Legionella pneumophila persists within biofilms formed by Klebsiella pneumoniae, Flavobacterium sp., and Pseudomonas fluorescens under dynamic flow conditions

Abstract

Legionella pneumophila, the agent of Legionnaires' disease pneumonia, is transmitted to humans following the inhalation of contaminated water droplets. In aquatic systems, L. pneumophila survives much of time within multi-organismal biofilms. Therefore, we examined the ability of L. pneumophila (clinical isolate 130 b) to persist within biofilms formed by various types of aquatic bacteria, using a bioreactor with flow, steel surfaces, and low-nutrient conditions. L. pneumophila was able to intercalate into and persist within a biofilm formed by Klebsiella pneumoniae, Flavobacterium sp. or Pseudomonas fluorescens. The levels of L. pneumophila within these biofilms were as much as 4 × 10(4) CFU per cm(2) of steel coupon and lasted for at least 12 days. These data document that K. pneumoniae, Flavobacterium sp., and P. fluorescens can promote the presence of L. pneumophila in dynamic biofilms. In contrast to these results, L. pneumophila 130 b did not persist within a biofilm formed by Pseudomonas aeruginosa, confirming that some bacteria are permissive for Legionella colonization whereas others are antagonistic. In addition to colonizing certain mono-species biofilms, L. pneumophila 130 b persisted within a two-species biofilm formed by K. pneumoniae and Flavobacterium sp. Interestingly, the legionellae were also able to colonize a two-species biofilm formed by K. pneumoniae and P. aeruginosa, demonstrating that a species that is permissive for L. pneumophila can override the inhibitory effect(s) of a non-permissive species.

Figure 1. Schematic of the bioreactor system.

(A) Representation of system set-up. Medium was pumped from the media carboy to the reactors by using a peristaltic pump. The bioreactors themselves were in a water bath that was maintained at 30°C by the use of an immersible aquarium heater. The bioreactors were kept spinning at a constant rpm over the course of the experiment. Liquid exited the bioreactors by gravity into a waste carboy. (B) Close-up view of the CDC biofilm reactor. The bioreactor has openings for 8 polypropylene rods. Each rod (four shown here, for clarity) contains three spaces for stainless steel coupons which can be removed to assay for bacterial CFU. At the center of the bioreactor is a stirring baffle that maintains constant shear stress. There is a spout located about 1/3 the length of the vessel from the bottom to allow for the exit of media.

Figure 2. Persistence of L. pneumophila in monospecies biofilms formed by K. pneumoniae, Flavobacterium sp., P. aeruginosa, or P. fluorescens.

A base biofilm of either K. pneumoniae strain DMDS 92-08-28a (□) (A), Flavobacterium sp. strain CDC-65 (Δ) (B), P. aeruginosa ATCC strain 7700 (◊) (C), or P. fluorescens ATCC strain 17569 (Δ) (D) pre-formed on stainless steel coupons was exposed to L. pneumophila strain 130b (▪) on day 3 and flow of 1∶100 R2A began at 1–2 ml/min. Each data point represents the averages and standard deviations of CFU obtained from the coupons within a single rod. The experiments shown are representative of four experiments for (A) and at least two for (B–D). In a repeat of the experiment using P. aeruginosa base biofilms, no legionellae were recovered at the initial sampling point.

Figure 3. Persistence of L. pneumophila in two-species biofilms formed by K. pneumoniae and Flavobacterium sp. or K. pneumoniae and P. aeruginosa.

A base biofilm of either K. pneumoniae strain DMDS 92-08-28a (□) and Flavobacterium sp. strain CDC-65 (◊) (A) or K. pneumoniae strain DMDS 92-08-28a (□) and P. aeruginosa ATCC strain 7700 (◊) (B) pre-formed on stainless steel coupons was inoculated with L. pneumophila strain 130b (▪) on day 3 and flow of 1∶100 R2A began at 1–2 ml/min. Each data point represents the averages and standard deviations of colony counts from a single rod, and the experiments shown here are representative of two experiments.

Figure 4. Persistence of L. pneumophila mutants in a K. pneumoniae biofilm.

A base biofilm of K. pneumoniae strain DMDS 92-08-28a pre-formed on stainless steel coupons was inoculated in (A) with either L. pneumophila wild-type strain 130b (▪), flaA mutant NU347(pMMB2002) (♦), or pilQ mutant NU278(pMMB2002) (▴) and in (B) with either L. pneumophila wild-type strain 130b (▪) or bbcB mutant NU388 (•) on day 3 and flow of 1∶100 R2A began at 1–2 ml/min. Each data point represents the averages and standard deviations of colony counts from a single rod, and the experiments presented are representative of two repeats.