临床常见病原菌的分布及耐药性分析

目的了解我院患者常见病原菌分布及其耐药情况,指导临床治疗,合理使用抗生素。方法将我院2006年~2008年分离的临床病原菌分布及其耐药情况进行回顾性分析。结果我院病原菌分布以革兰阴性杆菌为主,占40.9%,革兰阳性球菌占34.4%,真菌占24.7%,居于革兰阴性杆菌前3位的菌群是大肠埃希菌、变形杆菌属和克雷伯菌属;居于革兰阳性球菌前3位的菌群是凝固酶阴性葡萄球菌、肠球菌属和链球菌属;各菌群中抗菌药物的耐药率均有上升,部分菌株呈多重耐药。结论革兰阴性杆菌已成为医院感染的最常见病原菌,对抗生素耐药日益严重,医院应重视病原菌的耐药性监测,及时掌握细菌耐药的动态,指导临床合理使用抗生素,防止医院感染的发生。     

副溶血性弧菌的致病性及其快速检测

副溶血性弧菌(Vibrio parahaemolyticus,VP)是革兰阴性嗜盐性细菌,隶属弧菌科中的弧菌属,它是一种人畜共患病菌[1].     

住院患者革兰阴性杆菌的耐药性分析

目的了解临床分离的革兰阴性杆菌的分布及耐药性变迁。方法2001—2003年临床分离革兰阴性杆菌1710株。排除同一患者的重复菌株,按统一方案用K—B纸片扩散法,按美国国家临床实验室标准委员会（NCCLS）2000年版标准判读结果。结果住院患者最常见的临床分离菌株为铜绿假单胞菌（8．3％）、不动杆菌属（12．8％）、大肠埃希菌（9．5％）和沙雷菌属（13．6％）,革兰阴性杆菌占总分离率的62．4％。亚胺培南、头孢哌酮-舒巴坦和哌拉西林-他唑巴坦对革兰阴性杆菌抗菌活性好。产超广谱β-内酰胺酶（ESBLs）大肠埃希菌占其总株数的15．8％。结论近年来革兰阴性杆菌的耐药性呈上升趋势,特别对第3代头孢菌素耐药性上升最快,临床上应关注细菌耐药性变化,合理使用抗菌药物。     

血标本检出420株菌的菌谱分布及药敏分析

本文报告中山医科大学附一院从１９９５年１０月至１９９７年８月应用Ｂａｃｔ／Ａｌｅｒｔ血培养仪检测３６００份血液培养结果,阳性标本４１４份,其中６份为复数菌,菌株数４２０,其中革兰阳性球菌（Ｇ＋Ｃ）２３６株,革兰阴性杆菌（Ｇ－Ｂ）发酵菌７２株,Ｇ－Ｂ非发酵菌３４株,真菌５３株,革兰阳性杆菌（Ｇ＋Ｂ）１３株,革兰阴性球菌（Ｇ－Ｃ）２株,厌氧菌１０株。主要的细菌分离率由高到低为凝固酶阴性葡萄球菌（ＳＣＯＮ）１７８６％,金黄色葡萄球菌（ＳＡ）１２８６％,α—溶血性链球菌９５２％,肠球菌属９０５％,大肠埃希氏菌８１％,微球菌属６４３％,铜绿假单胞菌４２９％和沙门氏菌３１％。药敏结果显示Ｇ＋Ｃ中万古霉素的耐药率最低,Ｇ－Ｂ却对各种药物表现出不同的耐药率,其中对泰能和头胞噻甲肟的耐药率较低。     

肿瘤患者感染三种常见革兰阴性杆菌耐药性分析

目的分析肿瘤患者临床分离的革兰阴性杆菌分布及其主要菌株的耐药性,为指导临床合理用药提供依据。方法对2014年1月~2015年12月恶性肿瘤患者感染分离的1478株革兰阴性杆菌及其主要菌株的耐药性进行回顾性分析。结果分离的1478株革兰阴性杆菌中,以大肠埃希菌(568/1478)38.43%、肺炎克雷伯菌(338/1478)22.87%、铜绿假单胞菌(216/1478)14.61%3种为主。这3种革兰阴性杆菌均呈多药耐药性;除亚胺培南对革兰阴性杆菌有较好的抗菌活性外,其它药物均显示有一定的耐药性,其中对派拉西林的耐药率在91%以上,头孢他啶、头孢噻肟、复方新诺明等药物的耐药率在70%以上。结论肿瘤患者感染革兰阴性杆菌耐药现象严重,尤其是感染产超广谱β-内酰胺酶(ESBLs)的大肠埃希菌和肺炎克雷伯菌呈多重耐药,实验室应加强对革兰阴性杆菌耐药性的监测,以指导临床合理选用抗菌药物。     

重症监护病房革兰阴性杆菌耐药性分析

目的了解深圳市人民医院重症监护病房(ICU)革兰阴性杆菌的分布及其耐药性,指导临床合理用药。方法收集来自重症监护病房各类标本分离的革兰阴性杆菌540株,用VITEK AMS-60或VITEK-Ⅱ全自动微生物分析仪进行菌种鉴定,用K-B法进行药敏试验。结果ICU检出的革兰阴性杆菌以鲍曼不动杆菌、大肠埃希菌、铜绿假单胞菌和肺炎克雷伯菌为主,ESBLs阳性的大肠埃希菌和肺炎克雷伯菌比例为61.6%和51.8%,各类细菌对常用抗菌药物表现为严重耐药和多重耐药。结论该院ICU检出的革兰阴性杆菌以鲍曼不动杆菌、大肠埃希菌、铜绿假单胞菌和肺炎克雷伯菌为主,且呈现多重耐药性。     

肾移植术后体表微生物群的变化与感染的监测

本文对肾移植术后病人体表微生物群的变化进行了连续动态的观察。正常人体表微生物群主要为革兰阳性球菌、阴性球菌和阳性杆菌,感染发生前这些微生物群发生了不同程度的变化。24个病例中有22人发生了体表革兰阴性杆菌定植,其中有19人在革兰阴性杆菌定植后的2天～3周后并发了不同程度的泌尿道、肺部、伤口、血液和其它体液的同种细菌感染,并发严重感染的7例病人中有6例表现为全身性革兰阴性杆菌定植。绿脓杆菌、大肠杆菌、不动杆菌和沙雷氏菌是常在体表定植的致病菌。作者认为:皮肤正常菌群的改变和病原性革兰阴性杆菌定植是监测肾移植术后感染,尤其是严重感染的有效手段。病人的口咽部和腹股沟等处是引起感染的革兰阴性杆菌持续稳定的贮菌所,全身性同种革兰阴性杆菌定植是严重感染的征象。     

Vitek-AMS鉴定临床细菌的应用研究

目的探讨Vitek-AMS对临床细菌鉴定的应用价值。方法对玉溪市人民医院1999年至2008年临床分离11 537株细菌(临床株)和省、部级临床检验中心下发微生物学室间质量评价鉴定菌种(参考株)的Vitek-AMS鉴定结果作对比分析。结果 11 537株临床分离菌中,不能鉴定细菌8株(0.07%);除外传染病因子,鉴定到属细菌114株(1.62%),鉴定到种(含亚种、物生型)细菌6 908株(98.38%)。共有64属193种。细菌类型的分布革兰阴性杆菌革兰阳性球菌酵母菌革兰阳性杆菌厌氧菌革兰阴性球菌。用参考菌种作比较,Vitek鉴定种的符合率为83.08%(54/65),属的符合率为98.46%(64/65);其中革兰阴性杆菌符合率最高(100%,24/24),酵母菌类符合率最低(66.67%,14/21)。7种鉴定卡菌种的阳性检出率平均为56.80%(234/412),其中GPI卡最高(100%),NHI卡最低(13.33%);但应用机会最多是GNI+卡。Vitek-AMS检测葡萄球菌产β-内酰胺酶阳性率为88.89%,大肠埃希菌和肺炎克雷伯菌产ESBLs阳性率分别为59.74%和32.20%。结论 Vitek-AMS的应用为临床细菌学检验提供了一种高效、快速、可靠的实验方法;但对于个别菌种、细菌酶的检测必要时应以参考方法确认。     

684例胆道感染患者胆汁病原菌分布及耐药性分析

目的研究胆道感染患者胆汁病原菌的分布及其耐药性,为临床合理使用抗菌药物提供依据。方法对684例胆道感染患者胆汁进行培养,应用Micro Scan Walk Away40细菌鉴定及药敏分析系统对分离菌进行鉴定及药敏测定,用WHONET 5.6软件进行数据分析。结果从684例患者胆汁中分离出315株病原菌,革兰阴性杆菌占65.1%,革兰阳性球菌占34.3%,真菌占0.6%。革兰阴性杆菌对亚胺培南、美罗培南、头孢哌酮/舒巴坦、哌拉西林/他唑巴坦、左氧氟沙星、头孢吡肟、庆大霉素耐药率较低。肠球菌属、葡萄球菌属对喹诺酮类耐药率较低,未发现耐万古霉素、利奈唑胺的革兰阳性球菌。结论胆道感染仍以革兰阴性杆菌为主,病原菌分布广泛,耐药情况较严重,治疗使用抗生素需要进行病原菌检测及耐药性分析。     

革兰阴性杆菌产AmpC酶的初筛检测及意义

目的：探讨革兰阴性杆菌产AmpC酶的初筛检测及意义，并了解本院革兰阴性杆菌产生Am-pC酶的情况。方法：实验用头孢西丁三相试验进行检测。结果：从136株对第三代头孢菌素等多种抗生素耐药的革兰阴性杆菌中共检出46株产AmpC酶，检出率为33.8%，主要为肠杆菌属、铜绿假单胞菌、大肠埃希菌和肺炎克雷伯菌。结论：头孢西丁三相试验操作简便，可作为革兰阴性杆菌产AmpC酶的初筛检测，可为临床合理用药提供依据。     

Isolation and some properties of newly isolated oxalate-degrading Pandoraea sp. OXJ-11 from soil

AIMS: To isolate and characterize an oxalate-degrading Pandoraea sp. OXJ-11. METHODS AND RESULTS: A new bacterium Pandoraea sp. OXJ-11 was isolated from soil samples, which can grow in the medium with oxalate as the sole carbon and energy source. The isolate OXJ-11 is Gram-negative straight rod. It occurs singly and is motile by means of a double polar flagellum. Catalase is positive and nitrate is not reduced. It grows aerobically and the optimum growth temperature and the optimum pH are at 30 degrees C and pH 6.0, respectively. The polyphasic taxonomic data along with 16S rRNA sequence comparison demonstrate that the isolate OXJ-11 should belong to the genus Pandoraea and represent a new member in this family. CONCLUSIONS: Oxalate could be degraded and the oxalate-degrading enzyme activity was detected when the isolate OXJ-11 grew in the medium with oxalate as carbon source. SIGNIFICANCE AND IMPACT OF THE STUDY: Oxalate-degrading Pandoraea sp. OXJ-11 would be beneficial to the potential application in the control of sclerotinia stem rot in economically important plants caused by fungus Sclerotinia sclerotiorum, and in making plants resistant to the white mold disease by oxalate-degrading enzyme transgene.     

Use of the gyrB gene for the identification of Pandoraea species

The recently described genus Pandoraea consists of five named species and four unnamed genomospecies, several of which have been identified in clinical specimens including respiratory secretions from persons with cystic fibrosis. We investigated whether it is possible to distinguish species of the genus Pandoraea by means of restriction fragment length polymorphism (RFLP) analysis and direct sequencing of the gyrB gene. Sixty-seven Pandoraea isolates were included. Species-specific RFLP patterns were obtained following digestion of the PCR-amplified gyrB gene with MspI. Specificity of RFLP groupings was confirmed by direct sequencing of several representative isolates. Our results indicate that RFLP analysis and sequencing of the gyrB gene are useful for the identification of Pandoraea species. We also found that further taxonomic studies within the beta-Proteobacteria using the gyrB gene would benefit from the development of additional primers allowing more efficient amplification of the gyrB gene. Our data also indicate that the taxonomic status of Pandoraea genomospecies 2 should be reinvestigated.     

Induction of oxalate decarboxylase by oxalate in a newly isolated Pandoraea sp. OXJ-11 and its ability to protect against Sclerotinia sclerotiorum infection

Pandoraea sp. OXJ-11 has been shown to produce an oxalate decarboxylase. The enzyme could be induced by increasing the oxalate in the medium. An increasing concentration of yeast extract was able to stimulate the cell growth but could not increase the specific oxalate decarboxylase activity. The oxalate decarboxylase was produced maximally at 25-35 degrees C and pH 4.0-9.0, favoring its potential application in protection of host plants from oxalate-producing phytopathogens. The influence of glucose on the induction of oxalate decarboxylase by oxalate was examined, and it was found that glucose inhibited the production of the oxalate decarboxylase. Resistance results showed that Pandoraea sp. OXJ-11 was capable of suppressing Sclerotinia sclerotiorum infection on detached leaflets of Brassica napus plants.     

Evidence of species succession during chlorobenzene biodegradation

We have previously reported the disappearance of a specific strain degrading chlorobenzene from a functionally stable bioreactor. In the present work, we investigated this species succession and isolated a new dominant strain, identified as Pandoraea pnomenusa sp. strain MCB032. A specific 16S rRNA-targeted oligonucleotide probe was designed and validated to identify strain MCB032 using fluorescence in situ hybridisation (FISH). The results confirmed the presence of strain MCB032 in samples collected over time, and showed that it was primarily located within the biofilm. Denaturing gradient gel electrophoresis (DGGE) provided evidence that the species succession occurred early in the operating period. The application of these biomolecular tools highlighted the remarkable stability of this new strain during the 15 months of reactor operation. The succession was attributed to the competitive kinetic behaviour of strain MCB032, which exhibited faster growth (micro(max) = 0.34 h(-1)) and higher substrate affinity (K(s) = 0.35 mg L(-1)) than strain JS150. Finally, this study contributed to the characterisation of the recently established Pandoraea genus, an emerging group in the biodegradation field.     

Degradation of reactive dyes by ozonation and oxalic acid-assimilating bacteria isolated from soil

Ozonation and treatment of wastewaters with oxalic acid-assimilating bacterium was attempted for the complete degradation of reactive dyes. Oxalic acid-assimilating bacterium, Pandoraea sp. strain EBR-01, was newly isolated from soil under bamboo grove and was identified to be a member of the genus Pandoraea by physicochemical and biochemical tests including 16S rDNA sequence analysis. The bacterium was grown optimally at pH 7 and temperature of 30 degrees C under the laboratory conditions. Reactive Red 120 (RR120), Reactive Green 19 (RG19), Reactive Black 5 (RB5) and Remazol Brilliant Blue R (RBBR) were used in degradation experiments. At the initial reactive dye concentrations of 500 mg/l and the ozonation time of 80 min, it was confirmed that 75-90 mg/l oxalic acid was generated from reactive dyes by ozonation. Microbial treatment using EBR-01 greatly decreased the amount of oxalic acid in the mixture after 48 h, but it was not removed completely. TOC/TOC(0) of reactive dye solutions was also decreased to 80-90% and 20-40% by ozonation and microbial treatment using EBR-01, respectively. The study confirmed that consecutive treatments by ozone and microorganisms are efficient methods to mineralize reactive dyes.     

Isolation and molecular characterization of thiosulfate-oxidizing bacteria from an Italian rice field soil

In rice paddy soils an active cycling of sulfur compounds takes place. To elucidate the diversity of thiosulfate-oxidizing bacteria these organisms were enriched from bulk soil and rice roots by the most probable number method in liquid medium. From the MPN enrichment cultures 21 bacterial strains were isolated on solid mineral medium, and could be further shown to produce sulfate from thiosulfate. These strains were characterized by 16S rDNA analyses. The isolates were affiliated to seven different phylogenetic groups within the alpha- and beta-subclass of Proteobacteria. Two of these phylotypes were already described as S-oxidizers in this environment (Xanthobacter sp. and Bosea sp. related strains), but five groups represented new S-oxidizers in rice field soil. These isolates were closely related to Mesorhizobium loti, to Hydrogenophaga sp., to Delftia sp., to Pandoraea sp. or showed sequence similarity to a strain of Achromobacter sp.     

16S rRNA gene-based phylogenetic microarray for simultaneous identification of members of the genus Burkholderia

For cultivation-independent and highly parallel analysis of members of the genus Burkholderia , an oligonucleotide microarray (phylochip) consisting of 131 hierarchically nested 16S rRNA gene-targeted oligonucleotide probes was developed. A novel primer pair was designed for selective amplification of a 1.3 kb 16S rRNA gene fragment of Burkholderia species prior to microarray analysis. The diagnostic performance of the microarray for identification and differentiation of Burkholderia species was tested with 44 reference strains of the genera Burkholderia , Pandoraea , Ralstonia and Limnobacter . Hybridization patterns based on presence/absence of probe signals were interpreted semi-automatically using the novel likelihood-based strategy of the web-tool PhyloDetect. Eighty-eight per cent of the reference strains were correctly identified at the species level. The evaluated microarray was applied to investigate shifts in the Burkholderia community structure in acidic forest soil upon addition of cadmium, a condition that selected for Burkholderia species. The microarray results were in agreement with those obtained from phylogenetic analysis of Burkholderia 16S rRNA gene sequences recovered from the same cadmium-contaminated soil, demonstrating the value of the Burkholderia phylochip for determinative and environmental studies.     

Proteolytic activity in stored aerobic granular sludge and structural integrity

Aerobic granules lose stability during storage. The goal of this work was to highlight the main cause of stability loss for stored granules as intracellular protein hydrolysis. The quantity of extracellular proteins was noted to be significantly lower during granule storage, and protease enzyme activities were correspondingly higher in the cores of stored granules. The proteolytic bacteria, which secrete highly active protease enzymes, were for the first time isolated and characterized by analyzing 16S rDNA sequences. The proteolytic bacteria belonged to the genera Pseudomonas, Raoultella, Acinetobacter, Pandoraea, Klebsiella, Bacillus and uncultured bacterium, and were grouped into Proteobacteria, Enterobacteria and Firmicutes. The PB1 (Pseudomonas aeruginosa) strain, which exhibited very high proteolytic activity during the skim milk agar test, was located at the core regime with active protease enzymes, and was close to the obligate anaerobic strain Bacteroides sp. Hence, the extracellular proteins in stored granules were proposed to be hydrolyzed by enzymes secreted by proteolytic bacteria with the hydrolyzed products ultimately being used by nearby anaerobic strains. This process gradually digests the protein core, and eventually consumes the entire granule.     

Assessment of microbial diversity in effluent treatment plants by culture dependent and culture independent approaches

Microbial community structure of two distinct effluent treatment plants (ETPs) of pesticide and pharmaceutical industries was assessed and defined by (i) culture dependent and culture independent approaches on the basis of 16S rRNA gene sequencing, (ii) diversity index analysis - operational taxonomic units (OTUs). A total of 38 and 44 bacterial OTUs having 85-99% similarity with the closest match in the database were detected among pharmaceutical and pesticide sludge samples, respectively. Fifty percent of the OTUs were related to uncultured bacteria. These OTUs had a Shannon diversity index value of 2.09-2.33 for culturables and in the range of 3.25-3.38 for unculturables. The high species evenness values of 0.86 and 0.95 indicated the vastness of microbial diversity retrieved by these approaches. The dominant cultured bacteria indicative of microbial diversity in functional ETPs were Alcaligenes, Bacillus and Pseudomonas. Brevundimonas, Citrobacter, Pandoraea and Stenotrophomonas were specific to pesticide ETP and Agrobacterium, Brevibacterium, Micrococcus, Microbacterium, Paracoccus and Rhodococcus were specific to pharmaceutical ETP. These microbes can thus be maintained and exploited for efficient functioning and maintenance of ETPs.     

Has the Bacterial Biphenyl Catabolic Pathway Evolved Primarily To Degrade Biphenyl? The Diphenylmethane Case

In this work, we have compared the ability of Pandoraea pnomenusa B356 and of Burkholderia xenovorans LB400 to metabolize diphenylmethane and benzophenone, two biphenyl analogs in which the phenyl rings are bonded to a single carbon. Both chemicals are of environmental concern. P. pnomenusa B356 grew well on diphenylmethane. On the basis of growth kinetics analyses, diphenylmethane and biphenyl were shown to induce the same catabolic pathway. The profile of metabolites produced during growth of strain B356 on diphenylmethane was the same as the one produced by isolated enzymes of the biphenyl catabolic pathway acting individually or in coupled reactions. The biphenyl dioxygenase oxidizes diphenylmethane to 3-benzylcyclohexa-3,5-diene-1,2-diol very efficiently, and ultimately this metabolite is transformed to phenylacetic acid, which is further metabolized by a lower pathway. Strain B356 was also able to cometabolize benzophenone through its biphenyl pathway, although in this case, this substrate was unable to induce the biphenyl catabolic pathway and the degradation was incomplete, with accumulation of 2-hydroxy-6,7-dioxo-7-phenylheptanoic acid. Unlike strain B356, B. xenovorans LB400 did not grow on diphenylmethane. Its biphenyl pathway enzymes metabolized diphenylmethane, but they poorly metabolize benzophenone. The fact that the biphenyl catabolic pathway of strain B356 metabolized diphenylmethane and benzophenone more efficiently than that of strain LB400 brings us to postulate that in strain B356, this pathway evolved divergently to serve other functions not related to biphenyl degradation.