摘要用1.29×10⁷ CFUs肺炎杆菌滴鼻，引起小鼠呼吸道感染，未治疗组的动物大部分死亡。本文比较了左氧氟沙星、氧氟沙星和洛美沙星对小鼠呼吸道感染的治疗作用。结果发现感染20h后，40mg/kg的左氧氟沙星可使小鼠肺研磨液中的细菌数由每毫升10^9.29减少到10^5.84，其ED₅₀分别为氧氟沙星和洛美沙星的2.25和2.42倍，说明左氧氟沙星的疗效明显优于氧氟沙星和洛美沙星。

Experimental respiratory tract infection with Klebsiella pneumonia

in mice: chemotherapy with levofloxacin,

ofloxacin and lomefloxacin

Ma ZengshuanandLin Futian

(Institute of Medicinal Biotechnology, CAMS, PUMC；Beijing 100050)

ABSTRACTExperimental respiratory tract infection was induced in mice by intranasal instillation of 1.29×10⁷ CFUs Klebsiella pneumonia. Bacterial challenge directly resulted in deposition of 10^5.24 CFUs of the organisms in the lungs. The number of organisms in the lungs increased rapidly, and by 18h, a well-developed pneumonia was apparent. Most of the untreated mice died within two days after infection. The therapeutic effectiveness of levofloxacin, ofloxacin and lomefloxacin was compared with this established pneumonia model. For each compound, the CFUs decreased with the increase of dosage. The effectiveness of levofloxacin, however, was superior to those of the others. Administration of 40 mg of levofloxacin per kg can decrease the CFUs in the lungs from 10^9.29 to 10^5.84 at 20h after infection, and saved all infected animals. ED₅₀ of levofloxacin was 2.25 and 2.42 times that of ofloxacin and lomefloxacin respectively.

KEY WORDSExperimental respiratory tract infection；Klebsiella pneumonia；ED₅₀；Levofloxacin；Ofloxacin；Lomefloxacin

Introduction

Animal models of infectious disease bridge the gap between the in vitro characterization and the clinical evaluation of antimicrobial agents^［1］. Since the first report concerning intraperitoneal infection was published in 1935^［2］, systemic infection model has been the most commonly used method in the testing of potential antibiotics. However, it has several serious drawbacks which limit its further application. It doesn′t resemble the clinical infection course, nor does it reflect antibiotics′ penetrating characters in some special tissues. To overcome these disadvantages, various localized infection models were applied in this field. Experimental respiratory tract infection, as an important one of these models, has been widely used abroad in early evaluation of antimicrobial agents, but it is rarely applied in domestic experiments, especially in bacterial infections. Levofloxacin, ofloxacin and lomefloxacin are three newly developed fluroquinolones with excellent in vitro activity against Klebsiella pneumonia^［3～5］. These compounds also offer interesting therapeutic alternatives for the treatment of respiratory tract infections because their high rate of absorption after oral administration, prolonged half life and high tissue penetration^［6～8］. In the present study, the intranasal instillation method was used to establish a simple respiratory tract infection induced by K.pneumonia, and the effectiveness of levofloxacin, ofloxacin and lomefloxacin were examined by the established model.

Materials and methods

MiceMale mice weighing 18～20g were used, the strain was Kunming (Institute of Laboratory Animals, Peking Union Medical College).

Antibacterial compoundsLevofloxacin, ofloxacin and lomefloxacin tested were obtained from Zhumadian Pharmaceutical Factory, Xinchang Pharmaceutical Factory, Nanjing No.2 Pharmaceutical Factory, respec-tively.

OrganismsAll tested organisms are clinically isolated pathogenic bacteria, kindly provided by Department of Pharmacology of our institute.

Susceptibility testingThe MICs of levofloxacin, ofloxacin and lomefloxacin for the infected pathogens were determined by two-fold dilution method in M-H liquid culture medium. The MIC was defined as the lowest concentration to inhibit visible growth after 24h at 37℃.

Respiratory tract infection procedureThe model of pneumonia described by S.Goto et al was employed^［9］. Briefly, each mouse was anaesthetized by infection it with sodium pentobarbital (70 mg/kg body weight, Shanghai Chemical Agents Factory) before instilling 35μl suspension of 1.29×10⁷ CFUs of K.pneumonia 7. To prevent hypothermia, the anaesthetized mice were exposed to infrared light until they recovered from narcosis^［10］.

Bacteriological examination Mice were killed with ether at intervals from 0 to 30h. The whole lungs and one of the two kidneys were homogenized in 1.5 and 1.8ml of sterile physiological saline solution, respectively, using a tissue homogenizer. These homogenates were serially diluted 10-fold with saline solution, and 0.1 ml volumes of the various dilutions were inoculated onto M-H agar plates. The plates were incubated overnight at 37℃, and the colonies were counted. Bacterial counts were expressed as the log number of CFUs per ml homogenates. When no bacteria were detected in undiluted specimens, a value of 0 was assigned to calculate the geometric mean titer^［11］.

Histopathological examinationSpecimens obtained from some animals were fixed in Bouin′s solution, embedded in paraffin, stained by hematoxylin and erosin, and examined by optic microscopy.

TreatmentLevofloxacin, ofloxacin and lomefloxacin, dissolved in 0.5% carboxymethyl cellulose, were administrated orally in a fixed volume of 0.01 ml/g body weight at 6h after inoculation. The CFUs in the lungs (5 mice/group) were counted at 20h after infection, and the total number of mice surviving in each dosage (10 mice/group) was recorded at the seventh day. The amount of antibiotics (mg/kg) required for 50% survival of the animals (ED₅₀) and that for an eradication of challenged organisms from the lungs of 50% of the animals tested (CD₅₀), respectively, were calculated by Bliss method^［12］.

Results

Characteristics of the experimental respiratory tract infectionUnder sodium pentobarbital anesthesia, 1.29×10⁷ CFUs of K.pneumonia 7 were inoculated intranasally into the murine nares. The mice inoculated developed an acute pneumonia course. The mice showed no signs of faintness until 12h after instilling. By 18h, the mice became weak, and thereafter they exhibited a steady downhill course. The first case of death occurred after 36h, and most of the mice succumbed before 48h. The outcome of histopathological examination confirmed the course. At 12h, there was infiltration of inflammatory cells around the bronchi and the alveolar walls. Scattering abscess and dilatation of bronchi had happened and there had been neutrophils in the alveolar air spaces before 20h. Pulmonary edema with inflammation and dilated alveolar capillaries with congestion was found at 32h. Samples of 40h showed abscess around bronchi, pulmonary edema and congestion, while that in control group only showed slightly congestion and thickening of the alveolar walls at 40h.

After challenge, six mice each group were sacrificed at scheduled time, and the counts in the lungs and kidneys were quantitated at once (Fig.1). The mean CFUs/ml in t