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Short Communication
Characteristics of the Turkish Isolates of Francisella tularensis ¸aban Gürcan*, O˘guz Karabay1, Aynur Karadenizli2, Çi˘gdem Karagöl, Trakya University, Faculty of Medicine, Department of Microbiology and Clinical Microbiology, Edirne; 1˙Izzet Baysal University, Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology, Bolu; 2Kocaeli University, Faculty of Medicine, Department of Microbiology and Clinical Microbiology, Kocaeli, Turkey; and 3National Center of Infectious and Parasitic Diseases, Department of Microbiology, Sofia, Bulgaria (Received November 12, 2007. Accepted March 12, 2008) SUMMARY: In this study, cultures of patients with tularemia were evaluated, and antimicrobial susceptibilities
of two Francisella tularensis strains were tested by disk diffusion and E-test methods. A high-resolution multiple-
locus variable-number tandem repeat analysis (MLVA) comprising six variable-number tandem repeat loci was
applied to elucidate the genetic relatedness among Turkish and Bulgarian isolates which were isolated in a recent
outbreak. The patients were diagnosed in two outbreaks in two cities of Turkey in 2005 and 2006. A total of 16
samples from 12 patients were cultured, and PCR tests were carried out on 15 samples that were positive in five
lymph node aspirates and two soft tissue aspirates. F. tularensis was isolated from the lymph nodes of two
patients. Aminoglycosides, quinolones, chloramphenicole, tetracyclines, nitrofurantoin, and rifampicin inhibited
growth of the isolates. The Turkish isolates appeared to share a common MLVA pattern with one of the four
Bulgarian outbreak genotypes.
In recent years, tularemia outbreaks have been reported cystine, 1% glucose and 5% human blood in the presence or from some regions in Turkey (1). According to data of 2005, absence of 1,000 U/ml penicillin. If the bacteria were small, 431 confirmed tularemia cases were reported from 19 cities pale-staining, Gram-negative coccobacilli, without hemoly- of Turkey in 2005 (2). Oropharyngeal tularemia is the main sis, weakly catalase positive, as well as oxidase negative, they clinical form, and only one fatal case has been reported in were considered probable isolates of F. tularensis. Subse- Turkey (1). These data predict that Francisella tularensis quently, they were identified as F. tularensis by TaqMan PCR subsp. holarctica may be a dominant subspecies in Turkey, but this hypothesis had not been confirmed yet.
The antibiotic-free media for the culture defined above were In this study, cultures of patients with tularemia were evalu- used for the susceptibility tests. Bacteria were grown for 48 h ated, polymerase chain reaction (PCR) testing was applied at 37°C in atmospheric air, and the colonies were suspended to these samples and antimicrobial susceptibilities of two F.
in saline to achieve a turbidity equivalent to that of McFarland tularensis were tested by disk diffusion and the E-test meth- standard 1. The plates were inoculated with 100 l of a colony od. Additionally, a high-resolution multiple-locus variable- suspension. Ten to 15 min later, E-test strips (AB Biodisk, number tandem repeat analysis (MLVA) was applied to eluci- Solna, Sweden) and antibiotic disks (Becton Dickinson) were date the genetic relatedness among Turkish and Bulgarian placed on the plates with proper distancing among them.
isolates. The patients were diagnosed during outbreaks in two Minimum inhibitory concentrations (MICs) and zone diam- cities of Turkey in 2005 and 2006 (1). In clinically compatible eters were read after 48 h of incubation at 37°C in atmos- cases, a microagglutination test was performed on a V plate (1). BD Francisella tularensis antigen (Becton Dickinson, The isolation and characterization of Bulgarian isolates are Sparks, Md., USA) was used in the microagglutination tests described elsewhere (5). A high-resolution MLVA typing for the initial diagnosis. Two in-house antigens were prepared system comprising six variable number of tandem repeats from the two F. tularensis isolates cultured in the present (VNTR) loci was applied to the Turkish and Bulgarian iso- study, as previously described (3). The test results, which were lates. Some modifications were made to the MLVA protocol repeated by in-house antigens, were identical with results of described by Byström et al. (6). For compatibility with ALFexpressII sequencer (GE Healthcare Life Sciences, A total of 16 samples from 12 patients from villages of the Piscataway, N.J., USA) the Ft-M3, Ft-M6, Ft-M20, Ft-M21, Thrace region and the western Black Sea region of Turkey Ft-M22, and Ft-M24 primers were labeled with Cy5 instead were studied. Clinical samples were obtained by a suitable of 6-carboxytetramethylrhodamine (6,7). The primer concen- technique. PCR assays were performed as described elsewhere trations were adjusted as follows: Ft-M3 - 0.12 (4) in all samples except one lymph node aspirate sample.
M6 - 0.15 M, Ft-M20 - 0.05 M, Ft-M21 - 0.06 M, Ft- All samples were cultured on agar supplemented with 0.1% M22 - 0.03 M, and Ft-M24 - 0.5 M. The reaction volumewas 25 l with the following concentrations of PCR com- *Corresponding author: Mailing address: Trakya University, Fac- ulty of Medicine, Department of Microbiology and Clinical (50 mM KCl, 20 mM Tris-HCl, pH 8.4, Invitrogen Corp., Microbiology, 22030-Edirne, Turkey. Tel & Fax: +90-284-2361654, Carlsbad, Calif., USA), 5% DMSO (Merck KGaA, Darmstadt, Germany), 0.1 g/ l non-acetylated bovine serum albumin (Sigma-Aldrich, St. Louis, Mo., USA), 1 U Taq polymerase presented with a single allele. The two Turkish isolates were (Invitrogen). The cycling program was as described by found to be identical to each other, as well as to one of the Johansson et al. (7). Three microliters of PCR products was Bulgarian outbreak clusters (Fig. 1).
mixed with an equal volume of loading buffer (99.5% deion- Oropharyngeal tularemia is the most common clinical form ized formamide, 0.5% blue dextran), denatured for 5 min at of the disease, because almost all tularemia outbreaks are 94°C and loaded on an 8% ReproGel (GE Healthcare Life water-borne diseases in the Balkans and Anatolia (1). F.
Sciences). Separation was performed on an ALFexpressII tularensis enters the human body via the consumption of con- DNA sequencer (GE Healthcare Life Sciences). Electrophore- taminated water or food in oropharyngeal form. F. tularensis sis conditions were according to the manufacturer’s instruc- or its DNA were demonstrated in water specimens in Turkey tions. The gel processing and cluster analysis of the MLVA (1). Although one of 12 patients with oropharyngeal tularemia patterns were performed in Bionumerics v4.5 (Applied Maths in the present study had neurological symptoms (fever and NV, Kortrijk, Belgium) software. A dendrogram was gener- headache), the possibility of central nervous system infec- ated using categorical coefficient and unweighted pair-group tion was eliminated in this case by CSF assays.
with arithmetic means (UPGMA) algorithms.
Since the confirmation of tularemia by culture is generally All patients’ antibodies against F. tularensis were positive unsuccessful, this disease is often diagnosed with serological in 1/160 - 1/5,120 dilutions. Out of 16 samples, nine were lymph tests and molecular methods as well as clinical findings in node aspirates, three were throat swabs, two were aspirates Turkey and other parts of the world. Our cases were diag- from the soft tissue of the cheek, one was an oral lesion swab nosed within 8 weeks after the onset of symptoms by sero- and one was cerebrospidal fluid (CSF). Two F. tularensis logical tests. In the present study, the fact that 7 of 15 samples isolates were isolated from the lymph node aspirates of two were positive emphasizes the importance of molecular biol- patients living in the western Black Sea region in December ogy techniques for the early diagnosis of tularemia.
2005. PCR tests were positive in five lymph node aspirate Clinicians in Turkey have little experience in the use of samples and two soft tissue aspirates. Susceptibility zones culturing for the routine diagnosis of tularemia due to the high for these isolates were determined for aminoglycosides, degree virulence of the organism, the high risk of laboratory- quinolones, tetracyclines, chloramphenicol, nitrofurantoin, and acquired infection and the low sensitivity of the culture. F.
rifampicin by disk diffusion and E-tests, but no zone was seen tularensis was isolated in 10 (4.9%) out of 205 patients be- around beta-lactam antibiotics, erythromycin or co-trimoxazole tween 1988 and 1998 in Turkey (1). In the present study, two isolates were isolated from the lymph node aspirates of two A total of four genotypes were found among the nine in- patients living in the western Black Sea region of Turkey.
vestigated isolates (7 Bulgarian and 2 Turkish) (Fig. 1). The The Black Sea region is where most of the tularemia cases M3 marker appeared to be the most variable one, showing are reported in Turkey (1). There has been no isolation of F.
four different alleles among the nine isolates studied, followed tularensis isolates and no data collected regarding the anti- by M6 (3 alleles) and M20 (2 alleles). All other markers were microbial susceptibilities of F. tularensis isolates from the Table 1. Susceptibilities of antimicrobials for two Turkish isolates of Francisella tularensis by Penicillin, SAM, AMC, Piperacillin, PTZ, Oxacillin 1): Yazikara strains.
2): Nuhoren strains.
SAM, Ampicillin/sulbactam; AMC, Amoxicillin/clavulanate; PTZ, Piperacillin/tazobactam; NZ, nozone; ND, not determined.
Fig. 1. MLVA cluster analysis of Bulgarian and Turkish Francisella tularensis strains comprising six VNTR loci (M3 to M24). The corresponding fragment sizes (in base pairs) are given below each VNTR marker. Strains Schu4 and Japan wereincluded as outgroup subspecies for creation of the dendrogram.
western Black Sea region of Turkey so far. This study is the nomical and travel relations between the two countries may first to investigate this important issue.
explain the spreading this genotype. Also, immigrant birds A standardized broth dilution method and susceptibility or ticks on the birds may transport the causative agent from breakpoints for F. tularensis were defined for the suscepti- country to country, and infected mice may enter merchant bility tests. There is no consensus about diffusion methods, ships or vehicles and cause the distribution of isolates to other although diffusion tests have been applied in susceptibility countries. Another possibility is that the six-locus MLVA is studies. Tomaso et al. (8) and Ikaheimo et al. (9) harvested incapable of discriminating these very closely related iso- the bacteria with turbidity equivalent to that of McFarland lates. More in-depth analyses including more VNTR mark- standard 1 and 0.5 into the media of cysteine heart agar supple- ers and isolates are needed to confirm this unusual genetic mented with 10% sheep blood agar and medium of cystein heart agar with 2% hemoglobin, respectively. We testedsusceptibilities in the antibiotic-free medium mentioned for REFERENCES
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