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Journal of Clinical Microbiology, September 2005, p. 4921-4922, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4921-4922.2005

LETTER TO THE EDITOR

Potential Limitations of the 16S-23S rRNA Intergenic Region for Molecular Detection of Bartonella Species


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In the March 2005 issue of the Journal of Clinical Microbiology, Maggi and Breitschwerdt (5) evaluated the limitations of the 16S-23S rRNA intergenic region (ITS) for molecular detection of Bartonella species, one of the most important of which is Bartonella henselae. The authors warned us of contaminating Mezorhizobium spp. as a source of confusion, when previously published primers were used. They showed that the primer pair 983as and 321s has potential value for the detection of a range of Bartonella species on the basis of amplicon sizes, although some differed by as little as 7 bp and would be indistinguishable in agarose gels (5).

In previous publications, we and others (1, 2) have documented the presence of variable repeat regions in the 16S-23S ITS of certain B. henselae isolates. The size of the additional inserts varied from 15 bp to 45 bp (1, 2), and the difference between repeats in different collections implies that this may be a region of some diversity. In our own study, more than 10% of B. henselae isolates (human and feline) examined contained a variable repeat region (1). The primer pair 983as/321s amplifies a sequence which includes this region (GenBank accession no. L35101) (5), and we expect that this will be an important source of error (Table 1). Using the cited GenBank accession number, we derived a predicted ITS amplicon of 648 bp for B. henselae (Fig. 1). If 983as/321s amplicon sizes are used without further analysis, the range expected within B. henselae alone will potentially cause serious misidentifications (Table 1).


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  		TABLE 1. Predicted sizes for 983as/321s amplicons of B. henselae and other Bartonella species

 


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  		FIG. 1. B. henselae Houston 1 16S-23S ITS region (GenBank accession no. L35101). Marked primers are from previously published work (4, 5). This figure was produced with Gene Construction kit, v. 2.5 (Textco Inc.).

 
Drs. Maggi and Breitschwerdt are to be congratulated fortheir contribution, as this provides us with a robust single-step identification tool. However, we would further suggest, in the case of B. henselae at least, that a confirmatory target be chosen which has been validated within a standardized typing schema such as multilocus sequence typing (3).


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  1. Dillon, B., J. Valenzuela, R. Don, D. Blanckenberg, D. I. Wigney, R. Malik, A. J. Morris, J. M. Robson, and J. Iredell. 2002. Limited diversity among human isolates of Bartonella henselae. J. Clin. Microbiol. 40:4691-4699.[Abstract/Free Full Text]
  2. 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]
  3. Iredell, J., D. Blanckenberg, M. Arvand, S. Grauling, E. J. Feil, and R. J. Birtles. 2003. Characterization of the natural population of Bartonella henselae by multilocus sequence typing. J. Clin. Microbiol. 41:5071-5079.[Abstract/Free Full Text]
  4. Jensen, W. A., M. Z. Fall, J. Rooney, D. L. Kordick, and E. B. Breitschwerdt. 2000. Rapid identification and differentiation of Bartonella species using a single-step PCR assay. J. Clin. Microbiol. 38:1717-1722.[Abstract/Free Full Text]
  5. Maggi, R. G., 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]
Belinda Dillon
Jon Iredell*
Centre for Infectious Diseases and Microbiology
Level 3, ICPMR Bldg.
Westmead Hospital
Hawkesbury Road
Westmead, NSW 2145, Australia

* Phone: 61-2-98456255, Fax: 61-2-98915317, E-mail: joni{at}icpmr.wsahs.nsw.gov.au


Authors' Reply


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We thank Drs. Dillon and Iredell for the positive comments regarding our recent publication entitled "Potential Limitations of the 16S-23S rRNA Intergenic Spacer Regions for Molecular Detection of Bartonella Species." Our purpose was to alert others to the potential of nonspecific amplification of Mesorhizobium species using published primers. We agree that the substantial diversity among B. henselae variable repeat regions in the 16S-23S ITS region will limit the utility of this target for definitive determination of Bartonella species, as well as subspecies. We are currently finding a similar high degree of variability in the 16S-23S ITS region of Bartonella vinsonii berkhoffii isolates obtained from dogs and other animals. The primer set (321s and 983as) avoids nonspecific amplification of Mesorhizobium or closely related {alpha}-Proteobacteria species and should prove to be useful in the initial screening for Bartonella DNA in patient blood samples. As suggested, a confirmatory typing schema such as multilocus sequence typing should be used, particularly when an unexpected amplicon size is found in a reservoir host or in all instances when determining the Bartonella species in a patient sample.



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  		FIG. 1. B. henselae Houston 1 16S-23S ITS region (GenBank accession no. L35101). Marked primers are from previously published work (4, 5). This figure was produced with Gene Construction kit, v. 2.5 (Textco Inc.).

 

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  		TABLE 1. Predicted sizes for 983as/321s amplicons of B. henselae and other Bartonella species

 
Edward B. Breitschwerdt*
Ricardo G. Maggi
Intracellular Pathogens Research Laboratory
College of Veterinary Medicine
North Carolina State University
Raleigh, NC 27606

* Phone: (919) 513-8277
Fax: (919) 513-6336
E-mail: ed_breitschwerdt{at}ncsu.edu

Journal of Clinical Microbiology, September 2005, p. 4921-4922, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4921-4922.2005




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