| PART ONE Study on Epidemiologic of Escherichia Coli Isolates Producing Extended-Spectrum β-Lactamases (ESBLs) in ShandongBackgroudβ-lactamases are not only extensively used for the treatment of many kinds of infections, for example, pneumonia, urinary tract infections (UTI), and bloodstream infections (BSI), but also are frequently used as prophylaxis treatment beforesurgery. However, with the clinical use of extended-spectrum cephalosporins, drug resistance strains appeared which has become a stumbling blockon clinical treatment. In 1983, Extended-spectrum β-lactamases (ESBLs) were firstly isolated in Europe. Then, reports on ESBLs spread all over the world. There are several definitions for ESBLs, referring mainly enzymes produced by Gram-negative bacteria, including Escherichia Coli, klebsiellapneumonia, pseudomonas aeruginosa, acinetobacter and proteus mirabilis, and could degrade β-lactamases antibiotics with extended spectrum (such as penicillins, cephalosporin and aztreonam), being unable to hydrolyze cephamycin and carbapenems, while activities can be inhibited by β-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam. Due to their wider substrates for hydrolysis than broad-spectrum-lactamase (TEM-1, TEM-2 and SHV-1), the name of extended-specturmp-lactamases appears, which includes several genes, for example, TEM, SHV, CTX-M and OXA. Many hundreds of ESBLs have been found around the world. Although epidemic genotypes are different across different regions; those genotypes are generally evolved through TEM-1 or SHV-1 point mutations which can alter the amino acid sequence. By contrast, CTX-M is quite different with TEM and SHV, with homo logy being only 39%.ESBLs are mostly generated by enterobacteriaceae, especially E. Coli and K.pneumoniae. Although being common colonizers of the gastrointestinal tract, enterobacteriaceae could cause great infections in the central nervous system, lower respiratory tract, bloodstream, gastrointestinal and urinary tract.Due to environmental deviations across regions and different habits of prescribing, epidemic genotypes and characteristics of drug resistance vary with regions. Therefore, we firstly explore the drug resistance and genotypes of ESBLs-producing E. Coli isolates in Jinan, Shandong province. We reveal epidemiological characteristics and rules of ESBLs in the local area and provide guides for clinical therapy.We have collected 578 singlepatient E. Coli isolates based on samples of China from July 2010 to August 2014. We investigated the prevalence, resistance, and probable gene type of extended spectrum beta-lactamases (ESBLs) using minimum inhibitory concentrations (MICs) testing and polymerase chain reaction (PCR).Objective1. To investigate the epidemiological data of ESBLs-producing E. Coli in Jinan, Shandong.2. To explore the drug-resistance of ESBLs-producing E. Coli.3. To study genotypes of ESBLs-producing E. Coli of Jinan, Shandong province.Materials and Methods1.578 clinical isolates of E. Coli were collected from three hospitals in Jinan. The isolates were re-identified using Micrscan VITEK-2 system.2. The isolates of antimicrobial resistance were detected by Kirby-Bauer test.3. ESBLs producers were screened by initial screening test and phenotypic disk confirmation test according to Clinical and Laboratory Standard Institute (2013).4. Several primers were synthetic including TEM、SHV and CTX-M. And then, genotypes of ESBLs-producing were initially determined by the PCR.5. Taking sequence analysis ofthe amplification products.ResultsIn our samples, these strains were mainly isolated from patients in urinary surgery ward 220(38.06%) and ICU 128(22.15%). The main sources of ESBLs-producing E-coil were unire 252,wounds 84, sputa 76, genitalsecretionn56, hydrothorax and ascite52, blood40, bile18. A total of 360 ESBLs-producing isolates were detected phenotypically using the CLSI criteria for ESBLs screening and disk confirmation test, the detection rate being 62.28%(360/578). We found that much more ESBLs-producing isolates exited in urinary surgery ward and ICU and the detection rate were higher than the other wards.The ESBLs-producing isolates were often multidrug-resistant ESBLs producers have shown much higher rates of resistance than those non-ESBLs to ciprofloxacin (ESBLs vs non-ESBLs,79.4% vs 49.5%, P<0.05), gentamicin (73.6% vs 39.9%, P<0.05), co-trimoxazole (81.7% vs 54.6%, P<0.05), aztre-onam (81.4% vs 15.1%, P<0.05), and levofloxacin (91.9% vs 59.2%, P<0.05). The great majority of isolates were susceptible to Minocycline (ESBLs vs non-ESBLs,89.4% vs 96.3%, P<0.05). All isolates were susceptible to amikacin and imipenem (χ2 test for all groups).All 360 isolates with a ESBLs phenotype tested positive for blaCTX-M and blaTEM using the consensus primers.We found 303 isolates were positive for blaCTX-M, while 254 isolates were positive for blaTEM and 159 isolates were positive for blaSHV. TEM type gene sequencing analysis found that TEM-1 beta lactamase genotype was the main genotype, and we found only 12 TEM-135 spectrum beta lactamase genotype. For SHV genotype, the sequencing analysis showed that SHV-12 and SHV-2a spectrum beta lactamase genotype were the main genotype; for CTX-M genotype, the sequencing analysis showed that CTX-M-14 and CTX-15 spectrum beta lactamase genotypes were the main genotype; at the same time, we found CTX-M-3, CTX-M-98, CTX-M-142, CTX-M-65, CTX-M-55, CTX-M-27, and CTX-M-123 spectrum beta lactamase genotype in our sample. The last but the most important finding was that there were eight new TEM genotype strains for the first time, and then we need to take further tests and analyze the gene molecular biology characteristics.Conclusions1. The detectable rate of ESBLs was 62.28% in E. Coli isolates; we should pay more attention to it and take actions to control the prevalence of ESBLs.2. Most ESBLs-producing E. Coli isolates were multi-drug resistant, and the resistance rate was high to antibiotics which were often chosen in clinical. Therefore, we should select the appropriate antibiotics carefully.3. We had not yet found E. Coli isolates resistance to cabapenems. Therefore, our study suggested that carbapenems are the first drug of choice for serious infections due to ESBLs-producing bacteria.4. Among phenotypes of ESBLs-producing E-coil, genotype mostly was CTX-M, then TEM. Most of the strainscarry at least one genotype.5. The DNA sequeening result was that TEM-1, CTX-M-14 and SHV-12 spectrum beta lactamase genotype were the main genotypes in Shandong province.PART TWO The risk factors for infection by Escherichia Coli Isolates Producing Extended-Spectrum β-Lactamases (ESBLs)BackgroundESBLs-producing E. Coli isolates is a common bacteria existing in the communities and hospitals. ESBLs-producing E. Coli increased all over the world in recent years, not only increasing the economic burden of patients, but also affecting recovery of diseases. It suggested that the drug resistance gene was always mediated by resistance plasmid, which not only passed down drug resistance gene to their offspring, but also leaded to the horizontal transmission of resistance genes.The detection rate has been rapidly increased in different regions. For example, In Europe, the detection rates of ESBLs among E. Coli and K.pneumoniae were 17.6% and 38.9%, respectively; by contrast, the detection rate was 8.5% and 8.8% in America,5 and 0% in New Zealand,67% and 61% in China. The use of antibiotics has become more frequently in China, which was resulted from the loose management and prescribing habits for diseases. Researches on epidemic situation and related risk factors of ESBLs and multidrug resistance isolates contributed to improving awareness of disease prevention and providing instructions for treatment.Previous studies have found that usage of antibiotics before admission had been proved to be an independent risk factor for epidemic and infectionin hospitals and communities, especially usage of third-generation cephalosporins. Besides, risk factors of nosocomial infection included such as virulence operation, undergoing surgery, ICU, international travel and so on.Because of plasmids always carrying other antimicrobial resistant genes, most ESBLs-producing organisms might be resistant to fluororquinolones, aminogylcosides and macrolide, etc, which resulted in treatment failure. These resistance and long processing time needed rational drug usebased on doctors’ experience, so it is great important for acquiring local epidemic data.In this context, we explored the epidemiological characteristics in Shandong. It is imperative that risk factors of infections with ESBLs-producing organisms should be clearly identified so that effective strategies can be taken in advance to curtail the emergence and spread of these strains.ObjectiveBased on retrospective studies of patients affected by ESBLs-producing E. Coli in Qilu Hospital from December 2005 to December 2015, we will explore the incidence rates of community infection and hospital infection by ESBLs-producing E. Coli, and then evaluate related risk factors, in order to provide referrences for prevention and treatment. According to experimental results, we can evaluate effects of certain drugs so as to provide instructions for clinical treatment.Materials and Methods318 patients with ESBLs producing E. Coli isolates were identified over a period often years (from December 2005 to December 2015) in Qilu Hospital. We performed a retrospective, case-controlled study to evaluate risk factors for isolates of ESBLs-producing E. Coli and non-ESBLs-producing E. Coli isolates. We use the SPSS 22.0 software to analyze our sample. Use the chi-square statistical method and Logistic Regression analysis. In order to take statistical significance, we set P<0.05.Results1. Demographic characteristicIn our sample,214 patients (67.3%) were community infection, and the others were hospital infection. ESBLs-producing strains were identified in 202 patients (63.5%), non-ESBLs-producing strains 116(34.5%). The rates of ESBLs-producing E. Coli infections in communities and hospitals were 128(59.8%) and 74(71.2%), respectively.1.1 Age and gender distributionForty-four percent of the patients were male, and 56% were female. The mean age was 59.78±17.82 years. The average age of the men was 58.57±18.07 and the female was 60.72±17.04.1.2 Distribution departmentESBLs-producing E. Coli were more common in the urological department (58cases; 18.24%), endocrinology department 48(15.09%), ICU40 (12.59%). A high detection rate of ESBLs was observed in each ward, the average was 63.5%(202 /318). The rate of ESBLs was significantly higher in ICU82.5% (33/40) than other wards.2. Risk factors for ESBLs-producing E. Coli2.1 Community acquired infection (CAI)According to univariate analysis, previous treatment with antibiotics and hospitalization was more likely to be associated with isolation of ESBLs-producing organisms. Multivariate logistic regression analysis identified previous treatment with antibiotics (OR=5.957,95%CI:2.187-16.227, P=0.001) and hospitalization (OR=5.272,95%CI:2.072-13.415, P=0.001) as the independent risk factor for bacteremia due to ESBLs-producing pathogens.2.2 Nosocomial infectionAccording to univariate analysis, stay in the intensive care unit (P=0.021, OR5.185), use drainage tube (P=0.035, OR2.605) and previous treatment with cephalosporins (P=0.024, OR2.949) was more likely to be associated with isolation of ESBLs-producing organisms. Multivariate logistic regression analysis identified previous treatment with cephahosporins (P=0.036, OR2.944) as the independent risk factor for bacteremia due to ESBLs-producing pathogens.3. Treatment and outcome3.1 TreatmentBefore obtaining bacterial culture results, the empiric therapy of antibiotic treatment 274(86.16%), after analyzing antimicrobial susceptibility results,179 (63.93%) were sensitive antibiotics,95(36.07%) were drug resistance. According to antimicrobial susceptibility results,86 were adjusted,9 were not adjusted. The rest of 44 cases were not in the empiric therapy,38 based on antimicrobial susceptibility results,6 unused.3.2 Outcome3.2.1 In our study, empirical antibiotic therapy to patients with ESBLs-EC bacteraemia successed in 94 patients, and patients with non-ESBLs-EC bacteraemia was 85 (94/174 vs 85/100, P=0.000). However, we have found a similar mortality rate in patients with infection due to ESBLs producing E. Coli compared to non-ESBLs producing E. Coli (45/202 vs 25/116, P=0.881).3.2.2 Hospital stays of all cases were 18.54±11.00 days, ESBLs-producing strains 19.86±12.41 days, non-ESBLs-producing strains 16.25±7.45, P=0.003 of two groups in T-test. Outcome:70 occurred treatment failure, including carbapenems 3(3/48), β-lactamase inhibitor combinations 8(8/79), third generation cephalosporin 21(21/50), fluoroquinolones 11(11/72), initial untreatment 21(21/44). There was significant difference of rates of death between third generation cephalosporin and other antibiotics (21/50 vs 28/224, P=0.000). Patients treated with imipenem were more likely to survive while those receiving other antibiotics treatment tended to have a poorer outcome (3/48 vs 45/226, P=0.024)。ConclusionsBased on these results, we found that usage of antibiotics was the mainly independent risk factor of the emergence and infection of ESBLs-producing strains, especially cephalosporin, and hospitalization was another independent risk factor. As a consequence, we will not only exercise strict control over usage of antibiotics, but also take necessary actions prevent spread of ESBLs, such as strength environmental management, contact isolation, instrument disinfection, hand hygiene, and so on. |
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