Chemical disinfectants commonly used in the

poultry sector and their impact on Salmonella – an

outline of methods and results

Kim O. GradelDanish Veterinary Institute,

Århus, Denmark

SYMPOSIUM: DISINFECTION IN ANIMAL PRODUCTIONTuesday 18 November, 2003, Aarhus, Denmark

Disposition Resistance against disinfectants

Minimum inhibitory concentration (MIC) tests

Adaptation and de-adaptation tests Conclusions

Surface disinfection tests Materials and methods Results Conclusions

General conclusions

Chemical tests related to general disinfection factors

Micro-organism

Disinfectant Surroundings

Type”State/condition”

Organic matterTemperaturepHBiofilm

TypeConcentration

Resistance to disinfectants

Surface disinfection tests

Resistance against disinfectants

Hypothesis:There is an association between persistence of

Salmonella in poultry houses and the common use of a few types of disinfectants in these.

Aims: To see if minimum inhibitory concentrations

(MICs) against five commonly used disinfectants could be related to Salmonella persistence or use of disinfectants in Danish broiler houses.

To see if resistance against the five disinfectants could be introduced and maintained in the laboratory.

Resistance against disinfectants:

epidemiological tools

Since 1992, for all Danish broiler flocks/crop cycles:

samples for Salmonella have been submitted.

The use of disinfectants has been registered by a veterinarian visiting the flock.

All data have been registered in a central database.

Salmonella sources in broiler flocks (1/11/96-31/10/99)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

No

v-9

6

De

c-9

6

Jan

-97

Fe

b-9

7

Ma

r-9

7

Ap

r-9

7

Ma

y-9

7

Jun

-97

Jul-

97

Au

g-9

7

Se

p-9

7

Oct

-97

No

v-9

7

De

c-9

7

Jan

-98

Fe

b-9

8

Ma

r-9

8

Ap

r-9

8

Ma

y-9

8

Jun

-98

Jul-

98

Au

g-9

8

Se

p-9

8

Oct

-98

No

v-9

8

De

c-9

8

Jan

-99

Fe

b-9

9

Ma

r-9

9

Ap

r-9

9

Ma

y-9

9

Jun

-99

Jul-

99

Au

g-9

9

Se

p-9

9

Oct

-99

Month/year

No

. o

f fl

ocks

Unknown source

Other animals

Exotic, not PI

Ent/Typ, unknown source

Hatchery

PI-houses

Resistance against disinfectants:persistently Salmonella-infected

broiler houses

Salmonella serotypeNumber of crops with the same

Salmonella type12345

6-1011-2021-30> 30

 Enteritidis

 51

64 2    

Typhimurium2743122   

Tennessee 44112    

4.12:b:-132246451 

Infantis 923364   

Indiana112113    

Resistance against disinfectants:

persistence in broiler houses

No. of crops with the same Salmonella type Salmo- nella type 1 2 3 4 5

6-10

11-20

21-30

>30

Ent. 5 6 4 2

Typh. 2 7 4 3 1 2 2

Tenn. 4 4 1 1 2

4.12:b:- 1 3 2 2 4 6 4 5 1

Inf 9 2 3 3 6 4

Ind 1 1 2 1 1 3

•In this study, 67 and 21 broiler houses were represented with two or more than two isolates, respectively.

11 21 31 41 51Nr. 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 I alt1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 E E E E E E E E E E E E 212 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 E E E E E E E E 193 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 E E E E E E E 194 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 E E E E 175 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 E E E E E E E 176 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 E E E E E 157 E E E 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 E E E E E 158 1 1 1 1 1 1 1 1 1 1 1 1 1 1 E E E E E E 149 1 1 1 1 1 1 1 1 1 1 1 1 1 E E E E E E E E 1310 1 1 1 1 1 1 1 1 1 1 1 1 1 E E E E E E E E E E E E 1311 1 1 1 1 1 1 1 1 1 1 1 1 E E E E E 1212 1 1 1 1 1 1 1 1 1 1 1 1 E E E E E E 1213 1 1 1 1 1 1 1 1 1 1 1 E E E E E E E 1114 1 1 1 1 1 1 1 1 1 1 1 E E E E E E E E 1115 1 1 1 1 1 1 1 1 1 1 E E E E E 1016 1 1 1 1 1 1 1 1 1 E E E E 917 1 1 1 1 1 1 1 1 E E E E E E E E E E E E 818 1 1 1 1 1 1 1 1 E E E E E E E E E E E E E E E E E E E E E E E E E E E E 819 1 1 1 1 1 1 1 E E E E E E E E E E E E E 720 1 1 1 1 1 1 1 E E E E E E E E E 721 E E E E E E E 1 1 1 1 1 1 1 E E E E E E 722 1 1 1 1 1 1 1 E E E E E E E E E E E E E E E E E E E E E E E 723 1 1 1 1 1 1 E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E 624 1 1 1 1 1 1 E E E E E E E E E E E E E 625 1 1 1 1 1 1 E E E E E E E 626 E E E E E E E E E E 1 1 1 1 1 1 E E E E E E E E E E E E E E E E E 627 E E E E E E E E E E E E E E E E E E E E E E E E 1 1 1 1 1 1 E E E E E E 628 1 1 1 1 1 E E E E 529 1 1 1 1 1 E E E E 530 1 1 1 1 1 E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E 531 1 1 1 1 1 E E E E E 532 1 1 1 1 1 E E E E E E E E E E E E E E E 533 1 1 1 1 1 E E E E E E E 534 1 1 1 1 1 E E E E E E 535 1 1 1 1 E E E E 436 E E E E E E E E E E E E E E E E E E E E E E E E E E 1 1 1 1 E E E E E E 437 1 1 1 1 E E E E E 438 1 1 1 1 E E E E E E 439 1 1 1 1 E E E E E E E 4

Resistance against disinfectants:persistently Salmonella-infected broiler houses

First isolateselected

”Middle” isolateselected

Last isolateselected

Resistance against disinfectants:

sources of bacterial isolates

Country Type No. of isolates Source and descriptionDenmark S . Enteritidis 34 Danish broiler houses, “non-persistent type”Denmark S . Typhimurium 39 Danish broiler houses, “non-persistent type”Denmark S . Tennessee 24 Danish broiler houses, “non-persistent type”Denmark S . 4.12:b:- 81 Danish broiler houses, “persistent type”Denmark S . Infantis 61 Danish broiler houses, “persistent type”Denmark S. Indiana 17 Danish broiler houses, “persistent type”Denmark S . Senftenberg 13 Poultry sectorUK S. Choleraesuis NCTC 10653 1 Strain used in English disinfection testsUK S . Typhimurium, DT104 8 Pig and broiler farmsUK S . 4.12:d:- 4 Feed mill and hatcheryUK S . Senftenberg 4 HatcheryUK E. coli NCTC 10418 1 Control strainUK E. coli AG100 1 Control strainUK E. coli AG102 1 Control strain, mar mutant of E. coli AG100

Resistance against disinfectants: disinfectants

used in the study

Formaldehyde (24.5%) Glutaraldehyde (23%) and benzalkonium chloride (5%) compound (Bio Komplet Plus) Oxidising compound (blend of peroxygen

compounds) (Virkon S)

Phenol (30-45% high boiling tar acids) (FFS) Iodophor (FAM 30)

”Danish”disinfectants

”English”disinfectants

Resistance against disinfectants:

MIC-tests

Resistance against disinfectants:

adaptation and de-adaptation

MIC-testsMIC-tests

Resistance to disinfectants:MIC-tests (Salmonella

results)

Formalde-hyde

Glutarald./ BC

Oxidising Phenol Iodophor Country/ type

L H L H L H L H L H

DK/ Ent 34 0 14 20 28 6 13 21 13 21 DK/ Typ 39 0 20 19 36 3 19 20 22 17 DK/ Ten 9 15 6 18 8 16 4 20 2 22 DK/ 4.12:b:- 66 15 12 69 59 22 17 64 14 67 DK/ Inf 61 0 18 43 43 18 29 32 17 44 DK/ Ind 16 1 11 6 16 1 16 1 14 3 DK/ Senf 0 13 5 8 10 3 1 12 10 3 UK/ Chol 1 0 0 1 1 0 1 0 1 0 UK/ DT104 8 0 0 8 8 0 0 8 2 6 UK/ 4.12:d:- 4 0 0 4 4 0 0 4 0 4 UK/ Senf 0 4 0 4 0 4 0 4 0 4

Resistance to disinfectnats:Changes in MICs during the persistence period

Disinfectant No.of MIC-increases No.of MIC-decreasesFormaldehyde 5 9Oxidising compound 32 19Glutaraldehyde/BC 18 14Phenol 14 18Iodophor 24 22

• 67 broiler houses represented with two isolates.• 21 broiler houses represented with more than two isolates.• Generally, no significant changes (p = 0.30).• Oxidising compound has relatively most increases, but p = 0.10.

Resistance to disinfectants:more results (data not

shown)

•No associations between MICs and use of disinfectants in the preceding download period.

• Adaptation or de-adaptation did not alter any MICs beyond one doubling dilution, i.e. within normal biological variation.

Resistance to disinfectants:conclusions

• Resistance to commonly used disinfectants does not play a major role in persistence of Salmonella in Danish broiler houses.

• In the laboratory, it was not possible to adapt selected strains to the actual disinfectants.

Surface disinfection tests

Principles: Worst-case scenario surface disinfection tests simulating

conditions and disinfection procedures encountered in badly cleaned poultry houses, especially at low temperatures.

Factors: Isolates: S. Enteritidis (low MICs), S. Senftenberg (high MICs),

Enterococcus faecalis (putative indicator bacterium). Poultry house materials: Concrete flags, rusty feed chain

links, wooden dowels, jute egg belts. Organic matter: Feed, fats, egg yolk. Disinfectants: formalin, glutaraldehyde/benzalkomium

chloride, oxidising compound, water (control). Temperatures before and after disinfection: 6/11/20/30

and 6/11/30 oC, respectively. Disinfection time: 5, 15, 30 minutes.

Surface disinfection tests:design

1. High bacterial concentrations in organic matter.

2. Organic matter ”added to” poultry house materials.

3. 24 hours of drying.4. Disinfection for set time periods.5. 25 hours of drying.6. Traditional bacteriological procedures with

tenfold dilutions, i.e. a most probable number (”semi-quantitative”) method.

Surface disinfection tests:concrete flag with feed

Surface disinfection tests.feed chain links with feed

Surface disinfection tests: disinfection of concrete flags

Surface disinfection tests:results for concrete flagsesults for concrete flags

Cfu Gpf

TB TA DTS. Enteritidis S. Senftenberg

For G/B

Oks

Wa For G/B

Oks

Wa

L 10 20 11 30 0 0 0 1 0 0 0 2

L 20 20 11 30 0 0 4 ND 0 0 0 2

L 20 11 11 30 0 0 3 3 0 0 2 2

L 20 6 6 30 0 1 3 5 0 0 1 1

L 20 6 6 15 0 0 2 4 0 0 2 1

H 20 6 6 15 00 01 44 55 0 0 2 2

H 20 6 6 5 0 2 4 5 0 1 2 5

Surface disinfection tests: results for feed chain links

OM

TB TA DTS. Enteritidis S. Senftenberg Enterococcus

faecalis

For

G/B

Oks

Wa

For

G/B

Oks

Wa

For

G/B

Oks

Wa

FA30 30 30 43 32 00 55 12 11 00 32

6 6 30 55 55 55 55 35 35 44 44

FE

11 11 30 00 55 55 23 00 10 55 55

6 6 30 00 10 55 45 00 55 55 55

6 6 30 00 41 53 55 00 15 55 55

6 6 15 00 11 55 55 00 11 55 52

6 6 5 00 44 55 54 01 04 45 54

Surface disinfection tests:results for wooden dowels

OM

TB TA DTS. Enteritidis S. Senftenberg Enterococcus

faecalis

For

G/B

Oks

Wa

For

G/B

Oks Wa

For

G/B

Oks

Wa

FA30 30 30 21

0322

020

433

000

100

000 201

233

122

100

322

6 6 30 533

433

555

545

542

333

3>22

455

442

413

332

345

FE

11 11 30 000

110

111

111

000

000

100 122

000

111

232

545

6 6 30 000

000

111

443

000

000

000 211

122

100

112

453

6 6 15 001

010

111

232

000

000

000 111

012

001

112

545

6 6 5 210

010

222

333

000

100

212 133

112

111

334

555

Surface disinfection tests:results for jute egg belts

cfu

TB TA DTS. Enteritidis S. Senftenberg Enterococcus

faecalis

For G/B Oks

Wa

For

G/B

Oks

Wa

For

G/B

Oks

Wa

L 11 11 30 000

122 244

455

0 1 1 4

L 6 6 30 0 0 4 5 0 1 1 3

L 6 6 15 0 1 2 5 0 0 0 3

H 11 11 30 0 2 5 5 0 2 4 5 21 43 55 55

H 6 6 15 0 3 5 5 0 2 2 5

H 6 6 5 3 5 5 5 0 2 4 5 10 55 55 55

Surface disinfection tests: conclusions I

Efficacy (best first): formalin > glutaraldehyde/benzalkonium chloride > oxidising compound > water. Except: Feed chain links with fats at 30 oC

before and after disinfection, where the oxidising compound apparently was the most effective.

Formalin is better than glutaraldehyde/benzalkonium chloride at 6 oC, although it is often stated that formalin is only effective above 16 oC, whereas glutaraldehyde is effective down to 5 oC!

Surface disinfection tests: conclusions II

For the effective disinfectants (formalin and glutaraldehyde/benzalkonium chloride) there were no differences between the two Salmonella isolates, whereas S. Senftenberg was more susceptible than S. Enteritidis in tests with the oxidising compound and water, in spite of higher MICs for S. Senftenberg (for formalin and the oxidising compound).

In general, Enterococcus faecalis was more recalcitrant than the two Salmonella isolates, i.e. it is a putative indicator bacterium, e.g. in field trials.

Fats seem to be the best protectant for the bacteria among the types of organic matter used.

In general, there were few differences between the different poultry house materials.

General conclusions

For Salmonella, resistance to commonly used disinfectants does not seem to be an important aspect of persistence.

Results from unrealistic tests are difficult to extrapolate to realistic tests.

For Salmonella and other vegetative bacteria, results indicate that the surroundings are more important for the efficacy of disinfection than the type of bacteria.

Acknowledgments

Kirsten Holm, DVI, Århus, Denmark.

Luke Randall, Weybridge, UK.

Ian McLaren, Weybridge, UK.