Summary of Biology doctoral thesis: Antimicrobial resistance characteristics and related genes of multidrug-resistant Salmonella

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MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ……..….***………… NGUYEN THANH VIET ANTIMICROBIAL RESISTANCE CHARACTERISTICS AND RELATED GENES OF MULTIDRUG-RESISTANT Salmonella Major: Microbiology Code: 9420107 SUMMARY OF BIOLOGY DOCTORAL THESIS Hanoi – 2020 INTRODUCTION 1. The urgency of the thesis The burden of foodborne diseases is substantial. Each year, foodborne diseases cause almost 1 in 10 people fall ill and 33 million of healthy life years are lost. Foodborne diseases can be severe, especially for young children. Diarrhoeal diseases are the most common illnesses resulting from unsafe food, 550 million people falling ill each year, including 220 million children under the age of 5 years. Salmonella is 1 of the 4 key global causes of diarrhoeal diseases. The increasing rate of antibiotic resistance in Salmonella spp. poses a significant global concern and is a major threat to global health. Therefore, it is necessary to isolate and identify antibiotic resistance characteristics of Salmonella from food. Studying the antibiotic resistance characteristics of Salmonella will provide important information for the prevention, control of diseases as well as food contamination control and regulations on the use of antibiotics in treatment and animal husbandry in order to limit antibiotic resistance of bacteria. In Vietnam, there has been no research on identifying related gene categories of multidrug-resistant Salmonella isolated from food using Next-generation sequencing. Studying the expression genome of Salmonella, especially antibiotic resistance genes in multi‐ antibiotic resistant Salmonella isolates, provides insight into the molecular epidemiology of antibiotic resistance genes. More importantly, it is possible to detect new mutations in antibiotic resistance genes that cause antibiotic resistance in Salmonella. In addition, studying the expression genome could help to identify new gene groups that can cause antibiotic resistance in this bacterium. 1 According to the Food and Agriculture Organization of the United Nations, Vietnam is a country where consumption of beef, pork, and poultry has increased rapidly since 1993 and is expected to increase significantly in the next years. Therefore, this thesis has been implemented with the following objectives and content: 2. The objectives of the thesis • Identify Salmonella isolated from pork, beef, and chicken meat at the retail markets in Hanoi. • Determining antibiotic resistance characteristics of isolated Salmonella. • Analysis of related gene categories in multi-antibiotic resistant Salmonella. 3. The main contents of the thesis • Microbial culture, isolation and identification of Salmonella from retail meats. • Conducting antibiotic testing, then selecting multidrug resistant Salmonella. • Using next-generation sequencing to analyze the related gene categories in some of multi-antibiotic resistant Salmonella. Confirm some of the new finding results by Sanger sequencing Chapter 1. OVERVIEW 1.1. Biological characteristics of Salmonella Salmonella is a nonspore-forming rod-shaped, Gram-negative bacterium, most Salmonella strains are motile with peritrichous flagella, facultatively anaerobic bacilli. Salmonella can grow on some selective growth medium, such as XLD agar. On XLD agar, Salmonella species have red colonies, some with black centers. They are sensitive to heat and usually killed at temperatures over 70°C. 2 Salmonella possesses three major antigens: H (flagellar), O (somatic), and Vi antigens. The genus Salmonella is part of the Enterobacteriaceae. The genus comprises two species, S. bongori, and S. enterica, the latter of which is divided into six subspecies: I (enterica), II (salamae), IIIa (arizonae), IIIb (diarizonae), IV (houtenae) và VI (indica). 1.2. Genetic characteristics of Salmonella 1.2.1. Salmonella genome structure The genome size of Salmonella varies from 3.39 to 5.59 Mb. The number of genes average is 4,742. Salmonella contains from 1 to 2 plasmids, which vary in size from 2-200 kb. S. enterica needs about 3,499 genes and S. bongori needs about 3,368 genes for normal growth. Salmonella's genome is homologous from 65% to 99%. Salmonella strain has a large stable core, whilst there is an abundance of accessory genes, including the Salmonella pathogenicity islands (SPIs), transposable elements, phages, and plasmid DNA. The core and pan-genome of Salmonella were estimated to be around 2,800 and 10,000 gene families, respectively 1.2.2. Mechanisms of Antibiotic resistance in Salmonella The most common antimicrobials that Salmonella has developed resistance at the present are namely; aminoglycosides, βlactams, chloramphenicol, quinolones, tetracyclines, sulfonamides, and trimethoprim. Aminoglycosides. The Salmonella uses mechanisms such as expression of plasmid-mediated aminoglycoside modifying enzymes against aminoglycoside. These enzymes are categorized into three groups and are named based on reactions they perform, including acetyltransferases, phosphotransferases, and nucleotidyltransferases. 3 Beta-lactams. In Salmonella, the secretion of a beta-lactamase is the common mechanism of resistance to beta-lactams. These enzyme acts by hydrolyzing the structural rings of the B-lactam, by producing beta amino acids with no antimicrobial activity To date, there is well comprehended about more than 340 beta-lactamases resistance genes. Chloromphenicol (C). Chloramphenicol is specific and potent inhibitor of protein by binding to the peptidyltransferase center of the 50s ribosomal unit, thus preventing formation of peptide bonds. There are two mechanisms in which Salmonella resistance to chloramphenicol is conferred: (i) by the plasmid-located enzymes called chloramphenicol acetyltransferases or nonenzymatic chloramphenicol resistance gene cm1A and (ii) Efflux pump in which the antibiotic is removed. Quinolones. Salmonella resistance to quinolone has been classified into two mechanisms. The first is the two gyrA and gyrB, genes which encode for the subunits of DNA gyrase, and in the parC subunit of topisomerase IV. Also, the second mode of action involves changes in the AcrAB-TolC efflux system expression. However, it is an accumulation of multi-mutations that provides resistance, rather than one mutation. Tetracycline (TE). Tetracycline resistance in Salmonella can be attributed to the production of an energy dependent efflux pump to remove the antibiotic from within the cell. To date, 46 TE-resistance genes have been found. Sulfonamide and trimethoprime (SXT). These classes of antibiotics are bacteriostatic and it mode of action is by competitively inhibiting enzymes involved in the synthesis of tetrahydrofolic acid. 4 Sulfonamide inhibit dihyrdropteroate synthetase, while trimethoprim inhibits dihydrofolate reductase. The resistance of Salmonella to sulfonamide has been attributed to the presence of an extra sul gene. Beside, attributed to dhfr and dfr gene. 1.2.3. Relationship between drug resistance and gene mutations Although many mutations contributing to antibiotic resistance have been identified, the relationship between the mutations and the related phenotypic changes responsible for the resistance has yet to be fully elucidated. The relationship between a mutation and drug resistance is not always a simple one-to-one correspondence. Multiple mutations are often required to acquire high levels of resistance to a specific drug. Overall, the complex relationship between drug resistance acquisition, genetic alternations and global phenotypic changes remains unclear. 1.2.4. Bacterial efflux pumps Efflux pumps not only can expel a broad range of antibiotics owing to their poly-substrate specificity, but also drive the acquisition of additional resistance mechanisms by lowering intracellular antibiotic concentration and promoting mutation accumulation. Overexpression of multidrug efflux pumps have been increasingly found to be associated with clinically relevant drug resistance. 1.3. Contamination and antibiotic resistance of Salmonella in food 1.3.1. In the world There have been many different studies on Salmonella contamination rate in food which have been published. In general, the research results show that Salmonella strains are distributed differently depending on the geographical region and the food source. 5 The common serotypes vary by geographic region and their rates of antibiotic resistance in Salmonella are increasing each year. 1.3.2. In Vietnam Recent reports show that Vietnam food is contaminated Salmonella at different rates, including multiple antibiotic-resistant species. The prevalence of Salmonella in food and the rate of multiantibiotic resistance in Salmonella isolates are increasing every year. Therefore, it is necessary to isolate and determine the antibiotic resistance characteristics of Salmonella in each food type in a particular geographical region at different time periods. 1.4. Methods are commonly used in gene expression research Currently, there are four methods are used: Reverse transcription PCR (RT-PCR), Real-time PCR (qPCR), Microarray, and next generation sequencing (NGS) to study gene expression. Among the above techniques, the next generation sequencing has the most advantage, this technique can overcome the disadvantages of the remaining three techniques and is the only one capable of detecting new genes. Next generation sequencing NGS is a direct measurement of nucleic acid sequences present in the sample. There is the linear relationship between the number of sequences and the concentration of nucleic acid sequences present in the sample. Moreover, NGS is not dependent on the information of nucleic acid sequences and highly homologous genes that can be expressed in the sample. Thus new genes can be detected. Among the next generation sequencing technologies, the Illumina technology produces the most accurate data, the procedure is simple, and is widely applied in many different research fields. Currently, 6 more than 90% of the sequence data in the world is generated by Illumina technology. Chapter 2. MATERIAL AND METHODS 2.1. MATERIAL 2.1.1. Samples A total of 90 meat samples, including 30 porks (from TL-1 to TL-30), 30 chicken meats (from TG-1 to TG-30), and 30 beef samples (from TL-1 to TL-30). symbols from TB-1 to TB-30), randomly collected at 10 markets in Hanoi. 2.1.2. Culture media, chemicals, antibiotics, and kits Culture media, antibiotics, and kits are purchased from reputable companies globally. 2.1.3. Research equipment The equipments are used at prestigious lab in Vietnam, such as Vietnam Academy of Science and Technology, National Institute of Burn, Vietnam Medical Military University. 2.2. METHODS 2.2.1. Sampling Samples were collected according to TCVN 4833-2002, from 7-8 am in the winter season, from October to December 2016. 2.2.2. Identification of Salmonella Salmonella was detected according to ISO 6579: 2002. 2.2.3. Antibiotics susceptibility Salmonella’s antibiotics susceptibility was testing using Kirby-Bauer diffuse method. 2.2.4. Nex generation sequencing We choosed some of multidrug resistant Salmonella to transcriptome sequence using Illumina's technology. 7 2.2.5. Bioinformatics methods The sequence quality is checked by FastQC software. The adapter sequences and noise sequences were removed by Trimmomatic 0.32 software. The sequence at Q20 quality score was de novo assembled using Geneious R11 software. The de novo sequence was annotated by different databases such as RAST, PATRIC 3.5.2, BASys, and Geneious R11 software. Identification of antibiotic resistance genes using: ResFinder, ARG-ANNOT, CARD, and PATRIC 3.5.2. Identify the gene mutation resistance to quinolone, by ResFinder tool. 2.2.6. Confirm antimicrobial resistant gene mutations using Sanger sequencing To confirm the predicted results of antibiotic resistance gene mutations obtained from the next generation sequencing method, the new gene mutations related to quinolone resistance will be confirmed using Sanger sequencing from cDNA of the samples. 2.2.7. Statistical analyzed Using SPSS 16.0 software to calculate the χ2, Fisher exact test, p values. Chapter 3. RESULTS AND DISCUSSION 3.1. Isolate and identify the Salmonella serotype 3.1.1. Identify Salmonella results After non-selective enrichment, selective enrichment and biochemical confirmation, we have obtained Salmonella spp. from the research samples. The results are presented in Table 3.1 8 Table 3.1. Results of identify Salmonella in the research samples Positive Negative Total Sample (number) number rate (%) number rate (%) Chicken 30 11 36,7 19 63,3 Pork 30 9 30,0 21 70,0 Beef 30 5 16,7 25 83,3 Total 90 25 27,8 65 72,2 χ2 = 3,102; df = 2; p = 0,212 The list of Salmonella positive isolated sources is presented in Table 3.2 Table 3.2. List of samples positive for Salmonella 1 2 3 Sample sources Chicken (11 samples) Pork (9 samples ) Beef (5 samples ) Sample ID TG-1, 2, 3, 4, 5, 6, 14, 25, 28, 29, 30 TL-1, 2, 3, 4, 5, 15, 26, 29, 30 TB-1, 2, 3, 15, 29 The prevalence of Salmonella in this study was 27.8%, this result is in line to that of Do Ngoc Thuy et. al. (30%). However, this rate is lower than other studies such as Ta et. al. (48.7%), Nguyen et. al. (69.7%), Boomar et. al. (80%). This lower result may be due to collecting samples in the morning, when meat was fresh, limiting bacterial infection. Moreover, the time of sample collection is in the winter season, the low temperature and humidity, combined with the dry air, these factors pose inhibited to the growth of bacteria. Among Salmonella positive samples, chicken samples were the most prevalence (36.7%), followed by pork samples (30%), and beef samples were the lowest rate (16.7%). This result is in line with the research results of Do Ngoc Thuy et. al., Zhao et. al., Miranda et. al. However, this result is different from other studies such as Phan et. al. (pork, beef, chicken) and Nguyen et. al. (pork, chicken, beef). The Salmonella positive rate in chicken is often higher than pork, which 9
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