泌尿道致病性大腸桿菌於臺灣市售雞肉之盛行率及其於舒肥雞胸肉之熱失活預測模型

Abstract

泌尿道感染 (urinary tract infections, UTIs) 為多種微生物感染性疾病，其中泌尿致病性大腸桿菌 (uropathogenic Escherichia coli UPEC) 約佔 65-75%。近年來許多研究表明，家禽，尤其是雞肉，可能為人類腸道外致病性大腸桿菌 (extraintestinal pathogenic E. coli, ExPEC) 的主要宿主，而研究證據皆指向食物來源的泌尿致病性大腸桿菌會引發食源性泌尿道感染。因此，新興食源性泌尿道感染為不容忽視的食安議題。然而，據我們所知，目前關於臺灣市售肉品是否為泌尿致病性大腸桿菌宿主的文獻資料相對較少。舒肥法 (sous-vide) 是將真空包裝的食材，在穩定的低溫下長時間烹煮 (low-temperature long-time, LTLT)，使食物口感質地較佳並保留更多的營養成分。然而有研究發現沙門氏菌 (Salmonella spp.) 仍可以從舒肥烹煮過的雞肉中被分離。這表明低溫長時間烹煮過程可能無法滅活所有病原菌。鹽 (sodium chloride, NaCl) 是肉類在舒肥烹調過程中最廣泛使用的醃料之一。更重要的是研究指出由於鹽水溶液的低 pH 值和低水活性而具有抑菌作用，可延長肉類的保質期、減少細菌滋生與改善食物質地。如：研究表明2.5% NaCl溶液可抑制牛肉中O157:H7大腸桿菌的生長。這顯示舒肥食品仍存在微生物危害的食安問題，故舒肥烹調法應設置額外的措施，即鹽水醃漬，以確保即食舒肥產品的安全。 因此，本研究將著重於：(1) 調查臺灣市售肉類中泌尿致病性大腸桿菌的盛行率 (2) 從消費者的角度，選擇最具食源性泌尿道感染危害風險的肉類 (雞肉)，並以我國市售雞胸肉上仍存在泌尿致病性大腸桿菌的假設為前提，透過預測微生物學去建立熱失活模型，用以預測在不同溫度 (50、55、60及63°C) 舒肥烹調過程時有無使用鹽水醃漬對雞胸肉中泌尿致病性大腸桿菌的生長和存活情況的影響。本研究從臺灣的傳統市場和超市共收集了 65 個生肉樣品 (包含雞肉、豬肉和牛肉)。從樣品中分離出的大腸桿菌使用聚合酶鏈反應 (PCR) 被進一步鑑定 UPEC的特異性基因、系統分群基因與毒力因子。接著，將泌尿致病性大腸桿菌接種至雞胸肉以獲得存活曲線。同時額外建立58°C的存活曲線作為驗證，並以均方根誤差 (RMSE)、殘差平方和 (SSE)、調整後R平方 (Adjusted R2)、赤池資訊量準則 (AIC)、準確因子 (Af)、偏差因子 (Bf) 和可接受預測區域法 (APZ) 作為統計指標。 本研究結果顯示，臺灣市售肉類中存在泌尿致病性大腸桿菌，其中雞肉 (22.6%) 是受泌尿致病性大腸桿菌污染最多的肉類，其次是豬肉 (14.6%) 與牛肉 (13.5%)。從臺灣市售生肉中共分離了861 株大腸桿菌，其中156 株 (18.1%) 被鑑定為 UPEC。UPEC分離株中以系統分群 F 最佔優勢 (36.5%)，其次為 D (24.4%)和 B1 (11.5%)。在10個毒力基因中以fimH (69.2%)、traT (57.7%)、iutA (51.9%) 和ompT (46.8%) 為最常見。黏附素基因fimH主要分布在系統分群A、B1、E及F，而保護蛋白基因traT、ompT與鐵載體基因iutA則主要分布在系統分群B1、D、B2及A。在熱失活模型中，有無鹽水醃漬樣本的存活菌量皆會隨著加熱時間越久而下降。而鹽水醃漬是個有效的措施可在舒肥烹調過程時降低雞胸肉中 UPEC 菌量與其D 值。本實驗所建立的存活模式與驗證的存活曲線皆符合統計指標的標準，顯示此熱失活模型可準確進行預測。因此，本研究指出臺灣市售肉類存在泌尿致病性大腸桿菌污染的風險，並表明黏附素、鐵載體、保護蛋白等毒力因子對於UPEC能入侵泌尿系統並引起泌尿道感染扮演重要角色。而系統分群與毒力因子的分佈結果有助於了解我國泌尿致病性大腸桿菌生態並建立我國食源性泌尿道感染的流行病學資料庫，進而提出泌尿致病性大腸桿菌在即食舒肥雞胸肉之存活模式的預測公式 (the Linear model)，此模型可作為控制該類型產品的微生物危害並進行暴露評估的工具，來預防未來食源性泌尿道感染的發生。

Urinary tract infection (UTI) is known as a multi-microbial infectious disease, in which uropathogenic Escherichia coli (UPEC) accounts for about 65-75% of UTIs. Recent studies indicated that poultry products, especially chicken, could be a reservoir for human extraintestinal pathogenic E. coli (ExPEC) and UPEC was suspected the pathogen resulting in emerging foodborne UTIs. As a result, it is a food safety issue problem that should not be ignored. However, to our knowledge, there is no data on whether retail meats in Taiwan can be the reservoirs of UPEC. Sous-vide defines raw ingredients packaged in a vacuum under low-temperature long-time (LTLT) cooking, making food be better sensory quality and preserving more nutrients. Nevertheless, previous research found that Salmonella could still be isolated from sous-vide cooked chicken, suggesting that the LTLT process might not inactivate all kinds of pathogens. Salt (sodium chloride, NaCl) marinade is one of the most widely used poultry marinades during sous-vide cooking. More importantly, the studies have shown the efficacy of salt marinade in reduction pathogens among meat that can prolong the shelf life of meat, reducing bacterial growth as well as improving its texture. For example, Mukherjee et al. reported that a 2.5% NaCl solution can inhibit the growth of E. coli O157:H7 in ground beef. This means that safety concerns about the presence of microbial hazards in sous-vide food products still exist; hence, sous-vide treatment should apply extra hurdles as salt marinade for the assurance of these products’ safety. Therefore, the objectives of this study were proposed as follows: (1) investigate the prevalence of UPEC isolated from retail meats in Taiwan; (2) form the consumer’s perspective, choosing the meat (chicken) with the highest risk of foodborne UTI, assuming that UPEC remains on the retail chicken breast in Taiwan, and applying predictive microbiology to establish inactivation modeling of UPEC in chicken breast samples with or without salt marinade via sous-vide processing at different temperatures to evaluate the behavior of UPEC. A total of 65 raw meat samples (chicken, pork, and beef) were collected from traditional markets and supermarkets in Taiwan. The E. coli strains isolated from meat samples were further identified the expression of UPEC-specific, phylogenetic, and virulence genes by polymerase chain reaction (PCR). Then, the chicken breast meat was inoculated with a four-strain UPEC cocktail to obtain the survival curves via sous-vide processing at 50, 55, 60 and 63°C. Meanwhile, survival curves at 58°C were constructed for external validation and RMSE, SSE, adjusted R2, AIC, Af, Bf, and the APZ method (pRE) were used as the statistical indices. The results in the study showed that the presence of UPEC among various retail meat in Taiwan, which chicken (22.6%) was with the highest number of the meat contaminated with UPEC, followed by pork (14.6%) and beef (13.5%). All the 861 E. coli isolates from retail raw meat in Taiwan, 156 (18.1%) strains were identified as UPEC. In UPEC isolates, phylogenetic group F was predominant (36.5%), followed by D (24.4%), and B1 (11.5%). Among 10 virulence genes, fimH (69.2%), traT (57.7%), iutA (51.9%), and ompT (46.8%) genes were the most frequently observed. Adhesin gene of fimH was mainly detected in strains of phylogroups A, B1, E, and F, whereas protection protein genes of traT and ompT and siderophore gene of iutA were commonly found in groups B1, D, B2, and A. For inactivation models, the numbers of survival bacterial cells exhibit a decline with time in all treatments. Salt marination is an effective hurdle resulting in lower survival UPEC populations and D-values in the chicken breast during sous-vide cooking. All the models for all treatments fitted well with UPEC survival curves; while the Linear model fitted much better. In conclusion, retail meat in Taiwan has the risk of UPEC contamination. Our findings demonstrate the distribution of phylogenetic groups and virulence factors in UPEC isolates from retail raw meat in Taiwan. It also indicates an important role of adhesins, protection protein, and iron acquisition systems that allowed UPEC to enhance their capacity to colonize the genitourinary system and cause UTIs. The developed survival functions based on the Linear model of UPEC in sous-vide ready-to-eat (RTE) chicken breast can be a tool for controlling the microbial hazards and for exposure assessment to prevent future foodborne UTIs.