金黃色葡萄球菌空氣採樣方法效能評估-暴露艙研究

Abstract

研究指出金黃色葡萄球菌（Staphylococcus aureus）可透過直接接觸或空氣傳播造成人員感染，為醫院院內感染常見之病原菌；此外，在畜養家禽場所、紡織、麵粉與食品相關工作環境中亦發現Staphylococcus spp.為空氣中主要菌種之一。抗藥性金黃色葡萄球菌（Methicillin-resistant Staphylococcus aureus）之出現，亦導致在治療此細菌感染時變得更為棘手。 有效監測此致病菌在職業場所空氣中是否存在及其濃度值，將有助於釐清勞工暴露風險；然何種採樣策略最能有效捕集空氣中的S. aureus，目前仍不清楚。有鑑於此，本研究透過建置生物氣膠產生系統，評估不同採樣介質與生物氣膠採樣器針對S. aureus之採樣效能。在評估S. aureus培養基部分，係以Andersen 1-STG搭配TSA、MSA、BPA、CSA與CSM進行6分鐘之空氣採樣，而後以培養法分析並計算不同培養基之效能指標（R）。結果顯示，非選擇性培養基TSA之R值顯著高於四類選擇性培養基之R值（Kruskal Wallis test, p = 0.0092, Scheff&eacute; test, p < 0.05）；而選擇性培養基中，以MSA採樣效能最高，然與其他三種選擇性培養基並未達統計上顯著差異。而後以Andersen 1-STG搭配具較佳空氣採樣效能之TSA與MSA 評估時間因子對採樣效能之影響，結果顯示兩種培養基均於採樣3分鐘時具有最佳之採樣效能（Kruskal Wallis test, TSA：p = 0.008, MSA：p = 0.0447, Scheff&eacute; test, p < 0.05），而6至60分鐘採樣所得之R值彼此間則未達統計顯著差異。 評估S. aureus收集液部分，係以AGI-30搭配Tween 80 mixture、PBS與DW進行3分鐘之採樣。結果顯示，Tween 80 mixture之R值皆顯著高於其他兩種收集液之R值，而PBS之R值顯著高於DW之R值（Kruskal Wallis test, p = 0.0002, Scheff&eacute; test事後檢定, p < 0.05）；接著以AGI-30與BioSampler搭配三種收集液進行3、6與15分鐘空氣採樣以評估流失率。結果顯示，採樣時間自3分鐘延長至15分鐘時，流失率顯著上升（Kruskal Wallis test, p < 0.0001, Scheff&eacute; test, p < 0.05）。而後評估AGI-30與BioSampler搭配具較佳空氣採樣效能之Tween 80 mixture與PBS，於不同採樣時間下之採樣效能，結果顯示採樣器搭配Tween 80 mixture採樣6分鐘時之採樣效能顯著高於3分鐘之R值（Kruskal Wallis test, AGI-30：p = 0.0013, BioSampler：p = 0.0018, Scheff&eacute; test事後檢定, p < 0.05），大於15分鐘之R值即隨採樣時間延長而下降；採樣器搭配PBS採樣時，其採樣效能於不同採樣時間下彼此間皆具顯著差異（Kruskal Wallis test, AGI-30：p = 0.0014, BioSampler：p = 0.0011, Scheff&eacute; test事後檢定, p < 0.05），且隨採樣時間增加而下降。 將所有採樣方法之Ｒ值進行統計檢定，結果顯示，BioSampler 搭配Tween 80 mixture之採樣效能最佳，顯著高於其他五種採樣方法；BioSampler 搭配PBS以及AGI-30搭配Tween 80 mixture次之，而AGI-30搭配PBS之採樣效能最差（Kruskal Wallis test, p < 0.0001, Scheff&eacute; test事後檢定, p < 0.05）。 本研究進一步根據文獻與本實驗結果，對S. aureus實場空氣採樣策略之建議，依採樣目的分為兩項： 1. 若欲進行環境空氣中S. aureus濃度監測，本研究建議使用BioSampler搭配PBS進行前測。於經消毒控制之環境，建議前測時間為30與60分鐘，後續將收集液濃縮至0.2mL進行培養法分析。於無特定S. aureus污染源或未經消毒控制之環境，建議前測時間為6-60分鐘，其中採樣時間為6-15分鐘時，可將收集液濃縮至0.2 mL；若採樣時間延長為30-60分鐘時，可將收集液濃縮至0.2 mL、0.5mL或1mL後再進行培養法分析。此外，於空氣中S. aureus濃度較高之環境採樣時，則建議前測時間為3-60分鐘，收集液可濃縮至1mL或直接經適當稀釋以培養法分析。 2. 若欲進行空氣中可培養性S. aureus之定性分析，此時建議可以BioSampler搭配Tween 80 mixture進行30-60分鐘之空氣採樣，採樣中並定時補充收集液。 本研究成果預期未來可應用於實際職場環境中，用以有效監測空氣中致病菌，協助決策者採取適當的控制策略，減少院內感染並保護勞工健康。

Previous studies have indicated that Staphylococcus aureus may be spread via respirable-sized aerosols and transmitted by direct contact and airborne route. Staphylococcus spp. are revealed as the dominant bacteria in poultry houses and textile, flourmill and food-related factories. In particular, the emergence of methicillin- resistant S. aureus results in difficult-to-treat infections. However, limited research has been conducted to investigate the sampling methods for airborne S. aureus. To deal with this issue, a bioaerosol generation system was developed to evaluate the performance of various sampling methods for collecting airborne S. aureus. Sampling performance (R) of five agar plates for recovering airborne S. aureus was determined by 6-min sampling using Andersen 1-STG followed by the culture assay. Results showed that the non-selective agar TSA performed significantly better than the other four selective agar types (Kruskal Wallis test, p = 0.0092, Scheff&eacute; test, p < 0.05). For the four selective agar media, the MSA performed best but at no statistical significance compared with the other three. The effects of sampling time on the performance of Andersen 1-STG for collecting airborne S. aureus onto TSA and MSA showed that the R with 3 min-sampling was significantly greater than those of the other sampling times regardless of agar type (Kruskal Wallis test, TSA：p = 0.008, MSA：p = 0.0447, Scheff&eacute; test, p < 0.05), while there was no statistical difference in sampling performance among the sampling times between 6 and 60 min. In addition, the sampling performance of three collection liquid types for recovering airborne S. aureus was determined by 3-min sampling using AGI-30 followed by the same assay. It shows that no matter what kind of agar arranged, Tween 80 mixture performed significantly better than the other two liquid types (Kruskal Wallis test, p = 0.0002, Scheff&eacute; test, p < 0.05), PBS the second. As for the loss rate of three collection liquid types for sampling airborne S. aureus was determined by 3, 6 and 15-min sampling using AGI-30 and BioSampler. Results indicated that there was no statistical difference in loss rate among the three liquid types. Also, liquid-based samplers showed no statistical significance compared with each other. When sampling time was extended from 3min to 15 min, however, there was a statistical increasing in loss rate (Kruskal Wallis test, p < 0.0001, Scheff&eacute; test, p < 0.05). Moreover, the effects of sampling time on the performance of AGI-30 and BioSampler for recovering airborne S. aureus with Tween 80 mixture showed that the R with 6 min-sampling was significantly greater than those of the other sampling times, including 3 min-sampling (Kruskal Wallis test, AGI-30：p = 0.0013, BioSampler：p = 0.0018, Scheff&eacute; test, p < 0.05). The aforementioned results may be attributable to the proliferation of S. aureus collected in Tween 80 mixture. For the effects of sampling time on the performance of AGI-30 and BioSampler arranged with PBS, results showed that the performance of them decreased significantly with prolonged sampling time (Kruskal Wallis test, AGI-30：p = 0.0014, BioSampler：p = 0.0011, Scheff&eacute; test, p < 0.05). In summary, the performance of BioSampler arranged with Tween 80 mixture was the best. BioSampler and AGI-30 arranged with PBS and Tween 80 mixture, respectively, the second. Further, AGI-30 arranged with PBS gave the worst performance (Kruskal Wallis test, p < 0.0001, Scheff&eacute; test, p < 0.05). The advisable sampling strategy depends on the purpose of sampling. To monitor the concentration of airborne S. aureus in environment, the sampling carried out for 15 min with BioSampler equipped with PBS is recommended. Furthermore, to performe the qualitative analysis of airborne S. aureus, the sampling conducted for 30-60 min with BioSampler equipped with Tween 80 mixture is applicable. It is expected that this study will be able to be applied in occupational and residential environment to effectively identify and monitor concentration of airborne S. aureus.