生牛奶中分离大肠杆菌对季铵盐类消毒剂耐药性研究

目的为调查牛奶中大肠杆菌Escherichia coli的污染状况和对常见季铵盐类消毒剂的耐药情况,为大型养殖场季铵盐类消毒剂的合理使用提供指导。方法对从74份生牛奶样品中分离的45株大肠杆菌采用琼脂稀释法测定4种季铵盐类消毒剂[十六烷基三甲基溴化铵(CTAB)、苯扎氯铵(BC)、双十烷基二甲基氯化铵(DDAC)、氯代十六烷基吡啶(CPC)]的最小抑菌浓度(MIC),采用PCR法检测10种消毒剂耐药基因,并分析其MIC值与耐药基因之间的相关性。结果大肠杆菌对4种消毒剂表现出不同程度的抗性,所检测的4种消毒剂对大肠杆菌的MIC值均大于标准菌株,且比例为BC=DDACCTABCPC。大肠杆菌季铵盐类消毒剂的耐药基因检出率为ydgF(84.44%)ydgE(80.00%)sugE(c)(66.67%)emrE(40.00%)mdfA(37.78%)qacEΔ1(33.33%)qacE(17.78%)qacF(13.33%)qacG(11.11%)sugE(p)(4.44%)。分析大肠杆菌消毒剂基因检出情况与MIC值之间的关系发现,qacF基因的检出率与128 mg·L~(-1)的CPC之间差异有统计学意义。结论由此可见,牛奶中存在大肠杆菌污染严重的现象对季铵盐类消毒剂耐药情况不容乐观,需进一步规范季铵盐类消毒剂的使用。     

大熊猫源大肠杆菌及肺炎克雷伯氏菌对消毒剂耐药性研究

本实验对大熊猫肠道分离的88株大肠杆菌、32株肺炎克雷伯氏菌对季铵盐类消毒剂BC、CTPC、CTAB及DDAC的最小抑菌浓度(MIC)值进行测定,并扩增了消毒剂的耐药基因。结果显示,大肠杆菌对季铵盐类消毒剂MIC值为:BC的MIC值介于8~128 mg/L;CTPC的MIC值在32~256 mg/L之间;CTAB的MIC值为64~512 mg/L;DDAC的MIC值介于8~128 mg/L。肺炎克雷伯氏菌对季铵盐类消毒剂的耐药情况为:BC的MIC值介于16~512 mg/L;CTPC的MIC值在64~256 mg/L之间;CTAB的MIC值介于128~512 mg/L;DDAC的MIC值介于8~64 mg/L。可见,肺炎克雷伯氏菌对季铵盐类消毒剂的MIC值要大于大肠杆菌对季铵盐类消毒剂的MIC值。耐药基因检测结果表明,大肠杆菌季铵盐类消毒剂的染色体型耐药基因扩增率为68.18%~98.86%,最高为sug E(98.86%),emr E最低(68.18%),没有检测出qac E、qac F、qac G,检出率最高的可移动遗传元件介导耐药基因为qac EΔ1(19.31%)。肺炎克雷伯氏菌的染色体型耐药基因检出率为13.64%~28.41%,ydg E最高(28.14%),emr E检出率最低(13.64%),可移动遗传元件介导耐药基因sug E(p)检出率最高(6.82%),qac EΔ1、qac F、qac G基因未检出。测定大熊猫源大肠杆菌及肺炎克雷伯氏菌对消毒剂的耐药性,对圈养大熊猫消毒剂的规范使用,防控大熊猫细菌性疾病以及细菌对消毒剂耐药性有着重要意义。     

沙门氏菌对季铵盐类消毒剂的适应性耐受机制研究进展

沙门氏菌（Salmonella）是一种极其重要的食源性致病菌,具有耐高温、抗逆性强的特点,能在食品加工环境中长久存活。季铵盐类消毒剂在食品工业中的广泛应用使得沙门氏菌对其产生了适应性耐受和交叉耐受,为食品健康带来了极大的安全隐患。文章简要介绍了季铵盐类消毒剂的主要种类及其作用机理,分析并归纳了沙门氏菌对季铵盐类消毒剂的适应性耐受和交叉耐受现状,进一步从细胞膜生理特征、生物被膜形成、外排泵活性等角度,重点阐述了沙门氏菌对季铵盐类消毒剂的适应性耐受机制进展及新发现,并对未来研究方向进行了展望,旨在为季铵盐类消毒剂的合理使用和沙门氏菌的防控提供重要参考。     

鲍曼不动杆菌耐消毒剂基因检测

目的 了解深圳市人医院鲍曼不动杆菌抗季铵盐类消毒剂基因的检出率及基因型特点.方法 收集深圳市人民医院临床标本中分离鲍曼不动杆菌67株,PCR检测分离株的qacA/B、qacE△1、qacC、qacG和qacJ 5种抗季铵盐类消毒剂基因.结果 67株鲍曼不动杆菌中qacE△l基因阳性42株(62.6％),qacA/B与qacG基因各有一菌株阳性,未检测出qacC或qacJ基因阳性菌株.结论 深圳市人医院鲍曼不动杆菌中抗季铵盐类消毒剂基因检出率较高,主要为qacE△1型.     

复合季铵盐消毒剂含量的测定与滴定误差分析

研究复合季铵盐消毒剂含量的测定与滴定误差分析。探讨了四苯硼钠标准液配制及对滴定分析终点颜色的影响,并与单季铵盐定量分析方法进行了对比。结果表明:四苯硼钠两相滴定法适合于复合季铵盐定量分析,但要注意四苯硼钠标准液配制的正确性及滴定终点颜色的判断。     

评价两种消毒剂对IVF实验室微环境和妊娠结局的影响

目的在辅助生殖体外受精(in vitro fertilization,IVF)实验室的胚胎操作过程中,根据消毒剂对胚胎质量和妊娠结局的影响,评价季铵盐消毒剂是否比75%酒精消毒液更适合用于IVF实验室。方法回顾分析2016年1月至9月在我中心行IVF的619例新鲜移植周期,根据IVF实验室使用的消毒剂不同分为两组,A组383例为使用75%酒精消毒组,B组236例使用季铵盐组。对比两组的VOC浓度、获卵数、获卵率、受精率、多精率、卵裂率、优质胚胎率、优质囊胚形成率、着床率、临床妊娠率以及流产率。结果相比于A组,B组的VOC浓度显著降低;A、B两组的获卵数、获卵率、受精率、多精率、卵裂率、优质胚胎率、优质囊胚形成率和流产率的差异均不具有统计学意义(P0.05);但B组的着床率和妊娠率显著高于A组(P0.05)。结论两组对胚胎的体外培养质量没有显著性的影响,但使用季铵盐组相比于75%酒精组胚胎着床率和妊娠率显著提高。因此,季铵盐消毒剂比75%酒精更适合作为IVF实验室消毒剂。     

铜绿假单胞菌耐消毒剂基因检测

目的了解深圳市人民医院临床分离的铜绿假单胞菌耐季胺盐类消毒剂基因(qac)的检出率及基因型。方法收集深圳市人民医院近几年铜绿假单胞菌临床分离菌株63株,应用PCR法检测菌株的qacA/B、qacC、qacG、qacJ和qacE△1基因。结果63株铜绿假单胞菌中,qacE△1基因阳性51株(80.9%),qacA/B基因阳性5株(7.9%),其余的基因型未检出。结论我院临床分离的铜绿假单胞菌中耐季铵盐类消毒剂基因检出率较高,主要为qacE△1型。     

常用消毒剂对布鲁氏菌疫苗株的灭菌效果评估

【背景】布鲁氏菌是一种重要的人畜共患胞内寄生菌,是引起动物和人发生布鲁氏菌病(布病)的病原体。高效消毒剂杀灭环境中的布鲁氏菌是防控布病的关键措施。【目的】评价市面上常用消毒剂对布鲁氏菌的灭菌效果及差异。【方法】选用市面上9种不同类型的常用消毒剂,以高、中、低3种不同使用浓度分别研究其对牛种、羊种和猪种布鲁氏菌的杀灭效果。同时根据消毒剂的有效浓度通过最小抑菌浓度实验确定各消毒剂的最小抑菌浓度。【结果】除苯酚外,其余8种消毒剂,包括聚维酮碘、癸甲溴铵、苯扎溴铵、戊二醛、复合季铵盐、乙醇和2种商品化消毒剂等在推荐使用浓度下5 min内均能完全杀灭布鲁氏菌,碘酸制剂在0.1%低使用浓度下细菌存活率为0.000039%-0.000095%。苯酚按0.5%的推荐剂量,作用5 min后细菌存活率为0.000144%-0.000183%,20 min后,细菌存活率为0.000138%-0.000193%;按1%的使用剂量作用5 min后,猪种布鲁氏菌存活率为0.000125%,作用20min后的存活率下降至0.000038%。最小抑菌浓度研究结果表明,季铵盐类消毒剂其最小抑菌浓度大致在0.01563%-0.03125%之间,其余消毒剂的最小抑菌浓度结果与消毒剂最低推荐使用浓度基本一致。研究同时发现,不同种属布鲁氏菌对同一消毒剂的敏感性也存在一定差异。【结论】研究结果为生产实践中科学选择布鲁氏菌消毒剂提供了借鉴。     

细菌抵抗消毒剂及其对抗生素共耐药

消毒剂是一种可杀灭物体表面、器材设备、皮肤、空气和水源等传播媒介上携带的病原微生物的有机分子。它在体外能杀灭病原微生物,切断其传播途径,进而达到控制污染的目的,在生命安全防控中起着重要的作用。但是不合理地使用消毒剂导致细菌对消毒剂产生耐药。消毒剂耐药基因在不同种属间的水平转移加剧其传播风险,使消毒剂耐药情况进一步恶化。更令人担忧的是,细菌对消毒剂的耐药可能会导致对抗生素产生共耐药,给公共安全带来巨大的威胁。但目前为止,对消毒剂耐药以及共耐药的认识还不够全面。本文总结了关于细菌对消毒剂耐药的研究报道,对消毒剂的作用机制、细菌对消毒剂的耐药机制进行了论述,另外针对消毒剂耐药基因的传播以及细菌对消毒剂和抗生素的共耐药进行了综述,为减少消毒剂耐药性的产生和制定合理的消毒剂使用规范奠定基础。     

细菌抵抗消毒剂及其对抗生素共耐药

消毒剂是一种可杀灭物体表面、器材设备、皮肤、空气和水源等传播媒介上携带的病原微生物的有机分子。它在体外能杀灭病原微生物,切断其传播途径,进而达到控制污染的目的,在生命安全防控中起着重要的作用。但是不合理地使用消毒剂导致细菌对消毒剂产生耐药。消毒剂耐药基因在不同种属间的水平转移加剧其传播风险,使消毒剂耐药情况进一步恶化。更令人担忧的是,细菌对消毒剂的耐药可能会导致对抗生素产生共耐药,给公共安全带来巨大的威胁。但目前为止,对消毒剂耐药以及共耐药的认识还不够全面。本文总结了关于细菌对消毒剂耐药的研究报道,对消毒剂的作用机制、细菌对消毒剂的耐药机制进行了论述,另外针对消毒剂耐药基因的传播以及细菌对消毒剂和抗生素的共耐药进行了综述,为减少消毒剂耐药性的产生和制定合理的消毒剂使用规范奠定基础。     

Bacterial resistance to disinfectants containing quaternary ammonium compounds

Quaternary ammonium compounds (QAC) are widely used as disinfectants in both medical and food environments. Microbial contaminants are, therefore, regularly exposed to their action and the isolation from clinical and food sources of resistant bacteria continues to be reported in many countries. Resistance to QAC in clinical strains of staphylococci is encoded by one of at least three resistance genes, designated qacA, qacB and qacC. Using hybridisation analysis, we have shown that these QAC resistance genes are also distributed among staphylococcal strains in the food industry. In addition, we have discovered two new resistance determinants in these food isolates, which are now being characterised and sequenced. Although the general level of resistance of pure cultures is low, the resistant strains have originally been isolated after exposure to the recommended user concentration of a commercial brand of QAC.We have also studied resistance to QAC in pseudomonads isolated from the food industry. Their level of resistance is much higher than that found in staphylococci. About 30% of the collected strains were able to grow in 200 μg·ml⁻¹ benzalkonium chloride, the lowest recommended use concentration for this commonly used type of QAC.     

Changes in fatty acid composition of Pseudomonas aeruginosa ATCC 15442 induced by growth conditions: consequences of resistance to quaternary ammonium compounds

The effects of changes in the fatty acid composition of Pseudomonas aeruginosa induced by growth conditions on its resistance to two quaternary ammonium compounds (QAC) were investigated. The temperature and growth phase were the most influential parameters affecting the fatty acid composition of this bacterium. Furthermore, the formation of saturated fatty acids and cyclopropane fatty acids was stimulated by increasing the temperature, whereas the proportion of unsaturated fatty acids fell. The degree of saturation and the proportion of cyclopropane fatty acids increased in the course of the exponential and stationary phases. These modifications mostly concerned the inner membrane of the bacterium. Resistance of P. aeruginosa to both QAC tested was not significantly influenced by temperature and growth phase variations. Thus, resistance to the two QAC did not seem to be dependent on modifications of the fatty acid composition of the inner membrane.     

Effects of quaternary-ammonium-based formulations on bacterial community dynamics and antimicrobial susceptibility

Quaternary ammonium compounds (QACs) are widely used as adjuncts to hygiene in domestic cleaning products. Current concern that the increased use of such biocides in consumer products might contribute to the emergence of antibiotic resistance has led us to examine the effects of a QAC-containing domestic cleaning fluid on the population dynamics and antimicrobial susceptibility of domestic sink drain biofilm communities. QAC susceptibilities of numerically dominant, culturable drain bacteria (15 genera, 17 species) were determined in vitro before and after repeated QAC exposure (14 passages). A fully characterized drain microcosm was then exposed to short-term (12 days) and long-term (3 months) dosing with a QAC-containing domestic detergent (QD). QAC exposure of isolated cultures caused both increases (three species) and circa twofold decreases (six species) in QAC susceptibility. The susceptibility of Ralstonia sp. was considerably decreased following 14 consecutive QAC passages. Control drain microcosm biofilms maintained dynamic stability, as evidenced by culture and denaturing gradient gel electrophoresis (DGGE) analysis. Bacterial population densities were largely unaffected during short-term exposure to use levels of QD, although 50% QD caused circa 10-fold viability reductions. DGGE analysis supported these observations; identified the major microcosm genera as Pseudomonas, Pseudoalteromonas, Erwinia, and Enterobacter, and showed that aeromonads increased in abundance under 10 to 50% QD. Long-term exposure of the microcosms to QD did not significantly alter the pattern of antimicrobial susceptibility. These data demonstrate the recalcitrance of domestic drain biofilms toward QAC and that although repeated QAC exposure of drain isolates in pure culture results in susceptibility change in some test bacteria, such changes do not necessarily occur within complex communities.     

Resistance of bacteria from cooling waters to bactericides

Summary Bacteria from water cooling systems developed resistance to three different bactericides i.e. quarternary ammonium compound (QAC), isothiazolone and thiocarbamate. Resistance was induced by exposing isolates to increasing sublethal concentrations for a period of 10 weeks.Bacillus subtilis became resistant to 1000 mg l^–1 QAC. Cross-resistance was also detected, e.g. isothiazolone induced resistance to QAC and thiocarbamate.     

Survival of protozoa in cooling tower biocides

Protozoa from cooling towers may serve as hosts for legionellae, but such protozoa have not been examined with respect to effects of cooling tower biocides. In this study, two ciliate species,Tetrahymena sp andColpoda sp, and two amoebae species,Vannella miroides andAcanthamoeba hatchetti, were isolated from a cooling tower and tested for survival in the presence of four cooling tower biocides. The protozoa were exposed for 24 h to a thiocarbamate compound, an isothiazolone compound, quaternary ammonium compounds (QAC), and tributyltin neodecanoate with quarternary ammonium compounds (TBT/QAC). After exposure, cells were examined for viability. The highest concentration of each biocide in which cells could survive was compared to the manufacturers' recommended maintenance dosage (MRMD) of the biocides.Tetrahymena andColpoda survived concentrations within the range of the MRMD of thiocarbamate and QAC.Vannella andAcanthamoeba survived concentrations within the MRMD of thiocarbamate, isothiazolone, and QAC.Colpoda cysts andAcanthamoebae cysts remained viable after exposure to concentrations much greater than the MRMD of thiocarbamate, isothiazolone, and QAC. None of the protozoa in any stage could survive the MRMD of TBT/QAC. These results show that protozoa indigenous to cooling towers may survive the recommended concentration of certain biocides, and this information may be important in devising procedures for eradicating hosts for legionellae.     

Effects of Quaternary-Ammonium-Based Formulations on Bacterial Community Dynamics and Antimicrobial Susceptibility

Quaternary ammonium compounds (QACs) are widely used as adjuncts to hygiene in domestic cleaning products. Current concern that the increased use of such biocides in consumer products might contribute to the emergence of antibiotic resistance has led us to examine the effects of a QAC-containing domestic cleaning fluid on the population dynamics and antimicrobial susceptibility of domestic sink drain biofilm communities. QAC susceptibilities of numerically dominant, culturable drain bacteria (15 genera, 17 species) were determined in vitro before and after repeated QAC exposure (14 passages). A fully characterized drain microcosm was then exposed to short-term (12 days) and long-term (3 months) dosing with a QAC-containing domestic detergent (QD). QAC exposure of isolated cultures caused both increases (three species) and circa twofold decreases (six species) in QAC susceptibility. The susceptibility of Ralstonia sp. was considerably decreased following 14 consecutive QAC passages. Control drain microcosm biofilms maintained dynamic stability, as evidenced by culture and denaturing gradient gel electrophoresis (DGGE) analysis. Bacterial population densities were largely unaffected during short-term exposure to use levels of QD, although 50% QD caused circa 10-fold viability reductions. DGGE analysis supported these observations; identified the major microcosm genera as Pseudomonas, Pseudoalteromonas, Erwinia, and Enterobacter, and showed that aeromonads increased in abundance under 10 to 50% QD. Long-term exposure of the microcosms to QD did not significantly alter the pattern of antimicrobial susceptibility. These data demonstrate the recalcitrance of domestic drain biofilms toward QAC and that although repeated QAC exposure of drain isolates in pure culture results in susceptibility change in some test bacteria, such changes do not necessarily occur within complex communities.     

Quantitation and purification of quaternary ammonium compounds from halophyte tissue

A simple and sensitive spectrophotometric assay for quaternary ammonium compounds (QACs) based on Dragendorff's reagent is described. Although not specific for a particular QAC, the assay allows for rapid survey of tissue for QAC content. A separation method for QACs in halophyte tissue containing high levels of cations was developed using ion exchange resins and ninhydrin to remove the amino acid fraction.     

Bacterial disinfectant resistance—a challenge for the food industry

The focus on hygiene in the food industry has resulted in an increasing use of chemical disinfection and it has been speculated that this will impose a selective pressure and contribute to the emergence of disinfectant-resistant microorganisms. The frequency of strains with a low-level resistance to quaternary ammonium compounds (QAC) is relatively high for Listeria monocytogenes (10%), Staphylococcus spp. (13%) and Pseudomonas spp. (30%) and lower for lactic acid bacteria (1.5%) and coliforms (1%) isolated from food and food processing industry. In general, bacteria isolated after disinfection are more resistant and represent a potential food safety or food spoilage problem. Adaptation to disinfectants may be accompanied by cross-resistance to related disinfectants. We have recently found a genetic linkage between resistance to QAC and antibiotics in food associated staphylococci, and there is a growing concern about cross-resistance between antibiotics and disinfectants.Disinfectant resistance can in most cases be prevented by effective cleaning and disinfection procedures. More effort should be made to avoid build-up of resistance in food production environments.     

Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment

The impact of human activity on the selection for antibiotic resistance in the environment is largely unknown, although considerable amounts of antibiotics are introduced through domestic wastewater and farm animal waste. Selection for resistance may occur by exposure to antibiotic residues or by co-selection for mobile genetic elements (MGEs) which carry genes of varying activity. Class 1 integrons are genetic elements that carry antibiotic and quaternary ammonium compound (QAC) resistance genes that confer resistance to detergents and biocides. This study aimed to investigate the prevalence and diversity of class 1 integron and integron-associated QAC resistance genes in bacteria associated with industrial waste, sewage sludge and pig slurry. We show that prevalence of class 1 integrons is higher in bacteria exposed to detergents and/or antibiotic residues, specifically in sewage sludge and pig slurry compared with agricultural soils to which these waste products are amended. We also show that QAC resistance genes are more prevalent in the presence of detergents. Studies of class 1 integron prevalence in sewage sludge amended soil showed measurable differences compared with controls. Insertion sequence elements were discovered in integrons from QAC contaminated sediment, acting as powerful promoters likely to upregulate cassette gene expression. On the basis of this data, >1 × 10¹⁹ bacteria carrying class 1 integrons enter the United Kingdom environment by disposal of sewage sludge each year.