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Effect of PM10 on H influenzae and S pneumoniae
  1. A Adedeji1,
  2. T M A Weller1,
  3. J G Ayres2
  1. 1Department of Medical Microbiology, City Hospital, Birmingham B18 7QH, UK
  2. 2Department of Environmental and Occupational Medicine, University of Aberdeen, Aberdeen AB25 2ZP, UK; j.g.ayresabdn.ac.uk

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That air pollution, and specifically particles, are harmful to health is well accepted,1 causing direct effects such as lung inflammation resulting in exacerbations of lung and cardiac conditions2,3 and being associated with admissions for pneumonia. In the 1960s Lawther et al showed that ambient particles stimulated the growth of Haemophilus influenzae in vitro,4 suggesting a direct effect of particles on bacteria themselves. However, it is not known whether this remains so for modern ambient particles where the sources are different.

To address this we have assessed the effect of PM10 (particles essentially less than 10 μm in diameter) on the respiratory pathogens commonly associated with acute exacerbations of chronic obstructive pulmonary disease (COPD) and pneumonia. The effect of dilutions of extracts of PM10 on the growth of H influenzae and Streptococcus pneumoniae grown in liquid broth and the effect of PM10 on microbial growth kinetics of S pneumoniae was assessed.

Fresh isolates of H influenzae and S pneumoniae obtained from clinical specimens and the control strains H influenzae NCTC 11931 and S pneumoniae ATCC 49619 were used. Particles were collected on a tapered element oscillating microbalance situated in central Birmingham, representative of an urban background site. To obtain a usable sample the surface of the filter was wetted and rinsed with two sequential aliquots of 0.5 ml saline using a Gilson pipette until visual inspection showed no more particles coming off the filter. The two aliquots were combined and sonicated for 2 minutes to disperse the particles and aggregates. This procedure usually gives a yield of 50–300 μg/ml particles (Donaldson, personal communication). It is not known for certain how these concentrations relate to likely concentrations in the epithelial lining fluid, but this approach has been used in previous in vitro studies of inflammatory responses which have shown pro-inflammatory effects.

In the first experiment a 1:20 dilution of PM10 was made by adding 0.5 ml to 9.5 ml Iso sensitest broth (ISTA; Oxoid Ltd, Basingstoke, UK) supplemented with 5% horse blood and 20 μg/ml NAD. The same volume of normal saline was added to controls. Test and control bottles were inoculated with 0.5 ml of organism suspension at a density of 0.5 Mcfarland. A viable count was performed hourly for 5 hours while incubating at 37°C in 5% CO2 using the Miles and Misra technique.5 In the growth kinetic experiment equal volumes of PM10 solution and ISTA broth (supplemented with 5% lysed horse blood and 20 μg/ml NAD) were added to the first column of a sterile microtitre tray. Serial broth dilutions to a final dilution of 1:64 were performed. Control wells contained only broth and wells for sterility checks contained PM10 alone, broth alone and inoculum alone. Organism suspension, 50 μl S pneumoniae ATCC 49619, was added into each test and control column of the wells and incubated at 37°C in 5% CO2 for 5 hours. The Miles and Misra technique5 was used to estimate the viable count of organism in each well and the differences in log cfu/ml between test and control were plotted against serial dilutions of PM10. This test was repeated five times using the same strain to check for reproducibility.

In the first experiment the number of viable cells increased progressively and in the expected pattern over time (fig 1A and B), whether in the presence or absence of PM10, for both H influenzae and S pneumoniae. In the growth kinetics experiment the only consistent finding was an inhibition of growth at a PM10:broth medium dilution of 1:1 compared with the PM10 free control.

Growth of H influenzae and S pneumoniae is therefore neither inhibited nor promoted by incubation with PM10 at concentrations of diluted particles which are known to be able to exert pro-inflammatory effects in vitro. There was a constant inhibitory effect at a PM10 dilution of 1:1, possibly due to the particles themselves or to dilution of the broth by the added saline. These findings suggest that the association of air pollution with hospital admissions for exacerbations of COPD and for pneumonia is probably not mediated through direct promotion of bacterial growth. If particles alone are responsible for these effects, they are likely to be mediated by particles causing lung inflammation, thus encouraging penetration and growth of bacteria in the respiratory tract. Alternatively, gaseous pollutants may be responsible for the epidemiological findings, either directly or in conjunction with particles. This interactive mechanism is supported by the association of ambient nitrogen dioxide levels with admissions for croup, and is analogous to the potentiation of the airway response to inhaled allergen by both nitrogen dioxide6 and ozone.7 Finally, it is possible that the particles have an effect on bacterial virulence and toxin production rather than growth. This possibility has not been tested here but warrants further study.

Figure 1

Growth curve against time with and without PM10 solution for (A) S pneumoniae ATCC control strain and (B) H influenzae NCTC control strain.

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