In vitro and in vivo activities of piperacillin-tazobactam and meropenem at different inoculum sizes of ESBL-producing Klebsiella pneumoniae

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1 ORIGINAL ARTICLE BACTERIOLOGY In vitro and in vivo activities of piperacillin-tazobactam and meropenem at different inoculum sizes of ESBL-producing Klebsiella pneumoniae Y. Harada 1,2, Y. Morinaga 1,2, N. Kaku 1,2, S. Nakamura 2, N. Uno 1, H. Hasegawa 1, K. Izumikawa 2, S. Kohno 2,3 and K. Yanagihara 1,2 1) Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 2) Second Department of Internal Medicine, Nagasaki University Graduate School of Biomedical Sciences and 3) Global COE Program, Nagasaki University, Nagasaki, Japan Abstract The inoculum effect is a laboratory phenomenon in which the minimal inhibitory concentration (MIC) of an antibiotic is increased when a large number of organisms are exposed. Due to the emergence of extended-spectrum b-lactamase-producing Klebsiella pneumoniae (ESBL-Kpn) infections, the inoculum effect of ESBL-Kpn on b-lactams was studied in vitro and in vivo using an experimental model of pneumonia. The in vitro inoculum effect of 45 clinical ESBL-Kpn isolates on b-lactams was evaluated at standard (10 5 CFU/mL) and high (10 7 CFU/mL) organism concentrations. The MIC 50 of piperacillin-tazobactam, cefotaxime and cefepime was increased eight-fold or more and that of meropenem was increased two-fold. The in vivo inoculum effect was evaluated in an ESBL-Kpn pneumonia mouse model treated with bacteriostatic effect-adjusted doses of piperacillin-tazobactam (1000 mg/kg four times daily, %T > MIC; 32.60%) or meropenem (100 mg/kg twice daily, %T > MIC; 28.65%) at low/standard (10 4 CFU/mouse) and high (10 6 CFU/mouse) inocula. In mice administered a low inoculum, no mice died after treatment with piperacillin-tazobactam or meropenem, whereas all the control mice died. In contrast, in the high inoculum model, all mice in the piperacillin-tazobactam-treated group died, whereas all meropenem-treated mice survived and had a decreased bacterial load in the lungs and no invasion into the blood. In conclusion, meropenem was more resistant to the inoculum effect of ESBL-Kpn than piperacillin-tazobactam both in vitro and in vivo. In the management of severe pneumonia caused by ESBL-Kpn, carbapenems may be the drugs of choice to achieve a successful outcome. Keywords: Extended-spectrum b-lactamase, inoculum effect, Klebsiella pneumoniae, meropenem, piperacillin-tazobactam, pneumonia Original Submission: 6 February 2014; Revised Submission: 7 May 2014; Accepted: 7 May 2014 Editor: J.-M. Rolain Article published online: 11 May 2014 Clin Microbiol Infect 2014; 20: O831 O / Corresponding author: Y. Morinaga, Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto, Nagasaki , Japan y-morina@nagasaki-u.ac.jp Introduction The emergence of extended-spectrum b-lactamase (ESBL)-producing organisms, particularly Escherichia coli and Klebsiella pneumoniae, has been a major clinical concern. Because broad-spectrum penicillins and cephalosporins are often not effective against ESBL-producers, selecting the appropriate therapy for treatment of serious infections due to ESBL producers is a challenge. In fact, the mortality rate is reportedly higher when the causative bacteria produce ESBL or when the patients received inadequate therapy against ESBL producers [1 3]. Klebsiella pneumoniae pneumonia is often characterized by a rapid clinical course, leading to widespread systemic effects and death [3 5]. Because K. pneumoniae infections are commonly hospital or healthcare associated and primarily occur in patients with impaired host defences due to diabetes mellitus or alcoholism [4,5], these infections sometimes becomes severe. The recommended inoculum size for antimicrobial susceptibility testing based on the broth dilution method is c CFU/mL [6]. However, the in vitro effect of antibiotics is known to depend on the number of bacteria exposed [7,8]. The inoculum effect is a laboratory phenomenon in which the minimal inhibitory concentration (MIC) of an antibiotic is Clinical Microbiology and Infection ª2014 European Society of Clinical Microbiology and Infectious Diseases

2 O832 Clinical Microbiology and Infection, Volume 20 Number 11, November 2014 CMI significantly increased when the number of organisms in the inoculum is increased [9]. The inoculum effect has been observed for b-lactams against b-lactamase-producing bacteria [10,11]. However, the clinical significance of this laboratory phenomenon has yet to be elucidated. Therefore, strategies for successful treatment of pulmonary infections as well as bacteraemia due to ESBL-producing K. pneumoniae should be investigated [12]. In clinical settings, piperacillin-tazobactam and carbapenems have been widely used to treat patients with infections caused by ESBL producers. However, the therapeutic efficacy of piperacillin-tazobactam for patients with infections due to ESBL producers is controversial [13,14], whereas carbapenems produce favourable outcomes [1]. The in vitro activity of piperacillin-tazobactam has been called into question because of the substantial inoculum effect observed in dilution susceptibility tests using ESBL producers [15 17]. Although many studies about the in vitro inoculum effect on b-lactams have been reported [16,18,19], in vivo reports are limited. In a blood stream infection model with ESBL-producing E. coli, the high inoculum group showed piperacillin-tazobactam treatment failure [20]. In contrast, in a thigh infection model, carbapenems showed potent activity against ESBL-producing E. coli and K. pneumoniae [21]. To determine the efficacy of b-lactams in pulmonary infections caused by ESBL-producing K. pneumoniae, an in vivo study is required. In this study, the in vitro activity of b-lactams against ESBL-K. pneumoniae clinical isolates was evaluated at the standard and high inocula. In addition, the in vivo efficacies of meropenem and piperacillin-tazobactam were studied in a mouse model of pulmonary infection with different inoculum sizes of ESBL-producing K. pneumoniae. concentrations of bacteria were inoculated, the low/standard inoculum ( CFU/mL) and high inoculum ( CFU/mL). Genotyping of ESBL Plasmids were extracted from the bacteria by heat lysis as described previously [22]. For detecting ESBL genes, TEM, SHV and three CTX-M groups (CTX-M-1, CTX-M-2 and CTX-M-9) were targeted and each gene was amplified by polymerase chain reaction (PCR) [22]. The PCR products were analysed using 2% agarose gel electrophoresis and visualized by staining with ethidium bromide. Time-kill assays The bacteria were inoculated into 100 ll of Mueller-Hinton broth in a 96-well plate at the standard ( CFU/mL) and high ( CFU/mL) concentrations. Piperacillin-tazobactam or meropenem were added at concentrations of 19 and 169 MIC (for each strain) or 19, 49 and 169 MIC (for the experimental strain). After incubation with antibiotics at 37 C for 24 and 48 h, the bacteria were serially diluted and plated on Mueller-Hinton II broth agar plates. The visible colonies on the plates were counted. Animals Male, 6- to 8-week-old BALB/c mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). All animals were housed in a pathogen-free environment in the Laboratory Animal Center for Biomedical Science at Nagasaki University and were provided sterile food and water. The Ethics Review Committee for Animal Experimentation approved all experimental protocols used in this study. Materials and Methods Collection of clinical isolates ESBL-producing K. pneumoniae isolates were collected from patients at Nagasaki University Hospital during the period from January 2004 to December When several isolates were detected in a sample from the same patient, only one isolate was selected. A total of 45 isolates were analysed in this study. Antibiotic susceptibility testing Antibiotic susceptibility was determined using the broth microdilution method for penicillin, piperacillin-tazobactam, ceftazidime, cefotaxime, cefepime, cefpodoxime and meropenem according to the procedures outlined by Clinical and Laboratory Standards Institute (CLSI) [6]. Two different Pharmacokinetic studies Mice were treated with piperacillin (100 mg/kg) in combination with tazobactam (12.5 mg/kg) or with meropenem (100 mg/kg) in combination with cilastatin (100 mg/kg), a DHP-1 inhibitor. The plasma was separated by centrifugation and the isolated plasma of the meropenem-treated group was mixed with one-half volume of (N-morpholino)-propanesulfonic acid (MOPS) buffer (ph 5.5) for the purpose of stabilization of meropenem. The level of biologically active b-lactams in plasma was determined by the bioassay method with Micrococcus luteus ATCC9341 for piperacillin or with E. coli NIHJ for meropenem as the indicator organism. Standard curves were made for the antibiotics in pooled mouse plasma. The disk diffusion bioassay was performed in triplicate with 50 ll of plasma from b-lactam-administered mice by using 8-mm-diameter paper disks (cat. no ; Advantec, Tokyo, Japan). The

3 CMI Harada et al. Innoculum effects on ESBL-K. pneumoniae O833 lower limits of detection of piperacillin and meropenem were 0.5 and 0.06 lg/ml, respectively. The intra- and interday variations of both drugs were <15%. The pharmacokinetic parameters were calculated using a two-compartment model with the MULTI program [23]. The percentage of time that free-drug concentrations above the MIC was calculated using pharmacokinetic parameters, murine protein binding (10%) [24,25] and MICs. Preparation of bacteria and the mouse model of pulmonary infection Klebsiella pneumoniae, KEN-11 strain (positive for SHV and CTX-M-9 type ESBLs and positive for the string test [25,26]), was used. The MICs of piperacillin-tazobactam and meropenem were 1.0 and lg/ml, respectively. When evaluated at the high inoculum, the MICs of piperacillin-tazobactam and meropenem were 256 and 0.12 lg/ml, respectively. The bacteria were stored at 80 C in a Microbank system (Pro-Lab Diagnostics, Ontario, Canada) until use. To prepare inocula, a single colony of KEN-11 was preincubated in Luria-Bertani (LB) broth [26] containing 100 lg/ml penicillin at 37 C for 18 h with shaking at 250 rpm. After 8 h of additional incubation in fresh LB broth, the bacteria were adjusted to appropriate concentrations by turbidimetry. Mice were intratracheally infected with bacterial suspension (50 ll/mouse) under pentobarbital anaesthesia (40 mg/kg delivered by intraperitoneal injection). buffered formalin, and the paraffin-embedded sections were stained with hematoxylin-eosin. Analysis of bronchoalveolar lavage fluid After the pulmonary vasculature was flushed with 3 ml of normal saline via the right ventricle, lungs were lavaged three times with 1 ml of normal saline as described previously [28]. Statistical analysis Survival data are presented as Kaplan Meier curves. The data are expressed as the mean standard errors of the mean (SEM). Differences between the groups were analysed using Tukey s post-test followed by the Kruskal Wallis non-parametric test. A p value of <0.05 was considered statistically significant. Results In vitro inoculum effects of ESBL-producing Klebsiella pneumoniae clinical isolates on b-lactams We tested the susceptibilities of the clinical strains to seven b-lactams at low/standard and high inocula. A summary of ESBL genotypes is shown in Table 1. MIC shifting was observed for all agents and a greater than eight-fold increase in MIC 50 was observed for tazobactam-piperacillin, cefotaxime and cefepime (Table 2). Treatment protocol Meropenem and piperacillin-tazobactam were used for the in vivo study. Meropenem was used in combination with the same dose of cilastatin because carbapenems are hydrolyzed by murine DHP-1 [27]. The in vivo efficacy of the pharmacokinetically-adjusted treatment protocol was evaluated in the low ( CFU/mouse) and high ( CFU/mouse) inocula models. Treatment commenced 12 h after inoculation. Mice were treated four times a day with the same volume of the agents or saline. The mice were observed for up to 5 days after inoculation. Bacteriological and histopathological examination Mice were sacrificed at 36 and 84 h after inoculation. Blood was collected by right ventricular puncture using heparin-coated syringes. Aseptically-dissected lungs were homogenized using a homogenizer (AS One Co., Osaka, Japan). After culture of serially-diluted lung specimen on the Mueller-Hinton II agar, the visible colonies were counted. For the histopathological examination, the lung specimen was fixed in 10% Time-kill study of ESBL-Klebsiella pneumoniae clinical isolates Time-kill studies were performed at different drug concentrations. Of the 45 clinical strains, 21 were within the measurement range and were verifiable. At the standard inoculum, 19 MIC piperacillin-tazobactam did not decrease the number of viable bacteria, whereas the other conditions showed 3 log decreases after 24 h (169 MIC piperacillin-tazobactam, log; 19 MIC meropenem, log; 169 MIC meropenem, TABLE 1. Genotypes of ESBL-producing Klebsiella pneumoniae Genotype(s) TEM 1 SHV 16 CTX-M-9 1 TEM + SHV 6 TEM + CTX-M-9 2 SHV + CTX-M-1 1 SHV + CTX-M-2 4 SHV + CTX-M-9 4 TEM + SHV + CTX-M-1 4 TEM + SHV + CTX-M-9 3 Not detected 3 Total 45 Number of the isolates

4 O834 Clinical Microbiology and Infection, Volume 20 Number 11, November 2014 CMI TABLE 2. MICs of various antimicrobial agents against ESBL-producing Klebsiella pneumoniae clinical isolates at different inoculum sizes MIC 50 MIC 90 Antimicrobial agent Evaluated range SI (lg/ml) HI (lg/ml) MIC shift (HI/SI) SI (lg/ml) HI (lg/ml) MIC shift (HI/SI) Piperacillin <64 to > >1024 >2 >1024 >1024 Piperacillin-tazobactam <1 to> >1024 >1024 Ceftazidime <2 to> Cefotaxime <2 to> >512 >4 Cefpodoxime <2 to> >512 >2 Cefepime <1 to> >1024 >32 Meropenem <0.015 to > A summary of 45 clinical isolates. SI, standard inoculum ( CFU/mL); HI, high inoculum ( CFU/mL) log) (Fig. 1a). Similarly, at the high inoculum, verified conditions except for 19 MIC piperacillin-tazobactam decreased the number of viable bacteria (Fig. 1b). The sizes of the decreases after treatment with 169 MIC piperacillin-tazobactam and 19 and 169 MIC meropenem were , and log, respectively. Selection of an ESBL-producing Klebsiella pneumoniae strain for the pneumonia model The clinical isolates were prescreened for an in vivo experiment. Of the seven randomly selected isolates, only the KEN-11 strain was pneumotropic. KEN-11 was able to adapt to the mouse lung and consistently caused pneumonia. In vitro, a standard inoculum of KEN-11 was suppressed by antibiotics, except for 19 MIC piperacillin-tazobactam (Fig. 2a), but a high inoculum of KEN-11 was suppressed only by meropenem in a dose-dependent manner (Fig. 2b). The percentage of time above MIC (%T > MIC) that is required for bacteriostatic effects is 30 35% for penicillins [29] and 25 40% for carbapenems [30]. On the basis of pharmacokinetic analysis (Fig. 3), the dose of each agent was designed to exceed the bacteriostatic %T > MIC for the pathogen (piperacillin-tazobactam, 1000 mg/kg four times daily; meropenem, 100 mg/kg twice daily; Table 3). In vivo study of different inoculum sizes In the low inoculum model, all control mice died by 120 h, whereas all mice treated with antibiotics survived during the entire experimental period (n = 7, p <0.01 vs. the control, Fig. 4a). Next, the bacterial load in the lungs (Fig. 4b) and blood (Fig. 4c) was examined in the early (36 h) and late (84 h) phase of infection. Compared with the control group, the number of bacteria in mice of the piperacillin-tazobactam and meropenem-treated group was decreased. Bacteria were observed also in the blood of mice in the control group, whereas no bacteria were observed in the blood of mice in the other groups, except for two mice in the piperacillin-tazobactam group in the early phase. In the high inoculum model, all mice in the control and piperacillin-tazobactam-treated groups died, whereas all mice in the meropenem-treated group survived for the entire experimental period (p <0.01, vs. the control and piperacil- (a) (b) FIG. 1. Time-kill curves of ESBL-producing Klebsiella pneumoniae clinical isolates. Time-kill studies at the standard (a) and high (b) inocula were performed with different concentrations of piperacillin-tazobactam (filled circles) and meropenem (open circles) (n = 21). The isolates for which the MIC was out of measurement range were excluded. The log changes in the number of viable bacteria from the start of the experiment are shown. The detection limit of the colony count is 100 CFU/mL. When the number of the colony was less than the detection limit, the log change was considered as the number at the start of the experiment. The solid and dashed lines represent 19 MIC and 169 MIC, respectively. The error bars represent the standard error of the mean.

5 CMI Harada et al. Innoculum effects on ESBL-K. pneumoniae O835 (a) (b) FIG. 2. Time-kill curves for KEN-11. Time-kill studies for KEN-11 at the standard (a) and high (b) inocula were performed with different concentrations of piperacillin-tazobactam (filled circles) and meropenem (open circles). Solid, dashed and dotted lines represent 19, 49 and 169 MIC, respectively. TZP MEM FIG. 3. In vivo pharmacokinetic analysis of piperacillin and meropenem. Mouse serum concentrations of piperacillin (filled circles) and meropenem (open circles) were measured after intraperitoneal administration. Meropenem was administrated with the DHP-I inhibitor cilastatin. Error bars represent the standard error of the mean. TZP, piperacillin-tazobactam; MEM, meropenem. lin-tazobactam-treated groups) (n = 7, Fig. 4d). In the lungs (Fig. 4e), a significant decrease in the number of bacteria was observed in the early phase in the piperacillin-tazobactam-treated and meropenem-treated groups, compared with the control group. In the late phase, the surviving mice in the piperacillin-tazobactam-treated group had a high bacterial load in their lungs, compared with the meropenem group. In the blood, no bacteria were observed in the meropenem-treated mice (Fig. 4f). Evaluation of the in vivo inflammatory findings in mice with different inoculum sizes The severity of lung inflammation was evaluated by histopathological examination and cytological analysis of bronchoalveolar lavage fluid (BALF). In the low inoculum model, an influx of numerous inflammatory cells was observed in the alveoli of the control group, whereas fewer cells were observed in other groups (Fig. 5a). The number of neutrophils in BALF was significantly lower in the antibiotic-treated groups than in the control group (Fig. 5b). In the high inoculum model, piperacillin-tazobactam and meropenem suppressed the number of cells in the alveolar spaces in the early phase, and the levels appeared to be similar in the late phase (Fig. 5c). Treatment with piperacillin-tazobactam or meropenem significantly decreased the number of neutrophils in BALF compared with the number in the control group in the early phase (Fig. 5d). In the late phase, the number of neutrophils in the piperacillin-treated group increased slightly from that in the early phase. Discussion Infections caused by ESBL-producing K. pneumoniae have become a growing concern. Although their inoculum effect on b-lactams as an in vitro phenomenon is widely known, the potential utility of piperacillin-tazobactam and meropenem for TABLE 3. Pharmacokinetic parameters of piperacillin-tazobactam and meropenem and predicted values for in vivo treatment settings Selected pharmacokinetic parameters In vivo treatment settings Predicted 24 h%t > MIC (%) Antimicrobial agent AUC 0 (lgmin/ml) T 1/2 (min) CL (ml/min/kg) Vd (L/kg) MIC against KEN-11 (lg/ml) Dose setting (mg/kg) Predicted T > MIC (h) Once/day Twice/day Four times/day Piperacillin Meropenem AUC, area under the curve; T 1/2, elimination half-life; CL, clearance; Vd, volume of distribution.

6 O836 Clinical Microbiology and Infection, Volume 20 Number 11, November 2014 CMI (a) (b) (c) (d) (e) (f) FIG. 4. Survival and bacteriological examinations from the in vivo study. The inoculum effects were studied in vivo using a mouse model of pneumonia induced by ESBL-producing Klebsiella pneumoniae at the standard (a, b and c) and high (d, e and f) inocula. Mice were treated with saline (control, filled circles), piperacillin-tazobactam (open circles) or meropenem (gray circles) according to the pharmacokinetically-designed treatment protocol. (a and d) Kaplan Meier survival curves; *p <0.05 vs. the control, **p <0.01 vs. the control, and p <0.01 vs. piperacillin-tazobactam. Microbiological studies in the lungs (b and e) and the blood (c and f); *p <0.05 and **p <0.01. TZP, piperacillin-tazobactam; MEM, meropenem; N/A, not available. the treatment of severe pneumonia due to ESBL-producing K. pneumoniae is not well understood. In vitro study showed that piperacillin-tazobactam, cefotaxime and cefepime have the potential to be easily affected by the inoculum size of ESBL-producing K. pneumoniae. These findings were consistent with previous reports [16,31]. Because the MIC of cefepime tends to be increased substantially by the increased number of b-lactamase-producing bacteria [32,33], it can be ineffective for patients with bacteraemia caused by ESBL producers [34]. Although meropenem also showed a mild MIC shift, the shifted MIC of meropenem is still below the tissue concentration. Therefore, the in vitro results suggest that the antimicrobial activity of meropenem is stable at infection sites with high bacterial concentrations. The activities of b-lactams against ESBL producers can vary according to ESBL subgroups, such as TEM-, SHV- and CTX-M-type, and these subgroup ESBLs have different susceptibilities to piperacillin-tazobactam [35 37]. Our results were sorted by ESBL subgroup [22]; however, no clear differences were observed between the subgroups (data not shown). To evaluate this point, experiments using a greater number of isolates may be required. In the time-killing study, meropenem showed potent activity against the tested isolates but the activity of piperacillin-tazobactam seemed to be weak. This bactericidal activity of piperacillin-tazobactam was consistent with a previous report [38]. The inoculum effect on piperacillin-tazobactam has been shown to be associated with the release of large amounts of hydrolyzing b-lactamases from lysed bacteria [13]. In vivo, the efficacy of piperacillin-tazobactam was more sensitive to inoculum size than meropenem. Notably, our results suggest that the inoculum effect can have an influence on the outcome even if the causative strain shows very low MIC for piperacillin-tazobactam. The in vivo efficacies of antibiotics for different inoculum sizes have been reported for some animal models, such a pneutropenic mouse model of thigh infection [21] and a sepsis model [20]. The results in these previous reports and our results imply that carbapenems may be stable in vivo and have potent activities in heavy bacterial loads at the infection site. However, our results do not recommend the use of carbapenems against all infections caused by ESBL-producing K. pneumoniae, because carbapenem exposure may promote the selection and colonization of carbapenem-resistant variants such as Pseudomonas aeruginosa

7 CMI Harada et al. Innoculum effects on ESBL-K. pneumoniae O837 (a) (b) (c) (d) FIG. 5. Histopathological and BALF examinations of mice from the in vivo study. Haematoxylin-eosin staining of lung specimens (a and c) and the number of neutrophils in BALF (b and d) are shown. Experiments were performed using mice administered the standard (a and b) and high (c and d) inocula. Pathological data are shown for three representative mice in each group. Error bars represent the standard error of the mean. *p <0.05; TZP, piperacillin-tazobactam; MEM, meropenem; N/A, not available. and Acinetobacter baumannii [39,40]. In the case of ESBL-producing K. pneumoniae infections that are severe or have responded poorly to piperacillin-tazobactam, carbapenems can be considered as the drug of first choice. To reduce the risk of the increase of carbapenem-resistant strains, new combinations of penicillins and novel b-lactamase inhibitors that are insusceptible to the inoculum effect should be evaluated in our model. This study has some limitations. Our in vivo study was performed with only one strain because the number of K. pneumoniae isolates carrying ESBL and having affinity for the mouse lung were limited. From a pharmacokinetic point of view, it is not clear whether a dose schedule based on human pharmacokinetics is effective in mice. In addition, the plasma pharmacokinetics of antimicrobial agents in critically ill patients can differ substantially from that in clinically stable patients [41]. Therefore, clinical studies are needed to address these issues. In agreement with the in vitro data, the in vivo efficacy of meropenem for treating pneumonia due to ESBL-producing K. pneumoniae was not affected by inoculum size and was superior to that of piperacillin-tazobactam. In the management of severe pneumonia caused by ESBL-producing K. pneumoniae, carbapenems can be the first-choice drug for a successful cure. Acknowledgements We would like to thank K. Takemoto (Dainippon Sumitomo Pharma Co. Ltd., Osaka, Japan) for conducting the pharmacokinetic analyses. This research was supported by: a grant from Dainippon Sumitomo Pharma Co. Ltd.; a grant from Taisho Toyama Pharmaceutical; a Grant-in-Aid for Scientific Research (no to K. Y.) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology; a Grant-in-Aid for Young Scientists (B) (No and to Y. M.) from the Japan Society for the Promotion of Science; and a grant from the Global Centers of Excellence Program, Nagasaki University.

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