Khảo sát vi khuẩn escherichia coli sinh beta lactamase phổ rộng trên gà tại một số trại chăn nuôi ở đồng bằng sông cửu long

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL DISSERTATION Major: Veterinary Pathology and Treatment Code: 62 64 01 02 BUI THI LE MINH INVESTIGATION OF EXTENDED SPECTRUM BETA-LACTAMASE PRODUCING ESCHERICHIA COLI IN CHICKENS IN THE MEKONG DELTA Can Tho, 2018 THE DISSERTATION WAS COMPLETED AT CAN THO UNIVERSITY Supervisor: Assoc Prof Dr Luu Huu Manh The dissertation is defended in front of the University Examination Council in Can Tho University Place: ……………………….……, Can Tho University Time: ……………………………… Reviewer 1: ……………………………… Reviewer 2: ……………………………… Reviewer 3: ……………………………… Further information of the dissertation could be found at: Learning Resource Center of Can Tho University National Library of Vietnam THE LIST OF PUBLISHED WORKS RELATED TO THIS DISSERTATION A Published papers Bui Thi Le Minh, Luu Huu Manh, Nguyen Nhut Xuan Dung, 2016 Investigation on the presence of Escherichia coli producing extendedspectrum β-lactamase isolated from healthy chicken in Vinh Long province Veterinary Sciences and Techniques, 2: 22-27 Bui Thi Le Minh, Luu Huu Manh, Nguyen Nhut Xuan Dung, 2016 Investigation on CTX-M, TEM and SHV genes in extended-spectrum β-lactamase producing Escherichia coli from chickens in Tra Vinh province Journal of Science, Can Tho University, 42(b): 1-6 Bui Thi Le Minh, Luu Huu Manh, Nguyen Nhut Xuan Dung, 2016 Study on extended-spectrum β-lactamase producing Escherichia coli on chickens from slaughterhouses in Vinh Long province Journal of Science, Can Tho University, special issue 2: 1-5 Bui Thi Le Minh, Luu Huu Manh, Nguyen Nhut Xuan Dung, 2016 Prevalence of extended-spectrum β-lactamase producing Escherichia coli on sick chickens in Vinh Long province Journal of Science, Can Tho University, special issue 2: 6-10 Bui Thi Le Minh, Luu Huu Manh, Nguyen Nhut Xuan Dung, 2016 Study of extended spectrum beta-lactamase producing Escherichia coli isolated from sick chickens in Hau Giang province Proceeding of The 19th Federation of Asian Veterinary Associations Congress, September 6-9th 2016, Ho Chi Minh city Vietnam National University-Ho Chi Minh city Press, 148-150 Bui Thi Le Minh, Luu Huu Manh, Nguyen Nhut Xuan Dung, 2016 Occurrence of extended spectrum beta-lactamase producing Escherichia coli in chickens from slaughter houses in the Mekong delta of Vietnam Proceedings international conference on agriculture development in the context of international integration: Opportunities and challenges, December 7-8th 2016, Ha Noi Agriculture University Press, 198-202 Bui Thi Le Minh, Luu Huu Manh, Nguyen Nhut Xuan Dung, 2017 Occurrence of TEM, SHV and CTX-M genes in diarrhea chickens and antibiotic resistance Proceeding of 33th World Veterinary Congress, August 27-31 2017, Incheon-Korea Bui Thi Le Minh, Luu Huu Manh, Nguyen Nhut Xuan Dung, 2018 Prevalence of extended-spectrum beta-lactamase-producing Escherichia coli on chicken rearer in the Mekong Delta Journal of Science, Can Tho University, special issue 54: 1-5 B Scientific research has been accepted Bui Thi Le Minh (Coordinator) Investigation of extended-spectrum beta-lactamase producing Escherichia coli in chickens and antibiotic susceptibility testing Scientific research project grant Can Tho University, 2015, code number T2015 -62 Bui Thi Le Minh (Coordinator) Investigation on bla CTX-M, bla TEM and bla SHV genes in extended-spectrum beta-lactamase producing Escherichia coli isolated from sick chickens in Vinh Long province Scientific research project grant Can Tho University, 2016, code number T2016 -52 Bui Thi Le Minh (Coordinator) Investigation on TEM, SHV and CTX-M genes in extended-spectrum beta-lactamase producing Escherichia coli isolated from chicken farmers in the Mekong Delta Scientific research project grant Can Tho University, 2017, code number T2017-50 CHAPTER 1: INTRODUCTION 1.1 Necessity of thesis Colibacillosis is one of common infectious bacterial diseases of the broiler and layer industry (Raji et al., 2003) E coli are always found in the gastrointestinal of chickens and disseminated widely in faeces; therefore chickens are continuously exposed through contaminated faeces, water, dust and the environment E coli infection can result in several localized and systemic forms such as vaginitis, orchitis, yolk sac infection, diarrhea enteritis, meningitis, panophthalmitis, arthritis, respiratitis Colibacillosis can break out all chicken ages and cause high mortibity and morbility leading to economic losses on farmers (Barnes et al., 2008) Nowadays, using of antibiotics in feed, water frequently to prevent and treat diseases as well as improve growth of animals will result in selection depression of bacteria, exhibition of multi-resistance bacteria; therefore effects of antibiotic treatment will be slow (Linder, 2015) In recent years, antibiotic resistant bacteria has increased and antibiotic resistance is an notice issue to research deeply in the world, especially extended spectrum beta-lactamase producing E coli (ESBL producing E coli) Because ESBL producing E coli not only destroy most of beta-lactames but also multiresistant to other antibiotics including aminoglycoside, macrolide, chloramphenicol, tetracycline and fluoroquinolone (Lee, 2006) Several studies on ESBL producing E coli in humans and animals were carried out in many countries in the world and some papers were published For example, Blanc et al (2006) determined the presence of ESBL producing E coli in chickens Catalonia, Spain 59.8% In Belgium, Smet et al (2008) determined the occurrence of ESBL producing E coli in healthy week old chickens 45% The study of Costa et al (2009) on 76 chicken meat samples at slaughterhouses in South of Portugal the presence of ESBL producing E coli 38.2% Mamza et al (2010) studied on chickens in Maiduguri, Nigeria; the results showed that the presence of E coli in healthy chickens 4.3% (25/582), sick chickens 28.7% (64/223), the presence of ESBL producing E coli in chickens 11.1% (89/805) The study of Overdevest et al (2011) in Netherlands in 2009 showed 79.8% (68/89) chicken meat samples at stores were positive for ESBL producing E coli and 74.2% (23/31) patients from hospitals were positive for ESBL producing E coli In Vietnam, some studies of ESBL producing E coli on animals, animal food and environment were published: Van Thi Thu Hao et al (2008) investigated the presence of ESBL producing E coli in 30 chicken meat samples, 50 pork samples collected from markets and supermarkets around Ho Chi Minh City, 43 fecal samples of one month old chickens, the results showed that the occurrence of ESBL producing E coli in chicken meat samples, pork samples and fecal samples of chickens were 89.5%, 75% and 95%, respectively Vo Thanh Thin et al (2011) studied on beta-lactam resistant genes in E coli isolated from diarrhea piglets in the South Central Coast Region and Western Highlands of Vietnam; the results showed 115/184 E coli isolates produced at least one beta-lactamase type, beta-lactamases were encoded by TEM (61.96%) and SHV (0.54%) The results of Bui Thị Ba et al (2012) on E coli isolated from healthy cattle in the South Central Coast Region of Vietnam showed 64.70% (22/34) E coli isolates produced at least one beta-lactamase type, beta-lactamases were encoded by TEM (64.70%) and SHV (11.76%) Ho Thi Kim Hoa et al (2013) studied on 45 waste samples (15 fresh wastewater samples, 15 biogas wastewater samples and 15 chicken fecal samples); the results shoewed that the prevalence of TEM, SHV genes encoding extended spectrum beta-lactamase were 82.22% (37/45) and 8.89% (4/45) respectively and CTX-M gene was not detected The results of Vietnam and the world showed ESBL producing E coli were not only found in animals but also in farmers, animal food and environment Poultry husbandry of the Mekong Delta developed the industry chicken husbandry and the local chicken husbandry Chicken diseases, especially ESBL producing E coli, in the Mekong Delta were issues of concern for farmers and scientists bacause they were found in a lot of sample types and were multiresistant to antibiotics However, the information of ESBL producing E coli were limited Therefore, this study was carried out to investigate of the occurrence of ESBL producing E coli in chickens, risk factors and antibiotic resistant situation of them to have primary scientific data for studies in the future and to prevent colibacillosis in chickens effectively and to minimise transmission of antibiotic resistant bacteria from animal husbandry to humans 1.2 Objectives of research - Determination of the occurrence of ESBL producing E coli on chickens in households and industrial farms, risk factors including eggs, chicken rearers, chicken meat in slaughterhouses and environmental factors in households, farms and slaughterhouses in some provinces of the Mekong Delta - Determination of ESBL producing E coli resistance to common used antibiotics for chickens - Evaluation of the genetic correlation of CTX-M and TEM genes in ESBL producing E coli isolated from chickens, eggs, chicken rearers and husbandry environment 1.3 Innovativeness - The results have detemined the high occurrence of ESBL producing E coli on chickens in households and industrial farms Risk factors including eggs, chicken rearers, chicken meat in slaughterhouses and environmental factors in households, farms and slaughterhouses were also found the occurrence of ESBL producing E coli - ESBL producing E coli isolated from chickens were highly susceptible to aminkacin, fosformycin and colistin - The results of the dissertation have confirmed the genetic homologeneity of CTX-M and TEM genes in ESBL producing E coli isolated from chickens, eggs, chicken rearers and husbandry environment CHAPTER 2: MATERIALS AND METHODS 2.1 Contents of research The study was conducted contents of research: Content 1: Survey on the occurrence of ESBL producing E coli on chickens in households and industrial farms, eggs, chicken meats in slaughterhouses, chicken rearers and some factors in husbandry region and slaughterhouses The aim was to determine the prevalence of ESBL producing E coli on each subject of research to find the occurrence of them on chickens and risk factors Content 2: Investigate in antibiotic resistance of ESBL producing E coli to some antibiotics including beta-lactam and antibiotics commonly used in chickens The aim was to find available antibiotics for ESBL producing E coli and to help for the effective prevention and treatment of collibacillosis Content 3: Analysis of nucleotide sequences of some genes encoding extended-spectrum beta-lactamases and contruction of phylogenetic trees Determination of CTX-M and TEM genes encoding extended-spectrum beta-lactamases, analysis of nucleotide sequences of them and contruction of phylogenetic trees The aim was to confirm the genetic homologeneity of them in ESBL producing E coli isolated from chickens, eggs, husbandry environment and chicken rearers, to prevent transmission of antibiotic resistant bacteria in chicken husbandry as well as transmission of antibiotic resistant bacteria from animals to humans and to protect public health The study time: from 2014 to 2017 The location of sampling: in 304 housholds and 104 industrial chicken farms, 12 slaughterhouses in the Mekong delta including Vinh Long, Tra Vinh, Soc Trang and Hau Giang provinces The location of testing: isolation and identification of bacteria, antimicrobial susceptibility test and PCR reactions were carried out in Can Tho University, analyzing nucleotide sequences in Macrogen company, Korea 2.2 Materials The essential items including freezer, autoclave, dryer oven, incubator, vortex, UV-VIS spectrophotometer, PCR system Medium and chemical for incubation, isolation and identification of E coli; antibiotic paper disks; chemical using for PCR, analyzing genes 2.2 Methods 2.2.1 Sampling methods Samples were collected from areas including husbandry regions and slaughterhouses In husbandry regions, the samples were collected from households (50-180 chickens/household) and industrial farms (6.00014.000 chickens/farm) The total of 304 households and 104 farms were investigated Eighty households and farms (60 households and 20 farms) were collected egg, chicken, chicken rearer, and husbandry environment samples (including domestic water, feed and water in trough, air) to analyze the genetic correlation of CTX-M and TEM genes Besides, 12 slaughterhouses were examined At slaughterhouses, chicken and environment samples (domestic water, removing feather water, waste water, floor, air) were selected The samples were collected from Vinh long province (Binh Minh, Binh Tan, Mang Thit, Tra On, Tam Binh, Long Ho districts), Tra Vinh province (Tra Vinh, Tieu Can, Cang Long, Cau Ke, Cau Ngang, Chau Thanh districts), Soc Trang province (Ke Sach, Chau Thanh, Long Phu, Tran Đe, Nga Nam, Vinh Chau, Long Phu, My Tu districts) and Hau Giang province (Vi Thanh, Vi Thuy, Chau Thanh, Phung Hiep, Nga Bay districts) 2.2.1.1 The number of collected samples in households and farms Total of collected chickens in each province were calculated according to Cannon and Reo (1982), estimation of disease prevalence using simple random sampling method: n= Z2*P*(1- P)/d2 n: required sample size, P: expected prevalence, d: desired absolute precision With 95% level of confidence, P=50% and d=5%, total of samples in each provine were at least 384 chickens 2,040 healthy chickens and diarrhea chickens were collected in provinces Healthy chickens were collected fecal swabs from cloacae Each diarrhea chicken was collected samples including lung, liver, meat and feces Table 3.1: The number of collected chickens according to husbandry methods Husbandry methods Subjects of research Total Households Farms Broilers 858 338 1.196 Layers 626 218 844 Eggs 200 50 250 Chicken rearers 60 20 80 Husbandry environment (household or farm) 60 20 80 The rate of ESBL producing E coli isolated from chickens and farmers was sensitive and resistant to antibiotics The rate of ESBL producing E coli isolated from chickens and farmers was multiresistant to antibiotics The prevalence of TEM, SHV, CTX-M genes in chickens, egg, farmer, environmental factors of farmers and slaughterhouses Correlation level of CTX-M and TEM genes in ESBL producing E coli isolated from chickens, eggs, farmers, and environmental factors The percentage (%) was calculated: R=Total of positive samples*100/ Total of tested samples 2.3.7 Statistical analysis Differences of the rate were analyzed by Chi-square test Statistical analysis used Minitab version 16.0 software CHAPTER 3: RESULTS AND DISCUSSIONS 3.1 The occurrence of ESBL producing E coli in households and industrial farms 3.1.1 The occurrence of ESBL producing E coli in chickens according to location The results showed that the prevalence of ESBL producing E coli in chickens in the Mekong delta was 62.01% The prevalence of ESBL producing E coli on chickens in Hau Giang (44.44%) was slower than on chickens in Vinh Long (65.93%), Tra Vinh (68.73%) and Soc Trang (66.22%) significantly (P = 0.00) Some results in the world showed that the rate of chickens was positive ESBL producing E coli in Belgium 45% (Annemieke Smet et al., 2008), in Portugal 38.2% (Daniela Costa et al., 2009), in Maiduguri of Nigeria 4.3% (Sunday Akidarju Mamza et al., 2010), in Japan 40.43% (Midori Hiroi et al., 2012), in Switzerland 63.4% (Nadine Geser et al., 2012) 10 Table 3.1: The rate of ESBL producing E coli positive chickens The number of tested The number of positive Location samples (chicken) samples (chicken) Vinh Long 590 389 Tra Vinh 550 378 Soc Trang 450 298 Hau Giang 450 200 Total 2,040 1.265 % 65.93a 68.73a 66.22a 44.44b 62.01 Notice: The values in the same column with a,b different digits are different highly statistically significant (P4 Total The number of tested samples (broiler) 464 467 265 1,196 The number of positive samples (broiler) 301 316 188 805 % 64.87 67.67 70.94 67.31 3.1.4 The occurrence of ESBL producing E coli in eggs The results of study (Table 3.4) did not detect ESBL producing E coli in egg shell, egg white and egg yolk collected from layers in industrial farm For eggs collected from layers in households, ESBL producing E coli were not found in egg white and egg yolk but they were detected in egg shell (4.8%) Abubakar et al (2016) isolated ESBL producing E coli in 70 chicken eggs in Sokoto Metropolis of Nigeria The results showed that the occurrence of ESBL producing E coli in eggs was 5.7% (4/70) ESBL producing E coli were detected in egg shell 4.2% (3/70) and in mix of egg white and egg yolk 1.4% (1/70) Table 3.4: The rate of egg shell was infected ESBL producing E coli Husbandry method Households The number of tested samples (egg shell) The number of positive samples (egg shell) % 200 12 6.0 Farms 50 0.0 Total 250 12 4.8 3.1.5 The occurrence of ESBL producing E coli in husbandry environment ESBL producing E coli were confirmed in husbandry environment including domestic water, drinking water, feed and air The prevalance of ESBL producing E coli in air of housholds 20% was slower than in air of industrial farms 55% (P=0.03) 12 Table 3.5: The occurrence of ESBL producing E coli in environmental factors Samples Domestic water Water in trough Feed in trough Air Husbandry method The number of tested samples The number of positive samples Households Farms Households Farms Households Farms Households Farms 60 20 60 20 60 20 60 20 27 15 12 11 % 5.0 5.0 45.0 30.0 25.0 30.0 20.0b 55.0a Notice: The values in the same column with a,b different digits are different highly statistically significant (P