A multi-host approach to identify a transposon mutant of Pseudomonas aeruginosa LESB58 lacking full virulence.

OBJECTIVE: Pseudomonas aeruginosa is an opportunistic bacterial pathogen well known to cause chronic lung infections in individuals with cystic fibrosis (CF). Some strains adapted to this particular niche show distinct phenotypes, such as biofilm hyperproduction. It is necessary to study CF clinical P. aeruginosa isolates, such as Liverpool Epidemic Strains (LES), to acquire a better understanding of the key genes essential for in vivo maintenance and the major virulence mechanisms involved in CF lung infections. Previously, a library of 9216 mutants of the LESB58 strain were generated by signature-tagged mutagenesis (STM) and screened in the rat model of chronic lung infection, allowing the identification of 163 STM mutants showing defects in in vivo maintenance. RESULTS: In the present study, these 163 mutants were successively screened in two additional surrogate host models (the amoeba and the fruit fly). The STM PALES_11731 mutant was the unique non-virulent in the three hosts. A competitive index study in rat lungs confirmed that the mutant was 20-fold less virulent than the wild-type strain. This study demonstrated the pertinence to use a multi-host approach to study the genetic determinants of P. aeruginosa strains infecting CF patients.

Assessing Pseudomonas aeruginosa virulence using a nonmammalian host: Dictyostelium discoideum.

Dictyostelium discoideum, a soil amoeba, can be used as an alternative host to study the virulence of various bacterial species, including Pseudomonas aeruginosa. A simple quantitative test based on the ability of D. discoideum to grow on a bacterial lawn has been developed using this amoeba to assay the virulence of P. aeruginosa strains. The assay needs to be customized for the strains to be tested in order to be able to discriminate between virulent and avirulent P. aeruginosa strains. These steps are described in this protocol.

Insertion sequence AS5 (IS AS5 ) is involved in the genomic plasticity of Aeromonas salmonicida.

The genome of the fish pathogen Aeromonas salmonicida subsp salmonicida harbors a large number of insertion sequences (ISs), many of which are located on plasmids. In the present study, we analyzed the small plasmid profile of A. salmonicida strains to identify evidences of plasmid alterations. Ten out of 78 strains analyzed displayed an unconventional plasmid profile. However the HER1104 strain was unique, having a positive PCR signal for pAsal1 plasmid despite not carrying this plasmid. Instead, HER1104 was bearing a plasmid at higher molecular weight than pAsal1. We characterized this new larger plasmid, which we called pAsal1B since it is a derivative of pAsal1 containing one more complete IS (ISAS5) than the parental plasmid. An additional 96 bp relic of ISAS5 was also present in pAsal1B. These results propose that ISAS5 is another active mobile genetic element in A. salmonicida subsp salmonicida and provided further proof of the genomic plasticity of this bacterium.

Lipid composition of multilamellar bodies secreted by Dictyostelium discoideum reveals their amoebal origin.

When they are fed with bacteria, Dictyostelium discoideum amoebae produce and secrete multilamellar bodies (MLBs), which are composed of membranous material. It has been proposed that MLBs are a waste disposal system that allows D. discoideum to eliminate undigested bacterial remains. However, the real function of MLBs remains unknown. Determination of the biochemical composition of MLBs, especially lipids, represents a way to gain information about the role of these structures. To allow these analyses, a protocol involving various centrifugation procedures has been developed to purify secreted MLBs from amoeba-bacterium cocultures. The purity of the MLB preparation was confirmed by transmission electron microscopy and by immunofluorescence using H36, an antibody that binds to MLBs. The lipid and fatty acid compositions of pure MLBs were then analyzed by high-performance thin-layer chromatography (HPTLC) and gas chromatography (GC), respectively, and compared to those of amoebae as well as bacteria used as a food source. While the bacteria were devoid of phosphatidylcholine (PC) and phosphatidylinositol (PI), these two polar lipid species were major classes of lipids in MLBs and amoebae. Similarly, the fatty acid composition of MLBs and amoebae was characterized by the presence of polyunsaturated fatty acids, while cyclic fatty acids were found only in bacteria. These results strongly suggest that the lipids constituting the MLBs originate from the amoebal metabolism rather than from undigested bacterial membranes. This opens the possibility that MLBs, instead of being a waste disposal system, have unsuspected roles in D. discoideum physiology.

Draft genome sequence of the virulent strain 01-B526 of the fish pathogen Aeromonas salmonicida.

Aeromonas salmonicida is an important fish pathogen, mainly of salmonids. This bacterium causes a disease named furunculosis, which is particularly detrimental for the aquaculture industry. Here, we present the draft genome sequence of A. salmonicida 01-B526, a strain isolated from a brook trout that is more virulent than A. salmonicida reference strain A449, for which a genome sequence is available.

Amoeba host model for evaluation of Streptococcus suis virulence.

The Gram-positive bacterium Streptococcus suis is a major swine pathogen worldwide that causes meningitis, septicemia, and endocarditis. In this study, we demonstrate that the amoeba Dictyostelium discoideum can be a relevant alternative system to study the virulence of S. suis.

Alteration of virulence factors and rearrangement of pAsa5 plasmid caused by the growth of Aeromonas salmonicida in stressful conditions.

Aeromonas salmonicida, a fish pathogen, is the causative agent of furunculosis. It was already shown that growing this bacterium in stressful conditions such as temperature above 22°C might lead to virulence attenuation. Unfortunately, many veterinary microbiology services and reference centers still routinely cultivate A. salmonicida at 25°C. Here we tested the presence of virulence factors by growth on specific medium as well as the integrity of the pAsa5 plasmid, which bears an important virulence factor, the type III secretion system (TTSS), by PCR analysis in twenty strains, most of which were grown at 25°C in their laboratory of origin. The analysis revealed that strains, which encountered the more stressful growth conditions displayed the most frequent absence of A-layer protein and secreted proteolytic activity. Moreover, many strains had lost parts of the pAsa5 plasmid in which the TTSS region was almost always affected. To confirm the effect of stressful growth conditions on the plasmid, three strains with an intact pAsa5 were cultured at 25°C for two weeks. A low but significant fraction of the tested colonies displayed pAsa5 rearrangements. The rearrangement always affected the TTSS region and led to a loss of virulence in the Dictyostelium discoideum co-culture assay. These results demonstrate that the instability of pAsa5 did not lead to its complete loss as previously proposed but to a more complex rearrangement phenomenon and emphasizes the necessity to grow A. salmonicida in appropriate conditions to preserve the complete virulence of the bacterium.

Phase variation has a role in Burkholderia ambifaria niche adaptation.

Members of the Burkholderia cepacia complex (Bcc), such as B. ambifaria, are effective biocontrol strains, for instance, as plant growth-promoting bacteria; however, Bcc isolates can also cause severe respiratory infections in people suffering from cystic fibrosis (CF). No distinction is known between isolates from environmental and human origins, suggesting that the natural environment is a potential source of infectious Bcc species. While investigating the presence and role of phase variation in B. ambifaria HSJ1, an isolate recovered from a CF patient, we identified stable variants that arose spontaneously irrespective of the culture conditions. Phenotypic and proteomic approaches revealed that the transition from wild-type to variant types affects the expression of several putative virulence factors. By using four different infection models (Drosophila melanogaster, Galleria mellonella, macrophages and Dictyostelium discoideum), we showed that the wild-type was more virulent than the variant. It may be noted that the variant showed reduced replication in a human monocyte cell line when compared with the wild-type. On the other hand, the variant of isolate HSJ1 was more competitive in colonizing plant roots than the wild-type. Furthermore, we observed that only clinical B. ambifaria isolates generated phase variants, and that these variants showed the same phenotypes as observed with the HSJ1 variant. Finally, we determined that environmental B. ambifaria isolates showed traits that were characteristic of variants derived from clinical isolates. Our study therefore suggest that B. ambifaria uses phase variation to adapt to drastically different environments: the lung of patients with CF or the rhizosphere.

In situ detection of antibiotic-resistance elements in single Bacillus cereus spores.

Rapid detection of Bacillus spores is a challenging task in food and defense industries. In situ labeling of spores would be advantageous for detection by automated systems based on single-cell analysis. Determination of antibiotic-resistance genes in bacterial spores using in situ labeling has never been developed. Most of the in situ detection schemes employ techniques such as fluorescence in situ hybridization (FISH) that target the naturally amplified ribosomal RNA (rRNA). However, the majority of antibiotic-resistance genes has a plasmidic or chromosomal origin and is present in low copy numbers in the cell. The main challenge in the development of low-target in situ detection in spores is the permeabilization procedure and the signal amplification required for detection. This study presents permeabilization and in situ signal amplification protocols, using Bacillus cereus spores as a model, in order to detect antibiotic-resistance genes. The permeabilization protocol was designed based on the different layers of the Bacillus spore. Catalyzed reporter deposition (CARD)-FISH and in situ polymerase chain reaction (PCR) were used as signal amplification techniques. B. cereus was transformed with the high copy number pC194 and low copy number pMTL500Eres plasmids in order to induce resistance to chloramphenicol and erythromycin, respectively. In addition, a rifampicin-resistant B. cereus strain, conferred by a single-nucleotide polymorphism (SNP) in the chromosome, was used. Using CARD-FISH, only the high copy number plasmid pC194 was detected. On the other hand, in situ PCR gave positive results in all tested instances. This study demonstrated that it was feasible to detect antibiotic-resistance genes in Bacillus spores using in situ techniques. In addition, in situ PCR has been shown to be more sensitive and more applicable than CARD-FISH in detecting low copy targets.

Permeabilization and hybridization protocols for rapid detection of Bacillus spores using fluorescence in situ hybridization.

BACKGROUND: Fluorescence in situ hybridization (FISH) is not adapted for the detection of bacterial spores because of their resistance to conventional permeabilization treatments. Since spore-forming bacteria have important ecological, economical, and medical roles, their in situ detection needs to be improved. The aim of this study was to develop rapid and effective protocols to permeabilize Bacillus spores in order to apply the FISH technique. METHODS: Permeabilization protocols were developed for three species of Bacillus spores. Hybridization was performed using universal and specific probes. Surface structural analysis of the permeabilization treatments was performed using scanning electron microscopy. RESULTS: With the proposed protocols, Bacillus spores can be labeled in less than 1 h. The scanning microscopy showed some visible structural differences between the permeabilized spores compared to intact ones. CONCLUSION: For the first time, rapid and effective protocols to detect Bacillus spores by FISH were developed and can be applied to study Bacillus spores using in situ labeling within 1 h. Previously published in situ hybridization protocols have never reached or been close to the currently described rapidity. This work will contribute to the possibility of near real time detection of biological threats that may be present as spores.