Identification of Microorganisms: Old, New and Future Methods

A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Systems Microbiology".

Deadline for manuscript submissions: closed (30 September 2020) | Viewed by 75250

Special Issue Editors


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Guest Editor
1. Faculty of Medicine, University of Porto, 4099-002 Porto, Portugal
2. Institute for Research and Inovation in Health (i3S), University of Porto, 4099-002 Porto, Portugal
Interests: evidence-based medicine; phytochemistry; phytopharmacology; drug discovery; natural products biochemistry; bioactive molecules; functional foods; nutraceuticals; fungal and bacterial infections; resistance to antimicrobials
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Special Issue Information

Dear Colleagues,

Infections related to resistant and multidrug-resistant (MDR) pathogens are a significant and growing global threat, being also closely linked to increased morbidity, mortality, and healthcare costs. Indeed, by the year 2050, it is expected that 10 million people will die per year as a result of MDR infections, if an international and effective response is not achieved to address the problem.

This Special Issue, dedicated to the “Identification of Microorganisms: Old, New, and Future Methods”, is intended to cover all fields related to the identification, epidemiology, and detection of bacteria, fungi, parasites, and viruses. Original and review manuscripts reporting the development, evaluation, and validation of rapid diagnostic techniques that allow the fast identification of general pathogens or resistance patterns are required. In addition, research reporting the best tools and approaches to prevent and control pathogens’ spread is also welcome.

Dr. Natália Martins
Dr. Célia F. Rodrigues
Guest Editors

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Microorganisms is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • pathogens
  • infections
  • biofilms
  • resistant microorganisms
  • PCR
  • real-time PCR
  • chromogenic media
  • MALDI-TOF
  • LC-MS
  • microfluidics
  • synthetic biology
  • microorganism identification

Published Papers (6 papers)

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Research

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23 pages, 2525 KiB  
Article
A Practical Method to Implement Strain-Level Metagenomics-Based Foodborne Outbreak Investigation and Source Tracking in Routine
by Florence E. Buytaers, Assia Saltykova, Sarah Denayer, Bavo Verhaegen, Kevin Vanneste, Nancy H. C. Roosens, Denis Piérard, Kathleen Marchal and Sigrid C. J. De Keersmaecker
Microorganisms 2020, 8(8), 1191; https://doi.org/10.3390/microorganisms8081191 - 05 Aug 2020
Cited by 12 | Viewed by 4878
Abstract
The management of a foodborne outbreak depends on the rapid and accurate identification of the responsible food source. Conventional methods based on isolation of the pathogen from the food matrix and target-specific real-time polymerase chain reactions (qPCRs) are used in routine. In recent [...] Read more.
The management of a foodborne outbreak depends on the rapid and accurate identification of the responsible food source. Conventional methods based on isolation of the pathogen from the food matrix and target-specific real-time polymerase chain reactions (qPCRs) are used in routine. In recent years, the use of whole genome sequencing (WGS) of bacterial isolates has proven its value to collect relevant information for strain characterization as well as tracing the origin of the contamination by linking the food isolate with the patient’s isolate with high resolution. However, the isolation of a bacterial pathogen from food matrices is often time-consuming and not always successful. Therefore, we aimed to improve outbreak investigation by developing a method that can be implemented in reference laboratories to characterize the pathogen in the food vehicle without its prior isolation and link it back to human cases. We tested and validated a shotgun metagenomics approach by spiking food pathogens in specific food matrices using the Shiga toxin-producing Escherichia coli (STEC) as a case study. Different DNA extraction kits and enrichment procedures were investigated to obtain the most practical workflow. We demonstrated the feasibility of shotgun metagenomics to obtain the same information as in ISO/TS 13136:2012 and WGS of the isolate in parallel by inferring the genome of the contaminant and characterizing it in a shorter timeframe. This was achieved in food samples containing different E. coli strains, including a combination of different STEC strains. For the first time, we also managed to link individual strains from a food product to isolates from human cases, demonstrating the power of shotgun metagenomics for rapid outbreak investigation and source tracking. Full article
(This article belongs to the Special Issue Identification of Microorganisms: Old, New and Future Methods)
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14 pages, 1350 KiB  
Article
Exploiting the Advantages of Molecular Tools for the Monitoring of Fungal Indoor Air Contamination: First Detection of Exophiala jeanselmei in Indoor Air of Air-Conditioned Offices
by Xavier Libert, Camille Chasseur, Ann Packeu, Fabrice Bureau, Nancy H. Roosens and Sigrid C. J. De Keersmaecker
Microorganisms 2019, 7(12), 674; https://doi.org/10.3390/microorganisms7120674 - 10 Dec 2019
Cited by 5 | Viewed by 2349
Abstract
Today, indoor air pollution is considered a public health issue. Among the impacting pollutants, indoor airborne fungi are increasingly highlighted. Most of the monitoring protocols are culture-based, but these are unable to detect the uncultivable and/or dead fraction or species suppressed by fast-growing [...] Read more.
Today, indoor air pollution is considered a public health issue. Among the impacting pollutants, indoor airborne fungi are increasingly highlighted. Most of the monitoring protocols are culture-based, but these are unable to detect the uncultivable and/or dead fraction or species suppressed by fast-growing fungi, even though this fraction could impact health. Among the contaminants suspected to be part of this fraction, Exophiala jeanselmei is an interesting case study. Known to be pathogenic, this black yeast grows in humid environments such as air-conditioning systems, where it has been previously detected using classical culture-based methods. However, until now, this fungus was never detected in indoor air in contact with these air-conditioning systems. This study shows the first detection of E. jeanselmei in indoor air collected from offices in contact with contaminated air-conditioning reservoirs. While its presence in indoor air could not be demonstrated with culture-based methods, it was found by real-time PCR and massive parallel sequencing. The latter also allowed obtaining a broader view on the fungal diversity in the tested samples. Similar approaches were applied on water samples collected from the conditioning reservoirs to trace the source of contamination. The comparison of results obtained with both methods confirmed that the molecular tools could improve indoor air monitoring, especially of dead and/or uncultivable contaminants or when competition between species could occur. Full article
(This article belongs to the Special Issue Identification of Microorganisms: Old, New and Future Methods)
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15 pages, 3490 KiB  
Article
Development and Evaluation of a Duo Zaire ebolavirus Real-Time RT-PCR Assay Targeting Two Regions within the Genome
by Laurence Thirion, Remi N. Charrel, Yannik Boehmann, Iban Corcostegui, Hervé Raoul and Xavier de Lamballerie
Microorganisms 2019, 7(12), 652; https://doi.org/10.3390/microorganisms7120652 - 04 Dec 2019
Cited by 3 | Viewed by 2526
Abstract
Preparedness and response actions to mitigate Ebola virus disease (EVD) outbreaks rely on rapid diagnosis to be implemented locally to sort suspect patients attending health centers. Our aim was (i) to develop and evaluate an RT-qPCR assay combining primers and probes derived from [...] Read more.
Preparedness and response actions to mitigate Ebola virus disease (EVD) outbreaks rely on rapid diagnosis to be implemented locally to sort suspect patients attending health centers. Our aim was (i) to develop and evaluate an RT-qPCR assay combining primers and probes derived from two reference assays targeting different genomic regions; (ii) to study whether sensitivity and specificity of this dual-target assay were at least equal or better to the parental assays; (iii) to implement this dual-target assay onto the Cepheid GeneXpert open cartridge as a proof of principle for technological transfer aiming at bedsite testing locally. To do so, three home-made published RT-qPCR assays were selected to be compared with the RealStar® Filovirus Screen RT-PCR kit 1.0 (Altona Diagnostics, Hamburg, Germany), a technique that was largely deployed during the 2014–2015 West African EVD outbreak. Primers and probes sequences of the custom-made assays were analyzed in silico against a multiple sequence alignment, including >250 complete sequences corresponding to strains that have caused EVD epidemics in the past. Genomic RNA purified from the Mekambo strain of Zaire ebolavirus (EBOV) was used to study the sensitivity of the five methods. Based on these results, two in-house methods were selected and adapted to design the dual-target assay, which performances were compared to those of the parental assays using a synthetic RNA control. The dual-target assay showed better sensitivity and limit of detection (LoD95 at 0.4 copies/µL) than the parental methods (1.7 and 2.2 copies/µL). Ultimately, the dual-target assay was transferred onto the GeneXpert Flex-03 open cartridge, demonstrating a LoD95 at 0.75 copies/µL. Together these results indicate that EBOV dual-target assay has the potential to be used during EVD outbreak in the laboratory having performed molecular testing during the recent outbreaks. Full article
(This article belongs to the Special Issue Identification of Microorganisms: Old, New and Future Methods)
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Review

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30 pages, 874 KiB  
Review
Laboratory Diagnosis of Cutaneous and Visceral Leishmaniasis: Current and Future Methods
by Juliana Quero Reimão, Elizabeth Magiolo Coser, Monica Ran Lee and Adriano Cappellazzo Coelho
Microorganisms 2020, 8(11), 1632; https://doi.org/10.3390/microorganisms8111632 - 22 Oct 2020
Cited by 32 | Viewed by 14011
Abstract
Leishmaniasis is a neglected tropical disease with two main clinical forms: cutaneous and visceral leishmaniasis. Diagnosis of leishmaniasis is still a challenge, concerning the detection and correct identification of the species of the parasite, mainly in endemic areas where the absence of appropriate [...] Read more.
Leishmaniasis is a neglected tropical disease with two main clinical forms: cutaneous and visceral leishmaniasis. Diagnosis of leishmaniasis is still a challenge, concerning the detection and correct identification of the species of the parasite, mainly in endemic areas where the absence of appropriate resources is still a problem. Most accessible methods for diagnosis, particularly in these areas, do not include the identification of each one of more than 20 species responsible for the disease. Here, we summarize the main methods used for the detection and identification of leishmaniasis that can be performed by demonstration of the parasite in biological samples from the patient through microscopic examination, by in vitro culture or animal inoculation; by molecular methods through the detection of parasite DNA; or by immunological methods through the detection of parasite antigens that may be present in urine or through the detection of specific antibodies against the parasite. Potential new methods that can be applied for laboratory diagnosis of leishmaniasis are also discussed. Full article
(This article belongs to the Special Issue Identification of Microorganisms: Old, New and Future Methods)
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14 pages, 320 KiB  
Review
Detection and Identification of Bacillus anthracis: From Conventional to Molecular Microbiology Methods
by Aleksandra A. Zasada
Microorganisms 2020, 8(1), 125; https://doi.org/10.3390/microorganisms8010125 - 16 Jan 2020
Cited by 28 | Viewed by 13298
Abstract
Rapid and reliable identification of Bacillus anthracis is of great importance, especially in the event of suspected deliberate release of anthrax spores. However, the identification of B. anthracis is challenging due to its high similarity to closely related species. Since Amerithrax in 2001, [...] Read more.
Rapid and reliable identification of Bacillus anthracis is of great importance, especially in the event of suspected deliberate release of anthrax spores. However, the identification of B. anthracis is challenging due to its high similarity to closely related species. Since Amerithrax in 2001, a lot of effort has been made to develop rapid methods for detection and identification of this microorganism with special focus on easy-to-perform rapid tests for first-line responders. This article presents an overview of the evolution of B. anthracis identification methods from the time of the first description of the microorganism until the present day. Full article
(This article belongs to the Special Issue Identification of Microorganisms: Old, New and Future Methods)
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32 pages, 659 KiB  
Review
Advances in Chemical and Biological Methods to Identify Microorganisms—From Past to Present
by Ricardo Franco-Duarte, Lucia Černáková, Snehal Kadam, Karishma S. Kaushik, Bahare Salehi, Antonio Bevilacqua, Maria Rosaria Corbo, Hubert Antolak, Katarzyna Dybka-Stępień, Martyna Leszczewicz, Saulo Relison Tintino, Veruska Cintia Alexandrino de Souza, Javad Sharifi-Rad, Henrique Douglas Melo Coutinho, Natália Martins and Célia F. Rodrigues
Microorganisms 2019, 7(5), 130; https://doi.org/10.3390/microorganisms7050130 - 13 May 2019
Cited by 214 | Viewed by 37170
Abstract
Fast detection and identification of microorganisms is a challenging and significant feature from industry to medicine. Standard approaches are known to be very time-consuming and labor-intensive (e.g., culture media and biochemical tests). Conversely, screening techniques demand a quick and low-cost grouping of bacterial/fungal [...] Read more.
Fast detection and identification of microorganisms is a challenging and significant feature from industry to medicine. Standard approaches are known to be very time-consuming and labor-intensive (e.g., culture media and biochemical tests). Conversely, screening techniques demand a quick and low-cost grouping of bacterial/fungal isolates and current analysis call for broad reports of microorganisms, involving the application of molecular techniques (e.g., 16S ribosomal RNA gene sequencing based on polymerase chain reaction). The goal of this review is to present the past and the present methods of detection and identification of microorganisms, and to discuss their advantages and their limitations. Full article
(This article belongs to the Special Issue Identification of Microorganisms: Old, New and Future Methods)
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