Sandie Dauvergne

Comparison of three methods to study biofilm formation by clinical strains of Escherichia coli.

Biofilm formation seems to be a key factor in many bacterial infections, particularly those involving prosthetic implants or urinary catheters, where Escherichia coli is frequently involved. We have determined the ability to form biofilm in vitro of 34 E. coli isolates by 3 different methods (crystal violet staining, BioFilm Ring Test®, and resazurin assay) and tried to correlate biofilm production with phylogenetic background and with the presence of different genes involved in biofilm synthesis. Only 3 isolates (including positive control E. coli ATCC 25922) were classified as strong biofilm producers (1B1, 1D, and 1B2 = control) by the 3 methods, 2 isolates by 2 different methods, and 5 additional isolates by only 1 method. All isolates possessed the csgA gene belonging to the csgABC operon encoding curli, and its regulator csgD. By contrast, only 76% possessed pgaA gene which is part of the pgaABCD operon encoding a polysaccharide adhesin. Interestingly, one of the strong biofilm producers did not harbor pgaA. In the second part, we have selected 5 specific isolates to study the impact of various experimental conditions on biofilm formation. For all these isolates, biofilm production was decreased in anaerobiosis and increased in LB medium compared with brain heart infusion medium, but at various degrees for the different isolates. These results underline the problems encountered in comparing the different published studies using various methods to study biofilm formation in vitro and the great need of standardization.

Epidemiology of Escherichia coli clinical isolates producing AmpC plasmidic β-lactamase during a 5-year period in a French teaching Hospital ☆

We investigated the prevalence and epidemiology of AmpC plasmidic cephalosporinases in Escherichia coli clinical strains resistant to third-generation cephalosporins during a 5-year period at Nantes University Hospital, France (3100 beds). The prevalence and diversity of plasmidic cephalosporinase did not increase during the study period (0.09% of 25 861 E. coli isolates); only CMY-2 producers were detected (and 1 new variant, with a Y-to-C substitution at position 219). CMY-2-producing strains belonged to the 4 main phylogenetic groups and to 11 different sequence types. Three sequence types included more than 1 isolate (ST156, ST46, and ST354).

Occurrence of ST23 Complex Phylogroup A Escherichia coli Isolates Producing Extended-Spectrum AmpC β-Lactamase in a French Hospital

ABSTRACT Extended-spectrum AmpC β-lactamase (ESAC) Escherichia coli producers were investigated over a 5-year period. Eleven isolates presenting a strong ampC promoter and different strategic AmpC mutations, including two newly described modifications (A292V and an L-A-A insertion at 295), were characterized. All the isolates belonged to phylogenetic group A and to the ST23 complex.

MLST typing of Escherichia coli isolates overproducing AmpC β-lactamase

Escherichia coli is the major commensal aerobic bacterium of the digestive tract of humans and animals, but is also the most frequent human bacterial pathogen. As a consequence, resistance mechanisms carried by this species are of particular concern. One of these mechanisms is the overproduction of the chromosomal AmpC β-lactamase, leading to increased MICs of most β-lactams including third-generation cephalosporins. AmpC overproduction is caused by various mutations in the ampC promoter (–42, –32 and –11) increasing homology with the consensus promoter for RNA polymerase,

Pathogenic potential of Escherichia coli clinical strains from orthopedic implant infections towards human osteoblastic cells

Escherichia coli is one of the first causes of Gram-negative orthopedic implant infections (OII), but little is known about the pathogenicity of this species in such infections that are increasing due to the ageing of the population. We report how this pathogen interacts with human osteoblastic MG-63 cells in vitro, by comparing 20 OII E. coli strains to two Staphylococcus aureus and two Pseudomonas aeruginosa strains. LDH release assay revealed that 6/20 (30%) OII E. coli induced MG-63 cell lysis whereas none of the four control strains was cytotoxic after 4 h of coculture. This high cytotoxicity was associated with hemolytic properties and linked to hlyA gene expression. We further showed by gentamicin protection assay and confocal microscopy that the non-cytotoxic E. coli were not able to invade MG-63 cells unlike S. aureus strains (internalization rate <0.01% for the non-cytotoxic E. coli versus 8.88 ± 2.31% and 4.60 ± 0.42% for both S. aureus). The non-cytotoxic E. coli also demonstrated low adherence rates (<7%), the most adherent E. coli eliciting higher IL-6 and TNF-α mRNA expression in the osteoblastic cells. Either highly cytotoxic or slightly invasive OII E. coli do not show the same infection strategies as S. aureus towards osteoblasts.

Combined use of analytical high-performance liquid chromatography and cell morphology transformation assay to detect new protein phosphatase inhibitors of okadaic acid type

The search for new protein phosphatase inhibitors in shellfish contaminated by toxin-producing dinoflagellates generally relies on preliminary separation techniques followed by biological tests. To detect such substances without purifying them initially, we developed an approach based on a correlation of the results of two different analytical techniques applied to toxic extracts: high-performance liquid chromatography after derivation of the toxins and the cell morphology transformation assay on KB cells. Application of this protocol to stored frozen mussels showed a decrease in okadaic acid concentration during storage, with formation of degradation derivatives, some of which possessed notable protein phosphatase inhibition activity.