Detection of Extended-Spectrum Beta Lactamases and AmpC Beta Lactamases Producing Uropathogenic Escherichia coli in a Tertiary Care Hospital

Background: The multidrug resistant among uropathogenic E. coli has become a potential threat to global health. The aim of the current study to evaluate the antimicrobial activities of nitrofurantoin and fosfomycin along with other antimicrobials against Extended Spectrum β-Lactamases (ESBL) and AmpC producer isolates from the most common organism E. coli. Methods: A total of 6046 clean catch midstream urine samples were collected and processed in Microbiology department of tertiary care hospital. The antimicrobial susceptibility of E. coli isolates was initially screened by Kirby-Bauer disk diffusion method. The resistant isolates were confirmed to be ESBL and AmpC producers by their respective phenotypic confirmatory tests of combined disc method. Results: Out of 6046 patients there were 1855 E. coli positive patients. Maximum patients in the age group of 21-30 years were 51.5% followed by 31-40 years where patients were 26%. 64.4% E. coli were isolated from female patients and 35.6% from male patients. E. coli showed higher sensitivity towards, fosfomycin (100%), imipenem (100%), nitrofurantoin (84.1%), piperacillin and tazobactam (77.3%), amikacin (76.1%) and while they showed high degree resistance pattern against Penicillin, cotrimoxazole, ciprofloxacin, norfloxacin and 2nd and 3rd generation cephalosporin. Out of 1855 E. coli, multi drug resistance was seen in 520 E. coli isolates. ESBL production was observed among 50% of E. coli isolates by combined disk method. Out of 520 isolates, 150 isolates showed resistance to one or more extended-spectrum cephalosporins and cefoxitin by Kirby-Bauer disk diffusion method. These were selected and screened for ESBL and AmpC production. Among 150 cefoxitin-resistant isolates, AmpC phenotype was detected in 100 isolates (66.6%) by AmpC disc method. The overall occurrence of AmpC in the study was found to be 19.2%. Susceptibility of ESBL and AmpC producers to fosfomycin, imipenem, nitrofurantoin and amikacin were found to be 100%, 98.5%, 89% and 75% respectively. Conclusions: There is increased prevalence of ESBL and AmpC producing E. coli. Thus, early detection of ESBL and AmpC producer E. coli by simple phenotypic methods is necessary to avoid treatment failure, where molecular techniques are not available.

Detection of plasmid-mediated AmpC β-lactamase in Escherichia coli and Klebsiella pneumoniae.

Background: Detecting plasmid-mediated AmpC (pAmpC) β-lactamase-producing organism is important for optimal infection control and providing accurate and effective treatment option for physicians. Objectives: The aim of this study was to investigate the prevalence of pAmpC β-lactamase and compare the results of boronic acid (BA) disk test with other phenotypic tests detecting AmpC positive isolates. Materials and Methods: A total of 273 clinical isolates of Klebsiella pneumoniae (n: 82) and Escherichia coli (n: 191) were analysed. The presence of pAmpC β-lactamase was determined by BA disk test, cefoxitin (FOX) screening test, modified three dimensional test (M3DT), and multiplex polymerase chain reaction (PCR). Pulsed-field gel electrophoresis was performed to evaluate the genetic similarities between isolates. To detect extended spectrum β-lactamases (ESBL) in the presence of AmpC β-lactamase, ESBL confirmation test was carried out with and without BA solution. Results: Of the 273 strains tested, 127 strains were found FOX resistant, 114 were positive by M3DT, 108 were positive in BA disk test, and the multiplex PCR detected 24 pAmpC β-lactamase-positive isolate. The prevalence of AmpC-producing strains was 10.9% in E. coli and 3.6% in K. pneumoniae in the tested population by PCR. CIT and MOX group genes were predominant type in these strains. Conclusion: These results emphasize that clinical laboratories should consider testing the presence of pAmpC enzymes particularly in FOX-resistant isolates, and BA disk test will improve detection of this emerging resistance phenotype.

Comparison of the boronic acid disk potentiation test and cefepime-clavulanic acid method for the detection of ESBL among AmpC-producing Enterobacteriaceae.

Purpose: Extended spectrum β-lactamase (ESBL) and AmpC β-lactamase are important mechanisms of betalactam resistance among Enterobacteriaceae . The ESBL confirmation test described by Clinical Laboratory Standards Institute (CLSI) is in routine use. This method fails to detect ESBL in the presence of AmpC. Therefore, we compared two different ESBL detection methods against the CLSI confirmatory test. Materials and Methods: A total 200 consecutive clinical isolates of Enterobacteriaceae from various clinical samples were tested for ESBL production using (i) CLSI described phenotypic confirmatory test (PCT), (ii) boronic acid disk potentiation test and (iii) cefepime-CA disk potentiation method. AmpC confirmation was done by a modified three-dimensional test. Results: Among total 200 Enterobacteriaceae isolates, 82 were only ESBL producers, 12 were only AmpC producers, 55 were combined ESBL and AmpC producers, 14 were inducible AmpC producers and 37 isolates did not harboured any enzymes. The CLSI described PCT detected ESBL-producing organisms correctly but failed to detect 36.3% of ESBLs among combined enzyme producers. The boronic acid disk potentiation test reliably detected all ESBL, AmpC, and combined enzyme producers correctly. The cefepime-CA method detected all ESBLs correctly but another method of AmpC detection has to be adopted. Conclusion: The use of boronic acid in disk diffusion testing along with the CLSI described PCT enhances ESBL detection in the presence of AmpC betalactamases.

Preliminary study on AmpC β-lactamase and related drug-resistant mechanism of pathogenic bacteria in blood stream / 上海交通大学学报（医学版）

Objective To investigate the characteristics of strains of AmpC β-lactamase(AmpC enzyme)production in Dathogenic bacteria in blood stream and clinical presentations of the cases, and study the related ampC and ampD genes.Methods One hundred and eighty-one strains of gram negative bacillus in blood stream were collected,Cefoxitin screening test and three-dimensional test were performed for screening of strains of AmpC enzyme,production and those of AmpC enzyme hyperproduction retrospective analysis was condected in the strains with positive results.ampC and ampD gene PCR ampliftcation, sequencing and sequence analysis of positive strains were performed, and gene homology of ampC positive strains was analysed bv Rep-PCR. Results Among 181 strains in blood stream,strains of AmpC enzyme production were detected in 39 isolates by Cefoxitin screening test,with the detection rate of 21.5%(39/181).The detection rate of strains of AmpC enzyme hyperproduction by three-dimensional test was 43.6%(17/39).PCR revealed that the positive rates for ampC and ampD genes were 41%(16/39)and 56.4%(22/39),respectively.The ampC gene sequencing of 16 positive strains indicated that the homology was 98%to 100%by comparison with the GenBank,while the ampD gene sequencing of 2 strains of Enterobacter cloacae demonstrated that the suspected gene mutations existed in the carboxy-terminal of ampD gene. Conclusion The prevalence of drug-resistant pathogenic bacteria in blood stream in this study is due to nosocomial infection.The mutation of ampC gene is rare in the pathogenic bacteria in blood stream with production of AmpC enzyme,while the rate of gene mutation in Enterobacter cloacae is higher, and the deletion and amino acid substitutions in the carboxy-terminal of ampD is highly relevant to the depressed expression of AmpC enzyme.

Genotype identifications of ESBLs and AmpC β-Iactamase of a Enterococcus gallinarum isolated from chloebia gouidiae / 中国兽医学报

The genotypes of extended spectrum β-laetamases(ESBLs) and AmpC β-lactamases produced by Enterococcus gallinarum isolated from chloebia gouldiae were determined to elucidate the evolution mechanism of the resistant genes.The minimum inhibitory concentrations (MICs) of 18 antibacterial drugs against the Enterococcus gallinarum were detected with two dilution method,and the ESBLs and AmpC β-lactamases from the bacterium were amplified by PCR using the primers of TEM,SHV,CTX-M,ACC,CIT,DHA,EBC,FOX and MOX,respectively.The PCR products were cloned and then the cloned fragments were sequenced to identify their genotypes and subtypes.The bacterium was proved to be a ESBL-producing and AmpC β-lactamase-producing bacterium,showing severe resistant to the other drugs,except the third and forth cephalosporins,carbopenems and fosfomycin.Compared with that of AJ847364 (TEM-116),the sequence of the TEM-type was characterized by two nucleotide mutations (512T→A and 695A→C),which led to two mutations of amino acids(17111e→Lys and 232Lys→Thr),showing that the detected TEM-type was a new genotype,the sequence of the AmpC β-lactamase was similar to that of EF078894 (ACT-like type)with a 97% homology.The genotype of ESBLs of Enterococcus gallinarum was a new TEM-type derived from the TEM-type ESBLs of klebstella pneumoniae isolated from the same avian.The genotype of AmpC lactamase was ACT-type,which probably concerned with β-1actam antibiotics used.

Analysis of AmpC β-lactamase Gene in Pseudomonas aeruginosa / 华中科技大学学报（医学）（英德文版）

The gene and the amino acid sequence of the structural and regulatory region of the Pseudomonas aeruginosa with different resistance patterns were analyzed. Six strains with different resistance patterns were selected and the AmpC β-lactamase was identified. The objective gene fragment was amplified by colonies PCR. The sequences of the PCR-products were analyzed. The DNA sequence of the structural gene ampC and the regulatory genes ampR, ampD and ampE was detected. The 6 strains and the wild-type Pseudomonas aeruginosa are highly homogeneous in structural and regulatory region. Some new mutant points were found.