Free-living amoebae: what part do they play in healthcare-associated infections?

Free-living amoebae (FLA) are ubiquitous protozoa that do not require a host organism for survival. They are found in natural environments such as water or soil, and man-made environments including tap water or swimming pools, where they may interact with other micro-organisms, including bacteria, fungi and viruses. FLA can harbour micro-organisms including those found in hospital water systems, offering them protection against hostile conditions, providing a vehicle of dissemination, and enabling them to prepare for subsequent survival in macrophages. The interaction between Legionella pneumophila and FLA has been studied extensively; subsequent investigations have shown that FLA may serve as a reservoir for other bacteria including mycobacteria, Pseudomonas aeruginosa, Acinetobacter baumannii, or even fungi and viruses. Amoebae found in hospital water systems can serve as a reservoir of potential pathogens and thus be indirectly related to healthcare-associated infections.

Anti-Legionella activity of staphylococcal hemolytic peptides.

A collection of various Staphylococci was screened for their anti-Legionella activity. Nine of the tested strains were found to secrete anti-Legionella compounds. The culture supernatants of the strains, described in the literature to produce hemolytic peptides, were successfully submitted to a two step purification process. All the purified compounds, except one, corresponded to previously described hemolytic peptides and were not known for their anti-Legionella activity. By comparison of the minimal inhibitory concentrations, minimal permeabilization concentrations, decrease in the number of cultivable bacteria, hemolytic activity and selectivity, the purified peptides could be separated in two groups. First group, with warnericin RK as a leader, corresponds to the more hemolytic and bactericidal peptides. The peptides of the second group, represented by the PSMα from Staphylococcus epidermidis, appeared bacteriostatic and poorly hemolytic.

A sigma(54)-dependent PTS permease of the mannose family is responsible for sensitivity of Listeria monocytogenes to mesentericin Y105.

Sensitivity of Listeria monocytogenes to the bacteriocin mesentericin Y105 was previously shown to be dependent on the sigma(54) subunit of the RNA polymerase. This points towards expression of particular sigma(54)-dependent genes. The present study describes first, ManR, a new sigma(54)-associated activator, and second, Ell(t)(Man), a new sigma(54)-dependent PTS permease of the mannose family, both involved in sensitivity to mesentericin Y105, since interruption of their corresponding genes led to resistance of L. monocytogenes EGDe. Ell(t)(Man) is likely composed of three subunits encoded by the mpt operon (mptA, mptC and mptD genes). Interruption of either the proximal (mptA) or distal (mptD) gene led to resistance, supporting results obtained in Enterococcus faecalis. Accordingly, such PTS permeases of the mannose family should be involved in sensitivity of different target strains to mesentericin Y105. In L. monocytogenes, expression of the mpt operon is shown to be controlled by sigma(54) and ManR and to be induced by both glucose and mannose. The latter result indicates that these sugars are transported by the Ell(t)(Man) permease. Moreover, these sugars correlatively induce sensitivity of L. monocytogenes to mesentericin Y105, strongly favouring the primary role of Ell(t)(Man). MptD, a membrane subunit of Ell(t)(Man), presents an additional domain compared to most IID(Man) subunits described in data banks. An in-frame deletion of this domain in mptD led to resistance of L. monocytogenes, showing its connection with sensitivity and suggesting that it could be directly involved in the recognition of the target cell by mesentericin Y105. Taken together, the results of this work demonstrate that Ell(t)(Man) is prominent in sensitivity to mesentericin Y105 and could be a receptor for subclass IIa bacteriocins.

The rpoN gene of Enterococcus faecalis directs sensitivity to subclass IIa bacteriocins.

The final sigma 54 factor has been previously described to be involved in Listeria monocytogenes sensitivity to mesentericin Y105, a subclass IIa bacteriocin. Here, we identified the rpoN gene, encoding final sigma 54, of Enterococcus faecalis JH2-2 and showed that its interruption leads to E. faecalis resistance to different subclass IIa bacteriocins. Moreover, this rpoN mutant remained sensitive to nisin, a class I bacteriocin, suggesting that final sigma 54 is especially involved in sensitivity to subclass IIa bacteriocins.

Characterization of the mesB gene and expression of bacteriocins by Leuconostoc mesenteroides Y105.

Leuconostoc mesenteroides Y105, previously described for production of mesentericin Y105, an anti-Listeria bacteriocin, was shown to secrete a second bacteriocin. The latter was purified, and its molecular mass of 3446 Da, obtained by mass spectrometric analysis, indicates that this bacteriocin should be identical to mesenterocin 52B [Revol-Junelles et al., Lett Appl Microbiol 23:120, 1996]. This second bacteriocin produced by L. mesenteroides Y105 was named mesentericin B105. Its structural gene, mesB, was then localized by a reverse genetic approach, cloned, and sequenced. MesB was found on the pHY30 plasmid, next to mesY gene clusters. Curing experiments led to isolation of two L. mesenteroides Y105 derivatives, named L. mesenteroides Y29 and Y30. The latter had lost pHY30 plasmid, encoding bacteriocin determinants, therefore explaining its phenotype (MesY-, MesB-). On the contrary, Y29 derivative still harbors the pHY30 but did not produce any bacteriocin. Thus, its phenotype could likely result from a point mutation within a gene, probably encoding a protein involved in production of both mesentericin Y105 and mesentericin B105.

Characterisation of a new operon encoding a Zur-like protein and an associated ABC zinc permease in Listeria monocytogenes.

Metal ions uptake is mainly studied for iron, as it has often been implicated in bacterial virulence. Although Listeria monocytogenes virulence is expected to be controlled by the iron availability, little is known about such an uptake and its regulation. We describe the analysis of the first operon involved in metal ions uptake in L. monocytogenes. Its three ORFs encode respectively (1) an ABC protein, likely implicated in zinc uptake, (2) a hydrophobic membrane protein, generally associated with ABC proteins and (3) a ferric uptake regulator-like protein, that we named zinc uptake regulator, as it shows strong homologies with the zinc uptake regulator, a regulator of the zinc homeostasis in Bacillus subtilis. The expression of this operon is regulated by the zinc concentration.

The rpoN (sigma54) gene from Listeria monocytogenes is involved in resistance to mesentericin Y105, an antibacterial peptide from Leuconostoc mesenteroides.

To gain insight into the mode of action of mesentericin Y105, a bacteriocin bactericidal agent against Listeria monocytogenes, we undertook to identify the listerial factors mediating this susceptibility by using a genetic approach. Transposon mutants resistant to the bacteriocin were obtained. One of them corresponded to a transposon insertion in a gene (rpoN) encoding a putative protein (447 amino acids) with strong homologies to alternative transcriptional sigma54 factors, including that of Bacillus subtilis (38% identity). Complementation experiments with the wild-type rpoN gene demonstrated that the insertion in rpoN was responsible for the resistance phenotype in L. monocytogenes. Moreover, expression of the L. monocytogenes rpoN gene in an rpoN mutant strain of B. subtilis promoted transcription of a sigma54-dependent operon in the presence of the associated regulator. These results demonstrate that the L. monocytogenes rpoN gene encodes a new sigma54 factor.

Mesentericin Y105 gene clusters in Leuconostoc mesenteroides Y105.

Because of their potential usefulness as natural food preservatives, increased interest has focused on bacteriocins from lactic acid bacteria. Mesentericin Y105 is a small non-lantibiotic bacteriocin (class II) encoded within a 35 kb plasmid from Leuconostoc mesenteroides Y105 and it is active against Listeria monocytogenes. Using reverse genetic methodologies, an 8 kb DraII fragment has been cloned that contains the mesentericin Y105 structural gene, mesY, which encodes a precursor of the bacteriocin with a 24 amino acid N-terminal extension ending with a Gly-Gly motif upstream of the cleavage site, which is typical of class II bacteriocins. Four other putative genes are associated with mesY within two divergent putative operons. In addition to mesY, the first putative operon is predicted to encode a protein, similar to that encoded by ORF2 in the leucocin A operon, whose function remains to be elucidated. The second putative operon contains three ORFs, two of which, mesD and mesE, encode proteins that resemble ATP-dependent transporters and accessory factors, respectively. For three other class II bacteriocin systems (lactococcin A, pediocin PA-1, colicin V), these proteins have been shown to be involved in bacteriocin secretion independently of the general sec-dependent secretion pathway. The last putative gene (mesC) does not resemble any previously characterized gene. Results concerning the heterologous expression of the cloned mesY in Lactobacillus johnsonii NCK64 suggest that the maturation and secretion functions dedicated to lactacin F (another class II bacteriocin) are efficient for mesentericin Y105 as well. This characteristic may be of great interest in the development of industrial fermentation starters producing multiple bactericidal activities.

Membrane permeabilization of Listeria monocytogenes and mitochondria by the bacteriocin mesentericin Y105.

Mesentericin Y105, a bacteriocin produced by a Leuconostoc mesenteroides strain, dissipates the plasma membrane potential of Listeria monocytogenes and inhibits the transport of leucine and glutamic acid. It also induces an efflux of preaccumulated amino acids from cells. In addition, the bacteriocin uncouples mitochondria by increasing state 4 respiration and decreasing state 3 respiration. The bacteriocin inhibits ATP synthase and adenine nucleotide translocase of the organelle while the affinity of ADP for its carrier is not modified. The results suggest that mesentericin Y105 acts by inducing, directly or indirectly, pore formation in the energy-transducing membranes, especially those of its natural target.

Recent advances of flow cytometry in fundamental and applied microbiology.

This review focuses on the recent applications of flow cytometry (FCM) in microbiological research (1987-mid 1992). It tries to give a scope of the important breakthroughs which occurred in this field during this period. The technical difficulties of microorganism analysis by flow cytometry is briefly appraised. The significance and the limits of the different microbial cell parameters attainable by flow analyses are systematically evaluated: light scatter for cell size and structure, fluorescence measurements for quantification of cellular components, microbial antigen detection and cell physiological activity estimation. Emphasis is given on the new technological advances which appeared in the last two years. The second part of the review is devoted to the analysis of the usefulness of flow cytometric approach in the different fields of microbiology: fundamental studies in microbial physiology, differentiation, microbial ecology and aquatic sciences, medical microbiology, parasitology, microbial pharmacology and biotechnology.

Characterization and purification of mesentericin Y105, an anti-Listeria bacteriocin from Leuconostoc mesenteroides.

A Leuconostoc mesenteroides ssp. mesenteroides was isolated from goat's milk on the basis of its ability to inhibit the growth of Listeria monocytogenes. The antimicrobial effect was due to the presence in the culture medium of a compound, named mesentericin Y105, excreted by the Leuconostoc mesenteroides Y105. The compound displayed known features of bacteriocins from lactic acid bacteria. It appeared as a proteinaceous molecule exhibiting a narrow inhibitory spectrum limited to genus Listeria. The apparent relative molecular mass, as indicated by activity detection after SDS-PAGE, was 2.5-3.0 kDa. The bacteriocin was purified to homogeneity by a simple three-step procedure: a crude supernatant obtained from an early-stationary-phase culture in a defined medium was subjected to affinity chromatography on a blue agarose column, followed by ultrafiltration through a 5 kDa cut-off membrane, and finally by reverse-phase HPLC on a C4 column. Microsequencing of the pure bacteriocin and of tryptic fragments showed that mesentericin Y105 is a 36 amino acid polypeptide whose primary structure is close to that of leucocin A-UAL 187, which contains an extra residue at the C-terminus and displays only two differences in the overlapping sequence. However, unlike leucocin A-UAL 187, mesentericin Y105 displayed a bactericidal mode of action.

On-line visualization of the competitive behavior of antagonistic bacteria.

To study the interaction between cocultured Listeria monocytogenes and an antagonistic Leuconostoc strain producing an anti-Listeria bacteriocin, flow cytometry, a technique allowing on-line and real-time analysis, was used along with classical microbiological methods. Culture methods and flow cytometric measurements of the mixed culture over time point to a bactericidal action of the lactic acid-producing bacterial strain against L. monocytogenes cells.