Inflammatory thresholds and the species-specific effects of colonising bacteria in stable chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is characterised by airflow limitation and is associated with persistent airway and systemic inflammation, which increases during episodes of acute deterioration, known as exacerbations [1]-[3]. COPD is currently the third leading cause of death worldwide [4] and exacerbations contribute to the substantial morbidity and mortality, and the considerable health-economic burden associated with this disease [5].

Historically, the tracheobronchial tree and lung parenchyma in healthy, non-smoking individuals were described as sterile, using traditional, culture-based techniques. However, in COPD patients, potentially pathogenic microorganisms (PPMs) are frequently isolated from both sputum and bronchoscopic samples during periods of stability, termed lower airway bacterial colonisation (LABC) [6],[7]. Haemophilus influenzae (HI) is often the most commonly isolated PPM at stable and exacerbation states, with Streptococcus pneumoniae (SP), and Moraxella catarrhalis (MC) also frequently isolated [8],[9]. LABC has a detrimental effect on the natural history of COPD, with increased airway and systemic inflammation [7],[10]-[12], increased exacerbation frequency [6], and an accelerated lung function decline [13].

Previous LABC studies have used traditional, culture-based microbiological identification. However, as only 1% of bacteria can be cultured using traditional methods [14], there has been increasing interest in the use of culture-independent diagnostic techniques. Many of these rely on the analysis of the conserved bacterial 16S-rRNA gene. Studies using such techniques have demonstrated the presence of a wide range of bacterial species in healthy individuals, described as the core microbiome, which may become disrupted in disease states [15]. However, these techniques are expensive and time-consuming to perform and analyse, limiting their use to small sample numbers. The culture-independent quantitative polymerase chain reaction (qPCR) technique targeting commonly isolated PPMs in COPD; HI, SP, and MC, has been shown to be more discriminatory than culture, detecting a higher prevalence of PPMs at both stable and exacerbation states [16]. This technique is relatively inexpensive and can be used to examine much larger sample numbers.

We hypothesised that using this qPCR technique to accurately detect PPMs, airway and systemic inflammation would be related to both their presence and loads, and that a bacterial load threshold for increased inflammation would be observed, leading to worse health status. Furthermore, we hypothesised that as HI tends to persist in the airways, the higher inflammatory response seen in colonised patients would be attributable to HI rather than the other PPMs.

This is the first study to date to examine and validate the relationship between inflammation and airway PPM presence, load and species-effects using the culture-independent qPCR technique in a well-characterised cohort of stable COPD patients. We have demonstrated that the presence of sputum PPMs are associated with increased airway and systemic, as measured by fibrinogen, inflammatory response compared to samples without PPM detection. Increasing total bacterial load was associated with higher levels of airway but not systemic inflammation and importantly, we report an apparent inflammatory threshold. In addition, we have demonstrated a species-specific inflammatory response, with HI presence associated with significantly higher airway inflammatory response for all pathogen loads. However, there was discordance between health status and the airway inflammatory response.

Increasing qPCR total bacterial load was strongly associated with an increase in airway but not systemic inflammation. This was reflected by darker sputum colour which acts as a surrogate for MPO levels [21]. Importantly we have demonstrated an apparent total bacterial load threshold, above which airway inflammation is significantly higher than in samples without PPMs detected, or in those with lower loads, consistent with previous culture-based studies [10]. Detailed microbiomic studies in healthy, non-smoking individuals have demonstrated that a core pulmonary bacterial community exists which may include PPMs at low loads [15], and hence the detection of PPMs alone may not be pathological.

There is a long-standing debate as to the meaning of the term `colonisation’ [22]. This terminology suggests a benign process and one without host-pathogen interactions and consequences, which are key to the definition of infection. The importance of using a bacterial load threshold which is associated with a greater inflammatory response is that this avoids using purely the detection of a PPM to define colonisation, and thus enabling a more accurate definition which reflects the clear inflammatory consequences at higher bacterial loads. In this respect, it may be more appropriate to use the term ‘colonisation’ when PPMs are present, but in low loads and without inflammatory consequence, and `chronic airway infection’ when there are higher pathogen loads associated with a greater inflammatory response. We defined the threshold-effect using previous inflammatory data based on bacterial loads determined by culture [10]. However to date, there have been no direct comparisons of bacterial loads as measured by both culture and culture-independent microbiological techniques, and hence the bacterial load threshold measured by qPCR was an arbitrary measure. In our study, the colonised sample size may be underpowered to study this phenomenon in detail, and in particular with respect to the different PPMs. Therefore, further research is needed to investigate the presence of such a bacterial load threshold, using larger data sets, and different microbiological techniques, and to determine how different bacterial loads may affect this inflammatory threshold.

Systemic inflammation, as measured by fibrinogen but not CRP, was significantly higher in samples with PPMs detected than in those without, although the absolute difference was small. No relationship was observed between either total bacterial load or species-specific loads and systemic inflammation. The majority of previous studies investigating inflammatory response in stable COPD patients have focused on airway rather than systemic inflammation [6],[10],[11],[13]. When systemic inflammation has been measured in patients with PPM detection, the evidence for raised fibrinogen and CRP has been conflicting, and no clear association has been found with bacterial loads [7],[12]. However, systemic inflammation, and in particular raised fibrinogen, has been suggested as a possible link between COPD and associated cardiovascular events [23] and also as a potential biomarker to identify patients with a higher risk of mortality [24]. Therefore, the role of fibrinogen in the pathogenesis and clinical outcomes in COPD patients with bacterial colonisation is important area of research for future studies.

We demonstrated greater airway inflammation for all measured cytokines appears to be attributable to HI rather than other PPMs. Species-specific effects on airway inflammation have been alluded to in an in vitro study of MC [25], and also with HI in culture-based in vivo studies of patients with chronic bronchitis [10] and in stable COPD patients with HI colonisation [12]. However, these studies did not take into account the different pathogen loads. We have demonstrated that individual pathogens and mixed-PPM samples had significantly different loads, and at all bacterial loads, airway CXCL8, IL-1β, and MPO were significantly higher for HI.

HI is often the most prevalent PPM cultured at stable state in COPD patients [6],[11]-[13], and a key mechanism in its pathogenesis is its ability to adhere to the already damaged epithelium of the lower respiratory tract in COPD patients [26], and some studies have demonstrated that HI may reside between the epithelial and subepithelial tissues [27], evading mucosal immunity and worsening the underlying airway inflammation seen in COPD.

There was evidence in this study of discordance between inflammation and health status as measured by the SGRQ and CAT scores, and therefore targeting inflammation alone may not necessarily improve health status. However, a recent longitudinal study by Desai and colleagues demonstrated that daily symptoms, as measured by the Breathlessness, Cough and Sputum Scale (BCSS), were significantly higher during periods of colonisation compared to periods without [28]. PPM isolation in stable COPD has been shown to be associated with an increased exacerbation frequency [6] and faster decline in FEV₁ % predicted [13], both of which contribute significantly to health status [17]. Therefore, the lack of correlation between quality of life and colonisation in our study compared to the study by Desai and colleagues may be explained by the longitudinal nature of their study and different heath status questionnaires used. Further longitudinal studies are warranted to determine whether these important clinical outcomes are also species-specific, and thus whether targeting stable HI isolation by using long-term antibiotics or vaccination would provide clinical benefit.

PPM detection during stable COPD appears to be dynamic, with patients changing their colonisation status and which species, strain and load of PPMs are isolated, likely to result in waxing and waning of airway inflammation [13],[29]-[31]. This study was limited to cross-sectional analysis, with lower airway bacterial colonisation defined as being a single positive sputum sample. The inflammatory responses to bacterial loads as measured by molecular techniques have not been previously reported, but in view of the high diagnostic yield of this qPCR technique compared to culture, further studies exploring this dynamic process are likely to use similar techniques, and therefore the relationship between load and inflammation must first be explored. Unlike in other respiratory diseases, such as cystic fibrosis, where multiple positive sputum samples are required before patients are classified as `colonised’, there has been no such consensus in COPD research studies. Thus, further longitudinal studies must aim to address this key question as to whether single or multiple positive sputum samples define colonisation, so appropriate patients can be targeted for clinical trials.

Although qPCR may detect both viable and non-viable bacteria, a clear relationship between bacterial DNA load and airway inflammation is demonstrated. While this qPCR technique does not detect PPMs other than HI, SP, or MC, the corresponding routine culture data showed that only 2% of sputum samples had evidence of a PPM other than those that are able to be detected by this technique. However, routine bacterial culture only reports PPMs with a bacterial load greater than 10⁵ cfu/ml, and therefore some patients who were determined to be non-colonised, may indeed have PPMs present at stable state, contributing to the total bacterial load. Although total bacterial loads, as measured by quantitative bacterial culture and culture-independent techniques, such as 16S, are able to detect both recognised respiratory pathogens and commensal bacteria, these techniques also have recognised limitations. Quantitative culture has the inherent inaccuracy of visual interpretation of colony counts, and the interaction between non-respiratory pathogens, included in the total bacterial load as measured by 16S, and inflammation is not well characterised. However, despite these limitations, the high diagnostic yield of this qPCR technique highlights the strength of this technique and its potential utility in the clinical practice of microbiological study.

A further limitation in our study is that co-existing bronchiectasis was not assessed by CT scanning, although no patients had evidence of clinical bronchiectasis. Previous studies report up to 50-60% of stable COPD have radiological evidence of bronchiectasis, although these changes were generally mild [32],[33]. Secondary bronchiectasis may be more likely in COPD patients with airway PPM isolation as a result of the vicious cycle of inflammation and infection characteristic of LABC, and thus the availability of CT scans would be unlikely to alter the important findings from this study.

In conclusion, using the high diagnostic yield of qPCR to identify airway PPMs and their loads, we have demonstrated that the airway, but not systemic inflammatory response, appears to have an inflammatory threshold and is significantly higher with HI presence. Further observational and interventional evidence is required to understand the nature and significance of dynamic changes in HI and other PPMs.

RS, GCD and JAW were involved in the conception, hypothesis delineation and design of the study. RS, AJM, ARCP, BSK, DSG and SEB were involved in the acquisition of data. RS, AJM, ARCP, LED, PJB, GCD and JAW were involved in the analysis and interpretation of such information. RS, AJM, ARCP, LED, PJB, GCD and JAW were involved in writing the article. All authors read and approved the final manuscript.