Virulence patterns of Staphylococcus aureus strains from nasopharyngeal colonization.

BACKGROUND: The prevalence of nasopharyngeal colonization with Staphylococcus aureus can reach 20-30% among the population, which can lead to invasive infection. AIM: To investigate the prevalence of colonization among different age groups, and analyse S. aureus strain-specific virulence patterns. METHOD: For analysis of the prevalence of colonization, groups consisting of newborns, healthy volunteers aged 5-60 years, and nursing home residents aged >80 years were examined with nasopharyngeal swabs. After S. aureus was cultured, genetic analysis and phenotypic virulence testing were performed by cell-based assays. FINDINGS: Among 924 volunteers, the overall colonization rate was approximately 30%, with a peak in subjects aged 5-10 years (49%). Neonates and subjects aged >80 years showed different distributions of clonal clusters. Overall, the strains of all age groups exhibited virulence characteristics that can contribute to the development of infection. In particular, the neonatal strains exhibited a high incidence of toxin genes that resulted in increased cytotoxic effects compared with the other strains tested. CONCLUSIONS: Colonizing strains showed a virulence profile in all age groups, which may lead to the establishment of invasive infection. Consequently, decolonization measures could be considered for selected patients depending on the risk of infection.

Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain.

Host cell invasion is a major feature of Staphylococcus aureus and contributes to infection development. The intracellular metabolically active bacteria can induce host cell activation and death but they can also persist for long time periods. In this study a comparative analysis was performed of different well-characterized S. aureus strains in their interaction with a variety of host cell types. Staphylococcus aureus (strains 6850, USA300, LS1, SH1000, Cowan1) invasion was compared in different human cell types (epithelial and endothelial cells, keratinocytes, fibroblasts, osteoblasts). The number of intracellular bacteria was determined, cell inflammation was investigated, as well as cell death and phagosomal escape of bacteria. To explain strain-dependent differences in the secretome, a proteomic approach was used. Barrier cells took up high amounts of bacteria and were killed by aggressive strains. These strains expressed high levels of toxins, and possessed the ability to escape from phagolysosomes. Osteoblasts and keratinocytes ingested less bacteria, and were not killed, even though the primary osteoblasts were strongly activated by S. aureus. In all cell types S. aureus was able to persist. Strong differences in uptake, cytotoxicity, and inflammatory response were observed between primary cells and their corresponding cell lines, demonstrating that cell lines reflect only partially the functions and physiology of primary cells. This study provides a contribution for a better understanding of the pathomechanisms of S. aureus infections. The proteomic data provide important basic knowledge on strains commonly used in the analysis of S. aureus-host cell interaction.

Staphylococcus aureus develops increased resistance to antibiotics by forming dynamic small colony variants during chronic osteomyelitis.

OBJECTIVES: Staphylococcus aureus osteomyelitis often develops to chronicity despite antimicrobial treatments that have been found to be susceptible in in vitro tests. The complex infection strategies of S. aureus, including host cell invasion and intracellular persistence via the formation of dynamic small colony variant (SCV) phenotypes, could be responsible for therapy-refractory infection courses. METHODS: To analyse the efficacy of antibiotics in the acute and chronic stage of bone infections, we established long-term in vitro and in vivo osteomyelitis models. Antibiotics that were tested include ß-lactams, fluoroquinolones, vancomycin, linezolid, daptomycin, fosfomycin, gentamicin, rifampicin and clindamycin. RESULTS: Cell culture infection experiments revealed that all tested antibiotics reduced bacterial numbers within infected osteoblasts when treatment was started immediately, whereas some antibiotics lost their activity against intracellular persisting bacteria. Only rifampicin almost cleared infected osteoblasts in the acute and chronic stages. Furthermore, we detected that low concentrations of gentamicin, moxifloxacin and clindamycin enhanced the formation of SCVs, and these could promote chronic infections. Next, we treated a murine osteomyelitis model in the acute and chronic stages. Only rifampicin significantly reduced the bacterial load of bones in the acute phase, whereas cefuroxime and gentamicin were less effective and gentamicin strongly induced SCV formation. During chronicity none of the antimicrobial compounds tested showed a beneficial effect on bone deformation or reduced the numbers of persisting bacteria. CONCLUSIONS: In all infection models rifampicin was most effective at reducing bacterial loads. In the chronic stage, particularly in the in vivo model, many tested compounds lost activity against persisting bacteria and some antibiotics even induced SCV formation.

Molecular fingerprinting of Staphylococcus aureus isolated from patients with osteomyelitis in Argentina and clonal distribution of the cap5(8) genes and of other selected virulence genes.

The molecular fingerprinting of a collection of 94 Staphylococcus aureus isolates from patients with osteomyelitis in Argentina was performed. Twenty-three SmaI pulsed-field gel electrophoresis (PFGE) types and 37 spa types were identified. The isolates were assigned to 23 sequence types (STs). The proportion of methicillin-resistant S. aureus (MRSA) isolates was significantly higher among cap5 S. aureus (35/61) compared with cap8 S. aureus (8/33) isolates (p = 0.0025). Twenty-four of the 94 isolates carried the lukS-PV/lukF-PV genes, which were significantly associated to cap5 [(23/38) compared with cap8 S. aureus isolates (1/32) (p = 0.0001)]. Forty of the 94 isolates carried genes of the egc locus (seg/sei). The distribution of seg/sei genes among isolates was related to certain clones. Isolates of the four agr types were found in the S. aureus collection. Whereas agr I isolates were evenly distributed among cap5 and cap8 S. aureus isolates (32/61 and 14/33, respectively), the agr II group was composed of 29 cap5 S. aureus isolates and agr III was composed of 16 cap8 S. aureus isolates. Two clones originally associated to animals (ST 188, 7 isolates and ST 1796, 5 isolates) were associated with chronic osteomyelitis and lack of capsular polysaccharide (CP) production. Loss of CP production remains the single factor among those investigated that is associated with chronic osteomyelitis.

Characterization of a new variant of IS257 that has displaced the capsule genes within bovine isolates of Staphylococcus aureus.

Many bovine Staphylococcus aureus isolates from Argentina are nontypeable (NT), i.e., they do not produce serotype 5 or 8 capsular polysaccharides (CPs). Some of these NT strains have a deletion of the cap5(8) gene cluster mediated by a variant of IS257, now designated IScap. IScap showed 93% amino acid identity to S. aureus ORF49 but only 85% identity to IS431 from S. aureus N315 and 88% identity to an IS257-like element from bovine strain RF122. Thirty-six (53%) of 68 bovine isolates, drawn from a previously described S. aureus strain collection, carried some variant of IS257, including IScap. Of these 36 IS+ isolates, 6 were CP5+, 1 was CP8+, and 29 were NT. Forty-four of the 68 isolates were NT, and 24 of these 44 NT isolates (55%) exhibited IScap-mediated deletion of the cap5(8) gene cluster. IScap was not found among 20 human NT S. aureus isolates bearing the cap5HIJK genes, which suggests that IScap-mediated deletion of the capsule locus is restricted to bovine strains of S. aureus. We were unable to identify a precursor strain in which IScap flanked the cap5(8) capsule locus, nor were we able to select for deletion of the cap5(8) locus in vitro. Our results support the hypothesis that deletion of the cap5 locus occurred in the distant past and that the relative abundance of these NT strains may be a result of their ability to persist in subclinical mastitis infection in cows.

Lack of changes in serum prolactin, FSH, TSH, and estradiol after melatonin treatment in doses that improve sleep and reduce benzodiazepine consumption in sleep-disturbed, middle-aged, and elderly patients.

An open pilot study on the safety and efficacy of melatonin in the treatment of insomniac patients was conducted in 22 subjects (16 females), mean +/- S.D. age 60.1 +/- 9.5 years. All patients received 3 mg of gelatin melatonin capsules per os daily for 6 months, 30 min before expected sleep time. Twenty of 22 patients were on benzodiazepine treatment and they continued this treatment for part of or for the entire melatonin administration period. Serum concentrations of prolactin, follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), or estradiol were measured by radioimmunoassay (RIA) in morning samples at the beginning and after 6 months of melatonin administration, and standard clinical laboratory tests for blood components were performed. Urinary 6-sulphatoxymelatonin (aMT6s) excretion was measured by RIA before treatment. Serum concentrations of prolactin, FSH, TSH, or estradiol did not exhibit changes after 6 months of melatonin administration, nor were any indications of hematologic or blood biochemistry alteration found. Melatonin augmented significantly the quality and duration of sleep, and decreased sleep latency and the number of awakening episodes, as assessed from sleep logs filled by the patients (first 21 days) and from structured interviews performed by incumbent physicians (up to 6 months). Estimates of next-day function (i.e., alertness in the morning and during the day) also improved significantly during melatonin treatment. The observed effect lasted for the entire period examined (up to 6 months), with 22 out of 22 patients showing improved sleep at the end of treatment. The urinary excretion of aMT6s before starting administration of melatonin correlated negatively and significantly with age, but not with the intensity of sleep the disorder or the outcome of treatment. In 13 of 20 patients taking benzodiazepines together with melatonin, benzodiazepine use could be stopped, and in another four patients, benzodiazepine dose could be decreased to 25-66% of the initial dose. The results of this open, subacute administration trial indicate that melatonin is a safe and useful treatment for sleep disturbances in middle-aged or elderly patients, either by itself or together with benzodiazepines.

[Inhibitory and bactericidal activity and selection capacity of mutants resistant and tolerant to vancomycin in Staphylococcus aureus strains]. / Actividad inhibitoria, bactericida y capacidad de selección de mutantes resistentes y tolerantes de vancomicina en cepas de Staphylococcus aureus.

BACKGROUND: The aim of the present was to study the inhibitory and bactericide activity and risk of selection of mutants resistant or tolerant to vancomycin in strains of Staphylococcus aureus resistant to methicillin from out patient and hospitalary patients. MATERIALS AND METHODS: The minimum inhibitory concentration was obtained by the macrodilution method in liquid medium and by the Etest system. The minimum bactericide concentration was obtained by the macrodilution method in liquid medium. The selection of resistant or tolerant mutants was performed inoculating more than 5 x 10(6) UFC/ml in brain-heart agar plates and in tripteinsoya brought with different concentrations of vancomycin. RESULTS: Vancomycin showed greater inhibitory and bactericide activity in strains sensitive to methicillin of out patient and hospitalary origin with respect to strains resistant to methicillin with a minimum inhibitory concentration > 1 mg/l in 23.3, 20.0 and 70.0%, respectively and a minimum bactericide concentration of 90%, 8.16 and 64 mg/l. The incidence of tolerance was greater in the strains resistant to methicillin in comparison with the out patient and hospitalary strains sensitive to methicillin, since a minimum bactericide/inhibitory concentration greater than 4 was obtained in 63.3 in comparison with 20.0%. Only mutants with diminished sensitivity (minimum inhibitory concentration of 3 and 6 mg/l) or tolerant in strains resistant to methicillin could be selected. The increases in the minimum inhibitory concentrations were not stable, while the tolerant mutants preserved this characteristic after 20 subcultures. CONCLUSIONS: The in vitro selection of mutants with decreased sensitivity or tolerant was only obtained in strains resistant to methicillin. The changes in the minimum inhibitory concentrations were not stable in contrast to the increase in the minimum bactericide concentrations which remained unchanged. The differences in the selection of mutants with diminished sensitivity or tolerant among strains resistant and sensitive to methicillin found in this study were correlated with the differences in minimum inhibitory and bactericide concentrations and the relation between both in the clinical isolates.