Quinolone resistance among Salmonella Kentucky and Typhimurium isolates in Tunisia: first report of Salmonella Typhimurium ST34 in Africa and qnrB19 in Tunisia.

AIMS: Characterization of quinolone-resistant Salmonella Kentucky and Typhimurium isolates in Tunisia from various sources, detection of some plasmid-mediated quinolone resistance genes and the genetic relatedness. METHODS: A total of 1404 isolates of S. Kentucky (n = 1059)/S. Typhimurium (n = 345) from various sources from all over Tunisia were tested for quinolone resistance by disk diffusion method. Minimum inhibitory concentrations of nalidixic acid, ciprofloxacin and ofloxacin were determined. Quinolone-resistant isolates were screened for plasmid-mediated quinolone-resistance genes (qnrA,qnrB,qnrS, aac(6')-Ib-cr and qepA) by polymerase chain reaction (PCR). Mutations in the quinolone-resistance-determining regions of the gyrA and parC genes were detected by PCR and DNA sequencing. Pulsed-field gel electrophoresis and multilocus sequence typing were accomplished for isolates harbouring plasmid-mediated quinolone-resistance genes. RESULTS: According to our selection criteria (NAL = resistance phenotype; CIP = resistant with diameter 0, or intermediate), only 63 S. Kentucky/41 S. Typhimurium isolates were investigated: 49% (5/104) were multidrug resistant. Two S. Typhimurium isolates harboured qnrB19 with different PFGE profiles. A mutation was detected in the gyrA gene for each of these two isolates. MLST revealed the presence of ST313 and ST34, an endemic sequence type. CONCLUSION: Our study highlights the presence of quinolone multidrug-resistant Salmonella in humans and animals in Tunisia. This is the first report of S. Typhimurium ST34 in Africa and qnrB19 in Tunisia. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that describes not only the current epidemiological situation of the quinolone resistance in S. Kentucky and Typhimurium isolated from various sources and regions in Tunisia, but also, the genetic resistance determinants associated with phenotypic antibiotic resistance and the molecular mechanisms of their quinolone-resistance. Also, we provide the first report of S. Typhimurium ST34 in Africa, and the first report of qnrB19 in Salmonella in Tunisia.

Evaluation of antileishmanial, cytotoxic and antioxidant activities of essential oils extracted from plants issued from the leishmaniasis-endemic region of Sned (Tunisia).

In this study, we tested 10 essential oils (EOs) extracted from 10 plants issued from Sned region (Tunisia) to evaluate both their leishmanicidal effects against Leishmania major and L. infantum, and their cytotoxicity against murine macrophage cell line RAW 264.7 (ATCC, TIB-71). The antioxidant activity was also monitored by the DDPH method, while the chemical composition of active EO was assessed by GC-MS analysis. The results showed that the EOs obtained from Thymus hirtus sp. algeriensis (rich on monoterpenoids, especially linalool at 17.62% and camphor at 13.82%) is significantly active against both L. major and L. infantum, whereas Ruta chalepensis EO (rich on 2-undecanone at 84.28%) is only active against L. infantum. Both oil extracts showed low cytotoxicity towards murine macrophages. The characteristic ratios (IC80 Raw264.7 cells/IC50 L. infantum and IC80 Raw264.7 cells/IC50 L. major) were, respectively, 2.7 and 1.57 for T. hirtus sp. algeriensis, and 1.34 and 0.19 for R. chalepensis. However, when measuring the antioxidant effects (DDPH method), the two latter EOs presented a moderate 2,2-diphenyl-2-picrylhydrazyl hydrate scavenging effects compared to EOs from Eucaliptus globulus, Pinus halepensis, Pituranthos tortuosus, Rosmarinus officinalis, Tetraclinis articulata or to BHT.

[In vitro activity of daptomycin against strains isolated from diabetic foot ulcers]. / Activité in vitro de la daptomycine vis-à-vis de souches cliniques isolées de plaies du pied chez des diabétiques.

PURPOSE OF THE STUDY: The aim of this study was to evaluate the in vitro activity of daptomycin and other comparator agents against bacterial strains isolated from diabetic foot infections (DFI). PATIENTS AND METHODS: All diabetic patients hospitalized for a first episode of DFI (stage 2 to 4, according to the International Working Group of Diabetic Foot classification) were selected in Nîmes University hospital between June 2006 to August 2007. MIC were determined using E-test strip (AB Biodisk) and custom broth microdilution panels against bacterial strains isolated from foot samples. RESULTS: Two hundred strains were studied. Daptomycin was active against 99.5% of all the strains especially Streptococcus sp. (100%), Enterococcus sp. (100%), coagulase-negative Staphylococcus (100%) and methicillin-susceptible Staphylococcus aureus (100%). Exclusively, one methicillin-resistant S. aureus strain was not covered by this antibiotic. CONCLUSIONS: Daptomycin, a new broad spectrum antimicrobial agent against Gram-positive cocci, is qualified to belong to the therapeutic arsenal package of complicated skin and soft tissue infections in diabetic patients after microbial documentation.

An in silico immunological approach for prediction of CD8+ T cell epitopes of Leishmania major proteins in susceptible BALB/c and resistant C57BL/6 murine models of infection.

It is well established that MHC class II restricted-CD4 T cells are dominant during the development of immunity against Leishmania (L) in the C57BL/6-resistant mouse strain. However and in agreement with a number of previous observations indicating that specific CD8 T cells are primed during natural infection or vaccination in humans, a great deal of evidence obtained recently with the susceptible BALB/c murine model of infection by Leishmania major indicates that CD8 T cells participate in both pathogenesis and immunity to cutaneous leishmaniasis. Our goal herein was to identify in silico all parasitic peptides present in the whole L. major predicted proteome, using several public computational systems for the prediction of peptide binding to all MHC (histocompatibility complex-2) molecules in BALB/c and C57BL/6 mice (Syfpeithi, Rankpep, PRED(BALB/c) and Bimas). Peptides that were predicted to bind to different H2 molecules were then analysed for their homology with any of the murine proteins annotated so far, using the BLAST algorithm. Sets of selected peptides for each H2 molecule were defined by different prediction systems and compared to each other. Surprisingly, the results showed that a higher number of L. major peptides were predicted to bind H2 BALB/c molecules and very few or none to bind H2 C57BL/6 molecules. Our finding illustrates how a hybrid immuno-computational approach may be useful for biologists to target an in silico set of selected proteins to define potential candidate antigens for experimental vaccination with greater accuracy as well as a reduced number of T cell antigens.

TRAF1 is a negative regulator of TNF signaling. enhanced TNF signaling in TRAF1-deficient mice.

TNF receptor-associated factor 1 (TRAF1) is a unique TRAF protein because it lacks a RING finger domain and is predominantly expressed in activated lymphocytes. To elucidate the function of TRAF1, we generated TRAF1-deficient mice. TRAF1(-/-) mice are viable and have normal lymphocyte development. TRAF1(-/-) T cells exhibit stronger than wild-type (WT) T cell proliferation to anti-CD3 mAb, which persisted in the presence of IL-2 or anti-CD28 antibodies. Activated TRAF1(-/-) T cells, but not TRAF1(+/+) T cells, responded to TNF by proliferation and activation of the NF-kappa B and AP-1 signaling pathways. This TNF effect was mediated by TNFR2 (p75) but not by TNFR1 (p55). Furthermore, skin from TRAF1(-/-) mice was hypersensitive to TNF-induced necrosis. These findings suggest that TRAF1 is a negative regulator of TNF signaling.

V beta T cell repertoire of CD8+ splenocytes selected on nonpolymorphic MHC class I molecules.

In this work, we have studied the role of the MHC class Ib molecules in the selection and maintenance of CD8(+) T splenocytes. We have compared the CD8(+) T cell repertoires of wild-type, H-2K-deficient, H-2D-deficient, or double knockout C57BL/6 mice. We show that the different CD8(+) repertoires, selected either by class Ia and class Ib or by class Ib molecules only, use the various V alpha (AV) and V beta (BV) rearrangements in the same proportion and without biases in the CDR3 size distribution. Furthermore, we have estimated the size of the BV repertoire in the four different strains of mice. Interestingly, we have found that the BV repertoire size is proportional to the overall number of CD8(+) splenocytes. This observation implies that BV diversity is positively correlated with the number of CD8(+) cells, even when the number of CD8(+) splenocytes is dramatically reduced (90% in the double knockout mice).

The peripheral CD8 T cell repertoire is largely independent of the presence of intestinal flora.

While numerous studies have analyzed the shaping of T cell repertoires by self or foreign peptides, little is known on the influence of commensal self peptides derived from the intestinal flora (IF). Here, we have analyzed naive and immune repertoires in mice devoid of IF [germ-free (GF) mice]. First, by means of an extensive CDR3beta sequencing strategy, we show that the naive peripheral CD8 T cell repertoire does not exhibit a major imprint of IF antigens. Second, using MHC-peptide tetramers, CDR3beta length distribution analyses and TCR sequencing, we show that cytotoxic T lymphocyte (CTL) responses specific for two distinct epitopes are quasi-identical in normal and GF mice. Our findings indicate that, in general, peptides derived from the intestinal microflora have little if any influence on CTL responses in the mouse.

IkappaBepsilon-deficient mice: reduction of one T cell precursor subspecies and enhanced Ig isotype switching and cytokine synthesis.

Three major inhibitors of the NF-kappaB/Rel family of transcription factors, IkappaBalpha, IkappaBbeta, and IkappaBepsilon, have been described. To examine the in vivo role of the most recently discovered member of the IkappaB family, IkappaBepsilon, we generated a null allele of the murine IkappaBepsilon gene by replacement of all coding sequences with nlslacZ. Unlike IkappaBalpha nullizygous mice, mice lacking IkappaBepsilon are viable, fertile, and indistinguishable from wild-type animals in appearance and histology. Analysis of beta-galactosidase expression pattern revealed that IkappaBepsilon is mainly expressed in T cells in the thymus, spleen, and lymph nodes. Flow cytometric analysis of immune cell populations from the bone marrow, thymus, spleen, and lymph nodes did not show any specific differences between the wild-type and the mutant mice, with the exception of a reproducible 50% reduction of the CD44-CD25+ T cell subspecies. The IkappaBepsilon-null mice present constitutive up-regulation of IgM and IgG1 Ig isotypes together with a further increased synthesis of these two isotypes after immunization against T cell-dependent or independent Ags. The failure of observable augmentation of constitutive nuclear NF-kappaB/Rel-binding activity is probably due to compensatory mechanisms involving IkappaBalpha and IkappaBbeta, which are up-regulated in several organs. RNase-mapping analysis indicated that IL-1alpha, IL-1beta, IL-1Ra, and IL-6 mRNA levels are constitutively elevated in thioglycolate-elicited IkappaBepsilon-null macrophages in contrast to GM-CSF, G-CSF, and IFN-gamma, which remain undetectable.

Antibodies to human myelin proteins and gangliosides in patients with acute neuroparalytic accidents induced by brain-derived rabies vaccine.

Antibody responses to myelin antigens were analysed in 15 patients who developed acute neuroparalytic accidents (ANPA) during post-exposure rabies vaccination using a rabies vaccine prepared on brain tissues and in 30 individuals who were uneventfully vaccinated. High titers (> or = 100) of IgG and IgM antibodies to GM1 or GD1a gangliosides were detected by enzyme linked immunosorbent-assay (ELISA) in plasmas from ANPA patients but not in controls. These data suggest that antibodies to GM1 and GD1a gangliosides may play a pathogenic role in the demyelinating and/or inflammatory processes characteristic of rabies vaccine-induced acute neurologic complications.

Natural autoantibodies, IgG antibodies to tetanus toxoid and CD5+ B cells in patients with Mediterranean visceral leishmaniasis. The Leishmania Study Group.

Natural autoantibodies (NaAb) and IgG antibodies to tetanus toxoid (TT) were analysed in the sera of 38 children with active visceral leishmaniasis (VL) previously vaccinated with TT and in 30 healthy controls matched for sex and age. Patients exhibited high levels of NaAb to a panel of self antigens (tubulin, myosin, myoglobin, actin) contrasting to a low level of IgG to TT. Analysis of the circulating B cells in 26 untreated patients showed a low percentage of CD5+ per total B cells (3-66%, mean 36.6%) compared with 14 normal controls (17.8-66.6%, mean 52.7%) (P < 0.001). Evaluation of these parameters after antimonial therapy showed a significant decrease of the level of the NaAb (P < 0.0005), and a spontaneous increase of the level of the IgG to TT without any vaccine boosting (P < 0.01). In contrast, there was a significant increase in CD5+ B cells (P < 0.0005). This result suggests that CD5+ B cells may be sequestrated in parasitized lymphoid organs and may be released after remission. These findings show that the polyclonal B cell activation that occurs during active VL involves mainly B cells bearing NaAb and are in favour of a functional dichotomy of B cells.