Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We address the question of how biofilm organization affects antibiotic susceptibility. We established Escherichia coli biofilms with differential structural organization due to the presence of IncF plasmids expressing altered forms of the transfer pili in two different biofilm model systems. The mature biofilms were subsequently treated with two antibiotics with different molecular targets, the peptide antibiotic colistin and the fluoroquinolone ciprofloxacin. The dynamics of microbial killing were monitored by viable count determination, and confocal laser microscopy. Strains forming structurally organized biofilms show an increased bacterial survival when challenged with colistin, compared to strains forming unstructured biofilms. The increased survival is due to genetically regulated tolerant subpopulation formation and not caused by a general biofilm property. No significant difference in survival was detected when the strains were challenged with ciprofloxacin. Our data show that biofilm formation confers increased colistin tolerance to cells within the biofilm structure, but the protection is conditional being dependent on the structural organization of the biofilm, and the induction of specific tolerance mechanisms.

Global transcriptomic analysis via RNA-seq is often hampered by the high abundance of ribosomal (r)RNA in bacterial cells. To remove rRNA and enrich coding sequences, subtractive hybridization procedures have become the approach of choice prior to RNA-seq, with their efficiency varying in a manner dependent on sample type and composition. Yet, despite an increasing number of RNA-seq studies, comparative evaluation of bacterial rRNA depletion methods has remained limited. Moreover, no such study has utilized RNA derived from bacterial biofilms, which have potentially higher rRNA:mRNA ratios and higher rRNA carryover during RNA-seq analysis. Presently, we evaluated the efficiency of three subtractive hybridization-based kits in depleting rRNA from samples derived from biofilm, as well as planktonic cells of the opportunistic human pathogen Pseudomonas aeruginosa. Our results indicated different rRNA removal efficiency for the three procedures, with the Ribo-Zero kit yielding the highest degree of rRNA depletion, which translated into enhanced enrichment of non-rRNA transcripts and increased depth of RNA-seq coverage. The results indicated that, in addition to improving RNA-seq sensitivity, efficient rRNA removal enhanced detection of low abundance transcripts via qPCR. Finally, we demonstrate that the Ribo-Zero kit also exhibited the highest efficiency when P. aeruginosa/Staphylococcus aureus co-culture RNA samples were tested.

High rates of erythromycin resistance among Streptococcus pyogenes strains have been reported in Italy in the last few years. In this study, 370 erythromycin-resistant (MIC, > or = 1 microg/mL) Italian isolates of this species obtained in 1997-1998 from throat swabs from symptomatic patients were typed by analyzing SmaI macrorestriction fragment patterns by pulsed-field gel electrophoresis (PFGE). Among the typable isolates (n = 341; the genomic DNA of the remaining 29 isolates was not restricted by SmaI), 48 distinct PFGE types were recognized, of which 31 were recorded in only one isolate (one-strain types). Fifty-two percent of typable isolates fell into three type clusters and 75% into six, suggesting that erythromycin-resistant group A streptococci circulating in Italy are polyclonal, but the majority of them probably derives from the spread of a limited number of clones. In parallel experiments, the 370 test strains were characterized for the macrolide resistance phenotype: 80 were assigned to phenotype cMLS, 89 to phenotype iMLS-A, 33 to phenotype iMLS-B, 11 to phenotype iMLS-C, and 157 to phenotype M. There was a close correlation between these phenotypic data and the genotypic results of PFGE analysis, the vast majority of the isolates assigned to individual PFGE classes belonging usually to a single phenotype of macrolide resistance. All of the 29 untypable isolates belonged to the M phenotype. Further correlations were observed with tetracycline resistance.

The phenotypes and genetic determinants for macrolide resistance were determined for 167 erythromycinresistant Streptococcus pyogenes strains. A cMLS phenotype was shown in 18% of the erythromycin-resistant strains, while inducible resistance was apparent in 31% and the M phenotype was apparent in 50%. The emm gene type of this set of resistant isolates and that of 48 erythromycin-sensitive isolates were determined. emm2 and emm48 were recorded only in the resistant strains of the M phenotype, while approximately all of the strains harboring the emm22 gene had the cMLS phenotype. More than 80% of the emm89-positive strains had the iMLS phenotype, and the same portion of emm4 strains presented the M phenotype. emm3 is recorded only among sensitive strains. The distribution of frequencies of the genetic determinant for the virulence factor M protein was significantly different both among organisms of different types of resistance and between resistant and sensitive populations of S. pyogenes under study.

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.