Characterization of plasmid-mediated AmpC-β-lactamases in Klebsiella pneumoniae

• Main Authors: Meng-Ting Wu, 吳孟庭
• Other Authors: Chung-Yu Chang
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/71847885140760554918

碩士 === 高雄醫學大學 === 醫學研究所 === 96 === Klebsiella pneumoniae is one of the common pathogens causing community-acquired and nosocomial infections. It usually causes a wide spectrum of infections, including septicemia, pneumonia, urinary tract infection, meningitis and systemic infection. The high mortality still happened even if treatment of antibiotic. In the past 15 years, a new type of invasive K. pneumoniae disease has emerged in Taiwan that typically present as community-acquired primary liver abscess with metastatic meningitis and endophthalmitis. The high mortality rate and high incidence of K. pneumoniae pyogenic liver abscess in Taiwan make further investigation necessary. Improper use of various antibiotics for the treatment of diseases resulted in an increase of K. pneumoniae resistant to multiple drug. AmpC β-lactamases are cephalosporinases that confer resistance to a wide variety of β-lactam drugs and that may thereby create serious therapeutic problems. Currently, there are no recommendations available from the Clinical and Laboratory Standards Institute (CLSI) for detection of organisms producing AmpC β-lactamases（AmpCs）and often leaving clinicians with limited therapeutic options. Apart from R-plasmids and transposons, integron are known to be another mechanism for acquisition and dissemination of resistance genes among gram-negative bacteria. Therefore, this study attempts to detect AmpCs in K. pneumoniae. Class 1 integrons and plasmids were also characterized to further ascertain their roles in dissemination of AmpCs. A total of 620 cinical isolates of K. pneumoniae were collected from KMUH in 1993 and 2004. 47 K. pneumoniae isolates showing non-susceptible to cefoxitin (inhibition zone less than 18 mm) were tested further to confirm the presence of AmpC β-lactamases. AmpCs were confirmed in 17, 21 and 11 of the isolates by modified three-dimensional (M3D), boronic acid based disk test and PCR, respectively. Strains of positive for any one of the three tests included 25 K. pneumoniae isolates. PCR analysis revealed that blaACT-like、blaDHA-like and blaCMY-2-like were found in 1, 9 and 1 isolates, respectively. Among 25 AmpCs-producing K. pneumoniae isolates, antimicrobial susceptibility tests showed that susceptibility rate to cefepime and meropenem is 88 and 100%, respectively. 12-68% of the isolates were resistance to β-lactam/β-lactamase inhibitor combination, 3rd-cephalosporins, aminoglycosides, aztreonam, and ciprofloxacin. The percentage of class 1 integrons in these 25 isolates is 88%（22 islaotes）. The gene cassettes carried by integrons included those encoding resistance to trimethoprim (dfr5, 12 and 17), streptomycin (aadA1, 2, 5), erythromycin (ereA2), aminoglycoside-6’-N-acetyltransferase (aac(6’)-Il, aac(6’)IIc), rifampin (arr). Most of the class 1 integrons carried dfr12, orfF and aadA2 cassettes. Results from Southern hybridization pointed out there existed plasmids of different sizes. ampC genes such as blaDHA-like, blaACT-like and blaCMY-2-like were located on plasmids approximately of 130, 150 and 100 kb, respectively. Class 1 integrons were located on the plasmids ranging from 30 to 165 kb. In addition, conjugation experiments suggested that blaACT-like was located on non-transferable plasmid. On the other hand, an isolate producing CMY-2-like AmpCs possessed a plasmid of 100 kb involving in the transfer of the blaCMY-2 like gene, indicating the plasmid-mediated horizontal transfer of blaCMY-2 like gene. However, the transfer of ampC genes mediated by plasmids did not accompany class 1 integron except two isolates carrying unknown ampC genes. This study demonstrated that the boronic acid test is more sensitive to detect AmpCs in K. pneumoniae. AmpCs-producing K. pneumoniae isolates are often multidrug-resistant. Plasmid-mediated transfer of ampC genes can lead to the dissemination of antibiotic resistance to diverse bacterial populations. Thus, the ampC may play an important role in multi-resistance of K. pneumoniae.