MINIMUM INHIBITORY CONCENTRATIONS OF AND

ADAPTATION TO FIVE DISINFECTANTS COMMONLY USED AGAINST

SALMONELLA IN THE POULTRY INDUSTRY

Kim O. Gradel, Danish Veterinary Laboratory

Luke Randall, Veterinary Laboratories Agency Weybridge, England

Epidemiological background

Since 1992, for all Danish broiler flocks:Samples for Salmonella examination have been

submitted and results registered.Data, including the use of disinfectants in the

preceding download period, have been registered by a visiting veterinarian,.

All data are available in a central database.

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

Salmonella Infantis in persistently infected broiler houses

First isolateselected

”Middle” isolateselected

Last isolateselected

Aims of the study

To find minimum inhibitory concentrations (MICs) of five disinfectants for ”non-persistent” and ”persistent” Salmonella serotypes commonly isolated from Danish broiler houses.

To find MICs of five disinfectants for other Salmonella isolates from the poultry sector.

To see if selected strains can be adapted to the five disinfectants in the laboratory.

Sources of bacterial isolatesCountry 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

Disinfectants used in the study

Formaldehyde (24.5%)Glutaraldehyde (23%) and benzalkonium

chloride (5%) compoundOxidising compound (blend of peroxygen

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

”Danish”disinfectants

”English”disinfectants

MIC-tests (method)

Adaptation and deadaptation (method)Passages of isolate to increasingdisinfectant concentrations untilthere is no growth

MIC-tests MIC-tests

Six passages of isolate in brothwithout disinfectant

MIC-tests (Salmonella results)

• S. Tennessee is a ”non-persistent” serotype.• Among ”persistent” types from broiler houses, only S. 4.12:b:- has increased MICs to the iodophor, which is used rarely in Denmark.• S. Senftenberg may have a higher intrinsic resistance??

Low High Low High Low High Low High Low HighDenmark Enteritidis 34 0 28 6 14 20 13 21 13 21Denmark Typhimurium 39 0 36 3 20 19 19 20 22 17Denmark Tennessee 9 15 8 16 6 18 4 20 2 22Denmark 4.12:b:- 66 15 59 22 12 69 17 64 14 67Denmark Infantis 61 0 43 18 18 43 29 32 17 44Denmark Indiana 16 1 16 1 11 6 16 1 14 3Denmark Senftenberg 0 13 10 3 5 8 1 12 10 3UK Choleraesuis 1 0 1 0 0 1 1 0 1 0UK Typh., DT104 8 0 8 0 0 8 0 8 2 6UK 4.12:d:- 4 0 4 0 0 4 0 4 0 4UK Senftenberg 0 4 0 4 0 4 0 4 0 4

Country Salmonella type

Disinfectants (see text for designations) and MICsFormaldehyde Oxidising Glutarald./BC Phenol Iodophor

MIC-tests (results for E. coli control strains)

AG100 AG102 10418 AG100 AG102 10418 AG100 AG102 10418 AG100 AG102 10418 AG100 AG102 104180.008 8 11 1

0.015 23 20 30 12 6 2 2

0.03 3 1 5 17 25 28 26

0.06 13 13 1 24 26 1 1 4 12 6

0.125 14 16 19 1 4 17 23 6

0.25 11 1 1 25

0.5 1

MIC

Disinfectant and control strainFormaldehyde Oxidising Glutaraldehyde/BC Phenol Iodophor

• The mar response is apparently not involved in increased resistance.

More results (data not shown)

• No associations between increased MICs for the three disinfectants showing significant differences for some serotypes (formaldehyde, oxidising compound, iodophor).• No associations between MICs and use of disinfectants in the preceding download period.• Adaptation or deadaptation did not alter any MICs significantly.• No significant increases or decreases in MICs during the persistence periods (67 broiler houses with two isolates and 21 broiler houses with more than two isolates), either generally (p = 0.30) or for any individual 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.

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

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Unknown source

Other animals

Exotic, not PI

Ent/Typ, unknown source

Hatchery

PI-houses

One persistently Salmonella infected broiler houseDATE SALM1 SALM2 SALM3 DES1 DES2 DES3

24-feb-92 S.Enteritidis Formaldehyde Lime .28-apr-92 S.Enteritidis Formaldehyde . .08-sep-92 S.Infantis Formaldehyde . .10-nov-92 S.Infantis Orbivet (type??) . .17-maj-93 S.Infantis Formaldehyde . .13-sep-93 S.Typhimurium S.Infantis S.Schwarzengrund Formaldehyde . .15-nov-93 S.Typhimurium Orbivet (type??) . .08-mar-94 S.Typhimurium Orbivet (type??) . .25-okt-94 S.Infantis Orbivet (type??) . .28-dec-94 S.Infantis Formaldehyde Bases .28-feb-95 S.Infantis Orbivet (type??) . .19-apr-95 S.Infantis Orbivet (type??) . .14-jun-95 S.Infantis Formaldehyde . .17-aug-95 S.Infantis Glutaraldehyde/BC . .17-okt-95 S.Infantis Glutaraldehyde/BC . .30-nov-95 S.Infantis Formaldehyde Orbivet (type??) .25-jan-96 S.Infantis Glutaraldehyde/BC . .17-sep-96 S.Typhimurium S.Infantis Glutaraldehyde/BC . .05-nov-96 S.Infantis Glutaraldehyde/BC . .18-mar-97 S.Infantis S.Schwarzengrund Glutaraldehyde/BC . .15-maj-97 S.Infantis S.Schwarzengrund Glutaraldehyde/BC . .16-jul-97 S.Infantis Glutaraldehyde/BC . .

03-sep-97 S.Infantis Glutaraldehyde/BC . .20-jan-98 S.Hadar S.Infantis Bases Chloramin Glutaraldehyde/BC26-mar-98 S.Infantis Glutaraldehyde/BC Lime Bases

Use of disinfectants in Danish broiler houses

DISINFECTANT NO. OF BROILER FLOCKSBase 31No disinfection 177Formaldehyde 1227Glutaraldehyde/BC 4458Undefined 86Iodine compound 39Lime 165Chlor compound 101QAC 87Oxidising compound 595Unknown 416

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.