Multiplex PCR-based identification of Streptococcus canis, Streptococcus zooepidemicus and Streptococcus dysgalactiae subspecies from dogs.

Streptococcus canis (S. canis), Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) and Streptococcus dysgalactiae subspecies (S. dysgalactiae subspecies) are ß-haemolytic Gram positive bacteria infecting animals and humans. S. canis and S. zooepidemicus are considered as two of the major zoonotic species of Streptococcus, while more research is needed on S. dysgalactiae subspecies bacteria. In this work, a multiplex-PCR protocol was tested on strains and clinical samples to detect S. canis, S. dysgalactiae subspecies and S. equi subspecies bacteria in dogs. All strains were correctly identified as S. canis, S. equi subspecies or S. dysgalactiae subspecies by the multiplex-PCR. The main Streptococcus species isolated from symptomatic dogs were confirmed S. canis. The multiplex-PCR protocol described is a rapid, accurate and efficient method for identifying S. canis, S. equi subspecies and S. dysgalactiae subspecies in dogs and could be used for diagnostic purposes and for epidemiological studies.

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.

Characterization of levofloxacin non-susceptible clinical Streptococcus pyogenes isolated in the central part of Italy.

We investigated the prevalence, genetics, and clonality of fluoroquinolone non-susceptible isolates of Streptococcus pyogenes in the central part of Italy. S. pyogenes strains (n = 197) were isolated during 2012 from patients with tonsillopharyngitis, skin, wound or invasive infections and screened for fluoroquinolone non-susceptibility (resistance to norfloxacin and levofloxacin minimum inhibitory concentration (MIC) = 2 mg/L) following EUCAST guidelines. First-step topoisomerase parC and gyrA substitutions were investigated using sequencing analysis. Clonality was determined by pulsed field gel electrophoresis (PFGE; SmaI digestion) and by emm typing. The fluoroquinolone non-susceptible phenotype was identified in 18 isolates (9.1 %) and correlated with mutations in parC, but not in gyrA, the most frequent leading to substitution of the serine at position 79 with an alanine. Most of the fluoroquinolone non-susceptible isolates belonged to the emm-type 6, even if other emm-types were also represented (emm75, emm89, and emm2). A significant level of association was measured between PFGE and both emm type and substitutions in parC. The prevalence of fluoroquinolone non-susceptible Streptococcus pyogenes isolates in Italy is of concern and, although the well-known emm type 6 is dominant, other types are appearing and spreading.

Erythrocyte aggregation in patients with systemic lupus erythematosus.

Systemic Lupus Erythematosus (SLE) is an autoimmune, chronic inflammatory, non-organ specific disease. SLE patients present a high prevalence of thrombotic and arteriosclerotic disease. The aim of the present work was to study the erythrocyte aggregation kinetics, and the effect of plasma factors, namely, immunoglobulin and fibrinogen concentration, as well as cell factors such as deformability and erythrocyte membrane lipid fluidity on the erythrocyte aggregation, in SLE patients and healthy controls. The results show that SLE patients red blood cells aggregate at higher rate and the aggregates size are also greater than controls due to an increase of immunoglobulin and plasma fibrinogen. The negative correlation between aggregation parameters and rigidity index could point out that the altered deformability diminishes the erythrocyte aggregation. Correlation between rigidity index and anisotropy suggests that the decrease of membrane lipid fluidity might be a cause of deformability decrease. The erythrocyte aggregation increase in these patients could induce a decreased flow that might contribute to the thromboembolic process present in SLE patients.

Characterization of a Staphylococcus aureus strain showing high levels of biofilm formation isolated from a vascular graft: case report.

A methicillin-susceptible Staphylococcus aureus strain, SA-DZ1, was isolated from an infected bypass crossover graft. Its general microbiological features were reminiscent of those previously described for the wound Wiley strain. Removal of the prosthetic device was necessary to resolve the infection. SA-DZ1 grown under different conditions showed a very strong and distinctive biofilm-producing phenotype, which was also visualized by confocal laser scanning microscopy. The biofilm extracellular matrix was essentially polysaccharidic, as determined by differential growth and physicochemical tests. By Multi Locus Sequence Typing (MLST), SA-DZ1 was classified as st94, a single locus variant of st8. Several other genetic traits assayed by PCR, such as agr-type and the presence of gene encoding proteins involved in adhesion and virulence (e.g. ica operon), confirmed the identifying features of this clinical isolate.

Antibacterial activity and anti-biofilm effect of chitosan against strains of Streptococcus mutans isolated in dental plaque.

Streptococcus mutans is the major cause of dental plaque and is often associated with biofilm formation. The aim of this study is to evaluate the activity of a hydrosoluble derivative of chitosan against S. mutans biofilms in vitro and in vivo. Strains of S. mutans were isolated from the dental plaque of 84 patients enrolled in the study. The antibacterial activity of chitosan was determined by broth microdilutions. The effect of chitosan at different concentrations and exposure times on S. mutans biofilms at different phases of development was assessed by a clinical study using the classical "4-day plaque regrowth" experiment in adult volunteers. The MIC values of chitosan were between 0.5 and 2 g/L. Compared to distilled water, the chitosan solution significantly decreased the vitality of plaque microflora (p

Analysis of different genetic 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.

Distribution of mef(A)-containing genetic elements in erythromycin-resistant isolates of Streptococcus pyogenes from Italy.

In total, 124 Streptococcus pyogenes isolates were obtained from throat cultures of different symptomatic patients. All isolates showed M-phenotype macrolide resistance and contained the macrolide efflux gene mef(A). The isolates were screened for the presence and insertion site of mef(A)-containing genetic elements. In 25.8% of the isolates, mef(A) was found to be carried by elements belonging to the Tn1207.3/Phi10394.4 family inserted in the comEC gene, while 74.2% contained chimeric elements with a different genetic structure and chromosomal location, probably associated with the recently described 60-kb tet(O)-mef(A) element.

Strain-related virulence of the dominant Mycobacterium tuberculosis strain in the Canadian province of Manitoba.

SETTING: The Canadian province of Manitoba. OBJECTIVE: To confirm the putative hypervirulence observed in Mycobacterium tuberculosis Type1 strain and further characterize the progression and manifestation of pulmonary tuberculosis caused by this strain in comparison to other common clinical isolates from Manitoba. DESIGN: C3H and BALB/c mice were exposed to aerosols either of Type1, Type2, Type5, Type72 or H37Rv strain to study their respective survival profiles. Additionally, bacillary loads and lung histology were examined at 15 days post-exposure. RESULTS: In both mouse models, Type-1 infected mice succumbed to disease significantly earlier than other strains (p < or =0.0002). Differences between average log(10) CFU values between clinical isolates were less than 1log(10) difference. In C3H mice, the amount of granulomatous inflammation was highest in Type1 infected mice but not significantly different than all clinical strains. In contrast, BALB/c mice infected with Type1 induced the lowest amount of granulomatous inflammation compared to other clinical isolates. CONCLUSION: Our results indicate that although mice infected with the Type1 strain died significantly earlier than mice infected with other clinical strains in both C3H and BALB/c mice, the hypervirulence of the Type1 strain is not attributed to the growth rate of the organism, as differences in growth between clinical M. tuberculosis isolates were insignificant.

Translation initiation factor IF3: two domains, five functions, one mechanism?

Initiation factor IF3 contains two domains separated by a flexible linker. While the isolated N-domain displayed neither affinity for ribosomes nor a detectable function, the isolated C-domain, added in amounts compensating for its reduced affinity for 30S subunits, performed all activities of intact IF3, namely: (i) dissociation of 70S ribosomes; (ii) shift of 30S-bound mRNA from 'stand-by' to 'P-decoding' site; (iii) dissociation of 30S-poly(U)-NacPhe-tRNA pseudo- initiation complexes; (iv) dissociation of fMet-tRNA from initiation complexes containing mRNA with the non-canonical initiation triplet AUU (AUUmRNA); (v) stimulation of mRNA translation regardless of its start codon and inhibition of AUUmRNA translation at high IF3C/ribosome ratios. These results indicate that while IF3 performs all its functions through a C-domain-30S interaction, the N-domain function is to provide additional binding energy so that its fluctuating interaction with the 30S subunit can modulate the thermodynamic stability of the 30S-IF3 complex and IF3 recycling. The localization of IF3C far away from the decoding site and anticodon stem-loop of P-site-bound tRNA indicates that the IF3 fidelity function does not entail its direct contact with these structures.