H. Monteil

Bartonella tribocorum sp. nov. : a new Bartonella species isolated from the blood of wild rats

Two Bartonella strains from blood of two wild rats (Rattus norvegicus) living in a rural environment were isolated. These strains were distinct from all previously known Bartonella species based on phenotypic and genotypic characteristics. This new species is distinguished by its trypsin-like activity, the absence of the ability to hydrolyse proline and tributyrin, its 16S rRNA and citrate synthase gene sequences and by whole-DNA hybridization data. This new species, for which the name Bartonella tribocorum sp. nov. is proposed, seems to be genetically related to Bartonella elizabethae, an agent isolated in a case of human endocarditis. The type strain of Bartonella tribocorum sp. nov. is IBS 506T (CIP 105476T).

The structure of a Staphylococcus aureus leucocidin component (LukF-PV) reveals the fold of the water-soluble species of a family of transmembrane pore-forming toxins

BACKGROUND Leucocidins and gamma-hemolysins are bi-component toxins secreted by Staphylococcus aureus. These toxins activate responses of specific cells and form lethal transmembrane pores. Their leucotoxic and hemolytic activities involve the sequential binding and the synergistic association of a class S and a class F component, which form hetero-oligomeric complexes. The components of each protein class are produced as non-associated, water-soluble proteins that undergo conformational changes and oligomerization after recognition of their cell targets. RESULTS The crystal structure of the monomeric water-soluble form of the F component of Panton-Valentine leucocidin (LukF-PV) has been solved by the multiwavelength anomalous dispersion (MAD) method and refined at 2.0 A resolution. The core of this three-domain protein is similar to that of alpha-hemolysin, but significant differences occur in regions that may be involved in the mechanism of pore formation. The glycine-rich stem, which undergoes a major rearrangement in this process, forms an additional domain in LukF-PV. The fold of this domain is similar to that of the neurotoxins and cardiotoxins from snake venom. CONCLUSIONS The structure analysis and a multiple sequence alignment of all toxic components, suggest that LukF-PV represents the fold of any water-soluble secreted protein in this family of transmembrane pore-forming toxins. The comparison of the structures of LukF-PV and alpha-hemolysin provides some insights into the mechanism of transmembrane pore formation for the bi-component toxins, which may diverge from that of the alpha-hemolysin heptamer.

Ion channels and bacterial infection: the case of β-barrel pore-forming protein toxins of Staphylococcus aureus

Staphylococcus aureus strains causing human pathologies produce several toxins, including a pore‐forming protein family formed by the single‐component α‐hemolysin and the bicomponent leukocidins and γ‐hemolysins. The last comprise two protein elements, S and F, that co‐operatively form the active toxin. α‐Hemolysin is always expressed by S. aureus strains, whereas bicomponent leukotoxins are more specifically involved in a few diseases. X‐ray crystallography of the α‐hemolysin pore has shown it is a mushroom‐shaped, hollow heptamer, almost entirely consisting of β‐structure. Monomeric F subunits have a very similar core structure, except for the transmembrane stem domain which has to refold during pore formation. Large deletions in this domain abolished activity, whereas shorter deletions sometimes improved it, possibly by removing some of the interactions stabilizing the folded structure. Even before stem extension is completed, the formation of an oligomeric pre‐pore can trigger Ca2+‐mediated activation of some white cells, initiating an inflammatory response. Within the bicomponent toxins, γ‐hemolysins define three proteins (HlgA, HlgB, HlgC) that can generate two toxins: HlgA+HlgB and HlgC+HlgB. Like α‐hemolysin they form pores in planar bilayers with similar conductance, but opposite selectivity (cation instead of anion) for the presence of negative charges in the ion pathway. γ‐Hemolysin pores seem to be organized as α‐hemolysin, but should contain an even number of each component, alternating in a 1:1 stoichiometry.

Phylogeny of the Genus Corynebacterium Deduced from Analyses of Small-Subunit Ribosomal DNA Sequences

We determined almost complete small-subunit ribosomal DNA sequences of 50 reference strains belonging to the genera Corynebacterium, Rhodococcus, and Gordona and compared these sequences with previously published sequences. Three phylogenetic methods (the neighbor-joining, maximum-likelihood, and maximum-parsimony methods), as well as a bootstrap analysis, were used to assess the robustness of each topology which we obtained. The results of comparative phylogenetic analyses confirmed that the genera Corynebacterium, Dietzia, Gordona, Mycobacterium, Nocardia, Tsukamurella, and Turicella form a monophyletic taxon within the phylum containing the high-G+C-content gram-positive bacteria. The genus Corynebacterium appeared to be a monophyletic unit whose members could be divided into four major clusters. The validity of the genus Turicella is doubtful since members of this genus clearly belong to the genus Corynebacterium. The variability of chemotaxonomic characteristics within the genus Corynebacterium suggests that small-subunit ribosomal DNA sequence analysis is probably the most straightforward method for confirming that a bacterium belongs to this genus.

Bartonella bovis Bermond et al. sp. nov. and Bartonella capreoli sp. nov., isolated from European ruminants

Two novel species of Bartonella isolated from European ruminants are described. Bartonella capreoli sp. nov. was isolated from the blood of roe-deer (Capreolus capreolus) captured in Chizé, France. The type strain is IBS 193T (= CIP 106691T = CCUG 43827T). It is distinct from another European ruminant isolate that originated from a cow from a French herd of 430 dairy cattle. The latter isolate belongs to a novel species named Bartonella bovis Bermond et al. sp. nov. The type strain is strain 91-4T (= CIP 106692T = CCUG 43828T). The two bacteria appeared as small, fastidious, aerobic, oxidase-negative, gram-negative rods. Their biochemical properties were similar to those of members of the genus Bartonella. The sequences of the 16S rRNA and citrate synthase genes obtained from the two type strains were highly related to sequences of the different Bartonella species. Hybridization values when testing type strains of recognized Bartonella species, obtained with the nuclease/trichloroacetic acid method, support the creation of two novel species.

Species identities and antimicrobial susceptibilities of corynebacteria Isolated from various clinical sources

Over a 14-month period, 415 clinical isolates of coryneform gram-positive rods were recovered from various sources and identified to the species level according to recent identification schemes.Corynebacterium urealyticum, Corynebacterium striatum, Corynebacterium amycolatum, andCorynebacterium jeikeium predominated, accounting for 63% of all isolates.Corynebacterium accolens, Corynebacterium striatum, Corynebacterium argentoratense, Corynebacterium propinquum andCorynebacterium pseudodiphtheriticum were mostly recovered from the respiratory tract, whereasCorynebacterium afermentans, CDC group G, andCorynebacterium jeikeium were mainly isolated from blood. None of the isolates was identified asCorynebacterium diphtheriae orCorynebacterium xerosis. Ampicillin resistance was detected inCorynebacterium jeikeium (96%) andCorynebacterium urealyticum (99%) and varied amongCorynebacterium amycolatum (56%) and CDC group G (26%). These data emphasize the need for an accurate identification of coryneform organisms at the species level and for antimicrobial susceptibility testing of these organisms.

Flow Cytometric Determination of Panton-Valentine Leucocidin S Component Binding

ABSTRACT The binding of the S component (LukS-PV) from the bicomponent staphylococcal Panton-Valentine leucocidin to human polymorphonuclear neutrophils (PMNs) and monocytes was determined using flow cytometry and a single-cysteine substitution mutant of LukS-PV. The mutant was engineered by replacing a glycine at position 10 with a cysteine and was labeled with a fluorescein moiety. The biological activity of the mutant was identical to that of the native protein. It has been shown that LukS-PV has a high affinity for PMNs (Kd = 0.07 ± 0.02 nM, n = 5) and monocytes (Kd = 0.020 ± 0.003 nM,n = 3) with maximal binding capacities of 197,000 and 80,000 LukS-PV molecules per cell, respectively. The nonspecifically bound molecules of LukS-PV do not form pores in the presence of the F component (LukF-PV) of leucocidin. LukS-PV and HlgC share the same receptor on PMNs, but the S components of other staphylococcal leukotoxins, HlgA, LukE, and LukM, do not compete with LukS-PV for its receptor. Extracellular Ca2+ at physiological concentrations (1 to 2 nM) has only a slight influence on the LukS-PV binding, in contrast to its complete inhibition by Zn2+. The down-regulation by phorbol 12-myristate 13-acetate (PMA) of the binding of LukS-PV was blocked by staurosporine, suggesting that the regulatory effect of PMA depends on protein kinase C activation. The labeled mutant form of LukS-PV has proved very useful for detailed binding studies of circulating white cells by flow cytometry. LukS-PV possesses a high specific affinity for a unique receptor on PMNs and monocytes.

Bartonella birtlesii sp. nov., isolated from small mammals (Apodemus spp.)

Three strains isolated from Apodemus spp. were similar to Bartonella species on the basis of phenotypic characteristics. Futhermore, genotypic analysis based on sequence analysis of the 16S rRNA and gltA genes and on DNA-DNA hybridization showed that the three isolates represented a distinct and new species of Bartonella. The name Bartonella birtlesii is proposed for the new species. The type strain of B. birtlesii sp. nov. is IBS 325T (= CIP 106294T = CCUG 44360T).

High-performance liquid chromatography of antibiotics

High-performance liquid chromatographic (HPLC) monitoring of antimicrobial agents has recently become more widely used, and represents an interesting alternative to other methods. The methodology is characterized by good specificity and accuracy, and it is applicable to almost all antibiotics. This review first describes the successive steps to investigate for the development of an HPLC method for a new antibiotic, and how to make use of it. Particular emphasis is put on the problems related to the standardization of sample preparation and to the development of mobile phases for use with different molecules belonging to the same class. The second part of the review describes one or more HPLC techniques for a representative antibiotic of each major class.

Hospital routine analysis of penicillins, third-generation cephalosporins and aztreonam by conventional and high-speed high-performance liquid chromatography

A high-performance liquid chromatographic procedure for the measurement of fifteen beta-lactam antibiotics in body fluids is described, with special reference to high-speed techniques. The procedure involves a unique sample preparation before analysis for all the following fifteen compounds: benzylpenicillin, ampicillin, cloxacillin, ticarcillin, mezlocillin, azlocillin, piperacillin, cefotaxime and its desacetyl metabolite, cefsulodin, cefoperazone, cefmenoxime, ceftazidime, ceftriaxone and the monobactam aztreonam; thus all biological samples arriving at the laboratory can be treated in batch. Of these fifteen antibiotics, eleven can be chromatographed with the same type of mobile phase, which consists of a mixture of ammonium acetate and acetonitrile in various ratios. Three others need ionpairing chromatography because of their polarity, and ticarcillin requires citric acid. High-speed high-performance liquid chromatography seems to be particularly suitable for the routine analysis of beta-lactam antibiotics because columns equilibrate more rapidly, retention times are much shorter, detection limits are lower and the longer lifetime of columns reduces analysis costs.