Quantitative assessment of the blood-brain barrier opening caused by Streptococcus agalactiae hyaluronidase in a BALB/c mouse model Abstract Streptococcus agalactiae is a pathogen causing meningitis in animals and humans. However, little is known about the entry of S. agalactiae into brain tissue. In this study, we developed a BALB/c mouse model based on the intravenous injection of 尾-galactosidase-positive Escherichia coli M5 as an indicator of blood-brain barrier (BBB) opening. Under physiological conditions, the BBB is impermeable to E. coli M5. In pathological conditions caused by S. agalactiae, E. coli M5 is capable of penetrating the brain through a disrupted BBB. The level of BBB opening can be assessed by quantitative measurement of E. coli M5 loads per gram of brain tissue. Further, we used the model to evaluate the role of S. agalactiae hyaluronidase in BBB opening. The inactivation of hylB gene encoding a hyaluronidase, HylB, resulted in significantly decreased E. coli M5 colonization, and the intravenous injection of purified HylB protein induced BBB opening in a dose-dependent manner. This finding verified the direct role of HylB in BBB invasion and traversal, and further demonstrated the practicability of the in vivo mouse model established in this study. This model will help to understand the S. agalactiae鈥揾ost interactions that are involved in this bacterial traversal of the BBB and to develop efficacious strategies to prevent central nervous system infections. Introduction Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a Gram-positive, opportunistic pathogen that colonizes the gastrointestinal and genitourinary tracts of up to 50% of healthy adults1. It also causes septicemia, meningitis and pneumonia in neonates and is the main reason for significant morbidity in pregnant women, the elderly and immunocompromised adults2,3,4. S. agalactiae can also infect the mammary glands of cows, where it can survive for a long period of time, causing clinical or subclinical mastitis5. In addition, in recent years, it has been reported that S. agalactiae can cause meningoencephalitis in fish and bring losses to aquaculture6. Numerous outbreaks of S. agalactiae infections have been described in multiple fish farms, especially tilapia farms7,8,9. Since 2009, an outbreak of severe infectious GBS disease has occurred in tilapia farms in the south of China, causing tremendous economic losses due to high mortality in the infected fish10.The pathogenesis of meningitis caused by S. agalactiae has not been fully elucidated. It is well known that only pathogens entering the central nervous system (CNS) can cause meningitis. To gain access into the CNS, S. agalactiae must cross the blood-brain barrier (BBB), which is primarily comprised of a single layer of specialized brain microvascular endothelial cells (BMECs)11,12. This unique brain endothelial physiological barrier seals the CNS13,14,15, and regulates passage of molecules, nutrients, and infectious agents into the brain16. Pathogens can cross the BBB transcellularly, paracellularly and by a 鈥淭rojan-horse鈥?mechanism17. Some studies have demonstrated that S. agalactiae virulence factors contribute to the adherence and invasion of human BMECs (hBMECs), resulting in the activation of acute inflammatory responses that inevitably disrupt the integrity of the BBB. For example, the well-known virulence factor 尾-hemolysin/cytolysin stimulated the expression of genes linked to neutrophil recruitment and activation, such as IL-8 and ICAM-1, which act to facilitate neutrophil migration across polar hBMEC monolayers18. PilA, the pilus tip adhesin of GBS, had capability in promoting the expression of neutrophil chemokines IL-8, CXCL-1, CXCL-2, CCL-20 and IL-6 in brain endothelium, and therefore increased the permeability of the BBB19. Several investigations in animals have also shown that GBS can penetrate the CNS by crossing the BBB after a prolonged period of bacteremia20,21. Therefore, the BBB plays an important role in controlling the entry of pathogens into the brain.Increased permeability of the BBB can be seen in bacterial meningitis caused by S. agalactiae 22,23. However, how this bacterium crosses the BBB and enters the CNS is not clearly understood. Therefore, it is of crucial importance to characterize BBB permeability in order to better understand the pathogenesis of meningitis. In our previous study, the inactivation of hylB gene encoding a hyaluronidase, HylB, caused the significantly decreased brain bacterial counts in mice24. However, whether HylB directly acts on the BBB opening remains unclear. In this study, we sought to establish a model to evaluate BBB opening by screening a bacterial strain as an indicator, and used this model to quantitatively evaluate the direct role of HylB in S. agalactiae penetration across BBB.ResultsThe screening of an indicator strainTo make the counting easy, we aimed to find a bacterium with a colony morphology distinct from that of S. agalactiae. The results showed that among the M1 to M5 E. coli mutants, only the M5 strain generated characteristic blue clones on M63 media (Fig.聽1), indicating that it was 尾- galactosidase-positive.Figure 1The growth of E. coli M5 on an M63 plate. The bacteria formed blue colonies on M63 medium containing X-Gal.Full size image Determination of E. coli M5 virulence in miceTo determine whether E. coli M5 was virulent to mice, we performed bacterial infection in BALB/c mice. Interestingly, it was observed that none of the mice infected with 2鈥壝椻€?09鈥塁FU of E. coli M5 showed any signs of illness, and there was zero mortality throughout the experimental period of 7 d. The diet and mental state of the experimental mice were identical to those of the control group. The result indicated that E. coli M5 was avirulent to mice.Kinetics of E. coli presence in blood of miceTo investigate the rate of E. coli M5 metabolism in mice, the mice were injected intravenously with M5, and blood samples were collected from each mouse at intervals of 3鈥塵in, 5鈥塵in, 10鈥塵in, 30鈥塵in, 60鈥塵in and 120鈥塵in post-infection. The CFU enumeration results showed that the number of M5 cells in the blood increased from 6鈥壝椻€?05鈥塁FU/mL at 3鈥塵in to a peak value of approximately 2.5鈥壝椻€?06鈥塁FU/mL at 5鈥塵in and then decreased dramatically to 3鈥壝椻€?05鈥塁FU/mL at 10鈥塵in. After one hour, bacteria could hardly be detected, and they had clearly been removed from circulation within 2鈥塰ours after intravenous injection (Fig.聽2). No bacteria were detected in the brain at any time points.Figure 2Metabolism of E. coli M5 in mice. The recovered bacteria are expressed as CFU per milliliter of blood.Full size image Determination of a challenge concentration of S. agalactiae We first sought to determine whether the S. agalactiae GD201008-001 strain causes significant BBB permeability by intraperitoneal infection. It is important to determine an appropriate initial inoculation concentration for S. agalactiae to create an in vivo model. We chose values of 5-fold (50鈥塁FU) and 10-fold (100鈥塁FU) that of the median lethal dose (LD50鈥?lt;鈥?/sub>10鈥塁FU)25 of S. agalactiae to inoculate the mice. The result showed that with the duration of infection, the number of GD201008-001 cells increased in both the blood (Fig.聽3A) and the brain (Fig.聽3B), suggesting that GD201008-001 was capable of replicating within the bloodstream and spreading to the brain. GD201008-001 began to appear in the blood at 3鈥塰 post-infection, 3鈥塰 earlier than in the brain. The bacteria could be detected in the brain at 6鈥塰 post-infection when we injected 100鈥塁FU of S. agalactiae. However, we could not detect any bacteria until 12鈥塰 post-infection in the 50鈥塁FU group. This result suggests that the intraperitoneal infection with 100鈥塁FU of S. agalactiae is more appropriate to induce BBB opening in this mouse model.Figure 3Detection of S. agalactiae in blood and the brain tissues. (A) Bacterial loads in the blood; (B) bacterial loads in the brain. Groups of four BALB/c mice were inoculated with 50鈥塁FU or 100鈥塁FU of S. agalactiae and killed at different time points post-infection. The CFU of S. agalactiae in the blood and the brain were quantified and described as the mean and S.D. *P鈥?lt;鈥?.05, **P鈥?lt;鈥?.01 or ***P鈥?lt;鈥?.001 indicates significantly different bacterial loads between the two infection groups.Full size image Evaluation of BBB openingGroups of five mice were challenged with 100鈥塁FU of GD201008-001. At 3鈥塰, 6鈥塰, 9鈥塰 and 12鈥塰 after challenge, the indicator strain E. coli M5 was inoculated intravenously into each group. In determining the sampling time points, based on Fig.聽2, more M5 tracers were detected in the blood at 5鈥塵in. Therefore, 5鈥塵in after injection was selected to quantify the degree of BBB permeability. Our data showed that GD201008-001 began to appear in the brain 6鈥塰 post-infection, and with the duration of infection, the bacterial number increased (Fig.聽4). M5 could be detected 3鈥塰 after S. agalactiae inoculation, and the number of M5 had a similar increasing trend as GD201008-001 (Fig.聽5). This result suggests that E. coli M5 may be used as an appropriate indicator for describing the degree of BBB opening.Figure 4The changes in S. agalactiae loads in brain tissues of mice over time. The recovered S. agalactiae are expressed as CFU per gram of brain.Full size image Figure 5Evaluation of BBB opening caused by S. agalactiae. Mice were intraperitoneally infected with S. agalactiae, and the X-Gal-positive M5 were given by intravenous injection at different time points post infection. Five minutes later, the mice were killed, and the brains were removed for quantification of M5. The data are expressed as CFU per gram of brain.Full size image Detection of BBB opening caused by S. agalactiae hyaluronidasePrevious study showed that hyaluronidase contributed to S. agalactiae penetration into the CNS24. To verify the utility of this mouse model, the mice were infected with the wild-type S. agalactiae, 螖hylB and C螖hylB strains, and then E. coli M5 was administered to the mice. After intraperitoneal infection with 100鈥塁FU of S. agalactiae and its derivatives, the numbers of the three bacterial strains in the blood increased rapidly from 103鈥塁FU/mL at 3鈥塰 to鈥?gt;107鈥塁FU/mL at 15鈥塰 (Fig.聽6A). In the brains, the wild-type and the complemented strain C螖hylB were present 6鈥塰 post-infection, which was earlier than the mutant 螖hylB (Fig.聽6B). Compared with the wild-type and complemented strains, for the hylB mutant, the numbers were lower at each time point in the brains. As time passed, the number of bacteria in the brains increased to 105鈥塁FU/g at 15鈥塰 post-infection. E. coli M5 in the wild-type and C螖hylB groups could be detected in the brain tissues 6鈥塰 post-infection, and the increasing trend of the number of M5 cells was similar to that of S. agalactiae (Fig.聽6C). The sudden increase in the number of M5 at 15鈥塰 indicated that the BBB of the mice had opened to a great degree. However, in the 螖hylB group, the 螖hylB strain and M5 were not detected until 9鈥塰 post-infection, and the amount of M5 was significantly lower than that of the wild-type group at 9鈥塰 post-infection (P鈥?lt;鈥?.001). At 12鈥塰 and 15鈥塰, the difference in the number of M5 cells between the wild-type and 螖hylB groups reached a highly significant difference (P鈥?lt;鈥?.001). Although the number of M5 cells in the C螖hylB group was lower than that in the wild-type group, the difference was smaller than that with the 螖hylB group.Figure 6Evaluation of BBB opening in mice infected intraperitoneally with the S. agalactiae wild-type (WT), mutant (螖hylB), and complemented (C螖hylB) strains. Groups of 40 BALB/c mice were inoculated with 100 CFU S. agalactiae and its derivatives. At 3鈥塰, 6鈥塰, 9鈥塰, 12鈥塰, and 15鈥塰 post infection, the blood (A) and brain (B) tissues were collected from four mice of each groups for S. agalactiae quantification. Meanwhile, at each time point, another four mice from each group were injected intravenously with E. coli M5 (2鈥壝椻€?08鈥塁FU). Five minutes later, the mice were killed, and the brains were removed for quantification of M5 (C). Bacterial loads in the blood are expressed as CFU/mL, and those in the brains are expressed as CFU/g of tissue. *P鈥?lt;鈥?.05, **P鈥?lt;鈥?.01 or ***P鈥?lt;鈥?.001 indicates significantly different bacterial loads between the two infection groups.Full size image To further investigate the role of the hylB gene in BBB opening, the protein HylB, which is encoded by the hylB gene, was expressed and injected intravenously into the mice. An injection of 0.5鈥塵g/mL HylB did not cause BBB opening at 3鈥塰, as evidenced by the absence of E. coli M5 in brain, whereas M5 could enter and begin to accumulate in the brains of the 1.0鈥塵g/mL and 2.0鈥塵g/mL groups (Fig.聽7). There was a dose-dependent increase in the degree of BBB opening at each time point.Figure 7Evaluation of BBB opening in mice injected with different doses of hyaluronidase. Groups of four BALB/c mice were inoculated with 0.5, 1.0 or 2.0鈥塵g/ml of HylB, and at different time points post-infection, E. coli M5 (2鈥壝椻€?08鈥塁FU) was injected intravenously. Five minutes later, the mice were killed, and the brains were removed for quantification of M5. The data are expressed as CFU per gram of brain. *P鈥?lt;鈥?.05, **P鈥?lt;鈥?.01 or ***P鈥?lt;鈥?.001 indicates significantly different bacterial loads between the two infection groups.Full size image DiscussionMeningitis is the most common clinical syndrome of S. agalactiae infection. Bacterial penetration across the BBB and into the CNS is the first step in the development of meningitis26. Therefore, adequate BBB models need to be developed in order to characterize the properties of bacterial penetration into the CNS. The in vitro BBB model based on the culture of brain microvascular endothelial cells has been widely used to probe the potential role(s) of individual virulence determinants in the initial pathogenesis of CNS infection by S. agalactiae. For example, hBMEC has been used to determine the invasive roles of fibronectin binding protein A (SfbA)27, laminin-binding protein (Lmb)28 and the surface protein HvgA in GBS infection29. However, the in vitro model might not completely mimic the disease in animals or humans. It was reported that CovR-deficient GBS showed a decreased ability to invade the brain endothelium in vitro, but in vivo, this deletion mutant was more proficient in the induction of permeability and proinflammatory signaling pathways in the brain endothelium and in penetration of the BBB30. In contrast, a previous study on the major pilin subunit PilB reported that the pilB mutant was less virulent than its wild-type strain in the newborn mice model, whereas, in vitro, the mutant had the similar ability with the wild-type GBS in resistance to macrophage killing31. Therefore, the development of an in vivo model will be extremely helpful in the study of bacterial meningitis.The mouse has been widely used for investigating S. agalactiae virulence24,27,32. In recent years, some researchers have developed mouse models by using intravenous injection of exogenous tracers in mice and subsequent detection of extravasate molecules in the brain tissues to measure BBB permeability33,34. The tracers provide convenient morphological evidence for BBB opening; however, Saunders et al.35 reported that the exogenous tracers are not a satisfactory tool for studying blood-brain barrier dysfunction. For example, Evans blue, one of the most commonly used markers, has toxic properties and thus the dye detected in brain might be due to toxic effects on cerebral endothelial or ependymal cells. Also, Evans blue detected in brain is likely to be a mixture of dye bound to plasma proteins, dye bound to brain tissue and free dye. Therefore, it is unreliable to estimate the size of BBB impairment using Evans blue. In this study, we established a model to assess the degree of BBB opening using E. coli M5, which has strong 尾-galactosidase activity, as an indicator. The bacterial strain is capable of producing blue colonies when cultured on medium containing X-Gal, and cannot permeate an intact BBB. Any entry of this bacterium into and spread within the brain is indicative of a leaky BBB. Therefore, the extent of BBB integrity can be quantitatively assessed through the monitoring of E. coli M5 and the measurement of the bacterial number in brain tissue. The similar method has been reported in S. pneumonia by Tsao et al.26 Nevertheless, the parameters and criteria may be variable due to different bacterial species. In our study, this model was optimized for use in S. agalactiae, and demonstrated to be a powerful method for analyzing the BBB opening.Under physiological conditions, the BBB strictly regulates the entry of blood-borne substances into the brain. Brain inflammation can affect the permeability of the BBB directly via cytokine-mediated activation of metalloproteinases or tight junction disruption, or indirectly by promoting transmigration of leukocytes36. In S. agalactiae, hyaluronidase has been demonstrated to contribute to the bacterial invasion and the pathogenesis of meningitis in mice24. However, unlike the PilA and 尾-hemolysin/cytolysin which stimulate the release of pro-inflammatory cytokines18,19, hyaluronidase acts as an anti-inflammation factor instead. Our previous study demonstrated that compared to the wild-type S. agalactiae, the hyaluronidase- deficient mutant stimulated a significantly higher level of pro-inflammatory cytokines including IL-1尾, IL-6 and TNF-伪 in macrophages, whereas its mortality to zebrafish was lower24. Afterwards, a probable reason for this phenomenon was illustrated by Kolar et al.37. They revealed that GBS evades host immunity by degrading hyaluronan (HA) which is a component of extracellular matrix nearly in all tissues. HA is commonly cleaved into small fragments by tissue hyaluronidase in response to tissue injury. These small HA fragments are inflammatory factors that ligate to Toll-like receptor (TLRs) to elicit inflammatory response and repair the damaged tissue. However, bacterial hyaluronidase degrades pro-inflammatory HA fragments to the major end product disaccharides. HA disaccharides bind to TLR2/4 to block signaling elicited by host HA fragments and other TLR2/4 ligands, thus preventing GBS ligands from activating pro-inflammatory signaling cascades. Therefore we assume that hyaluronidase contributes to GBS meningitis by anti-inflammation and evasion of host immune. However, our recent study showed that intravenous injection of a purified hyaluronidase, HylB, induced acute lung and brain injury38. This led to us to speculate that HylB might play important role in BBB permeability. In order to evaluate this speculation, we first investigated the role of HylB in disrupting the BBB integrity using this model established in this study. Compared with the wild-type S. agalactiae, the inactivation of hylB resulted in decreased BBB opening throughout the infection. Although the presence of S. agalactiae in brain indicates the BBB opening, the use of E. coli M5 as an indicator excludes the possibility that the differential BBB integrity may be caused by different proliferation abilities in vivo between the wild-type and hylB mutant strains.To further determine whether HylB has a direct impact on BBB integrity, we intravenously injected the purified HylB protein into the mice. We found that the intravenous injection of HylB induced BBB opening in a dose-dependent manner. In the groups treated with 1鈥塵g/mL and 2鈥塵g/mL, the BBB was open 3鈥塰 post-infection, 3鈥塰 earlier than in the 0.5鈥塵g/mL group, and the number of E. coli M5 increased with the time of infection. In considering the dose of HylB protein, we tested a treatment of 3鈥塵g/mL HylB, but all the mice died 15鈥塰 post-infection. This finding indicated that HylB is one of the important virulence factors of S. agalactiae, which is in agreement with previous studies24,38. A similar role for hyaluronidase in inducing pneumococcal meningitis has also been reported by Zwijnenburg et al.39. The present investigation of HylB further demonstrates that using E. coli M5 as an indicator is an easy and reliable method for assessing BBB integrity and/or leakiness. In particular, the model could be more suitable to investigate the contribution of soluble bacterial virulence factors to BBB disruption.In this study, we used a piscine strain of S. agalactiae with an extremely high virulence to BALB/c mice. It is not clear why this piscine strain is so virulent and what genetic relationship exists between fish and human isolates. Our previous study has made a comparative genomic analysis among 15 S. agalactiae strains of different origins, and found that the Chinese piscine isolates GD201008-001 and ZQ0910 are phylogenetically distinct from the Latin American piscine isolates SA20-06 and STIR-CD-17, but are closely related to the human strain A90940. Additionally, a published study reported that a GBS isolate from a clinical case of human neonatal meningitis caused disease and death in Nile tilapia8. In this regard, it may be of interest to further investigate the pathogenic mechanisms of meningitis caused by different origins of S. agalactiae strains. This model established here could be a potentially useful tool for the investigation. Nevertheless, it will be imperative to demonstrate that the E. coli tracer works with other GBS and mouse strains that are widely used in the meningitis model.In summary, the present study developed a model that can quantify the degree of BBB opening caused by S. agalactiae, and used this model to demonstrate that hyaluronidase plays a direct role in BBB permeability.MethodsBacterial strains and growth conditions S. agalactiae strain GD201008-001, 尾-hemolysin/cytolysin positive, which belongs to serotype Ia, MLST type ST-7, was isolated from farmed tilapia with meningoencephalitis in Guangdong Province, China, in 201040. Its genome sequence has been deposited in the GenBank database under accession number CP003810. The S. agalactiae hylB deleted mutant strain 螖hylB and the complemented strain C螖hylB were constructed in the previous study24. All of the bacterial strains were grown using either Todd-Hewitt broth (THB) or agar (THA) (Becton Dickinson, MD, USA) or sheep blood agar plates at 37鈥壜癈.Screening for a 尾- galactosidase-positive E. coli strainTo describe the time and degree of BBB opening, an indicator bacterial strain was needed. Inspired by blue-white selection, five strains of engineered E. coli (numbered from M1 to M5) were grown overnight in the dark at 37鈥壜癈 in M63 basic medium (13.6% (w/v) KH2PO4, 0.4% (w/v) KOH, 0.2% (w/v) (NH4)2SO4, 0.1鈥塵M FeSO4) containing 1鈥塵M MgSO4, 0.2% (w/v) lactose, 0.002% (w/v) 5-bromo-4-chloro-3-indolyl-L-D- galactopyranoside (X-gal) and 0.002% (w/v) vitamin B1. The bacterial strains were screened for their ability to form blue colonies on M63 plates for use as an indicator in the study.Determination of E. coli M5 virulence in miceBALB/c mice (24鈥?6鈥塯, aged 5鈥? weeks) were bought from the Experimental Animal Center, Yangzhou University. The mice were divided into two groups with 10 mice for each group. The screened E. coli M5 were grown overnight in LB broth. A bacterial suspension of 50鈥壜礚 was transferred into 5鈥塵L LB and incubated at 37鈥壜癈 to allow the cells to reach mid-log phase growth. When the bacteria reached an OD600 of 0.6, they were harvested by centrifugation at 5000鈥壝椻€?i>g for 5鈥塵in. The cell pellets were washed twice with sterile phosphate-buffered saline (PBS) (pH 7.4) and re-suspended in PBS to a concentration of 2鈥壝椻€?010鈥塁FU/mL. One group of mice was injected intravenously with 100鈥壩糒 of bacterial suspension, whereas the other was injected with 100鈥壩糒 PBS and served as a control. The mice were observed until one week post infection.Kinetics of E. coli presence in blood of miceAs an indicator, E. coli M5 should be eliminated rapidly from the circulatory system. Based on five predetermined time points, BALB/c mice (24鈥?6鈥塯, aged 5鈥? weeks) were divided into five groups with eight mice for each group. Mid-log phase bacteria were washed twice with PBS, followed by re-suspension in PBS and adjustment of the concentration to 2鈥壝椻€?09鈥塁FU/mL. For each time point, five mice were used as the experimental group and were injected intravenously with 100鈥壩糒 of bacterial suspension, while another three were injected intravenously with 100鈥壩糒 of PBS. Blood samples and brains were obtained aseptically at 3鈥塵in, 5鈥塵in, 10鈥塵in, 30鈥塵in and 60鈥塵in post infection. Blood samples of 100鈥壩糒 were spread onto M63 plates. To avoid surface contamination, the organs were washed twice with PBS. Tissues were placed in 1鈥塵L of PBS and homogenized with a biological sample homogenizer (BioPrep-24, Ningbo Hinotek Instrument Co Ltd, China). Then, 100鈥壩糒 of homogenate that was either undiluted or diluted 10鈭?, 10鈭? and 10鈭? in PBS were plated on M63 plates. The M63 plates were incubated overnight at 37鈥壜癈. Colonies were counted and given as CFU/g for brain samples or CFU/mL for blood samples.Determination of the challenge concentration of S. agalactiae Our previous study has shown that the bacterial strain GD201008鈥?01 is highly virulent to BALB/c mice by intraperitoneal administration, with LD50 values of less than 10鈥塁FU25. Here, we chose two different doses of S. agalactiae, 50 and 100鈥塁FU (5- and 10-fold greater than the LD50), to find an applicable dose for this mouse model. BALB/c mice were divided into two groups with 16 mice for each group. One group received an intraperitoneal injection of 100鈥壩糒 of 500鈥塁FU/mL bacterial suspension, and the other received an injection of 1000鈥塁FU/mL. In each group, four mice were sacrificed every three hours to aseptically collect the blood and brain. Homogenized brain tissues and blood were plated onto THB plates for bacterial cell counting to determine tissue colonization. The experiments were repeated at least three times to ensure reproducibility. The data are expressed as CFU/g or CFU/mL per mouse.Evaluation of BBB openingBALB/c mice were divided into five groups with five mice for each group. The mice were infected with a predetermined dose of 100鈥塁FU of the strain GD201008-001. Control mice were injected with sterile PBS. Then, 2鈥壝椻€?08鈥塁FU of the indicator E. coli M5 in 100鈥壩糒 PBS was given intravenously at the specified time points (3鈥塰, 6鈥塰, 9鈥塰 and 12鈥塰), and 5鈥塵in later, the mice were sacrificed. To detect the degree of BBB opening, the brains were removed aseptically and homogenized in PBS. The homogenate was serially diluted and spread onto THB or M63 agar plates, then incubated overnight at 37鈥壜癈. The organ CFU enumeration of S. agalactiae and E. coli M5 were determined and expressed as the mean and S.D. per mouse.Detection of BBB opening caused by S. agalactiae hyaluronidaseTo investigate the effect of BBB opening caused by S. agalactiae hyaluronidase, we used the deficient mutant strain 螖hylB and the complemented strain C螖hylB constructed in our previous study24. One hundred and twenty mice were divided into three groups with 40 mice for each group. Mid-log phase S. agalactiae and its derivatives were washed twice in PBS and re-suspended in PBS to 1鈥壝椻€?03鈥塁FU/mL. The concentration of E. coli M5 was adjusted to 2鈥壝椻€?09鈥塁FU/mL. Three groups of mice were infected with 100鈥壜礚 of the wild-type S. agalactiae, 螖hylB or C螖hylB by intraperitoneal injection. At 3鈥塰, 6鈥塰, 9鈥塰, 12鈥塰, and 15鈥塰 post infection, groups of four mice were killed, and the blood and brain tissues were collected for S. agalactiae quantification. Meanwhile, another four mice from each group were respectively inoculated with 100鈥壜礚 of E. coli M5 by intravenous route at each time point as mentioned above. At 5鈥塵in post-inoculation with E. coli, the brains were aseptically removed and homogenized in PBS. The homogenates were serially diluted, spread onto THB plates for S. agalactiae counting or M63 plates for E. coli M5 counting and incubated overnight at 37鈥壜癈. The bacteria were counted and reported as CFU/g per mouse.To further determine the role of the hylB gene in the pathogenesis of BBB opening caused by S. agalactiae, we expressed HylB with good enzymatic activity, as described in our previous study38. Sixty mice were divided into three groups with 20 mice in each group. The three groups of the mice were intravenously injected with 200鈥壩糒 of HylB protein at a final concentration of 0.5鈥塵g/mL, 1.0鈥塵g/mL and 2.0鈥塵g/mL, respectively. Another 20 control mice were injected with 200鈥壩糒 of sterile PBS. Then, 100鈥壩糒 of the E. coli M5 (2鈥壝椻€?09鈥塁FU/mL) was inoculated intravenously at intervals of 3鈥塰, 6鈥塰, 9鈥塰, 12鈥塰 and 15鈥塰 post infection, and the brain tissues were sampled at 5鈥塵in post-infection with E. coli. The homogenates were serially diluted, spread onto M63 plates and incubated overnight at 37鈥壜癈 for E. coli M5 counting. The bacteria were counted and expressed as CFU/g per mouse.Statistical analysisData were collected and analyzed using MS Excel 2010 and SPSS Statistics version 20.0 software. Multiple comparisons were performed by analysis of variance (ANOVA) followed by Turkey鈥檚 multiple-comparison test, with P鈥?lt;鈥?.05 indicating a statistically significant difference and P鈥?lt;鈥?.01 indicating a highly significant difference. 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BMC Genomics 14, 775鈥?84 (2013).CAS聽 Article聽 PubMed聽 PubMed Central聽Google Scholar聽 Download referencesAcknowledgementsThis work was supported by the Natural Science Foundation of Jiangsu Province (BK20171376), Qing Lan Project of Jiangsu Province and the Priority Academic Program Development of Jiangsu Higher Educational Institutions (PAPD).Author informationAffiliationsCollege of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, ChinaSu Luo,聽Qing Cao,聽Ke Ma,聽Zhaofei Wang,聽Guangjin Liu,聽Chengping Lu聽 聽Yongjie LiuAuthorsSu LuoView author publicationsYou can also search for this author in PubMed聽Google ScholarQing CaoView author publicationsYou can also search for this author in PubMed聽Google ScholarKe MaView author publicationsYou can also search for this author in PubMed聽Google ScholarZhaofei WangView author publicationsYou can also search for this author in PubMed聽Google ScholarGuangjin LiuView author publicationsYou can also search for this author in PubMed聽Google ScholarChengping LuView author publicationsYou can also search for this author in PubMed聽Google ScholarYongjie LiuView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsY.J.L. and S.L. conceived the study and drafted the paper; S.L., Q.C., K.M., Z.F.W. and G.J.L. performed the experiments; C.P.L. provided valuable suggestions of the manuscript.Corresponding authorCorrespondence to Yongjie Liu.Ethics declarations Competing Interests The authors declare that they have no competing interests. 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To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and PermissionsAbout this articleCite this articleLuo, S., Cao, Q., Ma, K. et al. Quantitative assessment of the blood-brain barrier opening caused by Streptococcus agalactiae hyaluronidase in a BALB/c mouse model. Sci Rep 7, 13529 (2017). https://doi.org/10.1038/s41598-017-13234-1Download citationReceived: 26 April 2017Accepted: 20 September 2017Published: 19 October 2017DOI: https://doi.org/10.1038/s41598-017-13234-1 CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.