An optimized 16S–23S rRNA intergenic spacer region PCR for the detection and identification of Bartonella spp.

LETTER

The emerging genus Bartonella comprises 60 species with unknown medical relevance (National Center for Biotechnology Information, Taxonomy Browser, 5 December 2025 [1]). Some species are human pathogenic (e.g., B. henselae: cat scratch disease, bacillary angiomatosis; B. quintana: trench fever, endocarditis; and B. bacilliformis: Carrion’s disease); other species (e.g., B. alsatica: endocarditis and B. grahamii: lymphadenopathy) are only rarely described to cause human infections (2). Accurate diagnosis of Bartonella infections remains challenging due to unspecific clinical manifestations, difficulties in cultivation, genetic diversity within the genus, and frequent occurrence of low-level bacteremia in infected patients (3). Therefore, because of the fact that serological diagnostics have been developed only for B. henselae, B. quintana (4), and recently for B. bacilliformis (5), PCR-based assays have become indispensable for pathogen detection and differentiation. Pioneer work of Maggi and Breitschwerdt established the 16S–23S rRNA intergenic spacer (ITS) region as a valuable diagnostic target widely used in molecular diagnostics of Bartonella infections. This region is highly variable in sequence and length between Bartonella species and is embedded between conserved flanking regions, allowing the design of universal primers and enabling a reliable discrimination among closely related Bartonella species (6). Despite this great contribution to diagnostics, our results revealed that this PCR protocol with the described ITS primers fails to amplify particular species that were later fully sequenced or discovered because of numerous mismatches to the ITS target sequence (Fig. 1A). This limitation might lead to false-negative results in both clinical and epidemiological studies, ultimately impacting patient management or public health surveillance. Here, we aimed to optimize an ITS-PCR (targeting a sequence of the 16S–23S rRNA ITS) for improved diagnosis of clinically relevant Bartonella species.

Fig 1.

Primer binding sites and PCR amplification results (agarose gel electrophoresis) targeting the Bartonella ITS region. (A) Previously published primers (6) and (B) primers designed based on the consensus sequence established in this study were used. The consensus sequence of the ITS region and the corresponding primer sequences are shown in the subfigures, with mismatches indicated in red.

Genomic sequences from 16 Bartonella species (Table 1) were aligned using MAFFT v.7.490 (Geneious, Dotmatics, Auckland, New Zealand). Only 16S–23S rRNA ITS sites that were 100% conserved across all aligned sequences were considered for primer construction, covering the major pathogenic and novel emerging species (BsppITS-386s: 5′-GGTTAGAGCGCGCGCTTGATAAGC-3′ [3′ end of the 16S rRNA gene] and BsppITS-824as: 5′-CCTGCTTGCAAAGCAGGTGCTC-3′ [5′ end of the 23S rRNA gene]) generating PCR amplicons between 207 and 438 nucleotides.

TABLE 1.

Bartonella strains used in this study for the development of an optimized 16S–23S intergenic spacer region PCR

Bartonella strains	Accession no. of reference genomea	PCR amplicon size
B. alsatica IBS 382 (CIP105477)	CP058235	367
B. ancashensis A07	CP180576	311
B. bacilliformis KC583	CP000524	255
B. bacilliformis KC584	CP045671	255
B. birtlesii CIP106294	KE007210	407
B. capreoli CIP 106691	CADDZX010000017	207
B. chomelii A828	NZ_JACJIR010000013	219
B. doshiae ATCC 700133	CACVBE010000020	329
B. elizabethae ATCC 49927	NZ_LR134527	438
B. grahamii ATCC 700132	CP001562	352
B. henselae Houston 1	NZ_CP072903	438
B. henselae Marseille	NZ_CP072904	438
B. quintana JK31	KL411732	367
B. schoenbuchensis DSM 13525	NZ_CP154603	207
B. tribocorum CIP10476	AM260525	424
B. vinsonii ATCC 51672	CADEAK010000021	406

^a Reference genomes were taken from https://www.ncbi.nlm.nih.gov.

DNA was extracted from Bartonella cultures and 12 bacterial control strains (Table S1). PCR was performed under certified conditions (DIN EN ISO 15189:2024, certificate number D-ML-13102-01-00): initial denaturation (95°C/3 min), followed by 35 cycles (denaturation 94°C/20 s; annealing: 60°C/30 s, elongation: 72°C/40 s, final extension 72°C/1 min). Products were analyzed by agarose gel electrophoresis, demonstrating a robust amplification across all tested strains, including B. bacilliformis and B. ancashensis (Fig. 1B).

In silico, PCR products were aligned using MAFFT to assess sequence variability within the amplified ITS region to evaluate the potential for species-level discrimination. This in silico sequence analysis of the expected amplicons revealed unique DNA sequences except for B. capreoli and B. schoenbuchensis (Table S2), confirmed by Sanger sequencing (data not shown). Specificities of the old and new PCR protocols were evaluated using a broad selection of 12 clinically relevant bacterial strains (Fig. 2A; Table S1), demonstrating no unspecific amplicons between 207 and 438 bp, not even for the four closely related Brucella spp. PCR sensitivities were evaluated by spiking sterile human blood samples with DNA from B. henselae Marseille (final concentrations: 33 ng/µL to 3.3 × 10⁻⁶ ng/µL). DNA was extracted using the QIAsymphony DSP Virus/Pathogen Kit (Qiagen, Hilden, Germany) under certified conditions (DIN EN ISO 15189:2024), and the 16S–23S rRNA ITS region was amplified using both the old and new PCR protocol. Data revealed that both PCRs have a comparable sensitivity (3.3 × 10⁻³ ng/µL, Fig. 2B). As the 16S–23S rRNA ITS region is present twice in the genomes of Bartonella spp., it might represent a PCR target resulting in more sensitive PCRs compared to PCR targets with only one copy (16S-rDNA, ftsZ, gltA, groEL, ribC, and rpoB). This speculation, however, has not been addressed experimentally.

Fig 2.

Determination of (A) specificity and (B) sensitivity of the 16S–23S rRNA intergenic spacer region PCRs. (A) Control bacteria (see Table S1) were grown under standard laboratory conditions. Positive control: B. henselae Marseille, negative control: aqua dest. (dH₂O). (B) Sterile human blood samples were spiked with DNA from B. henselae Marseille at the indicated final concentrations (ranging from 33 to 3.3 × 10⁻⁶ ng/µL). Negative control: aqua dest. (dH₂O). Left panels: Primers 321s/983as (described in reference 6). Right panels: Primers BsppITS-386s/BsppITS-824as (described in this study).

Our study underscores the importance of periodically re-evaluating diagnostic primers in PCR-based diagnostic approaches as new bacterial species are identified. In regions where Bartonella-related illnesses are endemic, implementation of the optimized ITS primers could improve diagnostic accuracy, facilitate timely treatment, and support more reliable molecular epidemiology.

DATA AVAILABILITY

All data are provided within the article and the supplemental material.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/spectrum.02964-25.

Supplemental tables. spectrum.02964-25-s0001.pdf.

Tables S1 and S2.

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