Bartonellae are fastidious vector-borne Gram-negative bacteria responsible for multiple human diseases (Jin et al., 2023). The predominant Bartonelloses in humans are primarily attributed to Bartonella henselae, Bartonella bacilliformis, and Bartonella quintana. Infection with B. henselae can result in the prototypical cat-scratch disease (CSD), characterized by fever and regional lymphadenopathy, primarily affecting immunocompromised individuals and occasionally immunocompetent individuals, especially children and adolescents (Pulliainen and Dehio, 2012). In severe cases, B. henselae can lead to bacillary angiomatosis, a condition marked by vasoproliferative tumors affecting the skin and internal organs (Pulliainen and Dehio, 2012). Additionally, a broad spectrum of manifestations has been reported in association with persistent infection of B. henselae, including asymptomatic bacteremia, which is a distinctive characteristic of Bartonella (Massei et al., 2004; Breitschwerdt et al., 2008). The prevalence of B. henselae infection is worldwide, with a seroprevalence in China of up to 9.68% (Song Xiu et al., 2020). In the United States, there are estimated 12,500 cases of CSD annually, with the proportion of hospitalized CSD patients increasing from 3.5% in 2005–2007 to 4.2% in 2011–2013 (Nelson et al., 2016). Despite the prevalence, diagnosing B. henselae infections remains challenging. Antibody-based serological assays are widely applied for diagnosing B. henselae infection. However, these assays present several limitations, including cross-reactivity with other species and organisms, variability in antigen preparations, cutoff titers, and other factors (La Scola and Raoult, 1996; Massei et al., 2004).

Traditional diagnostic methods based on bacterial cultivation (Drummond et al., 2023; Koutantou et al., 2023), often face challenges due to the fastidious nature of B. henselae, which is difficult to culture and detect. This limitation can result in diagnostic difficulties, as in blood-culture-negative endocarditis (BCNE) (Okaro et al., 2017). While molecular techniques offer higher detection rates (Prudent et al., 2018; Sayed et al., 2022; Koutantou et al., 2023), they do not confirm the presence of live bacterial cells. Despite the well-established causative relationship between B. henselae and certain diseases, the difficulty of isolating B. henselae from some infected hosts, combined with the limitations of molecular detection methods, means that these techniques alone cannot fully satisfy Koch’s postulates. This difficulty underscores the importance of microbial culture, still considered the gold standard for detecting infectious pathogens. Higher recovery rates from clinical specimens have been achieved by the shell-vial technique, which involves the inoculation of clinical specimens onto confluent cell monolayers on coverslips within shell vials, followed by low-speed centrifugation to enhance bacterial attachment and penetration, with subsequent detection via immunofluorescence, and definitive bacterial identification by PCR (Lagier et al., 2015). In a study of endocarditis patients suspected of Bartonella spp. infection, the recovery rate was 44% using a shell vial culture assay, compared to only 4% when directly culturing on Columbia sheep blood agar plates (La Scola and Raoult, 1999). The improved sensitivity for recovering Bartonella isolates by shell-vial culture methods suggests that certain factors in the subculture system are essential for the growth of some Bartonella isolates, which may enter a viable but nonculturable (VBNC) state and have the potential to revert to a culturable state under appropriate conditions.

The VBNC state is a survival strategy employed by various bacterial species, allowing them to maintain cellular integrity and metabolic activity while remaining undetectable by standard culturing methods (Liu et al., 2023). VBNC bacteria can resuscitate under favorable conditions, potentially leading to recurrent infections and complicating treatment. For example, VBNC Listeria monocytogenes can resuscitate and cause listeriosis in vulnerable populations (Lotoux et al., 2022). VBNC cells also exhibit resistance to antibiotics and immune stress (Ayrapetyan et al., 2015b), contributing to persistent infections. The persistence of VBNC pathogens in hosts, especially intracellular pathogens like Bartonella, poses significant challenges for accurate diagnosis and risk assessment in clinical settings.

Given these challenges, this study aimed to determine whether B. henselae can enter a VBNC state and to characterize these cells in detail. We induced the VBNC state in B. henselae using high temperature and antibiotic treatment, mimicking conditions like fever and antimicrobial exposure, and developed a method for resuscitating B. henselae VBNC cells, which could greatly improve the diagnosis, detection, and treatment of bartonellosis.

Microbial cultivation has long been considered the gold standard for identifying pathogens in clinical samples. However, the existence of VBNC bacteria, which do not grow on routine culture media, complicates clinical diagnosis. For Bartonella species, cultivation on blood agar plates is critical for diagnosing infections (La Scola and Raoult, 1999; Okaro et al., 2017). However, cases of “culture-negative” endocarditis caused by B. henselae have been reported (Okaro et al., 2017). Furthermore, culture-based tests fail to detect all Bartonella in blood samples, with only 69% (11/16) of Bartonella-infected samples successfully cultured (Pitassi et al., 2015). Shell-vial culture techniques have improved the recovery rates of Bartonella from clinical specimens (Lagier et al., 2015), particularly compared to direct culture on blood agar plates (La Scola and Raoult, 1999), suggesting that Bartonella spp. may enter a viable but nonculturable (VBNC) state under certain conditions, which could be one of the causes for the failure in cultivation.

To prove that B. henselae can enter a VBNC state under specific conditions, B. henselae was cultivated at 38.8°C, a temperature equivalent to the body temperature of cats and dogs and the fever temperature in humans, to induce the VBNC state in this study. We found that B. henselae could enter a pure VBNC state after 19 days at 38.8°C, with no culturable cells detected ( Figure 1B ). The formation of VBNC cells was primarily indicated by discrepancies between the number of culturable cells and the total viable cell count. Culturable cells were routinely quantified through CFU counts on agar plates, while total viable cells were assessed using cell membrane integrity-based methods, such as PMA-qPCR (Kibbee and Ormeci, 2017; Lazou et al., 2019) and SYBR Green I/PI staining (Feng et al., 2018). The viability percentages measured by PMA-qPCR showed a linear correlation with those obtained using SYBR Green I/PI staining combined with plate reader or microscopic analysis ( Supplementary Figure S1 ). However, PMA-qPCR consistently reported lower viability compared to SYBR Green I/PI staining combined with microscopic observation ( Figure 1A ), indicating a higher signal from dead bacterial cells in PMA-qPCR. It is well-documented that extracellular DNA can lead to overestimations of viable bacterial counts in PMA-qPCR (Lovdal et al., 2011). Although washing bacterial cells may mitigate this bias, the additional washing and centrifugation steps can damage fragile B. henselae cells, further complicating the accurate assessment of viability. In this study, SYBR Green I/PI staining was favored for its faster and convenient sample processing, eliminating the need for cell washing and pelleting, and its minimal sample requirements (10 μL), making it an ideal method for the timely assessment of resuscitation.

Clinically used antibiotics, particularly bactericidal ones like gentamicin and streptomycin, also triggered the VBNC state ( Figure 3 ), converting the entire viable population into VBNC state after just 4 days. Over 100 bacterial species have been documented to enter a nonculturable state, with some species, such as Mycobacterium tuberculosis (Zhang et al., 2001), Vibrio vulnificus, and Legionella pneumophila, can resuscitate (Li et al., 2014). Given resuscitation is now a gold standard for verifying the VBNC state (Liu et al., 2023), we developed a specialized medium as described in the Methods section and successfully resuscitated VBNC cells generated by cultivation at 38.8°C or antibiotic treatment, which otherwise failed to grow on regular blood agar plates. The resuscitation method developed here would be promising to improve the diagnostic detection of B. henselae in clinical practice, though further studies in clinical settings are needed.

The viability assessment of bacterial cells in this study (PMA-qPCR assay and SYBR Green I/PI assay) relied on membrane integrity, which might misclassify dead cells with intact membrane as VBNC cells (Song and Wood, 2021). However, TEM analysis confirmed that B. henselae VBNC cells developed at 38.8°C maintain an intact cell wall, intact inner membrane, and dense cytosol ( Figure 2 ). Notably, substantial shrinkage of the inner membrane away from the cell wall was observed in a large portion of VBNC cells, highlighting its relevance under stress conditions. Similar morphology was reported in E. coli in response to nutrient depletion (Shi et al., 2021), and the presence of plasmolysis implies that bacterial cells were alive (Korber et al., 1996). The morphology changes of VBNC B. henselae need further study in the future.

The ability of B. henselae to enter the VBNC state likely contributes to the frequent failure to isolate the bacterium from hosts, posing challenges for clinical diagnosis and treatment. The generation of a pure VBNC state at an elevated temperature of 38.8°C in our study suggests that B. henselae may exist in the VBNC state within infected hosts, such as febrile humans, cats, and dogs, where the normal body temperatures in the latter animal hosts can range from 38.3 to 39.2°C. This hypothesis aligns with the difficulty of isolating B. henselae from infected animals (Diniz et al., 2013; Sayed et al., 2022). Additionally, it suggests that fever in patients caused by B. henselae infection may also promote the transition to the VBNC state.

VBNC state has been proposed as part of bacterial persistence (Zhang, 2014; Ayrapetyan et al., 2018) for bacterial survival in adverse environments, exhibiting significant convergence in their characteristics and molecular mechanisms, particularly in antibiotic tolerance (Ayrapetyan et al., 2015b). Previous studies demonstrated that stationary-phase cultures contain a large portion of drug-tolerant persisters in various bacterial species, including B. henselae (Li et al., 2019). This study found that pure B. henselae VBNC cells exhibited enhanced drug tolerance relative to persisters ( Figure 2E ), especially against anti-persister drugs like daptomycin and methylene blue (Li et al., 2019). Currently, the VBNC state and bacterial persistence are regarded as closely related phenomena along a shared ‘dormancy continuum’, rather than entirely distinct states (Ayrapetyan et al., 2015b). However, the mechanisms underlying drug tolerance in B. henselae during these two forms may be different and require further investigation. High-temperature exposure also significantly accelerated the formation of B. henselae persisters ( Figure 2F ), suggesting that B. henselae in infected individuals may be more drug-tolerant, posing substantial challenges for clinical treatment.

Furthermore, clinically used antibiotics were shown to induce the VBNC state in B. henselae. Bacterial antibiotics like gentamicin and streptomycin eliminated culturable cells within 4 days ( Figure 3A ), which is consistent with our previous findings (Li et al., 2019). However, viable cell counts remained high. This, combined with successful resuscitation, unearthed a novel insight: these clinically employed antibiotics can induce B. henselae into VBNC state, at least under in vitro conditions. Even bacteriostatic antibiotics like doxycycline and erythromycin, also converted a portion of B. henselae cells to VBNC state ( Figure 3B ). Therefore, the current antibiotic treatment for B. henselae-infected individuals may have paradoxical effect of killing some growing bacteria while promoting VBNC cell formation, which can contribute to persistent and/or chronic infection. These findings advance our understanding of the impact of antibiotic treatment for Bartonella infections, urging the need to develop more effective treatments to combat the persister forms of this emerging human pathogen, especially the VBNC form.

The molecular mechanisms underlying VBNC formation in bacteria remain incompletely understood (Liu et al., 2023), though key genes and pathways, including those related to stress response, membrane proteins, toxin-antitoxin systems, and cell division, have been identified (Dong et al., 2020). Proteomic analysis in this study revealed that 17.6% of identified proteins were significantly altered in B. henselae VBNC cells compared to culturable cells. Notably, up-regulated proteins involved in hemin and iron uptake, such as FhaB1 and the hemin-binding proteins (Sander et al., 2000), suggest an iron deficiency in VBNC cells. This iron deficiency is supported by the resuscitation process, where sheep blood, rich in heme iron, promoted the recovery of VBNC cells to a culturable state. However, the specific contribution of sheep blood in the resuscitation process is a complex issue. Future studies should focus on identifying the key factors involved in the resuscitation of B. henselae VBNC cells, including the evaluation of various iron components.

Beyond their role in facilitating hemin and/or iron uptake, the HecB/FhaC proteins and hemin-binding proteins are also responsible for mediating B. henselae adhesion to host cells (Franz and Kempf, 2011). More proteins involved in host cell adhesion and invasion were up-regulated in VBNC cells ( Figure 4 ; Supplementary Table S2 ; Supplementary Figure S2 ), including TrwL/J, VirB, and IalB. The Trw and VirB systems, which are Type 4 secretion system (T4SS), play critical roles in the adhesion of Bartonella to host cells (Jin et al., 2023), whereas IalB protein is implicated in erythrocyte invasion. The increased expression of these proteins in VBNC cells are likely responsible for facilitating the entry and invasion of B. henselae into host cells. FISH analysis in this study indeed confirmed that B. henselae in the VBNC state could invade HUVECs within 12-18 hours, whereas no invasion was observed for logarithmic-phase B. henselae during this same period ( Figure 4C ). Typically, actively growing bacteria require at least 24-30 hours to invade HUVECs (Dehio et al., 1997; Schmid et al., 2004). This suggests that VBNC B. henselae may have a faster invasion capability compared to its logarithmic-phase counterpart. This rapid invasion provides an effective strategy for countering environmental stress and evading the host immune clearance.

Another up-regulated protein in VBNC cells was BafA, a Bartonella-produced mitogen that exhibits pro-angiogenic activity through vascular endothelial growth factor receptor-2 (VEGFR-2) on the cell surface, resulting in the proliferation of Bartonella-infected vascular endothelial cells through activating the mitogen-activated protein kinase (MAPK) pathway (Tsukamoto et al., 2020). Other up-regulated proteins in VBNC cells included molecular chaperones and heat shock proteins ( Figure 4A ; Supplementary Figure S2 ), which are likely critical for maintaining the stability and folding of proteins in the VBNC state, thereby ensuring bacterial survival under unfavorable conditions. The down-regulation of cold shock proteins ( Figure 4A ) aligns with VBNC induction under high temperatures. SOD, an enzyme that eliminates reactive oxygen species, was also up-regulated, possibly aiding B. henselae VBNC cells survival at elevated temperatures.

Pathways involved in energy metabolism, lipid metabolism, and protein synthesis were up-regulated, while those related to the metabolism of nucleotides, amino acids, glycans, cofactors, and vitamins were down-regulated ( Figure 4B ). This metabolic shift suggests that B. henselae VBNC cells maintain essential functions for survival while down-regulating less critical pathways, characteristics of VBNC states (Liu et al., 2023) that share similarities with bacterial persister pathways (Zhang, 2014; Niu et al., 2024).

In conclusion, this study investigated B. henselae’s ability to enter the VBNC state and invade host cells efficiently. B. henselae can fully enter the VBNC state under fever-mimicking temperature (38.8°C) and antibiotic treatment, exhibiting enhanced drug tolerance and specific morphological and proteomic adjustments. The evidence suggests that bacteria in the VBNC state are not just a theoretical risk but may actively contribute to host pathogenesis, relapsing infections, and antibiotic failure. These findings underscore the diagnostic challenges posed by B. henselae in the VBNC state and emphasize the need for improving treatment strategies and developing effective culture methods to recover B. henselae isolates from patients.