David Lebeaux

From in vitro to in vivo Models of Bacterial Biofilm-Related Infections

The influence of microorganisms growing as sessile communities in a large number of human infections has been extensively studied and recognized for 30–40 years, therefore warranting intense scientific and medical research. Nonetheless, mimicking the biofilm-life style of bacteria and biofilm-related infections has been an arduous task. Models used to study biofilms range from simple in vitro to complex in vivo models of tissues or device-related infections. These different models have progressively contributed to the current knowledge of biofilm physiology within the host context. While far from a complete understanding of the multiple elements controlling the dynamic interactions between the host and biofilms, we are nowadays witnessing the emergence of promising preventive or curative strategies to fight biofilm-related infections. This review undertakes a comprehensive analysis of the literature from a historic perspective commenting on the contribution of the different models and discussing future venues and new approaches that can be merged with more traditional techniques in order to model biofilm-infections and efficiently fight them.

Starvation, Together with the SOS Response, Mediates High Biofilm-Specific Tolerance to the Fluoroquinolone Ofloxacin

High levels of antibiotic tolerance are a hallmark of bacterial biofilms. In contrast to well-characterized inherited antibiotic resistance, molecular mechanisms leading to reversible and transient antibiotic tolerance displayed by biofilm bacteria are still poorly understood. The physiological heterogeneity of biofilms influences the formation of transient specialized subpopulations that may be more tolerant to antibiotics. In this study, we used random transposon mutagenesis to identify biofilm-specific tolerant mutants normally exhibited by subpopulations located in specialized niches of heterogeneous biofilms. Using Escherichia coli as a model organism, we demonstrated, through identification of amino acid auxotroph mutants, that starved biofilms exhibited significantly greater tolerance towards fluoroquinolone ofloxacin than their planktonic counterparts. We demonstrated that the biofilm-associated tolerance to ofloxacin was fully dependent on a functional SOS response upon starvation to both amino acids and carbon source and partially dependent on the stringent response upon leucine starvation. However, the biofilm-specific ofloxacin increased tolerance did not involve any of the SOS-induced toxin–antitoxin systems previously associated with formation of highly tolerant persisters. We further demonstrated that ofloxacin tolerance was induced as a function of biofilm age, which was dependent on the SOS response. Our results therefore show that the SOS stress response induced in heterogeneous and nutrient-deprived biofilm microenvironments is a molecular mechanism leading to biofilm-specific high tolerance to the fluoroquinolone ofloxacin.

Methicillin-Resistant Coagulase-Negative Staphylococci in the Community: High Homology of SCCmec IVa between Staphylococcus epidermidis and Major Clones of Methicillin-Resistant Staphylococcus aureus

BACKGROUND Data on community spread of methicillin-resistant coagulase-negative staphylococci (MR-CoNS) are scarce. We assessed their potential role as a reservoir of staphylococcal cassette chromosome mec (SCCmec) IVa, the leading SCCmec subtype in community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). METHODS Nasal carriage of MR-CoNS was prospectively investigated in 291 adults at hospital admission. MR-CoNS were characterized by SCCmec typing, long-range polymerase chain reaction (PCR) for SCCmec IV, and multiple-locus variable-number tandem repeat analysis (MLVA) for Staphylococcus epidermidis (MRSE) strains. Three SCCmec IVa elements were fully sequenced. RESULTS The carriage rate of MR-CoNS was 19.2% (25.9% and 16.5% in patients with and patients without previous exposure to the health care system, respectively; P = .09). MR-CoNS strains (n = 83, including 58 MRSE strains with highly heterogeneous MLVA patterns) carried SCCmec type IVa (n = 9, all MRSE), other SCCmec IV subtypes (n = 9, including 7 MRSE), other SCCmec types (n = 15), and nontypeable SCCmec (n = 50). Long-range PCR indicated structural homology between SCCmec IV in MRSE and that in MRSA. Complete sequences of SCCmec IVa from 3 MRSE strains were highly homologous to those available for CA-MRSA, including major clones USA300 and USA400. CONCLUSIONS MR-CoNS are probably disseminated in the community, notably in subjects without previous exposure to the health care system. MRSE, the most prevalent species, may act as a reservoir of SCCmec IVa for CA-MRSA.

Therapeutic Drug Monitoring of Posaconazole: a Monocentric Study with 54 Adults

ABSTRACT Posaconazole is a potent broad-spectrum triazole antifungal. Little is known about the prevalence and risk factors for low plasma posaconazole concentrations (PPCs). We retrospectively reviewed all adult patients whose PPCs were measured after at least 5 days of treatment between April 2006 and July 2008 at the Hôpital Necker Enfants Malades. A low PPC was defined as a concentration lower than 500 ng/ml. Fifty-four patients were included: 36 receiving prophylactic (200 mg three times a day) and 18 receiving curative (400 mg twice a day) posaconazole therapy. The prevalence of low PPCs was 44% (16/36) in the prophylaxis group and 22% (4/18) in the curative-treatment group. In the prophylaxis group, low PPCs tended to be more frequent in cases of digestive disease (62.5% versus 30%; P = 0.051) and were significantly more frequent among patients with diarrhea (71.4% versus 27%; P = 0.009) or mucositis (100% versus 33%; P = 0.004). In the curative-treatment group, low PPCs were significantly more frequent in cases of diarrhea (75% versus 7%; P = 0.018). In the prophylaxis group, the only two patients who subsequently developed invasive fungal infections exhibited low PPCs. The only adverse event was hepatotoxicity for 2/54 patients (3.7%), which was not related to high plasma drug concentrations. In conclusion, low PPC is common, significantly more frequent in cases of diarrhea or mucositis, and potentially associated with subsequent invasive fungal infection. Therapeutic drug monitoring of posaconazole is therefore mandatory for immunosuppressed adults, at least for those with gastrointestinal disorders.

High prevalence of the arginine catabolic mobile element in carriage isolates of methicillin-resistant Staphylococcus epidermidis

BACKGROUND the arginine catabolic mobile element (ACME) associated with staphylococcal cassette chromosome mec (SCCmec) in the USA300 clone of community-acquired methicillin-resistant Staphylococcus aureus enhances its fitness and ability to colonize the host. Staphylococcus epidermidis may act as a reservoir of ACME for S. aureus. We assessed the diffusion of ACME in methicillin-resistant S. epidermidis (MRSE) isolates colonizing outpatients. METHODS seventy-eight MRSE strains isolated in outpatients from five countries were characterized by multilocus sequence typing (MLST) and SCCmec typing and screened for the arcA and opp3AB markers of ACME. ACME-arcA and ACME-opp3AB were sequenced. ACME type I from MRSE and USA300 were compared by long-range PCR (LR-PCR). RESULTS fifty-three (67.9%) MRSE strains carried an ACME element, including 19 (24.4%), 32 (41.0%) and 2 (2.6%) with ACME type I (arcA+/opp3AB+), II (arcA+/opp3AB-) and III (arcA-/opp3AB+), respectively. The prevalence of ACME did not differ between clonal complex 2 (42/60 strains) and other sequence types (11/18 strains, P = 0.7), with MLST data suggesting frequent intraspecies acquisition. ACME-arcA sequences were highly conserved, whereas ACME-opp3AB displayed 11 distinct allotypes. ACME was found in 14/29, 9/11 and 30/37 strains with type IV, type V and non-typeable SCCmec, respectively (P = 0.01). ACME was more frequently associated with ccrC than with ccrAB2 (82.4% versus 60.0%, P = 0.048). LR-PCR indicated structural homologies of ACME I between MRSE and USA300. CONCLUSIONS ACME is widely disseminated in MRSE strains colonizing outpatients and may contribute to their spread in a community environment with low antibiotic exposure, as suggested for USA300.

Full and Broad-Spectrum In Vivo Eradication of Catheter-Associated Biofilms Using Gentamicin-EDTA Antibiotic Lock Therapy

ABSTRACT Biofilms that develop on indwelling devices are a major concern in clinical settings. While removal of colonized devices remains the most frequent strategy for avoiding device-related complications, antibiotic lock therapy constitutes an adjunct therapy for catheter-related infection. However, currently used antibiotic lock solutions are not fully effective against biofilms, thus warranting a search for new antibiotic locks. Metal-binding chelators have emerged as potential adjuvants due to their dual anticoagulant/antibiofilm activities, but studies investigating their efficiency were mainly in vitro or else focused on their effects in prevention of infection. To assess the ability of such chelators to eradicate mature biofilms, we used an in vivo model of a totally implantable venous access port inserted in rats and colonized by either Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, or Pseudomonas aeruginosa. We demonstrate that use of tetrasodium EDTA (30 mg/ml) as a supplement to the gentamicin (5 mg/ml) antibiotic lock solution associated with systemic antibiotics completely eradicated Gram-positive and Gram-negative bacterial biofilms developed in totally implantable venous access ports. Gentamicin-EDTA lock was able to eliminate biofilms with a single instillation, thus reducing length of treatment. Moreover, we show that this combination was effective for immunosuppressed rats. Lastly, we demonstrate that a gentamicin-EDTA lock is able to eradicate the biofilm formed by a gentamicin-resistant strain of methicillin-resistant S. aureus. This in vivo study demonstrates the potential of EDTA as an efficient antibiotic adjuvant to eradicate catheter-associated biofilms of major bacterial pathogens and thus provides a promising new lock solution.

A Rat Model of Central Venous Catheter to Study Establishment of Long-Term Bacterial Biofilm and Related Acute and Chronic Infections

Formation of resilient biofilms on medical devices colonized by pathogenic microorganisms is a major cause of health-care associated infection. While in vitro biofilm analyses led to promising anti-biofilm approaches, little is known about their translation to in vivo situations and on host contribution to the in vivo dynamics of infections on medical devices. Here we have developed an in vivo model of long-term bacterial biofilm infections in a pediatric totally implantable venous access port (TIVAP) surgically placed in adult rats. Using non-invasive and quantitative bioluminescence, we studied TIVAP contamination by clinically relevant pathogens, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis, and we demonstrated that TIVAP bacterial populations display typical biofilm phenotypes. In our study, we showed that immunocompetent rats were able to control the colonization and clear the bloodstream infection except for up to 30% that suffered systemic infection and death whereas none of the immunosuppressed rats survived the infection. Besides, we mimicked some clinically relevant TIVAP associated complications such as port-pocket infection and hematogenous route of colonization. Finally, by assessing an optimized antibiotic lock therapy, we established that our in vivo model enables to assess innovative therapeutic strategies against bacterial biofilm infections.

pH-Mediated Potentiation of Aminoglycosides Kills Bacterial Persisters and Eradicates In Vivo Biofilms

BACKGROUND Limitations in treatment of biofilm-associated bacterial infections are often due to subpopulation of persistent bacteria (persisters) tolerant to high concentrations of antibiotics. Based on the increased aminoglycoside efficiency under alkaline conditions, we studied the combination of gentamicin and the clinically compatible basic amino acid L-arginine against planktonic and biofilm bacteria both in vitro and in vivo. METHODS Using Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli bioluminescent strains, we studied the combination of L-arginine and gentamicin against planktonic persisters through time-kill curves of late stationary-phase cultures. In vitro biofilm tolerance towards gentamicin was assessed using PVC 96 well-plates assays. Efficacy of gentamicin as antibiotic lock treatment (ALT) at 5 mg/mL at different pH was evaluated in vivo using a model of totally implantable venous access port (TIVAP) surgically implanted in rats. RESULTS We demonstrated that a combination of gentamicin and the clinically compatible basic amino acid L-arginine increases in vitro planktonic and biofilm susceptibility to gentamicin, with 99% mortality amongst clinically relevant pathogens, i.e. S. aureus, E. coli and P. aeruginosa persistent bacteria. Moreover, although gentamicin local treatment alone showed poor efficacy in a clinically relevant in vivo model of catheter-related infection, gentamicin supplemented with L-arginine led to complete, long-lasting eradication of S. aureus and E. coli biofilms, when used locally. CONCLUSION Given that intravenous administration of L-arginine to human patients is well tolerated, combined use of aminoglycoside and the non-toxic adjuvant L-arginine as catheter lock solution could constitute a new option for the eradication of pathogenic biofilms.

Clinical outcome after a totally implantable venous access port-related infection in cancer patients: a prospective study and review of the literature.

AbstractMorbidity and mortality after a totally implantable venous access port (TIVAP)-related infection in oncology patients have rarely been studied. We conducted this study to assess the incidence and factors associated with the following outcome endpoints: severe sepsis or septic shock at presentation, cancellation of antineoplastic chemotherapy, and mortality at week 12. We conducted a prospective single-center observational study including all adult patients with solid cancer who experienced a TIVAP-related infection between February 1, 2009, and October 31, 2010. Patients were prospectively followed for 12 weeks. Among 1728 patients receiving antineoplastic chemotherapy during the inclusion time, 72 had an episode of TIVAP-related infection (4.2%) and were included in the study (median age, 60 yr; range, 28–85 yr). The incidence of complications was 18% for severe sepsis or septic shock (13/72 patients), 30% for definitive cancellation of antineoplastic chemotherapy (14/46 patients who still had active treatment), and 46% for death at week 12 (33/72 patients). Factors associated with severe sepsis or septic shock were an elevated C-reactive protein (CRP) level and an infection caused by Candida species; 4 of the 13 severe episodes (31%) were due to coagulase-negative staphylococci (CoNS). Factors associated with death at week 12 were a low median Karnofsky score, an elevated Charlson comorbidity index, the metastatic evolution of cancer, palliative care, and an elevated CRP level at presentation. Hematogenous complications (that is, infective endocarditis, septic thrombophlebitis, septic pulmonary emboli, spondylodiscitis, septic arthritis, or organ abscesses) were found in 8 patients (11%). In conclusion, patients’ overall condition (comorbidities and autonomy) and elevated CRP level were associated with an unfavorable clinical outcome after a TIVAP-related infection. Candida species and CoNS were responsible for severe sepsis or septic shock.

Complications infectieuses liées aux chambres implantables : caractéristiques et prise en charge

Totally implanted access port-related infections are responsible for their own morbidity and mortality. Main risk factors of totally implanted access port-related infections are total parenteral nutrition, young age, difficulties during insertion, poor general status and neutropenia. Recent guidelines defined intravascular catheter-related infections. This relies on a strict clinical and microbiological work-up including simultaneous culture of blood drawn from the catheter and a peripheral vein. The search for local or general complications is mandatory: clinical and possibly echographic assessments are therefore needed. Depending on the context and the type of microorganism, this evaluation may include transoesophageal echocardiography and search for suppurative thrombosis in case of catheter-related bloodstream infection caused by Staphylococcus aureus. Indeed, intravascular complications in this setting are frequent. Catheter removal is mandatory in case of local complication (tunnel infection or port pocket abscess), septic thrombosis, infective endocarditis, osteomyelitis, septic shock or infection related to specific pathogens (S. aureus, Candida spp., Pseudomonas aeruginosa). Otherwise, retention of the catheter might be proposed given results from recent studies including antibiotic lock therapy associated with systemic antibiotic. Future studies must focus on defining more precisely the factors associated with salvage therapy failure including host, pathogens virulence factors and biofilm formation.