Post on 22-May-2018
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