scholarly journals Standard Sample Storage Conditions Have an Impact on Inferred Microbiome Composition and Antimicrobial Resistance Patterns

2021 ◽  
Author(s):  
Casper Sahl Poulsen ◽  
Rolf Sommer Kaas ◽  
Frank M. Aarestrup ◽  
Sünje Johanna Pamp
Keyword(s):  
Antimicrobial Resistance ◽  
Dna Isolation ◽  
Standard Sample ◽  
Storage Conditions ◽  
Library Preparation ◽  
Sample Storage ◽  
Sequencing Technology ◽  
Microbiome Composition ◽  
Resistance Patterns ◽  
Systematic Effects

Previous research has reported effects of DNA isolation, library preparation, and sequencing technology on metagenomics-based microbiome composition; however, the effect of biospecimen storage conditions has not been thoroughly assessed. We examined the effect of common sample storage conditions on metagenomics-based microbiome composition and found significant and, in part, systematic effects.

scholarly journals Standard Sample Storage Conditions Impact on Inferred Microbiome Composition and Antimicrobial Resistance Patterns

2021 ◽  
Author(s):  
Casper S. Poulsen ◽  
Rolf Sommer Kaas ◽  
Frank M. Aarestrup ◽  
Sünje Johanna Pamp
Keyword(s):  
Antimicrobial Resistance ◽  
Dna Sequencing ◽  
Storage Conditions ◽  
Systematic Effect ◽  
Sample Storage ◽  
Sequencing Data ◽  
Freezing And Thawing ◽  
Microbiome Composition ◽  
Freeze Thaw ◽  
Eukaryotic Microorganisms

Storage of biological specimens is crucial in the life and medical sciences. The storage conditions for samples can be different for a number of reasons, and it is unclear which effect this can have on the inferred microbiome composition in metagenomics analyses. Here, we assess the effect of common storage temperatures (deep freezer: -80°C, freezer: -20°C, fridge: 5°C, room temperature: 22°C) and storage times, (immediate sample processing: 0h, next day: 16h, over weekend: 64h, and longer term: 4, 8, 12 months), as well as repeated sample freezing and thawing (2-4 freeze-thaw cycles). We examine two different pig feces and sewage samples, unspiked and spiked with a mock community, and in triplicates, respectively, amounting to a total of 438 samples (777 Gbp; 5.1 billion reads). Storage conditions had a significant and systematic effect on the taxonomic and functional composition of microbiomes. Distinct microbial taxa and antimicrobial resistance classes were in some situations similarly effected across samples, while others were not, suggesting an impact of individual inherent sample characteristics. With an increasing number of freeze-thaw cycles, an increasing abundance of Firmicutes, Actinobacteria, and eukaryotic microorganisms was observed. We include recommendations for sample storage, and strongly suggest including more detailed information in the metadata together with the DNA sequencing data in public repositories to better facilitate meta-analyses and reproducibility of findings. IMPORTANCE: Previous research has reported effects of DNA isolation, library preparation, and sequencing technology on metagenomics-based microbiome composition; however, the effect of biospecimen storage conditions has not been thoroughly assessed. We examined the effect of common sample storage conditions on metagenomics-based microbiome composition and find significant and, in part, systematic effects. Repeated freeze-thaw cycles could be used to improve the detection of microorganisms with more rigid cell walls, including parasites. We provide a dataset that could also be used for benchmarking algorithms to identify and correct for batch effects. Overall, the findings suggest that all samples of a microbiome study should be stored in the same way. Furthermore, there is a need to mandate more detailed information about sample storage and processing published together with the DNA sequencing data at INSDC (ENA/EBI, NCBI, DDBJ) or other repositories.

scholarly journals Library preparation and sequencing platform introduce bias in metagenomics characterisation of microbial communities

2019 ◽  
Author(s):  
Casper S. Poulsen ◽  
Sünje J. Pamp ◽  
Claus T. Ekstrøm ◽  
Frank M. Aarestrup
Keyword(s):  
Microbial Communities ◽  
Dna Isolation ◽  
Meta Analysis ◽  
Library Preparation ◽  
Sample Processing ◽  
Fecal Samples ◽  
Sequencing Platform ◽  
Sequencing Technologies ◽  
Systematic Effects

AbstractNext generation sequencing technologies have become increasingly used to describe microbial communities. Metagenomics characterization of microbiomes is associated with minimal manipulation during sample processing, which includes sampling, storage, DNA isolation, library preparation and sequencing, before the raw data are obtained. Here we assess the effect of library preparation using a kit with a polymerase chain reaction (PCR) step (Nextera) and two PCR-free kits (NEXTflex and KAPA), and the effect of sequencing platform (HiSeq and NextSeq) on the description of microbial communities in pig feces and sewage. Two pig fecal samples were obtained from different farms and two sewage samples were collected as inlet water at a local wastewater treatment facility. Samples were processed to both perform DNA-isolation immediately upon arrival in the lab and after storage for 64 hours at −80°C, DNA isolation was performed in duplicate.We find that both library preparation and sequencing platform had systematic effects on the microbial community description. The effects were at a level that made differentiating between the two pig fecal samples difficult. The sewage samples represented two very different communities and were at all times distinguishable from each other. We find that library preparation and sequencing platform introduced more variation than freezing the samples. The community changes did not seem associated with contamination during processing and distinct patterns connected specific types of organisms with a processing method, but it was difficult to generalize between samples. This highlights the need for continuous validation of the effect of sample processing in different types of samples and that all processing steps need to be considered when comparing between studies. We believe standardization of sample processing is key to generate comparable data within a study and that comparisons of differently generated data, e.g. in a meta-analysis, should be performed cautiously.

Effect of Sample Storage Conditions on Body Burden Analysis of Organic Contaminants in Unionid Mussels

1992 ◽  
Vol 27 (4) ◽  
pp. 833-844 ◽  
Author(s):  
Micheline Hanna
Keyword(s):  
Organic Contaminants ◽  
Body Burden ◽  
Storage Conditions ◽  
The Body ◽  
Sample Storage ◽  
Pcb Congeners ◽  
Unionid Mussels ◽  
Group A ◽  
The Difference ◽  
Group B

Abstract In order to quantitatively assess the effect of sample storage conditions on the body burden analysis of organic contaminants, a comparative analysis was carried out on the unionid mussel Elliptic complanata. The mussels were divided into two groups, each with distinct storage conditions, while Group A was kept in the freezer at −20°C, Group B was kept in the refrigerator for five days at 5°C. All the compounds present in the control were also present in Group B samples. Analysis of the organic contaminants in each of these two groups showed that for total PCB concentrations, the two treatments were not significantly different; however when compared individually 6 of the 13 PCB congeners showed significant differences. The observed differences were relatively small for individual PCB congeners (7.1 to 15.3%), higher for chlorobenzenes (10.5 to 36.4%), and yet higher for HCE (44.1%); the difference for HCE, although large is nevertheless not significant, even if only marginally so.

The Causative Organisms of Bacterial Meningitis and their Antimicrobial Resistance Profiles in Iranian Children in 2011-2016

2020 ◽  
Vol 20 (2) ◽  
pp. 229-236
Author(s):  
Sepideh Keshavarz Valian ◽  
Shima Mahmoudi ◽  
Babak Pourakbari ◽  
Maryam Banar ◽  
Mohammad Taghi Haghi Ashtiani ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Bacterial Meningitis ◽  
Medical Center ◽  
Blood Cultures ◽  
Gram Positive ◽  
Gram Negative ◽  
Number Of Patients ◽  
Resistance Patterns ◽  
Iranian Children ◽  
Surgery Department

Objective: The study aimed to describe the identity and antimicrobial resistance patterns of the causative agents of bacterial meningitis in children referred to Children’s Medical Center (CMC) Hospital, Tehran, Iran. Methods: This retrospective study was performed at CMC Hospital during a six-year period from 2011 to 2016. The microbiological information of the patients with a diagnosis of bacterial meningitis was collected and the following data were obtained: patients’ age, sex, hospital ward, the results of CSF and blood cultures, and antibiotic susceptibility profiles of isolated organisms. Results: A total of 118 patients with bacterial meningitis were admitted to CMC hospital. Sixty-two percent (n=73) of the patients were male. The median age of the patients was ten months (interquartile range [IQR]: 2 months-2 years) and the majority of them (n=92, 80%) were younger than two years of age. The highest number of patients (n=47, 40%) were admitted to the surgery department. Streptococcus epidermidis was the most frequent isolated bacterium (n=27/127, 21%), followed by Klebsiella pneumoniae (n=20/127, 16%), and Staphylococcus aureus (n=16/127, 12.5%). Blood culture was positive in 28% (n=33/118) of patients. Ampicillin-sulbactam and imipenem were the most effective antibiotics against Gram-negative bacteria isolated from CSF cultures. In the case of Gram-positive organisms, ampicillinsulbactam, vancomycin, and linezolid were the best choices. Imipenem was the most active drug against Gram-negative blood pathogens. Also, ampicillin and vancomycin had the best effect on Gram-positive bacteria isolated from blood cultures. Conclusion: Results of this study provide valuable information about the antibiotic resistance profiles of the etiologic agents of childhood meningitis, which can be used for prescription of more effective empirical therapies.

Antimicrobial Resistance and Presence of Class 1 Integrons Among Different Serotypes of Salmonella spp. Recovered From Children with Diarrhea in Tehran, Iran

2020 ◽  
Vol 20 (2) ◽  
pp. 160-166
Author(s):  
Seyedeh Hanieh Eshaghi Zadeh ◽  
Hossein Fahimi ◽  
Fatemeh Fardsanei ◽  
Mohammad Mehdi Soltan Dallal
Keyword(s):  
Antibiotic Resistance ◽  
Antimicrobial Resistance ◽  
Multidrug Resistant ◽  
Salmonella Spp ◽  
Class 1 Integron ◽  
Class 1 Integrons ◽  
Food Borne ◽  
Class 1 ◽  
Resistance Patterns ◽  
C 30

Background: Salmonellosis is a major food-borne disease worldwide. The increasing prevalence of antimicrobial resistance among food-borne pathogens such as Salmonella spp. is concerning. Objective: The main objective of this study is to identify class 1 integron genes and to determine antibiotic resistance patterns among Salmonella isolates from children with diarrhea. Methods: A total of 30 Salmonella isolates were recovered from children with diarrhea. The isolates were characterized for antimicrobial susceptibility and screened for the presence of class 1 integron genes (i.e. intI1, sulI1, and qacEΔ1). Results: The most prevalent serotype was Enteritidis 36.7%, followed by Paratyphi C (30%), and Typhimurium (16.7%). The highest rates of antibiotic resistance were obtained for nalidixic acid (53.3%), followed by streptomycin (40%), and tetracycline (36.7%). Regarding class 1 integrons, 36.7%, 26.7%, and 33.3% of the isolates carried intI1, SulI, and qacEΔ1, respectively, most of which (81.8%) were multidrug-resistant (MDR). Statistical analysis revealed that the presence of class 1 integron was significantly associated with resistance to streptomycin and tetracycline (p = 0.042). However, there was no association between class 1 integron and other antibiotics used in this study (p > 0.05). Conclusion: The high frequency of integron class 1 gene in MDR Salmonella strains indicates that these mobile genetic elements are versatile among different Salmonella serotypes, and associated with reduced susceptibility to many antimicrobials.

scholarly journals Detection of extended spectrum beta-lactamase genes in Pseudomonas aeruginosa isolated from patients in rural Eastern Cape Province, South Africa

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mojisola C. Hosu ◽  
Sandeep D. Vasaikar ◽  
Grace E. Okuthe ◽  
Teke Apalata
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Resistance ◽  
Multidrug Resistant ◽  
High Rate ◽  
Infection Prevention And Control ◽  
Beta Lactamase ◽  
Eastern Cape ◽  
Extended Spectrum Beta Lactamase ◽  
Extended Spectrum ◽  
Resistance Patterns

AbstractThe proliferation of extended spectrum beta-lactamase (ESBL) producing Pseudomonas aeruginosa represent a major public health threat. In this study, we evaluated the antimicrobial resistance patterns of P. aeruginosa strains and characterized the ESBLs and Metallo- β-lactamases (MBL) produced. Strains of P. aeruginosa cultured from patients who attended Nelson Mandela Academic Hospital and other clinics in the four district municipalities of the Eastern Cape between August 2017 and May 2019 were identified; antimicrobial susceptibility testing was carried out against thirteen clinically relevant antibiotics using the BioMérieux VITEK 2 and confirmed by Beckman autoSCAN-4 System. Real-time PCR was done using Roche Light Cycler 2.0 to detect the presence of ESBLs; blaSHV, blaTEM and blaCTX-M genes; and MBLs; blaIMP, blaVIM. Strains of P. aeruginosa demonstrated resistance to wide-ranging clinically relevant antibiotics including piperacillin (64.2%), followed by aztreonam (57.8%), cefepime (51.5%), ceftazidime (51.0%), piperacillin/tazobactam (50.5%), and imipenem (46.6%). A total of 75 (36.8%) multidrug-resistant (MDR) strains were observed of the total pool of isolates. The blaTEM, blaSHV and blaCTX-M was detected in 79.3%, 69.5% and 31.7% isolates (n = 82), respectively. The blaIMP was detected in 1.25% while no blaVIM was detected in any of the strains tested. The study showed a high rate of MDR P. aeruginosa in our setting. The vast majority of these resistant strains carried blaTEM and blaSHV genes. Continuous monitoring of antimicrobial resistance and strict compliance towards infection prevention and control practices are the best defence against spread of MDR P. aeruginosa.

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