| BackgroundCholera is a kind of violent intestinal infectious diseases which is cause by Vibrio cholerae. It is a kind of international quarantine infectious diseases. It can cause pandemics for its acute onset, wide and fast spread characteristics. Since 1990s, there have been seven pandemics caused by V. cholerae. There are millions of deaths in the hundreds countries for the disease. Which has caused huge losses in terms of life and wealth. Cholera is transmitted by water, food and touching the things which had been stain by cholera in life. Last century, the waster were often been polluted by the feces. Once someone was infected with V. cholerae, the water was polluted also. People who used the polluted water would be infected. Therefore, the pandemics occurred. In the developing countries like Asia, Africa and South America where economics and sanitations are poor, cholera is sporadic or local outbreak nearly every year. Cholera is a major public health problem threatening people healthy in developing countries. Since the 1990s, our country carried on the patriotic public health campaign. V. cholerae transmitting through the water has been greatly improved and control. However, in recent years, the outbreak of the cholera is often caused by eating raw food which is polluted by the V. cholerae. Cholera remains a focused preventing intestinal infectious disease today.Early treatments including rehydration and antibiotics are effective for controlling it. Rehydration can help to recover the fluid lost. Sensitive antibiotic therapy can shorten the duration and severity of diarrhea in its acute stage. However, antibiotic resistance is common and severe with the antibiotic abused. Many researches had reported traditional antibiotic resistance such as tetracycline, chloramphenicol, penicillin and streptomycin in cholera treatment. As a consequence, it is changeling for clinical treatment and preventing the disease. So it is important to enhance monitoring the sensitivity of antibiotics. Only in this way can we provide a scientific evidence for early treatment and prevent the disease spreading.Cholera had been recorded in Guangdong province and it had been widely spread in the early time. Early in 1817, Cholera had been spread from India via Bangkok to Hongkong, Macao and Guangzhou. There were hundreds of cholera epidemic in coastal areas such as Guangzhou, Shantou, Beihai, Hainan from 1817 to 1947. The O1 El Tor strain which cause the 7th world wide pandemics raged firstly in our country yangjiang, Guangdong. Yangjiang became the place of 01 El Tor strain cholera originated. In the later time preventing 01 El Tor strain cholera spreading has been another goal.79386 people were infected and 1942 deaths by the cholera from 1961 to 2013. As a consequence, exploring the resistance of V. cholerae in Guangdong province is representative for understanding the resistance of V. cholerae in our country.In this study,360 clinical V. cholerae O1 ElTor isolates and 125 clinical V. cholerae 0139 collected over 50 years (1961 to 2013) were detected the drug resistance by micro broth dilution method. In order to understand the drug resistance of V. cholerae. Compare the characters of the resistance strain in different times to explore the changes of drug resistance and multidrug resistance of V. cholerae.We also conduct the resistance gene classification and pulsed field gel electrophoresis (PFGE) typing in multidrug resistant strains. Explore the mechanism of its resistance and the genetic relationship between the same pathogen strains and provide a scientific basis for the rational use of drugs and prevention and treatment of cholera in Guangdong province.Objective1. Compare the characters of the resistance strain in different times to explore the changes of drug resistance and multidrug resistance of V. cholerae and provide a scientific basis for the clinical treatment of cholera in Guangdong province.2. Resistance gene classification to study the resistance gene distribution, Explore the mechanism of its resistance3. Explore the relationship between resistance and genetic and provide basic data for future research of the cholera epidemic.Methods1. Strains:The strains were from Guangdong Provincial Disease Prevention and control center for microbial testing strains library. They were collected from the city across Guangdong province fromJanuary 1961 to December 2013.2. Method:A stratified random sampling method was used to extract the experimental strains; micro broth dilution method was used to detect the drug resistance; multiplex PCR method was used to detect the resistance genes; PFGE typing technique was used to study the genetic correlation between the strains.Contents1. We check out the historic surveillance data and reports to describe the overview of the cholera epidemic in Guangdong Province since 1961. Then we observed four distinct epidemic periods based on the different dominant serotype leading its epidemic,1961-1969,1978-1989,1990-2000,2001-2013, respectively. A stratified random sampling method according to the epidemic period was used to extract the experimental strains, a total of 485 strains were selected. Among them, 360 V. cholerae 01 strains and 125 V. cholerae 0139 strains. Recover, Identify and serotyping with all strains.2. Antibiotic susceptibility analysis of all tested strains was performed by the Minimum inhibitory concentration (MIC).To analysis antibiotic resistance profiles and multidrug resistance of V. cholerae, to explore the changes of drug resistance and multidrug resistance of V. cholerae.3. According to antibiotic resistance profiles, we chosed the MDR strains that resistant to three or more of the tested antibiotics simultaneously to detect the resistance gene and pulsed field gel electrophoresis (PFGE) typing. To explore the resistance gene distribution and the genetic relationships between strains.Results1. All the 360 V. cholerae O1 strains were susceptible to ceftriaxone and cefepime. Another ten antibiotics such as the imipenem, ciprofloxacin, doxycycline, chloramphenicol, azithromycin, gentamicin, nalidixic acid, ampicillin, amoxicillin and tetracycline showed a low level resistance to the V. cholerae 01 strains,which was lower than 10%. The resistant rate of cefoxitin, trimethoprim and sulfamethoxazole were 13.3%,14.7% and 24.4% respectively. However, a high rate of resistance to streptomycin was detected among V. cholerae 01 strains.85.3% of strains were resistant to streptomycin. Trend of antibiotic resistance among different periods of V.cholerae 01 strains showed that although all the tested 01 strains were susceptible to nalidixic acid, tetracycline, trimethoprim and sulfamethoxazole during 1961 to 1990, the resistant rate of these antibiotics increased rapidly after that. Especially the sulfamethoxazole, the resistant rate was up to 74.6% during 1991 to 1999. It’s worth noting that the resistant rate of streptomycin was as high as 78.1% during 1961 to 1964, increasing to 94.6% in recent years.2. All the 125 V. cholerae 0139 strains were sensitive to ceftriaxone, cefepime, imipenem and ciprofloxacin, and lower than 20% of strains were resistant to cefoxitin, azithromycin, doxycycline, amoxicillin and gentamicin. The rates of resistance to ampicillin, tetracycline and nalidixic acid were 34.4%,35.2% and 37.6% respectively. However, streptomycin had highly active against V. cholerae O139 strains with the resistant rate of 89.6%, followed by sulfamethoxazole (75.2%) and trimethoprim (67.2%).3. There were 25.9%(86/360) of 01 strains and 76.8 %(96/125) of O139 strains were resistant to three or more of the tested antibiotics. The multiple-drug resistance (MDR) rate of 0139 strains was higher than O1 strains (p<0.05). There were 36 MDR patterns among the 86 V. cholerae 01 strains. Furthermore,2 strains were resistant to nine antibiotics exhibiting the FOX+GEN+AMP+SXT+ SMX+ NAL+ STR+AZI+AMX pattern. Similarly, there were 36 MDR patterns among the 96 V. cholerae 0139 strains. Of these,6 strains were resistant to to ten antibiotics. Interesting, the 6 strains performed the same MDR pattern of TET+GEN+AMP+CHL+SXT+SMX+NAL+STR+AZI+AMX. The MDR rates in different epidemic period exhibited a statistically significant difference both of V. cholerae 01 and 0139 strains (p<0.05). The MDR rate showed an increasing trend with the passage of time.4. Detection of antibiotic resistance genes showed among the 86 O1 MDR strains, the positive rates of class I and class IV integrons and SXT were 33.7%,81.4% and 57.0%. Among the 960139 MDR strains, the positive rates of class 1 and class IV integrons and SXT were 22.9%,71.9% and 60.4%. However, no class Ⅱ and class III integrons was detected in any of these strains. Antibiotic resistance gene of dfrAl only existed specifically in the 01 strains, while dfrl8 only existed specifically in O139 strains. Another four resistance genes, including strA and strB (encoding streptomycin resistance), sulll (encoding sulfamethoxazole resistance) and floR (encoding chloramphenicol resistance) were widely distributed in 01 and 0139 multidrug resistant strains.5. Pulsed-field gel electrophoresis (PFGE) was used for subtyping of strains to investigate the clonality or genetic variation of V. cholerae 01 and 0139 strains.86 01 MDR strains were differentiated into 65 PFGE patterns.Un-weighted pair group method with arithmetic mean algorithm(UPGMA) analysis clusterer these PFGE patterns into six clusters.96 O139 MDR strains were differentiated into 74 PFGE patterns. UPGMA analysis clusterer these PFGE patterns into six clusters. There was no significant correlation between PFGE patterns and drug resistance patterns. For example, PFGE patterns of the strains which were highly resistant and multiply drug resistant had different genotypes. Only three clusters performed the same PFGE patterns with drug resistance patterns. It indicated the great genetic heterogeneity of V. cholerae 01 and 0139 strains and MDR strains were widely existed in Guangdong province.Conclusions1. V. cholerae 01 and 0139 showed high sensitivity to ceftriaxone, cefotaxime, imipenem and ciprofloxacin, can be used as the first-line antibiotics. In addition, the resistant to streptomycin, trimethoprime and sulfamethoxazole were common and serious.2. The multiple-drug resistance of V. cholerae 01 and 0139 in Guangdong province was common. The multiple-drug resistance rate was only 8.8% before 1990, after than, it was as high as 58.6%. The drug resistance patterns also changes and increases with the passage of time. Continuous monitoeing of the changing trends in antibiotic resistantce patterns of V. cholerae 01 and O139 strains may provide the basis of strategies for the control of new cholera pathogens.3. Class I and class IV integrons, SXT and another four resistance genes were widely distributed in 01 and O139 multidrug resistant strains, suggesting multiple-drug resistance of strains may related to resistant genes. Therefore, we should pay attention to strengthen the detection and analysis of drug resistance genes, in order to discover the variation and new resistance genes in time.4. PFGE patterns of the strains which were highly resistant and multiply drug resistant had different genotypes. It indicated the great genetic heterogeneity of V. cholerae O1 and O139 strains and MDR strains were widely existed in Guangdong province. |
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