Association between smoking and presence of Mycoplasma pneumoniae in circulation leukocytes

a Department of Cardiology, Medical Division, Oslo University Hospital, Norway b Department of Infectious Diseases, Medical Division, Oslo University Hospital, Norway c Department of Internal Medicine II, Medical University Vienna and the Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna, Austria d 3rd Medical Department for Cardiology and Emergency Medicine, Wilhelminenspital, Vienna, Austria


Introduction
Smoking is well-known major risk factor for premature mortality due to cancer, cardiovascular disease and chronic obstructive pulmonary disease [1].It currently causes 5 million deaths per year, and if present trends continue, 10 million smokers per year are projected to die by 2025.Nonetheless, the worldwide production and consumption of cigarettes is increasing [2].
Cigarette smoking also appears to be a major risk factor for respiratory tract and other systemic infections.Both active and passive cigarette smoke exposure increase the risk of infections [1].The morbidity and mortality of infectious diseases due to smoking are not widely appreciated by physicians.
It has been shown that community acquired pneumoniae (CAP) is associated with tobacco consumption [3].However, only limited data are available on the association between smoking and specific infectious agents [3].There is experimental evidence for a facilitating effect of smoking in infection with Streptococcus pneumoniae [4].To the best of our knowledge, there are no data showing an association between smoking and presence of Mycoplasma (M) pneumoniae in circulating leukocytes.
Infections with M pneumoniae have been associated with COPD exacerbation [5].A recent report described a detrimental interaction between M pneumoniae and cigarette smoke in a murine model, accelerating the onset of COPD [6].After infection with M pneumoniae, which usually occurs in the respiratory tract, this infectious agent can persist in circulating leukocytes and it is thereby suspected of fuelling chronic, systemic inflammation [7][8][9].
We raise the question of whether smoking is associated with the presence of M pneumoniae in circulating leukocytes.Furthermore, we studied whether the combination of smoking and M pneumoniae infection affects circulating levels of inflammatory biomarkers previously associated with chronic inflammatory conditions, such as atherosclerosis or COPD [10][11][12].

Subjects and study design
This is a cross sectional study.Subjects with stable coronary artery disease and with no history or current medication for COPD were recruited from the cardiology outpatient clinic.All subjects were of middle European ethnicity.At the time point of inclusion, all patients were asymptomatic for at least three months for their stable coronary artery disease.Patients were not included if there were clinical signs or treatment necessity for heart failure or use of nitrates was reported in the last three months.Patients were recruited between July 2004 and June 2006.Furthermore, we excluded subjects with concurrent malignant disease, severe kidney failure or who had suffered an acute coronary syndrome within the three months before enrolment.
Written informed consent was obtained from all participants.This study was approved by the Medical University of Vienna ethics committee, and complies with the declaration of Helsinki.The clinical data were entered into a computerised database.Baseline characteristics can be seen in table 1.

Definition of co-morbidities
Subjects were classified as having diabetes when under treatment for insulin-dependent or non-insulin-dependent diabetes mellitus or when they had fasting blood glucose levels ≥110 mg/dl or HbA1C levels ≥6%.Subjects were classified as smokers when they were smoking at the time of enrollment.

DNA isolation
All laboratory measurements were performed by investigators blinded to subject characteristics.Venous blood samples were drawn from the antecubital vein into EDTA tubes and DNA was extracted from whole blood using a commercially available DNA extraction kit (MagNA Pure LC DNA Isolation Kit, Roche, Basel, Switzerland) according to the manufacture's instructions.

Polymerase chain reaction
The method used for detection of M pneumoniae is described elsewhere [9].Briefly, the amplification conditions consisted of an initial denaturation step at 95 °C for 5 minutes, 30 cycles of 95 °C for 1 minute, 65 °C for 1 minute and 72 °C for 1 minute, a final elongation step at 72 °C for 7 minutes followed by a cooling phase to 4 °C.The design of the sense (5'-gcc acc ctg ggg ggc agt cag-3'; 2599-2619) and antisense (5'-gag tcg gga ttc ccc gcg gag g-3'; 2807-2729) primers resulted in an amplification product with the size of 209 base pairs.

Enzyme linked immunosorbent assays
Blood samples were drawn after overnight fasting (≥10 h) at inclusion.Serum was separated within one hour and prepared for c-reactive protein (CRP) analysis, performed by use of conventional methods.In addition, serum used for monocyte chemoattractant protein-1 (MCP-1) and complement factor 5a (C5a) was prepared and kept frozen at -70 ˚C until further analyses.Commercial Enzyme Linked Immunosorbent Assays were used for the MCP-1 and C5a analyses (R&D Systems Europe, Abingdon, Oxon, UK).

Statistical analysis
Sample size was calculated with an alpha set at 5% and a beta set at 80%, assuming that 10% difference in presence of M pneumoniae amongst smokers compared to nonsmokers would be of clinical relevance.Association between smoking and M pneumoniae infection was calculated using Fisher's exact test.Odds ratio (OR) for association between smoking and M pneumoniae infection was calculated with logistic regression.OR was adjusted for relevant covariates (age, gender and diabetes) in a multivariate regression model.For comparison of circulating levels of inflammatory markers in subjects with and without exposure to smoking and M pneumoniae infection, respectively, Kruskal-Wallis test or Bonferroni post hoc analysis was used for continuous data and the Pearson Chi squared test for categorical data where appropriate.A significance level of 0.05 was used throughout.The statistical analyses were performed with SPSS software, version 16.0 (SPSS Inc, Chicago).

Results
In accordance with previous findings, the prevalence of latent M pneumoniae infection in our study population was 66% [9,13].We found a significantly higher prevalence of M pneumoniae infections in smokers (83%) compared to non-smokers (59%).As can be seen from table 2, there We divided the study population (median age 64, 78% male) into four different groups, according to their respective exposure to smoking or presence of latent M pneumoniae infection.All inflammatory markers measured (leukocytes, CRP, MCP-1 and C5a) might have a tendency to be higher in individuals exposed to smoking and latent M pneumoniae infection (table 3) compared to unexposed individuals.However, this tendency was not statistically significant, maybe due to lack of power.At an alpha level of 5%, a post hoc power analysis revealed a beta of only 23.2% for a two tailed analysis, when comparing exposed to non-exposed individuals.

Discussion
The main finding of this study is that smoking is strongly associated with presence of M pneumoniae in circulating leukocytes.We did not find an effect on circulating levels of inflammatory cytokines, possibly due to the lack of power for this analysis.We are, therefore, formally not allowed to draw conclusions from this finding.However, this finding can be seen as hypothesis generating.Even though there was no statistically significant systemic inflammatory response, the local inflammation and remodelling in various tissues might still be affected as described previously [6].
To the best of our knowledge, this is the first description of an association between smoking and the presence of M pneumoniae in circulating leukocytes.This association could be relevant in maintaining chronic systemic inflammation by fuelling low grade inflammation in circulating leukocytes through the presence of this pathogen.A recent study showed that tobacco smoke and M pneumoniae increase oxidative stress synergistically in the lungs [14].Oxidative stress is a crucial component in inflammation in various organ systems.Through this interaction, the combination of two potentially pro-inflammatory stimuli, namely the presence of M pneumoniae and smoking, could lead to an increase in inflammation and oxidative stress in target organs of the circulating leukocytes analysed in this study.
One major limitation of this study is the low sample size in the subgroup analysis for the circulating inflammatory markers.This study was performed in a single medical centre.Thus, the external validity of its results is limited accordingly.

Funding / potential competing interests
This work was supported by the Ludwig Boltzmann Cluster for Cardiovascular Research and the Association for the Promotion of Research in Arteriosclerosis, Thrombosis and Vascular Biology.

Table 1
Clinical characteristics of the exposure groups (total n = 122); Median values (interquartile range) or proportions (%) are given.
n.s.-not significant Original article Swiss Med Wkly.2010;140:w13105 Swiss Medical Weekly • PDF of the online version • www.smw.ch

Table 2
Cross tabulation between smoking and presence of M. pneumoniae in circulating leukocytes.

Table 3
Circulating levels of inflammatory biomarkers in groups of differently exposed individuals.Median values (25, 75 percentiles) are given.No significant differences were detected.
Swiss Medical Weekly • PDF of the online version • www.smw.ch