Escherichia coli O157:H7 is difficult to acquire resistance to chlorine solution genetically

Yasuhiro Inatsu, Md Latiful Bari, Susumu Kawasaki, and Shinichi Kawamoto(Food Hygiene Laboratory, National Food Research Institute, NARO)

Taylor Farms (California, USA), 2007.8

The emergence and spread of microorganisms with reduced susceptibility to antimicrobial agents is a major public health problem.Chlorine water has been widely used to reduce the attached bacteria on the surface of food or sanitizing of the facilities. However, there is limited report for the development of acquired resistance of pathogen to chlorine. This study was designed to evaluate the possibility of acquired chlorine resistance to Escherichia coli (E.coli) O157:H7.No meaningful change was observed of bactericidal sensitivity during the repeatable cultivation. This study suggested that the possibility of emerging chlorine resistance of E. coli O157:H7 mutant would be a negligible risk. However, there is possibility of biofilm formation by the bacteria have shown to exhibit higher tolerance to the sanitizers.

Abstract

Vegetables can be a cause of outbreakFoods Pathogen Date Are a Patie n ts

Vege table salad Noroviru s 2002 .3 H iro sh ima 34

Bean sprou ts dre ssedw ith vin e gar E.co li 2 002 .4 Wakayama 202

Ligh tly fe rmen tedcucumbe r E.co li 2 002 .6 Fukuoka 102

Andes me ron Salmone lla 2 002 .9 Iwate 28

Po te to salad Noroviru s 2003 .5 Hokkaido 42

Apple salad Ye rs in ia 2 004 .7 Nara 40

Po te to salad Noroviru s 2004 .1 2 Yamagata 202

Japanese style d k imch i E.co li 2 005 .8 Ch iba 401

Swee t po te to Salmone lla 2 005 .1 2 Oo ita 67

Bo ile d sh redded radish Noroviru s 2006 .1 Hokkaido 106

Bo ile d gre en vege tabledre ssed w ith gratedse sami seed

Campylobac te r 2 006 .5 Kyo to 40

Bo ile d brac ken shoo t B . ce reu s 2006 .6 Nagano 22

Foods Pathogen Date Coun try Patie n ts

Icebe rg le ttu ce E.co li 2 003 .9 USA 40

Green on ion Hepatitis v iru s A 2003 .1 1 USA 490

Tomato Salmone lla 2 004 .6 USA 429

Icebe rg le ttu ce Salmone lla 2 004 .9 UK 372

Rucco la Salmone lla 2 004 .1 1 Norway 13

Icebe rg le ttu ce Hepatitis v iru s A 2005 .9-10 USA 60

Le ttuce salad E.co li 2 005 .1 0 USA 26

Bean sprou ts Salmone lla 2 005 .1 1 USA 379

Spin ach E.co li 2 0 06 .9-10 USA 199

Japan

4% of confirmed cases / patients caused by vegetable foods.(static of food poisoning, MHLW )

USA

20.4% of 138,622 casesin 1990-2003 relative tovegetables.

(CSPI Database)

Reduction of the surface attached bacteria by usingchlorine water is required.

Hydrophilic

Hydrophilic

hydrophobic

How bactericide kill bacteria?

Inhibition of intra cellar metabolism

Denature / disruption of cell wall membrane

Exposure of oxidativebactericide (NaClO etc)

Ionic form

Non-ionic form

Cl2 + H2OH+ + Cl-+HOCl

H+ + OCl-

HO

Cl(

%)

Ionic form

Non-ionic form

Similar chemical equilibrium is exist in the water solution of Ca(OCl)2 and electrolyzed (NaCl) water

Gram positive bacteria

Problems of the occurrence of drug resistance strain

“MRSA Note” (http://www.mrsanotes.com/)

“The why ? Files - Resisting Antibiotics ” (http://whyfiles.org/shorties/090antibio_resist/)

Microbiologically safe vegetables

Washing by chlorine water, etc.

Are bacteria exposed to sanitizermany times acquire drug resistance?

Are bacteria acquire resistance to sodium hypochlorite?

Combination of severalweak (natural) chemicalsand / or physical treatments

Resistant strainsSensitive strains

Prevent the occurrence of resistant strains Confirm the possibility

Enrichment cultivation(sodium hypochlorite)

* Viable cell counts* ATP measurement* Chemiluminescence assay

Hypochlorous acid (HOCl) resistant Salmonella was isolated from poultry abattoir (Mokgatla et al. 1998)

Compare the cellar activity

Experimental methodsE. coli O157:H7 CR-3 strain (from calf feces)

Introduce random mutation by NTG treatment

Wild type Mutant strain

(Enhance the genetically evolution)

Grown in 40 mL BHIWashing cell by PBS

Expose to 100 ppm NaClO(5min, 25oC, pH7.0)

Neutraize by Na2S2O3Washing cell by PBS

(Repeating times: 1-8)

Assay of Viability

Enrichment cultivation

H+ + NAD(P)H

NAD(P)+

酵素

生細胞メナジオン

酸素

スーパーオキシド

過酸化水素

発色試薬

メナジオンラジカル 発光

原理

インキュベーション 10分間

発光試薬

メナジオン溶液

自動分注

食材１０％乳剤 MILK

・・

37℃で増菌培養

分注

マイクロプレートに分注

０ １ ２ ３ ４ ５

計測時間（秒）

発光

強度

生菌の存在

約2秒以内に発光ピーク出現対照

検出

時間

(hr)

00

2

2 4

4

6

6

8

8●

●

●

●

●

●

Log CFU/ml緑茶飲料水中の大腸菌初発菌数と検出時間の関係

自動計測とデータ解析

測定 5秒間

H+ + NAD(P)H

NAD(P)+

酵素

生細胞メナジオン

酸素

スーパーオキシド

過酸化水素

発色試薬

メナジオンラジカル 発光

原理

インキュベーション 10分間

発光試薬

メナジオン溶液

自動分注

食材１０％乳剤 MILK

・・

37℃で増菌培養

分注

マイクロプレートに分注

０ １ ２ ３ ４ ５

計測時間（秒）

発光

強度

生菌の存在

約2秒以内に発光ピーク出現対照

発光試薬

メナジオン溶液

自動分注

発光試薬

メナジオン溶液

自動分注

発光試薬

メナジオン溶液

自動分注

食材１０％乳剤 MILKMILK

・・・・

37℃で増菌培養

分注

マイクロプレートに分注

０ １ ２ ３ ４ ５

計測時間（秒）

発光

強度

生菌の存在

約2秒以内に発光ピーク出現対照

０ １ ２ ３ ４ ５

計測時間（秒）

発光

強度

生菌の存在

約2秒以内に発光ピーク出現対照

検出

時間

(hr)

00

2

2 4

4

6

6

8

8●

●

●

●

●

●

Log CFU/ml緑茶飲料水中の大腸菌初発菌数と検出時間の関係

検出

時間

(hr)

00

2

2 4

4

6

6

8

8●

●

●

●

●

●

Log CFU/ml

検出

時間

(hr)

00

2

2 4

4

6

6

8

8●

●

●

●

●

●

Log CFU/ml緑茶飲料水中の大腸菌初発菌数と検出時間の関係

自動計測とデータ解析

測定 5秒間

Menadione catalyzed Chemiluminescence assay

Viable cell counts

ATP measurement

Chemiluminescenceassay

TSA - SMAC+3%NaClInjuredcells

RespirableEnzymeActivity

Control(PBS)

Optimization of NTG mutation treatment

1

2

3

4

5

6

7

8

9

10

After treatment（NB) After recovery（NB) After recovery（Rif)

Via

ble

cell （

log C

FU

/m

l）

NTG 0 ppm

NTG 50 ppm

NTG 100 ppm

NTG 200 ppm

Increasing NTG concentration reduced the viability of the total cells

3 log increase of mutation rate was achieved by using 50-100 mg/L NTG

Exposure to 50 mg/L NTG for 10 min at 25oC was adapted

Change of viable cell counts during enrichment cultivation

Mutant strain

2

3

4

5

6

7

8

9

10

1 2 3 4 5 6 7

Cycles of enrichment

Via

ble

cells

（lo

g C

FU

/m

L)

PBS (TSA)

PBS (SMAC)

NaClO (TSA)

NaClO (SMAC)

Enumerated by TSA

Enumerated by SMAC+3%NaCl

No significant change of bactericidal activity was observed

2

3

4

5

6

7

8

9

10

1 2 3 4 5 6 7

Cycles of enrichment

Via

ble

cells

（lo

g C

FU

/m

L)

PBS (TSA)

NaClO (TSA)

PBS (SMAC)

NaClO (SMAC)

Change of viable cell counts during enrichment cultivation

Wild type strain

Enumerated by TSA

Enumerated by SMAC+3%NaCl

Same as the case of mutant strain

0

1

2

3

4

5

6

7

8

9

10

NaClO-8 NaClO-1 PBS-1

Via

ble

cells (

log C

FU

/g),

CL

(lo

g c

ounts

/2s),

AT

P (

log R

LU

)

APC(SMAC)

APC(TSA)

CL

ATP

Comparison of the cellar viability before and after 8 times enrichment cultivation

The dropdown of menasione catalyzed chemiluminescence, cellar ATP level or viable cell counts by NaClO exposure did NOT change

ABPC 10 20.0 ± 0.4 19.6 ± 0.8CFX 30 25.3 ± 0.5 25.3 ± 0.8LMOX 30 27.2 ± 0.5 26.7 ± 0.4AZT 30 28.2 ± 0.4 28.6 ± 0.3IPM 10 30.4 ± 1.3 30.1 ± 0.5FOM 50 25.6 ± 0.6 25.6 ± 0.5NA 30 22.4 ± 0.4 22.9 ± 0.6TC 30 25.9 ± 0.4 26.1 ± 0.2SM 10 16.1 ± 0.4 15.9 ± 0.3GM 10 19.0 ± 1.0 19.1 ± 0.5

Beforenrichmentcultivation

(mm)

Afterenrichmentcultivation

(mm)

Diameter of diffusion zone (mm)

Antibiotics

weightin a

paperdisks(μg)

Comparison of the antibiotic resistance between the samples before and after enrichment cultivation

No significant change of antibiotic resistance was observed

Conclusion

It was suggested that E. coli O157:H7 is difficult to acquire genetic resistance even though the strain is routinely exposed by sodium hypochlorite.

Antibiotic

Target is specified

Oxidative sanitizer

Target is NOT specified

Why the effectiveness of chlorine water (NaClO) is said to reduce in the actual situation in food factories?

Possible reasons

Chlorine resistant biofilm is formed

Reduction of active chlorine by the contamination of organic material

Improvement of the general hygiene practices

(GHP) is required