Legionella spp. isolation and quantification from greywater.

Legionella, an opportunistic human pathogen whose natural environment is water, is transmitted to humans through inhalation of contaminated aerosols. Legionella has been isolated from a high diversity of water types. Due its importance as a pathogen, two ISO protocols have been developed for its monitoring. However, these two protocols are not suitable for analyzing Legionella in greywater (GW). GW is domestic wastewater excluding the inputs from toilets and kitchen. It can serve as an alternative water source, mainly for toilet flushing and garden irrigation; both producing aerosols that can cause a risk for Legionella infection. Hence, before reuse, GW has to be treated and its quality needs to be monitored. The difficulty of Legionella isolation from GW strives in the very high load of contaminant bacteria. Here we describe a modification of the ISO protocol 11731:1998 that enables the isolation and quantification of Legionella from GW samples. The following modifications were made:•To enable isolation of Legionella from greywater, a pre-filtration step that removes coarse matter is recommended.•Legionella can be isolated after a combined acid-thermic treatment that eliminates the high load of contaminant bacteria in the sample.

Method details

There are several ISO methods for Legionella isolation from water [1], [2]. However, none of them is suitable for Legionella isolation from GW samples due to the fact that GW is contaminated with a very high bacterial load. Thus, almost no data can be found regarding Legionella presence in GW.

Here we describe a modification of the ISO 11731:1998 protocol for Legionella isolation from GW. Our modified protocol allows the isolation of Legionella on GVPC selective Legionella medium (glycine–vancomycin–polymyxin–cycloheximide medium, Beckton Dickinson GmbH, Heidelberg, Germany) without the massive bacterial contamination that develops on the media when ISO 11731:1998 is applied.

Pre-filtration of the greywater sample

Filter a 100 ml GW sample (pre-filtration) to remove coarse matter, using a 100 μm pore size cell strainer (Becton Dickinson, USA) placed in one 50 ml tube (two 50 ml tubes are needed) (Fig. 1).

Fig. 1

(a) A 100 μm pore size cell strainer. (b) Pre-filtration of the greywater samples trough the 100 μm pore size cell strainer.

Filtration of the GW sample

The 100 ml pre-filtered GW sample is filtered again through a 0.2 μm cellulose nitrate filter using a vacuum filtration system attached to a 2511 Dry Vacuum Pump (WELCH, Germany) (Fig. 2).

Fig. 2

(a) A vacuum filtration system set-up: the filtration system comprised a stainless steel filter holder and its funnel attached by a rubber bung to a Buchner flask. The flask is connected by a rubber tube to a 2511 Dry Vacuum Pump (WELCH, Germany). (b) Zoom in on the 0.2 μm cellulose nitrate filter which is placed on the filter holder of the filtration system.

Bacteria resuspension in PBS

After filtration, the filter is placed into 10 ml phosphate buffered saline (PBS, pH 7.4 ± 0.2; NaCl 0.14 M; KCl 2.7 mM; Na2HPO4 10 mM, KH2PO4 1.8 mM) and vortexed for 10 min (Fig. 3).

Fig. 3

(a) The filter is folded before inserting it into the 10 ml PBS solution in a tube. (b) The filter inside the tube. The filter upper part must face the inside of the tube and it should overlap as less as possible. (c) Tubes are vortexed for 10 min using a 10 ml tube adaptor.

Combined acid–thermal treatment

Each sample is then subjected to a combined acid–thermal treatment as follows: 1 ml of the sample is centrifuged at 6000 × g for 10 min. For the acid treatment, 0.5 ml of the supernatant is replaced with 0.5 ml of acid buffer (HCl 27 mM; KCl 173 mM, pH 2.2). The sample is then vortexed and immediately subjected to thermal treatment for 30 min at 50 °C.

GVPC Legionella media inoculation

Following the ISO 11731:1998 recommendations, two 0.5 ml sub-samples are plated on a GVPC Legionella selective media immediately after the thermal treatment. The plates are incubated at 37 °C. Presumptive Legionella colonies are counted after 7 and 15 days of incubation.

Legionella identification

The presumptive Legionella colonies are then identified using a Legionella latex test (Oxoid, Basingstoke, UK). This test allows separate identification of Legionella pneumophila serogroup 1 and serogroups (2–14) and the detection of seven other Legionella species (L. longbeachae serogroups 1 and 2, L. bozemanii serogroup 1, L. dumoffii, L. gormanii, L. jordanis, L. micdadei and L. anisa).

Method validation: efficiency of the Legionella isolation protocol and limit of detection (LOD)

We used the method described above to successfully isolate and quantify Legionella along a one year GW monitoring campaign. The results of this study have been already published [3]. Briefly, a total of 16 greywater samples were analyzed. Legionella was isolated from 81% of the samples, with a mean of 1.2 × 105 cfu/l. Details about the efficiency and the limit of detection of this method can also be found in the mentioned publication. This method is highly aggressive, so the recovery rates of Legionella were very low (2.5%, SD = 1.5%) and the LOD established from this average recovery rate was 4.0 × 103 cfu/l. Nevertheless, the results were consistent. It should be noted that this modified methods is the only way to isolate Legionella from GW, as using the current ISO protocols does not allow the isolation of this bacteria.

Recommendations

This method is highly aggressive for the sampled bacteria, including Legionella. For that reason, the LOD of the method is high and the efficiency of Legionella isolation is low. We recommend using this method only with problematic samples in which Legionella can’t be isolated using the methods described in the ISO protocols 11731:1998 and 11731-2:2004 [1], [2] due to massive contamination with other bacterial species.