Anthrax is an infectious disease caused by Bacillus anthracis, a bioterrorism agent that develops resistance to clinically used antibiotics. Therefore, alternative mechanisms of action remain a challenge. Herein, we disclose deoxy glycosides responsible for specific carbohydrate-phospholipid interactions, causing phosphatidylethanolamine lamellar-to-inverted hexagonal phase transition and acting over B. anthracis and Bacillus cereus as potent and selective bactericides. Biological studies of the synthesized compound series differing in the anomeric atom, glycone configuration and deoxygenation pattern show that the latter is indeed a key modulator of efficacy and selectivity. Biomolecular simulations show no tendency to pore formation, whereas differential metabolomics and genomics rule out proteins as targets. Complete bacteria cell death in 10 min and cellular envelope disruption corroborate an effect over lipid polymorphism. Biophysical approaches show monolayer and bilayer reorganization with fast and high permeabilizing activity toward phosphatidylethanolamine membranes. Absence of bacterial resistance further supports this mechanism, triggering innovation on membrane-targeting antimicrobials.

This study suggests that macrolides usage is an important factor enhancing the spread of invasive erythromycin-resistant S. pneumoniae clones in Portugal. The study also makes a contribution to the understanding of spread of erythromycin-resistant clones in an international context.

BackgroundThe serine/threonine kinase StkP of Streptococcus pneumoniae is a major virulence factor in the mouse model of infection. StkP is a modular protein with a N-terminal kinase domain a C-terminal PASTA domain carrying the signature of penicillin-binding protein (PBP) and prokaryotic serine threonine kinase. In laboratory cultures, one target of StkP is the phosphoglucosamine mutase GlmM involved in the first steps of peptidoglycan biosynthesis. In order to further elucidate the importance of StkP in S. pneumoniae, its role in resistance to β-lactams has been assessed by mutational analysis in laboratory cultures and its genetic conservation has been investigated in isolates from infected sites (virulent), asymptomatic carriers, susceptible and non-susceptible to β-lactams.ResultsDeletion replacement mutation in stkP conferred hypersensitivity to penicillin G and was epistatic on mutations in PBP2X, PBP2B and PBP1A from the resistant 9V clinical isolate URA1258. Genetic analysis of 55 clinical isolates identified 11 StkP alleles differing from the reference R6 allele. None relevant mutation in the kinase or the PASTA domains were found to account for susceptibility of the isolates. Rather the minimal inhibitory concentration (MIC) values of the strains appeared to be determined by their PBP alleles.ConclusionThe results of genetic dissection analysis in lab strain Cp1015 reveal that StkP is involved in the bacterial response to penicillin and is epistatic on mutations PBP 2B, 2X and 1A. However analysis of the clinical isolates did not allow us to find the StkP alleles putatively involved in determining the virulence or the resistance level of a given strain, suggesting a strong conservation of StkP in clinical isolates.

Quinolone resistance is usually due to mutations in the genes encoding bacterial topoisomerases. However, different reports have shown that neither clinical quinolone resistant isolates nor in vitro obtained Stenotrophomonas maltophilia mutants present mutations in such genes. The mechanisms so far described consist on efflux pumps’ overexpression. Our objective is to get information on novel mechanisms of S. maltophilia quinolone resistance. For this purpose, a transposon-insertion mutant library was obtained in S. maltophilia D457. One mutant presenting reduced susceptibility to nalidixic acid was selected. Inverse PCR showed that the inactivated gene encodes RNase G. Complementation of the mutant with wild-type RNase G allele restored the susceptibility to quinolones. Transcriptomic and real-time RT-PCR analyses showed that several genes encoding heat-shock response proteins were expressed at higher levels in the RNase defective mutant than in the wild-type strain. In agreement with this situation, heat-shock reduces the S. maltophilia susceptibility to quinolone. We can then conclude that the inactivation of the RNase G reduces the susceptibility of S. maltophilia to quinolones, most likely by regulating the expression of heat-shock response genes. Heat-shock induces a transient phenotype of quinolone resistance in S. maltophilia.

Between 1997 and 2000 nasopharyngeal specimens were obtained from 466 children < or = 12 years old attending the Pediatric Emergency Department at S. Francisco Xavier Hospital, Lisbon, to evaluate risk factors for nasopharyngeal carriage of Haemophilus influenzae and Streptococcus pneumoniae and to characterize their phenotype and antimicrobial susceptibility. The attending pediatrician completed written questionnaires about the children's demographic and clinical histories. Over half the children (52.8%) carried H. influenzae and/or S. pneumoniae. Forty-one percent of these children had H. influenzae, 22.8% had S. pneumoniae and 36.2% had both. Risk factors identified for carriage of respiratory pathogens were: age below 3 years (p < 0.05), black race (p < 0.01), attending a daycare center (p < 0.05), and having a lower respiratory infection (p < 0.05). Asthmatic children were less likely to be carriers (p = 0.004). About two-thirds of H. influenzae isolates were susceptible to all antibiotics tested, 7.9% were beta-lactamase producers, 16.4% were nonsusceptible to trimethoprim, and 6.9% were intermediately resistant to clarithromycin. Over half (57.1%) of S. pneumoniae isolates were susceptible to all antibiotics tested, 21.1% were multiresistant, 23.3% were nonsusceptible to penicillin, and about 20% were resistant to macrolides. Low-level resistance to third-generation cephalosporins was detected in 2.3%. The data reflect the controversy surrounding risk factors of nasopharyngeal colonization. These may have significant implications on clinical practice and on antimicrobial strategies to prevent the appearance of further resistant strains. Our findings highlight the importance to investigate the relationship between asthma and carriage.

The rates of invasive pneumococcal disease (IPD), serotype distribution and antimicrobial susceptibility prior to and after the introduction of the heptavalent pneumococcal conjugate vaccine in Portuguese children were evaluated. The changes in incidence of IPD in children under 1 year old between the two periods of the study was not significant (P=0.53), despite the 21% decline. In children under 18 years old there was a 27.7% decrease in vaccine serotypes. All nonvaccine serotypes increased 71.4%. The decrease in vaccine serotypes was more impressive during the first year of life (-54.8%) than for children between 1 and 5 years of age (-19.1%). Among children under 1 year old, penicillin nonsusceptible isolates declined between the two periods of the study (47.2% vs. 25.0%) (P=0.03), as did those of cefotaxime and ceftriaxone nonsusceptible isolates. No changes were observed for isolates nonsusceptible to tetracycline and macrolides. The serotypes of these nonsusceptible isolates differed after the introduction of vaccine (P=0.01). Multiresistance increased 57.1% after the introduction of vaccine. Multiresistant isolates with vaccine serotype declined 42.9% (P<0.001), and nonvaccine serotypes appeared during the vaccination period (P<0.001). These findings suggest a replacement of vaccine serotypes by nonvaccine serotypes, mainly among nonsusceptible isolates.

Streptococcus pneumoniae is an important pathogen responsible for serious invasive diseases, including meningitis and septicemia. The spread of multidrug-resistant (MDR) pneumococci has become a worldwide problem, making treatment more difficult (18). Indeed, in addition to resistance to penicillin, resistance to other antibiotics, including erythromycin, tetracycline, and chloramphenicol, has emerged and is spreading (10).Since 1989, the National Institute of Health Dr. Ricardo Jorge reference laboratory has been continually monitoring the in vitro activity of antimicrobial agents against S. pneumoniae collected from invasive sources. This program for monitoring susceptibility to antibiotics in Portugal (ARSIP) provides a unique collection of Portuguese pneumococcal isolates. This national surveillance study reported that 4.6% of isolates were penicillin nonsusceptible in 1989, and this value remained generally stable until 1991 (6.4%) (32). Fully penicillin-resistant isolates (MIC of 2 g/ml) were reported for the first time in 1992 (0.8%) and made up 5.5% of isolates in the following year (32).Here, we describe the surveillance of pneumococci by the reference laboratory in Portugal. We report the in vitro activities of different antimicrobial agents used against S. pneumoniae isolated from invasive sources over 11 years (from 1994 to 2004). MATERIALS AND METHODSPatients and bacterial isolates. Between 1 January 1994 and 31 December 2004, the ARSIP survey conducted by the Antibiotic Resistance Unit (ARU) from the National Institute of Health Dr. Ricardo Jorge constantly monitored pneumococcal isolates from cases of invasive disease in various regions of Portugal. The national laboratory-based surveillance system collected 1,331 invasive pneumococcal strains, which were isolated in 24 bacteriology laboratories in hospitals and public health institutions. In the period 1994 to 1998, 12 hospitals participated in the study, and since 1999, 12 more hospitals have been added to the network. Isolates were included if they were nonrepetitive or consecutive blood, cerebrospinal fluid (CSF), or pleural fluid samples from patients with symptoms compatible with invasive pneumococcal disease. No changes were made to the methods of data collection during the study. Some isolates were from outpatients, but most were from patients hospitalized with communityacquired invasive pneumococci disease. Only one isolate per patient was considered. Patients over 15 years old were considered to be adults.Identification and serotyping. The isolates were sent at Ϫ20°C by hospital laboratories to the reference laboratory, ARU, in Trypticase soy broth (TSB; Oxoid, Basingstoke, England) containing 20% glycerol. On reception by the ARU, the purity of the pneumococcal isolate was checked using standard methods, and the isolate was then stored at Ϫ80°C in TSB containing 20% glycerol. Isolates were serotyped by dot blotting, the Quellung reaction, or both (11).Antimicrobial susceptibility testing. Susceptibility testing was performed by the aga...

A complete tfs plasticity zone cluster is a virulence factor that may be important for the colonization of H. pylori and to the development of severe outcomes of the infection with cagA-positive strains.