Approaches to analyse dynamic microbial communities such as those seen in cystic fibrosis lung

Abstract

Microbial communities play vital roles in many aspects of our lives, although our understanding of microbial biogeography and community profiles remains unclear. The number of microbes or the diversity of the microbes, even in small environmental niches, is staggering. Current microbiological methods used to analyse these communities are limited, in that many microorganisms cannot be cultured. Even for the isolates that can be cultured, the expense of identifying them definitively is much too high to be practical. Many recent molecular technologies, combined with bioinformatic tools, are raising the bar by improving the sensitivity and reliability of microbial community analysis. These tools and techniques range from those that attempt to understand a microbial community from their length heterogeneity profiles to those that help to identify the strains and species of a random sampling of the microbes in a given sample. These technologies are reviewed here, using the microbial communities present in the lungs of cystic fibrosis patients as a paradigm.

Long-Term Organic Nutrient Management Fosters the Eubacterial Community Diversity in the Indian Semi-arid Alfisol as Revealed by Length Heterogeneity–PCR

“…The hypervariable domains amplified are (i) from the start of V1 to the end of V2 (V1_V2), (ii) V1 alone, and (iii) V3 alone (Doud et al 2009). The V1_V2 region combination is used to assess the V2 region whose length is too short to be analyzed on its own.…”

“…LH-PCR uses the inherent length variations of these hypervariable regions to produce a DNA fingerprint or profile from a metagenomic DNA sample (Suzuki, Rappé, and Giovannoni 1998). This fluorescent-based PCR method is straightforward and, with optimization, highly reproducible (Mills et al 2007;Doud et al 2009Doud et al , 2010. Approaches based on 16S rRNA gene sequences and variable region lengths avoid the limitations of cultivability and have been used in the analyses of soil eubacteriome (Moreno et al 2006;Mills et al 2006;Entry et al 2008).…”

Designing Primers with Higher Taxonomic Distinguishability

“…Inevitably the universal primers have become too specific/biased, i.e., they fail to extract newly added species from the current database. Necessitated by particular projects, a number of taxon-specific primer pairs [11,12] have been developed and efforts have been made to improve complementarity of the majority of universal primers [13][14][15].…”

A Multi-Faceted Diagnostic Approach to Lung Infections in Patients with Cystic Fibrosis

“…Using the PERL script, the fungal ITS sequencing data was automatically compared against GenBank (Benson et al, 2008) The bacterial data were imported into Primer 5 (Primer-E, Ltd, Ivybridge, United Kingdom) and square root transformed to reduce the influence of the more common organisms (Grant & Ogilvie, 2003;Mills et al, 2003). To determine similarity between bacterial communities, a similarity matrix was calculated using the Bray-Curtis similarity index (Bray & Curtis, 1957;Doud et al, 2009;Mills et al, 2006;Moreno et al, 2006;Rees et al, 2004). Principal coordinate analysis (PCO) was performed on the Bray…”

“…The V1 region was amplified using fluorescent primer P1F-6FAM (5' -6FAM -GCG GCG TGC CTA ATA CAT GC -3') and reverse primer P2R (5' -TTC CCC ACG CGT TAC TCA CC -3') (Cocolin et al, 2001;Doud et al, 2009) and extension at 72°C, each for 1 minute and a final elongation at 72°C for 10 minutes.…”