scholarly journals Investigation of the Causes of Shigatoxigenic Escherichia coli PCR Positive and Culture Negative Samples

2020 ◽  
Vol 8 (4) ◽  
pp. 587
Author(s):  
Guerrino Macori ◽  
Siobhán C. McCarthy ◽  
Catherine M. Burgess ◽  
Séamus Fanning ◽  
Geraldine Duffy
Keyword(s):  
Escherichia Coli ◽  
Target Gene ◽  
Positive Culture ◽  
Induction Method ◽  
Negative Results ◽  
Specific Target Gene ◽  
Cultural Isolation ◽  
Two Samples ◽  
Free Particles ◽  
Culture Negative

Molecular methods may reveal the presence of pathogens in samples through the detection of specific target gene(s) associated with microorganisms, but often, the subsequent cultural isolation of the pathogen is not possible. This discrepancy may be related to low concentration of the cells, presence of dead cells, competitive microflora, injured cells and cells in a viable but non-culturable state, free DNA and the presence of free bacteriophages which can carry the target gene causing the PCR-positive/culture-negative results. Shiga-toxigenic Escherichia coli (STEC) was used as a model for studying this phenomenon, based on the phage-encoded cytotoxins genes (Stx family) as the detection target in samples through real-time qPCR. Stx phages can be integrated in the STEC chromosome or can be isolated as free particles in the environment. In this study, a combination of PCR with culturing was used for investigating the presence of the stx1 and stx2 genes in 155 ovine recto-anal junction swab samples (method (a)-PCR). Samples which were PCR-positive and culture-negative were subjected to additional analyses including detection of dead STEC cells (method (b)-PCR-PMA dye assay), presence of Stx phages (method (c)-plaque assays) and inducible integrated phages (method (d)-phage induction). Method (a) showed that even though 121 samples gave a PCR-positive result (78%), only 68 samples yielded a culturable isolate (43.9%). Among the 53 (34.2%) PCR-positive/culture-negative samples, 21 (39.6%) samples were shown to have STEC dead cells only, eight (15.1%) had a combination of dead cells and inducible stx phage, while two samples (3.8%) had a combination of dead cells, inducible phage and free stx phage, and a further two samples had Stx1 free phages only (3.8%). It was thus possible to reduce the samples with no explanation to 20 (37.7% of 53 samples), representing a further step towards an improved understanding of the STEC PCR-positive/culture-negative phenomenon.

Comparing Real-Time and Conventional PCR to Culture-Based Methods for Detecting and Quantifying Escherichia coli O157 in Cattle Feces

2014 ◽  
Vol 77 (2) ◽  
pp. 314-319 ◽  
Author(s):  
M. E. JACOB ◽  
J. BAI ◽  
D. G. RENTER ◽  
A. T. ROGERS ◽  
X. SHI ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Positive Culture ◽  
Feedlot Cattle ◽  
Escherichia Coli O157 ◽  
Conventional Pcr ◽  
Culture Methods ◽  
E Coli ◽  
Cattle Feces ◽  
Culture Negative

Detection of Escherichia coli O157 in cattle feces has traditionally used culture-based methods; PCR-based methods have been suggested as an alternative. We aimed to determine if multiplex real-time (mq) or conventional PCR methods could reliably detect cattle naturally shedding high (≥104 CFU/g of feces) and low (~102 CFU/g of feces) concentrations of E. coli O157. Feces were collected from pens of feedlot cattle and evaluated for E. coli O157 by culture methods. Samples were categorized as (i) high shedders, (ii) immunomagnetic separation (IMS) positive after enrichment, or (iii) culture negative. DNA was extracted pre- and postenrichment from 100 fecal samples from each category (high shedder, IMS positive, culture negative) and subjected to mqPCR and conventional PCR assays based on detecting three genes, rfbE, stx1, and stx2. In feces from cattle determined to be E. coli O157 high shedders by culture, 37% were positive by mqPCR prior to enrichment; 85% of samples were positive after enrichment. In IMS-positive samples, 4% were positive by mqPCR prior to enrichment, while 43% were positive after enrichment. In culture-negative feces, 7% were positive by mqPCR prior to enrichment, and 40% were positive after enrichment. The proportion of high shedder–positive and culture-positive (high shedder and IMS) samples were significantly different from mqPCR-positive samples before and after enrichment (P < 0.01). Similar results were observed for conventional PCR. Our data suggest that mqPCR and conventional PCR are most useful in identifying high shedder animals and may not be an appropriate substitute to culture-based methods for detection of E. coli O157 in cattle feces.

scholarly journals False-Positive Xpert MTB/RIF Results in Retested Patients with Previous Tuberculosis: Frequency, Profile, and Prospective Clinical Outcomes

2018 ◽  
Vol 56 (3) ◽  
Author(s):  
Grant Theron ◽  
Rouxjeane Venter ◽  
Liezel Smith ◽  
Aliasgar Esmail ◽  
Philippa Randall ◽  
...  
Keyword(s):  
False Positive ◽  
Positive Culture ◽  
Previous Treatment ◽  
Treatment Completion ◽  
Diagnostic Dilemma ◽  
Negative Results ◽  
Previously Treated Patients ◽  
Positive Results ◽  
Culture Negative

ABSTRACTGlobally, Xpert MTB/RIF (Xpert) is the most widely used PCR test for the diagnosis of tuberculosis (TB). Positive results in previously treated patients, which are due to old DNA or active disease, are a diagnostic dilemma. We prospectively retested sputum from 238 patients, irrespective of current symptoms, who were previously diagnosed to be Xpert positive and treated successfully. Patients who retested as Xpert positive and culture negative were exhaustively investigated (repeat culture, chest radiography, bronchoscopy with bronchoalveolar lavage, long-term clinical follow-up). We evaluated whether the duration since previous treatment completion, mycobacterial burden (the Xpert cycle threshold [CT] value), and reclassification of Xpert-positive results with a very low semiquantitation level to Xpert-negative results reduced the rate of false positivity. A total of 229/238 (96%) of patients were culture negative. Sixteen of 229 (7%) were Xpert positive a median of 11 months (interquartile range, 5 to 19 months) after treatment completion. The specificity was 93% (95% confidence interval [CI], 89 to 96%). Nine of 15 (40%) Xpert-positive, culture-negative patients reverted to Xpert negative after 2 to 3 months (1 patient declined further participation). Patients with false-positive Xpert results had a lower mycobacterial burden than patients with true-positive Xpert results (CT, 28.7 [95% CI, 27.2 to 30.4] versus 17.6 [95% CI, 16.9 to 18.2];P< 0.001), an increased likelihood of a chest radiograph not compatible with active TB (5/15 patients versus 0/5 patients;P= 0.026), and less-viscous sputum (15/16 patients versus 2/5 patients whose sputum was graded as mucoid or less;P= 0.038). All patients who initially retested as Xpert positive and culture negative (“Xpert false positive”) were clinically well without treatment after follow-up. The duration since the previous treatment poorly predicted false-positive results (a duration of ≤2 years identified only 66% of patients with false-positive results). Reclassifying Xpert-positive results with a very low semiquantitation level to Xpert negative improved the specificity (+3% [95% CI, +2 to +5%]) but reduced the sensitivity (−10% [95% CI, −4 to −15%]). Patients with previous TB retested with Xpert can have false-positive results and thus not require treatment. These data inform clinical practice by highlighting the challenges in interpreting Xpert-positive results, underscore the need for culture, and have implications for next-generation ultrasensitive tests.

scholarly journals Assessment of an enzyme immunoassay for the detection of salmonellas in foods and animal feeding stuffs

1987 ◽  
Vol 98 (3) ◽  
pp. 301-310 ◽  
Author(s):  
Lynne S. Todd ◽  
Diane Roberts ◽  
Barbara A. Bartholomew ◽  
R. J. Gilbert
Keyword(s):  
Enzyme Immunoassay ◽  
Positive Culture ◽  
Advantages And Disadvantages ◽  
Cultural Methods ◽  
Negative Results ◽  
Negative Culture ◽  
Animal Feeding ◽  
Culture Positive ◽  
Initial Results ◽  
Culture Negative

SUMMARYTheSalmonellaBio-EnzaBead Screening Kit, in its modified form with both the MOPC 467 and the 6H4 antibodies, was used for the detection of salmonellas in naturally contaminated foods and animal feeding stuffs in parallel with a traditional cultural procedure.Initial results showed an 82% agreement between the enzyme immunoassay (EIA) and cultural methods when using the criterion recommended by the manufacturer as a cut-off for all types of foods. By adjusting the cut-off for each type of food, the number of EIA positive, culture negative samples was reduced although the number of EIA negative, culture positive samples increased. The EIA may be more sensitive than the cultural methods as in many cases the EIA positive, culture negative results could be real positives which were not detected by the cultural methods.The screening kit provides a simple and convenient method for the detection of salmonella in foods and feeds and a presumptive positive result can be reported within 48 h. The advantages and disadvantages of the method are discussed.

scholarly journals UV Light Induces IS10 Transposition in Escherichia coli

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1173-1181
Author(s):  
Zehava Eichenbaum ◽  
Zvi Livneh
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Target Gene ◽  
Uv Light ◽  
Insertion Site ◽  
Donor Plasmid ◽  
Donor And Acceptor ◽  
Low Copy Number ◽  
Uv Induction ◽  
Target Plasmid

Abstract A new mutagenesis assay system based on the phage 434 cI gene carried on a low-copy number plasmid was used to investigate the effect of UV light on intermolecular transposition of IS10. Inactivation of the target gene by IS10 insertion was detected by the expression of the tet gene from the phage 434 PR promoter, followed by Southern blot analysis of plasmids isolated from TetR colonies. UV irradiation of cells harboring the target plasmid and a donor plasmid carrying an IS10 element led to an increase of up to 28-fold in IS10 transposition. Each UV-induced transposition of IS10 was accompanied by fusion of the donor and acceptor plasmid into a cointegrate structure, due to coupled homologous recombination at the insertion site, similar to the situation in spontaneous IS10 transposition. UV radiation also induced transposition of IS10 from the chromosome to the target plasmid, leading almost exclusively to the integration of the target plasmid into the chromosome. UV induction of IS10 transposition did not depend on the umuC and uvrA gene product, but it was not observed in lexA3 and ΔrecA strains, indicating that the SOS stress response is involved in regulating UV-induced transposition. IS10 transposition, known to increase the fitness of Escherichia coli, may have been recruited under the SOS response to assist in increasing cell survival under hostile environmental conditions. To our knowledge, this is the first report on the induction of transposition by a DNA-damaging agent and the SOS stress response in bacteria.

scholarly journals How Do Preoperative Antibiotics Affect Culture Yield in Diabetic Foot Infections?

2017 ◽  
Vol 4 (1) ◽  
Author(s):  
Heather Young ◽  
Whitney Miller ◽  
Randy Burnham ◽  
Susan Heard ◽  
Chrystal Berg ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Diabetic Foot ◽  
Gram Negative ◽  
Diabetic Foot Infection ◽  
Negative Results ◽  
Diabetic Foot Infections ◽  
Preoperative Antibiotics ◽  
The Impact ◽  
Culture Negative ◽  
Culture Yield

abstractThe impact of preoperative antibiotics on culture of diabetic foot infection samples has not been studied. We found that increasing exposure to preoperative antibiotics was associated with less frequent growth of streptococci and anaerobes and more culture-negative results. In contrast, the yield of Staphylococcus aureus and Gram-negative bacilli was unaffected.

Rapid diagnosis of influenza A infection by direct immunofluorescence of nasopharyngeal aspirates in adults

1979 ◽  
Vol 9 (6) ◽  
pp. 688-692
Author(s):  
J A Daisy ◽  
F S Lief ◽  
H M Friedman
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Virus Isolation ◽  
Fluorescent Antibody ◽  
Positive Culture ◽  
H3n2 Virus ◽  
Direct Immunofluorescence ◽  
The One ◽  
Immunofluorescent Test ◽  
Culture Negative

The efficacy for direct immunofluorescence of a commercial conjugate for influenza A virus prepared against whole A/Udorn (H3NS) virus was studied. The conjugate was specific for influenza A virus, but its sensitivity varied depending upon the strain of influenza A tested. Nasopharyngeal aspirates collected from 25 patients during an outbreak of influenza were examined for viral antigen with the conjugates and inoculated onto monkey kidney (MK) cells for virus isolation. Fifteen patients had isolates for influenza A/USSR/90/77 (H1N1); nasopharyngeal secretions were fluorescent antibody positive in 12. Fluorescent antibody was copositive with culture in 11/15 patients (73.3%) and conegative in 9/10 (90%). The one fluorescent antibody-positive, culture-negative patient had negative serology for influenza A and the fluorescent antibody result was considered to be a false positive. At a 1:10 dilution, the conjugate stained nasopharyngeal and MK cells infected with A/USSR (H1N1) 2 to 3+, whereas cells infected with H3N2 virus stained 4+. A conjugate made specifically against the ribonucleoprotein antigen, which is universal to all influenza A strains, may improve the sensitivity of the direct immunofluorescent test.

scholarly journals The microbiome of Escherichia coli and culture-negative nonsevere clinical mastitis: Characterization and associations with linear score and milk production

2019 ◽  
Vol 102 (1) ◽  
pp. 578-594 ◽  
Author(s):  
A.K. Vasquez ◽  
E.K. Ganda ◽  
M.B. Capel ◽  
S. Eicker ◽  
P.D. Virkler ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Milk Production ◽  
Clinical Mastitis ◽  
Culture Negative

scholarly journals Induction and control of the autolytic system of Escherichia coli

1982 ◽  
Vol 152 (1) ◽  
pp. 26-34
Author(s):  
M Leduc ◽  
R Kasra ◽  
J van Heijenoort
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Acid Synthesis ◽  
Induction Method ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Carbonyl Cyanide ◽  
And Control ◽  
First Time ◽  
Induced Autolysis

Various methods of inducing autolysis of Escherichia coli cells were investigated, some being described here for the first time. For the autolysis of growing cells only induction methods interfering with the biosynthesis of peptidoglycan were taken into consideration, whereas with harvested cells autolysis was induced by rapid osmotic or EDTA shock treatments. The highest rates of autolysis were observed after induction by moenomycin, EDTA, or cephaloridine. The different autolyses examined shared certain common properties. In particular, regardless of the induction method used, more or less extensive peptidoglycan degradation was observed, and 10(-2) M Mg2+ efficiently inhibited the autolytic process. However, for other properties a distinction was made between methods used for growing cells and those used for harvested cells. Autolysis of growing cells required RNA, protein, and fatty acid synthesis. No such requirements were observed with shock-induced autolysis performed with harvested cells. Thus, the effects of Mg2+, rifampicin, chloramphenicol, and cerulenin clearly suggest that distinct factors are involved in the control of the autolytic system of E. Coli. Uncoupling agents such as sodium azide, 2,4-dinitrophenol, and carbonyl-cyanide-m-chlorophenyl hydrazone used at their usual inhibiting concentration had no effect on the cephaloridine or shock-induced autolysis.

scholarly journals Elucidation of Regulatory Modes for Five Two-Component Systems in Escherichia coli Reveals Novel Relationships

mSystems ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Kumari Sonal Choudhary ◽  
Julia A. Kleinmanns ◽  
Katherine Decker ◽  
Anand V. Sastry ◽  
Ye Gao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Target Gene ◽  
Target Genes ◽  
Rna Seq ◽  
Content Type ◽  
E Coli ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

ABSTRACT Escherichia coli uses two-component systems (TCSs) to respond to environmental signals. TCSs affect gene expression and are parts of E. coli’s global transcriptional regulatory network (TRN). Here, we identified the regulons of five TCSs in E. coli MG1655: BaeSR and CpxAR, which were stimulated by ethanol stress; KdpDE and PhoRB, induced by limiting potassium and phosphate, respectively; and ZraSR, stimulated by zinc. We analyzed RNA-seq data using independent component analysis (ICA). ChIP-exo data were used to validate condition-specific target gene binding sites. Based on these data, we do the following: (i) identify the target genes for each TCS; (ii) show how the target genes are transcribed in response to stimulus; and (iii) reveal novel relationships between TCSs, which indicate noncognate inducers for various response regulators, such as BaeR to iron starvation, CpxR to phosphate limitation, and PhoB and ZraR to cell envelope stress. Our understanding of the TRN in E. coli is thus notably expanded. IMPORTANCE E. coli is a common commensal microbe found in the human gut microenvironment; however, some strains cause diseases like diarrhea, urinary tract infections, and meningitis. E. coli’s two-component systems (TCSs) modulate target gene expression, especially related to virulence, pathogenesis, and antimicrobial peptides, in response to environmental stimuli. Thus, it is of utmost importance to understand the transcriptional regulation of TCSs to infer bacterial environmental adaptation and disease pathogenicity. Utilizing a combinatorial approach integrating RNA sequencing (RNA-seq), independent component analysis, chromatin immunoprecipitation coupled with exonuclease treatment (ChIP-exo), and data mining, we suggest five different modes of TCS transcriptional regulation. Our data further highlight noncognate inducers of TCSs, which emphasizes the cross-regulatory nature of TCSs in E. coli and suggests that TCSs may have a role beyond their cognate functionalities. In summary, these results can lead to an understanding of the metabolic capabilities of bacteria and correctly predict complex phenotype under diverse conditions, especially when further incorporated with genome-scale metabolic models.

Concept and Types of Organizational Cultures of Hospitals

2015 ◽  
pp. 1279-1305
Author(s):  
Łukasz Sulkowski ◽  
Joanna Sulkowska
Keyword(s):  
Organizational Culture ◽  
Positive Culture ◽  
Corporate Identity ◽  
Two Dimensional ◽  
Organizational Cultures ◽  
Pilot Studies ◽  
Alternative Approach ◽  
Innovative Culture ◽  
Strong Culture ◽  
Culture Negative

This chapter sets out to analyze the problem of defining the concept of organizational culture as well as models and typologies used in reference materials. It presents various issues of organizational culture: paradigms of organizational culture, definitions of organizational culture, and two-dimensional typologies of organizational culture. The single-dimensional classifications present the following dichotomies: 1) weak culture – strong culture, 2) positive culture – negative culture, 3) pragmatic culture – bureaucratic culture, 4) introvert culture – extrovert culture, 5) conservative culture – innovative culture, 6) hierarchic culture – egalitarian culture, 7) individualist culture – collectivist culture. Furthermore, this chapter includes: multidimensional typologies of organizational culture, corporate identity – alternative approach to organizational culture and relations between culture, and structure, strategy, and organization setting. Moreover, based on the quality pilot study, it strives to explain peculiarity of this concept in relation to Polish hospitals. Results of pilot studies of organizational cultures of hospitals in Poland relate to four hospitals in Lodz Province.

Sign in / Sign up

Export Citation Format

Share Document