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.

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 beta-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 beta-lactamase in a French hospital.

Extended-spectrum AmpC beta-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.

Klebsiella pneumoniae clinical isolate coproducing SHV-2a, DHA-1, QnrB4, and AAC(6')-Ib-cr determinants in France.

We report on a Klebsiella pneumoniae clinical isolate coproducing bla(DHA-1), bla(SHV-2a), qnrB4, and aac(6')-Ib-cr genes. Molecular analysis demonstrated the presence of this combination on the same large plasmid. Despite a negative result for extended-spectrum beta-lactamase (ESBL) by Vitek2(R) system (bioMérieux, Marcy-l'Etoile, France), an ESBL was detected by a double-disk test. Phenotypic techniques and molecular analysis are key approaches to determine coresistance.

Most Escherichia coli strains overproducing chromosomal AmpC beta-lactamase belong to phylogenetic group A.

OBJECTIVES: To determine the phylogenetic group and the production of different virulence factors (VFs) of a collection of Escherichia coli strains overproducing their chromosomal AmpC cephalosporinase. METHODS: Fifty-five E. coli strains, isolated over a 12 year period, and previously identified as AmpC overproducers by increased MICs of third-generation cephalosporins without extended-spectrum beta-lactamase production (negative double-disc synergy test), were phylogrouped by multiplex PCR. As a comparison, 100 E. coli clinical isolates, susceptible to all beta-lactams, were also tested by the same method. The ampC promoter sequence was determined for all these isolates. ERIC-2 PCR (where ERIC stands for enterobacterial repetitive intergenic consensus) was used to compare the isolates. Search for virulence-associated genes (papG alleles, sfa/foc, hly and iucC) was performed by multiplex PCR for the 55 AmpC overproducers. RESULTS: Most of the AmpC overproducers (47/55) belonged to phylogenetic group A, correlated with a low prevalence of the main VFs in these strains. The - 32, -42 and - 11 mutations, responsible for AmpC overproduction, were usually associated with DNA polymorphisms at positions - 88, - 82, -18, +1 and + 58 in the ampC promoter. In the control susceptible isolates, these polymorphisms were detected in 13 ampC promoters (9 group B1 and 4 group A). These polymorphisms were never associated with the main phylogenetic group B2, representing 66% of the susceptible isolates. CONCLUSIONS: AmpC overproduction was clearly correlated with non-virulent commensal phylogenetic groups A and B1, and absence of the main E. coli VFs. Susceptible isolates harbouring the same sequence polymorphisms as AmpC overproducers also belonged to commensal phylogenetic groups.

Increased resistance to beta-lactams in a Klebsiella pneumoniae strain: role of a deletion downstream of the Pribnow box in the blaSHV-1 promoter.

Two hundred and five isolates of Klebsiella pneumoniae were collected from Nantes University Hospital in 2002. A new 30bp deletion was detected downstream of the -10 box of the SHV-1 promoter in a clinical K. pneumoniae isolate with a high amoxicillin/clavulanic acid minimum inhibitory concentration. Reverse transcription polymerase chain reaction revealed increased transcription of bla(SHV-1) mRNA. All conjugation mating assays failed. This new promoter was cloned upstream of the cat gene of the reporter plasmid pKK232-8 and compared with previously described bla(SHV-1) promoters. The deletion induced a 15-fold increase in promoter strength compared with the usual weak promoter. This study reports a new genetic event that increases bla(SHV-1) chromosomal gene expression, which may be of clinical relevance when associated with porin deficiency.