Report Pathogenic E. coli from pigs: virulence genes and ...€¦ · CODA-CODA ---CERVACERVA Veterinary and Agrochemical Research Centre Report Pathogenic E. coli from pigs: virulence

CODACODACODACODA----CERVACERVACERVACERVA

Veterinary and Agrochemical Research Centre

Report

Pathogenic E. coli from pigs: virulence genes and antibiotic

resistance, results from 2011

P. Butaye

Introduction

E. coli is still a major pathogen in several animal species, especially in food producing animals. In swine it

may cause neonatal diarrhoea, post weaning diarrhoea and oedema disease. Virulence genes are encoding

for different virulent characteristics. Many are known, though frequently in supposed pathogenic strains,

no virulence genes are found. This may be due to or unknown virulence genes or simply that the E. coli

strain isolated has nothing to do with the pathology or that E. coli is not involved in the pathology seen, but

for example a virus. It is impossible to differentiate between virulent and non-virulent E. coli when

performing general bacteriology (isolation and identification based on biochemical characteristics). For

isolates from pigs, one may rely only in part on haemolysis which is more often associated with

pathogenicity.

Vaccination is far from efficient against E. coli disease. Therefore antibiotic treatment is frequently

necessary. However, antibiotic resistance is an ever increasing problem. Diseases caused by E. coli are

difficult to treat, and this may be, in part due to the high level of resistance.

Due to the problems and the importance of the problems concerning E. coli in swine we are conducting

surveillance on prevalence of virulence genes and types of E. coli circulating, and their associated antibiotic

resistance genes. In this report we show results obtained on E. coli isolated from pigs showing symptoms

compatible with E. coli caused disease of the year 2011. The report contains also the cumulative data from

2005 on.

Materials and methods.

E. coli were isolated from diseased animals, showing symptoms that may be compatible with E. coli

infection. Strains were isolated and identified to the species level by DGZ (Dierengezondheid Vlaanderen)

and ARSIA (Association Régional de Santé et d’Identification Animale). Afterwards strains were sent to

CODA-CERVA for further investigation.


Scientific research at the service of safe food production and animal health

2/12

At CODA-CERVA, a multiplex PCR was performed for virulence genes. DNA was extracted from an overnight liquid culture. The culture was centrifuged at 14000 rpm for 5 min, the supernatant was removed and one ml of sterile distilled water was added. This suspension was heated at 95°C for 5 min, then centrifuged for 5 min at 14000 rpm and kept at –20°C. Table 1 shows the virulence genes tested for and primer sequences used. Amplification was done in

reaction volumes of 20µl containing 2 µl DNA template, 0.5µM of each primer, 2.5 U of Taq polymerase

(Ampli Taq Gold, Perkin Elmer, Norwalk, CT, US), 5 mM of MgCl2 (Perkin Elmer), 1X AmpliTaq gold buffer

(Perkin Elmer) and 0.2 mM of each dNTP (Pharmacia). Amplification was done in a GeneAmp PCR 9600

(Perkin Elmer) machine. PCR conditions were a first denaturation cycle for 3 minutes at 90°C, followed by

30 cycles of 1 minute 90°C, 1 minute 55°C increasing 3 seconds each cycle, 2 minutes 70°C and a final

extension cycle for 10 minutes at 70°C.

Positive control strains were E. coli B41 (F5, F41, Sta), E. coli 987P (F6, Sta, STb), E. coli E68 (F4, LT, STb) and

E. coli 107/86 (F18, Stx2e). E. coli HB101 was used as negative control. PCR products (20 µl PCR product and

3µl blue juice) were run in a 3% Nusieve 3.1 agarose gel in TBE buffer for 90 minutes at 120 Volt. Marker

VIII (Böhringer Ingelham) was used as size standard.

Antibiotic resistance was determined using Rosco tablets and according to the CLSI methods.



3/12

Table1. Primers used and expected size of PCR product.

Gene target Primer name Nucleotide sequence (3’→5’) Size (bp)

STb STb-1 TGCCTATGCATCTACACAAT 133

STb-2 CTCCAGCAGTACCATCTCTA

STaP StaP-1 CAACTGAATCACTTGACTCTT 158

StaP-2 TTAATAACATCCAGCACAGG

F5 (K99) K99-1 AATACTTGTTCAGGGAGAAA 230

K99-2 AACTTTGTGGTTAACTTCCT

LT LT-1 GGCGTTACTATCCTCTCTAT 272

LT-2 TGGTCTCGGTCAGATATGT

F18 F18-1 TGGTAACGTATCAGCAACTA 313

F18-2 ACTTACAGTGCTATTCGACG

F6 (987P) 987P-1 GTAACTCCACCGTTTGTATC 409

987P-2 AAGTTACTGCCAGTCTATGC

F4 (K88) K88-1 GAATCTGTCCGAGAATATCA 499

K88-2 GTTGGTACAGGTCTTAATGG

F41 F41-1 AGTATCTGGTTCAGTGATGG 612

F41-2 CCACTATAAGAGGTTGAAGC

STx2e STx2e-1 AATAGTATACGGACAGCGAT 733

STx2e-2 TCTGACATTCTGGTTGACTC



4/12

Results and discussion

Results are summarised in Table 2 to 5.

A total of one hundred thirty five pig strains were analysed in 2011, of which 133 were analysed for

virulence characteristics by PCR. In 76 of these (more than half) no virulence genes could be detected. The

most prevalent pathotype was ETEC. Few strains were positive for F41, F5 or F6 fimbrae. F4 was the most

frequently encountered adhesion factor, followed by F18. Of the ETEC associated toxins, STb was the most

prevalent. Haemolysis was seen in a little less than 60% of the strains. In approximately 10% (n=13) of the

haemolytic strains no virulence genes were detected. The other way round, in 7 (approx. 10% also) non

haemolytic strains, virulence genes were detected. There is a disagreement between virulence and

haemolysis, which means that when having a haemolytic strain, this does not necessary corresponds to

pathology, and when finding a non-haemolytic strain, it may be related to the disease anyhow.

The virulence profiles are shown in Table 4.



5/12

Table 2. Pathotypes found in pig E. coli strains in 2011.

Virulence Type N of strains % of strains

Colonising strains 5 3,8

Enterotoxinogene E. coli (ETEC) 35 26,3

Oedema disease (verotoxigenic E. coli, VTEC) 8 6,0

VTEC without attachment factor 0 0,0

No virulence genes detected 76 57,1

ETEC/VTEC 1 0,8

ETEC without attachment factor 8 6,0

Total 133

Table 3. Virulence genes detected in E. coli from diseased pigs in 2011.

Virulence gene Negative Positive % of the strains positive for the

gene

987P FIMBRIAE (F6) 131 2 1,5

F18 FIMBRIAE 106 27 20,3

F41 FIMBRIAE 131 2 1,5

K88 FIMBRIAE (F4) 116 17 12,8

K99 FIMBRIAE (F5) 131 2 1,5

LT TOXIN 112 21 15,8

SLT-IIV TOXIN 124 9 6,8

STb TOXIN 91 42 31,6

STa TOXIN 115 18 13,5

Haemolysis 72 63 46,7

No virulence gene detected 57 76 57,1



6/12

Table 4. Virulence profiles detected amongst the strains analysed in 2011.

Virulence profile Number of strains % of strains

F18 5 3,8

F18 LT STb 1 0,8

F18 STa STb 12 9,0

F18 VT 8 6,0

F18 VT STa STb 1 0,8

F4 LT STa STb 1 0,8

F4 LT STb 16 12,0

F41 LT 1 0,8

F41 LT STa STb 1 0,8

F5 F6 STb 2 1,5

LT STa STb 2 1,5

STa 1 0,8

STb 6 4,5

Negative 76 57,1

Antibiotic resistance in pig strains is shown in Table 5 and multi-resistance is shown in Table 6 and figure1.

Only 10 of the 135 strains tested (7.4%) were completely susceptible to all antibiotics tested (Figure 1,

Table 6). Resistance was lowest to apramycin and florfenicol, followed by the antibiotics gentamicin,

amoxicillin + clavulanic acid, enrofloxacin and neomycin. Ceftiofur resistance reached 17%. Most resistance

was found against ampicillin, tetracycline, and trimethoprim + sulphonamides. Strain resistant to

amoxicillin + clavulanic acid were not always resistant to ceftiofur (data not shown) and ceftiofur resistance

did not always match amoxicillin + clavulanic acid resistance indicating a high diversity and complex

composition of resistance mechanisms.

Most strains are resistant to 5 antibiotics, which represents the mathematical median. Few strains are

resistant to 9 or more antibiotics. One strain, in which no virulence genes were found was resistant to all

antibiotics tested. The most multi-resistant virulent strains were resistant to 9 different antibiotics (all 3

were ETEC).



7/12

Table 5. Antibiotic resistance in E. coli, isolated from pigs in 2011

Antibiotic Susceptible Resistant Total % Resistant

NEOMYCIN 114 20 134 14,9

TRIMETHOPRIM 45 89 134 66,4

GENTAMICIN 124 11 135 8,1

TETRACYCLINE 35 99 134 73,9

SULPHONAMIDE 32 103 135 76,3

STREPTOMYCIN 69 66 135 48,9

AMOXICILLIN + CLAVULANATE 118 16 134 11,9

APRAMYCIN 126 9 135 6,7

ENROFLOXACIN 117 17 134 12,7

CEFTIOFUR 112 23 135 17,0

FLORFENICOL 125 9 134 6,7

CHLORAMPHENICOL 96 39 135 28,9

AMPICILLIN 35 99 134 73,9

NALIDIXIC ACID 98 37 135 27,4

TRIMETHOPRIM SULPHONAMIDE 52 82 134 61,2

Figuur 1. Multi-resistance in E. coli from pigs in 2011.

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 8 9 10 11 12



8/12

Table 6. Multi-resistance in E. coli strains from pigs in 2011.

Number of antibiotics Number of strains % of strains Cumulative % of strains

0 10 7,4 7,4

1 4 3,0 10,4

2 9 6,7 17,0

3 11 8,1 25,2

4 24 17,8 43,0

5 33 24,4 67,4

6 15 11,1 78,5

7 14 10,4 88,9

8 8 5,9 94,8

9 3 2,2 97,0

10 3 2,2 99,3

11 0 0,0 99,3

12 1 0,7 100,0

Conclusions

A little more than 40% of the strains was proven to be pathogenic. Nearly 90% of these strains were

haemolytic, a characteristic associated with virulence characteristics. Compared to other years, this is a

high number, though it still indicates that haemolysis is not enough as a fast characterisation of virulence.

The other way round, the absence of haemolysis has been more associated with the absence of virulence

characteristics.

Resistance against some antibiotics is substantial. There are few antibiotics left against which resistance is

low. Strains are most frequently resistant to 5 antibiotics. They are rarely resistant to 1 or more antibiotics.

One strain was resistant to all antibiotics tested.



9/12

ADDENDUM: comparison of data obtained from 2005 to 2011

Table 1. Pathotypes found in pig E. coli strains in 2005-2011

% strains

Virulence Type 2005 2006 2007 2009 2010 2011

Colonising strains 8,5 22,3 15,8 10,8 1,8 3.8 Enterotoxinogenic E. coli without adhesines 2,7 0 0 14,7 6,5 6

Enterotoxinogene E. coli (ETEC) 33,7 32,4 36,4 43,1 20,6 26.3

Mixed ETEC / VTEC 2,0 0 0,4 2,9 3,5 0.8

Oedema disease (verotoxigenic E. coli, VTEC) 6,8 4,5 11,0 6,9 6,5 6

VTEC without attachment factor 0 3,6 3,5 0 2,4 0

No virulence genes detected 46,3 37,3 33,3 21,6 59,4 57.1

Total number of strains tested 294 247 228 102 170 133

Table 2. Virulence genes detected in E. coli from diseased pigs in 2005-2011

% strains positive for this gene

2005 2006 2007 2009 2010 2011

987P fimbriae (F6) 0,0 0 0 0,0 0,6 1,5

F18 fimbriae 24,5 33,2 33,2 38,2 8,2 20,3

F41 fimbriae 0,0 0,4 0,4 0,0 0 1,5

K88 fimbriae (F4) 26,5 27,9 27,9 25,5 14 12,8

K99 fimbriae (F5) 0,0 0 0 2,9 0 1,5

LT toxin 26,2 25,9 25,9 34,3 18,1 15,8

SLT-IIV toxin (vt2e) 10,2 8,1 8,1 13,7 6,4 6,8

STa toxin 16,0 10,1 10,1 27,5 28,7 31,6

STb toxin 32,0 29,6 29,6 53,9 11,1 13,5

Haemolysis 85,7 87,9 87,9 81,4 59,1 46,7

No virulence genes detected 46,3 37,3 37,3 21,6 59,4 57,1

There is no real evolution found in the different pathotypes encountered. Neither is there a long term

evolution in the distribution of virulence genes.



10/12

Table 3. Antibiotic resistance in E. coli, isolated from pigs in 2005-2011

% Resistance Antimicrobial 2005 2006 2007 2009 2010 2011 Amoxicillin + clavulanic acid 1,4 0,4 1,3 2,0 7,6 11.9

Ampicillin 63,1 68,4 75,4 82,4 80,7 73.9

Apramycin 7,8 2,4 7,5 7,8 5,3 6.7

Ceftiofur 0,7 2,0 2,6 9,8 19,3 17

Chloramphenicol 28,7 30,8 27,6 30,4 26,9 28.9

Enrofloxacin 4,1 2,8 6,1 7,8 12,9 12.7

Florfenicol 2,0 1,6 3,8 2,9 4,1 6.7

Gentamicin 2,7 1,6 2,6 10,8 8,8 8.1

Nalidixic acid 22,5 18,2 24,6 32,4 28,1 27.4

Neomycin 6,1 4,5 4,8 19,6 19,3 14.9

Tetracycline 75,4 85,8 79,4 80,4 71,3 73.9

Trimethoprim + Sulphonamide 68,9 62,8 75,9 73,8 75,4 61.2

Streptomycin NT NT NT 65,8 60,8 48.9

Sulphonamide NT NT NT 81,6 83,0 73.6

Trimethoprim NT NT NT 81,6 76,6 66.4



11/12

Figure 1. Evolution of resistance for the different antibiotics (2005-2011).

Figuur 2. Multi-resistance in E. coli from pigs in 2005-2011.

0

10

20

30

40

50

60

70

80

90

100

2005

2006

2007

2009

2010

2011

0

5

10

15

20

25

30

35

40

0 1 2 3 4 5 6 7 8 9 10 11 12

2005% (N=293)

2006% (N=247)

2007% (N=228)

2009% (N=102)

2010% (N=171)

2011 (n=135)



12/12

Table 5. Multi-resistance in E. coli strains from pigs in 2005-2011

% resistance

N antibiotics 2005 2006 2007 2009 2010 2011

0 8,5 9,7 7,5 5,9 7,6 7,4 1 13,3 10,5 8,8 2,9 2,9 3,0 2 18,4 17 13,6 10,8 2,9 6,7 3 29,4 30,8 35,1 19,6 6,4 8,1 4 16,4 20,6 14,9 16,7 12,9 17,8 5 7,2 7,7 14,0 16,7 25,1 24,4 6 4,8 3,2 3,5 15,7 18,1 11,1 7 1 0 2,2 6,9 10,5 10,4 8 1 0 0,4 2,0 6,4 5,9 9 0 0 0,0 2,0 4,7 2,2 10 0 0,4 0,0 1,0 2,3 2,2 11 0 0 0,0 0,0 0,0 0,0 12 0 0 0,0 0,0 0,0 0,7

After 7 years of surveillance, there is a clear and significant increase in resistance against ceftiofur and

amoxicillin + clavulanic acid (AMC). Because there is only in few cases cross resistance of the ceftiofur

resistant strains with AMC, most of the cephalosporin resistant strains seem to carry ESBLs. Confirmatory

tests are necessary to confirm this. The AMC resistant strains may be strains over-expressing the

chromosomal ampC ß-lactamase or may carry IRTs. A study on the genetic background of this resistance is

warranted.

For the fluoroquinolones, the aminoglycosides gentamicin and neomycin, and florfenicol a trend towards

higher resistance is obvious, although the resistance remains low. Statistical analysis of the data is

necessary. Against three, for pig industry therapeutically important antibiotics (ampicillin, tetracyclines and

sulfa-trimethoprim), resistance remains high with more than three quarter of the strains resistant. The

number of strains completely susceptible stays low and multi-resistance is at raise. Over a nearly 7 year

period, tendencies in resistance become clearer. Some antibiotics became more resistant, and the multi-

resistance has clearly increased from a median of 3 to 5 antibiotics. This urges for appropriate use of

antibiotics, in order to prevent an increase in resistance.

Documents

Report Pathogenic E. coli from pigs: virulence genes and ...€¦ · CODA-CODA ---CERVACERVA Veterinary and Agrochemical Research Centre Report Pathogenic E. coli from pigs: virulence