The Analysis On Antimicrobial Resistance And Molecular Typing Of Salmonella Enteritidis In Guangdong,2007-2013

BackgroundsSalmonella is a Gram-negative bacteria, belonging to Enterobacteriaceae genera, with multiple hosts and wide distribution, such as most animals and humans’ gastrointestinal tract, and often presents in water, eggs and its products, meat etc. According to the antigenic properties of O and H antigens classification, there are more than 2500 kinds of Salmonella serotypes, in which Salmonella typhimurium and Salmonella enteritidis are the most common zoonotic serotypes. Patients with Salmonella infection often occur diarrhea, abdominal pain, vomiting and other symptoms. Salmonella infections are mostly self-limiting disease. Patients can be self-healing, but sepsis and other complications requiring antibiotics.Currently, the treatment of Salmonella bacteria mainly uses fluoroquinolones and third generation cephalosporins in clinical cases. With the accumulation of time and frequency, it has been a considerable part of drug-resistant strains. According to the National Antibiotic Resistance Monitoring System (NARMS) latest reports indicate that 22.5% of non-typhoid Salmonella are resistant to at least one antibiotic. Salmonella enteritidis appeared multi-drug resistant (MDR) phenomenon.Plasmid-Mediated Quinolone Resistance (PMQR) is an important mechanism for drug-resistance of Salmonella. There have been many reports about three types of PMQR plasmid genes, qnr, aac (6’)-Ib-cr and qepA, encoding respective protein. PMQR mainly mediates low-level drug-resistance. But it can causing a high level of resistance, once it has accumulated enough mutations. PMQR-positive bacteria mainly are multi-drug resistant phenotype. Different regions have various distributions.The outbreaks of diarrhea caused by Salmonella contaminated food previously derived from point-souce distribution. However, it now become a regional inter-state (province) of sporadic outbreaks forms. So at present molecular subtyping technologies play important roles for outbreak identification and source tracking. Molecular typing methods generally include three principles:based on DNA sequence polymorphism analysis; based on PCR amplification of specific gene products, and based on restriction endonuclease analysis of bacterial DNA.Pulsed field gel electrophoresis (PFGE) is a technique for electrophoretic separation of high molecular weight linear DNA molecules which developed in the mid-1980s. PFGE is known as the "Gold Standard" for bacterial typing techniques of molecular biology. The principle is applied on an agarose gel, with alternating change of current pulse in electric field in the direction, time and current magnitude, so that the DNA molecule can separate effectively. Comparing the DNA molecule electrophoresis patterns, we can confirm its association. However, it will produce different patterns because of mutant restriction sites and different insertion sequences and some other factors. Generally, the experts believe that the less differences of the electrophoretic bands, the smaller differences in the genetic area, so the kinship in strains are more close, vice versa. PFGE is applied in the source-tracking study between strains, especially in outbreak cases.Multi-locus sequence typing (MLST) is a sequencing technology based on nucleic acid sequence. It has unparalleled advantages in inferring the phylogenetic relationship of strains by sequencing the change of specific gene sequences. MLST databases have the allele information of bacteria, so that it can make global standardization via Internet. Compared to other genotyping methods, MLST has high resolution level and comparability, and it is also fast to get results.Because of genetic stability, MLST cannot provide effective help for short-term epidemiological studies. So MLST is suitable for the genetic relationship research with a large population and a quite period of time.This study was designed to carry out the drug-resistant surveillance and genotyping of Salmonella enteritidis in Guangdong area. We collected the clinical isolates of Salmonella enteritidis during 2007 to 2013 in Global Salmonella Surveillance (GSS) to do drug susceptibility testing. According to the results and clinical practice, we chosed the strains which were resistant to II generation cephalosporins or III generation quinolones drugs to do PMQR detection and PFGE and MLST molecular typing research. This helps us to monitor the clinical treatment of high frequency antibiotics’tracking. And we can guide clinical use of antibiotics. Based on the genotyping data, we can analyze the phylogenetic relationship and molecular genetic characteristics of Salmonella enteritidis isolates. We can improve the capability of laboratory-based active surveillance, foodborne outbreaks identification and prevention by obtaining the baseline data and the molecular characteristic of Salmonella isolates with PFGE and MLST methods. We can provide scientific basis for controlling food-borne disease caused by Salmonella enteritidis.Objectives1.To investigate the infection situation and drug resistance status of Salmonella enteritidis among clinical diarrhea patients in Guangdong sentinel hospitals from 2007 to 2013. This study was also aimed to obtain the resistance spectrum and to analyze the characteristics of multi-drug resistance.2. To analyze the distribution and the epidemiological rule of PMQR genes in drug-resistant (fluoroquinolones and cephalosporins) Salmonella enteritidis strains in Guangdong.3. To analyze the aggregation and distribution of drug resistant strain patterns by PFGE and MLST typing, and to provide base data for outbreak surveillance and systematic analysis of epidemiology.Methods1. A total of 386 Salmonella enteritidis strains were collected from GSS. Broth dilution method was used to measure the minimum inhibitory concentration (MIC) according to the recommendation of Clinical and Laboratory Standards Institute (CLSI). We applied WHONET 5.3 to analyze the antibiotic sensitivity test data, and then we imported the numerical data into SPSS 13.0 software for statistical analysis, such as descriptive statistical analysis of three-dimension distribution, positive rate, as well as sensitivity to antibiotics and multidrug-resistant characteristics, etc.2. PMQR genes (qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxAB, aac (6’)-Ib-cr) were detected in the strains which were resistant to cephalosporins and fluoroquinolones drugs. We analyzed the distribution of PMQR gene in Guangdong and its effect of MIC values. Geometric mean of MIC values between groups were compared using unpaired t test after variables transforming; enumeration data between groups were compared using the chi-square test or Fisher’s exact test. Test level a= 0.05, with P <0.05 was considered statistically significant.3. According to international PulseNet standard operating program, Salmonella PFGE subtyping were completed of the above cephalosporins or fluoroquinolones-resistant Salmonella enteritidis isolates.Strains were digested by restriction endonuclease. After pulsed-field gel electrophoresis, gel image analysis system generated the image bands. BioNumerics software was adopted to identify and process image strips. H9812 strain was used as a molecular weight standard for calibration. The patterns were clustered by Unweighted Pair Group Method using Arithmetic average (UPGMA) and numbered by unified naming rules. Then the drug-resistant Salmonella enteritidis PFGE subtyping database was established.4. According to PulseNet MLST standard, we chosed seven loci (aroC, dnaN, hemD, hisD, purE, sucA, thrA) as house-keeping genes to amplify. After optimizing the reaction system and sequencing procedure, we obtained the results of sequencing typing (ST). Adding in strains’ epidemiological data, we analyzed the phylogenetic relationship and the association with resistance spectrum. We could evaluate the resolution and practice value of MLST method comprehensively by compared with PFGE.Results1. We collected 63,687 cases of sporadic cases of diarrhea from 2007 to 2013 in Guangdong.386 Salmonella enteritidis were isolated from the feces of the patients (detection rate of 6.06 ‰). Strains were mainly distributed in the age group of 1-4 years old(35.23%,136/386). The ratio of male to female of the 386 strains was 1:0.698. Epidemic season was from June to October. Strains were mainly from Guangzhou, Dongguan. They were sporadic, from the center of the Pearl River Delta city group. Regional detection rate was no significant difference.2. Broth dilution method’s results indicated that Salmonella enteritidis were likely to develop resistance to the eight antibiotic drugs. The resistant rate of Nalidixic acid was the highest (85.23%,329/386, MIC50=128μg/ml), followed by Tetracycline (30.83%,119/386, MIC50=2μg/ml).In this study, Cefoxitin (0.52%,2/386, MIC50=2μg/ml) and Ciprofloxacin (1.55%,6/386, MIC50=0.25μg/ml) had the lowest resistant rate. The 386 strains had a total of 27 antibiograms.43.78%(169/386) strains were single Nalidixic acid-resistant.63 strains were multi-drug resisitant (16.32%,63/386).3. According to drug susceptibility results, we screened 71 fluoroquinolone-resistant and cephalosporins-resistant in Salmonella enteritidis strains. PMQR genetic detection results were as follows:45 strains were PMQR positive strains (63.38%, 45/71), of which 35 strains had only one PMQR gene(49.30%,35/71). oqxAB gene had the highest detection rate(40.85%,29/71), followed by qnrC (9.86%,7/71). qepA was not detected. The geometric mean MIC values of chloramphenicol (CHL) and sulfa drugs (TMP/SMZ) were statistically significant between PMQR positive strains and PMQR negative strains.4. Xbal digested cluster analysis of the 71 drug-resistant Salmonella enteritidis were divided into two large clusters. PFGE patterns of similarity was 56% to 100%.71 strains divided into PFGE-XbaI 35 types. The maximum type had eight strains, and the minimum type had only one strain.5.71 drug-resistant Salmonella enteritidis strains had the same MLST results. Allele sequence were aroC5, dnaN2, hemD3, hisD7, purE6, sucA6, thrA11. Allelic spectrum was ST11, belonging to eBG4 type. It indicated that ST11 was the advantage type of drug-resistant Salmonella enteritidis in Guangdong Province.Conclusions1. Salmonella enteritidis, as the second largest advantage serotypes in Guangdong Province, was mainly susceptible to infect 1-4 year-old children, especially in summer and autumn. High incidence of cases were concentrated in the Pearl River Delta region. Nalidixic acid (NAL) had the highest drug-resistant rate. Multidrug resistant patterns of Salmonella enteritidis were diverse. Rational use of antibiotics and real-time monitoring can contribute to reducing the multidrug resistant strains.2. PMQR positive strains had a higher drug-resistant rate than PMQR negative strains. What’s more, PMQR positive strains were more likely to develop multi-drug resistance. This study cannot provide the statistical significant results of whether oqxAB would increase the risk of multi-drug resistant.3. MLST molecular typing results showed that the nucleotide sequence in housekeeping gene was highly conserved within the same serotype. The PFGE results indicated the low differentiation of Salmonella enteritidis. For Salmonella enteritidis, MLST and PFGE’s resolution were not enough to identification. We need to use some other higher-resolution molecular typing methods.