CF patients are typically subject to extended antibiotic regimes, but the drugs do not necessarily reach the entire lung at inhibitory concentrations CRT0066101 nmr [21]. Therefore, sub-inhibitory antibiotic exposure could be one factor that promotes P. aeruginosa diversification in the CF lung. Consequently, a better understanding of the responses of P. aeruginosa populations to these sub-inhibitory concentrations of antibiotics in the CF lung would allow clinicians to make better informed choices of antibiotic regimes. Although it is likely that most CF patients acquire P. aeruginosa infections from diverse environmental reservoirs and thus carry

their own unrelated strains, several multidrug-resistant “epidemic” strains capable of patient to patient transmission

have been identified [22]. The selleck LES is the most widespread transmissible strain of P. aeruginosa in the UK [23], and has also been reported in North America [24]. It has been detected in as many as 79% of adult CF patients in a Liverpool CF centre [25]. The high prevalence of LES in CF patients is a concern, given that chronic LES infection has been associated with a greater deterioration in pulmonary function and nutritional state [26] and increased antibiotic resistance [27]. In this study, we analysed P. aeruginosa LES populations in an artificial sputum medium (ASM) model Amylase that mimics CF sputum in terms of composition. Various groups have utilised ASM models to study, for example, gene expression patterns and the effects of bacteriophages [28–30]. P. aeruginosa, when cultured in ASM, forms biofilms and diversifies with respect to phenotype, in a manner that resembles behaviour in the CF lung [30]. We hypothesise that exposure to sub-inhibitory concentrations of antibiotics will drive bacterial diversification, possibly through a combination of antibiotic-induced mutagenesis or through the regulation of gene transcription [31–36]. Consequently, the objective

of this study was to test the hypothesis that exposure to sub-inhibitory concentrations of antibiotics has a role to play in promoting P. aeruginosa population diversification during growth in an ASM model. Results Sub-inhibitory antibiotics promote diversification of P. aeruginosa LESB58 The emergence of novel haplotypes was observed in all culture conditions, but the presence of sub-inhibitory concentrations of Quisinostat mouse certain antibiotics significantly increased both the number of novel haplotypes (p <0.01, LRT = 48.8, d.f. = 6) and the haplotype diversity found within populations (p < 0.01, F6,14 = 5.90) relative to control populations (Figures 1 and 2). However, some antibiotics contributed to this diversity more than others.

terreus isolates Fingerprints for all of the sequence-confirmed A. terreus isolates were generated using four ISSR primers

that were selected after initial screening as described above. GeneMapper v4.0 (Applied Biosystems, Carlsbad, CA) was used to assign fragment sizes to the PCR products. Fragments identified using GeneMapper software were converted HKI 272 to binary data with a “”0″” representing the absence and a “”1″” representing the presence of an allele. The binary strings of data representing the fingerprint generated by each primer were concatenated in Excel (Microsoft Corporation, Redmond, WA) to form a single, continuous, binary string incorporating the results from all primers. Alleles that appeared in all or fewer than 10% of isolates were excluded from the analysis. Phylogenetic trees and Bayesian clusters were generated from identical binary data sets. Phylogenetic Analysis of ISSR data Neighbor-joining (NJ) trees were generated by PAUP [Phylogenetic Analysis Using Parsimony (and Other Methods)] [15]. PHYLIP [Phylogeny Inference Package] [16] was used to produce the parsimony tree. Bayesian clustering was check details performed

using the program STRUCTURE [17]. Results Species Confirmation The ML tree was generated using 484 contiguous bases of aligned sequence from the calM locus of the 117 A. terreus isolates and additional reference section Terrei sequences acquired from GenBank. One hundred and thirteen isolates clustered with the reference A. Selleckchem CB-839 terreus isolates and four isolates, three from the Eastern United States and one from Italy, grouped with the A. alabamensis type isolate (Figure 1). Figure 1 Maximum

Liklihood Tree from Calmodulin Sequence of Aspergillus species. Maximum likelihood tree of partial nucleotide sequences of calmodulin gene region obtained for all isolates and reference A. terreus and A. alabamensis sequences from GenBank. A. alabamensis isolates and reference sequences are in bold. Bootstrap values above 50% from 1000 iterations are noted on nodes. ISSR Fingerprinting of the Global A. terreus Isolates On testing ten ISSR primers using a subset of IKBKE forty A. terreus isolates, it was found that four primers were suitable for generating robust fingerprints for A. terreus: three trinucleotide repeat flanking primers and a single tetranuclotide repeat flanking primer (ISSR 7, 9, 10 and 13 respectively) (Table 1). These four ISSR primers were used to generate fingerprints for all of the sequence-confirmed A. terreus isolates. The A. alabamensis isolates were not fingerprinted. ISSR subtyping of 113 A. terreus revealed 111 unique genotypes with only two isolates, both from the same center in the Eastern United States, demonstrating identical fingerprinting patterns. Data from the ISSR fingerprints were analyzed using three phylogenetic algorithms.