Figure 1.
NTUH-K1790N cps-PCR genotyping in K. pneumoniae.
(A) Comparison of the capsular polysaccharide synthesis (cps) regions of NK29 (Shu et al., 2009) and NTUH-K1790N strains. ORFs are shown by arrows, with the length as indicated below. Gray arrows show the conserved ORFs, and white arrows show the variable ORFs in different serotypes. The homology score between NK29 and NTUH-K1790N is compared using the sequence alignment program ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Black arrowheads refer to 5 pairs of PCR primers (a-e) used for cps genotyping. (B) cps-PCR genotyping of K. pneumoniae strains using primers as indicated: a. KN2-orf8-F and KN2-orf8-R; b. KN2-orf8-F2 and KN2-orf10-R; c. KN2-orf11-F and KN2-orf11-R; d. KN2-orf12-F and KN2-orf12-R; e. KN2-wzx-F and KN2-wzx-R; f. 23S rRNA as control for K. pneumoniae. K1 ref. presents the reference strain for the K1 serotype.G.
Figure 2.
Immunoblot serotyping of K. pneumoniae NTUH-K1790N, Ca0507, Ca0421, and C1975.
Rabbit anti-C1975 antiserum was used as the first antibody (1∶5000) and goat anti-rabbit IgG-HRP as the second antibody (1∶10 000). (A) The slot blot showing anti-C1975 antiserum reacts to the extracted capsular polysaccharides from strains NTUH-K1790N, C1975, Ca0421 and Ca0507. Ca0507Δorf8 presents the isogenic mutant with a deletion of gene orf8 in the cps. (B) The slot blot for the extracted capsular polysaccharides from 77 K serotype reference strains and a new type A1517. Sample1–9: K31 to K39; 10: C1975 as control. Negative reactions were observed in other reference strains and A1517 (data not shown).
Figure 3.
Bacterial host range of phage 0507-KN2-1.
(A) Upper panel showing spot tests of 0507-KN2-1 (109) on K. pneumoniae KN2 strains as indicated. Lower panel showing spot tests on Ca0507 isogenic mutant with a deletion of gene orf8 in cps (Ca0507Δorf8), K. aerogenes, E. coli 29522 and S. typhimurium ATCC14028. (B) Spot tests of 0507-KN2-1 (109) on K. pneumoniae 77 K serotype reference strains and a new type A1517. Ca0507 was used as a positive control (indicated by a white arrow). Negative reactions were observed in all reference strains and A1517 (data on K24-K77 and A1517 not shown).
Figure 4.
Characterization of phage 0507-KN2-1.
(A) Plaque morphology of 0507-KN2-1 on K. pneumoniae Ca0507. Scale bar, 3.5 mm. (B) TEM images of 0507-KN2-1 taken at 150 000 × magnification. Scale bars for both pictures, 100 nm. (C) Restriction digestion of 0507-KN2-1 genome. M: DNA marker; lane1: uncut genomic DNA, 2 µg; lane2: genomic DNA 2 µg incubated with HinPI1 at 37°C for 5 h.
Figure 5.
Bacteriophage-derived depolymerase specific for KN2 capsular polysaccharides.
(A) Immunoblot showing depolymerzation of KN2 extracted capsular polysaccharides by purified ORF96 proteins. The extracted capsular polysaccharides alone or incubated with ORF96 proteins (10 µg) at 37°C for 6 h were separated by SDS-PAGE, transferred onto a nitrocellulose membrane and detected using rabbit anti-C1975 antiserum (1∶5000) and goat anti-rabbit IgG-HRP (1∶10 000). Lane1: Ca0507Δorf8 alone; lane2: wild-type Ca0507 alone; lane3: wild-type Ca0507 plus ORF96 proteins. (B) A spot test showing ORF96 proteins with the depolymerase activity to KN2. Purified ORF96 proteins (1 µg) was spotted on the bacterial lawn and incubated at 37°C overnight. Decapsulation of KN2 K. pneumoniae NTUH-K1790N, Ca0507, Ca0421 and C1975 was observed. K1 ref. presents the reference strain of the K1 serotype. (C) Comparison of sensitivity of phage 0507-KN2-1 and capsular polysaccharide depolymerase. Spot tests with serial titration (counter-clockwise) of phage 0507-KN2-1 (upper panels) or purified ORF96 proteins (lower panels) on KN2 strains as indicated.