Figure 1.
Flow chart for STEC isolation (O157 and non-O157) and examples of typical colony morphologies.
The final isolation protocol (M3) incorporates the M1 and M2 methods and starts with enrichment in TSB and plating anti-O157 magnetic beads on three different media (“IMS”; media A, B, C) and direct plating of stx-positive enrichment broths on C-O157 (“PCR”; medium D). O157 suspect colonies appear as pale and steel blue colonies on SMAC and NT-RA, respectively. Suspect STEC colonies from any media are subcultured on LB and confirmed as either O157 or non-O157 STEC by PCR. Anti-O157 magnetic beads bind other bacteria present in enrichment broths of environmental samples, but, fortuitously, also many non-O157 STEC. Typical non-O157 STEC colonies are shown from enrichments growing on C-O157 (Indicator Media, panel D, blue colonies), NT-RA agar (panel B, pink colonies). Non-O157 STEC colonies expressing beta-galactosidase and hemolysin are indicated by blue colonies with a clearing zone of hemolysis on mSBA (panel C). The parts of the final method for isolating O157 and non-O157 STEC are shown by an orange box (O157), blue box (M1), green box (M2) and red box (M3).
Table 1.
Primers and probes.
Figure 2.
Colonies on C-O157 and NT-RA representative of colors and morphologies indicative of different STECs.
Colony colors and morphologies of selected STEC are shown on C-O157 (A-H) and NT-RA (I-Q). Letter designations refer to Table 5. It was noted that colony color changed occasionally depending on the density of colonies (see C, H, and I).
Table 2.
Sensitivity of three enrichment methods for recovery of E. coli O157 from environmental samples.
Figure 3.
Recovery of non-O157 STEC strains from enrichment samples with different RT-PCR Ct values for stx.
The fraction of samples positive for isolation of at least one non-O157 STEC strain based on threshold PCR Ct value (M1) is compared to the fraction positive with both the PCR and IMS methods (M2). A Ct <27 was selected as the value required for routine plating of an enrichment broth on C-O157. However, all enrichment broths, regardless of Ct value for stx, were treated with O157-IMS and beads were plated on NT-RA(IMS method) for attempted recovery of non-O157 STEC strains (M2).
Table 3.
RT-PCR with and without Environmental Master Mix (EMM) to detect stx genes in environmental sample enrichments spiked with STEC.
Figure 4.
Incidence of O157 and non-O157 STEC in samples processed by M1, M2 and M3.
Samples were processed over a 2.5 year period by M1 (IMS and NT-RA for O157 only+plating enrichment with Ct <26 for stx on C-O157), M2 (M1+ picking suspect STEC from NT-RA), M3 (M2+ mSBA).
Table 4.
Comparison of samples positive for O157 STEC and non-O157 STEC by M1, M2 and M3 at corresponding sequential periods.
Table 5.
Comparison of O-types and colony colors among STEC.
Table 6.
Incidence of virulence genes in all O157 and non-O157 STEC isolatesa.
Figure 5.
Venn diagram of samples positive for non-O157 STEC by NT-RA, C-O157 and mSBA.
The enrichment broths for 4,160 samples (sampling period Jan – Oct 2010) were processed by M3 corresponding to plating on C-O157 (PCR method; “C”), NT-RA agar (IMS method; “R”) and mSBA (IMS method; “B”). The figure shows the number of samples that were positive for non-O157 STEC on only 1 of the 3 media (R, C, B) and any combination of the 3 media (BC, RB, RC, RBC).
Figure 6.
Virulence genes and O-antigen genes in a subset of STEC strains.
A subset of strains isolated by the final prototype method (M3; all media) was analyzed by PCR as described previously (Quinones et al, 2012, Frontiers). This provided an opportunity to compare the types of strains isolated from samples exposed to all three media used for isolation of non-O157 STEC.
Figure 7.
Seasonal isolation of O157 and non-O157 STEC from cattle, pig and water samples.
The fractions of cattle, feral pig or water samples positive for at least one O157 (A) strain are shown for different months of the year. Each point represents the average fraction of positive enrichments, processed by the IMS method, for each month over a 30-month period from April 2008 until October 2010. Numbers adjacent to each point are the number of samples represented. Monthly rainfall averages are the average of 4 weather sites during the 30 month period (see Methods). Similarly, fractions of cattle, feral pig and water samples positive for a least one non-O157 STEC (B) and monthly rainfall totals are shown for a 10 month period (Jan 2010– Oct 2010) from the same 4 weather sites used in panel A. Letters to the left of the plots indicate significant (P<0.05) correlation between those plots with the same letter designation.
Figure 8.
Phylogeny of O157 STEC by 11-loci MLVA.
A minimal spanning tree was constructed of 278 MLVA types representing the O157 STEC strains isolated by M3. Node size indicates the relative number of isolates of that type; i.e. the smallest size node represents a single strain of that type. The nodes are color-coded by farm/ranch site code (Panel A) and by sample source (Panel B). Human clinical isolates EDL933 and Sakai are included for comparison only.
Figure 9.
Phylogeny of non-O157 STEC by 7-loci MLVA and ompA sequence analysis.
A minimal spanning tree was constructed of 286 MLVA/ompA types representing the non-O157 STEC strains isolated by M3. Node size indicates the relative number of isolates of that type; i.e. the smallest size node represents a single strain of that type. The nodes are color-coded by farm/ranch site code (Panel A) and by sample source (Panel B). Human clinical isolates RM12844 and RM12856 (OregonPublic Health, 2010) and RM14735 (Germany Fenugreek Outbreak strain, MA Dept. Public Health 2011) are included for comparison only.