Methanobrevibacter smithii Archaemia in Febrile Patients With Bacteremia, Including Those With Endocarditis

Abstract Background The spectrum of infections caused by methanogens remains to be described. We searched for methanogens in the blood of febrile patients using specific tools. Methods Blood culture samples routinely collected in patients with fever were prospectively screened by specific PCR assays for methanogens. Positive samples were observed by autofluorescence and electron microscopy, analyzed by metagenomics and cultured using previously developed methods. Blood culture bottles experimentally inoculated were used as controls. The presence of methanogens in vascular and cardiac tissues was assessed by indirect immunofluorescence, fluorescent in situ hybridization and PCR-based investigations. Results PCR detection attempted in 7,716 blood samples, was negative in all 1,312 aerobic bottles and 810 bacterial culture-negative anaerobic bottles. PCRs were positive in 27/5,594 (0.5%) bacterial culture-positive anaerobic bottles collected from 26 patients. Sequencing confirmed Methanobrevibacter smithii associated with staphylococci in 14 patients, Enterobacteriaceae in nine patients and streptococci in three patients. Metagenomics confirmed M. smithii in five samples, and M. smithii was isolated in broth from two samples; the genomes of these two isolates were sequenced. Blood cultures experimentally inoculated with Enterobacteriaceae, Staphylococcus epidermidis or Staphylococcus hominis yielded hydrogen, but no methane, authentifying observational data. Three patients diagnosed with infectious mitral endocarditis, were indisputably diagnosed by microscopy, PCR-based detections and culture: we showed M. smithii microscopically and by a specific PCR followed by sequencing method in two of three cardiovascular tissues. Conclusions Using appropriate laboratory methods, M. smithii is demonstrated as causing archaemia and endocarditis in febrile patients who are coinfected by bacteria.

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Fluorescent In Situ Hybridization Allows Rapid Identification of Microorganisms in Blood Cultures

Journal of Clinical Microbiology ◽

10.1128/jcm.38.2.830-838.2000 ◽

2000 ◽

Vol 38 (2) ◽

pp. 830-838 ◽

Cited By ~ 222

Author(s):

Volkhard A. J. Kempf ◽

Karlheinz Trebesius ◽

Ingo B. Autenrieth

Keyword(s):

In Situ Hybridization ◽

Blood Culture ◽

Fluorescent In Situ Hybridization ◽

Growth Index ◽

Rapid Identification ◽

Blood Cultures ◽

Oligonucleotide Probes ◽

Two Samples ◽

The Family

Using fluorescent in situ hybridization (FISH) with rRNA-targeted fluorescently labelled oligonucleotide probes, pathogens were rapidly detected and identified in positive blood culture bottles without cultivation and biotyping. In this study, 115 blood cultures with a positive growth index as determined by a continuous-reading automated blood culture system were examined by both conventional laboratory methods and FISH. For this purpose, oligonucleotide probes that allowed identification of approximately 95% of those pathogens typically associated with bacteremia were produced. The sensitivity and specificity of these probes were 100%. From all 115 blood cultures, microorganisms were grown after 1 day and identification to the family, genus, or species level was achieved after 1 to 3 days while 111 samples (96.5%) were similarly identified by FISH within 2.5 h. Staphylococci were identified in 62 of 62 samples, streptococci and enterococci were identified in 19 of 20 samples, gram-negative rods were identified in 28 of 30 samples, and fungi were identified in two of two samples. Thus, FISH is an appropriate method for identification of pathogens grown in blood cultures from septicemic patients.

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Association of blood culture with carbapenem use in pyogenic liver abscess: a two-center retrospective study

BMC Emergency Medicine ◽

10.1186/s12873-021-00442-2 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Shuangjun He ◽

Jie Yu ◽

Hairong Wang ◽

Lifeng Wang ◽

Yi Chen ◽

...

Keyword(s):

Blood Culture ◽

Liver Abscess ◽

Bacterial Culture ◽

Multivariate Analyses ◽

Tertiary Care ◽

Pyogenic Liver Abscess ◽

Extended Spectrum Beta Lactamase ◽

Tertiary Care Centers ◽

High Positivity Rate ◽

Positive Results

Abstract Background Highly empiric use of carbapenem in pyogenic liver abscess (PLA) is widespread problem. However, few studies have examined the association between blood culture and carbapenem use in patients with PLA in China. Thus, we conducted this observational study. Methods The data of patients diagnosed with PLA at two comprehensive tertiary care centers from 2014 to 2020 were retrospectively collected. Demographic and clinical data were analyzed, and univariate and multivariate analyses were performed to investigate the association between blood culture and carbapenem use. Subgroup analysis was conducted to explore whether the effect is different in sepsis. Results Blood culture was performed in 110 (46.0%) patients, of whom 44 (40.0%) patients had positive results for bacterial culture. Extended-spectrum beta-lactamase (ESBL)-positive blood culture isolates were detected in 8 (7.3%) patients. The positivity rate of blood culture in sepsis was higher than in non-sepsis (58.1% vs. 32.9%, P = 0.015). Fewer patients who had a blood culture received carbapenem treatment in comparison to patients without blood culture (19.1% vs. 31.8%, P = 0.026). Multivariate analysis showed that blood culture was independently associated with less carbapenem exposure (adjusted odds ratio [OR] = 0.33, 95% confidence interval [CI]: 0.16–0.68, P = 0.003), and this effect remained significant in the sepsis subgroup (adjusted OR = 0.17, 95% CI: 0.05–0.53, P = 0.002). Conclusion Blood culture had a high positivity rate and was associated with less carbapenem use in PLA, especially those who developed sepsis. More attention should be paid to performing early blood culture and less carbapenem use in PLA.

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Preliminary Study on In Situ Realtime Quantitation of Target Bacteria on the Principle of Flow Cytometry

Solid State Phenomena ◽

10.4028/www.scientific.net/ssp.262.224 ◽

2017 ◽

Vol 262 ◽

pp. 224-227

Author(s):

Gen Murakami ◽

Yuichi Sugai ◽

Kyuro Sasaki

Keyword(s):

Flow Cytometry ◽

Bacterial Culture ◽

Scattered Light ◽

Bacterial Suspension ◽

Culture Solution ◽

Bacterial Cells ◽

Monitoring Wells ◽

Measurement Principle ◽

Preliminary Study

In-situ realtime method that can monitor the target bacteria should be used to determine the real situation of the bacteria in deep parts of heaps in heap bioleaching plants. This study suggest to apply flow cytometry technology to in-situ realtime monitoring of target bacteria. Flow cytometry is a method that can rapidly quantify the bacterial cells in bacterial suspension based on the detection of lights that are emitted from bacterial cells. In this study, we estimated the possibility of the application of flow cytometry to the selective detection of target bacteria. The bacterial culture solution that had been diluted by water including other bacteria was provided for fluorescence spectral analysis and scattered light analysis that were functions of flow cytometry. Our target bacteria could be selectively detected by those analyses in this study, therefore, it was shown that the flow cytometry could be useful for detecting target bacteria selectively. Because the measurement principle of flow cytometry is quite simple, it can be expected to be installed into deep heaps through the monitoring wells and determine the dominance of target bacteria in-situ and realtime in the future.

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Enzootic Activity of Chlamydia in Farms Located in a Hotspot Area for Zoonosis Emergence

10.21203/rs.3.rs-1014196/v1 ◽

2021 ◽

Author(s):

Ezequiel Andres Vanderhoeven ◽

Jessica P. Mosmann ◽

Adrián Díaz ◽

Cecilia G. Cuffini

Keyword(s):

Bird Species ◽

Farm Animals ◽

Economic Losses ◽

Wild Mammals ◽

Specific Pcr ◽

Chlamydial Species ◽

Zoonotic Risk ◽

Two Samples ◽

In The Wild ◽

First Time

Abstract Chlamydias are obligated intracellular Gram-negative bacteria, considered important zoonotic pathogens, broadly present in several bird species and responsible for economic losses in animal production. We analyzed the presence of Chlamydial species with zoonotic risk in farm animals in a highly biodiverse area and with great human circulation, the Argentine, Brazil and Paraguay tri-border area. We surveyed nine farms in an area and nasally swabbed a total of 62 animals. DNA was extracted and specific PCR was performed to identify chlamydial species. We detected Chlamydia spp . in 6.5% (4/62) of the animals tested, positive samples belonged to cattle and none of them showed symptoms of respiratory disease nor had been diagnose with reproductive diseases. Specific nested PCR confirmed two samples belonged to C. pecorum and two to C. psittaci . We report for the first time Chlamydia circulation with zoonotic risk in the region. Surveys in birds and wild mammals could give a better understanding to know what Chlamydial species are circulating in the wild interface. The zoonotic potential should be taking into account as farm workers and the surrounding population could be silent carriers or have respiratory diseases being underdiagnosed, and therefore should be considered in the differential diagnoses.

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Laboratory Methods for the Detection of Chromosomal Structural Aberrations in Human and Mouse Sperm by Fluorescence In Situ Hybridization

Environmental Genomics - Methods in Molecular Biology ◽

10.1007/978-1-59745-548-0_14 ◽

2008 ◽

pp. 241-272 ◽

Cited By ~ 1

Author(s):

Francesco Marchetti ◽

Debby Cabreros ◽

Andrew J. Wyrobek

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Laboratory Methods ◽

Mouse Sperm ◽

Structural Aberrations ◽

Human And Mouse

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A case of death within 72 hours after community-acquired bloodstream infection caused by klebsiella variicola

10.21203/rs.3.rs-201872/v1 ◽

2021 ◽

Author(s):

Dali Long ◽

Yuhui Wang ◽

Jinlong Wang ◽

Sijie Mu ◽

Li Chen ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Rare Case ◽

Bacterial Culture ◽

Drug Susceptibility ◽

Klebsiella Pneumonia ◽

Gene Detection ◽

Laboratory Methods ◽

Short Period ◽

Genetic Detection ◽

Illness Progression

Abstract Klebsiella subspecies infection is widely misdiagnosed and underestimated in clinic. We report a patient who was admitted to hospital due to unknown high fever. Bacterial culture only revealed the klebsiella pneumonia. Broad-spectrum antibiotics failed to improve patient's symptoms, although these antibiotics are sensitive to klebsiella pneumonia according to the drug susceptibility results. Patient rapidly entered sepsis and subsequent sepsis shock, and died within 72 hours. The delayed PMseq-DNA Pro high throughput gene detection revealed the mixed infection of klebsiella pneumoniae and klebsiella variicola. This is a very rare case that patient suffered so rapid deterioration and died from bacterial infection within short period of time. Klebsiella variicola could contribute to rapid illness progression, while it was revealed by gene detection rather than classic laboratory methods. Which suggests that early genetic detection should be recommended in cases of complex infection.

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When time really is money: in situ quantification of the strobilurin resistance mutation G143A in the wheat pathogen Blumeria graminis f. sp. tritici

10.1101/2020.08.20.258921 ◽

2020 ◽

Author(s):

Kejal N Dodhia ◽

Belinda A Cox ◽

Richard P Oliver ◽

Francisco J Lopez-Ruiz

Keyword(s):

Fungicide Resistance ◽

Plant Pathogens ◽

Resistance Mutation ◽

Digital Pcr ◽

Blumeria Graminis ◽

Diagnostic Methods ◽

Sample Collection ◽

Specific Pcr ◽

Field Samples

AbstractBackgroundThere has been an inexorable increase in the incidence of fungicide resistance in plant pathogens in recent years. Control of diseases and the management of resistance would be greatly aided by rapid diagnostic methods. Quantitative allele specific PCR (ASqPCR) is an ideal technique for the analysis of fungicide resistance in the field as it can both detect and quantify the frequency of mutations associated with fungicide resistance. We have applied this technique to the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), an obligate biotrophic fungus that causes wheat powdery mildew and is responsible for up to 25% yield loss annually. In Australia, strobilurin resistant Bgt was first discovered in samples from Tasmania and Victoria in 2016. Molecular analysis revealed a nucleotide transversion in the cytochrome bc1 enzyme (cytb) complex, resulting in a substitution of alanine for glycine at position 143 (G143A) in Cytb.ResultsWe have developed an in-field ASqPCR assay that can quantify both the resistant (A143) and sensitive (G143) cytb alleles down to 1.67% in host and Bgt DNA mixtures within 90 min of sample collection. The in situ analysis of field samples collected during a survey in Tasmania revealed A143 frequencies ranging between 9-100%. We validated the analysis with a newly developed laboratory based digital PCR assay and found no significant differences between the two methods.ConclusionWe have successfully developed an in-field quantification method, for a QoI resistant allele, by pairing an ASqPCR assay on a lightweight qPCR instrument with a quick DNA extraction method. The deployment of this type of methodologies in the field can contribute to the effective in-season management of fungicide resistance.

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Clinical Evaluation of the iCubate iC-GPC Assay for Detection of Gram-Positive Bacteria and Resistance Markers from Positive Blood Cultures

Journal of Clinical Microbiology ◽

10.1128/jcm.00485-18 ◽

2018 ◽

Vol 56 (9) ◽

Cited By ~ 5

Author(s):

Paul A. Granato ◽

Melissa M. Unz ◽

Raymond H. Widen ◽

Suzane Silbert ◽

Stephen Young ◽

...

Keyword(s):

Blood Culture ◽

Staphylococcus Epidermidis ◽

Bloodstream Infections ◽

Blood Cultures ◽

Gram Positive ◽

Content Type ◽

Gram Positive Bacteria ◽

Sequencing Method ◽

Resistance Markers ◽

Gram Positive Cocci

ABSTRACT The iC-GPC Assay (iCubate, Huntsville, AL) is a qualitative multiplex test for the detection of five of the most common Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, and Enterococcus faecium) responsible for bacterial bloodstream infections, performed directly from positive blood cultures. The assay also detects the presence of the mecA, vanA, and vanB resistance determinants. This study comparatively evaluated the performance of the iC-GPC Assay against the Verigene Gram-positive blood culture (BC-GP) assay (Luminex Corp., Austin, TX) for 1,134 patient blood culture specimens positive for Gram-positive cocci. The iC-GPC Assay had an overall percent agreement with the BC-GP assay of 95.5%. Discordant specimens were further analyzed by PCR and a bidirectional sequencing method. The results indicate that the iC-GPC Assay together with the iCubate system is an accurate and reliable tool for the detection of the five most common Gram-positive bacteria and their resistance markers responsible for bloodstream infections.

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