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Institut für Medizinische Mikrobiologie, Virologie und Hygiene, Universität Rostock, D-18057 Rostock, Germany
Correspondence
Mardjan Arvand
mardjan.arvand{at}med.uni-rostock.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Domestic cats represent the main host and reservoir for B. henselae. Infected cats develop a relapsing bacteraemia of several months' duration during which B. henselae may be transmitted to other cats or humans by Ctenocephalides felis (cat flea) or cat scratch or bite injuries.
Identification of Bartonella species is usually based on molecular methods rather than biochemical reactions because of the fastidious and relatively inert nature of bartonellae. Analysis of the nucleotide sequence of the 16S rRNA gene was the first molecular method used for the identification of B. henselae (Relman et al., 1990). Bergmans et al. (1996) detected a 3 bp difference between the partial 16S rRNA sequences of clinical B. henselae isolates obtained from Dutch patients with cat scratch disease and divided the isolates into two distinct genotypes, 16S RNA type I and type II. Subsequently, isolates belonging to the 16S RNA types I or II were detected by different investigators in other countries (Birtles et al., 2002; Chang et al., 2002; Drancourt et al., 1996; Guptill et al., 2004; Melter et al., 2003; Sander et al., 1997). The 16S RNA types were designated 16S RNA alleles 1 and 2 and included in the MLST scheme for B. henselae (Iredell et al., 2003).
We have recently studied the molecular epidemiology of B. henselae isolates from different hosts and various geographical regions by MLST (Arvand et al., 2007). During that study and in subsequent investigations, a total of 191 B. henselae isolates were subjected to direct 16S rRNA gene sequencing. Three of these isolates could not be assigned to a distinct 16S RNA type by direct sequencing because of ambiguous nucleotides within a circumscribed region of the 16S rRNA gene. We hypothesized that these isolates may harbour two different copies of the 16S rRNA gene within their genome, leading to ambiguous base sequences within and downstream of the variable region. The atypical isolates were characterized by cloning and sequencing of the 16S rRNA gene, Southern blot analysis and hybridization, and type-specific 16S RNA-PCR, which has frequently been used in previous studies for discrimination of the 16S RNA variants. Our results demonstrate that the atypical B. henselae isolates harbour two different copies of the 16S rRNA gene in their genomes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The B. henselae isolates Ber-K186, Is-479 and Simba were isolated from blood cultures of naturally infected cats in Germany, Israel and the USA, respectively. The isolates were stored at –20 °C or –80 °C until use. Bacteria were grown on Columbia blood agar with 5 % sheep blood at 37 °C in 5 % CO2 for 7–14 days. A single colony of each isolate was passaged once on agar prior to isolation of bacterial DNA.
Direct 16S rRNA sequencing and MLST analysis.
Partial sequences of eight genetic loci (16S rRNA, batR, gltA, groEL, ftsZ, nlpD, ribC and rpoB) were determined by direct sequencing on both strands as described before (Arvand & Viezens, 2007; Iredell et al., 2003). The nucleotide sequences were analysed with the DNASTAR Lasergene software package 7. The isolates were assigned to alleles and sequence types (STs) as described previously (Arvand et al., 2007).
Cloning and sequencing of the 16S rRNA gene.
A 512 bp region of the 16S rRNA gene was amplified by PCR using the primers 16S-F (5'-AGAGTTTGATCCTGGYTCAG-3') and 16S-R (5'-CTTTACGCCCARTAAWTCCG-3'), which were originally described by Bergmans et al. (1996). Cloning was performed using the TOPO TA Cloning kit with the pCR2.1-TOPO vector (Invitrogen) in Escherichia coli; 5–9 clones of each isolate were sequenced (GATC Biotech).
Southern blotting and hybridization.
Bacteria were embedded in SeaPlaque GTG agarose and DNA was prepared following treatment with proteinase K and lysozyme as described for PFGE analysis (Arvand et al., 1998). The bacterial DNA was digested with HincII, or with HindIII and Eco81I simultaneously, at 37 °C according to the manufacturer's instructions. All restriction enzymes were purchased from Fermentas. Restriction digests were separated by electrophoresis in a 0.8 % agarose gel and transferred to a Biodyne B nylon membrane (Pall Gelman) by vacuum blotting. A DNA probe which was complementary to a 183 bp region of the B. henselae 16S rRNA gene corresponding to bases 1414428–1414610 of the B. henselae strain Houston-1 sequence (accession no. BX897699) was designed using the primers 16S-F and BH1 (5'-CCGATAAATCTTTCTCCCTAA-3'). The probe was labelled with dUTP by using the DIG Oligonucleotide Tailing kit (Roche Diagnostics). Hybridization was performed at 70 °C overnight and detection was performed with the CSPD chemiluminescence system (Roche Diagnostics). The B. henselae isolates Houston-1 (type I), Berlin-1 (type I), Urlly8 Marseille (type II), Berlin-2 (type II) and Audrey (type II) were included in different experiments as controls.
16S RNA type-specific PCR assay.
Type-specific PCR was performed using the primers 16S-F and BH1 or BH2 (5'-CCGATAAATCTTTCTCCAAAT-3') as described previously (Bergmans et al., 1996; Sander et al., 1998). Briefly, amplification was carried out in a 50 µl reaction volume with 3 min at 95 °C, 30 cycles of 20 s at 95 °C, 30 s at 57 °C (primers 16S-F and BH1) or 56 °C (primers 16S-F and BH2), 60 s at 72 °C, and a final extension of 5 min at 72 °C. The products were separated by electrophoresis in 2 % agarose, stained with ethidium bromide, and photographed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Therefore, we could not obtain overlapping sequences for those isolates.
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Determination of the copy number of the 16S rRNA gene by Southern blotting and hybridization
To determine the copy number of the 16S rRNA gene in the atypical isolates, chromosomal DNA of these isolates and reference strains was digested with HincII, which does not cleave within the 16S rRNA gene. The estimated size of the restriction digests that would contain the 16S RNA genes was 4110 and 4723 bp, as deduced from the sequence of the B. henselae Houston-1 strain. Hybridization was performed with a 183 bp 16S rRNA gene-specific probe. The isolates Ber-K186, IS-479 and Simba, and the reference strains Houston-1 and Urlly8, revealed two fragments, of approximately 4.1 and 4.7 kb (data not shown), indicating that all isolates harboured two copies of the 16S rRNA gene.
Evidence for two different 16S rRNA copies in a distinct B. henselae isolate by double digestion and Southern blotting
To confirm the presence of different 16S rRNA gene copies in the isolates Ber-K186, Is-479 and Simba, genomic DNA was subjected to double digestion with HindIII and Eco81I. The former enzyme has no restriction site within the 16S rRNA gene. Restriction of B. henselae DNA with HindIII should generate two fragments of approximately 3.7 and 2.7 kb that harbour the 16S rRNA gene copies, as deduced from the genome sequence of the Houston-1 isolate. Eco81I possesses a recognition site within the 16S RNA allele 1, but not in allele 2. Digestion of chromosomal DNA of a type I isolate with HindIII and Eco81I would yield four fragments, three of approximately 1.8 kb and one of approximately 0.8 kb, which contain the restriction fragments of the 16S rRNA gene. In contrast, digestion of chromosomal DNA of type II isolates with HindIII and Eco81I would yield two fragments of approximately 3.7 and 2.7 kb that harbour the two 16S rRNA gene copies. We hypothesized that the isolates with two different 16S rRNA gene copies would reveal a combination of type I and II patterns, presented schematically in Fig. 2(a) as option 1 and option 2. Hybridization was performed with the 183 bp 16S rRNA-specific probe as described above. As shown in Fig. 2(b), the isolates Ber-K186, Is-479 and Simba displayed a combined ‘type I and II’ pattern corresponding to option 2. In contrast, the reference strains Houston-1, which has 16S RNA allele 1, and Urlly8 and Audrey, which have 16S RNA allele 2, showed representative restriction patterns for type I or type II isolates, respectively.
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; Gurfield et al., 1997; Melter et al., 2003; Maruyama et al., 2001; Matar et al., 1999; Sander et al., 1997). We therefore tested whether the atypical B. henselae isolates could be assigned to a distinct 16S RNA type by using this assay. The isolates Ber-K186, Is-479 and Simba were subjected to 16S RNA type-specific PCR along with reference strains. The isolates with different 16S rRNA gene copies revealed positive results in both type I- and type II-specific PCR (Fig. 3). In contrast, the reference strains revealed a positive result in either type I- or type II-specific PCR. Thus, B. henselae isolates with two different 16S RNA alleles are recognized as double-positive isolates by the type-specific PCR assay.
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. They differed in one or more positions from each other, indicating that the isolates did not belong to the same ST. Since 16S rRNA is one of the genes included in the MLST scheme of B. henselae (Iredell et al., 2003), the isolates with different 16S rRNA gene copies could not be assigned to any ST.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Generally, multiple copies of rRNA genes in an organism are either identical or nearly identical in nucleotide sequence. Recent reports have shown that within some species, the multiple rRNA gene copies may show distinct sequence differences (Conville & Witebsky, 2005, 2007; Ninet et al., 1996; Reischl et al., 1998; Wang et al., 1997). Sequence diversity of the 16S rRNA gene in B. henselae was first described in 1996 (Bergmans et al., 1996). Two 16S RNA variants were detected among B. henselae isolates: type I represented by the Houston-1 strain, and type II represented by the Urlly8 and the Berlin-2 strain (Arvand et al., 2001; Drancourt et al., 1996). The delineation of B. henselae isolates into two 16S RNA types has been frequently used in previous studies and is also part of the MLST scheme for B. henselae (Chang et al., 2002; Guptill et al., 2004; Heller et al., 1997; Iredell et al., 2003; Maruyama et al., 2001; Melter et al., 2003). Recently, the complete genome sequence of the B. henselae Houston-1 strain became available and was shown to contain two identical copies of the 16S rRNA gene (Alsmark et al., 2004). In the present study, we demonstrate, we believe for the first time, the presence of two different 16S rRNA gene copies in the genomes of individual B. henselae isolates. The B. henselae isolates with different 16S rRNA gene copies were detected during a study on molecular epidemiology of B. henselae isolates from different hosts and geographical regions by MLST. The atypical isolates were originally isolated from distinct hosts in different countries and were therefore not epidemiologically related. The MLST allelic profiles of these isolates displayed one or more allelic differences, indicating that they were also not very closely related genetically. We conclude that our isolates with different 16S rRNA gene copies are not clonally related but rather have emerged independently from each other. A possible mechanism for the emergence of isolates with two different 16S RNA alleles is horizontal gene transfer. This could possibly occur when an arthropod vector or a feline host is simultaneously infected with different B. henselae strains harbouring distinct alleles. Other possible mechanisms such as mutation are less likely because the two alleles of the 16S RNA gene differ from each other in three nucleotides, which would require three simultaneous mutations at distinct positions within the 16S rRNA gene copy.
Theoretically, a hybrid-type isolate with two different 16S RNA copies may display two different genomic arrangements with allele 1 at position 1413128–1414619 and allele 2 at position 1583137–1584628 of the B. henselae Houston-1 sequence (accession no. BX897699), and vice versa. Restriction of these variants with HindIII and Eco81I would result in patterns presented as options 1 and 2 in Fig. 2(a)
. It is interesting that all three hybrid isolates detected in this study corresponded to option 2. Because of the limited number of the hybrid isolates in this study, we can only speculate whether this finding is a simple coincidence. Future studies on more hybrid isolates would help to elucidate this question.
The presence of two different 16S rRNA gene copies in a distinct B. henselae isolate has implications for future studies on B. henselae and probably other Bartonella species. When 16S rRNA gene sequencing is used for identification of Bartonella-like organisms to the species level, the investigators should be aware of the possibility that some B. henselae isolates may harbour different 16S rRNA gene copies, which might lead to equivocal nucleotide sequences by direct sequencing. Furthermore, it is possible that some previously described cases of double infection of a distinct host by two B. henselae isolates of 16S RNA types I and II, which were diagnosed by 16S RNA type-specific PCR, were in reality caused by a single isolate that harboured both 16S RNA alleles. It would be useful to subject those isolates to additional tests, e.g. direct sequencing of the 16S rRNA gene, MLST analysis or other genotyping methods, in order to differentiate between simultaneous infection by two different isolates and infection by one isolate that harbours two different copies of the 16S rRNA gene.
In summary, our data indicate that a small percentage of B. henselae isolates harbour two different copies of the 16S rRNA gene. These isolates have probably emerged by horizontal gene transfer. Investigators involved in sequence-based identification or typing of B. henselae should be aware of the existence of such atypical isolates.
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ACKNOWLEDGEMENTS |
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Edited by: P. E. Everest
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 18 March 2008;
revised 15 May 2008;
accepted 22 May 2008.
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