Stenotrophomonas maltophilia多重抗藥性的研究

Abstract

Stenotrophomonas maltophilia是一種嗜氧的革蘭氏陰性菌，為重要的院內感染致病菌，常見於免疫功能不全的病人。這隻菌的特徵是對各類結構不同的抗生素有高度的抗藥性，其中包括quinolones、aminoglycosides和β-lactams等。在之前的研究已知，S. maltophilia的多重抗藥性和有限的外膜通透性、integron，以及活躍的抗生素排出系統有關。S. maltophilia中具有SmeDEF及SmeABC兩種多重抗藥排出唧筒（multidrug efflux pumps）；SmeDEF已知在臨床菌株的抗藥性中扮演重要的角色，而SmeABC的角色尚未確定；直至目前對SmeDEF及SmeABC的相關研究還很缺乏。Integron具有一種site-specific recombinase-integrase可以抓取、移動gene cassettes(抗藥基因)，使得抗藥基因可藉此傳遞及散布。此外，外膜的蛋白質或脂多醣（LPS）結構上的改變也會影響藥物的感受性。而缺乏與LPS、rhamnolipids及褐藻酸鹽(alginate)合成有關的spgM基因的S. maltophilia對一些抗生素的敏感度會增加。形成生物膜的細菌對於抗生素的抗藥性比起浮游狀態的細菌更高，除此之外，S. maltophilia在特定的環境中會生成黑色素(melanin)，在其他細菌及微菌這是一種可以保護菌株免於各種不利生存壓力的物質，包括抗生物質。 為要了解及分析S. maltophilia的多重抗藥現象，我們分別收集臨床的抗藥菌株及對藥物敏感的菌株，首先利用聚合酶連鎖反應（Polymerase Chain Reaction，PCR）偵測integron的存在，發現抗藥菌株與敏感菌株帶有integron的比例有明顯的差異(82.5%及30%)，並且帶有integron的菌株比不帶integron的菌株更具有多重抗藥性的傾向；這意味著integron在S. maltophilia的多重抗藥性中可能扮演重要的角色。接著利用RT-PCR（反轉錄聚合酶連鎖反應）調查多重抗藥排出唧筒基因smeABC及smeDEF的表現情形，發現抗藥菌株高表現smeABC(57.5% VS 20%)及smeDEF(45% VS 10%)的比例皆明顯比敏感菌株高，並且高表現smeA及smeDEF的菌株有傾向對更多數目的抗生素產生抗藥，而臨床菌株中表現smeD (86%)的比例較smeA(66%)高，也暗示SmeDEF在S. maltophilia的表現及重要性可能高於SmeABC。在高表現spgM的菌株中，抗藥菌株的比例同樣比敏感菌株高，並且高表現spgM的菌株有傾向對更多數目的抗生素產生抗藥，不過上述的差異不及integron、smeDEF及smeABC大。在生物膜及黑色素的生成上，抗藥菌株的能力皆比敏感菌株強，並且有統計上的差異，不過生物膜及黑色素的表現如何造成抗藥與敏感菌株間的差異，還有待進一步的實驗研究。最後將本論文所探討的機制綜合來看，可以發現具有抗藥機制的數目越高，越有利於S. maltophilia表現多重抗藥性。

Stenotrophomonas maltophilia is an aerobic, nonfermentative gram-negative bacterium. The organism has increasingly emerged as an important nosocomial pathogen, particularly for immunocompromised patients. It is characterized by its hegh-level resistance to a variety of structurally unrelated antimicrobials, including quinolones, aminoglycosides and β-lactams. SmeDEF and SmeABC multidrug efflux pumps have been described in S. maltophilia.SmeDEF has been shown to play a role in the drug resistance of clinical strains. The SmeABC multidrug system is regulated by a two-component regulatory system encoded by the smeSR genes.Related studies about SmeDEF and SmeABC are still deficient. Integrons have the ability tocapture and mobilize gene cassettes via a site-specific recombinase that blongs to the integrase family.Besides, some structural change in the proteins or lipopolsaccharide correlate well with drug susceptibility. Mutants lacking spgM gene, which is responsible for the production of a phosphoglucomutase (PGM) associated with LPS and alginate biosynthesis in S. maltophilia, displayed a modest increase in susceptibility to several antimicrobial agents. It has been observed that the resistance of biofilms to antibiotics is increased compared with what is normally seen with planktonic cells, and cells become 10-1000 times more resistant to the effects of antibiotics when existing in a biofilm. When strains of S. maltophilia exist at a specific growth stage, they synthesize the melanins, which protect bacteria against stresses unfavorable for growth. In the present study, 40 clinical multidrug resistance strains (resistant to more than 7 drugs tested) and 30 susceptible strains (less than 5 drug) were collected to investigate the multidrug resistance in S. maltophilia. They were investigated the presence of integrons by PCR and found an obvious difference between the multidrug resistance strains and the susceptible strains in the rate of carrying class 1 integrons (82.5% and 30%, respectively), with an increasing tendency towards the multidrug resistance in carrying class 1 integrons.RT-PCR was then performed to assess and semi-quantify the expression of the Sme efflux pumps and PGM of S. maltophilia.It was found that the rate of multidrug resistance strains was higher than that of susceptible strains in hyperexpresion of smeABC (57.5% and 20%, respectively), smeDEF(45% and 10%) and spgM(75% and 60%), with an respective increasing tendency towards the multidrug resistance in hyperexpresion of these genes.Further, I found multidrug-resistant strains exhibit higher expression of SmeDEF (86%) than SmeABC (66%). This implies SmeDEF plays a more important role in multidrug resistance in S. maltophilia. Besides, the ability to form melanins and biofilms of the multidrug-resistant strains is also greater than that of the susceptible strains, and the difference is significant in statistics. We found the more above-mentioned mechanisms that strains possess, the more possibilities for strains to express multidrug resistance.