從患有尿道感染的狗與貓尿液檢體分離之ST131型大腸桿菌的特性分析

Abstract

ST131型大腸桿菌是在2008年，透過多重基因座定序分型法，被鑑定出來的大腸桿菌序列型別，這是一種在全球造成流行的新興多重耐藥性菌株。在人醫中，ST131型大腸桿菌是社區與醫院內的泌尿道感染以及敗血症的主要病原體。近年來，在健康人類、病患以及伴侶動物的排泄物以及感染部位中，都有檢測到此菌株的存在。此型別的大腸桿菌之所以能夠快速傳播的原因，是由於其抗生素耐藥性以及攜帶大量毒力因子的特性所導致。由回顧性研究中發現，ST131型大腸桿菌對於臨床治療中經常使用的氟喹諾酮類以及第三代頭孢子菌素等多種抗生素都具有耐藥性，這使得臨床治療上抗生素藥物的選擇受到限制。除此之外，該細菌也會攜帶許多與泌尿道致病性大腸桿菌相關的毒力因子。其抗生素耐藥性以及高致病性的特性，造成全世界廣泛地傳播，對公共衛生構成嚴重的隱憂。除此之外，飼養伴侶動物為現代人生活的一種趨勢，人類與伴侶動物之間的互動緊密，也可能成為重要傳播途徑之一。因此從公共衛生的觀點上，鑑定從伴侶動物中分離出的ST131型大腸桿菌，針對其檢出率、對藥物的感受性、攜帶的抗藥性基因型以及毒力基因等的分子特性及相關研究，顯得十分重要。本研究的對象是從2011年至2019年間，至國立臺灣大學生物資源暨農學院附設動物醫院就診，患有泌尿道感染的貓與狗，從其尿液檢體中所分離出的400株大腸桿菌。所有大腸桿菌分離株使用大腸桿菌系統發育群分類方法，鑑定出192株屬於B2組別 (192/ 400, 48.0%)，該組別是與ST131型大腸桿菌以及腸外致病性大腸桿菌最有相關的系統發育組別。所有B2組分離株透過鑑定是否在mdh 基因以及gyrB基因上產生相關的特異性單核苷酸多態性，共篩選出26株 (26/192, 13.5%) 的ST131型大腸桿菌分離株，而這些分離株也再經過多重基因座定序分型方法確認過。本研究針對篩選出來的所有ST131型大腸桿菌，進行一系列特性分析。利用多重聚合酶鏈鎖反應偵測包含黏附素、毒素、鐵離子獲取系統以及莢膜這五種不同類型的12種毒力基因，結果顯示最常檢測到的毒力基因是 fyuA (100.0%)，其次是 traT (92.3%)、kpsMT II (88.5%) 以及iutA (84.6%)。為了瞭解分離株對臨床治療藥物的感受性是如何，以最小抑菌濃度的方法，測試藥物敏感性試驗。在檢測的15種抗生素中，分離株都對美洛培南呈現敏感性，並且對阿莫西林和氨芐西林皆呈現耐藥性。針對會產生超廣譜乙內醯氨酶與AmpC乙內醯氨酶之ST131型大腸桿菌的表現型篩選以及基因型鑑定的結果顯示，一半的分離株會產生超廣譜乙內醯氨酶和/或AmpC乙內醯氨酶。在前者，鑑定出bla CTX-M-1、bla CTX-M-2、bla CTX-M-8以及bla CTX-M-9這4種bla基因組。而bla CIT組則是唯一檢測到的AmpC乙內醯氨酶bla基因組。以ST131型大腸桿菌分離株為提供者，對疊氮化鈉具有抵抗性的大腸桿菌J53菌株 (ATCC BAA-2730TM) 做為接受者，進行接合試驗，測試超廣譜乙內醯氨酶與AmpC乙內醯氨酶抗藥性基因在不同菌株之間的水平轉移能力。結果顯示，雖然大部分的bla基因可轉移至大腸桿菌J53菌株，但bla TEM-102、bla TEM-12、bla CTX-M-55、bla CTX-M-124 以及bla CTX-M-214基因型並沒有被成功轉移。本研究也利用質體複製子分型方法分析分離株所攜帶的Inc質體類型，發現最常見的質體類型是 IncF (100%) 以及IncB/O (69%)。FimH和O 抗原中廣泛存在的等位基因變異，在宿主與病原體之間的相互作用中，扮演重要的作用。因此，本研究也利用多重聚合酶鏈鎖反應，進行分離株O抗原與FimH譜系的分類。結果顯示，O25b-H30 (n=11, 42.3%) 以及O16-H41 (n=11, 42.3%) 為ST131型大腸桿菌分離株中最常見的譜系。總結來說，希望藉由這項研究，提供從狗與貓所分離出的ST131型大腸桿菌的重要資訊，以做為後續可能在公共衛生學上的比較依據。

Escherichia coli sequence type (ST) 131 is a globally distributed, emerging multidrug-resistant clone that was first identified in 2008 by multi-locus sequence typing (MLST). In medicine, E. coli ST131 is a major causative pathogen of urinary tract infections and sepsis in both outpatient and hospital settings. Recently, this strain has been detected in the feces and infection sites of healthy individuals, patients, and companion animals. The rapid spread of this E. coli type is attributed to its resistance to antibiotics and the possession of numerous virulence factors. Retrospective studies have shown that E. coli ST131 is resistant to many antibiotics such as fluoroquinolones (FQs) and 3rd generation cephalosporins (TGCs), which are commonly used in clinical treatment, limiting treatment options. In addition, E. coli ST131 contains a number of virulence factors associated with uropathogenic E. coli. The combination of antibiotic resistance and high pathogenicity has led to widespread dissemination and represents a serious public health problem. Moreover, the trend of keeping companion animals in modern life and the close interaction between humans and their pets could be a significant route of transmission. Therefore, from a public health perspective, identifying E. coli ST131isolated from companion animals and researching its prevalence, drug susceptibility, resistance genotypes, and virulence genes is of great importance. This study focuses on 400 E. coli strains isolated from the urine samples of cats and dogs with urinary tract infections, treated at the National Taiwan University Veterinary Hospital between 2011 and 2019. Using phylogenetic grouping methods, 192 strains (192/400, 48.0%) were identified as belonging to group B2, the phylogenetic group most closely associated with E. coli ST131 and extraintestinal pathogenic E. coli. Among these, 26 strains (26/192, 13.5%) were identified as E. coli ST131 by specific single nucleotide polymorphisms in the mdh and gyrB genes and further confirmed by multilocus sequence typing. The identified E. coli ST131 strains underwent a series of characterization analyses. Multiplex polymerase chain reaction was used to detect 12 virulence genes in five categories (adhesins, toxins, iron uptake systems, and capsules). The most frequently detected virulence gene was fyuA (100.0%), followed by traT (92.3%), kpsMT II (88.5%), and iutA (84.6%). Antimicrobial susceptibility was tested using the minimum inhibitory concentration method for 15 antibiotics, revealing that all isolates were sensitive to meropenem and resistant to amoxicillin and ampicillin. Phenotypic screening and genotypic identification of extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase production showed that half of the isolates produced ESBL and/or AmpC β-lactamases. Four bla gene groups—blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, and blaCTX-M-9—were identified among ESBL producers, while blaCIT was the only AmpC β-lactamase gene group detected. Conjugation experiments tested the horizontal transferability of ESBL and AmpC β-lactamase resistance genes from E. coli ST131 donors to azide-resistant E. coli J53 (ATCC BAA-2730TM) recipients. The results showed that while most bla genes could be transferred to E. coli J53, the blaTEM-102, blaTEM-12, blaCTX-M-55, blaCTX-M-124, and blaCTX-M-214 genotypes were not successfully transferred. Plasmid replicon typing revealed that the most common plasmid types were IncF (100%) and IncB/O (69%). The extensive allelic variation of the FimH and O antigens plays an important role in host–pathogen interactions. Therefore, the sublineages of E. coli ST131 isolates were determined by multiplex PCR-based O antigen typing and FimH subtyping. The present study found that the most common lineages of E. coli ST131 isolates were O25b-H30 (n=11, 42.3%) and O16-H41 (n=11, 42.3%). In summary, this study aims to provide crucial information about E. coli ST131 isolated from dogs and cats, serving as a basis for future public health comparisons.