Sugawara Etsuko, Kojima Seiji, Nikaido Hiroshi
Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
Department of Molecular and Cell Biology, University of California, Berkeley, California, USA
J Bacteriol. 2016 Nov 4;198(23):3200-3208. doi: 10.1128/JB.00590-16. Print 2016 Dec 1.
Klebsiella pneumoniae, one of the most important nosocomial pathogens, is becoming a major problem in health care because of its resistance to multiple antibiotics, including cephalosporins of the latest generation and, more recently, even carbapenems. This is largely due to the spread of plasmid-encoded extended-spectrum β-lactamases. However, antimicrobial agents must first penetrate the outer membrane barrier in order to reach their targets, and hydrophilic and charged β-lactams presumably diffuse through the porin channels. Unfortunately, the properties of K. pneumoniae porin channels are largely unknown. In this study, we made clean deletions of K. pneumoniae porin genes ompK35 and ompK36 and examined the antibiotic susceptibilities and diffusion rates of β-lactams. The results showed that OmpK35 and OmpK36 produced larger more permeable channels than their Escherichia coli homologs OmpF and OmpC; OmpK35 especially produced a diffusion channel of remarkably high permeability toward lipophilic (benzylpenicillin) and large (cefepime) compounds. These results were also confirmed by expressing various porins in an E. coli strain lacking major porins and the major multidrug efflux pump AcrAB. Our data explain why the development of drug resistance in K. pneumoniae is so often accompanied by the mutational loss of its porins, especially OmpK35, in addition to the various plasmid-carried genes of antibiotic resistance, because even hydrolysis by β-lactamases becomes inefficient in producing high levels of resistance if the bacterium continues to allow a rapid influx of β-lactams through its wide porin channels.
In Gram-negative bacteria, drugs must first enter the outer membrane, usually through porin channels. Thus, the quantitative examination of influx rates is essential for the assessment of resistance mechanisms, yet no such studies exist for a very important nosocomial pathogen, Klebsiella pneumoniae We found that the larger channel porin of this organism, OmpK35, produces a significantly larger channel than its Escherichia coli homolog, OmpF. This makes unmodified K. pneumoniae strains more susceptible to relatively large antibiotics, such as the third- and fourth-generation cephalosporins. Also, even the acquisition of powerful β-lactamases is not likely to make them fully resistant in the presence of such an effective influx process, explaining why so many clinical isolates of this organism lack porins.
肺炎克雷伯菌是最重要的医院病原体之一，由于其对多种抗生素耐药，包括最新一代头孢菌素，最近甚至对碳青霉烯类抗生素耐药，正成为医疗保健领域的一个重大问题。这主要归因于质粒编码的超广谱β-内酰胺酶的传播。然而，抗菌剂必须首先穿透外膜屏障才能到达其靶点，亲水性和带电荷的β-内酰胺类药物可能通过孔蛋白通道扩散。不幸的是，肺炎克雷伯菌孔蛋白通道的特性在很大程度上尚不清楚。在本研究中，我们对肺炎克雷伯菌孔蛋白基因ompK35和ompK36进行了完全缺失，并检测了β-内酰胺类药物的抗生素敏感性和扩散速率。结果表明，与大肠杆菌同源物OmpF和OmpC相比，OmpK35和OmpK36产生的通道更大且通透性更高；特别是OmpK35产生了一个对亲脂性（苄青霉素）和大分子（头孢吡肟）化合物具有显著高通透性的扩散通道。在缺乏主要孔蛋白和主要多药外排泵AcrAB的大肠杆菌菌株中表达各种孔蛋白，也证实了这些结果。我们的数据解释了为什么肺炎克雷伯菌耐药性的产生除了各种携带质粒的抗生素耐药基因外，常常伴随着其孔蛋白尤其是OmpK35的突变性缺失，因为如果细菌继续允许β-内酰胺类药物通过其宽阔的孔蛋白通道快速流入，即使β-内酰胺酶水解也难以产生高水平的耐药性。
在革兰氏阴性菌中，药物通常必须通过孔蛋白通道首先进入外膜。因此，对流入速率进行定量检测对于评估耐药机制至关重要，但对于一种非常重要的医院病原体肺炎克雷伯菌，尚无此类研究。我们发现，该菌较大的孔蛋白OmpK35产生的通道比其大肠杆菌同源物OmpF大得多。这使得未修饰的肺炎克雷伯菌菌株对相对较大的抗生素，如第三代和第四代头孢菌素更敏感。此外，即使获得强大的β-内酰胺酶，在存在如此有效的流入过程的情况下，也不太可能使其完全耐药。这就解释了为什么该菌如此多的临床分离株缺乏孔蛋白。
