scholarly journals Confirmation of psaA in All 90 Serotypes of Streptococcus pneumoniae by PCR and Potential of This Assay for Identification and Diagnosis

2000 ◽  
Vol 38 (1) ◽  
pp. 434-437
Author(s):  
Katherine E. Morrison ◽  
Derrick Lake ◽  
Jennifer Crook ◽  
George M. Carlone ◽  
Edwin Ades ◽  
...  
Keyword(s):  
United States ◽  
Streptococcus Pneumoniae ◽  
Bacterial Species ◽  
The United States ◽  
Culture Collection ◽  
Pcr Assay ◽  
Gene Encoding ◽  
Atcc Strain ◽  
Viridans Group Streptococci ◽  
Culture Positive

ABSTRACT The gene encoding the pneumococcal surface adhesin A (PsaA) protein, psaA , was confirmed in all Streptococcus pneumoniae serotypes by a newly developed PCR ( psaA PCR) assay. Eighty-nine of the 90 serotypes amplified produced an 838-bp fragment; the exception was a serotype 16F strain acquired from the American Type Culture Collection (ATCC). Analysis of 20 additional 16F strains from the United States and Brazil showed that the gene was amplified in all 16F strains, implying that the serotype 16F ATCC strain must be a variant. The specificity of the assay was verified by the lack of signal from analysis of heterologous bacterial species ( n = 30) and genera ( n = 14), including viridans group streptococci. The potential of the assay for clinical application was shown by its ability to detect pneumococci in culture-positive nasopharyngeal specimens. Demonstration of psaA in all 90 serotypes and lack of amplification of heterologous organisms suggest that this assay could be a useful tool for detection of pneumococci and diagnosis of disease.

scholarly journals Identification of the psaA Gene, Coding for Pneumococcal Surface Adhesin A, in Viridans Group Streptococci other than Streptococcus pneumoniae

2001 ◽  
Vol 8 (5) ◽  
pp. 895-898 ◽  
Author(s):  
Isabel Jado ◽  
Asunción Fenoll ◽  
Julio Casal ◽  
Amalia Pérez
Keyword(s):  
Streptococcus Pneumoniae ◽  
Bacterial Species ◽  
Open Reading Frame ◽  
Gene Encoding ◽  
Reading Frame ◽  
Gene Coding ◽  
Pcr Products ◽  
Streptococcal Species ◽  
Viridans Group Streptococci ◽  
High Degree

ABSTRACT The gene encoding the pneumococcal surface adhesin A (PsaA) protein has been identified in three different viridans group streptococcal species. Comparative studies of the psaA gene identified in different pneumococcal isolates by sequencing PCR products showed a high degree of conservation among these strains. PsaA is encoded by an open reading frame of 930 bp. The analysis of this fragment inStreptococcus mitis, Streptococcus oralis, andStreptococcus anginosus strains revealed a sequence identity of 95, 94, and 90%, respectively, to the corresponding open reading frame of the previously reported Streptococcus pneumoniae serotype 6B strain. Our results confirm thatpsaA is present and detectable in heterologous bacterial species. The possible implications of these results for the suitability and potential use of PsaA in the identification and diagnosis of pneumococcal diseases are discussed.

scholarly journals A Multiplex PCR Assay to Detect and Differentiate Select Agent Strains of Ralstonia solanacearum

Plant Disease ◽  
2015 ◽  
Vol 99 (3) ◽  
pp. 333-341 ◽  
Author(s):  
Michael J. Stulberg ◽  
Jonathan Shao ◽  
Qi Huang
Keyword(s):  
United States ◽  
Multiplex Pcr ◽  
Ralstonia Solanacearum ◽  
Species Complex ◽  
Bacterial Species ◽  
The United States ◽  
Pcr Assay ◽  
Plant Dna ◽  
Multiplex Pcr Assay ◽  
Verification Methods

Ralstonia solanacearum race 3 biovar 2 strains are considered select agents by the U.S. government because they are not endemic to the United States and have the potential to cause brown rot in our potato production fields. Simple and accurate methods are needed for quick identification prior to more discriminating but time-consuming verification methods. We developed a multiplex PCR assay that identifies R. solanacearum species complex strains, signals whether the strain detected is a select agent, and controls for false negatives associated with PCR inhibition or unsuccessful DNA extractions in one reaction. We identified unique sequences of non-phage-related DNA for the R. solanacearum species complex strains, and for select agent strains, using in silico genome subtraction. We also designed and included an internal plant DNA control assay. Our multiplex PCR assay correctly identified 90 R. solanacearum species complex strains and 34 select agent strains, while not recognizing five out-group bacterial species. Additionally, the multiplex PCR assay facilitated the detection of plant DNA and R. solanacearum from infected tomato, potato, geranium, and tobacco plants. Our rapid, accurate, and reliable detection assay can help government officials make timely and appropriate recommendations to exclude this bacterium from the United States.

scholarly journals Prevalence of Pathogenic Yersinia enterocolitica Strains in Pigs in the United States

2005 ◽  
Vol 71 (11) ◽  
pp. 7117-7121 ◽  
Author(s):  
Saumya Bhaduri ◽  
Irene V. Wesley ◽  
Eric J. Bush
Keyword(s):  
United States ◽  
Yersinia Enterocolitica ◽  
Culture Method ◽  
The United States ◽  
Contamination Rate ◽  
Pcr Assay ◽  
Fecal Samples ◽  
Pork Production ◽  
Food Borne ◽  
Culture Positive

ABSTRACT Yersinia enterocolitica is considered an important food-borne pathogen impacting the pork production and processing industry in the United States. Since this bacterium is a commensal of swine, the primary goal of this study was to determine the prevalence of pathogenic Y. enterocolitica in pigs in the United Sates using feces as the sample source. A total of 2,793 fecal samples were tested for its presence in swine. Fecal samples were collected from late finisher pigs from 77 production sites in the 15 eastern and midwestern pork-producing states over a period of 27 weeks (6 September 2000 to 20 March 2001). The prevalence of ail-positive Y. enterocolitica was determined in samples using both a fluorogenic 5′ nuclease PCR assay and a culture method. The mean prevalence was 13.10% (366 of 2,793 fecal samples tested) when both PCR- and culture-positive results were combined. Forty-one of 77 premises (53.25%) contained at least one fecal sample positive for the ail sequence. The PCR assay indicated a contamination rate of 12.35% (345/2,793) compared to 4.08% (114/2,793) by the culture method. Of the 345 PCR-positive samples, 252 were culture negative, while of the 114 culture-positive samples, 21 were PCR negative. Among 77 premises, the PCR assay revealed a significantly (P < 0.05) higher percentage (46.75%, n = 36 sites) of samples positive for the pathogen (ail sequence) than the culture method (22.08%, n = 17 sites). Thus, higher sensitivity, with respect to number of samples and sites identified as positive for the PCR method compared with the culture method for detecting pathogenic Y. enterocolitica, was demonstrated in this study. The results support the hypothesis that swine are a reservoir for Y. enterocolitica strains potentially pathogenic for humans.

scholarly journals Need for Annual Surveillance of Antimicrobial Resistance in Streptococcus pneumoniae in the United States: 2-Year Longitudinal Analysis

2001 ◽  
Vol 45 (4) ◽  
pp. 1037-1042 ◽  
Author(s):  
Daniel F. Sahm ◽  
James A. Karlowsky ◽  
Laurie J. Kelly ◽  
Ian A. Critchley ◽  
Mark E. Jones ◽  
...  
Keyword(s):  
United States ◽  
Streptococcus Pneumoniae ◽  
Antimicrobial Resistance ◽  
Antimicrobial Agents ◽  
The United States ◽  
Multidrug Resistant ◽  
Fluoroquinolone Resistance ◽  
Resistance Patterns ◽  
Changing Patterns ◽  
Resistance To Penicillin

ABSTRACT Although changing patterns in antimicrobial resistance inStreptococcus pneumoniae have prompted several surveillance initiatives in recent years, the frequency with which these studies are needed has not been addressed. To approach this issue, the extent to which resistance patterns change over a 1-year period was examined. In this study we analyzed S. pneumoniaeantimicrobial susceptibility results produced in our laboratory with isolates obtained over 2 consecutive years (1997–1998 and 1998–1999) from the same 96 institutions distributed throughout the United States. Comparison of results revealed increases in resistant percentages for all antimicrobial agents studied except vancomycin. For four of the agents tested (penicillin, cefuroxime, trimethoprim-sulfamethoxazole, and levofloxacin), the increases were statistically significant (P < 0.05). Resistance to the fluoroquinolone remained low in both years (0.1 and 0.6%, respectively); in contrast, resistance to macrolides was consistently greater than 20%, and resistance to trimethoprim-sulfamethoxazole increased from 13.3 to 27.3%. Multidrug resistance, concurrent resistance to three or more antimicrobials of different chemical classes, also increased significantly between years, from 5.9 to 11%. The most prevalent phenotype was resistance to penicillin, azithromycin (representative macrolide), and trimethoprim-sulfamethoxazole. Multidrug-resistant phenotypes that included fluoroquinolone resistance were uncommon; however, two phenotypes that included fluoroquinolone resistance not found in 1997–1998 were encountered in 1998–1999. This longitudinal surveillance study of resistance inS. pneumoniae revealed that significant changes do occur in just a single year and supports the need for surveillance at least on an annual basis, if not continuously.

Assessment of Laboratory Performance With Streptococcus pneumoniae Antimicrobial Susceptibility Testing in the United States

1999 ◽  
Vol 123 (4) ◽  
pp. 285-289 ◽  
Author(s):  
Gary V. Doern ◽  
Angela B. Brueggemann ◽  
Michael A. Pfaller ◽  
Ronald N. Jones
Keyword(s):  
United States ◽  
Streptococcus Pneumoniae ◽  
Antimicrobial Agents ◽  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
The United States ◽  
National Committee ◽  
In Vitro Susceptibility ◽  
Susceptibility Tests

Abstract Objective.—To assess the performance of clinical microbiology laboratories in the United States when conducting in vitro susceptibility tests with Streptococcus pneumoniae. Methods.—The results of a nationwide College of American Pathologists Proficiency Survey test sample, in which susceptibility testing of an isolate of S pneumoniae was performed, were assessed with respect to precision and accuracy. Results.—Wide variability was noted among participating laboratories with both minimum inhibitory concentration procedures and disk diffusion susceptibility tests when both methods were applied to S pneumoniae. Despite this high degree of variation, categorical interpretive errors were uncommon. Numerous laboratories reported results for antimicrobial agents that are not recommended by the National Committee for Clinical Laboratory Standards for tests with S pneumoniae. Conclusions.—Current susceptibility testing practices with S pneumoniae in the United States indicate limited precision and a tendency for laboratories to test and report results obtained with antimicrobial agents of questionable therapeutic value against this organism. Continued efforts to standardize susceptibility testing of S pneumoniae in the United States are warranted. In addition, modifications of existing interpretive criteria may be necessary.

scholarly journals The pCS20 PCR assay for Ehrlichia ruminantium does not cross-react with the novel deer ehrlichial agent found in white-tailed deer in the United States of America

2004 ◽  
Vol 71 (2) ◽  
Author(s):  
S.M. Mahan ◽  
B.H. Simbi ◽  
M.J. Burridge
Keyword(s):  
United States ◽  
High Specificity ◽  
Pcr Primers ◽  
The United States ◽  
Rrna Gene ◽  
The Novel ◽  
Pcr Assay ◽  
Ehrlichia Ruminantium ◽  
Cowdria Ruminantium ◽  
Specificity And Sensitivity

White-tailed deer are susceptible to heartwater (Ehrlichia [Cowdria] ruminantium infection) and are likely to suffer high mortality if the disease spreads to the United States. It is vital, therefore, to validate a highly specific and sensitive detection method for E. ruminantium infection that can be reliably used in testing white-tailed deer, which are reservoirs of antigenically or genetically related agents such as Ehrlichia chaffeensis, Anaplasma (Ehrlichia) phagocytophilum (HGE agent) and Ehrlichia ewingii. Recently, a novel but as yet unnamed ehrlichial species, the white-tailed deer ehrlichia (WTDE), has been discovered in deer populations in the United States. Although the significance of WTDE as a pathogen is unknown at present, it can be distinguished from other Ehrlichia spp. based on 16S rRNA gene sequence analysis. In this study it was differentiated from E. ruminantium by the use of the pCS20 PCR assay which has high specificity and sensitivity for the detection of E. ruminantium. This assay did not amplify DNA from the WTDE DNA samples isolated from deer resident in Florida, Georgia and Missouri, but amplified the specific 279 bp fragment from E. ruminantium DNA. The specificity of the pCS20 PCR assay for E. ruminantium was confirmed by Southern hybridization. Similarly, the 16S PCR primers (nested) that amplify a specific 405-412 bp fragment from the WTDE DNA samples, did not amplify any product from E. ruminantium DNA. This result demonstrates that it would be possible to differentiate between E. ruminantium and the novel WTDE agent found in white tailed deer by applying the two respective PCR assays followed by Southern hybridizations. Since the pCS20 PCR assay also does not amplify any DNA products from E. chaffeensis or Ehrlichia canis DNA, it is therefore the method of choice for the detection of E. ruminantium in these deer and other animal hosts.

scholarly journals Levofloxacin-Resistant Invasive Streptococcus pneumoniae in the United States: Evidence for Clonal Spread and the Impact of Conjugate Pneumococcal Vaccine

2004 ◽  
Vol 48 (9) ◽  
pp. 3491-3497 ◽  
Author(s):  
Mathias W. R. Pletz ◽  
Lesley McGee ◽  
James Jorgensen ◽  
Bernard Beall ◽  
Richard R. Facklam ◽  
...  
Keyword(s):  
United States ◽  
Streptococcus Pneumoniae ◽  
The United States ◽  
Fluoroquinolone Resistance ◽  
Active Efflux ◽  
Vaccine Serotypes ◽  
Drug Classes ◽  
Control And Prevention ◽  
Clonal Spread ◽  
The Impact

ABSTRACT The emergence of fluoroquinolone resistance in sterile-site isolates of Streptococcus pneumoniae is documented in this study characterizing all invasive levofloxacin-resistant (MIC, ≥8 mg/liter) S. pneumoniae isolates (n = 50) obtained from the Centers for Disease Control and Prevention Active Bacterial Core Surveillance from 1998 to 2002. Resistance among all isolates increased from 0.1% in 1998 to 0.6% in 2001 (P = 0.008) but decreased to 0.4% in 2002, while resistance among vaccine serotypes continued to increase from 0.3% in 1998 to 1.0% in 2002, suggesting that fluoroquinolones continue to exert selective pressure on these vaccine serotypes. Only 22% of resistant isolates were not covered by the conjugate vaccine serogroups. Multilocus sequence typing revealed that 58% of resistant strains were related to five international clones identified by the Pneumococcal Molecular Epidemiology Network, with the Spain23F-1 clone being most frequent (16% of all isolates). Thirty-six percent of the isolates were coresistant to penicillin, 44% were coresistant to macrolides, and 28% were multiresistant to penicillin, macrolides, and fluoroquinolones. Fifty percent of the isolates were resistant to any three drug classes. Ninety-four percent of the isolates had multiple mutations in the quinolone resistance-determining regions of the gyrA, gyrB, parC, and parE genes. In 16% of the isolates, there was evidence of an active efflux mechanism. An unusual isolate was found that showed only a single parE mutation and for which the ciprofloxacin MIC was lower (2 mg/liter) than that of levofloxacin (8 mg/liter). Our results suggest that invasive pneumococcal isolates resistant to levofloxacin in the United States show considerable evidence of multiple resistance and of clonal spread.

scholarly journals Emergence of high rates of antimicrobial resistance among viridans group streptococci in the United States.

1996 ◽  
Vol 40 (4) ◽  
pp. 891-894 ◽  
Author(s):  
G V Doern ◽  
M J Ferraro ◽  
A B Brueggemann ◽  
K L Ruoff
Keyword(s):  
United States ◽  
Antimicrobial Resistance ◽  
Blood Culture ◽  
Rank Order ◽  
The United States ◽  
Medical Centers ◽  
Streptococcus Milleri ◽  
Intermediate Resistance ◽  
High Level ◽  
Viridans Group Streptococci

Three hundred fifty-two blood culture isolates of viridans group streptococci obtained from 43 U.S. medical centers during 1993 and 1994 were characterized. Included were 48 isolates of "Streptococcus milleri," 219 S. mitis isolates, 29 S. salivarius isolates, and 56 S. sanguis isolates. High-level penicillin resistance (MIC, > or = 4.0 micrograms/ml) was noted among 13.4% of the strains; for 42.9% of the strains, penicillin MICs were 0.25 to 2.0 micrograms/ml (i.e., intermediate resistance). In general, amoxicillin was slightly more active than penicillin. The rank order of activity for five cephalosporins versus viridans group streptococci was cefpodoxime = ceftriaxone > cefprozil = cefuroxime > cephalexin. The percentages of isolates resistant (MIC, > or = 2 micrograms/ml) to these agents were 15, 17, 18, 20, and 96, respectively. The rates of resistance to erythromycin, tetracycline, and trimethoprim-sulfamethoxazole were 12 to 38%. Resistance to either chloramphenicol or ofloxacin was uncommon (i.e., < 1%). In general, among the four species, S. mitis was the most resistant and "S. milleri" was the most susceptible.

scholarly journals First Report of Leaf Blight and Stem Canker of Pachysandra terminalis Caused by Pseudonectria pachysandricola in Korea

Plant Disease ◽  
2012 ◽  
Vol 96 (2) ◽  
pp. 287-287
Author(s):  
K. S. Han ◽  
J. H. Park ◽  
S. E. Cho ◽  
H. D. Shin
Keyword(s):  
United States ◽  
Its Region ◽  
Stem Canker ◽  
The United States ◽  
Culture Collection ◽  
Leaf Blight ◽  
First Report ◽  
Cornell University ◽  
Plastic Bags ◽  
Young Leaves

Pachysandra terminalis Siebold & Zucc., known as Japanese pachysandra, is a creeping evergreen perennial belonging to the family Buxaceae. In April 2011, hundreds of plants showing symptoms of leaf blight and stem canker with nearly 100% incidence were found in a private garden in Suwon, Korea. Plants with the same symptoms were found in Seoul in May and Hongcheon in August. Affected leaves contained tan-to-yellow brown blotches. Stem and stolon cankers first appeared as water soaked and developed into necrotic lesions. Sporodochia were solitary, erumpent, circular, 50 to 150 μm in diameter, salmon-colored, pink-orange when wet, and with or without setae. Setae were hyaline, acicular, 60 to 100 μm long, and had a base that was 4 to 6 μm wide. Conidiophores were in a dense fascicle, not branched, hyaline, aseptate or uniseptate, and 8 to 20 × 2 to 3.5 μm. Conidia were long, ellipsoid to cylindric, fusiform, rounded at the apex, subtruncate at the base, straight to slightly bent, guttulate, hyaline, aseptate, 11 to 26 × 2.5 to 4.0 μm. A single-conidial isolate formed cream-colored colonies that turned into salmon-colored colonies on potato dextrose agar (PDA). Morphological and cultural characteristics of the fungus were consistent with previous reports of Pseudonectria pachysandricola B.O. Dodge (1,3,4). Voucher specimens were housed at Korea University (KUS). Two isolates, KACC46110 (ex KUS-F25663) and KACC46111 (ex KUS-F25683), were accessioned in the Korean Agricultural Culture Collection. Fungal DNA was extracted with DNeasy Plant Mini DNA Extraction Kits (Qiagen Inc., Valencia, CA). The complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced using ABI Prism 337 automatic DNA sequencer (Applied Biosystems, Foster, CA). The resulting sequence of 487 bp was deposited in GenBank (Accession No. JN797821). This showed 100% similarity with a sequence of P. pachysandricola from the United States (HQ897807). Isolate KACC46110 was used in pathogenicity tests. Inoculum was prepared by harvesting conidia from 2-week-old cultures on PDA. Ten young leaves wounded with needles were sprayed with conidial suspensions (~1 × 106 conidia/ml). Ten young leaves that served as the control were treated with sterile distilled water. Plants were covered with plastic bags to maintain a relative humidity of 100% at 25 ± 2°C for 24 h. Typical symptoms of brown spots appeared on the inoculated leaves 4 days after inoculation and were identical to the ones observed in the field. P. pachysandricola was reisolated from 10 symptomatic leaf tissues, confirming Koch's postulates. No symptoms were observed on control plants. Previously, the disease was reported in the United States, Britain, Japan, and the Czech Republic (2,3), but not in Korea. To our knowledge, this is the first report of P. pachysandricola on Pachysandra terminalis in Korea. Since this plant is popular and widely planted in Korea, this disease could cause significant damage to nurseries and the landscape. References: (1) B. O. Dodge. Mycologia 36:532, 1944. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , September 24, 2011. (3) I. Safrankova. Plant Prot. Sci. 43:10, 2007. (4) W. A. Sinclair and H. H. Lyon. Disease of Trees and Shrubs. 2nd ed. Cornell University Press, Ithaca, NY, 2005.

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