Genetic characterization of Bartonella henselae samples isolated from stray cats by multi-locus sequence typing

Background Bartonella henselae is one of the most commonly identified Bartonella species associated with several human diseases. Although B. henselae was detected in humans and cats in Turkey, they have not been genotyped previously. Therefore, this study aimed to genotype B. henselae samples (n = 44) isolated from stray cats using the multi-locus sequence typing (MLST) method. For this aim, eight different housekeeping markers were amplified by nested PCR and then sequenced to reveal sequence types (STs) of B. henselae samples. Results Allelic profiles obtained from 40 B. henselae isolates (90.9%) were compatible with available allelic profiles in the MLST online database. However, allelic profiles obtained from the remaining 4 B. henselae isolates (9.1%) were incompatible with the database. Among B. henselae isolates with compatible allelic profiles, 5 different STs including ST1, ST5, ST9, ST35 and ST36 were identified according to the B. henselae MLST online database. ST35 was the most prevalent ST with a prevalence rate of 29.5% (13/44), followed by ST36 with a prevalence rate of 22.7% (10/44). In addition, ST5 (16%, 7/44) and ST9 (18.2%, 8/44) were also among the prevalent STs. The prevalence of ST1 was 4.5% (2/44). For B. henselae isolates with incompatible allelic profiles, we recommended a new ST called ST38. Conclusion The present study genotyped B. henselae samples isolated from stray cats in Turkey for the first time and ST1, ST5, ST9, ST35, and ST36 as well as a new sequence type named ST38 were identified among these B. henselae isolates. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-023-03748-4.


Background
Bartonella spp.are vector-borne pathogens that successfully infect many mammals including humans.Of the 38 defined Bartonella species, at least 18 have been stated to be zoonotic [1].The most common species associated with human diseases are Bartonella henselae (B.henselae), B. clarridgeiae, B. quintana, and B. bacilliformis [2,3].Cat-scratch disease is caused by B. henselae and B. clarridgeiae, whereas trench fever disease is caused by B. quintana.Both diseases are known as bartonellosis, and symptoms include fever, bacteremia, bacillary angiomatosis, and endocarditis.Sporadic cases of endocarditis in humans have also been associated with B. koehlerae, B. elizabethae, and B. alsatica [3][4][5].
The main reservoir is the domestic cat for B. henselae, B. clarridgeiae, and B. koehlerae [5] while cat fleas (Ctenocephalides felis) is their natural vector.Additionally, B. rochalimae, B. elizabethae, B. quintana and B. grahamii have been found in cats [6].Although Bartonella species can infect cats, no symptoms generally occur.However, B. henselae infection has been linked to uveitis and endocarditis in cats, and some symptoms including lymphadenopathy, fever, and neurological signs have been reported in experimentally infected cats [7,8].To date, different seroprevalence rates varying from 0 to 80% for Bartonella spp. in cats have been reported [9,10].Our previous study also detected a prevalence rate of 12.5% in stray cats for Bartonella spp.by a nested PCR targeting the 16-23 S internal transcribed spacer gene (ITS) [11].
To reveal the genetic diversity of B. henselae samples detected in humans or cats, genotyping studies have been performed by multi-locus sequence typing (MLTS) where 16 S rRNA, batR, ftsZ, gltA, groEL, nlpD, ribC, and rpoB genes are analyzed.Until now, 37 sequence types (STs) have been identified [1] and among these STs, some of them such as ST1, ST2, ST5 and ST8 have been associated with human diseases while others including ST6 and ST7 were mainly detected in cats.In addition, ST5 and S9 have also been associated with feline infection in Spain [12].
Although several studies investigating the presence of Bartonella spp. in cats have been conducted in Turkey, genotype profiles of B. henselae isolates are not revealed.Therefore, this study aimed to genotype B. henselae isolates previously detected in stray cats living in İzmir, Turkey [11] using MLST analysis.
Allelic profiles obtained from 40 B. henselae isolates (90.9%) were compatible with available allelic profiles in the database and STs of these samples were successfully identified.However, allelic profiles obtained from the remaining 4 B. henselae isolates (9.1%) were incompatible with the database and thus their STs could not be defined.All B. henselae isolates with incompatible allelic profiles had the same allelic profile [16 S rRNA (allele 2), batR (allele 7), groEL (allele 1), ftsZ (allele 1), gltA (allele 1), nlpD (allele 1), ribC (allele 1), and rpoB (allele 1)] (Table 1).Depending on this result, we recommended a new sequence type called ST38.Chromatogram images belonging to these B. henselae isolates were given in Additional file 1:S2.

Discussion
To date, in many studies conducted in different regions/ countries, B. henselae isolates detected in humans or cats were genotyped to reveal their genetic diversity [1,[13][14][15].Thanks to these studies, the number of STs within B. henselae isolates has increased from 7 to 37 [13,14].Although B. henselae isolates were detected in humans or cats in Turkey, they have not been genotyped before and thus, genotype profiles of these isolates are not known.In our previous study, the prevalence of Bartonella spp. in stray cats living in İzmir, Turkey were found to be 12.05% and among these positive Bartonella spp., B. henselae, B. clarridgeiae and B. koehlerae were identified [11].In this study, previously identified B. henselae isolates were genotyped by MLST targeting 16 S rRNA, batR, ftsZ, gltA, groEL, nlpD, ribC, and rpoB genes.According to the MLST results, ST1, ST5, ST9, ST35 and ST36 were detected among analyzed B. henselae isolates.The presence of ST1 and ST5 in stray cats was one of the important findings of this study in terms of their medical and veterinary importance because both of them have been associated with Cat Scratch Disease in humans and ST5 has been associated with feline infection [14].The results of this study was comparable with previous studies and shows that ST1, ST5 and ST9 were frequently detected in cats [1,[12][13][14][15][16][17][18].For example, of the 31 B. henselae samples isolated from domestic cats in Japan, 28 (90.3%) were identified as ST1 [17].In another study, among 39 B. henselae samples isolated from cats in German, 10 (25.6%) were detected to be ST5 while 3 (7.69%)were detected to be ST1 [15].In a study conducted in Spain, of the 21 B. henselae samples isolated from cats, 15 (71.4%) were identified as ST5 [12].In a different study conducted in Argentina, of the 12 B. henselae samples isolated from cats, 7 (58.3%)were detected to be ST1 [18].In Brazil, of the 12 B. henselae samples isolated from domestic cats, 11 (91.6%) were detected to be ST1 whereas 1 (8.3%) was found to be ST5 [1].In a different study conducted in the same region, ST9 also was detected among B. henselae samples isolated from cats [14].In a comprehensive study analyzing B. henselae samples isolated from cats in different regions such as Europe, USA and Australia, ST1 was detected with a prevalence rate of 17.1% while ST5 was detected with a prevalence rate of 20.9% [16].In addition to these STs including ST1, ST5 and ST9, ST35 and ST36 were also reported to be detected in Spain [1].Surprisingly, the allelic profile belonging to four B. henselae samples 16 S rRNA (allele 2), batR (allele 7), groEL (allele 1), ftsZ (allele 1), gltA (allele 1), nlpD (allele 1), ribC (allele 1), and rpoB (allele 1)] (Table 1) was incompatible when compared with the available allelic profile present in MLST database and thus this strain was recommended as a new sequence type called ST38 in this study.Likewise, numerous new STs were detected in different regions in previous studies [12,[14][15][16][17]19].These previous results and our results support the idea that more studies are required to fully understand molecular epidemiology of B. henselae isolates.
Fig. 2 The SplitsTree results of B. henselae isolates.The SplitsTree showed that all of ST36 isolates detected in this study were clustered with reference ST36 isolate detected in Spain.Surprisingly, all of ST9 isolates were also detected to cluster with reference ST9 isolate detected in Germany contrary to PHYLOViZ result.However, results belonging to ST1, ST5, ST35 as well as B. henselae isolates with new allelic profile (circled) called as ST38 were same with results obtained from PHYLOViZ.
Fig. 1 The PHYLOViZ results of B. henselae isolates.According to results of PHYLOViZ, many of ST35 isolates detected in this study except samples 482 and 380 were found to cluster with reference ST35 isolate detected in Spain.Among ST36 isolates, many of ST36 isolates detected in this study except samples 766 and 812 were found to cluster with reference ST36 isolate detected in Spain.Of the ST5 isolates detected in this study, many of them except sample 559 were found to cluster with ST5 isolate detected in New Zealand.ST9 isolates detected in this study were detected to have a higher variation and four of them were clustered with reference ST9 isolate detected in Germany whereas the remaining four ST9 isolates were closely clustered with reference ST9.One of ST1 isolates were clustered with reference ST1 detected in New Zealand whereas the other was closely clustered with reference ST1.B. henselae isolates (circled) with incompatible allelic profiles and called as ST38 were detected to cluster close to ST35 detected in Spain Stray cats are animals whose population is on the rise due to owned cats either escaped or dumped on the street, and uncontrolled breeding of cats on the street.Some of these cats are imported and some come to the country by immigration from neighboring countries.The coexistence of these street-found or runaway cats facilitates the transmission of pathogens they carry to one another.Therefore, the import, migration, and mixing events observed in stray cats are thought to be potential mechanisms that can explain the ST diversity detected in Bartonella species and the presence of same predominant STs in diverse regions.

Conclusion
This study genotyped for the first time B. henselae samples isolated from stray cats living in İzmir, Turkey using MLST method.ST1, ST5, ST9, ST35, ST36 as well as a new ST called ST38 were detected among B. henselae isolates.Depending on these results, it was thought that there is a wide B. henselae genetic diversity in Turkey and new studies analyzing more B. henselae isolates can be helpful to reveal new STs in stray cats in Turkey.

B. henselaeisolates
A total of 44 B. henselae isolates previously detected in stray cats by sequencing ITS region [11] were used for genotyping by MLST analysis.

MLST analysis
During MLST analysis, eight different housekeeping markers, including 16 S rRNA, batR, ftsZ, gltA, groEL, nlpD, ribC, and rpoB loci were amplified by nested PCR and then sequenced for revealing STs of B. henselae isolates as previously described [13].Briefly, each gene was amplified from B. henselae positive DNA samples using their specific primer pairs by a nested PCR.In the first reaction, the 25 µl reaction volume consisted of 1 µl template DNA, 1 µl of each primer (10 µM), 12.5 µl PCR master mix (GeneMark, Taichung, Taiwan) and 9.5 µl distilled water.In the second reaction, the same reaction condition was used except that 1 µl PCR product obtained from the first reaction was used as a template.During gene amplification, PCR was performed using the following calculated-control protocol: 5 min initial denaturation step at 96 •C, followed by 40 cycles of 10 s at 96 •C, 10 s at 55 •C, and 50 s at 72 •C, and a final extension of 10 min at 72 •C.After amplification, PCR products were visualized on 1% agarose gel, purified by the Qiaquick PCR Purification Kit (Qiagen, USA) and sequenced.Following this, the obtained sequences were analyzed by comparison with allelic profiles in the MLST database (https://pubmlst.org/organisms/Bartonellahenselae).The minimum spanning trees were created with the PHYLOViZ online platform (https://online.phyloviz.net/index#)using goeBURST algorithm along with reference examples from the database [20].In addition, to determine the genetic distance between individuals, distance analysis (Split-Network) with Neighbor-net [21] method was created using the SplitsTree 4.11.3 program [22].For the genetic analyses of B. henselae isolates, Split-sTree distance analysis was used previously by Furquim et al. [14], and the minimum spanning trees analysis by Dias et al. [1] and by Furquim et al. [14].Reference samples containing a reference from each ST used during analyses were presented in Additional file 1:S1.

Table 1
Allelic profiles of 44 B. henselae samples detected in stray cats according to the MLST database *indicates that B. henselae isolate has an incompatible allelic profile.These isolates have been called as ST38 in this study.