Cefazolin Plus Minocycline Against a Clinical Isolate of Vibrio Vulnificus: in Vitro and Animal Studies

Jpn. J. Infect. Dis., 63, 16-18, 2010

Original Article Cefazolin Plus Minocycline against a Clinical Isolate of Vibrio vulnificus: In Vitro and Animal Studies Hung-Jen Tang1, Wen-Chien Ko3, Yin-Ching Chuang1,2, Chi-Chung Chen2, and Shyh-Ren Chiang1* 1Department of Medicine and 2Department of Medical Research, Chi Mei Medical Center, Tainan; and 3Department of Medicine, National Cheng Kung University Medical College, Tainan, Taiwan, Republic of China (Received July 13, 2009. Accepted November 2, 2009)

SUMMARY: Cefotaxime plus minocycline has been shown to have synergistic activity against Vibrio vulnificus; however, the clinical role of cefazolin in combination with minocycline in immunocompromised hosts has not been established. Therefore, antimicrobial susceptibility of the V. vulnificus clinical isolate Vv05191 was studied by the agar dilution method. Antibacterial activity of cefazolin, minocycline, and a combination of the two drugs was investigated by time-kill studies in vitro and further examined for therapeutic efficacy in a murine model. When cefazolin at a combination of 4 mg/L (1/2 × MIC) was combined with minocycline at a concentra- tion of 0.03 mg/L (1/2 × MIC), sustained inhibitory activity was noted until 24 h. In BALB/cByJ mice with cyclophosphamide-induced neutropenia, an inoculum of 1.5×108 CFU caused death within 96 h when the infected mice were treated by cefazolin (400 mg/kg every 3 h), while 6.3% of mice survived when treated by minocycline (4 mg/kg stat, then 2 mg/kg every 12 h). However, 62.5% of mice survived for 96 h when mice were treated by cefazolin (400 mg/kg every 3 h) plus minocycline (4 mg/kg stat, then 2 mg/kg every 12 h) (P = 0.002, log rank test). In conclusion, cefazolin in combination with minocycline exhibits in vitro synergistic antibacterial activity against V. vulnificus and provides a therapeutic advantage in neutropenic mice with V. vulnificus infection.

of both drugs were purchased from Sigma-Aldrich (St. Louis, INTRODUCTION Mo., USA). The drugs were incorporated into agars in a series Vibrio vulnificus is primarily associated with a severe, dis- of concentrations in two-fold dilution, ranging from 0.00375 tinctive soft tissue infection or septicemia, or both, especially to 256 mg/L. Minocycline was dissolved in 0.1 N NaOH solu- in immunocompromised patients with conditions such as liver tion, and cefazolin was dissolved in sterile water. Escherichia cirrhosis, adrenal insufficiency, malignancy, or diabetes (1– coli ATCC 25922 was used in each run as a control. 4). Most V. vulnificus isolates are in vitro susceptible to a Bacterial strains: A clinical isolate of V. vulnificus, Vv- variety of antibiotics, including newer fluoroquinolones (5– 05191, was randomly selected from a cirrhotic case with 10). Two previous in vitro and in vivo studies by Chuang et lethal V. vulnificus bacteremia and necrotizing fasciitis. The al. have demonstrated that combination therapy of cefotaxime organism was stored at –70°C in Protect Bacterial Preservers and minocycline was more advantageous than either anti- (Technical Service Consultants Ltd., Heywood, Lancashire, biotic alone for the treatment of severe experimental murine England) before use. V. vulnificus infections (11,12). Inhibitory activities of cefazolin, minocycline, and a In a previous animal study of V. vulnificus infection, combination of the two drugs in time-kill studies: Bacte- immunocompetent mice were experimentally treated by a rial concentrations were diluted to around 5.0 × 105 CFU/ third-generation cephalosporin combined with minocycline mL in 25 mL of fresh Mueller-Hinton broth. The drug con- (12). However, most lethal V. vulnificus infections occur in centrations of minocycline or cefazolin in the following time- immunocompromised patients. When reviewing a published kill studies were adjusted to the 1/2 or 1 × MIC of each clinical study (4), we found some successful experiences in drug as indicated. Each flask was incubated at 37°C. Bacte- treating V. vulnificus infections with cefazolin alone or in rial counts were measured at selected time intervals of 0, 2, combination with other drugs. Therefore, in the present study, 4, 6, 8, 12, 24, 30, 36, and 48 h by enumerating the colonies we examined the in vitro susceptibility of V. vulnificus to in 10-fold serially diluted specimens of 100-μl aliquots plated cefazolin and minocycline, to evaluate the efficacy of com- on nutrient agar (Difco Laboratories, Detroit, Mich., USA). bination therapy in a neutropenic mouse model. All experiments were carried out in duplicate. In vivo mouse study: Female, pathogen-free, inbred BALB/cByJ mice (Animal Center, National Science Coun- MATERIALS AND METHODS cil, Taipei, Taiwan), weighing on average 20 g (6–10 weeks Minimal inhibitory concentration (MIC): The MIC val- old), were used throughout this study. A bacterial suspension ues of minocycline and cefazolin were measured by the agar in a volume of 0.1 mL was delivered intraperitoneally into dilution method as previously described by National Commit- each mouse. Minocycline was administered intraperitoneally tee for Clinical Laboratory Standards (13). Standard powders at doses of 4 mg/kg stat and then 2 mg/kg every 12 h, and cefazolin was administered at doses of 400 mg/kg every 3 h, *Corresponding author: Mailing address: Department of Medical as previously described (14–16). Cefazolin was administered Research, Chi Mei Medical Center, No. 901 Chung-Hwa Road, at 10 times the human dose (40 mg/kg) every 3 h due to the Yung-Kang City, Tainan, Taiwan 710. Tel: +886-6-281-2811 ext. high body surface area and metabolic rate of mice and our in 52605, Fax: +886-6-251-7849, E-mail: [email protected] vivo pharmacokinetics data (data not shown). Intraperitoneal

16 injection of cyclophosphamide (Sigma) at daily doses of 100 of magnitude in comparison with either drug alone at 24 h. mg/kg for 3 days was used to induce neutropenia in mice Therapeutic efficacy of combination therapy in the (17). murine model: Two rounds of animal experiments were There were four experimental groups, including the control performed, and the initial inoculum was 4.5×108 and 1.5× group (no antimicrobial agent was given), and the cefazolin, 108 CFU, respectively. Neutropenia was induced at round 2. minocycline, and cefazolin-minocycline treatment groups. Neutrophil counts in three normal mice at 7–8 weeks old were There were 9 or 16 mice in each group, and the experiments 2,750/ul, 3,140/ul, and 2,980/ul, respectively, and in mice were performed twice, one in non-neutropenic mice and one receiving cyclophosphamide were 35/ul, 55/ul, and 20/ul, in neutropenic mice. In performing the animal experiments respectively (Table 1). Without antimicrobial therapy or with we complied with all relevant national guidelines of the Re- cefazolin therapy, no mouse survived at 5 days after intra- public of China and with the Chi-Mei Foundation Medical peritoneal inoculation of Vv05191. Of non-neutropenic mice Center Animal Use Policy. treated by minocycline (2 mg/kg every 12 h) in combination Statistical methods: Data analyses were performed with with cefazolin (400 mg/kg every 3 h), the survival rate was SPSS for Windows 10.0 (SPSS Inc., Chicago, Ill., USA). The similar to that of mice treated by minocycline (77.8% versus log-rank test was applied to compare the effect between dif- 66.7%, P = 0.73). However, the survival rate of neutropenic ferent groups. A P value of less than 0.05 was considered to mice treated by the combination regimen was significantly be statistically significant. higher than that of neutropenic mice treated by minocycline only (62.5% versus 6.3%, P = 0.002) (Table 1). RESULTS DISCUSSION Time-kill studies: The MIC of cefazolin and minocycline for Vv05191 was 8 and 0.06 mg/L, respectively. When The clinical course of septicemic patients with V. vulnificus Vv05191 at an inoculum of 5×105 CFU/mL was incubated is often rapid progression, and over 50% of such patients die with minocycline at the concentration of 1/2 × MIC (0.03 within 48 h of hospitalization (4). The previous study showed mg/L) or 1 × MIC (0.06 mg/L), the bacterial growth was that cefotaxime combined with minocycline was in vitro inhibited temporarily, but resumed at 12 or 24 h, respectively active against V. vulnificus and was effective in a mouse model (Figure 1). At the cefazolin concentration of 1 × MIC (8 mg/ (12). Clinical experiences also support the use of minocycline L), there was sustained bactericidal activity lasting for 48 h. plus cefotaxime for severe V. vulnificus infection (4). Due to With the combination of cefazolin and minocycline, both at the broad spectrum and higher cost of cefotaxime, it was our the concentration of 1/2 × MIC, the inhibitory activity lasted intent to determine whether cefazolin has similar inhibitory for 24 h and was synergistically bactericidal, defined as a activity against V. vulnificus. Moreover, since severe V. reduction of viable bacterial colonies by at least two orders vulnificus infection often occurrs in immunocompromised patients, it is important to investigate the therapeutic effect of cefazolin plus minocycline in neutropenic animals before its application in clinical practice. The in vitro antibacterial effects of cefazolin and cefotaxime against V. vulnificus, as reflected by the time-kill method, were greatly different. With cefotaxime at the concentration of 1 × MIC (0.03 mg/L) in an earlier report, the number of bacterial colonies returned to the initial inoculum within 8 h (11), but in the present study with cefazolin at 8 mg/L (1 × MIC) there was sustained inhibitory activity for at least 48 h. Such a result is probably related to the different bacterial strains and mouse species tested. Despite of the obvious dif- ference in in vitro antibacterial activity between cefotaxime and cefazolin, the therapeutic efficacy of both drugs for mice infected by a large inoculum of V. vulnificus was poor, i.e., all infected mice died at 96 h (12). In time-kill studies, some differences in antibacterial ef- fects between minocycline and cefazolin were noted. At the MIC, the inhibitory effect of minocycline persisted for about Fig. 1. Time-kill curves of a clinical isolate of Vibrio vulnificus, co- cultivated with minocycline (0.03 mg/L, 1/2 × MIC or 0.06 mg/L, 24 h, but the inhibitory effect of cefazolin alone persisted for 1 × MIC), cefazolin (4 mg/L, 1/2 × MIC or 8 mg/L, 1× MIC), or a more than 48 h. At the 1/2 × MIC of both drugs, synergism combination of the two drugs (minocycline 0.03 mg/L + cefazolin 4 was found and the inhibitory effect persisted for 24 h. In the mg/L). mouse model, the survival rate of the combination group

Table 1. Survival rates of BALB/c mice intraperitoneally infected with Vibrio vulnificus 05191 at 96 h after treatment with saline (control), cefazolin, minocycline, or a combination of the two drugs Initial inocula Cyclophosphamide- Surviving/Total mouse no. (%) (CFU) induced neutropenia Control Cefazolin Minocycline Cefazolin + minocycline

4.5 × 108 No 0/9 (0) 0/9 (0) 6/9 (66.7) 7/9 (77.8) 1.5 × 108 Yes 0/16 (0) 0/16 (0) 1/16 (6.3) 10/16 (62.5)1) 1): Cefazolin + minocycline group versus minocycline group. P = 0.002.

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