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

Contribution of Exogenous Genetic Elements to the Group A Streptococcus Metagenome

Variation in gene content among strains of a bacterial species contributes to biomedically relevant differences in phenotypes such as virulence and antimicrobial resistance. Group A Streptococcus (GAS) causes a diverse array of human infections and sequelae, and exhibits a complex pathogenic behavior. To enhance our understanding of genotype-phenotype relationships in this important pathogen, we determined the complete genome sequences of four GAS strains expressing M protein serotypes (M2, M4, and 2 M12) that commonly cause noninvasive and invasive infections. These sequences were compared with eight previously determined GAS genomes and regions of variably present gene content were assessed. Consistent with the previously determined genomes, each of the new genomes is ∼1.9 Mb in size, with ∼10% of the gene content of each encoded on variably present exogenous genetic elements. Like the other GAS genomes, these four genomes are polylysogenic and prophage encode the majority of the variably present gene content of each. In contrast to most of the previously determined genomes, multiple exogenous integrated conjugative elements (ICEs) with characteristics of conjugative transposons and plasmids are present in these new genomes. Cumulatively, 242 new GAS metagenome genes were identified that were not present in the previously sequenced genomes. Importantly, ICEs accounted for 41% of the new GAS metagenome gene content identified in these four genomes. Two large ICEs, designated 2096-RD.2 (63 kb) and 10750-RD.2 (49 kb), have multiple genes encoding resistance to antimicrobial agents, including tetracycline and erythromycin, respectively. Also resident on these ICEs are three genes encoding inferred extracellular proteins of unknown function, including a predicted cell surface protein that is only present in the genome of the serotype M12 strain cultured from a patient with acute poststreptococcal glomerulonephritis. The data provide new information about the GAS metagenome and will assist studies of pathogenesis, antimicrobial resistance, and population genomics

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

Activity of ceftibuten, cefaclor, azithromycin, clarithromycin, erythromycin and telithromycin against Streptococcus pyogenes clinical isolates with different genotypes and phenotypes

Background: The growing number of macrolide-resistant strains of Streptococcus pyogenes represents an increasing worldwide problem. Macrolide resistance in S. pyogenes is mediated by several different genes, which determine different levels of resistance to macrolides, lincosamides and streptogramin B (MLS). Methods:This study compared the in vitro antimicrobial activity of azithromycin, clarithromycin, erythromycin, ceftibuten, cefaclor, and telithromycin against 287 strains of S. pyogenes by the broth microdilution method. All strains were characterized both phenotypically and genotypically for erythromycin resistance and most of them have been M-typed by means of PCR. Results: Ceftibuten and cefaclor showed the best antimicrobial activity, while MIC values for telithromycin were higher against constitutively MLS (cMLS)-resistant strains rather than against the other phenotypes. Conclusion: Oral cephalosporins retain the best activity against S. pyogenes; showing good activity except for cMLS-resistant strains, telithromycin is a valid alternative to these antimicrobials

Characterisation of biofilm-forming Staphylococcus epidermidis clinical isolates

Objective: Staphylococcus epidermidis is one of the bacterial species mainly implicated in foreign body associated infections. We have characterized several S.epidermidis clinical isolates for their ability to form biofilms and their resistance to antibiotics. Methods: 76 S.epidermidis strains isolated from implantable medical devices have been collected from hospitals of Central Italy. Susceptibility to penicillin, methicillin, erythromycin and tetracycline has been determined in vitro by E-test according to NCCLS guidelines. The specific antibiotic resistance determinants have been checked by PCR (blaZ, mecA, ermA, ermB, ermC, msrA, tetK and tetM). The ability to form biofilms has been determined: (i) by PCR, detecting genes specific for attachment and biofilm development (icaADBC operon, aap, and atlE); (ii) by Congo Red Agar (CRA) plate test to assay the production of polisaccaridic intercellular adhesin (PIA); (iii) by crystal violet (CV) stain to determine the biofilm biomass development on polystyrene microtiter plates; (iv) and by CSLM microscopy observations to investigate biofilm structure. Results: 94% of the strains under study was resistant to penicillin, 87% to methicillin, 72% to erythromycin and 25% to tetracycline. On the side of biofilm-specific genes detection, 66% of strains was positive to ica operon genes, 82% possessed atlE gene, and 42% aap determinant. In 89% of the population, the CRA test confirmed the correlation between the presence of ica genes and slime expression. The CV assay classified the quasitotality of our strains (97%) as biofilm producers on plastic surface. In addition, the distribution of optical density values (OD540) obtained after CV stain, showed a significant statistical difference in biofilm biomass development between the ica- ADBC-positive strains and the icaADBC-negative ones. Finally, a correlation, although not always present, has been observed between ability of the strains to develop in a high-structureted biofilm and specific biofilm-formation determinants. Conclusions: The investigated bacterial population shows a very high and alarming level of resistance to all tested drugs. Although the specific determinants for biofilms formation are not always present, nevertheless all the strains are able to develop in sessile form showing that different and not still identified factors could work together in the formation and organization of staphylococcal complex microbial communities

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

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