The author expresses his heartfelt appreciation to all the laboratory assistants of Department of Microbiology and Veterinary Public Health, Chattogram Veterinary and Animal Sciences University, Chattogram. Escherichia coli (E. coli), a gram-negative bacterium that has a significant impact on human and animal health (Salehi and Bonab, 2006).
Brief history of the development of antibiotics
Knowledge about the mechanism of action of antimicrobial agents is important to understand the resistance mechanism developed by the microbes. Mechanism of action of different antimicrobial agents along with their target sites are summarized in Table 2.1.
Different categories of E. coli with their pathogenic potential
Faecal contamination of water and other food, as well as cross-contamination during food preparation (with beef and other meat products, contaminated surfaces and kitchen utensils), will also lead to infection. Visiting farms and other places where the public may come into direct contact with farm animals has also been identified as an important risk factor for STEC infection.
Antimicrobials used as chemoprophylaxis in animals to control enteric bacterial infections
STEC has also been isolated from bodies of water (such as ponds and streams), wells and cisterns, and has been found to survive in manure and water trough sediments for months. Prophylactic antibiotics may be considered for short-term relief for those individuals at high risk of those enteric pathogens.
Antimicrobial resistance
Antimicrobial drugs are widely used in livestock farming to increase production, treat infectious diseases and as growth promoters (Bien et al., 2015). Tetracycline, β-lactams and macrolides are the most common antibiotic groups used for veterinary purposes (Li et al., 2012). Due to their lower cost and higher antimicrobial activity, tetracycline antibiotics are widely used as veterinary drugs for the prevention and treatment of various infectious diseases.
An individual relying on prophylactic antibiotics will need to carry an alternate antibiotic to use if severe diarrhea develops despite prophylaxis. The risks associated with the use of prophylactic antibiotics must be weighed against the benefit of using rapid and early self-treatment with antibiotics when moderate to severe clinical conditions develop, shortening the duration of the disease. In poultry and livestock, mass administration of antibiotics is often practiced during transport or movement of young animals, during dry cow therapy in dairy cows, and in the prevention of respiratory and intestinal diseases when animals have been subjected to severe stressful conditions.
When animals are given an antibiotic that is closely related to an antibiotic used in human medicine, cross-resistance occurs and disease-causing bacteria become resistant to the drug used in human medicine. The consensus of the world's veterinary and medical experts is that it is dangerous and unjustifiable to use antibiotics related to drugs that are crucial in human medicine. Any use of antibiotics can increase selection pressure in a bacterial population, causing vulnerable bacteria to die, increasing the relative number of resistant bacteria and allowing further growth.
Mechanisms involved behind the emergence of antimicrobial resistance in bacterial pathogens
- Resistance to β-lactam antibiotics
- Resistance to tetracyclines
- Resistance to aminoglycosides
- Resistance to quinolones and fluroquinolones
- Resistance to sulfonamides and trimethoprim
The penicillins are one of the most widely used antibiotics in developing countries because of their easy availability and relatively low cost. Resistance to β-lactams in many bacteria is usually due to the hydrolysis of the antibiotic by a β-lactamase or the modification of PBPs or cellular permeability. Tetracyclines are another of the many widely used antimicrobials in both human and veterinary medicine in developing countries because of their availability and low cost as well as low toxicity and broad spectrum of activity.
Drug efflux occurs via an export protein from the major promoter superfamily (MFS). Mutations in gyrA cause changes in the conformation of the binding site that may be important for quinolone-DNA gyrase interaction. Changes in the cell envelope of gram-negative bacteria, particularly in the outer membrane, have been associated with reduced uptake and increased resistance to fluroquinolones, which has not been demonstrated in gram-positive bacteria.
Sulfonamide resistance in gram-negative bacilli generally arises from the acquisition of one of the two genes sul1 and sul2 that encode the production of the enzyme dihydropteroate synthase, which is not inhibited by the drug (Enne et al., 2001). Tetracyclines interfere with the initiation step of protein synthesis by inhibiting the binding of aminoacyl tRNA to the A site of the ribosome (Chopra et al., 2001). In addition, tetracyclines bind, or at least protrude, in the P site upon change in ribosome conformation in the posttranslocational state and can modify the ribosome conformation at the level of the head of the 30S subunit and the interface side of the 50S subunit. .
WHO’s prioritization of multi-drug resistant bacterial pathogens
7S protein and 16S RNA show the greatest affinity for tetracyclines and are therefore the main targets. While tigecycline increases the number of bindings to the target 16S RNA, the drug does not affect the ribosome protection mechanism (Olson et al., 2006). Together with the adjacent hydrophobic part, it makes the molecule less susceptible to efflux, with the notable exception of resistance nodulation cell division (RND)-type efflux pumps constitutively expressed by P.
The WHO list is divided into three categories according to the urgent need for new antibiotics: critical, high and medium priority.
Priority 1: Critical
MDR E. coli in poultry and poultry farm environment
The problem of antimicrobial drug resistance in veterinary pathogens, mainly in poultry, is compounded by the uncontrolled use of uncontrolled antimicrobial drugs in developing countries such as Bangladesh.
MDR E. coli in different food animals
Of the isolates, 39.74% showed multidrug resistance (resistant to 3 to 8 classes of antimicrobials) (Islam et al., 2016b).
Prevalence of multi-drug resistant E. coli in humans in different parts of the world
Co-resistance was frequent for penicillin, cephalosporin and quinolone (Purohit et al. conducted a retrospective study to find out the prevalence of MDR pathogens in a teaching hospital in Oman. The proportion of livestock workers associated with multi-drug resistance was also higher than the rates in restaurant workers (Cho et al., 2012) Street food contains different types and numbers of microorganisms that cause foodborne illness in humans.
Prevalence of multi-drug resistant E. coli of environmental origin in different parts of the world
FAO’s goals on AMR
Study area
A total of 405 meat samples were collected, including 215 breast muscles and 190 liver, of which 225 samples were collected from super stores and 180 samples from the live bird market.
Sample source and type
Sample collection, transportation, and processing procedure
Preservation of the isolates
- Isolation and identification of E. coli
- Molecular identification of E. coli
- Screening of antimicrobial resistance pattern of E. coli isolates against a panel of antimicrobials
Bright pink colored large colonies produced on a MacConkey agar plate were suspected to be the growth of E. After completion of the incubation period, colonies from blood agar were used for DNA extraction to be used for polymerase chain reaction (PCR). All phenotypically positive isolates in blood agar were subjected to molecular identification with species-specific multiplex PCR in the thermal cycler (DLAB, USA) using primers for the uidA gene and flanking region of the uspA gene.
Proportions of different reagents used for PCR for two different resistance genes are given in table 3.3. All PCR reactions were performed on a thermal cycler (DLAB Scientific Inc., USA) in Molecular Microbiology lab under DMVPH, CVASU following the cycling conditions mentioned in Table 3.4. Eight antimicrobials from seven different groups (β-lactam antibiotics, .26 tetracyclines, polymyxins, aminoglycosides, quinolones, sulfonamides, and penicillins) of public health drugs were selected for the CS test.
Procedure of CS test
For CS testing, 26 tetracyclines, polymyxins, aminoglycosides, quinolones, sulfonamides and penicillins) of drugs important to public health were selected. The 27 inoculations were done to avoid clumping of cells inside the test tube using the vortex machine. Then the bacterial suspension was adjusted to the turbidity of the McFarland standard 0.5 (equivalent to a growth of 1-2×108CFU/ml).
The swab was then streaked over the entire dry surface of Mueller Hinton agar three times by rotating the plate approx. 60 degrees.
Polymerase chain reaction (PCR) to test for the presence of tetracycline resistant isolates
- Sub-culturing on blood agar
- DNA extraction from the isolates
- PCR reactions
- Visualization of PCR products by Agar Gel Electrophoresis
All the molecular examination of the isolates for tet genes was performed with PCR machine name DLAB Scientific, USA in DMPH-CVASU. Proportions of different reagents used for PCR for two different resistance genes are given in table 3.8. 30 PCR was run on a thermal cycler (DLAB TC1000-G thermal cycler, China) following the cycling conditions mentioned in Table 3.9.
Then the agarose mixture was cooled to 50°C in a water bath and a drop of ethidium bromide was added to the mixture. The gel casting tray was assembled by sealing the ends of the gel chamber with tape and placing the appropriate number of ridges in the gel tray. The agarose-TAE buffer mixture was poured into the gel tray and kept for 20 min at room temperature to solidify, after which the combs were removed and the gel was moved into an electrophoresis tank filled with 1X TAE buffer and kept until the gel was completely submerged.
Finally, the gel was examined using a UV transilluminator for image acquisition and analysis. All data from the CS test results are recorded and sorted (by sample and market type) in Microsoft excel 2019 for statistical analysis. Data were then analyzed in STATA-13 to obtain prevalence and 95% confidence interval (CI).
Prevalence of E. coli in different sources
Antimicrobial resistance pattern of E. coli isolates of different sources
Multi-drug resistant (MDR) Escherichia coli originated from clinical and environmental sources in Ismailia-Egypt. Enterotoxin profiling and antibiogram of Escherichia coli isolated from poultry faeces in Dhaka district of Bangladesh. Prevalence of multidrug-resistant enteropathogenic and enteroinvasive Escherichia coli isolated from children with and without diarrhea in the Northeast Indian population.
Comparison of antimicrobial resistance in Escherichia coli strains isolated from healthy poultry and pig farms using antibiotics in Korea. Isolation of multidrug-resistant Escherichia coli O157 from the caecal contents and carcasses of goats in the Somali region of Ethiopia. Isolation of multidrug-resistant Escherichia coli O157 from goats in the Somali region of Ethiopia: a cross-sectional slaughterhouse-based study.
High prevalence of antibiotic resistance in pathogenic Escherichia coli from large and small scale poultry farms in Bangladesh. Prevalence, characteristics and antibiogram profiles of Escherichia coli isolated from apparently healthy chickens in Mymensingh, Bangladesh. Distribution and transferability of tetracycline resistance determinants in Escherichia coli isolated from meat and meat products.
Antibiotic susceptibility pattern of Escherichia coli strains isolated from chickens with colisepticemia in Tabriz province, Iran. Characterization of multidrug-resistant Escherichia coli and Salmonella isolated from food-producing animals in Northeast India.
