血平板针尖样及云雾状菌落分离与鉴定

采集98份临床标本接种血平板培养,40份生长出针尖样及云雾状菌落。经分离鉴定38株为细菌L型,其中金黄色葡萄球菌（Staphyloeoccusaureus）居首位,占52．1％（20／38）．其返视性与L型平板分离相比较差。但对抗生素的敏感性则无差别。表明血平板可用于分离细菌L型,并保持了传统L型的特性。     

1332例发热患者血液标本菌群及血清学分析

目的：探讨发热患者血液标本中菌群的种类及其分布情况,为临床的诊断与治疗提供实验依据。方法：采用常规细菌学分离培养和L型高渗培养法从1332例感染症患者的血液标本中分离细菌及L型。结果：1332例感染标本共检出细菌486株,检出率为36．5％,检出的细菌包括沙门菌,葡萄球菌,肠道杆菌及奈瑟菌,149例标本中检出细菌L型29株,检出率为19．5％。其中返祖的细菌包括葡萄球菌和奈瑟菌。结论：沙门菌和葡萄球菌及其L型是引起血液感染的常见病原菌。     

湿型外耳道分泌物的菌群结构及其药物敏感性分析

人的外耳道中存在种类繁多的微生物。为了解健康人湿型外耳道分泌物中的细菌数量、种类及其对药物敏感性,收集了38例无耳道疾病健康人左、右耳的湿型外耳道分泌物76份。采用分离培养的方法进行平板涂布、菌落计数及鉴定,分析其菌群数量及种类;通过纸片扩散法(Kirby-Bauer纸片扩散法)对分离的细菌进行药物敏感性试验,并利用头孢硝噻吩纸片检测青霉素耐药株产β-内酰胺酶情况。结果显示,88.3%的湿型外耳道分泌物的菌群数量在1×10² CFU/mL～1×10⁶ CFU/mL;共检出细菌20种,其中球菌占97.1%,主要为葡萄球菌;杆菌为2.9%。分离率前4位的细菌为头葡萄球菌、耳葡萄球菌、人葡萄球菌和表皮葡萄球菌,对临床常用的妥布霉素、米诺环素、万古霉素和呋喃妥因等抗生素敏感率达90.0%以上;对复方新诺明和青霉素耐药率相对较高,但低于50.0%。分离得到的132株葡萄球菌中,25株对青霉素耐药,耐药株的β-内酰胺酶阳性率为44.0%。结果提示,从健康人湿型外耳道分泌物中分离的细菌种类多样,以葡萄球菌为主,对临床常用的抗生素有较高的敏感性,但对青霉素、复方新诺明等具有一定耐药性。     

美洲大蠊肠道内生细菌的分离及其初步抑菌活性研究

分离鉴定美洲大蠊肠道内生细菌,并初步研究了其抑菌活性。采用平板稀释法对美洲大蠊肠道内生细菌进行分离纯化,分离菌株通过形态学观察和16S rDNA基因序列BLAST比对进行种属鉴定。以金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌、肺炎克雷伯菌、大肠埃希氏菌4株细菌为对象,采用牛津杯法初步鉴定各分离菌株的抑菌活性。BLAST比对分析结果表明:从15只美洲大蠊肠道内分离鉴定出125株内生细菌,分属12科20属。初步抑菌活性显示:部分菌株对金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌、肺炎克雷伯菌、大肠埃希氏菌有明显抑菌活性。美洲大蠊肠道内生菌种类繁多,而且较多菌株具有抑菌活性。     

空气环境中几种细菌检测与16S rRNA测序鉴定

采集空气环境微生物,根据菌落和革兰氏染色特征,分离5株细菌。提取分离株DNA,采用设计的16SrRNA引物扩增DNA片段;纯化PCR产物,进行测序反应并再纯化,上机读序。拼接与校对DNA序列,在NCBI网站进行Blast,鉴定细菌型别。5株菌株分别是表皮葡萄球菌、头状葡萄球菌、施氏假单胞菌、溶血葡萄球菌和大肠杆菌;其中溶血葡萄球菌是致病菌,其他4株是条件致病菌。这提示,应警惕空气环境中致病菌的感染。     

金黄色萄葡球菌L型特异性抗体的免疫组化试验在妇科感染性疾病诊断上的应用

本文对49例慢性子宫内膜炎、慢性宫颈炎等妇科疾病的病理切片,用金黄色葡萄球菌细菌型及L型的特异性杭体作酶免疫组化染色(PAP法)证明在金黄色葡萄球菌引起的各种妇科感染中,细菌型及L型混合感染占60％,单独细菌型感染占28.9％,单独L型感染占11.1％。本研究证明了金葡菌变异为L型后,有特异性杭原出现,并证明细菌L型的特异性抗原有一定的免疫学诊断价值。     

万古霉素耐药金黄色葡萄球菌生物学特性

目的观察金黄色葡萄球菌对万古霉素耐药后生物学特性的变化,探讨有效方法,为临床正确诊断奠定基础。方法使用万古霉素体外诱导将1株h-VRSA逐步诱导为VISA;观察原代菌及其诱导菌的形态结构、培养特性和生化反应结果并与标准菌株ATCC 29213比较,了解金黄色葡萄球菌对万古霉素耐药后生物学特性变化规律;比较手工法、仪器法和PCR检测法的细菌鉴定结果。结果金黄色葡萄球菌对万古霉素耐药后主要生物学特性改变为:细胞壁增厚、细胞表面粗糙,有结节状凸起、菌落变小,溶血环和色素消失,血浆凝固酶、甘露醇、甘露糖等生化反应由阳性转为阴性。生物学特性改变可导致手工法和仪器法细菌鉴定结果错误,但对PCR检测鉴定结果没有影响。结论金葡菌对万古霉素耐药后生物学特性改变可导致常规方法细菌鉴定结果错误,应引起临床的重视。     

慢性前列腺炎与细菌L型的相关性研究

目的：观察慢性前列腺炎与细菌L型感染的关系,探索慢性前列腺炎延不愈,反复发作的机制,提供临床用药的参考。方法：对108例慢性前列腺炎患者的前列腺液进行普通培养和细菌L型的同步培养,并对检出的L型菌株进行耐药性检测。结果：普通培养检出细菌28株,阳性率为26．3％,L型细菌检出20株,阳性率为19．1％,总阳性率为45．4％,阳性率显著提高（P＜0．01）;检出的L型细菌对β－内酰胺抗生素呈高度耐药性,其他作用于细菌蛋白质合成的药物,其敏感性与原菌相似或升高。结论：细菌L型的感染与慢性前列腺炎的发生,发展具有一定的相关性;L型细菌具有特有的抗生素耐药机制。     

135例足癣患者微生物学调查及药敏分析

目的研究重庆地区足癣患者足部真菌、细菌分布以及药物敏感情况。方法对135例经临床和真菌镜检确诊的足癣患者病灶取样行真菌及细菌培养,用VITEK2系统进行细菌鉴定及药敏分析。结果 135例足癣患者中真菌培养阳性128例,其中红色毛癣菌126例,石膏样小孢子菌及白念珠菌各1例。细菌培养阳性129例,共分离出致病菌株147株,主要为金黄色葡萄球菌(25株)及凝固酶阴性葡萄球菌(114株),94株致病菌中,72株(76.60%)对1种及以上抗生素耐药。结论目前重庆地区足癣患者病原菌以红色毛癣菌为主,细菌病原菌主要为金黄色葡萄球菌及凝固酶阴性葡萄球菌,其中较多细菌对数种常见抗生素耐药,在临床治疗中需予以重视。     

安徽不同地区奶站牛奶中金黄色葡萄球菌及其肠毒素污染状况评估

目的评定安徽地区各奶站牛奶中金黄色葡萄球菌以及肠毒素的污染情况。方法通过从安徽省不同地区30所奶站采集乳样,进行乳源性金黄色葡萄球菌的分离与生化鉴定,并采用PCR技术对分离出的菌株进行金黄色葡萄球菌肠毒素血清型鉴定。结果安徽省30个奶站中有4个地区奶站的牛奶中污染金黄色葡萄球菌;从污染牛奶中共分离出5株金黄色葡萄球菌,检出率为16.7%。经鉴定,所分离出的金黄色葡萄球菌中2株为肠毒素A型,1株为肠毒素C型,2株为同时产肠毒素A和肠毒素C。结论安徽省不同地区奶站中的牛奶污染的金黄色葡萄球菌产肠毒素类型以肠毒素A为主。     

L-Form Induction, Morphology, and Development in Two Related Strains of Erysipelothrix rhusiopathiae

Two related strains of Erysipelothrix rhusiopathiae, one the parent and the other an L-form revertant, were studied for their propensity or ability to produce L-forms under the influence of penicillin. The parent strain produced L-forms in nutrient solid media in an osmolarity range between 0.85 and 5.0% NaCl concentration whereas the revertant strain did so between 0.5 and 3.0% NaCl concentration. When various hyperosmolar media were tried without penicillin, recovery of L-forms from the revertant strain was optimal at a salt concentration of 2.0%, whereas the parent strain occasionally produced a few L-forms on 3.0% salt medium only. The process of penicillin-induced transformation from bacteria to L-form followed an unusual morphological sequence, beginning with beading of the bacterial body, followed by disintegration into granules from which the L-form colony derived. No large bodies were seen during the initial process of L-form induction, but they evolved later from the original granules and had the potential to reproduce L-type growth. The spontaneous development of L-forms in hyperosmolar media had a different morphological sequence starting with elongation of the bacteria into filaments which later developed polar and central dilatations from which granules and L-type growth developed. The differences in biological behavior between these related bacterial strains suggest that the revertant strain developed new properties, probably of genetic origin. Consequently, the assumption that L-forms revert to the "parent" bacteria may not always be justified. It can be made only after the biological properties of the parent and the revertant organisms have been properly identified.     

慢性肾盂肾炎L型细菌感染及耐药分析

目的了解L型细菌在慢性肾盂肾炎的感染及耐药状况。方法对71例患者清洁中段尿做普通细菌培养(B型)、L型细菌培养(L型)及耐药分析。结果细菌阳性率为77.5%,其中单独L型阳性率为49.3%、B型与L型混合感染为15.5%,而B型阳性率仅为12.7%。主要是大肠埃希菌,其次是葡萄球菌;青霉素及头孢噻肟均有较高的耐药率(88.9%及73.6%)。结论L型细菌在慢性肾盂肾炎感染中占主导,β-内酰胺类药物有较高的耐药性,临床治疗应据药敏结果合理选择及时调整抗生素。     

Polyacrylamide Gel Identification of Bacterial L-Forms and Mycoplasma Species of Human Origin

Polyacrylamide gel electrophoretic patterns of acidified phenol extracts prepared from whole cells can be used for the identification of bacterial L-forms and Mycoplasma species of human origin. Ten human Mycoplasma serotypes and eight L-forms belonging to five different genera were studied. The gel patterns were sufficiently distinct and reproducible that it was possible not only to identify L-forms at the genus level (group with streptococci) and different Mycoplasma serotypes but also to differentiate between the two of them. The parentage of L-forms of Streptobacillus moniliformis L1, Listeria monocytogenes, Streptococcus MG, and Staphylococcus aureus Smith strain was established by relating their gel patterns directly to parent bacteria. It was found that an L-form designated S. moniliformis An (ATCC 14220) was actually an L-form of Proteus. In addition, it was shown electrophoretically that no relationship existed between the Streptococcus MG L-form and M. pneumoniae. The applicability of this method as a diagnostic and taxonomic tool for the differentiation of L-forms and mycoplasmas is discussed.     

尿路感染患者尿中细菌L型培养的意义

目的通过对尿路感染患者尿标本中的细菌（普通型和L型）检测,以确定L型菌检测的临床价值。方法采用一般培养法和特殊（L型菌）培养法对临床标本检测。结果364份标本中一般培养法检出细菌112株,阳性率为30．8％,而特殊培养法共培养细菌及L型菌共162株,细菌总检出率为44．5％,其中单独检出L型菌50株。结论细菌在一定条件下可演变成L型菌,增加临床标本中L型菌的培养可明显提高尿路感染的细菌阳性检出率。     

Identification with the aid of various serological reactions of L forms of streptococci isolated from the live organism]

Growth inhibition, agglutination, precipitation, and passive hemagglutination tests were used for the identification of the L-forms of streptococci isolated from the organism of experimental rabbits both after the infection with the L-forms of streptococci and with the streptococci of group A. The tests were positive not only with the antiserum of homologous, but also of heterologous strains of the L-form of streptococcus, group A. The L-form cultures isolated from the experimental animals failed to differ from the laboratory strain of the L-forms of streptococcus, group A, by serological properties.     

Permanent alterations of the L-forms of proteus and salmonella under various conditions

L-forms obtained from three strains of Proteus and from one strain of Salmonella have been kept for 15 to 20 years by weekly or monthly transfers on agar plates containing penicillin. The morphology and growth requirements of these strains have changed. They now grow abundantly on the surface of agar and in broth. The cultures consist of large bodies, small granules, and transitional forms. These organisms are more resistant to distortion and stain more deeply than organisms of the usual L-forms. In broth and to a lesser extent on agar, branching filaments develop, on the ends of which both the large, round organisms and small organisms are produced. The filaments are a transitional stage in the development of the cultures. Usual bacillary forms were not present in the culture and did not appear in successive transfers in the absence of penicillin. Bacilli reappeared on exposure of the L cultures to the influence of a spore-bearing bacillus. A similar transformation of L-forms has also been observed developing within a short time in recently isolated A and B type L cultures of one Proteus strain during the process of reversion to the bacterial form. The altered cultures are fixed in a stage of transition between the B type L-form and the regular bacteria.     

Generic Identification of L-Forms by Polyacrylamide Gel Electrophoretic Comparison of Extracts from Parent Strains and Their Derived L-Forms

A polyacrylamide gel electrophoretic method was used for identification of L-forms in the genera Streptococcus, Staphylococcus, and Proteus, by comparing phenol-acetic acid-water extracts of homologous parent L-form pairs to one another and to other pairs. The method requires only milligram quantities of material for analysis. The standard patterns for the strains used in this study are shown as pictures of the gels and as densitometric tracings of appropriate gels. They show that, despite occasional minor differences in some organisms, the gel electrophoretic patterns of homologous L-forms and bacterial pairs are sufficiently similar-as well as sufficiently dissimilar from patterns of other genera-to permit generic identification of an unknown L-form by reference to patterns derived from possible parental bacteria.     

Induction, growth and antibiotic production of Streptomyces viridifaciens L-form bacteria

AIMS: To induce, cultivate and investigate the characteristics of L-form bacteria derived from the filamentous actinomycete Streptomyces viridifaciens. METHODS AND RESULTS: L-forms were induced in a liquid medium supplemented with lysozyme and penicillin. A stable culture which no longer required inducing agents but could still revert, was obtained by the twelfth subculture. The specific growth rate of stable L-forms was faster (0.751) than unstable L-forms (0.361). After the exponential growth phase, the cell diameter continued to increase, as did the percentage of vacuoles. Morphologically, the L-forms appeared as spherical bodies with no signs of differentiation and were sensitive to osmotic stress, indicating removal of the cell wall. The L-forms produced secondary metabolites although much lower levels of antibiotic were assayed in the L-forms compared with the cell walled forms. CONCLUSION: Stable L-form bacteria were induced from S. viridifaciens and their growth characterized. The L-forms produced secondary metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Stable Streptomyces L-forms were induced and have potential as biocontrol agents.     

An ELISA for the detection of Bacillus subtilis L-form bacteria confirms their symbiosis in strawberry

AIMS: To develop an ELISA for the detection of antigens derived from stable Bacillus subtilis L-form bacteria and to detect these in plants injected with L-form bacteria. METHODS AND RESULTS: A sandwich ELISA was developed and its specificity was investigated using L-forms and cell-walled forms of B. subtilis, different Bacillus species and a range of bacteria isolated from glasshouse-grown strawberry plants. The detection limits of the ELISA were approximately 10(3) viable cells ml(-1) for L-forms compared with 10(7) viable cells ml(-1) for cell-walled forms. Results showed that L-forms survived and moved within strawberry tissues injected with L-form bacteria. CONCLUSION: An ELISA that selectively detects B. subtilis L-form bacteria was developed and shown to confirm the presence of L-forms in plants. SIGNIFICANCE AND IMPACT OF THE STUDY: This will be a valuable rapid method to further studies on L-form plant interactions.     

Presence and synthesis of cholesterol in stable staphylococcal L-forms.

The sterol which was present in two strains of a stable staphylococcal L-form was analyzed by gas-liquid chromatography and combined gas-liquid chromatography-mass spectrometry. The retention time of the sterol on gas-liquid chromatography was the same as that of authentic cholesterol. Analysis of the sterol by mass spectrometry showed a molecular ion at an m/e of 386 and the same patterns of major ions above an m/e of 145 as those of authentic cholesterol. As a result, the sterol in staphylococcal L-form was identified as cholesterol. A parent strain and its L-forms were cultured in medium containing [14C]acetate, and the synthesis of cholesterol was examined. In the L-forms, 0.52% of the total lipid radioactivity was found in cholesterol fraction, whereas no significant radioactivity was detected in the cholesterol fraction of the parent strain, indicating that staphylococcal L-forms have acquired the capacity to synthesize cholesterol.