Marie-Laure Joly-Guillou

High content screening identifies decaprenyl-phosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors.

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2′ epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.

Curbing methicillin-resistant Staphylococcus aureus in 38 French hospitals through a 15-year institutional control program.

BACKGROUND The Assistance Publique-Hôpitaux de Paris (AP-HP) institution administers 38 teaching hospitals (23 acute care and 15 rehabilitation and long-term care hospitals; total, 23 000 beds) scattered across Paris and surrounding suburbs in France. In the late 1980s, the proportion of methicillin resistance among clinical strains of Staphylococcus aureus (MRSA) reached approximately 40% at AP-HP. METHODS A program aimed at curbing the MRSA burden was launched in 1993, based on passive and active surveillance, barrier precautions, training, and feedback. This program, supported by the strong commitment of the institution, was reinforced in 2001 by a campaign promoting the use of alcohol-based hand-rub solutions. An observational study on MRSA rate was prospectively carried out from 1993 onwards. RESULTS There was a significant progressive decrease in MRSA burden (-35%) from 1993 to 2007, whether recorded as the proportion (expressed as percentage) of MRSA among S aureus strains (41.0% down to 26.6% overall; 45.3% to 24.2% in blood cultures) or incidence of MRSA cases (0.86 down to 0.56 per 1000 hospital days). The MRSA burden decreased more markedly in intensive care units (-59%) than in surgical (-44%) and medical (-32%) wards. The use of ABHR solutions (in liters per 1000 hospital days) increased steadily from 2 L to 21 L (to 26 L in acute care hospitals and to 10 L in rehabilitation and long-term care hospitals) following the campaign. CONCLUSION A sustained reduction of MRSA burden can be obtained at the scale of a large hospital institution with high endemic MRSA rates, providing that an intensive program is maintained for a long period.

Evaluation of a strategy of screening multiple anatomical sites for methicillin-resistant Staphylococcus aureus at admission to a teaching hospital.

We compared the sensitivity of screening with nasal culture alone with that of a multiple-site screening method for the identification of carriers of methicillin-resistant Staphylococcus aureus at hospital admission. If nasal cultures alone had been used during the 1-year study, 27.0% of carriers of methicillin-resistant S. aureus would have been missed, which corresponds to 560 theoretical isolation days. If rectal screening had not been used, 431 theoretical isolation days would have been missed, and, if axillary screening had not been used, 99 theoretical isolation days would have been missed.

An outbreak of imipenem-resistant Acinetobacter baumannii in critically ill surgical patients.

OBJECTIVE To describe an outbreak of imipenem-resistant Acinetobacter baumannii (IR-Ab) and the measures for its control, and to investigate risk factors for IR-Ab acquisition. DESIGN An observational and a case-control study. SETTING A surgical intensive care unit (ICU) in a university tertiary care hospital. METHODS After admission to the ICU of an IR-Ab-positive patient, patients were prospectively screened for IR-Ab carriage upon admission and then once a week. Environmental cleaning and barrier safety measures were used for IR-Ab carriers. A case-control study was performed to identify factors associated with IR-Ab acquisition. Cases were patients who acquired IR-Ab. Controls were patients who were hospitalized in the ICU at the same time as cases and were exposed to IR-Ab for a similar duration as cases. The following variables were investigated as potential risk factors: baseline characteristics, scores for severity of illness and therapeutic intervention, presence and duration of invasive procedures, and antimicrobial administration. RESULTS Beginning in May 1996, the outbreak involved 17 patients over 9 months, of whom 12 acquired IR-Ab (cases), 4 had IR-Ab isolates on admission to the ICU, and 1 could not be classified. Genotypic analysis identified two different IR-Ab isolates, responsible for three clusters. Ten of the 12 nosocomial cases developed infection. Control measures included reinforcement of barrier safety measures, limitation of the number of admissions, and thorough environmental cleaning. No new case was identified after January 1997. Eleven of the 12 cases could be compared to 19 controls. After adjustment for severity of illness, a high individual therapeutic intervention score appeared to be a risk factor for IR-Ab acquisition. CONCLUSION The outbreak ended after strict application of control measures. Our results suggest that high work load contributes to IR-Ab acquisition.

Control and Outcome of a Large Outbreak of Colonization and Infection with Glycopeptide- Intermediate Staphylococcus aureus in an Intensive Care Unit

BACKGROUND Glycopeptide-intermediate Staphylococcus aureus (GISA) is emerging as a cause of nosocomial infection and outbreaks of infection and colonization in intensive care units (ICUs). We describe an outbreak of GISA colonization/infection and the ensuing control measures in an ICU and investigate outcomes of the affected patients. METHODS We describe an outbreak of GISA colonization and infection that affected 21 patients in a medical ICU at a tertiary care teaching hospital, as well as the measures taken to eradicate the GISA strain. RESULT Recognition of the outbreak was difficult. Infections, all of which were severe, were diagnosed in 11 of 21 patients. Patient isolation and barrier precautions failed when used alone. Addition of a stringent policy of restricted admissions, twice daily environmental cleaning, and implementation of hand decontamination with a hydroalcoholic solution led to outbreak termination. This was associated with increases in workload, despite a marked decrease in the number of admissions. CONCLUSION This first description of a large outbreak of GISA colonization and infection underlines the importance of routine GISA-strain detection when methicillin-resistant S. aureus is isolated. Outbreak control may be difficult to achieve.

Reservoirs of Acinetobacter baumannii outside the hospital and potential involvement in emerging human community-acquired infections.

The objective of the present report was to review briefly the potentially community-acquired Acinetobacter baumannii infections, to update information on the reservoirs of A. baumannii outside the hospital, and to consider their potential interactions with human infections. Most reports on potentially community-acquired A. baumannii have been published during the last 15 years. They concern community-acquired pneumonia, infections in survivors from natural disasters, and infected war wounds in troops from Iraq and Afghanistan. Although the existence of extra-hospital reservoirs of A. baumannii has long been disputed, the recent implementation of molecular methods has allowed the demonstration of the actual presence of this organism in various environmental locations, in human carriage, in pets, slaughter animals, and human lice. Although the origin of the A. baumannii infections in soldiers injured in Southwestern Asia is difficult to determine, there are some arguments to support the involvement of extra-hospital reservoirs in the occurrence of community-acquired infections. Overall, the emergence of community-acquired A. baumannii infections could be associated with interactions between animals, environment, and humans that are considered to be potentially involved in the emergence or re-emergence of some infectious diseases.

The virulence variability of different Acinetobacter baumannii strains in experimental pneumonia.

OBJECTIVES Our objective was to compare the virulence of 5 strains of Acinetobacter baumannii by using a mouse model of pneumonia. METHODS Six-week old female C3H/HeN mice were used. The pneumonia was inducted by intra-tracheal inoculation of 5.10(6) bacteria. Spontaneous outcome was evaluated by mortality, mice weight variations, and a clinical score. Bacterial counts in lungs, spleen and blood, and inflammatory response in lungs (dosages of tumor necrosis factor-alpha and macrophage inflammatory protein-2) were also measured. Lastly, a histological examination of lungs was performed for 3 strains, giving a histological score. RESULTS Global mortality varied from 13% to 79% (P<10(-4)). Bacterial counts in lungs within the 4 days following inoculation varied significantly according to different strains. The evolution curves of bacterial counts were also different. There was a significant correlation between the clinical score and mortality (P<0.05) but not between bacterial counts in lungs and mortality. The increase of pro-inflammatory mediator production in lungs and the histological score also varied according to strains. CONCLUSIONS These results demonstrate the variability of the virulence between strains, and suggest that bacterial proliferation is not the only virulence factor responsible for the pathogenesis in A. baumannii pneumonia.

First report of blaNDM-1-producing Acinetobacter baumannii isolated in Lebanon from civilians wounded during the Syrian war

OBJECTIVES The emergence of carbapenem-resistant Acinetobacter baumannii has been observed worldwide. We describe the first detection of A. baumannii carrying the blaNDM-1 gene in Lebanon, isolated from Syrian patients wounded during the civil war. METHODS Four carbapenem-resistant A. baumannii strains isolated in 2012 in the Tripoli Government Hospital, Lebanon, from civilians wounded during the Syrian war, were analysed. Susceptibility was determined by disk diffusion testing, and resistance to carbapenems was confirmed by Etest. The presence of blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, blaOXA-143-like, and blaNDM was investigated by PCR. Clonal relationships were studied by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and blaOXA-51 sequence-based typing. RESULTS All isolates harboured the blaNDM-1 gene and were negative for other tested carbapenemases. They all belonged to the sequence type 85 and formed a single cluster by PFGE. Finally, blaOXA-51-like gene sequencing revealed the presence of the blaOXA-94 variant in all four isolates. CONCLUSION These findings show that Syria constitutes a reservoir for NDM-1-producing bacteria. These results also highlight the need for effective measures to stop the threatening spread of such strains.

Lipid-based liquid crystals as carriers for antimicrobial peptides : Phase behavior and antimicrobial effect

The number of antibiotic-resistant bacteria is increasing worldwide, and the demand for novel antimicrobials is constantly growing. Antimicrobial peptides (AMPs) could be an important part of future treatment strategies of various bacterial infection diseases. However, AMPs have relatively low stability, because of proteolytic and chemical degradation. As a consequence, carrier systems protecting the AMPs are greatly needed, to achieve efficient treatments. In addition, the carrier system also must administrate the peptide in a controlled manner to match the therapeutic dose window. In this work, lyotropic liquid crystalline (LC) structures consisting of cubic glycerol monooleate/water and hexagonal glycerol monooleate/oleic acid/water have been examined as carriers for AMPs. These LC structures have the capability of solubilizing both hydrophilic and hydrophobic substances, as well as being biocompatible and biodegradable. Both bulk gels and discrete dispersed structures (i.e., cubosomes and hexosomes) have been studied. Three AMPs have been investigated with respect to phase stability of the LC structures and antimicrobial effect: AP114, DPK-060, and LL-37. Characterization of the LC structures was performed using small-angle X-ray scattering (SAXS), dynamic light scattering, ζ-potential, and cryogenic transmission electron microscopy (Cryo-TEM) and peptide loading efficacy by ultra performance liquid chromatography. The antimicrobial effect of the LCNPs was investigated in vitro using minimum inhibitory concentration (MIC) and time-kill assay. The most hydrophobic peptide (AP114) was shown to induce an increase in negative curvature of the cubic LC system. The most polar peptide (DPK-060) induced a decrease in negative curvature while LL-37 did not change the LC phase at all. The hexagonal LC phase was not affected by any of the AMPs. Moreover, cubosomes loaded with peptides AP114 and DPK-060 showed preserved antimicrobial activity, whereas particles loaded with peptide LL-37 displayed a loss in its broad-spectrum bactericidal properties. AMP-loaded hexosomes showed a reduction in antimicrobial activity.

Measurement and interpretation of hand hygiene compliance rates: importance of monitoring entire care episodes.

Our objective was to assess the importance of monitoring hand hygiene compliance (HHC) during series of successive contacts with patients or surroundings for measurement and interpretation of the compliance rates. A direct observational study of HHC was performed in four intensive care units (ICUs) and four healthcare settings with non-intensive care wards (NICWs). Hand hygiene (HH) opportunities were differentiated into two categories: extra-series opportunities (ESOs) (before or after a single contact, and before the first contact or after the last contact of a series of successive contacts) or as intra-series opportunities (ISOs) (from the opportunity following the first contact to the opportunity preceding the last in the same series). In all, 903 opportunities of HH were performed in ICUs and 760 in NICWs. The proportion of ISOs was 46.0% in ICUs and 22.9% in NICWs. The overall HHC was significantly higher in NICWs than in ICUs (61.2% vs 47.5%, P<0.00001). The HHC was significantly higher for ESOs than for ISOs (67.7% vs 28.5%, P<0.00001). The HHC for ISOs was significantly higher in ICUs (32.2% vs 19.0%, P<0.005). If the distribution of categories of HH opportunities observed in NICWs had been the same as in ICUs, the overall HHC would have been similar in NICWs (46.4%) and in ICUs (47.5%). Monitoring HHC during entire care episodes in series of successive contacts is necessary to avoid a strong overestimation of the overall compliance rates. Concurrently, comparison of compliance data should take into account the proportion of ISOs included in the evaluation study.