Pulsed field gel electrophoresis of group A streptococci

Here we describe the protocols to perform PFGE analysis of chromosomal DNA from the bacterial species Streptococcus pyogenes (group A streptococcus, GAS) after digestion with the restriction enzyme SmaI. Large parts of the procedures are suitable for application to DNA digested with other restriction enzymes as well. We have put an effort to present extensions to solve possible limitations to the discriminatory power of the method in the specific case of S. pyogenes

Antimicrobial Activity of Single And Combined Extracts of Medicinal Plants From Cameroon

Selected Cameroonian plants have been investigated for their antifungal and antibacterial activity against five species: four bacteria, namely S. aureus, S. pyogenes, S. mutans, Pseudomonas aeruginosa and the yeast C. albicans. The solvents used for plant extraction were either ethanol or water. The in vitro antimicrobial activity was performed by disk diffusion and microdilution method. The aqueous extracts showed no activity whereas the ethanolic extracts showed a significant antibacterial activity, which may be associated to the high content in tannins shown by some of the extracts. In conclusion, this work adds to the accumulating evidences supporting the development of new alternatives to antibiotics based on the use of natural products

Molecular cloning and biochemical characterization of Xaa-Pro dipeptidyl-peptidase from Streptococcus mutans and its inhibition by anti-human DPP IV drugs

Streptococcus mutans harbours an intracellular, human DPP IV-analogous enzyme Xaa-Pro dipeptidyl-peptidase (EC 3.4.14.11). According to previous reports, an extracellular isozyme in S. gordonii and S. suis has been associated with virulence. Speculating that even an intracellular form may aid in virulence of S. mutans, we have tried to purify, characterize and evaluate enzyme inhibition by specific inhibitors. The native enzyme was partially purified by ion-exchange and gel filtration chromatography. Owing to low yield, the enzyme was overexpressed in Lactococcus lactis and purified by affinity chromatography. The recombinant enzyme (rSm-XPDAP) had a specific activity of 1070 U mg-1, while the Vmax and Km were 7 μM min-1 and 89 ± 7 μM (n = 3), respectively. The serine protease inhibitor phenylmethylsulphonyl fluoride and a DPP IV-specific inhibitor diprotin A proved to be active against rSm-XPDAP. As a novel approach, the evaluation of the effect of anti-human DPP IV (AHD) drugs on rSm-XPDAP activity found saxagliptin to be effective to some extent (Ki = 129 ± 16 μM), which may lead to the synthesis and development of a new class of antimicrobial agents

Analysis of meticillin-susceptible and meticillin-resistant biofilm-forming Staphylococcus aureus from catheter infections isolated in a large Italian hospital

Several characteristics were analysed in 37 Staphylococcus aureus isolates from nosocomial catheter infections: the PFGE profile after SmaI digestion of chromosomal DNA, the ability to form a biofilm on a polystyrene surface, antibiotic susceptibility patterns (penicillin, oxacillin, erythromycin, tetracycline, clindamycin, telithromycin, gentamicin, ciprofloxacin, quinupristin/dalfopristin, rifampicin, vancomycin and linezolid), and the presence of genetic determinants of antibiotic resistance and biofilm formation. All strains but three (92 %) were able to grow on a plastic surface as a biofilm. An almost complete association was found between phenotypes and genotypic traits of antibiotic resistance, whilst PFGE profiling showed the highly polyclonal composition of the set of strains under study. Sixteen isolates (43 %) were meticillin-resistant and were subjected to staphylococcal cassette chromosome mec (SCCmec) and cassette chromosome recombinase (ccr) complex type determination by multiplex PCR. Only a subgroup of six strains belonged to the archaic clone PFGE type and bore the SCCmec/ccrAB type I structure. Among the remaining strains some presented small rearrangements of the SCCmec/ccrAB genetic locus, whilst others could barely be traced back to a known structural type. These observations suggest that, at the local level and at a particular site of infection, S. aureus may show great genetic variability and escape the general rule of expansion of the S. aureus pandemic clones

Analysis of different genetics traits and their association with biofilm formation in Staphylococcus epidermidis isolates from central venous catheter infections

The aim of the present study was to characterize clinical isolates of Staphylococcus epidermidis, one of the bacterial species most often implicated in foreign-body-associated infections, for their ability to form biofilms and for the presence of mecA and IS256 element. Sixty-seven Staphylococcus epidermidis clinical isolates, obtained from implantable medical devices, were investigated. Overall, 70% of the strains were positive for ica operon genes, 85% possessed atlE, and 46% contained aap. In 89% of the population, the Congo red agar test confirmed the correlation between the presence of ica genes and slime expression. Almost all of the strains could be classified as biofilm producers by both the crystal violet assay and microscopy. The bacterial population studied showed a very high frequency of strains positive for mecA as well as for the IS256 element. Although well-structured biofilms have been previously observed only in those strains possessing genes belonging to the ica operon, this study demonstrates that strains lacking specific biofilm-formation determinants can be isolated from catheters and can form a biofilm in vitro. Hence, different and yet-to-be identified factors may work together in the formation and organization of complex staphylococcal microbial communities and sustain infections associated with implanted medical devices

Decline in macrolide resistance rates among Streptococcus pyogenes causing pharyngitis in children isolated in Italy

Macrolides are often used to treat group A streptococcus (GAS) infections, but their resistance rates reached high proportions worldwide. The aim of the present study was to give an update on the characteristics and contemporary prevalence of macrolide-resistant pharyngeal GAS in Central Italy. A total of 592 isolates causing pharyngitis in children were collected in the period 2012-2013. Clonality was assessed by emm typing and pulsed-field gel electrophoresis (PFGE) for all macrolide-resistant strains and for selected susceptible isolates. Genetic determinants of resistance were screened by polymerase chain reaction (PCR). Forty-four GAS were erythromycin-resistant (7.4 %). Among them, 52.3 % and 50 % were clindamycin- and tetracycline-resistant, respectively. erm(B)-positive isolates (52.3 %) expressed the constitutive cMLSB phenotype. mef(A) and its associated M phenotype were recorded in 40.9 % of the cases. The remaining erm(A)-positive isolates expressed the iMLSB phenotype. Seventeen tetracycline-resistant isolates carried tet(M) and five isolates carried tet(O). Twenty-five emm types were found among all strains, with the predominance of emm types 12, 89, 1, and 4. Eleven emm types and 12 PFGE clusters characterized macrolide-resistant strains, with almost two-thirds belonging to emm12, emm4, and emm11. Macrolide-susceptible and -resistant emm types 12, 89, 11, and 4 shared related PFGE profiles. There was a dramatic decline in macrolide resistance in Central Italy among pharyngeal GAS isolates in 2012-2013 when compared to previous studies from the same region (p < 0.05), although macrolide consumption remained stable over the past 15 years. We observed a decrease in the proportion of macrolide-resistant strains within emm types commonly associated with macrolide resistance in the past, namely emm12, 1, and 89

Francisella-like endosymbionts, potentially harmful to human health, are transported by the universally distributed species of the ciliate Euplotes.

Genome analyses of wild-type strains of two ecologically separated Euplotes species, E. raikovi living in temperate sea waters and E. petzi living in the polar seas, revealed that both host bacteria in their cytoplasm. These bacteria have been identified with facultative intracellular gamma-proteobacteria of the genus Francisella, which includes a number of closely related species well known as extremely infectious to a great variety of organisms. Francisella tularensis, with its four subspecies, is a specialized intracellular pathogen capable of infecting both invertebrate and vertebrate hosts, humans included; F. noatunensis is the etiological agent of the fish disease known as francisellosis, and its two subspecies well adapt to different temperatures of their hosts; the Francisella-like endosymbionts Wolbachia persica, together with the freely living generalists F. philomiragia and F. novicida cause diseases in humans with a compromised immune system. The Francisella endosymbionts of E. raikovi and E. petzi have been successfully isolated and their genomes completely sequenced. They are genetically distant from one another and form two different clades in the Francisella phylogenetic tree, which are distinct from the all other well-established Francisella clades. The finding that Francisella has equally colonized polar and temperate-water species provides evidence that this bacterium is more common and widespread than previously hypothesized, and confirms that free-living Euplotes species and ciliates in general, with their worldwide distribution, may represent a natural reservoir of Francisella in every aquatic environment

Structure/activity virtual screening and in vitro testing of small molecule inhibitors of 8-hydroxy-5-deazaflavin:NADPH oxidoreductase from gut methanogenic bacteria

Abstract Virtual screening techniques and in vitro binding/inhibitory assays were used to search within a set of more than 8,000 naturally occurring small ligands for candidate inhibitors of 8-hydroxy-5-deazaflavin:NADPH oxidoreductase (FNO) from Methanobrevibacter smithii, the enzyme that catalyses the bidirectional electron transfer between NADP+ and F420H2 during the intestinal production of CH4 from CO2. In silico screening using molecular docking classified the ligand-enzyme complexes in the range between − 4.9 and − 10.5 kcal/mol. Molecular flexibility, the number of H-bond acceptors and donors, the extent of hydrophobic interactions, and the exposure to the solvent were the major discriminants in determining the affinity of the ligands for FNO. In vitro studies on a group of these ligands selected from the most populated/representative clusters provided quantitative kinetic, equilibrium, and structural information on ligands' behaviour, in optimal agreement with the predictive computational results