A specific polymerase chain reaction test for the identification of Streptococcus pneumoniae

Using an approach based on the comparison of arbitrary primer polymerase chain reaction (PCR) genomic profiles from oral streptococci and Streptococcus pneumoniae strains, we identified a 434-bp genomic fragment apparently specific for S. pneumoniae. From the nucleotidic sequence of this common fragment, a pair of primers was designed and tested on a set of strains comprising the major Streptococcus species. One species, S. anginosus, gave an amplification product of the same length as S. pneumoniae. Sequence comparison of the S. anginosus and S. pneumoniae amplicons revealed several variations which were used to define a new set of primers giving a 181-bp S. pneumoniae-specific fragment. The amplified fragment contains the 5' terminal part of a gene encoding a putative sugar-specific permease and an intergenic sequence. The PCR test was evaluated on 257 strains of invasive S. pneumoniae corresponding to clinical isolates and on 153 non-pneumoniae oral streptococci strains; in addition, 3 S. pseudopneumoniae strains were tested. With these primers, an amplification product was only obtained with the S. pneumoniae strains. Moreover, the test was successfully evaluated on 10 atypical S. pneumoniae strains related to pneumococcal diseases. In this study, we therefore established the capacity of a simple PCR test to discriminate S. pneumoniae from other Streptococci (including S. pseudopneumoniae), thus allowing rapid and accurate diagnosis.     

A comparison of the activity of tigecycline against multiresistant clinical isolates of Staphylococcus aureus and Streptococcus agalactiae

We evaluated the activity of several antibiotics against 225 clinical isolates of Staphylococcus aureus and 252 isolates of Streptococcus agalactiae. Only tigecycline, glycopeptides, and linezolid were active against all the isolates of S. aureus, whereas the beta-lactams were also active against S. agalactiae. Tigecycline could be a good alternative to ampicillin in the treatment of group B Streptococcus infections in patients allergic to beta-lactam.     

BMY-28100, a new oral cephalosporin: antimicrobial activity against nearly 7,000 recent clinical isolates, comparative potency with other oral agents, and activity against beta-lactamase producing isolates

The antimicrobial activity of BMY-28100 was tested against approximately 7,000 bacterial pathogens in a multicenter, multiphased collaborative investigation. The BMY-28100 spectrum and antimicrobial potency was most similar to that of cefaclor and superior to that of cephalexin among currently available cephalosporins. Species that had greater than or equal to 90% of clinical strains inhibited by BMY-28100 (less than or equal to 8.0 micrograms/ml) were: Citrobacter diversus, Escherichia coli, Klebsiella spp., Proteus mirabilis, Salmonella spp., Branhamella catarrhalis, Haemophilus influenzae, Neisseria gonorrhoeae, N. meningitidis, methicillin-susceptible Staphylococcus supp., Streptococcus pneumoniae, S. pyogenes, S. agalactiae, S. bovis, serogroup C and G streptococci, Listeria monocytogenes and gm-positive anaerobes. BMY-28100 inhibited 9% more of the 6286 fresh clinical isolates at less than or equal to 8.0 micrograms/ml than cefaclor at the same concentration. BMY-28100 was generally bactericidal, but MICs for some species were markedly increased when an inoculum concentration of 10(7) CFU/ml was used. Strains producing plasmid-mediated beta-lactamases (TEM, OXA, SHV, HMS) were susceptible to BMY-28100, cefaclor, and cefuroxime. BMY-28100 was less active against strains producing chromosomal-mediated beta-lactamases (Types I and IV). BMY-28100 was not hydrolyzed significantly by the tested plasmid-mediated beta-lactamases, but was destroyed by Type I cephalosporinases and Klebsiella K1 enzymes.     

Evaluation of an extended diagnostic PCR assay for detection and verification of the common causes of bacterial meningitis in CSF and other biological samples

A seminested polymerase chain reaction (PCR)-based diagnostic assay was evaluated for detection and verification of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, Steptococcus agalactiae and Listeria monocytogenes in cerebrospinal fluid (CSF) and other biological samples. A general bacterial amplicon from the 16S rRNA gene was amplified in a first step, and species-specific regions in a second. The detection level was 4 fg DNA/reaction, corresponding to about one bacterial genome per reaction tube. Sample preparations (Dynabeads DNA DIRECT kit) were assayed from 140 bacterial strains suspended in saline. In CSF the detection level for bacteria was 10(3)CFU ml-1for N. meningitidis, H. influenzae and S. pneumoniae, 10(4)CFU ml-1for Escherichia coli and 10(5)CFU ml-1for S. agalactiae and L. monocytogenes. The detection levels for these bacteria were the same in the other tested biological samples, like blood with or without culture media. Clinical CSF samples were evaluated from 71 patients with proven bacterial meningitis, as were 61 CSF samples from individuals without bacterial meningitis. The diagnostic sensitivity of the assay in detecting bacteria in general was 0.97, and for the specific species in the clinical CSF samples 0.87-0.94. The specificity was 1.0 for detecting bacteria in general. Some cross-reactions were noted within the streptococcus group. The PCR results were verified by banding patterns of Hae III digested PCR products.     

Comparison of PCR-ELISA and LightCycler real-time PCR assays for detecting Salmonella spp. in milk and meat samples

In a previous study, we reported the performance of a PCR assay amplifying 285-bp of the invA gene of Salmonella spp. through an international ring-trial involving four participating laboratories [Int. J. Food Microbiol. 89 (2003) 241]. Based on the validated set of primers and recent advancements in PCR technology, we have designed two specific PCR assays for detecting Salmonella spp. We have compared PCR-enzyme-linked immunosorbent assay (PCR-ELISA) and LightCycler real-time PCR assay (LC-PCR) with the standard ISO 6579 bacteriological reference method. The two PCR tests incorporated an internal amplification control (IAC) co-amplified with the invA gene of Salmonella to monitor potential PCR inhibitors and ensure successful amplification. The selectivity study involved 84 Salmonella and 44 non-Salmonella strains and the samples tested were represented by 60 artificially-contaminated samples of fish, minced beef and raw milk, and 92 naturally-contaminated milk and meat samples. When using either PCR-ELISA or LC-PCR assays, only Salmonella strains were detected. PCR-ELISA and LC-PCR assays gave with pure Salmonella cultures the same detection limit level of 10(3)CFU/ml, which corresponds respectively to 50 and 10 cells per PCR tube. Data on artificially contaminated samples indicated that both PCR methods were able to detect after enrichment less than five Salmonella cells in 25 g of food, giving 100% concordance with the ISO 6579 reference method. The results on naturally contaminated samples demonstrated that despite certain inhibition problems, LC-PCR and PCR-ELISA assays were highly specific and sensitive, and provide a powerful tool for detection of Salmonella in food samples.     

实时荧光定量聚合酶链反应检测血清2型猪链球菌的研究

目的 基于TaqMan-MGB探针实时荧光定量聚合酶链反应(Real.time PCR)技术,建立针对血清2型猪链球菌的快速检测方法.方法 针对血清2型猪链球菌的csp2J基因序列,应用Beacon Designer 7.0,设计了引物和TaqMan-MGB探针,建立Real-time PCR检测方法;把目的 片段克隆...     

Lincomycin resistance gene lnu(D) in Streptococcus uberis

Streptococcus uberis UCN 42, isolated from a case of bovine mastitis, was intermediately resistant to lincomycin (MIC = 2 microg/ml) while remaining susceptible to clindamycin (MIC = 0.06 microg/ml) and erythromycin. A 1.1-kb SacI fragment was cloned from S. uberis UCN 42 total DNA on plasmid pUC 18 and introduced into Escherichia coli AG100A, where it conferred resistance to both clindamycin and lincomycin. The sequence analysis of the fragment showed the presence of a new gene, named lnu(D), that encoded a 164-amino-acid protein with 53% identity with Lnu(C) previously reported to occur in Streptococcus agalactiae. Crude lysates of E. coli AG100A containing the cloned lnu(D) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl(2). Mass spectrometry experiments demonstrated that the lnu(D) enzyme catalyzed adenylylation of clindamycin. A domain conserved in deduced sequences of lincosamide O-nucleotidyltransferases Lnu(A), Lnu(C), LinA(N2), and Lin(D) and in the aminoglycoside nucleotidyltransferase ANT(2') was identified.     

Bactericidal activity of cefepime and ceftriaxone tested against Streptococcus pneumoniae

The bactericidal activities of cefepime and ceftriaxone were assessed by testing a contemporary collection of 50 Streptococcus pneumoniae strains. Minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively) of cefepime and ceftriaxone were determined, and time-kill studies were performed on 14 selected strains (10 penicillin-resistant, 2-intermediate, and 2-susceptible). Cefepime and ceftriaxone showed essentially identical potency (MIC50, 1 microg/mL and MIC90, 2 microg/mL, for both compounds) and MBC values (MBC50, 1 microg/mL for both). MBC/MIC ratios were < or = 4 for cefepime and < or = 8 for ceftriaxone on 48 (96.0%) strains, and 2 strains (4.0%) displayed MBC/MIC ratios > or = 32 (tolerance) to the 2 cephalosporins. Time-kill curves corroborated the MBC/MIC studies. Cefepime and ceftriaxone bactericidal activity (> or = 3 log10 CFU/mL reduction in inoculum) was demonstrable after 24 h of exposure to 8x MIC for 13 (92.9%) of 14 strains, whereas 1 strain showed approximately 2 log10 CFU/mL reduction. In conclusion, our results indicate that cefepime and ceftriaxone exhibit comparable potency and bactericidal activities when tested against contemporary pneumococcal strains with varying penicillin susceptibility patterns. Both parenteral cephems offer alternative therapeutic choices for the treatment of invasive pneumococcal infections.     

TaqMan荧光定量PCR检测流感嗜血杆菌和肺炎链球菌方法的建立及应用

目的 建立TaqMan荧光定量PCR检测方法 ,用于流感嗜血杆菌和肺炎链球菌的检测和鉴别诊断.方法 针对流感嗜血杆菌种属特异性基因bexA和肺炎链球菌种属特异性基因lytA,设计合成引物和TaqMan探针,研究不同引物和探针荧光定量PCR检测的特异性和灵敏度,确定标本检测中循环阈值(Ct)的临界值(cut-off值).将荧光定量PCR、乳胶凝集和细菌培养3种检测方法 同时应用于278份细菌性脑膜炎患者脑脊髓液标本的检测.结果 bexA基因引物和探针能特异性检测流感嗜血杆菌a、b、c、d血清型的菌株,检测灵敏度为每个反应10个基因组DNA拷贝;lytA基因引物和探针能特异性检测肺炎链球菌常见致病的血清型肺炎链球菌菌株,检测灵敏度为每个反应90个基因组DNA拷贝.通过荧光定量PCR方法 ,278份脑脊髓液标本中共检测出 4份流感嗜血杆菌阳性和7份肺炎链球菌阳性,其中各有2份培养出相应的病原菌,另有1份流感嗜血杆菌和2份肺炎链球菌乳胶凝集结果 阳性.结论 TaqMan荧光定苗PCR方法 能特异地检测和鉴定流感嗜血杆菌和肺炎链球菌,具有较高的灵敏度和快速检测的特点,能提高临床流感嗜血杆菌和肺炎链球菌感染患者标本的阳性检出率.     

泌尿生殖道分离无乳链球菌药物敏感性分析

目的了解泌尿生殖道无乳链球菌感染及耐药状况,为临床用药提供依据。方法对临床送检的泌尿生殖道标本常规培养鉴定,并对分离出的无乳链球菌进行纸片扩散法药敏试验,对红霉素耐药、克林霉素敏感的菌株做D试验检测。结果144株无乳链球菌（尿77株,前列腺液36株,阴道分泌物31株）药敏结果显示,对万古霉素、利奈唑胺、青霉素和头孢曲松的耐药率最低,全部144株无乳链球菌中未发现耐药株;左氧氟沙星的耐药率较低,为16．2％;红霉素和克林霉素耐药率较高,分别为56．2％和53．5％。D试验阳性率为26．7％。结论从泌尿生殖道分离的无乳链球菌对青霉素、氨苄西林和头孢菌素类抗菌药物的敏感性较高,但对大环内酯类和克林霉素已有一定的耐药。     

环介导等温扩增检测肺炎链球菌方法的建立及临床应用

目的 建立环介导等温扩增(LAMP)快速检测肺炎链球菌的方法.方法 根据美国国家生物信息中心(GenBank)上提交的肺炎链球菌R6株的lytA基因序列(登录号AF2)08540)设计特异LAMP引物,以盐酸胍一苯甲醇法提取阳性血培养瓶中细菌DNA,然后以LAMP技术扩增lytA基因,反应条件为63℃45 min,采用肉眼观察浊度和琼脂糖电泳的方法来观察结果.结果 在196份阳性血培养瓶中,采用LAMP法检测到肺炎链球菌16株,与传统方法比较,其检测肺炎链球菌的灵敏度和特异度均达到100%,且检测时间缩短为1 h左右.模拟标本的检测结果显示该方法的最低检测限为102CFU/ml.结论 该方法灵敏度高,特异性强,操作方便快速,适合从临床标本中检测肺炎链球菌.     

Antimicrobial activities of tosufloxacin against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella branhamella catarrhalis isolated from otolaryngological infectious diseases

In 2003, the Japan Society for Infectious Diseases in Otolaryngology conducted its third nationwide survey of clinical isolates from otolaryngological infectious diseases. We selected three primary causative organisms of otolaryngological infectious diseases, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella Branhamella catarrhalis, and evaluated their sensitivities to tosufloxacin (TFLX), a new oral quinolone, because the survey revealed a rise in drug-resistant strains, suggesting potential problems with the antibiotics commonly used against these organisms. The minimum inhibitory concentration (MIC)₉₀ values of TFLX against S. pneumoniae, H. influenzae, and M. catarrhalis were 0.25 μg/ml, ≤0.06 μg/ml, and ≤0.06 μg/ml respectively, and TFLX was shown to be as effective as or superior to other new quinolones. In addition, TFLX showed sufficient antimicrobial effects against frequently detected drug-resistant bacteria such as penicillin-resistant S. pneumoniae (PRSP) and β-lactamase-negative, ampicillin-resistant strains of H. influenzae (BLNAR). Furthermore, only a few strains of bacteria showed resistance to TFLX.     

In vitro activities of tigecycline against erythromycin-resistant Streptococcus pyogenes and Streptococcus agalactiae: mechanisms of macrolide and tetracycline resistance

The activity of tigecycline was tested against erythromycin-resistant streptococci (107 Streptococcus pyogenes and 98 Streptococcus agalactiae strains). The presence of erythromycin and tetracycline resistance genes was determined by PCR. Among S. pyogenes strains the most prevalent gene was mef(A) (91.6%). The erm(B) gene was the most prevalent (65.3%) among S. agalactiae strains. Tigecycline proved to be very active against all the isolates tested (MIC at which 90% of the isolates tested were inhibited, 0.06 micro g/ml), including those resistant to tetracycline.     

泌尿生殖道分离无乳链球菌药物敏感性分析

目的了解泌尿生殖道无乳链球菌感染及耐药状况,为临床用药提供依据。方法对临床送检的泌尿生殖道标本常规培养鉴定,并对分离出的无乳链球菌进行纸片扩散法药敏试验,对红霉素耐药、克林霉素敏感的菌株做D试验检测。结果144株无乳链球菌(尿77株,前列腺液36株,阴道分泌物31株)药敏结果显示,对万古霉素、利奈唑胺、青霉素和头孢曲松的耐药率最低,全部144株无乳链球菌中未发现耐药株;左氧氟沙星的耐药率较低,为16.2%;红霉素和克林霉素耐药率较高,分别为56.2%和53.5%。D试验阳性率为26.7%。结论从泌尿生殖道分离的无乳链球菌对青霉素、氨苄西林和头孢菌素类抗菌药物的敏感性较高,但对大环内酯类和克林霉素已有一定的耐药。     

血清群6肺炎链球菌分子分型与耐药特征研究

目的 比较不同方法鉴别血清群6肺炎链球菌不同血清型的能力,了解血清群6肺炎链球菌的耐药性与分子特征。 方法 采用荚膜肿胀分型方法与聚合酶链反应(PCR)分型方法鉴别33株血清群6肺炎链球菌的血清型及相应的基因型;检测菌株对6种常用抗生素的敏感性;采用脉冲场凝胶电泳(PFGE)方法分析不同血清型菌株的基因组特征。 结果 PCR分型方法与传统的血清学分型方法结果一致。全部菌株对阿奇霉素均耐药,对利福平及左氧氟沙星均敏感;6B型菌株呈现较高的多重耐药特征。根据PFGE带型特征,所有菌株可分为2个簇,6C菌株之间、6B菌株之间带型呈现较高相似性。 结论 PCR分型方法可用于鉴别血清群6菌株的不同血清型;菌株耐药情况严重,且大多数菌株呈多重耐药特征;PFGE对鉴别肺炎链球菌不同菌株间的差异有较高的分辨率。     

Streptococcus suis in Hong Kong

Streptococcus suis was isolated from 6.1% of raw pork meat from 3 of the 6 wet markets in 6 districts in Hong Kong. S. suis was particularly isolated in sites from the tongue, tonsil, bone, and tail, but not from lean meat/minced pork or internal organs. Isolates were confirmed by polymerase chain reaction using S. suis-specific primers, did not belong to serotype 2 using serotype 2-specific antiserum, and were clustered closely with other known serotypes by phylogenetic analysis. Ten strains from patients admitted to Hong Kong hospitals with sepsis or meningitis in the past 10 years all belonged to type 2, with closely related pulsed-field gel electrophoresis types that were distinct from the S. suis strains isolated from pork in this study. These methods may serve as useful tools in studying and enhancing our understanding of these infections in Hong Kong.     

反向斑点杂交快速检测肺炎链球菌

目的:建立一种利用DNA探针快速检测肺炎链球菌的反向斑点杂交方法.方法:在肺炎链球菌特异的自溶素基因序列内设计引物,聚合酶链反应合成1段263 bp的DNA探针,然后用生物素标记的细菌DNA与该探针行反向斑点杂交,并将该方法应用于痰样本的检测.结果:所合成的探针具有高度特异性,与其他细菌间无交叉反应,该方法能检测出10 ng细菌DNA.50份痰标本肺炎链球菌培养阳性者8份,杂交阳性者12份,PCR阳性者18份.结论:反向斑点杂交具有快速、敏感、特异的优点,对肺炎链球菌的快速诊断有重要意义.     

肺炎链球菌呼吸道感染分离株的耐药性

目的：研究肺炎链球菌呼吸道感染分离株的耐药性及其传播情况。方法：肺炎链球菌分离株进行药物敏感试验和血清学分型,并结合BOX PCR、脉冲场电泳(pulse—field gel electrophoresis,PFGE)和青霉素结合蛋白基因(penicilin binding proteingene,pbp)指纹等分子生物学方法分析菌株间亲缘关系。结果：呼吸道感染分离株128株中有青霉素低度耐药株(penicillin intermediated Streptococcus pneurnoniae,PISP)10株,未发现青霉素高度耐药株(penicillin resistant Streptococcus pneumoniae,PRSP)。lll株存活菌株分属25种血清型,主要为23F、3、19F、14、9V、6A、6B等。9株存活PISP中有6种PFGE谱型,2株PFGE谱型相同菌株的耐药谱和血清型相仿;2株不同PFGE谱型菌株pbp指纹相似,对青霉素和头孢曲松敏感性相似;上述呼吸道感染分离PISP中未发现与世界主要流行耐药克隆代表株DNA指纹或pbp基因相似菌株。结论：上述肺炎链球菌呼吸道感染分离株青霉素耐药率尚低,存在青霉素耐药克隆和基因的传播,其中尚未发现世界主要流行耐药克隆。     

Variation in the attachment of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> to human pharyngeal epithelial cells after treatment with S-carboxymethylcysteine

S-carboxymethylcysteine (S-CMC) is a mucolytic agent that can prevent respiratory infection by decreasing the attachment of respiratory pathogens to human pharyngeal epithelial cells (HPECs). Streptococcus pneumoniae is a major cause of respiratory infections. A previous study revealed that treatment of S. pneumoniae with S-CMC caused a decrease in the attachment of this bacterium to HPECs. In the present study we found that the effect of S-CMC varied according to hosts and strains. S-CMC treatment altered the surface structure of S. pneumoniae, resulting in a decrease of attachment, without affecting the virulence of the bacteria.     

Antibiotic Susceptibility of Streptococcus bovis and Other Group D Streptococci Causing Endocarditis

Seventy-four strains of Streptococcus bovis and 35 strains of enterococci (Streptococcus faecalis and its varieties, Streptococcus faecium and Streptococcus durans), most of which were isolated from patients with endocarditis, were tested for their susceptibility to penicillin, ampicillin, erythromycin, cephalothin, vancomycin, methicillin, tetracycline, chloramphenicol, kanamycin, streptomycin, and gentamicin. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) were determined by a microtiter broth dilution technique. All of these organisms are group D streptococci, but the S. bovis strains are not enterococci. On the basis of both MIC and MBC, the S. bovis strains were much more susceptibile in general to antibiotics then were the enterococcal strains. For the S. bovis strains, the lowest MICs were obtained with penicillin, ampicillin, and erythromycin, and the lowest MBCs with penicillin and ampicillin. Although these antibiotics were also the most active against the enterococci, the MICs and MBCs were much higher than obtained with the S. bovis strains. Gentamicin was the most active aminoglycoside. On the basis of in vitro susceptibility results, the S. bovis strains resemble the viridans streptococci rather than enterococci.