Phenotypic Heterogeneity in Attachment of Marine Bacteria toward Antifouling Copolymers Unraveled by AFM.

Up to recent years, bacterial adhesion has mostly been evaluated at the population level. Single cell level has improved in the past few years allowing a better comprehension of the implication of individual behaviors as compared to the one of a whole community. A new approach using atomic force microscopy (AFM) to measure adhesion forces between a live bacterium attached via a silica microbead to the AFM tipless cantilever and the surface has been recently developed. The objectives of this study is to examine the bacterial adhesion to a surface dedicated to ship hulls at the population and the cellular level to understand to what extent these two levels could be correlated. Adhesion of marine bacteria on inert surfaces are poorly studied in particular when substrata are dedicated to ship hulls. Studying these interactions in this context are worthwhile as they may involve different adhesion behaviors, taking place in salty conditions, using different surfaces than the ones usually utilized in the literacy. FRC (fouling release coatings)-SPC (self-polishing coatings) hybrids antifouling coatings have been used as substrata and are of particular interest for designing environmentally friendly surfaces, combining progressive surface erosion and low adhesion properties. In this study, a hybrid coating has been synthetized and used to study the adhesion of three marine bacteria, displaying different surface characteristics, using microplate assays associated with confocal scanning laser microscopy (CSLM) and AFM. This study shows that the bacterial strain that appeared to have the weakest adhesion and biofilm formation abilities when evaluated at the population level using microplates assays and CSLM, displayed stronger adhesion forces on the same surfaces at the single cell level using AFM. In addition, one of the strains tested which presented a strong ability to adhere and to form biofilm at the population level, displayed a heterogeneous phenotypic behavior at the single cell level. Therefore, these results suggest that the evaluation of adhesion at the population level cannot always be correlated with adhesion forces measured individually by AFM and that some bacteria are prone to phenotypic heterogeneity among their population.

Analysis of Streptococcus agalactiae pan-genome for prevalence, diversity and functionality of integrative and conjugative or mobilizable elements integrated in the tRNA(Lys CTT) gene.

Streptococcus agalactiae is the first cause of invasive infections in human neonates and is also a major bovine and fish pathogen. High genomic diversity was observed in this species that hosts numerous mobile genetic elements, in particular elements transferable by conjugation. This works aims to evaluate the contribution of these elements to GBS genome diversity. Focusing on genomic islands integrated in the tRNA(Lys) (CTT) gene, a known hotspot of recombination, an extensive in silico search was performed on the sequenced genome of 303 strains of S. agalactiae isolated from different hosts. In all the isolates (except 9), whatever their origin (human, bovine, camel, dog, gray seal, dolphin, fish species or bullfrog), this locus carries highly diverse genomic islands transferable by conjugation such as integrative and conjugative elements (ICEs), integrative and mobilizable elements (IMEs), CIs-mobilizable elements (CIMEs) or composite elements. Transfer of an ICE from an ST67 bovine strain to a phylogenetically distant ST23 human isolate was obtained experimentally indicating that there was no barrier to ICE transfer between strains from different hosts. Interestingly, a novel family of putative IMEs that site-specifically integrate in the nic site of oriT of ICEs belonging to Tn916/ICESt3 superfamily was detected in silico. These elements carry an antibiotic resistance gene (lsa(C)) already described to confer cross-resistance to lincosamides, streptogramins A and pleuromutilins. Further work is needed to evaluate the impact of these IMEs on the transfer of targeted ICEs and the mobility and the dissemination of these IMEs.

Conjugative transfer and cis-mobilization of a genomic island by an integrative and conjugative element of Streptococcus agalactiae.

Putative integrative and conjugative elements (ICEs), i.e., genomic islands which could excise, self-transfer by conjugation, and integrate into the chromosome of the bacterial host strain, were previously identified by in silico analysis in the sequenced genomes of Streptococcus agalactiae (M. Brochet et al., J. Bacteriol. 190:6913-6917, 2008). We investigated here the mobility of the elements integrated into the 3' end of a tRNA(Lys) gene. Three of the four putative ICEs tested were found to excise but only one (ICE_515_tRNA(Lys)) was found to transfer by conjugation not only to S. agalactiae strains but also to a Streptococcus pyogenes strain. Transfer was observed even if recipient cell already carries a related resident ICE or a genomic island flanked by attL and attR recombination sites but devoid of conjugation or recombination genes (CIs-Mobilizable Element [CIME]). The incoming ICE preferentially integrates into the 3' end of the tRNA(Lys) gene (i.e., the attR site of the resident element), leading to a CIME-ICE structure. Transfer of the whole composite element CIME-ICE was obtained, showing that the CIME is mobilizable in cis by the ICE. Therefore, genomic islands carrying putative virulence genes but lacking the mobility gene can be mobilized by a related ICE after site-specific accretion.

Characterization of a new CAMP factor carried by an integrative and conjugative element in Streptococcus agalactiae and spreading in Streptococci.

Genetic exchanges between Streptococci occur frequently and contribute to their genome diversification. Most of sequenced streptococcal genomes carry multiple mobile genetic elements including Integrative and Conjugative Elements (ICEs) that play a major role in these horizontal gene transfers. In addition to genes involved in their mobility and regulation, ICEs also carry genes that can confer selective advantages to bacteria. Numerous elements have been described in S. agalactiae especially those integrated at the 3' end of a tRNA(Lys) encoding gene. In strain 515 of S. agalactiae, an invasive neonate human pathogen, the ICE (called 515_tRNA(Lys)) is functional and carries different putative virulence genes including one encoding a putative new CAMP factor in addition to the one previously described. This work demonstrated the functionality of this CAMP factor (CAMP factor II) in Lactococcus lactis but also in pathogenic strains of veterinary origin. The search for co-hemolytic factors in a collection of field strains revealed their presence in S. uberis, S. dysgalactiae, but also for the first time in S. equisimilis and S. bovis. Sequencing of these genes revealed the prevalence of a species-specific factor in S. uberis strains (Uberis factor) and the presence of a CAMP factor II encoding gene in S. bovis and S. equisimilis. Furthermore, most of the CAMP factor II positive strains also carried an element integrated in the tRNA(Lys) gene. This work thus describes a CAMP factor that is carried by a mobile genetic element and has spread to different streptococcal species.

Site-specific accretion of an integrative conjugative element together with a related genomic island leads to cis mobilization and gene capture.

Genomic islands, flanked by attachment sites, devoid of conjugation and recombination modules and related to the integrative and conjugative element (ICE) ICESt3, were previously found in Streptococcus thermophilus. Here, we show that ICESt3 transfers to a recipient harbouring a similar engineered genomic island, CIMEL3catR3, and integrates by site-specific recombination into its attachment sites, leading to their accretion. The resulting composite island can excise, showing that ICESt3 mobilizes CIMEL3catR3, in cis. ICESt3, CIMEL3catR3, and the whole composite element can transfer from the strain harbouring the composite structure. The ICESt3 transfer to a recipient bearing CIMEL3catR3, can also lead to retromobilization, i.e. its capture by the donor. This is the first demonstration of specific conjugative mobilization of a genomic island in cis and the first report of ICE-mediated retromobilization. CIMEL3catR3, would be the prototype of a novel class of non-autonomous mobile elements (CIMEs: CIs mobilizable elements), which hijack the recombination and conjugation machinery of related ICEs to excise, transfer and integrate. Few genome analyses have shown that CIMEs could be widespread and have revealed internal repeats that could result from accretions in numerous genomic islands, suggesting that accretion and cis mobilization have a key role in evolution of genomic islands.