| Objectives: The main aim of this study was to detect the resistance of 80 strains of clinically isolated Klebsiella pneumoniae to 18 commonly used antibiotics, and analyze the clinical characteristics of these 80 pateints with Klebsiella pneumoniae infection in our hospital. Double-disk synergy method was used to detect the extended Spectrum Beta-lactamase(ESBLs) of Klebsiella pneumoniae. Polymerase Chain Reaction(PCR) method was used to amplify ESBLs associated drug resistance genes in ESBLs positive Klebsiella pneumoniae. The clinical data of 80 patients with Klebsiella pneumoniae infection were carried on statistical and epidemiological analysis to investigate the association between the drug resistance and ESBLs-producing genes and explore the characteristics of distribution and drug resistance of Klebsiella pneumoniae in our hospital, so as to provide reference for clinical medication.Methods: Eighty Klebsiella pneumoniae isolates were collected from the clinical laboratory of Affiliated Hospital of North Sichuan Medical College between Sep 2014 and Sep 2015. All samples came from different patients and different parts of patients. The minimum inhibitory concentration(MIC) values of 18 antibiotic agents against eighty strains of Klebsiella pneumoniae were determined by two-fold agar dilution method. Double disc synergy method was used to screen ESBLs positive Klebsiella pneumoniae. The total DNA of the above ESBLs positive strains was extracted. PCR method was used to amplified ESBLs associated genes including CTX-M-1, 2, 7, 9, 15 and 16, as well as TEM and SHV. All the clinical data from patients with Klebsiella pneumoniae infection, such as gender, age, the source isolates, the characteristics of clinical medication, and other related information, were collected to analyze the relationship between the characteristics of drug resistance and ESBLs related genes.Results: The average age was 50.13(years). The female/male ratio in these 80 patients was 39 to 41. All Klebsiella pneumoniae strains were collected from the patients with pulmonary infection accounting for 50%(40/80), septicemia(12.5%), urinary tract infection(10%), peritoneal cavity infection(10%), perianal abscess(10%), infection of biliary tract(6.25%) and encephalitis(2.5%), and from different clinical departments including general surgery and pediatric surgery accounting for 22.5%(18/80), department of respiratory and gastroenterology accounting for 20%(16/80), department of anorectal surgery and thoracic surgery accounting for 12.5%(10/80), department of pediatrics and neonatal department accounting for 10%(8/80), urinary surgery and neurosurgery accounting for 7.5%(6/80) and with diffused distribution in other clinical departments. Patients with underlying diseases accounted for 16.25%(13/80). The proportion of single antibiotic usage was 28.75%(23/80), while the proportion of two antibiotics combined and three combined antibiotics usage was 71.25%(57/80). The average hospital stay was 20.3 days, and 78.75%(63/80) patients were cured in the hospital. The highest resistant rate of Klebsiella pneumoniae was ampicillin(85.0%). The second was first-generation cephalosporin(42.5%), followed by second-generation cephalosporin(35%), ceftazidime(26.25%), ceftriaxone(25%), cefoperazone/tazobactam(15%) and cefoperazone/sulbacta(13.75%). The resistant rate of monocyclic β-lactam antibiotics was 3.25%. The resistant rates of fluoroquinolone antibiotics such as ciprofloxacin and levofloxacin were 21.25% and 20%, respectively, aminoglycoside antibiotics such as amikacin(22.5%) and gentamicin(37.5%), macrolides antibiotics such as azithromycin(31%). The resistant rate of sulfonamide antibiotics such as compound sulfamethoxazole was 21.25%. Notably, the resistant rate of carbapenem antibiotics such as biapenem was 3.75%. Twenty strains among the 80 isolated strains of Klebsiella pneumoniae tested positive for ESBLs production(25%); 13(65%) of these produced SHV ESBLs, 12(60%) produced CTX-M-9 ESBLs. The detection ratios of CTX-M-1, TEM, CTX-M-2, CTX-M-16 and CTX-M-7 in these 20 strains were 35%, 25%, 20%, 20% and 20%, respectively. The lowest detected gene was CTX-M-15(10%).The resistance rates of 20 strains of ESBLs positive Klebsiella pneumoniae to 18 commonly used antibiotics were 90% for Cefazolin, 70% for Ampicillin, 65% for Cefuroxime, 55% for Compound Sulfamethoxazole Tablets, 50% for Ceftazidime, 45% for Gentamicin, 45% for Azithromycin, 45% for Aztreonam, 40% for Ceftriaxone, 30% for Ciprofloxacin, 30% for Levofloxacin, 15% for Piperacillin and Sulbactam, 15% for Mezlocillin and Sulbactam, 15% for Amikacin, 5% for Cefoperazone Sodium and Tazobactam Sodium, 5% for Cefoperazone and Sulbactam, 5% for Imipenem, 0% for Biapenem. Multiple-drug resistant Klebsiella pneumoniae accounted for 90%(18/20) of all 20 strains of ESBLs positive Klebsiella pneumoniae. One strain of Klebsiella pneumoniae which was resistant to a class of antibiotics accounted for 5%. Another strain of Klebsiella pneumoniae which was resistant to two classes of antibiotics accounted for 5%. The resistance rates of 18 strains of ESBLs positive Klebsiella pneumoniae to 18 commonly used antibiotics were 94.4% for Cefazolin, 72.22% for Ampicillin, 66.67% for Cefuroxime, 61.11% for Compound Sulfamethoxazole Tablets, 55.56% for Ceftazidime, 50% for Gentamicin, 50% for Aztreonam, 44.44% for Ceftriaxone, 44.44% for Azithromycin, 33.33% for Ciprofloxacin, 33.33% for Levofloxacin, 16.67% for Piperacillin and Sulbactam, 16.67% for Mezlocillin and Sulbactam, 16.67% for Amikacin, 5.56% for Cefoperazone Sodium and Tazobactam Sodium, 5.56% for Cefoperazone and Sulbactam, 5.56% for Imipenem, 0% for Biapenem.The subtypes of 20 ESBLs producing Klebsiella pneumoniae were SHV-ESBLs accounting for 66.67%(12/18), CTX-M-9 accounting for 50%(9/18), CTX-M-1 accounting for 33.33%(6/18), TEM accounting for 27.78%(5/18), CTX-M-2 accounting for 22.22%(4/18), CTX-M-7 accounting for 11.11%(2/18), CTX-M-15 accounting for 11.11%(2/18), and CTX-M-16 accounting for 11.11%(2/18). The resistance rates of 13 strains of SHV-ESBLs positive Klebsiella pneumoniae to 18 commonly used antibiotics were 61.54% for Ampicillin, 15.38% for Piperacillin and Sulbactam, 23.08% for Mezlocillin and Sulbactam, 100% for Cefazolin, 61.54% for Cefuroxime, 53.85% for Ceftazidime, 53.85% for Ceftriaxone, 7.69% for Cefoperazone Sodium and Tazobactam Sodium, 7.69% for Cefoperazone and Sulbactam, 7.69% for Imipenem, 7.69% for Biapenem, 23.08% for Ciprofloxacin, 15.38% for Levofloxacin, 53.85% for Gentamicin, 0.00% for Amikacin, 38.46% for Azithromycin, 61.54% for Compound Sulfamethoxazole Tablets, and 46.15% for Aztreonam.The resistance rates of 12 strains of CTX-M-9-ESBLs positive Klebsiella pneumoniae to 18 commonly used antibiotics were 66.67% for Ampicillin, 8.33% for Piperacillin and Sulbactam, 8.33% for Mezlocillin and Sulbactam, 91.67% for Cefazolin, 75.00% for Cefuroxime, 41.67% for Ceftazidime, 41.67% for Ceftriaxone, 0.00% for Cefoperazone Sodium and Tazobactam Sodium, 0.00% for Cefoperazone and Sulbactam, 0.00% for Imipenem, 0.00% for Biapenem, 16.67% for Ciprofloxacin, 25.00% for Levofloxacin, 41.67% for Gentamicin, 8.33% for Amikacin, 50.00% for Azithromycin, 58.33% for Compound Sulfamethoxazole Tablets, and 33.33% for Aztreonam. The remaining subtypes of ESBLs positive Klebsiella pneumoniae such as CTX-M-1, CTX-M-2, CTX-M-7, CTX-M-15, CTX-M-16, and TEM also showed different resistance rates to 18 commonly used antibiotics.Conclusions:1. Eighty strains of Klebsiella pneumoniae were mainly collected from the Department of general surgery, the Department pediatric surgery, the Department of anorectal surgery, and the Department of thoracic surgery.The disease of patients is more common in lung infectioin, followed by septicemia, urinary tract infection, peritoneal cavity infection, perianal abscess, infection of biliary tract, and encephalitis. It can be seen from the results that the resistance rates of Klebsiella pneumoniae to penicillin, first-generation cephalosporin, and second-generation cephalosporin were higher than other antibiotics in Nanchong city. At present, carbapenems were the most effective antibiotics treating Klebsiella pneumoniae infection, but strains characterized with drug-resistance to carbapenems antibiotics have emerged, for example, in this study the resistance rate of these 80 strains of Klebsiella pneumoniae to Biapenem was 3.75%. The resistance rate to monobactams such as Aztreonam was 3.25%. Therefore, we should choose the sensitive antibiotics according to the results of drug sensitivity tests as soon as possible, so as to prevent the emergence of multiple-drug resistant Klebsiella pneumoniae.2. In the 80 strains of Klebsiella pneumoniae clinically isolated from our hospital, ESBLs positive strains accounted for 25%(20/80). For 20 strains of ESBLs producing Klebsiella pneumoniae, the resistance rate ofCefazolin is 90%, it was the highest among the 18 antibiotics. Ampicillin was the second highest(resistance rate: 70%). Among 18 antibiotics tested, third generation cephalosporins contanining beta lactamase inhibitor and carbapenems had relatively lower resistance rates.There were two main subtypes of ESBLs positive Klebsiella pneumoniae: SHV-ESBLs and CTX-M-9-ESBLs. However, the subtype of TEM-ESBLs positive Klebsiella pneumoniae accounted for 25%(5/20), and the resistance rate of such a type to penicillin with beta lactamase inhibitor had reached 25%, which may be attributed to the variation of Klebsiella pneumoniae genome under long-term stress of antibiotics.3. Multiple-drug resistant Klebsiella pneumoniae accounted for 90%(18/20) among 20 ESBLs positive Klebsiella pneumoniae, which suggests that the acquisition of multiple-drug resistance is closely associated with the emergence of ESBLs. For these 18 Klebsiella pneumoniae, the antibiotics with highest resistance rate were Cefazolin, Ampicillin, and Cefuroxime; with relatively lower resistance rate were Cefoperazone Sodium and Tazobactam Sodium, Cefoperazone and Sulbactam; with 100% sensitivity was Biapenem. Notably, the resistance rate to Imipenem was 5.56%. The constituent ratios of SHV, CTX-M-9, CTX-M-1 and TEM were 66.67%, 50.00%, 33.33% and 27.78%, repectively. The emergence of new ESBLs producing strains may be the leading cause of diversity of drug resistance in Klebsiella pneumoniae infection.4. The resistance rates of all subtypes of ESBLs producing Klebsiella pneumoniae to 18 antibiotics differ with each other. For SHV-ESBLs, CTX-M-9 ESBLs and CTX-M-1 ESBLs, the antibiotics with high resistance rates were Ampicillin, Cefazolin, Cefuroxime, Ceftazidime, Ceftriaxone and Compound Sulfamethoxazole Tablets; with low resistance rates were Piperacillin and Sulbactam, Mezlocillin and Sulbactam, Cefoperazone Sodium and Tazobactam Sodium, Cefoperazone and Sulbactam, as well as Imipenem. However, the resistance rate of SHV-ESBLs to Imipenem was up to 7.69%. The resistance rates of CTX-M-2-ESBLs, CTX-M-7-ESBLs to Ceftazidime, Levofloxacin, Gentamicin and Aztreonam were relatively low. The resistance rate of CTX-M-16-ESBLs to Gentamicin and Aztreonam was up to 50%. The resistance rate of CTX-M-15-ESBLs to Gentamicin and Aztreonam was up to 100%. Additionally, the resistance rate of TEM-ESBLs to antibiotics containing beta lactamase inhibitor such as Piperacillin and Sulbactam, Piperacillin and Sulbactam, as well as aminoglycosides such as Amikacin was up to 20%. We can flexibly select sensitive antibiotics according to the characteristics of different subtypes of ESBLs producing Klebsiella pneumoniae in the process of treating Klebsiella pneumoniae infection, and then adjust the medication plan according to the variation of ESBLs, the results of drug sensitivity tests and the treating effects.5. In order to prevent the outbreak of the resistant Klebsiella pneumoniae and the emergence of multiple-drug resistant strains, we should investigate the clinical risk factors of ESBLs positive Klebsiella pneumoniae and take effective control measures. Meanwhile, we shold analyze the genotype of Klebsiella pneumoniae by the method of molecular biology, and select effective antibiotics according to the characteristics of resistance and ESBLs. It is of great significance to explore the mechanisms by which the Klebsiella pneumoniae aquire resistance and the transmission of resistance genes, so as to reduce or prevent the emergence and dissemination of new resistant strains or multiple-drug resistant Klebsiella pneumoniae. |
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