Acquired antibiotic resistance in lactic acid bacteria from food

Acquired antibiotic resistance, i.e. resistance genes located on conjugative or mobilizable plasmids and transposons can be found in species living in habitats (e.g. human and animal intestines) which are regularly challenged with antibiotics. Most data are available for enterococci and enteric lactobacilli. Raw material from animals (milk and meat) which are inadvertantly contaminated with fecal matters during production will carry antibiotic resistant lactic acid bacteria into the final fermented products such as raw milk cheeses and raw sausages. The discovered conjugative genetic elements of LAB isolated from animals and food are very similar to elements studied previously in pathogenic streptococci and enterococci, e.g. -type replicating plasmids of the pAM1, pIP501-family, and transposons of the Tn916-type. Observed resistance genes include known genes like tetM, ermAM, cat, sat and vanA. A composite 29'871 bp resistance plasmid detected in Lactococcus lacti s subsp. lactis isolated from a raw milk soft cheese contains tetS previously described in Listeria monocytogenes, cat and str from Staphylococcus aureus. Three out of five IS elements on the plasmid are almost or completely identical to IS1216 present in the vanA resistance transposon Tn1546. These data support the view that in antibiotic challenged habitats lactic acid bacteria like other bacteria participate in the communication systems which transfer resistance traits over species and genus borders. The prevalence of such bacteria with acquired resistances like enterococci is high in animals (and humans) which are regularly treated with antibiotics. The transfer of antibiotic resistant bacteria from animals into fermented and other food can be avoided if the raw substrate milk or meat is pasteurized or heat treated. Antibiotic resistance traits as selectable markers in genetic modification of lactic acid bacteria for different purposes are presently being replaced, e.g. by metabo lic traits to generate food-grade vectors.     

3种淡水环境中低核酸含量细菌的滤过性

【目的】增加低核酸含量(LNA)细菌与过滤性细菌之间的认识。【方法】采用流式细胞技术(FCM)、变性梯度凝胶电泳(DGGE)及统计学分析研究3种典型淡水环境中细菌群落与滤过性。【结果】LNA细菌与过滤性细菌在数量上具有很好的相关性(y=0.646x+22.42,R2=0.984,P0.01),细菌的滤过性不仅与细菌大小有关,还与细胞整体形状及其伸缩性有关;细菌群落组织与LNA细菌比重呈负相关性,而与HNA细菌呈正相关性。【结论】0.45μm膜过滤对水样微生物群落中的LNA细菌具有极强的筛选效果;细菌群落组织与其基于流式细胞技术测定的基因含量具有密切联系。     

市售酸奶中乳酸菌的鉴定与耐药性

[目的]检测市售酸奶中乳酸菌的种类及其耐药情况.[方法]收集10种来自5个不同厂家的酸奶,通过细菌基因组重复基因外回文序列-PCR (repetitive extragenic palindromic-PCR,rep-PCR)结合16S rRNA同源性分析的方法对分离的乳酸菌进行基因分型和菌种鉴定.利用药敏纸片扩散法(K-B法)对分离的乳酸菌进行针对7种抗生素的药敏实验,用PCR特异性扩增结合测序的方法检测每个样品中不同基因型菌株的耐药基因(包括红霉素耐药基因erm A、erm B和四环素耐药基因tetM、tetK、tet S、tetQ、tetO、tetL、tetW).[结果]10种市售酸奶中分离到100株乳酸菌.其中,德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckii ssp.bulgaricus)23株,干酪乳杆菌(Lactobacillus casei)26株,嗜热链球菌(Streptococcus thermophilus)30株,嗜酸乳杆菌(Lactobacillus acidophilus)5株,植物乳杆菌(Lactobacillus plantarum)6株,副干酪乳杆菌(Lactobacillus paracasei)10株.药敏实验发现所有100株乳酸菌均对链霉素和庆大霉素耐药,42株对万古霉素耐药,没有菌株对头孢氨苄,四环素,红霉素以及土霉素耐药.在28株经过16S rRNA测序的乳酸菌中检测到5种不同的耐药基因,在8株乳酸菌中检测到erm B基因,4株检测到tetK基因,2株菌检测到tetL基因,4株菌检测到tet M基因,2株菌检测到tet O基因,没有检测到erm A,tet S,tet Q,tet W基因.28株乳酸菌中有15株(53.57%)检测到耐药基因,其中有4株L delbrueckii ssp.bulgaricus检测到2-3种不同的耐药基因.[结论]本研究在市售酸奶中除了检测到商品标签上标注的L.delbrueckii ssp.bulgaricus和S.thermophilus以外,还检测到商标上没有标注的乳酸菌;作为常用发酵剂的德氏乳杆菌保加利亚亚种和嗜热链球菌更容易检测到耐药基因;分离得到的乳酸菌均对红霉素和四环素敏感却检测到相应的耐药基因,再一次证明了没有耐药表型的菌株也可能携带耐药基因.     

Combating antibiotic resistance in bacteria

Combinations of certain antibiotics select against resistant strains of bacteria. This finding may provide a strategy of combating antibiotic resistant bacteria.     

Production of hydrolases by lactic acid bacteria and bifidobacteria and their antibiotic resistance

It was demonstrated that bifidobacteria and lactic acid bacteria B. adolescentis and Lactobacillus sp. synthesized extracellular enzymes cleaving glycoside bonds in the molecules of dextran, pectic acid, and soluble starch. The maximal production of extracellular β-galactosidase by B. adolescentis 91-BIM and 94-BIM at a rate of 0.08 and 0.03 U/mg per h was observed during the exponential growth phase at 5 and 12 h of cultivation, respectively. The cultures of bifidobacteria retained 60–70% of β-galactosidase and α-amylase activities after six months of storage. The bifidobacterium strains studied were resistant to amphotericin and aminoglycosides (gentamicin, kanamycin, and netromycin). The lactam antibiotics (ampicillin, benzylpenicillin, bicillin 3, bicillin 5, and carbenicillin), the preparations inhibiting protein synthesis at the level of ribosomes (lincomycin), RNA polymerase inhibitors (rifampin), cephalosporin, and Maxipime inhibited the growth of bifidobacteria. Rifampin, erythromycin, amphotericin, Maxipime, Fortum, doxycycline, levomycetin, streptomycin, and the aminoglycosides netromycin, gentamicin, and kanamycin did not have an effect on the growth of Lactobacillus sp., whereas semisynthetic derivatives of penicillin, carbenicillin and ampicillin, inhibited its growth as well as Oxamp and lincomycin. The lactam antibiotics benzylpenicillin, bicillin 3, and bicillin 5 inhibited the growth of lactic acid bacilli by 30–90%.     

Production of hydrolases by lactic acid bacteria and bifidobacteria and their antibiotic resistance

It was demonstrated that bifidobacteria and lactic acid bacteria B. adolescentis and Lactobacillus sp. synthesized extracellular enzymes cleaving glycoside bonds in the molecules of dextran, pectic acid, and soluble starch. The maximal production of extracellular beta-galactosidase by B. adolescentis 91-BIM and 94-BIM at a rate of 0.08 and 0.03 U/mg h was observed during the exponential growth phase at 5 and 12 h of cultivation, respectively. The cultures of bifidobacteria retained 60-70% of beta-galactosidase and alpha-amylase activities after six months of storage. The bifidobacterium strains studied were resistant to amphotericin and aminoglycosides (gentamicin, kanamycin, and netromycin). The lactam antibiotics (ampicillin, benzylpenicillin, bicillin 3, bicillin 5, and carbenicillin), the preparations inhibiting protein synthesis at the level of ribosomes (lincomycin), RNA polymerase inhibitors (rifampin), cephalosporin, and Maxipime inhibited the growth of bifidobacteria. Rifampin, erythromycin, amphotericin, Maxipime, Fortum, doxycycline, levomycetin, streptomycin, and the aminoglycosides netromycin, gentamicin, and kanamycin did not have an effect on the growth of Lactobacillus sp., whereas semisynthetic derivatives of penicillin, carbenicillin and ampicillin, inhibited its growth as well as Oxamp and lincomycin. The lactam antibiotics benzylpenicillin, bicillin 3, and bicillin 5 inhibited the growth of lactic acid bacilli by 30-90%.     

Molecular characterization of intrinsic and acquired antibiotic resistance in lactic acid bacteria and bifidobacteria

The minimum inhibitory concentrations (MICs) of 6 different antibiotics (chloramphenicol, clindamycin, erythromycin, streptomycin, tetracycline and vancomycin) were determined for 143 strains of lactic acid bacteria and bifidobacteria using the Etest. Different MICs were found for different species and strains. Based on the distribution of these MIC values, most of the strains were either susceptible or intrinsically resistant to these antibiotics. However, the MIC range of some of these antibiotics showed a bimodal distribution, which suggested that some of the tested strains possess acquired antibiotic resistance. Screening for resistance genes was performed by PCR using specific primers, or using a DNA microarray with around 300 nucleotide probes representing 7 classes of antibiotic resistance genes. The genes identified encoded resistance to tetracycline [tet(M), tet(W), tet(O) and tet(O/W)], erythromycin and clindamycin [erm(B)] and streptomycin [aph(E) and sat(3)]. Internal portions of some of these determinants were sequenced and found to be identical to genes described in other bacteria. All resistance determinants were located on the bacterial chromosome, except for tet(M), which was identified on plasmids in Lactococcus lactis. The contribution of intrinsic multidrug transporters to the antibiotic resistance was investigated by cloning and measuring the expression of Bifidobacterium breve genes in L. lactis.     

新疆发酵乳品中乳酸菌的分离鉴定及其耐药性

目的对新疆传统发酵乳品中乳酸菌进行分离鉴定并检测其耐药性。方法利用传统形态学鉴定法和生化鉴定等方法对新疆发酵乳中乳酸菌进行鉴定,采用纸片扩散法对分离鉴定的菌进行耐药性分析。结果从新疆发酵乳品中共分离出8株乳酸菌,经鉴定分别为瑞士乳杆菌(Lactobacillus helveticus)、植物乳杆菌(Lactobacillus plantarum)、马乳酒样乳杆菌(Lactobacillus kefianofaciens)、乳酸乳球菌(Lactococcus lactis)、副干酪乳杆菌(Lactobacillus paracasei)、副干酪乳杆菌类坚韧亚种(Lactobacillus paracasei subsp.tolerans)、哈尔滨乳杆菌(Lactobacillus harbinensis)、希氏乳杆菌(Lactobacillus hilgardii),并且发现8株乳酸菌对万古霉素、庆大霉素、阿莫西林、多西环素、环丙沙星、阿奇霉素、头孢他啶、头孢孟多具有一定敏感性。结论新疆发酵乳品中以乳杆菌居多,对常见抗生素具有一定的敏感性。     

Recent investigations and updated criteria for the assessment of antibiotic resistance in food lactic acid bacteria

The worldwide use, and misuse, of antibiotics for about sixty years in the so-called antibiotic era, has been estimated in some one to ten million tons, a relevant part of which destined for non-therapeutic purposes such as growth promoting treatments for livestock or crop protection. As highly adaptable organisms, bacteria have reacted to this dramatic change in their environment by developing several well-known mechanisms of antibiotic resistance and are becoming increasingly resistant to conventional antibiotics. In recent years, commensal bacteria have become a cause of concern since they may act as reservoirs for the antibiotic resistance genes found in human pathogens. In particular, the food chain has been considered the main route for the introduction of animal and environment associated antibiotic resistant bacteria into the human gastrointestinal tract (GIT) where these genes may be transferred to pathogenic and opportunistic bacteria. As fundamental microbial communities in a large variety of fermented foods and feed, the anaerobe facultative, aerotolerant lactic acid bacteria (LAB) are likely to play a pivotal role in the resistance gene exchange occurring in the environment, food, feed and animal and human GIT. Therefore their antibiotic resistance features and their genetic basis have recently received increasing attention. The present article summarises the results of the latest studies on the most typical genera belonging to the low G + C branch of LAB. The evolution of the criteria established by European regulatory bodies to ensure a safe use of microorganisms in food and feed, including the assessment of their antibiotic resistance is also reviewed.     

Studies on the nucleic acid of human bladder carcinoma

DNA was isolated from different histopathologic types and grades of human bladder carcinoma. The isolated DNA was submitted to quantitative determination and base composition analysis. A pilot study was done on the effect of gamma irradiation as a physical mutagen on characteristics of DNA in the examined tissues. Identity in the genetic components in the urinary bilharziasis snails and the human bladder cancer was observed. The same was observed in both intestinal bilharziasis snails and the cancerous intestinal tissues.     

革兰阴性细菌耐药特性的双聚类分析

目的通过双聚类分析了解临床分离的革兰阴性菌的耐药特征。方法采用K-B法对临床分离的113株细菌进行药物敏感试验,用软件WHONET 5.6和MATLAB对药敏试验结果进行统计分析。结果传统耐药分析方法表明113株细菌总体耐药率较低,其中大肠埃希菌、铜绿假单胞菌和鲍曼不动杆菌的耐药率则较高,而肺炎克雷伯菌耐药率较低。通过双聚类分析,所有菌株被聚为三大类,I类菌株占23.0%,耐药率最高,几乎对18种药物都耐药,细菌种类以肺炎克雷伯菌、铜绿假单胞菌、鲍曼不动杆菌为主;Ⅱ类菌株占56.6%,耐药率普遍较低,以肺炎克雷伯菌为主;Ⅲ类菌株占20.4%,菌株的耐药率介于I和Ⅱ类之间,包括肺炎克雷伯菌、铜绿假单胞菌和大肠埃希菌。其中Ⅱ大类,根据所耐抗生素的不同又分为Ⅱ-A、Ⅱ-B、Ⅱ-C三个亚类,每一亚类都具有相似的耐药特点。结论本实验收集的革兰阴性菌株根据耐药特征被聚为Ⅰ、Ⅱ、Ⅲ类,耐药程度为IⅢⅡ,双聚类分析法有利于快速找到具有相同耐药特征的菌株以及不同菌株之间的耐药差别。     

Effect of chlorination on antibiotic resistance profiles of sewage-related bacteria

A total of 1,900 lactose-fermenting bacteria were isolated from raw sewage influent and chlorinated sewage effluent from a sewage treatment plant, as well as from chlorinated and neutralized dilute sewage, before and after a 24-h regrowth period in the laboratory. Of these isolates, 84% were resistant to one or more antibiotics. Chlorination of influent resulted in an increase in the proportion of bacteria resistant to ampicillin and cephalothin, the increase being most marked after regrowth occurred following chlorination. Of the other nine antibiotics tested, chlorination resulted in an increased proportion of bacteria resistant to some, but a decrease in the proportion resistant to the remainder. Multiple resistance was found for up to nine antibiotics, especially in regrowth populations. Identification of about 5% of the isolates showed that the highest proportion of Escherichia coli fell in untreated sewage. Some rare and potentially pathogenic species were isolated from chlorinated and regrowth samples, including Yersinia enterocolitica, Yersinia pestis, Pasteurella multocida, and Hafnia alvei. Our results indicate that chlorination, while initially lowering the total number of bacteria in sewage, may substantially increase the proportions of antibiotic-resistant, potentially pathogenic organisms.