Nghiên cứu đặc điểm kháng kháng sinh và gen liên quan ở các chủng salmonella đa kháng tt tiếng anh

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 The urgency of the thesis The burden of foodborne diseases is substantial Each year, foodborne diseases cause almost 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 years Salmonella is of the 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 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: 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 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 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 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) 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 to 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 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 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 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, more than 90% of the sequence data in the world is generated by Illumina technology Chapter 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 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 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 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 30,0 21 70,0 Beef 30 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 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 through the consumption of food The rate of antibiotic resistance in this study (52%) is lower than the results of some studies in Vietnam (62.2%) and Japan (89.9%) The rate of multi-resistance (36%) is lower than some other research results such as Nguyen et al (41.1%) and Katoh et al (90.2%) This difference may be due to the overuse of antibiotics in animal husbandry and treatment between nations, which increases selective pressures, resulting in emergence different rates of antimicrobial resistance Salmonella The number of samples in this study is small (25 samples) Therefore, further research on resistance rates and multiple antibiotic resistance should be conducted with a larger number of samples 3.2.2 The number of each Salmonella resistant to antibiotics according to the isolation source Determining the rate of antibiotic resistance of Salmonella according to isolated sources helps to evaluate in detail their antibiotic resistance characteristics by food source The results showed that Salmonella isolates from pork were most resistant to antibiotics, with 66.7% (6/9 strains), of which 44.4% (4/9 strains) were multi-antibiotic resistant Following by the isolated from chicken with the rate of 36.4% (4/11 strains) of which 27.3% (3/11 strains) are resistant There is only one S Typhimurium from beef that is antibiotic resistant and is multi-resistant S Typhimurium accounts for a large proportion of all three isolates (11 strains), but only strains of antibiotic resistance are also multi-resistant 3.2.3 Number of Salmonella resistant to each antibiotic according to the isolation source The determination of Salmonella antibiotic resistance according to isolated sources helps to evaluate in detail their antibiotic 11 resistance characteristics by food source Accordingly, all Salmonella isolated from chicken were antibiotic resistant (except CAZ) No strains were isolated from pork and beef resistant to CIP Salmonella isolated from three meat sources that are resistant to AM, STR, C, TE and SXT, among which isolates from pork have the highest rate of antibiotic resistance All Salmonella isolates from beef are sensitive to CAZ, GN and CIP Salmonella was isolated from pork resistant to AM, STR, and TE with the largest percentage Salmonella from chicken is the most C resistant, especially the CIP resistant strains were found only in chicken Beef is the least contaminated Salmonella and these strains are most susceptible to antibiotics 3.2.4 Antibiotic resistance phenotypic Base upon antibiotic susceptibility results, we have identified the antibiotic resistance pattern of Salmonella studied Two common phenotypes of antibiotic resistance are TE, STR, AM (2/9), and C, TE, SXT, STR, AM (3/9) TE, STR, AM phenotypes are only found in Salmonella isolated from pork The two common antibiotic resistant phenotypes in this study differ from the antibiotic resistance phenotypes published in the Miranda et al., Kim et al studies From the above results it can be said that the common patterns of antibiotic resistance are different between the studies Identifying the antibiotic resistance phenotype in the research samples is important This phenotypic result combined with genomic analysis results will show antibiotic resistance genes, mutations related to antibiotic resistance phenotypes From the phenotypic results, we have obtained multi-resistant Salmonella, of which S Typhimurium is the most common Pork is the most isolated source of 12 multi-resistant Salmonella (5 serovars), followed by chicken (3 serovars), beef (1 serovar) Antibiotic resistance rates, multidrug resistance rates, and antibiotic resistance patterns are different among published studies This difference, according to some researchers, may be due to the overuse of antibiotics in treatment and animal husbandry, increasing the selection pressure on bacteria leading to the emergence of different strains of multi-antibiotic resistant Salmonella by region geography 3.3 Results of analyzing gene categories in some multidrug resistance Salmonella Transcriptome sequencing is rarely used to identify antibiotic resistance genes Instead, researchers often use the entire whole genome sequencing Transcriptome sequencing allows us to study the function of genes better than DNA sequencing In this study, we want to focus on the functional genome of bacteria This is important because the unnecessary genes will not be expressed and tend to be lost or degraded Moreover, the whole genome sequencing technique could not distinguish the genes that were inactivated in the genome and other related functions of that gene In addition, in our opinion, the expression gene will be related to the phenotype, particular resistant or sensitive phenotypes, higher than the non-expression gene We have sequenced transcriptome of multidrug resistant Salmonella using method which was published by Marcelino et al (the author also sequenced the transcriptome of bacteria in bird gut in Australia to identify of antibiotic resistance genes) It is very important that antibiotic resistance genes are also expressed in antibiotic sensitive bacteria under normal culture conditions Therefore, in this study we did not use antibiotic sensitive strains for comparison 13 Instead, we use the same phenotype that is sensitive and resistant to each antibiotic in the strains to compare From that, we predict which genes, or mutations, might be related to antibiotic resistance in this bacterium In order to analyze the gene groups in multidrug-resistant Salmonella, several resistant serovars should be selected to sequence transcriptome However, in the framework of this thesis, we selected only strains according to the criteria of high infection rate, resistance to as many antibiotics as possible and by isolated sources, including S Derby, S Give, S Indiana, S Typhimurium S384, S Typhimurium S360, and S Typhimurium S181 After extracted RNA from research samples, we conducted quality control by concentration measurement at OD260/280, and electrophoresis Results showed that these RNA samples were of good quality This sample was then synthesized cDNA and tested for integrity The results showed that the RIN (RNA Integrity Number) was above 8.0, qualified for sequencing The results are as follows: 3.3.1 Number of read From the raw sequence results, we conducted trimming sequence by Trimmomatic software: this yield total of 160,043,486 read, total number of read at Q20 score is 146,080,642 The number of read is highest in Sal and at least in Sal The read sequences at Q20 will be used for de novo assembly and used for further analysis 3.3.2 De novo assembly Results of de novo assembly showed that the transcriptome size ranged from 4.69 Mb (Sal 4) to 5.1 Mb (Sal 11), GC values around 52%, N50 values are high and L50 values are low This result is similar to the sequencing result of S Derby 07CR553 published by 14 Kérouanton et al From that, it can be concluded that the sequence of research samples is of good quality, eligible for subsequent analysis 3.3.3 Genes annotation To avoid the missing annotation genes, gene analysis tools have been used as many as possible The number of genes detected in these tools is different The findings of these tools' genes differ because of their different methods of analysis, and there is currently no standard method for annotating genes accepted among researchers around the world The number of coding sequences found by different databases is shown in Table 3.3 Table 3.3 Coding sequences in research samples Number of coding sequences Gene analysis tool Sal RAST 4.807 4.917 5.154 5.097 5.357 5.000 PATRIC 4.807 4.917 5.154 5.049 5.357 5.000 BASys 4.973 5.100 5.336 5.253 5.566 5.209 Genious R11 4.400 4.513 5.022 5.207 5.304 5.309 Sal Sal Sal Sal 11 Sal 12 In 2017, Baek et al announced that many genes that encode proteins less than 100 amino acids undetectable when annotating the bacterial genome Therefore, further studies of the above genes are needed in the research samples 3.3.4 Gene categories analysis in multidrug resistant Salmonella In addition to housekeeping genes We have identified important gene categories expressed in multi-resistant Salmonella: 3.3.4.1 Antibiotic resistance genes Results of antibiotic resistance genes and antibiotic resistance phenotype are presented in Table 3.4 Accordingly, a total of 107 15 antibiotic resistance genes (list of genes not shown in this summary) Including 22 β-lactam resistance genes, 46 aminoglycoside resistance genes, quinolone resistance genes, phenicol resistance genes, cycline resistance genes, sulfonamide resistance genes, trimethoprim resistance genes Furthermore, we have found 12 antibiotic resistance genes which resistant to macrolides, rifamycin, fosfomycin, lincosamide, polymyxin, and peptides The number and diversity of antibiotic resistance genes in this study are similar to the results of the study by Saskia et al (2018) A total of 42 phenotypes were identified from antibiotic susceptibility results There are 29 antibiotic resistant phenotypes have expression of antibiotic resistance genes There are 12 antibioticsensitive phenotypes have expression of antibiotic resistance gene The only sensitive phenotype is that there is no expression of the antibiotic resistance gene, Sal is susceptible to SXT and there is no expression of SXT resistance gene Thus, the genotype and phenotype accordant is (29 + 1) / 42 = 71.4% And the genotype and antibiotic resistance phenotype not accordant is 12/42 = 28.6% The correlation between genotype and phenotype is similar to the study results of Owen et al., 2017 (72.7%) Our research results are lower than those of some other authors like McDermott et al, 2016 (99%), Zankari et al., 2013 (99.74%) The genotypes and phenotypes not accordant in this study can be explained by the inadequate expression of antibiotic resistance genes, due to the multiple antibiotic resistance mechanisms involved in resistance to one antibiotic, and the other mechanisms of antibiotic resistance have not been found 16 Table 3.4 Summarize results of antibiotic resistance genes and antibiotic resistance phenotype in the research samples KS Mẫu nghiên cứu (Kiểu hình kháng kháng sinh/gen kháng kháng sinh) Sal (R) blaOXA-1 blaTEM family, PBPE** (R) aac family* aph family* ant family*, kdpE Sal (R) blaTEM family (S) aac(6')-Iy kdpE Sal (R) blaTEM family PBPE** (S) aac (6')-Iy, aac6-Iy, aadA8, aadA17; kdpE Sal (R) blaTEM family (R) aac family* aph family* ant family*, kdpE (R) aac(6')Ib-cr, gyrA, gyrB, parC (R) aac(6')-Iy kdpE (R) aac (6')-Iaa, aac3-IIa aadA17, aadA8b aph3-IIa, kdpE (S) qnrS1, qnr-S3, qnr-S5, gyrB, parC (R) aac family* aph family* kdpE (S) gyrA, gyrB, parC (S) floR, cmlA1 (S) cmlA1 (R) sul1, sul2, dfrA12 (R) tet(A), tet(B), tet(C), tet(R) (R) sul2, dfrA14, dfrA5 (R) tet(A) SXT (R) floR, cmlA1, cmlA5, cat2 (R) tet(A), tet(B), tet(C), tet(M), tet(R), tet(S) (R) sul2, sul3, dfrA12 (R) floR, cmlA1 TE (R) floR, cmlA1, catB4, catB8 (R) tet(A), tet(R) (R) aac (6')-Iy, aac6-Iy aadA8, aadA17 kdpE (S) qnr-S1, qnr-S3, gyrA, gyrB, parC, parE (R) floR, cmlA1, cmlA5, cat2 (R) tet(A), tet(M), tet(R), tet(S) (R) sul2, sul3, dfrA12 Sal 12 (R) blaTEM family PBPE** (S) aac(6')-Iaa, aac6Iaa, aph(6)-Id, aph(3'')-Ib strA, strB, kdpE (R) aac(6')-Iaa, aac6-Iaa aph(6)-Id, aph(3'')-Ib strA, strB, kdpE (S) gyrA, gyrB, parC AM GN STR CIP C catB3, (S) gyrA, gyrB, parC (R) tet(A), et(M), tet(S) (S) (S) aac (6')-Iaa, aac3-IIa, aadA17, aadA8b aph3-IIa, kdpE Sal 11 (R) blaTEM family PBPE** (R) aac family* aph family* kdpE (S) sul2 Abbreviation: *Aac (Acetylation) family; Aph (Phosphorylation) family; Ant (Adenylylation) family; ** Penicillin Binding Protein E coli KS (kháng sinh), AM (ampicillin), GN (gentamycin), STR (streptomycin), CIP (ciprofloxacin), C (chloramphenicol), TE (tetracyclin), SXT (sulfamethoxazol/trimetoprim) 17 3.3.4.2 Quinolone resistance gene mutations Quinolone is commonly used in the treatment of Salmonella infections in humans For food-borne Salmonella, quinolone resistance is the most concerned and has been mentioned in the list of the most important antibiotics in the field of medicine in 2016 by WHO One of the quinolone resistance mechanisms in Salmonella is caused by mutations of the gyrA, gyrB and parC genes Thus, we found mutations of these genes, the results are presented in Table 3.5 Table 3.5 Results of the quinolone resistance gene mutations Gene mutations Samples SR gyrA parC Sal R Sal S Sal S Sal S T255S; N395S; S469A; A620T Sal 11 S T255S; N395S; S469A; A620T Sal 12 S T255S; N395S; S469A; A620T parE S83F;D87G T57S; S80R; T255S; A628S T57S; T255S; N395S S83Y T57S; T255S; A352V S592N Abbreviation: A (Alanine), N (Asparagine), R (Arginine), S (Serine), T (Threonine), V (Valine), SR (susceptibility results), R (resistant), S (sensitive) The results of Table 3.5 show that the list of mutations was identified: S83F, S83Y, D87G, S80R, T57S, T255S, N395S, S469A, A620T, A628S, S592N To date, there are no reports of mutations A628S, T255S, N395S, S469A, and A620T have been published However, the mutations T255S, N395S, S469A, A620T were identified in CIP-sensitive strains Sal 6, Sal 7, Sal 8, Sal 11, and Sal 18 12 proving that these mutations have no role in CIP resistance in research samples The parC mutation (A628S) may have a role in CIP resistance because it appears only in Sal 4, the only CIP resistant strain This result is not only new but also has high scientific significance, paving the way for further research on antibiotic resistance in Salmonella 3.3.4.3 The gene categories involved in the efflux pumps We identified the expression of 41 efflux pumps-related genes, including 37 genes in S Typhimurium S181, 25 genes in S Typhimurium S384, 27 genes in S Typhimurium S360, 27 genes in S Give, 23 genes in S Derby, 26 genes in S Indiana Efflux pumps were detected in research samples belonging to families: MFS (MefB, EmrAB, tetA, tetB, MdtD), SMR (QacE), and RND (AcrAB-TolC, AcrAD-TolC, AcrEF-TolC, MdtABC-TolC, MexPQ-OpmE) Understanding of efflux pumps is essential for the development of interventions to limit antibiotic resistance in Salmonella Some strains of Salmonella are susceptible to antibiotics but still have expression of efflux pumps, like AcrAB-TolC, AcrEF-TolC, and MdfA in C-sensitive strains AcrD in GN-sensitive strains MexPQ-OpmE in CIP-sensitive and C-sensitive strains Therefore these channels not play a role in resistant to these antibiotics Some antibiotic resistant strains have the expression of efflux pumps, like AcrD in AM, STR-resistant strains, MexPQ-OpmE in TEresistance strains, EmrAB in TE, STR and AM-resistant strains Therefore, we predicted that these channels may be related to resistance to the respective antibiotics Further studies on the drug dispensing canal system in Salmonella need to be conducted in the future 19 New finding the role of the above efflux pumps on antibiotic resistance is the novelty of the thesis 3.3.4.4 Analysis results of some other gene categories In addition to the antibiotic resistance gene category and the genes related to efflux pumps, we identified many other gene groups related to antibiotic resistance and bacterial toxins The number of genes in each of these functional categories is shown in Table 3.6 Table 3.6 Expression of related gene groups Gene category Number of genes Sal Sal Sal Sal Sal 11 Sal 12 Toxin genes 118 124 125 130 131 133 flagellar 38 38 38 38 38 38 Cell wall and LPS 52 39 48 52 51 51 Response to Selenium 2 2 2 Response to Tellurite 3 3 3 2 2 2 12 24 22 27 26 26 21 21 21 21 21 21 Response to Formaldehyde Mobile elements, phage and prophage ABC transporter Abbreviation: Sal (S Indiana), Sal (S Derby), Sal (S Give), Sal (S Typhimurium S360), Sal 11 (S Typhimurium S384), Sal 12 (S Typhimurium S181) Toxic gene categories To date, about 200 toxin-related genes in Salmonella have been discovered, however, the function and action mechanism of most of these genes have not been fully elucidated In this study, a total of 134 toxin genes were identified The number and diversity of these 20 genes are similar to those of other publications, for example, by Figueiredo et al (114 genes), Card et al (105 genes) The presence of the toxic gene indicates that all six multidrug resistant Salmonella strains are virulent These genes play a role in the pathogenesis and may have a role in antibiotic resistance in Salmonella This is the first study to determine the expression of all virulence genes in multidrug resistant Salmonlla strains in Vietnam The gene categories involved in flagellar synthesis Flagellar plays an important role in producing the toxin of bacteria, which is often mixed in the outer membrane protein when an antigen is isolated to make a Salmonella vaccine Understanding the expression genes associated with flagellar will be very helpful for purifying this component For example, inactivating these genes will create pure outer membrane vesicles, as antigens to make the vaccine This is also one of the new contributions of the thesis The gene categories involved in the synthesis of LPS and cell walls Apart from S Typhi, LPS of other Salmonella has not been fully studied The lipidA synthesis genes, an important component of the LPS, involved in resistance to polymixin antibiotics were also expressed in all six study samples LPS and the outer membrane protein form the outer vesicles, which are immune stimulating and are considered important components for making vaccines The results of this study contribute in part to the knowledge of LPS-encoded genes, which is a reference source for further studies Genetic group responds to toxic substances In addition to selenium, tellurite is a highly toxic element, and formaldehyde is also found in six Salmonella samples, which is an alarming fact The results of this study show that extensive research is 21 needed to shed more light on the original sources of these substances and the extent of contamination of this compound in foodstuffs in Hanoi Therefore, controlling the origin and food processing process is necessary Mobile elemetnts group, phage and prophage Phage usually carries toxic genes, forming toxins of Salmonella A number of bacteriophages that exist in Salmonella can be used as a marker for rapid diagnostic We have identified the Gifsy2 phage in all samples, so this could be a marker for quick diagnosis of Salmonella However, the number of research samples is small (6 samples), so it is necessary to conduct research on a larger number of samples to confirm the above problem The gene group involved in the ABC transport channel The ABC transport channel produces toxins in many bacteria Their ability to transport is related to therapeutic antibiotic resistance However, in most Salmonella species, the function of some components of this transport channel such as sapABCDF has not been fully elucidated In S Typhimurium, sapABCDF produces a toxin that helps bacteria resist peptide antibiotics 3.4 Confirm antimicrobial resistant gene mutations using Sanger sequencing Base on the predicted results by the next generation sequencing method on the new mutation in parC gene (A628S) possibly related to quinolone resistance, we conducted a Sanger sequencing of parC gene containing the above mutation to confirm the results The Sanger gene sequencing results showed that only the Sal sample at position 1882 of the parC gene showed a mutation that 22 changed nucleotide G→T, this mutation altered amino acid A628S, this result was consistent with the results of the analysis by the next generation sequencing method The A628S mutation may be a factor that changes the function of the parC gene, making the gene resistant to antibiotics in Sal We have not seen any announcement of this mutation yet However, to confirm whether the parC gene mutation (A628S) is related to quinolone antibiotic resistance, further studies are needed CONCLUSIONS To conclude Isolated and identified Salmonella strains from pork, beef, and chicken at retail markets in Hanoi Samples contaminated with Salmonella were 27.8%, different serovars have been found, the most common serovar was S Typhimurium Identified antibiotic resistance characteristics of Salmonella isolated The antibiotic resistance rate of Salmonella was 52%, the rate of multidrug resistant Salmonella was 36% The highly resistant antibiotics are STR and TE (44%) In contrast, 100% of strains are sensitive to CAZ The common of antibiotic resistance phenotype are TE, STR, AM, and C, TE, SXT, STR, AM Analyzed the gene categories of multidrug resistant Salmonella These include the groups of genes related to antibiotic resistance and the groups of genes related to bacterial toxins Base these results we have identified: • A new mutation in the parC gene (A628S) is possibly associated with quinolone resistance in Salmonella • Identified genes related to efflux pumps that may play a role in resistance to antibiotics in Salmonella such as: AcrD in 23 AM, STR and GN resistance, EmrAB in TE and STR and AM resistance, and MexPQ-OpmE in TE resistance Recommendation Studies on the role of the parC gene mutation (A628S) in response to quinolone resistance Studies on the role and function of the some efflux pumps in Salmonella, especially the role of AcrD in AM, STR and GN resistance, EmrAB in TE, STR and AM resistance, MexPQ-OpmE in TE resistance Studies to evaluate the role of small-sized genes, coding for proteins less than 100 amino acids undetectable in the study samples NEW FINDINGS OF THE THESIS Identify a new mutation point in the parC gene (A628S) possibly related to quinolone antibiotic resistance in Salmonella Proposing the role of the efflux pumps in resistance to some antibiotics in Salmonella such as AcrD in AM, STR and GN resistance, EmrAB channel in TE, STR and AM resistance, MexPQ-OpmE in TE resistance 24 LIST OF WORKS HAS BEEN PUBLISHED Minh Ngoc Nghiem, Viet Thanh Nguyen, Eui-Bae Jeung, Thuy Thi Bich Vo Alternate antimicrobial resistance genes in multidrug resistant Salmonella spp isolated from retail meats in Vietnam using RNA-sequencing analysis (2019) Journal of Food Safety, SCI, Q2 ID: JFS12707, 07 October 2019 https://doi.org/10.1111/jfs.12707 Minh Ngoc Nghiem, Viet Thanh Nguyen, Thu Thi Hoai Nguyen, Ton Dang Nguyen, Thuy Thi Bich Vo (2017) Antimicrobial resistance gene expression associated with multidrug resistant Salmonella spp isolated from retail meat in Hanoi, Vietnam International Microbiology, SCIE, Q3 20(2): 85-93 Nguyễn Thanh Việt, Nghiêm Ngọc Minh, Võ Thị Bích Thủy (2018) Nghiên cứu đặc điểm kháng kháng sinh vi khuẩn Salmonella phân lập từ mẫu thịt lợn, thịt bò, thịt gà chợ bán lẻ Hà Nội Tạp chí Cơng nghệ sinh học 3(16): 553-564 Nguyễn Thanh Việt, Nguyễn Thanh Tùng, Võ Thị Bích Thủy, Nghiêm Ngọc Minh (2019) Nghiên cứu xác định gen liên quan đến tổng hợp lipopolysaccharide chủng Salmonella typhimurium đa kháng công nghệ giải trình tự gen hệ Tạp chí Y học Thực hành 4(1094): 52-56 Nguyen Thanh Viet, Nghiem Ngọc Minh, Nguyen Duc Hieu, Dang Thi Quynh, Vo Thi Bich Thuy (2018) Single nucleotide polymorphism discovery in multi-drug resistant Salmonella spp isolated from retail meats in noi, vietnam using rna-seq technology The 7th International Conference in Vietnam on the Development of Biomedical Engineering: 58 Nguyễn Thanh Việt, Võ Thị Bích Thủy (2018) Nghiên cứu xác định gen mã hóa kênh bơm thải thuốc chủng Salmonella spp đa kháng kháng sinh công nghệ giải trình tự hệ (2018) Hội nghị Công nghệ sinh học toàn quốc 2018: 965-971 ... (2019) Nghiên cứu xác định gen liên quan đến tổng hợp lipopolysaccharide chủng Salmonella typhimurium đa kháng công nghệ giải trình tự gen hệ Tạp chí Y học Thực hành 4(1094): 52-56 Nguyen Thanh... Thị Bích Thủy (2018) Nghiên cứu xác định gen mã hóa kênh bơm thải thuốc chủng Salmonella spp đa kháng kháng sinh công nghệ giải trình tự hệ (2018) Hội nghị Công nghệ sinh học toàn quốc 2018:... results of antibiotic resistance genes and antibiotic resistance phenotype in the research samples KS Mẫu nghiên cứu (Kiểu hình kháng kháng sinh/ gen kháng kháng sinh) Sal (R) blaOXA-1 blaTEM