| Klebsiella pneumoniae is one of the most common gram-negative bacteria responsible for hospital-acquired pneumonia. As abuse of antibiotics, multidrug resistance Klebsiella pneumoniae (MRKP) has steadily grown beyond the control worldwide. It has become more difficult to treat due to limited clinical treatment options and a serious threat to our health. Bacteriophages, or phages, are viruses that specifically attack and kill their host bacteria, which exist widely in nature. As the side effects of the antibiotics and superiorities of bacteriophage, it has gained increased attention as an alternative to antibiotic for the control of bacterial infectious diseases avoid the spreading of multi-resistant bacteria. The purpose of our study is to apply lytic bacteriophage to treat pneumonia caused by MRKP in mice in order to provide a potential alternative for the control of pneumonia induced by MRKP.By using plate screening techniques, the bacterial strain was isolated from phlegm of a pneumonia patient in hospital. It was identified by morphological observation, physiological and biochemical indexes test, sequence similarity analysis of16S rRNA genes, drug sensitive test and biofilm formation test. A multidrug resistant Klebsiella pneumoniae was isolated and named as KP1513. The isolated strain was used as host to isolate and purify bacteriophages via double-layer plate method. Morphology of the isolated phage was observed by transmission electron microscope. One-step growth curve, the sensitive to chloroform, the host range, pH and temperature stabilities were performed to characterize bacteriophages. A lytic bacteriophage, phage1513was isolated and it had a clear plaque with a halo and was classified as Siphoviridae by transmission electron microscopy. It owned host specificity and were insensitive to chloroform. It existed stable at a pH range of5-10and a temperature less than40℃. One-step growth curve showed that it had a short latent period of30min and an estimated burst size of264PFU/cell. Genome sequencing and function gene analysis showed that phage1513had a genome of49462bp,72code genes,4functional modules. It had close relationship with Tunalinkvirus. In addition, there was no antibiotic resistant genes and virus genes in the genome. Its lytic function was related to holin and endolysin.Lytic efficacy of phage1513in vitro was carried out by measuring the development of co-cultures’optical density at650nm and showed that phage1513quickly infected and lysed the host, it could also inhibit the formation of biofilm. Intranasal administration of a single dose of2×109PFU/mouse at2h after KP1513inoculation was able to protect mice against lethal pneumonia. However, phage1513couldn’t protect mice24h before KP1513infection. In a sublethal pneumonia model, mice treated with phage showed a lower level of K. pneumonia burden in the lungs compared with untreated control. And these mice suffered from less body weight loss, and showed lower levels of inflammatory cytokines (interleukin-6[IL-6] and tumor necrosis factor [TNF-α]) in the lungs. Lung lesions conditions were also obviously improved by phage therapy compared with control.This was the first separation and research for the phage of multidrug resistant Klebsiella pneumonia strain, investigated the biological property, and assessed the efficacy in vitro and in vivo. The results of this study suggested that phage1513had great potential to be used as an alternative to antibiotic for the treatment of pneumonia caused by multidrug resistance K. pneumoniae. |
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