Contamination of the cell sorter fluidics system with the water-borne bacterium Burkholderia cepacia

One of the purposes of flow cytometry (FCM) is to isolate and collect cells of interest from preparations composed of a large number of cell populations. Droplets containing single cells are given a positive or negative charge, depending on whether the cell is fluorescent or not. The droplets are then deflected by an electric field into collecting tubes according to their charge and the collected cells are subjected to a subsequent experiment directly or after additional culture.

For many applications, the collected cell samples should be sterile, and therefore an aseptic sheath flow system is required. Thus, cell sorter must be carefully maintained with regard to sterility for sorting. The ways for maintenance of the sterility include a filter system equipped in the fluid line and a sterilization procedure using a bleach solution and ethanol. The sterilization procedure should be regularly applied to the cell sorter.

As greater number of cell sorters have been employed in research institutes, the number of researchers involved in cell sorter is increasing. It should be noted that some of the users occasionally lack knowledge regarding sterile cell sorting. In fact, in some institutes, including our university, the sterilization procedure is not regularly applied to the cell sorter since the cell sorter is used only for the analytical purpose, e.g., the determination of phenotype of patients' lymphocytes. Under such conditions, we once experienced a severe bacterial contamination in sorted cell samples. In this communication, we report the outline of this event to draw attention to the users in the institutes where the significance of the regular maintenance is not recognized very well.

The genome of influenza C virus consists of seven RNA segments. The packaging mechanism, by which one set of the genome is incorporated into a progeny virus, remains unclear (1). To step forward to the elucidation of the mechanism, we generated influenza C virus-like particles (VLPs) harboring one or two types of artificial gene segments (Fig. 1A). The 11 plasmid DNAs expressing artificial viral RNA genomes (GFP-vRNA and DsRed2-vRNA) and nine viral proteins were transfected into 293T cells as described previously (2). At 48 h posttransfection, the cells were trypsinized and suspended in Opti-MEM (Lifetechnologies, CA) with 1% FCS (1 × 10⁶/ml), and the cell suspension was subjected to FCM (BD FACSAria Cell Sorter, Becton Dickinson, San Jose, CA). As a result, 25.3% of the cells expressed fluorescences (GFP or DsRed2), and among the fluorescence-positive cells, 11.4% expressed both GFP and DsRed2 (data not shown). To elucidate the packaging mechanism, the transfected 293T cells expressing both GFP and DsRed2 were sorted into a sterile tube containing Opti-MEM according to the manufacture's instruction, and then the sorted 293T cells were cultured in a 35-mm Petri dish to generate VLPs. However, the Opti-MEM became cloudy on the next day although penicillin and streptomycin were added to the medium, which forced us to stop culturing and to discontinue the subsequent experiments. Gram staining revealed that the medium was contaminated with gram-negative bacilli, and the bacteria were subsequently identified as glucose-nonfermentative Burkholderia cepacia (data not shown).

Figure 1. A: Schematic diagram of the project. The 11 plasmid DNAs for the expression of artificial viral RNA genomes and viral proteins were transfected into 293T cells. In the transfected 293T cells, the viral RNA genomes (GFP-vRNA and DsRed2-vRNA) were replicated and transcribed by the RNA polymerase complex composed of the PB2, PB1, P3, and NP proteins. At 48 h posttransfection, the transfected cells were subjected to FCM, and the cells expressing both GFP and DsRed2 were sorted into a sterile plastic tube. B: Fluidics system of FACSAria. The fluidics system is composed of sheath flow and sample flow. The sheath fluid in the sheath tank is sent to plenum reservoir and then to the cuvette flow cell. Asterisk indicates the apparatus from which the contaminated bacteria were detected (see text).

To solve the problem, we exchanged all the filters and tubes for the sheath fluid (FACSFlow®), ethanol, bleach solution, and distilled water (d.w.). However, the sheath fluid collected at the sample collection chamber was found to be contaminated two days after exchange. This finding indicated that the fluidics system is contaminated with a considerable amount of bacteria, and therefore, we examined all the materials involved in the fluidics system. As a result, although the sheath fluid in the sheath tank was negative, bacterial contamination was detected in the following samples: the d.w. in the tank container, smear samples from the d.w. filter, and from the plenum reservoir (Fig. 1B). The bacteria B. cepacia was identified in the sample obtained from the d.w. tank.

Based on these observations, we concluded that the source of the contamination is the d.w. in the d.w. tank container, and the contaminated bacteria in the tank were expanded to the whole fluidics system, resulting in the contamination of the sorted samples. Therefore, we exchanged all the materials involved in the fluidics system, e.g., tanks, filters, tubes, and fluids (sheath fluid, ethanol, bleach solution and d.w.). Furthermore, we developed an in-house disinfection protocol in the case of exchanging d.w. in the tank container, which has kept the fluidics system clean as of today.

B. cepacia was first described by Walter Burkholder in 1949 (3). It was originally named Pseudomonas cepacia, and was later changed to its current name. B. cepacia can be commonly found in the environment (e.g. water and soil) and can survive for prolonged periods in moist environments, although it is an important human pathogen that often causes pneumonia in immunocompromised individuals with underlying lung disease (4). It is intrinsically resistant to most antimicrobial or disinfectant agents (5). Considering these characteristics, we proposed the following hypothesis: a small amount of B. cepacia in the environment was initially introduced into the d.w. tank container four months before this event when the FACSAria was newly set in our university, and the contaminated bacteria grew under the moist environment in the tank and then expanded to the filter and tubing systems. This hypothesis is supported by the fact (i) that the smear test for surrounding area of the d.w. supplier in the laboratory was positive for B. cepacia (data not shown), (ii) that the application of sterilization procedure was totally dependent on voluntary work by the users, (iii) that all users involved in the FACSAria during the four months used the machine only for analytical purposes, not for cell sorting, and (iv) that therefore, the sterilization procedure for aseptic sorting had not been applied to the system during the period since the users did not recognize the significance of the regular maintenance to keep the cell sorter sterile. In conclusion, the contamination event was attributed to the users' lack of knowledge for sterile sorting. Thus, we showed the significance of the regular maintenance of cell sorters particularly for beginners and the bacteria commonly present in the environment as a possible contamination source in the present article.