Bartonella henselae Persistence within Mesenchymal Stromal Cells Enhances Endothelial Cell Activation and Infectibility That Amplifies the Angiogenic Process.

Some bacterial pathogens can manipulate the angiogenic response, suppressing or inducing it for their own ends. In humans, Bartonella henselae is associated with cat-scratch disease and vasculoproliferative disorders such as bacillary angiomatosis and bacillary peliosis. Although endothelial cells (ECs) support the pathogenesis of B. henselae, the mechanisms by which B. henselae induces EC activation are not completely clear, as well as the possible contributions of other cells recruited at the site of infection. Mesenchymal stromal cells (MSCs) are endowed with angiogenic potential and play a dual role in infections, exerting antimicrobial properties but also acting as a shelter for pathogens. Here, we delved into the role of MSCs as a reservoir of B. henselae and modulator of EC functions. B. henselae readily infected MSCs and survived in perinuclearly bound vacuoles for up to 8 days. Infection enhanced MSC proliferation and the expression of epidermal growth factor receptor (EGFR), Toll-like receptor 2 (TLR2), and nucleotide-binding oligomerization domain-containing protein 1 (NOD1), proteins that are involved in bacterial internalization and cytokine production. Secretome analysis revealed that infected MSCs secreted higher levels of the proangiogenic factors vascular endothelial growth factor (VEGF), fibroblast growth factor 7 (FGF-7), matrix metallopeptidase 9 (MMP-9), placental growth factor (PIGF), serpin E1, thrombospondin 1 (TSP-1), urokinase-type plasminogen activator (uPA), interleukin 6 (IL-6), platelet-derived growth factor D (PDGF-D), chemokine ligand 5 (CCL5), and C-X-C motif chemokine ligand 8 (CXCL8). Supernatants from B. henselae-infected MSCs increased the susceptibility of ECs to B. henselae infection and enhanced EC proliferation, invasion, and reorganization in tube-like structures. Altogether, these results indicate MSCs as a still underestimated niche for persistent B. henselae infection and reveal MSC-EC cross talk that may contribute to exacerbate bacterium-induced angiogenesis and granuloma formation.

Bartonella infections diagnosed in the French reference center, 2014-2019, and focus on infections in the immunocompromised.

We studied retrospectively 651 PCR-confirmed Bartonella infections diagnosed at the French reference center for bartonellosis from 2014 to 2019. The most common form was cat-scratch disease (89%) followed by endocarditis (9%). Disseminated forms (2%) mainly presented as bacillary angiomatosis or peliosis hepatis in solid organ transplant recipients.

Effect of different drugs and drug combinations on killing stationary phase and biofilms recovered cells of Bartonella henselae in vitro.

BACKGROUND: Bartonella henselae is a Gram-negative bacterium transmitted to humans by a scratch from cat in the presence of ectoparasites. Humans infected with B. henselae can result in various clinical diseases including local lymphadenopathy and more serious systemic disease such as persistent bacteremia and endocarditis. The current treatment of persistent B. henselae infections is not very effective and remains a challenge. To find more effective treatments for persistent and biofilm Bartonella infections, in this study, we evaluated a panel of drugs and drug combinations based on the current treatment and also promising hits identified from a recent drug screen against stationary phase and biofilm recovered cells of B. henselae. RESULTS: We evaluated 14 antibiotics and 25 antibiotic combinations for activity against stationary phase B. henselae (all antibiotics were at 5 µg/ml) and found that ciprofloxacin, gentamicin, and nitrofurantoin were the most active agents, while clofazimine and miconazole had poor activity. Drug combinations azithromycin/ciprofloxacin, azithromycin/methylene blue, rifampin/ciprofloxacin, and rifampin/methylene blue could rapidly kill stationary phase B. henselae with no detectable CFU after 1-day exposure. Methylene blue and rifampin were the most active agents against the biofilm B. henselae after 6 days of drug exposure. Antibiotic combinations (azithromycin/ciprofloxacin, azithromycin/methylene blue, rifampin/ciprofloxacin, rifampin/methylene blue) completely eradicated the biofilm B. henselae after treatment for 6 days. CONCLUSIONS: These findings may facilitate development of more effective treatment of persistent Bartonella infections in the future.

Combined Application of Immunohistochemistry and Warthin-Starry Silver Stain on the Pathologic Diagnosis of Cat Scratch Disease.

PURPOSE: Cat scratch disease (CSD) is an infectious disorder caused primarily by the bacterium Bartonella henselae (B. henselae). Immunohistochemistry (IHC) and Warthin-Starry silver stain (WS) are considered to be indispensable to diagnose CSD in combination with morphologic characteristics. In this study, we retrieved and reviewed 46 cases of paraffin-embedded lymphadenitis with histologic and/or clinical suspicion of CSD between 2014 and 2018, and detected B. henselae by IHC and WS, respectively, and evaluated the application significance of IHC and WS for the detection of B. henselae and validated their values in the pathologic diagnosis of CSD. MATERIALS AND METHODS: B. henselae was detected by IHC and WS; validation of 2 methods for detecting B. henselae was evaluated by sensitivity, specificity, false-positive rate, false-negative rate, precision, negative predictive value, and agreement rate. RESULTS: Microscopically, suppurative granulomas and/or multiple stellate microabscesses were observed in the accessory cortex of lymph nodes, especially near the subcapsule. Our results showed that 80.4% (37/46) of cases were positive for B. henselae by IHC, manifesting mainly punctuate, granular, or linear to outline the shape of bacteria. However, the positive rate of B. henselae by the WS method was 52.2% (24/46). There was a significant difference between IHC and WS (P=0.023). Moreover, a positive percentage of B. henselae was 97.8% (45/46), which was detected by the combined application of IHC and WS. The combination of IHC and WS exhibited high sensitivity (97.8%) and good agreement rate (86.5%). CONCLUSION: The combined application of the IHC and WS method may have important clinical advantages, which is with the highest sensitivity and agreement rate for pathologic diagnosis of CSD.

Cats as a reservoir of Bartonella henselae for dogs.

Bartonellosis is a disease affecting a variety of animals. Many Bartonella infections are zoonotic, including cat scratch disease. Within the genus Bartonella are 45 species, of which more than 10 can infect cats and dogs. Companion animals serve as reservoirs for several zoonotic species of Bartonella, and may also serve as sentinels for zoonotic Bartonella species harbored by wildlife. The aim of this study was to determine the frequency of the occurrence of Bartonella spp. DNA in dogs from households where cats with clinical bartonellosis were kept. The presence of DNA with 99-100% compliance of the nucleotide sequence with the sequence of the Bartonella DNA isolated from cats was demonstrated in the body of 10% of tested dogs. The results indicate that cats serve as a Bartonella reservoir for dogs, and the dogs can play the same role with regard to humans.

Immunity of fleas (Order Siphonaptera).

The immune response of arthropod vectors plays a key role in the spread and transmission of vector-borne diseases. Although fleas transmit several human pathogens (e.g., Bartonella henselae, Rickettsia felis, R. typhi, and Yersinia pestis), few studies have examined how these vectors respond to infection. In hematophagous arthropods, imbibed pathogens must survive the hostile environment of blood meal digestion, which includes proteolytic digestive enzymes, protease inhibitors and expression of genes associated with protection of epithelial linings. Additionally, insect epithelial cells exhibit local immune defense against ingested pathogens by producing antimicrobial peptides and reactive oxygen species. This review details these and other aspects of insect immunity as it relates to fleas, with an emphasis on the gut immune response to two blood-borne pathogens, R. typhi and Y. pestis.

Bartonella henselae in small Indian mongooses (Herpestes auropunctatus) from Grenada, West Indies.

Many mammals are established hosts for the vector borne bacterial genus, Bartonella. Small Indian mongooses (Herpestes auropunctatus) have only been reported as a possible host for Bartonella henselae in southern Japan. Confirming Bartonella presence in mongooses from other regions in the world may support their role as potential reservoirs of this human pathogen. Specifically, documenting Bartonella in Caribbean mongooses would identify a potential source of zoonotic risk with mongoose-human contact in the New World. Using serological and molecular techniques, we investigated B. henselae DNA and specific antibody prevalence in 171 mongooses from all six parishes in Grenada, West Indies. Almost a third (32.3%, 54/167) of the tested mongooses were B. henselae seropositive and extracted DNA from 18/51 (35.3%) blood pellets were PCR positive for the citrate synthase (gltA) and/or the ß subunit of RNA polymerase (rpoB) genes. All sequences were identical to B. henselae genotype I, as previously reported from Japan. This study confirms the role of small Indian mongooses as a natural reservoir of B. henselae in the New World.

Prevalence of Bartonella spp. by culture, PCR and serology, in veterinary personnel from Spain.

BACKGROUND: The genus Bartonella includes fastidious, facultative intracellular bacteria mainly transmitted by arthropods and distributed among mammalian reservoirs. Bartonella spp. implicated as etiological agents of zoonoses are increasing. Apart from the classical Bartonella henselae, B. bacilliformis or B. quintana, other species (B. elizabethae, B. rochalimae, B. vinsonii arupensis and B. v. berkhoffii, B. tamiae or B. koehlerae, among others) have also been associated with human and/or animal diseases. Laboratory techniques for diagnosis (culture, PCR assays and serology) usually show lack of sensitivity. Since 2005, a method based on a liquid enrichment Bartonella alphaproteobacteria growth medium (BAPGM) followed by PCRs for the amplification of Bartonella spp. has been developed. We aimed to assess culture, molecular and serological prevalence of Bartonella infections in companion animal veterinary personnel from Spain. METHODS: Each of 89 participants completed a questionnaire. Immunofluorescence assays (IFA) using B. vinsonii berkhoffii (genotypes I, II and III), B. henselae, B. quintana and B. koehlerae as antigens were performed. A cut-off of 1:64 was selected as a seroreactivity titer. Blood samples were inoculated into BAPGM and subcultured onto blood agar plates. Bartonella spp. was detected using conventional and quantitative real-time PCR assays and DNA sequencing. RESULTS: Among antigens corresponding to six Bartonella spp. or genotypes, the lowest seroreactivity was found against B. quintana (11.2%) and the highest, against B. v. berkhoffii genotype III (56%). A total of 27% of 89 individuals were not seroreactive to any test antigen. Bartonella spp. IFA seroreactivity was not associated with any clinical sign or symptom. DNA from Bartonella spp., including B. henselae (n = 2), B. v. berkhoffii genotypes I (n = 1) and III (n = 2), and B. quintana (n = 2) was detected in 7/89 veterinary personnel. PCR and DNA sequencing findings were not associated with clinical signs or symptoms. No co-infections were observed. One of the two B. henselae PCR-positive individuals was IFA seronegative to all tested antigens whereas the other one was not B. henselae seroreactive. The remaining PCR-positive individuals were seroreactive to multiple Bartonella spp. antigens. CONCLUSIONS: High serological and molecular prevalences of exposure to, or infection with, Bartonella spp. were found in companion animal veterinary personnel from Spain. More studies using BAPGM enrichment blood culture and PCR are needed to clarify the finding of Bartonella PCR-positive individuals lacking clinical symptoms.

Infections by pathogens with different transmission modes in feral cats from urban and rural areas of Korea.

In this study, we examine prevalences of three infectious pathogens with different transmission modes (Bartonella henselae, hemoplasma, and Toxoplasma gondii) in feral cats from urban and rural habitats. Infection status of the three pathogens in blood samples (n = 117) was determined through molecular or serological diagnostic methods. Overall prevalence of hemoplasma, Toxoplasma gondii, and Bartonella henselae was 47.9%, 50%, and 35.7%, respectively. Comparing the two habitats, only seroprevalence of Bartonella henselae was significantly higher in urban cats. Based on the results, we discuss how pathogens with distinct transmission modes may show different prevalence between urban and rural habitat types.

Bartonella henselae infection presenting with a picture of adult-onset Still's disease.

We report a patient with a clinical picture of suggestive for adult-onset Still's Disease (ASOD) due to Bartonella infection. A 42-year-old immunocompetent man was admitted with fever, rash, arthralgia and sore throat. As his clinical picture suggested ASOD except unusual skin manifestation, we treated him on steroid and ibuprofen. His fever and constitutional symptoms responded immediately within 24hrs of commencing therapy, yet rash and leukocytosis remained. Meanwhile, Bartonella infection was proved by culture of bone marrow. Minocyclin treatment started combined with hydroxychloroquine sulfate and the patient discharged with overall improvement.

Characterization of the general stress response in Bartonella henselae.

Bacteria utilize a general stress response system to combat stresses from their surrounding environments. In alpha-proteobacteria, the general stress response uses an alternate sigma factor as the main regulator and incorporates it with a two-component system into a unique regulatory circuit. This system has been described in several alpha-proteobacterial species, including the pathogens Bartonella quintana and Brucella abortus. Most of the studies have focused on characterizing the PhyR anti-anti-sigma factor, the NepR anti-sigma factor, and the alternate sigma factor. However, not enough attention is directed toward studying the role of histidine kinases in the general stress response. Our study identifies the general stress response system in Bartonella henselae, where the gene synteny is conserved and both the PhyR and alternate sigma factor have similar sequence and domain structures with other alpha-proteobacteria. Our data showed that the general stress response genes are up-regulated under conditions that mimic the cat flea vector. Furthermore, we showed that both RpoE and PhyR positively regulate this system and that RpoE also affects transcription of genes encoding heme-binding proteins and the gene encoding the BadA adhesin. Finally, we identified a histidine kinase, annotated as BH13820 that can potentially phosphorylate PhyR.

Cat scratches, not bites, are associated with unipolar depression--cross-sectional study.

BACKGROUND: A recent study performed on 1.3 million patients showed a strong association between being bitten by a cat and probability of being diagnosed with depression. Authors suggested that infection with cat parasite Toxoplasma could be the reason for this association. METHOD: A cross sectional internet study on a non-clinical population of 5,535 subjects was undertaken. RESULTS: The subjects that reported having been bitten by a dog and a cat or scratched by a cat have higher Beck depression score. They were more likely to have visited psychiatrists, psychotherapists and neurologists in past two years, to have been previously diagnosed with depression (but not with bipolar disorder). Multivariate analysis of models with cat biting, cat scratching, toxoplasmosis, the number of cats at home, and the age of subjects as independent variables showed that only cat scratching had positive effect on depression (p = 0.004). Cat biting and toxoplasmosis had no effect on the depression, and the number of cats at home had a negative effect on depression (p = 0.021). CONCLUSIONS: Absence of association between toxoplasmosis and depression and five times stronger association of depression with cat scratching than with cat biting suggests that the pathogen responsible for mood disorders in animals-injured subjects is probably not the protozoon Toxoplasma gondii but another organism; possibly the agent of cat-scratched disease - the bacteria Bartonella henselae.

Analysis of Endothelial Adherence of Bartonella henselae and Acinetobacter baumannii Using a Dynamic Human Ex Vivo Infection Model.

Bacterial adherence determines the virulence of many human-pathogenic bacteria. Experimental approaches elucidating this early infection event in greater detail have been performed using mainly methods of cellular microbiology. However, in vitro infections of cell monolayers reflect the in vivo situation only partially, and animal infection models are not available for many human-pathogenic bacteria. Therefore, ex vivo infection of human organs might represent an attractive method to overcome these limitations. We infected whole human umbilical cords ex vivo with Bartonella henselae or Acinetobacter baumannii under dynamic flow conditions mimicking the in vivo infection situation of human endothelium. For this purpose, methods for quantifying endothelium-adherent wild-type and trimeric autotransporter adhesin (TAA)-deficient bacteria were set up. Data revealed that (i) A. baumannii binds in a TAA-dependent manner to endothelial cells, (ii) this organ infection model led to highly reproducible adherence rates, and furthermore, (iii) this model allowed to dissect the biological function of TAAs in the natural course of human infections. These findings indicate that infection models using ex vivo human tissue samples ("organ microbiology") might be a valuable tool in analyzing bacterial pathogenicity with the capacity to replace animal infection models at least partially.

Coexistence of Bartonella henselae and B. clarridgeiae in populations of cats and their fleas in Guatemala.

Cats and their fleas collected in Guatemala were investigated for the presence of Bartonella infections. Bartonella bacteria were cultured from 8.2% (13/159) of cats, and all cultures were identified as B. henselae. Molecular analysis allowed detection of Bartonella DNA in 33.8% (48/142) of cats and in 22.4% (34/152) of cat fleas using gltA, nuoG, and 16S-23S internal transcribed spacer targets. Two Bartonella species, B. henselae and B. clarridgeiae, were identified in cats and cat fleas by molecular analysis, with B. henselae being more common than B. clarridgeiae in the cats (68.1%; 32/47 vs 31.9%; 15/47). The nuoG was found to be less sensitive for detecting B. clarridgeiae compared with other molecular targets and could detect only two of the 15 B. clarridgeiae-infected cats. No significant differences were observed for prevalence between male and female cats and between different age groups. No evident association was observed between the presence of Bartonella species in cats and in their fleas.

Did Bartonella henselae contribute to the deaths of two veterinarians?

Bartonella henselae, a flea-transmitted bacterium, causes chronic, zoonotic, blood stream infections in immunocompetent and immunocompromised patients throughout the world. As an intra-erythrocytic and endotheliotropic bacterium, B. henselae causes a spectrum of symptomatology ranging from asymptomatic bacteremia to fever, endocarditis and death. Veterinary workers are at occupational risk for acquiring bartonellosis. As an emerging, and incompletely understood, stealth bacterial pathogen, B. henselae may or may not have been responsible for the deaths of two veterinarians; however, recent evidence indicates that this genus is of much greater medical importance than is currently appreciated by the majority of the biomedical community.

Reprogramming of myeloid angiogenic cells by Bartonella henselae leads to microenvironmental regulation of pathological angiogenesis.

The contribution of myeloid cells to tumour microenvironments is a decisive factor in cancer progression. Tumour-associated macrophages (TAMs) mediate tumour invasion and angiogenesis through matrix remodelling, immune modulation and release of pro-angiogenic cytokines. Nothing is known about how pathogenic bacteria affect myeloid cells in these processes. Here we show that Bartonella henselae, a bacterial pathogen causing vasculoproliferative diseases (bacillary angiomatosis), reprogrammes human myeloid angiogenic cells (MACs), a pro-angiogenic subset of circulating progenitor cells, towards a TAM-like phenotype with increased pro-angiogenic capacity. B. henselae infection resulted in inhibition of cell death, activation of angiogenic cellular programmes and induction of M2 macrophage polarization. MACs infected with B. henselae incorporated into endothelial sprouts and increased angiogenic growth. Infected MACs developed a vascular mimicry phenotype in vitro, and expression of B. henselae adhesin A was essential in inducing these angiogenic effects. Secretome analysis revealed that increased pro-angiogenic activities were associated with the creation of a tumour-like microenvironment dominated by angiogenic inflammatory cytokines and matrix remodelling compounds. Our results demonstrate that manipulation of myeloid cells by pathogenic bacteria can contribute to microenvironmental regulation of pathological tissue growth and suggest parallels underlying both bacterial infections and cancer.

Bartonella henselae infection-associated vasculitis and crescentic glomerulonephritis leading to renal allograft loss.

Bartonella henselae (BH) is the main cause of cat scratch disease (CSD), which more typically presents as a self-limited localized suppurative lymphadenopathy in immunocompetent individuals. In contrast, immunocompromised patients commonly have systemic disease with life-threatening complications. In addition to the angioproliferative lesions, such as bacillary angiomatosis, an increasing number of immune post-infectious complications are being recognized with BH infections, including glomerulonephritis, vasculitis, hemophagocytic syndrome, and neurological problems. We report the case of a renal transplant recipient who developed CSD in the second year post transplantation. In addition to prolonged fever and generalized lymphadenopathy and splenomegaly requiring differentiation from a post-transplant lymphoproliferative disorder, the course was complicated by the development of dermal leukocytoclastic vasculitis and pauci-immune necrotizing and crescentic glomerulonephritis, which led to failure of the renal graft. Glomerulonephritis as a complication of CSD has never been described in a kidney allograft, to our knowledge. Awareness of the diverse clinical symptoms associated with BH, including granulomatous/suppurative lesions and other less common complications can lead to more rapid and accurate diagnosis. Also, as recommended by the current guidelines, a thorough history of pet ownership should be part of the clinical evaluation before and after transplantation for all transplant recipients.

The efficacy of a selamectin (Stronghold ®) spot on treatment in the prevention of Bartonella henselae transmission by Ctenocephalides felis in cats, using a new high-challenge model.

Bartonella henselae is the causative agent of cat scratch disease in humans, which is recognized as an emerging zoonotic disease. Ctenocephalides felis is the main vector, and transmission of B. henselae infection between cats and humans occurs mainly through infected flea feces. Control of feline infestation with this arthropod vector therefore provides an important strategy for the prevention of infection of both humans and cats. In the present study, a new challenge model is used to evaluate the efficacy of selamectin (Stronghold(®) spot on) in the prevention of B. henselae transmission by C. felis. In this new challenge model, domestic cats were infected by direct application of B. henselae-positive fleas. The fleas used for infestation were infected by feeding on blood that contained in vitro-cultured B. henselae. The direct application of the fleas to the animals and the use of different B. henselae strains ensured a high and consistent challenge. Two groups of six cats were randomly allocated on pre-treatment flea counts to either control (untreated cats) or the selamectin-treated group with one pipette per cat according to the label instruction. Stronghold (selamectin 6 % spot on solution) was administered on days 0 and 32. On days 3, 10, 19, 25, and 31, each cat was infested by direct application of 20 fleas that fed on blood inoculated with B. henselae. Polymerase chain reaction (PCR) on pooled fleas confirmed that the fleas were infected. Blood samples were collected from each cat on days -3 (prior to flea infestation and treatment), 9, 17, 24, 30, 37, and 44 and assayed for B. henselae antibodies using an indirect immunofluorescence (IFA), for the presence of bacteria by bacterial culture and for B. henselae DNA presence by PCR. Cats were also assessed on a daily basis for general health. There were no abnormal health observations during the study and none of the animals required concomitant treatment. None of the cats displayed any clinical signs of bartonellosis during the study. In the untreated group, all cats became bacteremic within 17 to 44 days. None of the selamectin-treated cats became positive during the study. It was concluded that Stronghold(®) spot on administered to cats was efficacious in the prevention of the transmission of B. henselae by fleas to cats in a high-challenge model.

[Lyme borreliosis and co-infections. Place of Anaplasma phagocytophilum and Bartonella henselae]. / Borréliose de Lyme et co-infections. Place d'Ana- plasma phagocytophilum et de Bartonella henselae.

Lyme borreliosis (LB) is certainly the most common infection transmitted through the bite of Ixodes in Northern Hemisphere. These ticks are also able to transmit other microorganisms such as the bacteria Anaplasma phagocytophilum (Ap) and Bartonella henselae (Bh), with the latter discovered fairly recently, leading to diferent clinical presentations often close to those of LB. The aim of this study was to evaluate the frequency of co-infection by either of these bacteria in patients with LB, particularly when a treatment with beta-lactam antibiotic was only partially effective. Of these patients, on the basis of serological data, 8.07% were simultaneously contaminated by Bh, 6.83% by Ap and 4.96% were co-infected by Bh and Ap. Since the choice of an antibiotic should take into account the specificities of these germs and especially their intracellular proliferation, these results should be considered in selecting treatment.