Molecular genetic techniques for typing of - Springer Link

ISSN 0891-4168, Molecular Genetics, Microbiology and Virology, 2007, Vol. 22, No. 1, pp. 7–15. © Allerton Press, Inc., 2007. Original Russian Text © M.Yu. Kirillov, A. P. Markov, I.V. Lopyrev, V.N. Pankratova, S.A. Levitskii, V.N. Bashkirov, G.B. Smirnov, A.N. Kruglov, V.A. Osadchaya, G.P. Frolova, G.V. Barmina, O.A. Morozova, M.Y. Kosoy, 2007, published in Molekulyarnaya Genetika, Mikrobiologiya i Virusologiya, 2007, No. 1, pp. 8–14.

Molecular Genetic Techniques for Typing of Bartonella Isolates M. Yu. Kirillova, A. P. Markova, I. V. Lopyreva, V. N. Pankratova a, S. A. Levitskiia, V. N. Bashkirova, G. B. Smirnova, A. N. Kruglovb, V. A. Osadchayab, G. P. Frolovab, G. V. Barminab, O. A. Morozovab, and M. Y. Kosoyc a

Gamaleya Research Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, ul. Gamalei 18, Moscow, 123098 Russia b Sechenov Moscow Medical Academy, ul. Trubetskaya 8, kor. 2, Moscow, 119992 Russia c Centers for Disease Control and Prevention, Fort Collins, CO, USA Received December 28, 2005

Abstract—Primer sets for PCR detection of four housekeeping genes of Bartonella spp. isolated from clinical material were developed and tested. We tested a strategy of the specific restriction fragment length polymorphism typing of bartonella species using as an example two strains isolated for the first time in Russia from patients with endocarditis and fever of uncertain origin. The results of typing were confirmed by sequencing of the obtained amplicons. The results of sequencing confirmed the affiliation of these isolates to the subspecies B. vinsonii subsp. arupensis. The necessity of molecular epidemiological analysis of bartonellosis in Russia was substantiated. DOI: 10.3103/S0891416807010028

ing to various reports, nine Bartonella species with different virulence properties are pathogenic to humans [6]. In addition to the above-mentioned species, this genus includes a number of nonpathogenic species, such as B. doshiae, B. taylorii, B. talpae, and B. peromysci [4]. Natural reservoirs of Bartonella species are small rodents, members of the cat family (cats, pumas), and dogs [6, 14]. The pathogen usually (but not necessarily) produces an asymptomatic infection and prolonged bacteremia. At present, the natural reservoir of B. quintana is still uncertain; humans are the only known reservoir of the microorganism. It is suggested that various bloodsucking arthropods (mosquitoes, fleas, lice, and ticks) transmit the agent from wild and domestic animals serving as reservoir hosts. At any rate, contacts with bloodsucking arthropods are believed to be one of the risk factors associated with Bartonella infections [6].

The first human case of an infectious disease caused by the Bartonella species B. quintana (formerly Rochalimaea quintana) was reported early in the 20th century [24]. Only in the last decade of the twentieth century, according to the results of sequencing of some housekeeping genes, was this pathogen placed in the separate genus Bartonella, along with B. henselae, the pathogen that causes cat scratch disease, and B. bacilliformis, the agent of Carrion’s disease, which has been documented only in the Andes [7, 8]. At about the same time, B. elizabethae, a new species of pathogens presumably responsible for infective endocarditis, which was also placed in the genus Bartonella, was described [9]. Isolation of these bacteria from bacillary angiomatosis patients [19, 22] was the principal factor contributing to our current understanding of the pathogenicity of Bartonella. Further investigations made it possible to reveal other common symptoms of bartonellosis, such as fever, bacteremia, skin lesions, endocarditis, and benign lymphadenopathy. Central nervous system abnormalities and liver disorders, as well as damage to eye and bone tissue, occur more rarely [2]. The chronic forms of bartonellosis, which mostly strike patients with immunodeficiency, are accompanied by prolonged bacteremia (in spite of long-term antibiotic therapy) due to the intracellular localization of a part of the pathogen population. At present, bartonelloses unite a group of human diseases caused by gram-negative aerobic facultatively intracellular bacteria that require hemin or products of erythrocyte destruction for growth. In 1993, bartonellae were assigned to the family Bartonellaceae (the α-2 subclass of Proteobacteria), phylogenetically close to the genus Brucella. Accord-

A worldwide search for Bartonella species in natural populations of rodents has demonstrated a great abundance of these bacteria among rodents in various countries (United States, Bolivia, Paraguay, Canada, Poland, etc.). The species composition of these microorganisms in populations of bloodsucking insects has been characterized [12]. A wide range of modern techniques, including bacteriological methods, ELISA, and PCR, was applied. On the contrary, no systematic investigations of Bartonella species have been carried out in Russia up till now. Studies of the distribution and diversity of genetic variants of the pathogen, as well as of the host–pathogen specificity within wild and domestic animal populations, have only begun in our country [1]. Reports of successful 7

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KIRILLOV et al.

isolation of B. quintana and B. henselae from arthropods are scarce [17, 20]. The aim of the present work was to develop a method for reliable typing of Bartonella infections in patients with septic endocarditis and fever of uncertain origin using modern molecular genetic techniques. Taking into account that, so far, investigations of this kind have not been carried out in Russia, this paper may be of interest to medical bacteriologists. The paper is concerned with the development of systems for detection of the three most “popular” Bartonella housekeeping genes using a two-round (nested) PCR technique and with application of the generated PCR products for identification of two clinical Bartonella isolates. MATERIALS AND METHODS The patients were chosen from among patients with fever of uncertain origin. Cultivation methods. Blood samples (5 ml) were taken from all the patients. The obtained samples were inoculated into Vero E6 cell cultures, incubated in a 10% CO2 atmosphere for 5 days, and subcultured on a solid nutrient medium (brain heart infusion agar supplemented with 5% sheep blood). Blood samples taken from the patients were simultaneously cultivated in biphase systems (agar/brain heart infusion broth). Bacterial cultures of microorganisms growing in the environment or the human body under various conditions were obtained from the collections of the Tarasevich State Research Institute of Standardization and Control of Biomedical Preparations and the Gamaleya Research Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences. To assess the specificity of the primers used, DNAs from the following cultures were used: Ach. laidlavii; Bif. adolescentis; Bif. bifidum; Bif. breve; Bord. bronchisepica; Bord. parapertussis; Bord. pertussis; Burkh. cepacia; Can. albicans; Can. tropicales; Chl. pneumoniae; Chl. trachomatis; Chl. psittaci; CMV; Esch. coli B and K, Gard. vaginalis; Haem. influenzae a; Haem. influenzae b; Haem. influenzae d; HSV2; Mycob. tuberculosis; Mycob. bovis; Mycob. gastri; Mycob. avium; Mycob. smegmatis; Myc. hominis; Myc. genitalium; N. gonorrhoeae; N. meningitidis (group A); Ps. putida; Ps. aeruginosa; S. typhimurium; St. aureus; Str. faecalis; Str. viridans; Str. pneumoniae; streptococci of the groups A, B, C, D, E, F, and G; U. urealyticum; Y. enterocolitica; and Y. pestis EV. To assess the sensitivity of the primers used, DNAs isolated from the following cultures were used: B. elizabethae, B. clarridgeiae, B. grahamii, B. henselae, and B. vinsonii subsp. arupensis. The DNA samples were obtained from the collection of the Centers for Disease Control and Prevention, Fort Collins, Colorado (United States).

The DNAs were isolated from the cell cultures and blood samples using a reagent kit (ZAO LAGIS, Moscow) according to the manufacturer’s instructions. The technique was based on the method of Boom et al. [5] and involved cell lysis in 100 µl 5 M guanidine thiocyanate and sorption of nucleic acids on porous glass. After washing of the sorbent, DNA was eluted in 100 µl of TE buffer. At the stage of testing and optimization of primer sets, DNA preparations of Bartonella strains were added to blood samples taken from six to eight healthy volunteers in a ratio of 1 : 10 in order to produce conditions close to those of clinical specimens. Amplification was carried out using a universal PCR buffer (ammonium sulfate buffer, ZAO LAGIS), supplemented with 10 πM of each primer, 2 U of Taq polymerase, 2.5 mM of MgCl2, and 100 µM of nucleotide triphosphates, in a total volume of 25 µl, in a Tercyc PCR Cycler (DNA-Technology, Moscow). The first round of amplification was for 41 cycles. The thermocycler was programmed to denature DNA at 94°C for 10 s, 55°C for 10 s, and 72°C for 10 s with preliminary incubation at 94°C for 3 min and final elongation for 3 min. The universal primers proposed by Lu et al. [15] were used during the first round of amplification of the 16S rRNA gene fragment under the same amplification conditions. The second round of amplification was performed under similar conditions with an annealing temperature of 60°C. The first-round PCR product was diluted 1:100 and used as the matrix for the second round of amplification. The reaction product was assayed by electrophoresis on a 1.2% agarose gel. The restriction analysis of amplicons was performed using restriction enzymes manufactured by Fermentas (Lithuania) according to the manufacturer’s instructions. To be certain of the restriction completeness, the reaction was performed for 16 h. The reaction products were electrophoresed on a 2% agarose gel supplemented with 1% Synergel (Diversified Biotech, United States) to improve gel resolution. To determine the sizes of the obtained restriction fragments, we used GeneRuler molecular weight markers (50bp DNA Ladder or 100bp DNA Ladder (Fermentas)). The results were recorded with a ViTran gel documentation system (Biokom, Moscow), which includes a transilluminator, darkroom, CCD camera, and software. Determination of nucleotide sequences of the PCR products. The obtained amplicons (400 pg) were isolated from the agarose gel with an elution kit (GENOMED, Germany) and subjected to cyclic sequencing. The thermal program was 30 s at 96°C, 10 s at 60°C, and 4 min at 72°C (25 cycles). The amplicons were analyzed in an Applied Biosystems 373 automatic sequencer using fluorescence-labeled transcription terminators. Comparison of the determined nucleotide sequences with sequences within the GenBank database, as well as the determination of Bartonella species, was performed using the BLAST2 software (National Center for Biotechnology Information) [3].

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Table 1. Sequences of the primers used Code U518_F U1513_R S003o_F S003o_R F001o_F F001o_R F001i_F F001i_R R002o_F R002o_R R001i_F R001i_R G003o_F G004_R G004i_F G004i_R

5' CCA ATC ATG GGC TGG ATG GTT CAC AAT GCC CGT GGT TAT ACT TTC TTT

GCA GGY TAG TTT CTA AGA GGG CTG TGC AGA AGG TGT TAC CGA ACA AGG

GCC TAC GCG TGG TGT CCT CAA GTC GTG AAG AAG CTC AGA TGA GGT CAC

GCG CTT GAT AGA CAA GTA GCG AGT TTC TTT AAG CAG TCC CCA CCC GCT

Amplicon size

3' sequence GTA GTT ATT TTA AGG ATG GCT CAT AGG AGC CGT GTT GCA AAA AAC TCA

ATA ACG TAA GCT CTG GAA GTA AGC AGT AGC TGC GTC ACA CCC TCT TTA

CG ACT GTC CGA AAC GCA GAG AAA TTG ATA GTT ATT GAG ATA TGC GCT

TC AGA C G ACA C GAA TGT AGC ATC A ACG TGA TCA AAT A CGC CCA

RESULTS AND DISCUSSION Development of primer sets for PCR analysis of DNA from Bartonella species. At the first stage, we assessed the specificity of the previously described primer pairs (theoretically, according to the results of comparison of the determined nucleotide sequences with sequences within the GenBank database; the BLAST2 software package), as well as the reaction efficiency (the ratio between the calculated and applied annealing temperatures, the number of nucleotide substitutions in relation to the sequences of certain Bartonella genes, ∆G, and the ability to form secondary structures). On the basis of the data obtained, we selected the most appropriate primer pairs for detection of the five generally recognized target genes of Bartonella spp.: the 16S rRNA gene [21], the citrate synthase gene (gltA) [18], the gene encoding the cell division protein (ftsZ) [10], and the genes encoding the heat shock protein (groEL) [16] and riboflavin synthase (ribC) [13]. This number of targets is associated with the “extended gold standard” for detection of difficultto-cultivate or uncultivable microorganisms. Based on the results of this analysis, we drew a conclusion about the potential clinical application of the above-mentioned primer pairs. At the second stage of our investigation, we designed our own primer pairs for PCR detection of the same genes. For this purpose, we retrieved all the gene sequences that showed extensive homology (more than 200 bp; more than 70%) to the studied gene from the GenBank database using the NCBI BLAST2 software. The retrieved sequences were then aligned using the ClustalX 1.83 software package [23]

C AGT G GA TAT CCA

Target gene

PCR round

995

16S rDNA

1

715

16S rDNA

2

453

ftsZ

1

339

ftsZ

2

480

ribC

1

330

ribC

2

470

gltA

1

324

gltA

2

and visualized with the GeneDoc multiple sequence alignment editor (v. 2.6.002; Karl Nicholas, 2000). According to the alignment results, we selected the regions that contained the greatest number of conserved base pairs within the genus, as well as the greatest number of base pairs that differed from the sequences of these target genes in other bacterial species. We used such regions for calculating primer pairs using the Gene Runner software package (v. 3.05; Hastings Software, Inc., 1994). When developing internal primers, we excluded the sequences of the 16S rRNA, gltA, ftsZ, and ribC target genes of the species that have no medical significance from the analysis. This made it possible to reveal the most extensive regions of the target genes conserved for B. bacilliformis, B. clarridgeiae, B. elizabethae, B. grahamii, B. henselae, B. quintana, B. tribocorum, and B. vinsonii. The internal primers developed were located within these regions, shared by most Bartonella species. Based on the performance characteristics of nested PCR, the annealing temperature of internal primers has to be greater than that of external primers, and, therefore, internal primers must be longer than external ones. This enhances the possibility of the formation of secondary structures. The characteristics of the primer pairs developed and of the amplicons obtained with these primers are summarized in Table 1. It should be noted that, during design of internal primers, their specificity was taken into account. In this connection, the choice of genus-specific nondegenerate primers with a high annealing temperature was very limited. Thus, we had to introduce additional nucleotide substitutions to prevent the assembly of stable homo- and heterodimers. All the internal primers used contained such substitutions.


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Table 2. The most informative restriction profiles of the studied Bartonella amplicons ftsZ 339 bp Pattern no. HhaI

Species

1

2

3

4

5

6

7

8

9

339 (nr)

208 131

203 136

203 131 5

224 115

194 145

145 131 58 5

143 115 81

C Va W

QD

ST

P R Tr

Vb Vv

H

G

143 127 60 5 2+2 E

Pattern no.

Ba

1

2

3

4

5

6

HinfI

339 (nr)

283 56

218 65 56

SW

206 65 56 12 D

159 124 56

Species

200 65 56 18 T

G

E Ba R Tr

C H P Q Va Vb Vv

ribC 331 bp Pattern no. DdeI

Species Pattern no. MboI

Species

1

2

3

4

5

6

7

331 (nr)

218 113

197 134

Ba C G

E

A Bi D H K S

197 129 5 Q

197 71 63 Va Vb Vv

197 76 58 T

197 99 35 Bo

1

2

3

4

5

6

7

8

9

331 (nr)

270 61

261 70

223 108

187 144

187 137 7

144 108 79

144 126 61

C

D

A

E

H T Vv

Ba Bo Bi S Va Vb

G

K

137 126 61 7 Q

gltA 324 bp Pattern no. HhaI Species Pattern no. MboI

Species

1

2

3

4

324 (nr) EGHKQ

296 28 DT

243 81 H Va Vb Vv

169 155 S

1

2

3

4

5

188 96 43

164 136 24

136 114 74

136 90 74 24

D

Ba

HS

K Vv Vb

93 90 74 43 24 E G Q T Va

Note: A – B. alsatica; Ba – B. bacilliformis; Bi – B. birtlesii; Bo – B. bovis; C – B. clarridgeiae; D – B. doshiae; E – B. elizabethae; G − B. grahamii; H – B. henselae; K – B. koehlerae; P – phoceensis; Q – B. quintana; R – B. rattimassiliensis; S – B. schoenbuchensis; T – B. taylorii; Tr – B. tribocorum; Va – B. vinsonii subsp. arupensis; Vb – B. vinsonii subsp. berkhoffii; Vv – B. vinsonii subsp. vinsonnii; W – B. weissii. MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY

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HhaI

A Ba Bi C D E G H K P Q R S T Tr VaVbVv W

11 HinfI

L

400 300 81 82

200 150

Nr

Nr

81 82

100 70 50 20 10 1

Fig. 2. Fragment of the electrophoretogram of the ftsZ restriction patterns in Bartonella reference strains. The studied Bartonella species are designated as in Table 2. L, GeneRuler 50 bp DNA Ladder; HhaI and HinfI, restriction endonucleases used.

(b) A Ba Bi Bo C D E G H K Q S T Va Vb Vv

400 300 200 150 100 70 50

20 10 1 Fig. 1. A schematic depiction of restriction patterns of the ftsZ/HhaI (a) and ribC/MboI (b) amplicons. The studied Bartonella species are designated as in Table 2. (a) The patterns C–Va–W, Q–D, S–T, P–R–Tr, and Vb–Vv coincide. The patterns E, G, and K are difficult to differentiate in gel slabs at low resolution. (b) Similar difficulties are encountered with, for example, pattern nos. 2 and 3 (D and A).

In order to increase the specificity and sensitivity of our experiment, two-round PCR was carried out. All the target genes used in the analysis are housekeeping genes. They are present in all bacterial genomes and show high homology of nucleotide sequences. The use of a single primer pair does not guarantee absolute specificity; therefore, the application of two primer pairs significantly increases the process reliability. Blood, being the main clinical material that we analyzed, has a strong inhibitory effect on PCR. As the method sensitivity increases, the amplification process becomes hundreds of times less efficient. After the second round of PCR, the concentration of inhibitors decreases and the product obtained during the firstround PCR amplification can be visualized by gel electrophoresis.

At the next stage, we proved in practice the efficiency of the above-mentioned primer pairs. When using each primer pair, we assessed the specificity and sensitivity of amplification, as well as the presence of artifacts (we used external and internal pairs in both one- and two-round amplification systems). The primers were tested for specificity to DNA from pure cultures of microorganisms growing in the environment or the human body under various conditions and four DNA samples from the blood of healthy human subjects without apparent bartonellosis symptoms. Also, the primer sensitivity was tested with serial tenfold dilutions (in TE buffer and a human DNA solution prepared after one round of DNA replication) of various Bartonella cultures. The results are not presented due to the size of the final table. The obtained results demonstrated that the properties of our test system, such as sensitivity, specificity, and the absence of amplification artifacts in analysis of DNA from pure cultures or generation of large amounts of ballast DNA, are very similar to previously described analogues. The sensitivity of the two-round process when applied to test systems (pure blood supplemented with DNA from Bartonella spp.) is roughly tens of bacterial cells per reaction. DNA typing of Bartonella spp. with restriction fragment length polymorphism (RFLP) analysis. We selected the RFLP method for the preliminary screening of the species affiliation of natural and clinical Bartonella isolates because this method is less costly than sequencing. High numbers of interspecific variations (9.4–21.8% of nucleotide substitutions in the studied region of the ribC gene, 6.9–12.1% of substitutions in the studied region of the ftsZ gene, and 3.1–15.9% in that of the gltA gene) were already detected at the stage of sequence alignment. Similar data on interspecific variations were obtained during the alignment of amplicon sequences: 7.6–21.8% of nucleotide substitutions


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KIRILLOV et al. FtsZ HhaI

G1tA HinfI

81 82 E Nr 81 82 E

L

HhaI

Ribc MboI

81 82 E Nr 81 82 E

DdeI

AluI

L

81 82 E Nr 81 82 E

Fig. 3. Fragment of the electrophoretogram of the restriction typing of the ftsZ, gltA, and ribC gene amplicons of the clinical Bartonella isolates MMA81 and MMA82 (81 and 82). Designated are amplicons subjected to restriction enzyme digestion and the restriction endonucleases used. E, corresponding B. elizabethae amplicons (restriction control); Nr, corresponding B. elizabethae amplicons without restriction; L, GeneRuler 100 bp DNA Ladder. Explanations in the text.

in the studied region of the ribC gene, 6.8–13.6% of substitutions in the studied region of ftsZ, and 3.1– 16.1% in the gltA region. Evidently, at such a level of variability, one would expect that interspecific differences would be revealed during the restriction analysis of amplicons. At the first stage of this study, we generated restriction maps for the obtained internal amplicons of the used target genes of all Bartonella species available in GenBank (the Vector NTI 9.0.0 software package, 1994–2003 InforMax). For each gene fragment, we selected the most informative restriction enzymes and generated tables of restriction profiles (Table 2). In fact, we theoretically succeeded in obtaining several sets of profiles characteristic of one to three Bartonella species for two genes, ftsZ and ribC. We planned to use such restriction endonucleases as Hhal and MboI (for the ftsZ and ribC fragments, respectively) for screening of the obtained amplicons. Unfortunately, we failed to choose an enzyme for the gltA gene that would make it possible to achieve appropriate results. Moreover, some profiles are difficult to discern in agarose electrophoresis due to low resolution (for example, there are no differences between 145 and 143; 131 and 127 or 136; and 58 and 60 bp, ftsZ/HhaI). In Table 2, these profiles are situated in neighboring columns. As an illustration, we modeled the most informative profiles using the Vector NTI 9.0.0 software package. The results of “electrophoresis” in a 4% polyacrylamide gel are shown in Figs. 1a (ftsZ/HhaI) and 1b (ribC/MboI). As can be seen, some profiles can be discerned only due to the schematic nature of the illustration (high resolution, the absence of band “overload,” and the presence of bands smaller than 60 bp). Hence, RFLP analysis of amplicons enables an obtained amplicon to be assigned to a group consisting of two to six species. It should be noted that it is possible to detect a mixed infection induced by Bartonella species belong-

ing to different profiles provided that the restriction process has been completed [1]. To determine the species affiliation of members of “multispecies” or nondifferentiable profiles, we planned to perform restriction by the chosen enzymes (HinfI for ftsZ and DdeI for ribC, Table 2), or, if they were uninformative, by enzymes unique for each member. To test the approach developed, we subjected the amplicons obtained from the control DNAs to restriction enzyme digestion in accordance with the proposed scheme. In all cases, we were able to generate the correct profiles. Figure 2 shows a fragment of the final gel slab: for the ftsZ amplicon, the enzymes HinfI (three profiles) and HhaI (four profiles). According to the results of the two restriction procedures, all the five Bartonella species were identified. Identification of clinical Bartonella isolates. At the Sechenov Moscow Medical Academy (MMA), we isolated two strains of Bartonella from blood samples taken from several 20- and 30-year-old female patients by culturing on Vero E6 cell cultures. The antibody titers against B. vinsonii were determined in obtained sera. Both one- and two-round PCR assays of the isolated DNA, designated as MMA81 and MMA82, respectively, with all the primer sets devised showed positive reactions irrespective of the negative results obtained from the control DNA isolations and the negative control PCR reaction. RFLP typing was performed according to the proposed scheme (Fig. 3). The profiles HinfI-6 (characteristic of C, H, P, Q, Va, Vb, and Vv) and HinfI-1 (characteristic of C, Va, and W) were obtained through sequence analysis of ftsZ amplicons. The species was recognized as C or Va. The profiles HhaI-3 (characteristic of H, Va, Vb, and Vv) and MboI-5 (characteristic of E, G, Q, T, and Va) were obtained through sequence


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analysis of gltA amplicons. The species was recognized as Va; however, due to the fact that the sequence of this gene fragment of B. clarridgeiae is not known, this species cannot be excluded. Analysis of ribC amplicons was performed based on the results obtained. For the DdeI restriction enzyme, we obtained profile no. 5, unique to only three subspecies of B. vinsonii (we failed to obtain a B. clarridgeiae restriction enzyme; Table 2). The species was recognized as Va. To improve the reliability of the results obtained, the ribC amplicon was subjected to digestion with the enzyme AluI, which produces 133 and 198 bp fragments from the A, Q, Va, and Vv amplicons. The obtained profiles coincided with the predicted restriction profiles since two bands (130 and 200 bp) can be seen (Fig. 3). To be absolutely sure, we subjected the ribC amplicon to digestion with the following restriction endonucleases: AatII, producing 247 + 84 bp fragments from the amplicons Va and Vv, and MnlI, producing 175 + 85 + 71 bp fragments only from Va amplicons. In Fig. 4, we can see two bands, one about 180 bp long and another wide band of about 80 bp, coinciding with the predicted profiles (Fig. 4). According to the results of the RFLP analysis of the clinical Bartonella isolates from MMA clinics, strains MMA81 and MMA82 could be recognized as B. vinsonii subsp. arupensis. These results could be improved only by sequencing. According to the sequencing results, the sequence similarities of the ftsZ gene fragments in strains MMA81 and MMA82 and B. vinsonii subsp. arupensis were 99.28 and 99.67%, respectively (Fig. 5a). By comparison, the sequence similarities between the ftsZ gene fragment of MMA81 and the sequences of the closest Bartonella species, B. vinsonii subsp. vinsonii and B. vinsonii subsp. berkhoffii, were only 97.2 and 96.8%, respectively. The sequence similarities between the ribC gene fragments of strains MMA81 and MMA82 and B. vinsonii subsp. arupensis were 98.96 and 96.58%, respectively (Fig. 5b). The sequence similarities between the gltA gene fragments of strains MMA81 and MMA82 and B. vinsonii subsp. arupensis were 98.68 and 98.64%, respectively (Fig. 5c). Hence, the ribC, ftsZ, and gltA amplicons of both clinical isolates were, in fact, closest to the sequences of B. vinsonii subsp. arupensis. The first case of human infection caused by this Bartonella species in the United States was reported in 1999, when a patient with atypical fever was described [25], although this species can often be found in rodents Peromyscus leucopus. Recently, an endocarditis case caused by this species has been reported [11]. In Russia, B. vinsonii subsp. arupensis was detected and isolated for the first time from patients with atypical fever and endocarditis. Our data support the opinion that B. vinsonii subsp. arupensis should be included in the list of dangerous pathogens that can cause the above-mentioned diseases. Further investigations into bartonellosis cases in Russia

13

AatII

L

H

G Va Q

E

MnlI

Fig. 4. Fragment of the electrophoretogram of the additional typing of the ribC amplicons of the clinical Bartonella isolates MMA81 and MMA82 (81 and 82, respectively). Nr, B. elizabethae amplicons without restriction; AatII and MnlI, applied restriction endonucleases; L, GeneRuler 100 bp DNA Ladder. Explanations in the text.

should be aimed at determining of the role of rodents as natural reservoirs of this infection and of bloodsucking arthropods as potential Bartonella spp. carriers. We have developed and tested our own primer sets for PCR analysis of four housekeeping genes of Bartonella spp. isolated from clinical material. Having analyzed the results of comparative studies, we have arrived at the conclusion that the PCR test systems we developed are very similar to previously described analogues. A scheme of RFLP typing of Bartonella spp. has been developed and tested. The results of typing were confirmed by sequencing of the obtained amplicons. Pure cultures of B. vinsonii subsp. arupensis were isolated for the first time in Russia from patients with atypical fever and suspected endocarditis.


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B. birtlesii B. schoenbuchensis B. weissii B. bacilliformis B. clarridgelae

B. grahamii B. tribocorum B. elizabethae B. rattimassiliensis B. taylorii B. alsatica B. phoceensis MMA81 [DQ334267] MMA82 [DQ334268] B. vinsonii arupensis B. vinsonii berkhoffii B. vinsonii vinsonii B. henselae B. koehlerae B. quintana B. doshiae

0.1 (b)

0.1

B. birtlesii B. schoenbuchensis B. bovis B. bacilliformis B. clarridgelae

B. doshiae B. taylorii B. henselae B. koehlerae B. quintana B. vinsonii arupensis MMA82 [DQ334269] MMA81 B. vinsonii berkhoffii B. vinsonii vinsonii B. elizabethae B. grahamii B. alsatica

(c)

MMA81 [DQ334265] MMA82 [DQ334266] B. vinsonii arupensis B. vinsonii berkhoffii B. vinsonii vinsonii B. taylorii B. elizabethae B. grahamii

B. henselae B. koehlerae B. quintana B. doshiae B. bacilliformis

0.01

B. schoenbuchensis

Fig. 5. Dendrogram of the studied sequences of the amplicons of Bartonella spp. and the clinical Bartonella isolates MMA81 and MMA82. (a) ftsZ; (b) ribC; (c) gltA. Square brackets enclose numbers of items available in the GenBank database. MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY

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2007