scholarly journals Identification and Elimination of the Clinically Relevant Multi-Resistant Environmental Bacteria Ralstonia insidiosa in Primary Cell Culture

2020 ◽  
Vol 8 (10) ◽  
pp. 1599
Author(s):  
Dennis Nurjadi ◽  
Sébastien Boutin ◽  
Katja Schmidt ◽  
Melinda Ahmels ◽  
Daniel Hasche
Keyword(s):  
Cell Culture ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Resistant Bacteria ◽  
Rrna Gene ◽  
Whole Genome ◽  
Black Dot ◽  
Microscopic Appearance ◽  
Ralstonia Insidiosa

In times of spreading multidrug-resistant bacteria, species identification and decontamination of cell cultures can be challenging. Here, we describe a mobile cell culture contaminant with “black dot”-like microscopic appearance in newly established irreplaceable hybridoma cell lines and its identification. Using 16S rRNA gene sequencing, species-specific PCRs, whole genome sequencing (WGS), and MALDI-TOF mass spectrometry, the contaminant was identified as the ubiquitous environmental and clinically relevant Gram-negative bacterium Ralstonia insidiosa (R. insidiosa), a strong biofilm producer. Further characterizations by transmission electron microscopy (TEM) and biochemical API test were not conclusive. Whole genome sequencing of our R. insidiosa isolate revealed numerous drug-resistance determinants. Genome-wide comparison to other Ralstonia species could not unambiguously designate our isolate to R. insidiosa (<95% average nucleotide identity) suggesting a potential novel species or subspecies, closely related to R. insidiosa and R. pickettii. After determining the antibiotic susceptibility profile, the hybridoma cell culture was successfully decontaminated with ciprofloxacin without affecting antibody production.

scholarly journals Whole-Genome Sequencing ofAggregatibacterSpecies Isolated from Human Clinical Specimens and Description ofAggregatibacter kilianiisp. nov.

2018 ◽  
Vol 56 (7) ◽  
Author(s):  
May Murra ◽  
Lisbeth Lützen ◽  
Aynur Barut ◽  
Reinhard Zbinden ◽  
Marianne Lund ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Whole Genome ◽  
Content Type ◽  
Clinical Specimens ◽  
Phylogenetic Cluster ◽  
Invasive Infections ◽  
Rrna Gene Sequencing

ABSTRACTAggregatibacterspecies are commensal bacteria of human mucosal surfaces that are sometimes involved in serious invasive infections. During the investigation of strains cultured from various clinical specimens, we encountered a coherent group of 10 isolates that could not be allocated to any validly named species by phenotype, mass spectrometry, or partial 16S rRNA gene sequencing. Whole-genome sequencing revealed a phylogenetic cluster related to but separate fromAggregatibacter aphrophilus. The meanin silicoDNA hybridization value for strains of the new cluster versusA. aphrophiluswas 56% (range, 53.7 to 58.0%), whereas the average nucleotide identity was 94.4% (range, 93.9 to 94.8%). The new cluster exhibited aggregative properties typical of the genusAggregatibacter. Key phenotypic tests for discrimination of the new cluster from validly namedAggregatibacterspecies are alanine-phenylalanine-proline arylamidase,N-acetylglucosamine, and β-galactosidase. The nameAggregatibacter kilianiiis proposed, with PN_528 (CCUG 70536Tor DSM 105094T) as the type strain.

scholarly journals Enterobacter cloacae Complex Sequence Type 171 Isolates Expressing KPC-4 Carbapenemase Recovered from Canine Patients in Ohio

2018 ◽  
Vol 62 (12) ◽  
Author(s):  
Joshua B. Daniels ◽  
Liang Chen ◽  
Susan V. Grooters ◽  
Dixie F. Mollenkopf ◽  
Dimitria A. Mathys ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Companion Animals ◽  
Enterobacter Cloacae ◽  
Sequence Type ◽  
Resistant Bacteria ◽  
Whole Genome ◽  
Content Type ◽  
Complex Sequence ◽  
Enterobacter Cloacae Complex

ABSTRACT Companion animals are likely relevant in the transmission of antimicrobial-resistant bacteria. Enterobacter xiangfangensis sequence type 171 (ST171), a clone that has been implicated in clusters of infections in humans, was isolated from two dogs with clinical disease in Ohio. The canine isolates contained IncHI2 plasmids encoding blaKPC-4. Whole-genome sequencing was used to put the canine isolates in phylogenetic context with available human ST171 sequences, as well as to characterize their blaKPC-4 plasmids.

scholarly journals Recommendations To Address the Difficulties Encountered When Determining Linezolid Resistance from Whole-Genome Sequencing Data

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Alicia G. Beukers ◽  
Henrik Hasman ◽  
Kristin Hegstad ◽  
Sebastiaan J. van Hal
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
23S Rrna ◽  
Whole Genome Sequencing Data ◽  
Rrna Gene ◽  
Whole Genome ◽  
Sequencing Data ◽  
Content Type ◽  
Numbering System ◽  
Linezolid Resistance

ABSTRACT Mutations associated with linezolid resistance within the V domain of 23S rRNA are annotated using an Escherichia coli numbering system. The 23S rRNA gene varies in length, nucleotide sequence, and copy number among bacterial species. Consequently, this numbering system is not intuitive and can lead to confusion when mutation sites are being located using whole-genome sequencing data. Using the mutation G2576T as an example, we demonstrate the difficulties associated with using the E. coli numbering system.

scholarly journals How Do We Find the Source of Foodborne Superbug Outbreaks?

2021 ◽  
Vol 9 ◽  
Author(s):  
Joy Scaria ◽  
Shruti Menon ◽  
Maristela Rovai
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Food Supply ◽  
Supply System ◽  
New Method ◽  
Resistant Bacteria ◽  
Whole Genome ◽  
Testing Methods ◽  
Antibiotic Resistant ◽  
The World

Lately, there have been more foodborne “superbug” outbreaks than ever before, which creates a problem because superbugs are antibiotic-resistant bacteria that are difficult to treat. To reduce such outbreaks, better ways of finding the source of the infection are needed. Superbugs, such as Salmonella are often transmitted through food. The world’s food supply system has become so complex that it is often difficult to find the source of an outbreak with older testing methods. A new method called whole genome sequencing (WGS) has now been developed to track superbug infections. Using WGS, it is now possible to identify the source of an outbreak in one country that may be transmitted through food imported from the opposite side of the world. Good outbreak tracing methods help scientists make better predictions about outbreaks. Finding the source of an outbreak early on can lead to better containment and lower costs.

scholarly journals Reliable Identification of Environmental Pseudomonas Isolates Using the rpoD Gene

2020 ◽  
Vol 8 (8) ◽  
pp. 1166
Author(s):  
Léa Girard ◽  
Cédric Lood ◽  
Hassan Rokni-Zadeh ◽  
Vera van Noort ◽  
Rob Lavigne ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Low Cost ◽  
Rrna Gene ◽  
Whole Genome ◽  
Comprehensive Overview ◽  
Rpod Gene ◽  
The 16S Rrna Gene

The taxonomic affiliation of Pseudomonas isolates is currently assessed by using the 16S rRNA gene, MultiLocus Sequence Analysis (MLSA), or whole genome sequencing. Therefore, microbiologists are facing an arduous choice, either using the universal marker, knowing that these affiliations could be inaccurate, or engaging in more laborious and costly approaches. The rpoD gene, like the 16S rRNA gene, is included in most MLSA procedures and has already been suggested for the rapid identification of certain groups of Pseudomonas. However, a comprehensive overview of the rpoD-based phylogenetic relationships within the Pseudomonas genus is lacking. In this study, we present the rpoD-based phylogeny of 217 type strains of Pseudomonas and defined a cutoff value of 98% nucleotide identity to differentiate strains at the species level. To validate this approach, we sequenced the rpoD of 145 environmental isolates and complemented this analysis with whole genome sequencing. The rpoD sequence allowed us to accurately assign Pseudomonas isolates to 20 known species and represents an excellent first diagnostic tool to identify new Pseudomonas species. Finally, rpoD amplicon sequencing appears as a reliable and low-cost alternative, particularly in the case of large environmental studies with hundreds or thousands of isolates.

scholarly journals Whole-Genome Sequencing of Bacterial Pathogens: the Future of Nosocomial Outbreak Analysis

2017 ◽  
Vol 30 (4) ◽  
pp. 1015-1063 ◽  
Author(s):  
Scott Quainoo ◽  
Jordy P. M. Coolen ◽  
Sacha A. F. T. van Hijum ◽  
Martijn A. Huynen ◽  
Willem J. G. Melchers ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Health Care Professionals ◽  
Resistant Bacteria ◽  
Whole Genome ◽  
Nosocomial Outbreak ◽  
Icu Patients ◽  
Outbreak Management ◽  
Sequencing Technologies ◽  
Analysis Tools

SUMMARY Outbreaks of multidrug-resistant bacteria present a frequent threat to vulnerable patient populations in hospitals around the world. Intensive care unit (ICU) patients are particularly susceptible to nosocomial infections due to indwelling devices such as intravascular catheters, drains, and intratracheal tubes for mechanical ventilation. The increased vulnerability of infected ICU patients demonstrates the importance of effective outbreak management protocols to be in place. Understanding the transmission of pathogens via genotyping methods is an important tool for outbreak management. Recently, whole-genome sequencing (WGS) of pathogens has become more accessible and affordable as a tool for genotyping. Analysis of the entire pathogen genome via WGS could provide unprecedented resolution in discriminating even highly related lineages of bacteria and revolutionize outbreak analysis in hospitals. Nevertheless, clinicians have long been hesitant to implement WGS in outbreak analyses due to the expensive and cumbersome nature of early sequencing platforms. Recent improvements in sequencing technologies and analysis tools have rapidly increased the output and analysis speed as well as reduced the overall costs of WGS. In this review, we assess the feasibility of WGS technologies and bioinformatics analysis tools for nosocomial outbreak analyses and provide a comparison to conventional outbreak analysis workflows. Moreover, we review advantages and limitations of sequencing technologies and analysis tools and present a real-world example of the implementation of WGS for antimicrobial resistance analysis. We aimed to provide health care professionals with a guide to WGS outbreak analysis that highlights its benefits for hospitals and assists in the transition from conventional to WGS-based outbreak analysis.

scholarly journals Metagenomic survey and whole genome sequencing of antimicrobial resistant bacteria in the sewage of a Japanese hospital

2020 ◽  
Author(s):  
Miwa Katagiri ◽  
Makoto Kuroda ◽  
Tsuyoshi Sekizuka ◽  
Norihide Nakada ◽  
Yukitaka Ito ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genome Analysis ◽  
Therapeutic Agents ◽  
Resistant Bacteria ◽  
Whole Genome ◽  
Comparative Genome Analysis ◽  
Comparative Genome ◽  
Hospital Sewage

Abstract Background The dissemination of antimicrobial-resistant bacteria (ARB) and the transfer of antimicrobial resistance genes (ARGs) are a threat to public health. Antibiotics are indispensable therapeutic agents essential for the treatment of infectious diseases; however, inappropriate use of antibiotics leads to the emergence of ARB. It is established that hospitals are closely involved in the spread of antimicrobial resistance (AMR), which impedes antibiotic treatment and subsequently increases mortality. In addition, excrement of patients or healthy carriers of ARB are discharged from the hospital sewage through the wastewater treatment plant (WWTP) into the rivers, causing an AMR burden on the environment.Method Metagenomic analysis was performed on the hospital sewage samples, followed by whole genome sequencing of the extended spectrum β-lactamase (ESBL)-producing organisms (EPOs). A comparative genome analysis was also performed between EPO isolates from sewage and clinical isolates.Results Metagenomic analysis showed that the hospital sewage tanks had bacterial flora corresponding to the human gut. During the study period, the hospital was relocated to a newly constructed building with new sewage tanks; however, the presence of ARB/ARGs in the new hospital sewage tanks became markedly equivalent to that of the old hospital within one month. The ESBL blaCTX−M and carbapenemase blaIMP genes were not much detected in the original hospital sewage samples by metagenome analysis, but selection on CHROMagar ESBL increased the sensitivity to detect those β-lactamase genes. Comparative genome analysis between sewage and clinical EPO isolates revealed partial similarity; however, most EPO isolates exhibited a notable difference (≥ 50) in single nucleotide variations based on core-genome phylogeny. This result suggests that only some of the sewage EPO isolates were originated from the clinical patient. Therapeutic agents in the hospital sewage were analyzed and the concentration of levofloxacin and clarithromycin was 0.0325 and 0.0135 µg/mL, respectively.Conclusions Whole genome analysis between sewage and clinical isolates suggested that healthy or asymptomatic carriers may be involved in the contamination of hospital sewage. Moreover, the hospital sewage tank may serve as a hotspot for the horizontal transfer of ARGs under the selective pressure of antimicrobial agents. Therefore, ARB monitoring in hospital sewage is expected to detect the presence of carriers prior to nosocomial ARB outbreaks. In addition, hospital wastewater should be treated suitably to bring ARB below detectable levels to reduce the environmental AMR burden.

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