Viviane Cassisa

Seasonal and regional dynamics of M. ulcerans transmission in environmental context: deciphering the role of water bugs as hosts and vectors.

Background Buruli ulcer, the third mycobacterial disease after tuberculosis and leprosy, is caused by the environmental mycobacterium M. ulcerans. Various modes of transmission have been suspected for this disease, with no general consensus acceptance for any of them up to now. Since laboratory models demonstrated the ability of water bugs to transmit M. ulcerans, a particular attention is focused on the transmission of the bacilli by water bugs as hosts and vectors. However, it is only through detailed knowledge of the biodiversity and ecology of water bugs that the importance of this mode of transmission can be fully assessed. It is the objective of the work here to decipher the role of water bugs in M. ulcerans ecology and transmission, based on large-scale field studies. Methodology/Principal Findings The distribution of M. ulcerans-hosting water bugs was monitored on previously unprecedented time and space scales: a total of 7,407 water bugs, belonging to large number of different families, were collected over one year, in Buruli ulcer endemic and non endemic areas in central Cameroon. This study demonstrated the presence of M. ulcerans in insect saliva. In addition, the field results provided a full picture of the ecology of transmission in terms of biodiversity and detailed specification of seasonal and regional dynamics, with large temporal heterogeneity in the insect tissue colonization rate and detection of M. ulcerans only in water bug tissues collected in Buruli ulcer endemic areas. Conclusion/Significance The large-scale detection of bacilli in saliva of biting water bugs gives enhanced weight to their role in M. ulcerans transmission. On practical grounds, beyond the ecological interest, the results concerning seasonal and regional dynamics can provide an efficient tool in the hands of sanitary authorities to monitor environmental risks associated with Buruli ulcer.

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.

Antifungal and Antibacterial Metabolites from a French Poplar Type Propolis

During this study, the in vitro antifungal and antibacterial activities of different extracts (aqueous and organic) obtained from a French propolis batch were evaluated. Antifungal activity was evaluated by broth microdilution on three pathogenic strains: Candida albicans, C. glabrata, and Aspergillus fumigatus. Antibacterial activity was assayed using agar dilution method on 36 Gram-negative and Gram-positive strains including Staphylococcus aureus. Organic extracts showed a significant antifungal activity against C. albicans and C. glabrata (MIC80 between 16 and 31 µg/mL) but only a weak activity towards A. fumigatus (MIC80 = 250 µg/mL). DCM based extracts exhibited a selective Gram-positive antibacterial activity, especially against S. aureus (SA) and several of its methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) strains (MIC100 30–97 µg/mL). A new and active derivative of catechin was also identified whereas a synergistic antimicrobial effect was noticed during this study.

Use of Fine-Needle Aspiration for Diagnosis of Mycobacterium ulcerans Infection

ABSTRACT Noninvasive methods for the bacteriological diagnosis of early-stage Mycobacterium ulcerans infection are not available. It was recently shown that fine-needle aspiration (FNA) could be used for diagnosing M. ulcerans infection in ulcerative lesions. We report that FNA is an appropriate sampling method for diagnosing M. ulcerans infection in nonulcerative lesions.

Antibacterial action of lipid nanocapsules containing fatty acids or monoglycerides as co-surfactants

Lipid nanocapsules (LNCs) are a new generation of biomimetic nanocarriers obtained via a phase inversion temperature method and have an oily core of medium-chain triglycerides that is surrounded by a shelf containing a lipophilic surfactant (lecithin) and a hydrophilic surfactant macrogol 15-hydroxystearate. The aim of the present study was to produce LNCs with antibacterial activity by replacing lecithin with other lipophilic surface active compounds, namely medium-chain fatty acids and their 1-monoglycerides, which are known to have antimicrobial properties. Fatty acids and monoglycerides were found to affect the properties of LNCs, such as particle size and zeta potential. Incorporation of a co-surfactant decreased significantly particle size (p⩽0.0039). Furthermore, incorporation of either lecithin or fatty acids with at least 10 carbon atoms yielded LNCs with the zeta potential significantly more negative than that of LNCs composed solely of triglycerides and macrogol 15 hydroxystearate (p⩽0.0310). Moreover, they were capable of decreasing the phase inversion temperature. The activity of the LCNs against Gram-positive S. aureus, including a methicillin-resistant strain, increased with increases in the length of the hydrocarbon tail. Monoglyceride-LNCs were found to be more active than the corresponding fatty acids. The opposite behaviour was observed for Gram-negative bacteria, whereby only caproic acid- and caprylic acid-LNCs were found to be active against these organisms. The monoglyceride-LNCs were bactericidal, and they killed in a time-dependent manner. Fatty acid-LNCs killed in a concentration-dependent manner. A haemolysis assay was performed to obtain preliminary information on the safety of the tested LNCs. In the case of fatty acid-LNCs, the concentrations at which bacterial growth was inhibited were similar to the haemolytic concentrations. However, monoglyceride-LNCs showed antibacterial action at concentrations much lower than those at which haemolysis was observed. In conclusion, monoglyceride-LNCs are promising candidates as carriers for the encapsulation of antibacterial agents, particularly against S. aureus.

Discordance in the minimal inhibitory concentrations of ertapenem for Enterobacter cloacae: Vitek 2 system versus Etest and agar dilution methods

Our objective was to compare the ertapenem minimal inhibitory concentrations (MICs) for Enterobacter cloacae isolates categorized intermediate or resistant to ertapenem when measured with the Vitek 2 system, with the MICs for these isolates when measured by two methods performed in agar medium: the Etest and agar plate dilution method (APDM). Overall, 50 E. cloacae isolates were included in the study. The mean MIC of ertapenem was 2.92±1.77μg/ml according to the Vitek 2 system, 0.94±0.84μg/ml according to the Etest strips, and 0.93±0.62μg/ml according to the APDM. Furthermore, the MICs determined by the Vitek 2 system were higher than the MICs determined by the two other methods for 96% of strains. Lastly, according to the Etest strips and APDM, 42% of E. cloacae were susceptible to ertapenem. No carbapenemase was identified by the screening method used. Using the Vitek 2 system to determine ertapenem MICs for E. cloacae can have potential consequences in terms of additional carbapenemase-detecting tests and antimicrobial therapy. It would be interesting to determine if the Vitek 2 system is more effective for the detection of carbapenemase producers with low-level carbapenem resistance than the two methods performed in agar medium.

Synergistic interactions between antimicrobial peptides derived from plectasin and lipid nanocapsules containing monolaurin as a cosurfactant against Staphylococcus aureus

Development of effective antibacterial agents for the treatment of infections caused by Gram-positive bacteria resistant to existing antibiotics, such as methicillin-resistant Staphylococcus aureus (MRSA), is an area of intensive research. In this work, the antibacterial efficacy of two antimicrobial peptides derived from plectasin, AP114 and AP138, used alone and in combination with monolaurin-lipid nanocapsules (ML-LNCs) was evaluated. Several interesting findings emerged from the present study. First, ML-LNCs and both plectasin derivatives showed potent activity against all 14 tested strains of S. aureus, independent of their resistance phenotype. Both peptides displayed a considerable adsorption (33%–62%) onto ML-LNCs without having an important impact on the particle properties such as size. The combinations of peptide with ML-LNC displayed synergistic effect against S. aureus, as confirmed by two methods: checkerboard and time-kill assays. This synergistic interaction enables a dose reduction and consequently decreases the risk of toxicity and has the potential of minimizing the development of resistance. Together, these results suggest that ML-LNCs loaded with a plectasin derivative may be a very promising drug delivery system for further development as a novel antibacterial agent against S. aureus, including MRSA.

Antibacterial activity of antipsychotic agents, their association with lipid nanocapsules and its impact on the properties of the nanocarriers and on antibacterial activity

Bacterial antibiotic resistance is an emerging public health problem worldwide; therefore, new therapeutic strategies are needed. Many studies have described antipsychotic compounds that present antibacterial activity. Hence, the aims of this study were to evaluate the in vitro antibacterial activity of antipsychotics belonging to different chemical families, to assess the influence of their association with lipid nanocapsules (LNCs) on their antimicrobial activity as well as drug release and to study the uptake of LNCs by bacterial cells. Antibacterial activity was evaluated against Gram-positive Staphylococcus aureus and Gram negative Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii by minimum inhibitory concentration (MIC) assay, and the capability of killing tested microorganisms was evaluated by time kill assay. LNCs were prepared by phase inversion method, and the antipsychotic agents were incorporated using pre-loading and post-loading strategies. Only phenothiazines and thioxanthenes showed antibacterial activity, which was independent of antibiotic-resistance patterns. Loading the nanocarriers with the drugs affected the properties of the former, particularly their zeta potential. The release rate depended on the drug and its concentration—a maximum of released drug of less than 40% over 24 hours was observed for promazine. The influence of the drug associations on the antibacterial properties was concentration-dependent since, at low concentrations (high nanocarrier/drug ratio), the activity was lost, probably due to the high affinity of the drug to nanocarriers and slow release rate, whereas at higher concentrations, the activity was well maintained for the majority of the drugs. Chlorpromazine and thioridazine increased the uptake of the LNCs by bacteria compared with blank LNCs, even below the minimum inhibitory concentration.

Effect of faecal microbiota transplantation on mouse gut colonization with carbapenemase-producing Escherichia coli.

Sir, We were very interested to read the recent systematic review and meta-analysis carried out by Bar-Yoseph et al. and published in this journal. This paper highlighted that a significant proportion of carbapenem-resistant Enterobacteriaceae carriers remained colonized for up to 1 year in the healthcare setting, with some potential consequences in terms of cross-transmissions and infections. Carbapenemase-producing Enterobacteriaceae (CPE) represent a major and growing public health threat. Very few options remain for the treatment of CPE infections, and infection control measures play a major role in reducing transmission. Selective digestive decontamination of patients with CPE carriage raised the risk of decolonization-associated drug resistance and poor effectiveness. Therefore, non-antibiotic strategies are of particular interest to avoid any risk of cumulative antibiotic resistance. Faecal microbiota transplantation (FMT) appears to be a promising approach to restore a normal intestinal microbiota and has been widely used successfully in recurrent Clostridium difficile infections and to eradicate colonization with CPE or ESBL-producing Enterobacteriaceae. The aim of this exploratory study was to evaluate the effectiveness of FMT against CPE colonization in a murine model of CPE digestive carriage. Ethics approval (reference 2015041415088410/APAFIS513) was obtained from the Ethics Committee for Animal Experimentation of Pays-de-la-Loire, France. Experiments were conducted according to the European directives concerning the use of animals in research (86/609/EEC). A murine model of CPE colonization was first implemented. Eight-week-old female Swiss mice were exposed to a combination of antibiotics to disrupt their gut microbiome. Oral administration of ceftriaxone (25 mg/kg), metronidazole (25 mg/kg) and vancomycin (50 mg/kg) was performed once daily from day 1 to day 5. A New Delhi metallo-b-lactamase (NDM-1)producing Escherichia coli (5 10 cfu/mL) was orally administered on each of days 4, 5 and 8 during and after the end of antibiotic exposure. Mice were then randomly assigned on day 9 to receive 200 lL of saline solution or FMT once daily for 3 days. FMT was performed using a saline solution of pooled stools at 50 mg/mL collected from the same mice before treatment. Stool cultures were performed on selective medium for CPE detection (Chrom ID CARBA, bioMérieux, France). Commensal flora was also studied on chromogenic medium for Enterobacteriaceae (UTI, Oxoid, UK) and blood agar with colistin and nalidixic acid under aerobic and anaerobic conditions (CNA agar medium, bioMérieux). Bacterial identifications were performed using MALDI-TOF MS (Vitek MS, bioMérieux). Faecal concentrations of CPE in the two groups were compared after treatment by using a Wilcoxon–Mann–Whitney test. A total of 30 mice (15 in each group) were followed for 2 weeks after FMT or placebo administration. Each mouse was individually housed to prevent cross-contamination. The mean CPE stool concentration before treatment was similar in the two groups (2 10 and 2.3 10 cfu/mL for the FMT group and the placebo group, respectively; P1⁄40.86). Before antibiotic exposure, mouse digestive flora contained 2.2 10 lactobacilli, 2.2 10 anaerobes (mainly Bacteroides spp. and Clostridium spp.), 10 Enterobacteriaceae and 6.8 10 enterococci. Antibiotic exposure was associated with a complete disappearance of Enterobacteriaceae and anaerobes, and with a significant reduction in Enterococcus and Lactobacillus populations (mean reduction of 99% and 91% from baseline, respectively). Mice recovered most of these bacteria (except Enterobacteriaceae) on day 8, before the beginning of treatment (FMT versus placebo). Six days after treatment (day 15), the mean CPE concentration was significantly lower among FMT-treated mice compared with the placebo group (8.6 10 and 1.4 10 cfu/mL, respectively; P1⁄40.021) (Figure 1). This difference persisted on day 22 (7.2 10 and 6.5 10 cfu/mL; P1⁄40.044). No adverse event was recorded during the protocol. In our murine model of CPE colonization compared with placebo, FMT was associated with a slight reduction in CPE carriage, reaching statistical significance but remaining insufficient from a clinical point of view. These results are not similar to a previous evaluation of FMT in a murine model of vancomycin-resistant Enterococcus faecium colonization. Indeed, in that model transplanted mice demonstrated an eradication of vancomycin-resistant E. faecium 15 days after FMT. Interestingly, the mice recovered a normal digestive flora with a low level of Enterobacteriaceae within a few days following the last administration of antibiotics. Therefore, the FMT was probably more effective in reinforcing the bacterial diversity than in providing high bacterial loads in the digestive tract. The absence of data from previous control studies on CPE carriage and the low number of mice included in our experiment limit the interpretation of our results. To our knowledge, our model is the first experimental controlled study of the effectiveness of FMT against CPE carriage. Therefore, a strict comparison with other bacterial colonization models cannot be done. FMT appears to be a potential candidate to accelerate transiently the decrease in CPE load in the digestive tract without any risk of increasing antibiotic resistance. However, because of the slight effect observed in our study, additional experiments are needed to confirm these preliminary data.

Synergistic Effect of Combinations Containing EDTA and the Antimicrobial Peptide AA230, an Arenicin-3 Derivative, on Gram-Negative Bacteria

The worldwide occurrence of resistance to standard antibiotics and lack of new antibacterial drugs demand new strategies to treat complicated infections. Hence, the aim of this study was to examine the antibacterial activities of an antimicrobial peptide, arenicin-3 derivative AA230, and ethylenediaminetetraacetic acid (EDTA) as well as the two compounds in combination against Gram-negative bacteria. AA230 showed strong antibacterial activity against all of the studied standard strains and clinical isolates, with minimum inhibitory concentrations ranging between 1 µg/mL and 8 µg/mL. AA230 exhibited a bactericidal mode of action. EDTA inhibited the growth of Acinetobacter baumannii at 500–1000 µg/mL. Strains of Acinetobacter baumannii were found to be more susceptible to EDTA than Pseudomonas aeruginosa or Escherichia coli. The antibacterial effects of both AA230 and EDTA were independent of the antibiotic resistance patterns. Indifference to synergistic activity was observed for AA230 and EDTA combinations using checkerboard titration. In time-kill studies, a substantial synergistic interaction between AA230 and EDTA was detected against all of the tested strains. The addition of EDTA enabled a 2–4-fold decrease in the AA230 dose. In conclusion, AA230 could have potential applications in the treatment of infections caused by Gram-negative organisms, and its effect can be potentiated by EDTA.