In Pseudomonas aeruginosa, N-acylhomoserine lactone signals regulate the expression of several hundreds of genes, via the transcriptional regulator LasR and, in part, also via the subordinate regulator RhlR. This regulatory network termed quorum sensing contributes to the virulence of P. aeruginosa as a pathogen. The fact that two supposed PAO1 wild-type strains from strain collections were found to be defective for LasR function because of independent point mutations in the lasR gene led to the hypothesis that loss of quorum sensing might confer a selective advantage on P. aeruginosa under certain environmental conditions. A convenient plate assay for LasR function was devised, based on the observation that lasR mutants did not grow on adenosine as the sole carbon source because a key degradative enzyme, nucleoside hydrolase (Nuh), is positively controlled by LasR. The wild-type PAO1 and lasR mutants showed similar growth rates when incubated in nutrient yeast broth at pH 6.8 and 37°C with good aeration. However, after termination of growth during 30 to 54 h of incubation, when the pH rose to > 9, the lasR mutants were significantly more resistant to cell lysis and death than was the wild type. As a consequence, the lasR mutant-to-wild-type ratio increased about 10-fold in mixed cultures incubated for 54 h. In a PAO1 culture, five consecutive cycles of 48 h of incubation sufficed to enrich for about 10% of spontaneous mutants with a Nuh ؊ phenotype, and five of these mutants, which were functionally complemented by lasR ؉ , had mutations in lasR. The observation that, in buffered nutrient yeast broth, the wild type and lasR mutants exhibited similar low tendencies to undergo cell lysis and death suggests that alkaline stress may be a critical factor providing a selective survival advantage to lasR mutants.

No abstract

The goal of this study was to estimate the distribution of pathogens, as well as their antimicrobial resistance pattern, in cows affected by clinical or subclinical mastitis in the Rhône-Alpes region of France. A total of 1770 samples were taken between January 2007 and March 2008, leading to the identification of 1631 bacterial isolates. Streptococcus uberis (22.1%), Escherichia coli (16%), and coagulase-positive staphylococci (15.8%) were identified as the major causative agents of clinical mastitis, whereas coagulase-positive staphylococci (30.2%), coagulase-negative staphylococci (13.7%), and Streptococcus dysgalactiae (9.3%) were predominantly implicated in subclinical mastitis. Yet, in both types of mastitis, about 20% of all cases were due to a large number of different bacterial species that were isolated at a low frequency (<5%), which cannot be considered as minor (e.g., Klebsiella spp.) or noncontagious (e.g., Corynebacterium spp.). The overall proportion of antibiotic resistance was low, except for penicillin G in staphylococci, as well as for macrolides and tetracycline in streptococci. Yet, these resistance proportions were much lower than those reported in human medicine. Besides providing up-to-date information on mastitis in France, this survey also indicates the prudent use of antibiotics by veterinarians. As a result, this study suggests that the risk of transmission of resistant bacteria from milk or milk products to human is very limited, even in case of consumption of raw milk. However, it also confirms the fact that attention must be maintained to avoid any emergence of such resistant bacteria.

We investigated the evolution and epidemiology of a novel livestock-associated methicillin-resistant Staphylococcus aureus strain, which colonizes and infects urban-dwelling Danes even without a Danish animal reservoir. Genetic evidence suggests both poultry and human adaptation, with poultry meat implicated as a probable source.

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryoelectron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.Gram-positive bacteria possess a cell envelope composed of a plasma membrane and a thick network of peptidoglycan, secondary acids, and proteins, which maintains cell shape and provides resistance to osmotic pressure (39). Electron microscopy studies performed on fixed (either chemically or by freezing) and dehydrated specimens favored the widely accepted model of the cell envelope consisting of the peptidoglycan network in direct contact with the plasma membrane (4). Nevertheless, the existence of a periplasmic space separated from the cytoplasm and the outer medium by a plasma membrane and a thick cell wall has been suggested by previous biochemical studies (3,14,25). Recent data obtained with cryo-electron microscopy of vitreous sections (CEMOVIS), a method that allows the observation of biological specimens in the closest-to-native state, challenged the classical model (23,24). CEMOVIS consists of cooling the specimen at high pressure so rapidly that water cannot crystallize and instead becomes vitreous. Water stays liquid, but its viscosity dramatically increases (10). It is then possible to make thin sections at low temperatures and observe them using a cryo-electron microscope (1). Consequently, water does not need to be removed from the sample, and molecular aggrega...

Bovine isolates of Streptococcus agalactiae (n ‫؍‬ 76), Streptococcus dysgalactiae subsp. dysgalactiae (n ‫؍‬ 32), and Streptococcus uberis (n ‫؍‬ 101) were analyzed for the presence of different integrative and conjugative elements (ICEs) and their association with macrolide, lincosamide, and tetracycline resistance. The diversity of the isolates included in this study was demonstrated by multilocus sequence typing for S. agalactiae and pulsedfield gel electrophoresis for S. dysgalactiae and S. uberis. Most of the erythromycin-resistant strains carry an ermB gene. Five strains of S. uberis that are resistant to lincomycin but susceptible to erythromycin carry the lin(B) gene, and one has both linB and lnuD genes. In contrast to S. uberis, most of the S. agalactiae and S. dysgalactiae tetracycline-resistant isolates carry a tet(M) gene. A tet(S) gene was also detected in the three species. A Tn916-related element was detected in 30 to 50% of the tetracycline-resistant strains in the three species. Tetracycline resistance was successfully transferred by conjugation to an S. agalactiae strain. Most of the isolates carry an ICE integrated in the rplL gene. In addition, half of the S. agalactiae isolates have an ICE integrated in a tRNA lysine (tRNA Lys ) gene. Such an element is also present in 20% of the isolates of S. dysgalactiae and S. uberis. A circular form of these ICEs was detected in all of the isolates tested, indicating that these genetic elements are mobile. These ICEs could thus also be a vehicle for horizontal gene transfer between streptococci of animal and/or human origin.

Crushed seeds of the Moringa oleifera tree have been used traditionally as natural flocculants to clarify drinking water. We previously showed that one of the seed peptides mediates both the sedimentation of suspended particles such as bacterial cells and a direct bactericidal activity, raising the possibility that the two activities might be related. In this study, the conformational modeling of the peptide was coupled to a functional analysis of synthetic derivatives. This indicated that partly overlapping structural determinants mediate the sedimentation and antibacterial activities. Sedimentation requires a positively charged, glutaminerich portion of the peptide that aggregates bacterial cells. The bactericidal activity was localized to a sequence prone to form a helix-loop-helix structural motif. Amino acid substitution showed that the bactericidal activity requires hydrophobic proline residues within the protruding loop. Vital dye staining indicated that treatment with peptides containing this motif results in bacterial membrane damage. Assembly of multiple copies of this structural motif into a branched peptide enhanced antibacterial activity, since low concentrations effectively kill bacteria such as Pseudomonas aeruginosa and Streptococcus pyogenes without displaying a toxic effect on human red blood cells. This study thus identifies a synthetic peptide with potent antibacterial activity against specific human pathogens. It also suggests partly distinct molecular mechanisms for each activity. Sedimentation may result from coupled flocculation and coagulation effects, while the bactericidal activity would require bacterial membrane destabilization by a hydrophobic loop.Diverse communities in the world traditionally use different natural agents from animal or vegetal sources as raw water additives to produce drinking water. Systematic studies have shown that among the different plant-derived materials tested, Moringa oleifera seeds seem to be one of the most effective primary water treatments (17, 28). Water-soluble proteins released from the crushed seed kernels function as natural flocculating agents, which have been proposed to bind and crosslink particles suspended in a colloidal structure, forming larger sedimenting particles (17,18,29). Microorganisms are generally attached to solid particles in raw water samples, and treatment employing Moringa seed powder can remove over 90% of the bacterial load (35). Unexpectedly, a study on the sedimentation effect of the seed-derived peptide termed Flo indicated also that it mediates bacterial disinfection, being able to kill antibiotic-resistant bacteria, including several human pathogens (62).The cationic nature of the Flo polypeptide is characteristic of a wider group of cationic peptide antibiotics that are usually less than 10 kDa in size and display an overall net positive charge. Cationic antimicrobial peptides (AMPs) make part of the innate immunity response, which is the first line of defense against pathogens. Most of the AMPs are expressed constitu...

No abstract