# Identification of Novel Zoonotic Activity of Bartonella spp., France

Muriel Vayssier Taussat, Sara Moutailler, Frangoise Femenia, Philippe Raymond, Olivier Croce, Bernard La Scola, Pierre-Edouard Fournier, Didier Raoult

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## To cite this version:

Muriel Vayssier Taussat, Sara Moutailler, Frangoise Femenia, Philippe Raymond, Olivier Croce, et al.. Identification of Novel Zoonotic Activity of Bartonella spp., France. Emerging Infectious Diseases, 2016, 22 (3), pp.457-462. 10.3201/eid2203.150269. hal-01459542

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# Identification of Novel Zoonotic Activity of *Bartonella* spp., France

Muriel Vayssier-Taussat, Sara Moutailler, Françoise Féménia, Philippe Raymond, Olivier Croce, Bernard La Scola, Pierre-Edouard Fournier, Didier Raoult

Certain *Bartonella* species are known to cause afebrile bacteremia in humans and other mammals, including *B. quintana*, the agent of trench fever, and *B. henselae*, the agent of cat scratch disease. Reports have indicated that animal-associated *Bartonella* species may cause paucisymptomatic bacteremia and endocarditis in humans. We identified potentially zoonotic strains from 6 *Bartonella* species in samples from patients who had chronic, subjective symptoms and who reported tick bites. Three strains were *B. henselae* and 3 were from other animal-associated *Bartonella* spp. (*B. doshiae*, *B. schoenbuchensis*, and *B. tribocorum*). Genomic analysis of the isolated strains revealed differences from previously sequenced *Bartonella* strains. Our investigation identified 3 novel *Bartonella* spp. strains with human pathogenic potential and showed that *Bartonella* spp. may be the cause of undifferentiated chronic illness in humans who have been bitten by ticks.

Bartonella spp. cause varied and multifaceted human diseases, including cat scratch disease (*B. henselae*), Carrion's disease (*B. bacilliformis*), trench fever (*B. quintana*), endocarditis (*B. quintana* and *B. henselae*) (1,2), bacillary angiomatosis (*B. quintana* and *B. henselae*), and hepatic peliosis (*B. henselae*). Bartonella spp. can also cause prolonged intra-erythrocytic bacteremia in both humans and animals (3): in humans, *B. quintana*, *B. bacilliformis*, and *B. rochalimae* are known pathogens, and in animals, *B. henselae*, *B. clarridgeiae*, and *B. koehlerae* have been identified in felids; *B. grahamii*, *B. taylorii*, *B. doshiae*, *B. birtlesii*, and others in rodents; and *B. bovis*, *B. chomelii*, *B. schoenbuchensis*, in ruminants. In humans, chronic bacteremia caused by *B. quintana* causes few obvious symptoms apart from generalized fatigue and nonspecific leg pain (1,4).

It has been assumed that each *Bartonella* species infected 1 or a few closely related mammalian reservoir hosts, in which infection caused long-lasting bacteremia.

Author affiliations: Agence Nationale de Sécurité Sanitaire de l'Alimentation, de l'Environnement et du Travail, Maisons Alfort, France (M. Vayssier-Taussat, S. Moutailler, F. Féménia); Chronimed, Minnetonka, Minnesota, USA (P. Raymond); Aix-Marseille Université, Marseille, France (O. Croce, B. La Scola, P.-E. Fournier, D. Raoult)

DOI: http://dx.doi.org/10.3201/eid2203.150269

Nonreservoir hosts were considered incidentally infected without bacteria being detected in blood. Recently, these assumptions has been contradicted by studies describing animal-associated *Bartonella* spp. indirectly associated with bacteremia and a spectrum of diverse symptoms in immune-competent persons who had contact with animals, arthropods, or both, which are natural routes of *Bartonella* transmission (5–7). In some cases, the source of infection remains unknown; ticks have been suggested as a possible source of animal-associated *Bartonella* infection in humans (6,8–10).

Related to a patient's history of tick bites, it is common for physicians to suspect Lyme disease, some rickettsial diseases, or tickborne encephalitis. However, in many cases, the diagnosis is not confirmed by serologic or DNA-based tests. In recent years, alternate interpretations of Lyme disease serology have flourished, leading to considerable discord between formal institutions for infectious disease and patient advocacy associations. Thus, unexplained symptoms after tick bites have become an issue of increasing importance for patients and their physicians (11,12).

In this context, we screened for the presence of *Bartonella* in the blood of patients reporting tick bites and with unexplained and aspecific symptoms. Here we report the isolation and genomic sequencing of 6 *Bartonella* strains obtained by blood culture from 66 patients. Three strains were identified as *B. henselae*, and 3 other strains were identified as different animal-associated species (*B. doshiae*, *B. tribocorum*, and *B. schoenbuchensis*).

## Methods

### Patients

During January–June 2013, we conducted a study of a cohort of 66 French patients who had consulted their doctors for chronic symptoms appearing after a tick bite. The entire study protocol was approved by the ethics committee of the Institut Federatif de Recherche 48 under reference 13-022-1.

All patients associated symptom onset with tick bites that occurred during 2008–2012 (Table 1). At symptom onset, local doctors were consulted, and serologic tests for Lyme borreliosis were performed. All patient samples
<table><caption>**Table 1.** Patients whose blood cultures were positive for *Bartonella* spp. that had no previously known zoonotic activity, France</caption><thead><tr><th>Case-patient no./age, y/sex</th><th><i>Bartonella</i> spp.</th><th>Tick bite date</th><th>Pets</th><th>Wild animal contact</th><th>Main complaints</th><th>Bacteremia, CFU/mL</th></tr></thead><tbody><tr><td>1/49/F</td><td><i>B. henselae</i></td><td>Multiple since 2008</td><td>Cats, dogs, horses</td><td>Rats, fish</td><td>Fatigue, muscle pain, headache</td><td>50</td></tr><tr><td>2/58/M</td><td><i>B. henselae</i></td><td>2011</td><td>Birds, rabbits</td><td>No</td><td>Fatigue, muscle pain</td><td>70</td></tr><tr><td>3/47/F</td><td><i>B. henselae</i></td><td>2012</td><td>Dog, hamster</td><td>No</td><td>Fatigue, generalized pain, insomnia</td><td>80</td></tr><tr><td>4/45/F</td><td><i>B. doshiae</i></td><td>2009</td><td>No</td><td>No</td><td>Fatigue, blurred vision, arthralgia</td><td>50</td></tr><tr><td>5/64/M</td><td><i>B. tribocorum</i></td><td>2012</td><td>Dog</td><td>Game animals (hunter)</td><td>Fatigue, muscle pain, headache</td><td>60</td></tr><tr><td>6/40/F</td><td><i>B. schoenbuchensis</i></td><td>2011</td><td>No</td><td>No</td><td>Fatigue, muscle pain, fever</td><td>850</td></tr></tbody></table>

tested were seronegative for Lyme borreliosis bacteria; however, since that time, their symptoms had become chronic. The patients completed information forms giving informed consent for the use of their samples in the study. All of the patients lived in the countryside, where ticks were abundant and contact with wild animals was possible. The patients reported that they had not undergone antibacterial drug treatment for ≥3 months before the study.

We collected blood samples from each patient in EDTA-containing sample tubes. For a control population, we used anticoagulated blood samples from 70 anonymous healthy blood donors from Paris (France). All samples (control and patients) were tested simultaneously.

## Bartonella Isolation from Blood

To specifically isolate *Bartonella* spp., 100 mL of blood samples from patients or healthy donors were directly plated onto sheep blood agar plates and incubated at 35°C in a humidified atmosphere with 5% CO₂ for 45 days. The plates were assessed daily from days 7–45 before the culture was deemed negative (i.e., absence of colony in the absence of contamination) (1). Colony-forming units (CFU) were counted and bacteremia (UFD/mL of blood) evaluated.

## Genome Sequencing, Assembly, and Analysis

We extracted genomic DNA from each isolated strain by using the EZ1 automated extraction system (QIAGEN, Hilden, Germany), following the manufacturer's recommendations. Bacterial genomic DNA was sequenced by using the Nextera XT DNA sample prep kit (Illumina Inc., San Diego, CA, USA) and a 2×250 paired-end protocol with the MiSeq pyrosequencer (Illumina), according to the manufacturer's instructions. We aligned each genome by using Mira version 3.2 software in the mapping mode (13). The resulting contigs were combined by using Opera version 1.2 (14) and GapFiller (15) software. Finally, the genomic assemblies were improved with manual refinement by using the CLC Genomics version 4.7.2 software package (CLC Bio, Aarhus, Denmark). Noncoding genes and miscellaneous features were predicted by using RNAmmer (16) and ARAGORN (17). Coding DNA sequences were

predicted by using Prodigal (18), and functional annotation was achieved by using BLAST+ (19) and HMMER3 (20) against the UniProtKB database (21). Coding DNA sequences were also annotated by using the Clusters of Orthologous Groups databases (22) with blastp (http://blast.ncbi.nlm.nih.gov/Blast.cgi) default parameters.

Single-nucleotide polymorphisms (SNPs) among genomes were identified by using SNIT software (23). SNPs were searched in regions exhibiting >95% nt sequence identity and the SNIT software was used with default parameters except for the Tandem Repeat Finder filter for avoiding ambiguous SNPs in repeat regions. We also performed in silico DNA-DNA hybridization (DDH) between *Bartonella* strains by using GGDC software (24).

## Taxonomic Classification

To determine the taxonomic classification of the 6 isolates, we used previously proposed criteria (25) in which the *gltA* and *rpoB* gene sequences from each strain were compared to those of validated published *Bartonella* species. These criteria classify *Bartonella* isolates within a particular species if they share >96% and 95.4% nucleotide sequence similarity for the *gltA* and *rpoB* genes, respectively (25). In our study, *gltA* and *rpoB* sequences were retrieved from the genomes.

## Results

### Bartonella spp. Isolation

*Bartonella* spp. were isolated by prolonged culture from blood samples of 6 of the 66 patients who reported chronic symptoms following a tick bite. In contrast, samples from the 70 healthy blood donors remained negative after 45 days of incubation.

Bacteremia in the *Bartonella* infected patients increased from 50 to 850 CFU/mL. For 1 patient (case-patient 2), we had access to 2 blood samples that were taken at a 1-month interval. *B. henselae* was grown from the 2 samples, with similar bacteremia (50 and 60 CFU/mL, respectively), suggesting chronic bacteremia.

The case-patients who tested positive for *Bartonella* (Table 1) reported tick bites occurred 1–5 years before
blood samples were collected. All of them live in the coun-
tryside, in contrast to the healthy blood donors, who were
all from Paris, France. The main complaint of the case-pat-
tients was chronic fatigue, but they also reported other sub-
jective or nonspecific symptoms (or both), such as head-
aches and myalgia. A qualifying characteristic of the 70
healthy blood donors was absence of chronic fatigue. Even
though potential exposure to ticks is difficult to evaluate,
because the anonymous blood donors all lived in Paris, we
assumed they were not likely to have frequent tick expo-
sure or wild animal contact.

Taxonomic Classification

Of the 6 *Bartonella* isolates from this study, 3 (MVT01, MVT02, and MVT03) were classified within the *B. hense-laë* species on the basis of both their phylogenetic position and *gltA* and *rpoB* sequence similarities (Figure; Tables 1, 2). The isolates from samples from case-patient 2 at a 1-month interval shared 100% identity, based on *gltA* and *rpoB* gene comparison. Isolates MVT04, MVT05, and MVT07 were classified within the *B. tribocorum*, *B. doshiae*, and *B. schoenbuchensis* species, respectively.

The assembly data and main genomic characteristics
of each isolated strain are summarized in Table 3; in silico
DDH values and SNP numbers are described in Table 4
(http://wwwnc.cdc.gov/EID/article/22/3/15-0269-T4.htm).

All studied strains displayed a similar genomic content when compared with reference genomes. The GGDC software we used proposes that DDH values >70% could classify isolates in the same species. Here, intraspecies values ranged from 80.3% to 100% (Table 4). Nevertheless, from 10 to 1,938 SNPs were identified among *B. henselae* isolates MVT01, MVT02, and MVT03 and from 693 to 2,093 SNPs when comparing these strains to *B. henselae* Hoxton-1 (Table 4), confirming that each strain was unique and did not result from cross-contamination or contamination from laboratory strains.

Of note, *B. tribocorum* isolate MVT04 and *B. schoen-
buchensis* isolate MVT07 were the only 2 that exhibited
plasmids. However, when compared with reference strain
m07a, MVT04 and MVT07 carried a large plasmid and not
the small plasmid homologous to the cryptic pBGR plas-
mid harbored by *B. grahamii* (26).

Discussion

In this study, animal-associated *Bartonella* isolates were in-
dividually cultured from the blood of patients who had been
bitten by ticks and reported subjective symptoms, whereas
no strains were isolated from healthy blood donors. This re-
port describes the isolation of 3 different animal-associated
*Bartonella* species from human samples, highlighting their
potential novel zoonotic properties. Moreover, we found

<figure><img src="image_3.png" /></figure>

**Figure.** *rpoB* gene-based phylogenetic tree showing the relationships of 6 *Bartonella* isolates (underlined). Briefly, *rpoB* nucleotide sequences were aligned by using ClustalW software (http://www.clustal.org/clustal2/), and phylogenetic relationships were inferred by using the maximum-likelihood strategy and MEGA software (http://www.megasoftware.net). Bootstrap values above 70%, obtained from 500 analyses, are indicated at the nodes. Scale bar represents a 2% nucleotide sequence divergence.
<table><caption>**Table 2.** Nucleotide similarity of 6 *Bartonella* isolates from patients in France expressing novel zoonotic activity and their most phylogenetically similar published validated species*</caption><thead><tr><th>Isolate</th><th>gltA, %</th><th>rpoB, %</th><th>Species</th></tr></thead><tbody><tr><td>MVT01</td><td>100</td><td>100</td><td><i>B. henselae</i></td></tr><tr><td>MVT02</td><td>100</td><td>100</td><td><i>B. henselae</i></td></tr><tr><td>MVT03</td><td>99.7</td><td>99.6</td><td><i>B. henselae</i></td></tr><tr><td>MVT04</td><td>100</td><td>100</td><td><i>B. tribocorum</i></td></tr><tr><td>MVT05</td><td>98.7</td><td>100</td><td><i>B. doshiae</i></td></tr><tr><td>MVT07</td><td>100</td><td>99.9</td><td><i>B. schoenbuchensis</i></td></tr></tbody></table>

* *gltA* and *rpoB* sequences were obtained from genomic sequences. Genome sequences of *B. henselae* strains MVT01, MVT02, and MVT03 were deposited in GenBank under accession numbers HG965802, NZ_LN879429, and HG969191, respectively; the genome sequence of *B. tribocorum* strain MVT04 was deposited in GenBank under accession numbers HG969192 and HG969193; the genome sequence of *B. doshiae* strain MVT05 was deposited in GenBank under accession numbers CCBL010000001-CCBL010000013; the genome sequence of *B. schoenbuchensis* strain MVT07 was deposited in GenBank under accession numbers HG977193-HG977197; the genome sequences of the reference strains *B. henselae* strain Houston-1, *B. tribocorum* strain CIP 105476, *B. doshiae* strain NCTC 12862, and *B. schoenbuchensis* strain m07a are available in GenBank under accession numbers NC_005956, NC_010161, and NC_010160, NZ_JH725094 to NZ_JH725100 and NZ_KB915627-NZ_KB915629, NZ_CMO01846-NZ_CMO01845, respectively.

that zoonotic *Bartonella* spp. can be detected in the blood ofafebrile patients, as has been shown for human-specific *B. quintana* and *B. bacilliformis* and as was recently reported for *Candidatus* Bartonella ancashi (27). Chronic bacteremia caused by infection by *Bartonella* spp. is well-described in many mammals, including humans (4,28). The *Bartonella*-mammalian host association is considered to be species-specific and attributable to co-evolution between host and pathogen (28). However, we show that animal-associated species can also chronically infect human blood, highlighting the possibility of host shift despite apparent host specificity (28,29).

This work is similar to that of E.B. Breitschwerdt et al. (5–7), who also recovered zoonotic *Bartonella* spp. from human samples using an in-house technique based on results of blood pre-enrichment followed by PCR detection of *Bartonella* spp.; members of the same team have investigated many cases of persons who had nonspecific symptoms, including arthralgia, muscle pain, fatigue, headaches, visual blurring, neurocognitive symptoms, and in 2 case-patients, hemangioendothelioma (30). In total, *B. henselae* DNA was detected in 47 cases (5,30–33); *B. koehlerae* (another common agent of feline bacteremia) DNA in 96 cases, including 2 co-infected with *B. henselae* (31,32,34,35); and *B. vinsonii* *berkhoffii* (an agent of canine bacteremia and endocarditis) DNA in 24 cases (31–34), including 16 case-patients with *B. henselae* and 2 cases of *B. melophagi* (36). These results have been questioned because minute levels of contamination can result in false positives by PCR. Therefore, we deliberately avoided PCR to overcome this problem, and the resulting strain isolation was consequently straightforward and indisputable. These isolates (Table 2) have been archived in our collection (Collection de Souches de l'Unité des Rickettsies, World Data Center for Microorganisms no. 875, http://www.mediterranee-infection.com/article.php?laref=14& titre=collection-de-souches) and are available upon request under references B546, B547, B548, B549, B550, and B551 for isolates MVT01, MVT02, MVT03, MVT04, MVT05, and MVT07, respectively.

Our findings also confirm studies identifying zoonotic *Bartonella* in the blood of patients with nonspecific complaints. Among them, *B. henselae* is well known worldwide

<table><caption>**Table 3.** Assembly information and main characteristics of 6 sequenced *Bartonella* genomes from patients in France expressing novel zoonotic activity</caption><thead><tr><th rowspan="2">Genome characteristics</th><th colspan="6">Species and isolate identification</th></tr><tr><th><i>B. henselae</i><br>MVT01</th><th><i>B. henselae</i><br>MVT02</th><th><i>B. henselae</i><br>MVT03</th><th><i>B. tribocorum</i><br>MVT04</th><th><i>B. doshiae</i><br>MVT05</th><th><i>B. schoenbuchensis</i><br>MVT07</th></tr></thead><tbody><tr><td>GenBank accession nos.</td><td>HG965802</td><td>NZ_LN879429</td><td>HG969191</td><td>HG969192-HG969193</td><td>CCBL010000001-CCBL010000013</td><td>HG977193-HG977197</td></tr><tr><td>Size, bp</td><td>1,902,535</td><td>1,905,383</td><td>1,975,503</td><td>2,609,404</td><td>1,919,109</td><td>1,734,324</td></tr><tr><td>No. contigs</td><td>1</td><td>1</td><td>1</td><td>2</td><td>13</td><td>5</td></tr><tr><td>Average read coverage</td><td>87</td><td>94</td><td>110</td><td>46</td><td>15</td><td>41</td></tr><tr><td>Average read length, trimmed</td><td>183</td><td>190</td><td>192</td><td>194</td><td>168</td><td>193</td></tr><tr><td>Total no. reads, trimmed</td><td>946,882</td><td>1,034,894</td><td>1,263,492</td><td>738,522</td><td>261,0852</td><td>666,371</td></tr><tr><td>Total no. predicted genes</td><td>1,659</td><td>1,658</td><td>1,726</td><td>2,335</td><td>1,720</td><td>1,574</td></tr><tr><td>Protein-coding genes</td><td>1,603</td><td>1,602</td><td>1,668</td><td>2,279</td><td>1,654</td><td>1,519</td></tr><tr><td>rRNA operons</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td></tr><tr><td>tRNAs</td><td>43</td><td>43</td><td>45</td><td>43</td><td>53</td><td>41</td></tr><tr><td>Other RNAs</td><td>7</td><td>7</td><td>7</td><td>7</td><td>9</td><td>8</td></tr><tr><td>GC% content</td><td>38.18</td><td>38.18</td><td>38.09</td><td>38.84</td><td>37.82</td><td>35.58</td></tr><tr><td>Plasmid</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>1</td></tr><tr><td>Genome used as a reference for assembly (accession nos.)</td><td><i>B. henselae</i><br>Houston-1<br>(NC_005956)</td><td><i>B. henselae</i><br>Houston-1<br>(NC_005956)</td><td><i>B. henselae</i><br>Houston-1<br>(NC_005956)</td><td><i>B. tribocorum</i><br>CIP 105476<br>(NC_010161, NC_010160)</td><td><i>B. doshiae</i><br>NCTC 12862<br>(NZ_JH725094-NZ_JH725100)</td><td><i>B. schoenbuchensis</i><br>m07a (NZ_KB915627-NZ_KB915629, NZ_CMO01846, NZ_CMO01845)</td></tr></tbody></table>
as a zoonotic agent infecting both cats and their fleas and
has also been found in ticks (10). *B. henselae* has been de-
tected in the blood of a patient without apparent symptoms
4 months after recovering from cat scratch disease. For
this particular case, the sequence of manifestation of cat
scratch disease, then bacteremia, followed by endocarditis
was proposed because it has been known to occur for *B. quintana* bacteremia. One of the case-patients in this study
owns a cat and may have been infected by this pet.

The 3 other animal-associated species we detected
should now be considered zoonotic *Bartonella* spp. *B. doshiae* and *B. tribocorum* are both rodent-associated species; in France and worldwide, these species have mainly
been recovered from rats (*Microtus agentiis* for *B. doshiae* and *Rattus rattus* for *B. tribocorum*). *B. schoenbuchensis* is
normally found in deer, elk, and cattle (37,38).

The zoonotic agents we isolated from patients from France have also been detected in animals in France. Similarly, in the United States and Thailand, *Bartonella* species known to be prevalent in animals have also been identified in humans: (*B. henselae*, *B. vinsonii* berkhoffii, and *B. koehlerae* in the United States (33,35) and *B. tribocorum* and *B. rattimassiliensis* in Thailand (39). Therefore, the zoonotic *Bartonella* species discovered in humans in this study generally appear to be related to the prevalence among animals.

The significance of these *Bartonella* spp. in the gen-
esis of the clinical picture is difficult to determine. Bar-
tonella spp. are present in ticks, and we have previously
reported Bartonella infections following tick bites, such
as SENLAT (scalp eschar and neck lymphadenopathy af-
ter tick bite [40]). However, the causal link between the
conditions observed here, Bartonella and tick bite, cannot
yet be concretely established, especially for persons with
tick bites occurring up to 5 years previously, which intro-
duces innumerable potential confounding exposures within
the same period, including bites by other arthropods. For
instance, 1 of the 3 patients with B. henselae bacteremia re-
ported contact with cats; this contact was a more plausible
source of infection than tick bites. Furthermore, it is crucial
to determine whether Bartonella played a notable role in
the observed pathologies, because treatment for chronic
Bartonella bacteremia (as for B. quintana) is particularly
arduous and may require 6 weeks of doxycycline treatment
together with 3 weeks of gentamicin, as these are the only
antimicrobial drugs known to be effective in eradication of
Bartonella (1). Many Bartonella spp. can also cause endo-
carditis, including B. quintana and B. henselae; therefore,
reports of rare cases of endocarditis attributed to zoonotic
Bartonella such as B. kohlerae, B. alsatica, Candidatus B.
mayotimonensis, B. vinsonii, or B. elizabethae may actu-
ally be the final manifestation of asymptomatic bacteremia,
similar to that reported by our infected patients (28).

In summary, our major finding is the isolation of zoo-
notic *Bartonella* other than *B. quintana* in the blood of pa-
tients with poorly qualified syndromes. These results in-
dicate that zoonotic *Bartonella* spp. infection may cause
undifferentiated chronic illness in humans.

Acknowledgments

We thank Evelyne Le Naour and Martine Cotte for their techni-
cal support, Lionel Arnaud and the Institut National de Transfu-
sion Sanguine for providing blood samples from anonymous
blood donors, and the physicians from the Chronimed network
who participated in patient recruitment.

This work was partially funded by the French National Institute of Agronomical Research and by the European Union grant FP7-261504 EDENext and is catalogued by the EDENext Steering Committee as EDENext422 (http://www.edenext.eu). The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. This work was also supported by the COST action TD1303 (EurNegVec).

Dr. Vayssier-Taussat is a senior scientist at the French
National Institute of Agronomical Research, where she leads
a research team involved in the study of Bartonella and other
vectorborne pathogens.

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