從患有尿道感染的寵物所分離出的具有超廣譜乙內醯氨酶與pAmpC的肺炎克雷伯氏菌之分離率及特性

Abstract

當包括細菌、病毒、真菌和寄生蟲等各種類型的微生物發生進化變化，使其對治療產生抗藥性時，就會出現所謂抗生素抗藥性的問題。這種現象對治療細菌感染的症狀上產生了極大的挑戰，也增加了疾病傳播、患病和死亡的風險。第三代頭孢菌素是用於對抗嚴重人類疾病的重要抗生素。頭孢菌素是屬於乙內醯氨類的抗生素，通過抑制細菌細胞壁的合成達到殺菌的作用。然而，有些細菌已經發展出可以產生乙內醯氨酶，用來分解乙內醯氨分子，使抗生素失效。肺炎克雷伯氏菌是一種屬於腸桿菌科的革蘭氏陰性、伺機性細菌，是造成肺炎、尿道感染、腦膜炎和血液感染等常見院內感染的原因。除了大腸桿菌外，肺炎克雷伯氏菌也是伴侶動物細菌性尿道感染的重要因子。基於從患有尿道感染的伴侶動物所分離出攜帶有超廣譜乙內醯氨酶，以及由質體所攜帶的AmpC的肺炎克雷伯氏菌，其在臺灣的盛行率與細菌相關的研究資訊有限，因此，本研究目的將分析自 2014 年至 2019 年間，從國立臺灣大學附設動物醫院就診之犬貓的尿液樣本內，分離出的 70 株肺炎克雷伯氏菌中的多重抗藥性基因，並進行闡述。初步使用CHROMagar ESBL培養基，篩選出帶有超廣譜乙內醯氨酶的肺炎克雷伯氏菌，隨後再採用由臨床和實驗室標準協會所頒定的表現型鑑定方法加以確認。利用聚合酶鏈鎖反應檢測這些分離株中的ESBL編碼基因，其中有19個分離株帶有屬於 blaCTX-M-1、blaCTX-M-9 和 blaSHV 組別的ESBL基因，而隸屬於 blaTEM、blaCTX-M- 2、blaCTX-M-8、blaCTX-M-25的組別之基因均未檢測到。 另外，利用相同的實驗方法檢測可發現70個肺炎克雷伯菌分離株中，有56個分離株存在pAmpC編碼基因，分別為blaCIT和blaDHA。總體而言，在所有樣本中有81% (57/70) 的分離株帶有ESBL和/或pAmpC基因。利用多重基因座序列分型法分析57株肺炎克雷伯氏菌在流行病學上的相關性，發現最普遍的序列類型是ST11 (7/57)、ST15 (6/57)和ST655 (6/57)。此外，使用聚合酶鏈鎖反應檢測 9 個毒力基因來評估每株具有抗藥性的肺炎克雷伯氏菌分離株中的 ESBL 和/或 pAmpC之毒力特徵。結果顯示主要的毒力基因型包括了wabG、uge、entB、mrkD和fimH。為測試超廣譜乙內醯氨酶基因以及由質體所攜帶的AmpC基因是否存在轉移性，使用大腸桿菌J53菌株作為接受體進行接合試驗，分別測試41株細菌，其中有43.9% (18/41) 的分離株之抗藥性基因成功轉移至大腸桿菌J53菌株中。由聚合酶鏈鎖反應的結果顯示，15株轉化接合子攜帶超廣譜乙內醯氨酶基因以及由質體所攜帶的AmpC基因，而3株轉化接合子僅攜帶質體所攜帶的AmpC基因。然而，有些 blaCIT基因並沒有成功轉移。最後，以抗微生物製劑敏感性試驗，評估57株肺炎克雷伯氏菌分離株對不同類別抗菌藥物的敏感性。含有ESBL基因的菌株對大多數乙內醯胺類和氟喹諾酮類抗生素表現出較高的耐藥性，但對胺基醣苷類和磺胺類抗生素的耐受程度相對較低。有趣的是，7% (4/57) 的分離株對屬於碳青黴烯類抗生素中的亞胺培南具有耐藥性，但這是一種僅允許人類使用的抗微生物藥物，而這種可能存在於人類和伴侶動物之間的細菌耐藥基因的傳播能力，即可能構成人類醫學的重大威脅。後續我們將收集更多來自國內動物醫院中，造成伴侶動物的尿道感染之肺炎克雷伯氏菌的臨床檢體，其所帶有的ESBL和pAmpC基因進行更詳細的闡述，期望能建構出臺灣關於伴侶動物的肺炎克雷伯氏菌之抗生素抗藥性基因資料庫。

Antimicrobial resistance (AMR) occurs when various types of microbes, including bacteria, viruses, fungi, and parasites, undergo evolutionary changes that render them resistant to modern medicine. This phenomenon poses significant challenges in the treatment of infections and increases the risk of disease spread, illness, and mortality. Third-generation cephalosporins (3GCs) are the most common antimicrobial drugs currently used to combat severe human diseases. Cephalosporins belong to the β-lactam class of antimicrobials and work by inhibiting the synthesis of bacterial cell walls. However, certain bacteria have developed the ability to produce β-lactamase enzymes such as extended-spectrum β-lactamase (ESBL) and plasmid-encoded AmpC (pAmpC) that break down β-lactam molecules, rendering the antimicrobial drugs ineffective. Klebsiella pneumoniae, a gram-negative opportunistic bacterium that belongs to the Enterobacteriaceae family, is a common cause of nosocomial infections such as pneumonia, urinary tract infections (UTIs), meningitis, and bloodstream infections. Apart from E. coli, K. pneumoniae is also a significant contributor of bacterial UTIs in companion animals. This study aims to characterise the prevalence of ESBL and pAmpC-producing K. pneumoniae in companion animals with UTI in Taiwan. Seventy K. pneumoniae isolates were obtained from the urine of dogs and cats admitted to the National Taiwan University Veterinary Hospital between 2014 and 2019. The ESBL-producing K. pneumoniae were initially identified using CHROMagar ESBL and subsequently phenotypically screened using a confirmatory disc test specified by the Clinical and Laboratory Standards Institute (CLSI). Polymerase chain reaction (PCR) was then used to detect ESBL-encoding genes from these isolates and it was found that 19 contained ESBL genes from the blaCTX-M-1, blaCTX-M-9, and blaSHV groups, while the blaTEM, blaCTX-M-2, blaCTX-M-8, and blaCTX-M-25 groups were not detected. PCR was also used to detect pAmpC-encoding genes in the 70 K. pneumoniae isolates and it was found that blaCIT and blaDHA groups were present in 56 isolates. Overall, ESBL and/or pAmpC genes were detected in 81% (57/70) of the isolates. Multi-locus sequence typing (MLST) was used to gain epidemiological insights into the relatedness of the 57 isolates, with the most prevalent sequence types being ST11 (7/57), ST15 (6/57), and ST655 (6/57). Furthermore, the virulence profiles of each ESBL and/or pAmpC-producing K. pneumoniae were assessed using PCR to detect 9 virulence genes. The primary virulence genotype identified included wabG, uge, entB, mrkD, and fimH. To observe the transferability of ESBL and pAmpC genes, a conjugation test was performed using an E. coli J53 strain as the recipient. Of the 41 isolates that were tested, 43.9% (18/41) were able to transfer resistant genes to the E. coli J53 strain. The PCR results showed that 15 of the transconjugates carried ESBL and pAmpC genes, while 3 carried only pAmpC genes. However, some blaCIT genes were not successfully transferred. Finally, an antimicrobial susceptibility test was conducted to evaluate the susceptibilities of the 57 K. pneumoniae isolates to different classes of antimicrobial drugs. The isolates that contained ESBL genes exhibited high resistance to most β-lactams and fluoroquinolones, but showed comparatively lower rates of resistance to aminoglycosides and sulfonamides. Interestingly, 7% (4/57) of the isolates were resistant to imipenem, a carbapenem, an antimicrobial approved for human use only. The spread of resistant genes between humans and companion animals could pose a significant threat to human medicine. In the future, we might collect more clinical samples from other animal hospitals across the country to further contribute to a database on ESBL and pAmpC genes carried by K. pneumoniae that cause UTI in companion animals in Taiwan.