examining the food environment impacts prevalence and antibiotic profile of extended spectrum beta lactamase esbl producing escherichia coli isolated from pork and chicken

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURAL AND FORESTRY EI PHYU WIN EXAMINING THE FOOD ENVIRONMENT IMPACT: PREVALENCE AND ANTIBIOTIC PROFILE OF EXTENDED SPECTRUM BETA LACTAMASE ES

Introduction

Research Rationale

"Food poisoning" is a term used to describe an acute illness that develops after consuming dangerous food or beverages "Food poisoning" is a term used to describe an acute condition that develops after ingesting or drinking deadly food Microbial contamination of pork can happen at any point, including during pig slaughter, retail distribution, and home handling (Dang-Xuan et al., 2016)

One of the main worldwide health issues today is the rapid rise in antibiotic resistance to widely used drugs in human medicine and animal husbandry (Rousham et al., 2018) Improved water supply and good sanitation can lessen the incidence of gastrointestinal disorders As a result, monitoring fecal contamination levels and disease outbreak prevention are crucial from a public health and economic perspective Even in developed nations, outbreaks of water and food-borne illnesses continue to be common

Consumers of pork are worried about concerns related to food safety, particularly those involving chemical and microbiological risks (Ha et al., 2019) Monitoring for fecal indicator bacteria is required for drinking water, ground water, and recreational water Since identification and enumeration of many different types of pathogenic organisms are sometimes challenging due to their low numbers and particular growth indication, these bacteria are employed to suggest the presence of pathogens in the environment

Fecal bacteria, which affects linked disorders and is the largest cause of morbidity and mortality globally, is still a widespread and ongoing issue Of these, it is estimated that

The incidence of water and food-borne infections has negative effects on the economy and society (medical expenses, lost production, decreased tourism, etc.) Most severe cases of foodborne illness affect the very young, the very elderly, pregnant women, and people with impaired immune systems (Braden & Tauxe, 2013)

Manure and other animal wastes, wastewater from slaughterhouses, and effluent from wastewater treatment facilities can all contribute to pathogenic E coli contamination of the environment It is crucial to comprehend the environmental factors that affect E coli's ability to survive include low or fluctuating energy source levels, high to low oxygen levels, fluctuating and frequently extreme temperature ranges, low pH, and/or high osmolarity

According to estimates made by the United States, Department of Agriculture's Economic Research Service (USDA-ERS), in 2001, illnesses brought on by five main bacterial infections in the country had a negative impact on local and state economy as well as public health The majority of evaluations have solely addressed microbiological contamination or food safety procedures (Vo et al., 2015) With 1.2 million fatalities per year, infections due to antimicrobial resistance (AMR) have surpassed all other causes of death around the globe (Murray et al., 2022)

This foodborne pathogen has an infectious dosage of 10-100 bacterial cells, which can cause a variety of extra-intestinal or intestinal disorders that might develop into stages that endanger human life (Rani et al., 2021).The United States Environmental Protection Agency (USEPA) compared the prevalence of bacteria to swimming-related gastroenteric disorders at beaches throughout the east coast of the country to establish the most accurate indication of fecal contamination According to their findings, the largest link between illness occurrence at freshwater and marine beaches was found to be between enterococci and E coli In order to evaluate fecal loading in freshwater systems and associated health implications, counting E coli was advised In other nations, E coli is also utilized as a sign of fecal contamination

Both biotic and abiotic variables have the potential to affect E coli growth and survival in their natural habitats (Rochelle-Newall et al.2015) Temperature, the availability of water and nutrients, pH, and sun radiation are examples of abiotic variables Other microorganisms' existence and E coli's capacity to consume resources, outcompete other microbes, and build biofilms in natural habitats are examples of biotic factors

According to Hedberg et al., (1999), there are more than 76 million foodborne illnesses, 320,000 hospital admissions, and 5000 fatalities per year in the US According to the World Health Organization, acute diarrheal illnesses brought on by several foodborne pathogens resulted in the deaths of almost 1.5 million individuals worldwide (Pires et al., 2021)

In particular, global research (Lazarus et al., 2015) found that eating poultry meat is a significant source of bacteria that produce ESBL Multidrug resistance (MDR) is frequently associated with ESBL-producing bacteria and concurrent resistance to additional classes of antibiotic drugs (World Health Organization., 2020)

Since local producers cannot sustain the high production expenses, the bulk (79%) of these poultry foods are imported from foreign nations (Zamani et al., 2021) In Iran or of E coli O157 serogroup in various dietary samples (Pakbin et al., 2021) One of the most effective methods to manage and stop any outbreaks brought on by this virus has been reported to be quantitative prevalence examination of various food samples (Laidlaw et al., 2019).

Research’s Objectives

This study aimed to generate information on the prevalence and antibotic profile of Extended Spectrum Beta Lactamase (ESBL) producing E.coli isolated from pork and chicken meat Specifically, a To isolate and determine of the prevalence of E coli bacteria on samples of fresh chicken and pork at some traditional markets in Dong Anh district, Hanoi city b To determine the antibiotic resistance of E coli strains isolated from chicken and pork c To determine the phenotype and genotype of ESBLs.

Research Questions and Hypothesis

Questions :Why we need to study and investigate the prevalence of bacteria relate with

ESBL producing E.coli from pork and chicken meat causing disease around the world?

Hypothesis: Based on the study findings, people may better understand the risk of eating meat contaminated with E.coli bacteria and the issue of E.coli bacteria that are antibiotic resistant The management of E coli bacterium contamination in meat is also addressed, along with suggestions for the responsible use of antibiotics in animals for treating E coli related to illnesses To aid in the development of vaccines and biological products to control bacteria for use in both animals and people, provide antibiotic resistant of E coli strains that generate ESBL So, the purpose of this study will be to learn more about ESBLs and to examine the antibiotic resistance of ESBL-producing E coli and their genotype as a source of references for more research.

Scope and Limitations

Through the isolation of pork and chicken meat, this study will only be able to gather data on the prevalence and antibiotic profile of Extended Spectrum Beta Lactamase (ESBL) producing Escherichia coli from the traditional market in Hanoi, Vietnam.

Definition

The list of terminology used for this study and their accompanying definitions is provided here solely for the purpose of comprehending these complex and scientifically specialized words

Bacterial strain - A collection of organism that share genetic traits with other members of their species but do not belong to the same species Within a single species, microorganisms like viruses, bacteria, and fungus can have several strains

Biochemistry test - A battery of blood tests known as the biochemical profile are used to assess the ability of many vital organs and systems, including the liver and kidneys, to operate These tests can be performed on an empty stomach or not, and a complete blood count (CBC) is typically included

Bacterial colony - A collection of bacterial cells that have developed from a single mother cell is known as a bacterial colony Depending on the kind of bacteria, a single mother cell reproduces to generate a collection of genetically similar cells that come together to form a colony with a variety of morphologies

Gram staining: This test looks for bacteria in the throat, lungs, genitalia, and skin wounds— areas where an infection is likely to have occurred Gram stains can also be used to examine specific bodily fluids like blood or urine for microorganisms

Proliferation - Binary fission, a mechanism that causes bacteria to divide into two daughter cells, is the basis of bacterial growth The daughter cells that arise are genetically identical to the original cell, assuming no event takes place As a result, germs start to proliferate Not often do the two daughter cells from the division survive

PCR reaction - A common method for making millions to billions of copies of a specific

DNA sample fast is the polymerase chain reaction This method enables researchers to amplify a very small DNA sample to a size large enough to conduct in-depth study

Biochemical indicator - A substance that is frequently detected in blood or urine is a biochemical indication The nutritional state of an individual can be determined through biochemical markers of food and nutrition.

Literature Review

Overview of food poisoning

2.1.1 The concept of food poisoning and food-borne diseases

Acute illness known as "food poisoning" results from ingesting or drinking deadly food A food-borne illness is one that is contracted by consuming or drinking food that has been contaminated with pathogens This includes illnesses brought on by toxins, infections from microbes, and parasitic infections Escherichia coli is a well-known indicator that any food or water sample has been contaminated by feces (Welinder-Olsson & Kaijser, 2005).This is true regardless of the source of the sample

Large populations of E coli are present in the intestines and are discharged in the urine and feces Consequently, bacteria that contaminate food are spread by flies from feces or when the person making the food does not wash their hands after using the bathroom Also, because veterinary hygiene standards are not met during the killing of animals, meat products may become contaminated with the E coli bacteria, endangering human health at the (2-20 hours) incubation time According to (Teophilo et al., 2002) Escherichia coli can result in a urinary infection, fever, vomiting, diarrhea, severe stomach discomfort, hemorrhagic colitis, haemolytic-uremic syndrome, and even death

Those who are poisoned experience excruciating stomach discomfort, frequent loose stools, and infrequent vomiting It's possible to have a mild fever In severe circumstances, the patient can experience a high fever, exhaustion, and limb perspiration frequently discovered in human guts A person can contract the virus from feces on their hands or by ingesting infected water or food The virus has an extremely low threshold for human infection

Worms are the third agent Entamoeba hystolytica, roundworms, whipworms, hookworms, helminths, small liver flukes, lung flukes, swine tapeworms, and bovine tapeworms are the most prevalent parasites in food-borne illnesses

2.1.3 Food poisoning is caused by the food itself being poisonous

Toxins in food include cyanhydric acid, which is present in bamboo shoots and cassava, Solamin, which is present in sprouted potatoes, mycotoxins, amnesiac shellfish poisoning (ASP), diarrheal shellfish poisoning (DSP), neurotoxic shellfish poisoning (NSP), and paralytic shellfish poisoning (PSP), which is present in some seafood, shrimp, and can cause paralysis of the muscles Pathogenic Escherichia coli, Salmonella spp., Campylobacter spp., Staphylococcus aureus, Clostridium perfringens, Listeria monocytogenes, Shigella spp., and Cronobacter sakazakii are the most prevalent foodborne bacterial pathogens (Demirci et al., 2020)

The majority of foodborne illnesses, according to Mann et al (1983), are thought to have microbial origins, according to the World Health Organization (WHO), which claims that food is the primary cause of nearly 50% of all human deaths worldwide today

2.1.4 Food poisoning brought on by microbial poisons and contamination

The most frequent cause of food contamination is bacteria Bacteria are responsible for 50-60% of food poisoning incidents in Vietnam, according to statistics (according to the Food Safety Department - Ministry of Health) The most well-known bacteria include:

Bacteria eating protein-rich foods infected with staphylococcus bacteria is a typical cause of Staphylococcus aureus

Staphylococcal bacteria are prevalent in the air and water, are found on the skin and throat of infected people in large numbers, and are easily spread by unsanitary food preparation and storage practices The uncontrolled use of antibiotics on these farms, however (Paintsil et al., 2021) could lead to the development of drug-resistant bacteria in the community, notably ESBL-producing E coli (Carattoli, 2011)

Poisoning can result from consuming food that has been tainted by staphylococci or their toxins A meal's effects can start to show anywhere between 30 minutes and 4 hours later Patients frequently vomit recent meals, have several watery bowel movements, are tired, and if exposed to staph toxins, may also experience headaches and unconsciousness Dehydration and electrolyte loss make it simple to pass away if the illness is not addressed in a timely manner A novel strain of E coli led to a food poisoning incident (Dams-Chahin et al., 2022) that was primarily confined to northern Germany

Of the approximately 4000 afflicted individuals, 53 perished Six women, including a 4-year-old girl, died in Sapporo and Ebetsu as a result of a severe outbreak of food poisoning in Hokkaido, according to an article in the Japan Times Online on August 20,

2012 as a result of consuming salted cabbage tainted with E coli germs When they ate Chinese pickled cabbage prepared in late July by a firm in Sapporo, about 103 people experienced the same symptoms

Cases of food poisoning are increasing Nowadays, there are 6,000 fatalities and

175 out of every 1000 persons have food illness, on average Food poisoning and other food-borne illnesses have been on the rise globally and are getting increasingly widespread

As a result, each nation is having a harder time preventing and treating this issue, which poses a significant challenge for all of humanity

Numerous E coli serotypes have been discovered and isolated from environmental, dietary, and clinical samples The most prevalent serotype of pathogenic Escherichia coli strains recognized as enterohemorrhagic and shiga-toxin-producing bacterial foodborne pathogens is reported to be 0157 (Rani et al., 2021).The first E coli 0157 outbreaks were documented in the US states of Oregon and Michigan in 1982 After eating hamburgers at nearby eateries, patients who had severe bloody diarrhea and abdominal cramps had the organism isolated (Lim et al., 2010).

The overview of food poisoning in Vietnam

Food poisoning is currently a serious problem in Vietnam that worries the entire population Notwithstanding the State's extensive collection of laws, regulations, and guidelines, there are still many gaps in the administration, oversight, and planning of local implementation

According to WHO, Vietnam has more than 3 million cases of food poisoning annually, resulting in losses of more than 200 million USD In our nation, there have been more incidents of food poisoning in recent years Statistics show that Vietnam experiences 250-500 incidents of food poisoning annually, resulting in 7,000-10,000 victims and 100-

The Food Safety Bureau reports that between 2010 and 2019, more than 47,400 persons were sick nationwide in 1,556 cases of food poisoning, 271 of whom passed away, and roughly 40,190 of whom required hospitalization Specifically in 2020, as of May 31, the entire nation has registered 48 incidents of food poisoning, leading to more than 870 illnesses, 824 hospitalizations, and 22 fatalities

An rise of 18 cases, an increase of 17 deaths, and an increase of 11 cases (29.7%) of food poisoning when compared to the same period in 2019 According to an analysis of 1,604 cases of poisoning that were reported from 2010 to 2020, bacteria (which account for 38.7%), natural toxins (which account for 28.4%), and chemicals (which account for 12%) are the leading causes of poisoning

This foodborne pathogen has an infectious dosage of 10-100 bacterial cells, which can cause a variety of extra-intestinal or intestinal disorders that might develop into stages that endanger human life (Rani et al., 2021) As advised by the Centers for Disease Control (CDC), a number of tactics have been researched in the past to manage and stop the spread of E coli O157 (Singha et al., 2022)

One of the most effective methods to manage and stop any outbreaks brought on by this virus has been reported to be quantitative prevalence examination of various food samples (Laidlaw et al., 2019) In Iran or other areas of the world, there have been few investigations on the quantitative prevalence of E coli O157 serogroup in various dietary samples (Pakbin et al., 2021)

More than 52% of these outbreaks were related to consuming food, 9% to drinking animal-to-animal contact It's intriguing to notice that the majority of these outbreaks were linked to consuming tainted salads, unpasteurized milk, and undercooked animal items So, according to (Heiman et al., 2015) it is one of the most significant foodborne bacterial infections

Eighty-eight (88) people were hospitalized on January 24, 2019, after eating bread stuffed with cold pork, rolls, egg sauce, pate, ham, sour food, and raw vegetables that they had purchased from a bakery at address 236 Van Tien Dung, Hoa Xuan Ward, Cam Le District, Da Nang

Theodore Escherich (1857- 1911), a biologist, originally isolated Escherichia coli in 1885 from the feces of young infants The genus Escherichia and species Escherichia coli are members of the kingdom Bacteria, the phylum Proteobacteria, the class Gamma proteobacteria, the order Enterobacteriales, and the family Enterobacteriaceae (“Bergey’s Manual® of Systematic Bacteriology,” 2005) As a typical example of the enterobacteriaceae family, the cultivar Escherichia was chosen Many species, including

E coli, E adecarboxylase, E blattae, E fergusonni, E hermani, and E vulneris, are included in this variety

In which E coli, which makes up 80% of the aerobic bacteria parasites in the digestive system of adults, plays the key function and is chosen as the typical type of the genus Escherichia coli (Spence, 1974)

E coli bacteria are widespread in the environment and are particularly prevalent in warm-blooded animals' intestinal tracts E coli is found in the human body in places including the intestines and respiratory system In the first few hours following delivery,

E coli enters the digestive tract of both humans and animals It often enters the posterior part of the intestine but sporadically enters the stomach or small intestine It is frequently discovered in the mucous membranes of numerous other body areas The majority of E coli strains can live innocuously in the gastrointestinal tracts of healthy people and animals, but E coli is also responsible for a range of illnesses in both people and animals

E coli is a small, rod-shaped bacteria with dimensions of 2-3 x 0.6 mm Spherical bacilli can sometimes be found in short chains or standing alone in the body Bacteria that are 4 to 8 long can occasionally be discovered in the culture media; these kinds are common in older cultures Because of the body hairs, the majority of E coli are mobile, but some are not

The Enterobacteriaceae family contains the rod-shaped, gram-negative, facultative anaerobe, non-sporulating bacterium Escherichia coli, which is a natural component of both humans and animals' gut microbiota Additionally, water and soil frequently contain large amounts of it (Denamur et al., 2021)

The bacteria E coli may contain thyroid tissue but may not produce spores Gram- negative bacteria can stain uniformly or with darker ends and lighter centers The tissue border is not apparent when the tissue is young, but it can be seen if bacteria are extracted from the mucus colony and stained E coli cells have a nucleus when fixed with osmic acid and viewed under an electron microscope

Figure 2.1 E coli bacteria have the diameter of 2-3 mm and are spherical,moist, transparent, pale white and slightly convex after 24hrs on gram staining

Antibiotics

Effectives antibiotics (both in vitro and in vivo) kill harmful microorganisms or prevent their growth, with bacteria, fungus and actinomycetes accounting for the majority of cases Due to their resistance to penicillin, broad-spectrum cephalosporins, and monobactams, bacteria that produce extended spectrum b-lactamases (ESBLs) posses a severe threat to human and veterinary healthcare Infections brought on by Enterobacteriales are routinely treated with beta-lactam antibiotics in both human and veterinary medicine (Nüesch-Inderbinen & Stephan, 2016)

The roots of the medications in this class are broken because the bonds in the chemical formula containing the lactamin group are so weak This group consists of penicillin G is a slow acting penicillin similar to quinine penicillin and procain penicillin

- Natural penicillins: Penicillin V, Penicillin K, and others Oxacillin, Cloxacillin, Ampicillin, and Amoxicillin are semi-synthetic penicillins

- Cephalosporins, such as Cephaloridin, Cephalotin, and Cephacetril, which are natural and semi-synthetic or synthetic

Common antibiotics in this group: Streptomycin, Gentamycin, Neomycin, Kanamycin

Includes natural tetracyclines, oxytetracyclines, chlortetracyclines and synthetic tetracyclines

Includes: Erythromycin, Lincomycin, Lindamycin, Tylosin, Spiramycin, Rifamycin

Common drugs: Bacitracin, Colistin, Polymycin

2.3.8 Sulfamides and Nitrofurantoin, a class of chemotherapeutic medicines with an antibiotic-like action that can be totally manufactured, have a weaker bacteriostatic impact than the groups previously discussed The quinolone group, which includes the well-known medicines Ciprofloxacin, Norfloxacin, Ofloxacin, and Enrofloxacin, was entirely manufactured by humans over the course of three generations

• Also, patients are split into two groups based on the strength of the antibiotics: bactericidal antibiotic group and bacteriostatic antibiotic group

• The bactericidal antibiotic class, which includes medications with modes of release of yeast autolyza Drug classes such as -lactamin, streptomycin, neomycin, framomycin, colistin, kanamycin, vancomycin, and bacitracin fall within this category

• A class of antibiotics known as bacteriostatics inhibits the generation of bacterial proteins by binding to ribosomal subunits 30, 50, and 70 Members of this group include sulphonamides, tetracyclines, chloramphenicol, erythromycin, novobiocin, and tiamulin.

Mechanism of action of antibiotics

2.4.1 Antibiotics that inhibit bacterial cell membrane synthesis

Certain antibiotics work by preventing the bacterial cell wall's ability to synthesize mucopeptides The possible risk of antimicrobial-resistant bacteria from animals or animal products spreading to humans in sub-Saharan African nations has been established in the past (Falgenhauer et al., 2019)

Antibiotics in this category include lactamine and cephalosporin The peptide chains that bacteria produce to create cell membranes share the same structure as penicillin and ò-lactamine derivatives In nations with low resources, poor sanitation, in addition to the abuse of antibiotics, is anticipated to have a considerable impact on the development and spread of antibiotic resistance (Aslam et al., 2018)

Bacteria accidentally build stable, irreversible compounds with certain chemicals during the synthesis of bacterial cell membranes Transvascular membrane formation is hampered The impact of mucopeptide molecules on transport is another way that antibiotics work They have the result of impairing the main membrane's function in bacterial cells According to reports from Nigeria, blaTEM, blaCTX-M, and blaSHV were the three most common genes (Mohammed et al., 2016) The 30 chemicals that make up this category include Polymicin B, Colistin, Bacitracin, Anbomycin, Vancomycin, and Ristomycin

Aminoglycoside group: antibiotics that stop protein synthesis and inhibit and kill bacteria by immobilizing the 30s component of the 70s ribosome The most prevalent blaCTX-M gene linked to human infections is blaCTX-M-15 (Irrgang et al., 2017) The prevalence of antibiotic use and availability in Ghana may be reflected in the drug resistance found in this study (Nkansa et al., 2020) To treat disorders outside of the digestive tract, the medication must be administered intravenously

Tetracycline prevents RNA aminoacyl-transferase from binding to the receptor site on the ribosome (subunit) position 30S, hence inhibiting protein synthesis Phenicol group: this category of medications slows protein synthesis by inhibiting the activity of peptidyl- transferase in the ribosome's 50s subunit

Macrolide group: Macrolides are big medicinal compounds with a 14-16 element macrocyclic lactone nucleus Erythromycin binds to the 23s rRNA in the 50s ribosomal subunit, which prevents translocation during protein synthesis

- Lincosamide group: The medication attaches to the ribosome's 50s subunit and stops the synthesis of peptides required for protein synthesis

- Fusiic acid, a steroid-like compound that suppresses protein synthesis by building a stable combination with guanosin diphosphate, the splicing factor, and ribosomes

2.4.3 Antibiotics that inhibit nucleic acid synthesis

Several medications, including imidazoles, quinolones (norfloxacin, enrofloxacin), nitrofurantoin, furazolidone, and rifamycin, have an impact on the division and synthesis of chromosomes in bacterial cells These medications are extremely hazardous and should only be used in human medicine

- Biosynthesis - Sulfonamide: The medicine competes with PABA (para aminobenzoic), which is important in the metabolism of folic acid, because it has a structure comparable to PABA (which is a precursor for nucleic acid synthesis) making use of alkaline bacteria (Le et al., 2015a)

-Trimethoprim: inhibits the enzyme dihydrofolate reductase, affects folic acid synthesis, bacteriostatic effect.

The phenomenon of antibiotic resistance

2.5.1 The concept of antibiotic resistance

A person or a type of bacteria is said to be resistant if it can survive and procreate in an environment where the concentration of antibiotics is higher than the concentration at which the organism's reproduction is inhibited 59 percent of the strains were ciprofloxacin-resistant when found in Ghana on poultry meat (Eibach et al., 2018) The huge population of people from the same culture or belonging to the same species Since ciprofloxacin use in smallholder farms is less prevalent in this study area, this difference in resistance might be explained (Paintsil et al., 2021)

There are two primary categories of the phenomena of bacterial antibiotic resistance: - Inherent resistance: Natural bacteria already contain specific enzymes or compounds that are impervious to the effects of antibiotics Bacteria develop acquired drug resistance throughout the course of their lives

2.5.2 Mechanism of transmission of antibiotic resistance in bacteria

Contact or transmission between resistant bacteria and susceptible bacteria is what leads to antibiotic resistance Among the most frequent and clinically relevant ESBL genes, blaCTX-M positive Clinical isolates of E coli from both people and animals tend to have this genotype (Paintsil et al., 2021)

Bacterial factor R can be transmitted between them when there is a resistance factor: plasmid.The ability of bacteria to transfer resistance genes horizontally (horizontal transfer), across bacteria of the same generation, or between bacteria of different species, is the primary cause of the formation of drug resistance in bacteria

There are three ways that resistance genes can spread horizontally:

- Transformation: the transfer of a portion of bare DNA from a donor cell to a recipient cell

- Transduction is the process by which a bacteriophage moves a fragment of DNA from a donor cell to a recipient cell

- Conjugation is the interaction of two cells that results in the transfer of a portion of DNA from one cell to another The process of conjugation is regarded as being the most significant one among them

2.5.3 Mechanism of Antibiotic Resistance Development

To remove antibiotics from the cell pump, bacteria develop a specialized pump This is situated on the membrane of the bacterial cell Incompatibility group FII plasmids, also referred to as "epidemic resistance plasmid ) because of their propensity to pick up additional resistance genes and their high potential for horizontal gene transfer, frequently contain blaCTX-M-15 (Cantón et al., 2006)

Resistance genes are created by bacteria By breaking the ò-lactam ring or by altering the structure and target of an antibiotic, the resistance plasmids created in the protoplasm will kill or inactivate the antibiotic Infected patients from two hospitals in Ghana were also found to have bla CTX-M-15 dominance (Mahazu et al., 2022)

Then, using the three processes stated above, but mostly by conjugation, these resistant plasmids will be passed down to the following generation or transported horizontally between bacteria Through conjugation, resistance can be passed from one cell to another by moving an identical copy of the plasmid Circular DNA makes up plasmids, which function independently of bacterial chromosomes

Many microorganisms found in the interior and external habitats of animals and humans play a significant role in the horizontal transfer of resistance among members of the same generation Antibiotic use has produced a climate conducive to the survival of bacteria with resistance genes Hence, these strains serve as a vehicle for the transfer and spread of resistance genes to all bacterial strains, including dangerous ones

2.5.4 Situation of antibiotic use in livestock in Vietnam

Because antibiotic resistance has made it difficult to treat infections in cattle and poultry, the phenomenon of bacterial antibiotic resistance has been brought up by local researchers The unchecked use of antibiotics which includes taking many medicines at once in an effort to treat the disease more effectively is the origin of this phenomena

Yet, this is also the reason why "drug resistance" occurs The use of antibiotics as growth promoters in poultry feed is another contributing factor.The basic basis for the use of antibiotics and veterinary drugs is human development through animal husbandry Breeders frequently increase dosages and treatment durations at random

In Vietnam, 75% of antibiotics are used in agriculture, compared to 60% globally, according to the department of animal health under the Ministry of Agriculture and Rural Development In agriculture, antibiotics are routinely utilized against approved practices, utilizing both prohibited and discouraged compounds It is concerning that antibiotic residues found in meat and seafood are contributing to human antibiotic resistance

The BlaCMY2 gene, which is resistant to Cephalosporin antibiotics, the Florenicol gene, which is resistant to the Florfenicol group, the DfA17 gene, which is resistant to Trimethoprim, and the dA5 gene, which is resistant to Spectinomycin are the most common genes for drug resistance

The rise of multi-drug resistance E coli bacteria is a result of the concurrent use of antibiotics and broad-spectrum antibiotics Moreover, the strain has the ability to spread resistance to numerous other bacteria, including Proteus, Klebsiella pneumonia, and Salmonella spp.The most resistant strain of E.coli to tetracycline, ampicillin, and streptomycin was found in chicken, followed by pig and poultry meat

The rate of resistance to ampicillin, sulfonamides, tetracyclines, ciprofloxacin, nalidixic acid, and cefotaxime among E coli isolates from chicken meat in Denmark is

16%, 15%, 13%, 4%, 4%, and 0.6%, respectively, according to the report of the antimicrobial resistance monitoring program of EFSA 2010

Human isolates of antibiotic-resistant E coli strains have grown recently, particularly those resistant to third-generation cephalosporins (ceftazidime and cefotaxime), a new generation of lactam antibiotics This group of bacteria's ability to manufacture broad-spectrum -lactamases (ESBLs) and other enzymes is the basis for their mechanism of antibiotic resistance other beta-lactamases, such AmpC

Overview of Beta-Lactamase Wide Special Activities

2.6.1 History and genetics of broad-spectrum beta-lactamases

Ampicillin, the first generation of broad-spectrum penicillin developed in 1961, was efficient against both Gram-positive cocci and Gram-negative bacilli When Temoneria, an ampicillin-resistant E coli strain, was identified from a blood patient in Athens, Greece, in 1963, the yeast was given the designation TEM-1 Because TEM 1 had been altered with an amino acid, TEM-2 was found on E coli bacteria in 1965 The gene for the enzyme beta-lactamase was found in the bacteria Klebsiella pneumoniae in 1974 This enzyme, which was given the designation SHV-1, contains many more amino acid modifications than TEM-1 and TEM-2 (Sulphyryl Variable)

Bacteria are becoming resistant to penicillin, ampicillin, and first-generation cephalosporins since the appearance of TEM-1, TEM-2, and SHV-1 The development of broad-spectrum beta-lactam antibiotics in the early 1980s, such as second- and third- infections caused by TEM-1, TEM-2, and SHV-1 Although Nevertheless, mutations in TEM-1, TEM-2, and SHV-1, which alter certain anine acids, result in a broad-spectrum beta-lactamase enzyme that can degrade these novel medicines

ESBLs (Extended Spectrum Beta-Lactamase) first showed up The first instance of ESBLs was discovered in a cefotaxime-resistant Klebsiella ozaenae strain called SHV-2 in Germany in 1983 Masumato discovered SHV in 1986 in Japan where E coli developed non-TEM cefotaxime-resistant ESBLs under the name CTX-M-1 Importantly, CTX-M promotes the degradation of the majority of third- and fourth-generation cephalosporin antibiotics by bacteria (Paterson & Bonomo, 2005).

Method for the detection of broad-spectrum beta-lactamase producing Bacteria

2.7.1.Microclinical methods a Double-disk synergy test

This technique relies on ESBLs that can break down broad-spectrum cephalosporin antibiotics but are inhibited by clavulanic acid, which causes the bacterial inhibition zone to develop and enlarge surrounding amoxicillin-clavulanate-coated paper plates Amoxicillin clavulanate and antibiotic interaction inhibition A cefotaxime 30g plate (and/or ceftriaxone and/or ceftazidime and/or aztreonam) and an amoxicillin-clavulanate dish (containing 10 g clavulanate) were placed 30 mm apart for the tests to be conducted on the a gar

The test is considered positive when the sterile ring of the antibiotic disc extends toward the junction of the amoxicillin-clavulanate disc and a bottleneck or keyhole antibacterial zone forms This is a simple, useful, and accurate method for identifying

ESBLs, but on sometimes it may be necessary to adjust the distance between the placements of paper plates with antibiotics on them (Shash et al., 2019) b Combied disk method

The goal of the test is to determine if the discs ceftazidime 30g and ceftazidime/clavulanic acid 30/10 g, or cefotaxime 30g and cefotaxime/clavulanic acid 30/10 g, have a synergistic effect The strain was determined to carry the type ESBL model if the antibacterial ring diameter of the cephalosporin/clavulanic acid disc is 5 mm compared to the ring diameter antibacterial activity of a cephalosporin disc (CLSI, 2015) c Method using E-test ESBL paper tape

Two-way E-test paper tapes are used in this technique Antibiotics known as broad- spectrum cephalosporins (CTX, CAZ) were present in low to high dilutions in half of the samples The remaining portion contains a broad-spectrum cephalosporin with a 4 g/ml clavulanic acid component When the MIC value of the medicine being tested is decreased by more than three steps of dilution (ratio of MIC 8 in the presence of clavulanic acid), the ESBL test is deemed positive (Cormican et al., 1996)

Fundamentals A template strand, or a starting sequence of DNA, serves as the basis for the Polymerase Chain Reaction (PCR), a method of multiple DNA synthesis that amplifies and doubles this template's copy number into millions of copies through activation action of the polymerase enzyme along with two primers designed specifically for this DNA fragment

The PCR reaction consists of multiple cycles of continuous reactions, each cycle having three stages:

• Denaturation stage: bonds at a high temperature of 90-95°C The DNA molecule's hydrogen is cut, causing the two strands to split apart

• Annealing stage: The temperature is decreased (to between 50 and 68 °C) after the two DNA strands have separated, allowing the primer to cling to the remaining DNA strand If the annealing temperature is set incorrectly, the primer may only partially or randomly bind to the template DNA

• Elongation phase: The DNA polymerase enzyme may catalyze the synthesis of adding nucleotides at the end of the primer, which causes the primers to be stretched to form a new strand, at a temperature of 60 to 72°C When PCR results are subjected to electrophoresis, the negative charge of the DNA molecules causes them to migrate toward the anode in an electric field The pace of this migration is dependent on the mass of the DNA molecule

Component DNA Template (DNA template): Used as a template for the primers to attach to the complementary site with it This is followed by the participation of the required components, and the fusion reaction results in the formation of the new DNA fragment and the one in between the two primer binding sites are the same

The primer is a small, 14-35 nucleotide oligonucleotide fragment that can complementarily pair with a strand of the template DNA fragment When DNA polymerase is activated, this primer segment is prolonged to create a new strand

DNA polymerase: is a DNA synthesis enzyme that catalyzes the synthesis of new

DNA fragments that are copies of the original DNA sequence Enzyme commonly used is Taq polymerase with high heat resistance

PCR buffer: ensure the necessary ingredients for the enzyme DNA polymerase acts like MgCl2, KCl and Tris dNTPs - deoxynucleoside triphosphate: consisting of 4 types of free nucleotides (A, T, G,

X), are basic components, considered as "bricks" that make up the structure of DNA

This PCR technique innovation allows for the simultaneous amplification of numerous target DNA fragments utilizing a variety of distinct primer pairings in an answer

PART III MATERIALS AND METHODS 3.1 Subjects, Location and Time of Research

Samples were collected according to TCVN 4833-1:2002, TCVN 4833-2:2002 Samples were randomly collected at chicken (n) and pork (n) meat in traditional markets in Dong Anh district, Hanoi city between December 2022 and June 2023 Samples are contained in sterile zip bags, filled with information, refrigerated in an ice box and transported to the Laboratory of Bacteriology, Veterinary Hospital, Department of Veterinary Medicine, Gia Lam, Hanoi for E coli isolation The time between sampling and sample processing should not exceed 24 hours

Figure 3.1 Meat stalls at some markets in Dong Anh District

3.1.1 The guideline of the TCVN 4833-1:2002, TCVN 4833-2:2002

MEAT AND MEAT PRODUCTS - GET PURPOSE AND PREPARATION OF TRIAL SAMPLES

Meat and meat Products - Sampling and preparation of test samples

TCVN 4833 - 1: 2002 is completely equivalent to ISO 3100 - 1: 1991;

TCVN 4833 - 1: 2002 is compiled by the Technical Committee for standards TCVN/TC/F8 Meat and meat products, proposed by the General Department of Standards, Metrology and Quality, and issued by the Ministry of Science and Technology

Meat and meat products - Sampling and sample preparation

1.1 This standard gives general guidelines and specifies procedures for the primary sampling of meat and meat products

1.2 The differences between the sampling procedures for different types of products are as follows:

(a) Consignment or batches of meat, or meat products processed, or packaged in individual units of various sizes (e.g sausages, vacuum bagged ground meat, thinly sliced sausages) or pieces of meat weighing no more than 2 kg

(b) Carcasses, cuts or cured meats weighing more than 2 kg (e.g bacon loins, bacon ribs, boneless and boneless fresh and frozen meat, beef ribs or loin, pork ribs, lamb carcasses, venison), and mechanically separated or dried meat

1.3 The size and commercial value of such meat products may require secondary sampling, using only a portion of the primary sample, to achieve the purpose of the sampling

1.4 This sampling procedure is generally for commercial use In special cases, for example for food monitoring, other procedures may be necessary

ISO 7002 : 1986, Agricultural food Products - Layout for a Standard method of sampling from a lot

In this International Standard, the definitions given in ISO 7002 apply

Sampling should be performed by a sampler authorized by the relevant parties and properly trained in the appropriate technique That person must work independently and not accept third party interference The sampler may be assisted by others, but the sampler is primarily responsible Samplers and assistants take appropriate measures to avoid contaminating both the consignment (or lot) and the sample unit (eg, washing hands before handling the material to be sampled)

4.2 Representative of the relevant party

If possible, representatives of interested parties should be present when sampling is conducted

Laboratory samples should be accompanied by a sampling record signed by the sampler and by representatives of the interested parties, if present The minutes include the following information:

(a) the name and address of the person taking the sample;

(b) names and addresses of representatives of the parties concerned;

(c) the place, date, sampling point and time of sampling;

(d) the nature and origin of the consignment or consignment;

(e) the quantity and number of units constituting the consignment or consignment;

(g) identification of freight trains, railcars or trucks, as appropriate;

(j) the date of arrival of the shipment or consignment;

(k) the name and address of the seller;

(l) the name and address of the purchaser;

(m) the number and date of the bill of lading or contract;

(o) the number of sample units for each lot;

(q) the number and designation of the lot from which the sample units were drawn;

(s) where the sample unit is sent

The report should also include any details regarding the conditions or circumstances that may affect the sampling, for example the condition of the packages and the environmental conditions around them (temperature and humidity of the air) ), product and sample unit temperatures, methods of sterilizing equipment and sample containers, and any other special information relevant to the material to be sampled

The laboratory sample must be sealed and labeled The seal shall be affixed so that the sample or label cannot be removed without destroying the seal

Materials & Methods

Research Methods

Figure 3.10 TSI agar status before (left) and after (right) inoculation of E coli

Presumptive E coli colonies with sequential IMViC test results of ++ are E coli Carry out bacterial culture in 20% Glycerol medium then stored at -20°C

3.3.1 Methods of determination of antibiotic resistance by dilution on agar method Principles

Antibiotics are diluted at different concentrations from low to high nutrient agar medium The antibiotic sensitivity of the tested bacterial strains was assessed based on the ability of the bacteria to grow on the medium containing the antibiotic at that concentration The lowest value of antibiotic concentration capable of inhibiting bacteria is called the minimum inhibitory concentration, abbreviated as MIC (Minimum Inhibitor Concentration)

E coli strains isolated as a sample from chicken and pork Cage plate, test tube, inoculation chamber, analytical balance, alcohol lamp, pipette, whisker were used as the instruments and equipments Muller Hinton Agar (MHA) were used as a chemicals and reagents Powdered antibiotics were stored in a separate box at dry, in the refrigerator at a temperature of about 80°C or -20°C Solvent for mixing antibiotics were used for Luria- Bertani Broth (LB) medium, Distilled water, International strain Escherichia coli ATCC

Prepare bacterial suspension: E coli colonies on TSA agar were inoculated into LB broth, cultured at 37º C for 4-6 hours to reach a concentration of 107 CFU/ml

Figure 3.11 E coli strains grown in LB medium

Prepare antibiotic - added agar plates

Antibiotics were diluted on Mueller Hinton Agar nutrient agar (MHA) at different concentrations according to the guidelines of the Clinical and Laboratory Standards Institute (Clinical Laboratory & Institute, 2015)

- Add (3-5) liter of diluted bacterial suspension onto tonic agar plates pre-prepared antibiotic supplement

- Leave the agar plate with the lid open, wait 3-5 minutes for the jelly to dry completely

- Put in 37°C incubator for incubation

Figure 3.12 Placing the bacterial suspension on the agar plate

Results: After 20 hours of incubation, remove the agar plates from the incubator Observe with the naked eye and evaluate the growth ability of bacterial strains on different concentrations, thereby determining the MIC value of bacterial strains with antibiotics

Figure 3.13 Read the results of antibiotic susceptibility of the bacterial strains after 20 hours of incubation

Based on the MIC value, the antibiotic susceptibility of the bacterial strains will classified according to different levels: sensitive (susceptible), resistant to intermediate (intermediate), resistant according to the guidelines of the Institute of Clinical Standards and Laboratories (CLSI) and European Commission for Drug Sensitivity Te (EUCAST)

Table 3.1 The antibiotics used in the study and the sensitivity scores were limited according to the standards of the Clinical and Laboratory Standards Institute (CLSI, 2018) and the European Commission for Drug Sensitivity Testing (EUCAST, 2015)

Group Antibiotic Abbreviations MIC (àg/ml)

Methods of Detection of E coli strains with ESBL phenotype…

The selected strains of E coli resistant to ceftazidime were tested for their ability to produce ESBLs by a combination method (Synergy test) according to the guidelines of the Clinical and Laboratory Standards Institute (Humphries et al., 2018) Specifically as follows: Use a sterile cotton swab to spread a thin layer of bacterial suspension (108 CFU/mL; turbidity equivalent to 0.5 McFarland) on the surface of Mueller Hinton agar, let the agar surface dry for 10 minutes at room temperature Use sterile forceps to pick up a plate impregnated with antibiotics ceftazidime (30 g/l), clavulanic acid (10 àg), cefotaxime (30 àg/l), cefotaxime (30 àg/l) + clavulanic acid (10 àg) will be placed on Mueller Hinton agar and incubated at 37°C for 16-18 h

The next day if the sterile ring diameter of the plate will be observed, if the difference between clavulanic acid and the plate without clavulanic acid will be greater than 5 mm, the conclusion will be positive (synergy test +) and the tested bacterial strain will be capable of producing ESBLs Conversely, if the diameter of the sterile ring of the dish with clavulanic acid compared with the dish without clavulanic acid is less than 5 mm, then the conclusion of a negative association (synergy test -), means that the strain of bacteria tested does not have clavulanic acid ability to produce ESBLs

3.5 Methods of Detection of genes encoding ESBLs

Strains with the ESBL phenotype were tested for the presence of genes encodes the enzyme ESBL a Preparation of sample and primer DNA

The DNA of these strains was extracted by boiling method

Primer pairs and thermal cycling for PCR to detect coding genes ESBLs (Le et al., 2015b)

Table 3.2 Sequence of primer pairs for PCR reaction

The composition of PCR reaction and thermal cycling are described in Table 3.3

Table 3.3 Components of the PCR reaction

Ingredient Concentration Volume (àl )2.5 dNTPs 0.2mM 0.75

PCRdis buffer 1X 2.5 Taq polymerase 1U/àl 2

Table 3.4 PCR reaction heat cycle

PCR products after amplification will be analyzed by electrophoresis on 1.5% agarose gel in 1X TAE buffer Conduct electrophoresis at 75V, 300A, when the position of the colored line runs to 2/3 of the gel then stop the electrophoresis process

Figure 3.14 Place PCR products into gel

Stain the gel with Ethidium Bromide for 30 minutes and photograph with a scanner

Results & Discussion

The prevalence of E coli conditions on chicken and pork in some changes in

One of the sanitary indicator microorganisms to gauge the state of the situation is

E coli fresh food hygiene, particularly for animal-derived meals In this study, we looked at 80 samples of chicken and 80 samples of pork that were purchased at local markets in the Dong Anh neighborhood of central Hanoi We also looked at 80 samples of E coli strains that had been isolated and bred

The results are summarized in Tables 4.1 and 4.2

Table 4.1 Prevalence of E coli from pork and chicken meat

Meat Number of samples Number of positive samples Ratio

Table (4.1) is prevalence test findings revealed that 113 (70.63%) of a total of 160 meat samples tested positive for E coli In particular, E coli was found in 58/80 (72.50%) chicken samples and 55/80 (68.75%) pork samples In contrast to the findings of certain research by some authors, our analysis found a larger proportion of e coli contamination of chicken is more serious than the pork

The cross-contamination during the preparation of pork, and suggests that unhygienic handling of pig at the street food stall or kitchen might increase its microbial contamination (Dang-Xuan et al., 2018) According to (Cardinale et al., 2015) Taiwan and the Philippines (8-12%), Thailand (7.7%), and Madagascar (5-10%), this incidence was significantly greater

Especially, the direct animal contact was linked to a greater probability of carrying ESBL producers among pig slaughterhouse employees (Dohmen et al., 2017) highlighting the significance of transmission along the pork food chain Our findings differ from those of other research due to geographical diversity, sample size and timing, detection strategy, and test accuracy Avoid eating pig and poultry from the grocery store without being under stringent hygienic monitoring

Table 4.2 Prevalence of E coli infection in chicken pork meat in some Dong

No (%) of positive Chợ Châu

The findings in Table 4.2 demonstrate that there is a very high incidence of E coli infection on meat in markets, with a rate of 55% When purchasing samples of meat at the markets, we saw that the majority of the booths selling meat did not maintain proper food hygiene and safety The potential of E coli contamination from the table to the meat is quite high since meat is sold on shops made of wood, cardboard, stone, and stainless steel, none of which are routinely cleaned and disinfected Pork, and chicken are all offered together, which increases the possibility of cross-contamination When handling meat, vendors at meat booths do not use gloves or masks

Infected materials that were ingested and resulted in a number of cases of food poisoning also included modest levels of contamination, according to (Franz et al., 2019) According to (Kim et al., 2020) This foodborne pathogen produced a variety of intestinal and extraintestinal diseases in humans, including acute bloody diarrhea

Through human consumption of various foods contaminated with bovine feces (such as vegetables contaminated with animal-based agricultural fertilizers), undercooked meat, and unpasteurized dairy products, cattle have been identified as the most significant reservoir of E coli serogroup 0157 that causes zoonotic diseases (Gutema et al., 2021)

Figure 4.4 Meat is sold on unsecured towels, cloths paperboards hygiene protection

In addition, dangerous industrial conditions and equipment may contribute to the high risk of E coli infections in chicken and pig sold in markets, as well as meat that has been tainted from the slaughterhouse Even the slaughter procedure is not done adequately in these facilities Lack of distinction between the phases of slaughtering and inappropriate removal of intestines and viscera cause bacterial contamination of carcasses, which lowers the quality of the finished product

Additionally, the high level of microbial contamination in meat is also a result of the touch between the customer and seller's hands during the meat selection process In

Figure 4.1 Chicken is slaughtered at the market butchered on unsanitary ground

Figure 4.2 Pork and poultry are sold together, there is a risk of cross- contamination

Figure 4.3 Meat is not stored at low temperature with water or soap after use This practice does not guarantee that all bacteria will be removed from the tools, and when these tools are used frequently, microbial contamination increases Our study did not aim to assess the level of E coli contamination in chicken and pork; rather, it looked primarily at the E coli contamination of retail chicken and pork

(1981) noted that due to unsanitary conditions during slaughter, transportation, and consumption, meat is commonly discovered to be infected When compared to groceries (40%) and hypermarkets (20%) (Iyer et al., 2013) study reported a high frequency of E coli in open butcher shops (65%) These findings are closely related

As a result, the figures above may not accurately represent the degree of E coli infection in the meat of E coli is a signal, though, and meat infected with it indicates that there may also be excrement and other harmful bacteria present Many different strains of

E coli are also capable of spreading disease Additionally, E coli bacteria frequently have a large number of antibiotic resistance genes that can spread to other bacteria Consequently, eating meat that has been exposed to E coli bacteria puts customers' health at danger.

Antibiotic Resistance of E coli Isolated from Chicken and Pork

In this work, we looked at E coli strains that had been identified using 14 antibiotics that are often used in animal agriculture The results are shown in Tables 4.3 and 4.4

Table 4.3 Percentage of E coli isolated from chicken and pork antibiotic resistance

Group No Type of antibiotic

No of resistant strains Ratio (%)

No of resistant strains Ratio (%)

No of resistant strains Ratio (%)

Amoxicillin 54 93.10 51 92,73 105 92.92 Macrolides Erythromycin 56 96.55 52 94.55 108 95.58 Tetracyclines Tetracycline 58 100.00 54 98.18 112 99.12

Ceftazidime 23 39.66 20 36.36 43 38.05 Quinolones Nalidic acid 50 80.21 45 81.82 95 84.07 Fluoroquinolones Ciprofloxacin 53 91.38 42 76.36 95 84.07

Enrofloxacin 52 89.66 40 72.73 92 81.42 Phenicols Chloramphenicol 53 91.38 48 87.27 101 89.38 Sulfonamides Sulfamethoxazole/

Figure 4.5 Rate of E coli isolated from chicken and pork resistant to antibiotics

From the results from Table 4.3 and Figure 4.5, It demonstrates that the majority (99.12%) of E coli bacteria recovered from meat are Tetracycline-resistant, followed by Erythromycin (95.58%), Sulfamethoxazole/Trimethoprim (95.58%), and Streptomycin, Ampicillin (92.04%), Amoxicillin (92.92%), and 93.81% The lowest rates of E coli resistance to ceftazidime (38.05%) and cefotaxime (42.48%) were observed Look in E coli bacteria obtained from chicken and pork often exhibit similar rates of resistance

Figure 4.6 Results from ability test Figure 4.7 Results from ability test of E coli

E coli resistance strains to Cefotaxime resistance to Tetracycline

According to Figure 4.6 and 4.7, it demonstrated that the study on the antibiotic resistance of E coli isolated from chicken and pork in the Northern Delta in 2004 revealed that E coli isolated from chicken meat had high rates of resistance to common antibiotics like streptomycin, ampicillin, tetracycline, and chloramphenicol but remained sensitive to gentamycin

Tetracycline, Sulfamethoxazole/Trimethoprim, and Streptomycin make up the majority (>80%) (Li et al., 2019) reported that the prevalence of E coli resistant to tetracycline was 99.1%, ampicillin was 97.6%, and sulfamethoxazole/trimethoprim was 96.1% when compared to other research conducted throughout the world E coli isolated from meat displayed extremely high rates of resistance to tetracycline and sulfamethoxazole/trimethoprim, with 84.9% and 88.4%, respectively, according to research by (Parvin et al., 2020)

As a consequence, the findings of our study are in line with those of the research conducted by the authors mentioned above as well as the trend that E coli antibiotic resistance is on the rise Our opinion is that the high rate of E coli resistance to antibiotics like Tetracycline, Erythromycin, and Amoxicillin is due to the fact that these drugs are old and are still frequently used to treat cattle and poultry diseases as well as to prevent diseases and promote long - term growth in animals

Additionally, the problem of unchecked antibiotic usage in cattle has helped to promote E coli bacteria's resistance to antibiotics Possibly because they are new generation antibiotics and aren't yet commonly used in cattle, ceftazidime and cefotaxime new generation antibiotics E coli 0157 has been cited as one of the world's greatest difficulties and top concerns in relation to public health and food safety (Abreham et al., 2019)

Today, E coli had resistance rates to these antibiotic classes that were on average greater than 40% These findings show the level of antibiotic resistance in E coli and offer a crucial benchmark for determining risk and managing multidrug-resistant bacterial control

Table 4.4 Results of testing for multi-antibiotic resistance of E coli strains isolated from chicken and pork

Chicken (nX) Pork (nU) Total (n3) No of models/ image resistance

Figure 4.8 Percentage of E coli resistant to the number of antibiotics

Table 4.4 and Figure 4.8 show that only 3 strains which account for the lowest percentage of 2.65% are resistant from 0 to 4 antibiotics While 37 strains resistant from 5 to 9 antibiotics account for the percentage rate (32.74%) with 24 multi-resistant phenotypes, there are 73 strains resistant to most antibiotics (from 10 to 14 medications), accounting for the highest rate (64.60%) Nine bacteria in particular are totally resistant to all 14 antibiotics

Le Van Tao et al (1993) observed that 12 E coli strains are multi-resistant to seven medicines, 32% are multi-resistant to six medications, 40% are multi-resistant to five medications, and 10% are multi-resistant to five medications 6% were multi-resistant to 3 medications and 4 medicines The findings of a short research by Hoang Mai Phuong et al (2008) show that 80.1% of E coli isolated from food are antibiotic-resistant, and 61.5% are resistant to at least one drug and two or more antibiotics

According to Tran Thi Thuy Giang et al (2014), from January 1 to June 2014, researchers at the Pasteur Institute in Ho Chi Minh City examined the rate of infection, the rate of antibiotic resistance, and the rapid screening of beta-lactamase-producing strains from 60 strains of E coli isolated on 270 food samples and 1716 drinking water samples

As a result, 76.7% of people were resistant to at least one antibiotic, and 65% were resistant to two or more

According to Truong Ha Thai et al (2017), 30 of the 46 E coli strains that were isolated from chicken eggs and tested positive for resistance to 12 different antibiotics: Three strains are resistant to eight forms of resistance antibiotics, six strains to nine types, seven strains to ten types, and three strains to eleven types Six strains were resistant to seven types of resistance development

Our study's findings differ and are more significant than those reported above for a variety of reasons, including sample type, study duration, and sample size Our findings show that the prevalence of multi-drug resistant E coli bacteria is on the rise when compared to earlier investigations The aforementioned findings further demonstrate the complexity of E coli bacteria's multi-resistant phenotypes E coli is becoming more and more multi-antibiotic resistant; in addition to being resistant to several different antibiotics at once, they also exhibit a wide variety of complicated multi-resistant phenotypes, which makes it challenging to select and mix medications to treat the condition

The usage of antibiotic resistance is growing for a variety of reasons, and E coli bacteria are resistant to numerous medicines One of the causes is the unchecked use of antibiotics, which has led to the "resistance" to the drug phenomenon This is because numerous antibiotics are administered at once in an effort to treat the disease more efficiently In truth, breeders frequently use antibiotics on cattle and poultry farms for a longer period of time than allowed by the manufacturer, arbitrarily increasing the dose

The addition of extra antibiotics to animal feed with the intention of increasing weight growth is another contributing factor Additionally, the use of antibiotics in livestock is not strictly regulated by the appropriate authorities, and the problem of antibiotic resistance has not received enough attention As a result, there is a major misuse of antibiotics, which makes it challenging to prevent and cure animal illnesses.

Determination of ESBL Genotype and Phenotype

A total of 42 (97.67%) E coli strains (strains resistant to ceftazidime) were possessed from 43 sample of E coli strains, which is the ESBL phenotype (ESBL-EC) The widespread use of cephalosporin medicines in human and veterinary medicine is the inescapable factor causing the rise of E coli strains able to produce ESBL According to (Le et al., 2015b) study, 58.7% of chicken samples sold in Vietnam's traditional markets have ESBL-EC According to (Leverstein-van Hall et al., 2011a) 94% of the E coli strains identified from chicken in the Netherlands were able to generate ESBL (Egea et al., 2012) study's findings revealed that 93.3% of chicken samples in Spain tested positive for ESBL-

Figure 4.9 ESBL phenotypic test results

There are several genes that produce various ESBL enzymes, but these three genes are frequently detected in ESBL-EC, we concentrated on blaCTX-M, blaTEM, and blaSHV in this investigation 48.84% (21/43) of the ESBL-EC strains in the investigation were revealed by the gene expressing ESBL, according to the data In this study, 11.63% (5/43) of participants had the blaSHV gene, 18.60% (8/43) had the blaTEM gene, and 20.93% (9/43) had both the blaCTX-M and blaTEM genes

This finding is in line with earlier research that identified the blaCTX-M gene to be the most frequently present gene in ESBL-EC strains (Egea et al., 2012) (Leverstein-van Hall et al., 2011b)found that up to 92.3% of ESBL-EC strains contained the gene encoding blaCTX-M, 59.9% had the blaTEM gene, and just 2.1% carried the blaSHV gene when they looked at the genotype of ESBL-EC strains isolated from meat in Vietnam The CTX-

M gene is present in all ESBL-EC strains recovered from frozen chicken in Bangladesh, according to a research by (Parvin et al., 2020)

Figure 4.10: PCR image of bacterial DNA extracted from isolated E coli colonies of chicken and pork meat collected from four retail market in the Dong Anh neighborhood of central Ha Noi (Strain1, 2, 3 - Escherichia coli-positive control) molecular marker Strain 3

372 bp (bla TEM) 588bp (bla CTX-M

Conclusion & Recommendation

Conclusion

5.1.1 Prevalence of E coli bacteria in chicken and pork

Seventy-six percent of the 160 tested samples of chicken and pork meat tested positive for E coli 55/80 (68.75%) of the pork samples and 58/80 (72.5%) of the poultry samples were positive ESBL E coli

5.1.2 Antibiotic resistance of E coli strains found in chicken and pork

Tetracycline had the greatest rate of resistance among E coli organisms found in meat (99.12%), followed by Sulfamethoxazole/ Trimethoprime (92.04%), Erythromycin (95.58%), Streptomycin (93.81%), Amoxicillin (92.92%), and Ampicillin ((92.92%) Ceftazidime and Cefotaxime had the lowest rates of E coli resistance, respectively

(38.05% and 42.48%) E coli bacteria obtained from chicken and pork often exhibit similar rates of resistance

The lowest percentage of 2.65% was caused by just 3 bacteria being resistant to 0-

4 antibiotics While 37 strains resistant to 5 to 9 antibiotics account for the percentage rate (32.74%) with 24 multi-resistant phenotypes, there are 73 strains resistant to most antibiotics (from 10 to 14 medications), accounting for the highest rate (64.60%) Nine bacteria in particular are totally resistant to all 14 antibiotics

5.1.3 Calculation of ESBL-producing E coli in chicken and pork

A total of 42 (97.67%) E coli strains (strains resistant to ceftazidime) were possessed from 43 sample of E coli strains, which is the ESBL phenotype (ESBL-EC) Of which, 11.63% (5/43) contained the blaSHV gene, 48.84% (21/43) of ESBL-EC strains carry the blaCTX-M gene, 18.60% (8/43) carried the blaTEM gene, and 20.93% (9/43) had the blaSHV gene, both the blaCTX-M and blaTEM genes.

Recommendation

We offer the following recommendations and remedies based on the aforementioned research findings:

5.2.1 Recommendations and solutions to reduce and prevent E coli contamination of meat: Management agency: oversee the operation of slaughterhouses; improve food quality inspection and assessment; promoting and informing customers on the need of selecting healthy and safe food Slaughterhouses for animals and commercial establishments: closely adhere to laws governing the production and distribution of food that are safe and hygienic

To produce goods that are safe for customers' health, producers should make use of modern technology Business establishments: are required to uphold standards for food safety and hygiene, as well as to assure the cleanliness of instruments for sale, protective gear, and the butcher's health Consumers: It is important to consider the product's origin when purchasing fresh meat products, and to ensure that the meat is completely cooked throughout processing

5.2.2 Measures to reduce and control the E coli bacteria from becoming more resistant to antibiotics

• Informing farmers about the advantages of antibiotics and the risks associated with using them in the improper type, dose, amount, or for an extended period of time

• Control and strictly regulate the use of antibiotics in animal facilities, as well as their and frequent inspection to provide deterrent punishments A livestock law, as well as investments in tools and training for laboratories to advance animal husbandry, are requiredin the future detection of antibiotic residues that is quick and precise

• To prevent and manage infections and the spread of germs, it is required to construct breeding facilities that are biosecure and to restrict small-scale livestock production To substitute antibiotics, you could seek for herbs or products made from benevolent microorganisms Control the operation of abattoirs, where microorganisms are easily infected; must completely prohibit the use of antibiotics in livestock and aquaculture; and traceability from slaughterhouses to livestock establishments, to feed mills, to antibiotic importers Tighten the free sale of antibiotics in the direction of only selling antibiotics according to doctor's prescription, moving toward the use of antibiotics for therapeutic purposes

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