2.1 Percent occurrence of E. coli contamination
Out of a total of 147 pure isolates of E. coli obtained in the entire study from the workers’ hands, butchering knives, slaughtering tables, floors and effluent water at the different abattoir locations, the highest number of isolated organisms were obtained from the Sekona-1 slaughter slab-type abattoir followed by Almaleek batch abattoir and Sekona-2 slaughter slab-type abattoir in descending order of percent occurrence with actual values ranging from 46.3%, 27.9% and 25.8% respectively (Table 2). When typical contamination sites, namely, workers’ hands, butchering knives, slaughtering tables and floors were compared for degree of contamination for the selected abattoir types, results showed that the Sekona-2 slaughter slab consistently had the lowest contaminants followed by the Almaleek batch type abattoir and Sekona-1 slaughter slab in increasing degree of contamination (Table 3).
2.2 Banding patterns from the multiplex assays of pcr products
Figures 1a and b show the banding patterns of pcr products of representative samples from the multiplex assay for the presence of stx1, stx2, eae and hly genes when these were run on 2% agarose gel. The banding pattern was varied, indicating that some of the isolates possess up to four of the virulence genes, while some others did not show any evidence of possessing any of these genes. Conversely, none of the samples showed any bands for the multiplex assay for the presence of O157 and O111 genes (Figures 2a and b). These results were confirmed through serotyping as none of the strains showed any visible agglutination with the O157 latex reagent. The caused a visible agglutination with the seroscreen latex reagent for detecting the 6 common nonO157STEC and were therefore identified as belonging to any of the non-O157 serotypes.
Furthermore, the results of the banding pattern revealed that all the sampled locations exhibited the presence of E coli strains possessing a combination of the stx1, stx2, eae and hly genes. However, the only isolate with 4 out of the total of 6 genes considered was Se1-5-W5. This isolate which was obtained from the effluent water from the Sekona abattoir was sequenced and the phylogenetic analysis of the sequencing result is presented in Figures 3.
2.3 Sequencing and BLAST analysis of representative sample
Following BLAST analysis, the sequence showed 100% coverage and 96.46% percentage identity with Escherichia coli O113:H21 (GenBank Accession number: CP031892.1) strain from Canada. From evolutionary analyses, the strain from Nigeria, sequenced in this study, is evolutionarily distant when compared with the publicly available sequences from Nigeria (Fig 3). The isolated 16S rRNA sequence was subsequently submitted to GenBank and registered with accession number MW463885.
Table 1: The PCR Primers
Primer
|
Sequence (5’–3’)
|
Specificity1
|
Amplicon size (bp)
|
Assay 1
|
stx1F
stx1R
|
ATAAATCGCCATTCGTTGACTAC
AGAACGCCCACTGAGATCATC
|
nt 454–633 of A subunit coding region of stx
|
180
|
stx2F
stx2R
|
GGCACTGTCTGAAACTGCTCC
TCGCCAGTTATCTGACATTCTG
|
nt 603–857 of A subunit coding region of stx2 (including stx2 variants)
|
255
|
eaeAF
eaeAR
|
GACCCGGCACAAGCATAAGC
CCACCTGCAGCAACAAGAGG
|
nt 27–410 of eaeA (this region is conserved between EPEC and STEC)
|
384
|
hlyAF
hlyAR
|
GCATCATCAAGCGTACGTTCC
AATGAGCCAAGCTGGTTAAGCT
|
nt 70–603 of EHEC hlyA
|
534
|
Assay 2
|
O157F
O157R
|
CGGACATCCATGTGATATGG
TTGCCTATGTACAGCTAATCC
|
nt 393–651 of rfbEO157:H7
|
259
|
O111F
O111R
|
TAGAGAAATTATCAAGTTAGTTCC
ATAGTTATGAACATCTTGTTTAGC
|
nt 24–429 of ORF 3.4 of E. coli O111 rfb region
|
406
|
1 nt, nucleotide; ORF, open reading frame12.
Table 2: Percent occurrence of E coli isolates found in the various meat abattoir types
Location
|
Abattoir type
|
*Total number of pure isolates
|
Almaleek
|
Batch
|
41 (27.9)**
|
Sekona - 1
|
Slaughter slab
|
68 (46.3)
|
Sekona-2
|
Slaughter slab
|
38 (25.8)
|
Total
|
|
147(100)
|
*This number represents the total number of distinct isolates obtained from the workers’ hands, butchering knives, slaughtering tables and slaughtering floors. **Percentage of the total number of isolates observed for all sample sites shown in parentheses.
Table 3: Total colony counts (TCC of E coli colonies expressed in log10cfu/ml) sampled from workers’ hands, butchering knives, slaughtering tables, floors and effluent water at selected abattoirs.
Sample
Location
|
Abattoir
Type
|
Workers’
Hands
|
Butchering Knives
|
Slaughter Tables
|
Slaughtering Floors
|
Effluent Water
|
Total
|
Almaleek
|
Batch
|
15(10)
|
2(1.5)
|
17(11)
|
4(2.7)
|
8(5.5)
|
46(31)
|
Sekona-1
|
Slaughter
Slab
|
11(8)
|
2(1.5)
|
13(9)
|
25(17)
|
12(8)
|
63(43)
|
Sekona-2
|
Slaughter
Slab
|
12(8)
|
3(2)
|
3(2)
|
12(8.1)
|
8(5.5)
|
38(26)
|
Total
|
|
38(26)
|
7(5)
|
33(22)
|
41(28)
|
28(19)
|
147(100)
|
**Percentage of the TCC values are shown in parentheses.
Table 4: Grouping of E coli isolates based on the banding pattern of pcr products on 2% gel electrophoresis. The expected band sizes were stx 1 – 180; stx 2- 255; eae- 384; hlyA- 534; O157 – 259 and O111 – 406 bps. Where genes were indicated based on banding pattern, a positive mark (+) was used, where absent, negative (-) mark was inserted.
S/N
|
Isolate ID
|
Source
|
Stx-1
|
Stx-2
|
eae
|
hlyA
|
O157
|
O111
|
1
|
Alh-A3-3
|
Almaleek- Hands
|
-
|
-
|
-
|
-
|
-
|
-
|
2
|
Alh-B3-5
|
,,
|
+
|
+
|
+
|
-
|
-
|
-
|
3
|
Alh-A3-5
|
,,
|
+
|
+
|
+
|
-
|
-
|
-
|
4
|
Alh-C3-1
|
,,
|
+
|
+
|
+
|
-
|
-
|
-
|
5
|
Alh-B5-4
|
,,
|
+
|
+
|
+
|
-
|
-
|
-
|
6
|
Alh-A3-4
|
,,
|
+
|
+
|
+
|
-
|
-
|
-
|
7
|
Se2h-C1-1
|
Sekona- Hands
|
+
|
-
|
-
|
-
|
-
|
-
|
8
|
Se1h-A5-1
|
,,
|
-
|
-
|
+
|
-
|
-
|
-
|
9
|
Se1h-C3-4
|
,,
|
-
|
+
|
+
|
-
|
-
|
-
|
10
|
Se1h-A1-4
|
,,
|
-
|
+
|
+
|
-
|
-
|
-
|
11
|
Se1h-A1-4
|
,,
|
+
|
-
|
+
|
-
|
-
|
-
|
12
|
Se1h-B3-5
|
,,
|
+
|
-
|
-
|
-
|
-
|
-
|
13
|
Se1FA3-4
|
Sekona- Floor
|
-
|
+
|
+
|
-
|
-
|
-
|
14
|
Se2FC1-2
|
,,
|
+
|
-
|
+
|
+
|
-
|
-
|
15
|
Se2FC1-5
|
,,
|
-
|
-
|
+
|
-
|
-
|
-
|
16
|
Se1F3-1
|
,,
|
+
|
-
|
+
|
-
|
-
|
-
|
17
|
Se2FA3-3
|
,,
|
-
|
-
|
-
|
-
|
-
|
-
|
18
|
Se1EC1-3
|
,,
|
-
|
-
|
-
|
-
|
-
|
-
|
19
|
Se1FA3-1
|
,,
|
-
|
-
|
-
|
-
|
-
|
-
|
20
|
Se2FC5-3
|
,,
|
-
|
-
|
-
|
-
|
-
|
-
|
21
|
Se1FC1-3
|
,,
|
-
|
-
|
-
|
-
|
-
|
-
|
22
|
AlFA-3
|
Almaleek- Floor
|
-
|
-
|
-
|
-
|
-
|
-
|
23
|
AlfC5-3
|
,,
|
-
|
+
|
+
|
-
|
-
|
-
|
24
|
AlFC3-4
|
,,
|
-
|
+
|
+
|
-
|
-
|
-
|
25
|
AlF-3-2
|
,,
|
+
|
+
|
+
|
-
|
-
|
-
|
26
|
Al-3-W3
|
Almaleek- Water
|
+
|
+
|
+
|
-
|
-
|
-
|
27
|
Al-3-W4
|
,,
|
-
|
+
|
+
|
-
|
-
|
-
|
28
|
Al-1-W2
|
,,
|
-
|
-
|
+
|
-
|
-
|
-
|
29
|
Se2-3-W3
|
Sekona- Water
|
-
|
-
|
-
|
-
|
-
|
-
|
30
|
Se2-5-W1
|
,,
|
-
|
-
|
-
|
-
|
-
|
-
|
31
|
Se2-3W1
|
,,
|
+
|
+
|
+
|
-
|
-
|
-
|
32
|
Se1-5-W5
|
,,
|
+
|
+
|
+
|
+
|
-
|
-
|
33
|
ALTB-1
|
Almaleek- Table
|
-
|
+
|
+
|
-
|
-
|
-
|
34
|
AlTC-5
|
,,
|
-
|
+
|
+
|
-
|
-
|
-
|
35
|
AlTA-5
|
,,
|
+
|
-
|
-
|
-
|
-
|
-
|
36
|
AlTC-1
|
,,
|
-
|
-
|
-
|
-
|
-
|
-
|
37
|
ALTB-5
|
,,
|
-
|
-
|
-
|
-
|
-
|
-
|
38
|
Se2TC-3-4
|
Sekona- Table
|
-
|
+
|
+
|
-
|
-
|
-
|
39
|
Se1TC-3-3
|
,,
|
-
|
+
|
+
|
-
|
-
|
-
|
40
|
Se2-TB-1-4
|
,,
|
-
|
-
|
-
|
-
|
-
|
-
|
41
|
Se1TB-3-1
|
,,
|
-
|
-
|
-
|
-
|
-
|
-
|
42
|
Se1TA-3-2
|
,,
|
-
|
-
|
-
|
-
|
-
|
-
|
43
|
Alk-B3-1
|
Almaleek- Knife
|
-
|
-
|
-
|
-
|
-
|
-
|
44
|
AlkB3-2
|
,,
|
-
|
-
|
-
|
-
|
-
|
-
|
45
|
Se2K-C3-2
|
Sekona- Knife
|
-
|
-
|
+
|
-
|
-
|
-
|
46
|
SeikA-3-2
|
,,
|
-
|
-
|
+
|
-
|
-
|
-
|
47
|
Se2K-BC-2
|
,,
|
-
|
-
|
-
|
-
|
-
|
-
|
48
|
SeiKB5-5
|
,,
|
-
|
-
|
-
|
-
|
-
|
-
|
49
|
Se2K-A3-3
|
,,
|
-
|
-
|
-
|
-
|
-
|
-
|
50
|
Al-1-W1
|
Almaleek-Water
|
-
|
-
|
-
|
-
|
-
|
-
|