Table_3_Surveillance of Extended-Spectrum β-Lactamase-, Cephalosporinase- and Carbapenemase-Producing Gram-Negative Bacteria in Raw Milk Filters and Healthy Dairy Cattle in Three Farms in Île-de-France, France.XLSX

The aim of this work was to test a surveillance protocol able to detect extended-spectrum β-lactamase (ESBL)-, cephalosporinase (AmpC)- and carbapenemase (CP)-producing gram-negative bacteria in three conveniently chosen dairy farms with known prior occurrences of ESBL- and CP-producing strains. The protocol was applied monthly for a year. At each visit, 10 healthy lactating dairy cows were rectally swabbed, and raw milk filters (RMFs) were sampled in two of the three farms. Bacterial isolation was based on a first screening step with MacConkey agar supplemented with 1 mg/L cefotaxime and commercial carbapenem-supplemented media. We failed to detect CP-producing strains but showed that ESBL-Escherichia strains, found in one farm only (13 strains), were closely associated with multi-drug resistance (12 out of 13). The limited number of conveniently selected farms and the fact that RMFs could not be retrieved from one of them limit the validity of our findings. Still, our results illustrate that ESBL-status changes monthly based on fecal swabs and negative herds should be qualified as “unsuspected” as proposed by previous authors. Although surveillance of farm statuses based on RMF analysis could theoretically allow for a better sensitivity than individual swabs, we failed to illustrate it as both farms where RMFs could be retrieved were constantly negative. Determination of CP herd-level status based on RMFs and our surveillance protocol was hindered by the presence of intrinsically resistant bacteria or strains cumulating multiple non-CP resistance mechanisms which means our protocol is not specific enough for routine monitoring of CP in dairy farms.

本研究旨在验证一套监测方案，该方案可在3个经便利选取且既往存在产超广谱β-内酰胺酶（extended-spectrum β-lactamase, ESBL）和产碳青霉烯酶（carbapenemase, CP）菌株的奶牛场中，检测产超广谱β-内酰胺酶、头孢菌素酶（cephalosporinase, AmpC）及碳青霉烯酶的革兰氏阴性菌。该方案以月度为采样周期，连续监测一整年。每次采样时，对10头健康泌乳奶牛进行直肠拭子采样；同时在3个奶牛场中的2个内采集生鲜乳过滤器（raw milk filters, RMFs）样本。细菌分离流程首先采用添加1 mg/L头孢噻肟的麦康凯琼脂（MacConkey agar）以及商品化碳青霉烯类补充培养基完成初筛。本研究未检出产碳青霉烯酶菌株，但发现仅在1个奶牛场中分离到的产ESBL埃希菌菌株（共13株）与多重耐药性显著相关，13株菌株中有12株表现为多重耐药。本研究存在一定局限性：便利选取的奶牛场样本量有限，且其中1个奶牛场无法获取生鲜乳过滤器样本，这限制了本研究结果的可信度与外推性。尽管如此，本研究结果表明：基于粪便拭子检测的产ESBL菌株定植状态存在月度波动，且此前有学者提出，应将检测结果为阴性的牛群归类为"unsuspected"状态。尽管从理论层面而言，基于生鲜乳过滤器分析的牛群状态监测灵敏度优于个体拭子采样，但本研究未证实这一优势：可获取生鲜乳过滤器样本的2个奶牛场的检测结果始终为阴性。基于生鲜乳过滤器分析与本研究监测方案的牛群产CP状态判定受到干扰，原因是存在固有耐药菌或同时积累多种非碳青霉烯酶耐药机制的菌株，这意味着本监测方案的特异性不足以支撑奶牛场产碳青霉烯酶的常规监测工作。