Isolation and Identification of Klebsiella pneumoniae using API-20E analytical system and conventional PCR assay

Background. The term “persisters” refers to a small bacterial population that persists during treatment with high antibiotic concentration or dose in the absence of genetic resistance. The present study was designed to investigate the transcriptional response in indigenous Klebsiella pneumoniae under the ciprofloxacin stress. Methods. Isolation and identification of K. pneumoniae were carried out through standard microbiological protocols. The characterization of quinolone resistance was performed by estimating the quinolone susceptibility testing, MIC estimation, and detecting the QRDR and PMQR. Transcriptional response of the isolates to ciprofloxacin was determined using qPCR. Results. Among 34 isolates, 23 (67%) were resistant to ciprofloxacin. Both QRDR (gyrA and gyrB) and PMQR (qnrA, qnrB, and qnrS) were detected in the isolates, and all were found resistant to ciprofloxacin. The mRNA levels of both mutS and euTu under the influence of ciprofloxacin were significantly increased. On ciprofloxacin exposure, the mRNA levels of the DNA damage response element (mutS) were raised in a time-dependent fashion. K. pneumoniae showed high-level resistance to ciprofloxacin in the presence of mutations in QRDR and PMQR genes. Conclusion. The transcriptional response revealed the upregulation of DNA repair and protein folding elements (mutS and euTu) in ciprofloxacin stress and delayed cell division. The ciprofloxacin was found to trigger various stress responses in a time- and concentration-dependent manner.

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Use of "EP"(Peroxidase) allele in soybean varietal characterization

Journal of Seed Science ◽

10.1590/2317-1545v36n4996 ◽

2014 ◽

Vol 36 (4) ◽

pp. 465-470

Author(s):

Bárbara Panoff Valário ◽

Cláudio Cavariani ◽

José de Barros França-Neto ◽

Elisa Serra Negra Vieira ◽

Juliana Pereira Bravo ◽

...

Keyword(s):

Plant Production ◽

Pcr Analysis ◽

Agarose Gel Electrophoresis ◽

Conventional Pcr ◽

Chain Reaction ◽

Pcr Assay ◽

Molecular Biology Technique ◽

Soybean Cultivars ◽

Colorimetric Test

The aim of this work was to evaluate the use of the molecular biology technique of PCR (Polimerase Chain Reaction) in the characterization of soybean cultivars. The study was performed at the Department of Plant Production, Faculty of Agricultural Sciences/ UNESP and Institute of Bioscience, Botucatu-SP. Fourteen commercial soybean cultivars were used, of which six were selected as positive reaction to peroxidase (BRS 320, BRS 284, BRS 232, BRS 7860RR, BRSMG 760SRR, BRS295RR), four as negative reaction (BRS 326, BRS 8160RR, BRSMG 800A (NutriSoy), BRS Valiosa RR) and four as double reaction (BRSGO 8060, BRS 270RR, FTS Campo Mourão and BRS 239). Thus, the results attained by the traditional biochemical colorimetric test for the 14 cultivars were compared with the conventional PCR assay. For PCR analysis, DNA was extracted from whole seeds and the primers were tested, and subsequently PCR and agarose gel electrophoresis were performed. The combination of primers prx9 + prx10 confirmed the use of the PCR reaction to characterize soybean cultivars considered doubtful by conventional colorimetric text.

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Isolation and Identification of Rickettsia massiliae from Rhipicephalus sanguineus Ticks Collected in Arizona

Applied and Environmental Microbiology ◽

10.1128/aem.00122-06 ◽

2006 ◽

Vol 72 (8) ◽

pp. 5569-5577 ◽

Cited By ~ 111

Author(s):

Marina E. Eremeeva ◽

Elizabeth A. Bosserman ◽

Linda J. Demma ◽

Maria L. Zambrano ◽

Dianna M. Blau ◽

...

Keyword(s):

Spotted Fever ◽

Rhipicephalus Sanguineus ◽

Vero Cells ◽

The United States ◽

Spotted Fever Group ◽

Pcr Assay ◽

Isolation And Identification ◽

Cytotoxic Effects ◽

Rickettsia Massiliae ◽

Genotypic Characteristics

ABSTRACT Twenty Rhipicephalus sanguineus ticks collected in eastern Arizona were tested by PCR assay to establish their infection rate with spotted fever group rickettsiae. With a nested PCR assay which detects a fragment of the Rickettsia genus-specific 17-kDa antigen gene (htrA), five ticks (25%) were found to contain rickettsial DNA. One rickettsial isolate was obtained from these ticks by inoculating a suspension of a triturated tick into monolayers of Vero E6 monkey kidney cells and XTC-2 clawed toad cells, and its cell culture and genotypic characteristics were determined. Fragments of the 16S rRNA, GltA, rOmpA, rOmpB, and Sca4 genes had 100%, 100%, 99%, 99%, and 99%, respectively, nucleotide similarity to Rickettsia massiliae strain Bar29, previously isolated from R. sanguineus in Catalonia, Spain (L. Beati et al., J. Clin. Microbiol. 34:2688-2694, 1996). The new isolate, AZT80, does not elicit cytotoxic effects in Vero cells and causes a persistent infection in XTC-2 cells. The AZT80 strain is susceptible to doxycycline but resistant to rifampin and erythromycin. Whether R. massiliae AZT80 is pathogenic or infectious for dogs and humans or can cause seroconversion to spotted fever group antigens in the United States is unknown.

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Improved Primers for the Specific Detection of Leifsonia xyli subsp. xyli in Sugarcane Using a Conventional PCR Assay

Plant Disease ◽

10.1094/pdis-12-18-2221-re ◽

2019 ◽

Vol 103 (12) ◽

pp. 3251-3258

Author(s):

Sheng-Ren Sun ◽

Jun-Lü Chen ◽

Yao-Yao Duan ◽

Na Chu ◽

Mei-Ting Huang ◽

...

Keyword(s):

Pathogen Detection ◽

Dna Amplification ◽

High Yield ◽

Conventional Pcr ◽

Pcr Assay ◽

Saccharum Spp ◽

Sensitivity Tests ◽

Field Samples ◽

Pcr Assays ◽

Higher Sensitivity

Ratoon stunting disease (RSD), one of the most important diseases of sugarcane, is caused by the bacterium Leifsonia xyli subsp. xyli (Lxx). Lxx infects sugarcane worldwide and RSD results in high yield losses and varietal degeneration. It is highly challenging to diagnose RSD based on visual symptomatology because this disease does not exhibit distinct external and internal symptoms. In this study, a novel Lxx-specific primer pair Lxx-F1/Lxx-R1 was designed to detect this pathogen using a conventional PCR assay. These primers were then compared with four published Lxx-specific primers and one universal Leifsonia generic primer pair LayF/LayR. Sugarcane leaf samples were collected from Saccharum spp. hybrids in commercial fields (315 samples) and from germplasm clones of five Saccharum species and Erianthus arundinaceus (216 samples). These samples were used for comparative field diagnosis with six conventional PCR assays. Sensitivity tests suggested that the PCR assay with primers Lxx-F1/Lxx-R1 had the same detection limit (1 pg of Lxx genomic DNA) as the primer pairs Cxx1/Cxx2 and CxxITSf#5/CxxITSr#5 and had 10-fold higher sensitivity than the primer pairs Pat1-F2/Pat1-R2, LayF/LayR, and C2F/C2R. Comparison of PCR assays revealed that natural Lxx-infection incidence (6.1%) in field sample evaluation identified by Lxx-F1/Lxx-R1 primers was higher than incidences (0.7 to 3.0%) determined by other primer pairs. Moreover, no nonspecific DNA amplification occurred within these field samples with Lxx-F1/Lxx-R1 primers, unlike with the primer pairs Cxx1/Cxx2 and LayF/LayR. Diverse Leifsonia strains were identified by PCR detection with LayF/LayR primers in the field samples, whereas whether these Leifsonia strains were pathogenic to sugarcane requires further research. Our investigations revealed that the PCR assay with the newly designed primers Lxx-F1/Lxx-R1 could be widely used for RSD diagnosis and Lxx-pathogen detection with satisfactory sensitivity and specificity.

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Development and application of a triplex real-time PCR assay for simultaneous detection of avian influenza virus, Newcastle disease virus, and duck Tembusu virus

BMC Veterinary Research ◽

10.1186/s12917-020-02399-z ◽

2020 ◽

Vol 16 (1) ◽

Author(s):

Xiyu Zhang ◽

Ming Yao ◽

Zhihui Tang ◽

Daning Xu ◽

Yan Luo ◽

...

Keyword(s):

Influenza Virus ◽

Standard Deviation ◽

Real Time ◽

Real Time Pcr ◽

Virus Isolation ◽

Simultaneous Detection ◽

Conventional Pcr ◽

Pcr Assay ◽

Duck Tembusu Virus ◽

Relative Standard

Abstract Background Pathogens including duck-origin avian influenza virus (AIV), duck-origin Newcastle disease virus (NDV) and duck Tembusu virus (DTMUV) posed great harm to ducks and caused great economic losses to the duck industry. In this study, we aim to develop a triplex real-time polymerase chain reaction (PCR) assay to detect these three viruses as early as possible in the suspicious duck flocks. Results The detection limit of the triplex real-time PCR for AIV, NDV, and DTMUV was 1 × 101 copies/μL, which was at least 10 times higher than the conventional PCR. In addition, the triplex assay was highly specific, and won’t cross-react with other duck pathogens. Besides, the intra-day relative standard deviation and inter-day relative standard deviation were lower than 4.44% for these viruses at three different concentrations. Finally, a total of 120 clinical samples were evaluated by the triplex real-time PCR, the conventional PCR and virus isolation, and the positive rates for these three methods were 20.83, 21.67, 19.17%, respectively. Taking virus isolation as the gold standard, the diagnostic specificity and positive predictive value of the three viruses were all above 85%, while the diagnostic sensitivity and negative predictive value of the three viruses were all 100%. Conclusion The developed triplex real-time PCR is fast, specific and sensitive, and is feasible and effective for the simultaneous detection of AIV, NDV, and DTMUV in ducks.

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Molecular Detection of Burkholderia cepacia in Toiletry, Cosmetic, and Pharmaceutical Raw Materials and Finished Products

Journal of AOAC International ◽

10.1093/jaoac/83.4.963 ◽

2000 ◽

Vol 83 (4) ◽

pp. 963-966 ◽

Cited By ~ 13

Author(s):

Luis Jimenez ◽

Stacey Smalls

Keyword(s):

Burkholderia Cepacia ◽

Raw Materials ◽

Pcr Detection ◽

Proteinase K ◽

Tween 20 ◽

Pcr Assay ◽

Isolation And Identification ◽

Standard Methods ◽

Microbiological Methods ◽

Dna Primers

Abstract A polymerase chain reaction (PCR) assay was developed and compared with standard methods for rapid detection of Burkholderia cepacia, a major industrial contaminant, in cosmetic and pharmaceutical raw materials and finished products. Artificially contaminated samples were incubated for 24 h in trypticase soy broth containing 4% Tween 20 and 0.5% soy lecithin. DNA was extracted from each sample using a proteinase K-tris-EDTA-Tween 20 treatment at 35°C. The extracted DNA was added to Ready-To-Go PCR beads and specific DNA primers for B. cepacia. The B. cepacia DNA primers coded for a 209-base pair (bp) fragment of the 16S rRNA ribosomal gene. No DNA amplification was observed in samples that were not spiked with B. cepacia. However, all contaminated samples showed the specific 209-bp fragment for B. cepacia. There was a 100% correlation between standard methods and the PCR assay. Standard microbiological methods required 5–6 days for isolation and identification of spiked microorganisms, whereas PCR detection and identification was completed in 27 h. PCR detection of B. cepacia allows for rapid quality evaluation of cosmetic and pharmaceutical raw materials and finished products.

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Clinical Impact of a PCR Assay for Rapid Identification of Klebsiella pneumoniae in Blood Cultures

Journal of Clinical Microbiology ◽

10.1128/jcm.00568-07 ◽

2007 ◽

Vol 46 (1) ◽

pp. 377-379 ◽

Cited By ~ 9

Author(s):

A. Neuberger ◽

I. Oren ◽

H. Sprecher

Keyword(s):

Klebsiella Pneumoniae ◽

Rapid Identification ◽

Blood Cultures ◽

Clinical Impact ◽

Pcr Assay

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Development of a multiplex PCR assay for identification of Klebsiella pneumoniae hypervirulent clones of capsular serotype K2

Journal of Medical Microbiology ◽

10.1099/jmm.0.081448-0 ◽

2014 ◽

Vol 63 (12) ◽

pp. 1608-1614 ◽

Cited By ~ 21

Author(s):

Suzanne Bialek-Davenet ◽

Alexis Criscuolo ◽

Florent Ailloud ◽

Virginie Passet ◽

Marie-Hélène Nicolas-Chanoine ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Multiplex Pcr ◽

Sensu Stricto ◽

Phylogenetic Group ◽

Pcr Assay ◽

Closely Related Species ◽

Multiplex Pcr Assay ◽

Genomic Analyses ◽

Invasive Infections ◽

Serotype K2

Hypervirulent Klebsiella pneumoniae isolates of capsular serotype K2 (hvKP-K2) that cause community-acquired invasive infections represent several unrelated clones, which all belong to phylogenetic group KpI. These clones can be recognized using multilocus sequence typing and genomic analyses, but no rapid method currently exists to differentiate them. In this work, a multiplex PCR assay was developed to identify three hvKP-K2 groups: (i) sequence type (ST)86; (ii) ST380 and ST679 (i.e. clonal group 380); and (iii) ST65 and ST375. A specific genetic marker, Kp50233, allowing K. pneumoniae sensu stricto (corresponding to phylogroup KpI) to be distinguished from closely related species, was included in the assay. This PCR assay will be useful in better defining the epidemiology and clinical features of emerging virulent K. pneumoniae clones.

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Development of Isothermal LAMP Assay for Detection of Intimin Gene (eae) in E. coli Associated with Piglet Diarrhea

Indian Journal of Animal Research ◽

10.18805/ijar.b-4173 ◽

2020 ◽

Author(s):

Sanjeev Kumar ◽

Jagan Mohanarao Gali ◽

T.K. Dutta ◽

P. Roychoudhury ◽

P.K. Subudhi

Keyword(s):

Bacterial Species ◽

False Negative ◽

Acute Diarrhoea ◽

Lamp Assay ◽

Dna Amplification ◽

Analytical Sensitivity ◽

Conventional Pcr ◽

Isolation And Identification ◽

Reliable Technique ◽

E Coli

Background: Diarroeagenic Escherichia coli (DEC) including enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) is associated with acute diarrhoea in children and young animals. The virulence is associated with attaching and effacing lesions encoded by eaeA gene is considered as marker for EPEC and EHEC. Laboratory diagnosis of such infections is carried out by traditional bacteriological techniques and by conventional PCR assays. Those techniques often provide false negative result and at the same time are costly as well as difficult to perform in the field level. The loop-mediated isothermal amplification (LAMP) is a new generation DNA amplification assay is developed for detection of eaeA gene in E. coli isolated from diarrhoeic piglets.Methods: Samples were collected from diarrhoeic piglets for isolation and identification of E. coli. eaeA gene was detected by conventional PCR using specific primers in all the isolates. LAMP assay was standardized for detection of eaeA gene. Analytical sensitivity of LAMP was evaluated using 10 fold serially diluted E. coli genomic DNA. The specificity of the LAMP assay was determined by evaluating the cross reactivity with 19 other enteric and non-enteric bacterial species. Standardized LAMP was applied for detection of eaeA gene in the field isolates.Result: A total of 37 (24.67%) isolates were recorded as positive for eaeA gene by conventional PCR, while 49 (32.67%) isolates were recorded as positive for eaeA gene by LAMP assay. The LAMP assay was 10 times more sensitive than conventional PCR. LAMP assay was found to be more sensitive, specific, cost effective, user friendly and reliable technique over conventional PCR, which can be applied for screening of the clinical isolates for confirmation of EPEC and/or EHEC.

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Species distribution and antimicrobial susceptibility profile of gram-negative bacteria in hospitalized cancer patients

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.e20730 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. e20730-e20730

Author(s):

H. M. Ashour ◽

A. El-Sharif

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Cancer Patients ◽

Solid Tumor ◽

Klebsiella Pneumonia ◽

Gram Negative Bacteria ◽

Isolation And Identification ◽

Tumor Patients ◽

Acinetobacter Species ◽

Gram Negative

e20730 Background: Nosocomial infections pose significant threats to hospitalized patients, especially the immunocompromised ones, such as cancer patients. Methods: This study examined the microbial spectrum of gram-negative bacteria in various infection sites in patients with leukemia and solid tumors. The antimicrobial resistance patterns of the isolated bacteria were studied. Results: The most frequently isolated gram-negative bacteria were Klebsiella pneumonia (31.2%) followed by Escherichia coli (22.2%). We report the first-time isolation and identification of a number of less-frequent gram negative bacteria (Chromobacterium violacum, Burkholderia cepacia, Kluyvera ascorbata, Stenotrophomonas maltophilia, Yersinia pseudotuberculosis, and Salmonella arizona). Most of the gram-negative isolates from RTI, GITI, UTI, and BSI were obtained from leukemic patients. All gram-negative isolates from SI were obtained from solid-tumor patients. In both leukemic and solid-tumor patients, gram-negative bacteria causing UTI were mainly Escherichia coli and Klebsiella pneumoniae, while gram-negative bacteria causing RTI were mainly Klebsiella pneumoniae. Escherichia coli was the main gram-negative pathogen causing BSI in solid-tumor patients and GITI in leukemic patients. Isolates of Escherichia coli, Klebsiella, Enterobacter, Pseudomonas, and Acinetobacter species were resistant to most antibiotics tested. There was significant imipenem-resistance in Acinetobacter (40.9%), Pseudomonas (40%), and Enterobacter (22.2%) species, and noticeable imipinem-resistance in Klebsiella (13.9%) and Escherichia coli (8%). Conclusions: This is the first study to report the evolution of imipenem-resistant gram-negative strains in Egypt. Mortality rates were higher in cancer patients with nosocomial Pseudomonas infections than any other bacterial infections. Policies restricting antibiotic consumption should be implemented to avoid the evolution of newer generations of antibiotic resistant-pathogens. No significant financial relationships to disclose.

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