Characterization of erythromycin-resistant Streptococcus pneumoniae isolates causing invasive diseases in Chinese children

Background  Erythromycin-resistant Streptococcus pneumoniae isolates that causing invasive pneumococcal diseases (IPD) in Chinese children remain uncharacterized. This study aims to identify the resistance genes associated with erythromycin resistance and to determine the genetic relationships of IPD isolates in Chinese children.

Methods  A total of 171 S. pneumoniae strains were isolated from 11 medical centers in China from 2006 to 2008. All the isolates were characterized via serotyping and antibiotic susceptibility determination. The erythromycin-resistant isolates were further characterized via ermB and mefA gene detection, multi-locus sequence typing analysis, and pulsed-field gel electrophoresis.

Results  A total of 164 (95.9%) isolates showed resistance to erythromycin, of which 162 strains with high high-level resistance (MIC ≥ 256 μg/ml). A total of 104 (63.4%) isolates carry the ermB gene alone, whereas 59 (36.0%) harbor both ermB and mefA genes. Of the 59 strains, 54 were of serotypes 19A and 19F and were identified as highly clonal and related to the Taiwan^19F-14 clone.

Conclusions The erythromycin resistance rate in IPD isolates is significantly high and is predominantly mediated by the ermB gene. Isolates that carry both ermB and mefA genes are predominantly of serotypes 19A and 19F.

     

E-cadherin mediates adhesion and endocytosis of Aspergillus fumigatus blastospores in human epithelial cells

Background  Aspergillus fumigatus (A. fumigatus) is a ubiquitous saprophytic fungus responsible for the majority of invasive mold infections in patients undergoing chemotherapy, organ transplantation or with persistent neutropenia. This study aimed to determine the role of E-cadherin for adhesion and endocytosis of A. fumigatus blastospores in the human epithelial cell line A549.

Methods  A. fumigatus blastospores were incubated with the total protein of A549 to investigate the binding of E-cadherin and blastospores followed by an affinity purification procedure. After establishing the adhesion model, the adhesion and endocytosis of A. fumigatus blastospores by A549 cells were evaluated by down-regulating E-cadherin of A549 cells using blocking antibody or small interfering RNA (siRNA).

Results  E-cadherin was adhered to the surface of A. fumigatus blastospore. Adhesion and endocytosis of the blastospores were reduced by blocking or down-regulating E-cadherin in A549 cells.

Conclusions  E-cadherin is a receptor for adhesion and endocytosis of A. fumigatus blastospores in epithelial cells. This may open a new approach to treat this fungal infection.

     

Three-dimensional cultures of human endometrial cells on Matrigel mimic in vivo morphology

Background  The regulation of endometrial physiology and morphogenesis by the paracrine effectors has been well established using in vivo studies. A more complete understanding of the endometrial function has been delayed due, in part, to a lack of appropriate culture models. In this study, we aimed to simulate the in vivo three-dimensional (3-D) growth pattern of endometrial cells using a 3-D in vitro culture system.

Methods  Isolated endometrial epithelial cells, stromal cells and RL95-2 cells were seeded into culture chambers coated with the extracellular matrix Matrigel and observed using light microscopy. Fluorescence staining and immunohistochemistry were used to assess the morphology.

Results  Depending on the culture conditions, epithelial cells and RL95-2 cells formed multicellular structures on Matrigel; stromal cells remained individually distinguishable or grew together to form 3-D lattice-like structures.

Conclusions  Matrigel provided a good microenvironment for culturing endometrial cells. The cells cultured in the Matrigel-coated chambers closely resembled those seen in vivo.

     

Taxonomic analysis of cryptococcus species complex strain S8012 revealed Cryptococcus gattii with high heterogeneity on the genetics

Background  Initially, Cryptococcus (C.) neoformans was previously divided into two varieties comprising C. neoformans var. neoformans and C. neoformans var. gattii. Currently, taxonomic studies defined C. neoformans as C. species complex, which contains C. neoformans var. neoformans (serotype D), the hybrid isolates (serotype AD), C. neoformans var. grubii (serotype A) and C. gattii (serotypes B and C). However, Liao and his team once isolated a unique C. gattii isolate, namely strain S8012 with unique phenotype from cerebrospinal fluid (CSF) of a 43-year-old male patient in the Shanghai Changzheng Hospital and described as C. neoformans var. shanghaiensis in 1980s. The aim of this study was to explore the genetic background and polymorphism of Chinese clinical C. gattii isolates.

Methods  S8012 was analyzed as representative strain using the M13-polymerase chain reaction (PCR) fingerprinting pattern and multilocus sequence analysis including internal transcribed spacers of rDNA (ITS region), the intergenic spacer 1 regions (IGS1), RPB1, RPB2, CNLAC1, and TEF1 genes.

Results  The PCR fingerprinting pattern results showed strain S8012 belonged to molecular types VGI, and phylogenetic analysis suggested strain S8012 was grouped into the cluster of C. gattii environmental isolates originated from Eucalyptus camaldulensis trees in Australia.

Conclusion  C. gattii isolates from Chinese patients expresses high polymorphism on the phenotype, and molecular type VGI isolates from China have a close genetic relationship with the C. gattii isolates from Australia.

     

Genetic diversity analysis of Penicillium marneffei isolated from AIDS patients in Guangdong, China using randomly amplified polymorphic DNA

Background  Penicillium marneffei (P. marneffei) is an emerging pathogenic fungus that can cause invasive mycosis in patients with AIDS. The epidemiological features of P. marneffei infection in AIDS patients in Guangdong province remain unclear so far. This study aimed to investigate the genetic diversity within a population of 163 P. marneffei isolates obtained from AIDS patients and search for the dominant clinical strains in Guangdong province.

Methods  One hundred and sixty-three P. marneffei isolates obtained from AIDS patients in Guangdong province during January 2004 and December 2009 were studied by randomly amplified polymorphic DNA (RAPD) using two random primers (H2 and H22). The degree of similarity between samples was calculated through similarity coefficients from RAPD fragment data and the dendrogram was assessed using the unweighted pair group method with arithmetic mean (UPGMA).

Results  Two primers showed a high degree of discrimination and good stability. Primer H2 yielded eight different patterns (H2-1 to H2-8) among 163 isolates with the discriminatory power being 0.413. Primer H22 identified seven types (H22-1 to H22-7) among 163 isolates with the discriminatory power being 0.467. Genetic similarity coefficients based on RAPD data among 163 P. marneffei isolates ranged from 0.681 to 0.957, 61.96% of which were no less than 0.83. The discriminatory power of the two primers was 0.524. One hundred and sixty-three P. marneffei isolates were clustered into nine distinct groups (groups I to IX) at the similarity coefficient value of 0.83 and group I was the most common, including 101 strains (61.96%).

Conclusion  The RAPD analyses could provide important information as to the degree of genetic diversity and the relationship among clinical P. marneffei isolates, revealing genetic polymorphism and dominant strains.

     

Reversal of multidrug resistance in renal cell carcinoma by short hairpin RNA targeting MDR1 gene

Background  Over-expression of P-glycoprotein (P-gp), encoded by the MDR1 gene, confers multidrug resistance (MDR) in renal cell carcinoma (RCC) and is a major reason for unsuccessful chemotherapy. This study aimed to determine the effct of RNA interference (RNAi) on the reversal of MDR in human RCC.

Methods  We designed and selected one short hairpin RNA (shRNA) targeting MDR1 gene, which is stably expressed from integrated plasmid and transfected by lentivirus fluid in human RCC A498 cell.

Results  The MDR1-targeted RNAi resulted in decreased MDR1 gene mRNA level (P <0.001), almost abolished P-gp expression and reversed MDR to different chemotherapy drugs in the RCC A498 cell line.

Conclusion  MDR could be reversed by RNAi in human RCC A498 cell line, which may be used for clinical application in future.

     

Mapping and localization of susceptible genes in asthma

Objective  To elucidate the development of mapping and localization of susceptible genes on chromosomes to asthma related phenotypes.

Data sources  Published articles about susceptibility genes for asthma related phenotypes were selected using PubMed.

Study selection  Using methods of candidate gene positional clone and genome-wide scan with linkage and association analysis to determine the location in the genome of susceptibility genes to asthma and asthma related phenotypes.

Results  There are multiple regions in the genome harboring susceptibility genes to asthma and asthma related phenotypes, including chromosomes 5, 11, 12, 6, 2, 3, 13, 7, 14, 9, 19 and 17. Many of these regions contain candidate genes involved in asthma development and progression. Some susceptible genes may affect the phenotype expression or response to therapy. In addition, the interaction of multiple genes with the environment may contribute to the susceptibility to asthma.

Conclusions  As an essential step toward cloning the susceptible genes to asthma, fine mapping and localization on chromosomes are definitely needed. Novel powerful tools for gene discovery and the integration of genetics, biology and bioinformatics should be pursued.

     

Proteomic analysis for detecting serum biomarkers related to smoking in humans

Background  Smoking is the leading cause of death in the world. This study focused on the difference of the serum proteomic profiling between healthy smokers and nonsmokers in order to find smoking-specific serum biomarkers.

Methods  Pattern-based proteomic profiling of 100 serum samples (from 50 Chinese male smokers and 50 matched nonsmokers) was performed through magnetic bead fractionation coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF-MS) and resulting data were statistically analyzed by Ciphergen ProteinChip software 3.0.2.

Results  We found 72 serum peaks were significantly different between smokers and nonsmokers (P <0.05). Marker peaks of mass-to-charge ratio (m/z) 3159.13, 7561.03 and 9407.32 were smoking-specific.

Conclusion  The preliminary data suggested that smoking-specific serum biomarkers could be detected in humans.

     

Use of the laser speckle flowgraphy in posterior fundus circulation research

Objective  To review articles aiming to present an overview of the principles, progress, uses and limitations of laser speckle flowgraphy (LSFG) in posterior fundus circulation research.

Data sources  The data used in this review was obtained mainly from the studies reported in PubMed using the key terms “laser speckle”, “ocular blood flowmetry” and “retinal imaging”.

Study selection  Relevant literatures on studies of LSFG were selected.

Results  LSFG is a unique, noninvasive imaging instrument to quantitatively visualize posterior fundus circulation in vivo. This review delineates the LSFG principles and development, demonstrates its extensive applicability for measurement of retina, choroid and optic nerve head circulation, compares it with other retinal imaging technologies and discusses unresolved issues.

Conclusions  LSFG is a noninvasive, two-dimensional objective diagnostic technique that has become a powerful method for the clinical and scientific assessment of posterior fundus circulation. Further studies may help to develop a more comprehensive evidence-based measurement and facilitate the correlation with other methods for chorioretinal circulation assessment.

     

Serological and molecular capsular typing, antibiotic susceptibility and multilocus sequence typing of Streptococcus pneumoniae isolates from invasive and non-invasive infections

Background Streptococcus pneumoniae (S. pneumoniae) is a major causative agent of severe infections, including sepsis, pneumonia, meningitis, and otitis media, and has become a major public health concern. We report the pneumococcal serotype and sequence type (ST) distribution, and antimicrobial resistance of 39 S. pneumoniae strains from seven hospitals in China.

Methods Blood/cerebrospinal fluid (CSF) and sputum isolates from patients were analyzed to determine S. pneumoniae serotypes by polymerase chain reaction (PCR) and the Neufeld Quellung reaction, the multilocus sequence types (MLST) by PCR and sequencing, and susceptibility to antimicrobial agents by the VITEK Gram Positive Susceptibility Card.

Results A total of 39 isolates were collected including 21 blood/CSF and 18 sputum isolates. Conventional serotyping by the Quellung reaction required 749 reactions. In contrast, PCR based typing needed only 106 PCR reactions. The most frequent serotypes from the blood/CSF isolates were 14 (38.1%), 19A (14.3%), 23F (9.5%), and 18C (9.5%). In the sputum isolates the most frequent serotypes were 19F (33.3%), 23F (16.7%), 19A (11.1%), and 3 (11.1%). The incidence of penicillin resistance in the blood/CSF and sputum isolates was 66.7% and 55.6%, respectively. Statistical analysis showed that patients ≤5 years old had a higher resistance to penicillin when they compared with the patients ≥65 years old (P=0.011). Serotypes 14, 19A and 19F were significantly associated with penicillin resistance (P <0.001). ST320, ST271, and ST876 isolates showed high resistant rates to several antibiotics including penicillin (P=0.006). All of the isolates of serotype 19A were resistant to both penicillin and erythromycin, and they were all multi-drug resistant (MDR) isolates.

Conclusions The specificity and sensitivity of multiplex-PCR are good, and this method represents a substantial savings of time and money, and can be widely used in the laboratory and clinical practice. Data from this research showed an extremely high prevalence of penicillin resistance and an increasing prevalence of multi-drug resistant (MDR) rate in S. pneumoniae. A distinctive emergence of serotype 19A was observed which was also associated with the increasing prevalence of antimicrobial resistance. Therefore, nationwide surveillance of pneumococcal resistance and serotypes is strongly warranted.

     

侵袭性肺炎链球菌148株血清型、耐药性及分子分型研究

目的 研究临床分离的侵袭性肺炎链球菌血清型分布、耐药性及分子流行病学特点,为抗生素的应用和免疫预防提供参考依据.方法 收集2005年1月至2008年8月全国15个地区148株来自血液、脑脊液等侵袭性感染部位的肺炎链球菌.采用琼脂稀释法测定青霉素等抗生素对148株肺炎链球菌的最低抑菌浓度(MIC);采用简易棋盘式肺炎链球菌分型系统和荚膜肿胀实验进行血清分型;多位点序列分型(MIST)技术对53株19群菌株进行基因分型,了解其与国际流行株的关系.结果 血清分型共检出20个血清型/群,主要集中在19A、19F、3、23F、5、6、14和9血清型/群,共计105株,占70.9%,以19A(22.3%,33/148)、19F(16.9%,25/148)最常见,其次为3群(7.4%,11/148)和23F(6.8%,10/148).7价疫苗(PCV7)在2岁以下儿童中的覆盖率为33.3%(12/36).PCV7相关血清型肺炎链球菌对阿莫西林/克拉维酸、红霉素等耐药率均明显高于非疫苗相关血清型菌株(P<0.05).53株19群肺炎链球菌MIST分析共检出9种序列型(ST),其中以ST320(28/53,52.8%)和ST271(12/53,22.6%)为主.结论 我国侵袭性肺炎链球菌血清分型以19A、19F、3和23F血清型为主.侵袭性肺炎链球菌耐药情况严重.     

上海市791名健康儿童222株肺炎链球菌携带株研究

目的 了解健康儿童肺炎链球菌携带株的流行病学特征。方法 测定来自上海5所幼儿园（代号分别为JR、PL、SY、WS、ZF）791名健康儿童鼻咽部分离的222株肺炎链球菌健康儿童携带株的耐药谱和血清型,并以PFGE、BOX、PGR和pbp基因指纹等分子生物学方法分析菌株间亲缘关系。结果 222株携带株中有青霉素低度耐药株32株,未发现青霉素高度耐药株,全部菌株包括22种血清型,常见为23F（25.7%),6A（13.1%)、19F（10.8%)、6B（8.1%)等;青霉素低度耐药株包括8种血清型,主要为23F（50%）、19A和19F（各占15.6%)。32株青霉素低度耐药菌株可分为8种BOXPCR谱型和7种PFGE谱型。PFGE谱型A菌株的耐药谱,血清型以及BOXPCR和pbp基因指纹高度一致。自幼儿园SY的儿童分离的肺炎链球菌青霉素耐药率31.8%,远高于平均水平,该园21株青霉素耐药菌株中20株属于PFGE谱型A或B。结论 上海5所幼儿园健康儿童肺炎链球菌携带株青霉素耐药率总体尚较低。但个别幼儿园具有较高耐药率,可能与青霉素耐药克隆的流行有关。     

广西玉林市住院患者肺炎链球菌的血清型分布及耐药性分析

目的 分析住院患者肺炎链球菌分离株的血清型分布、耐药率及耐药基因携带,评估疫苗对本地区肺炎链球菌血清型的覆盖率,为临床合理使用抗菌药物提供参考。方法 收集2015年1月—2019年12月广西玉林市第一人民医院住院患者送检标本分离的非重复肺炎链球菌150株,进行血清分型及抗菌药物敏感性试验。用PCR方法检测pbp2b、ermB、tetM三种耐药基因的携带率。结果 150株肺炎链球菌经PCR技术分型率为93.1%,经荚膜肿胀试验分型率为100%,共分出19种血清型,以19F和6B为主。儿童血清型以19F、6B和15A为主;成人血清型以19F、14和23F为主。PCV7、PCV10、PCV13和PPV23疫苗的覆盖率依次分别为36.8%、42.1%、57.9%和68.4%。血清型为19F、6B、3和23F的菌株对抗菌药物的耐药率较高。肺炎链球菌对青霉素的敏感率大于96.0%。侵袭性与非侵袭性菌株中耐药率有显著差异的抗菌药物为青霉素、莫西沙星和左氧氟沙星。菌株同时携带ermB和tetM两种耐药基因占96.0%,pbp2b、ermB、tetM三种耐药基因与耐药表型的一致率>98.0%。共检...     

2000-2005年自北京儿童医院门诊患儿鼻咽部分离的19群肺炎链球菌的耐药性变化

目的明确2000—2005年分离的北京儿童19群肺炎链球菌的抗生素敏感性分布及年代变化特点。方法采用简易棋盘式肺炎链球菌分型系统以荚膜肿胀试验进行血清分型。E—test法检测分离株对青霉素等6种抗生素的最低抑菌浓度（minimum inhibitory concentrations,MIC）。结果2002—2003年和2004—2005年肺炎链球菌19群在分离的青霉素不敏感肺炎链球菌中所占比例明显升高,青霉素、头孢克洛和头孢呋辛钠敏感率明显下降,MIC50、MIC90和最大MIC值的水平升高。从MIC值分布看,19群分离株对阿莫西林／克拉维酸和头孢曲松的敏感性下降,但仍处于判定标准的敏感范围。结论2000—2005年自北京儿童医院门诊患儿鼻咽部分离的肺炎链球菌19群对β内酰胺类抗生素耐药性增强,耐药株增多。     

武汉地区肺炎链球菌的红霉素耐药性分析及血清分型研究

目的 研究武汉地区肺炎链球菌的红霉素耐药率、耐药表型及肺炎链球菌血清分型分布情况.方法 以琼脂稀释法测定红霉素、青霉素及其他大环内酯类等抗菌药物对304株肺炎链球菌的最低抑菌浓度(MIC),采用荚膜肿胀试验进行血清分型,以双纸片法测定红霉素耐药菌株的耐药表型.结果 304株肺炎链球菌红霉素耐药率高达84.21%(256/304).红霉素耐药菌株中,cMLSB型耐药占98.44%(252/256),M型耐药仅占1.56%(4/256).血清分型涉及20个血清型、群,主要集中在19、23、6、15和14血清群,其中PNSSP集中分布在6、19、23和未分型血清群.结论 武汉地区红霉素不敏感肺炎链球菌的发生率较高,其耐药表型以cMLSB型为主,双纸片法对此耐药表型的检测有很好的预见性.血清分型,尤其多重耐药菌株以6、19和23血清群为主,推荐采用疫苗免疫预防肺炎链球菌感染.     

Nasal carriage of Streptococcus pneumoniae among children in Beijing

目的　了解北京地区儿童鼻部携带的肺炎链球菌对抗生素的敏感性以及血清型分布 ,分析鼻部携带青霉素非敏感肺炎链球菌的危险因素。方法　用纸片扩散法检测肺炎链球菌对红霉素 ,复方新诺明 ,氯霉素和四环素的敏感性 ;E -试验确定青霉素 ,头孢呋新 ,头孢噻肟 ,安灭菌和亚胺培南的最小抑菌浓度 ;Quellung反应确定肺炎链球菌的血清型。结果　未发现对青霉素和头孢呋新高度耐药的肺炎链球菌 ,但对青霉素和头孢呋新中度耐药的肺炎链球菌分别占 8 2 %和 2 1%。所有菌株均对头孢噻肟 ,安灭菌和亚胺培南敏感。对红霉素 ,复方新诺明和四环素耐药的肺炎链球菌特别多 ,分别占 72 % 70 %和 79%。所有肺炎链球菌中 ,有 5种血清型 (19,6 ,14 ,2 3,17)共占 5 4 7% ,不定型占 2 0 6 %。既往中耳炎病史是鼻部携带青霉素非敏感肺炎链球菌的危险因素。结论　连续监测肺炎链球菌对抗生素的敏感性具有重要意义。需要进行更大规模的研究以明确目前的 7价或 9价肺炎链球菌结合疫苗是否适合中国儿童     

儿童肺炎链球菌的血清型及对抗生素的耐药性

目的　对本校儿科医院住院病例进行肺炎链球菌的血清型和耐药性监测。方法　收集因呼吸道感染而住院的病例痰标本和病史资料 ,同时采集部分患儿的血标本 ,分离与鉴定标本中的肺炎链球菌。荚膜肿胀试验进行血清分型 ;E test测定对青霉素的最低抑菌浓度。结果　共收集到 10 13例患儿的资料 ,分离出 112株肺炎链球菌。 19F、2 3F、6A、14和 6B等 5种血清型占 81.3% ( 91/112 ) ,其他类型的血清型占 11.6 % ( 13/112 )。另有 7.1% ( 8/112 )的肺炎链球菌不能分型。只有 1例血标本检测到肺炎链球菌 ,这可能与患儿入院前的抗菌素广泛使用有关。对青霉素中度和高度耐药的肺炎链球菌比例分别为 5 0 .9%和 8.0 % ;对氨苄青霉素、头孢唑林、头孢克洛、头孢曲松、红霉素、氯林可霉素和氯霉素的耐药率分别为 5 8.6 %、6 .6 %、2 5 .0 %、6 .6 %、85 .7%、6 6 .7%和 2 8.2 %。在 6 6例对青霉素耐药的菌株中 ,以 9F血清型最为多见 ,其次为 2 3F和 14。结论　肺炎链球菌是导致上海地区儿童呼吸道感染的重要病原菌 ,主要由 5种血清型组成 ,该菌株对各类抗菌素的耐药性普遍较高     

沙门菌携带者分离株血清学及PFGE分型研究

目的建立在健康携带者分离的沙门菌的血清型及PFGE分子型别图谱库,探讨其多态相关性关系。方法将预防性健康体检分离获得的21株沙门菌鉴定培养,进行血清分型;将确定后的菌株用限制性内切酶Xba I消化酶切其基因组DNA,在脉冲场凝胶电泳仪进行DNA电泳,所得的分子型别图谱用Bionumerics5.1软件建立图谱数据库,并进行聚类分析。结果21株沙门菌分属于O：4（B）、O：9（D1）和O：3,10（E1）3个菌群,8种血清型;以德尔卑沙门菌为主,共7株占所有分离株的占33.3%,其次为阿贡纳沙门菌19.1%（4/21）,鼠伤寒沙门菌14.3%（3/21）。经比对分析,18株沙门菌分为15个PFGE型别,菌株间的相似值在53.9%～100%之间,同一血清型别的沙门菌可对应多个PFGE型别。结论沙门菌的PFGE型别呈现多样性,脉冲场凝胶电泳对从业人员体检沙门菌有较高的分型能力,可为沙门菌的日常监测和相关疫情的处理提供重要的技术支撑。