Détection des légionelles dans les échantillons environnementaux par méthode culturale : alternative à la norme AFNOR NF T90-431

Detection of Legionella sp. in environnemental samples using plate culture method : alternative to AFNOR NF T90-431

Centre d’Analyses Environnementales, 1, place de Turenne, 94417 St Maurice Cedex

Résumé

Actuellement la recherche et le dénombrement de Legionella sp. et L. pneumophila dans les eaux s’effectuent selon la norme AFNOR NF T90-431 (2003). Par défaut, cette méthode est également appliquée à d’autres échantillons environnementaux liquides. Cependant 5,3 à 14,2 % des échantillons d’eau de tours aéroréfrigérantes et 6 à 40 % des échantillons issus de stations d’épuration ne peuvent être analysés par cette méthode car les milieux de croissance sont envahis par une flore bactérienne et/ou fongique qui masque ou inhibe la croissance des légionelles. Afin d’améliorer la détection de Legionella sp. et L. pneumophila dans les matrices complexes, nous avons notamment évalué plusieurs traitements de dispersion et de décontamination de l’échantillon, ce qui nous permet de proposer finalement une alternative à la méthode AFNOR NF T90-431 (2003). Cette méthode alternative repose sur une dispersion de l’échantillon à l’aide d’un Pulsifier^® suivie d’un traitement de décontamination réalisé avant l’ensemencement de l’échantillon sur géloses sélectives. Le protocole mis en place permet d’améliorer la détection des légionelles dans des échantillons issus de stations d’épuration. Il a ainsi permis de mettre en évidence la présence de légionelles dans 64,3 % des échantillons analysés contre seulement 28,6 % par la méthode AFNOR NF T90-431 (2003).

Abstract

Legionella species are ubiquitous gram-negative bacteria found in environmental waters and some species are pathogenic to humans causing Legionnaires’ disease or Pontiac fever. The mode of transmission for these diseases is the inhalation of aerosols containing Legionella sp. and especially L. pneumophila. Several outbreaks of legionellosis associated with aerosols generated from contaminated water by air conditioners, cooling towers and shower heads have been described around the world. Palmer et al. (1993) and Roll and Fujioka (1995) have shown that sewage treatment plants may provide an environmental reservoir for Legionella sp. In waste water treatment plant, some process may involve aerosolization and exposure of people to Legionella sp. potentially present in sewage or sludge, but few information is available on the occurrence of this bacteria in this type of sample. The conventional plate culture method described in the standard AFNOR NF T90431 (2003) is a very important technique for the detection of Legionella sp. because it is the currently used standard method and it provides strains for epidemiological studies. At the moment, the standard AFNOR NF T90-431 (2003) concerning the detection and enumeration of Legionella sp. and L. pneumophila on waters is also used to detect these bacteria on sewage and sludge. Several authors have reported the poor accuracy of this plate count method for some water samples due to the overgrowth of non-target microorganisms and this phenomenon is more important for complex matrices containing a high level of background flora. The accurate detection of Legionella sp. and L. pneumophila is made impossible for 6 to 40 % of samples from waste water treatment plants. In order to improve the cultural detection of Legionella sp. and L. pneumophila in such matrices, several ways have been studied and the investigations conducted were divided into four main parts. The first phase was a comparison of dispersion treatment. The second phase deals with reduction of the overgrowth by using heat and acid treatment. The third phase consists in increasing the selectivity of GVPC agar by addition of antibacterial or antifungal agents. Finally the protocol proposed was compared with the standard AFNOR NF T90-431 (2003). Different types of dispersion treatment have been compared including homogenization using sonication, pulsifying directly or after a ten fold dilution in PBS or PBS Tween or PBS Triton X 100. Best results have been obtained by using Pulsifier^®. Four different treatments of decontamination have been compared corresponding to acidification at pH 2.7, heating at 50 °C for 30 min, heating at 50 °C for 30 min followed by acidification at pH 2.7, and acidification at pH 2.7 and heating at 50 °C realized simultaneously for 30 min. Best results have been obtained by using acidification and heating simultaneously. Three modified GVPC agar were compared for their ability to reduce the bacterial overgrowth. Two modified GVPC agar were compared for their ability to reduce the mold overgrowth. According to the results obtained, we propose a protocol for detection and enumeration of Legionella sp. and L. pneumophila which consists of a dispersion of sample using a Pulsifier^® followed by decontamination treatment of samples prior to inoculation on selective agar. Detection of Legionella sp. has been performed on fourteen samples of sewage sludge with the standard AFNOR NF T90-431 (2003) and the protocol proposed. First results obtained on natural activated sludge samples have shown that among 14 samples of sludge, the presence of Legionella sp. have been detected on 28.6% and 64.3% respectively with the standard AFNOR NF T90-431 (2003) and the new method developed. The two methods tested gave results that depended on the concentrations of Legionella sp. With regard to the samples containing low concentrations of Legionella sp. (< 5.10 4 cfu.L -1), the new protocol allowed the highest recovery percentage. With increasing concentrations, the differences between the both techniques became less evident.