Biofilm Induced Tolerance towards Antimicrobial Peptides

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

PCR M Typing: a New Method for Rapid Typing of Group A Streptococci

A new approach for the M-typing of Streptococcus pyogenes is reported. Oligonucleotide primers were used in a PCR to amplify the N-terminal region of the emm gene. The presence of the PCR amplification product is associated with the corresponding M serotype. This technique offers potential advantages over other molecular typing methods

emm Gene Distribution among Erythromycin-Resistant and -Susceptible Italian Isolates of Streptococcus pyogenes

The phenotypes and genetic determinants for macrolide resistance were determined for 167 erythromycin-resistant 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

Impact of nutritional factors on in vitro PK/PD modelling of polymyxin B against various strains of Acinetobacter baumannii

International audienceThe main objective of this study was to assess the effect of rich artificial cation-adjusted Mueller-Hinton broth (CAMHB) on the growth of three strains of Acinetobacter baumannii (ATCC 19606 and two clinical strains), either susceptible or resistant to polymyxin B (PMB), and on PMB bactericidal activity. A pharmacokinetic (PK)/pharmacodynamic (PD) modelling approach was used to characterize the effect of PMB in various conditions.Time-kill experiments were performed using undiluted CAMHB or CAMHB diluted to 50%, 25% and 10%, with or without Ca2+ and Mg2+ compensation (known to affect PMB activity), and with PMB concentrations ranging from 0.25 to 256 mg/L based on the strain's MIC. For each strain, time-kill replicates were modelled using NONMEM.Unexpectedly, dilution of CAMHB by up to 10-fold did not affect the growth rate of any of the three strains in the absence of PMB. However, the bactericidal activity of PMB increased with medium dilution, resulting in a reduction in the apparent bacterial regrowth of the various strains observed after a few hours. Data for each strain were well characterized by a PK/PD model, with two bacterial subpopulations with different susceptibility to PMB (more susceptible and less susceptible). The impact of medium dilution and cation compensation showed relatively high, unexplained between-strain variability. Further studies are needed to characterize the mechanism underlying the medium dilution effect

Genetic Diversity of Cell-Invasive Erythromycin-Resistant and -Susceptible Group A Streptococci Determined by Analysis of the RD2 Region of the prtF1 Gene

The RD2 region of the internalization-associated gene prtF1, which encodes the fibronectin-binding repeat domain type 2 of protein F1, plays a crucial role in the entry of group A streptococci (GAS) into epithelial cells. A molecular study of the variability of the RD2 region was carried out with 77 independent Italian GAS, 66 erythromycin resistant (ER) and 11 erythromycin susceptible (ES), which had previously been investigated for the association between erythromycin resistance and ability to enter human respiratory cells. The amplicons obtained from PCR analysis of the RD2 region were consistent with a number of RD2 repeats ranging from one to five, more frequently four (n = 30), three (n = 27), and one (n = 18). A new method to type cell-invasive GAS (RD2 typing) was developed by combining PCR analysis of the RD2 region and restriction analysis of PCR products with endonucleases HaeIII, DdeI, and HinfI. Overall, 10 RD2 types (a to j) were distinguished (all detected among the 66 ER isolates, four detected among the 11 ES isolates). Comparison and correlation of RD2 typing data with the genotype and phenotype of macrolide resistance and with data from PCR M typing and SmaI macrorestriction analysis allowed us to identify 41 different clones (31 among the 66 ER isolates and 10 among the 11 ES isolates). Three major clones accounted for 40% of the isolates (47% of ER strains). Some ES isolates appeared to be related to ER isolates with identical combinations of RD2 type and emm type. While simultaneous use of different typing methods is essential for a thorough investigation of GAS epidemiology, RD2 typing may be especially helpful in typing cell-invasive GAS