Polymicrobial Nature of Chronic Diabetic Foot Ulcer Biofilm Infections Determined Using Bacterial Tag Encoded FLX Amplicon Pyrosequencing (bTEFAP)

Abstract

BackgroundDiabetic extremity ulcers are associated with chronic infections. Such ulcer infections are too often followed by amputation because there is little or no understanding of the ecology of such infections or how to control or eliminate this type of chronic infection. A primary impediment to the healing of chronic wounds is biofilm phenotype infections. Diabetic foot ulcers are the most common, disabling, and costly complications of diabetes. Here we seek to derive a better understanding of the polymicrobial nature of chronic diabetic extremity ulcer infections.Methods and FindingsUsing a new bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) approach we have evaluated the bacterial diversity of 40 chronic diabetic foot ulcers from different patients. The most prevalent bacterial genus associated with diabetic chronic wounds was Corynebacterium spp. Findings also show that obligate anaerobes including Bacteroides, Peptoniphilus, Fingoldia, Anaerococcus, and Peptostreptococcus spp. are ubiquitous in diabetic ulcers, comprising a significant portion of the wound biofilm communities. Other major components of the bacterial communities included commonly cultured genera such as Streptococcus, Serratia, Staphylococcus and Enterococcus spp.ConclusionsIn this article, we highlight the patterns of population diversity observed in the samples and introduce preliminary evidence to support the concept of functional equivalent pathogroups (FEP). Here we introduce FEP as consortia of genotypically distinct bacteria that symbiotically produce a pathogenic community. According to this hypothesis, individual members of these communities when they occur alone may not cause disease but when they coaggregate or consort together into a FEP the synergistic effect provides the functional equivalence of well-known pathogens, such as Staphylococcus aureus, giving the biofilm community the factors necessary to maintain chronic biofilm infections. Further work is definitely warranted and needed in order to prove whether the FEPs concept is a viable hypothesis. The findings here also suggest that traditional culturing methods may be extremely biased as a diagnostic tool as they select for easily cultured organisms such as Staphylococcus aureus and against difficult to culture bacteria such as anaerobes. While PCR methods also have bias, further work is now needed in comparing traditional culture results to high-resolution molecular diagnostic methods such as bTEFAP.

Vertical Distribution of Functional Potential and Active Microbial Communities in Meromictic Lake Kivu

“…The specificity of the amplification products was further confirmed by melting curve analyses, and the expected sizes of the amplified fragments were checked on a 1 % agarose gel. Overall, average efficiencies for February (RS) September (DS) 1 6 S Ta g -E n c o d e d F L X -T i t a n i u m A m p l i c o n Pyrosequencing Bacterial and archaeal tag-encoded FLX amplicon pyrosequencing (bTEFAP and aTEFAP, respectively), analyses by means of a Roche 454 FLX instrument with titanium reagents, were performed at the Research and Testing Laboratory (Lubbock, TX, USA) as described previously [37,38]. The PCR primers for FLX amplicon pyrosequencing were chosen to span the variable V1-V3 regions in the 16S rRNA gene: 27 F (5′-GAGTTTGATCNTGGCTCAG-3′) and 519R (5′-GWNTTACNGCGGCKGCTG-3′) for bacteria and V3-V4 regions ARCH 349 F (5′-GYGCASCAGKCGMGAAW-3′) and ARCH 806R (5′-GGACTACVSGGGTATCTAAT-3′) for archaea.…”

Reducing bias in bacterial community analysis of lower respiratory infections

“…Initial generation of the sequencing library involved a one-step PCR of 30 cycles, using a mixture of Hot Start and HotStar high-fidelity taq polymerase, as described previously (Dowd et al, 2008). Tag-encoded FLX amplicon pyrosequencing analyses utilised Roche 454 FLX instrument (Roche, Branford, CT, USA) with Titanium reagents, titanium procedures performed at the Research and Testing Laboratory (Lubbock, TX, USA) using RTL protocols (www.researchand testing.com).…”

“…Sequences with ambiguous base calls and sequences with homopolymers 46 bp were removed. Further, any non-bacterial ribosome sequences and chimeras were removed using Black Box Chimera Check software (B2C2) (Gontcharova et al, 2010), as has been described previously (Dowd et al, 2008). To determine the identity of bacteria in the remaining sequences, sequences were de-noised, assembled into OTU clusters at 97% identity and queried using a distributed NET algorithm that utilises Blastn þ (Kraken BLAST; www.krakenblast.com) against a database of high-quality 16S bacterial sequences.…”

“…To determine the identity of bacteria in the remaining sequences, sequences were de-noised, assembled into OTU clusters at 97% identity and queried using a distributed NET algorithm that utilises Blastn þ (Kraken BLAST; www.krakenblast.com) against a database of high-quality 16S bacterial sequences. Using a NET and C# analysis pipeline, the resulting BLASTn þ outputs were compiled, and data reduction analysis was performed as described previously (Dowd et al, 2008).…”

Diabetic foot ulcers and biofilm formation- The culprits

“…This is significantly different from the results of the majority of studies, in which DFUs are polymicrobial in nature. [13][14][15][16][17][18][19][20][21][22] However, some studies have shown lower than expected rates of polymicrobial infection. [1,20,23] Monomicrobial nature of infection is associated with the duration of the ulcer.…”