外科手术切口感染大肠埃希菌的耐药性分析

目的分析外科手术切口中大肠埃希菌的感染率及耐药性,为临床合理用药提供科学依据。方法对2009年6月至2010年9月外科患者手术切口标本中分离的大肠埃希菌耐药情况进行分析,并对产超广谱β-内酰胺酶（ESBLs）菌株进行表型确证试验。结果 140株大肠埃希菌中,产ESBLs菌株的检出率为42.14%;产ESBLs菌株对一～四代头孢菌素、广谱青霉素、氟哇诺酮类及氨基糖苷类抗菌药物具有较高的耐药率;非产ESBLs菌株对青霉素类以外的抗菌药物具有较高的敏感率;产ESBLs菌株对大多数抗菌药物的耐药率明显高于非产ESBLs菌株（P〈0.05）。结论外科手术切口术后大肠埃希菌的感染较严重,且产ESBLs菌株多药耐药明显,临床应加强监测与控制。     

Mycotoxin production from fungi isolated from grapes

AIMS: In order to assess the potential for producing mycotoxins, fungi were isolated from wine producing grapes. METHODS AND RESULTS: The isolates were identified and Penicillium expansum, the most well recognized mycotoxin producer, was analysed for mycotoxin production by TLC. Many of the strains produced patulin and/or citrinin, often depending on whether they were grown on a grape or yeast extract sucrose media. CONCLUSION: Citrinin was produced by all strains grown in the yeast extract sucrose medium, but only one strain (from 51) was able to produce this compound in grape juice medium. Patulin was produced in the yeast extract medium by 20 strains and in grape juice medium by 33 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of mycotoxins in wine producing grapes is discussed. Grapes contamination with patulin seems not to contribute to wine contamination, and no ochratoxin producing fungi was identified.     

小儿呼吸道感染肺炎克雷伯杆菌耐药性和基因型研究

目的探讨临海地区小儿呼吸道感染肺炎克雷伯杆菌产超β-内酰胺酶（ESBLs和AmpC酶）的耐药性及耐药基因型分布情况。方法采用VITEK-60型全自动细菌鉴定仪鉴定细菌,按CLSI推荐的确证试验检测ESBLs和K-B纸片法测定药敏结果;采用头孢西丁纸片扩散法筛选产AmpC酶阳性菌株,采用PCR检测AmpC酶基因,并对产物进行测序分析基因型。结果113株肺炎克雷伯杆菌ESBLs和AmpC酶总检测率分别为29．20％和18．58％,其中单产ESBLs、单产AmpC酶和同产AmpC酶＋ESBLs检出率分别为23．01％、12．39％和6．19％;AmpC酶阳性菌株的耐药基因型：16株为DHA-1型,5株为ACT-1型。药敏试验：所分离的肺炎克雷伯杆菌对亚胺培南全部敏感,对喹诺酮类耐药率很低,对大多β-内酰胺类抗生素耐药率较高,并且产酶株的耐药性明显高于非产酶株,耐药现象在同产ES-BLS和AmpC酶菌株中更为严重。结论临海地区小儿呼吸道分离的肺炎克雷伯杆菌产ESBLs和AmpC酶检出率较高;AmpC酶以DHA-1基因型流行为主,产酶株呈现出高度多重耐药性。     

不同产地美花石斛内生细菌分离及促生潜力比较

对采自广西、云南和广东的美花石斛(Dendrobium loddigesii Rolfe)野生植株根、茎和叶内的内生细菌进行分离并测定其促生特性,采用扩增核糖体DNA限制性酶切分析(ARDRA)与UPGMA聚类分析相结合的方法对内生细菌菌株进行分类并确定优势属;在此基础上,对具有解磷、解钾和产生长素能力的菌株进行促生潜力评价。结果显示:从不同产地美花石斛植株不同部位共分离得到67株内生细菌菌株,其分布呈现出组织与地区的特异性;其中,来源于广西的植株中菌株数量最多(42株),分离自茎的菌株数量最多(34株)。67株内生细菌菌株可分为31个ARDRA簇,经16S rDNA序列比对鉴定为12个属,包括假单胞菌属(Pseudomonas)、微杆菌属(Microbacterium)、肠杆菌属(Enterobacter)、芽孢杆菌属(Bacillus)、鞘氨醇单胞菌属(Sphingomonas)、葡萄球菌属(Staphylococcus)、嗜冷杆菌属(Psychrobacter)、短波单胞菌属(Brevundimonas)、涅斯捷连科氏菌属(Nesterenkonia)、副球菌属(Paracoccus)、泛菌属(Pantoea)和沙雷氏菌属(Serratia),其中芽孢杆菌属、微杆菌属和肠杆菌属为优势属;来源于广西的植株中内生细菌的丰度与多样性均高于其他两地。在67株内生细菌菌株中,有30株菌株具有解无机磷和解有机磷的双重能力、22株具有解钾能力、24株具有产生长素能力,其中仅8株菌株兼具3种促生能力。组培实验结果显示:在培养基中接种1×106CFU·mL-1芽孢杆菌DLB20菌株,对株高2~3 cm和3~4 cm的美花石斛试管苗生长有促进作用,且更有利于株高3~4 cm试管苗的生根,表明具有解磷、解钾和产生长素能力的菌株对美花石斛试管苗有一定的促生潜力。     

新疆尉犁县黑湖嗜盐嗜碱菌的分离及系统发育学分析

【目的】从新疆尉犁县黑湖中筛选分离获得嗜盐嗜碱微生物,并对筛选获得的微生物进行种属鉴定。【方法】采用传统分离鉴定技术,进行形态和生理生化特性研究和基于16S r RNA基因的序列分析。【结果】从样品中分离获得可培养嗜盐嗜碱菌25株,对其进行鉴定。根据生理生化特征、16S r RNA基因序列测定和系统发育分析表明,25株菌分布在古菌Halorubrum、Haloarcula、Natrialba、Halohasta和Halopiger等5个属。其中优势菌群为Halorubrum,次优势菌群为Natrialba。其中DH-66(KU663028)属于Halopiger属,16S r RNA基因序列同源性与该属的模式菌株Halopiger aswanensis 56T同源性最高,为95.75%,预示为潜在的新种(新种鉴定将另行报道)。25株嗜盐嗜碱菌生长条件实验表明,这些菌适应Na Cl的浓度范围为15%-30%、最适浓度为20%-25%,生长的p H范围为7.0-13.0、最适p H为9.0-10.0。各种水解酶类的分析表明,在分离的25株菌中产淀粉酶的菌有5株占20%、产蛋白酶的菌有4株占16%、产酯酶可水解吐温20的菌有15株占60%、可水解吐温40的有7株占28%、可水解吐温80的有4株占16%、产过氧化氢酶的菌有14株占56%。9株菌同时能产4种酶,2株菌同时能产3种酶。表明了嗜盐嗜碱菌产酶的多样性。19株菌硝酸盐还原为阳性。【结论】揭示了新疆尉犁县黑湖嗜盐嗜碱菌生理生化特性的多样性和系统发育多样性,而且蕴藏着较丰富的新的微生物类群,亟待系统研究和进一步开发利用。     

Comparative characteristics of penicillin-binding proteins of representatives of the genus Streptomyces

Penicillin-binding proteins (PBPs) in Strepomyces strains producing clavulanic acid and beta-lactamase and in Streptomyces strains not producing these compounds were studied comparatively. In S. clavuligerus, the organism producing clavulanic acid, there were detected 3 PBPs in the membrane fraction. S. griseus, the organism producing beta-lactamase, contained 6 PBP. In S. cacaoi and S. olivaceus, organisms producing neither beta-lactams nor beta-lactamase, there were detected 5 and 4 PBP, respectively. The set of the PBP in the organism producing clavulanic acid varied during fermentation. In a variant of S. clavuligerus isolated after protoplasting of the mycelial cells and their regeneration the content of the electrophoretically most mobile PBP lowered. The PBP of S. clavuligerus did no show any high affinity to other beta-lactams such as methicillin and ampicillin tested as competing agents of 14C-benzylpenicillin.     

Serogroups of Escherichia coli strains producing cytotoxic necrotizing factors CNF1 and CNF2

The serogroups of 396 necrotizing Escherichia coli of human and bovine origin isolated in Spain between 1979 and 1991 have been determined. The 270 cytotoxic necrotizing factor strains belonged to 22 different O serogroups; however, 84% (226 of 270) were of one of seven serogroups (O2, O4, O6, O14, O22, O75 and O83). Although necrotizing E. coli producing cytotoxic necrotizing factor 2 belonged to 28 different serogroups, only six of them (O1, O3, O15, O55, O88 and O123) accounted for 60% (76 of 126) of cytotoxic necrotizing factor 2 strains. Furthermore, only 3% (4 of 126) of cytotoxic necrotizing factor 2 strains belonged to serogroups most common among strains producing cytotoxic necrotizing factor 1. The majority of necrotizing E. coli producing cytotoxic necrotizing factor 1 were obtained from human extraintestinal infections, whereas cytotoxic necrotizing factor 2 strains were isolated from stools of healthy and diarrhoeic calves.     

湘江河岸土壤中高产甲壳素脱乙酰酶菌株的筛选及鉴定

【目的】甲壳素脱乙酰酶(CDA)是将天然甲壳素生物转化为可商品化利用的壳聚糖的关键酶。本文旨在从湘江河岸的土壤中筛选可高产CDA的新菌株。【方法】以甲壳素为唯一碳源,利用4'-硝基乙酰苯胺为显色剂,通过变色圈法进行产CDA菌株初筛,产酶活性分析复筛;通过形态学和ITS区序列特征对菌株进行鉴定。【结果】从湘江(长沙段)河岸边的土壤中分离出的117株菌株中筛选到可产CDA的菌株30株,其中4株具有较强产CDA的能力。进一步经发酵产酶分析验证,菌株A1具有较强的产CDA能力,其胞外CDA酶活高达13.21 U/m L。结合形态学和ITS区序列特征,菌株A1初步鉴定为层生镰孢菌。【结论】从湘江河岸边的土壤中筛选到可高产CDA的菌株A1,具有较好的开发应用前景。     

Use of the protoplast fusion method in the selection of Streptomyces griseus--the producer of the streptothricin antibiotic grisin

An effective method for protoplast fusion in S. griseus producing grisin was developed. The method requires the use of polyethylene glycol with a molecular weight of 1000. It was demonstrated that protoplasts formed most effectively in this organism, when the mycelium of the streptomycete previously treated with ultrasound in the process of its growth was used for the treatment with lysozyme. The efficacy of protoplast regeneration in the strains with the use of the modified hypertonic medium R2MD was 25-75 per cent. The possibility of using the protoplast fusion method for constructing phage resistant strains producing kormogrisin was shown.     

Production by fungi of the genera Aspergillus, Acremonium, Verticillium of extracellular proteases which coagulate blood plasma and lyse blood clots

Among 24 fungal strains belonging to the genera Aspergillus, Acremonium and Verticillium, eight strains synthesized proteases with the coagulating activity. The most active strains producing such proteases were found among Aspergillus species. The fibrinolytic activity was detected in 12 strains of the fungi. Proteases with the fibrinolytic activity differed in their sensitivity to plasma inhibitors and the least sensitive proteases were found in A. niger and A. flavus. Some fungal strains synthesized proteases with the fibrinolytic activity exerting an indirect action. Such enzymes activated the conversion of blood profibrinolysin into fibrinolysin in a manner similar to that of staphylokinase, urokinase and trypsin.     

Diarrhea in neonatal piglets caused by K99+ST+ enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli strains were isolated from the feces of 34.4% of 64 diarrheaic neonatal piglets on seven farms. Of a total of 518 isolates, 86 (16.6%) were enterotoxigenic and grouped into four phenotypes: K99+ST+ (K99 pilus antigen and heat-stable enterotoxin producing, 36 strains), ST+ (37 strains), K88+LT+ (K88 pilus antigen and heat-labile enterotoxin producing, 11 strains), and K88+ST+ (2 strains). K99+ST+ and ST+ isolates showed multiple drug resistance and most of those (58.3% and 56.8%, respectively) belonged to O serogroup 101. A K99+ST+ isolate proved to be capable of inducing diarrhea and death in hysterectomy-produced colostrum-deprived piglets when orally inoculated.     

A novel small acid soluble protein variant is important for spore resistance of most Clostridium perfringens food poisoning isolates

Clostridium perfringens is a major cause of food poisoning (FP) in developed countries. C. perfringens isolates usually induce the gastrointestinal symptoms of this FP by producing an enterotoxin that is encoded by a chromosomal (cpe) gene. Those typical FP strains also produce spores that are extremely resistant to food preservation approaches such as heating and chemical preservatives. This resistance favors their survival and subsequent germination in improperly cooked, prepared, or stored foods. The current study identified a novel alpha/beta-type small acid soluble protein, now named Ssp4, and showed that sporulating cultures of FP isolates producing resistant spores consistently express a variant Ssp4 with an Asp substitution at residue 36. In contrast, Gly was detected at Ssp4 residue 36 in C. perfringens strains producing sensitive spores. Studies with isogenic mutants and complementing strains demonstrated the importance of the Asp 36 Ssp4 variant for the exceptional heat and sodium nitrite resistance of spores made by most FP strains carrying a chromosomal cpe gene. Electrophoretic mobility shift assays and DNA binding studies showed that Ssp4 variants with an Asp at residue 36 bind more efficiently and tightly to DNA than do Ssp4 variants with Gly at residue 36. Besides suggesting one possible mechanistic explanation for the highly resistant spore phenotype of most FP strains carrying a chromosomal cpe gene, these findings may facilitate eventual development of targeted strategies to increase killing of the resistant spores in foods. They also provide the first indication that SASP variants can be important contributors to intra-species (and perhaps inter-species) variations in bacterial spore resistance phenotypes. Finally, Ssp4 may contribute to spore resistance properties throughout the genus Clostridium since ssp4 genes also exist in the genomes of other clostridial species.     

Correlation of Biological to Chromatographic Data for Two Mycotoxins Elaborated by Fusarium

Thirty-seven identified strains of Fusarium, most of them isolated from fescue grass, were tested for their ability to elaborate mycotoxins in laboratory culture. The presence of the toxins was determined by infrared light, thin-layer chromatography, mouse toxicity, fungistatic effects, and phytotoxic properties. A good correlation was demonstrated between T-2 toxin detection by thin-layer chromatography and inhibition of Rhodotorula rubra by culture extracts. All of the strains producing either butenolide or T-2 toxin were toxic to mice with but one exception; those producing T-2 toxin inhibited growth of the yeast.     

ICU患者肠道定植产ESBLs大肠埃希菌基因同源性研究

目的研究ICU患者肠道定植产ESBLs大肠埃希菌基因的同源性,为制定控制多重耐菌医院感染措施提供流行病学和分子生物学依据。方法对ICU患者肛拭子分离出的80株产ESBLs大肠埃希菌做药敏试验,对药敏结果表型完全相同的分成组,对筛选出的药敏表型完全相同的菌株采用Diversilab自动化重复序列（REP）-PCR（repetitive—element sequence—basedPCR）分型系统,分析肠道定植产ESBLs大肠埃希菌基因的同源性。结果80株菌株中有56株未找到药敏结果相同的菌株,未做基因同源性检测;有7组2株药敏表型结果完全相同,有2组3株药敏表型结果完全相同,有1组4株药敏表型结果完全相同,10组药敏表型结果完全相同的菌株做基因同源性检测;7组2株药敏表型结果完全相同只有1组基因有同源性,2组3株药敏表型结果完全相同只有1组中的2株基因有同源性,1组4株药敏表型结果完全相同基因无同源性。5株来源于不同组别药敏表型结果不完全相同的菌株基因具有同源性;具有同源性的菌株患者分别来源于不同时间和不同地点的患者。结论肠道定植产ESBLs大肠埃希菌大部分药敏结果不完全相同;药敏结果完全相同的菌株基因不一定有同源性,药敏结果不完全相同的菌株基因少部分有同源性。     

ICU病房产超广谱β-内酰胺酶肺炎克雷伯菌的监测和耐药性分析

了解产超广谱β-内酰胺酶(ESBLs)肺炎克雷伯菌的耐药性,为临床合理使用抗菌药物提供依据。采用自动化细菌鉴定仪(法国生物梅里埃公司Vitek-2和BD Phonenix 100)进行细菌鉴定和药敏试验,应用WHO-NET5.4软件进行耐药性分析。ICU病房共分离出100株肺炎克雷伯菌,其中产ESBLs株48株,占48%,药敏结果显示产ESBLs菌株的耐药率普遍高于不产ESBLs株,产ESBLs株对氨苄西林、头孢呋辛、头孢唑啉等全部耐药,对头孢曲松、头孢噻肟的耐药率也大于80%,对氨基糖苷类的庆大霉素、四环素,对喹诺酮类的环丙沙星、左氧氟沙星以及磺胺类中的复方新诺明耐药率较高(均大于55%),对哌拉西林/他唑巴坦、阿米卡星、头孢西丁等耐药率较低,对亚胺培南、美洛培南高度敏感。产ESBLs肺炎克雷伯菌耐药现象严重,及时监测产ESBLs菌的发生率及其耐药趋势,为临床合理应用抗菌药物,延缓细菌耐药性的产生,控制医院感染具有重要意义。     

血流感染患者大肠埃希菌产超广谱β-内酰胺酶及耐药性分析

目的分析血流感染患者大肠埃希菌产超广谱β-内酰胺酶（Extended—Spectrum Beta Lactamases,ESBLs）的现状及其耐药特征,为临床合理使用抗菌药物提供依据。方法对浙江省上虞市人民医院2011年1月至2012年12月住院患者血培养分离的96株大肠埃希菌,采用纸片扩散表型确证试验进行ESBLs检测,用K．B法做药敏试验。结果血培养的大肠埃希菌分离率2011年、2012年分别为19．48％、17．47％。大肠埃希菌产ESBLs的检出率2011年、2012年分别为60．00％、60．78％。产ESBLs菌株对多种抗菌药物的耐药率显著高于不产ESBLs菌株。无论大肠埃希菌是否产ESBLs,碳青霉烯类抗生素均具有很高的敏感率。结论血流感染患者分离的大肠埃希菌产ESBLs比率高,产ESBLs菌株对多种抗菌药物耐药性高。可经验性使用碳青霉烯类抗生素治疗大肠埃希菌所致的血流感染。     

红曲霉桔霉素的检测方法及红曲霉产桔霉素的判别方法*

建立了红曲霉真菌毒素桔霉素的HPLC检测方法。用谷氨酸和葡萄糖为唯一氮、碳源的培养基(MSG)液态摇瓶培养及红曲米固态培养,对30多株红曲霉产桔霉素的情况进行了普查,发现大多数红曲霉菌种可产生桔霉素。红曲霉的培养状态及条件对桔霉素的产生有重大影响。红曲霉菌种是否产桔霉素,可根据MSG培养基摇瓶发酵液中是否含有桔霉素来初步定性判断,为准确判断红曲霉菌是否产桔霉素,可采用多种培养法综合判断。     

九孔鲍消化道及养殖水体中异养细菌胞外产物的分析

从汕尾健生鲍鱼场养殖水体及鲍鱼消化道中分离筛选到26株异养细菌,分析了其胞外产物,并采用API条带对其进行了种类鉴定。结果表明,就整体而言,成鲍消化道菌株产蛋白酶、淀粉酶、明胶酶和溶血能力均高于养殖水体的菌株,而产脂肪酶和卵磷脂酶菌株的比例则低于后者。无论是消化道还是养殖水体,均存在着分泌胞外产物能力极强的菌株,且大部分为鞘氨醇单胞菌。因此,在该养殖环境中,鞘氨醇单胞菌应视为一种主要的条件致病菌,同时消化道和养殖水体均应视为潜在致病菌的源泉。此外,在考虑细菌对水产经济动物的致病作用时,除了优势种类外,还应从菌群结构的角度出发,考虑整个细菌群落胞外产物的作用。     

Vitek—AMS系统检测超广谱β—内酰胺酶的评价

目的：评价Vitek-AMS系统检测超广谱β-内酰胺酶（ESBLs)方法的敏感性和特性。方法：用Vitek－32仪器法、双纸片协同试验法检测45株ESBLs阳性和45株ESBLs阴性菌株,并将结果进行比较。结果：Vitek－32仪器法检出45株ESBLs阳性株中的43株,而双纸片协同试验法仅检出38株,45株ESBLs阴性株用两法检测均为阴性。Vitek－AMS仪器法检测ESBLs的方法敏感性为95．5％,特异性为100％,而双纸片协同试验法分别为84．4％和100％。结论：Vitek－AMS系统检测ESBLs的方法敏感性和特异性均优于双纸片同试验法,且操作简便,有条件的实验室可将其作为初筛ESBLs的首选方法。     

Molecular genetic characteristics of the B. pertussis strains isolated in different periods of the pertussis epidemic process

Despite the fact that the mass immunization of the children population with the DPTs vaccine has been carried out in the Russian Federation since 1959, the pertussis infection persists to be one of the pressing problems for the children population. Although the vaccination coverage of the children population with pertussis vaccines is high in Russia, at present time the pertussis incidence rates are increasing among schoolchildren and remain high among infants younger than 12 months old. Many researchers believe that the variability of the genetic structure of the pertussis causative agent may be one of the causes of increasing pertussis incidence rates. This investigation provides the molecular genetic characteristics of 97 B. pertussis strains isolated in pertussis patients in Moscow in different periods of pertussis epidemic process since the 1950s up to present time. It shows the changes in the structures of genes, which are encoding the main protective antigens of the pertussis microbe that are the pertussis toxin (ptxS1) and the pertactin (pm). The structurre of the ptxS1 and pm gene of the B. pertussis vaccine strains was compared with the structures of these genes in the B. pertussis strains isolated from the pertussis patients at present time and also in past years. All B. pertussis strains isolated in the prevaccination period (1948-1959) and most strains (95%) isolated during the first twenty years of the mass immunization in Russia are characterized by the presence of the so called "vaccine" alleles of the pertussis toxin and pertactin genes that are ptxS1 B or ptxS1 D and pm 1 alleles that corresponds to the genetic structure of the vaccine producing strains. In the early 1970s the B. pertussis strains of another toxin and pertactin genetic structures with so-called "non-vaccinal" alleles ptxS1 A and pm 3 (pm 2 since 1980s) began to appear. The B. pertussis strains with "non-vaccinal" alleles have completely displaced the "old" strains. At present time in Moscow the pertussis disease is caused by the B. pertussis strains bearing ptxS1 A and pm 2 or pm 3 alleles of pertussis toxin and pertactin genes. There was no correlation between the genotype and serotype. Thus, the structure of the B. pertussis toxin and pertactin genes in strains which have been isolated since the 1980s up to now differs from the structure of these genes in strains which are used for producing DPTs vaccine. The data obtained in this investigation suggest that the genetic structure specificity of circulating B. pertussis strains that are producing the disease at present time should be used as one of the criteria for selecting vaccine producing strains.