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Differential effects of frozen storage on the molecular detection of bacterial taxa that inhabit the nasopharynx.

Kwambana BA, Mohammed NI, Jeffries D, Barer M, Adegbola RA, Antonio M - BMC Clin Pathol (2011)

Bottom Line: There was substantial heterogeneity among subjects with respect to the effect of freezing on the number of operational taxonomic units (OTUs) detected.A strong interaction between sex and the effect of freezing was found, whereby there was no significant change observed for males while the mean number of OTUs significantly declined among female infants following frozen storage.Although frozen storage of biological samples is often necessary for archiving and logistic purposes, the potential effects on the number of taxa (composition) detected in microbial community studies are significant and should not be overlooked.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bacterial Diseases Programme, Medical Research Council Laboratories (UK), Banjul, The Gambia. mantonio@mrc.gm.

ABSTRACT

Background: Frozen storage often precedes metagenomic analysis of biological samples; however, the freezing process can have adverse effects on microbial composition. The effect of freezing on the detection of bacteria inhabiting the infant nasopharynx, a major reservoir of bacterial pathogens, was investigated.

Methods: 16S ribosomal RNA (rRNA) gene-based terminal restriction fragment length polymorphism (T-RFLP) analysis of nasopharyngeal (NP) swabs from twelve Gambian infants was employed. NP swabs were analysed within hours of collection and then after 30 days of storage at -70°C.

Results: There was substantial heterogeneity among subjects with respect to the effect of freezing on the number of operational taxonomic units (OTUs) detected. Nevertheless, the mean number of OTUs decreased after frozen storage and the relative abundance for 72% of the OTUs changed by less than 0.5% after deep frozen storage. There were differences in the odds of detection and relative abundance of OTUs matched with Moraxella sp., Haemophilus sp./Burkholderia sp., and Pseudomonas sp. A strong interaction between sex and the effect of freezing was found, whereby there was no significant change observed for males while the mean number of OTUs significantly declined among female infants following frozen storage.

Conclusions: Although frozen storage of biological samples is often necessary for archiving and logistic purposes, the potential effects on the number of taxa (composition) detected in microbial community studies are significant and should not be overlooked. Moreover, genetic factors such as sex may influence the integrity of nucleic acids during the freezing process.

No MeSH data available.


Related in: MedlinePlus

Bar Graph showing the relative distribution of the bacterial OTUs detected before and after frozen storage of NP swabs at -70°C amongst male and female infants by 16S rRNA-based T-RFLP. Partial 16S rRNA gene sequences from infant nasopharyngeal clone libraries were BLASTED to identify the microbes (>97% sequence similarity) and in silico T-RFLP analysis was used to match them to the OTUs.
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Figure 3: Bar Graph showing the relative distribution of the bacterial OTUs detected before and after frozen storage of NP swabs at -70°C amongst male and female infants by 16S rRNA-based T-RFLP. Partial 16S rRNA gene sequences from infant nasopharyngeal clone libraries were BLASTED to identify the microbes (>97% sequence similarity) and in silico T-RFLP analysis was used to match them to the OTUs.

Mentions: The effect of freezing on the relative abundance of OTUs was investigated. For more than 70% of the OTUs, there was less than a 0.5% change in relative abundance after freezing, see Figure 2. 28% of the OTUs had 0.6% to 18.6% shifts in relative abundance post frozen storage, with half showing an increase and the other half showing a decrease in relative abundance. The relative distributions of OTUs were compared for male and female infants as well as before and after freezing, see Figure 3. The relative distributions of bacterial taxa were comparable between male and female infants as well as before and after freezing. The relative proportions of some major taxonomic groups including Haemophilus sp., Staphylococcus sp. Moraxella sp., and Firmicutes were comparable before and after freezing for both sexes, see Figure 3. However, the relative proportions of some OTUs including the OTU 392 bp, the 309 bp OTU (Pseudomonas sp), the 216 bp OTU (Rothia sp), the 278 bp OTU (Acinetobacter sp.) and OTUs with relative abundance less than 1% showed some change before and after freezing amongst the infants.


Differential effects of frozen storage on the molecular detection of bacterial taxa that inhabit the nasopharynx.

Kwambana BA, Mohammed NI, Jeffries D, Barer M, Adegbola RA, Antonio M - BMC Clin Pathol (2011)

Bar Graph showing the relative distribution of the bacterial OTUs detected before and after frozen storage of NP swabs at -70°C amongst male and female infants by 16S rRNA-based T-RFLP. Partial 16S rRNA gene sequences from infant nasopharyngeal clone libraries were BLASTED to identify the microbes (>97% sequence similarity) and in silico T-RFLP analysis was used to match them to the OTUs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037839&req=5

Figure 3: Bar Graph showing the relative distribution of the bacterial OTUs detected before and after frozen storage of NP swabs at -70°C amongst male and female infants by 16S rRNA-based T-RFLP. Partial 16S rRNA gene sequences from infant nasopharyngeal clone libraries were BLASTED to identify the microbes (>97% sequence similarity) and in silico T-RFLP analysis was used to match them to the OTUs.
Mentions: The effect of freezing on the relative abundance of OTUs was investigated. For more than 70% of the OTUs, there was less than a 0.5% change in relative abundance after freezing, see Figure 2. 28% of the OTUs had 0.6% to 18.6% shifts in relative abundance post frozen storage, with half showing an increase and the other half showing a decrease in relative abundance. The relative distributions of OTUs were compared for male and female infants as well as before and after freezing, see Figure 3. The relative distributions of bacterial taxa were comparable between male and female infants as well as before and after freezing. The relative proportions of some major taxonomic groups including Haemophilus sp., Staphylococcus sp. Moraxella sp., and Firmicutes were comparable before and after freezing for both sexes, see Figure 3. However, the relative proportions of some OTUs including the OTU 392 bp, the 309 bp OTU (Pseudomonas sp), the 216 bp OTU (Rothia sp), the 278 bp OTU (Acinetobacter sp.) and OTUs with relative abundance less than 1% showed some change before and after freezing amongst the infants.

Bottom Line: There was substantial heterogeneity among subjects with respect to the effect of freezing on the number of operational taxonomic units (OTUs) detected.A strong interaction between sex and the effect of freezing was found, whereby there was no significant change observed for males while the mean number of OTUs significantly declined among female infants following frozen storage.Although frozen storage of biological samples is often necessary for archiving and logistic purposes, the potential effects on the number of taxa (composition) detected in microbial community studies are significant and should not be overlooked.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bacterial Diseases Programme, Medical Research Council Laboratories (UK), Banjul, The Gambia. mantonio@mrc.gm.

ABSTRACT

Background: Frozen storage often precedes metagenomic analysis of biological samples; however, the freezing process can have adverse effects on microbial composition. The effect of freezing on the detection of bacteria inhabiting the infant nasopharynx, a major reservoir of bacterial pathogens, was investigated.

Methods: 16S ribosomal RNA (rRNA) gene-based terminal restriction fragment length polymorphism (T-RFLP) analysis of nasopharyngeal (NP) swabs from twelve Gambian infants was employed. NP swabs were analysed within hours of collection and then after 30 days of storage at -70°C.

Results: There was substantial heterogeneity among subjects with respect to the effect of freezing on the number of operational taxonomic units (OTUs) detected. Nevertheless, the mean number of OTUs decreased after frozen storage and the relative abundance for 72% of the OTUs changed by less than 0.5% after deep frozen storage. There were differences in the odds of detection and relative abundance of OTUs matched with Moraxella sp., Haemophilus sp./Burkholderia sp., and Pseudomonas sp. A strong interaction between sex and the effect of freezing was found, whereby there was no significant change observed for males while the mean number of OTUs significantly declined among female infants following frozen storage.

Conclusions: Although frozen storage of biological samples is often necessary for archiving and logistic purposes, the potential effects on the number of taxa (composition) detected in microbial community studies are significant and should not be overlooked. Moreover, genetic factors such as sex may influence the integrity of nucleic acids during the freezing process.

No MeSH data available.


Related in: MedlinePlus