Jean-Philippe Lavigne

Dosage effect on uropathogenic Escherichia coli anti-adhesion activity in urine following consumption of cranberry powder standardized for proanthocyanidin content: a multicentric randomized double blind study

BackgroundIngestion of cranberry (Vaccinium macrocarpon Ait.) has traditionally been utilized for prevention of urinary tract infections. The proanthocyanidins (PACs) in cranberry, in particular the A-type linkages have been implicated as important inhibitors of primarily P-fimbriated E. coli adhesion to uroepithelial cells. Additional experiments were required to investigate the persistence in urine samples over a broader time period, to determine the most effective dose per day and to determine if the urinary anti-adhesion effect following cranberry is detected within volunteers of different origins.MethodsTwo separate bioassays (a mannose-resistant hemagglutination assay and an original new human T24 epithelial cell-line assay) have assessed the ex-vivo urinary bacterial anti-adhesion activity on urines samples collected from 32 volunteers from Japan, Hungary, Spain and France in a randomized, double-blind versus placebo study. An in vivo Caenorhabditis elegans model was used to evaluate the influence of cranberry regimen on the virulence of E. coli strain.ResultsThe results indicated a significant bacterial anti-adhesion activity in urine samples collected from volunteers that consumed cranberry powder compared to placebo (p < 0.001). This inhibition was clearly dose-dependent, prolonged (until 24 h with 72 mg of PAC) and increasing with the amount of PAC equivalents consumed in each cranberry powder regimen. An in vivo Caenorhabditis elegans model showed that cranberry acted against bacterial virulence: E. coli strain presented a reduced ability to kill worms after a growth in urines samples of patients who took cranberry capsules. This effect is particularly important with the regimen of 72 mg of PAC.ConclusionsAdministration of PAC-standardized cranberry powder at dosages containing 72 mg of PAC per day may offer some protection against bacterial adhesion and virulence in the urinary tract. This effect may offer a nyctohemeral protection.

Membrane Permeability and Regulation of Drug “Influx and Efflux” in Enterobacterial Pathogens

In Enterobacteriaceae, membrane permeability is a key in the level of susceptibility to antibiotics. Modification of the bacterial envelope by decreasing the porin production or increasing the expression of efflux pump systems has been reported. These phenomena are frequently associated with other resistance mechanisms such as alteration of antibiotics or modification of the drug targets, in various clinical isolates showing a Multi Drug Resistant phenotype (MDR). In Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae and Salmonella enterica several genes and external factors are involved in the emergence of MDR isolates. These bacterial isolates exhibit a noticeable reduction of functional porins per cell due to a decrease, a complete shutdown of synthesis, or the expression of an altered porin and a high expression of efflux systems (e.g. overexpression of the pump). The combined action of these mechanisms during an infection confers a significant decrease in bacterial sensitivity to antibiotherapy ensuring dissemination and colonization of the patient and favours the acquisition of additional mechanisms of resistance. MarA and ramA are involved in a complex regulation cascade controlling membrane permeability and actively participate in the triggering of the MDR phenotype. Mutations in regulator genes have been shown to induce the overproduction of efflux and the down-regulation of porin synthesis. In addition, various compounds such as salicylate, imipenem or chloramphenicol are able to activate the MDR response. This phenomenon has been observed both in vitro during culture of bacteria in the presence of drugs and in vivo during antibiotic treatment of infected patients. These effectors activate the expression of specific global regulators, marA, ramA, or target other genes located downstream in the regulation cascade.

Serum procalcitonin and C-reactive protein concentrations to distinguish mildly infected from non-infected diabetic foot ulcers: a pilot study

Aims/hypothesisInfection of diabetic foot ulcers is common; at early stages it is difficult to differentiate between non-infected ulcers (or those colonised with normal flora) and ulcers infected with virulent bacteria that lead to deterioration. This pilot study aimed to assess the diagnostic accuracy of inflammatory markers as an aid to making this distinction.MethodsWe included 93 diabetic patients who had an episode of foot ulcer and had not received antibiotics during the 6 months preceding the study. Ulcers were classified as infected or uninfected, according to the Infectious Diseases Society of America–International Working Group on the Diabetic Foot classification. Diabetic patients without ulcers (n = 102) served as controls. C-reactive protein (CRP), orosomucoid, haptoglobin and procalcitonin were measured together with white blood cell and neutrophil counts. The diagnostic performance of each marker, in combination (using logistic regression) or alone, was assessed.ResultsAs a single marker, CRP was the most informative for differentiating grade 1 from grade 2 ulcers (sensitivity 0.727, specificity 1.000, positive predictive value 1.000, negative predictive value 0.793) with an optimal cut-off value of 17 mg/l. In contrast, white blood cell and neutrophil counts were not predictive. The most relevant combination derived from the logistic regression was the association of CRP and procalcitonin (AUC 0.947), which resulted in a significantly more effective determination of ulcer grades, as shown by comparing receiver operating characteristic curves.Conclusions/interpretationMeasurement of only two inflammatory markers, CRP and procalcitonin, might be of value for distinguishing between infected and non-infected foot ulcers in subgroups of diabetic patients, to help ensure the appropriate allocation of antibiotic treatment. Nevertheless, external validation of the diagnostic value of procalcitonin and CRP in diabetic foot ulcers is needed before routine use can be recommended.

In‐vitro and in‐vivo evidence of dose‐dependent decrease of uropathogenic Escherichia coli virulence after consumption of commercial Vaccinium macrocarpon (cranberry) capsules

This study evaluated the antibacterial efficacy of the consumption of cranberry capsules vs. placebo in the urine of healthy volunteers. A first double-blind, randomised, crossover trial involved eight volunteers who had followed three regimens, with or without cranberry, with a wash-out period of at least 6 days between each regimen. Twelve hours after consumption of cranberry or placebo hard capsules, the first urine of the morning was collected. Different Escherichia coli strains were cultured in the urine samples. Urinary antibacterial adhesion activity was measured in vitro using the human T24 epithelial cell-line, and in vivo using the Caenorhabditis elegans killing model. With the in-vitro model, 108 mg of cranberry induced a significant reduction in bacterial adherence to T24 cells as compared with placebo (p <0.001). A significant dose-dependent decrease in bacterial adherence in vitro was noted after the consumption of 108 and 36 mg of cranberry (p <0.001). The in-vivo model confirmed that E. coli strains had a reduced ability to kill C. elegans after growth in the urine of patients who consumed cranberry capsules. Overall, these in-vivo and in-vitro studies suggested that consumption of cranberry juice represents an interesting new strategy to prevent recurrent urinary tract infection.

Efflux Pump, the Masked Side of ß-Lactam Resistance in Klebsiella pneumoniae Clinical Isolates

Background β-lactamase production and porin decrease are the well-recognized mechanisms of acquired ß-lactam resistance in Klebsiella pneumoniae isolates. However, such mechanisms proved to be absent in K. pneumoniae isolates that are non susceptible to cefoxitin (FOX) and succeptible to amoxicillin+clavulanic acid in our hospital. Assessing the role of efflux pumps in this β-lactam phenotype was the aim of this study. Methodology/Findings MICs of 9 β-lactams, including cloxacillin (CLX), and other antibiotic families were tested alone and with an efflux pump inhibitor (EPI), then with both CLX (subinhibitory concentrations) and EPI against 11 unique bacteremia K. pneumoniae isolates displaying the unusual phenotype, and 2 ATCC strains. CLX and EPI-dose dependent effects were studied on 4 representatives strains. CLX MICs significantly decreased when tested with EPI. A similar phenomenon was observed with piperacillin+tazobactam whereas MICs of the other β-lactams significantly decreased only in the presence of both EPI and CLX. Thus, FOX MICs decreased 128 fold in the K. pneumoniae isolates but also16 fold in ATCC strain. Restoration of FOX activity was CLX dose-dependent suggesting a competitive relationship between CLX and the other β-lactams with regard to their efflux. For chloramphenicol, erythromycin and nalidixic acid whose resistance was also due to efflux, adding CLX to EPI did not increase their activity suggesting differences between the efflux process of these molecules and that of β-lactams. Conclusion This is the first study demonstrating that efflux mechanism plays a key role in the β-lactam susceptibility of clinical isolates of K. pneumoniae. Such data clearly evidence that the involvement of efflux pumps in ß-lactam resistance is specially underestimated in clinical isolates.

Virulence Potential and Genomic Mapping of the Worldwide Clone Escherichia coli ST131

Recently, the worldwide propagation of clonal CTX-M-15-producing Escherichia coli isolates, namely ST131 and O25b:H4, has been reported. Like the majority of extra-intestinal pathogenic E. coli isolates, the pandemic clone ST131 belongs to phylogenetic group B2, and has recently been shown to be highly virulent in a mouse model, even though it lacks several genes encoding key virulence factors (Pap, Cnf1 and HlyA). Using two animal models, Caenorhabditis elegans and zebrafish embryos, we assessed the virulence of three E. coli ST131 strains (2 CTX-M-15- producing urine and 1 non-ESBL-producing faecal isolate), comparing them with five non-ST131 B2 and a group A uropathogenic E. coli (UPEC). In C. elegans, the three ST131 strains showed intermediate virulence between the non virulent group A isolate and the virulent non-ST131 B2 strains. In zebrafish, the CTX-M-15-producing ST131 UPEC isolates were also less virulent than the non-ST131 B2 strains, suggesting that the production of CTX-M-15 is not correlated with enhanced virulence. Amongst the non-ST131 B2 group isolates, variation in pathogenic potential in zebrafish embryos was observed ranging from intermediate to highly virulent. Interestingly, the ST131 strains were equally persistent in surviving embryos as the non-ST131-group B2 strains, suggesting similar mechanisms may account for development of persistent infection. Optical maps of the genome of the ST131 strains were compared with those of 24 reference E. coli strains. Although small differences were seen within the ST131 strains, the tree built on the optical maps showed that these strains belonged to a specific cluster (86% similarity) with only 45% similarity with the other group B2 strains and 25% with strains of group A and D. Thus, the ST131 clone has a genetic composition that differs from other group B2 strains, and appears to be less virulent than previously suspected.

Turkey: a further country concerned by community-acquired Escherichia coli clone O25-ST131 producing CTX-M-15

OBJECTIVES The aim of this study was to assess the frequency and diversity of extended-spectrum beta-lactamases (ESBLs) produced by exclusively community-acquired Escherichia coli isolates in Izmir (Turkey) and to search for isolates producing CTX-M-15 and belonging to the pandemic clone E. coli O25-ST131. METHODS The patients with E. coli urinary tract infections (UTIs) and no hospitalization in the last 12 months, and no transfer from hospital, no stay in nursing home and no antimicrobial treatment in the previous 3 months were prospectively included over a 1 year period. Those E. coli detected positive for ESBL were characterized and compared with a representative of E. coli clone O25-ST131 with regard to bla genes, antibiotic resistance, phylogenetic groups, PFGE profiles and virulence factor genes (n = 17). O serotyping, multilocus sequence typing (MLST) and AmpC typing were performed to confirm that the Turkish isolate belonged to the clone O25-ST131. RESULTS Among the 3108 UTIs diagnosed, 82 (2.6%) were due to community E. coli isolates and followed the strict inclusion criteria. Seventeen of them (21%) produced an ESBL, of which CTX-M-15 was predominant (53%). These ESBL-positive isolates, distributed equally into three phylogenetic groups, displayed 13 PFGE profiles and three clusters. A Turkish CTX-M-15-producing isolate as a member of the clone ST131 was suggested by a high similarity of its PFGE profile to that of the clone representative and was confirmed by O serotyping, AmpC typing and MLST. CONCLUSIONS This study describes the community emergence of CTX-M-15-producing E. coli isolates, including an isolate of clone O25-ST131, in Turkey.

The Th17/Treg Ratio, IL-1RA and sCD14 Levels in Primary HIV Infection Predict the T-cell Activation Set Point in the Absence of Systemic Microbial Translocation

Impairment of the intestinal barrier and subsequent microbial translocation (MT) may be involved in chronic immune activation, which plays a central role in HIV pathogenesis. Th17 cells are critical to prevent MT. The aim of the study was to investigate, in patients with primary HIV infection (PHI), the early relationship between the Th17/Treg ratio, monocyte activation and MT and their impact on the T-cell activation set point, which is known to predict disease progression. 27 patients with early PHI were included in a prospective longitudinal study and followed-up for 6 months. At baseline, the Th17/Treg ratio strongly negatively correlated with the proportion of activated CD8 T cells expressing CD38/HLA-DR or Ki-67. Also, the Th17/Treg ratio was negatively related to viral load and plasma levels of sCD14 and IL-1RA, two markers of monocyte activation. In untreated patients, the Th17/Treg ratio at baseline negatively correlated with CD8 T-cell activation at month 6 defining the T-cell activation set point (% HLA-DR+CD38+ and %Ki-67+). Soluble CD14 and IL-1RA plasma levels also predicted the T-cell activation set point. Levels of I-FABP, a marker of mucosal damages, were similar to healthy controls at baseline but increased at month 6. No decrease in anti-endotoxin core antibody (EndoCAb) and no peptidoglycan were detected during PHI. In addition, 16S rDNA was only detected at low levels in 2 out 27 patients at baseline and in one additional patient at M6. Altogether, data support the hypothesis that T-cell and monocyte activation in PHI are not primarily driven by systemic MT but rather by viral replication. Moreover, the “innate immune set point” defined by the early levels of sCD14 and IL-1RA might be powerful early surrogate markers for disease progression and should be considered for use in clinical practice.

Epidemiology of carbapenemase-producing Enterobacteriaceae and Acinetobacter baumannii in Mediterranean countries.

The emergence and global spread of carbapenemase-producing Enterobacteriaceae and Acinetobacter baumannii are of great concern to health services worldwide. These β-lactamases hydrolyse almost all β-lactams, are plasmid-encoded, and are easily transferable among bacterial species. They are mostly of the KPC, VIM, IMP, NDM, and OXA-48 types. Their current extensive spread worldwide in Enterobacteriaceae is an important source of concern. Infections caused by these bacteria have limited treatment options and have been associated with high mortality rates. Carbapenemase producers are mainly identified among Klebsiella pneumoniae, Escherichia coli, and A. baumannii and still mostly in hospital settings and rarely in the community. The Mediterranean region is of interest due to a great diversity and population mixing. The prevalence of carbapenemases is particularly high, with this area constituting one of the most important reservoirs. The types of carbapenemase vary among countries, partially depending on the population exchange relationship between the regions and the possible reservoirs of each carbapenemase. This review described the epidemiology of carbapenemases produced by enterobacteria and A. baumannii in this part of the world highlighting the worrisome situation and the need to screen and detect these enzymes to prevent and control their dissemination.

Phosphonate monolayers functionalized by silver thiolate species as antibacterial nanocoatings on titanium and stainless steel

Titanium and stainless steel substrates were modified by grafting with mercaptododecylphosphonic acid (MDPA) followed by reaction with silver nitrate (AgNO3), in order to investigate the potential of phosphonate self-assembled monolayers functionalized by silver thiolate species as antibacterial nanocoatings for inorganic biomaterials. The samples were characterized by Fourier transform infrared (FTIR) spectroscopy in grazing-incidence mode, water contact angle measurements, and X-ray photoelectron spectroscopy (XPS). The influence of the surface modification on bacterial adhesion and biofilm growth was investigated in vitro using Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus strains. The stability of the monolayer in blood-mimicking medium was examined. Despite their very low silver content, MDPA + AgNO3 monolayers strongly decreased bacterial adhesion (>99.9% reduction in the number of viable adherent bacteria) and biofilm formation in comparison to the bare substrates.