多重抗藥綠膿桿菌內跳躍子Tn6001之特性及 甲苯胺藍對多重抗藥綠膿桿菌光動力殺菌之探討

Abstract

在台灣，水解carbapenem藥物的VIM-type metallo-β-lactamase (MBL) blaVIM-3常發現於多重抗藥的綠膿桿菌內。本論文發現一與Tn501相似的跳躍子命名為Tn6001，此跳躍子的基本結構包含了左側的tnpA和tnpR以及右側的orfB、orfC和orfD基因，此結構與跳躍子Tn1404* 或Tn1403的結構相似顯示其來源可能是來自同一種的跳躍子。這個跳躍子內帶有class 1 integron In450其所帶有的抗藥基因卡匣包含了六個抗藥基因：blaVIM-3 (carbapenemases)、orf2 (推測與fosfomycin有關之抗藥基因)、兩個相連之aacA4 (與aminoglycoside有關之抗藥基因, aminoglycoside 6'–acetyltransferase)、aadB (與aminoglycoside有關之抗藥基因, aminoglycoside 2'-adenylyltransferase)和另一個aacA4。In450的結構與In4的家族類似含有五端保留片段(5'-conserved segment (5'-CS))、三端保留片段(3'-CS segment)和一個IS6100，序列分析發現In450插入到tnpR基因上游的res site內造成site I的缺失以及僅剩部分的site II。這個發現與In4和In28的情形不同，此兩個integorn插入res site會在integron兩側產生5bp重複的序列。以限制酶I-CeuI分析脈衝式電泳的結果發現blaVIM-3基因的座落於染色體之上，此外由脈衝式電泳配合南方點墨法發現在同病人身上所分離出來且前後相差三個月的兩株臨床菌株，在脈衝式電泳分析下只有一個band的不同。且造成此band的差異是由Tn6001的傳遞造成的，此結果可以推測跳躍子Tn6001是可在臨床菌株內傳遞的。 為了瞭解台灣區域對carbapenem抗藥的臨床菌株對其他抗生素的抗藥情形，本論文分析了316株從台大醫院分離對carbapenem抗藥的菌株(2000-2005年)以及26株從六間醫學中心分離的多重抗藥菌株(2003年三月到八月)。結果發現，在台大醫院只對colistin呈現敏感性的菌株佔有6% (18/316)而全抗藥型的菌株則有4% (13/316)。而六間醫學中心分離的多重抗藥菌株則有35% (9/26)的菌株對colistin呈現中度抗藥性(intermediately-resistant)。在跳躍子Tn6001的調查中發現，只對colistin呈現敏感性的菌株帶有跳躍子Tn6001的比率為33% (6/18)而全抗藥型的菌株則有38% (5/13)帶有跳躍子Tn6001。以脈衝式電泳分析這些菌株發現多屬於不同的基因型顯示在台灣綠膿桿菌中造成抗藥性的原因可能是由於跳躍子Tn6001的傳遞所引起的。 在世界各地，多重抗藥性的綠膿桿菌的感染都是治療上一個嚴重的問題，目前發現綠膿桿菌對於後線藥物carbapenem開始具有抗藥性，所以科學家開始評估一些古老但是仍然有殺菌效果的藥物像是colistin，然而在早期會放棄使用這個藥物是因為之前的研究發現colisitn會造成nephrotoxicity和neurotoxicity。所以發展新的治療方式就顯的刻不容緩，在本實驗中我們評估以甲苯胺藍為光感物質的光動力殺菌方式對多重抗藥性綠膿桿菌的殺菌效果。結果發現對於難以治療的多重抗藥性綠膿桿菌光動力治療的方式可以有效殺死4-6 logs的細菌。此外我們也發現這樣光動力殺菌的方式皆會造成多重抗藥性和敏感型綠膿桿菌的染色體片段化，對於細菌體內的蛋白質也會由於氧化壓力造成損傷。此結果顯示甲苯胺藍可以有效的穿過細菌細胞膜在紅光照射下可以有效造成細菌體內染色體與蛋白質的傷害。雖然有11株多重抗藥性菌株分析帶有過量表現的efflux幫浦可以將甲苯胺藍打出細菌體外，但是在最小抑菌濃度光動力殺菌的實驗下，這些菌株與其他菌株的最小抑菌濃度並沒有顯著的差別。綜上所述，以光動力殺菌用來治療多重抗藥性綠膿桿菌是相當有效的方式值得再繼續深入研究，以期能克服光穿透力不足以及其他技術上的問題。

The VIM-type metallo-β-lactamase (MBL) blaVIM-3 was found in multidrug- resistant (MDR) Pseudomonas aeruginosa clinical isolates from Taiwan. We identified a Tn501-related (class II) transposon, termed Tn6001, which carries a class 1 integron In450-associated multiple drug resistance genes including blaVIM-3. Tn6001 backbone which contains the tnpA, tnpR at the region of left-end and orfB, orfC, orfD at the region of right-end is very similar to that found in Tn1404* or Tn1403. In450 gene cassette contains the following six genes: the blaVIM-3 (resistance to carbapenems), orf2 (putative fosfomycin resistance gene), two copies of aacA4 (resistance to aminogylcoside), aadB (resistance to aminogylcoside), and another copy of aacA4. In450 is related to the In4-like family which contains the 5'-conserved segment (5'-CS), 3'-CS segment and one copy of IS6100. Sequence analysis revealed that In450 is integrated upstream of the tnpR gene in the site II of the res site and lacks the site I. This finding is unlike In4 and In28 with part of res site on each side and with a 5 bp duplication of backbone sequences. I-CeuI digested pulsed-field gel electrophoresis (PFGE) analysis revealed the blaVIM-3 gene is located on the chromosome. Further, hybridization with the blaVIM-3 revealed only one band difference between an imipenem susceptible and an imipenem resistant isolate recovered from the same patient, suggesting that the gene element might have transferred horizontally. Subsequently, we investigated the antibiotic and MBLs surveillance of 316 non-duplicate isolates of carbapenem intermediately-resistant or resistant P. aeruginosa at the National Taiwan University Hospital (NTUH) from 2000 to 2005 and 26 MDR P. aeruginosa isolates collected from different parts of Taiwan from March to August 2003. Overall, 6% (18/316) colistin-only- sensitive (COS) P. aeruginosa and 4% (13/316) pan-drug-resistant (PDR) P. aeruginosa were found in NTUH and 35% (9/26) MDR P. aeruginosa isolates from six hospitals were found colistin intermediately-resistant. 33% (6/18) COS P. aeruginosa and 38% (5/13) PDR P. aeruginosa isolates carried the Tn6001 which contained a blaVIM-3-harboring integron In450 were found in different genotypes at NTUH. These finding suggest that mobile element Tn6001 might have transferred horizontally and cause the resistant genes delivery in Taiwan. MDR Pseudomonas aeruginosa infection is becoming a critical problem worldwide. Currently only limited therapeutic options are available for the treatment of infections caused by MDR P. aeruginosa, therefore the development of new alternative treatments may be needed. In this study, we evaluated the antibacterial photodynamic inactivation effect on MDR P. aeruginosa using toluidine blue O (TBO) as photosensitizer followed by red light irradiation. We demonstrated 4 to 6 logs of killing effect in MDR P. aeruginosa clinical isolates. We also found that TBO caused DNA fragmentation and protein carbonylation in both MDR and susceptible P. aeruginosa clinical isolates. These results suggest that TBO is able to permeate through the membrane and subsequently induces photodamages to DNA and proteins after red light irradiation. Although eleven strains of MDR P. aeruginosa contain an active efflux pump which can pump TBO out, the efficacy of PDT among these resistant strains did not differ from those susceptible strains as shown by minimum lethal drug concentrations (MLC) assay. In summary, antimicrobial photodynamic therapy with TBO showed the same efficacy between efflux pump-positive MDR P. aeruginosa strains and non-MDR strains.