Propuesta de Reforma al artículo 239 del Reglamento a la Ley Orgánica

From the PCR-DGGE bacterial community of all COPD subjects some qualitative observations can be made. Intense banding in the upper section of the gel at the top of the gradient suggests that the lungs of the COPD subjects are heavily colonised with bacteria that have a low GC-ratio genomic content such as H. influenzae, M. catarrhalis, P. aeruginosa, and S. pneumoniae (Fig. 5.1). Indeed, this has previously been shown in patients suffering from acute exacerbations in COPD as these same bacterial species were isolated from positive culture from expectorated sputa in 59 patients (Larsen et al., 2009). However, in this cohort, sampling strategy involved lower airway BAL from the RLL, not expectorated sputum. Additionally, our patient cohort on presentation at clinic was free of the symptoms associated with those of acute exacerbations.

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It has been hypothesised that initial colonisation of pathogenic organisms in patients with COPD in the lower airways elicits an immune response to the pathogen in the host whilst inducing an acute exacerbation. However, acquisition of the same bacterial strain at a later encounter induces a less frequent and severe exacerbation strongly indicating that despite the impairment of the lung innate immune defences, there is still an adaptive immune response in play mediating partial protection against respiratory pathogens (Sethi et al., 2004). A study recently published, described that the lower airways in healthy individuals, i.e., control subjects in their patient cohorts respectively, were not sterile, challenging the bronchial tree sterility hypothesis (Hilty et al., 2010) reported by other authors (Stockley, 1998, Wilson et al., 1996). Hilty, et al., (2010) also analysed the microbial communities in 5 COPD patients, in which pathogenic Proteobacteria, particularly Haemophilus spp. were highly prevalent (Hilty et al., 2010). The sampling strategy adopted by Hilty, et al., (2010) utilised disposable sheathed cytology brushes and sampled the left upper and right lower lobes of the subject’s lungs allowing for a more extensive analysis of the lower airways. Our preliminary data from the PCR-DGGE profile (Fig. 5.1) would support these findings of a microbial community which is present in the LRT of COPD individuals, in particular low GC-count bacterial species like the above.

Execution of a PCR-DGGE approach in this patient cohort yielded the detection of 23 putative bacterial taxa across the 11 COPD individuals enrolled into this study, with a maximum of 88 bacterial taxa detected in total (Fig. 5.1). The use of DGGE in ascertaining these bacterial taxa are reputable as no sequencing data was obtained from any of the co- migrated bands against the 16S standard ladder (SL) in each of the COPD subjects profiled. This is against the observed four bacterial species (H. influenzae, M. catarrhalis, S.

pneumoniae, and C. indologenes) isolated from the BAL specimens using conventional

microbiological techniques. However, despite the greater detection of microbial species using DGGE to investigate the bacterial community in our COPD cohort, this is insignificant to our own metagenomic analysis in which 1,799 unique OTUs were generated from 454- pyrosequencing. This is in addition to several recently culture-independent studies investigating the lower airways of COPD patients in which the number of OTUs and sequence reads was far greater than both the PCR-DGGE and conventional microbiology approach presented here. Hilty, et al., (2010) identified ~ 3,000 sequence reads in their study (n = 5), Erb-Downward, et al., (2011) averaged ~12,000 sequences per sample (n = 14), Sze,

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et al., (2012) identified > 1,400 OTUs (n = 8), and finally, Cabrera-Rubio, et al., (2012)

generated ~ 1,033 sequences from the 16S rRNA gene metagenomic analysis corresponding to ~ 500 bacterial species per sample (n = 6).

Comparing the bacterial organisms in the 16S SL against the bacterial taxa present in the DGGE profile (Fig. 5.1) several confounding factors can be demonstrated. In preparing the 16S SL via PCR-DGGE of the bacterial isolates, an apparent observation was the heterogeneous nature of the 16S rRNA gene leading to multiple bands migrating through the denaturing gradient by an individual bacterial isolate. This finding has already been demonstrated, indicating that many bacterial species possess several copy numbers of the 16S rRNA gene which can lead to an overestimation of the bacterial community (Dahllöf et al., 2000, Nübel et al., 1996). Additionally, when using 16S rRNA PCR-DGGE technique for microbial community analysis it has also been shown that amplicons from different bacterial species can migrate at the same rate (Jackson et al., 2000). This co-migration of different bacterial species can pose problems for the excision of bands for Sanger sequencing applications ― i.e., the retrieval of clean DNA sequences from individual bands (Muyzer and Smalla, 1998). No sequencing data was generated from our cohort using PCR-DGGE technique in both the bacterial and fungal communities investigated. Construction of the 16S SL by PCR-DGGE has revealed the above caveats, thus an accurate depiction of the microbial community detected in our COPD cohort is putative at best.

Other drawbacks of using the 16S rRNA PCR-DGGE approach adopted here also need to be taken into consideration. The employment of a ‘semi-nested’ approach for PCR amplification of the V3 region could have led to additional biasing factors in the final DGGE profile produced (Fig. 5.1). Furthermore, the attachment of a GC-clamp to the 5′-end of either the forward or reverse primers tends to lower PCR amplification efficiency in addition to increasing the risk of artefact generation and heteroduplex formation (Ferris and Ward, 1997, Lee et al., 1996, Nocker et al., 2007, Ruano and Kidd, 1992). A critical issue in using PCR- DGGE technique is its resolution, i.e., the maximum number of different DNA fragments in a microbial community that can be separated out in the denaturing gradient (Muyzer and Smalla, 1998). Bacterial populations that constitute ≥ 1 % of the total community can be detected by PCR-DGGE as previously shown (Murray et al., 1996, Muyzer et al., 1993), however, the rDNA amplicons obtained in PCR-DGGE mediated studies only reveals the

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predominant species present in the community (Muyzer and Smalla, 1998), thus the total population analysed is misrepresented or biased. Nevertheless, despite these inherent limitations, DGGE is a useful technique in comparing community structural changes in response to perturbations or to identify differences observed when comparing bacterial communities in disparate micro-habitats (van der Gast et al., 2008). This would have been invaluable if the BAL samples from our COPD cohort were followed longitudinally as opposed to a cross-sectional enrolment. To date, no present study using a culture-independent approach has addressed this issue.

Traditional culture-based techniques using bacterial morphology suffer from several drawbacks such as the inability of selective media to isolate fastidious and anaerobic bacteria (Staley and Konopka, 1985), in addition to the difficulty of differentiating colonial morphology on plates after swarming with the mucoid phenotype of P. aeruginosa (van Belkum et al., 2000). An over-reliance in routine diagnostic microbiology for assessment of the aetiology of microbial infections in COPD is concerning because of the differentiation between colonisation and infection in the host (Lentino and Lucks, 1987). In spite of these limitations, the conventional microbiology data presented here is strongly corroborated by several culture-dependent lower airway investigations in which both stable and exacerbating COPD patients were examined by using quantitative microbial culture methods on BAL samples (Cabello et al., 1997, Soler et al., 1999, Soler et al., 1998). As well as the established microbial agents associated with COPD exacerbations, the identification of C. indologenes in CS#6 is unusual as infection with this organism is rare as it is an environmental microbe (Hsueh et al., 1996). However, in a previous study, C. indologenes has been implicated in causing pneumonia in mechanically ventilated patients in a critical care unit (Bonton et al., 1993), but there was no report of any patients enrolled into this study as being diagnosed with COPD. A recent retrospective study has been published in which Chen, et al., (2012) investigated 125 reported cases of C. indologenes being isolated from clinical samples. From their findings, C. indologenes was isolated in 91 cases of pneumonia, of which 19 patients presented with COPD as an underlying disease (Chen et al., 2012). The role of C.

indologenes in stable and exacerbated COPD patients has not been fully investigated yet.

As a comparison between culture-dependent and –independent methods, a recent study investigated clinically stable CF and COPD patients whose expectorated sputum samples

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were then inoculated onto six different media. Using terminal restriction fragment length polymorphism (T-RFLP) profiling technique Rogers, et al., (2009) also harvested the bacterial growth on these plates for both RNA and DNA extraction, in addition to RNA and DNA being directly extracted from the same sputum sample. The authors concluded that when comparing conventional microbiology and T-RFLP, the former method was shown to be highly selective for the isolation of a small group of recognised respiratory tract pathogens. The use of T-RFLP from both the RNA and DNA directly harvested, in addition to the RNA and DNA extracted from the bacterial growth on diagnostic media, provided a more comprehensive profile of the bacterial species identified confirming that many bacterial taxa are present in sputum samples and that these may be clinically relevant (Rogers et al., 2009b). From the study conducted by Rogers, et al., (2009) and the PCR-DGGE approach we employed against our conventional microbiology data, there is partial corroboration, although between out study and Rogers, et al., (2009) there is a distinct difference in the patient sampling methodologies and a lack of sequencing data. Furthermore, unlike that of Rogers, et

al., (2005) only DNA was extracted from the clinical BAL samples, not RNA. Because of

this factor, the profiling of the bacterial community represented in our COPD cohort does not indicate which bacterial taxa are metabolically active. This limitation was addressed in a previous study using extracted RNA to detect the presence of metabolically active bacteria in CF sputum samples using reverse transcribed 16S rRNA yielding cDNA amplicons which were analysed by reverse transcription T-RFLP (Rogers et al., 2005b).

The identification of microbial communities has been demonstrated for several years in CF using culture-independent methodologies reported in numerous studies (Rogers et al., 2005b, Rogers et al., 2003, Rogers et al., 2004, Rogers et al., 2006, Harris et al., 2007, Bittar et al., 2008) revealing the potential true number of bacterial isolates in CF infected individuals. The preliminary data generated from this small COPD cohort using a 16S rRNA PCR-DGGE methodology suggests that a microbial community in the lower airways is perpetuating host inflammatory responses and provides evidence supporting the vicious circle hypothesis (Murphy and Sethi, 1992). However, further analysis is required to indicate whether the bacterial genera in the lower bronchioles represent a core-microbiota in COPD patients as seen recently in CF (van der Gast et al., 2011). Investigations of this core- microbiota in different respiratory disease states such as COPD, CF and non- CF

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bronchiectasis could prove valuable for additional studies in the lower airways of individuals afflicted with these diseases.