Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Woolley, M. W. Articles by Wetherall, B. L. Search for Related Content PubMed PubMed Citation Articles by Woolley, M. W. Articles by Wetherall, B. L.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, June 2007, p. 2040-2043, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.00175-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Analysis of the First Australian Strains of Bartonella quintana Reveals Unique Genotypes

Mark W. Woolley,^* David L. Gordon, and Bruce L. Wetherall

Department of Microbiology and Infectious Diseases, School of Medicine, Flinders University and Flinders Medical Centre, Bedford Park, South Australia, Australia 5042

Received 23 January 2007/ Returned for modification 2 March 2007/ Accepted 4 April 2007

Top ABSTRACT TEXT REFERENCES

 
Bartonella quintana is increasingly recognized as a cause of clinical disease in various geographical locations. We characterized three Australian strains associated with endocarditis, using established molecular-typing techniques, the 16S/23S rRNA intergenic spacer (ITS) region, and multispacer typing (MST). All strains examined demonstrated novel ITS and/or MST genotypes. Further characterization of Australian strains is required to determine whether there is an association between genotype and geographical location.

Top ABSTRACT TEXT REFERENCES

 
Bacteria of the genus Bartonella are considered emerging pathogens, as many new species and subspecies have been recognized in humans and other mammals in recent years. The 1984 edition of Bergey's Manual of Systematic Bacteriology lists only one species of Bartonella (B. bacilliformis) (13) and two species of Rochalimaea (R. quintana and R. vinsonii) (18), which were later included in the Bartonella genus. The genus now comprises 20 species or subspecies. Nine of these species have been implicated as causative agents of human disease, including B. quintana, which was originally recognized as the agent of trench fever during World War I. The spectrum of diseases associated with this organism now includes bacillary angiomatosis (12), native- and prosthetic-valve endocarditis (5, 7), chronic asymptomatic bacteremia, and relapses of illness in people with risk factors such as homelessness and alcoholism (3).

Bartonella quintana is a fastidious organism that is distributed worldwide and is transmitted to humans via several arthropod vectors. Although the human body louse is the primary vector (12), B. quintana has also been detected in the human head louse (17), fleas from gerbils (11) and cats (15), the human flea Pulex irritans (14), Ixodes pacificus ticks in California (1), and cat dental pulp (8), suggesting that other vectors and sources of the organism may also be responsible for its transmission.

Infection with B. quintana has not been previously reported in Australia. However, over the past 3 years we have identified one case of culture-positive and two cases of culture-negative endocarditis due to B. quintana. All patients (two Caucasians, one indigenous Australian) were from northern and central Australia and had confirmed infective endocarditis with aortic valve involvement, severe aortic incompetence, and cardiac failure requiring valve replacement. In this study, the three strains identified in isolates from the endocarditis patients were investigated for their relationships to genotypes described in other areas of the world.

Conventional microbiological techniques were used for microscopy and culture of aortic valve tissue material and for presumptive identification of the cultured strain (1300/02) as a Bartonella species. Briefly, cultures showed growth on chocolate agar after 2 weeks of incubation in 5% CO₂ at 35°C, with typical colonial appearance, Gram stain morphology, and biochemical inertness, except for the production of leucine aminopeptidase and glycyl-tryptophane arylamidase.

Genomic DNA was prepared from this isolate, using the Wizard genomic DNA purification kit (Promega Corporation, Madison, WI). For the culture-negative strains, DNA from aortic valve tissue was prepared by using a genomic DNA extraction miniprep system (Viogene, Sijhih Taipei, Taiwan) for fresh tissue (strain IMVS/04) or a xylene-based method for paraffin-embedded tissue (strain 8896/04) (http://cc.ucsf.edu/people/waldman/Protocols/paraffin.html).

The hypervariable 16S/23S intergenic spacer (ITS) region of Bartonella species was amplified by PCR, using primers 321s and 983as as previously described (10). As a positive control, DNA from a quality assurance strain of B. henselae was included. Both culture-positive and culture-negative DNA extracts yielded an amplicon size of 564 bp, identifying the isolates as B. quintana. The B. henselae positive control sample yielded the correct amplicon size of 648 bp (2).

To establish the relationship among the B. quintana strains and to compare them with those reported from other geographical areas, two separate genotyping methods were employed, as described elsewhere (4, 6). Using primers BQF and BQR, the variable 260-bp segment between positions 882 and 1141 of the ITS region was amplified (6). The PCR products were purified, using the Geneclean spin kit (Q-BIOgene, Carlsbad, CA), before being included in two sequencing reactions (DNA sequencing kit; Perkin-Elmer) that incorporated either primer BQF or BQR. Nucleotide sequences were determined with an ABI PRISM 310 genetic analyzer (Perkin-Elmer). ClustalW was used to align sequences to produce consensus sequences of specific PCR targets, and BlastN was used to compare these sequences with those stored in GenBank. The B. quintana strains were assigned genotypes according to variable single-nucleotide polymorphisms (SNPs) occurring at the correct ITS nucleotide positions: 938, 1018, 1037, and 1082 (Fig. 1). These SNPs correspond to the 860-, 940-, 959-, and 1004-bp positions described by Houpikian and Raoult (6). The cultured B. quintana isolate (1300/02) was identical to the previously described ITS genotype III on the basis of matching nucleotides at the SNP positions. The uncultured B. quintana strains (8896/04 and IMVS/04) revealed an additional nucleotide substitution at position 950 bp with variable SNP profiles not previously reported, generating new ITS genotypes designated here as IV and V (Fig. 1).

View larger version (9K): [in this window] [in a new window]

FIG. 1. Schematic diagram of the B. quintana hypervariable rRNA ITS region amplified by primers BQF and BQR, showing nucleotide positions of SNPs and resulting genotypes. The strains examined in this study are in bold. Other strains representing established genotypes and with GenBank accession numbers are from Fournier et al. (5). Genotypes marked with a superscript "a" are newly described.

To further characterize the strains, the multispacer-typing (MST) genotyping method of Foucault et al. was employed (4). In their study, sequences of a series of noncoding spacer regions from 81 B. quintana isolates derived from France, Yugoslavia, Russia, Oklahoma, England, and Burundi were assessed for variability and hence discriminatory capacity. Five genotypes were distinguished and depended on the combined SNP profiles of two spacers (336 and 894).

In our study, spacers 336 and 894 were amplified by PCR and the nucleotide sequence was determined. The nucleotide substitution and deletion profiles for spacers 336 and 894 of our strains and comparative sequences are shown in Fig. 2. Strain 8896/04 showed a unique spacer 336 sequence profile, here designated type 3, while both the 1300/02 and IMVS/04 strains were identical to spacer 336 sequence types 1 and 2, respectively (4). At spacer 894, all three B. quintana strains showed the same, unique sequence profile different from that of the four sequence types previously documented and classified here as type 5. By combining the different sequence types for spacers 336 and 894, we classified the strains examined in this study as new genotypes 6, 7, and 8. A total of eight genotypes, including the newly described genotypes and the five already described, can now be defined (4) (Table 1). To monitor for contamination, appropriate negative controls, including separately cultured B. henselae and water extractions and a no-template control, ensured B. quintana specificity. To exclude sequencing artifacts, PCR using Vent DNA polymerase (3'5' exonuclease) was performed, and sequencing in both directions on two separate occasions gave identical results.

View larger version (11K): [in this window] [in a new window]

FIG. 2. Schematic diagram of spacers 336 and 894, showing SNP positions and corresponding sequence types. The strains examined in this study are in bold. Other strains representing established sequence types and with a GenBank accession number are from Foucault et al. (3). Sequence types marked with a superscript "a" are newly described.

View this table: [in this window] [in a new window]

TABLE 1. MST genotypes of B. quintana derived from spacer 336 and 894 sequence types

Relatively few strains of B. quintana have been subjected to genotyping studies. To date, the greatest number of strains have originated in France, where ITS genotypes I, II, and III and MST genotypes 1, 2, and 3 were found (4, 6). A comparison of the ITS and MST genotypes of our strains to all previously reported genotypes from different countries reveals the unique profiles of strains found in Australia to date (Table 2). One strain (1300/02) was ITS genotype III, the second-most-common genotype found among homeless bacteremic patients and body lice in France; however, its MST genotype of 6 was unique. No other previously observed ITS or MST genotypes were found among our strains.

View this table: [in this window] [in a new window]

TABLE 2. Comparison of combined results of ITS and MST genotyping methods for Australian B. quintana strains and genotypes found in other countries

B. quintana, an emerging pathogen, is increasingly being detected globally and from different sources and hosts. Epidemiological studies to establish genetic relationships among strains from both local and more-widespread areas have been hampered by the lack of isolates and a suitably reproducible and discriminatory typing technique. Various genotyping techniques, including pulsed-field gel electrophoresis (PFGE) (16), 16S-23S rRNA ITS typing (6), and MST (4), have been utilized to discriminate among B. quintana strains. PFGE was found to be an inappropriate molecular-typing tool for B. quintana, due to frequent genomic rearrangements (4). The natural diversity found within the 16S-23S ITS region, as described previously, provides a more sensitive means of differentiating strains of several Bartonella species (6). However, due to the low heterogeneity found in this region, relatively few genotypes have been identified for B. quintana (6). MST, which interrogates multiple intergenic and pseudogene sequences genome-wide, appears to be more discriminatory due to the greater diversity included within these noncoding regions (4, 9). We applied both the ITS and MST techniques to three Australian strains and detected further heterogeneity, despite the relatively conserved B. quintana genome (4, 6). Our strains, representing three novel genotypes, were all derived from aortic valve tissues from patients with endocarditis, and our results are indicative of the difficulty in assigning disease associations to specific genotypes.

Despite the small number of strains examined, our results demonstrate clear diversity among B. quintana isolates from different geographic locations. Analyses of further strains may shed more light on the ecology of B. quintana and the potential clinical significance of these variations.

Nucleotide sequence accession numbers. Newly encountered genotypes for the 260-bp region between bp 882 and 1141 of 16S-23S ITS and sequence types for the MST spacers 336 and 894 have been submitted to GenBank under the following accession numbers: 16S-23S ITS genotype IV, EF532789; 16S-23S ITS genotype V, EF532790; MST spacer 336, sequence type 3, EF532791; and MST spacer 894, sequence type 5, EF532792.

 
We thank Rolf Wise (Institute of Medical and Veterinary Science, Adelaide) for providing a DNA extract of cardiac valve tissue previously shown to be positive for B. quintana by PCR.

 
* Corresponding author. Mailing address: Department of Microbiology and Infectious Diseases, Flinders Medical Centre, Flinders Drive, Bedford Park, S.A., Australia 5042. Phone: 61-8-8204 4392. Fax: 61-8-8204 4733. E-mail: mark.woolley{at}fmc.sa.gov.au

Published ahead of print on 11 April 2007.

Top ABSTRACT TEXT REFERENCES

 

1
1. Chang, C. C., B. B. Chomel, R. W. Kasten, V. Romano, and N. Tietze. 2001. Molecular evidence of Bartonella spp. in questing adult Ixodes pacificus ticks in California. J. Clin. Microbiol. 39:1221-1226.[Abstract/Free Full Text]
2
2. Dillon, B., and J. Iredell. 2005. Potential limitations of the 16S-23S rRNA intergenic region for molecular detection of Bartonella species. J. Clin. Microbiol. 43:4921-4922.[Free Full Text]
3
3. Foucault, C., K. Barrau, P. Brouqui, and D. Raoult. 2002. Bartonella quintana bacteremia among homeless people. Clin. Infect. Dis. 35:684-689.[CrossRef][Medline]
4
4. Foucault, C., B. La Scola, H. Lindroos, S. G. E. Andersson, and D. Raoult. 2005. Multispacer typing technique for sequence-based typing of Bartonella quintana. J. Clin. Microbiol. 43:41-48.[Abstract/Free Full Text]
5
5. Fournier, P. E., H. Lelievre, S. J. Eykyn, J. L. Mainardi, T. J. Marrie, F. Bruneel, C. Roure, J. Nash, D. Clave, E. James, C. Benoit-Lemercier, L. Deforges, H. Tissot-Dupont, and D. Raoult. 2001. Epidemiologic and clinical characteristics of Bartonella quintana and Bartonella henselae endocarditis: a study of 48 patients. Medicine 80:245-251.[CrossRef][Medline]
6
6. Houpikian, P., and D. Raoult. 2001. 16S/23S rRNA intergenic spacer regions for phylogenetic analysis, identification, and subtyping of Bartonella species. J. Clin. Microbiol. 39:2768-2778.[Abstract/Free Full Text]
7
7. Klein, J. L., S. K. Nair, T. G. Harrison, I. Hunt, N. K. Fry, and J. S. Friedland. 2002. Prosthetic valve endocarditis caused by Bartonella quintana. Emerg. Infect. Dis. 8:202-203.[Medline]
8
8. La, V. D., L. Tran-Hung, G. Aboudharam, D. Raoult, and M. Drancourt. 2005. Bartonella quintana in domestic cat. Emerg. Infect. Dis. 11:1287-1289.[Medline]
9
9. Li, W., B. B. Chomel, S. Maruyama, L. Guptil, A. Sander, D. Raoult, and P. Fournier. 2006. Multispacer typing to study the genotypic distribution of Bartonella henselae populations. J. Clin. Microbiol. 44:2499-2506.[Abstract/Free Full Text]
10
10. Maggi, R. C., and E. B. Breitschwerdt. 2005. Potential limitations of the 16S-23S rRNA intergenic region for molecular detection of Bartonella species. J. Clin. Microbiol. 43:1171-1176.[Abstract/Free Full Text]
11
11. Marié, J. L., P. E. Fournier, J. M. Rolain, S. Briolant, B. Davoust, and D. Raoult. 2006. Molecular detection of Bartonella quintana, B. elizabethae, B. koehlerae, B. doshiae, B. taylorii, and Rickettsia felis in rodent fleas collected in Kabul, Afghanistan. Am. J. Trop. Med. Hyg. 74:436-439.[Abstract/Free Full Text]
12
12. Maurin, M., R. Birtles, and D. Raoult. 1997. Current knowledge of Bartonella species. Eur. J. Clin. Microbiol. Infect. Dis. 16:487-506.[CrossRef][Medline]
13
13. Ristic, M., and J. P. Kreier. 1984. Bartonellaceae, p. 717-718. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams and Wilkins Co., Baltimore, MD.
14
14. Rolain, J. M., O. Bourry, B. Davoust, and D. Raoult. 2005. Bartonella quintana and Rickettsia felis in Gabon. Emerg. Infect. Dis. 11:1742-1744.[Medline]
15
15. Rolain, J. M., M. Franc, B. Davoust, and D. Raoult. 2003. Molecular detection of Bartonella quintana, B. koehlerae, B. henselae, B. clarridgeiae, Rickettsia felis, and Wolbachia pipientis in cat fleas, France. Emerg. Infect. Dis. 9:338-342.[Medline]
16
16. Roux, V., and D. Raoult. 1995. Inter- and intraspecies identification of Bartonella (Rochalimaea) species. J. Clin. Microbiol. 33:1573-1579.[Abstract]
17
17. Sasaki, T., S. K. Poudel, H. Isawa, T. Hayashi, N. Seki, T. Tomita, K. Sawabe, and M. Kobayashi. 2006. First molecular evidence of Bartonella quintana in Pediculus humanus captis (Phthiraptera: Pediculidae), collected from Nepalese children. J. Med. Entomol. 43:110-112.[CrossRef][Medline]
18
18. Weiss, E., and J. W. Moulder. 1984. Rochalimaea, p. 698-701. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams and Wilkins Co., Baltimore, MD.

---

Journal of Clinical Microbiology, June 2007, p. 2040-2043, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.00175-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Woolley, M. W. Articles by Wetherall, B. L. Search for Related Content PubMed PubMed Citation Articles by Woolley, M. W. Articles by Wetherall, B. L.