (genotypes 21. E. in whom brucellosis most probably resulted from traditional food habits. Esendal. G. Identification of Brucella species isolated from proven brucellosis patients in Izmir. Microbiol. 2006. Clin. García-Yoldi. Clin.. MLVA-16Orsay typing enabled us to identify the source of laboratory-acquired brucellosis in a laboratory worker who was exposed to Brucella while processing a blood culture specimen. Clin. Kilic.. Papadimitriou. M. Chemother. 4. 17. MLVA-16Orsay proved to be highly discriminatory among related human Brucella isolates that could not be differentiated by conventional microbiological methods. Lancet Infect. The present findings also confirmed relapses. 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