Abstract：
Phage therapy has shown great promise in the treatment of bacterial infections. However, the effectiveness of phage therapy is compromised by the inevitable emergence of phage-resistant strains. In this study, a phage-resistant carbapenem-resistant Klebsiella pneumoniae strain SWKP1711R, derived from parental carbapenem-resistant K. pneumoniae strain SWKP1711 was identified. The mechanism of bacteriophage resistance in SWKP1711R was investigated and the molecular determinants causing altered growth characteristics, antibiotic resistance, and virulence of SWKP1711R were tested. Compared to SWKP1711, SWKP1711R showed slower growth, smaller colonies, filamentous cells visible under the microscope, reduced production of capsular polysaccharide (CPS) and lipopolysaccharide, and reduced resistance to various antibiotics accompanied by reduced virulence. Adsorption experiments showed that phage vB_kpnM_17-11 lost the ability to adsorb onto SWKP1711R, and the adsorption receptor was identified to be bacterial surface polysaccharides. Genetic variation analysis revealed that, compared to the parental strain, SWKP1711R had only one thymine deletion at position 78 of the open reading frame of the lpcA gene, resulting in a frameshift mutation that caused alteration of the bacterial surface polysaccharide and inhibition of phage adsorption, ultimately leading to phage resistance. Transcriptome analysis and quantitative reverse transcriptase PCR revealed that genes encoding lipopolysaccharide synthesis, ompK35, blaTEM-1, and type II and Hha-TomB toxin-antitoxin systems, were all downregulated in SWKP1711R. Taken together, the evidence presented here indicates that the phenotypic alterations and phage resistance displayed by the mutant may be related to the frameshift mutation of lpcA and altered gene expression. While evolution of phage resistance remains an issue, our study suggests that the reduced antibiotic resistance and virulence of phage-resistant strain derivatives might be beneficial in alleviating the burden caused by multidrug-resistant bacteria.
摘要：
噬菌体疗法在治疗细菌感染方面显示出巨大的希望。然而,噬菌体治疗的有效性因不可避免的噬菌体抗性菌株的出现而受到损害。在这项研究中,一株耐噬菌体耐碳青霉烯类肺炎克雷伯菌(CRKP)菌株SWKP1711R,鉴定了源自亲本CRKP菌株SWKP1711。研究了SWKP1711R中噬菌体抗性的机制，并研究了导致生长特性改变的分子决定因素，抗生素耐药性,并对SWKP1711R进行了毒力测试。与SWKP1711相比，SWKP1711R的生长速度较慢，较小的殖民地，在显微镜下可见的丝状细胞，减少荚膜多糖（CPS）和脂多糖（LPS）的产生，对各种抗生素的耐药性降低，同时毒力降低。吸附实验表明,噬菌体vB_kpnM_17-11失去了对SWKP1711R的吸附能力,吸附受体被鉴定为细菌表面多糖。遗传变异分析显示,与亲本菌株相比，SWKP1711R在lpcA基因的开放阅读框的第78位只有一个胸腺嘧啶缺失，导致移码突变，导致细菌表面多糖的改变和噬菌体吸附的抑制，最终导致噬菌体抗性。转录组分析和定量逆转录酶PCR(qRT-PCR)显示,ompK35，blaTEM-1和II型和Hha-TomB毒素抗毒素（TA）系统，在SWKP1711R中均下调。一起来看,此处提供的证据表明，突变体显示的表型改变和噬菌体抗性可能与lpcA的移码突变和基因表达改变有关。虽然噬菌体抗性的进化仍然是一个问题，我们的研究表明，噬菌体耐药菌株衍生物的抗生素耐药性和毒力降低可能有助于减轻多药耐药细菌造成的负担。
