The present results showed that zinc frequently inhibited biofilms formed by typical EAEC strains isolated from diarrheic PND-1186 in vitro children, indicating a possible buy AZD0530 explanation for its efficient use in the management of diarrhea. Conclusions Previously, we reported that typical EAEC strains negative for the AAF fimbriae were statistically associated with persistent diarrhea [9], indicating the occurrence of other adhesion factors among wild-type typical EAEC strains. Here, the results indicate that putative F pili may work as central adhesion factor during the biofilm formation by typical

EAEC strains. Moreover, putative F pili engage typical EAEC strains in forming mixed biofilms increasing the overall bacterial adhesion when diarrhea-isolated aggregative C. freundii is present. Methods Bacterial strains During a case-control study focusing on the epidemiology of EAEC [9], the biofilm-forming aggregative C. freundii (EACF) strain 205 was isolated from a child (aged 13 months) on the fifth day of a mucous diarrhea that presented, on average, 15 evacuations per day. A typical EAEC strain was isolated concomitantly from the same child (strain 205-1, genotype CVD432+AggR+AAF-I+PilS-Pic+). The diffusely adherent C. freundii strain

047 was isolated from a healthy child (aged 21 months) together with the atypical EAEC strain 047-1 (CVD432-AggR-AAF-PilS-Pic+). Typical EAEC strain 340-1, which shares with EAEC 205-1 the same genotype (CVD432+AggR+AAF-I+PilS-Pic+), was isolated from a persistent (lasting ≥ 14 days) mucous diarrhea Tanespimycin ic50 affecting a child aged 3 months. This strain was chosen based upon its shared genotype with EAEC 205-1. Forty three typical EAEC strains negative for the AAF alleles I and II and isolated during the same study from children up to 5 years of age were used to why evaluate the role of putative pili F and the effect of zinc on the single biofilm formation. Prototype EAEC strains 042 [40] and 17-2 [41] were also used for the assays. Bacterial

strains were preserved at -20°C in Luria Bertani (LB) broth with 15% glycerol. Unless otherwise stated, bacterial strains were cultured in LB broth at 37°C for 18 h with constant agitation (200 rpm). Primers and PCR conditions Primers were designed in order to detect multiple alleles of the agn43 gene. Agn43-oxy primers detect alleles harbored by prototype strains of E. coli K12 (Genbank accession numbers: NC_000913, AC_000091, NC_010473 and NC_012759) whose transcription is under the control of the oxyR locus. The forward primer (5′-CGATCGATAAGCTAATAATAACC-3′) targets the locus oxyR (nucleotide position 2069371..2069393 in the Genbank sequence NC_000913) while the reverse primer (5′-GAAGACCACCACTGGTGACA-3′) recognizes the region encoding α43 subunit (position: 2069903..2069922). Additionally to agn43-oxy primers, oligonucleotides were designed to detect agn43-like loci harbored by uropathogenic E.