Detection limits of Legionella pneumophila in environmental samples after co-culture with Acanthamoeba polyphaga

The genus Legionella includes approximately 53 species[1], with Legionella pneumophila being the most common human pathogenic species and causing 90% of all outbreaks of Legionnaires’ disease (LD) in Europe[2]. Legionella species are ubiquitous microorganisms, occurring predominantly in aquatic environments, freshwaters and hot water systems[2], soils, potting soils[3], and composts[4]. Cooling towers, whirlpool spas and shower faucets could be the sources of contaminated bioaerosols, the inhalation of which is generally considered to cause LD outbreaks[2].

A variety of culture methods to detect Legionella species are used to analyze environmental samples[5]. Experience of the laboratory staff in Legionella identification mostly influences and limits the sensitivity of the method[2]. For clinical samples, for instance, the sensitivity and specificity of culture for respiratory secretions are approximately 42.8% and 100%, respectively[5, 6]. The standard detection method (ISO/DIS 11731) for Legionella in environmental samples consists of inoculating samples on selective glycine–vancomycin–polymyxin B–cycloheximide (GVPC) agar or on non-selective buffered-charcoal-yeast-extract (BCYE)[5, 7]. Limitations of the plating method are prolonged incubation periods[5, 8]; bacterial losses due to sample centrifugation or filtration and decontamination steps[8]; presence of contaminating microorganisms that may interfere with Legionella growth, thus decreasing sensitivity; and presence of Legionella cells as viable but not cultivable (VBNC) organisms[9]. The sensitivity of the culture method for samples with low Legionella counts (e.g. bioaerosols and rain) may be enhanced with an efficient enrichment or concentration step; correspondingly, samples with a rich and diverse flora (e.g. soils and composts) should be decontaminated before culture to inhibit growth of concurrent microorganisms[5], because the use of selective media cannot completely inhibit the growth of moulds, bacteria and yeasts[5].

Free-living amoebae (FLA) have long been used to enhance isolation of amoeba-resistant bacteria[10] and already more than 20 years ago Rowbotham proposed to use amoebal enrichment (co-culture) to recover Legionella from natural habitats and clinical specimens[11].

Co-culture aims to enrich the bacteria present in the specimen by exposing them to viable host amoebae[12]. The relative numbers of amoebae used for enrichment is important because too few amoebae may be destroyed before infection[13] and too many may encyst before spread, because L. pneumophila is able to penetrate trophozoites but not cysts[13]. Using co-culture, Legionella bacteria could be easily detected even in samples with high contaminant loads[12]. Macrophages have also been employed for enrichment steps[11].

L. pneumophila serogroup 1 strains are known to grow inside Acanthamoeba (A. castellanii and A. polyphaga) and Naegleria[14]. Non-pneumophila strains, e.g. L. anisa[12], L. drancourtii[15], L. micdadei[16], have also been isolated by co-culture with A. polyphaga.

Because of its sensitivity, the co-culture has the potential of improving bacterial yields in surveys of environmental samples with low Legionella counts or containing contaminating microorganisms. Co-culture has been described as the method of choice for the isolation of Legionella species, but no investigations have so far been carried out to compare the recovery efficiency for Legionella by co-culture with that of conventional culturing methods. In addition, the efficiency of recovery and the detection limit of Legionella after co-culture with A. polyphaga are not known.

In the present work, we utilized L. pneumophila as a model organism to study the interactions with A. polyphaga which, together with A. castellanii, is one of two FLA frequently used in co-culture experiments. We used trophozoites of the A. polyphaga because this species can be easily infected with L. pneumophila and can be effortlessly grown in vitro[13, 14].

This study aimed to determine the detection limits of co-culture with A. polyphaga compared to conventional culturing methods for L. pneumophila in compost and air samples.

This study demonstrates that the detection limit of co-culture is lower than that of conventional culture and allows detecting Lp1 in compost and air samples when the concentration is as low as 10⁵ cells in 1 m³ air and 10⁶ in 1 g compost samples.

The recovery of Lp1 from the compost by co-culture was significantly higher than with culture alone: the co-culture method showed a 3 logs higher sensitivity, with a detection limit of 10² in 1 g (culture: 10⁵ in 1 g compost) (Figure 1), similarly the recovery of Lp1 from the air (Figure 2) by co-culture was 3 log units higher, with a detection limit of 10³ Lp1 cells in 1 m³ air (culture: 10⁶ cells in 1 m³ air).

Recovery from air and compost samples by conventional culture were approximately one log unit lower, compared to the theoretical recovery by 100% efficiency. By contrast, the recovery by co-culture from both compost and air were at least 2 logs higher compared to the theoretical recovery by 100% efficiency (Figure 1 and Figure 2).

An important limitation of this, as well as of previous, similar studies, is the lack of quantification of the amplification power by amoebae. In fact, only Legionella cells that grow on GVPC agar after interaction with A. polyphaga can be counted. The amount of Legionella cells that are present as free cells in the supernatant and the cells that are not phagocyted by the amoeba cannot be assessed. Entry/uptake of Legionella by the amoebae, the ability of Legionella to replicate within and to escape from the amoebal cytoplasm cannot be reliably quantified using standard methods[20]. We further observed that co-culture needs longer incubation periods than culture. We do not tested the recovery of Legionella from spiked samples without acid treatment, we are aware that this causes a dilution of samples, but for non-sterile compost samples the recovery of Legionella without acid treatment is not possible due to overgrowth of contaminant flora. Nevertheless, our study shows that co-culture, on the average, allows detecting smaller amounts of Legionella cells in a given substrate.

Culture, however, yields apparently a better picture of the biodiversity of Legionella spp. in compost (Table 1); in fact, more species were recovered from each sample, whereas only one or two species per sample were enriched by co-culture (Additional file1). Up to now, in Switzerland and in Europe mainly L. pneumophila was isolated from compost[4, 22], in contrast to Australia and Japan where L. longbeachae was frequently isolated from compost by the conventional culture method[3, 23].

Co-culture allowed enriching Lp1 by up to 6 log units from the starting bacterial cells number; the method is thus potentially useful in environmental monitoring, in particular when low Legionella loads are expected (e.g. bioaerosol, rain and water). The presumptive concentration of Legionella bacteria in the bioaerosols of composting facility is between 0 to 10³Legionella per m³.

The detection of Legionella in environmental samples such as soil and compost is hampered by the presence of other microorganisms (mould and bacteria) that grow on selective media and may interfere with the Legionella growth, leading to an underestimation of the effective number of Legionella present in the sample[4]. PCR allows quantification, but the amplification of DNA of dead cells present in a sample makes the interpretation of results difficult; PCR is not an alternative for a reliable quantification of Legionella in environmental samples because humic acids present in the samples may inhibit the reaction[24, 25]. PCR has also been used to detect Legionella spp. in clinical samples, but sensitivity varies greatly (30-90%) depending on the type of specimen studied. In addition, the design of generic Legionella spp. primers is difficult[26].

Previous studies reported that the use of co-culture has allowed the isolation of L. pneumophila when plating on BCYE agar plates did not yield any colonies; it has also allowed isolation of several fastidious Legionella species from clinical stool[27] and sputum samples[12], as well as from environmental samples such as floating biofilms[28]. Co-culture allows the recovery of VBNC cells[14, 29] or of some Legionella species not growing onto BCYE agar[12], such as Legionella-like amoebal pathogens (LLAP)[30] or L. pneumophila in pulmonary specimens[31]. According to Descours et al. (2012) the amoebic co-culture was effective to isolate Legionella spp. from respiratory samples contaminated with other microorganisms even if the type of sample impacted on the performance of culture and co-culture[31].

The use of co-culture is thus potentially useful to detect Legionella spp. in clinical samples with a low degree of contamination by Legionella spp., but the long incubation period needed is a strong negative aspect of the method.

Further studies are needed to test different amoebal strains susceptibilities to various Legionella species. The detection of Legionella in environmental samples is still commonly carried out by conventional culture, but co-culture should be considered whenever there is a need to detect Legionella or VBNC expected to be present at concentrations below 10⁵ – 10⁶ cells, in particular when working with air samples.