Transposon mutagenesis provides a direct selection for mutants and is an extremely powerful technique to analyze genetic functions in a variety of prokaryotes. Transposon mutagenesis of Mycobacterium tuberculosis has been limited in part because of the inefficiency of the delivery systems. This report describes the development of conditionally replicating shuttle phasmids from the mycobacteriophages D29 and TM4 that enable efficient delivery of transposons into both fast-and slow-growing mycobacteria. These shuttle phasmids consist of an Escherichia coli cosmid vector containing either a miniTn10(kan) or Tn5367 inserted into a nonessential region of the phage genome. Thermosensitive mutations were created in the mycobacteriophage genome that allow replication at 30°C but not at 37°C (

BackgroundNothing is known about the epidemiology and resistance mechanisms of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE) in Burkina Faso. The objective of this study was to determine ESBL-PE prevalence and to characterize ESBL genes in Burkina Faso.MethodsDuring 2 months (June-July 2014), 1602 clinical samples were sent for bacteriologic investigations to the microbiology laboratories of the tree main hospitals of Burkina Faso. Isolates were identified by mass spectrometry using a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) BioTyper. Antibiotic susceptibility was tested using the disk diffusion method on Müller-Hinton agar. The different ESBL genes in potential ESBL-producing isolates were detected by PCR and double stranded DNA sequencing. Escherichia coli phylogenetic groups were determined using a PCR-based method.ResultsESBL-PE frequency was 58 % (179 strains among the 308 Enterobacteriaceae isolates identified in the collected samples; 45 % in outpatients and 70 % in hospitalized patients). The CTX-M-1 group was dominant (94 %, CTX-M-15 enzyme), followed by the CTX-M-9 group (4 %). ESBL producers were more often found in E. coli (67.5 %) and Klebsiella pneumoniae (26 %) isolates. E. coli isolates (n = 202; 60 % of all Enterobacteriaceae samples) were distributed in eight phylogenetic groups (A = 49, B1 = 15, B2 = 43, C = 22, Clade I = 7, D = 37, F = 13 and 16 unknown); 22 strains belonged to the sequence type ST131. No association between a specific strain and ESBL production was detected.ConclusionsThis report shows the alarming spread of ESBL genes in Burkina Faso. Public health efforts should focus on education (population and healthcare professionals), surveillance and promotion of correct and restricted antibiotic use to limit their dissemination.

Among the seven species characterized within the genus Veillonella, three (Veillonella dispar, Veillonella parvula and Veillonella atypica) have so far been isolated from human flora and during infectious processes. Sequencing and analysis of 16S rDNA (rrs) has been described as the best method for identification of Veillonella strains at the species level since phenotypic characteristics are unable to differentiate between species. rrs sequencing for the three species isolated from humans showed more than 98 % identity between them. Four rrs copies were found in the reference strains and in all the clinical isolates studied. The sequences of each rrs were determined for the clinical strain ADV 360.1, and they showed a relatively high level of heterogeneity (1?43 %). In the majority of cases, polymorphic positions corresponded to nucleotides allowing differentiation between the three species isolated from humans. Moreover, variability observed between rrs copies was higher than that between 16S rDNA sequences of V. parvula and V. dispar. Phylogenetic analysis showed that polymorphism between rrs copies affected the position of strain ADV 360.1 in the tree. Variable positions occurred in stems and loops belonging to variable and hypervariable regions of the 16S rRNA secondary structure but did not change the overall structure of the 16S rRNA. PCR-RFLP experiments performed on 27 clinical isolates of Veillonella sp. suggested that inter-rrs heterogeneity occurs widely among the members of the genus Veillonella. These results, together with the lack of phenotypic criteria for species differentiation, give preliminary arguments for unification of V. dispar and V. parvula.

A panel of 28 insertion mutants of the human immunodeficiency virus type 1 (HIV-1) Gag precursor (Pr55Gag) was constructed by linker-insertion mutagenesis and expressed in recombinant baculovirus-infected insect cells. One set of 14 mutants carried the normal N-myristylation signal; the other set constituted their non-N-myristylated counterparts. The mutants were characterized with respect to (i) assembly and extracellular release of membrane-enveloped budding Gag particles, (ii) intracellular assembly and nuclear transport of Gag cores, (iii) specific processing of P655(ag by HIV-1 protease in vivo, and (iv) binding of Pr55Gag to an HIV-1 genomic RNA probe in Northwestern blotting. Insertions within the region between amino acid residues 209 and 334 in the CA domain appeared to be the most detrimental to Gag particle assembly and release of Gag into the external medium, whereas a narrower window, between residues 209 and 241, was found to be critical for secretion of soluble Pr55Ga. Differences in Pr55(;'a processing in vivo and RNA binding in vitro between N-myristylated and non-N-myristylated Gag mutants suggested a major conformational role for the myristylated N terminus of Gag precursor. In coinfection experiments using wild-type Gagand mutant Gag-expressing recombinants, a transdominant negative effect on Gag particle assembly and release was observed for insertions located in two separate domains, the matrix and nucleocapsid.

We conducted a molecular epidemiology study on 120 Mycobacterium tuberculosis isolates from patients presenting pulmonary tuberculosis (TB) in Burkina Faso. Classical antibiogram studies and genetic characterization, using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing and spoligotyping, were applied after culture. Molecular analysis of specific signatures showed that all TB cases reported in this study were caused by M. tuberculosis and identified no Mycobacterium bovis or Mycobacterium africanum isolates. This result is unexpected, as M. africanum strains were reportedly the etiologic agent in 20% of TB cases 2 decades ago. The comparison of spoligotypes from Burkina Faso with an international spoligotype database (SpolDB4) showed that the majority of isolates belong to major clades of M. tuberculosis (Haarlem, 9%; Latin American-Mediterranean, 30%; and T, 20%). The predominant group of isolates (30%) corresponds to spoligotype 61, described in Cameroon as the "Cameroon family." In Burkina Faso, as in Cameroon, this family could be associated with recent transmission of TB, suggesting a recent expansion in West Africa. Our data suggest a low level of primary drug resistance that may be a positive result of the Directly Observed Therapy Shortcourse program. Besides, based on spoligotyping plus MIRU-VNTR, data showed a high number of clusters in our sample, suggesting a high level of recent TB transmission in Burkina Faso. Nevertheless, an important genetic polymorphism was observed in this country, reflecting an endemicity situation where the control of TB would have less impact in the main towns.

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The determinants for HIV-1 particle morphology were investigated using various deletion and insertion mutants of the Gap precursor protein (Gag) expressed in baculovirus-infected cells and ultrastructural analysis of membrane-enveloped Gag particles under the electron microscope. Five discrete regions were found to influence the size, the variability in dimension, and the sphericity of the particles: (i) the matrix (MA) N-terminal domain, within residues 10-21, the junctions of (ii) MA-CA (capsid), (iii) CA-spacer peptide SP1 and (iv) nucleocapsid (NC)-SP2, and (v) the p6gag C-terminus. Internal regions (ii), (iii), and (iv) contained HIV-1 protease cleavage sites separating major structural domains. No particle assembly was observed for am276, a MA-CA polyprotein mutant lacking the C-terminal third of the CA domain. However, MA-CA domains including the MHR (residues 277-306), or downstream sequence to CA residue 357, resulted in the assembly into tubular or filamentous structures, suggesting a helical symmetry of Gag packing. Mutant amb374, derived from amb 357 by further addition of the heptadecapeptide motif HKARVLAEAMSQVTNSA, overlapping the CA-SP1 junction and the SP1 domain, showed a drastic change in the pattern of Gag assembly, compared to amb357, with formation of spherical particles. These data suggested a novel function for the spacer domain SP1, acting as a spherical shape determinant of the Gag particle which would negatively affect the helical symmetry of assembly of the Gag precursor molecules conferred by the MHR and the downstream CA sequence, within residues 307-357.

OBJECTIVETo extend our previous work on evaluating the use of oligonucleotide arrays to discriminate colonization from infection owing to Staphylococcus aureus in diabetic foot ulcers (DFUs).RESEARCH DESIGN AND METHODSPatients admitted to 14 French diabetic foot departments for a DFU were screened for entry into the study. At admission, ulcers were classified based on clinical examination according to the Infectious Diseases Society of America system. Only patients with monomicrobial culture for S. aureus were included. In persons with an uninfected ulcer, a second wound bacterial specimen was obtained 1 month later. Using oligonucleotide arrays, S. aureus resistance and virulence genes were determined, and each isolate was affiliated to a clonal complex (CC).RESULTSS. aureus was initially isolated from 75 uninfected and 120 infected ulcers; 35 were methicillin resistant. A total of 44 (59%) strains from uninfected DFUs belonged to CC5/CC8 clones vs. 6 (5%) from infected DFUs (P < 0.001). During follow-up, 57 (76%) of uninfected DFUs healed or had a favorable outcome; the strain in 49 (86%) of them belonged to CC5/CC8. Conversely, 18 (24%) had a poor outcome but not a single strain belonged to CC5/CC8 clone. Moreover, lukDE was significantly associated with a favorable outcome of the wound.CONCLUSIONSAs suggested by our previous study, the use of DNA arrays appears to be a promising technique that might help distinguishing uninfected from infected wounds, predicting ulcer outcome and then contributing to a more adequate use of antibiotics.