Jacques Frère

VBNC Legionella pneumophila cells are still able to produce virulence proteins

Legionella pneumophila is the agent responsible for legionellosis. Numerous bacteria, including L. pneumophila, can enter into a viable but not culturable (VBNC) state under unfavorable environmental conditions. In this state, cells are unable to form colonies on standard medium but are still alive. Here we show that VBNC L. pneumophila cells, obtained by monochloramine treatment, were still able to synthesize proteins, some of which are involved in virulence. Protein synthesis was measured using (35)S-labeling and the proteomes of VBNC and culturable cells then compared. This analysis allowed the identification of nine proteins that were accumulated in the VBNC state. Among them, four were involved in virulence, i.e., the macrophage infectivity potentiator protein, the hypothetical protein lpl2247, the ClpP protease proteolytic subunit and the 27 kDa outer membrane protein. Others, i.e., the enoyl reductase, the electron transfer flavoprotein (alpha and beta subunits), the 50S ribosomal proteins (L1 and L25) are involved in metabolic and energy production pathways. However, resuscitation experiments performed with Acanthamoeba castellanii failed, suggesting that the accumulation of virulence factors by VBNC cells is not sufficient to maintain their virulence.

Passage through Tetrahymena tropicalis enhances the resistance to stress and the infectivity of Legionella pneumophila

Legionella pneumophila is a gram-negative bacterium prevalent in fresh water which accidentally infects humans and is responsible for the disease called legionellosis. Intracellular growth of L. pneumophila in Tetrahymena is inconsistent; in the species Tetrahymena tropicalis stationary-phase forms (SPFs) of L. pneumophila differentiate into mature intracellular forms (MIFs) without apparent bacterial replication and are expelled from the ciliate as pellets containing numerous MIFS. In the present work, we tested the impact of L. pneumophila passage through T. tropicalis. We observed that MIFs released from T. tropicalis are more resistant to various stresses than SPFs. Under our conditions, MIFs harboured a higher gentamicin resistance, maintained even after 3 months as pellets. Long-term survival essays revealed that MIFs survived better in a nutrient-poor environment than SFPs, as a reduction of only about 3 logs was observed after 4 months in the MIF population, whereas no cultivable SPFs were detected after 3 months in the same medium, corresponding to a loss of about 7 logs. We have also observed that MIFs are significantly more infectious in human pneumocyte cells compared with SPFs. These results strongly suggest a potential role of ciliates in increasing the risk of legionellosis.

Heterologous expression of bacteriocins using the mesentericin Y105 dedicated transport system by Leuconostoc mesenteroides

Mesentericin Y105 (MesY105) is a class IIa anti-Listeria bacteriocin, produced by Leuconostoc (Ln.) mesenteroides Y105 and with potential food grade application. This bacterium produces a second bacteriocin, mesentericin B105 (MesB105), that does not belong to the same class. To study secretion of bacteriocins by the use of the MesY105 dedicated transport system (DTS), plasmids were constructed for heterologous expression by Ln. mesenteroides. pFBYC04 (Microbiology 144 (1998) 2845) harbours two divergent operons required for MesY105 secretion, i.e. the mesYI operon, encoding pre-MesY105 and immunity, respectively, and the mesCDE operon for secretion. A pFBYC04 derivative, pDMJF01 was constructed by divergent PCR to remove the mesY gene. Ln. mesenteroides DSM20484(pDMJF01) was unable to produce MesY105. The mesYI operon and mesB, mesH and mesF genes, encoding pre-MesB105, MesB105 immunity and a putative protein with unknown function, respectively, were cloned independently into a compatible pDMJF01 plasmid to produce, respectively, pDMJF:YI and pDMJF:BHF. DSM20484 transformed independently with these plasmids was unable to secrete any bacteriocin. MesY105 and MesB105 secretion was observed for DSM20484(pDMJF01) harbouring both pDMJF:YI and pDMJF:BHF. This indicates that the MesY105 DTS permits the transport of MesB105. MesY105 secretion machinery was used to secrete pediocin PA-1 (PedPA-1) by DSM20484 by an in-frame gene fusion strategy where the gene portions corresponding to the MesY105 leader peptide and the mature PedPA-1 were ligated. Thus, MesY105 secretion machinery appears to be a useful tool for secretion of class II bacteriocins by Leuconostoc.

Effects of Disinfection on Legionella spp., Eukarya, and Biofilms in a Hot Water System

ABSTRACT Legionella species are frequently detected in hot water systems, attached to the surface as a biofilm. In this work, the dynamics of Legionella spp. and diverse bacteria and eukarya associated together in the biofilm, coming from a pilot scale 1 system simulating a real hot water system, were investigated throughout 6 months after two successive heat shock treatments followed by three successive chemical treatments. Community structure was assessed by a fingerprint technique, single-strand conformation polymorphism (SSCP). In addition, the diversity and dynamics of Legionella and eukarya were investigated by small-subunit (SSU) ribosomal cloning and sequencing. Our results showed that pathogenic Legionella species remained after the heat shock and chemical treatments (Legionella pneumophila and Legionella anisa, respectively). The biofilm was not removed, and the bacterial community structure was transitorily affected by the treatments. Moreover, several amoebae had been detected in the biofilm before treatments (Thecamoebae sp., Vannella sp., and Hartmanella vermiformis) and after the first heat shock treatment, but only H. vermiformis remained. However, another protozoan affiliated with Alveolata, which is known as a host cell for Legionella, dominated the eukaryal species after the second heat shock and chemical treatment tests. Therefore, effective Legionella disinfection may be dependent on the elimination of these important microbial components. We suggest that eradicating Legionella in hot water networks requires better study of bacterial and eukaryal species associated with Legionella in biofilms.

Development of a pilot-scale 1 for Legionella elimination in biofilm in hot water network: heat shock treatment evaluation.

Aims:  (i) To develop an analytical tool in order to evaluate the effectiveness of anti‐Legionella treatment in biofilm and (ii) study the fate of Legionella populations in water and biofilm after applying a heat shock treatment.