Mupirocin-induced mutations in ileS in various genetic backgrounds of methicillin-resistant Staphylococcus aureus.

Topical mupirocin is widely used for the decolonization of methicillin-resistant Staphylococcus aureus (MRSA) carriers. We evaluated the capacity of various MRSA clonotypes to develop mutations in the ileS gene associated with low-level mupirocin resistance. Twenty-four mupirocin-sensitive MRSA isolates from a variety of genotypes (determined by a multilocus variable-number tandem-repeat assay) were selected. Mupirocin MICs were determined by Etest. The isolates were then incubated in subinhibitory concentrations of mupirocin for 7 to 14 days. Repeat MIC determinations and sequencing of the ileS gene were then performed. Doubling times of isolates exposed to mupirocin and of unexposed isolates were compared. We found that exposure to mupirocin led to rapid induction of low-level resistance (MICs of 8 to 24 µg/ml) in 11 of 24 (46%) MRSA isolates. This phenomenon was observed in strains with diverse genetic backgrounds. Various mutations were detected in 18 of 24 (75%) MRSA isolates. Acquisition of mutations appeared to be a stepwise process during prolonged incubation with the drug. Among the five isolates exhibiting low-level resistance and the highest MICs, four tested sensitive after incubation in the absence of mupirocin but there was no reversion to the susceptible wild-type primary sequence. Resistance was not associated with significant fitness cost, suggesting that MRSA strains with low-level mupirocin resistance may have a selective advantage in facilities where mupirocin is commonly used. Our findings emphasize the importance of the judicious use of this topical agent and the need to closely monitor for the emergence of resistance.

Characterization of Streptococcus tigurinus small-colony variants causing prosthetic joint infection by comparative whole-genome analyses.

Small-colony variants (SCVs) of bacteria are associated with recurrent and persistent infections. We describe for the first time SCVs of Streptococcus tigurinus in a patient with a prosthetic joint infection. S. tigurinus is a novel pathogen of the Streptococcus mitis group and causes invasive infections. We sought to characterize S. tigurinus SCVs using experimental methods and find possible genetic explanations for their phenotypes. The S. tigurinus SCVs were compared with the wild-type (WT) isolate using phenotypic methods, including growth under different conditions, autolysis, and visualization of the cell ultrastructure by use of transmission electron microscopy (TEM). Furthermore, comparative genome analyses were performed. The S. tigurinus SCVs displayed reduced growth compared to the WT and showed either a very stable or a fluctuating SCV phenotype. TEM analyses revealed major alterations in cell separation and morphological abnormalities, which were partially explained by impaired autolytic behavior. Intriguingly, the SCVs were more resistant to induced autolysis. Whole-genome sequencing revealed mutations in the genes involved in general cell metabolism, cell division, stringent response, and virulence. Clinically, the patient recovered after a 2-stage exchange of the prosthesis. Comparative whole-genome sequencing in clinical strains is a useful tool for identifying novel genetic signatures leading to the most persistent bacterial forms. The detection of viridans streptococcal SCVs is challenging in a clinical laboratory due to the small colony size. Thus, it is of major clinical importance for microbiologists and clinicians to be aware of viridans streptococcal SCVs, such as those of S. tigurinus, which lead to difficult-to-treat infections.

Whole-Genome Sequences of Streptococcus tigurinus Type Strain AZ_3a and S. tigurinus 1366, a Strain Causing Prosthetic Joint Infection.

Streptococcus tigurinus, a novel member of the Streptococcus mitis group, was recently identified as a causative agent of invasive infections. We report the complete genome sequences of the S. tigurinus type strain AZ_3a and S. tigurinus strain 1366. The genome sequences assist in the characterization of virulence determinants of S. tigurinus.

Bacterial diversity in oral samples of children in niger with acute noma, acute necrotizing gingivitis, and healthy controls.

BACKGROUND: Noma is a gangrenous disease that leads to severe disfigurement of the face with high morbidity and mortality, but its etiology remains unknown. Young children in developing countries are almost exclusively affected. The purpose of the study was to record and compare bacterial diversity in oral samples from children with or without acute noma or acute necrotizing gingivitis from a defined geographical region in Niger by culture-independent molecular methods. METHODS AND PRINCIPAL FINDINGS: Gingival samples from 23 healthy children, nine children with acute necrotizing gingivitis, and 23 children with acute noma (both healthy and diseased oral sites) were amplified using "universal" PCR primers for the 16 S rRNA gene and pooled according to category (noma, healthy, or acute necrotizing gingivitis), gender, and site status (diseased or control site). Seven libraries were generated. A total of 1237 partial 16 S rRNA sequences representing 339 bacterial species or phylotypes at a 98-99% identity level were obtained. Analysis of bacterial composition and frequency showed that diseased (noma or acute necrotizing gingivitis) and healthy site bacterial communities are composed of similar bacteria, but differ in the prevalence of a limited group of phylotypes. Large increases in counts of Prevotella intermedia and members of the Peptostreptococcus genus are associated with disease. In contrast, no clear-cut differences were found between noma and non-noma libraries. CONCLUSIONS: Similarities between acute necrotizing gingivitis and noma samples support the hypothesis that the disease could evolve from acute necrotizing gingivitis in certain children for reasons still to be elucidated. This study revealed oral microbiological patterns associated with noma and acute necrotizing gingivitis, but no evidence was found for a specific infection-triggering agent.

Analysis of the salivary microbiome using culture-independent techniques.

BACKGROUND: The salivary microbiota is a potential diagnostic indicator of several diseases. Culture-independent techniques are required to study the salivary microbial community since many of its members have not been cultivated. METHODS: We explored the bacterial community composition in the saliva sample using metagenomic whole genome shotgun (WGS) sequencing, the extraction of 16S rRNA gene fragments from metagenomic sequences (16S-WGS) and high-throughput sequencing of PCR-amplified bacterial 16S rDNA gene (16S-HTS) regions V1 and V3. RESULTS: The hierarchical clustering of data based on the relative abundance of bacterial genera revealed that distances between 16S-HTS datasets for V1 and V3 regions were greater than those obtained for the same V region with different numbers of PCR cycles. Datasets generated by 16S-HTS and 16S-WGS were even more distant. Finally, comparison of WGS and 16S-based datasets revealed the highest dissimilarity.The analysis of the 16S-HTS, WGS and 16S-WGS datasets revealed 206, 56 and 39 bacterial genera, respectively, 124 of which have not been previously identified in salivary microbiomes. A large fraction of DNA extracted from saliva corresponded to human DNA. Based on sequence similarity search against completely sequenced genomes, bacterial and viral sequences represented 0.73% and 0.0036% of the salivary metagenome, respectively. Several sequence reads were identified as parts of the human herpesvirus 7. CONCLUSIONS: Analysis of the salivary metagenome may have implications in diagnostics e.g. in detection of microorganisms and viruses without designing specific tests for each pathogen.