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Orthopaedic-implant infections by Escherichia coli: molecular and phenotypic analysis of the causative strains.

OBJECTIVES Little is known about Escherichia coli Orthopaedic Implant Infections (OII) pathogenesis. Thus, we compared 30 clinical strains isolated in this context with 30 clinical strains of faecal origin, in order to identify phenotypic and genetic features related to E. coli OII. METHODS Phylogenetic analysis and detection of 19 virulence genes were performed by PCR. Ability to form biofilm was studied using the crystal violet reference method and the innovative BioFilm Ring Test(®). RESULTS Most of the OII isolates (56.7%) belonged to the virulence-associated phylogenetic group B2, but did not present a specific set of virulence factors. S fimbriae was the only adhesin significantly associated with OII isolates. Isolates varied greatly in their ability to form biofilm but OII isolates did not produce significantly more biofilm in vitro than isolates of faecal origin, whatever the method used. CONCLUSIONS Neither a specific pathogenic signature nor an increased ability to form biofilm in vitro was detected in E. coli strains isolated from OII. Nevertheless, genetic properties of these isolates could provide a clue to their origin. Hence, we found that virulence factors of uropathogenic strains and urological disorders were frequently detected among our OII cohort.

Nosocomial outbreak of carbapenem-resistant Enterobacter cloacae highlighting the interspecies transferability of the blaOXA-48 gene in the gut flora

Sir, The emergence and dissemination of carbapenemases (KPC, VIM, IMP, NDM or OXA-48) among Enterobacteriaceae is a serious concern worldwide as it raises the problem of the lack of therapeutic options linked to frequent co-resistance. In November 2010, French guidelines were published to control the spread of carbapenemase-producing Enterobacteriaceae from patients repatriated and travellers hospitalized in French hospitals. However, we report the in vivo interspecies transferability of the OXA-48 carbapenemase by the investigation and management of a nosocomial outbreak in France. In April 2011, an elderly patient (Patient A) was transferred from Agadir (Morocco) to the internal medicine unit at Nantes University Hospital, France, for the treatment of a hip prosthetic joint infection. Upon admission, contact precautions were immediately adopted. A rectal swab inoculated on CHROMagarTM KPC medium (CHROMagar, Paris, France) revealed the gastrointestinal carriage of Enterobacter cloacae and Escherichia coli, both resistant to ertapenem and positive for blaOXA-48 by PCR. Therefore, a weekly colonization surveillance was performed on all patients hospitalized in the unit, and led to the discovery of OXA-48-producing E. cloacae in 3/54 patients (B, C and D) without recent history of travel. Furthermore, rectal swabs performed for Patients A and B found two OXA-48-producing Klebsiella pneumoniae isolates (Figure 1). The time between admission to the unit and the first positive culture varied between 3 and 16 days for the three secondary patients. However, Patient D, with a first negative screening, was transferred to the intensive care unit, and was detected as a carrier 3 days after re-admission to the internal medicine unit. We cannot exclude the possibility that this patient was colonized during the first stay in the internal medicine unit. OXA-48-producing E. cloacae isolates were detected intermittently in this patient (Figure 1). None of the four carriers developed infection. Active surveillance was continued until the last colonized patient was discharged. All isolates were resistant to ertapenem (range of MICs, 2 to ≥32 mg/L) and exhibited intermediate susceptibility or susceptibility to imipenem (range of MICs, 0.38–6 mg/L) and meropenem (range of MICs, 0.25–0.5 mg/L) according to the EUCAST guidelines 2011. Molecular testing showed that all E. cloacae isolates harboured the blaCTX-M-15 ESBL gene, while both E. coli and K. pneumoniae isolates were susceptible to third-generation cephalosporins and did not present any of the additional b-lactamases searched for (blaTEM, blaSHV apart from blaSHV-1, and blaCTX-M). The E. coli and K. pneumoniae isolates did not yield subcultures when plated on a CHROMagarTM ESBL medium (CHROMagar, Paris, France). Although other authors reported poor growth of E. coli strains, and underlined difficulties in differentiating colonies of E. cloacae and K. pneumoniae, in our experience the CHROMagarTM KPC medium was useful. The OXA-48 producing E. coli isolate from Patient A yielded a few small pink colonies, whereas the OXA-48producing K. pneumoniae isolate showed better growth, with large navy blue colonies easily distinguishable from the steel blue colonies of the OXA-48-producing E. cloacae isolate. All E. cloacae isolates showed indistinguishable PFGE patterns. According to PFGE and multilocus sequence typing (MLST; http://www.pasteur.fr/recherche/genopole/PF8/mlst/ Kpneumoniae.html) analyses, K. pneumoniae isolates were not clonally related [one new sequence type (ST) and one ST152]. The E. coli isolate belonged to ST38 (MLST, http://mlst.ucc.ie/ mlst/dbs/Ecoli). The blaOXA-48 gene was transferred by conjugation 4 to a rifampicin-resistant E. coli J53-2 from the E. cloacae, K. pneumoniae and E. coli isolates, while transfer of the blaCTX-M-15 gene from the E. cloacae isolates failed. Extraction of plasmids revealed that E. cloacae isolates carried two plasmids (60 and 165 kb), whereas E. coli, both K. pneumoniae isolates and all blaOXA-48-positive transconjugants carried a single plasmid that co-migrated with the 60 kb plasmid of E. cloacae isolates. The blaOXA-48 gene was part of the plasmid-borne Tn1999.2 transposon, since an insertion sequence IS1999 interrupted by an IS1R was detected by PCR mapping upstream of the blaOXA-48 gene. 2 This is the first report of a patient colonized with three enterobacterial isolates (E. cloacae, E. coli and K. pneumoniae) harbouring the blaOXA-48 gene. The emergence of this gene has been linked to the spread of a peculiar Tn1999-type transposon, but also to the dissemination of specific clones. Poirel et al. indicated that the same strain of OXA-48-producing E. coli, belonging to ST38, had been imported from Egypt and Turkey into France. In our study, Patient A carried an ST38-type E. coli, but the strain did not display an ESBL phenotype, as previously described. The discovery of the OXA-48 carbapenemase in several enterobacteria of the index case’s gastrointestinal flora rather suggested the possibility of an in vivo transfer of the OXA-48-encoding plasmid. This was confirmed by the isolation of another OXA-48-producing K. pneumoniae isolate in Patient B. In the gut, selection of resistant strains has been associated with a biological fitness cost and often reflects the impact of antimicrobial selection pressure. Previous exposure to fluoroquinolones or antipseudomonal penicillins has been described as a risk factor for acquisition of Research letters

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.

Detection of clonally related Escherichia coli isolates producing different CMY β-lactamases from a cystic fibrosis patient

OBJECTIVES This study reports details on Escherichia coli isolates recovered from a cystic fibrosis (CF) patient in order to understand how this pathogen adapts to and resists broad-spectrum antipseudomonal therapy in this context. METHODS Five E. coli isolates were obtained from various clinical samples (airways, urine or dialysis catheter) over a 7 month period covering a double-lung transplantation. All isolates were analysed in terms of clonality [enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus sequence typing], virulence profiles (phylogroup and search for 15 virulence genes), growth patterns (morphotype, biofilm-forming ability and growth rate), hypermutability and antimicrobial susceptibility, with molecular characterization of β-lactamases and porins. RESULTS The five isolates shared similar ERIC-PCR profiles and sequence types (ST1193) and exhibited the same virulence profile. The respiratory isolates were strong mutators, exhibited mucoid or small-colony morphotypes, exhibited strong biofilm-forming ability and grew slowly compared with the others. All isolates were highly resistant to ceftazidime. The respiratory isolates showed reduced susceptibility to cefepime and high resistance to aztreonam. The patient had received a 31 day course of ceftazidime/aztreonam until transplantation. All isolates harboured the same wild-type chromosomal AmpC. A CMY-2 enzyme was detected in the non-respiratory isolates. The respiratory isolates harboured L293S and V211A/L293S new CMY-2 variants, which were designated CMY-94 and CMY-95, respectively. OmpF porin loss was observed in the non-respiratory isolates. CONCLUSIONS Our study shows that, similarly to Pseudomonas aeruginosa, E. coli can undergo phenotypic and genomic changes in the CF context. For the first time, we identified an in vivo expanded-spectrum evolution of the CMY-2 β-lactamase, during bacterial persistence in the CF lung.

Subversion of human intestinal mucosa innate immunity by a Crohn's disease-associated E. coli

Adherent-invasive Escherichia coli (AIEC), associated with Crohn’s disease, are likely candidate contributory factors in the disease. However, signaling pathways involved in human intestinal mucosa innate host response to AIEC remain unknown. Here we use a 3D model of human intestinal mucosa explant culture to explore the effects of the AIEC strain LF82 on two innate immunity platforms, i.e., the inflammasome through evaluation of caspase-1 status, and NFκB signaling. We showed that LF82 bacteria enter and survive within a few intestinal epithelial cells and macrophages, without altering the mucosa overall architecture. Although 4-h infection with a Salmonella strain caused crypt disorganization, caspase-1 activation, and mature IL-18 production, LF82 bacteria were unable to activate caspase-1 and induce IL-18 production. In parallel, LF82 bacteria activated NFκB signaling in epithelial cells through IκBα phosphorylation, NFκBp65 nuclear translocation, and TNFα secretion. In addition, NFκB activation was crucial for the maintenance of epithelial homeostasis upon LF82 infection. In conclusion, here we decipher at the whole-mucosa level the mechanisms of the LF82-induced subversion of innate immunity that, by maintaining host cell integrity, ensure intracellular bacteria survival.

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.

Outbreak caused by Proteus mirabilis isolates producing weakly expressed TEM-derived extended-spectrum β-lactamase in spinal cord injury patients with recurrent bacteriuria.

Abstract We performed a retrospective extended-spectrum β-lactamase (ESBL) molecular characterization of Proteus mirabilis isolates recovered from urine of spinal cord injury patients. A incorrectly detected TEM-24-producing clone and a new weakly expressed TEM-derived ESBL were discovered. In such patients, ESBL detection in daily practice should be improved by systematic use of a synergy test in strains of P. mirabilis resistant to penicillins.

Vitek2® system: a reliable tool to detect qnr determinants in Enterobacteriaceae without quinolone resistance-determining region modifications

Qnr determinants have now been found worldwide. In 2008, among 6150 Enterobacteriaceae, 12 isolates belonging to different bacterial species showing an unusual quinolone resistance pattern on Vitek2 system were investigated. Of 12, 11 harbored a qnr gene. Without QRDR modifications, qnr genes can be detected based on a Vitek2 resistance phenotype.