A novel chromatography system to isolate active ribosomes from pathogenic bacteria

We have developed a novel chromatography for the rapid isolation of active ribosomes from bacteria without the use of harsh conditions or lengthy procedures that damage ribosomes. Ribosomes interact with an alkyl linker attached to the resin, apparently through their RNA component. Examples are given with ribosomes from Escherichia coli, Deinococcus radiodurans, and with clinical isolates of Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA). The ribosomes obtained by this method are unusually intact, so that highly active ribosomes can now be isolated from the clinical isolates, enabling significantly improved in vitro functional assays that will greatly assist the discovery and development of new ribosomally targeted antibiotics.     

闭环式护理对小儿细菌性肺炎症状 和呼吸道菌群的影响

摘要：目的 探究细菌性肺炎患儿实行闭环式护理管理模式后患儿临床症状、呼吸道菌群及疾病复发的情况。方法 选取2016年5月至2018年11月我院收治的113例细菌性肺炎患儿为研究对象,随机分为观察组（n=55）和对照组（n=58）。两组患儿均给予抗生素治疗及常规护理。观察组患儿在常规护理基础上应用闭环式护理。观察两组患儿各主要临床症状缓解时间及住院时间、治疗有效率、治疗前及治疗1周后呼吸道菌群分布情况及治疗结束后6个月内细菌性肺炎复发率。结果 观察组患儿体温恢复时间、咳嗽及咳痰缓解时间及住院时间均短于对照组（均P<0.05）。观察组患儿呼吸道革兰阳性菌中以肺炎链球菌（48.39%,15/31）、金黄色葡萄球菌（38.71%,12/31）为主,革兰阴性菌中以肺炎克雷伯菌（31.82%,21/66）、大肠埃希菌（34.85%,23/66）为主。观察组患儿治疗1周后呼吸道革兰阳性菌分布比例（33.33%,12/36）低于对照组（37.04%,20/54）。治疗1周后观察组患儿呼吸道细菌总检出数低于对照组（P<0.05）。观察组患儿治疗结束后6个月内复发率为3.64%（2/55）,对照组为12.07%（7/58）,二者比较差异无统计学意义（P>0.05）。观察组患儿治疗有效率为89.09%（49/55）,高于对照组的72.41%（42/58）。结论 细菌性肺炎患儿呼吸道菌群以肺炎链球菌、金黄色葡萄球菌、肺炎克雷伯菌及大肠埃希菌为主。应用闭环式护理管理模式后,可明显改善细菌性肺炎患儿各临床症状,缩短症状缓解时间,抑制呼吸道革兰阳性菌群生长,但对治疗后复发率影响不大。     

Induction of murine B cell proliferation and immunoglobulin synthesis by some bacterial ribosomes

Ribosomal preparations from Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, and Streptococcus pneumoniae were investigated with respect to their activating capacity towards murine lymphoid cells. The proliferation of BALB/c spleen cells was induced in a dose-dependent fashion (from 1 to 100 micrograms/ml) by ribosomes of K. pneumoniae, H. influenzae, and S. pyogenes with a peak activity at 48 or 72 hr of culture. The majority of the blast cells induced by these ribosomal preparations were positive for surface-immunoglobulin (S-Ig) and negative for Thy 1.2. Furthermore, K. pneumoniae, H. influenzae, and S. pyogenes ribosomes induced the synthesis of IgM and some IgA. Cell proliferation and induction of IgM production were also demonstrated with the 3 ribosomal preparations using spleen cells from athymic nude (nu+/nu+) mice, Lyb-5-defective CBA/N spleen cells, B cell-enriched and T cell-depleted BALB/c spleen cell suspensions, as well as spleen cells from the Ips gene-deficient C3H/HeJ strain. Cell culture supernatants contained specific anti-ribosome IgM antibodies. Antibodies of other specificities (anti-sheep erythrocytes) were also demonstrated in supernatants from K. pneumoniae-stimulated cultures. Evidence against a possible role of contamination of K. pneumoniae and H. influenzae ribosomes by lipopolysaccharide- or lipid A-associated proteins in this effect is discussed. Ribosomes from S. pneumoniae did not induce 3H-thymidine incorporation nor Ig production. None of the 4 ribosomal preparations was found to stimulate T cell blastogenesis or to induce interleukin-2 production by naive BALB/c spleen cells. Finally, ribosomes from H. influenzae, S. pyogenes, S. pneumoniae but not those of K. pneumoniae stimulated interleukin-1 production by adherent spleen cells, from BALB/c mice.     

Tamarin model of pneumococcal bacteremia

Tamarins (Saguinus labiatus) were utilized to study host defenses against pneumococcal bacteremia. Tamarins had a poor antibody response to immunization with varying doses of pneumococcal capsular polysaccharide (PCP) vaccine (2 of 15 positive) or to infection with serotype 7F Streptococcus pneumoniae (2 of 14 positive). Tamarins were protected against challenge with a lethal dose of serotype 7F S. pneumoniae if the bacteria were preopsonized with human immune globulin intravenous or if the tamarins were injected with the immune globulin 30 min before challenge. There was minimal protection utilizing a mouse monoclonal anti-type 7F PCP antibody.     

丽水市中心医院2017年临床分离菌耐药情况

摘要：目的 了解2017年丽水市中心医院临床分离病原菌的分布情况和耐药特征。方法 收集丽水市中心医院2017年全年临床分离病原菌,采用K-B纸片法及Vitek2-Compact系统进行药物敏感性试验,采用WHONET 5.6软件进行数据分析。结果 共分离到病原菌5 928株,其中革兰阳性菌1 875株（31.63%）,革兰阴性菌4 053株（68.37%）。革兰阳性菌中以金黄色葡萄球菌、凝固酶阴性葡萄球菌、屎肠球菌、粪肠球菌、肺炎链球菌为多见。革兰阴性菌中以大肠埃希菌、肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌、流感嗜血杆菌为多见。肠杆菌科细菌对碳青霉烯类抗菌药、头孢哌酮/舒巴坦、哌拉西林/他唑巴坦、头孢西丁敏感性较高。产超广谱β内酰胺酶大肠埃希菌和肺炎克雷伯菌的检出率分别为53.5%和23.6%。鲍曼不动杆菌复合群对碳青霉烯类抗菌药、喹诺酮类、头孢吡肟的耐药率>70%。铜绿假单胞菌对阿米卡星、头孢他啶、头孢吡肟、哌拉西林/他唑巴坦的耐药率<15%。葡萄球菌属对喹诺酮类、红霉素、四环素类药物的耐药率>30%,未检到万古霉素和利奈唑胺的耐药株。耐甲氧西林金黄色葡萄球菌和凝固酶阴性葡萄球菌的检出率分别为43.6%和68.5%。结论 细菌耐药情况日益严重,特别是多重耐药及泛耐药细菌的不断增加给临床治疗带来了极大的困难,因此须定期监测细菌耐药情况以预防耐药菌的产生和传播流行。     

Use of surface-enhanced laser desorption/ionization -time of flight to explore bacterial proteomes

Surface-enhanced laser desorption/ionization (SELDI)-time of flight is a recent technology that allows proteomic analysis with limited material requirements. This characteristic makes it a valuable technique for microbiologists handling problematic samples, such as low cell number cultures. We compared three simple procedures for protein extraction from bacteria for compatibility with the ProteinChip Array; we also determined the amount of protein required for each analysis. The protocol for the SELDI analysis was evaluated by generating protein expression profiles of a Streptococcus pneumoniae strain grown in different conditions and those of different strains of the same species. The protocol also was successfully applied to a wide range of Gram positive and negative bacteria. The results of this study suggest the appropriateness of this technology for microorganism protein profiling as complementary or alternative to two-dimensional gel electrophoresis.     

深圳市儿童社区获得性肺炎细菌病原学及其耐药性

目的研究儿童社区获得性肺炎细菌病原学及其耐药性特征,指导临床合理应用抗菌药物。方法对2006年2月至2007年3月1年期间住院的5岁及5岁以下社区获得性肺炎病人,进行深部呼吸道吸引物细菌培养,并且检测分离菌株对常用抗菌药物的耐药性。结果1441例病人中,722例检出细菌共761株,分离阳性率为50．1％,分离菌依次为肺炎克雷伯菌170株（22．3％）、大肠埃希菌130株（17．1％）、肺炎链球菌89株（11．7％）、金黄色葡萄球菌63株（8．3％）及流感和副流感嗜血杆菌60株（7．9％）。耐甲氧西林金黄色葡萄球菌（MRSA）检出率为15．9％;对青霉素不敏感的肺炎链球菌（包括PISP和PRSP）检出率为84．3％;肺炎克雷伯菌、大肠埃希菌、粘质沙雷菌和阴沟肠杆菌产ESBLs的检出率分别为31．2％、46．2％、94．8％和16．8％;流感嗜血杆菌和副流感嗜血杆菌对氨苄西林的耐药率为36％和40％;铜绿假单胞菌和鲍曼复合不动杆菌对亚胺培南的耐药率分别为10．7％和13．2％。结论在深圳儿童社区获得性肺炎的分离菌中,革兰阴性菌明显多于革兰阳性菌,分离菌依次为肺炎克雷伯菌、大肠埃希菌、肺炎链球菌、金黄色葡萄球菌及流感和副流感嗜血杆菌。分离细菌对常用抗菌药物的耐药性较为严重。     

MALDI-TOF MS对肺炎链球菌鉴定与分型的应用

目的探讨MALDI-TOF MS对肺炎链球菌鉴定和质谱分型的应用价值。方法收集2009年1月至2013年5月温州医科大学附属第二医院临床分离的112株肺炎链球菌标本,采用Optochin敏感试验和全自动细菌分析仪对收集的菌株进行鉴定验证,并用Microflex MALDI-TOF质谱仪进行分析鉴定。根据质谱图的相似性进行细菌同源聚类树分析并构建质谱分型模型,采用荚膜肿胀试验对参与分型的菌株进行血清型比较。结果除20株不符合检测条件之外,92株临床菌株和1株标准株经质谱分析均为肺炎链球菌,选取的60株菌株以0.5的差异水平,将60株肺炎链球菌分为18个质谱型别,在这些菌株的血清分型中有19F、19A、23F、23A、3和14六个血清型别,分布于不同的MALDI-TOF MS分型中,其中19F有18株,占30%(18/60),分布在6种不同的MALDI-TOF MS分型中,也有3型血清型较为集中地分布于相应的MALDI-TOF MS一个型别里。结论 MALDI-TOF MS能快速、准确、简便地鉴定肺炎链球菌,且能达到种的水平。对比血清型,按照0.5差异水平,建立的18个质谱分型部分的型别与血清型有一致性,但也存有差异。     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats.

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent. faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium-like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum, shared characteristics with Ent. columbae. The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol. Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent.faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium -like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum , shared characteristics with Ent. columbae.
The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol.
Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.     

内蒙古地区临床危重患者三种常见感染细菌耐药基因的检测及耐药性分析

目的:探讨内蒙古地区临床危重患者常见感染细菌耐药基因的检测及耐药性相关因素,以便临床合理运用抗菌药物,为病原菌感染的预防和控制提供依据。方法:选取2010年1月至2013年1月在我院重症监护室治疗的病例中检测出的215株细菌为研究对象,运用相关的检测手段分析细菌的耐药性和耐药基因情况。结果:经过临床的检测后得出大肠埃希菌、肺炎克雷伯菌、鲍氏不动杆菌分别为85株、55株和75株,其中产ESBLs大肠埃希菌54株,非产ESBLs大肠埃希菌31株;产ESBLs肺炎克雷伯菌15株,非产ESBLs肺炎克雷伯菌40株。大肠埃希菌、肺炎克雷伯菌、鲍氏不动杆菌对美罗培南、亚胺培南的敏感性最高,且在产与非产ESBLs菌株耐药上比较有差异性(P0.05);产与非产ESBLs菌株耐药基因检测方面比较无明显差异性(P0.05)。结论:大肠埃希菌、肺炎克雷伯菌、鲍氏不动杆菌均存在多重耐药情况,且耐药与喹诺酮耐药机制有一定的相关性。     

重症肺炎新生儿支气管肺泡灌洗液 病原菌分布及耐药性

目的研究重症肺炎新生儿支气管肺泡灌洗液的病原菌分布和耐药性。方法选择2016年4月至2018年4月在本院呼吸科治疗的新生儿268例,其中符合重症肺炎诊断标准的患儿142例,归为重症肺炎组;不符合重症肺炎诊断标准的患儿126例,归为对照组。检测患儿肺泡灌洗液病原菌分布情况和耐药情况。结果重症肺炎组患儿肺炎克雷伯菌、流感嗜血菌、铜绿假单胞菌、阴沟肠杆菌、大肠埃希菌、金黄葡萄球菌、溶血葡萄球菌、表皮葡萄球菌、肺炎链球菌、草绿链球菌检出率明显高于对照组。肺炎克雷伯菌对亚胺培南,美罗培南的耐药性为0.0%,大肠埃希菌对亚胺培南,美罗培南,阿米卡星的耐药性为0.0%,阴沟肠杆菌对亚胺培南,美罗培南,左氧氟沙星的耐药性为0.0%,肺炎链球菌对万古霉素的耐药性为0.0%,金黄葡萄球菌对万古霉素的耐药性为0.0%。结论新生儿重症肺炎患者病原菌以革兰阴性菌为主,亚胺培南、美罗培南、万古霉素可以用于治疗新生儿重症肺炎,但由于其毒副作用较大,应严格把握适应症。     

口腔常见致龋菌荧光光谱特征的初步研究

目的采用三维荧光光谱分析技术对口腔常见致龋菌荧光光谱特征进行初步分析。方法选择口腔常见致龋菌变形链球菌及远缘链球菌,对其进行复苏和培养,运用三维荧光光谱分析技术对其菌液进行荧光学光谱检测。结果变形链球菌及远缘链球菌的三维荧光光谱图相似,均出现2个荧光峰,最佳激发波长分别位于230nm和280nm,最佳发射波长相同,均为340nm。结论成功获得口腔常见致龋菌(变形链球菌及远缘链球菌)的固有荧光三维光谱图,为致龋菌荧光评价技术的应用提供参考。     

Evolution of Streptococcus pneumoniae and its close commensal relatives

Streptococcus pneumoniae is a member of the Mitis group of streptococci which, according to 16S rRNA-sequence based phylogenetic reconstruction, includes 12 species. While other species of this group are considered prototypes of commensal bacteria, S. pneumoniae is among the most frequent microbial killers worldwide. Population genetic analysis of 118 strains, supported by demonstration of a distinct cell wall carbohydrate structure and competence pheromone sequence signature, shows that S. pneumoniae is one of several hundred evolutionary lineages forming a cluster separate from Streptococcus oralis and Streptococcus infantis. The remaining lineages of this distinct cluster are commensals previously collectively referred to as Streptococcus mitis and each represent separate species by traditional taxonomic standard. Virulence genes including the operon for capsule polysaccharide synthesis and genes encoding IgA1 protease, pneumolysin, and autolysin were randomly distributed among S. mitis lineages. Estimates of the evolutionary age of the lineages, the identical location of remnants of virulence genes in the genomes of commensal strains, the pattern of genome reductions, and the proportion of unique genes and their origin support the model that the entire cluster of S. pneumoniae, S. pseudopneumoniae, and S. mitis lineages evolved from pneumococcus-like bacteria presumably pathogenic to the common immediate ancestor of hominoids. During their adaptation to a commensal life style, most of the lineages gradually lost the majority of genes determining virulence and became genetically distinct due to sexual isolation in their respective hosts.     

Autolysins are direct involved in the bactericidal effect caused by penicillin in wild type and in tolerant pneumococci

The two pneumococcal autolytic enzymes (an N-acetylmuramoyl-L-alanine amidase and an endo-beta-1,4-N-acetylglucosaminidase) are directly involved in the penicillin-induced killing of Streptococcus pneumoniae. The activity of these lytic enzymes was efficiently controlled in tolerant mutants under physiological conditions.     

细菌自然转化的分子机制及生物学功能研究进展

基因的水平转移在细菌的进化中起着非常重要的作用。自然界中的细菌之间主要通过3种机制进行基因水平转移:由噬菌体介导的转导、接合转移和自然转化。自然转化是指自然感受态的细菌能够自发地从外界环境中摄取DNA分子并整合到自身基因组上的过程。该现象首先发现于肺炎链球菌,目前至少有83种细菌被发现具有发生自然转化的能力,其中革兰氏阳性菌以肺炎链球菌(Streptococcus pneumoniae,S. pneumoniae)为代表,革兰氏阴性菌以奈瑟氏菌(Neisseria)为代表,对其自然转化机制的研究和认识较为清楚,但不同细菌之间自然转化的机制有所差异。自然转化的生物学功能一直以来有以下几种推测:获取营养、修复DNA损伤、生物进化,而近年来对此认识争论不休。本文将详细描述细菌自然转化的分子机制,并对其主要的生物学功能争论焦点进行评述,以期对细菌自然转化有更深入的理解和认识。     

Minimizing the release of proinflammatory and toxic bacterial products within the host: a promising approach to improve outcome in life-threatening infections

Various bacterial components (e.g., endotoxin, teichoic and lipoteichoic acids, peptidoglycans, DNA) induce or enhance inflammation by stimulating the innate immune system and/or are directly toxic in eukariotic cells (e.g., hemolysins). When antibiotics which inhibit bacterial protein synthesis kill bacteria, smaller quantities of proinflammatory or toxic compounds are released in vitro and in vivo than during killing of bacteria by beta-lactams and other cell-wall active drugs. In general, high antibiotic concentrations liberate lower quantities of bacterial proinflammatory or toxic compounds than concentrations close to the minimum inhibitory concentration. In animal models of Escherichia coli Pseudomonas aeruginosa and Staphylococcus aureus peritonitis/sepsis and of Streptococcus pneumoniae meningitis, a lower release of proinflammatory bacterial compounds was associated with a reduced mortality or neuronal injury. Pre-treatment with a bacterial protein synthesis inhibitor reduced the strong release of bacterial products usually observed during treatment with a beta-lactam antibiotic. Data available strongly encourage clinical trials comparing antibiotic regimens with different release of proinflammatory/toxic bacterial products. The benefit of the approach to reduce the liberation of bacterial products should be greatest in patients with a high bacterial load.     

Quantitation of bacterial adherence by image analysis.

In studies of the adherence of pathogenic bacteria to host eukaryotic cells in vitro, the counting of the bacteria is often challenging, especially if many experiments are involved. We developed a method to use digital imaging and computer-aided recognition for the quantitation of bacteria attached to cultured cells. We employed an immunocytochemical method to stain the bacteria and leave the hosts cells relatively unstained. We describe this method for use with five species of bacteria, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, and Chlamydia pneumoniae. To demonstrate an application of this method, we studied the attachment of H. influenzae and S. pneumoniae to target epithelial cell lines derived from the respiratory tract.     

Resistance mechanism of chloramphenicol in Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis.

The chloramphenicol resistance of Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis isolated from clinical materials was proved to be due to an inactivating enzyme produced by these bacteria. The inactivated products of chloramphenicol were identified as 1-acetoxy, 3-acetoxy and 1,3-diacetoxy derivatives by thin-layer chromatography and infrared spectroscopy. The responsible enzyme was thus confirmed to be chloramphenicol acetyltransferase. The enzyme was inducible. It was partially purified by ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-150. The enzymes obtained from S. haemolyticus, S. pneumoniae and S. faecalis have been compared with the conclusion that they are identical with respect to molecular weight (approximately 75,000-80,000), optimum pH and heat stability.