At 24, 48, 72, and 96 h post-inoculation, mice were euthanized and the eyes removed and fixed in 4% formalin in PBS (pH 7.2). After hemotoxylin and eosin (H&E) staining, eye sections were examined using a light microscope. Statistical analysis Experimental data were analyzed with SPSS and comparisons made using Student’s t-test. Differences with a P-value less than 0.05 were considered statistically significant. Results Detection of ipaH, ial, and set1B in S. flexneri clinical isolates The ipaH gene was detected in all 86 S. flexneri clinical isolates,

whereas ial was detected in 45 isolates (52.3%), and set1B detected in 69 isolates (80.2%). Amplicons for PF-6463922 in vitro ipaH, ial and set1B were not seen from E. coli ATCC

25922 samples. All three genes were detected in the SF301 positive control. BIBW2992 HeLa cell invasion of S. flexneri clinical isolates Nine isolates in our study contained all three genes, one SF68 CFTRinh-172 cell line isolate contained ipaH and set1B, one SF51 isolate had ipaH and ial, and one SF36 isolate contained only ipaH (Table 2). The nine isolates that contained all three virulence-associated genes demonstrated high invasive ability in HeLa cells (>200 plaques/well). In HeLa cells, SF68 and SF36 were less invasive resulting in 4 and 2 plaques/well, respectively. SF51 lacked set1B but retained ial, and showed a decrease in invasiveness (20 plaques/well; Table 2). Using PCR through and nucleotide sequencing, it was shown that SF51 lacked the entire pic gene on PAI-1, but harbored sigA and other significant open reading frames (Figure 1). Table 2 Invasion of HeLa cells by S. flexneri clinical isolates as determined by plaque formation tests Gene detected by

mPCR Number of strains Plaque formation number (per well)* ipaH ial set1B + + + 9 >200±16 + + – 1 (SF51) 20±5 + – + 1 (SF68) 4±2 + – - 1 (SF36) 2±1 * Values represent the mean ± standard deviation of three wells. Figure 1 Amplicons from SF51 int , orf30 , sigA and pic . Specific amplicons for (A) int; (B) orf30; (C) sigA; and (D) pic. The target genes int, orf30 and sigA were amplified from SF51 DNA but pic was not. All four target genes were amplified from the SF301 positive control. The sequence of all PCR products was confirmed by nucleic acid sequencing. HeLa cell invasion of SF301 and mutants The growth curves for SF51, SF301-∆ pic, SF301, SF301-∆ pic/pPic and SF51/pPic were similar under aerobic growth conditions (Figure 2).