The present results showed that zinc frequently inhibited biofilms formed by typical EAEC strains isolated from diarrheic click here children, indicating a possible 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+AZD5582 in vivo 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 PI3K Inhibitor Library manufacturer 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 BCKDHB 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.